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Sample records for salmon sperm dna

  1. Spectroscopic study on the interaction of eugenol with salmon sperm DNA in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Bi Shuyun, E-mail: sy_bi@sina.com [College of Chemistry, Changchun Normal University, Changchun 130032 (China); Yan Lili; Wang Yu; Pang Bong; Wang Tianjiao [College of Chemistry, Changchun Normal University, Changchun 130032 (China)

    2012-09-15

    Fluorescence spectra, absorption spectra, melting temperature, ionic strength effect, and viscosity experiments were described that characterize the interaction of eugenol with salmon sperm DNA in vitro. Eugenol was found to bind but weakly to DNA, with binding constants of 4.23 Multiplication-Sign 10{sup 3}, 3.62 Multiplication-Sign 10{sup 3} and 2.47 Multiplication-Sign 10{sup 3} L mol{sup -1} at 18, 28 and 38 Degree-Sign C respectively. The Stern-Volmer plots at different temperatures suggested that the quenching type of fluorescence of eugenol by DNA was a static quenching. Both the relative viscosity and the melting temperature of DNA were increased by the addition of eugenol. The changes of ionic strength had no affect on the binding. In addition, the binding constant of eugenol with single stranded DNA (ssDNA) was larger than that of eugenol with double stranded DNA (dsDNA). These results revealed that the binding mode of eugenol to DNA was intercalative binding. The thermodynamic parameters {Delta}H, {Delta}G and {Delta}S were also obtained according to the Van't Hoff equations, which suggested that hydrogen bond or van der Waals force might play an important role in a binding of eugenol to DNA. Based on the theory of the Foerster energy transference, the binding distance between DNA and eugenol was determined as 4.40 nm, indicating that the static fluorescence quenching of eugenol by DNA was also a non-radiation energy transfer process. - Highlights: Black-Right-Pointing-Pointer DNA quenched the fluorescence of eugenol. Black-Right-Pointing-Pointer Binding constant, binding site and binding force were determined. Black-Right-Pointing-Pointer Binding mode of eugenol to DNA was intercalative. Black-Right-Pointing-Pointer Energy transfer occurred between eugenol and DNA.

  2. Sperm traits in farmed and wild Atlantic salmon Salmo salar.

    Science.gov (United States)

    Camarillo-Sepulveda, N; Hamoutene, D; Lush, L; Burt, K; Volkoff, H; Fleming, I A

    2016-02-01

    Differences in sperm metabolism and morphology between wild and non-local farmed Atlantic salmon Salmo salar were assessed by measuring metabolic enzyme activities and length of sperm flagella. No differences were observed between wild and farmed S. salar sperm with regards to cell counts or any of the biochemical variables assessed. Flagella of sperm cells were significantly longer in wild than farmed S. salar; however, this did not result in higher energy levels or different fertilization rates. © 2015 The Fisheries Society of the British Isles.

  3. Raman spectroscopic study of plasma-treated salmon DNA

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Geon Joon; Kim, Yong Hee; Choi, Eun Ha [Plasma Bioscience Research Center, Kwangwoon University, Seoul 139-701 (Korea, Republic of); Kwon, Young-Wan [Department of Chemistry, Korea University, Seoul 136-701 (Korea, Republic of)

    2013-01-14

    In this research, we studied the effect of plasma treatment on the optical/structural properties of the deoxyribonucleic acid (DNA) extracted from salmon sperm. DNA-cetyltrimethylammonium (CTMA) films were obtained by complexation of DNA with CTMA. Circular dichroism (CD) and Raman spectra indicated that DNA retained its double helical structure in the solid film. The Raman spectra exhibited several vibration modes corresponding to the nuclear bases and the deoxyribose-phosphate backbones of the DNA, as well as the alkylchains of CTMA. Dielectric-barrier-discharge (DBD) plasma treatment induced structural modification and damage to the DNA, as observed by changes in the ultraviolet-visible absorption, CD, and Raman spectra. The optical emission spectra of the DBD plasma confirmed that DNA modification was induced by plasma ions such as reactive oxygen species and reactive nitrogen species.

  4. SPERM DNA INTEGRITY IN BUFFALO, BULL AND STALLION

    OpenAIRE

    Serafini, Rosanna

    2015-01-01

    The interest in sperm DNA integrity evaluation and its relationship to subfertility and infertility loaded to development of several sperm DNA assays. The aim of this study was to compare several sperm DNA assays in buffaloes, bulls and stallions, and to identify the relationships between those DNA assays and traditional sperm features. In Italian Mediterranean Buffalo (IMB) bulls traditional sperm features (motility, viability, acrosome integrity and morphology), sperm DNA integrity (neutral...

  5. Tenacity of Exogenous Human Papillomavirus DNA in Sperm Washing

    OpenAIRE

    Brossfield, Jeralyn E.; Philip J. Chan; Patton, William C.; King, Alan

    1999-01-01

    Purpose:Sperm cells have been shown to take up exogenous DNA readily. The hypothesis was that sperm washing would remove exogenous viral DNA infecting sperm cells. The objective was to compare three types of sperm washing procedures for their capacity to remove exogenous human papillomavirus (HPV) DNA from infected sperm.

  6. Sperm fractions obtained following density gradient centrifugation in human ejaculates show differences in sperm DNA longevity

    Directory of Open Access Journals (Sweden)

    Jaime Gosálvez

    2014-06-01

    Conclusion: 1 Unnecessary incubation of spermatozoa prior to artificial insemination or in vitro fertilization, should be avoided, since sperm DNA longevity is significantly reduced after ex vivo sperm handling and 2 Although sperm selection by DCG significantly reduces the baseline levels of SDF of sperm in Fraction 3, sperm DNA longevity in this fraction was ultimately lower following 24 h incubation when compared to sperm recovered from non-centrifuged NSS.

  7. Sperm DNA damage measured by comet assay.

    Science.gov (United States)

    Simon, Luke; Carrell, Douglas T

    2013-01-01

    Measurement of sperm DNA damage is a useful tool in the evaluation of male infertility, as the sperm nucleus lacks protection against oxidative stress and is vulnerable to oxidation-mediated DNA damage. The Comet assay or single-cell gel electrophoresis is a relatively simple and sensitive method for measuring strand breaks in DNA in individual sperm. During this procedure, sperm cells are embedded in a thin layer of agarose on a microscope slide and lysed with detergent under high salt conditions. This process removes protamines and histones allowing the nucleus to form a nucleoid-like structure containing supercoiled loops of DNA. Alkaline pH conditions result in unwinding of double-stranded DNA, and subsequent electrophoresis results in the migration of broken strands towards the anode, forming a comet tail, when observed under fluorescence microscope. The amount of DNA in the head and tail is reflected by its fluorescent intensity. The relative fluorescence in the tail compared with its head serves as a measure of the level of DNA damage. In this chapter, we describe the alkaline version of the Comet assay, which is highly sensitive for measuring single- and double-strand DNA breaks.

  8. DNA integrity in sexed bull sperm assessed by neutral Comet assay and sperm chromatin structure assay.

    Science.gov (United States)

    Boe-Hansen, Gry B; Morris, Ian D; Ersbøll, Annette K; Greve, Torben; Christensen, Preben

    2005-04-01

    During the production of sex-sorted spermatozoa from bull semen, the cells are exposed to a number of potential hazards including: dilution, centrifugation, incubation, exposure to DNA stains and laser light. These factors may affect the survival capacity and fertilization potential of the sperm. The objective of this study was to determine whether sex-sorted bull spermatozoa have more DNA damage than sperm from conventional processed bull semen. Two methods were used to determine DNA integrity: the neutral Comet assay (NCA) and the sperm chromatin structure assay (SCSA). The NCA showed that the conventional samples had a higher tail moment (TM) (P sperm and that cell sorting by flow cytometry improves the integrity of the sperm cell population. Additionally the results from the SCSA indicated that the sex-sorted sperm had less homogenous sperm chromatin. In the future assessment of sperm DNA integrity may be used to select bulls for sperm sex sorting and optimizing sperm sex sorting procedures.

  9. Tenacity of exogenous human papillomavirus DNA in sperm washing.

    Science.gov (United States)

    Brossfield, J E; Chan, P J; Patton, W C; King, A

    1999-07-01

    Sperm cells have been shown to take up exogenous DNA readily. The hypothesis was that sperm washing would remove exogenous viral DNA infecting sperm cells. The objective was to compare three types of sperm washing procedures for their capacity to remove exogenous human papillomavirus (HPV) DNA from infected sperm. Prewashed sperm were equally divided and sperm in one portion were exposed to L1 HPV DNA fragments for 30 min at 37 degrees C. Untreated washed sperm served as the control. After transfection, the sperm were washed by either centrifuge, two-layer Isolate colloid wash, or test-yolk buffer procedures. Sperm parameters were measured on a Hamilton Thorn HTM-C analyzer. Sperm DNA were extracted and polymerase chain reaction (PCR) was carried out targeting the L1 consensus gene of HPV and the designated sentinel gene, 17q21 spanning the D17S855 gene. Amplified products were analyzed in 2% agarose gel electrophoresis. PCR analyses detected the consensus L1 HPV gene in sperm after they were processed through either of the three procedures. Controls were negative for the L1 gene. Extracted DNA were verified by PCR amplification of 17q21 spanning the D17S855 gene. Transfected sperm had higher percentages of total motility and progression compared with the control. Centrifuged, washed, transfected sperm exhibited a greater curvilinear velocity and hyperactivation. The data showed that washing would not remove exogenous HPV DNA from sperm cells. The viral DNA was tenaciously bound to the sperm, suggesting an internalization into the sperm. The viral DNA also increased the motility of the sperm by affecting the velocity and progression of the sperm, which suggested either an increase in metabolism, an enhancement of the calcium-regulated motility mechanism, or an artifact of PCR reagents. More studies are needed to elucidate the mechanism of DNA stimulated sperm motility.

  10. Salmon redd identification using environmental DNA (eDNA)

    Science.gov (United States)

    Pilliod, David S.; Laramie, Matthew B.

    2016-06-10

    IntroductionThe purpose of this project was to develop a technique to use environmental DNA (eDNA) to distinguish between redds made by Chinook salmon (Oncorhynchus tshawytscha) and redds made by Coho salmon (O. kisutch) and to distinguish utilized redds from test/abandoned redds or scours that have the appearance of redds. The project had two phases:Phase 1. Develop, test, and optimize a molecular assay for detecting and identifying Coho salmon DNA and differentiating it from Chinook salmon DNA.Phase 2. Demonstrate the efficacy of the technique.Collect and preserve water samples from the interstitial spaces of 10 known redds (as identified by expert observers) of each species and 10 gravel patches that do not include a redd of either species.Collect control samples from the water column adjacent to each redd to establish background eDNA levels.Analyze the samples using the developed molecular assays for Coho salmon (phase I) and Chinook salmon (Laramie and others, 2015).Evaluate whether samples collected from Chinook and Coho redds have significantly higher levels of eDNA of the respective species than background levels (that is, from gravel, water column).Evaluate whether samples collected from the interstitial spaces of gravel patches that are not redds are similar to background eDNA levels.The Sandy River is a large tributary of the Columbia River. The Sandy River meets the Columbia River approximately 23 km upstream of Portland, Oregon. The Sandy River Basin provides overlapping spawning habitat for both Chinook and Coho salmon.Samples provided by Portland Water Bureau for analysis were collected from the Bull Run River, Sixes Creek, Still Creek, Arrah Wanna Side Channel, and Side Channel 18.

  11. Should we evaluate and treat sperm DNA fragmentation?

    Science.gov (United States)

    Agarwal, Ashok; Cho, Chak-Lam; Esteves, Sandro C

    2016-06-01

    The clinical utility of sperm DNA fragmentation tests needs to be revisited in light of increasing evidence of detrimental effect of sperm DNA damage on reproductive outcomes. Current evidence supports the association between high sperm DNA fragmentation and poor outcomes with regards to natural conception and intrauterine insemination. The relationship between high sperm DNA fragmentation and impaired outcomes after in-vitro fertilization and intracytoplasmic sperm injection are more equivocal. However, recent studies indicate that poor sperm chromatin content is associated with an increased risk of early pregnancy loss after in-vitro fertilization and intracytoplasmic sperm injection. Several strategies are proposed to alleviate sperm DNA fragmentation and/or select sperm with higher quality chromatin content for assisted reproductive techniques. The intake of oral antioxidants, varicocele repair, use of recurrent ejaculations alone or combined with micromanipulation-based sperm selection techniques, and the use of testicular sperm for intracytoplasmic sperm injection have been attempted with promising results. Sperm DNA fragmentation tests provide clinically relevant information for natural conception and artificial reproduction independent of those derived from conventional semen parameters. The increasing knowledge of paternal factors on pregnancy outcome and the improvement in treatment strategies should prompt routine evaluation of sperm DNA fragmentation in infertile couples.

  12. Role of Disulfide Bonds on DNA Packaging Forces in Bull Sperm Chromatin.

    Science.gov (United States)

    Hutchison, James M; Rau, Donald C; DeRouchey, Jason E

    2017-11-07

    Short arginine-rich proteins called protamines mediate the near crystalline DNA packaging in most vertebrate sperm cells. Protamines are synthesized during spermiogenesis and condense the paternal genome into a transcriptionally inactive state in late-stage spermatids. Protamines from eutherian mammals, including bulls and humans, also contain multiple cysteine residues that form intra- and interprotamine sulfur-sulfur bonds during the final stages of sperm maturation. Although the cross-linked protamine network is known to stabilize the resulting nucleoprotamine structure, little is known about the role of disulfide bonds on DNA condensation in the mammalian sperm. Using small angle x-ray scattering, we show that isolated bull nuclei achieve slightly lower DNA packing densities compared to salmon nuclei despite salmon protamine lacking cysteine residues. Surprisingly, reduction of the intermolecular sulfur-sulfur bonds of bull protamine results in tighter DNA packing. Complete reduction of the intraprotamine disulfide bonds ultimately leads to decondensation, suggesting that disulfide-mediated secondary structure is also critical for proper protamine function. Lastly, comparison of multiple bull collections showed some to have aberrant x-ray scattering profiles consistent with incorrect disulfide bond formation. Together, these observations shed light on the biological functions of disulfide linkages for in vivo DNA packaging in sperm chromatin. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  13. Decreased Fecundity and Sperm DNA Methylation Patterns

    Science.gov (United States)

    Jenkins, Timothy G.; Aston, Kenneth I.; Meyer, Tyson D.; Hotaling, James M.; Shamsi, Monis B.; Johnstone, Erica B.; Cox, Kyley J.; Stanford, Joseph B.; Porucznik, Christina A.; Carrell, Douglas T.

    2016-01-01

    Objective To evaluate the relationship between epigenetic patterns in sperm and fecundity. Design Prospective study of couples trying to conceive, utilizing semen samples collected through the HOPE study, at the University of Utah. Setting Academic Andrology and IVF Laboratory Patients DNA methylation alterations associated with fecundity were analyzed in 124 semen samples. 27 semen samples from couples who conceived within 2 months of attempting a pregnancy and a total of 29 semen samples from couples who were unable to achieve a pregnancy within 12 months were analyzed to identify regions of interest. Interventions None. Main Outcome Measures Genome-wide assessment of differential sperm DNA methylation and standard semen analysis. Results No differences in sperm count, sperm morphology, or semen volume were observed between the patients achieving a pregnancy within 2 months of study time and those not obtaining a pregnancy within 12 months. However, using data from the Human Methylation 450k array analysis we did identify 2 genomic regions with significantly decreased (FDR <0.01) methylation and 3 genomic regions with significantly increased methylation in the “failure-to-conceive” group. Interestingly, the only two sites where decreased methylation was associated with reduced fecundity are at closely related genes known to be expressed in sperm, HSPA1L and HSPA1B. Conclusions Our data suggest that there are genomic loci where DNA methylation alterations are associated with decreased fecundity. We have thus identified candidate loci for future study to verify these results and investigate the causative or contributory relationship between altered sperm methylation and decreased fecundity. PMID:26453269

  14. The relationship between sperm viability and DNA fragmentation rates.

    Science.gov (United States)

    Samplaski, Mary K; Dimitromanolakis, Apostolos; Lo, Kirk C; Grober, Ethan D; Mullen, Brendan; Garbens, Alaina; Jarvi, Keith A

    2015-05-14

    In humans, sperm DNA fragmentation rates have been correlated with sperm viability rates. Reduced sperm viability is associated with high sperm DNA fragmentation, while conversely high sperm viability is associated with low rates of sperm DNA fragmentation. Both elevated DNA fragmentation rates and poor viability are correlated with impaired male fertility, with a DNA fragmentation rate of >30% indicating subfertility. We postulated that in some men, the sperm viability assay could predict the sperm DNA fragmentation rates. This in turn could reduce the need for sperm DNA fragmentation assay testing, simplifying the infertility investigation and saving money for infertile couples. All men having semen analyses with both viability and DNA fragmentation testing were identified via a prospectively collected database. Viability was measured by eosin-nigrosin assay. DNA fragmentation was measured using the sperm chromosome structure assay. The relationship between DNA fragmentation and viability was assessed using Pearson's correlation coefficient. From 2008-2013, 3049 semen analyses had both viability and DNA fragmentation testing. A strong inverse relationship was seen between sperm viability and DNA fragmentation rates, with r=-0.83. If viability was ≤50% (n=301) then DNA fragmentation was ≥ 30% for 95% of the samples. If viability was ≥75% (n=1736), then the DNA fragmentation was ≤30% for 95% of the patients. Sperm viability correlates strongly with DNA fragmentation rates. In men with high levels of sperm viability≥75%, or low levels of sperm viability≤ 30%, DFI testing may be not be routinely necessary. Given that DNA fragmentation testing is substantially more expensive than vitality testing, this may represent a valuable cost-saving measure for couples undergoing a fertility evaluation.

  15. Sperm DNA fragmentation, recurrent implantation failure and recurrent miscarriage

    Directory of Open Access Journals (Sweden)

    Carol Coughlan

    2015-01-01

    Full Text Available Evidence is increasing that the integrity of sperm DNA may also be related to implantation failure and recurrent miscarriage (RM. To investigate this, the sperm DNA fragmentation in partners of 35 women with recurrent implantation failure (RIF following in vitro fertilization, 16 women diagnosed with RM and seven recent fathers (control were examined. Sperm were examined pre- and post-density centrifugation by the sperm chromatin dispersion (SCD test and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assay. There were no significant differences in the age of either partner or sperm concentration, motility or morphology between three groups. Moreover, there were no obvious differences in sperm DNA fragmentation measured by either test. However, whilst on average sperm DNA fragmentation in all groups was statistically lower in prepared sperm when measured by the SCD test, this was not seen with the results from the TUNEL assay. These results do not support the hypothesis that sperm DNA fragmentation is an important cause of RIF or RM, or that sperm DNA integrity testing has value in such patients. It also highlights significant differences between test methodologies and sperm preparation methods in interpreting the data from sperm DNA fragmentation tests.

  16. Sperm DNA assays and their relationship to sperm motility and morphology in bulls (Bos Taurus).

    Science.gov (United States)

    Serafini, Rosanna; Romano, Juan E; Varner, Dickson D; Di Palo, Rossella; Love, Charles C

    2015-08-01

    The relationship among sperm DNA assays in bulls with different sperm motility and morphology measures has not been reported. The objectives of the present study were to (1) describe Comet assay measures and examine their repeatability (inter- and intra-assay); (2) compare sperm DNA quality assays (i.e., Sperm Chromatin Structure Assay-SCSA; alkaline and neutral Comet assays and Sperm Bos Halomax assay-SBH) in two groups of bulls selected on either greater and lesser sperm motility and morphology (greater compared with lesser); (3) determine the relationship among DNA assays and sperm motility and morphology values. Inter-assay repeatability was greater for the neutral Comet assay as compared to the alkaline Comet assay. Intra-assay repeatability was greater than inter-assay repeatability for both Comet assays. Comet assay dimension measures and percentage tail DNA were the most repeatable for both Comet assays. Among sperm DNA quality assays, only SCSA measures and neutral Comet assay Ghosts (% Ghosts), head diameter and area, and comet area were different between greater and lesser sperm quality groups (PComet head measures (diameter, area, and intensity) and positively with percentage Ghosts (Psperm morphology and sperm motility. The neutral Comet assay was more appropriate for sperm evaluation than the alkaline Comet assay for distinguishing among groups with different sperm quality. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Pregnancy prediction by free sperm DNA and sperm DNA fragmentation in semen specimens of IVF/ICSI-ET patients.

    Science.gov (United States)

    Bounartzi, Theofania; Dafopoulos, Konstantinos; Anifandis, George; Messini, Christina I; Koutsonikou, Chrysoula; Kouris, Spyros; Satra, Maria; Sotiriou, Sotirios; Vamvakopoulos, Nicholas; Messinis, Ioannis E

    2016-04-01

    The purpose of this study was to evaluate the predictive value of free sperm plasma DNA (f-spDNA) and sperm DNA fragmentation (SDF), in semen specimens from men undergoing in vitro fertilization/intracytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET) treatments. Fifty-five semen samples were evaluated during 55 consecutive IVF/ICSI-ET cycles. F-spDNA was determined by conventional quantitative real-time PCR-Sybr green detection approach, while evaluation of sperm DNA damage was performed using the sperm chromatin dispersion (SCD) assay. While f-spDNA only correlated with total sperm count, SDF correlated with many semen parameters (including sperm concentration, total sperm count and the per cent of non-progressive sperm). Neither SDF nor the proportion of sperm with small or no halos correlated with f-spDNA. Interestingly, smoking status correlated with f-spDNA but not with SDF. Although these two factors seem to interact for the prediction of pregnancy, receiver-operating characteristics (ROC) analysis revealed that SDF had a stronger predictive value (AUC = 0.7, p  0.05). SDF and f-spDNA may not be associated together but they interact at a significant level in order to exert their actions on pregnancy outcome. Among the two markers, SDF appears to have stronger and significantly predictive value for pregnancy success.

  18. No association between body mass index and sperm DNA integrity

    DEFF Research Database (Denmark)

    Bandel, I; Bungum, Mona; Richtoff, J

    2015-01-01

    STUDY QUESTION: Is overweight associated with impaired sperm DNA integrity? SUMMARY ANSWER: High body mass index (BMI) is not associated with impaired sperm DNA integrity as assessed by the DNA Fragmentation Index (DFI). WHAT IS KNOWN ALREADY: Previous studies, based on fewer subjects and including...

  19. An immunochemical assay to detect DNA damage in bovine sperm

    NARCIS (Netherlands)

    Schans, G.P. van der; Haring, R.; Dijk- Knijnenburg, H.C.M. van; Bruijnzeel, P.L.B.; Daas, N.H.G. den

    2000-01-01

    An immunochemical assay has been developed to detect oxidative damage in bovine sperm DNA. Sperm DNA contains a large amount of oxidative damage as a result of exposure to exogenous agents, but damage also can caused by normal metabolic processes and the absence of DNA repair in the later stages of

  20. DNA fragmentation and sperm head morphometry in cat epididymal spermatozoa.

    Science.gov (United States)

    Vernocchi, Valentina; Morselli, Maria Giorgia; Lange Consiglio, Anna; Faustini, Massimo; Luvoni, Gaia Cecilia

    2014-10-15

    Sperm DNA fragmentation is an important parameter to assess sperm quality and can be a putative fertility predictor. Because the sperm head consists almost entirely of DNA, subtle differences in sperm head morphometry might be related to DNA status. Several techniques are available to analyze sperm DNA fragmentation, but they are labor-intensive and require expensive instrumentations. Recently, a kit (Sperm-Halomax) based on the sperm chromatin dispersion test and developed for spermatozoa of different species, but not for cat spermatozoa, became commercially available. The first aim of the present study was to verify the suitability of Sperm-Halomax assay, specifically developed for canine semen, for the evaluation of DNA fragmentation of epididymal cat spermatozoa. For this purpose, DNA fragmentation indexes (DFIs) obtained with Sperm-Halomax and terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) were compared. The second aim was to investigate whether a correlation between DNA status, sperm head morphology, and morphometry assessed by computer-assisted semen analysis exists in cat epididymal spermatozoa. No differences were observed in DFIs obtained with Sperm-Halomax and TUNEL. This result indicates that Sperm-Halomax assay provides a reliable evaluation of DNA fragmentation of epididymal feline spermatozoa. The DFI seems to be independent from all the measured variables of sperm head morphology and morphometry. Thus, the evaluation of the DNA status of spermatozoa could effectively contribute to the completion of the standard analysis of fresh or frozen semen used in assisted reproductive technologies. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Vitrification of neat semen alters sperm parameters and DNA integrity.

    Science.gov (United States)

    Khalili, Mohammad Ali; Adib, Maryam; Halvaei, Iman; Nabi, Ali

    2014-05-06

    Our aim was to evaluate the effect of neat semen vitrification on human sperm vital parameters and DNA integrity in men with normal and abnormal sperm parameters. Semen samples were 17 normozoospermic samples and 17 specimens with abnormal sperm parameters. Semen analysis was performed according to World Health Organization (WHO) criteria. Then, the smear was provided from each sample and fixed for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Vitrification of neat semen was done by plunging cryoloops directly into liquid nitrogen and preserved for 7 days. The samples were warmed and re-evaluated for sperm parameters as well as DNA integrity. Besides, the correlation between sperm parameters and DNA fragmentation was assessed pre- and post vitrification. Cryopreserved spermatozoa showed significant decrease in sperm motility, viability and normal morphology after thawing in both normal and abnormal semen. Also, the rate of sperm DNA fragmentation was significantly higher after vitrification compared to fresh samples in normal (24.76 ± 5.03 and 16.41 ± 4.53, P = .002) and abnormal (34.29 ± 10.02 and 23.5 ± 8.31, P neat ejaculates has negative impact on sperm parameters as well as DNA integrity, particularly among abnormal semen subjects. It is, therefore, recommend to process semen samples and vitrify the sperm pellets.

  2. Effect of varicocelectomy on sperm functional characteristics and DNA methylation.

    Science.gov (United States)

    Tavalaee, M; Bahreinian, M; Barekat, F; Abbasi, H; Nasr-Esfahani, M H

    2015-10-01

    In individuals with varicocele, DNA is damaged due to high level of oxidative stress, and varicocelectomy can overcome this effect. Damaged DNA is less liable to DNA methylation, and antioxidant therapy appears to have the potential to reduce sperm oxidative stress and DNA damage and thereby maintain DNA methylation, while effect of varicocelectomy on DNA methylation patterns has remained unclear. In the light of these considerations, we aimed to examine the effect of varicocelectomy on sperm DNA methylation and functional characteristics. Fifty-two men with left-sided varicocele (grade II &III) were included. Sperm parameters, DNA fragmentation, protamine deficiency, oxidative stress and global DNA methylation were evaluated before and 3 months after surgery. Our data show that sperm concentration, percentages of spermatozoon with abnormal morphology, DNA fragmentation, protamine deficiency and oxidative stress significantly improved after surgery. Percentage of sperm motility, global DNA methylation and intensity of DNA methylation also improved after surgery, although the differences were not significant when compared with before surgery. Categorisation of individuals to subgroups revealed that improvement of DNA methylation appears to take place in oligozoospermic individuals, which are more severely affected by state of varicocele. However, this is a preliminary study, and further studies are required to solidify this conclusion. © 2014 Blackwell Verlag GmbH.

  3. Sperm DNA damage in relation to lipid peroxidation following ...

    African Journals Online (AJOL)

    ... were no consistent associations between post-thaw sperm LPO and sperm quality characteristics. It could be suggested that the increased LPO of membrane phospholipids is associated with higher susceptibility of boar spermatozoa to cryo-induced DNA damage. Keywords: Comet assay measurements, cryopreservation ...

  4. Use of testicular sperm for intracytoplasmic sperm injection in men with high sperm DNA fragmentation: a SWOT analysis.

    Science.gov (United States)

    Esteves, Sandro C; Roque, Matheus; Garrido, Nicolás

    2018-01-01

    Spermatozoa retrieved from the testis of men with high levels of sperm DNA fragmentation (SDF) in the neat semen tend to have better DNA quality. Given the negative impact of SDF on the outcomes of Assisted Reproductive Technology (ART), an increased interest has emerged about the use of testicular sperm for intracytoplasmic sperm injection (Testi-ICSI). In this article, we used a SWOT (strengths, weaknesses, opportunities, and threats) analysis to summarize the advantages and drawbacks of this intervention. The rationale of Testi-ICSI is bypass posttesticular DNA fragmentation caused by oxidative stress during sperm transit through the epididymis. Hence, oocyte fertilization by genomically intact testicular spermatozoa may be optimized, thus increasing the chances of creating a normal embryonic genome and the likelihood of achieving a live birth, as recently demonstrated in men with high SDF. However, there is still limited evidence as regards the clinical efficacy of Testi-ICSI, thus creating opportunities for further confirmatory clinical research as well as investigation of Testi-ICSI in clinical scenarios other than high SDF. Furthermore, Testi-ICSI can be compared to other laboratory preparation methods for deselecting sperm with damaged DNA. At present, the available literature supports the use of testicular sperm when performing ICSI in infertile couples whose male partners have posttesticular SDF. Due to inherent risks of sperm retrieval, Testi-ICSI should be offered when less invasive treatments for alleviating DNA damage have failed. A call for continuous monitoring is nonetheless required concerning the health of generated offspring and the potential complications of sperm retrieval.

  5. High resolution DNA content measurements of mammalian sperm

    Energy Technology Data Exchange (ETDEWEB)

    Pinkel, D.; Lake, S.; Gledhill, B.L.; Van Dilla, M.A.; Stephenson, D.; Watchmaker, G.

    1982-01-01

    The high condensation and flat shape of the mammalian sperm nucleus present unique difficulties to flow cytometric measurement of DNA content. Chromatin compactness makes quantitative fluorescent staining for DNA difficult and causes a high index of refraction. The refractive index makes optical measurements sensitive to sperm head orientation. We demonstrate that the optical problems can be overcome using the commercial ICP22 epiillumination flow cytometer (Ortho Instruments, Westwood, MA) or a specially built cell orientating flow cytometer (OFCM). The design and operation of the OFCM are described. Measurements of the angular dependence of fluorescence from acriflavine stained rabbit sperm show that it is capable of orienting flat sperm with a tolerance of +-7/sup 0/. Differences in the angular dependence for the similarly shaped bull and rabbit sperm allow discrimination of these cells. We show that DNA staining with 4-6 diamidino-2-phenylindole (DAPI) or an ethidium bromide mithramycin combination allows resolution of the X and Y populations in mouse sperm. They have also been successful with sperm from the bull, ram, rabbit, and boar. Reliable results with human sperm are not obtained. The accuracy of the staining and measurement techniques are verified by the correct determination of the relative content of these two populations in sperm from normal mice and those with the Cattanach (7 to X) translocation. Among the potential uses of these techniques are measurement of DNA content errors induced in sperm due to mutagen exposure, and assessment of the fractions of X and Y sperm in semen that may have one population artifically enriched.

  6. Sperm DNA fragmentation affects epigenetic feature in human male pronucleus.

    Science.gov (United States)

    Rajabi, H; Mohseni-Kouchesfehani, H; Eslami-Arshaghi, T; Salehi, M

    2018-02-01

    To evaluate whether the sperm DNA fragmentation affects male pronucleus epigenetic factors, semen analysis was performed and DNA fragmentation was assessed by the method of sperm chromatin structure assay (SCSA). Human-mouse interspecies fertilisation was used to create human male pronucleus. Male pronucleus DNA methylation and H4K12 acetylation were evaluated by immunostaining. Results showed a significant positive correlation between the level of sperm DNA fragmentation and DNA methylation in male pronuclei. In other words, an increase in DNA damage caused an upsurge in DNA methylation. In the case of H4K12 acetylation, no correlation was detected between DNA damage and the level of histone acetylation in the normal group, but results for the group in which male pronuclei were derived from sperm cells with DNA fragmentation, increased DNA damage led to a decreased acetylation level. Sperm DNA fragmentation interferes with the active demethylation process and disrupts the insertion of histones into the male chromatin in the male pronucleus, following fertilisation. © 2017 Blackwell Verlag GmbH.

  7. DNA topoisomerase II enzyme activity appears in mouse sperm ...

    African Journals Online (AJOL)

    Jane

    2011-08-22

    Aug 22, 2011 ... Full Length Research Paper. DNA topoisomerase II enzyme activity ... examine the presence of DNA topoisomerase II (top 2) activity in sperm heads. The initial percentage motile of male A was ..... topoisomerase 2 is required for segregation of daughter molecules at termination of DNA replication. Proc.

  8. Papillomavirus DNA in sperm from infertile patients

    Directory of Open Access Journals (Sweden)

    William Gennari

    2011-12-01

    Full Text Available The Human Papillomaviruses (HPV are causative agents of sexually transmitted disease that affect both men and women (6, 7.The genus includes more than 150 types divided in according to different tropism for skin surfaces and paved mucosal epithelia. Although HPV infection has a very high incidence in both sexes, HPV infection in men is often neglected because of its transitory nature and its lack of clinical relevance.The HPV infection in males was found to be borne by the anal region, perineum, scrotum, urethra and glans. The persistence of the virus in these sites of infection has been linked both to male infertility and to the development of neoplasia in genital areas and not. In addition, several studies have documented the presence of HPV in the semen but with conflicting results regarding the location of the virus in the various components of semen (5, 9,10. The objective of this study was to highlight the presence of HPV DNA in the sperm of patients waiting for a Medically Assisted Procreation and to evaluate if there is a correlation between the semen parameters (motility, concentration and morphology of spermatozoa and HPV infection.

  9. Comparative analysis of three sperm DNA damage assays and sperm nuclear protein content in couples undergoing assisted reproduction treatment.

    Science.gov (United States)

    Simon, L; Liu, L; Murphy, K; Ge, S; Hotaling, J; Aston, K I; Emery, B; Carrell, D T

    2014-05-01

    Is there an association between sperm DNA damage, measured by three different assays, sperm nuclear protein content and clinical outcomes in assisted reproduction treatment (ART)? Sperm DNA damage measured by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) and the Comet assay were significantly associated with ART outcomes in our single institution study. Abnormal protamine expression is known to be associated with sperm DNA damage and male infertility. A number of studies have shown a significant relationship between sperm DNA damage and ART outcomes. To date, there are no large studies providing direct comparisons of DNA damage tests within the same study population. Thus, the prognostic value for each method remains unknown. Cross-sectional study of 238 men from infertile couples undergoing ART at the University Center for Reproductive Medicine, Utah, USA, between April 2011 and March 2013. Sperm from men undergoing ART were tested for DNA damage using the alkaline Comet assay, TUNEL and flow cytometric chromatin evaluation (FCCE) assays. Histone retention was analysed using the aniline blue staining method, whereas protamine content (proteins P1 and P2) and ratio were analysed using acid urea gel electrophoresis. The prognostic value of each sperm DNA test to predict clinical pregnancy was calculated. Histone retention was associated with sperm DNA damage (P sperm with DNA damage was significantly higher in sperm from non-pregnant couples compared with that from pregnant couples, as measured by TUNEL assay (15.04 ± 1.16% versus 8.79 ± 0.56%; P Comet assay (72.79 ± 2.49% versus 55.86 ± 2.29%; P sperm count (P = 0.013), men's age (P = 0.037), maturity (P = 0.049) and blastocyst quality (P = 0.012). Histone retention and sperm DNA damage measured by Comet and TUNEL assays were associated with fertilization rate (P sperm is one part of the equation; there are also a number of female factors that have the potential to influence ART

  10. DNA topoisomerase II enzyme activity appears in mouse sperm ...

    African Journals Online (AJOL)

    Sperm suspensions of 4 male mice (A, B, C and D), having an initial motility grade of 3.5 were used to examine the presence of DNA topoisomerase II (top 2) activity in sperm heads. The initial percentage motile of male A was 75%, male B was 80%, male C was 70% and male D was 60%. Top 2 activity was examined by ...

  11. Sperm DNA fragmentation in couples with unexplained recurrent spontaneous abortions.

    Science.gov (United States)

    Khadem, N; Poorhoseyni, A; Jalali, M; Akbary, A; Heydari, S T

    2014-03-01

    The aim of the present study was to evaluate the degree of sperm DNA fragmentation in couples with idiopathic recurrent spontaneous abortion (RSA) and in those with no history of infertility or abortion. In this cohort study, 30 couples with RSA and 30 fertile couples as control group completed the demographic data questionnaires, and their semen samples were analysed according to World Health Organization (WHO) standards (September 2009-March 2010) for evaluation of sperm DNA fragmentation, using sperm chromatin dispersion (SCD) technique. The percentage of morphologically normal sperm was significantly lower in RSA patients compared with control group (51.50 ± 11.60 versus 58.00 ± 9.05, P = 0.019), but not in other parameters. Additionally, the level of abnormal DNA fragmentation in the RSA group was significantly higher than in the control group (43.3% versus 16.7%, P = 0.024). Our results indicated a negative correlation between the number of sperm with progressive motility and DNA fragmentation (r = -0.613; P fragmentation and poor motility than those of the control group, indicating a possible relationship between idiopathic RSA and DNA fragmentation. © 2012 Blackwell Verlag GmbH.

  12. Human papillomavirus DNA detection in sperm using polymerase chain reaction.

    Science.gov (United States)

    Olatunbosun, O; Deneer, H; Pierson, R

    2001-03-01

    To detect human papillomavirus (HPV) in semen and find if sperm washing removes HPV DNA. Amplification by nested polymerase chain reaction (PCR) was used to detect viral DNA sequences in semen samples from 85 volunteers. Forty-five men had historical or clinical evidence of genital HPV infection (study group) and 40 were healthy, clinically HPV-negative semen donors. We detected HPV DNA in the sperm cells of 24 of 45 subjects (53%) with past or current HPV infections in contrast to three of 40 healthy subjects (8%) (P HPV in 21 of 32 subjects (66%) with identifiable lesions and six of 53 (11%) without them (P sperm cells with HPV reduced cellular HPV DNA below detectable levels in only two cases. HPV is present in sperm cells from infected and apparently healthy subjects, and sperm washing does not eliminate the risk of HPV transmission to recipients. We suggest that HPV DNA testing should be done on the semen of prospective donors, and those with positive tests should be excluded from donation.

  13. Effect of transfection and co-incubation of bovine sperm with exogenous DNA on sperm quality and functional parameters for its use in sperm-mediated gene transfer.

    Science.gov (United States)

    Arias, María Elena; Sánchez-Villalba, Esther; Delgado, Andrea; Felmer, Ricardo

    2017-02-01

    Sperm-mediated gene transfer (SMGT) is based on the capacity of sperm to bind exogenous DNA and transfer it into the oocyte during fertilization. In bovines, the progress of this technology has been slow due to the poor reproducibility and efficiency of the production of transgenic embryos. The aim of the present study was to evaluate the effects of different sperm transfection systems on the quality and functional parameters of sperm. Additionally, the ability of sperm to bind and incorporate exogenous DNA was assessed. These analyses were carried out by flow cytometry and confocal fluorescence microscopy, and motility parameters were also evaluated by computer-assisted sperm analysis (CASA). Transfection was carried out using complexes of plasmid DNA with Lipofectamine, SuperFect and TurboFect for 0.5, 1, 2 or 4 h. The results showed that all of the transfection treatments promoted sperm binding and incorporation of exogenous DNA, similar to sperm incorporation of DNA alone, without affecting the viability. Nevertheless, the treatments and incubation times significantly affected the motility parameters, although no effect on the integrity of DNA or the levels of reactive oxygen species (ROS) was observed. Additionally, we observed that transfection using SuperFect and TurboFect negatively affected the acrosome integrity, and TurboFect affected the mitochondrial membrane potential of sperm. In conclusion, we demonstrated binding and incorporation of exogenous DNA by sperm after transfection and confirmed the capacity of sperm to spontaneously incorporate exogenous DNA. These findings will allow the establishment of the most appropriate method [intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF)] of generating transgenic embryos via SMGT based on the fertilization capacity of transfected sperm.

  14. Cytometric analysis of shape and DNA content in mammalian sperm

    Energy Technology Data Exchange (ETDEWEB)

    Gledhill, B.L.

    1983-10-10

    Male germ cells respond dramatically to a variety of insults and are important reproductive dosimeters. Semen analyses are very useful in studies on the effects of drugs, chemicals, and environmental hazards on testicular function, male fertility and heritable germinal mutations. Sperm were analyzed by flow cytometry and slit-scan flow analysis for injury following the exposure of testes to mutagens. The utility of flow cytometry in genotoxin screening and monitoring of occupational exposure was evaluated. The technique proved valuable in separation of X- and Y-chromosome bearing sperm and the potential applicability of this technique in artificial insemination and a solution, of accurately assessing the DNA content of sperm were evaluated-with reference to determination of X- and Y-chromosome bearing sperm.

  15. Sperm DNA quality evaluated by comet assay and sperm chromatin structure assay in stallions after unilateral orchiectomy.

    Science.gov (United States)

    Serafini, R; Varner, D D; Bissett, W; Blanchard, T L; Teague, S R; Love, C C

    2015-09-15

    Unilateral orchiectomy (UO) may interfere with thermoregulation of the remaining testis caused by inflammation surrounding the incision site, thus altering normal spermatogenesis and consequently sperm quality. Two measures of sperm DNA quality (neutral comet assay and the sperm chromatin structure assay [SCSA]) were compared before UO (0 days) and at 14, 30, and 60 days after UO to determine whether sperm DNA changed after a mild testis stress (i.e., UO). The percent DNA in the comet tail was higher at 14 and 60 days compared to 0 days (P comet tail measures (i.e., length, moment, migration) were higher at all time periods after UO compared to 0 days (P sperm DNA quality using both the neutral comet assay and the SCSA, which was not identified using traditional measures of sperm quality. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Oxidative DNA damage in mouse sperm chromosomes: Size matters.

    Science.gov (United States)

    Kocer, Ayhan; Henry-Berger, Joelle; Noblanc, Anais; Champroux, Alexandre; Pogorelcnik, Romain; Guiton, Rachel; Janny, Laurent; Pons-Rejraji, Hanae; Saez, Fabrice; Johnson, Graham D; Krawetz, Stephen A; Alvarez, Juan G; Aitken, R John; Drevet, Joël R

    2015-12-01

    Normal embryo and foetal development as well as the health of the progeny are mostly dependent on gamete nuclear integrity. In the present study, in order to characterize more precisely oxidative DNA damage in mouse sperm we used two mouse models that display high levels of sperm oxidative DNA damage, a common alteration encountered both in in vivo and in vitro reproduction. Immunoprecipitation of oxidized sperm DNA coupled to deep sequencing showed that mouse chromosomes may be largely affected by oxidative alterations. We show that the vulnerability of chromosomes to oxidative attack inversely correlated with their size and was not linked to their GC richness. It was neither correlated with the chromosome content in persisting nucleosomes nor associated with methylated sequences. A strong correlation was found between oxidized sequences and sequences rich in short interspersed repeat elements (SINEs). Chromosome position in the sperm nucleus as revealed by fluorescent in situ hybridization appears to be a confounder. These data map for the first time fragile mouse sperm chromosomal regions when facing oxidative damage that may challenge the repair mechanisms of the oocyte post-fertilization. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. No increased sperm DNA fragmentation index in semen containing human papillomavirus or herpesvirus

    DEFF Research Database (Denmark)

    Kaspersen, Maja Døvling; Bungum, Mona; Fedder, Jens

    2013-01-01

    It remains unknown whether human papillomaviruses (HPVs) or human herpesviruses (HHVs) in semen affect sperm DNA integrity. We investigated whether the presence of these viruses in semen was associated with an elevated sperm DNA fragmentation index. Semen from 76 sperm donors was examined by a PCR......-based hybridization array that identifies all HHVs and 35 of the most common HPVs. Sperm DNA integrity was determined by the sperm chromatin structure assay. HPVs or HHVs, or both, were found in 57% of semen samples; however, sperm DNA fragmentation index was not increased in semen containing these viruses....

  18. Sperm DNA damage has a negative association with live-birth rates after IVF.

    Science.gov (United States)

    Simon, L; Proutski, I; Stevenson, M; Jennings, D; McManus, J; Lutton, D; Lewis, S E M

    2013-01-01

    Sperm DNA damage has a negative impact on pregnancy rates following assisted reproduction treatment (ART). The aim of the present study was to examine the relationship between sperm DNA fragmentation and live-birth rates after IVF and intracytoplasmic sperm injection (ICSI). The alkaline Comet assay was employed to measure sperm DNA fragmentation in native semen and in spermatozoa following density-gradient centrifugation in semen samples from 203 couples undergoing IVF and 136 couples undergoing ICSI. Men were divided into groups according to sperm DNA damage. Following IVF, couples with sperm DNA fragmentation had a live-birth rate of 33%; in contrast, couples with >50% sperm DNA fragmentation had a much lower live-birth rate of 13%. Following ICSI, no significant differences in sperm DNA damage were found between any groups of patients. Sperm DNA damage was also associated with low live-birth rates following IVF in both men and couples with idiopathic infertility: 39% of couples and 41% of men with idiopathic infertility have high sperm DNA damage. Sperm DNA damage assessed by the Comet assay has a close inverse relationship with live-birth rates after IVF. Sperm DNA damage has a negative impact on assisted reproduction treatment outcome, in particular, on pregnancy rates. The aim of the present study was to examine the relationship between sperm DNA fragmentation and live-birth rates after IVF and intracytoplasmic sperm injection (ICSI). The alkaline Comet assay was employed to measure sperm DNA fragmentation in native semen and in spermatozoa following density-gradient centrifugation in semen samples from 203 couples undergoing IVF and 136 couples undergoing ICSI. Men were divided into groups according to sperm DNA damage and treatment outcome. Following IVF, couples with sperm DNA fragmentation had a live birth rate of 33%. In contrast, couples with >50% sperm DNA fragmentation had a much lower live-birth rate of 13% following IVF. Following ICSI, there were

  19. Sperm preparation: DNA damage by comet assay in normo- and teratozoospermics.

    Science.gov (United States)

    Ahmad, Laiq; Jalali, Samina; Shami, Sajjad Aslam; Akram, Zertashia

    2007-01-01

    The present study was carried out on semen samples of human fertile and infertile subjects, teratozoospermics (TZs) and idiopathics (IDs), with neat semen and sperm prepared by swim up or Percoll density gradient centrifugation procedures. Sperm morphology analysis revealed that only head and midpiece defects in TZs and IDs were significantly (P sperm DNA damage compared to fertile subjects. Fertile subjects with sperm prepared from neat and Percoll density gradient centrifugation exhibited a comet tail DNA percentage of 20% and 15%, respectively. The TZs and IDs infertile subjects had higher levels of comet tail DNA of 33% and 25% and 25% and 19%, respectively. A significant (F = 24.01; P = 0.0059) decrease in mean comet head DNA percentage or sperm DNA integrity was observed in neat samples from fertile and infertile subjects by Repeated Measures ANOVA. In Percoll prepared samples from fertile, TZs, and IDs, there was a significant increase in sperm DNA integrity. Similarily, there was a decrease in abnormal sperm morphology in swim up and Percoll prepared sperm compared to neat samples. The Percoll density gradient centrifugation procedure yields sperm with an increase in sperm DNA integrity relative to swim up. Sperm DNA damage of TZs with both sperm preparation methods was significantly (P sperm with improved DNA integrity. In conjunction with semen analysis, the assessment of nuclear integrity improves the characterization of the semen sample and may be used as a tool for allocating the patients to specific assisted reproductive treatments.

  20. Sperm DNA fragmentation and its role in wildlife conservation.

    Science.gov (United States)

    Gosálvez, Jaime; Holt, William V; Johnston, Stephen D

    2014-01-01

    Until about 20 years ago, sperm assessment in the laboratory was focused on motility, morphology and acrosomal integrity. Then came the gradual realisation that, because the main objective of a spermatozoon is to deliver an intact genetic payload of DNA to the egg, being able to check DNA quality of spermatozoa would be equally important, if not more so. Research over the last two decades has therefore led to the development of several techniques for reliably detecting DNA strand breaks, and the more recent focus has been directed towards understanding the fertility implications of DNA damage. It is now clear that evolutionary history has played an important role in determining the stability of sperm DNA under stressful conditions, and that the nature of the DNA-protein interactions also influence the extent to which fertility is affected by both technical procedures involved in sperm preservation and the basic biology of the species concerned. Here we present an overview of the principles involved in DNA assessment and also provide some cases studies that illustrate the influences of species diversity.

  1. Short communication Sperm DNA damage in relation to lipid ...

    African Journals Online (AJOL)

    Leyland Fraser

    2017-03-08

    Mar 8, 2017 ... spermatozoa, particularly for assisted reproductive techniques (Thomson et al., 2010), even though it has been reported that the repeated cryo-cycle might compromise sperm DNA integrity and viability (Liu et al.,. 2016). Consistent significant correlations were found between the comet assay parameters ...

  2. Environmental car exhaust pollution damages human sperm chromatin and DNA.

    Science.gov (United States)

    Calogero, A E; La Vignera, S; Condorelli, R A; Perdichizzi, A; Valenti, D; Asero, P; Carbone, U; Boggia, B; De Rosa, N; Lombardi, G; D'Agata, R; Vicari, L O; Vicari, E; De Rosa, M

    2011-06-01

    The adverse role of traffic pollutants on male fertility is well known. Aim of this study was to evaluate their effects on sperm chromatin/DNA integrity. To accomplish this, 36 men working at motorway tollgates and 32 unexposed healthy men (controls) were enrolled. All of them were interviewed about their lifestyle. Hormone, semen samples, and environmental and biological markers of pollution were evaluated. Sperm chromatin and DNA integrity were evaluated by flow cytometry following propidium iodide staining and TUNEL assay, respectively. LH, FSH, and testosterone serum levels were within the normal range in tollgate workers. Sperm concentration, total sperm count, total and progressive motility, and normal forms were significantly lower in these men compared with controls. Motorway tollgate workers had a significantly higher percentage of spermatozoa with damaged chromatin and DNA fragmentation, a late sign of apoptosis, compared with controls. A significant direct correlation was found between spermatozoa with damaged chromatin or fragmented DNA and the length of occupational exposure, suggesting a time-dependent relationship. This study showed that car exhaust exposure has a genotoxic effect on human spermatozoa. This may be of relevant importance not only for the reproductive performance of the men exposed, but also for the offspring health.

  3. Mitochondrial DNA variation in chinook salmon and chum salmon detected by restriction enzyme analysis of polymerase chain reaction products

    Science.gov (United States)

    Cronin, M.; Spearman, R.; Wilmot, R.; Patton, J.; Bickman, J.

    1993-01-01

    We analyze intraspecific mitochondrial DNA variation in chinook salmon from drainages in the Yukon River, the Kenai River, and Oregon and California rivers; and chum salmon from the Yukon River and vancouver Island, and Washington rivers. For each species, three different portions of the mtDNA molecule were amplified seperately using the polymerase chain reaction and then digested with at least 19 restrictions enzymes. Intraspecific sequence divergences between haplotypes were less than 0.01 base subsitution per nucleotide. Nine chum salmon haplotypes were identified. Yukon River chum salmon stocks displayed more haplotypes (8) occurred in all areas. Seven chinook salmon haplotypes were identified. Four haplotypes occurred in the Yukon and Kenai rviers and four occured in the Oregon/California, with only one haplotype shared between the regions. Sample sizes were too small to quantify the degree of stock seperation among drainages, but the patterns of variation that we observed suggest utility of the technique in genetic stock identification.

  4. Assessment of sperm DNA fragmentation in stallion (Equus caballus) and donkey (Equus asinus) using the sperm chromatin dispersion test.

    Science.gov (United States)

    Cortés-Gutiérrez, E I; Crespo, F; Serres-Dalmau, C; Gutiérrez de las Rozas, A L; Dávila-Rodríguez, M I; López-Fernández, C; Gósalvez, J

    2009-10-01

    Sperm DNA fragmentation (sDF) is an important parameter to assessing sperm quality. Information about sperm quality is not available for donkeys, especially in some breeds at risk of extinction. The objectives of this research were to test the four commercial variants of sperm chromatin dispersion test (SCD; sperm Halomax test), originally developed to assess sDF in boars, bulls, rams and stallions, in order to scrutinize their applicability in the study of sDF in a donkey breed at risk of extinction (Zamorano-Leonesa), for which there is no specific test available to analyze sperm at present. Only the SCD test, originally developed for stallions, produced stable and consistent results, and was deemed suitable to assess DNA fragmentation in sperm samples from donkeys. Image analysis was used to compare differences between the SCD methodology applied to stallion and donkey semen samples processed under the same experimental conditions. The extent of SCD in the SCD test was approximately 20% lower in donkey sperm than in stallion sperm. Yet, the ratio of chromatin sperm dispersion achieved in fragmented and unfragmented nuclei did not differ significantly between species. These data suggest that a similar protein depletion treatment can cause differences in protein removal in equivalent cells from different species and that sperm chromatin may be organized differently in stallions and donkeys.

  5. Synthesis, spectroscopic characterization and in vitro antimicrobial, anticancer and antileishmanial activities as well interaction with Salmon sperm DNA of newly synthesized carboxylic acid derivative, 4-(4-methoxy-2-nitrophenylamino)-4-oxobutanoic acid

    Science.gov (United States)

    Sirajuddin, Muhammad; Ali, Saqib; McKee, Vickie; Ullah, Hameed

    2015-03-01

    This paper stresses on the synthesis, characterization of novel carboxylic acid derivative and its application in pharmaceutics. Carboxylic acid derivatives have a growing importance in medicine, particularly in oncology. A novel carboxylic acid, 4-(4-methoxy-2-nitrophenylamino)-4-oxobutanoic acid, was synthesized and characterized by elemental analysis, FT-IR, NMR (1H, and 13C), mass spectrometry and single crystal X-ray structural analysis. The structure of the title compound, C11H12N2O6, shows the molecules dimerised by short intramolecular Osbnd H⋯O hydrogen bonds. The compound was screened for in vitro antimicrobial, anticancer, and antileishmanial activities as well as interaction with SS-DNA. The compound was also checked for in vitro anticancer activity against BHK-21, H-157 and HCEC cell lines, and showed significant anticancer activity. The compound was almost non-toxic towards human corneal epithelial cells (HCEC) and did not show more than 7.4% antiproliferative activity when used at the 2.0 μg/mL end concentration. It was also tested for antileishmanial activity against the promastigote form of leishmania major and obtained attractive result. DNA interaction study exposes that the binding mode of the compound with SS-DNA is an intercalative as it results in hypochromism along with minor red shift. A new and efficient strategy to identify pharmacophores sites in carboxylic acid derivative for antibacterial/antifungal activity using Petra, Osiris and Molinspiration (POM) analyses was also carried out.

  6. Frozen-thawed rhinoceros sperm exhibit DNA damage shortly after thawing when assessed by the sperm chromatin dispersion assay.

    Science.gov (United States)

    Portas, T; Johnston, S D; Hermes, R; Arroyo, F; López-Fernadez, C; Bryant, B; Hildebrandt, T B; Göritz, F; Gosalvez, J

    2009-09-15

    This study reports on the successful validation (via in situ nick translation and neutral comet assay) of the equine Sperm-Halomax kit as an appropriate methodology for the assessment of sperm DNA fragmentation in three species of rhinoceros. Rhinoceros sperm nuclei with fragmented DNA (validated using in situ nick translation) were evident as large halos with dispersed DNA fragments, whereas those with nonfragmented DNA displayed small halos of nondispersed DNA within the microgel. There was a high correlation (r) of 0.974 (R(2) value=0.949; PSperm Chromatin Dispersion test (SCDt) and the neutral comet assay. Application of the SCDt to determine the DNA fragmentation dynamics of rhinoceros (n=6) sperm frozen in liquid nitrogen vapor and incubated postthaw at 37 degrees C for up to 48 h to mimic in vitro conditions in the female reproductive tract, revealed an increase (P=0.001) in DNA damage, as soon as 4h after the start of incubation. Linear regression equations were calculated for all six rhinoceroses over the first 6h of incubation and revealed individual animal variation. Freshly collected and incubated (37 degrees C) rhinoceros (n=3) sperm had no increase in the basal level of DNA fragmentation for up to 48 h, indicating that the cryopreservation of rhinoceros sperm in liquid nitrogen vapor, as used in this study, appeared to result in freeze-thaw DNA damage.

  7. Human sperm sex chromosome disomy and sperm DNA damage assessed by the neutral comet assay.

    Science.gov (United States)

    McAuliffe, M E; Williams, P L; Korrick, S A; Dadd, R; Marchetti, F; Martenies, S E; Perry, M J

    2014-10-10

    Is there an association between human sperm sex chromosome disomy and sperm DNA damage? An increase in human sperm XY disomy was associated with higher comet extent; however, there was no other consistent association of sex chromosome disomies with DNA damage. There is limited published research on the association between sex chromosome disomy and sperm DNA damage and the findings are not consistent across studies. We conducted a cross-sectional study of 190 men (25% ever smoker, 75% never smoker) from subfertile couples presenting at the Massachusetts General Hospital Fertility Clinic from January 2000 to May 2003. Multiprobe fluorescence in situ hybridization for chromosomes X, Y and 18 was used to determine XX, YY, XY and total sex chromosome disomy in sperm nuclei using an automated scoring method. The neutral comet assay was used to measure sperm DNA damage, as reflected by comet extent, percentage DNA in the comet tail, and tail distributed moment. Univariate and multiple linear regression models were constructed with sex chromosome disomy (separate models for each of the four disomic conditions) as the independent variable, and DNA damage parameters (separate models for each measure of DNA damage) as the dependent variable. Men with current or past smoking history had significantly greater comet extent (µm: regression coefficients with 95% CI) [XX18: 15.17 (1.98, 28.36); YY18: 14.68 (1.50, 27.86); XY18: 15.41 (2.37, 28.45); Total Sex Chromosome Disomy: 15.23 (2.09, 28.38)], and tail distributed moment [XX18: 3.01 (0.30, 5.72); YY18: 2.95 (0.24, 5.67); XY18: 3.04 (0.36, 5.72); Total Sex Chromosome Disomy: 3.10 (0.31, 5.71)] than men who had never smoked. In regression models adjusted for age and smoking, there was a positive association between XY disomy and comet extent. For an increase in XY disomy from 0.56 to 1.47% (representing the 25th to 75th percentile), there was a mean increase of 5.08 µm in comet extent. No other statistically significant

  8. Sperm DNA: organization, protection and vulnerability: from basic science to clinical applications--a position report

    National Research Council Canada - National Science Library

    Barratt, C.L; Aitken, R.J; Bjorndahl, L; Carrell, D.T; Boer, P. de; Kvist, U; Lewis, S.E; Perreault, S.D; Perry, M.J; Ramos, L; Robaire, B; Ward, S; Zini, A

    2010-01-01

    ... (for details see Appendix). Its focus is on methods for detecting sperm DNA damage and potential application of new knowledge about sperm chromatin organization, vulnerability and repair to improve the diagnosis and treatment...

  9. [DNA sperm methylation in assisted reproductive techniques].

    Science.gov (United States)

    Benchaïb, M; Ajina, M; Braun, V; Niveleau, A; Guérin, J-F

    2006-09-01

    In the last few years, many tests were developed to study the fertilizing properties of the spermatozoa. However none of them was useful to obtain a prognostic factor. Indeed, the integrity of the spermatic DNA is also necessary to a successful fertilization for obtaining a pregnancy. DNA integrity could be evaluated by the measurement of the level of DNA methylation. Indeed, in the mammals, the methylation of the ADN is involved in diverse processes amongst them the regulation of the genome expression during the embryonic development. The objective of this study is to evaluate the impact of the level of methylation of the spermatic DNA in the success of in vitro fertilization (IVF), in terms of rate of fertilization, quality of the embryos and rate of pregnancy. The immunostaining of the 5-methylecytosine, then the quantification by image analysis or with flow cytometry, allowed an objective evaluation of the level of total methylation of spermatic DNA. Our data show that the level of DNA methylation influences neither the fertilization rate nor the embryos quality. On the other hand, the rate of pregnancy is decreased if the total level of DNA methylation is lower than a threshold value. The level of spermatic DNA methylation represents a new parameter of spermatic maturation.

  10. Processes involved in assisted reproduction technologies significantly increase sperm DNA fragmentation and phosphatidylserine translocation.

    Science.gov (United States)

    Balasuriya, A; Serhal, P; Doshi, A; Harper, J C

    2014-03-01

    Sperm preparation techniques in assisted reproduction technologies (ART) are potential generators of exogenous stresses that cause additional DNA damage. DNA fragmentation tests, such as the sperm chromatin structure assay, involve freezing sperm samples in the absence of cryoprotectant. Thermal, oxidative stress (OS) and freezing are detrimental to sperm DNA fragmentation and phosphatidylserine (PS) translocation. The primary aim of this study was to subject mature sperm to environmental insults that normally occur during ART. We tested the hypotheses that OS, thermal stress and freeze-thawing caused sperm nuclear and membrane damage and that a positive correlation exists between PS translocation and DNA fragmentation. Sperm DNA integrity deteriorates in semen samples from men with advancing age and a sperm concentration of <15 m ml(-1) . The significant increase in sperm DNA fragmentation at 37 °C after merely 1 h is important clinically as semen liquefaction and short-term sperm storage in an ART cycle involve incubating samples at this temperature. Freezing without a cryoprotectant significantly increases the level of sperm nuclear damage, so it is important not to freeze neat semen prior to DNA fragmentation testing. This study highlights the importance of minimising the production of exogenous stresses during sperm preparation in ART. © 2012 Blackwell Verlag GmbH.

  11. Comparison of DNA fragmentation of frozen-thawed epididymal sperm of dogs using Sperm Chromatin Structure Analysis and Sperm Chromatin Dispersion test.

    Science.gov (United States)

    Ortiz, I; Urbano, M; Dorado, J; Morrell, J M; Al-Essawe, E; Johannisson, A; Hidalgo, M

    2017-12-01

    The aim of this study was to compare sperm DNA fragmentation of frozen-thawed epididymal sperm of dogs using the SCSA (Sperm Chromatin Structure Assay) and SCDt (Sperm Chromatin Dispersion test). For this purpose, epididymis from neutered dogs were minced and incubated in a Tris-based extender. The recovered sperm were frozen in a two-step cooling protocol with Tris-based, egg yolk extender and increasing glycerol concentrations, and stored in liquid nitrogen. After thawing, each replica was incubated at 38°C for 24h. Sperm DNA fragmentation index (sDFi) was assessed by SCSA and SCDt at 0, 3, 6 and 24h of incubation and compared within treatments. The relationship and agreement between techniques were evaluated by Pearson's coefficient and Intraclass Correlation Coefficient (ICC). The results were expressed as mean±standard error of the mean (SEM). Both techniques indicated there was a significant increase of DNA fragmentation after 24h of incubation. Moderate correlation (r=0.65; P0.05) was found between SCSA and SCDt. The lack of agreement indicates that SCSA and SCDt measure different aspects of DNA fragmentation. Four halo morphologies were detected after 24h of incubation using the SCDt: un-fragmented DNA with a small halo, fragmented DNA with large halo and two new halo presentations never described before for dog sperm: receding sperm with a disappearing halo and "bald" sperm without chromatin dispersion halo around the core. Sperm without a halo of chromatin dispersion are not described by the manufacturer and are similar to un-fragmented sperm, which could lead to erroneous results when using the SCDt. Further studies with different incubation periods and including the new morphologies described in this study should be performed. In conclusion, although SCSA and SCDt can evaluate the changes in the sperm DNA fragmentation dynamics of frozen-thawed epididymal dog sperm, these provided different findings on sperm DNA fragmentation. Copyright © 2017

  12. [Type of sperm DNA strand breaks in infertile men and its clinical implication].

    Science.gov (United States)

    Wei, Ren-xiong; Chen, Jian-wei; Huang, Ji-hong; Zhang, Xiao-xia; Cui, Yun

    2015-07-01

    To observe the characteristics of sperm single-stranded DNA breaks (SSB) and double-stranded DNA breaks (DSB) in infertile men, explore the association of DSB with male infertility, and provide a new observation index and idea for the diagnosis and treatment of the disease. This study involved 60 infertile men (infertility group) and 30 normal healthy males with infertile wives (control group). We comparatively analyzed the seminal parameters of the two groups, determined sperm concentration and viability using the computer aided sperm analysis system, measured the sperm survival rate by hypoosmotic swelling (HOS) test, examined sperm morphology by Diff-Quick staining, and detected sperm DNA damage by two-tail comet assay. Nine two-tail comet models were established for detecting sperm DNA integrity. Comparisons between the fertility and control groups showed that the sperm DNA fragmentation index (DFI) was (33.8 ± 13.1) vs (16.3 ± 7.9)% (P 0.05), the SSB-DFI/DFI was (56.8 ± 32.4) vs (91.4 ± 27.8)% (P 0.05); DFI was correlated negatively with the percentage of progressively motile sperm, sperm survival rate, and the percentage of morphologically normal sperm (P sperm concentration (P > 0.05); both DSB-DFI and DSB-DFI/DFI showed a negative correlation with sperm concentration, sperm survival rate, and the percentages of progressively motile sperm and morphologically normal sperm (P sperm DNA strand damage is of much reference value for the assessment of male fertility.

  13. The effect of sperm DNA damage on assisted reproduction outcomes. A review.

    Science.gov (United States)

    Agarwal, A; Allamaneni, S S R

    2004-06-01

    The quality of sperm DNA is very important in maintaining the reproductive potential of men. Sperm DNA is resistant to many types of insults that occur during its journey from the testis to time it reaches the oocyte for fertilization. During natural conception, a selection process occurs that allows only sperm with intact DNA to fertilize an oocyte. When assisted reproductive techniques (ART) are used, some or all of this selection process is bypassed. As a result, sperm with damaged DNA may fertilize an oocyte. Damage to sperm nuclear DNA negatively affects assisted and natural fertility. Increasingly, sperm DNA is being recognized as an independent measure of sperm quality that may have better diagnostic and prognostic capabilities than standard sperm parameters. This review summarizes the available evidence for the role of sperm DNA damage in assisted fertility. Two important facts are obvious from the available evidence: 1) men with spermatozoal DNA damage appears to have a decreased ability to father offspring, 2) spermatozoa with abnormal DNA can fertilize an oocyte, which may progress to a live birth. Infertile men who are planning to undergo ART procedures with their partner should have their sperm evaluated for DNA damage. The results of this evaluation may be used to counsel the couple about their chances for live birth and for genomic abnormalities in their offspring.

  14. Interpreting sperm DNA damage in a diverse range of mammalian sperm by means of the two-tailed comet assay.

    Science.gov (United States)

    Cortés-Gutiérrez, Elva I; López-Fernández, Carmen; Fernández, José Luis; Dávila-Rodríguez, Martha I; Johnston, Stephen D; Gosálvez, Jaime

    2014-01-01

    Key ConceptsThe two-dimensional Two-Tailed Comet assay (TT-comet) protocol is a valuable technique to differentiate between single-stranded (SSBs) and double-stranded DNA breaks (DSBs) on the same sperm cell.Protein lysis inherent with the TT-comet protocol accounts for differences in sperm protamine composition at a species-specific level to produce reliable visualization of sperm DNA damage.Alkaline treatment may break the sugar-phosphate backbone in abasic sites or at sites with deoxyribose damage, transforming these lesions into DNA breaks that are also converted into ssDNA. These lesions are known as Alkali Labile Sites "ALSs."DBD-FISH permits the in situ visualization of DNA breaks, abasic sites or alkaline-sensitive DNA regions.The alkaline comet single assay reveals that all mammalian species display constitutive ALS related with the requirement of the sperm to undergo transient changes in DNA structure linked with chromatin packing.Sperm DNA damage is associated with fertilization failure, impaired pre-and post- embryo implantation and poor pregnancy outcome.The TT is a valuable tool for identifying SSBs or DSBs in sperm cells with DNA fragmentation and can be therefore used for the purposes of fertility assessment. Sperm DNA damage is associated with fertilization failure, impaired pre-and post- embryo implantation and poor pregnancy outcome. A series of methodologies to assess DNA damage in spermatozoa have been developed but most are unable to differentiate between single-stranded DNA breaks (SSBs) and double-stranded DNA breaks (DSBs) on the same sperm cell. The two-dimensional Two-Tailed Comet assay (TT-comet) protocol highlighted in this review overcomes this limitation and emphasizes the importance in accounting for the difference in sperm protamine composition at a species-specific level for the appropriate preparation of the assay. The TT-comet is a modification of the original comet assay that uses a two dimensional electrophoresis to allow for

  15. Interpreting sperm DNA damage in a diverse range of mammalian sperm by means of the two-tailed comet assay

    Science.gov (United States)

    Cortés-Gutiérrez, Elva I.; López-Fernández, Carmen; Fernández, José Luis; Dávila-Rodríguez, Martha I.; Johnston, Stephen D.; Gosálvez, Jaime

    2014-01-01

    Key Concepts The two-dimensional Two-Tailed Comet assay (TT-comet) protocol is a valuable technique to differentiate between single-stranded (SSBs) and double-stranded DNA breaks (DSBs) on the same sperm cell.Protein lysis inherent with the TT-comet protocol accounts for differences in sperm protamine composition at a species-specific level to produce reliable visualization of sperm DNA damage.Alkaline treatment may break the sugar–phosphate backbone in abasic sites or at sites with deoxyribose damage, transforming these lesions into DNA breaks that are also converted into ssDNA. These lesions are known as Alkali Labile Sites “ALSs.”DBD–FISH permits the in situ visualization of DNA breaks, abasic sites or alkaline-sensitive DNA regions.The alkaline comet single assay reveals that all mammalian species display constitutive ALS related with the requirement of the sperm to undergo transient changes in DNA structure linked with chromatin packing.Sperm DNA damage is associated with fertilization failure, impaired pre-and post- embryo implantation and poor pregnancy outcome.The TT is a valuable tool for identifying SSBs or DSBs in sperm cells with DNA fragmentation and can be therefore used for the purposes of fertility assessment. Sperm DNA damage is associated with fertilization failure, impaired pre-and post- embryo implantation and poor pregnancy outcome. A series of methodologies to assess DNA damage in spermatozoa have been developed but most are unable to differentiate between single-stranded DNA breaks (SSBs) and double-stranded DNA breaks (DSBs) on the same sperm cell. The two-dimensional Two-Tailed Comet assay (TT-comet) protocol highlighted in this review overcomes this limitation and emphasizes the importance in accounting for the difference in sperm protamine composition at a species-specific level for the appropriate preparation of the assay. The TT-comet is a modification of the original comet assay that uses a two dimensional electrophoresis to

  16. Use of laptop computers connected to internet through Wi-Fi decreases human sperm motility and increases sperm DNA fragmentation.

    Science.gov (United States)

    Avendaño, Conrado; Mata, Ariela; Sanchez Sarmiento, César A; Doncel, Gustavo F

    2012-01-01

    To evaluate the effects of laptop computers connected to local area networks wirelessly (Wi-Fi) on human spermatozoa. Prospective in vitro study. Center for reproductive medicine. Semen samples from 29 healthy donors. Motile sperm were selected by swim up. Each sperm suspension was divided into two aliquots. One sperm aliquot (experimental) from each patient was exposed to an internet-connected laptop by Wi-Fi for 4 hours, whereas the second aliquot (unexposed) was used as control, incubated under identical conditions without being exposed to the laptop. Evaluation of sperm motility, viability, and DNA fragmentation. Donor sperm samples, mostly normozoospermic, exposed ex vivo during 4 hours to a wireless internet-connected laptop showed a significant decrease in progressive sperm motility and an increase in sperm DNA fragmentation. Levels of dead sperm showed no significant differences between the two groups. To our knowledge, this is the first study to evaluate the direct impact of laptop use on human spermatozoa. Ex vivo exposure of human spermatozoa to a wireless internet-connected laptop decreased motility and induced DNA fragmentation by a nonthermal effect. We speculate that keeping a laptop connected wirelessly to the internet on the lap near the testes may result in decreased male fertility. Further in vitro and in vivo studies are needed to prove this contention. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  17. Double-stranded DNA breaks hidden in the neutral Comet assay suggest a role of the sperm nuclear matrix in DNA integrity maintenance

    OpenAIRE

    Ribas-Maynou, J.; Gawecka, J.E.; Benet, J.; Ward, W.S.

    2013-01-01

    We used a mouse model in which sperm DNA damage was induced to understand the relationship of double-stranded DNA (dsDNA) breaks to sperm chromatin structure and to the Comet assay. Sperm chromatin fragmentation (SCF) produces dsDNA breaks located on the matrix attachment regions, between protamine toroids. In this model, epididymal sperm induced to undergo SCF can religate dsDNA breaks while vas deferens sperm cannot. Here, we demonstrated that the conventional neutral Comet assay underestim...

  18. Interpreting sperm DNA damage in a diverse range of mammalian sperm by means of the two-tailed comet assay

    OpenAIRE

    Elva I eCortes-Gutierrez; Carmen eLopez-Fernandez; Jose Luis Fernandez; Martha I Davila-Rodriguez; Stephen eJohnston; Jaime eGosalvez

    2014-01-01

    Key Concepts The two-dimensional Two-Tailed Comet assay (TT-comet) protocol is a valuable technique to differentiate between single-stranded (SSBs) and double-stranded DNA breaks (DSBs) on the same sperm cell. Protein lysis inherent with the TT-comet protocol accounts for differences in sperm protamine composition at a species-specific level to produce reliable visualization of sperm DNA damage. Alkaline treatment may break the sugar–phosphate backbone in abasic sites or at sites wi...

  19. The effect of flash-freezing temperature on stallion sperm DNA structure.

    Science.gov (United States)

    Serafini, R; Varner, D D; Bissett, W; Blanchard, T L; Teague, S R; Love, C C

    2017-06-01

    The effect of flash-freezing storage temperature on stallion sperm DNA has not been evaluated. Commonly, sperm are flash-frozen at various temperatures to preserve sperm DNA prior to analysis. It is unclear whether the temperature at which sperm are frozen and stored may affect the results of DNA assays. In this study, the neutral comet assay was used to evaluate the effect of flash-freezing storage temperature (freezer [-60 °C], dry ice [-78.5 °C], liquid nitrogen [-196 °C]) compared to fresh sperm DNA structure. In addition, intra- and inter-assay and intra- and inter-stallion variabilities were determined. All comet tail measures were higher following any flash-freezing method, as compared to fresh sperm DNA (P  0.05). For most comet variables, intra- and inter-assay variabilities were comet head length (HL) and width (CW) were less variable as compared to comet tail values, i.e., % comet tail DNA (T-DNA), tail length (TL), tail moment (OTM), and tail migration (TM). Certain comet tail values in fresh (% T-DNA, and OTM) and flash-frozen sperm (OTM, % T-DNA, TL, and TM) were correlated to the Sperm Chromatin Structure Assay (SCSA) variable, COMP-α t . The comet tail measures were negatively correlated to % morphologically normal sperm (P sperm motility were not correlated to any morphologic sperm feature in this group of stallions (P > 0.05). While significant differences in the structure of the sperm DNA were identified in the flash-frozen as compared to the fresh sperm DNA with the neutral comet assay, it cannot be assumed that these changes are fertility limiting. Copyright © 2017. Published by Elsevier Inc.

  20. Paternal DNA damage resulting from various sperm treatments persists after fertilization and is similar before and after DNA replication.

    Science.gov (United States)

    Yamauchi, Yasuhiro; Riel, Jonathan M; Ward, Monika A

    2012-01-01

    In spite of its highly condensed state, sperm DNA is vulnerable to damage that can originate from oxidative stress, the activity of sperm-specific nucleases, or both. After fertilization, in the oocyte, paternal chromatin undergoes dramatic changes, and during this extensive remodeling, it can be both repaired and degraded, and these processes can be linked to DNA synthesis. Here, we analyzed sperm response to damage-inducing treatments both before and after fertilization and before or after zygotic DNA replication. Epididymal mouse spermatozoa were either frozen without cryoprotection (FT) or treated with detergent Triton X-100 coupled with dithiothreitol (TX+DTT) to induce DNA damage. Fresh, untreated sperm served as control. Immediately after preparation, spermatozoa from 3 groups were taken for comet assay, or for intracytoplasmic sperm injection into prometaphase I oocytes to visualize prematurely condensed single-chromatid chromosomes, or into mature metaphase II oocytes to visualize chromosomes after DNA replication. Comet assay revealed increased DNA fragmentation in treated sperm when compared with control, with FT sperm more severely affected. Chromosome analysis demonstrated paternal DNA damage in oocytes injected with treated, but not with fresh, sperm, with FT and TX+DTT groups now yielding similar damage. There were no differences in the incidence of abnormal paternal karyoplates before and after DNA synthesis in all examined groups. This study provides evidence that subjecting sperm to DNA damage-inducing treatments results in degradation of highly condensed sperm chromatin when it is still packed within the sperm head, and that this DNA damage persists after fertilization. The difference in DNA damage in sperm subjected to 2 treatments was ameliorated in the fertilized oocytes, suggesting that some chromatin repair might have occurred. This process, however, was independent of DNA synthesis and took place during oocyte maturation.

  1. Paternal DNA damage resulting from various sperm treatments persists after fertilization and is similar prior and after DNA replication1

    Science.gov (United States)

    Yamauchi, Yasuhiro; Riel, Jonathan M.; Ward, Monika A.

    2012-01-01

    In spite of its highly condensed state sperm DNA is vulnerable to damage that can originate from oxidative stress and/or the activity of sperm-specific nucleases. After fertilization, in the oocyte, paternal chromatin undergoes dramatic changes and during this extensive remodeling it can be both repaired and degraded, and these processes can be linked to DNA synthesis. Here, we analyzed sperm response to damage-inducing treatments both before and after fertilization, and prior or after zygotic DNA replication. Epididymal mouse spermatozoa were either frozen without cryoprotection (FT) or treated with detergent Triton X-100 coupled with dithiothreitol (TX+DTT) to induce DNA damage. Fresh, untreated sperm served as control. Immediately after preparation spermatozoa from three groups were taken for comet assay, or for intracytoplasmic sperm injection (ICSI) into prometaphase I (proMI) oocytes to visualize prematurely condensed single chromatid chromosomes, or into mature metaphase II (MII) oocytes to visualize chromosomes after DNA replication. Comet assay revealed increased DNA fragmentation in treated sperm when compared to control, with FT sperm more severely affected. Chromosome analysis demonstrated paternal DNA damage in oocytes injected with treated but not with fresh sperm, with FT and TX+DTT groups now yielding similar damage. There were no differences in the incidence of abnormal paternal karyoplates prior and after DNA synthesis in all examined groups. This study provides evidence that subjecting sperm to DNA damage inducing treatments results in degradation of highly condensed sperm chromatin when it is still packed within the sperm head, and that this DNA damage persists after fertilization. The difference in DNA damage in sperm subjected to two treatments was ameliorated in the fertilized oocytes suggesting that some chromatin repair might have occurred. This process, however, was independent of DNA synthesis, and took place during oocyte maturation

  2. Raman Spectroscopy of DNA Packaging in Individual Human Sperm Cells distinguishes Normal from Abnormal Cells

    Energy Technology Data Exchange (ETDEWEB)

    Huser, T; Orme, C; Hollars, C; Corzett, M; Balhorn, R

    2009-03-09

    Healthy human males produce sperm cells of which about 25-40% have abnormal head shapes. Increases in the percentage of sperm exhibiting aberrant sperm head morphologies have been correlated with male infertility, and biochemical studies of pooled sperm have suggested that sperm with abnormal shape may contain DNA that has not been properly repackaged by protamine during spermatid development. We have used micro-Raman spectroscopy to obtain Raman spectra from individual human sperm cells and examined how differences in the Raman spectra of sperm chromatin correlate with cell shape. We show that Raman spectra of individual sperm cells contain vibrational marker modes that can be used to assess the efficiency of DNA-packaging for each cell. Raman spectra obtained from sperm cells with normal shape provide evidence that DNA in these sperm is very efficiently packaged. We find, however, that the relative protein content per cell and DNA packaging efficiencies are distributed over a relatively wide range for sperm cells with both normal and abnormal shape. These findings indicate that single cell Raman spectroscopy should be a valuable tool in assessing the quality of sperm cells for in-vitro fertilization.

  3. Sperm-mediated DNA transfer to cells of the uterus and embryo.

    Science.gov (United States)

    Chan, P J

    2000-06-01

    There is a paucity of information about sperm-mediated transmission of exogenous DNA to implanting embryos and cells of the reproductive tract. Preliminary experiments established that sperm has the capacity to actively take in exogenous DNA derived from HPV. In addition, blastocysts also take up exogenous HPV DNA, but in contrast to sperm, the process appears passive. DNA-carrying sperm migrating in an artificial glass tube or excised mouse bicornuate uteri transfected the blastocysts at the remote position using a flip-flop mechanism. There were preferential transmission of the types of HPV DNA but this was not attributed to the gene sequence or the size of the DNA fragments. The internalized DNA became undetectable unless continuous sperm bombardment or pricking took place. Mycoplasma vectors offer a novel way to enhance the transfection of blastocyst with exogenous DNA.

  4. CASA derived human sperm abnormalities: correlation with chromatin packing and DNA fragmentation

    National Research Council Canada - National Science Library

    Sivanarayana, T; Krishna, Ch Ravi; Prakash, G Jaya; Krishna, K Murali; Madan, K; Rani, B Sireesha; Sudhakar, G; Raju, G A. Rama

    2012-01-01

    The present study was undertaken to evaluate the effects of morphokinetic abnormalities of human spermatozoa on chromatin packing and DNA integrity and possible beneficial effects of sperm selection...

  5. Measuring Sperm DNA Fragmentation and Clinical Outcomes of Medically Assisted Reproduction: A Systematic Review and Meta-Analysis

    NARCIS (Netherlands)

    Cissen, Maartje; Wely, Madelon van; Scholten, Irma; Mansell, Steven; Bruin, Jan Peter de; Mol, Ben Willem; Braat, Didi; Repping, Sjoerd; Hamer, Geert

    2016-01-01

    Sperm DNA fragmentation has been associated with reduced fertilization rates, embryo quality, pregnancy rates and increased miscarriage rates. Various methods exist to test sperm DNA fragmentation such as the sperm chromatin structure assay (SCSA), the sperm chromatin dispersion (SCD) test, the

  6. Mouse zygotes respond to severe sperm DNA damage by delaying paternal DNA replication and embryonic development.

    Directory of Open Access Journals (Sweden)

    Joanna E Gawecka

    Full Text Available Mouse zygotes do not activate apoptosis in response to DNA damage. We previously reported a unique form of inducible sperm DNA damage termed sperm chromatin fragmentation (SCF. SCF mirrors some aspects of somatic cell apoptosis in that the DNA degradation is mediated by reversible double strand breaks caused by topoisomerase 2B (TOP2B followed by irreversible DNA degradation by a nuclease(s. Here, we created zygotes using spermatozoa induced to undergo SCF (SCF zygotes and tested how they responded to moderate and severe paternal DNA damage during the first cell cycle. We found that the TUNEL assay was not sensitive enough to identify the breaks caused by SCF in zygotes in either case. However, paternal pronuclei in both groups stained positively for γH2AX, a marker for DNA damage, at 5 hrs after fertilization, just before DNA synthesis, while the maternal pronuclei were negative. We also found that both pronuclei in SCF zygotes with moderate DNA damage replicated normally, but paternal pronuclei in the SCF zygotes with severe DNA damage delayed the initiation of DNA replication by up to 12 hrs even though the maternal pronuclei had no discernable delay. Chromosomal analysis of both groups confirmed that the paternal DNA was degraded after S-phase while the maternal pronuclei formed normal chromosomes. The DNA replication delay caused a marked retardation in progression to the 2-cell stage, and a large portion of the embryos arrested at the G2/M border, suggesting that this is an important checkpoint in zygotic development. Those embryos that progressed through the G2/M border died at later stages and none developed to the blastocyst stage. Our data demonstrate that the zygote responds to sperm DNA damage through a non-apoptotic mechanism that acts by slowing paternal DNA replication and ultimately leads to arrest in embryonic development.

  7. Exposure to Perfluoroalkyl Substances and Sperm DNA Global Methylation in Arctic and European Populations

    DEFF Research Database (Denmark)

    Leter, Giorgio; Consales, Claudia; Eleuteri, Patrizia

    2014-01-01

    exposure and sperm DNA global methylation endpoints could be detected. However, since weak but statistically significant associations of different PFASs with DNA hypo- and hyper-methylation were found in some of the studied populations, effects of PFASs on sperm epigenetic processes cannot be completely...

  8. Genetic Variation in DNA of Coho Salmon from the Lower Columbia River : Final Report 1993.

    Energy Technology Data Exchange (ETDEWEB)

    Fobes, Stephen; Knudsen, Kathy; Allendorf, Fred

    1993-04-01

    The goal of this project was to develop techniques to provide the information needed to determine if Lower Columbia River coho salmon represent a 'species' under the Endangered Species Act. Our report features two new nuclear DNA approaches to the improved detection of genetic variation: (1) Studies of DNA-level genetic variation for two nuclear growth hormone genes; (2) Use of arbitrary DNA primers (randomly amplified polymorphic DNA, or 'RAPD' primers) to detect variation at large numbers of nuclear genes. We used the polymerase chain reaction (PCR) to amplify variable sections (introns) of two growth hormone genes (GH-I and G/f-Z) in several salmonid species. Coho salmon had three DNA length variants for G/-I intron C. Restriction analysis and sequencing provided valuable information about the mode of evolution of these DNA sequences. We tested segregation of the variants in captive broods of coho salmon, and demonstrated that they are alleles at a single Mendelian locus. Population studies using the GH-1 alleles showed highly significant frequency differences between Lower Columbia River and Oregon Coast coho salmon, and marginal differences among stocks within these regions. These new markers are adequately defined and tested to use in coho salmon population studies of any size. The nature of the variation at GH-1 (Variable Number Tandem Repeats, or 'VNTRs') suggests that more genetic variants will be found in coho salmon from other areas. GH-2 intron C also showed length variation in coho salmon, and this variation was found to be sex-linked. Because PCR methods require minute amounts of tissue, this discovery provides a technique to determine the gender of immature coho salmon without killing them. Chinook salmon had restriction patterns and sequence divergences similar to coho salmon. Thus, we expect that sex linkage of GH-2 alleles predates the evolutionary divergence of Pacific salmon species, and that gender testing with

  9. Temperature variable and the efficiency of sperm mediated transfection of HPV16 DNA into cells.

    Science.gov (United States)

    Kadze, Ruslana; Chan, Philip J; Jacobson, John D; Corselli, Johannah U; King, Alan

    2002-09-01

    To pretreat sperm at various temperatures before exposure to human papillomavirus (HPV) 16 DNA fragments and to assess the efficiency of HPV carrier sperm to transfect cumulus cells. Cumulus cells from follicular aspirates were obtained, pooled and divided into culture dishes containing Sybr Gold-stained HPV DNA carrying sperm that were either pretreated at 4 degree C, 37 degree C or 40 degree C (n = 5). The cells were incubated in 5% CO(2) in air mixture at 37 degree C for 24 hours. The efficiency of sperm to take up fluorescent HPV DNA was determined at hour 0. After incubation, cumulus cell viability was assessed using the eosin method and the percentages of fluorescent cumulus cells determined. Over half of all the cumulus cells became fluorescent with the highest percentage in the 37 degree C group. Sperm pretreated at 4 degree C had the greatest amount of HPV DNA fragments. Total sperm motility was similar for the 3 pretreatment groups. There were no differences in cumulus viability among the groups. Sperm pretreated at 37 degree C transferred the greatest amount of fluorescent HPV DNA fragments to the cumulus cells. The HPV DNA was observed in the nuclear and cytoplasmic compartments. The data suggested the possibility of sperm as a vector for the transmission of HPV DNA to the cumulus cells surrounding ovulated oocytes, which might lead to early implantation failures.

  10. The etiologies of sperm DNA abnormalities in male infertility: An assessment and review

    Directory of Open Access Journals (Sweden)

    Soheila Pourmasumi

    2017-07-01

    Full Text Available The sperm DNA damage may occur in testis, genital ducts, and also after ejaculation. Mechanisms altering chromatin remodeling are abortive apoptosis and oxidative stress resulting from reactive oxygen species. Three classifications of intratesticular, post-testicular, and external factors have been correlated with increased levels of sperm DNA damage which can affect the potential of fertility. Alcohol consumption may not increase the rate of sperm residual histones and protamine deficiency; however, it causes an increase in the percentage of spermatozoa with DNA fragmentation and apoptosis. In a medical problem as spinal cord injury, poor semen parameters and sperm DNA damage were reported. Infection induces reactive oxygen species production, decreases the total antioxidant capacity and sperm DNA fragmentation or antigen production that lead to sperm dysfunctions and DNA fragmentation. While reactive oxygen species generation increases with age, oxidative stress may be responsible for the age-dependent sperm DNA damage. The exposing of reproductive organs in older men to oxidative stress for a long time may produce more DNA-damaged spermatozoa than youngers. Examining the sperm chromatin quality in testicular cancer and Hodgkin’s lymphoma patients prior to chemotherapy demonstrated the high incidence of DNA damage and low compaction in spermatozoa at the time of diagnosis. In chemotherapy cycles with genotoxic agents in cancer patients, an increase in sperm DNA damage was shown after treatment. In overall, those factors occurring during the prenatal or the adult life alter the distribution of proteins associated with sperm chromatin induce changes in germ cells which can be detected in infertile patients.

  11. Interpreting sperm DNA damage in a diverse range of mammalian sperm by means of the two-tailed comet assay

    Directory of Open Access Journals (Sweden)

    Elva I eCortes-Gutierrez

    2014-11-01

    Full Text Available Sperm DNA damage is associated with fertilization failure, impaired pre-and post- embryo implantation and poor pregnancy outcome. A series of methodologies to assess DNA damage in spermatozoa have been developed but most are unable to differentiate between single-stranded DNA breaks (SSBs and double-stranded DNA breaks (DSBs on the same sperm cell. The two-dimensional Two-Tailed Comet assay (TT-comet protocol highlighted in this review overcomes this limitation and emphasizes the importance in accounting for difference in sperm protamine composition at a species-specific level for the appropriate preparation of the assay. The TT-comet is a modification of the original comet assay that uses a two dimensional electrophoresis to allow for the simultaneous evaluation of relatively high numbers of DSBs and SSBs in mammalian spermatozoa. Here we have compiled a retrospective overview of how the TT-comet assay has been used to investigate the structure and function of sperm DNA across a diverse range of mammalian species (eutheria, metatheria and prototheria. When conducted as part of the TT-comet assay, we discuss how (a the alkaline comet single assay has been used to help understand the constitutive and transient changes in DNA structure associated with chromatin packing, (b the capacity of the TT-comet to differentiate between the presence of SSBs and DSBs (c and the possible implications of SSBs or DSBs for the assessment of infertility.

  12. Comparison of three different techniques of human sperm DNA isolation for methylation assay.

    Science.gov (United States)

    Yuan, Hong-fang; Kuete, Martin; Su, Li; Yang, Fan; Hu, Zhi-yong; Tian, Bo-zhen; Zhang, Hui-ping; Zhao, Kai

    2015-12-01

    Human sperm DNA is an important genetic and epigenetic material, whose chromatin structure differs from that of somatic cells. As such, conventional methods for DNA extraction of somatic cells may not be suitable for obtaining sperm DNA. In this study, we evaluated and compared three sperm DNA extraction techniques, namely, modified guanidinium thiocyanate method (method A), traditional phenol-chloroform method (method B), and TianGen kit method (method C). Spectrophotometry and agarose gel electrophoresis analyses showed that method A produced DNA with higher quantity and purity than those of methods B and C (Psperm DNA methylation assay further indicated that methods A and B were effective, and the former yielded higher quantitative accuracy. In conclusion, the modified guanidinium thiocyanate method provided high quality and reliable results and could be an optimal technique for extracting sperm DNA for methylation assay.

  13. Different Levels of DNA Methylation Detected in Human Sperms after Morphological Selection Using High Magnification Microscopy

    Directory of Open Access Journals (Sweden)

    Nino Guy Cassuto

    2016-01-01

    Full Text Available Objective. To analyze DNA methylation levels between two groups of spermatozoa taken from the same sample, following morphological selection by high magnification (HM at 6100x microscopy. A prospective study was conducted and studied 876 spermatozoa from 10 randomly selected men. Sperm morphology was characterized at HM according to criteria previously established. High-scoring Score 6 and low-scoring Score 0 sperm were selected. Sperm DNA methylation level was assessed using an immunoassay method targeting 5-methylcytosine residues by fluorescence microscopy with imaging analysis system to detect DNA methylation in single spermatozoon. Results. In total, 448 S6 spermatozoa and 428 S0 spermatozoa were analyzed. A strong relationship was found between sperm DNA methylation levels and sperm morphology observed at HM. Sperm DNA methylation level in the S6 group was significantly lower compared with that in the S0 group (p<10-6, OR = 2.4; and p<0.001, as determined using the Wilcoxon test. Conclusion. Differences in DNA methylation levels are associated with sperm morphology variations as observed at HM, which allows spermatozoa with abnormal levels to be discarded and ultimately decrease birth defects, malformations, and epigenetic diseases that may be transmitted from sperm to offspring in ICSI.

  14. The presence of human papillomavirus in semen does not affect the integrity of sperm DNA.

    Science.gov (United States)

    Cortés-Gutiérrez, E I; Dávila-Rodríguez, M I; Fernández, J L; de la O-Pérez, L O; Garza-Flores, M E; Eguren-Garza, R; Gosálvez, J

    2017-03-06

    It remains unknown whether human papillomaviruses (HPVs) in semen affect sperm DNA integrity. We investigated whether the presence of these viruses in semen was associated with an elevated sperm DNA fragmentation index. Semen samples of 22 normozoospermic patients undergoing infertility treatment, nine fertile donors and seven fertile men with a risk of HPV infection (genital warts or condylomas) were included in the study. The samples were examined by an INNO-LiPA test PCR-based reverse hybridisation array that identifies 28 types of HPVs as simple or multiple infections. Sperm DNA integrity was determined by sperm chromatin dispersion assay (SCD). Our preliminary findings demonstrate an increase in HPV infection in infertile men with respect to fertile men. However, the sperm DNA fragmentation index was not increased in semen containing these viruses. © 2017 Blackwell Verlag GmbH.

  15. The effects of male age on sperm DNA damage in healthy non-smokers

    Energy Technology Data Exchange (ETDEWEB)

    Schmid, T; Eskenazi, B; Baumgartner, A; Marchetti, F; Young, S; Weldon, R; Anderson, D; Wyrobek, A

    2006-03-08

    The trend for men to have children at older ages raises concerns that advancing age may increase the production of genetically defective sperm, increasing the risks of transmitting germ-line mutations. We investigated the associations between male age and sperm DNA damage and the influence of several lifestyle factors in a healthy non-clinical group of 80 non-smokers (age: 22-80) with no known fertility problems using the sperm Comet analyses. The average percent of DNA that migrated out of the sperm nucleus under alkaline electrophoresis increased with age (0.18% per year, p=0.006); but there was no age association for damage measured under neutral conditions (p=0.7). Men who consumed >3 cups coffee per day had {approx}20% higher % tail DNA under neutral but not alkaline conditions compared to men who consumed no caffeine (p=0.005). Our findings indicate that (a) older men have increased sperm DNA damage associated with alkali-labile sites or single-strand DNA breaks, and (b) independent of age, men with substantial daily caffeine consumption have increased sperm DNA damage associated with double-strand DNA breaks. DNA damage in sperm can be converted to chromosomal aberrations and gene mutations after fertilization increasing the risks for developmental defects and genetic diseases among offspring.

  16. The effect of two cryopreservation methods on human sperm DNA damage.

    Science.gov (United States)

    Liu, Taixiu; Gao, Jianfang; Zhou, Niya; Mo, Min; Wang, Xiaogang; Zhang, Xi; Yang, Huan; Chen, Qing; Ao, Lin; Liu, Jinyi; Cui, Zhihong; Cao, Jia

    2016-06-01

    Several methods are currently available for selection when conducting sperm cryopreservation, however, these methods might cause different degrees of damage on sperm DNA. The aim of the this study is to compare the effects of storage at -80 °C (in ultra-low temperature refrigerator) and at -196 °C (in liquid nitrogen) on sperm DNA damage, thus to provide a reference for choosing the right method according to different aims. We randomly collected 28 semen samples from college students of Chongqing city. The samples stored at -80 °C were neat semen samples and the samples stored at -196 °C were mixed with additional cryoprotectants. Each sample was subjected to two freezing-thawing cycles, and the sperm DNA damage levels of fresh and thawed samples were measured by single cell gel electrophoresis (SCGE) and sperm chromatin structure assay (SCSA). Both SCGE and SCSA assays showed cryopreservation induced significant damage to sperm DNA. However, storage at -196 °C lead to more severe damage to sperm DNA than storage at -80 °C measured by SCSA. Sperm DNA damage increased simultaneously with the higher frequency of freezing-thawing cycles. We concluded that storage of neat semen samples at -80 °C had milder damage to sperm DNA than storage at -196 °C mixed with cryoprotectants. To avoid additional sperm DNA damage, repeated freezing and thawing should be prevented. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Relation between serum xenobiotic induced receptor activities and sperm DNA damage and sperm apoptotic markers in European and Inuit populations

    DEFF Research Database (Denmark)

    Long, Manhai; Stronati, Alessanda; Bizzaro, Davide

    2007-01-01

    Persistent organic pollutants (POPs) can interfere with hormone activities and are suspected as endocrine disrupters involved in disorders, e.g. reproductive disorders. We investigated the possible relation between the actual integrated serum xenoestrogenic, xenoandrogenic and aryl hydrocarbon...... receptor (AhR) activities, and the sperm DNA damage and sperm apoptotic markers of 262 adult males (54 Inuits from Greenland, 69 from Warsaw (Poland), 81 from Sweden, and 58 from Kharkiv (Ukraine)) exposed to different levels of POPs. Xenobiotic-induced receptor activities were determined by receptor......-mediated luciferase reporter gene expression. Sperm DNA damage was measured using terminal deoxynucleotidyl transferase-driven dUTP nick labeling assay (TUNEL) and pro- (Fas) and anti-apoptotic (Bcl-xL) markers were determined by immune methods. Different features of xenobiotic-induced receptor activity in serum...

  18. Types, Causes, Detection and Repair of DNA Fragmentation in Animal and Human Sperm Cells

    Directory of Open Access Journals (Sweden)

    Rosa Roy

    2012-10-01

    Full Text Available Concentration, motility and morphology are parameters commonly used to determine the fertilization potential of an ejaculate. These parameters give a general view on the quality of sperm but do not provide information about one of the most important components of the reproductive outcome: DNA. Either single or double DNA strand breaks can set the difference between fertile and infertile males. Sperm DNA fragmentation can be caused by intrinsic factors like abortive apoptosis, deficiencies in recombination, protamine imbalances or oxidative stress. Damage can also occur due to extrinsic factors such as storage temperatures, extenders, handling conditions, time after ejaculation, infections and reaction to medicines or post-testicular oxidative stress, among others. Two singular characteristics differentiate sperm from somatic cells: Protamination and absence of DNA repair. DNA repair in sperm is terminated as transcription and translation stops post-spermiogenesis, so these cells have no mechanism to repair the damage occurred during their transit through the epididymis and post-ejaculation. Oocytes and early embryos have been shown to repair sperm DNA damage, so the effect of sperm DNA fragmentation depends on the combined effects of sperm chromatin damage and the capacity of the oocyte to repair it. In this contribution we review some of these issues.

  19. DNA flow cytometry of human spermatozoa: consistent stoichiometric staining of sperm DNA using a novel decondensation protocol.

    Science.gov (United States)

    Kovács, Tamás; Békési, Gyöngyi; Fábián, Akos; Rákosy, Zsuzsa; Horváth, Gábor; Mátyus, László; Balázs, Margit; Jenei, Attila

    2008-10-01

    Rapid flow cytometric measurement of the frequency of aneuploid human sperms is in increasing demand but development of an exploitable method is hindered by difficulties of stoichiometric staining of sperm DNA. An aggressive decondensation protocol is needed after which cell integrity still remains intact. We used flow cytometry to examine the effect of lithium diiodosalicylate (LIS, chaotropic agent) on fluorescence intensity of propidium iodide-treated human spermatozoa from 10 normozoospermic men. When flow cytometric identification of diploid spermatozoa was achieved, validation was performed after sorting by three-color FISH. In contrast with the extremely variable histograms of nondecondensed sperms, consistent identification of haploid and diploid spermatozoa was possible if samples were decondensed with LIS prior to flow cytometry. A 76-fold enrichment of diploid sperms was observed in the sorted fractions by FISH. A significant correlation was found between the proportion of sorted cells and of diploid sperms by FISH. Application of LIS during the preparation of sperm for flow cytometry appears to ensure the stoichiometric staining of sperm DNA, making quantification of aneuploid sperm percentage possible. To our knowledge this is the first report in terms of separating spermatozoa with confirmedly abnormal chromosomal content. High correlation between the proportion of cells identified as having double DNA content by flow cytometry and diploid sperm by FISH allows rapid calculation of diploidy rate. Copyright 2008 International Society for Advancement of Cytometry.

  20. Pentoxifylline increase sperm motility in devitrified spermatozoa from asthenozoospermic patient without damage chromatin and DNA integrity.

    Science.gov (United States)

    Nabi, Ali; Khalili, Mohammad Ali; Fesahat, Farzaneh; Talebi, Alireza; Ghasemi-Esmailabad, Saeed

    2017-06-01

    The freeze-thaw process results in reduced motility, viability and fertilization potential of human spermatozoa. So, a variety of substances were evaluated in order to enhance human sperm resistance to the stress of cryopreservation, such as Pentoxifylline (PTX) for improving the Intracytoplasmic sperm injection (ICSI) outcomes. The aim was to investigate the effect of PTX on sperm parameters and chromatin/DNA integrity of asthenozoospermic semen post vitrification. A total of 30 semen specimens were obtained from infertile men with asthenozoospermia. The cryoprotectant-free vitrification was performed for the samples after assessment of sperm parameters. After warming, each sample was exposed for 30 min to 3.6 mmol/l PTX in experimental group and the control group without any treatment apposing at 37 °C for 30 min in regard, to repeat all in vitro analysis (sperm parameters and DNA integrity assay). Regardless of the vitrification devastating impacts on sperm parameters, incubation of post vitrified samples with PTX increased the rate of progressive motility (P integrity of asthenozoospermic sperm samples. The data showed that PTX was able to improve sperm movement without any adverse effects on sperm chromatin/DNA integrity in vitrification program. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Adducts in sperm protamine and DNA (deoxyribonuclease) vs. mutation frequency

    Energy Technology Data Exchange (ETDEWEB)

    Sega, G.A.

    1990-01-01

    In mammals, variability in the genetic sensitivity of different germ-cell stages to mutagens could be the result of how much chemical reaches the different stages, what molecular targets may be affected in the different stages and whether or not repair of lesions occurs. In the mouse, several mutagens have been found that cause their greatest genetic damage in late-spermatid and early-spermatozoa stages and that also bind very strongly to the protamine in these stages. Chemicals which are less genetically damaging to these stages have been found to have much less affinity for protamine. Furthermore, the level of chemical binding to DNA in late-spermatid and early-spermatozoa stages has not been correlated with the level of induced genetic damage, although DNA breakage in these sensitive stages has been shown to increase. This DNA damage is believed to indirectly result from chemical binding to sulfhydryl groups in protamine which prevents normal chromatin condensation within the sperm nucleus. 16 refs., 5 figs.

  2. Sperm quality and DNA integrity of coke oven workers exposed to polycyclic aromatic hydrocarbons.

    Science.gov (United States)

    Jeng, Hueiwang Anna; Pan, Chih-Hong; Chao, Mu-Rong; Chiu, Chien-Chih; Zhou, Guodong; Chou, Chon-Kit; Lin, Wen-Yi

    2016-11-18

    The objective of this study was to assess sperm quality and deoxyribonucleic acid (DNA) integrity of coke oven workers exposed to polycyclic aromatic hydrocarbons (PAHs) as compared to control subjects. The coke oven workers (N = 52) and administrative staff (N = 35) of a steel plant served as the exposed and control groups, respectively. Exposure to PAHs was assessed by measuring 1-hydroxypyren. Analysis of sperm quality (concentration, motility, vitality, and morphology) was performed simultaneously with sperm DNA integrity analysis, including DNA fragmentation, denaturation, bulky DNA adducts, and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo). A questionnaire was conducted to collect demographic and potential confounding data. The coke oven workers had lower percentages of sperm motility, vitality and normal morphology than the control group, but the difference was not significant. For DNA integrity, the coke oven workers had significantly higher concentrations of bulky DNA adducts and 8-oxo-dGuo than the control subjects (p = 0.009 and p = 0.048, respectively). However, DNA fragmentation percentages did not significantly increase as compared to those in the subjects from the control group (p = 0.232). There was no correlation between sperm quality parameters and DNA integrity indicators. Occupational exposure of the coke oven workers to PAHs was associated with decreased sperm DNA integrity. Int J Occup Med Environ Health 2016;29(6):915-926.

  3. SPERM MORPHOLOGICAL ABNORMALITIES AS INDICATORS OF DNA FRAGMENTATION AND FERTILIZATION IN ASSISTED REPRODUCTION

    Directory of Open Access Journals (Sweden)

    Barbara Dariš

    2018-02-01

    Full Text Available Background. To determine the relationship between sperm morphological abnormalities, DNA fragmentation and fertilization rate in IVF and ICSI. Methods. Sperm samples from 10 IVF and 20 ICSI cycles were analyzed. Morphology was assessed according to strict criteria, and DNA fragmentation was measured by terminal deoxynucleotidyl transferase (TdT-mediated fluorescein-dUTP nick end labelling (TUNEL using a flow cytometry. Results. There was a significant difference in the amount of morphological abnormalities between sperm samples with low (< 20 % and high (≥ 20 % degree of DNA fragmentation. The percentages of amorphous heads (10 vs. 4 % and overall head abnormalities (42 vs. 30 % were significantly higher in sperm samples with elevated degree of DNA fragmentation. No correlation was found between sperm DNA fragmentation and fertilization rate after IVF and ICSI. When the predominant morphological abnormality in sperm samples was determined, a negative correlation was found between the percentage of spermatozoa with elongated heads and fertilization rate in ICSI (r = –0.45, P < 0.05. The fertilization rate after IVF was lower in the case of acrosomal abnormalities (35.3 %, compared to the cases of other predominant morphological abnormalities. Conclusions. Head abnormalities, especially amorphous heads, are related to elevated degree of DNA fragmentation. Predominant abnormal form in sperm samples, such as elongated heads and acrosomal abnormalities, may affect fertilization in ART.

  4. Sperm DNA damage has a negative effect on early embryonic development following in vitro fertilization

    Directory of Open Access Journals (Sweden)

    Wei-Wei Zheng

    2018-01-01

    Full Text Available Sperm DNA damage is recognized as an important biomarker of male infertility. To investigate this, sperm DNA damage was assessed by the sperm chromatin dispersion (SCD test in semen and motile spermatozoa harvested by combined density gradient centrifugation (DGC and swim-up in 161 couples undergoing in vitro fertilization (IVF. Semen analysis and sperm DNA damage results were compared between couples who did or did not achieve pregnancy. The sperm DNA damage level was significantly different between the two groups (P < 0.05 and was negatively correlated with IVF outcomes. Logistic regression analysis confirmed that it was an independent predictor for achieving clinical pregnancy. The effects of different levels of sperm DNA damage on IVF outcomes were also compared. There were significant differences in day 3 embryo quality, blastocyst formation rate, and implantation and pregnancy rates (P < 0.05, but not in the basic fertilization rate between the two groups. Thus, sperm DNA damage as measured by the SCD appears useful for predicting the clinical pregnancy rate following IVF.

  5. The ability of sperm selection techniques to remove single- or double-strand DNA damage

    Science.gov (United States)

    Enciso, María; Iglesias, Miriam; Galán, Isabel; Sarasa, Jonás; Gosálvez, Antonio; Gosálvez, Jaime

    2011-01-01

    A wide variety of techniques for the preparation of sperm are currently available, of which the most commonly employed are density–gradient centrifugation (DGC) and swim-up (SUP). To date, these methods appear to be effective in selecting functional sperm for assisted reproduction techniques (ART), but they may have negative effects on sperm DNA. In this study, the ability of these semen processing techniques to eliminate spermatozoa containing single- and double-strand DNA damage was assessed by the two-tailed comet assay and the sperm chromatin dispersion test in 157 semen samples from patients seeking assisted reproduction treatment. Our results indicated that SUP and DGC are equally efficient in eliminating spermatozoa containing double-strand DNA damage and sperm with highly damaged (degraded) DNA, as characterized by the presence of both single- and double-strand DNA breaks. However, DGC is more efficient than SUP in selecting spermatozoa that are free from single-strand DNA damage. Future studies should characterise the importance of the various types of DNA damage and examine the sperm processing protocols used in each laboratory to determine their ability to eliminate DNA damage and hence, prevent the potential transmission of genetic mutations via ART. PMID:21725332

  6. Flow cytometry of DNA in mouse sperm and testis nuclei

    Energy Technology Data Exchange (ETDEWEB)

    Meistrich, M.L. (Univ. of Texas, Houston); Lake, S.; Steinmetz, L.L.; Gledhill, B.L.

    1978-01-01

    Mutations that occur in spermatogenic cells may be expressed as changes in DNA content, but developmentally-dependent alteration of its staining properties complicates the quantitation of DNA in individual germ cells. These alterations have been studied with flow cytometric techniques. Nuclei from mouse testis cells and sperm were stained by the acriflavine--Feulgen method. The fluorescence intensity frequency distribution of nuclei of testis cells was characterized by 2 major and 5 minor peaks. Nuclei sorted from the various peaks with a fluorescence-activated cell sorter were identified microscopically. These data were confirmed by generation of peaks with nuclei prepared from cell suspensions enriched in specific cell types. One of the major peaks corresponded to round spermatid nuclei. The other major peak, located at 0.6 of the fluorescence intensity of the round nuclei, corresponded to elongated spermatid nuclei. Purified nuclei of epididymal and vas deferens spermatozoa displayed asymmetric fluorescence distributions. A minor peak at 0.8 the intensity of the round spermatid nuclei was tentatively assigned to elongating spermatids. 2 of the minor peaks, located at 1.7 and 2.0 times the fluorescence intensity of the round nuclei, corresponded to clumps of 2 haploid and diploid nuclei.

  7. Acridine Orange and Flow Cytometry: Which Is Better to Measure the Effect of Varicocele on Sperm DNA Integrity?

    Directory of Open Access Journals (Sweden)

    Essam-Elden M. Mohammed

    2015-01-01

    Full Text Available We evaluated the effect of varicocelectomy on semen parameters and levels of sperm DNA damage in infertile men. A total of 75 infertile men with varicocele and 40 fertile men (controls were included in this study. Semen analysis and sperm DNA damage expressed as the DNA fragmentation index using acridine orange staining and chromatin condensation test by flow cytometry were assessed before and 6 months after varicocelectomy. The patients were also followed up for 1 year for pregnancy outcome. Semen parameters were significantly lower in varicocele patients compared to controls (P<0.05. Mean percentages of sperm DNA fragmentation and sperm DNA chromatin condensation in patients were significantly higher than those in controls (P<0.05. After varicocelectomy, sperm DNA fragmentation improved significantly, whereas sperm chromatin condensation was not significantly changed. In 15 out of 75 varicocele patients, clinical pregnancy was diagnosed; those with positive pregnancy outcome had significant improvement in sperm count, progressive sperm motility, and sperm DNA fragmentation, but there was no significant difference in sperm DNA condensation compared to negative pregnancy outcome patients. We concluded from this study that acridine orange stain is more reliable method than flow cytometry in the evaluation of sperm DNA integrity after varicocelectomy.

  8. Comparison of four methods to evaluate sperm DNA integrity between mouse caput and cauda epididymidis

    Science.gov (United States)

    Pérez-Cerezales, Serafín; Miranda, Alberto; Gutiérrez-Adán, Alfonso

    2012-01-01

    It is well known that transit through the epididymis involves an increase in the compaction of sperm chromatin, which acquires fully condensed status at the caput epididymidis. The purpose of this study was to compare the terminal deoxyribonucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) assay, the comet assay, the sperm chromatin structure assay (SCSA) and the sperm chromatin dispersion (SCD) test by analysing spermatozoa from the caput and cauda epididymidis in order to demonstrate the ability of each technique to discriminate between different degrees of sperm maturity related to chromatin compaction and DNA fragmentation. Our results suggest that some populations of DNA-fragmented spermatozoa associated with immature sperm can only be identified using the comet assay and the SCSA but not with the SCD test or the TUNEL assay. PMID:22002436

  9. Disruption of Maternal DNA Repair Increases Sperm-DerivedChromosomal Aberrations

    Energy Technology Data Exchange (ETDEWEB)

    Marchetti, Francesco; Essers, Jeroun; Kanaar, Roland; Wyrobek,Andrew J.

    2007-02-07

    The final weeks of male germ cell differentiation occur in aDNA repair-deficient environment and normal development depends on theability of the egg to repair DNA damage in the fertilizing sperm. Geneticdisruption of maternal DNA double-strand break repair pathways in micesignificantly increased the frequency of zygotes with chromosomalstructural aberrations after paternal exposure to ionizing radiation.These findings demonstrate that radiation-induced DNA sperm lesions arerepaired after fertilization by maternal factors and suggest that geneticvariation in maternal DNA repair can modulate the risk of early pregnancylosses and of children with chromosomal aberrations of paternalorigin.

  10. Repeated Assessment by High-Throughput Assay Demonstrates that Sperm DNA Methylation Levels Are Highly Reproducible

    Science.gov (United States)

    Cortessis, Victoria K.; Siegmund, Kimberly; Houshdaran, Sahar; Laird, Peter W.; Sokol, Rebecca Z.

    2011-01-01

    Objective To assess reliability of high-throughput assay of sperm DNA methylation. Design Observational study comparing DNA methylation of sperm isolated from three divided and twelve longitudinally collected semen samples. Setting Academic Medical Center Patients One man undergoing screening semen analysis during evaluation of the infertile couple and two healthy fertile male volunteers. Interventions Spermatozoa were separated from seminal plasma and somatic cells using gradient separation. DNA was extracted from spermatozoa, and DNA methylation was assessed at 1,505 DNA-sequence specific sites. Main Outcome Measures Repeatability of sperm DNA methylation measures, estimated by correlation coefficients. Results DNA methylation levels were highly correlated within matched sets of divided samples (all r≥0.97) and longitudinal samples (average r=0.97). Conclusions The described methodology reliably assesses methylation of sperm DNA at large numbers of sites. Methylation profiles were consistent over time. High-throughput assessment of sperm DNA methylation is a promising tool for studying the role of epigenetic state in male fertility. PMID:22035967

  11. Sperm DNA fragmentation as a result of ultra-endurance exercise training in male athletes.

    Science.gov (United States)

    Vaamonde, D; Algar-Santacruz, C; Abbasi, A; García-Manso, J M

    2018-02-01

    Intensive sports practice seems to exert negative effects on semen parameters; in order to assess these effects, the objective of this study was to assess semen, including DNA fragmentation, and hormone parameters in elite triathletes. Twelve high-level triathletes preparing for a National Triathlon Championship participated in the study. The qualitative sperm parameters analysed were volume, sperm count, motility, morphology and DNA fragmentation; when needed, additional testing was performed. Assessed hormones were testosterone (T), cortisol (C) and testosterone-cortisol ratio (T/C). Maximum oxygen consumption and training characteristics were also assessed. Hormonal values and physical semen parameters were within normal ranges. DNA fragmentation showed high values (20.4 ± 6.1%). Round cells in semen were higher than normal (2.8 ± 1.5 million/ml), with the presence of macrophages. Correlations were found for several parameters: concentration of round cells positively correlated with progressive sperm motility (p = .01) and sperm morphology (p = .02); contrarily, the correlation found with DNA fragmentation was negative (p = .04). Sperm DNA fragmentation and the T/C ratio, however, were correlated in a positive manner (p = .03). As evidenced by the observed results, sperm DNA fragmentation is affected by high-level sports practice; therefore, high loads of endurance training could potentially interfere with the athlete's fertility potential. © 2017 Blackwell Verlag GmbH.

  12. Isolating Sperm from Cell Mixtures Using Magnetic Beads Coupled with an Anti-PH-20 Antibody for Forensic DNA Analysis.

    Science.gov (United States)

    Zhao, Xing-Chun; Wang, Le; Sun, Jing; Jiang, Bo-Wei; Zhang, Er-Li; Ye, Jian

    2016-01-01

    Vaginal swabs taken in rape cases usually contain epithelial cells from the victim and sperm from the assailant and forensic DNA analysis requires separation of sperm from these cell mixtures. PH-20, which is a glycosylphosphatidylinositol-anchored hyaluronidase located on the head of sperm, has important functions in fertilization. Here we describe a newly developed method for sperm isolation using anti-PH-20 antibody-coupled immunomagnetic beads (anti-PH-20 IMBs). Optical microscopy and scanning electron microscopy showed the IMBs recognized the head of sperm specifically and exhibited a great capacity to capture sperm cells. However, we found it necessary to incubate the IMB-sperm complex with DNase I before sperm lysis in order to remove any female DNA completely. We compared the sensitivity of anti-PH-20 IMBs in sperm and epithelial cell discrimination to those coated with a different anti-sperm antibody (anti-SP-10, anti-ADAM2 or anti-JLP). Only the anti-PH-20 IMBs succeeded in isolating sperm from cell mixtures at a sperm/epithelial cell ratio of 103:105. Further, our method exhibited greater power and better stability for sperm isolation compared to the traditional differential lysis strategy. Taken together, the anti-PH-20 IMB method described here could be effective for the isolation of sperm needed to obtain a single-sourced DNA profile as an aid to identifying the perpetrator in sexual assault cases.

  13. Relationship between Potential Sperm Factors Involved in Oocyte Activation and Sperm DNA Fragmentation with Intra-Cytoplasmic Sperm Injection Clinical Outcomes.

    Science.gov (United States)

    Tavalaee, Marziyeh; Kiani-Esfahani, Abbas; Nasr-Esfahani, Mohammad Hossein

    2017-01-01

    The present study aimed to simultaneously evaluate the association between expression of three potential factors [post-acrosomal sheath WW domain-binding protein (PAWP), phospholipase Cζ (PLCζ), and truncated form of the kit receptor (TR-KIT)] as candidates of oocyte activation with fertilization rate and early embryonic development. In this experimental study, semen samples were collected from 35 intra-cytoplasmic sperm injection (ICSI) candidates and analyzed according to World Health Organization criteria (2010). Each sample was divided into two parts. The first part was processed for insemination by density-gradient centrifugation (DGC) and the second part was prepared for assessment of sperm morphology (Papanicolaou staining), DNA fragmentation [transferase dUTP nick end labeling (TUNEL)], and three Sperm-borne oocyte-activating factor (s) (SOAFs)-PLCζ, PAWP, and TR-KIT. Significant positive correlations existed between the percentages of PLCζ, PAWP, and TR-KIT with fertilization rate. In addition, significant negative correlations existed between the percentage of DNA fragmentation with the percentages of PLCζ and PAWP. We did not find a relationship between percentages of PLCζ, PAWP, and TR-KIT with embryo quality and pregnancy rate (P>0.05). There was a significant negative correlation between percentage of DNA fragmentation with fertilization and embryo quality. Oocyte activation was associated with the studied sperm factors (PAWP, PLCζ, and TR-KIT). These factors might hold the potential to be considered as diagnostic factors in the assessment of semen samples to evaluate their potential to induce oocyte activation. In addition, we observed a significant association between DNA fragmentation with fertilization, as well as embryo quality and expression of PAWP and PLCζ, which indicated that men with high degrees of DNA fragmentation might require artificial oocyte activation. Whether such action should take place, and its cost and benefits should

  14. Effect of cryopreservation on the sperm DNA fragmentation dynamics of the bottlenose dolphin (Tursiops truncatus).

    Science.gov (United States)

    Sánchez-Calabuig, M J; López-Fernández, C; Johnston, S D; Blyde, D; Cooper, J; Harrison, K; de la Fuente, J; Gosálvez, J

    2015-04-01

    Sperm DNA fragmentation is one of the major causes of infertility; the sperm chromatin dispersion test (SCDt) evaluates this parameter and offers the advantage of species-specific validated protocol and ease of use under field conditions. The main purpose of this study was to evaluate sperm DNA fragmentation dynamics in both fresh and post-thaw bottlenose dolphin sperm using the SCDt following different cryopreservation protocols to gain new information about the post-thaw differential sperm DNA longevity in this species. Fresh and cryopreserved semen samples from five bottlenose dolphins were examined for sperm DNA fragmentation dynamics using the SCDt (Halomax(®)). Sperm DNA fragmentation was assessed immediately at collection and following cryopreservation (T0) and then after 0.5, 1, 4, 8, 24, 48 and 72 h incubation at 37°C. Serially collected ejaculates from four dolphins were frozen using different cryopreservation protocols in a TES-TRIS-fructose buffer (TTF), an egg-yolk-free vegetable lipid LP1 buffer (LP1) and human sperm preservation medium (HSPM). Fresh ejaculated spermatozoa initially showed low levels of DNA fragmentation for up to 48 h. Lower Sperm DNA fragmentation (SDF) was found in the second fresh ejaculate compared to the first when more than one sample was collected on the same day (p < 0.05); this difference was not apparent in any other seminal characteristic. While there was no difference observed in SDF between fresh and frozen-thawed sperm using the different cryopreservation protocols immediately after thawing (T0), frozen-thawed spermatozoa incubated at 37°C showed an increase in the rate of SDF after 24 h. Sperm frozen in the LP1(℗) buffer had higher levels (p < 0.05) of DNA fragmentation after 24- and 48-h incubation than those frozen in TTF or HSPM. No correlation was found between any seminal characteristic and DNA fragmentation in either fresh and/or frozen-thawed samples. © 2015 Blackwell Verlag GmbH.

  15. Sperm DNA damage-the effect of stress and everyday life factors.

    Science.gov (United States)

    Radwan, M; Jurewicz, J; Merecz-Kot, D; Sobala, W; Radwan, P; Bochenek, M; Hanke, W

    2016-07-01

    The clinical significance of sperm DNA damage lies in its association with natural conception rates and also might have a serious consequence on developmental outcome of the newborn. The aim of the present study is to determine whether stress and everyday life factors are associated with sperm DNA damage in adult men. The study population consisted of 286 men who attended the infertility clinic for diagnostic purposes and who had normal semen concentration of 20-300 m ml(-1) or with slight oligozoospermia (semen concentration of 15-20 m ml(-1)) (WHO, 1999). Participants were interviewed and provided a semen sample. The sperm chromatin structure assay was assessed using flow cytometry. In the present study, we found evidence for a relationship between sperm DNA damage parameters and everyday life factors. High and medium level of occupational stress and age increase DNA fragmentation index (P=0.03, P=0.004 and P=0.03, respectively). Other lifestyle factors that were positively associated with percentage of immature sperms (high DNA stainability index) included: obesity and cell phone use for more than 10 years (P=0.02 and P=0.04, respectively). Our findings indicate that stress and lifestyle factor may affect sperm DNA damage. Data from the present study showed a significant effect of age, obesity, mobile phone radiation and occupational stress on sperm DNA damage. As DNA fragmentation represents an extremely important parameter indicative of infertility and potential outcome of assisted reproduction treatment, and most of the lifestyle factors are easily modifiable, the information about factors that may affect DNA damage are important.

  16. Evaluation of potential protein biomarkers in patients with high sperm DNA damage.

    Science.gov (United States)

    Behrouzi, Bahar; Kenigsberg, Shlomit; Alladin, Naazish; Swanson, Sonja; Zicherman, Jonathan; Hong, Seok-Ho; Moskovtsev, Sergey I; Librach, Clifford L

    2013-06-01

    The laboratory evaluation of male infertility remains an essential area of research as 40-60% of infertility cases are attributable to male-related factors. Current sperm analysis methods add only partial information on sperm quality and fertility outcomes. The specific underlying cause of infertility in most cases is unknown, while a proportion of male infertility could be caused by molecular factors such as the absence or abnormal expression of some essential sperm proteins. The objective of this study was to screen for associations between sperm protein profiles and sperm concentration, motility, and DNA fragmentation index in patients undergoing fertility evaluation in a clinical setting. Based on those parameters, semen samples were categorized as either normal or abnormal. We screened 34 semen samples with various abnormal parameters and compared them to 24 normal control samples by using one dimensional (1-D) gel electrophoresis and mass-spectrometry. In this study, we anticipated to establish a normal sperm parameter profile which would be compared to abnormal sperm samples and reveal candidate proteins. Our preliminary results indicate that no normal uniform profile could be established, which affirms the complexity of male fertility and confirms the limitations of standard semen analysis. Four main protein groups were identified in correlation with abnormal DNA fragmentation and/or motility. The first group included sperm nuclear proteins such as the SPANX (sperm protein associated with the nucleus on the X chromosome) isoforms and several types of histones. The second group contained mitochondria-related functions and oxidative stress proteins including Mitochondrial Ferritin, Mitochondrial Single-Stranded DNA Binding Protein, and several isoforms of Peroxiredoxins. Two other protein groups were related to sperm motility such as microtubule-based flagellum and spindle microtubule as well as proteins related to the ubiquitin-proteasome pathway. Further

  17. Assessment of DNA integrity (COMET assay) in sperm cells of boron-exposed workers.

    Science.gov (United States)

    Duydu, Yalçin; Başaran, Nurşen; Ustündağ, Aylin; Aydin, Sevtap; Undeğer, Ulkü; Ataman, Osman Yavuz; Aydos, Kaan; Düker, Yalçin; Ickstadt, Katja; Waltrup, Britta Schulze; Golka, Klaus; Bolt, Hermann M

    2012-01-01

    An extension of a male reproductive study conducted in a boric acid/borate production zone at Bandırma, Turkey, is presented. The relation between DNA-strand breaks (COMET assay, neutral and alkaline version) in sperm cells and previously described sperm quality parameters was investigated in boron-exposed males. A correlation between blood boron levels and mean DNA-strand breaks in sperm was weak, and DNA-strand breaks in sperm were statistically not different between control and exposed groups. Therefore, increasing boron exposures had no additional contribution in addition to already pre-existing DNA-strand breaks in the sperm cells. Weak but statistically significant correlations between DNA-strand breaks and motility/morphology parameters of sperm samples were observed in the neutral version of the COMET assay, while correlations between the same variables were statistically not significant in the alkaline version. A likely reason for these negative results, even in highly exposed humans, is that experimental exposures that had led to reproductive toxicity in animals were significantly higher than any boron exposures, which may be reached under realistic human conditions.

  18. Evaluation of urinary metal concentrations and sperm DNA damage in infertile men from an infertility clinic.

    Science.gov (United States)

    Zhou, Yan; Fu, Xiao-Ming; He, Dong-Liang; Zou, Xue-Min; Wu, Cheng-Qiu; Guo, Wei-Zhen; Feng, Wei

    2016-07-01

    This study aimed to examine associations between urinary metal concentrations and sperm DNA damage. Thirteen metals [arsenic (As), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), lead (Pb), manganese (Mn), molybdenum (Mo), mercury (Hg), nickel (Ni), selenium (Se), and zinc (Zn)] were detected in urine samples of 207 infertile men from an infertility clinic using inductively coupled plasma mass spectrometry, and also, sperm DNA damage (tail length, percent DNA tail, and tail distributed moment) were assessed using neutral comet assay. We found that urinary Hg and Ni were associated with increasing trends for tail length (both p for trendsperm DNA damage. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Micronutrients intake is associated with improved sperm DNA quality in older men.

    Science.gov (United States)

    Schmid, Thomas E; Eskenazi, Brenda; Marchetti, Francesco; Young, Suzanne; Weldon, Rosana H; Baumgartner, Adolf; Anderson, Diana; Wyrobek, Andrew J

    2012-11-01

    To investigate whether lifestyle factors such as increased dietary intake of micronutrients reduce the risks of sperm DNA damage, and whether older men benefit more than younger men. Cross-sectional study design with equalized assignments into age groups. National laboratory and university. Nonclinical group of 22-80-year-old nonsmoking men (n = 80) who reported no fertility problems. Sperm DNA damage measured by alkaline and neutral DNA electrophoresis (i.e., sperm Comet assay). Sociodemographics, occupational exposures, medical and reproductive histories, and lifestyle habits were determined by questionnaire. The average daily dietary and supplement intake of micronutrients (vitamin C, vitamin E, b-carotene, zinc, and folate) was determined using the 100-item Modified Block Food Frequency Questionnaire (FFQ). Men with the highest intake of vitamin C had approximately 16% less sperm DNA damage (alkaline sperm Comet) than men with the lowest intake, with similar findings for vitamin E, folate, and zinc (but not β-carotene). Older men (>44 years) with the highest vitamin C intake had approximately 20% less sperm DNA damage compared with older men with the lowest intake, with similar findings for vitamin E and zinc. The older men with the highest intake of these micronutrients showed levels of sperm damage that were similar to those of the younger men. However, younger men (micronutrients surveyed. Men with higher dietary and supplement intake of certain micronutrients may produce sperm with less DNA damage, especially among older men. This raises the broader question of how lifestyle factors, including higher intakes of antioxidants and micronutrients, might protect somatic as well as germ cells against age-associated genomic damage. Copyright © 2012. Published by Elsevier Inc.

  20. Evaluation of effect of selected trace elements on dynamics of sperm dna fragmentation

    Directory of Open Access Journals (Sweden)

    Artur Wdowiak

    2015-12-01

    Full Text Available Introduction: Lead and cadmium can lead to negative effects on sperm chromatin DNA integrity. Copper, zinc and selenium are essential components of many enzymes which are important for reproduction. The aim of this research was to evaluate the influence of lead, cadmium, zinc, copper and selenium on the dynamics of semen DNA fragmentation.Material and methods: The present study concerned 85 fertile and 131 infertile men aged 25-35. DNA fragmentation in the samples was determined after 3 h, 6 h and 12 h. The Pb, Cd, Cu, Zn, and Se measurements were performed by the electrothermal-atomic absorption spectrometry method.Results: We found that sperm DNA fragmentation was a dynamic process which was intensified with an increase in the level of lead in seminal plasma. The levels of lead and cadmium were higher in seminal plasma of infertile men, compared to fertile men. The levels of zinc, copper and selenium in seminal plasma were higher in men with proven fertility, compared to infertile men, and did not exert a significant effect on the dynamics of sperm DNA fragmentation. The level of cadmium had no significant effect on intensification of sperm DNA fragmentation in time.Discussion: Reports in the literature which concern the effect of trace elements on human reproduction are equivocal. The present study confirmed an unfavourable effect, especially that of lead, on the dynamics of sperm DNA fragmentation; however, these studies need to be expanded and continued in the future.

  1. Genetic structure of European populations of Salmo salar L (Atlantic salmon) inferred from mitochondrial DNA

    DEFF Research Database (Denmark)

    Eg Nielsen, Einar; Hansen, Michael Møller; Loeschcke, V.

    1996-01-01

    The genetic relationships between the only natural population of Atlantic salmon (Salmo salar L.) in Denmark and seven other European salmon populations were studied using RFLP analysis of PCR amplified mitochondrial DNA segments. Six different haplotypes were detected by restriction enzyme...... analyses of the NADH dehydrogenase 1 segment, employing four endonucleases. Significant genetic differentiation was observed among populations. A hierarchical analysis of the distribution of the mtDNA variability revealed that only a small part was distributed among geographical groups within the study...

  2. Improving the resolution of cryopreserved X- and Y-sperm during DNA flow cytometric analysis with the addition of Percoll to quench the fluorescence of dead sperm

    NARCIS (Netherlands)

    Stap, J.; Hoebe, R. A.; Merton, J. S.; Haring, R. M.; Bakker, P. J.; Aten, J. A.

    1998-01-01

    The most effective method to control the sex of offspring is by separating X- from Y-bearing sperm on the basis of their DNA content. Sperm can be stained with Hoechst 33342 and efficiently sexed using a flow cytometer/cell sorter. However, applying this established assay to cryopreserved bovine

  3. The effect of sperm DNA fragmentation on miscarriage rates: a systematic review and meta-analysis.

    Science.gov (United States)

    Robinson, Lynne; Gallos, Ioannis D; Conner, Sarah J; Rajkhowa, Madhurima; Miller, David; Lewis, Sheena; Kirkman-Brown, Jackson; Coomarasamy, Arri

    2012-10-01

    Is there an association between high levels of sperm DNA damage and miscarriage? Miscarriage rates are positively correlated with sperm DNA damage levels. Most ejaculates contain a subpopulation of sperm with DNA damage, also referred to as DNA fragmentation, in the form of double or single-strand breaks which have been induced in the DNA prior to or following ejaculation. This DNA damage may be particularly elevated in some subfertile men, hence several studies have examined the link between sperm DNA damage levels and conception and miscarriage rates. A systematic review and meta-analysis of studies which examined the effect of sperm DNA damage on miscarriage rates was performed. Searches were conducted on MEDLINE, EMBASE and the Cochrane Library without any language restrictions from database inception to January 2012. We used the terms 'DNA damage' or 'DNA fragmentation' combined with 'miscarriage', 'abortion' or 'pregnancy' to generate a set of relevant citations. Data extraction was performed by two reviewers. Study quality was assessed using the Newcastle-Ottawa Scale. Meta-analysis of relative risks of miscarriage was performed with a random effects model. Subgroup analyses were performed by the type of DNA damage test, whether the sperm examined were prepared or from raw semen and for pregnancies resulting from IVF or ICSI treatment. We identified 16 cohort studies (2969 couples), 14 of which were prospective. Eight studies used acridine orange-based assays, six the TUNEL assay and two the COMET assay. Meta-analysis showed a significant increase in miscarriage in patients with high DNA damage compared with those with low DNA damage [risk ratio (RR) = 2.16 (1.54, 3.03), P < 0.00001)]. A subgroup analysis showed that the miscarriage association is strongest for the TUNEL assay (RR = 3.94 (2.45, 6.32), P < 0.00001). There is some variation in study characteristics, including the use of different assays and different thresholds for DNA damage and the

  4. [Impact of sperm DNA and acrosome integrity and acrosome reaction rate on outcomes of rescue intracytoplasmic sperm injection].

    Science.gov (United States)

    He, Yongzhi; Li, Dawen; Cheng, Junping; Huo, Zhongchao; Huang, Hongyi; Xiao, Xin

    2016-01-01

    Objective To explore the effects of sperm DNA integrity rate, acrosome integrity rate and acrosome reaction rate on the outcomes of rescue intracytoplasmic sperm injection (ICSI). This retrospective analysis was conducted among 97 infertile couples receiving rescue ICSI due to failure of in vitro fertilization procedures in our Reproductive Medicine Center. Of these 97 women, 41 had clinical pregnancy and 56 did not, and the effects of sperm DNA integrity rate (estimated by DNA fragmentation index, DFI), acrosome integrity rate and acrosome reaction rate on rescue ICSI outcomes were analyzed. No significant difference was found in paternal age, testosterone value, testicular volume, FSH, female patient' age or the number of eggs retrieved between the two groups (P>0.05), but the infertility years was significantly shorter in the pregnancy group than in the non-pregnancy group (Prate and cleavage rate were similar between the two groups (P>0.05), but the good embryo rate was significantly higher in the pregnancy group (Preaction rate did not differ significantly between the two groups (P>0.05), but the acrosome integrity rate was significantly higher in the pregnancy group (Prate, acrosome integrity or acrosome reaction rate were not correlated with the fertilization rate, cleavage rate or good embryo rate (P>0.05). The pregnancy rate, twin and single fetus rates were 42.3%, 10.3% and 32.0% in this cohort after recue ICSI, respectively. Rescue ICSI is an effective treatment after failed in vitro fertilization procedure, and sperm acrosome integrity rate is associated with the outcome of rescue ICSI.

  5. Evaluation of DNA Integrity of Cryopreserved Boer Goat (Capra hircus) Sperm Using Comet Assay at Various pH Conditions

    OpenAIRE

    Ismail Iswadi Mohd; Fazly Ann Zainalabidin; Mohamed Norhazilah; Mohd Padzil Rahman; Mazni Othman Abas; Fatimah Ibrahim Siti

    2013-01-01

    A very wide range of temperature changes during cryopreservation process reported cause complications to the sperm. One of it is DNA damage on the sperm which has been identified during the freezing and thawing process. Comet assay is a useful tool in sperm DNA integrity evaluation. Alkaline and neutral comet assay were able to differentiate DNA single- and double-strand breaks. The objective of this study was to evaluate the DNA integrity of post-thaw Boer goat (Capra hircus) sperm using com...

  6. Induced lipid peroxidation in ram sperm: semen profile, DNA fragmentation and antioxidant status.

    Science.gov (United States)

    Hamilton, Thais Rose dos Santos; de Castro, Letícia Signori; Delgado, Juliana de Carvalho; de Assis, Patrícia Monken; Siqueira, Adriano Felipe Perez; Mendes, Camilla Mota; Goissis, Marcelo Demarchi; Muiño-Blanco, Teresa; Cebrián-Pérez, José Álvaro; Nichi, Marcílio; Visintin, José Antonio; D'Ávila Assumpção, Mayra Elena Ortiz

    2016-04-01

    Action of reactive oxygen species, protamination failures and apoptosis are considered the most important etiologies of sperm DNA fragmentation. This study evaluated the effects of induced lipid peroxidation susceptibility on native semen profile and identified the mechanisms involved in sperm DNA fragmentation and testicular antioxidant defense on Santa Ines ram sperm samples. Semen was collected from 12 adult rams (Ovis aries) performed weekly over a 9-week period. Sperm analysis (motility, mass motility, abnormalities, membrane and acrosome status, mitochondrial potential, DNA fragmentation, lipid peroxidation and intracellular free radicals production); protamine deficiency; PRM1, TNP1 and TNP2 gene expression; and determination of glutathione peroxidase (GPx), glutathione reductase, catalase (CAT) and superoxide dismutase activity and immunodetection in seminal plasma were performed. Samples were distributed into four groups according to the sperm susceptibility to lipid peroxidation after induction with ascorbate and ferrous sulfate (low, medium, high and very high). The results were analyzed by GLM test and post hoc least significant difference. We observed an increase in native GPx activity and CAT immunodetection in groups with high susceptibility to induced lipid peroxidation. We also found an increase in total sperm defects, acrosome and membrane damages in the group with the highest susceptibility to induced lipid peroxidation. Additionally, the low mitochondrial membrane potential, susceptible to chromatin fragmentation and the PRM1 mRNA were increased in the group showing higher susceptibility to lipid peroxidation. Ram sperm susceptibility to lipid peroxidation may compromise sperm quality and interfere with the oxidative homeostasis by oxidative stress, which may be the main cause of chromatin damage in ram sperm. © 2016 Society for Reproduction and Fertility.

  7. The effect of environmental exposure to pyrethroids and DNA damage in human sperm.

    Science.gov (United States)

    Jurewicz, Joanna; Radwan, Michał; Wielgomas, Bartosz; Sobala, Wojciech; Piskunowicz, Marta; Radwan, Paweł; Bochenek, Michał; Hanke, Wojciech

    2015-01-01

    The present study was designed to investigate whether environmental exposure to pyrethroids was associated with sperm DNA damage. Between January 2008 and April 2011 286 men under 45 years of age with a normal sperm concentration of 15-300 10(6)/ml [WHO 2010] were recruited from an infertility clinic in Lodz, Poland. Participants were interviewed and provided urine, saliva, and semen samples. The pyrethroids metabolites: 3-phenoxybenzoic acid (3PBA), cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (CDCCA), trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (TDCCA), and cis-2,2-dibromovinyl-2,2-dimethylcyclopropane-carboxylic acid (DBCA) were analyzed in the urine using a validated gas chromatography ion-tap mass spectrometry method. Sperm DNA damage was assessed using a flow cytometry based on sperm chromatin structure assay (SCSA). A positive association was observed between CDCCA >50th percentile and the percentage of medium DNA fragmentation index (M DFI) and percentage of immature sperms (HDS) (p = 0.04, p = 0.04 respectively). The level of 3PBA >50th percentile in urine was positively related to the percentage of high DNA fragmentation index (H DFI) (p = 0.03). The TDCCA, DBCA levels, and the sum of pyrethroid metabolites were not associated with any sperm DNA damage measures. Our results suggest that environmental pyrethroid exposure may affect sperm DNA damage measures index indicated the reproductive effects of pyrethroid exposure on adult men. In view of the importance of human reproductive health and the widespread usage of pyrethroids, it is important to further investigate these correlations.

  8. High resolution DNA flow cytometry of boar sperm cells in identification of boars carrying cytogenetic aberrations

    DEFF Research Database (Denmark)

    Larsen, Jacob; Christensen, Knud; Larsen, Jørgen K

    2004-01-01

    -resolution DNA flow cytometry on DAPI-stained sperm cell nuclei (CV animals judged normal by their fertility statistics and a series of seven animals with known reciprocal translocations, a model...... for identifying sperm cells from cytogenetically aberrant animals was proposed. This model was applied to a series of 50 uncharacterized animals. The model successfully identified a mosaic or chimaeric carrier of an aberrant X chromosome. However, implementation of this technique for screening purposes would...

  9. Modern human sperm freezing: Effect on DNA, chromatin and acrosome integrity

    Directory of Open Access Journals (Sweden)

    Tahereh Rahiminia

    2017-08-01

    Conclusion: Sperm in Vapour was healthier in terms of DNA, chromatin and acrosome integrity. In contrast of higher motility and normal morphology; DNA, chromatin and acrosome integrity were decreased in Vit. However, these findings were more acceptable in SSV or Vapour.

  10. Human semen quality and sperm DNA damage assessed by comet assay in clinical groups.

    Science.gov (United States)

    Ramzan, Muhammad Haris; Ramzan, Muhammad; Khan, Muhammad Mumtaz; Ramzan, Faiqah; Wahab, Fazal; Khan, Muhammad Aslam; Jillani, Musharraf; Shah, Mohsin

    2015-01-01

    About 10%-15% of couples around the world suffer from infertility. Male infertility is responsible directly or indirectly in approximately 60% of cases. A deficiency in semen is the most common cause of male infertility. The study included 180 male subjects aged 18-50 years with 26 fertile and 154 infertile. The infertile subjects were further subdivided according to the WHO guidelines of semen analysis (2010) into different clinical groups. Sperm DNA damage was estimated using a neutral comet assay. Plasma gonadotropin and testosterone levels were measured using a chemiluminescence assay. The results of the study revealed no significant differences, in semen volume, pH, and liquefaction time between the fertile and all infertile groups. However, sperm concentration, sperm vitality, and sperm motility were significantly lower in all infertile groups as compared to the fertile males. The morphological forms of the sperm and its DNA fragmentation varied significantly between the fertile and infertile males. Reproductive hormone levels were observed to be significantly lower in the infertile than in the fertile males. Sperm DNA fragmentation was higher in all of the infertile subjects as compared to the fertile ones. Reproductive hormone levels varied significantly between the infertile patients and the fertile ones.

  11. Relationships between seminal plasma metals/metalloids and semen quality, sperm apoptosis and DNA integrity.

    Science.gov (United States)

    Wang, Yi-Xin; Wang, Peng; Feng, Wei; Liu, Chong; Yang, Pan; Chen, Ying-Jun; Sun, Li; Sun, Yang; Yue, Jing; Gu, Long-Jie; Zeng, Qiang; Lu, Wen-Qing

    2017-05-01

    This study aimed to investigate the relationships between environmental exposure to metals/metalloids and semen quality, sperm apoptosis and DNA integrity using the metal/metalloids levels in seminal plasma as biomarkers. We determined 18 metals/metalloids in seminal plasma using an inductively coupled plasma-mass spectrometry among 746 men recruited from a reproductive medicine center. Associations of these metals/metalloids with semen quality (n = 746), sperm apoptosis (n = 331) and DNA integrity (n = 404) were evaluated using multivariate linear and logistic regression models. After accounting for multiple comparisons and confounders, seminal plasma arsenic (As) quartiles were negatively associated with progressive and total sperm motility using multivariable linear regression analysis, which were in accordance with the trends for increased odds ratios (ORs) for below-reference semen quality parameters in the logistic models. We also found inverse correlations between cadmium (Cd) quartiles and progressive and total sperm motility, whereas positive correlations between zinc (Zn) quartiles and sperm concentration, between copper (Cu) and As quartiles and the percentage of tail DNA, between As and selenium (Se) quartiles and tail extent and tail distributed moment, and between tin (Sn) categories and the percentage of necrotic spermatozoa (all Ptrendmale reproductive health, whereas Zn may be beneficial to sperm concentration. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Influence of extended incubation time on Human sperm chromatin condensation, sperm DNA strand breaks and their effect on fertilisation rate.

    Science.gov (United States)

    Ahmed, I; Abdelateef, S; Laqqan, M; Amor, H; Abdel-Lah, M A; Hammadeh, M E

    2018-02-14

    The purpose of this study was to determine influence of extended incubation time on sperm chromatin condensation and DNA strand breaks and their effect on fertilisation rate. Forty couples undergoing ICSI therapy were included. Semen was prepared by PureSperm gradient centrifugation and divided into two parts. The first part (G1) was used immediately for ICSI, whereas the second part (G2) was kept in the incubator at 37°C, 5% and 90% Humidity for 5 hr, and thereafter, the capacitated spermatozoa were used for ICSI. The TUNEL test and chromomycin CMA3 were used to evaluate the DNA strand breaks and chromatin condensation respectively. The percentage of condensed chromatin was 73.92 ± 12.70 in the group 1 and 81.13 ± 10.31% in group 2 (p = .001). However, the double-strand breaks were 11.15 ± 8.67% in G.1 and 16.30 ± 11.12% in G.2. (p = .001). Fertilisation rate in the (Group 1) was 62.45% and 69.17% in (Group 2). There was a positive correlation between condensed chromatin and fertilisation rate (r = 0.846, p = .001) and a negative correlation with DNA double-strand breaks (r = -0.802; p = .001). In conclusion, the prolonged sperm incubation (5 hr) leads to a higher chromatin condensation and to a significantly increased number of DNA strands double breaks with no influence on fertilisation rates. © 2018 Blackwell Verlag GmbH.

  13. [Values of the sperm deformity index, acrosome abnormity rate, and sperm DNA fragmentation index of optimized sperm in predicting IVF fertilization failure].

    Science.gov (United States)

    Jiang, Wei-jie; Jin, Fan; Zhou, Li-ming

    2016-02-01

    To investigate the values of the sperm deformity index (SDI), acrosome abnormity rate (AAR), and DNA fragmentation index (DFI) of optimized sperm in the prediction of fertilization failure (fertilization rate fertilization (IVF). We selected 695 cycles of conventional IVF for pure oviductal infertility in this study, including 603 cycles of normal fertilization and 92 cycles of fertilization failure. On the day of oocyte retrieval, we examined sperm morphology, acrosome morphology, and DNA fragmentation using the Diff-Quik, PSA-FITC and SCD methods. We established the joint predictor (JP) by logistic equation and analyzed the values of different parameters in predicting fertilization failure with the receiver operating characteristic (ROC) curve. The fertilization rate was negatively correlated with SDI (r = - 0.07; P = 0.03), AAR (r = -0.49; P fertilization group were 1.24 ± 0.20, (7.75 ± 2.28)%, and (7.87 ± 3.15)%, and those in the fertilization failure group were 1.42 ± 0.15, (12.02 ± 3.06)%, and (13.32 ± 4.13)%, respectively, all with statistically significant differences between the two groups (P fertilization failure ( OR = 2.68, 14.11, and 3.85; P = 0.01, fertilization failure were approximately 1.45, 10%, and 12%. The SDI, AAR and DFI of optimized sperm are closely associated with the fertilization rate, and all have the value for predicting fertilization failure in IVF. The AAR is more valuable than the other single predictors, but JP is more effective than the AAR.

  14. Deteksi Kerusakan DNA Spermatozoa Semen Segar dan Semen Beku Sapi Menggunakan Pewarnaan Toluidine Blue (DETECTION OF SPERM DNA DAMAGE INFRESH AND FROZEN SEMEN USING TOLUIDINE BLUE STAINING

    Directory of Open Access Journals (Sweden)

    Langgeng Priyanto

    2015-05-01

    Full Text Available Sperm DNA integrity is very important in the success of fertilization and embryo development.Toluidine blue (TB is a sensitive staining to examine the chromatin structure of sperm. The aim of thisstudy was to detect the sperm DNA damage before and after freezing using TB. Semen was obtained fromeight superior bulls (two Brahman, two Ongole, two Simental and two Limosin belong to Lembang ArtificialInsemination Centre. Semen was collected twice a week using artificial vagina, then was evaluatedmacrocopically and microscopically after collected, including sperm motility, viability, plasma membraneintegrity (MI and acrosome intact (AI, sperm concentration, abnormality and sperm DNA integrity. Thesemen that been used in this study showed the total motility was more than 70%, sperm concentrationwas more than 1000x106, and the sperm abnormality was below 20%. The result showed that the qualityof semen after freezing processed was decreased significantly (P<0.05 on percentage of sperm motility,viability, MI and AI, whereas there was no different on DNA integrity (P>0.05. Sperm DNA integrity offresh and frozen semen were 93.91±4.77% and 92.06 ±2.41% respectively. The decrease of DNA integritywas low (1.84% compared to motility (28.3%, viability (21.6%, MI (14.1%, and AI (11.8%. In conclusion,toluidine blue can be used to detect of DNA damage and the freezing process will not decrease the DNAintegrity.

  15. Inter- and intra-laboratory standardization of TUNEL assay for assessment of sperm DNA fragmentation.

    Science.gov (United States)

    Ribeiro, S; Sharma, R; Gupta, S; Cakar, Z; De Geyter, C; Agarwal, A

    2017-05-01

    One of the challenges with the sperm DNA fragmentation results is the inconsistency and the large variability in the results obtained by different techniques. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay quantifies the incorporation of fluoresceinated dUTP into single- and double-strand DNA breaks by labeling the 3'-OH terminal with TdT. The goal of this study was optimize the TUNEL protocol for assessment of sperm DNA fragmentation by standardization of the method and comparison of the data across two reference laboratories (i) at Basel, Switzerland and (ii) Cleveland Clinic, Ohio, USA. Semen samples from 31 subjects grouped into three cohorts. Sperm DNA fragmentation was data measured by two experienced operators at two different laboratories using identical semen samples, assay kit, protocol and acquisition settings using identical flow cytometers (BD Accuri C6). No significant differences were observed between the duplicates in any of the experiments performed. By including an additional washing step after fixation in paraformaldehyde, a high correlation was seen between the two laboratories (r = 0.94). A strong positive correlation was observed between the average sperm DNA fragmentation rates (r = 0.719). The mean sperm DNA fragmentation measured in each laboratory was similar. Both flow cytometers were identical in their settings and performance. This inter- and intra-laboratory study establishes that TUNEL is a reproducible assay when utilizing a standardized staining protocol and flow cytometer acquisition settings. Standardization and consensual guidelines for TUNEL validate the assay and establishes TUNEL as a robust test for measuring sperm DNA fragmentation especially in a multicenter setting. © 2017 American Society of Andrology and European Academy of Andrology.

  16. Sperm DNA adducts impair fertilization during ICSI but not during IVF.

    Directory of Open Access Journals (Sweden)

    Piotr Widłak

    2008-04-01

    Full Text Available Many studies emphasize the influence of the status of spermatozoal nucleus on fertilization, mainly with regard to DNA fragmentation. This study was undertaken to analyze the influence of DNA adducts content in spermatozoa on fertilization during assisted reproduction. Ovarian hyperstimulation, oocyte retrieval and laboratory work-up in 61 IVF (in vitro fertilization and 118 ICSI (intracytoplasmic sperm injection first cycles were performed according to the same protocol. Semen analysis was made according to WHO Manual (1999. DNA adducts assay in spermatozoa was performed by 32Ppostlabeling method. In total 331 fertilizable oocytes were obtained during IVF and 659 during ICSI. Both groups differed significantly by sperm count, motility and morphology but not by the concentration of DNA adducts in spermatozoa (0.0306 +/- 0.0217 in IVF versus 0.0373 +/- 0.0321 in ICSI. The fertilization rate during IVF was significantly influenced by sperm count (p=0.0002 and motility (p=0.0037 but not by DNA adducts concentration (p=0.30528, whereas during ICSI was positively influenced by sperm motility (p=0.04669 and negatively by DNA adducts concentration (p=0.00796. DNA adducts concentration in spermatozoa significantly negatively influences fertilization rate during ICSI, but not during IVF.

  17. SALMON SOFT ROE DNA ON BLOOD CELLS SECRETION OF CYTOKINES IN HEALTHY DONORS

    Directory of Open Access Journals (Sweden)

    L. N. Fedjanina

    2005-01-01

    Full Text Available Abstract. Salmon soft roe DNA influence on healthy donors blood cells secretion of early hemopoietic factors (IL-3, GM-CSF, TNFα as well as biologically active substance influence on cytokine balance of Тh1 and Тh2 responses (IFNγ, IL-10 in vitro was studied. It is established, that DNA has modulatory effect on secretion of all investigated cytokines - IL-3, GM-CSF, TNFα, INFγ and IL-10 by blood cells of healthy donors, increases their initially low concentration, reduces initially high and does not have essential influence at an average level of their secretion. Under action of DNA IFNγ level (stimulation index=3,3 increases more significantly than IL-10 level (stimulation index =1,9. Thus, salmon soft roe DNA possesses immunomodulatory properties.

  18. Exposure to persistent organic pollutants and sperm DNA methylation changes in Arctic and European populations.

    Science.gov (United States)

    Consales, Claudia; Toft, Gunnar; Leter, Giorgio; Bonde, Jens Peter E; Uccelli, Raffaella; Pacchierotti, Francesca; Eleuteri, Patrizia; Jönsson, Bo A G; Giwercman, Aleksander; Pedersen, Henning S; Struciński, Paweł; Góralczyk, Katarzyna; Zviezdai, Valentyna; Spanò, Marcello

    2016-04-01

    Persistent organic pollutants (POPs), such as PCBs (polychlorinated biphenyls) and DDT [1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane], are environmental contaminants with potential endocrine disrupting activity. DNA methylation levels in peripheral blood lymphocytes have been associated with serum concentrations of POPs in Greenland Inuit and Korean populations. Greenland Inuits are characterized by the highest worldwide POP levels. In this cross-sectional study we evaluated the relationship between serum POP concentrations and DNA methylation levels in sperm of non-occupationally exposed fertile men from Greenland, Warsaw (Poland), and Kharkiv (Ukraine). Serum levels of PCB-153 [1,2,4-trichloro-5-(2,4,5-trichlorophenyl)benzene], as a proxy of the total PCBs body burden, and of p,p'-DDE [1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene], the main metabolite of DDT were measured. Sperm DNA methylation level was assessed globally by flow cytometric (FCM) immunodetection of 5-methyl-cytosines and at specific repetitive DNA sequences (Alu, LINE-1, Satα) by PCR-pyrosequencing after bisulfite conversion. Multivariate linear regression analysis was applied to investigate correlations between serum POP concentrations and DNA methylation. No consistent associations between exposure to POPs and sperm DNA methylation at repetitive DNA sequences were detected. A statistically significant global decrease in methylation was associated with exposure to either POP by FCM analysis. This is the first study to investigate environmental exposure to POPs and DNA methylation levels considering sperm as the target cells. Although POP exposure appears to have a limited negative impact on sperm DNA methylation levels in adult males, the global hypomethylation detected by one of the methods applied suggests that further investigation is warranted. © 2016 Wiley Periodicals, Inc.

  19. One abstinence day decreases sperm DNA fragmentation in 90 % of selected patients.

    Science.gov (United States)

    Pons, Isabel; Cercas, Rosa; Villas, Celia; Braña, Cristina; Fernández-Shaw, Sylvia

    2013-09-01

    The aim of this prospective descriptive study was to evaluate the efficacy of reducing sexual abstinence as a strategy to decrease sperm DNA fragmentation. Men with one or more of the following characteristics were included in the study: older than 44, smoking more than 10 cigarettes per day, with a body mass index over 25, diabetes mellitus, varicocele, a previous chemotherapy treatment, severe oligozoospermia, prostatitis, cryptorchidism, having a partner with recurrent miscarriage and/or implantation failure, poor embryo morphology and/or fertilization failure. Patients were asked to produce a semen sample after 3 to 7 abstinence days which was subjected to a sperm DNA fragmentation test. When DNA fragmentation was above or equal to 30 %, it was considered to be altered. Patients with increased DNA fragmentation were asked to produce another semen sample following a "one abstinence day protocol". This protocol required producing up to three semen samples with 1 day of abstinence and measuring sperm DNA fragmentation. Four hundred and sixteen patients produced a first semen sample after a sexual abstinence of 3 to 7 days. Sperm DNA fragmentation was altered in 46 samples (11.1 %). Thirty five patients with increased DNA fragmentation samples completed the "one abstinence day protocol". DNA fragmentation decreased to normal values in one of the three attempts in 91.4 % of the patients: 81.3 % in the first attempt, 12.5 % in the second try and 6.3 % in the third. This approach could be a simple, low-cost and effective way to decrease sperm DNA damage to normal values.

  20. ReProComet: a new in vitro method to assess DNA damage in mammalian sperm.

    Science.gov (United States)

    Cordelli, Eugenia; Fresegna, Anna Maria; D'Alessio, Alessia; Eleuteri, Patrizia; Spanò, Marcello; Pacchierotti, Francesca; Villani, Paola

    2007-10-01

    The increasing request of chemical safety assessment demands for the validation of alternative methods to reduce the resort to animal experimentation. Methods that evaluate reproductive toxicity are among those requiring the largest use of animals. Presently, no validated in vitro alternative exists for the assessment of reproductive toxicity. Mammalian sperm are sensitive targets of DNA-reactive chemicals, which form premutagenic adducts. Here, we propose a new method based on comet assay to detect DNA damage induced by potential germ cell mutagens in bull sperm available from assisted reproduction practices. In somatic cells, chemical-induced adducts can be revealed by comet assay that detects DNA breaks produced during adduct repair. Mature sperm, however, are devoid of repair enzymes, and adducts are processed only after fertilization. For this reason, comet assay is not sensitive to detect DNA lesions induced in sperm by most chemicals. To overcome such limitation, we developed a modified comet assay based on the addition of a protein extract from HeLa cells to agarose-embedded sperm on microscopic slides. To test the method, sperm were treated in vitro with methyl methanesulfonate (MMS) or melphalan (MLP) and comet assay was conducted both with and without protein supplementation. No effect of MMS or MLP was detected without protein supplementation; on the contrary, a clear-cut dose-dependent effect was measured after addition of the cell extract. These results represent a proof of concept of a novel in vitro mutagenicity test on sperm that could offer a promising approach to complement previously validated in vivo germ cell genotoxicity assays.

  1. Relationships between sperm DNA fragmentation, sperm apoptotic markers and serum levels of CB-153 and p,p'-DDE in European and Inuit populations

    DEFF Research Database (Denmark)

    Stronati, A; Manicardi, G C; Cecati, M

    2006-01-01

    Persistent organochlorine pollutants (POPs) are suspected to interfere with hormone activity and the normal homeostasis of spermatogenesis. We investigated the relationships between sperm DNA fragmentation, apoptotic markers identified on ejaculated spermatozoa and POP levels in the blood of 652....... Sperm DNA fragmentation was measured by using the TUNEL assay, whereas immunofluorescence methods were utilized for detecting pro-apoptotic (Fas) and anti-apoptotic (Bcl-xL) markers. Both TUNEL assay and apoptotic markers were statistically differed across the four populations. No correlation between...... neither sperm DNA fragmentation nor apoptotic sperm parameters and the large variations in POPs exposure was observed for the separate study groups. However, considering the European populations taken together, we showed that both %TUNEL positivity and Bcl-xL were related to CB-153 serum levels, whereas...

  2. Widespread epigenetic abnormalities suggest a broad DNA methylation erasure defect in abnormal human sperm.

    Directory of Open Access Journals (Sweden)

    Sahar Houshdaran

    2007-12-01

    Full Text Available Male-factor infertility is a common condition, and etiology is unknown for a high proportion of cases. Abnormal epigenetic programming of the germline is proposed as a possible mechanism compromising spermatogenesis of some men currently diagnosed with idiopathic infertility. During germ cell maturation and gametogenesis, cells of the germ line undergo extensive epigenetic reprogramming. This process involves widespread erasure of somatic-like patterns of DNA methylation followed by establishment of sex-specific patterns by de novo DNA methylation. Incomplete reprogramming of the male germ line could, in theory, result in both altered sperm DNA methylation and compromised spermatogenesis.We determined concentration, motility and morphology of sperm in semen samples collected by male members of couples attending an infertility clinic. Using MethyLight and Illumina assays we measured methylation of DNA isolated from purified sperm from the same samples. Methylation at numerous sequences was elevated in DNA from poor quality sperm.This is the first report of a broad epigenetic defect associated with abnormal semen parameters. Our results suggest that the underlying mechanism for these epigenetic changes may be improper erasure of DNA methylation during epigenetic reprogramming of the male germ line.

  3. Sperm DNA fragmentation abnormalities in men from couples with a history of recurrent miscarriage.

    Science.gov (United States)

    Leach, Mikaela; Aitken, Robert J; Sacks, Gavin

    2015-08-01

    Previous studies have described an association between sperm with DNA damage and a history of recurrent miscarriage (RM), although it is not clear whether there is benefit in screening for sperm DNA fragmentation and to what extent DNA fragmentation impacts upon RM. To identify what proportion of couples experiencing RM are affected by DNA fragmentation abnormalities. In this retrospective study, between 2008 and 2013, couples with a history of recurrent miscarriage (≥3 first trimester miscarriages) were investigated comprehensively for known causes (karyotype, uterine, antiphospholipid syndrome, thrombophilia) and also by semen analysis, including DNA fragmentation [sperm chromatin structure analysis (SCSA)]. Statistical analysis was performed on SPSS software with significance taken as P fragmentation index (DFI) of 9.50%. Normal levels were found in 70.5% of men (DFI 30%). Couples with otherwise unexplained recurrent miscarriage had significantly higher DFI than those with other causes identified on routine screening (P = 0.012). In couples experiencing RM, 30% (32/108) of men had sperm with high levels of DNA fragmentation (DFI > 15%). This may be a contributing factor to the clinical syndrome of RM, and future clinical trials of therapies for these couples are warranted. © 2015 The Royal Australian and New Zealand College of Obstetricians and Gynaecologists.

  4. Sperm nuclear DNA fragmentation and its association with semen quality in Greek men.

    Science.gov (United States)

    Evgeni, E; Lymberopoulos, G; Touloupidis, S; Asimakopoulos, B

    2015-12-01

    Due to the limitations of conventional semen analysis in predicting a man's fertility potential, sperm DNA fragmentation was recently introduced as a novel marker of sperm quality. This prospective study was undertaken to investigate the associations between conventional seminal parameters and DNA fragmentation in Greek men. A total of 669 subject data were evaluated in two groups, normozoospermic (n = 184) and non-normozoospermic (n = 485), according to the WHO 2010 (WHO Laboratory Manual for the Examination and Processing of Human Semen, 5th edn. World Health Organization), reference limits. For all the subjects, semen volume, sperm concentration, total count, rapid and total progressive motility and morphology were recorded following the WHO 2010 methods and DNA fragmentation was assessed by the sperm chromatin dispersion assay. An inverse correlation was established between DNA fragmentation and all conventional seminal parameters except semen volume in men with seminal profiles below the reference limits, with statistical significance for rapid and total progressive motility. Normozoospermic men exhibited lower levels of DNA fragmentation than their non-normozoospermic counterparts, even though the values were not always below 30%. DNA fragmentation testing and traditional semen analysis should therefore be considered as complementary diagnostic tools in a comprehensive evaluation of male infertility. © 2015 Blackwell Verlag GmbH.

  5. Alkaline and neutral Comet assay profiles of sperm DNA damage in clinical groups.

    Science.gov (United States)

    Ribas-Maynou, J; García-Peiró, A; Abad, C; Amengual, M J; Navarro, J; Benet, J

    2012-03-01

    The analysis of sperm DNA fragmentation has become a new marker to predict male infertility, and many techniques have been developed. The sperm Comet assay offers the possibility of differentiating single- and double-stranded DNA (ssDNA and dsDNA) breaks, which could have different effects on fertility. The objective of this study was to perform a descriptive characterization of different groups of patients, such as those with asthenoteratozoospermic (ATZ) with or without varicocele, oligoasthenoteratozoospermic (OATZ) or balanced chromosome rearrangements, as compared with fertile donors. The Comet assay was used to investigate sperm samples for ssDNA and dsDNA breaks. The analysis of alkaline and neutral Comet assays in different groups of patients showed different sperm DNA damage profiles. Most fertile donors presented low values for ssDNA and dsDNA fragmentation (low-equivalent Comet profile), which would be the best prognosis for achieving a pregnancy. OATZ, ATZ and ATZ with varicocele presented high percentages of ssDNA and dsDNA fragmentation (high-equivalent Comet assay profile), ATZ with varicocele being associated with the worst prognosis, due to higher levels of DNA fragmentation. Rearranged chromosome carriers display a very high variability and, interestingly, two different profiles were seen: a high-equivalent Comet assay profile, which could be compatible with a bad prognosis, and a non-equivalent Comet assay profile, which has also been found in three fertile donors. Comet assay profiles, applied to different clinical groups, may be useful for determining prognosis in cases of male infertility.

  6. Effects of reduced seminal enzymatic antioxidants on sperm DNA fragmentation and semen quality of Tunisian infertile men.

    Science.gov (United States)

    Atig, Fatma; Kerkeni, Abdelhamid; Saad, Ali; Ajina, Mounir

    2017-03-01

    To evaluate levels of sperm DNA fragmentation and enzymatic antioxidant status in seminal plasma of Tunisian fertile and infertile men in order to assess the effects of seminal oxidative stress on sperm DNA integrity and semen quality. Semen samples from 100 infertile patients (40 oligoasthenoteratozoospermics, 31 teratozoospermics and 29 asthenozoospermics) and 50 fertile men (controls) were analyzed for DNA fragmentation by TUNEL assay and biochemical parameters. Seminal antioxidant activities (Superoxide dismutase, Glutathione peroxidase and Catalase) and malondialdehyde concentrations were measured spectrophotometrically. Sperm DNA fragmentation and malondialdehyde levels in infertile groups were more elevated than controls. Nevertheless, the activities of the antioxidant enzymes were significantly lower in abnormal groups compared to normozoospermics. Sperm DNA fragmentation was closely and positively correlated to malondialdehyde levels (r = 0.37, P = 0.008); meanwhile, reduced seminal antioxidant profile was negatively associated to sperm DNA fragmentation. Interestingly, we noted also that sperm DNA fragmentation was negatively correlated to sperm motility (r = -0.54, P fragmentation can be due to the impaired seminal enzymatic antioxidant profile and increased Lipid peroxidation. Our results sustain that the evaluation of sperm DNA fragmentation and seminal oxidative biomarkers in infertile men is recommended as a consistent prognostic tool for male infertility assessment.

  7. Transfection of the inner cell mass and lack of a unique DNA sequence affecting the uptake of exogenous DNA by sperm as shown by dideoxy sequencing analogues.

    Science.gov (United States)

    Cabrera, M; Chan, P J; Kalugdan, T H; King, A

    1997-02-01

    The purpose of this study was to determine whether exogenous DNA internalized into blastocysts after transference from DNA-carrier sperm are localized at the inner cell mass or trophoblast cells and to identify differences in uptake of exogenous DNA fragments by sperm due to unique DNA sequences. Mouse blastocysts at the hatching stage were exposed to migrating human sperm cells carrying exogenous DNA fragments synthesized from the E6-E7 conserved gene regions of human papillomavirus (HPV) types 16 and 18. After an interaction period of 2 hr, the transfected blastocysts were washed several times to remove extraneous sperm and the blastocysts were dissected into groups of cells derived from the inner cell mass and trophoblasts. The cells were analyzed by polymerase chain reaction (PCR) for the presence of HPV DNA fragments. In the second part of the experiment, thawed donor (N = 10) sperm cells were pooled, washed, and divided into two fractions. The first (control) fraction was added with formalin and further divided and added with a 35S-radiolabeled G, A, T, or C sequencing mixture. The second fraction was similarly treated but the formalin step was omitted from the treatment. After an hour of incubation at 37 degrees C, the sperm specimens were washed several times by centrifugation and DNA extracted by the GeneReleaser method. The extracted DNA were processed on sequence gels, and the autoradiographs analyzed. Mouse blastocysts transfected by carrier sperm with DNA from HPV types 16 and 18 showed localization of the HPV DNA to both the inner cell mass and trophoblast cells. Negative controls consisting of untreated human sperm and untreated mouse blastocysts did not reveal any evidence of HPV DNA. The positive sperm control generated expected DNA fragments from HPV types 16 and 18. In the second experiment, the intensities of the DNA fragments in the G, A, T, and C columns from low to high molecular weights were not different from the positive control bands

  8. Effects of Cinnamon (C. zeylanicum) Bark Oil Against Taxanes-Induced Damages in Sperm Quality, Testicular and Epididymal Oxidant/Antioxidant Balance, Testicular Apoptosis, and Sperm DNA Integrity.

    Science.gov (United States)

    Sariözkan, Serpil; Türk, Gaffari; Güvenç, Mehmet; Yüce, Abdurrauf; Özdamar, Saim; Cantürk, Fazile; Yay, Arzu Hanım

    2016-01-01

    The aim of this study was to investigate whether cinnamon bark oil (CBO) has protective effect on taxanes-induced adverse changes in sperm quality, testicular and epididymal oxidant/antioxidant balance, testicular apoptosis, and sperm DNA integrity. For this purpose, 88 adult male rats were equally divided into 8 groups: control, CBO, docetaxel (DTX), paclitaxel (PTX), DTX+PTX, DTX+CBO, PTX+CBO, and DTX+PTX+CBO. CBO was given by gavage daily for 10 weeks at the dose of 100 mg/kg. DTX and PTX were administered by intraperitoneal injection at the doses of 5 and 4 mg/kg/week, respectively, for 10 weeks. DTX+PTX and DTX+PTX+CBO groups were treated with DTX during first 5 weeks and PTX during next 5 weeks. DTX, PTX, and their mixed administrations caused significant decreases in absolute and relative weights of all reproductive organs, testosterone level, sperm motility, concentration, glutathione level, and catalase activity in testicular and epididymal tissues. They also significantly increased abnormal sperm rate, testicular and epididymal malondialdehyde level, apoptotic germ cell number, and sperm DNA fragmentation and significantly damaged the histological structure of testes. CBO consumption by DTX-, PTX-, and DTX+PTX-treated rats provided significant ameliorations in decreased relative weights of reproductive organs, decreased testosterone, decreased sperm quality, imbalanced oxidant/antioxidant system, increased apoptotic germ cell number, rate of sperm with fragmented DNA, and severity of testicular histopathological lesions induced by taxanes. In conclusion, taxanes cause impairments in sperm quality, testicular and epididymal oxidant/antioxidant balance, testicular histopathological structure, and sperm DNA integrity, and long-term CBO consumption protects male reproductive system of rats.

  9. Multiple Determinations of Sperm DNA Fragmentation Show That Varicocelectomy Is Not Indicated for Infertile Patients with Subclinical Varicocele

    Directory of Open Access Journals (Sweden)

    Agustín García-Peiró

    2014-01-01

    Full Text Available Varicocele is one of the most common causes of low semen quality, which is reflected in high percentages of sperm cells with fragmented DNA. While varicocelectomy is usually performed to ameliorate a patient’s fertility, its impact on sperm DNA integrity in the case of subclinical varicocele is poorly documented. In this study, multiple DNA fragmentation analyses (TUNEL, SCD, and SCSA were performed on semen samples from sixty infertile patients with varicocele (15 clinical varicoceles, 19 clinical varicoceles after surgical treatment, 16 subclinical varicoceles, and 10 subclinical varicoceles after surgical treatment. TUNEL, SCD, and SCSA assays all showed substantial sperm DNA fragmentation levels that were comparable between subclinical and clinical varicocele patients. Importantly, varicocelectomy did improve sperm quality in patients with clinical varicocele; however, this was not the case in patients with subclinical varicocele. In summary, although infertile patients with clinical and subclinical varicocele have similar sperm DNA quality, varicocelectomy should only be advised for patients with clinical varicocele.

  10. Multiple Determinations of Sperm DNA Fragmentation Show That Varicocelectomy Is Not Indicated for Infertile Patients with Subclinical Varicocele

    Science.gov (United States)

    García-Peiró, Agustín; Ribas-Maynou, Jordi; Oliver-Bonet, María; Navarro, Joaquima; Checa, Miguel A.; Nikolaou, Alexandros; Amengual, María J.; Abad, Carlos; Benet, Jordi

    2014-01-01

    Varicocele is one of the most common causes of low semen quality, which is reflected in high percentages of sperm cells with fragmented DNA. While varicocelectomy is usually performed to ameliorate a patient's fertility, its impact on sperm DNA integrity in the case of subclinical varicocele is poorly documented. In this study, multiple DNA fragmentation analyses (TUNEL, SCD, and SCSA) were performed on semen samples from sixty infertile patients with varicocele (15 clinical varicoceles, 19 clinical varicoceles after surgical treatment, 16 subclinical varicoceles, and 10 subclinical varicoceles after surgical treatment). TUNEL, SCD, and SCSA assays all showed substantial sperm DNA fragmentation levels that were comparable between subclinical and clinical varicocele patients. Importantly, varicocelectomy did improve sperm quality in patients with clinical varicocele; however, this was not the case in patients with subclinical varicocele. In summary, although infertile patients with clinical and subclinical varicocele have similar sperm DNA quality, varicocelectomy should only be advised for patients with clinical varicocele. PMID:24967335

  11. Modern human sperm freezing: Effect on DNA, chromatin and acrosome integrity.

    Science.gov (United States)

    Rahiminia, Tahereh; Hosseini, Akram; Anvari, Morteza; Ghasemi-Esmailabad, Saeed; Talebi, Ali Reza

    2017-08-01

    Presence of vitrification method in sperm freezing and the introduction of solid surface vitrification beside rapid freezing in vapour, opens an easy and safe way to help infertility centres. While the effects of cryopreservation on motility, morphology and viability of sperm are documented, the question of the probable alteration of sperm DNA, chromatin and acrosome integrity after freezing and thawing procedures in different methods is still controversial. Normal sample were collected according to WHO strict criteria. Sperm suspensions were mixed 1:1 with 0.5 M sucrose and divided into four equal aliquots for freezing: fresh, nitrogen direct immersion vitrification (Vit), solid surface vitrification (SSV) and in vapour (Vapour). Sperm suspensions were transferred into a 0.25 ml sterile plastic. Then straw was inserted inside the 0.5 ml straw. For thawing, the straws were immersed in a 42 °C water bath. Beside the sperm parameters, we assessed the acrosome reaction by double staining, chromatin integrity by toluidine blue (Tb) and chromomycin A3 (CMA3) and DNA integrity by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) respectively. In progressive motility, the highest rate occurred in Vit (39.9 ± 13.3). Moreover, the lowest rate of immotile sperm was in Vit (32.7 ± 16.3). In normal morphology, the group Vit was similar to the fresh, while SSV and Vapour were significantly different from the fresh. The percentage of acrosome-reacted sperms was more in Vit (81.3 ± 10.2) than the fresh group. TUNEL+ results showed that DNA fragmentation was significantly increased in Vit (p-value = 0.025). While in SSV and Vapour results were comparable to fresh. There was a significant correlation between TUNEL+ and normal morphology, TB, CMA3 and presence of intact acrosome. Sperm in Vapour was healthier in terms of DNA, chromatin and acrosome integrity. In contrast of higher motility and normal morphology; DNA, chromatin and acrosome

  12. Detection and mapping of mtDNA SNPs in Atlantic salmon using high throughput DNA sequencing

    Directory of Open Access Journals (Sweden)

    Olafsdottir Gudbjorg

    2011-04-01

    Full Text Available Abstract Background Approximately half of the mitochondrial genome inherent within 546 individual Atlantic salmon (Salmo salar derived from across the species' North Atlantic range, was selectively amplified with a novel combination of standard PCR and pyro-sequencing in a single run using 454 Titanium FLX technology (Roche, 454 Life Sciences. A unique combination of barcoded primers and a partitioned sequencing plate was employed to designate each sequence read to its original sample. The sequence reads were aligned according to the S. salar mitochondrial reference sequence (NC_001960.1, with the objective of identifying single nucleotide polymorphisms (SNPs. They were validated if they met with the following three stringent criteria: (i sequence reads were produced from both DNA strands; (ii SNPs were confirmed in a minimum of 90% of replicate sequence reads; and (iii SNPs occurred in more than one individual. Results Pyrosequencing generated a total of 179,826,884 bp of data, and 10,765 of the total 10,920 S. salar sequences (98.6% were assigned back to their original samples. The approach taken resulted in a total of 216 SNPs and 2 indels, which were validated and mapped onto the S. salar mitochondrial genome, including 107 SNPs and one indel not previously reported. An average of 27.3 sequence reads with a standard deviation of 11.7 supported each SNP per individual. Conclusion The study generated a mitochondrial SNP panel from a large sample group across a broad geographical area, reducing the potential for ascertainment bias, which has hampered previous studies. The SNPs identified here validate those identified in previous studies, and also contribute additional potentially informative loci for the future study of phylogeography and evolution in the Atlantic salmon. The overall success experienced with this novel application of HT sequencing of targeted regions suggests that the same approach could be successfully applied for SNP mining

  13. Microchip-based cell lysis and DNA extraction from sperm cells for application to forensic analysis.

    Science.gov (United States)

    Bienvenue, Joan M; Duncalf, Natalie; Marchiarullo, Daniel; Ferrance, Jerome P; Landers, James P

    2006-03-01

    The current backlog of casework is among the most significant challenges facing crime laboratories at this time. While the development of next-generation microchip-based technology for expedited forensic casework analysis offers one solution to this problem, this will require the adaptation of manual, large-volume, benchtop chemistry to small volume microfluidic devices. Analysis of evidentiary materials from rape kits where semen or sperm cells are commonly found represents a unique set of challenges for on-chip cell lysis and DNA extraction that must be addressed for successful application. The work presented here details the development of a microdevice capable of DNA extraction directly from sperm cells for application to the analysis of sexual assault evidence. A variety of chemical lysing agents are assessed for inclusion in the extraction protocol and a method for DNA purification from sperm cells is described. Suitability of the extracted DNA for short tandem repeat (STR) analysis is assessed and genetic profiles shown. Finally, on-chip cell lysis methods are evaluated, with results from fluorescence visualization of cell rupture and DNA extraction from an integrated cell lysis and purification with subsequent STR amplification presented. A method for on-chip cell lysis and DNA purification is described, with considerations toward inclusion in an integrated microdevice capable of both differential cell sorting and DNA extraction. The results of this work demonstrate the feasibility of incorporating microchip-based cell lysis and DNA extraction into forensic casework analysis.

  14. Exploring the use of environmental DNA to determine the species of salmon redds

    Science.gov (United States)

    Strobel, Burke; Laramie, Matthew; Pilliod, David

    2017-01-01

    Annual redd counts are used to monitor the status and trends of salmonid populations, but methods to easily and reliably determine which of sympatric species made specific redds are lacking. We explored whether environmental DNA (eDNA) analysis might prove useful for determining the species of salmon redds. We collected eDNA samples from the interstitial spaces of redds of Chinook Salmon Oncorhynchus tshawytscha, redds of Coho Salmon O. kisutch, and areas of undisturbed gravel (n = 10, each), as well as from the water column adjacent to each of those sites in the Sandy River basin, Oregon, USA during the fall of 2013. The concentrations of Chinook and Coho eDNA were quantified within each sample using real-time PCR. The water in the interstitial spaces of redds contained significantly higher eDNA concentrations of the species that made the redd than (1) the other species and (2) the adjacent water column. In contrast, neither Chinook nor Coho eDNA was significantly more concentrated than the other in the water from the interstitial spaces of undisturbed gravel. The interstitial water of undisturbed gravel contained significantly higher eDNA concentrations of Coho than the adjacent water column. In contrast, Chinook eDNA concentration was similar in the interstitial water of undisturbed gravel and the adjacent water column. Both species’ redds had significantly higher concentrations of their respective species’ eDNA than did undisturbed gravel, but conclusions were confounded by differences in the timing and locations of sampling. This initial investigation highlights the potential value and some of the complexity of using eDNA analysis to indicate redd species.

  15. A systematic review and meta-analysis to determine the effect of sperm DNA damage onin vitrofertilization and intracytoplasmic sperm injection outcome.

    Science.gov (United States)

    Simon, Luke; Zini, Armand; Dyachenko, Alina; Ciampi, Antonio; Carrell, Douglas T

    2017-01-01

    Sperm DNA damage is prevalent among infertile men and is known to influence natural reproduction. However, the impact of sperm DNA damage on assisted reproduction outcomes remains controversial. Here, we conducted a meta-analysis of studies on sperm DNA damage (assessed by SCSA, TUNEL, SCD, or Comet assay) and clinical pregnancy after IVF and/or ICSI treatment from MEDLINE, EMBASE, and PUBMED database searches for this analysis. We identified 41 articles (with a total of 56 studies) including 16 IVF studies, 24 ICSI studies, and 16 mixed (IVF + ICSI) studies. These studies measured DNA damage (by one of four assays: 23 SCSA, 18 TUNEL, 8 SCD, and 7 Comet) and included a total of 8068 treatment cycles (3734 IVF, 2282 ICSI, and 2052 mixed IVF + ICSI). The combined OR of 1.68 (95% CI: 1.49-1.89; P sperm DNA damage affects clinical pregnancy following IVF and/or ICSI treatment. In addition, the combined OR estimates of IVF (16 estimates, OR = 1.65; 95% CI: 1.34-2.04; P sperm DNA damage has a negative effect on clinical pregnancy following IVF and/or ICSI treatment.

  16. Deteksi Kerusakan DNA Spermatozoa Semen Segar dan Semen Beku Sapi Menggunakan Pewarnaan Toluidine Blue (DETECTION OF SPERM DNA DAMAGE INFRESH AND FROZEN SEMEN USING TOLUIDINE BLUE STAINING

    Directory of Open Access Journals (Sweden)

    Langgeng Priyanto

    2015-05-01

    Full Text Available Sperm DNA integrity is very important in the success of fertilization and embryo development.Toluidine blue (TB is a sensitive staining to examine the chromatin structure of sperm. The aim of thisstudy was to detect the sperm DNA damage before and after freezing using TB. Semen was obtained fromeight superior bulls (two Brahman, two Ongole, two Simental and two Limosin belong to Lembang ArtificialInsemination Centre. Semen was collected twice a week using artificial vagina, then was evaluatedmacrocopically and microscopically after collected, including sperm motility, viability, plasma membraneintegrity (MI and acrosome intact (AI, sperm concentration, abnormality and sperm DNA integrity. Thesemen that been used in this study showed the total motility was more than 70%, sperm concentrationwas more than 1000x106, and the sperm abnormality was below 20%. The result showed that the qualityof semen after freezing processed was decreased significantly (P0.05. Sperm DNA integrity offresh and frozen semen were 93.91±4.77% and 92.06 ±2.41% respectively. The decrease of DNA integritywas low (1.84% compared to motility (28.3%, viability (21.6%, MI (14.1%, and AI (11.8%. In conclusion,toluidine blue can be used to detect of DNA damage and the freezing process will not decrease the DNAintegrity.

  17. Potential importance of transition metals in the induction of DNA damage by sperm preparation media.

    Science.gov (United States)

    Aitken, R J; Finnie, J M; Muscio, L; Whiting, S; Connaughton, H S; Kuczera, L; Rothkirch, T B; De Iuliis, G N

    2014-10-10

    What are the mechanisms by which the preparation of spermatozoa on discontinuous density gradients leads to an increase in oxidative DNA damage? The colloidal silicon solutions that are commonly used to prepare human spermatozoa for assisted reproduction technology (ART) purposes contain metals in concentrations that promote free radical-mediated DNA damage. Sporadic reports have already appeared indicating that the use of colloidal silicon-based discontinuous density gradients for sperm preparation is occasionally associated with the induction of oxidative DNA damage. The cause of this damage is however unknown. This study comprised a series of experiments designed to: (i) confirm the induction of oxidative DNA damage in spermatozoa prepared on commercially available colloidal silicon gradients, (ii) compare the levels of damage observed with alterative sperm preparation techniques including an electrophoretic approach and (iii) determine the cause of the oxidative DNA damage and develop strategies for its prevention. The semen samples employed for this analysis involved a cohort of >50 unselected donors and at least three independent samples were used for each component of the analysis. The setting was a University biomedical science laboratory. The major techniques employed were: (i) flow cytometry to study reactive oxygen species generation, lipid peroxidation and DNA damage, (ii) computer-aided sperm analysis to measure sperm movement and (iii) inductively coupled mass spectrometry to determine the elemental composition of sperm preparation media. Oxidative DNA damage is induced in spermatozoa prepared on PureSperm(®) discontinuous colloidal silicon gradients (P colloidal silicon gradients can result in oxidative DNA damage. The results are of immediate relevance to the development of safe, effective protocols for the preparation of spermatozoa for ART purposes. The study was funded by the Australian Health and Medical Research Council. One of the authors (R

  18. Sperm DNA: organization, protection and vulnerability: from basic science to clinical applications--a position report.

    NARCIS (Netherlands)

    Barratt, C.L.; Aitken, R.J.; Bjorndahl, L.; Carrell, D.T.; Boer, P. de; Kvist, U.; Lewis, S.E.; Perreault, S.D.; Perry, M.J.; Ramos, L.; Robaire, B.; Ward, S.; Zini, A.

    2010-01-01

    This article reports the results of the most recent in a series of EHSRE workshops designed to synthesize the current state of the field in Andrology and provide recommendations for future work (for details see Appendix). Its focus is on methods for detecting sperm DNA damage and potential

  19. High resolution DNA flow cytometry of boar sperm cells in identification of boars carrying cytogenetic aberrations

    DEFF Research Database (Denmark)

    Larsen, Jacob; Christensen, Knud; Larsen, Jørgen K

    2004-01-01

    The cytogenetic quality of boars used for breeding determines the litter outcome and thus has large economical consequences. Traditionally, quality controls based on the examination of simple karyograms are time consuming and sometimes give uncertain results. As an alternative, the use of high-re......-resolution DNA flow cytometry on DAPI-stained sperm cell nuclei (CV...

  20. Low folate in seminal plasma is associated with increased sperm DNA damage

    NARCIS (Netherlands)

    Boxmeer, Jolanda C.; Smit, Marij; Utomo, Elaine; Romijn, Johannes C.; Eijkemans, Marinus J. C.; Lindemans, Jan; Laven, Joop S. E.; Macklon, Nick S.; Steegers, Eric A. P.; Steegers-Theunissen, Regine P. M.

    Objective: To determine associations between vitamin B status, homocysteine (tHcy), semen parameters, and sperm DNA damage. Design: Observational study. Setting: A tertiary referral fertility clinic. Patient(s): Two hundred fifty-one men of couples undergoing in vitro fertilization (IVF) or

  1. The Comet assay for detection of DNA damage in canine sperm.

    Science.gov (United States)

    Pereira, A F; Borges, P; Fontbonne, A; Cardoso, L; Gaivão, I; Martins-Bessa, A

    2017-12-01

    Sperm DNA integrity is a fundamental prerequisite in fertilization and embryo development. Among DNA integrity tests, the Comet assay is an accurate and sensitive test for the detection of sperm oxidative damage. The aim of this work was to evaluate sperm oxidative damage using the Comet assay and to study the correlation between Comet and routine assays for the evaluation of semen quality. Dogs were divided in two groups: group A (n = 6), comprising dogs with abnormal spermiogram, that is astheno-, terato- or oligoasthenoteratozoospermic (OAT); and group B (n = 8), comprising normospermic dogs. The distribution of sperm oxidative damage was significantly different between the two groups (p = .001): group A-median: 31.55%, interquartile range (IQR): 30.18-38.01; group B-median: 0.90%, IQR: 0.65-1.96. The correlation between oxidative damage and abnormal morphology was high (r = .846; p sperm quality, the Comet assay has ample potential for clinical and research purposes in dogs. © 2017 Blackwell Verlag GmbH.

  2. Sperm DNA Integrity Assessment: A New Tool in Diagnosis and Treatment of Fertility

    Directory of Open Access Journals (Sweden)

    Mona Bungum

    2012-01-01

    Full Text Available Infertility affects 15% of all couples. Although male infertility factors with reduced semen quality are contributing to about half of all involuntary childlessness, the value of standard semen parameters in prediction of fertility in vivo and choice of proper method for assisted reproduction is limited. In the search for better markers of male fertility, during the last 10 years, assessment of sperm DNA integrity has emerged as a strong new biomarker of semen quality that may have the potential to discriminate between infertile and fertile men. Sperm DNA Fragmentation Index (DFI as assessed by the flow cytometric Sperm Chromatin Structure Assay (SCSA can be used for evaluation of sperm chromatin integrity. The biological background for abnormal DFI is not completely known, but clinical data show that DFI above 30% is associated with very low chance for achieving pregnancy in natural way or by insemination, but not in vitro. Already when the DFI is above 20%, the chance of natural pregnancy may be reduced, despite other sperm parameters being normal. Thus this method may explain a significant proportion of cases of unexplained infertility and can be beneficial in counselling involuntary childless couples need of in vitro fertilisation.

  3. Additional deleterious effects of alcohol consumption on sperm parameters and DNA integrity in diabetic mice.

    Science.gov (United States)

    Pourentezari, M; Talebi, A R; Mangoli, E; Anvari, M; Rahimipour, M

    2016-06-01

    The aim of this study was to survey the impact of alcohol consumption on sperm parameters and DNA integrity in experimentally induced diabetic mice. A total of 32 adult male mice were divided into four groups: mice of group 1 served as control fed on basal diet, group 2 received streptozotocin (STZ) (200 mg kg(-1) , single dose, intraperitoneal) and basal diet, group 3 received alcohol (10 mg kg(-1) , water soluble) and basal diet, and group 4 received STZ and alcohol for 35 days. The cauda epididymidis of each mouse was dissected and placed in 1 ml of pre-warm Ham's F10 culture medium for 30 min. The swim-out spermatozoa were analysed for count, motility, morphology and viability. Sperm chromatin quality was evaluated with aniline blue, toluidine blue, acridine orange and chromomycin A3 staining. The results showed that all sperm parameters had significant differences (P alcohol and diabetes can cause abnormalities in sperm parameters and chromatin quality. In addition, alcohol consumption in diabetic mice can intensify sperm chromatin/DNA damage. © 2015 Blackwell Verlag GmbH.

  4. New approach to assess sperm DNA fragmentation dynamics: Fine-tuning mathematical models.

    Science.gov (United States)

    Ortiz, Isabel; Dorado, Jesús; Morrell, Jane; Gosálvez, Jaime; Crespo, Francisco; Jiménez, Juan M; Hidalgo, Manuel

    2017-01-01

    Sperm DNA fragmentation (sDF) has been proved to be an important parameter in order to predict in vitro the potential fertility of a semen sample. Colloid centrifugation could be a suitable technique to select those donkey sperm more resistant to DNA fragmentation after thawing. Previous studies have shown that to elucidate the latent damage of the DNA molecule, sDF should be assessed dynamically, where the rate of fragmentation between treatments indicates how resistant the DNA is to iatrogenic damage. The rate of fragmentation is calculated using the slope of a linear regression equation. However, it has not been studied if sDF dynamics fit this model. The objectives of this study were to evaluate the effect of different after-thawing centrifugation protocols on sperm DNA fragmentation and elucidate the most accurate mathematical model (linear regression, exponential or polynomial) for DNA fragmentation over time in frozen-thawed donkey semen. After submitting post-thaw semen samples to no centrifugation (UDC), sperm washing (SW) or single layer centrifugation (SLC) protocols, sDF values after 6 h of incubation were significantly lower in SLC samples than in SW or UDC. Coefficient of determination (R2) values were significantly higher for a second order polynomial model than for linear or exponential. The highest values for acceleration of fragmentation (aSDF) were obtained for SW, followed by SLC and UDC. SLC after thawing seems to preserve longer DNA longevity in comparison to UDC and SW. Moreover, the fine-tuning of models has shown that sDF dynamics in frozen-thawed donkey semen fit a second order polynomial model, which implies that fragmentation rate is not constant and fragmentation acceleration must be taken into account to elucidate hidden damage in the DNA molecule.

  5. Impaired protamination and sperm DNA damage in a Nellore bull with high percentages of morphological sperm defects in comparison to normospermic bulls

    Directory of Open Access Journals (Sweden)

    J.T. Carreira

    2015-04-01

    Full Text Available The routine semen evaluation assessing sperm concentration, motility and morphology, does not identify subtle defects in sperm chromatin architecture. Bulls appear to have stable chromatin, with low levels of DNA fragmentation. However, the nature of fragmentation and its impact on fertility remain unclear and there are no detailed reports characterizing the DNA organization and damage in this species. The intensive genetic selection, the use of artificial insemination and in vitro embryo production associated to the cryopreservation process can contribute to the chromatin damage and highlights the importance of sperm DNA integrity for the success of these technologies. Frozen-thawed semen samples from three ejaculates from a Nellore bull showed high levels of morphological sperm abnormalities (55.8±5.1%, and were selected for complementary tests. Damage of acrosomal (76.9±8.9% and plasma membranes (75.7±9.3% as well as sperm DNA strand breaks (13.8±9.5% and protamination deficiency (3.7±0.6% were significantly higher compared to the values measured in the semen of five Nellore bulls with normospermia (24.3±3.3%; 24.5±6.1%; 0.6±0.5%; 0.4±0.6% for acrosome, plasma membrane, DNA breaks and protamine deficiency, respectively (P<0.05. Motility and percentage of spermatozoa with low mitochondrial potential showed no differences between groups. This study shows how routine semen analyses (in this case morphology may point to the length and complexity of sperm cell damage emphasizing the importance of sperm function testing.

  6. Interaction mode between methylene blue-Sm(III complex and herring sperm DNA

    Directory of Open Access Journals (Sweden)

    Li-sheng Ding

    2012-12-01

    Full Text Available Spectroscopic and viscosity methods were applied to investigate the interaction between methylene blue (MB-Sm(III complex and herring sperm DNA by using acridine orange as a spectral probe in Tris-HCl buffer (pH 7.40. By means of molar ratio method, the binding ratios between MB-Sm(III and DNA were determined as nSm(III:nMB =1:3, nMB-Sm(III complex:n DNA = 8:1, and the apparent molar absorption coefficient of εMB-Sm(III-DNA was found to be 7.01×105 M-1•cm-1. The bonding constants at different temperatures Kθ292K = 7.35×105 M-1, Kθ310K = 1.25×105 M-1 were obtained by double reciprocal method. Thermodynamic function computation demonstrates that ΔrSmӨ is the primary driving power of the interaction between MB-Sm(III and herring sperm DNA. By combination analysis of the Scatchard method, circular dichroism spectroscopy and viscosity method, it suggests that the interaction mode between MB-Sm(III complex and herring sperm DNA are a mixed binding, which contains partial intercalation and electrostatic interaction.DOI: http://dx.doi.org/10.4314/bcse.v26i3.8

  7. Evaluation of DNA Integrity of Cryopreserved Boer Goat (Capra hircus Sperm Using Comet Assay at Various pH Conditions

    Directory of Open Access Journals (Sweden)

    Ismail Iswadi Mohd

    2013-05-01

    Full Text Available A very wide range of temperature changes during cryopreservation process reported cause complications to the sperm. One of it is DNA damage on the sperm which has been identified during the freezing and thawing process. Comet assay is a useful tool in sperm DNA integrity evaluation. Alkaline and neutral comet assay were able to differentiate DNA single- and double-strand breaks. The objective of this study was to evaluate the DNA integrity of post-thaw Boer goat (Capra hircus sperm using comet assay. The semen was collected using artificial vagina technique and cryopreserved in liquid nitrogen by slow-freezing cryopreservation protocol. DNA integrity of the sperm was evaluated using both neutral and alkaline comet assay immediately after thawing process. Tail moment and Olive tail moment were significantly higher under neutral condition (pH10 compared to alkaline condition (pH13 on post-thawed sperm (p<0.05. As a conclusion, cryopreservation of Boer goat sperm produces DNA double-strand breaks. The optimum detection of DNA strand breaks is at pH10.

  8. Differential effects of human papillomavirus DNA types on p53 tumor-suppressor gene apoptosis in sperm.

    Science.gov (United States)

    Lee, Cathy A; Huang, Christopher T F; King, Alan; Chan, Philip J

    2002-06-01

    Sperm DNA undergoes apoptotic fragmentation when exposed to HPV DNA. Details of the specific gene regions targeted by HPV in sperm are lacking. The objective of this study was to determine the integrity of exons 5 and 8 of the p53 gene in sperm exposed to HPV DNA. Washed sperm were exposed to either HLA-DQA1 (control) or HPV type 6b/11, 16, 18, 31, or 33 DNA fragments for 24 h at 37 degrees C. The integrity of sperm p53 exons 5 and 8 was assessed using a novel DNA disc chip assay based on comparative genomic hybridization. Fragmentation of exon 5 occurred after exposure to HPV DNA type 18. In contrast, only exon 8 was affected by HPV type 16. HPV DNA from type 31 or 33 was without effect on the p53 exons. Sperm motility but not hyperactivation was reduced in all HPV groups. The data suggest that different HPV types preferentially degrade different exons of important genes. Decreased motility but not hyperactivation in HPV-exposed sperm suggests retention of some fertilizing capacity and the possibility of transmitting virus-destabilized genes through fertilization.

  9. Indices of methylation in sperm DNA from fertile men differ between distinct geographical regions.

    Science.gov (United States)

    Consales, C; Leter, G; Bonde, J P E; Toft, G; Eleuteri, P; Moccia, T; Budillon, A; Jönsson, B A G; Giwercman, A; Pedersen, H S; Ludwicki, J K; Zviezdai, V; Heederik, D; Spanò, M

    2014-09-01

    Which are the main determinants, if any, of sperm DNA methylation levels? Geographical region resulted associated with the sperm methylation status assessed on genome-wide repetitive sequences. DNA methylation level, assessed on repetitive sequences from peripheral blood lymphocyte, can vary with age, gender, alcohol consumption and white blood cell counts. A cross-sectional study. Individual data were collected from 269 young healthy men of proven fertility living in three geographical regions: Inuits from Greenland, Caucasians from Warsaw (Poland) and Kharkiv (Ukraine). Semen samples were collected between May 2002 and February 2004 and aliquots were immediately frozen. We estimated sperm DNA global methylation level (DGML) in two ways. First DNA methylation in repetitive DNA sequences (LINE-1, Satα and Alu) was quantified by PCR pyrosequencing after bisulfite conversion and second by flow cytometry (FCM) using fluorescently labeled monoclonal antibodies anti-5-methylcytosine. We analyzed whether personal characteristics and habits, body mass index, semen quality parameters, sperm chromatin integrity, biomarkers of accessory gland function and the plasma concentration of reproductive hormones were associated with sperm DNA methylation levels in men. Associations were evaluated by analysis of variance and linear regression analyses. The geographical location emerged as the main determinant when using the methylation level in repetitive sequences. FCM DGML results were not associated with those from repetitive sequence analysis. No other consistent associations between methylation markers and the assessed variables were identified across countries. The methods used are only surrogates of the actual sperm methylome and the methylation levels at individual specific loci were not explored. Sperm DGML is relatively independent from semen quality parameters and is a new candidate biomarker for epidemiological studies of the impact of environmental contaminants on male

  10. DNA fragmentation in frozen sperm of Equus asinus: Zamorano-Leonés, a breed at risk of extinction.

    Science.gov (United States)

    Cortés-Gutiérrez, E I; Crespo, F; Gosálvez, A; Dávila-Rodríguez, M I; López-Fernández, C; Gósalvez, J

    2008-05-01

    The dynamics of sperm DNA fragmentation (sDF) and sperm viability were analyzed in frozen-thawed sperm samples of Equus asinus (Zamorano-Leonés), a breed at risk of extinction. Sperm DNA fragmentation was assessed using an adaptation of the sperm chromatin dispersion test developed for stallions in five different frozen samples. Sperm were thawed and incubated at different temperatures (37 degrees C, 25 degrees C, and 4 degrees C) and sDF was assessed at different times and compared. The mean sDF after thawing at the beginning of the experiment was 18.20+/-14.77% and did not differ significantly from the results of a neutral comet assay (22.0+/-19.34%). The tendency in the sDF of all donkeys indicated that sperm DNA is more sensitive to breakage when incubated at 37 degrees C than when incubated at 25 degrees C or 4 degrees C. Interestingly, the tendency was not the same when different animals were compared, and differences in sDF dynamics were established among individuals. sDF correlated negatively with sperm viability in some individuals but not in others. From a conservation perspective, sDF analysis may offer a new way to assess sperm quality in endangered breeds in order to identify and select the best semen samples for artificial reproduction purposes. In particular, we recommend for artificial insemination the use of semen samples with a slow increase in sDF with time after thawing.

  11. Association between the MTHFR-C677T isoform and structure of sperm DNA.

    Science.gov (United States)

    Cornet, Dominique; Cohen, Marc; Clement, Arthur; Amar, Edouard; Fournols, Laetitia; Clement, Patrice; Neveux, Paul; Ménézo, Yves

    2017-10-01

    The aim of this study is to evaluate whether the MTHFR contribution to male decreased fertility can be attributable to anomalies in sperm nucleus DNA structure in relation to defective methylation. The presence of MTHFR C677T, contributing at most for male infertility, was determined from a venous blood sample, using real-time polymerase chain reaction (PCR). Sperm DNA fragmentation (SDF) and sperm nucleus decondensation index (SDI) measurements were performed using acridine orange and flow cytometry. SDF and SDI of men MTHFR C677T heterozygous or homozygous were compared to a general population of hypo-fertile patients RESULTS: SDF is not increased either in homozygous or heterozygous carriers of MTHFR C677T. In contrast, SDI is increased with a higher incidence in homozygous (p = 0.0006) than in heterozygous (p = 0.029) patients when compared with the control population. Using a critical threshold of 20% for either SDI or SDF assayed with our technique, the percentage of patients with results higher than this value is not significant with respect to fragmentation (0.128), but is significantly increased for decondensation (0.0003). Defective methylation linked to MTHFR may contribute to sperm pathogenesis via increased SDI. After DNA structure analysis, especially SDI, treatment with 5-methyl tetrahydrofolate (MTHF), the metabolite downstream from the action of MTHFR, should be recommended as a therapeutic approach. Patients with a high SDI should be tested for MTHFR isoforms as part of a healthcare policy.

  12. Double-stranded DNA breaks hidden in the neutral Comet assay suggest a role of the sperm nuclear matrix in DNA integrity maintenance.

    Science.gov (United States)

    Ribas-Maynou, J; Gawecka, J E; Benet, J; Ward, W S

    2014-04-01

    We used a mouse model in which sperm DNA damage was induced to understand the relationship of double-stranded DNA (dsDNA) breaks to sperm chromatin structure and to the Comet assay. Sperm chromatin fragmentation (SCF) produces dsDNA breaks located on the matrix attachment regions, between protamine toroids. In this model, epididymal sperm induced to undergo SCF can religate dsDNA breaks while vas deferens sperm cannot. Here, we demonstrated that the conventional neutral Comet assay underestimates the epididymal SCF breaks because the broken DNA ends remain attached to the nuclear matrix, causing the DNA to remain associated with the dispersion halo, and the Comet tails to be weak. Therefore, we term these hidden dsDNA breaks. When the Comet assay was modified to include an additional incubation with sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) after the conventional lysis, thereby solubilizing the nuclear matrix, the broken DNA was released from the matrix, which resulted in a reduction of the sperm head halo and an increase in the Comet tail length, exposing the hidden dsDNA breaks. Conversely, SCF-induced vas deferens sperm had small halos and long tails with the conventional neutral Comet assay, suggesting that the broken DNA ends were not tethered to the nuclear matrix. These results suggest that the attachment to the nuclear matrix is crucial for the religation of SCF-induced DNA breaks in sperm. Our data suggest that the neutral Comet assay identifies only dsDNA breaks that are released from the nuclear matrix and that the addition of an SDS treatment can reveal these hidden dsDNA breaks.

  13. Methamidophos alters sperm function and DNA at different stages of spermatogenesis in mice

    Energy Technology Data Exchange (ETDEWEB)

    Urióstegui-Acosta, Mayrut; Hernández-Ochoa, Isabel [Departamento de Toxicología, CINVESTAV-IPN, D.F. (Mexico); Sánchez-Gutiérrez, Manuel [Instituto de Ciencias de la Salud, Universidad Autónoma del Estado de Hidalgo, Hidalgo (Mexico); Piña-Guzmán, Belem [Instituto Politécnico Nacional-UPIBI, D.F. (Mexico); Rafael-Vázquez, Leticia; Solís-Heredia, M.J.; Martínez-Aguilar, Gerardo [Departamento de Toxicología, CINVESTAV-IPN, D.F. (Mexico); Quintanilla-Vega, Betzabet, E-mail: mquintan@cinvestav.mx [Departamento de Toxicología, CINVESTAV-IPN, D.F. (Mexico)

    2014-09-15

    Methamidophos (MET) is a highly toxic organophosphate (OP) pesticide that is widely used in developing countries. MET has male reproductive effects, including decreased fertility. We evaluated MET effects on sperm quality, fertilization and DNA integrity, exploring the sensitivity of different stages of spermatogenesis. Adult male mice received MET (3.75 or 5 mg/kg-bw/ip/day/4 days) and were euthanized 1, 28 or 45 days post-treatment (dpt) to evaluate MET's effects on epididymal maturation, meiosis or mitosis, respectively. Spermatozoa were obtained from the cauda epididymis–vas deferens and were evaluated for sperm quality, acrosome reaction (AR; Coomassie staining), mitochondrial membrane potential (by JC-1), DNA damage (comet assay), oxidative damage (malondialdehyde (MDA) production), in vitro fertilization and protein phosphorylation (immunodetection), and erythrocyte acetylcholinesterase (AChE) activity. At 1-dpt, MET inhibited AChE (43–57%) and increased abnormal cells (6%). While at 28- and 45-dpt, sperm motility and viability were significantly reduced with an increasing MET dose, and abnormal morphology increased at 5 mg/kg/day/4 days. MDA and mitochondrial activity were not affected at any dose or time. DNA damage (OTM and %DNA) was observed at 5 mg/kg/day/4 days in a time-dependent manner, whereas both parameters were altered in cells from mice exposed to 3.75 mg/kg/day/4 days only at 28-dpt. Depending on the time of collection, initial-, spontaneous- and induced-AR were altered at 5 mg/kg/day/4 days, and the fertilization capacity also decreased. Sperm phosphorylation (at serine and tyrosine residues) was observed at all time points. Data suggest that meiosis and mitosis are the more sensitive stages of spermatogenesis for MET reproductive toxicity compared to epididymal maturation. - Highlights: • Methamidophos alters sperm cell function at different stages of spermatogenesis. • Testicular stages of spermatogenesis are more sensitive to

  14. Exposure to persistent organic pollutants and sperm DNA methylation changes in Arctic and European populations

    DEFF Research Database (Denmark)

    Consales, Claudia; Toft, Gunnar; Leter, Giorgio

    2016-01-01

    Persistent organic pollutants (POPs), such as PCBs (polychlorinated biphenyls) and DDT [1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane], are environmental contaminants with potential endocrine disrupting activity. DNA methylation levels in peripheral blood lymphocytes have been associated with serum...... concentrations of POPs in Greenland Inuit and Korean populations. Greenland Inuits are characterized by the highest worldwide POP levels. In this cross-sectional study we evaluated the relationship between serum POP concentrations and DNA methylation levels in sperm of non-occupationally exposed fertile men from...... Greenland, Warsaw (Poland), and Kharkiv (Ukraine). Serum levels of PCB-153 [1,2,4-trichloro-5-(2,4,5-trichlorophenyl)benzene], as a proxy of the total PCBs body burden, and of p,p'-DDE [1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene], the main metabolite of DDT were measured. Sperm DNA methylation level...

  15. Single-cell pulsed-field gel electrophoresis to detect the early stage of DNA fragmentation in human sperm nuclei.

    Science.gov (United States)

    Kaneko, Satoru; Yoshida, Joji; Ishikawa, Hiromichi; Takamatsu, Kiyoshi

    2012-01-01

    Single-cell pulsed-field gel electrophoresis (SCPFGE) with dual electrode pairs was developed to detect the early stage of DNA fragmentation in human sperm. The motile sperm were purified by the commonly used density-gradient centrifugation technique and subsequent swim-up. The sperm were embedded in a thin film of agarose containing bovine trypsin (20 µg/mL) and were then lysed. Prior to SCPFGE, proteolysis of DNA-binding components, such as protamine and the nuclear matrix was essential to separate the long chain fibers from the fibrous and granular fragments derived from a single nucleus. The overall electrophoretic profiles elucidated the course of DNA fragmentation. A few large fibrous fragments were observed at the beginning of the process, however, as the fragmentation advanced, the long chain fibers decreased and shortened, and, conversely, the granular fragments increased until finally almost all the DNA was shredded. Although the ejaculate contained sperm with heterogeneous stages, the purified motile sperm exhibited several dozens of uniformly elongated fibers arising from the tangled DNA at the origin, whereas a part of these fibers gave rise to fibrous fragments beyond the tip of the elongated fibers, and their numbers and sizes varied among the sperm. Conventional intra-cytoplasmic sperm injection (ICSI) usually depends on intra-operative light microscopic observation to select a sperm for injection. The present results revealed that sperm motility could not give full assurance of DNA integrity. SCPFGE is likely to serve an important role in the preoperative differential diagnosis to determine the competence of the sperm population provided for injection.

  16. Single-cell pulsed-field gel electrophoresis to detect the early stage of DNA fragmentation in human sperm nuclei.

    Directory of Open Access Journals (Sweden)

    Satoru Kaneko

    Full Text Available Single-cell pulsed-field gel electrophoresis (SCPFGE with dual electrode pairs was developed to detect the early stage of DNA fragmentation in human sperm. The motile sperm were purified by the commonly used density-gradient centrifugation technique and subsequent swim-up. The sperm were embedded in a thin film of agarose containing bovine trypsin (20 µg/mL and were then lysed. Prior to SCPFGE, proteolysis of DNA-binding components, such as protamine and the nuclear matrix was essential to separate the long chain fibers from the fibrous and granular fragments derived from a single nucleus. The overall electrophoretic profiles elucidated the course of DNA fragmentation. A few large fibrous fragments were observed at the beginning of the process, however, as the fragmentation advanced, the long chain fibers decreased and shortened, and, conversely, the granular fragments increased until finally almost all the DNA was shredded. Although the ejaculate contained sperm with heterogeneous stages, the purified motile sperm exhibited several dozens of uniformly elongated fibers arising from the tangled DNA at the origin, whereas a part of these fibers gave rise to fibrous fragments beyond the tip of the elongated fibers, and their numbers and sizes varied among the sperm. Conventional intra-cytoplasmic sperm injection (ICSI usually depends on intra-operative light microscopic observation to select a sperm for injection. The present results revealed that sperm motility could not give full assurance of DNA integrity. SCPFGE is likely to serve an important role in the preoperative differential diagnosis to determine the competence of the sperm population provided for injection.

  17. Maternal exposure to a mixture of persistent organic pollutants (POPs) affects testis histology, epididymal sperm count and induces sperm DNA fragmentation in mice.

    Science.gov (United States)

    Khezri, Abdolrahman; Lindeman, Birgitte; Krogenæs, Anette K; Berntsen, Hanne F; Zimmer, Karin E; Ropstad, Erik

    2017-08-15

    Persistent organic pollutants (POPs) are widespread throughout the environment and some are suspected to induce reproductive toxicity. As animals and humans are exposed to complex mixtures of POPs, it is reasonable to assess how such mixtures could interact with the reproductive system. Our aim is to investigate how maternal exposure to a mixture of 29 different persistent organic pollutants, formulated to mimic the relative POP levels in the food basket of the Scandinavian population, could alter reproductive endpoints. Female mice were exposed via feed from weaning, during pregnancy and lactation in 3 exposure groups (control (C), low (L) and high (H)). Testicular morphometric endpoints, epididymal sperm concentration and sperm DNA integrity were assessed in adult male offspring. We found that the number of tubules, proportion of tubule compartments and epididymal sperm concentration significantly decreased in both POP exposed groups. Epididymal sperm from both POP exposed groups showed increased DNA fragmentation. It is concluded that maternal exposure to a defined POP mixture relevant to human exposure can affect testicular development, sperm production and sperm chromatin integrity. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Human Sperm Cryopreservation: Update on Techniques, Effect on DNA Integrity, and Implications for ART

    Directory of Open Access Journals (Sweden)

    Marlea Di Santo

    2012-01-01

    Full Text Available Cryopreservation of human spermatozoa—introduced in the 1960's—has been recognized as an efficient procedure for management of male fertility before therapy for malignant diseases, vasectomy or surgical infertility treatments, to store donor and partner spermatozoa before assisted reproduction treatments and to ensure the recovery of a small number of spermatozoa in severe male factor infertility. Despite the usefulness of it, cryopreservation may lead to deleterious changes of sperm structure and function: while the effects of cryopreservation on cells are well documented, to date there is no agreement in the literature on whether or not cryopreservation affects sperm chromatin integrity or on the use of a unique and functional protocol for the freezing-thawing procedure. Therefore, sperm cryopreservation is an important component of fertility management and much of its successful application seems to affect the reproductive outcome of assisted reproduction technologies (ART: appropriate use of cryoprotectants before and sperm selection technologies after cryopreservation seem to have the greatest impact on preventing DNA fragmentation, thus improving sperm cryosurvival rates.

  19. Stability of the human sperm DNA methylome to folic acid fortification and short-term supplementation.

    Science.gov (United States)

    Chan, D; McGraw, S; Klein, K; Wallock, L M; Konermann, C; Plass, C; Chan, P; Robaire, B; Jacob, R A; Greenwood, C M T; Trasler, J M

    2017-02-01

    Do short-term and long-term exposures to low-dose folic acid supplementation alter DNA methylation in sperm? No alterations in sperm DNA methylation patterns were found following the administration of low-dose folic acid supplements of 400 μg/day for 90 days (short-term exposure) or when pre-fortification of food with folic acid and post-fortification sperm samples (long-term exposure) were compared. Excess dietary folate may be detrimental to health and DNA methylation profiles due to folate's role in one-carbon metabolism and the formation of S-adenosyl methionine, the universal methyl donor. DNA methylation patterns are established in developing male germ cells and have been suggested to be affected by high-dose (5 mg/day) folic acid supplementation. This is a control versus treatment study where genome-wide sperm DNA methylation patterns were examined prior to fortification of food (1996-1997) in men with no history of infertility at baseline and following 90-day exposure to placebo (n = 9) or supplement containing 400 μg folic acid/day (n = 10). Additionally, pre-fortification sperm DNA methylation profiles (n = 19) were compared with those of a group of post-fortification (post-2004) men (n = 8) who had been exposed for several years to dietary folic acid fortification. Blood and seminal plasma folate levels were measured in participants before and following the 90-day treatment with placebo or supplement. Sperm DNA methylation was assessed using the whole-genome and genome-wide techniques, MassArray epityper, restriction landmark genomic scanning, methyl-CpG immunoprecipitation and Illumina HumanMethylation450 Bead Array. Following treatment, supplemented individuals had significantly higher levels of blood and seminal plasma folates compared to placebo. Initial first-generation genome-wide analyses of sperm DNA methylation showed little evidence of changes when comparing pre- and post-treatment samples. With Illumina HumanMethylation450 BeadChip arrays

  20. Correlation of spermiogram profiles with DNA damage in sperm cells of infertile men: a comet assay study.

    Science.gov (United States)

    Tug, Niyazi; Sandal, Suleyman; Ozelgun, Berna; Yilmaz, Bayram

    2011-01-01

    We have investigated a relationship between DNA damage in sperm and spermiogram profiles in the infertile men. Twenty-one non-smoking infertile men Sperm samples were collected and evaluated according to WHO guidelines. DNA damage of sperm cells was assessed using neutral comet assay. Fifty cells per slide and two slides per sample were scored to evaluate DNA damage. The cells were visually classified into four categories based on DNA migration such as undamaged (UD), little damage (LD), moderate damage (MD) and significant damage (SD). Total comet scores (TCS) were calculated as: 1×UD + 2×LD + 3×MD + 4×SD. There was a negative correlation between the percentage of slow- and in situ-motion sperm cells in spermiograms and TCS (p sperm cells and TCS was negative (p sperm motility parameters are negatively correlated. We suggest that evaluation of sperm DNA by the neutral comet assay may be valuable to use in fertility research.

  1. Concentrations of environmental DNA (eDNA) reflect spawning salmon abundance at fine spatial and temporal scales

    Science.gov (United States)

    Tillotson, Michael D.; Kelly, Ryan P.; Duda, Jeff; Hoy, Marshal S.; Kralj, James; Quinn, Thomas P.

    2018-01-01

    Developing fast, cost-effective assessments of wild animal abundance is an important goal for many researchers, and environmental DNA (eDNA) holds much promise for this purpose. However, the quantitative relationship between species abundance and the amount of DNA present in the environment is likely to vary substantially among taxa and with ecological context. Here, we report a strong quantitative relationship between eDNA concentration and the abundance of spawning sockeye salmon in a small stream in Alaska, USA, where we took temporally- and spatially-replicated samples during the spawning period. This high-resolution dataset suggests that (1) eDNA concentrations vary significantly day-to-day, and likely within hours, in the context of the dynamic biological event of a salmon spawning season; (2) eDNA, as detected by species-specific quantitative PCR probes, seems to be conserved over short distances (tens of meters) in running water, but degrade quickly over larger scales (ca. 1.5 km); and (3) factors other than the mere presence of live, individual fish — such as location within the stream, live/dead ratio, and water temperature — can affect the eDNA-biomass correlation in space or time. A multivariate model incorporating both biotic and abiotic variables accounted for over 75% of the eDNA variance observed, suggesting that where a system is well-characterized, it may be possible to predict species' abundance from eDNA surveys, although we underscore that species- and system-specific variables are likely to limit the generality of any given quantitative model. Nevertheless, these findings provide an important step toward quantitative applications of eDNA in conservation and management.

  2. Human semen cryopreservation: a sperm DNA fragmentation study with alkaline and neutral Comet assay.

    Science.gov (United States)

    Ribas-Maynou, J; Fernández-Encinas, A; García-Peiró, A; Prada, E; Abad, C; Amengual, M J; Navarro, J; Benet, J

    2014-01-01

    Sperm cryopreservation is widely used for both research and reproduction purposes, but its effect on sperm DNA damage remains controversial. Sperm DNA fragmentation (SDF) has become an important biomarker to assess male infertility. In particular, the differentiation between single- and double-stranded DNA fragmentation (ssSDF and dsSDF) has clinical implications for male infertility where ssSDF is associated with reduced fertility, whereas dsSDF is associated with increased risk of miscarriage. In this study, semen samples from 30 human males have been analysed in both fresh and cryopreserved using the alkaline and neutral Comet assays. Results show an increase of about 10% of ssSDF, assessed by the alkaline Comet assay, regardless of the male fertility status. Neutral Comet analysis of dsSDF does not show any statistical increase when comparing fresh and cryopreserved samples in any of the patient groups. Results support previous reports that oxidative stress is the major effector in DNA damage during sample cryopreservation, as, on one hand, ssSDF has previously been related to oxidative damage and, on the other hand, we have not found any effect on dsSDF. Therefore, there might be a slight risk of decreased fertility after using a freezed sample, but no evidence for increased miscarriage risk from cryopreserved spermatozoa should be expected. © 2013 American Society of Andrology and European Academy of Andrology.

  3. Single-Cell Pulsed-Field Gel Electrophoresis to Detect the Early Stage of DNA Fragmentation in Human Sperm Nuclei

    OpenAIRE

    Satoru Kaneko; Joji Yoshida; Hiromichi Ishikawa; Kiyoshi Takamatsu

    2012-01-01

    Single-cell pulsed-field gel electrophoresis (SCPFGE) with dual electrode pairs was developed to detect the early stage of DNA fragmentation in human sperm. The motile sperm were purified by the commonly used density-gradient centrifugation technique and subsequent swim-up. The sperm were embedded in a thin film of agarose containing bovine trypsin (20 µg/mL) and were then lysed. Prior to SCPFGE, proteolysis of DNA-binding components, such as protamine and the nuclear matrix was essential to ...

  4. A systematic review and meta-analysis to determine the effect of sperm DNA damage on in vitro fertilization and intracytoplasmic sperm injection outcome

    Directory of Open Access Journals (Sweden)

    Luke Simon

    2017-01-01

    Full Text Available Sperm DNA damage is prevalent among infertile men and is known to influence natural reproduction. However, the impact of sperm DNA damage on assisted reproduction outcomes remains controversial. Here, we conducted a meta-analysis of studies on sperm DNA damage (assessed by SCSA, TUNEL, SCD, or Comet assay and clinical pregnancy after IVF and/or ICSI treatment from MEDLINE, EMBASE, and PUBMED database searches for this analysis. We identified 41 articles (with a total of 56 studies including 16 IVF studies, 24 ICSI studies, and 16 mixed (IVF + ICSI studies. These studies measured DNA damage (by one of four assays: 23 SCSA, 18 TUNEL, 8 SCD, and 7 Comet and included a total of 8068 treatment cycles (3734 IVF, 2282 ICSI, and 2052 mixed IVF + ICSI. The combined OR of 1.68 (95% CI: 1.49-1.89; P < 0.0001 indicates that sperm DNA damage affects clinical pregnancy following IVF and/or ICSI treatment. In addition, the combined OR estimates of IVF (16 estimates, OR = 1.65; 95% CI: 1.34-2.04; P < 0.0001, ICSI (24 estimates, OR = 1.31; 95% CI: 1.08-1.59; P = 0.0068, and mixed IVF + ICSI studies (16 estimates, OR = 2.37; 95% CI: 1.89-2.97; P < 0.0001 were also statistically significant. There is sufficient evidence in the existing literature suggesting that sperm DNA damage has a negative effect on clinical pregnancy following IVF and/or ICSI treatment.

  5. Increased Sperm DNA Damage in Experimental Rat Varicocele Model and The Beneficial Effect of Varicocelectomy

    Directory of Open Access Journals (Sweden)

    Metin İshak Öztürk

    2012-01-01

    Full Text Available Background: Varicocele, the abnormal dilatation of the veins in the pampiniform plexusis commonly seen in infertile patients. In this study, we aim to examine sperm DNAdamage after the creation of experimental varicocele in rats and to observe the changeof this damage after a varicocelectomy.Materials and Methods: In this experimental study, a total of 30 adult male Wistar albinorats were divided into three groups. The 10 rats in group 1 underwent a sham operation, anexperimental varicocele was created in both the10 rats in group 2 and the 10 rats in group 3 (atotal of 20 rats. While the rats of group 2 were sacrificed after four weeks, the rats in group 3underwent a varicocelectomy after four weeks and were sacrificed four weeks after the varicocelectomyto observe its effects. Sperm DNA fragmentation was assessed with a Halomax®kit. The DNA Fragmentation Index (DFI was calculated and the groups were compared accordingto their DFI. Statistical analysis was performed using the Mann-Whitney U test.Results: Median sperm DFI was 17.6 (range: 7.6 in the right testicle and 18.3 (range: 6.8in the left testicle in the control group; 30.7 (range: 8.8 in the right testicle and 31.8 (range:9.6 in the left testicle in the varicocele group; 27.1 (range: 8.1 in the right testicle and 28.6(range: 8.9 in the left testicle in the varicocelectomy group. DNA damage in both right andleft testicles was statistically significant between the three groups (p<0.05.Conclusion: The results of this study show that varicocele leads to increased spermDNA damage and this damage is decreased by varicocelectomy.

  6. Urinary Concentrations of Parabens and Serum Hormone Levels, Semen Quality Parameters, and Sperm DNA Damage

    Science.gov (United States)

    Meeker, John D.; Yang, Tiffany; Ye, Xiaoyun; Calafat, Antonia M.; Hauser, Russ

    2011-01-01

    Background Parabens are commonly used as antimicrobial preservatives in cosmetics, pharmaceuticals, and food and beverage processing. Widespread human exposure to parabens has been recently documented, and some parabens have demonstrated adverse effects on male reproduction in animal studies. However, human epidemiologic studies are lacking. Objective We investigated relationships between urinary concentrations of parabens and markers of male reproductive health in an ongoing reproductive epidemiology study. Methods Urine samples collected from male partners attending an infertility clinic were analyzed for methyl paraben (MP), propyl paraben (PP), butyl paraben (BP), and bisphenol A (BPA). Associations with serum hormone levels (n = 167), semen quality parameters (n = 190), and sperm DNA damage measures (n = 132) were assessed using multivariable linear regression. Results Detection rates in urine were 100% for MP, 92% for PP, and 32% for BP. We observed no statistically significant associations between MP or PP and the outcome measures. Categories of urinary BP concentration were not associated with hormone levels or conventional semen quality parameters, but they were positively associated with sperm DNA damage (p for trend = 0.03). When urinary BPA quartiles were added to the model, BP and BPA were both positively associated with sperm DNA damage (p for trend = 0.03). Assessment of paraben concentrations measured on repeated urine samples from a subset of the men (n = 78) revealed substantial temporal variability. Conclusions We found no evidence for a relationship between urinary parabens and hormone levels or semen quality, although intraindividual variability in exposure and a modest sample size could have limited our ability to detect subtle relationships. Our observation of a relationship between BP and sperm DNA damage warrants further investigation. PMID:20876036

  7. Effect of low density lipoprotein on DNA integrity of freezing-thawing boar sperm by neutral comet assay.

    Science.gov (United States)

    Jiang, Zhong-Liang; Li, Qing-Wang; Li, Wen-Ye; Hu, Jian-Hong; Zhao, Hong-Wei; Zhang, Shu-Shan

    2007-06-01

    A modified protocol of neutral comet assay was utilized to assess the effect of low density lipoprotein (LDL) on the DNA integrity of boar freezing-thawing semen. The results demonstrated that the method was high sensitive and easier manipulation and LDL significantly protected sperm DNA integrity (psperm DNA in cryopreservation 0 day and 30 days (p>0.05).

  8. Sperm DNA damage measured by the alkaline Comet assay as an independent predictor of male infertility and in vitro fertilization success.

    Science.gov (United States)

    Simon, Luke; Lutton, Deborah; McManus, Joanne; Lewis, Sheena E M

    2011-02-01

    To evaluate sperm DNA fragmentation and semen parameters to diagnose male factor infertility and predict pregnancy after IVF. Prospective study. Academic research laboratory. Seventy-five couples undergoing IVF and 28 fertile donors. Sperm DNA fragmentation was measured by the alkaline Comet assay in semen and sperm after density gradient centrifugation (DGC). Binary logistic regression was used to analyze odds ratios (OR) and relative risks (RR) for IVF outcomes. Semen parameters and sperm DNA fragmentation in semen and DGC sperm compared with fertilization rates, embryo quality, and pregnancy. Men with sperm DNA fragmentation at more than a diagnostic threshold of 25% had a high risk of infertility (OR: 117.33, 95% confidence interval [CI]: 12.72-2,731.84, RR: 8.75). Fertilization rates and embryo quality decreased as sperm DNA fragmentation increased in semen and DGC sperm. The risk of failure to achieve a pregnancy increased when sperm DNA fragmentation exceeded a prognostic threshold value of 52% for semen (OR: 76.00, CI: 8.69-1,714.44, RR: 4.75) and 42% for DGC sperm (OR: 24.18, CI: 2.89-522.34, RR: 2.16). Sperm DNA testing by the alkaline Comet assay is useful for both diagnosis of male factor infertility and prediction of IVF outcome. Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  9. Obesity-induced sperm DNA methylation changes at satellite repeats are reprogrammed in rat offspring

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    Neil A Youngson

    2016-01-01

    Full Text Available There is now strong evidence that the paternal contribution to offspring phenotype at fertilisation is more than just DNA. However, the identity and mechanisms of this nongenetic inheritance are poorly understood. One of the more important questions in this research area is: do changes in sperm DNA methylation have phenotypic consequences for offspring? We have previously reported that offspring of obese male rats have altered glucose metabolism compared with controls and that this effect was inherited through nongenetic means. Here, we describe investigations into sperm DNA methylation in a new cohort using the same protocol. Male rats on a high-fat diet were 30% heavier than control-fed males at the time of mating (16-19 weeks old, n = 14/14. A small (0.25% increase in total 5-methyl-2Ͳ-deoxycytidine was detected in obese rat spermatozoa by liquid chromatography tandem mass spectrometry. Examination of the repetitive fraction of the genome with methyl-CpG binding domain protein-enriched genome sequencing (MBD-Seq and pyrosequencing revealed that retrotransposon DNA methylation states in spermatozoa were not affected by obesity, but methylation at satellite repeats throughout the genome was increased. However, examination of muscle, liver, and spermatozoa from male 27-week-old offspring from obese and control fathers (both groups from n = 8 fathers revealed that normal DNA methylation levels were restored during offspring development. Furthermore, no changes were found in three genomic imprints in obese rat spermatozoa. Our findings have implications for transgenerational epigenetic reprogramming. They suggest that postfertilization mechanisms exist for normalising some environmentally-induced DNA methylation changes in sperm cells.

  10. Administration of high dose of methamphetamine has detrimental effects on sperm parameters and DNA integrity in mice

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    Mojdeh Sabour

    2017-08-01

    Full Text Available Background: Methamphetamine (MA was shown to have harmful effects on male reproductive system. Objective: To investigate probable effects of daily administration of MA on sperm parameters and chromatin/DNA integrity in mouse. Material and Methods: Thirty-five NMRI male mice were divided into five groups including low, medium, and high dosage groups which were injected intraperitoneally with 4, 8 and 15 mg/kg/day for 35 days, respectively. Normal saline was injected in sham group and no medications were used in control group. Then, the mice were killed and caudal epididymis of each animal was cut and placed in Ham’s F10 medium for sperm retrieval. To evaluate sperm chromatin abnormalities, the aniline blue, toluidine blue and chromomycine A3 were used. For sperm DNA integrity and apoptosis, the acridine orange, sperm chromatin dispersion, and TUNEL assay were applied. For sperm morphology, Papanicolaou staining was done Results: Normal morphology and progressive motility of spermatozoa decreased in medium and high dosage groups in comparison with the control group (p=0.035. There was a significant increase in rate of aniline blue, toluidine blue, and chromomycine A3 positive spermatozoa in high dosage group. In a similar manner, there was an increase in rates of acridine orange, TUNEL and sperm chromatin dispersion positive sperm cells in high dosage group with respect to others. Conclusion: MA abuse in a dose-dependent manner could have detrimental effects on male reproductive indices including sperm parameters and sperm chromatin/DNA integrity in mice.

  11. Atrazine in sub-acute exposure results in sperm DNA disintegrity and nuclear immaturity in rats

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    Rajab-Ali Sadrkhanloo

    2012-03-01

    Full Text Available This study was designed to evaluate the detrimental effect of atrazine (ATR on germinal epitheliums (GE cytoplasmic carbohydrate (CH and unsaturated fatty acids (UFA ratio and to clarify the effect of ATR on serum levels of FSH, LH, testosterone and inhibin-B (INH-B. The impact of ATR exposure on total antioxidant capacity (TAC, sperm DNA packing and integrity were also investigated. Seventy two Wistar rats were used. The rats in control group received vehicle and the animals in test groups received 100, 200 and 300 mg kg-1 BW of ATR orally on daily bases for 12, 24 and 48 days. In ATR-received groups the spermatogenesis cell were presented with dense reactive sites for lipidophilic staining associated with faint cytoplasmic CH accumulation. Dissociated germinal epithelium, negative tubular and repopulation indexes were manifested. The serum levels of testosterone, FSH, LH and INH-B decreased by 85% after 48 days exposure to high dose of ATR. TAC was reduced in a time- and dose-dependent manner. The sperm DNA damage was marked in animals which exposed to high dose of ATR (72.50 ± 2.25% and the percentage of nuclear immature sperm increased up to 83.40 ± 0.89%. In conclusion, ATR not only induced its detrimental effect on the endocrine function of the testes and pituitary gland but also affected the cytoplasmic CH ratio and consequently leads to inadequate energy supplement in spermatogenesis cells. Therefore the imbalanced oxidative stress occurs in testicular tissue, which in turn enhances the sperm DNA disintegrity and nuclear immaturity.

  12. In Vitro Effect of Cell Phone Radiation on Motility, DNA Fragmentation and Clusterin Gene Expression in Human Sperm

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    Adel Zalata

    2015-04-01

    Full Text Available Background: Use of cellular phones emitting radiofrequency electromagnetic field (RF-EMF has been increased exponentially and become a part of everyday life. This study aimed to investigate the effects of in vitro RF-EMF exposure emitted from cellular phones on sperm motility index, sperm DNA fragmentation and seminal clusterin (CLU gene expression. Materials and Methods: In this prospective study, a total of 124 semen samples were grouped into the following main categories: i. normozoospermia (N, n=26, ii. asthenozoospermia (A, n=32, iii. asthenoteratozoospermia (AT, n=31 and iv. oligoasthenoteratozoospermia (OAT, n=35. The same semen samples were then divided into two portions non-exposed and exposed samples to cell phone radiation for 1 hour. Before and immediately after exposure, both aliquots were subjected to different assessments for sperm motility, acrosin activity, sperm DNA fragmentation and CLU gene expression. Statistical differences were analyzed using paired t student test for comparisons between two sub-groups where pAT>A>N groups, respectively (p<0.05. Conclusion: Cell phone emissions have a negative impact on exposed sperm motility index, sperm acrosin activity, sperm DNA fragmentation and seminal CLU gene expression, especially in OAT cases.

  13. In vitro effect of cell phone radiation on motility, DNA fragmentation and clusterin gene expression in human sperm.

    Science.gov (United States)

    Zalata, Adel; El-Samanoudy, Ayman Z; Shaalan, Dalia; El-Baiomy, Youssef; Mostafa, Taymour

    2015-01-01

    Use of cellular phones emitting radiofrequency electromagnetic field (RF-EMF) has been increased exponentially and become a part of everyday life. This study aimed to investigate the effects of in vitro RF-EMF exposure emitted from cellular phones on sperm motility index, sperm DNA fragmentation and seminal clusterin (CLU) gene expression. In this prospective study, a total of 124 semen samples were grouped into the following main categories: i. normozoospermia (N, n=26), ii. asthenozoospermia (A, n=32), iii. asthenoteratozoospermia (AT, n=31) and iv. oligoasthenoteratozoospermia (OAT, n=35). The same semen samples were then divided into two portions non-exposed and exposed samples to cell phone radiation for 1 hour. Before and immediately after exposure, both aliquots were subjected to different assessments for sperm motility, acrosin activity, sperm DNA fragmentation and CLU gene expression. Statistical differences were analyzed using paired t student test for comparisons between two sub-groups where pAT>A>N groups, respectively (pnegative impact on exposed sperm motility index, sperm acrosin activity, sperm DNA fragmentation and seminal CLU gene expression, especially in OAT cases.

  14. Impact of the Z potential technique on reducing the sperm DNA fragmentation index, fertilization rate and embryo development.

    Science.gov (United States)

    Duarte, Carlos; Núñez, Víctor; Wong, Yat; Vivar, Carlos; Benites, Elder; Rodriguez, Urso; Vergara, Carlos; Ponce, Jorge

    2017-12-01

    In assisted reproduction procedures, we need to develop and enhance new protocols to optimize sperm selection. The aim of this study is to evaluate the ability of the Z potential technique to select sperm with intact DNA in non-normospermic patients and evaluate the impact of this selection on embryonic development. We analyzed a total of 174 human seminal samples with at least one altered parameter. We measured basal, post density gradients, and post density gradients + Z potential DNA fragmentation index. To evaluate the impact of this technique on embryo development, 54 cases were selected. The embryo development parameters evaluated were fertilization rate, cleavage rate, top quality embryos at the third day and blastocysts rate. We found significant differences in the study groups when we compared the sperm fragmentation index by adding the Z potential technique to density gradient selection vs. density gradients alone. Furthermore, there was no significant difference in the embryo development parameters between the low sperm fragmentation index group vs. the moderate and high sperm fragmentation index groups, when selecting sperms with this new technique. The Z potential technique is a very useful tool for sperm selection; it significantly reduces the DNA fragmentation index and improves the parameters of embryo development. This technique could be considered routine for its simplicity and low cost.

  15. Mechanistic investigation of ROS-induced DNA damage by oestrogenic compounds in lymphocytes and sperm using the comet assay.

    Science.gov (United States)

    Cemeli, Eduardo; Anderson, Diana

    2011-01-01

    Past research has demonstrated that oestrogenic compounds produce strand breaks in the DNA of sperm and lymphocytes via reactive oxygen species (ROS). In the current investigation, sperm and lymphocytes were treated in vitro with oestrogenic compounds (diethylstilboestrol, progesterone, 17β-oestradiol, noradrenaline and triiodotyronine) and several aspects of DNA damage were investigated. Firstly, mediation of DNA damage by lipid peroxidation was investigated in the presence of BHA (a lipid peroxidation blocker). BHA reduced the DNA damage generated by 17β-oestradiol and diethylstilboestrol in a statistically significant manner. No effects were observed for sperm. Secondly, the presence of oxidized bases employing FPG and EndoIII were detected for lymphocytes and sperm in the negative control and after 24 h recovery in lymphocytes but not immediately after treatment for both cell types. The successful detection of oxidized bases in the negative control (untreated) of sperm provides an opportunity for its application in biomonitoring studies. DNA repair at 24 h after exposure was also studied. A nearly complete recovery to negative control levels was shown in lymphocytes 24 h recovery after oestrogenic exposure and this was statistically significant in all cases. Rapid rejoining of DNA, in a matter of hours, is a characteristic of DNA damaged by ROS.

  16. Mechanistic Investigation of ROS-Induced DNA Damage by Oestrogenic Compounds in Lymphocytes and Sperm Using the Comet Assay

    Directory of Open Access Journals (Sweden)

    Diana Anderson

    2011-04-01

    Full Text Available Past research has demonstrated that oestrogenic compounds produce strand breaks in the DNA of sperm and lymphocytes via reactive oxygen species (ROS. In the current investigation, sperm and lymphocytes were treated in vitro with oestrogenic compounds (diethylstilboestrol, progesterone, 17β-oestradiol, noradrenaline and triiodotyronine and several aspects of DNA damage were investigated. Firstly, mediation of DNA damage by lipid peroxidation was investigated in the presence of BHA (a lipid peroxidation blocker. BHA reduced the DNA damage generated by 17β-oestradiol and diethylstilboestrol in a statistically significant manner. No effects were observed for sperm. Secondly, the presence of oxidized bases employing FPG and EndoIII were detected for lymphocytes and sperm in the negative control and after 24 h recovery in lymphocytes but not immediately after treatment for both cell types. The successful detection of oxidized bases in the negative control (untreated of sperm provides an opportunity for its application in biomonitoring studies. DNA repair at 24 h after exposure was also studied. A nearly complete recovery to negative control levels was shown in lymphocytes 24 h recovery after oestrogenic exposure and this was statistically significant in all cases. Rapid rejoining of DNA, in a matter of hours, is a characteristic of DNA damaged by ROS.

  17. Production of ROS and its effects on mitochondrial and nuclear DNA, human spermatozoa, and sperm function

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    Hardi Darmawan

    2007-06-01

    Full Text Available Over the past few decades many researchers studying the causes of male infertility have recently focused on the role played by reactive oxygen species (ROS – highly reactive oxidizing agents belonging to the class of free radicals. If ROS levels rise, oxidative stress (OS occurs, which results in oxygen and oxygen derived oxidants, and in turn increases the rates of cellular damage. In human, ROS are produced by a variety of semen components, and antioxidants in the seminal fluid keep their level balance. Small amounts of ROS help spermatozoa acquire their necessary fertilizing capabilities. Many researches showed that ROS attack DNA integrity in the sperm nucleus by causing base modification, DNA strand breaks, and chromatin cross linking. The DNA damage induced excessive levels of ROS and might accelerate the process of germ cell apoptosis leading to a decline in sperm counts associated with male infertility. This paper will review the molecular (cellular origins of ROS in human semen, how ROS damage sperm nuclear DNA, and how such DNA damage contributes to male infertility. Increased ROS production by spermatozoa is associated with a decreased mitochondrial membrane potential (MMP, which is an important indicator of functional integrity of the spermatozoa. Germ cell apoptosis is essential for normal spermatogenesis and its dysregulation may lead to male infertility. Thus, understanding the causes and mechanisms of germ cell apoptosis is of major importance in preventing male reproductive problems. Levels of apoptosis in mature spermatozoa that were significantly correlated with levels of seminal ROS determined by chemiluminescence assay indicate the linkage between ROS and male fertility problems. (Med J Indones 2007; 16:127-33 Keywords: Apoptosis, infertility, free radicals

  18. Effect of sperm DNA fragmentation on embryo development: clinical and biological aspects.

    Science.gov (United States)

    Alvarez Sedó, Cristian; Bilinski, Melina; Lorenzi, Daniela; Uriondo, Heydy; Noblía, Felicitas; Longobucco, Valeria; Lagar, Estefanía Ventimiglia; Nodar, Florencia

    2017-12-01

    The aim of this study was to investigate the effect of sperm DNA fragmentation on fertilization rate, embryo development (blastulation rate), and pregnancy outcomes for ICSI cycles performed in a cohort of couples using donor eggs and to assess the remaining embryos that were not transferred or frozen for apoptotic markers. Eighty-two women (egg recipients) were included in the study (2016) were included in the study. The recipients' mean age was 41.8±5.1 y/o (36-49), while the egg donors' mean age was 30.8±2.1 y/o (27-33). Even though donor egg cycles with frozen sperm samples are performed regularly in our center, 35 cycles were done using fresh sperm samples. The mean age of the males involved in the procedure was 40.1±5.2 y/o. Fertilization, blastulation, and pregnancy rates were assessed. The patients were divided into two groups, TUNEL fragmentation and blastulation rate. High levels of DNA fragmentation were associated with low blastulation and pregnancy rates (per transfer); however, fertilization rate was not affected. Samples with higher levels of DNA fragmentation were associated with higher levels of DNA fragmentation in blastomeres without activating the apoptotic pathway (9.1% vs. 15.9%) (pfragmentation activated the apoptotic pathway in higher levels than samples with TUNEL fragmentation was negatively correlated with blastulation and pregnancy rates even in good quality oocytes. High levels of DNA damage promote embryo arrest and induce the activation of the apoptotic pathway.

  19. A simple comet assay for archived sperm correlates DNA fragmentation to reduced hyperactivation and penetration of zona-free hamster oocytes.

    Science.gov (United States)

    Chan, P J; Corselli, J U; Patton, W C; Jacobson, J D; Chan, S R; King, A

    2001-01-01

    To correlate sperm variables with sperm DNA fragmentation, as assessed by using a modified alkaline comet assay for sperm smears. The comet assay was adapted for fixed sperm smears (59 cases), and the level of DNA fragmentation was determined. Clinical and academic research environment. 59 patients undergoing fertility treatment. Sperm samples leftover from IVF procedures were fixed and processed for the comet assay. Sperm head DNA density and sperm variables. A correlation was observed between increased sperm head DNA fragmentation and decreased penetration of zona-free hamster oocytes. Heat-induced hyperactive motility decreased as DNA fragmentation increased. The DNA fragmentation did not correlate with percentages of intact acrosome, normality, maturity, and strict normal morphology. The advantages of the comet assay for archived cells include simplicity, low intraassay coefficient of variation, and low performance cost; in addition, DNA analysis can be carried out at leisure. Low DNA damage was associated with higher hyperactivation and oocyte penetration, suggesting that failed fertilization was linked to compromised DNA integrity in the sperm. Exploration of compounds to repair damaged DNA is warranted.

  20. The spectrum of DNA damage in human sperm assessed by single cell gel electrophoresis (Comet assay) and its relationship to fertilization and embryo development.

    Science.gov (United States)

    Morris, I D; Ilott, S; Dixon, L; Brison, D R

    2002-04-01

    The integrity of sperm DNA is important for the success of natural or assisted fertilization, as well as normal development of the embryo, fetus and child. ICSI, by bypassing sperm selection mechanisms, increases the risk of transmitting damaged DNA and the significance of this requires investigation. DNA damage in sperm from an unselected group of 60 men undergoing IVF treatment was measured by single cell gel electrophoresis (Comet assay) and correlated with semen and treatment cycle parameters. Wide spectra of sperm DNA damage were found both within and between men but no specific subgroups were identified. Semen and treatment cycle parameters were not different in men grouped according to high or low sperm DNA damage. However, regression analysis showed that DNA damage was positively associated with age (29-44 years), abnormal sperm and motility and negatively associated with sperm concentration. In ICSI cycles DNA damage was positively associated with impairment of post-fertilization embryo cleavage. This study contributes to the evidence of DNA damage within sperm. High loads of DNA damage measured by the Comet assay were predictive of failure of embryo development after ICSI. As it is likely that sperm with DNA damage contributed to successful fertilization and in-vitro development, potential adverse effects remain to be clarified.

  1. The Society for Translational Medicine: clinical practice guidelines for sperm DNA fragmentation testing in male infertility

    Science.gov (United States)

    Cho, Chak-Lam; Majzoub, Ahmad; Esteves, Sandro C.

    2017-01-01

    Sperm DNA fragmentation (SDF) testing has been emerging as a valuable tool for male fertility evaluation. While the essential role of sperm DNA integrity in human reproduction was extensively studied, the clinical indication of SDF testing is less clear. This clinical practice guideline provides recommendations of clinical utility of the test supported by evidence. It is intended to serve as a reference for fertility specialists in identifying the circumstances in which SDF testing should be of greatest clinical value. SDF testing is recommended in patients with clinical varicocele and borderline to normal semen parameters as it can better select varicocelectomy candidates. Outcomes of natural pregnancy and assisted reproductive techniques (ART) can be predicted by result of SDF tests. High SDF is also linked with recurrent pregnancy loss (RPL) and failure of ART. Result of SDF testing may change the management decision by selecting the most appropriate ART with the highest success rate for infertile couples. Several studies have demonstrated the benefit in using testicular instead of ejaculated sperm in men with high SDF, oligozoospermia or recurrent in vitro fertilization (IVF) failure. Infertile men with modifiable lifestyle factor may benefit from SDF testing by reinforcing risk factor modification and monitoring patient’s progress to intervention. PMID:29082206

  2. Epididymis seleno-independent glutathione peroxidase 5 maintains sperm DNA integrity in mice

    Science.gov (United States)

    Chabory, Eléonore; Damon, Christelle; Lenoir, Alain; Kauselmann, Gary; Kern, Hedrun; Zevnik, Branko; Garrel, Catherine; Saez, Fabrice; Cadet, Rémi; Henry-Berger, Joelle; Schoor, Michael; Gottwald, Ulrich; Habenicht, Ursula; Drevet, Joël R.; Vernet, Patrick

    2009-01-01

    The mammalian epididymis provides sperm with an environment that promotes their maturation and protects them from external stresses. For example, it harbors an array of antioxidants, including non-conventional glutathione peroxidase 5 (GPX5), to protect them from oxidative stress. To explore the role of GPX5 in the epididymis, we generated mice that lack epididymal expression of the enzyme. Histological analyses of Gpx5–/– epididymides and sperm cells revealed no obvious defects. Furthermore, there were no apparent differences in the fertilization rate of sexually mature Gpx5–/– male mice compared with WT male mice. However, a higher incidence of miscarriages and developmental defects were observed when WT female mice were mated with Gpx5-deficient males over 1 year old compared with WT males of the same age. Flow cytometric analysis of spermatozoa recovered from Gpx5-null and WT male mice revealed that sperm DNA compaction was substantially lower in the cauda epididymides of Gpx5-null animals and that they suffered from DNA oxidative attacks. Real-time PCR analysis of enzymatic scavengers expressed in the mouse epididymis indicated that the cauda epididymidis epithelium of Gpx5-null male mice mounted an antioxidant response to cope with an excess of ROS. These observations suggest that GPX5 is a potent antioxidant scavenger in the luminal compartment of the mouse cauda epididymidis that protects spermatozoa from oxidative injuries that could compromise their integrity and, consequently, embryo viability. PMID:19546506

  3. Measuring Sperm DNA Fragmentation and Clinical Outcomes of Medically Assisted Reproduction: A Systematic Review and Meta-Analysis.

    Science.gov (United States)

    Cissen, Maartje; Wely, Madelon van; Scholten, Irma; Mansell, Steven; Bruin, Jan Peter de; Mol, Ben Willem; Braat, Didi; Repping, Sjoerd; Hamer, Geert

    2016-01-01

    Sperm DNA fragmentation has been associated with reduced fertilization rates, embryo quality, pregnancy rates and increased miscarriage rates. Various methods exist to test sperm DNA fragmentation such as the sperm chromatin structure assay (SCSA), the sperm chromatin dispersion (SCD) test, the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay and the single cell gel electrophoresis (Comet) assay. We performed a systematic review and meta-analysis to assess the value of measuring sperm DNA fragmentation in predicting chance of ongoing pregnancy with IVF or ICSI. Out of 658 unique studies, 30 had extractable data and were thus included in the meta-analysis. Overall, the sperm DNA fragmentation tests had a reasonable to good sensitivity. A wide variety of other factors may also affect the IVF/ICSI outcome, reflected by limited to very low specificity. The constructed hierarchical summary receiver operating characteristic (HSROC) curve indicated a fair discriminatory capacity of the TUNEL assay (area under the curve (AUC) of 0.71; 95% CI 0.66 to 0.74) and Comet assay (AUC of 0.73; 95% CI 0.19 to 0.97). The SCSA and the SCD test had poor predictive capacity. Importantly, for the TUNEL assay, SCD test and Comet assay, meta-regression showed no differences in predictive value between IVF and ICSI. For the SCSA meta-regression indicated the predictive values for IVF and ICSI were different. The present review suggests that current sperm DNA fragmentation tests have limited capacity to predict the chance of pregnancy in the context of MAR. Furthermore, sperm DNA fragmentation tests have little or no difference in predictive value between IVF and ICSI. At this moment, there is insufficient evidence to recommend the routine use of sperm DNA fragmentation tests in couples undergoing MAR both for the prediction of pregnancy and for the choice of treatment. Given the significant limitations of the evidence and the

  4. Measuring Sperm DNA Fragmentation and Clinical Outcomes of Medically Assisted Reproduction: A Systematic Review and Meta-Analysis.

    Directory of Open Access Journals (Sweden)

    Maartje Cissen

    Full Text Available Sperm DNA fragmentation has been associated with reduced fertilization rates, embryo quality, pregnancy rates and increased miscarriage rates. Various methods exist to test sperm DNA fragmentation such as the sperm chromatin structure assay (SCSA, the sperm chromatin dispersion (SCD test, the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labelling (TUNEL assay and the single cell gel electrophoresis (Comet assay. We performed a systematic review and meta-analysis to assess the value of measuring sperm DNA fragmentation in predicting chance of ongoing pregnancy with IVF or ICSI. Out of 658 unique studies, 30 had extractable data and were thus included in the meta-analysis. Overall, the sperm DNA fragmentation tests had a reasonable to good sensitivity. A wide variety of other factors may also affect the IVF/ICSI outcome, reflected by limited to very low specificity. The constructed hierarchical summary receiver operating characteristic (HSROC curve indicated a fair discriminatory capacity of the TUNEL assay (area under the curve (AUC of 0.71; 95% CI 0.66 to 0.74 and Comet assay (AUC of 0.73; 95% CI 0.19 to 0.97. The SCSA and the SCD test had poor predictive capacity. Importantly, for the TUNEL assay, SCD test and Comet assay, meta-regression showed no differences in predictive value between IVF and ICSI. For the SCSA meta-regression indicated the predictive values for IVF and ICSI were different. The present review suggests that current sperm DNA fragmentation tests have limited capacity to predict the chance of pregnancy in the context of MAR. Furthermore, sperm DNA fragmentation tests have little or no difference in predictive value between IVF and ICSI. At this moment, there is insufficient evidence to recommend the routine use of sperm DNA fragmentation tests in couples undergoing MAR both for the prediction of pregnancy and for the choice of treatment. Given the significant limitations of the evidence and

  5. Association between sperm DNA integrity and seminal plasma antioxidant levels in health workers occupationally exposed to ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Dayanidhi; Salian, Sujith Raj; Kalthur, Guruprasad; Uppangala, Shubhashree; Kumari, Sandhya [Division of Clinical Embryology, Department of Obstetrics and Gynecology, Kasturba Medical College, Manipal University, Manipal 576104 (India); Challapalli, Srinivas [Department of Radiotherapy, Kasturba Medical College, Mangalore (India); Chandraguthi, Shrinidhi Gururajarao [Department of Radiotherapy and Oncology, Kasturba Medical College, Manipal (India); Jain, Navya; Krishnamurthy, Hanumanthappa [National Centre for Biological Sciences, Bangalore (India); Kumar, Pratap [Department of Obstetrics and Gynecology, Kasturba Medical College, Manipal University, Manipal (India); Adiga, Satish Kumar, E-mail: satish.adiga@manipal.edu [Division of Clinical Embryology, Department of Obstetrics and Gynecology, Kasturba Medical College, Manipal University, Manipal 576104 (India)

    2014-07-15

    There is a paucity of data regarding the association between occupational radiation exposure and risk to human fertility. Recently, we provided the first evidence on altered sperm functional characteristics, DNA damage and hypermethylation in radiation health workers. However, there is no report elucidating the association between seminal plasma antioxidants and sperm chromatin integrity in occupationally exposed subjects. Here, we assessed the seminal plasma antioxidants and lipid peroxidation level in 83 men who were occupationally exposed to ionizing radiation and then correlated with the sperm chromatin integrity. Flow cytometry based sperm chromatin integrity assay revealed a significant decline in αt value in the exposed group in comparison to the non-exposed group (P<0.0001). Similarly, both total and reduced glutathione levels and total antioxidant capacity in the seminal plasma were significantly higher in exposed group than the non-exposed group (P<0.01, 0.001 and 0.0001, respectively). However, superoxide dismutase level and malondialdehyde level, which is an indicator of lipid peroxidation in the seminal plasma, did not differ significantly between two groups. The total antioxidant capacity (TAC) and GSH level exhibited a positive correlation with sperm DNA integrity in exposed subjects. To conclude, this study distinctly shows that altered sperm chromatin integrity in radiation health workers is associated with increase in seminal plasma antioxidant level. Further, the increased seminal plasma GSH and TAC could be an adaptive measure to tackle the oxidative stress to protect genetic and functional sperm deformities in radiation health workers. - Highlights: • Seminal plasma antioxidants were measured in men occupationally exposed to radiation. • Sperm chromatin integrity was significantly affected in the exposed group. • Glutathione and total antioxidant capacity was significantly higher in exposed group. • Sperm DNA damage in exposed subjects

  6. Sperm DNA integrity testing: big halo is a good predictor of embryo quality and pregnancy after conventional IVF.

    Science.gov (United States)

    Tandara, M; Bajić, A; Tandara, L; Bilić-Zulle, L; Šunj, M; Kozina, V; Goluža, T; Jukić, M

    2014-09-01

    Sperm DNA integrity is a sperm functional parameter of male fertility evaluation. Two parameters of sperm DNA integrity were observed: DNA damage expressed as DNA fragmentation index (DFI) and percentage of the DNA undamaged spermatozoa expressed as big halo. Halosperm test was used for sperm DNA integrity determination. The aim of this study was to evaluate which DNA integrity parameter is better as an embryo quality and pregnancy prognostic parameter after the conventional IVF. We evaluated two embryo groups (positive and negative group) according to the 3rd day cumulative embryo score. Big halo and DFI, as we expected, showed good correlation (r = -0.69; p quality. ROC curves comparison of DFI and big halo revealed the AUC value for big halo to be significantly higher (DFI AUC = 0.71 vs. big halo AUC = 0.83; p = 0.025) than for DFI. Big halo was found to be the only independent predictor of embryo quality. Sperm DNA integrity both parameters are good prognostic parameters of embryo quality after the conventional IVF where big halo seems to be better. ROC analyses show DFI and big halo as significant prognostic parameters for achieved pregnancy (AUC ± SE for DFI was 0.67 ± 0.06 and 0.75 ± 0.06 for big halo). To our knowledge, this is the first study demonstrating the correlation between sperm DNA undamaged rate expressed as big halo parameter and semen characteristics as well as the influence on fertilization rate, embryo quality and pregnancy in conventional IVF. © 2014 American Society of Andrology and European Academy of Andrology.

  7. Findings on sperm alterations and DNA fragmentation, nutritional, hormonal and antioxidant status in an elite triathlete. Case report

    Directory of Open Access Journals (Sweden)

    D. Vaamonde

    2014-12-01

    Conclusions: In this high-intensity endurance athlete, sperm parameters, mainly sperm morphology and DNA fragmentation, are altered. Further knowledge is needed with regards nutritional antioxidant intake and other dietetic strategies oriented toward avoiding oxidative damage in semen of high-performance triathletes. Moreover, adequate nutritional strategies must be found and nutritional advice given to athletes so as to palliate or dampen the effects of exercise on semen quality.

  8. Meiotic segregation and sperm DNA fragmentation in Tunisian men with dysplasia of the fibrous sheath (DFS) associated with head abnormalities.

    Science.gov (United States)

    Ghedir, H; Mehri, A; Mehdi, M; Brahem, S; Saad, A; Ibala-Romdhane, S

    2014-09-01

    Dysplasia of the Fibrous Sheath (DFS) is a primitive flagellar pathology for which a broad spectrum of ultrastructural flagellar abnormalities has been described responsible for a severe to total asthenozoospermia. To this phenotype other morphological abnormalities including cephalic and abnormalities in nuclear structure can be associated that could compromise embryonic development in case of use of Assisted Reproductive Technology (ART). The aim of this study was to evaluate the level of DNA fragmentation and aneuploidy rate in ejaculated spermatozoa of Tunisian men presented with DFS sperm defect associated to high percentage of head abnormalities and to compare the results with those from fertile men. Sperm DNA fragmentation was evaluated by the terminal desoxynucleotidyl transferase mediated deoxyuridine triphosphate biotin nick-end labelling (TUNEL) assay. The study of meiotic segregation was performed by Fluorescence in situ hybridization (FISH) for chromosomes X, Y and 18. The mean DNA fragmentation index was significantly higher in patients compared to the control group. FISH revealed a significantly higher incidence of sperm aneuploidies compared with controls. All patients showed elevated frequencies of sex chromosomes disomy, disomy 18 and diploidy. In some cases of syndromic teratozoospermia due to sperm tail structural abnormalities, such as DFS, other morphological cephalic abnormalities may be associated. In these cases we have demonstrated impaired sperm nuclear quality which will affect the results in ICSI. Hence the interest of a thorough study of the sperm nucleus in these forms of infertility in order to predict the chances of success in ART.

  9. Interaction of drug based copper(II) complexes with Herring Sperm DNA and their biological activities

    Science.gov (United States)

    Patel, Mohan N.; Patel, Chintan R.; Joshi, Hardik N.

    2012-11-01

    Square pyramidal Cu(II) complexes with NS donor ligand and ciprofloxacin have been synthesized and characterized using analytical and spectral techniques. The synthesized complexes have been tested for their antimicrobial activity using double dilution technique in terms of minimum inhibitory concentration (MIC) and colony forming unit (CFU). The DNA binding ability of the complexes with Sperm Herring DNA has been performed using absorption titration and viscosity measurement. The nuclease activity of complexes with plasmid DNA (pUC19) has been carried out using agarose gel electrophoresis technique. Synthesized complexes have been tested for their SOD mimic activity using NBT/NADH/PMS system. The cytotoxic properties of metal complexes have been evaluated using brine shrimp lethality bioassay.

  10. ABO genotyping of suspects from sperm DNA isolated from postcoital samples in sex crimes.

    Science.gov (United States)

    Sasaki, M; Shiono, H

    1996-03-01

    In sexual assaults against women, one key to identifying the suspect is ABO phenotyping or the typing of other polymorphic markers of the seminal fluid in the victim's vagina. However, ABO phenotyping is frequently unsuccessful, since mixtures of fluids cannot be separated to be subjected to conventional methods for the detection of antibody or antigen material. We therefore studied ABO blood group genotyping of sperm DNA isolated from contaminating vaginal fluid by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Seminal samples of genotypes OO, AO, BO and AB were experimentally mixed with vaginal fluid (OO, AO, BO and AB), and were successfully separated and genotyped by this method. In practice, we also separated and genotyped the seminal DNA of suspects from contaminated postcoital vaginal fluid obtained in 4 sexual assaults. These forensic samples were easily separated and completely genotyped. This reliable ABO genotyping method by PCR-RFLP, using separated sperm DNA, should be of value in forensic identification in sexual assaults.

  11. Effect of Incubation Time and Vitamin E Supplementation on Sperm Motility, Viability and DNA Fragmentation in Asthenoteratozoospermic Samples

    Directory of Open Access Journals (Sweden)

    Ali Asghar Ghafarizadeh

    2017-09-01

    Full Text Available Abstract Background: In Asthenoteratozoospermic‎ men, low motility, defected DNA and highly oxidative stress in ‎sperm ‎‎cause ‎poor‎ assisted reproductive techniques (ART outcomes. The aim of this study was to determine the effect of Vitamin E (Vit E, as a potent antioxidant, on sperm motility, viability and DNA integrity at different times of in vitro incubation (after 2, 4 and 6-h to improve asthenoteratozoospermic semen samples for ART. Materials and Methods: Asthenoteratozoospermic semen samples of 50 volunteers were collected and examined. Each sample was divided into two groups of control and vitamin E (2mM and kept in the 37 °C and 6 % CO2 for 2, 4 and 6 hours. After this incubation, sperm motility, viability and sperm DNA fragmentation (SCD were evaluated in each group. Data were analyzed using repeated measurement of ANOVA and T-test. The means were considered significantly different at p<0.05. Results:Significant decrease in total and progressive motility and viability as well as significant increase in sperm DNA damage (after 6h of incubation were found in control group vs. the control group before incubation (p<0.05. The sperm motility and viability was significantly higher in vitamin E group compared to untreated control group (p<0.05. Our results also showed that DNA fragmentation significantly was lower after 6h of vitamin E treatment (p<0.05. Conclusion: In vitro supplementation of vitamin E in asthenoteratozoospermia semen samples may protect spermatozoa from maltreatment effect of ROS during sperm sampling via keeping enzymatic and antioxidant process in optimum condition.

  12. Association between Urinary Polycyclic Aromatic Hydrocarbon Metabolites and Sperm DNA Damage: A Population Study in Chongqing, China

    Science.gov (United States)

    Han, Xue; Zhou, Niya; Cui, Zhihong; Ma, Mingfu; Li, Lianbing; Cai, Min; Li, Yafei; Lin, Hui; Li, Ying; Ao, Lin; Liu, Jinyi; Cao, Jia

    2011-01-01

    Background Polycyclic aromatic hydrocarbons (PAHs), a class of the most ubiquitous environmental contaminants, may reduce male reproductive functions, but the data from human population studies are very limited. Objectives We designed this study to determine whether environmental exposure to PAHs contributes to the alteration in semen quality, sperm DNA damage, and apoptosis in the general male human population. Methods We measured urinary levels of four PAH metabolites and assessed semen quality, sperm apoptotic markers with Annexin V assay, and sperm DNA damage with comet assay in 232 men from Chongqing, China. Results We found that increased urinary 2-hydroxynaphthalene (2-OHNa) levels were associated with increased comet parameters, including the percentage of DNA in the tail (tail%) [β coefficient = 13.26% per log unit 2-OHNa (micrograms per gram creatinine); 95% confidence interval (CI), 7.97–18.55]; tail length (12.25; 95% CI, 0.01–24.52), and tail distribution (7.55; 95% CI, 1.28–18.83). The urinary level of 1-hydroxypyrene was associated only with increased tail% (5.32; 95% CI, 0.47–10.17). Additionally, the increased levels of four urinary PAH metabolites were significantly associated with decreased vital Annexin V negative sperm counts. However, there was no significant association between urinary PAH metabolite levels and human semen parameters or morphology of the sperm samples. Conclusions Our data indicate that the environmental level of PAH exposure is associated with increased sperm DNA damage but not with semen quality. These findings suggest that exposure to PAHs may disrupt sperm DNA and thereby interfere with human male fertility. PMID:21147605

  13. Fabrication of natural DNA-containing organic light emitting diodes

    Science.gov (United States)

    Gomez, Eliot F.; Spaeth, Hans D.; Steckl, Andrew J.; Grote, James G.

    2011-09-01

    The process of creating natural DNA-containing bio-organic light emitting diodes is a fascinating journey from salmon fish to the highly-efficient BiOLED. DNA from salmon sperm is used as a high-performance electron blocking layer, to enhance the efficiency of the BiOLED over its conventional OLED counterpart. An overview of the BiOLED fabrication process and its key steps are presented in this paper.

  14. Evaluation of sperm DNA quality in men presenting with testicular cancer and lymphoma using alkaline and neutral Comet assays.

    Science.gov (United States)

    Kumar, K; Lewis, S; Vinci, S; Riera-Escamilla, A; Fino, M-G; Tamburrino, L; Muratori, M; Larsen, P; Krausz, C

    2018-01-01

    Despite more cancers in young men over the past two decades, improvements in therapies give a greater chance to live full lives following treatment. Sperm genomic quality is variable following cancer diagnosis, so its assessment is important if sperm cryopreservation is being considered. Here, we evaluated DNA damage using two DNA damage assays: an alkaline and for the first time, a neutral Comet assays in men presenting with testicular cancer (n = 19 for alkaline and 13 for neutral group) and lymphoma (n = 13 for alkaline and 09 for neutral group) compared with fertile donors (n = 20 for alkaline and 14 for neutral group). No significant differences were observed in any semen analysis parameters. In contrast, sperm DNA damage was higher in men with testicular cancer than in donors as assessed by both the alkaline (12.4% vs. 37.4%, p Comet assays. Similar trends were observed in men with lymphoma. Here, sperm DNA damage was higher using both the alkaline (35.0% vs. 12.4%) and neutral (10.7% against 7.5% (p Comet assays. Moreover, the DNA strand breaks (particularly double-strand breaks) were significantly more prominent in men with cancer having abnormal seminal parameters than normozoospermic ones. This study showed that sperm DNA testing using alkaline and neutral Comet assays is more sensitive than semen analysis in detecting impaired sperm quality in men presenting with cancer. It may provide a useful adjunct when considering storage prior to cancer investigations and assisted reproductive techniques (ART)-based treatment. © 2017 American Society of Andrology and European Academy of Andrology.

  15. The antimalarial agent artesunate causes sperm DNA damage and hepatic antioxidant defense in mice.

    Science.gov (United States)

    Singh, Supriya; Giri, Anirudha; Giri, Sarbani

    2015-01-01

    Artesunate is an artemisinin derivative effective against multidrug resistant malaria. We analyzed the effects of artesunate 40 mg/kg b.w. as a single dose (ART1) or 13.3mg/kg b.w. for 3 days at 24h intervals (ART2) on mice spermatozoa at morphological and molecular level, and hepatic antioxidant status following 24h and 35 days following exposures in vivo. Artesunate significantly reduced epididymal sperm count and increased the frequency of sperms with abnormal head morphology following 24h of exposure. Comet assay analysis revealed significant increase in DNA strand breaks in spermatozoa evidenced by about 3-fold increase in comet tail DNA and up to 10-fold increase in Olive tail moment following 35 days of artesunate treatment. The damage index was significantly higher in the treated groups (40.27 ± 6.62 and 37.07 ± 5.35 for ART1 and ART2 respectively) as compared to the control group (16.13 ± 3.21) indicating the genotoxic effect of artesunate. The significant reduction in GSH, SOD and increase in lipid peroxidation indicate involvement of oxidative mechanisms in artesunate induced toxicity in mice. The present study suggests that artesunate has the potential to breach the testis-blood barrier and cause toxicity to male germ cells which may have implications in male reproductive toxicity. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Polycyclic aromatic hydrocarbons exposure decreased sperm mitochondrial DNA copy number: A cross-sectional study (MARHCS) in Chongqing, China.

    Science.gov (United States)

    Ling, Xi; Zhang, Guowei; Sun, Lei; Wang, Zhi; Zou, Peng; Gao, Jianfang; Peng, Kaige; Chen, Qing; Yang, Huan; Zhou, Niya; Cui, Zhihong; Zhou, Ziyuan; Liu, Jinyi; Cao, Jia; Ao, Lin

    2017-01-01

    Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental pollutants that have adverse effects on the male reproductive function. Many studies have confirmed that PAHs preferentially accumulate in mitochondria DNA relative to nuclear DNA and disrupt mitochondrial functions. However, it is rare whether exposure to PAHs is associated with mitochondrial damage and dysfunction in sperm. To evaluate the effects of PAHs on sperm mitochondria, we measured mitochondrial membrane potential (MMP), mitochondrial DNA copy number (mtDNAcn) and mtDNA integrity in 666 individuals from the Male Reproductive Health in Chongqing College Students (MARHCS) study. PAHs exposure was estimated by measuring eight urinary PAH metabolites (1-OHNap, 2-OHNap, 1-OHPhe, 2-OHPhe, 3-OHPhe, 4-OHPhe, 2-OHFlu and 1-OHPyr). The subjects were divided into low, median and high exposure groups using the tertile levels of urinary PAH metabolites. In univariate analyses, the results showed that increased levels of 2-OHPhe, 3-OHPhe, ∑Phe metabolites and 2-OHFlu were found to be associated with decreased sperm mtDNAcn. After adjusting for potential confounders, significantly negative associations of these metabolites remained (p = 0.039, 0.012, 0.01, 0.035, respectively). Each 1 μg/g creatinine increase in 2-OHPhe, 3-OHPhe, ∑Phe metabolites and 2-OHFlu was associated with a decrease in sperm mtDNAcn of 9.427%, 11.488%, 9.635% and 11.692%, respectively. There were no significant associations between urinary PAH metabolites and sperm MMP or mtDNA integrity. The results indicated that the low exposure levels of PAHs can cause abnormities in sperm mitochondria. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. The effect of sperm DNA fragmentation on live birth rate after IVF or ICSI: a systematic review and meta-analysis.

    Science.gov (United States)

    Osman, A; Alsomait, H; Seshadri, S; El-Toukhy, T; Khalaf, Y

    2015-02-01

    A systematic review and meta-analysis was conducted to evaluate the relationship between the extent of sperm DNA damage and live birth rate (LBR) per couple and the influence of the method of fertilization on treatment outcome. Searches were conducted on MEDLINE, EMBASE and Cochrane Library. Six studies were eligible for inclusion in the meta-analysis. Overall, LBR increased signficantly in couples with low sperm DNA fragmentation compared with those with high sperm DNA fragmentation (RR 1.17, 95% CI 1.07 to 1.28; P = 0.0005). After IVF and intracytoplasmic sperm injection (ICSI), men with low sperm DNA fragmentation had significantly higher LBR (RR 1.27, 95% CI 1.05 to 1.52; P = 0.01) and (RR 1.11, 95% CI 1.00 to 1.23, P = 0.04), respectively. A sensitivity analysis showed no statistically significant difference in LBR between low and high sperm DNA fragmentation when ICSI treatment was used (RR 1.08, 95% CI 0.39 to 2.96; P = 0.88). High sperm DNA fragmentation in couples undergoing assisted reproduction techniques is associated with lower LBR. Well-designed randomized studies are required to assess the role of ICSI over IVF in the treatment of men with high sperm DNA fragmentation. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  18. Evidence that single-stranded DNA breaks are a normal feature of koala sperm chromatin, while double-stranded DNA breaks are indicative of DNA damage.

    Science.gov (United States)

    Zee, Yeng Peng; López-Fernández, Carmen; Arroyo, F; Johnston, Stephen D; Holt, William V; Gosalvez, Jaime

    2009-08-01

    In this study, we have used single and double comet assays to differentiate between single- and double-stranded DNA damage in an effort to refine the interpretation of DNA damage in mature koala spermatozoa. We have also investigated the likelihood that single-stranded DNA breakage is part of the natural spermiogenic process in koalas, where its function would be the generation of structural bends in the DNA molecule so that appropriate packaging and compaction can occur. Koala spermatozoa were examined using the sperm chromatin dispersion test (SCDt) and comet assays to investigate non-orthodox double-stranded DNA. Comet assays were conducted under 1) neutral conditions; and 2) neutral followed by alkaline conditions (double comet assay); the latter technique enabled simultaneous visualisation of both single-stranded and double-stranded DNA breaks. Following the SCDt, there was a continuum of nuclear morphotypes, ranging from no apparent DNA fragmentation to those with highly dispersed and degraded chromatin. Dispersion morphotypes were mirrored by a similar diversity of comet morphologies that could be further differentiated using the double comet assay. The majority of koala spermatozoa had nuclei with DNA abasic-like residues that produced single-tailed comets following the double comet assay. The ubiquity of these residues suggests that constitutive alkali-labile sites are part of the structural configuration of the koala sperm nucleus. Spermatozoa with 'true' DNA fragmentation exhibited a continuum of comet morphologies, ranging from a more severe form of alkaline-susceptible DNA with a diffuse single tail to nuclei that exhibited both single- and double-stranded breaks with two comet tails.

  19. Characterization of Nuclease Activity in Human Seminal Plasma and its Relationship to Semen Parameters, Sperm DNA Fragmentation and Male Infertility.

    Science.gov (United States)

    Fernandez-Encinas, Alba; García-Peiró, Agustí; Ribas-Maynou, Jordi; Abad, Carlos; Amengual, María José; Navarro, Joaquima; Benet, Jordi

    2016-01-01

    Some studies have shown that complementary biomarkers are needed in semen analysis to provide a more accurate diagnosis for couples with infertility problems. To our knowledge no study has been done to determine the relationships among nuclease activity in seminal plasma, semen parameters, sperm DNA fragmentation and male infertility. A total of 94 semen samples were collected according to WHO 2010 semen analysis parameters. Samples were analyzed using the single radial enzyme diffusion method for nuclease activity in seminal plasma, and alkaline and neutral Comet assay for sperm DNA fragmentation. Samples were obtained from 11 fertile donors with proven fertility, 17 patients with normozoospermia in an infertile couple, and 16 patients with asthenozoospermia, 19 with teratozoospermia, 21 with asthenoteratozoospermia and 10 with azoospermia. Nuclease activity analyzed in seminal plasma was higher in patients than in controls. It correlated with sperm motility and morphology, and sperm DNA fragmentation measured by the alkaline Comet assay. No correlation with sperm DNA fragmentation was measured by the neutral Comet assay. ROC curves to determine male infertility revealed 0.658 sensitivity, 0.727 specificity and 0.705 cm(2) AUC for the single radial enzyme diffusion method, 0.918, 1 and 0.994 cm(2) for the alkaline Comet assay, and 0.917, 0.250 and 0.373 cm(2), respectively, for the neutral Comet assay. Nuclease activity in seminal plasma corrected by sperm count is a good variable to predict male infertility. Results indicate that it could be a useful complementary parameter for male infertility diagnosis. Copyright © 2016 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

  20. Exposures that may affect sperm DNA integrity: Two decades of follow-up in a pregnancy cohort

    DEFF Research Database (Denmark)

    Håkonsen, L B; Spano, M; Bonde, J P

    2012-01-01

    Prenatal lifestyle exposures are linked to alterations in conventional semen characteristics. Sperm DNA integrity is another marker of semen quality shown to be altered in mice prenatally exposed to chemicals. From a Danish pregnancy cohort established in 1984-1987, sons were selected for a follo...

  1. The effects of pyridaben pesticide on the DNA integrity of sperms and early in vitro embryonic development in mice

    Directory of Open Access Journals (Sweden)

    Ghodrat Ebadi Manas

    2013-01-01

    Full Text Available Background: Pyridaben, a pyridazinone derivative, is a new acaricide and insecticide for control of mites and some insects such as white flies, aphids and thrips. Objective: This study was designed to elucidate how pyridaben can affect the sperms' morphological parameters, its DNA integrity, and to estimate the effect of various quantities of pyridaben on in vitro fertilization rate. Materials and Methods: In this study, 80 adult male Balb/C strain mice were used. Animals were divided into control and two test groups. Control group received distilled water. The test group was divided into two subgroups, viz, high dose (212 mg/kg/day and low dose (53 mg/kg/day and they received the pyridaben, orally for duration of 45 days. The spermatozoa were obtained from caudae epididymides on day 45 in all groups. Sperm viability, protamin compression (nuclear maturity, DNA double-strand breaks, and in vitro fertilizing (IVF ability were examined. Results: The pyridaben treatment provoked a significant decrease in sperm population and viability in epididymides. The data obtained from this experiment revealed that, the pyridaben brings about negative impact on the sperm maturation and DNA integrity in a time-dependent manner, which consequently caused a significant (p<0.05 reduction in IVF capability. Embryo developing arrest was significantly (p<0.05 higher in treated than the control group. Conclusion: Theses results confirmed that, the pyridaben is able to induce DNA damage and chromatin abnormalities in spermatozoa which were evident by low IVF rate.

  2. Episodic air pollution is associated with increased DNA fragmentation in human sperm without other changes in semen quality

    Energy Technology Data Exchange (ETDEWEB)

    Rubes, J.; Selevan, S.G.; Evenson, D.P.; Zudova, D.; Vozdova, M.; Zudova, Z.; Robbins, W.A.; Perreault, S.D. [US EPA, Research Triangle Park, NC (United States)

    2005-10-01

    This study examined potential associations between exposure to episodes of air pollution and alterations in semen quality. The air pollution, resulting from combustion of coal for industry and home heating in the Teplice district of the Czech Republic, was much higher during the winter than at other times of year with peaks exceeding US air quality standards. Young men from Teplice were sampled up to seven times over 2 years allowing evaluation of semen quality after periods of exposure to both low and high air pollution. Routine semen analysis (sperm concentration, motility and morphology) and tests for sperm aneuploidy and chromatin integrity were performed, comparing measurements within each subject. Exposure was classified as high or low based on data from ambient air pollution monitoring. Using repeated measures analysis, a significant association was found between exposure to periods of high air pollution (at or above the upper limit of US air quality standards) and the percentage of sperm with DNA fragmentation according to sperm chromatin structure assay (SCSA). Other semen measures were not associated with air pollution. It is concluded that exposure to intermittent air pollution may result in sperm DNA damage and thereby increase the rates of male-mediated infertility, miscarriage, and other adverse reproductive outcomes.

  3. Comparison of post-thaw DNA integrity of boar spermatozoa assessed with the neutral comet assay and Sperm-Sus Halomax test kit.

    Science.gov (United States)

    Fraser, L; Parda, A; Filipowicz, K; Strzeżek, J

    2010-10-01

    In this study, we tested the hypothesis whether the neutral Comet assay (NCA) and the Sperm-Sus-Halomax (SSH) test kit could provide similar measurements of post-thaw DNA fragmentation of boar spermatozoa. Whole ejaculates or sperm-rich fractions of boar semen were frozen in an extender containing lactose, lipoprotein fractions isolated from ostrich egg yolk (LPFo), glycerol (lactose-LPFo-G) or in a standard boar semen extender (K3), without the addition of cryoprotective substances. In all boars, both the NCA and SSH test showed similar levels of post-thaw sperm DNA fragmentation in samples of the same ejaculates, regardless of the ejaculate collection procedure and extender. Yet, the levels of post-thaw sperm DNA damage, detected by the NCA and SSH test, were more accentuated in spermatozoa frozen in the absence of cryoprotective substances. Both the NCA and SSH detected variations among individual boars in terms of post-thaw sperm DNA fragmentation. Agreement between the measurements of the NCA and SSH was confirmed by scatter plots of differences, suggesting that the DNA integrity tests could detect the same sperm populations, which were susceptible to cryo-induced DNA damage. The findings of this study indicate that the NCA and the SSH test are effective in detecting similar levels of sperm DNA fragmentation and reinforce their importance in the assessment of frozen-thawed boar semen quality. © 2009 Blackwell Verlag GmbH.

  4. Electromagnetic and optical characteristics of Nb5+-doped double-crossover and salmon DNA thin films

    Science.gov (United States)

    Babu Mitta, Sekhar; Reddy Dugasani, Sreekantha; Jung, Soon-Gil; Vellampatti, Srivithya; Park, Tuson; Park, Sung Ha

    2017-10-01

    We report the fabrication and physical characteristics of niobium ion (Nb5+)-doped double-crossover DNA (DX-DNA) and salmon DNA (SDNA) thin films. Different concentrations of Nb5+ ([Nb5+]) are coordinated into the DNA molecules, and the thin films are fabricated via substrate-assisted growth (DX-DNA) and drop-casting (SDNA) on oxygen plasma treated substrates. We conducted atomic force microscopy to estimate the optimum concentration of Nb5+ ([Nb5+]O = 0.08 mM) in Nb5+-doped DX-DNA thin films, up to which the DX-DNA lattices maintain their structures without deformation. X-ray photoelectron spectroscopy (XPS) was performed to probe the chemical nature of the intercalated Nb5+ in the SDNA thin films. The change in peak intensities and the shift in binding energy were witnessed in XPS spectra to explicate the binding and charge transfer mechanisms between Nb5+ and SDNA molecules. UV-visible, Raman, and photoluminescence (PL) spectra were measured to determine the optical properties and thus investigate the binding modes, Nb5+ coordination sites in Nb5+-doped SDNA thin films, and energy transfer mechanisms, respectively. As [Nb5+] increases, the absorbance peak intensities monotonically increase until ˜[Nb5+]O and then decrease. However, from the Raman measurements, the peak intensities gradually decrease with an increase in [Nb5+] to reveal the binding mechanism and binding sites of metal ions in the SDNA molecules. From the PL, we observe the emission intensities to reduce them at up to ˜[Nb5+]O and then increase after that, expecting the energy transfer between the Nb5+ and SDNA molecules. The current-voltage measurement shows a significant increase in the current observed as [Nb5+] increases in the SDNA thin films when compared to that of pristine SDNA thin films. Finally, we investigate the temperature dependent magnetization in which the Nb5+-doped SDNA thin films reveal weak ferromagnetism due to the existence of tiny magnetic dipoles in the Nb5+-doped SDNA

  5. Investigating the effects of dietary folic acid on sperm count, DNA damage and mutation in Balb/c mice

    Energy Technology Data Exchange (ETDEWEB)

    Swayne, Breanne G.; Kawata, Alice [Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario, K1A 0K9 (Canada); Behan, Nathalie A. [Nutrition Research Division, Food Directorate, Health Products and Food Branch, Health Canada, Ottawa, Ontario, K1A 0K9 (Canada); Williams, Andrew; Wade, Mike G. [Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario, K1A 0K9 (Canada); MacFarlane, Amanda J. [Nutrition Research Division, Food Directorate, Health Products and Food Branch, Health Canada, Ottawa, Ontario, K1A 0K9 (Canada); Yauk, Carole L., E-mail: carole.yauk@hc-sc.ga.ca [Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario, K1A 0K9 (Canada)

    2012-09-01

    To date, fewer than 50 mutagens have been studied for their ability to cause heritable mutations. The majority of those studied are classical mutagens like radiation and anti-cancer drugs. Very little is known about the dietary variables influencing germline mutation rates. Folate is essential for DNA synthesis and methylation and can impact chromatin structure. We therefore determined the effects of folic acid-deficient (0 mg/kg), control (2 mg/kg) and supplemented (6 mg/kg) diets in early development and during lactation or post-weaning on mutation rates and chromatin quality in sperm of adult male Balb/c mice. The sperm chromatin structure assay and mutation frequencies at expanded simple tandem repeats (ESTRs) were used to evaluate germline DNA integrity. Treatment of a subset of mice fed the control diet with the mutagen ethylnitrosourea (ENU) at 8 weeks of age was included as a positive control. ENU treated mice exhibited decreased cauda sperm counts, increased DNA fragmentation and increased ESTR mutation frequencies relative to non-ENU treated mice fed the control diet. Male mice weaned to the folic acid deficient diet had decreased cauda sperm numbers, increased DNA fragmentation index, and increased ESTR mutation frequency. Folic acid deficiency in early development did not lead to changes in sperm counts or chromatin integrity in adult mice. Folic acid supplementation in early development or post-weaning did not affect germ cell measures. Therefore, adequate folic acid intake in adulthood is important for preventing chromatin damage and mutation in the male germline. Folic acid supplementation at the level achieved in this study does not improve nor is it detrimental to male germline chromatin integrity.

  6. Paternal sperm DNA methylation associated with early signs of autism risk in an autism-enriched cohort.

    Science.gov (United States)

    Feinberg, Jason I; Bakulski, Kelly M; Jaffe, Andrew E; Tryggvadottir, Rakel; Brown, Shannon C; Goldman, Lynn R; Croen, Lisa A; Hertz-Picciotto, Irva; Newschaffer, Craig J; Fallin, M Daniele; Feinberg, Andrew P

    2015-08-01

    Epigenetic mechanisms such as altered DNA methylation have been suggested to play a role in autism, beginning with the classical association of Prader-Willi syndrome, an imprinting disorder, with autistic features. Here we tested for the relationship of paternal sperm DNA methylation with autism risk in offspring, examining an enriched-risk cohort of fathers of autistic children. We examined genome-wide DNA methylation (DNAm) in paternal semen biosamples obtained from an autism spectrum disorder (ASD) enriched-risk pregnancy cohort, the Early Autism Risk Longitudinal Investigation (EARLI) cohort, to estimate associations between sperm DNAm and prospective ASD development, using a 12-month ASD symptoms assessment, the Autism Observation Scale for Infants (AOSI). We analysed methylation data from 44 sperm samples run on the CHARM 3.0 array, which contains over 4 million probes (over 7 million CpG sites), including 30 samples also run on the Illumina Infinium HumanMethylation450 (450K) BeadChip platform (∼485 000 CpG sites). We also examined associated regions in an independent sample of post-mortem human brain ASD and control samples for which Illumina 450K DNA methylation data were available. Using region-based statistical approaches, we identified 193 differentially methylated regions (DMRs) in paternal sperm with a family-wise empirical P-value [family-wise error rate (FWER)] syndrome gene cluster. These results were consistent among the 75 probes on the Illumina 450K array that cover AOSI-associated DMRs from CHARM. Further, 18 of 75 (24%) 450K array probes showed consistent differences in the cerebellums of autistic individuals compared with controls. These data suggest that epigenetic differences in paternal sperm may contribute to autism risk in offspring, and provide evidence that directionally consistent, potentially related epigenetic mechanisms may be operating in the cerebellum of individuals with autism. © The Author 2015; all rights reserved

  7. Sperm DNA fragmentation induced by cryopreservation: new insights and effect of a natural extract from Opuntia ficus-indica.

    Science.gov (United States)

    Meamar, Mehrdad; Zribi, Nassira; Cambi, Marta; Tamburrino, Lara; Marchiani, Sara; Filimberti, Erminio; Fino, Maria Grazia; Biggeri, Annibale; Menezo, Yves; Forti, Gianni; Baldi, Elisabetta; Muratori, Monica

    2012-08-01

    To analyze the effect of cryopreservation on sperm DNA fragmentation (SDF) in two cytometric sperm populations, PI(brighter) and PI(dimmer), and to test the effects of Opuntia ficus-indica (OFI) extracts, which contain antioxidants and flavanoids, and of resveratrol on cryopreservation of human semen. In vitro prospective study. Institutional study. Twenty-one normozoospermic men undergoing semen analysis for couple infertility. Cryopreservation using the routine method in the presence of OFI extracts or resveratrol. Measurement of SDF by TUNEL/PI flow cytometric method to evaluate sperm motility (by automated motion analysis, CASA system) and viability (by eosin/nigrosin staining) in the two populations of sperm PI(br) and PI(dim). Cryopreservation induced an increase of SDF only in the PI(br) sperm population. The increase was negatively dependent on the basal values of SDF in the same population. Addition of OFI extracts and resveratrol to the cryopreservation medium slightly but statistically significantly reduced SDF in the PI(br) population without affecting the deleterious effect of cryopreservation on sperm motion parameters or viability. The increase of SDF in the PI(br) population, which is unrelated to semen quality, suggests that caution must be taken in using cryopreserved semen, as morphologically normal and motile sperm may be damaged. The addition of substances with multifunctional properties such as OFI extracts to cryopreservation medium is only slightly effective in preventing the dramatic effects on SDF. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  8. Protective effects of Opuntia ficus-indica extract on ram sperm quality, lipid peroxidation and DNA fragmentation during liquid storage.

    Science.gov (United States)

    Allai, Larbi; Druart, Xavier; Öztürk, Mehmet; BenMoula, Anass; Nasser, Boubker; El Amiri, Bouchra

    2016-12-01

    The present study aimed to assess the phenolic composition of the acetone extract from Opuntia ficus indica cladodes (ACTEX) and its effects on ram semen variables, lipid peroxidation and DNA fragmentation during liquid storage at 5°C for up to 72h in skim milk and Tris egg yolk extenders. Semen samples from five rams were pooled extended with Tris-egg yolk (TEY) or skim milk (SM) extenders containing ACTEX (0%, 1%, 2%, 4% and 8%) at a final concentration of 0.8×109 sperm/ml and stored for up to 72h at 5°C. The sperm variables were evaluated at different time periods (8, 24, 48 and 72h). Sperm total motility and viability were superior in TEY than in SM whereas the progressive motility, membrane integrity, abnormality and spontaneous lipid peroxidation were greater in SM compared to TEY (P<0.05). The results also indicated that the inclusion of 1% ACTEX in the SM or TEY extender increased the sperm motility, viability, membrane integrity, and decreased the abnormality, lipids peroxidation up to 72h in storage compared to control group. Similarly, even at 72h of storage, 1% ACTEX can efficiently decrease the negative effects of liquid storage on sperm DNA fragmentation (P<0.05). In conclusion, SM and TEY supplemented with 1% of ACTEX can improve the quality of ram semen. Further studies are required to identify the active components in ACTEX involved in its effect on ram sperm preservation. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Sperm DNA damage output parameters measured by the alkaline Comet assay and their importance.

    Science.gov (United States)

    Simon, L; Aston, K I; Emery, B R; Hotaling, J; Carrell, D T

    2017-03-01

    The alkaline Comet assay has shown high diagnostic value to determine male reproductive health and prognostic ability to predict ART success. Here, spermatozoon was analysed in 47 fertile donors and 238 patients, including 132 couples undergoing ART [semen was collected: Group I - within 3 months of their treatment (n = 79); and Group II - 3 months prior to their treatment (n = 53)]. We introduce four Comet distribution plots (A, B1, B2 and C) by plotting the level of DNA damage (x-axis) and percentage of comets (y-axis). Fertile donors had low mean DNA damage, olive tail moment and per cent of spermatozoa with damage and increased type A plots. Comet parameters were associated with clinical pregnancies in Group I. About 66% of couples with type A distribution plot were successful after ART, whereas couples with type B1, B2 and C distribution plots achieved 56%, 44% and 33% pregnancies respectively. The efficiency of the Comet assay was due to complete decondensation process, where the compact sperm nuclear DNA (28.2 ± 0.2 μm 3 ) is decondensed to ~63 μm 3 (before lysis) and ~1018 μm 3 (after lysis). A combinational analysis of all the Comet output parameters may provide a comprehensive evaluation of patient's reproductive health as these parameters measure different aspects of DNA damage within the spermatozoa. © 2016 Blackwell Verlag GmbH.

  10. Evaluation of a modified comet assay to detect DNA damage in mammalian sperm exposed in vitro to different mutagenic compounds.

    Science.gov (United States)

    Villani, Paola; Spanò, Marcello; Pacchierotti, Francesca; Weimer, Marc; Cordelli, Eugenia

    2010-08-01

    The final stages of male gametogenesis are sensitive targets of DNA-reactive chemicals, most of which form adducts. Comet assay is a widely applied genotoxicity test that reveals DNA adducts through breaks formed during repair processes. However, sperm cells are essentially devoid of repair enzymes and comet assay is poorly sensitive in detecting chemically induced DNA lesions in sperm. To overcome such limitation, in a previous paper we proposed a modified protocol for comet assay. In this work we further tested the method treating bull sperm with additional mutagens (diethylsulfate, mitomycin C, bleomycin and colchicine) in parallel with the standard comet assay. No treatment-related increase of DNA migration was ever detected with the standard protocol. A dose-dependent effect of diethylsulfate, was obtained with the modified assay. As expected, the mitotic poison colchicine resulted negative even by the modified assay. Results with the other two compounds were consistent with their mechanism of action. Copyright 2009 Elsevier Inc. All rights reserved.

  11. Drug-Delivery System Based on Salmon DNA Nano- and Micro-Scale Structures.

    Science.gov (United States)

    Lee, Yunwoo; Dugansani, Sreekantha Reddy; Jeon, So Hee; Hwang, Soon Hyoung; Kim, Jae-Hyun; Park, Sung Ha; Jeong, Jun-Ho

    2017-08-29

    Microneedles, fabricated by nano-moulding technology show great promise in the field of drug delivery by enabling the painless self-administration of drugs in a patient-friendly manner. In this study, double-stranded salmon DNA (SDNA) was used as both a drug-delivery vehicle and structural material with a microneedle system. SDNA is non-toxic and demonstrates good mechanical robustness, mouldability, biocompatibility, bio-absorbability, and binding affinity with drug molecules for bio-functional applications. Benign fabrication conditions to protect temperature-sensitive biomolecules are used to produce SDNA structures of various sizes with a high aspect ratio (4: 1). Unlike existing dissolving microneedle structure materials, the special binding characteristics of doxorubicin hydrochloride, anti-cancer drug molecules, and SDNA demonstrate the stability of drug-molecule encapsulation via UV-absorption and photoluminescence analyses. Based on COMSOL simulation and in vitro analysis of the stratum corneum of porcine skin, the mechanical functionality of SDNA microneedles was evaluated in vitro by penetrating the stratum corneum of porcine skin. The SDNA microneedle dissolved and drug permeation was assessed using rhodamine, a drug surrogate. Owing to its many beneficial characteristics, we anticipate that the SDNA microneedle platform will serve as an effective alternative for drug delivery.

  12. Integrity of human sperm DNA assessed by the neutral comet assay and its relationship to semen parameters and clinical outcomes for the IVF-ET program.

    Science.gov (United States)

    Chi, Hee-Jun; Chung, Da-Yeon; Choi, Soon-Young; Kim, Jong-Hyun; Kim, Gi-Young; Lee, Jae-Seok; Lee, Hee-Sun; Kim, Myung-Hee; Roh, Sung-Il

    2011-03-01

    To explore potential relationships between sperm DNA integrity and both semen parameters and clinical outcomes. Semen analysis of 498 samples was performed according to the 2010 criteria of the World Health Organization. The sperm DNA fragmentation Index (DFI) of the semen samples was assessed using a neutral comet assay. Sperm DFI showed a significant correlation with semen parameters, including the patient's age, sperm viability, motility, morphology, and number of leukocytes (psperm DFI values for asthenozoospermic (15.2%), oligoteratozoospermic (18.3%), asthenoteratozoospermic (17.5%), and oligoasthenoteratozoospermic semen samples (21.3%) were significantly higher than that observed in normozoospermic semen samples (10.5%, psperm DFI value of 14% was used as a threshold of sperm DFI in assessing whether DNA was highly damaged. In 114 IVF-ET cycles, the fertilization rate of the sperm DFI sperm DFI assessed using the comet assay was shown to improve the quality of the semen evaluation. To evaluate the precise effect of ICSI on pregnancy rates in the patients who demonstrate high sperm DFI values, further study is necessary.

  13. Comparison of four methods to evaluate sperm DNA integrity between mouse caput and cauda epididymidis

    OpenAIRE

    Pérez-Cerezales, Serafín; Miranda, Alberto; Gutiérrez-Adán, Alfonso

    2011-01-01

    It is well known that transit through the epididymis involves an increase in the compaction of sperm chromatin, which acquires fully condensed status at the caput epididymidis. The purpose of this study was to compare the terminal deoxyribonucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) assay, the comet assay, the sperm chromatin structure assay (SCSA) and the sperm chromatin dispersion (SCD) test by analysing spermatozoa from the caput and cauda epididymidis in order to demo...

  14. HPV‐DNA sperm infection and infertility: from a systematic literature review to a possible clinical management proposal

    National Research Council Canada - National Science Library

    Foresta, C; Noventa, M; De Toni, L; Gizzo, S; Garolla, A

    2015-01-01

    ... ) sperm infection on male fertility, impairment of sperm parameters, and possible alteration of sperm nuclear status and to identify a possible effective management of infertile men with HPV sperm infection...

  15. The relationship between environmental exposures to phthalates and DNA damage in human sperm using the neutral comet assay.

    OpenAIRE

    Duty, Susan M; Singh, Narendra P; Silva, Manori J; Barr, Dana B; Brock, John W; Ryan, Louise; Herrick, Robert F; Christiani, David C; Hauser, Russ

    2003-01-01

    Phthalates are industrial chemicals widely used in many commercial applications. The general population is exposed to phthalates through consumer products as well as through diet and medical treatments. To determine whether environmental levels of phthalates are associated with altered DNA integrity in human sperm, we selected a population without identified sources of exposure to phthalates. One hundred sixty-eight subjects recruited from the Massachusetts General Hospital Andrology Laborato...

  16. Impact of extensive laparoscopic venous disconnection on the recurrence rate and sperm DNA quality in infertile varicocele patients

    Science.gov (United States)

    Abdelaziz, Alsayed Saad; Burham, Waleed Ahmed; Aboelsaad, Ahmed Yosef; Badran, Yaser Ali; Ahmed, Abul-Fotouh Abdel-Maguid

    2015-01-01

    Introduction: Although there are many literature examining the possible effects of varicocelectomy on classic semen parameters and recurrence rate, few published articles have examined the effect of conventional laparoscopic varicocelectomy on sperm DNA integrity. Objectives: The objective was to evaluate the effect of extensive laparoscopic venous disconnections on the recurrence rate and sperm DNA damage in varicocele patients. Methods: Totally, 54 patients with varicocele underwent extensive laparoscopic venous disconnections were assessed by clinical evaluation, duplex scan, semen analysis, and sperm DNA fragmentation assay before surgery and after 6 and 12 months following surgery. Results: No intra- or post-operative complications were observed and out of 54 patients preoperatively complained from varicocele 2 (3.7%) patients' have recurrence during the follow-up period for 12 months. Out of 54 patients complaining from male infertility, 14 patients success to get pregnancy after 6 months with pregnancy rates of 25.92% and 22 (40.74%) after 12 months, and 28 patients (51.85%) had a preoperative DNA fragmentation index (DFI) >30%, decreased following surgery below 30% in 19 (35.18%) patients after 6 months, and 11 (20.37%) after 12 months, and the percentage of sperm with DFI > 30% was significantly decreased after 6 and 12 months, respectively (38.4 ± 10.6 vs. 31.3 ± 12.4, [P < 0.001] at 6 months, and 22.9 ± 13.2, [P < 0.001] after 1-year). Other spermatic parameter was significantly improved. Conclusions: An extensive laparoscopic venous disconnection was significantly decreasing the recurrence rate, DFI and improving normal semen parameters and fertility. PMID:26692670

  17. Serum levels of lycopene, beta-carotene, and retinol and their correlation with sperm DNA damage in normospermic and infertile men

    Directory of Open Access Journals (Sweden)

    Taiebeh Ghyasvand

    2015-12-01

    Full Text Available Background: Oxidative stress in reproductive system leads to sperm DNA damage and sperm membrane lipid peroxidation and may play an important role in the pathogenesis of male infertility, especially in idiopathic cases. Antioxidants such as carotenoids function against free radical damages. Objective: The aim of this study was to determine the levels of lycopene, beta-carotene and retinol in serum and their relationship with sperm DNA damage and lipid peroxidation in infertile and normospermic males. Materials and Methods: Sixty two infertile men and 71 normospermic men participated in this study. Blood and semen samples were collected from all subjects. Sperm DNA damage was measured using TUNEL method. Carotenoids, retinol, and malonedildehyde in serum were also determined. Results: DNA fragmentation was higher in infertile group comparing to control group. Serum levels of lycopene, beta-carotene and, vitamin A in infertile men were significantly lower than normospermic men (p< 0.001, =0.005, and =0.003 respectively. While serum MDA was not significantly different between two groups, MDA in seminal plasma of infertile men was significantly higher than control group (p< 0.001. Conclusion: We concluded that lycopene, beta-carotene, and retinol can reduce sperm DNA fragmentation and lipid peroxidation through their antioxidant effect. Therefore the DNA fragmentation assay and determination of antioxidants factors such as lycopene, beta-carotene and retinol, along with sperm analysis can be useful in diagnosis and treatment of men with idiopathic infertility.

  18. Associations of urinary metal levels with serum hormones, spermatozoa apoptosis and sperm DNA damage in a Chinese population.

    Science.gov (United States)

    Wang, Yi-Xin; Sun, Yang; Huang, Zhen; Wang, Peng; Feng, Wei; Li, Jin; Yang, Pan; Wang, Mu; Sun, Li; Chen, Ying-Jun; Liu, Chong; Yue, Jing; Gu, Long-Jie; Zeng, Qiang; Lu, Wen-Qing

    2016-09-01

    Exposure to metals, including essential and nonessential elements, is widespread and may be associated with male reproductive health. To examine whether environmental exposure to metals contributes to reproductive hormone changes, spermatozoa apoptosis and sperm DNA damage in a Chinese population. Eighteen metals (aluminum, arsenic, antimony, chromium, cobalt, copper, cadmium, iron, lead, manganese, molybdenum, nickel, selenium, tin, tungsten, thallium, uranium and zinc) were analyzed in two urine samples collected a few hours apart from male partners of couples attending an infertility clinic. Multivariable linear regression models were used to assess the cross-sectional associations of average urinary metal levels with serum hormones (n=511), spermatozoa apoptosis measures (n=460) and sperm DNA damage parameters (n=516). We found significant inverse dose-dependent trends of urinary tin quartiles with total testosterone (T), and tin, nickel, zinc and molybdenum with the ratio of total T to luteinizing hormone (total T/LH ratio) (all Ptrendmetals, and they persisted when the metals were modeled as continuous variables in a cubic spline analysis. There were no significant associations between urinary metals and sperm DNA damage after adjustment for multiple testing. Environmental exposure to tin, nickel, zinc and molybdenum may be associated decreased total T or total T/LH ratio; manganese may induce spermatozoa apoptosis, while iron may be important for living spermatozoa. However, additional prospective research is needed to corroborate these findings in the general population. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Assessment of the DNA Damage in Human Sperm and Lymphocytes Exposed to the Carcinogen Food Contaminant Furan with Comet Assay

    Directory of Open Access Journals (Sweden)

    Dilek Pandir

    2015-10-01

    Full Text Available ABSTRACTThe aim of this work was to assess the damage of DNA in human blood cell and spermin vitro under the influence of furan. These cells were administered 0-600 μM of furan at 37 and 32oC for 30 and 60 min, respectively. A significant increase in tail DNA%, tail length and moment indicating DNA damage was observed at increasing doses when compared to the controls. The treatment with 300 and 600 μM of furan showed a maximum increase of 86.74 ± 2.43 and 93.29 ± 8.68 compared to the control tail DNA% in lymphocytes. However, only 600 μM of furan showed a maximum increase of 94.71 ± 6.24 compared to the control tail DNA% in sperm. The results suggested that furan caused DNA damage in lymphocytes at increasing doses, but appeared to have not the same effect on human sperm at the low doses. Genotoxic activity had an impact on the risk assessment of furan formed on the food for human cells. Therefore, it would be important to further investigate these properties of furan as the food mutagen.

  20. Clinical and prognostic significance of human papillomavirus DNA in the sperm or exfoliated cells of infertile patients and subjects with risk factors.

    Science.gov (United States)

    Foresta, Carlo; Pizzol, Damiano; Moretti, Afra; Barzon, Luisa; Palù, Giorgio; Garolla, Andrea

    2010-10-01

    To evaluate human papillomavirus (HPV) sperm infection and its correlation with sperm parameters in infertile patients and subjects with risk factors. Cross-sectional clinical study. Andrology and microbiology sections at a university hospital. A cohort of 290 subjects: 26 with genital warts, 66 with HPV positive partners, 108 infertile patients, and 90 fertile controls. Semen analysis, sperm culture, polymerase chain reaction, and fluorescence in situ hybridization (FISH) for HPV detection. Statistical analysis was performed with a two-tailed Student's t-test. The prevalence of HPV semen infection found in all groups was as follows: patients with genital warts, 53.8%; infected partners, 40.9%; infertile patients, 10.2%, fertile controls, 2.2%. Infertile patients had a higher HPV DNA prevalence in sperm cells than the other groups. The results of HPV investigation were compared with sperm parameters and the results of FISH analysis. Sperm motility was more frequently reduced in infected samples and in particular when the infection was present in the sperm. This study demonstrated a very high prevalence of infection in the semen of patients with risk factors for HPV. In each group of subjects, HPV seems to be preferentially located in sperm or exfoliated cells, with different effects on sperm motility. Copyright © 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  1. [HYPERBARIC OXYGEN THERAPY IN THE TREATMENT OF MALE INFERTILITY ASSOCIATED WITH INCREASED SPERM DNA FRAGMENTATION AND REACTIVE OXYGEN SPECIES IN SEMEN].

    Science.gov (United States)

    Metelev, A Yu; Bogdanov, A B; Ivkinl, E V; Mitrokhin, A A; Vodneva, M M; Veliev, E I

    2015-01-01

    The aim of this study was to explore the potential of hyperbaric oxygenation (HBO) for reduction of sperm DNA fragmentation level and reactive oxygen species (ROS) in semen. The study included 90 men with idiopathic infertility. Patients of the treatment group (n = 60) underwent HBO before the vitro fertilization (IVF) procedure. In the control group (n = 30) IVF was carried out without prior cours of HBO. Sperm DNA fragmentation analysis was carried out using the TUNEL assay, the level of ROS in the ejaculate was measured by chemiluminescence. HBO treatment resulted in a significant decrease in the mean level of sperm DNA fragmentation from 33.2 ± 7.5 to 11.9 ± 5.9%, and the median ROS in sperm from 0.89 to 0.39 mV/s (p treatment group men and in 36.7% (11/30) of the control group (p treatment of men with idiopathic infertility.

  2. Frozen-thawed spermatozoa from oligozoospermic ejaculates are susceptible to in situ DNA fragmentation in polyvinylpyrrolidone-based sperm-immobilization medium.

    Science.gov (United States)

    Salian, Sujit Raj; Kalthur, Guruprasad; Uppangala, Shubhashree; Kumar, Pratap; Adiga, Satish Kumar

    2012-08-01

    To elucidate the effect of sperm immobilization media that are and are not based on polyvinylpyrrolidone (PVP) on the DNA integrity of fresh and frozen-thawed spermatozoa during standard intracytoplasmic sperm injection (ICSI) conditions. Experimental prospective study. Embryology research laboratory. Forty-six ejaculates from normozoospermic and oligozoospermic men. Assessment of sperm DNA fragmentation by single-cell gel electrophoresis assay. DNA integrity of fresh and frozen-thawed spermatozoa from normozoospermic and oligozoospermic ejaculates exposed to PVP-based and non-PVP-based media. Exposure of fresh and frozen thawed spermatozoa from normozoospermic and oligozoospermic ejaculates to PVP-based medium in an ICSI dish for 30 minutes statistically significantly increased the DNA fragmentation. In contrast, the extent of DNA fragmentation in non-PVP-based medium did not statistically significantly differ from control. A PVP-based medium can induce a statistically significant amount of sperm DNA fragmentation in an ICSI dish, and frozen-thawed sperm from oligozoospermic ejaculates are more susceptible to in situ DNA fragmentation. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  3. Increasing the luminous efficiency of an MEH-PPV based PLED using salmon DNA and single walled carbon nanotube

    Energy Technology Data Exchange (ETDEWEB)

    Madhwal, Devinder, E-mail: devindermadhwal@gmail.co [Department of Electronic Science, University of Delhi South campus, Benito Juarez Road, New Delhi 110021 (India); Singh, Inderpreet; Kumar, Jitender [Department of Electronic Science, University of Delhi South campus, Benito Juarez Road, New Delhi 110021 (India); Bhatia, C.S. [Department of Electrical and Computer Engineering, National University of Singapore (Singapore); Bhatnagar, P.K.; Mathur, P.C. [Department of Electronic Science, University of Delhi South campus, Benito Juarez Road, New Delhi 110021 (India)

    2011-07-15

    The combined effect of a salmon deoxyribonucleic acid (DNA)-based electron blocking layer and a single walled carbon nanotube (SWCNT) composite-based electron transport layer on the performance of a poly[2-methoxy-5-(2'-ethyl-hexyloxy)-1,4-phenylene vinylene](MEH-PPV) polymer light emitting diode (PLED) has been examined. The SWCNT network in the composite layer improves electron injection from cathode and the DNA blocks these high mobility electrons at the electron blocking layer-polymer interface, leading to high luminance from the device. The luminous efficiency of the PLED is increased {approx}20 times compared to that of a PLED using only MEH-PPV. - Highlights: {yields} We report fabrication of a high luminous efficiency MEH-PPV based polymer LED. {yields} Salmon DNA-CTMA layer is used to block injected electrons in the polymer layer. {yields} MEH-PPV-SWCNT composite is used to transport electrons in the polymer layer. {yields} The luminous efficiency of the polymer LED thereby improves about 20 times.

  4. The relationship between environmental exposures to phthalates and DNA damage in human sperm using the neutral comet assay.

    Science.gov (United States)

    Duty, Susan M; Singh, Narendra P; Silva, Manori J; Barr, Dana B; Brock, John W; Ryan, Louise; Herrick, Robert F; Christiani, David C; Hauser, Russ

    2003-07-01

    Phthalates are industrial chemicals widely used in many commercial applications. The general population is exposed to phthalates through consumer products as well as through diet and medical treatments. To determine whether environmental levels of phthalates are associated with altered DNA integrity in human sperm, we selected a population without identified sources of exposure to phthalates. One hundred sixty-eight subjects recruited from the Massachusetts General Hospital Andrology Laboratory provided a semen and a urine sample. Eight phthalate metabolites were measured in urine by using high-performance liquid chromatography and tandem mass spectrometry; data were corrected for urine dilution by adjusting for specific gravity. The neutral single-cell microgel electrophoresis assay (comet assay) was used to measure DNA integrity in sperm. VisComet image analysis software was used to measure comet extent, a measure of total comet length (micrometers); percent DNA in tail (tail%), a measure of the proportion of total DNA present in the comet tail; and tail distributed moment (TDM), an integrated measure of length and intensity (micrometers). For an interquartile range increase in specific gravity-adjusted monoethyl phthalate (MEP) level, the comet extent increased significantly by 3.6 micro m [95% confidence interval (95% CI), 0.74-6.47]; the TDM also increased 1.2 micro m (95% CI, -0.05 to 2.38) but was of borderline significance. Monobutyl, monobenzyl, monomethyl, and mono-2-ethylhexyl phthalates were not significantly associated with comet assay parameters. In conclusion, this study represents the first human data to demonstrate that urinary MEP, at environmental levels, is associated with increased DNA damage in sperm.

  5. Influence of Selected Extenders for Liquid Storage at 4 degrees C of Breeding Chinchilla (Chinchilla lanigera) Semen on Sperm DNA Integrity.

    Science.gov (United States)

    Niedbała, Piotr; Szeleszczuk, Olga; Kuchta-Gładysz, Marta; Joneczek, Maria; Dobrzyńska, Małgorzata; Maj, Dorota

    2015-01-01

    The influence of two commercial and two laboratory oriented extenders on survival rate and DNA integrity of chinchilla (Chinchilla lanigera) sperm was determined during liquid storage. Semen was collected using an electroejaculator from 6 adult male chinchillas. Ejaculates (n = 16) were diluted with extenders to obtain a concentration of 40 x 10 (3) sperm/5 μl. After dilution the semen samples were stored at 4"C. The percent motility, progressive motility, and morphology were assessed conventionally, whereas DNA integrity was evaluated by Single Cell Gel Electrophoresis (comet) assay at 0 (just after dilution), 24, 48 and 72 h. Conventional assessment of sperm quality showed that commercial extenders are characterized by the lowest sperm survival parameters out of the investigated extenders. In commercial extenders spermatozoa lost their capacity for progressive motility compared to laboratory extenders. After 24 h storage, from 21.67% to 30% of motile sperms were observed in commercial extender whereas total sperm motility was 63.33% (41.67% with progressive motility) in samples in which stallion semen extender was used. After 72 h storage, 10% of sperm were motile in stallion semen extender while no sperm movement was observed in tubes containing the commercial extender. Furthermore, a lower percentage of damaged spermatozoa in laboratory oriented extenders was demonstrated. It was also stated that along with the extended time of semen storage at 4 degrees C, commercial extenders lost their protective action. An analysis of DNA content in the heads of sperm cells and tail moment (TM) showed that the most useful extender for liquid preservation of chinchilla semen was the extender for stallions.

  6. The effects of male age on sperm DNA damage in healthy non-smokers

    National Research Council Canada - National Science Library

    Schmid, T E; Eskenazi, B; Baumgartner, A; Marchetti, F; Young, S; Weldon, R; Anderson, D; Wyrobek, A J

    The trend for men to have children at older age raises concerns that advancing age may increase the production of genetically defective sperm, increasing the risks of transmitting germ-line mutations...

  7. Protective effect of alpha glucosyl hesperidin (G-hesperidin) on chronic vanadium induced testicular toxicity and sperm nuclear DNA damage in male Sprague Dawley rats.

    Science.gov (United States)

    Vijaya Bharathi, B; Jaya Prakash, G; Krishna, K M; Ravi Krishna, C H; Sivanarayana, T; Madan, K; Rama Raju, G A; Annapurna, A

    2015-06-01

    The study was conducted to evaluate the vanadium-induced testicular toxicity and its effect on sperm parameters, sperm nuclear DNA damage and histological alterations in Sprague Dawley rats and to assess the protective effect of G-hesperidin against this damage. Treatment of rats with vanadium at a dose of 1 mg kg bw(-1) for 90 days resulted in significant reduction in serum testosterone levels, sperm count and motility. Further, a parallel increase in abnormal sperm morphology and adverse histopathological changes in testis was also associated with vanadium administration when compared to normal control. Moreover, sperm chromatin dispersion assay revealed that vanadium induces sperm nuclear DNA fragmentation. A marked increase in testicular malondialdehyde levels and decreased activity of antioxidant enzymes such as superoxide dismutase and catalase indicates vanadium-induced oxidative stress. Co-administration of G-hesperidin at a dose of 25 and 50 mg kg bw(-1) significantly attenuated the sperm parameters and histological changes by restoring the antioxidant levels in rat testis. These results suggested that vanadium exposure caused reduced bioavailability of androgens to the tissue and increased free radical formation, thereby causing structural and functional changes in spermatozoa. G-hesperidin exhibited antioxidant effect by protecting the rat testis against vanadium-induced oxidative damage, further ensures antioxidant potential of bioflavonoids. © 2014 Blackwell Verlag GmbH.

  8. Sperm DNA fragmentation index does not correlate with blastocyst aneuploidy or morphological grading.

    Directory of Open Access Journals (Sweden)

    Itai Gat

    Full Text Available High DNA fragmentation index (DFI may be associated with poor outcome after IVF. Our aim was to determine whether DFI impacts blastocyst quality or clinical outcome. This retrospective study included 134 couples who underwent 177 IVF-ICSI and pre-implantation genetic screening (PGS cycles during January 1st, 2014-March 31st, 2016 and had documented previous DFI. Group 1 (DFI>30% encompassed 25 couples who underwent 36 cycles; Group 2 (DFI 15-30% included 45 couples and 57 cycles; group 3 (DFI<15% included 64 couples and 83 cycles. Male partners within group 1 were older (45.1 compared to 40.6 and 38.3 years, respectively, p<0.05, had higher BMI (32.4 compared to 26.6 and 25.8 respectively, p<0.05 and lower sperm count and motility (46*106/ml and 35.5%, respectively compared to groups 2 (61.8*106/ml and 46.6%, respectively and 3 (75.8*106/ml and 55.1%, respectively, p<0.05. Female parameters including ovarian reserve and response and embryo development were similar. Total numbers of biopsied blastocysts were 116, 175 and 259 in groups 1, 2 and 3, respectively. PGS for 24 chromosomes revealed comparable euploidy rate of 46-50.4%, with a similar morphological classification. No significant differences were found regarding pregnancy rates or pregnancy loss. It seems that DFI doesn't correlate with blastocyst aneuploidy or morphological grading.

  9. Characterisation of QTL-linked and genome-wide restriction site-associated DNA (RAD markers in farmed Atlantic salmon

    Directory of Open Access Journals (Sweden)

    Houston Ross D

    2012-06-01

    Full Text Available Abstract Background Restriction site-associated DNA sequencing (RAD-Seq is a genome complexity reduction technique that facilitates large-scale marker discovery and genotyping by sequencing. Recent applications of RAD-Seq have included linkage and QTL mapping with a particular focus on non-model species. In the current study, we have applied RAD-Seq to two Atlantic salmon families from a commercial breeding program. The offspring from these families were classified into resistant or susceptible based on survival/mortality in an Infectious Pancreatic Necrosis (IPN challenge experiment, and putative homozygous resistant or susceptible genotype at a major IPN-resistance QTL. From each family, the genomic DNA of the two heterozygous parents and seven offspring of each IPN phenotype and genotype was digested with the SbfI enzyme and sequenced in multiplexed pools. Results Sequence was obtained from approximately 70,000 RAD loci in both families and a filtered set of 6,712 segregating SNPs were identified. Analyses of genome-wide RAD marker segregation patterns in the two families suggested SNP discovery on all 29 Atlantic salmon chromosome pairs, and highlighted the dearth of male recombination. The use of pedigreed samples allowed us to distinguish segregating SNPs from putative paralogous sequence variants resulting from the relatively recent genome duplication of salmonid species. Of the segregating SNPs, 50 were linked to the QTL. A subset of these QTL-linked SNPs were converted to a high-throughput assay and genotyped across large commercial populations of IPNV-challenged salmon fry. Several SNPs showed highly significant linkage and association with resistance to IPN, and population linkage-disequilibrium-based SNP tests for resistance were identified. Conclusions We used RAD-Seq to successfully identify and characterise high-density genetic markers in pedigreed aquaculture Atlantic salmon. These results underline the effectiveness of RAD

  10. An investigation of the potential effect of sperm nuclear vacuoles in human spermatozoa on DNA fragmentation using a neutral and alkaline Comet assay.

    Science.gov (United States)

    Pastuszek, E; Kiewisz, J; Skowronska, P; Liss, J; Lukaszuk, M; Bruszczynska, A; Jakiel, G; Lukaszuk, K

    2017-03-01

    Presence of vacuoles and degree of sperm DNA damage are considered to be the basic factors used for the assessment of sperm fertilization capacity. We aimed to investigate the link between these two parameters. According to our knowledge, this is the first study where the Comet assay was used to assess the degree of DNA fragmentation of sperm categorized by Motile Sperm Organelle Morphology Examination (MSOME) Grades. Semen samples from 10 patients were assessed. Spermatozoa were graded into four MSOME groups according to the Vanderzwalmen's criteria. A total of 3930 motile spermatozoa were selected one-by-one using an inverted microscope and transferred onto two different slides. The degree of DNA fragmentation was analyzed by alkaline and neutral Comet assay. Results of the neutral Comet assay showed that Grade I spermatozoa (absence of vacuoles) presented significantly lower dsDNA fragmentation level (mean: 3.13 ± 1.17%) than Grade II (maximum of two small vacuoles; mean: 10.34 ± 2.65%), Grade III (more than two small vacuoles or at least one large vacuole; mean: 23.88 ± 8.37%), and Grade IV (large vacuoles associated with abnormal head shapes or other abnormalities; mean: 36.94 ± 7.78%; p Comet assay showed that Grade I spermatozoa had significantly lower DNA (ssDNA + dsDNA) fragmentation level (mean: 8.33 ± 3.62%) than Grade III (mean: 25.64 ± 9.15%) and Grade IV (mean: 40.10 ± 9.10%, p  0.05). Probably, the vacuoles may be responsible for double strand DNA breaks rather than single strand DNA breaks (only 2.39% spermatozoa in MSOME Grade II, 1.76% in III, and 3.16% in IV has single strand breaks). The results demonstrate that lower MSOME grading correlates with lower sperm DNA fragmentation. Therefore, the observation of sperm nuclear vacuoles using real-time optical microscopy without precise DNA fragmentation examination is not sufficient for optimal sperm selection for intracytoplasmic sperm injection. © 2017 American Society of

  11. Development of a one-tube extraction and amplification method for DNA analysis of sperm and epithelial cells recovered from forensic samples by laser microdissection.

    Science.gov (United States)

    Meredith, Melanie; Bright, Jo-Anne; Cockerton, Sarah; Vintiner, Sue

    2012-01-01

    Laser microdissection can be used in forensic casework to isolate specific cell types from mixtures of biological samples. Extraction of DNA from selected cells is still required prior to STR amplification. Because of the relatively pristine nature of the recovered cells, laser microdissection is more sensitive than more traditional methods of DNA analysis, theoretically resulting in DNA profiles from less cellular material. A one-tube extraction and amplification method minimises loss of DNA through liquid transfers and reduces the potential for contamination events occurring. In this paper, the development of a one-tube method for the effective extraction of DNA from laser microdissected sperm and epithelial cells is described. The performance of the in-house method was compared to that of a commercial DNA extraction kit for extraction of DNA from sperm and the downstream compatibility with STR amplification was determined for both sperm and epithelial samples. Full Identifiler™ profiles after 28 amplification cycles were obtained from as few as 15 epithelial cells and 30 sperm. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  12. Mechanistic pathway for controlled extraction of guest molecule bound to herring sperm DNA using α-cyclodextrin

    Science.gov (United States)

    Jaffer, S. Syed; Ghosh, Prasun; Purkayastha, Pradipta

    2011-05-01

    trans-2-[4-(Dimethylamino)styryl]benzothiazole (DMASBT) is known to have dual emitting states where the locally excited (LE) state is responsible for fluorescence in less polar environment and in polar milieu fluorescence is from the twisted intramolecular charge transfer (TICT) state. This compound also undergoes minor groove binding to herring sperm DNA (hsDNA) evidenced by the absorption spectra before and after the binding process and an effect on DMASBT fluorescence by an anionic quencher. The binding occurs efficiently in a 1:1 manner, i.e. one guest molecule binds to one site on the hsDNA. Instead of following the DNA twist, the aromatic part seems to project outward. Thus, the bound molecule can be successfully extracted out from the DNA in a controlled way by the hydrophobic cavity of α-cyclodextrin (α-CD). The extraction starts even with a low concentration of α-CD and increases as the concentration is increased. Absorption, steady-state and time resolved fluorescence spectroscopic methods have been employed to explore the mechanistic pathway of binding of DMASBT to hsDNA. The mechanistic approach toward controlled extraction of the guest molecules from hsDNA by α-CD is reported and is expected to serve a significant purpose in treatment of drug overdose.

  13. Sexing Mammalian Sperm - Where Do We Go from Here?

    National Research Council Canada - National Science Library

    SEIDEL, Jr, George E

    2012-01-01

    The only commercially viable method of sexing mammalian sperm is to use a flow cytometer to measure sperm DNA content via fluorescence of the DNA-bound fluorophore Hoechst 33342, and then sort sperm...

  14. Serum concentrations of estradiol and free T4 are inversely correlated with sperm DNA damage in men from an infertility clinic

    Science.gov (United States)

    Meeker, John D.; Singh, Narendra P.; Hauser, Russ

    2009-01-01

    Sperm DNA damage adversely affects male fertility and contributes to poorer embryo development and lower pregnancy rates. Endogenous hormones are critical to spermatogenesis and maintenance of male reproductive function, and likely play an important role in human sperm DNA integrity, but this relationship is not fully understood. The present study measured serum hormone levels and sperm DNA damage with the neutral comet assay in 362 male partners of infertile couples. When adjusting for sperm concentration and other potential confounding variables in multiple linear regression, serum estradiol and free T4 levels were inversely associated with sperm DNA damage. Among other statistically significant associations that were observed, an interquartile range (IQR) increase in estradiol was associated with a 6.3% decline (95% confidence interval −9.7%, −2.9%) in comet extent and a 16.2% (−22.4%, −9.2%) decline in the percentage of DNA in the comet tail (Tail%), while an IQR increase in free T4 was associated with a 24.4% (−31.5%, −17.4%) decline in Tail%. Likewise, in multiple logistic regression, men in the highest estradiol quartile had an 81% reduced risk of having a comet extent value in the highest quartile compared to men in the lowest estradiol quartile. Men in the highest free T4 quartile had 92% decreased odds of being categorized in the highest Tail% quartile compared to men in the lowest free T4 quartile. These results suggest estradiol and free T4 may have a protective effect against sperm DNA damage, but future mechanistic and epidemiologic studies are needed to confirm these findings. PMID:18359755

  15. Condensyl® and decreases sperm DNA damage which is a risk factor for male infertility: evaluation of a health claim pursuant to Article 14 of Regulation (EC) No 1924/2006

    DEFF Research Database (Denmark)

    Sjödin, Anders Mikael

    2017-01-01

    on the scientific substantiation of a health claim related to ‘Condensyl® and decreases sperm DNA damage. High sperm DNA damage is a risk factor for male subfertility/infertility’. Condensyl® is a fixed combination of opuntia fruit dry extract, N-acetyl cysteine, zinc, nicotinamide, vitamins B2, B6, B12 and E...

  16. Sperm DNA Integrity and Meiotic Behavior Assessment in an Infertile Male Carrier of a 9qh+++ Polymorphism

    Directory of Open Access Journals (Sweden)

    A. García-Peiró

    2011-01-01

    Full Text Available Although several reports on male infertility suggest a relationship between chromosome 9 polymorphisms and infertility, the effects on the phenotype have not been extensively reported. In this study, an infertile patient was found to carry a 9qh+++ chromosome. The flow cytometric TUNEL assay and SCD test have been applied to characterize sperm DNA integrity. In order to assess its meiotic behaviour, synapsis, recombination, and aneuploidy, analyses have been also performed. Sperm DNA fragmentation (SDF was 77.81% and 87% for the TUNEL and SCD tests, respectively. Ninety-two percent of pachytene cells analyzed showed meiotic abnormalities. The mean number of MLH1 foci per pachytene in the control group was higher (49 than the mean found in the 9qh+++ patient (38 (P<.0001. In spermatozoa, significant increases of disomy rates were observed for chromosome 18 and for the sex chromosomes (P<.0001. These disturbances could be present in other male carriers of a less marked 9qh+.

  17. [Preparative isolation of hexa-, hepta-, octa-, nona- and decapyrimidine oligonucleotides from hydrolysates of depurinated herring sperm DNA].

    Science.gov (United States)

    Schott, H

    1984-02-03

    The pyrimidine oligonucleotides (dT)5; (dT)6 and the mixtures of sequence isomers (dC, dT5); (dC2, dT5); (dC3, dT4); (dC4, dT3); (dC4, dT4); (dC3, dT5); (dC2, dT6); (dC4, dT5); (dC3, dT6); (dC2, dT7); (dC5, dT5); (dC4, dT6) and (dC3, dT7) with or without terminal phosphate groups have been isolated on a preparative scale from hydrolysates of depurinated herring sperm DNA by the following procedure. Herring sperm DNA (1 kg) is chemically depurinated and partially hydrolysed to a mixture of pyrimidine nucleotides. The partial hydrolysate is first separated into a low- and a high-molecular-weight pyrimidine nucleotide mixture by column chromatography on DEAE-cellulose. The mixture of high-molecular-weight pyrimidine nucleotides is subsequently fractionated on QAE-Sephadex. Impurities which are not fully removed by column chromatography are separated by paper chromatography. The compositions of the mixtures of sequence isomers are determined from the data of column, paper and homochromatography; from absorption characteristics and by enzymatic degradation.

  18. Effect of amifostine on sperm DNA fragmentation and testes after radioiodine treatment

    Directory of Open Access Journals (Sweden)

    Sen Cigdem Cebi

    2017-12-01

    Full Text Available Introduction: Radioactive iodine (RAI is commonly used for the treatment of hyperthyroidism caused by Graves’ disease or thyroid nodules. However, information available on the impact of RAI therapy on male gonadal function is scarce. This study aimed to determine any possible damage to testicular tissue and sperm quality caused by RAI therapy, and the radioprotective effect of amifostine against such damage.

  19. DNA fragmentation dynamics allows the assessment of cryptic sperm damage in human: Evaluation of exposure to ionizing radiation, hyperthermia, acidic pH and nitric oxide

    Energy Technology Data Exchange (ETDEWEB)

    Santiso, Rebeca; Tamayo, Maria [Laboratorio de Genetica Molecular y Radiobiologia, Centro Oncologico de Galicia, Doctor Camilo Veiras 1, 15009-A Coruna (Spain); Genetics Unit, INIBIC-Complejo Hospitalario Universitario A Coruna (CHUAC), As Xubias, 84, 15006-A Coruna (Spain); Gosalvez, Jaime [Genetics Unit, Facultad de Biologia, Universidad Autonoma de Madrid, Ciudad Universitaria de Cantoblanco, 28049 Madrid (Spain); Johnston, Steve [School of Agriculture and Food Science, University of Queensland, Gatton 4343 (Australia); Marino, Alfonso [Servicio de Oncologia Radioterapica, Centro Oncologico de Galicia, Doctor Camilo Veiras 1, 15009-A Coruna (Spain); Fernandez, Carlos; Losada, Carlos [Servicio de Radiofisica, Centro Oncologico de Galicia, Doctor Camilo Veiras 1, 15009-A Coruna (Spain); Fernandez, Jose Luis, E-mail: Jose.Luis.Fernandez.Garcia@sergas.es [Laboratorio de Genetica Molecular y Radiobiologia, Centro Oncologico de Galicia, Doctor Camilo Veiras 1, 15009-A Coruna (Spain); Genetics Unit, INIBIC-Complejo Hospitalario Universitario A Coruna (CHUAC), As Xubias, 84, 15006-A Coruna (Spain)

    2012-06-01

    Sperm DNA fragmentation (SDF) is not a static seminal parameter, since the longevity of sperm DNA decreases progressively with time following ejaculation or thawing. While the dynamics of SDF is a species-specific characteristic, in the case of humans, there is still significant variation within patients. To evaluate the suitability of the dynamic SDF assay to assess the adverse effects of agents that cause genetic damage, fresh semen samples from different donors were exposed in vitro to (1) increasing acute doses of ionizing radiation, (2) elevated temperature (41 Degree-Sign C and 45 Degree-Sign C), (3) acidic pH (pH 4) and (4) the nitric oxide (NO) donor sodium nitroprusside (SNP). Sperm DNA fragmentation was analyzed after an incubation period of chronic (24 h), or acute (1 h) exposure to each treatment followed by incubation at 37 Degree-Sign C over a period of 24 h. SDF was assessed using the sperm chromatin dispersion (SCD) test. Dynamic SDF for each treatment was analyzed using Kaplan-Meier survival curves. All agents, except for ionizing radiation, accelerated SDF kinetics following chronic exposure over a 24 h period. Transient exposure to NO and heat but not acidic pH increased the basal (T0) level of SDF. Despite the removal of the three toxicants, the remaining sperm following acute exposure showed a decrease in their expected DNA longevity. It is concluded that the assessment of sperm DNA fragmentation dynamics is an effective methodological approach for revealing latent damage associated with toxicants that is not initially expressed following a single initial observation of SDF.

  20. Increased luminance of MEH-PPV and PFO based PLEDs by using salmon DNA as an electron blocking layer

    Energy Technology Data Exchange (ETDEWEB)

    Madhwal, Devinder, E-mail: devindermadhwal@gmail.co [Material Science Laboratory, Department of Electronic Science, University of Delhi South campus, Benito Juarez Road, New Delhi-110021 (India); Rait, S.S.; Verma, A.; Kumar, Amit; Bhatnagar, P.K.; Mathur, P.C. [Material Science Laboratory, Department of Electronic Science, University of Delhi South campus, Benito Juarez Road, New Delhi-110021 (India); Onoda, M. [Department of Electrical Engineering and Computer Science, University of Hyogo, Himeji (Japan)

    2010-02-15

    The effect of salmon DNA-CTMA as an electron blocking layer (EBL) has been examined on the performance of MEH-PPV and PFO-based light emitting diodes. Though the turn-on voltage increases with incorporation of EBL, a significant increase in luminance and luminous efficiency for both the devices is observed. The EBL improves the device performance by blocking electrons at the EBL-polymer interface, thereby increasing the recombination probability of electrons and holes. The luminance of the MEH-PPV based Bio-LED increases to 100 cd/m{sup 2} from 30 cd/m{sup 2} while a corresponding increase for the PFO based LED is to 160 cd/m{sup 2} from 80 cd/m{sup 2} with and without EBL, respectively.

  1. The application of alkaline lysis and pressure cycling technology in the differential extraction of DNA from sperm and epithelial cells recovered from cotton swabs.

    Science.gov (United States)

    Nori, Deepthi V; McCord, Bruce R

    2015-09-01

    This study reports the development of a two-step protocol using pressure cycling technology (PCT) and alkaline lysis for differential extraction of DNA from mixtures of sperm and vaginal epithelial cells recovered from cotton swabs. In controlled experiments, in which equal quantities of sperm and female epithelial cells were added to cotton swabs, 5 min of pressure pulsing in the presence of 0.4 M NaOH resulted in 104 ± 6% recovery of female epithelial DNA present on the swab. Following the pressure treatment, exposing the swabs to a second 5-min alkaline treatment at 95 °C without pressure resulted in the selective recovery of 69 ± 6% of the sperm DNA. The recovery of the vaginal epithelia and sperm DNA was optimized by examining the effect of sodium hydroxide concentration, incubation temperature, and time. Following the alkaline lysis steps, the samples were neutralized with 2 M Tris (pH 7.5) and purified with phenol-chloroform-isoamyl alcohol to permit downstream analysis. The total processing time to remove both fractions from the swab was less than 20 min. Short tandem repeat (STR) analysis of these fractions obtained from PCT treatment and alkaline lysis generated clean profiles of female epithelial DNA and male sperm DNA for 1:1 mixtures of female and male cells and predominant male profiles for mixtures up to 5:1 female to male cells. By reducing the time and increasing the recovery of DNA from cotton swabs, this new method presents a novel and potentially useful procedure for forensic differential extractions.

  2. Sperm protamine mRNA ratio and DNA fragmentation index represent reliable clinical biomarkers for men with varicocele after microsurgical varicocele ligation.

    Science.gov (United States)

    Ni, Kai; Steger, Klaus; Yang, Hao; Wang, Hongxiang; Hu, Kai; Chen, Bin

    2014-07-01

    We investigated whether the sperm protamine-1/2 mRNA ratio and DNA fragmentation index are reliable biomarkers in patients with clinical varicocele. We performed a prospective study in 42 subfertile patients with left clinical varicocele and 10 normozoospermic healthy donors with proven fertility. All patients and female partners were seen 3 and 6 months after varicocelectomy for fertility examination. Real-time quantitative reverse transcriptase-polymerase chain reaction and SCSA® were performed to analyze the sperm protamine-1/2 mRNA ratio and DNA fragmentation index. The protamine-1/2 mRNA ratio and DNA fragmentation index were significantly higher in the preoperative group than in the control group (p fragmentation index was significantly lower than in the preoperative group (p fragmentation index did not differ (p >0.05). However, significant differences were present preoperatively according to varicocele severity in the protamine-1/2 mRNA ratio and DNA fragmentation index (p 0.05). The protamine-1/2 mRNA ratio was strongly related to preoperative and postoperative sperm concentration (Rs -0.238, p fragmentation index (Rs 0.293, p 0.05). The sperm protamine-1/2 mRNA ratio and DNA fragmentation index can effectively be used to evaluate male fertility. Male infertility due to varicocele may be associated with protamine deficiency and sperm DNA damage. The post-varicocelectomy protamine-1/2 mRNA ratio and DNA fragmentation index are associated with the post-varicocelectomy pregnancy rate. Copyright © 2014 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

  3. Male fertility status is associated with DNA methylation signatures in sperm and transcriptomic profiles of bovine preimplantation embryos.

    Science.gov (United States)

    Kropp, Jenna; Carrillo, José A; Namous, Hadjer; Daniels, Alyssa; Salih, Sana M; Song, Jiuzhou; Khatib, Hasan

    2017-04-05

    Infertility in dairy cattle is a concern where reduced fertilization rates and high embryonic loss are contributing factors. Studies of the paternal contribution to reproductive performance are limited. However, recent discoveries have shown that, in addition to DNA, sperm delivers transcription factors and epigenetic components that are required for fertilization and proper embryonic development. Hence, characterization of the paternal contribution at the time of fertilization is warranted. We hypothesized that sire fertility is associated with differences in DNA methylation patterns in sperm and that the embryonic transcriptomic profiles are influenced by the fertility status of the bull. Embryos were generated in vitro by fertilization with either a high or low fertility Holstein bull. Blastocysts derived from each high and low fertility bulls were evaluated for morphology, development, and transcriptomic analysis using RNA-Sequencing. Additionally, DNA methylation signatures of sperm from high and low fertility sires were characterized by performing whole-genome DNA methylation binding domain sequencing. Embryo morphology and developmental capacity did not differ between embryos generated from either a high or low fertility bull. However, RNA-Sequencing revealed 98 genes to be differentially expressed at a false discovery rate fertility bull derived embryos, and 33 genes were upregulated in low fertility derived embryos. Expression of the genes CYCS, EEA1, SLC16A7, MEPCE, and TFB2M was validated in three new pairs of biological replicates of embryos. The role of the differentially expressed gene TFB2M in embryonic development was further assessed through expression knockdown at the zygotic stage, which resulted in decreased development to the blastocyst stage. Assessment of the epigenetic signature of spermatozoa between high and low fertility bulls revealed 76 differentially methylated regions. Despite similar morphology and development to the blastocyst

  4. An evaluation of DNA damage in human lymphocytes and sperm exposed to methyl methanesulfonate involving the regulation pathways associated with apoptosis.

    Science.gov (United States)

    Habas, Khaled; Najafzadeh, Mojgan; Baumgartner, Adolf; Brinkworth, Martin H; Anderson, Diana

    2017-10-01

    Exposure to DNA-damaging agents produces a range of stress-related responses. These change the expression of genes leading to mutations that cause cell cycle arrest, induction of apoptosis and cancer. We have examined the contribution of haploid and diploid DNA damage and genes involved in the regulation of the apoptotic process associated with exposure, The Comet assay was used to detect DNA damage and quantitative RT-PCR analysis (qPCR) to detect gene expression changes in lymphocytes and sperm in response to methyl methanesulfonate. In the Comet assay, cells were administered 0-1.2 mM of MMS at 37 °C for 30 min for lymphocytes and 32 °C for 60 min for sperm to obtain optimal survival for both cell types. In the Comet assay a significant increase in Olive tail moment (OTM) and % tail DNA indicated DNA damage at increasing concentrations compared to the control group. In the qPCR study, cells were treated for 4 h, and RNA was isolated at the end of the treatment. qPCR analysis of genes associated with DNA stress responses showed that TP53 and CDKN1A are upregulated, while BCL2 is downregulated compared with the control. Thus, MMS caused DNA damage in lymphocytes at increasing concentrations, but appeared not to have the same effect in sperm at the low concentrations. These results indicate that exposure to MMS increased DNA damage and triggered the apoptotic response by activating TP53, CDKN1A and BCL2. These findings of the processing of DNA damage in human lymphocytes and sperm should be taken into account when genotoxic alterations in both cell types are produced when monitoring human exposure. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. DNA fragmentation and membrane damage of bocachico Prochilodus magdalenae (Ostariophysi: Prochilodontidae sperm following cryopreservation with dimethylsulfoxide and glucose

    Directory of Open Access Journals (Sweden)

    José Gregorio Martínez

    Full Text Available The endangered bocachico Prochilodus magdalenae is a native freshwater fish of Colombia, the most captured species locally and one of the most important species for ex-situ conservation (germplasm banks. The aim of this study was to examine the effect of three concentrations of Dimethylsulfoxide (DMSO (5%, 10%, 15% and three of glucose (305, 333, 361 mM in the extender on spermatic DNA fragmentation (F-DNA (by Halomax®, Chromatin dispersion and membrane damage (D-Me (by eosin-nigrosin staining. After assessment of sperm quality by computer analysis of motility, one part of semen from males was diluted separately with three parts of extender and filled into 0.5 ml straws. Freezing was carried out in liquid nitrogen vapor dry shipper for 30 minutes and thawed at 60ºC for 8 seconds in a water bath and evaluated for the percentage of cells found with F-DNA and D-Me. The results demonstrated that cryopreservation causes greater F-DNA (13.62 ± 1.6% to 28.91 ± 3.25 and D-Me (24.27 ± 1.1% to 58.33 ± 2.81% when compared with pre-freezing semen (PFS (6.71 ± 1.54% and 2.34 ± 0.5%, respectively for F-DNA and D-Me. A significant interaction was found between DMSO and glucose concentration in this experiment. Use of extender: 10% DMSO + 305 mM glucose + 12% chicken egg yolk and, 10% DMSO + 333 mM glucose + 12% chicken egg yolk, allow for lower F-DNA and D-Me during cryopreservation of bocachico semen. A high correlation between F-DNA and D-Me was found (r = 0.771.

  6. Effect of semen extender supplementation with cysteine on postthaw sperm quality, DNA damage, and fertilizing ability in the common carp (Cyprinus carpio).

    Science.gov (United States)

    Öğretmen, Fatih; İnanan, Burak Evren; Kutluyer, Filiz; Kayim, Murathan

    2015-06-01

    Amino acids have an important biological role for prevention of cell damage during cryopreservation. The objective of this study is to determine the effects of cysteine on postthaw sperm motility, duration of sperm motility, DNA damage, and fertility in the common carp (Cyprinus carpio). Sperm collected from 10 individuals was cryopreserved in extenders containing different cysteine concentrations (2.5, 5, 10, and 20 mM). Semen samples diluted at the ratio of 1:9 by the extenders were subjected to cryopreservation. After dilution, the semen was aspirated into 0.25-mL straws; the straws were placed on the tray, frozen in nitrogen vapor, and plunged into liquid nitrogen. DNA damage was evaluated by comet assay after cryopreservation. Our results indicated that an increase in the concentration of cysteine caused a significant increase in the motility rate and duration of sperm in the common carp (C carpio; P < 0.05). Comparing all concentrations of cysteine, the best concentration of cysteine was 20 mM. Higher postthaw motility (76.00 ± 1.00%) and fertilization (97.00 ± 1.73%) rates were obtained with the extender at the concentration of 20 mM. Supplementation of the extender with cysteine was increased the fertilization and hatching rate and decreased DNA damage. Consequently, cysteine affected the motility, fertilization, and DNA damage positively, and extenders could be supplemented with cysteine. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Calcium phosphate nanoparticles: a study of their synthesis, characterization and mode of interaction with salmon testis DNA.

    Science.gov (United States)

    Banik, Milon; Basu, Tarakdas

    2014-02-28

    Calcium phosphate nanoparticles (CPNPs) are presently emerging as a second generation vector for efficient delivery and stabilization of nucleic acids inside cells, although the detailed mode of interaction between CPNPs and DNA is still obscure. This study discloses some features of the interaction. For this study, we synthesized CPNPs by a modified co-precipitation method and characterized the particles by different techniques such as dynamic light scattering, X-ray diffraction, electron dispersive spectroscopy, Fourier transform infra-red spectroscopy, differential thermal and thermo-gravimetric analysis, and atomic force, scanning and transmission electron microscopy. The characterization studies showed that the nanoparticles were spherical in shape, about 45 nm in size and were composed of the hydroxyapatite form of calcium phosphate; almost 90% of the starting materials were converted to nanoparticles (NPs). The different aspects of the interaction between CPNPs and salmon testis DNA were investigated using techniques such as UV-Vis spectrophotometry, circular dichroism, Fourier transform infra-red spectrometry, thermal denaturation, microviscometry, agarose gel electrophoresis, cyclic voltammetry and atomic force microscopy. The results revealed that CPNPs interacted with DNA with ~1 : 3.3 stoichiometry with a binding constant of the order of 10(4) M(-1) through groove-interacting mode and a single nanoparticle covered about 6.2 base pairs of the DNA chain. Moreover, the binding interaction was spontaneous, cooperative, exothermic and enthalpy-driven and some electrostatic nature of the binding was also evident; however, the non-polyelectrolyte contribution was dominant. The binding interaction finally caused an increase in the melting temperature of DNA from 70.8 °C to 75 °C and alteration of its secondary structure from the naturally occurring B-form to C-form.

  8. In vitro evaluation of baseline and induced DNA damage in human sperm exposed to benzo[a]pyrene or its metabolite benzo[a]pyrene-7,8-diol-9,10-epoxide, using the comet assay.

    Science.gov (United States)

    Sipinen, V; Laubenthal, J; Baumgartner, A; Cemeli, E; Linschooten, J O; Godschalk, R W L; Van Schooten, F J; Anderson, D; Brunborg, G

    2010-07-01

    Exposure to genotoxins may compromise DNA integrity in male reproductive cells, putting future progeny at risk for developmental defects and diseases. To study the usefulness of sperm DNA damage as a biomarker for genotoxic exposure, we have investigated cellular and molecular changes induced by benzo[a]pyrene (B[a]P) in human sperm in vitro, and results have been compared for smokers and non-smokers. Sperm DNA obtained from five smokers was indeed more fragmented than sperm of six non-smokers (mean % Tail DNA 26.5 and 48.8, respectively), as assessed by the alkaline comet assay (P sperm cells to B[a]P (10 or 25 microM) caused moderate increases in DNA fragmentation which was independent of addition of human liver S9 mix for enzymatic activation of B[a]P, suggesting some unknown metabolism of B[a]P in ejaculates. In vitro exposure of samples to various doses of B[a]P (with or without S9) did not reveal any significant differences in sensitivity to DNA fragmentation between smokers and non-smokers. Incubations with the proximate metabolite benzo[a]pyrene-r-7,t-8-dihydrodiol-t9,10-epoxide (BPDE) produced DNA fragmentation in a dose-dependent manner (20 or 50 microM), but only when formamidopyrimidine DNA glycosylase treatment was included in the comet assay. These levels of DNA fragmentation were, however, low in relation to very high amounts of BPDE-DNA adducts as measured with (32)P postlabelling. We conclude that sperm DNA damage may be useful as a biomarker of direct exposure of sperm using the comet assay adapted to sperm, and as such the method may be applicable to cohort studies. Although the sensitivity is relatively low, DNA damage induced in earlier stages of spermatogenesis may be detected with higher efficiencies.

  9. Is passive transmission of non-viral vectors through artificial insemination of sperm-DNA mixtures sufficient for chicken transgenesis?

    Science.gov (United States)

    Chaparian, Shahram; Abdulahnejad, Ahad; Rashidi, Farzad; Toghyani, Majid; Gheisari, Abbasali; Eghbalsaied, Shahin

    2016-06-17

    DNA uptake in the post-acrosomal region of the spermatozoa takes place exclusively in immotile spermatozoa that are naturally unable to fertilize eggs. The present study aimed to assess whether passive transmission of non-viral vectors to the surrounding areas of chicken embryos could be an alternate mechanism in chicken sperm-mediated gene transfer. First, the presence of nucleases in rooster seminal plasma was evaluated. Semen ejaculates from five roosters were centrifuged and the supernatant was incubated with pBL2 for 1 h. A robust nuclease cocktail was detected in the rooster semen. To overcome these nucleases, plasmid-TransIT combinations were incubated with semen for 1 h. Incubation of exogenous DNA in the lipoplex structure could considerably bypass the semen nuclease effect. Then, intravaginal insemination of 1 × 10(9) sperm mixed with lipoplexes (40 µg pBL2:40 µl TransIT) was carried out in 15 virgin hens. Neither the epithelial tissue from the inseminated female reproductive tracts nor the produced embryos following artificial insemination showed the transgene. To remove any bias in the transgene transmission possibility, the plasmid-TransIT admixture was directly injected in close vicinity of the embryos in newly laid eggs. Nonetheless, none of the produced fetuses or chicks carried the transgene. In conclusion, the results of the present study revealed a nuclease admixture in rooster seminal plasma, and passive/active transmission of the non-viral vector into close vicinity of the chicken embryo was inefficient for producing transgenic chicks.

  10. Determination of DNA damage after exposure to inhalation anesthetics in human peripheral lymphocytes and sperm cells in vitro by comet assay.

    Science.gov (United States)

    Kaymak, C; Kadioglu, E; Coskun, E; Basar, H; Basar, M

    2012-12-01

    In this study, genotoxic activities of four halogenated anesthetics (halothane, isoflurane, sevoflurane and desflurane) were investigated in human peripheral blood lymphocytes (PBLs) and sperm cells in vitro by alkaline comet assay. For this purpose, sperm or lymphocyte suspension was exposed to different concentrations (0.1 mM, 1 mM, 10 mM and 100 mM) of anesthetic agents and 1% dimethyl sulfoxide (DMSO) or phosphate-buffered saline (PBS) as controls. The DNA strand breaks as well as alkali-labile sites were measured as percentage tail intensity with comet assay. The results of this study demonstrate that all analyzed drugs were capable of inducing DNA damage on PBLs in a dose-dependent manner in vitro. However, the results in sperm cells were slightly different since we did not observe any genotoxic effect for desflurane in any of the exposure doses, and the genotoxic effect of halothane was not dose dependent. This experimental study points out to the presence of DNA damage after exposure to halogenated anesthetics in both PBLs and sperm cells, although this effect seems to be higher in PBLs.

  11. Evaluation of zearalenone and alpha-zearalenol toxicity on boar sperm DNA integrity.

    Science.gov (United States)

    Tsakmakidis, Ioannis A; Lymberopoulos, Aristoteles G; Khalifa, Tarek A A; Boscos, Constanten M; Saratsi, Aikaterini; Alexopoulos, Costas

    2008-07-01

    This study aimed at investigating the in vitro effects of zearalenone (zen) and alpha-zearalenol (alpha-zen) on motility and nuclear chromatin integrity (NCI) of boar spermatozoa. Mycotoxins were tested, at levels ranging from 10 to 30 microg ml(-1) of diluted semen. Four boars were used for semen collection (eight replicates per boar, four per mycotoxin). After the addition of zen or alpha-zen, semen samples were incubated for 4 h at 38.5 degrees C, 5% CO(2) and 96% humidified air. Motility and NCI were assessed at 0 and 4 h of incubation. No significant differences were noticed in motility among the experimental groups (P > 0.05) for all tested boars. Chromatin instability was significantly higher (P < 0.05) in spermatozoa of only one boar treated with zen and alpha-zen independently of the dose. In conclusion, under our experimental conditions, zen and alpha-zen did not affect the motility of boar sperm, whereas the effects of these toxins on sperm NCI were individual-dependent. 2008 John Wiley & Sons, Ltd

  12. Characterization of a human glycoprotein with a potential role in sperm-egg fusion: cDNA cloning, immunohistochemical localization, and chromosomal assignment of the gene (AEGL1)

    Energy Technology Data Exchange (ETDEWEB)

    Hayashi, Masaru; Fujimoto, Seiichiro; Takano, Hiroko [Hokkaido Univ. School of Medicine, Sapporo (Japan)] [and others

    1996-03-05

    Acidic epididymal glycoprotein (AEG), thus far identified only in rodents, is one of the sperm surface proteins involved in the fusion of the sperm and egg plasma membranes. In the present study, we describe the isolation and characterization of cDNA encoding a human glycoprotein related to AEG. Although this protein, designated ARP (AEG-related protein), is not the ortholog of rodent AEG, it resembles AEG in that it is an epididymal secretory glycoprotein that binds to the postacrosomal region of the sperm head. The fact that no AEG mRNA can be detected in the human epididymis suggests that ARP might be the functional counterpart of rodent AEG. The gene encoding ARP (AEGL1) was mapped by fluorescence in situ hybridization to 6p21.1-p21.2. This result indicates that AEGL1 and the mouse gene for AEG are located in the chromosomal segments with conserved syntenies. 43 refs., 6 figs.

  13. Experience in the use of docosahexaenoic acid (BrudiPlus in patients with increased sperm DNA fragmentation index in Acad. V.I. Kulakov Research Center for Obstetrics, Gynecology and Perinatology

    Directory of Open Access Journals (Sweden)

    A. Yu. Popova

    2015-01-01

    Full Text Available Male factor is the reason of infertility in almost half of marriages. Infertile men have the percentage of sperm with violations of DNA integrity of over 30 %; with that, healthy fertile men have that indicator of less than 15 %. Understanding of importance of damages of sperm DNA is growing with distribution ofauxiliary reproductive technologies. As of today, these consequences have not been studies yet, and the therapeutic effect of intake of antioxidants has not direct correlation with the sperm DNA fragmentation level. Docosahexaenoic acid is one of the most valuable omega-3 polyunsaturated fatty acids for human health. Docosahexaenoic acid is the main component of the brain gray matter, retina, testes, and sperm cell membranes. In connection with that, a study was held the purpose of which was to assess the effect of the nutraceutical enzymatic docosahexaenoic acid triglyceride (BrudiPlus in high concentrations on damaged sperm DNA of patients with idiopathic pathozoospermia. 40 patients with idiopathic pathozoospermia and the level of DNA fragmentation over the statutory value took part in this study. The following positive results were received: intake of BrudiPlus allowed decreasing sperm DNA damages and improving of antioxidant system of sperm

  14. Male and Couple Fertility Impairment due to HPV-DNA Sperm Infection: Update on Molecular Mechanism and Clinical Impact—Systematic Review

    Directory of Open Access Journals (Sweden)

    Salvatore Gizzo

    2014-01-01

    Full Text Available Recent evidences identify Human Papillomavirus (HPV sperm infection as a possible cause of male and couple infertility. It acts through different mechanisms at various steps of human conception and early gestational development. We performed a systematic review to assess the role of HPV semen infection on male and couple infertility. Analysis of available and eligible data does not permit us to fund clear evidences about clinical impact of HPV infection on fertility, although sperm parameters impairment is the most widely recognized effect. Regarding biomolecular implications, the available data are often conflicting. More studies are required to define the role of HPV sperm infection in clinical practice. The great majority of evidences are obtained by in vitro studies and this fact represents a limitation for the clinical management of HPVDNA sperm infection. Understanding the biological significance of HPV-DNA semen infection could permit us to explain most of the idiopathic male and couple infertility, leading to a better management of infertile men and a better timing for sperm banking storage before ART cycles.

  15. Male and couple fertility impairment due to HPV-DNA sperm infection: update on molecular mechanism and clinical impact--systematic review.

    Science.gov (United States)

    Gizzo, Salvatore; Ferrari, Bruno; Noventa, Marco; Ferrari, Emanuele; Patrelli, Tito Silvio; Gangemi, Michele; Nardelli, Giovanni Battista

    2014-01-01

    Recent evidences identify Human Papillomavirus (HPV) sperm infection as a possible cause of male and couple infertility. It acts through different mechanisms at various steps of human conception and early gestational development. We performed a systematic review to assess the role of HPV semen infection on male and couple infertility. Analysis of available and eligible data does not permit us to fund clear evidences about clinical impact of HPV infection on fertility, although sperm parameters impairment is the most widely recognized effect. Regarding biomolecular implications, the available data are often conflicting. More studies are required to define the role of HPV sperm infection in clinical practice. The great majority of evidences are obtained by in vitro studies and this fact represents a limitation for the clinical management of HPVDNA sperm infection. Understanding the biological significance of HPV-DNA semen infection could permit us to explain most of the idiopathic male and couple infertility, leading to a better management of infertile men and a better timing for sperm banking storage before ART cycles.

  16. Male and Couple Fertility Impairment due to HPV-DNA Sperm Infection: Update on Molecular Mechanism and Clinical Impact—Systematic Review

    Science.gov (United States)

    Gizzo, Salvatore; Ferrari, Bruno; Noventa, Marco; Ferrari, Emanuele; Patrelli, Tito Silvio; Gangemi, Michele; Nardelli, Giovanni Battista

    2014-01-01

    Recent evidences identify Human Papillomavirus (HPV) sperm infection as a possible cause of male and couple infertility. It acts through different mechanisms at various steps of human conception and early gestational development. We performed a systematic review to assess the role of HPV semen infection on male and couple infertility. Analysis of available and eligible data does not permit us to fund clear evidences about clinical impact of HPV infection on fertility, although sperm parameters impairment is the most widely recognized effect. Regarding biomolecular implications, the available data are often conflicting. More studies are required to define the role of HPV sperm infection in clinical practice. The great majority of evidences are obtained by in vitro studies and this fact represents a limitation for the clinical management of HPVDNA sperm infection. Understanding the biological significance of HPV-DNA semen infection could permit us to explain most of the idiopathic male and couple infertility, leading to a better management of infertile men and a better timing for sperm banking storage before ART cycles. PMID:24783196

  17. The influence of ginger (Zingiber officinale on human sperm quality and DNA fragmentation: A double-blind randomized clinical trial

    Directory of Open Access Journals (Sweden)

    Jalil Hosseini

    2016-08-01

    Full Text Available Background: Although the effectiveness of ginger as an antioxidant agent has been exploited, little human research has been conducted on its activity on male reproductive functions. Objective: This study was designed to investigate the effects of ginger (Zingiber officinale on sperm DNA fragmentation (SDF in infertile men. Materials and Methods: This randomized double-blind, placebo-controlled trial with a 1:1 allocation was performed on 100 infertility treatment candidates who were admitted to Royan Institute for Reproductive Biomedicine, Tehran, Iran. Patients were randomly assigned to receive one of two treatments: ginger and placebo. Patients were given a 3-month oral treatment (members received capsules containing 250 mg of ginger powder twice a day in ginger and a placebo in other group. Before and after treatment, standardized semen samples were obtained to determine sperm concentration, motility, and SDF according to World Health Organization. Results: There was no significant difference between two groups regarding SDF at baseline (53.48. 95%CI: 37.95-69.02 in cases and (56.75, 95%CI: 40.01-73.5 in controls. The average positive percentage of SDF in patients receiving ginger (17.77, 95%CI: 6.16-29.39 was lower compared with placebo (40.54, 95%CI: 23.94-57.13 after three month of treatment (p=0.02. In multivariate analysis, SDF was significantly lower in patients receiving ginger compared with placebo (mean difference: 3.21, 95%CI: 0.78-5.63, p=0.009. There were no significant differences between two groups regarding to semen parameters. Conclusion: The present study has demonstrated that ginger in a controlled study of efficacy was effective in decreasing SDF in infertile men.

  18. The influence of ginger (Zingiber officinale) on human sperm quality and DNA fragmentation: A double-blind randomized clinical trial.

    Science.gov (United States)

    Hosseini, Jalil; Mardi Mamaghani, Azar; Hosseinifar, Hani; Sadighi Gilani, Mohammad Ali; Dadkhah, Farid; Sepidarkish, Mahdi

    2016-08-01

    Although the effectiveness of ginger as an antioxidant agent has been exploited, little human research has been conducted on its activity on male reproductive functions. This study was designed to investigate the effects of ginger (Zingiber officinale) on sperm DNA fragmentation (SDF) in infertile men. This randomized double-blind, placebo-controlled trial with a 1:1 allocation was performed on 100 infertility treatment candidates who were admitted to Royan Institute for Reproductive Biomedicine, Tehran, Iran. Patients were randomly assigned to receive one of two treatments: ginger and placebo. Patients were given a 3-month oral treatment (members received capsules containing 250 mg of ginger powder twice a day in ginger and a placebo in other group). Before and after treatment, standardized semen samples were obtained to determine sperm concentration, motility, and SDF according to World Health Organization. There was no significant difference between two groups regarding SDF at baseline (53.48. 95%CI: 37.95-69.02) in cases and (56.75, 95%CI: 40.01-73.5) in controls. The average positive percentage of SDF in patients receiving ginger (17.77, 95%CI: 6.16-29.39) was lower compared with placebo (40.54, 95%CI: 23.94-57.13) after three month of treatment (p=0.02). In multivariate analysis, SDF was significantly lower in patients receiving ginger compared with placebo (mean difference: 3.21, 95%CI: 0.78-5.63, p=0.009). There were no significant differences between two groups regarding to semen parameters. The present study has demonstrated that ginger in a controlled study of efficacy was effective in decreasing SDF in infertile men.

  19. Sperm design and sperm function

    National Research Council Canada - National Science Library

    Aurelio F Malo; Montserrat Gomendio; Julian Garde; Barbara Lang-Lenton; Ana J Soler; Eduardo R.S Roldan

    2006-01-01

    .... Sperm swimming velocity is a key determinant of male fertilization success, but previous efforts to identity which sperm phenotypic traits are associated with swimming velocity have been unsuccessful...

  20. Production of live foals via intracytoplasmic injection of lyophilized sperm and sperm extract in the horse.

    Science.gov (United States)

    Choi, Y H; Varner, D D; Love, C C; Hartman, D L; Hinrichs, K

    2011-10-01

    Work with lyophilized sperm helps delineate the factors required for successful fertilization. We investigated the use of lyophilized sperm in equine embryo production. In Experiment 1, sperm DNA fragmentation index was not affected by three freeze/thaw or lyophilization cycles. In Experiment 2, oocytes injected with lyophilized sperm or with sperm from a treatment in which lyophilized sperm were suspended in sperm cytoplasmic extract (SE) yielded blastocyst development rates of 0 and 28% respectively (P fertilization with lyophilized sperm in a non-laboratory animal species.

  1. Lack of an association between environmental exposure to polychlorinated biphenyls and p,p'-DDE and DNA damage in human sperm measured using the neutral comet assay.

    Science.gov (United States)

    Hauser, R; Singh, N P; Chen, Z; Pothier, L; Altshul, L

    2003-12-01

    Chlorinated organic chemicals, such as polychlorinated biphenyls (PCB), hexachlorobenzene (HCB), dichlorodiphenyl trichloroethane (DDT), and dichlorodiphenyl dichloroethene (DDE, the most stable daughter compound of DDT) are persistent lipophilic compounds found in a large portion of the general population. To explore the hypothesis that environmental exposure to these compounds is associated with altered DNA integrity in human sperm, a study of 212 male partners of a sub-fertile couple who presented to the Massachusetts General Hospital Andrology Laboratory was conducted. The neutral single cell microgel electrophoresis assay (comet assay) was used to assess DNA integrity in sperm. VisComet image analysis software was used to measure total comet length, the proportion of DNA present in the comet tail, and tail distributed moment, an integrated measure of length and intensity. In the regression analyses, there were no statistically significant consistent associations between the comet assay parameters and any of the individual PCB congeners, sum of PCB, or p,p'-DDE. These results suggest that there are not strong relationships between adult levels of these chlorinated organic compounds and sperm DNA damage as measured by the comet assay.

  2. HPV-DNA sperm infection and infertility: from a systematic literature review to a possible clinical management proposal.

    Science.gov (United States)

    Foresta, C; Noventa, M; De Toni, L; Gizzo, S; Garolla, A

    2015-03-01

    The objectives of this study were to investigate the implications of human papillomavirus (HPV) sperm infection on male fertility, impairment of sperm parameters, and possible alteration of sperm nuclear status and to identify a possible effective management of infertile men with HPV sperm infection. We employed a systematic review and clinical management proposal at the Centers for Reproductive and Health care for treating infertile male patients with HPV infection. Literature search was carried out in electronic databases in the last two decades. We focused our attention on: (i) HPV sperm prevalence (ii) HPV-related alteration of sperm parameters; (iii) molecular mechanisms of HPV semen infection and infertility. The main outcome measures were HPV prevalence in infertile male patients and semen parameters. The prevalence of HPV sperm infection ranges between 2 and 31% in men from general population and between 10 and 35.7% in men affected by unexplained infertility. The presence of HPV in semen is associated with an impairment of sperm motility and the presence of anti-sperm antibodies. The molecular mechanisms underlying impairment of sperm motility apparatus need further evaluations. A greater attention should be applied to assess HPV sperm infection, particularly in men undergoing assisted reproduction techniques cycle for male infertility or sperm banking. It would be useful to perform HPV test and fluorescent in situ hybridization analysis for HPV in semen from these patients both at first admission, to define the possible presence and localization of semen infection, and after 6 months, to assess the possible virus clearance retrieval on normal sperm parameters. © 2014 American Society of Andrology and European Academy of Andrology.

  3. Altered DNA methylation patterns of the H19 differentially methylated region and the DAZL gene promoter are associated with defective human sperm.

    Directory of Open Access Journals (Sweden)

    Bo Li

    Full Text Available DNA methylation disturbance is associated with defective human sperm. However, oligozoospermia (OZ and asthenozoospermia (AZ usually present together, and the relationship between the single-phenotype defects in human sperm and DNA methylation is poorly understood. In this study, 20 infertile OZ patients and 20 infertile AZ patients were compared with 20 fertile normozoospermic men. Bisulfate-specific PCR was used to analyze DNA methylation of the H19-DMR and the DAZL promoter in these subjects. A similar DNA methylation pattern of the H19-DMR was detected in AZ and NZ(control, with only complete methylation and mild hypomethylation(0.05. However, the methylation pattern of severe hypomethylation (>50% unmethylated CpGs and complete unmethylation was only detected in 5 OZ patients, and the occurrence of these two methylation patterns was 8.54±10.86% and 9±6.06%, respectively. Loss of DNA methylation of the H19-DMR in the OZ patients was found to mainly occur in CTCF-binding site 6, with occurrence of 18.15±14.71%, which was much higher than that in patients with NZ (0.84±2.05% and AZ (0.58±1.77% (P20% methylated clones in the DAZL promoter only in infertile patients, there was no significant difference between the AZ and OZ patients in the proportion of moderately-to-severely hypermethylated clones (p>0.05. In all cases, global sperm genome methylation analyses, using LINE1 transposon as the indicator, showed that dysregulation of DNA methylation is specifically associated with the H19-DMR and DAZL promoter. Therefore, abnormal DNA methylation status of H19-DMR, especially at the CTCF-binding site 6, is closely associated with OZ. Abnormal DNA methylation of the DAZL promoter might represent an epigenetic marker of male infertility.

  4. Superior protection conferred by inactivated whole virus vaccine over subunit and DNA vaccines against salmonid alphavirus infection in Atlantic salmon (Salmo salar L.).

    Science.gov (United States)

    Xu, Cheng; Mutoloki, Stephen; Evensen, Øystein

    2012-06-06

    Salmonid alphavirus 3 (SAV-3) is an emerging pathogen in Norwegian salmon farming and causes severe annual losses. We studied the immunogenicity and protective ability of subunit and DNA vaccines based on E1 and E2 spike proteins of salmonid alphavirus subtype 3 (SAV-3), and compared these to an experimental inactivated, whole virus (IWV) vaccine in Atlantic salmon. The antigens were delivered as water-in-oil emulsions for the subunit and inactivated vaccines and non-formulated for the DNA vaccines. The IWV and the E2 subunit prime-boost groups had circulating neutralizing antibodies at challenge, correlating with high protection against lethal challenge and 3-log(10) reduction of virus titer in heart for the IWV group. Prime-boost with E1 subunit vaccine also conferred significant protection against mortality, but did not correlate with neutralizing antibody levels. Protection against pathology in internal organs was only seen for the IWV group. Prime-boost with E1 and E2 DNA vaccines showed marginal protection in terms of reduction of viral replication in target organs and protection against mortality was not different from controls. The IWV group showed significant upregulation of IFNγ and IL2 mRNA expression at 4 weeks post challenge possibly indicating that other mechanisms in addition to antibody responses play a role in mediating protection against infection. This is the first report comparing the immunogenicity and protection against mortality for IWV vaccines and spike protein subunit and DNA vaccines against salmonid alphavirus infection in Atlantic salmon. The IWV vaccine has superior immunogenicity over sub-unit and DNA vaccines. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. Elucidation of Listeria monocytogenes Contamination Routes in Cold-Smoked Salmon Processing Plants Detected by DNA-Based Typing Methods

    Science.gov (United States)

    Fonnesbech Vogel, Birte; Huss, Hans Henrik; Ojeniyi, Bente; Ahrens, Peter; Gram, Lone

    2001-01-01

    The contamination routes of Listeria monocytogenes in cold-smoked salmon processing plants were investigated by analyzing 3,585 samples from products (produced in 1995, 1996, 1998, and 1999) and processing environments (samples obtained in 1998 and 1999) of two Danish smokehouses. The level of product contamination in plant I varied from 31 to 85%, and no L. monocytogenes was found on raw fish (30 fish were sampled). In plant II, the levels of both raw fish and product contamination varied from 0 to 25% (16 of 185 raw fish samples and 59 of 1,000 product samples were positive for L. monocytogenes). A total of 429 strains of L. monocytogenes were subsequently compared by random amplified polymorphic DNA (RAPD) profiling, and 55 different RAPD types were found. The RAPD types detected on the products were identical to types found on the processing equipment and in the processing environment, suggesting that contamination of the final product (cold-smoked salmon) in both plants (but primarily in plant I) was due to contamination during processing rather than to contamination from raw fish. However, the possibility that raw fish was an important source of contamination of the processing equipment and environment could not be excluded. Contamination of the product occurred in specific areas (the brining and slicing areas). In plant I, the same RAPD type (RAPD type 12) was found over a 4-year period, indicating that an established in-house flora persisted and was not eliminated by routine hygienic procedures. In plant II, where the prevalence of L. monocytogenes was much lower, no RAPD type persisted over long periods of time, and several different L. monocytogenes RAPD types were isolated. This indicates that persistent strains may be avoided by rigorous cleaning and sanitation; however, due to the ubiquitous nature of the organism, sporadic contamination occurred. A subset of strains was also typed by using pulsed-field gel electrophoresis and amplified fragment length

  6. Phase, current, absorbance, and photoluminescence of double and triple metal ion-doped synthetic and salmon DNA thin films

    Science.gov (United States)

    Chopade, Prathamesh; Reddy Dugasani, Sreekantha; Reddy Kesama, Mallikarjuna; Yoo, Sanghyun; Gnapareddy, Bramaramba; Lee, Yun Woo; Jeon, Sohee; Jeong, Jun-Ho; Park, Sung Ha

    2017-10-01

    We fabricated synthetic double-crossover (DX) DNA lattices and natural salmon DNA (SDNA) thin films, doped with 3 combinations of double divalent metal ions (M2+)-doped groups (Co2+-Ni2+, Cu2+-Co2+, and Cu2+-Ni2+) and single combination of a triple M2+-doped group (Cu2+-Ni2+-Co2+) at various concentrations of M2+ ([M2+]). We evaluated the optimum concentration of M2+ ([M2+]O) (the phase of M2+-doped DX DNA lattices changed from crystalline (up to ([M2+]O) to amorphous (above [M2+]O)) and measured the current, absorbance, and photoluminescent characteristics of multiple M2+-doped SDNA thin films. Phase transitions (visualized in phase diagrams theoretically as well as experimentally) from crystalline to amorphous for double (Co2+-Ni2+, Cu2+-Co2+, and Cu2+-Ni2+) and triple (Cu2+-Ni2+-Co2+) dopings occurred between 0.8 mM and 1.0 mM of Ni2+ at a fixed 0.5 mM of Co2+, between 0.6 mM and 0.8 mM of Co2+ at a fixed 3.0 mM of Cu2+, between 0.6 mM and 0.8 mM of Ni2+ at a fixed 3.0 mM of Cu2+, and between 0.6 mM and 0.8 mM of Co2+ at fixed 2.0 mM of Cu2+ and 0.8 mM of Ni2+, respectively. The overall behavior of the current and photoluminescence showed increments as increasing [M2+] up to [M2+]O, then decrements with further increasing [M2+]. On the other hand, absorbance at 260 nm showed the opposite behavior. Multiple M2+-doped DNA thin films can be used in specific devices and sensors with enhanced optoelectric characteristics and tunable multi-functionalities.

  7. Nickel (II) Ions Interaction with Polynucleotides and DNA of Different GC Composition

    OpenAIRE

    Bregadze, Vasil G.; Khutsishvili, Irina G.; Melikishvili, Sophie Z.; Melikishvili, Zaza G.

    2009-01-01

    The goal of the work was to study the role of GC alternative dimmers in the binding of DNA with Ni (II) ions. The method of ultraviolet difference spectroscopy has been applied to investigate Ni (II) ions interactions with DNA extracted from Clostridium perfringens, Mice liver (C3HA line), Calf thymus, Salmon sperm, Herring sperm, E.coli, Micrococcus luteus and polynucleotides Poly (dA-dT)xPoly (dA-dT), Poly (dG)x Poly (dC), Poly (dG-dC)xPoly (dG-dC). It is shown that Ni (II) ions at outer-sp...

  8. Role of Extracellular DNA during Biofilm Formation by Listeria monocytogenes

    DEFF Research Database (Denmark)

    Harmsen, Morten; Lappann, Martin; Knøchel, S

    2010-01-01

    Listeria monocytogenes is a food-borne pathogen that is capable of living in harsh environments. It is believed to do this by forming biofilms, which are surface-associated multicellular structures encased in a self-produced matrix. In this paper we show that in L. monocytogenes extracellular DNA...... cell biofilm assays. However, it was also demonstrated that in a culture without eDNA, neither Listeria genomic DNA nor salmon sperm DNA by itself could restore the capacity to adhere. A search for additional necessary components revealed that peptidoglycan (PG), specifically N-acetylglucosamine (NAG...

  9. Genetic variation among major sockeye salmon populations in Kamchatka peninsula inferred from SNP and microsatellite DNA analyses

    DEFF Research Database (Denmark)

    Khrustaleva, A.M.; Limborg, Morten; Seeb, J. E.

    Sockeye salmon samples from six populations from Kamchatka Peninsula were tested for polymorphism at six microsatellite (STR) and forty-five single nucleotide polymorphism (SNP) loci. These populations included the five largest populations in the region. Statistically significant genetic differen......Sockeye salmon samples from six populations from Kamchatka Peninsula were tested for polymorphism at six microsatellite (STR) and forty-five single nucleotide polymorphism (SNP) loci. These populations included the five largest populations in the region. Statistically significant genetic...

  10. Reduced sperm DNA longevity is associated with an increased incidence of still born; evidence from a multi-ovulating sequential artificial insemination animal model.

    Science.gov (United States)

    Johnston, Stephen D; López-Fernández, Carmen; Arroyo, Francisca; Gosálbez, Altea; Cortés Gutiérrez, Elva I; Fernández, Jose-Luis; Gosálvez, Jaime

    2016-09-01

    Using a rabbit model, we assessed the influence of sperm DNA longevity on female reproductive outcomes. Semen was collected from 40 bucks, incubated at 38 °C for 24 h, and the rate of sperm DNA fragmentation (rSDF) was determined using the sperm chromatin dispersion assay. Males were allocated into high rSDF (>0.5 units of increase per hour) or low rSDF (artificially inseminated into the same doe to reduce female factor variability, and pregnancy outcomes were recorded. While there was no difference in SDFs between rSDF groups immediately after collection (T0), differences were significant after 2 h of incubation; SDFs determined at collection and rSDF behaved as independent characters (Pearson correlation = 0.099; P = 0.542). Following artificial insemination, the rate of stillborn pups was significantly higher in does inseminated by males with a high rSDF (14/21) compared to those with low rSDF (15/6); (contingency χ(2) 5.19; p = 0.022). The risk of stillborn when low rSDF rabbits were used for insemination was 0.16, but increased to 0.36 when high rSDF animals were used (odds ratio = 2.85; 95 % confidence interval = 1.4-2.7). Dynamic assessment of SDF coupled with natural multiple ovulation, high fecundity of the rabbit and control over female factor influence, provided a useful experimental model to demonstrate the adverse effect of reduced sperm DNA longevity on reproductive outcome.

  11. Generational comparisons (F1 versus F3) of vinclozolin induced epigenetic transgenerational inheritance of sperm differential DNA methylation regions (epimutations) using MeDIP-Seq.

    Science.gov (United States)

    Beck, Daniel; Sadler-Riggleman, Ingrid; Skinner, Michael K

    2017-07-01

    Environmentally induced epigenetic transgenerational inheritance of disease and phenotypic variation has been shown to involve DNA methylation alterations in the germline (e.g. sperm). These differential DNA methylation regions (DMRs) are termed epimutations and in part transmit the transgenerational phenotypes. The agricultural fungicide vinclozolin exposure of a gestating female rat has previously been shown to promote transgenerational disease and epimutations in F3 generation (great-grand-offspring) animals. The current study was designed to investigate the actions of direct fetal exposure on the F1 generation rat sperm DMRs compared to the F3 transgenerational sperm DMRs. A protocol involving methylated DNA immunoprecipitation (MeDIP) followed by next-generation sequencing (Seq) was used in the current study. Bioinformatics analysis of the MeDIP-Seq data was developed and several different variations in the bioinformatic analysis were evaluated. Observations indicate needs to be considered. Interestingly, the F1 generation DMRs were found to be fewer in number and for the most part distinct from the F3 generation epimutations. Observations suggest the direct exposure induced F1 generation sperm DMRs appear to promote in subsequent generations alterations in the germ cell developmental programming that leads to the distinct epimutations in the F3 generation. This may help explain the differences in disease and phenotypes between the direct exposure F1 generation and transgenerational F3 generation. Observations demonstrate a distinction between the direct exposure versus transgenerational epigenetic programming induced by environmental exposures and provide insights into the molecular mechanisms involved in the epigenetic transgenerational inheritance phenomenon.

  12. [Preparative isolation of tri-, tetra-, penta- and hexapyrimidine nucleotides from hydrolysates of depurinated herring sperm DNA (author's transl)].

    Science.gov (United States)

    Schott, H

    1979-04-21

    The pyrimidine nucleotides p(dC)3p, p(dT)3p and p(dT)4p and mixtures of the sequence isomers p(dC3, dT), (dC3, dT)p; p(dC3, dT)p; p(dC2, dT2)p; p(dC, dT3)p; p(dC3, dT2)p; p(dC2, dT3); p(dC2, dT3)p; p(dC, dT4)p; p(dC4, dT2); p(dC3, dT3); p(dC3, dT3)p and p(dC2, dT4)p have been isolated on a preparative scale from hydrolysates of depurinated herring sperm DNA. The DNA hydrolysate is first separated into a high- and a low-molecular weight pyrimidine nucleotide mixture by column chromatography at pH 7.5 on DEAE-cellulose. The high-molecular-weight pyrimidine nucleotide mixture is further separated into four peaks on QAE-Sephadex at pH 7.5. The second peak is re-chromatographed on QAE-Sephadex at pH 3.5. Pyrimidine nucleotides containing predominantly cytidylic acid units may thus be separated from these with predominantly thymidylic acid units. Subsequent separation according to number of phosphate groups at pH 7.5 on QAE-Sephadex yields products of 70--93% purity. In a final separation step, the pyrimidine nucleotides and mixtures of sequence isomers are once again chromatographed on QAE-Sephadex with 7 M urea at pH 7.5. The products thus obtained are generally chromatographically pure. Impurities which are not fully removed by column chromatography are separated by paper chromatography. The structure of the isolated DNA fragments and the composition of the mixtures of sequence isomers are determined from the chromatographic data, absorption characteristics and by enzymatic degradation.

  13. Double stranded sperm DNA breaks, measured by Comet assay, are associated with unexplained recurrent miscarriage in couples without a female factor.

    Science.gov (United States)

    Ribas-Maynou, Jordi; García-Peiró, Agustín; Fernandez-Encinas, Alba; Amengual, Maria José; Prada, Elena; Cortés, Pilar; Navarro, Joaquima; Benet, Jordi

    2012-01-01

    It is known that sperm samples from recurrent pregnancy loss (RPL) couples have an increase in their sperm DNA fragmentation (SDF), but no studies have been performed in order to identify differences between single stranded SDF (ssSDF) and double stranded SDF (dsSDF) in these patients. This could be relevant because the type of DNA damage could have different effects. Semen samples were classified attending their clinical status: 25 fertile donors and 20 RPL patients with at least two unexplained first trimester miscarriages. SDF was analysed using alkaline and neutral Comet assay, SCD test and pulsed-field gel electrophoresis (PFGE), and ROC analysis including data from 105 more infertile patients (n = 150) was performed to establish predictive threshold values. SDF for alkaline and neutral Comet, and the SCD test was analysed in these categories of individuals. Data revealed the presence of two subgroups within fertile donors. The values obtained were 21.10±9.13, 23.35±10.45 and 12.31±4.31, respectively, for fertile donors with low values for both ssSDF and dsSDF; 27.86±12.64, 80.69±12.67 and 12.43±5.22, for fertile donors with low ssSDF and high dsSDF; and 33.61±15.50, 84.64±11.28 and 19.28±6.05, for unexplained RPL patients, also showing a low ssSDF and high dsSDF profile. This latter profile was seen in 85% of unexplained RPL and 33% of fertile donors, suggesting that it may be associated to a male risk factor for undergoing RPL. ROC analysis regarding recurrent miscarriage set the cut-off value at 77.50% of dsDNA SDF. PFGE for low ssSDF and high dsSDF profile samples and positive controls treated with DNase, to induce dsDNA breaks, showed a more intense band of about 48 kb, which fits the toroid model of DNA compaction in sperm, pointing out that some nuclease activity may be affecting their sperm DNA in RPL patients. This work identifies a very specific SDF profile related to the paternal risk of having RPL.

  14. Double stranded sperm DNA breaks, measured by Comet assay, are associated with unexplained recurrent miscarriage in couples without a female factor.

    Directory of Open Access Journals (Sweden)

    Jordi Ribas-Maynou

    Full Text Available It is known that sperm samples from recurrent pregnancy loss (RPL couples have an increase in their sperm DNA fragmentation (SDF, but no studies have been performed in order to identify differences between single stranded SDF (ssSDF and double stranded SDF (dsSDF in these patients. This could be relevant because the type of DNA damage could have different effects. Semen samples were classified attending their clinical status: 25 fertile donors and 20 RPL patients with at least two unexplained first trimester miscarriages. SDF was analysed using alkaline and neutral Comet assay, SCD test and pulsed-field gel electrophoresis (PFGE, and ROC analysis including data from 105 more infertile patients (n = 150 was performed to establish predictive threshold values. SDF for alkaline and neutral Comet, and the SCD test was analysed in these categories of individuals. Data revealed the presence of two subgroups within fertile donors. The values obtained were 21.10±9.13, 23.35±10.45 and 12.31±4.31, respectively, for fertile donors with low values for both ssSDF and dsSDF; 27.86±12.64, 80.69±12.67 and 12.43±5.22, for fertile donors with low ssSDF and high dsSDF; and 33.61±15.50, 84.64±11.28 and 19.28±6.05, for unexplained RPL patients, also showing a low ssSDF and high dsSDF profile. This latter profile was seen in 85% of unexplained RPL and 33% of fertile donors, suggesting that it may be associated to a male risk factor for undergoing RPL. ROC analysis regarding recurrent miscarriage set the cut-off value at 77.50% of dsDNA SDF. PFGE for low ssSDF and high dsSDF profile samples and positive controls treated with DNase, to induce dsDNA breaks, showed a more intense band of about 48 kb, which fits the toroid model of DNA compaction in sperm, pointing out that some nuclease activity may be affecting their sperm DNA in RPL patients. This work identifies a very specific SDF profile related to the paternal risk of having RPL.

  15. Double Stranded Sperm DNA Breaks, Measured by Comet Assay, Are Associated with Unexplained Recurrent Miscarriage in Couples without a Female Factor

    Science.gov (United States)

    Ribas-Maynou, Jordi; García-Peiró, Agustín; Fernandez-Encinas, Alba; Amengual, Maria José; Prada, Elena; Cortés, Pilar; Navarro, Joaquima; Benet, Jordi

    2012-01-01

    It is known that sperm samples from recurrent pregnancy loss (RPL) couples have an increase in their sperm DNA fragmentation (SDF), but no studies have been performed in order to identify differences between single stranded SDF (ssSDF) and double stranded SDF (dsSDF) in these patients. This could be relevant because the type of DNA damage could have different effects. Semen samples were classified attending their clinical status: 25 fertile donors and 20 RPL patients with at least two unexplained first trimester miscarriages. SDF was analysed using alkaline and neutral Comet assay, SCD test and pulsed-field gel electrophoresis (PFGE), and ROC analysis including data from 105 more infertile patients (n = 150) was performed to establish predictive threshold values. SDF for alkaline and neutral Comet, and the SCD test was analysed in these categories of individuals. Data revealed the presence of two subgroups within fertile donors. The values obtained were 21.10±9.13, 23.35±10.45 and 12.31±4.31, respectively, for fertile donors with low values for both ssSDF and dsSDF; 27.86±12.64, 80.69±12.67 and 12.43±5.22, for fertile donors with low ssSDF and high dsSDF; and 33.61±15.50, 84.64±11.28 and 19.28±6.05, for unexplained RPL patients, also showing a low ssSDF and high dsSDF profile. This latter profile was seen in 85% of unexplained RPL and 33% of fertile donors, suggesting that it may be associated to a male risk factor for undergoing RPL. ROC analysis regarding recurrent miscarriage set the cut-off value at 77.50% of dsDNA SDF. PFGE for low ssSDF and high dsSDF profile samples and positive controls treated with DNase, to induce dsDNA breaks, showed a more intense band of about 48 kb, which fits the toroid model of DNA compaction in sperm, pointing out that some nuclease activity may be affecting their sperm DNA in RPL patients. This work identifies a very specific SDF profile related to the paternal risk of having RPL. PMID:23028579

  16. DNA fragmentation and membrane damage of bocachico Prochilodus magdalenae (Ostariophysi: Prochilodontidae sperm following cryopreservation with dimethylsulfoxide and glucose

    Directory of Open Access Journals (Sweden)

    José Gregorio Martínez

    2012-09-01

    Full Text Available The endangered bocachico Prochilodus magdalenae is a native freshwater fish of Colombia, the most captured species locally and one of the most important species for ex-situ conservation (germplasm banks. The aim of this study was to examine the effect of three concentrations of Dimethylsulfoxide (DMSO (5%, 10%, 15% and three of glucose (305, 333, 361 mM in the extender on spermatic DNA fragmentation (F-DNA (by Halomax®, Chromatin dispersion and membrane damage (D-Me (by eosin-nigrosin staining. After assessment of sperm quality by computer analysis of motility, one part of semen from males was diluted separately with three parts of extender and filled into 0.5 ml straws. Freezing was carried out in liquid nitrogen vapor dry shipper for 30 minutes and thawed at 60ºC for 8 seconds in a water bath and evaluated for the percentage of cells found with F-DNA and D-Me. The results demonstrated that cryopreservation causes greater F-DNA (13.62 ± 1.6% to 28.91 ± 3.25 and D-Me (24.27 ± 1.1% to 58.33 ± 2.81% when compared with pre-freezing semen (PFS (6.71 ± 1.54% and 2.34 ± 0.5%, respectively for F-DNA and D-Me. A significant interaction was found between DMSO and glucose concentration in this experiment. Use of extender: 10% DMSO + 305 mM glucose + 12% chicken egg yolk and, 10% DMSO + 333 mM glucose + 12% chicken egg yolk, allow for lower F-DNA and D-Me during cryopreservation of bocachico semen. A high correlation between F-DNA and D-Me was found (r = 0.771.O curimba Prochilodus magdalenae, é uma espécie nativa de água doce da Colômbia ameaçada de extinção, sendo a mais capturada localmente e uma das mais importantes para a conservação ex-situ (bancos de germoplasma. O objetivo deste estudo foi avaliar o efeito de três concentrações de dimetilsulfóxido (DMSO (5%, 10%, 15% e três de glicose (305, 333, 361 mM no diluente sobre a fragmentação do ADN espermático (F-DNA (através de Halomax®, dispersão da cromatina e danos em

  17. Seasonal fluctuation in the secretion of the antioxidant melatonin is not associated with alterations in sperm DNA damage

    Directory of Open Access Journals (Sweden)

    Gunilla Malm

    2017-01-01

    Full Text Available A high sperm DNA fragmentation index (DFI is associated with reduced fertility. DFI is influenced by the balance between reactive oxygen species and antioxidants. A circannual variation in melatonin, an antioxidant and free radical scavenger, could thus impact semen quality and fertility. The association between the major melatonin metabolite, urine 6-sulfatoxymelatonin (aMT6s, and DFI was analyzed in 110 Oslo men (south of the Arctic Circle and 86 Tromsoe men (north of the Arctic Circle. Two semen analyses, summer and winter, and four urine samples (early/late summer; early/late winter, were analyzed. The associations between aMT6s in urine and DFI were characterized in a cross-sectional and longitudinal manner using correlation analysis and linear regression. Regardless of season and location, no significant correlations between aMT6s and DFI were observed. The correlation coefficients for associations between changes over time (early winter-early summer in aMT6s and DFI were for the total cohort: rho = −0.08 (P = 0.322, for the Oslo cohort: rho = −0.07 (P = 0.485, and for the Tromsoe cohort: rho = −0.14 (P = 0.273, respectively. Similar results were seen when comparing late winter and late summer. There was no any statistically significant correlation between changes over time in aMT6s and DFI for men with DFI below and above the median value (10%, respectively. The seasonal variation in melatonin excretion seems not to have any impact on DFI.

  18. Secondary structure of protamine in sperm nuclei: an infrared spectroscopy study

    Directory of Open Access Journals (Sweden)

    Suau Pedro

    2011-03-01

    Full Text Available Abstract Background Protamines are small basic proteins that condense the DNA in mature spermatozoa. Typical protamines are of simple composition and very arginine-rich, usually in the range of 60-80%. Arginine residues are distributed in a number of stretches separated by neutral amino acids. We have used Fourier transform infrared spectroscopy (FTIR to gain access for the first time to the secondary structure of protamines in sperm nuclei. This technique is particularly well suited to the study of DNA-bound protamine in whole nuclei since it is not affected by turbidity. Results We show that DNA -bound salmon (salmine and squid protamines contain α-helix, β-turns and a proportion of other structures not stabilized by intramolecular hydrogen bonding. No β-sheet was observed. In salmine, the α-helix amounted to ~20%, while in squid protamine it reached ~40%. In contrast, the structure not stabilized by intermolecular hydrogen bonding was more abundant in salmine (~40% than in squid protamine (~20%. Both protamines contained ~40% β-turns. The different helical potential of salmine and squid protamine was confirmed by structure predictions and CD in the presence of trifluoroethanol. Conclusion DNA-bound protamine in sperm nuclei contains large amounts of defined secondary structure stabilized by intramolecular hydrogen bonding. Both salmine and squid protamine contain similar amounts of β-turns, but differ in the proportions of α-helix and non-hydrogen bonded conformations. In spite of the large differences in the proportions of secondary structure motifs between salmon and squid protamines, they appear to be equally efficient in promoting tight hexagonal packing of the DNA molecules in sperm nuclei.

  19. Elucidation of Listeria monocytogenes contamination routes in cold-smoked salmon processing plants detected by DNA-based typing methods

    DEFF Research Database (Denmark)

    Vogel, Birte Fonnesbech; Huss, Hans Henrik; Ojeniyi, B.

    2001-01-01

    The contamination routes of Listeria monocytogenes in cold- smoked salmon processing plants were investigated by analyzing 3,585 samples from products (produced in 1995, 1996, 1998, and 1999) and processing environments (samples obtained in 1998 and 1999) of two Danish smokehouses. The level...... of product contamination in plant I varied from 31 to 85%, and no L. monocytogenes was found on raw fish (30 fish were sampled). In plant II, the levels of both raw fish and product contamination varied from 0 to 25% (16 of 185 raw fish samples and 59 of 1,000 product samples were positive for L......, suggesting that contamination of the final product (cold-smoked salmon) in both plants (but primarily in plant I) was due to contamination during processing rather than to contamination from raw fish. However, the possibility that raw fish was an important source of contamination of the processing equipment...

  20. Use of sperm DNA integrity as a marker for exposure to contamination in Palaemon serratus (Pennant 1777): Intrinsic variability, baseline level and in situ deployment.

    Science.gov (United States)

    Erraud, Alexandre; Bonnard, Marc; Chaumot, Arnaud; Geffard, Olivier; Duflot, Aurélie; Forget-Leray, Joëlle; Le Foll, Frank; Geffard, Alain; Xuereb, Benoit

    2018-04-01

    In a previous study, the Comet assay was optimized for Palaemon serratus prawns in order to propose a biomarker for sperm quality in this species. However, better knowledge of its basal level and its natural variability, related to intrinsic biotic and environmental abiotic factors, is required before any relevant use of this biomarker in the field. To fulfill this goal, the present study proceeded in three steps: (i) the temporal variability of DNA integrity was followed monthly in a reference population over a 2-year period, (ii) the correlation between the main intrinsic biotic (i.e. size, weight and molting stage) and abiotic factors (i.e. water temperature) were recorded in the field, and the basal DNA integrity was assessed in order to scrutinize any confounding influence of factors unrelated to toxic response, (iii) the baseline level was used to discriminate biomarker response among different stations displaying contrasting contamination levels. The results of the two-year monitoring in the reference population revealed no correlation between the levels of spermatozoa DNA damage and temperature, body size, weight or molting stage. Only a slight variability between monthly samplings was detected. On the basis of these field-collected data, we defined a reference distribution (i.e. 52.6 ± 5.6 A.U) with a threshold value (i.e. 61.7 A.U). Finally, this threshold value proved its relevance to discriminate among stations with contrasting pollution levels around the Seine Bay. Indeed, the results suggest significant DNA damage in populations nearest the Seine estuary, a major source of contaminants in the Bay, and a lower effect in populations further away from the estuary. The overall conclusion was that the Comet assay on P. serratus spermatozoa could be a useful tool for the monitoring of the toxicological print within sperm and main globally the contamination exposure of crustaceans in marine waters. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Sperm whale population structure in the eastern and central North Pacific inferred by the use of single-nucleotide polymorphisms, microsatellites and mitochondrial DNA.

    Science.gov (United States)

    Mesnick, Sarah L; Taylor, Barbara L; Archer, Frederick I; Martien, Karen K; Treviño, Sergio Escorza; Hancock-Hanser, Brittany L; Moreno Medina, Sandra Carolina; Pease, Victoria L; Robertson, Kelly M; Straley, Janice M; Baird, Robin W; Calambokidis, John; Schorr, Gregory S; Wade, Paul; Burkanov, Vladimir; Lunsford, Chris R; Rendell, Luke; Morin, Phillip A

    2011-03-01

    We use mitochondrial DNA (mtDNA) (400 bp), six microsatellites and 36 single-nucleotide polymorphisms (SNPs), 20 of which were linked, to investigate population structure of sperm whales (Physeter macrocephalus) in the eastern and central North Pacific. SNP markers, reproducible across technologies and laboratories, are ideal for long-term studies of globally distributed species such as sperm whales, a species of conservation concern because of both historical and contemporary impacts. We estimate genetic differentiation among three strata in the temperate to tropical waters where females are found: California Current, Hawai`i and the eastern tropical Pacific. We then consider how males on sub-Arctic foraging grounds assign to these strata. The California Current stratum was differentiated from both the other strata (P < 0.05) for mtDNA, microsatellites and SNPs, suggesting that the region supports a demographically independent population and providing the first indication that males may exhibit reproductive philopatry. Comparisons between the Hawai`i stratum and the eastern tropical Pacific stratum are not conclusive at this time. Comparisons with Alaska males were statistically significant, or nearly so, from all three strata and individuals showed mixed assignment to, and few exclusions from, the three potential source strata, suggesting widespread origin of males on sub-Arctic feeding grounds. We show that SNPs have sufficient power to detect population structure even when genetic differentiation is low. There is a need for better analytical methods for SNPs, especially when linked SNPs are used, but SNPs appear to be a valuable marker for long-term studies of globally dispersed and highly mobile species. © 2011 Blackwell Publishing Ltd.

  2. The effect of bovine serum albumin and fetal calf serum on sperm quality, DNA fragmentation and lipid peroxidation of the liquid stored rabbit semen.

    Science.gov (United States)

    Sarıözkan, Serpil; Türk, Gaffari; Cantürk, Fazile; Yay, Arzu; Eken, Ayşe; Akçay, Aytaç

    2013-08-01

    The aim of the present study was to determine the effects of the bovine serum albumin (BSA) and fetal calf serum (FCS) on sperm quality, DNA fragmentation and lipid peroxidation of liquid stored rabbit semen stored up to 72 h at 5 °C. Ejaculates were collected from five New Zealand male rabbits by artificial vagina and pooled at 37 °C following evaluation. Each pooled ejaculate was split into three equal experimental groups and diluted to a final concentration of approximately 40 × 10(6)sperm/ml (single step dilution), in an Eppendorf tube, with the Tris based extender containing BSA (5mg/ml), FCS (10%) or no additive (control) at 37 °C, cooled to 5 °C and stored for up to 72 h. The extender supplemented with BSA and FCS did not improve the percentages of motility and acrosomal abnormality during 48 h compared to the control. The additives BSA and FCS had a significant effect in the maintaining of plasma membrane integrity between 48 and 72 h storage period, compared to the control (P<0.01). The supplementation of BSA and FCS had a protective effect on motility (P<0.05), plasma membrane integrity (P<0.01) and acrosomal integrity (P<0.01) at 72 h compared to the control. The supplementations with BSA and FCS led to a reduction in DNA damage of rabbit sperm at 48 and 72 h during storage period, compared to the control (P<0.001). Although supplementation of BSA and FCS caused significant (P<0.01) decreases in malondialdehyde (MDA) level at 48 h and 72 h, they significantly (P<0.01) increased the glutathione peroxidase (GPx) antioxidant activity up to 72 h when compared to the control group. In conclusion, BSA and FCS supplementation to liquid stored rabbit semen provide a protection for spermatozoa against cool storage-induced DNA damage and plasma membrane integrity by their antioxidative properties. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. Etiology and Evaluation of Sperm Chromatin Anomalies

    Directory of Open Access Journals (Sweden)

    Marziyeh Tavalaee

    2008-01-01

    Full Text Available Evidence suggests that human sperm chromatin anomalies adversely affect reproductive outcomesand infertile men possess substantially amount of sperm with chromatin anomalies than fertilemen.Routine semen analysis evaluates parameters such as sperm motility and morphology, but doesnot examine the nuclear DNA integrity of spermatozoa. It has been suggested that altered nuclearchromatin structure or damaged DNA in spermatozoa could modify the special cellular functionsof human spermatozoa, and thereby affect the fertility potential. Intra-cytoplasmic sperm injection(ICSI bypass the barriers to fertilization for such a sperm, then the effect of chromatin anomalies onthe development remains a concern. Therefore, it is essential to develop and use accurate diagnostictests, which may provide better prognostic capabilities than the standard sperm assessments. Thisreview discusses our current understanding of the structure and organization of sperm DNA,the different procedures for assessment of sperm chromatin anomalies including comet assay,Chromomycin A3 (CMA3, sperm chromatin structure assay (SCSA, acridine orange test (AOT,terminal TdT-mediated dUTP-nick-end labelling (TUNEL assay, aniline blue and sperm chromatindispersion (SCD test and the impact of chromatin anomalies on reproductive outcome.

  4. Organic salmon

    DEFF Research Database (Denmark)

    Ankamah Yeboah, Isaac; Nielsen, Max; Nielsen, Rasmus

    The year 2016 is groundbreaking for organic aquaculture producers in EU, as it represents the deadline for implementing a full organic life cycle in the aquaculture production. Such a shift induces production costs for farmers and if it should be profitable, they must receive higher prices....... This study identifies the price premium on organic salmon in the Danish retail sale sector using consumer panel scanner data for households by applying the hedonic price model while permitting unobserved heterogeneity between households. A premium of 20% for organic salmon is found. Since this premium...... is closer to organic labeled agriculture products than to ecolabelled capture fisheries products, it indicates that consumers value organic salmon as an agriculture product more than fisheries product....

  5. Multiple genes for functional 6 fatty acyl desaturases (Fad) in Atlantic salmon (Salmo salar L.): gene and cDNA characterization, functional expression, tissue distribution and nutritional regulation.

    Science.gov (United States)

    Monroig, Oscar; Zheng, Xiaozhong; Morais, Sofia; Leaver, Michael J; Taggart, John B; Tocher, Douglas R

    2010-09-01

    Fish are the primary source in the human food basket of the n-3 long-chain polyunsaturated fatty acids, eicosapentaenoate (EPA; 20:5n-3) and docosahexaenoate (DHA; 22:6n-3), that are crucial to the health of higher vertebrates. Atlantic salmon are able to synthesize EPA and DHA from 18:3n-3 through reactions catalyzed by fatty acyl desaturases (Fad) and elongases of very long chain fatty acids. Previously, two cDNAs encoding functionally distinct Delta5 and Delta6 Fads were isolated, but screening of a genomic DNA library revealed the existence of more putative fad genes in the Atlantic salmon genome. In the present study, we show that there are at least four genes encoding putative Fad proteins in Atlantic salmon. Two genes, Delta6fad_a and Delta5fad, corresponded to the previously cloned Delta6 and Delta5 Fad cDNAs. Functional characterization by heterologous expression in yeast showed that the cDNAs for both the two further putative fad genes, Delta6fad_b and Delta6fad_c, had only Delta6 activity, converting 47 % and 12 % of 18:3n-3 to 18:4n-3, and 25 and 7 % of 18:2n-6 to 18:3n-6, for 6Fad_b and Delta6fad_c, respectively. Both 6fad_a and 6fad_b genes were highly expressed in intestine (pyloric caeca), liver and brain, with 6fad_b also highly expressed in gill, whereas 6fad_c transcript was found predominantly in brain, with lower expression levels in all other tissues. The expression levels of the 6fad_a gene in liver and the 6fad_b gene in intestine were significantly higher in fish fed diets containing vegetable oil compared to fish fed fish oil suggesting up-regulation in response to reduced dietary EPA and DHA. In contrast, no significant differences were found between transcript levels for 6fad_a in intestine, 6fad_b in liver, or 6fad_c in liver or intestine of fish fed vegetable oil compared to fish fed fish oil. The observed differences in tissue expression and nutritional regulation of the fad genes are discussed in relation to gene structures and fish

  6. Myoinositol: does it improve sperm mitochondrial function and sperm motility?

    Science.gov (United States)

    Condorelli, Rosita A; La Vignera, Sandro; Bellanca, Salvatore; Vicari, Enzo; Calogero, Aldo E

    2012-06-01

    To evaluate whether an improvement in mitochondrial membrane potential was associated with sperm motility amelioration and greater sperm recovery after the swim-up procedure. A second purpose was to evaluate the effects of myoinositol (MYO) on sperm apoptosis, quality of chromatin compaction, and DNA integrity. Spermatozoa from 20 normozoospermic men and 20 patients with oligo-astheno-teratozoospermia were incubated in vitro with 2 mg/mL of MYO or phosphate-buffered saline as a control for 2 hours. After this incubation period, sperm motility was evaluated. Flow cytometry was used to analyze the mitochondrial membrane potential, phosphatidylserine externalization, chromatin compactness, and DNA fragmentation. We also evaluated the total number of motile spermatozoa recovered after swim-up after incubation with MYO or phosphate-buffered saline. MYO significantly increased the percentage of spermatozoa with progressive motility in both normozoospermic men and patients with oligo-astheno-teratozoospermia. Motility improvement in the latter group was associated with a significant increase in the percentage of spermatozoa with high mitochondrial membrane potential. MYO had no effects on mitochondrial function in spermatozoa from normozoospermic men. Sperm phosphatidylserine externalization, chromatin compactness, and DNA fragmentation were unaffected by MYO in both groups. After incubation with MYO, the total number of spermatozoa recovered after swim-up had improved significantly in both groups. These data show that MYO increases sperm motility and the number of spermatozoa retrieved after swim-up in both normozoospermic men and patients with abnormal sperm parameters. In patients with oligo-astheno-teratozoospermia, the improvement in these parameters was associated with improved sperm mitochondrial function. These findings support the use of MYO in both in vivo- and in vitro-assisted reproductive techniques. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. The effect of cholesterol loaded cyclodextrins on post-thawing quality of buffalo semen in relation to sperm DNA damage and ultrastructure.

    Science.gov (United States)

    Ezz, Mohamed Aboul; Montasser, Abd Elmonem; Hussein, Mamdouh; Eldesouky, Ashraf; Badr, Magdy; Hegab, Abd Elraouf; Balboula, Ahmed; Zaabel, Samy M

    2017-03-01

    The cryopreservation of germ cells is a major tool for the propagation of animals with desired genetic traits. Although cryopreservation of spermatozoa in some animals is effective, its effectiveness is variable. For example, cryopreservation efficiency of buffalo bull spermatozoa remains very poor. In this study, we evaluated sperm DNA damage and ultrastructure in buffalo bull spermatozoa vitrified in the presence or absence of cholesterol-loaded cyclodextrins (CLC). Our results showed that cryopreserved buffalo spermatozoa had elevated levels of deteriorated plasma and mitochondrial membranes, which are the likely causes of DNA damage after vitrification. Accordingly, the levels of the activity of Alanine Aminotransferase (ALT), Alkaline phosphatase (ALP) and Aspartate Aminotransferase (AST) were also elevated following exposure of buffalo bull spermatozoa to a cycle of freezing-thawing. Importantly, supplementation of Tris-Egg Yolk-Glucose (TEYG) extender with (CLC) improved the quality of buffalo spermatozoa following cryopreservation. This protective effect of CLC is likely due to decreasing mitochondrial and plasma membrane deterioration with subsequent inhibition of DNA damage. These results suggest that cholesterol loss is the likely reason for poor semen quality in buffaloes following cryopreservation, and provide evidence that manipulating lipid content during cryopreservation is a promising strategy to improve the quality of buffalo semen. Copyright © 2016. Published by Elsevier Urban & Partner Sp. z o.o.

  8. Production of trout offspring from triploid salmon parents.

    Science.gov (United States)

    Okutsu, Tomoyuki; Shikina, Shinya; Kanno, Megumi; Takeuchi, Yutaka; Yoshizaki, Goro

    2007-09-14

    Many salmonids have become at risk of extinction. For teleosts whose eggs cannot be cryopreserved, developing techniques other than egg cryopreservation to save genetic resources is imperative. In this study, spermatogonia from rainbow trout were intraperitoneally transplanted into newly hatched sterile triploid masu salmon. Transplanted trout spermatogonia underwent spermatogenesis and oogenesis in male and female recipients, respectively. At 2 years after transplantation, triploid salmon recipients only produced trout sperm and eggs. With use of these salmon as parents, we successfully produced only donor-derived trout offspring. Thus, by transplanting cryopreserved spermatogonia into sterile xenogeneic recipients, we can generate individuals of a threatened species.

  9. Studies on the Interaction Mechanism of 1,10-Phenanthroline Cobalt(II Complex with DNA and Preparation of Electrochemical DNA Biosensor

    Directory of Open Access Journals (Sweden)

    Shiying Wang

    2006-10-01

    Full Text Available Fluorescence spectroscopy and ultraviolet (UV spectroscopy techniques coupled with cyclic voltammetry (CV were used to study the interaction between salmon sperm DNA and 1,10-Phenanthroline cobalt(II complex, [Co(phen2(Cl(H2O]Cl·H2O, where phen = 1,10-phenanthroline. The interaction between [Co(phen2(Cl(H2O]+ and double-strand DNA (dsDNA was identified to be intercalative mode. An electrochemical DNA biosensor was developed by covalent immobilization of probe single-strand DNA (ssDNA related to human immunodeficiency virus (HIV on the activated glassy carbon electrode (GCE. With [Co(phen2(Cl(H2O]+ being the novel electrochemical hybridization indicator, the selectivity of ssDNA-modified electrode was investigated and selective detection of complementary ssDNA was achieved using differential pulse voltammetry (DPV.

  10. Chirality of sulforhodamine dye molecules incorporated in DNA thin films

    Science.gov (United States)

    Steckl, A. J.; Spaeth, H.; Singh, K.; Grote, J.; Naik, R.

    2008-11-01

    Thin films formed from salmon sperm DNA reacted with a cationic surfactant (CTMA-Cl) included up to 25 wt % fluorescent molecule sulforhodamine (SRh). SRh effect on DNA chirality and vice versa was investigated by circular dichroism (CD) spectroscopy. The CD signals at 250-265 nm indicate that DNA chirality was maintained or enhanced. Induced CD (iCD) signal at 580-610 nm indicates that SRh is chiral in DNA/CTMA. iCD signal from both solutions and thin films generally increases with SRh concentration. The chirality induced in SRh molecules and the absence of significant DNA reduction in chirality are clear indicators of strong binding to DNA/CTMA.

  11. Sperm-mediated host-derived DNA transfer as a new mechanism for immune system evasion of sexually transmitted genital tract pathogens.

    Science.gov (United States)

    Landau, Eliahu Yuval; Wainrach, Bezalel

    2012-09-01

    Over one century of extensive efforts directed towards investigating the immune response and the immuno-protection associated with sexually transmitted infections have failed to produce any effective vaccines against most of the major pathogens, among them Neisseria gonorrhea, herpes simplex virus type 2, and Chlamydia trachomatis. Attempts to design and develop protective vaccines against them have also yielded disappointing results. It has long been felt that there might be another yet undiscovered complicating factor, in addition to the recognized difficulties, which might be impeding the development of successful vaccines. Unlike the other body organs and systems, the genital tract and the elements found within it (e.g., spermatozoa) are endowed with unique features, some of which are associated with inherent DNA transferability skills as physiologically required from such an environment. We hypothesize that there is a novel evasion mechanism that involves an unusual sperm-mediated host-derived DNA transfer by which sexually transmitted genital tract microorganisms can express brand new chimeric antigens and epitopes and, by doing so, thus evade the surveillance of the immune system. This hypothesis may describe what would be the long-awaited breakthrough in the search for a vaccine against sexually transmitted infections. It may also assist in developing better-designed vaccines in general, and may have implications on other microorganism-related challenges (e.g., antibiotic resistance). Copyright © 2012 Elsevier Ltd. All rights reserved.

  12. Cytometry of mammalian sperm

    Energy Technology Data Exchange (ETDEWEB)

    Gledhill, B.L.

    1983-10-11

    Male germ cells respond dramatically to a variety of insults and are important reproductive dosimeters. Semen analyses are very useful in studies on the effects of drugs, chemicals, and environmental hazards on testicular function, male fertility and heritable germinal mutations. The accessibility of male cells makes them well suited for analytical cytology. We might automate the process of determining sperm morphology but should not do so solely for increased speed. Rather, richer tangible benefits will derive from cytometric evaluation through increased sensitivity, reduced subjectivity, standardization between investigators and laboratories, enhanced archival systems, and the benefits of easily exchanged standardized data. Inroads on the standardization of assays for motility and functional integrity are being made. Flow cytometric analysis of total DNA content of individual sperm is an insensitive means to detect exposure to reproductive toxins because of the small size and low frequency of the DNA content errors. Flow cytometry can be applied to determine the proportions of X- and Y-sperm in semen samples.

  13. Sperm Production Rate, Gonadal and Extragonadal Sperm ...

    African Journals Online (AJOL)

    Five healthy West African Dwarf (WAD) rams, 1.5 to 2.5 years of age and weighing between 15 kg to 20 kg were used to determine daily sperm production, gonadal and exragonadal sperm reserves. Gonadal and extragonadal sperm reserves were estimated by the haemocytometric method, while the daily sperm production ...

  14. [Optimization of sperm alkaline single-cell gel electrophoresis].

    Science.gov (United States)

    Deng, Shuang; Fan, Lang; Wu, Xi-yan; Zhu, Yan; Xu, Ke-qian

    2015-02-01

    To investigate the main factors that influence the results of sperm alkaline single-cell gel electrophoresis (SCGE), optimize the conditions, and standardize its procedures. Using alkaline SCGE, we detected the DNA fragments of sperm treated with different concentrations of H2O2 and determined the influences of the number of agarose gel layers, pH during DNA unwinding and electrophoresis, the time of DNA unwinding and electrophoresis, and cumulative sperm number on the results of sperm alkaline SCGE. Then we optimized the procedures, analyzed the repeatability of the optimized method, and examined 40 semen samples using the method. Three agarose gel layers could reduce the background. The optimal pH during DNA unwinding and electrophoresis was 10, and the best times for DNA unwinding and electrophoresis were 40 min and 30 min, respectively. Fifty sperm were adequate to ensure the reliability of the results. Based on the percentage of tail DNA, the intra- and inter-assay repeatabilities of the optimized sperm alkaline SCGE were 3.12% and 7.13%, and by the DNA damage score, they were 2.38% and 6.09%, respectively. Sperm DNA fragments were significantly increased in the infertile patients with oligoasthenoteratozoospermia as compared with healthy fertile males (P sperm alkaline SCGE, highly repeatable and easy to be standardized, can be applied to the clinical detection of sperm DNA fragmentation in infertile men.

  15. A Strengths-Weaknesses-Opportunities-Threats (SWOT) analysis on the clinical utility of sperm DNA fragmentation testing in specific male infertility scenarios

    Science.gov (United States)

    Agarwal, Ashok; Cho, Chak-Lam; Majzoub, Ahmad

    2017-01-01

    Background Sperm DNA fragmentation (SDF) is recognized as a leading cause of male infertility because it can impair the paternal genome through distinct pathophysiological mechanisms. Current evidence supports SDF as a major factor in the pathophysiology of several conditions, including varicocele, unexplained infertility, assisted reproductive technology failure, and environmental lifestyle factors, although the mechanisms involved have not been fully described yet. Measurement of the levels of DNA fragmentation in semen provides valuable information on the integrity of paternal chromatin and may guide therapeutic strategies. A recently published clinical practice guideline (CPG) highlighted how to use the information provided by SDF testing in daily practice, which triggered a series of commentaries by leading infertility experts. These commentaries contained an abundance of information and conflicting views about the clinical utility of SDF testing, which underline the complex nature of SDF. Methods A search of papers published in response to the CPG entitled “Clinical utility of sperm DNA fragmentation testing: practice recommendations based on clinical scenarios” was performed within the Translational Andrology and Urology (TAU) website (http://tau.amegroups.com/). The start and end dates for the search were May 2017 and August 2017, respectively. Each commentary meeting our inclusion criteria was rated as “supportive without reservation”, “supportive with reservation”, “not supportive” or “neutral”. We recorded whether articles discussed either SDF characteristics as a laboratory test method or clinical scenarios, or both. Subsequently, we extracted the particulars from each commentary and utilized the ‘Strengths-Weaknesses-Opportunities-Threats’ (SWOT) analysis to understand the perceived advantages and drawbacks of SDF as a specialized sperm function method in clinical practice. Results Fifty-eight fertility experts from six

  16. A Strengths-Weaknesses-Opportunities-Threats (SWOT) analysis on the clinical utility of sperm DNA fragmentation testing in specific male infertility scenarios.

    Science.gov (United States)

    Esteves, Sandro C; Agarwal, Ashok; Cho, Chak-Lam; Majzoub, Ahmad

    2017-09-01

    Sperm DNA fragmentation (SDF) is recognized as a leading cause of male infertility because it can impair the paternal genome through distinct pathophysiological mechanisms. Current evidence supports SDF as a major factor in the pathophysiology of several conditions, including varicocele, unexplained infertility, assisted reproductive technology failure, and environmental lifestyle factors, although the mechanisms involved have not been fully described yet. Measurement of the levels of DNA fragmentation in semen provides valuable information on the integrity of paternal chromatin and may guide therapeutic strategies. A recently published clinical practice guideline (CPG) highlighted how to use the information provided by SDF testing in daily practice, which triggered a series of commentaries by leading infertility experts. These commentaries contained an abundance of information and conflicting views about the clinical utility of SDF testing, which underline the complex nature of SDF. A search of papers published in response to the CPG entitled "Clinical utility of sperm DNA fragmentation testing: practice recommendations based on clinical scenarios" was performed within the Translational Andrology and Urology ( TAU ) website (http://tau.amegroups.com/). The start and end dates for the search were May 2017 and August 2017, respectively. Each commentary meeting our inclusion criteria was rated as "supportive without reservation", "supportive with reservation", "not supportive" or "neutral". We recorded whether articles discussed either SDF characteristics as a laboratory test method or clinical scenarios, or both. Subsequently, we extracted the particulars from each commentary and utilized the 'Strengths-Weaknesses-Opportunities-Threats' (SWOT) analysis to understand the perceived advantages and drawbacks of SDF as a specialized sperm function method in clinical practice. Fifty-eight fertility experts from six continents and twenty-two countries contributed

  17. Nuclear microscopy of sperm cell elemental structure

    Energy Technology Data Exchange (ETDEWEB)

    Bench, G.S.; Balhorn, R.; Friz, A.M.; Freeman, S.P.H.T.

    1994-09-28

    Theories suggest there is a link between protamine concentrations in individual sperm and male fertility. Previously, biochemical analyses have used pooled samples containing millions of sperm to determine protamine concentrations. These methods have not been able to determine what percentage of morphologically normal sperm are biochemically defective and potentially infertile. Nuclear microscopy has been utilized to measure elemental profiles at the single sperm level. By measuring the amount of phosphorus and sulfur, the total DNA and protamine content in individual sperm from fertile bull and mouse semen have been determined. These values agree with results obtained from other biochemical analyses. Nuclear microscopy shows promise for measuring elemental profiles in the chromatin of individual sperm. The technique may be able to resolve theories regarding the importance of protamines to male fertility and identify biochemical defects responsible for certain types of male infertility.

  18. Sperm cells as vectors in the production of transgenic animals

    Energy Technology Data Exchange (ETDEWEB)

    Prince, R.M.

    1993-04-28

    Transgenic animals are used in industry and in biomedical research in order to provide in vivo experimental model systems. Sperm cells have been reported used as vectors in the production of transgenic animals before, however no approach has of yet proven to be successful. Fertilizing eggs with genetically modified sperm would be advantageous in that sperm are readily accessible and stable, and eggs can be fertilized by modified sperm cells in vivo. Recent elucidations regarding the unique manner of DNA packaging in sperm chromatin by protamines has provided us with the insight for developing a method of introducing foreign DNA into sperm which is likely to succeed where others have failed. We have developed a method for mimicking the in vivo system of sperm chromatin toroid subunits in vitro, concentrating these toroids, and fluorescent visualization. Our present work concerns development of a method to successfully deliver DNA across the cell membranes and into the nucleus.

  19. [Sperm as (epigenetic) carrier of obesity?].

    Science.gov (United States)

    Jordan, Bertrand

    2016-03-01

    A demonstration of (somewhat) specific differential RNA content and DNA methylation in sperm from obese individuals opens the possibility of epigenetic transmission to offspring. © 2016 médecine/sciences – Inserm.

  20. Adverse effect of paroxetine on sperm

    National Research Council Canada - National Science Library

    Tanrikut, Cigdem; Feldman, Adam S; Altemus, Margaret; Paduch, Darius A; Schlegel, Peter N

    2010-01-01

    .... Paroxetine administration for 5 weeks. Serum hormone levels, semen analyses, percent sperm DNA fragmentation, and questionnaire assessment of sexual function assessed before, during, and 1 month after drug administration...

  1. Comprehensive analysis of sperm DNA fragmentation by five different assays: TUNEL assay, SCSA, SCD test and alkaline and neutral Comet assay.

    Science.gov (United States)

    Ribas-Maynou, J; García-Peiró, A; Fernández-Encinas, A; Abad, C; Amengual, M J; Prada, E; Navarro, J; Benet, J

    2013-09-01

    Sperm DNA fragmentation (SDF) is becoming an important test to assess male infertility. Several different tests are available, but no consensus has yet been reached as to which tests are most predictive of infertility. Few publications have reported a comprehensive analysis comparing these methods within the same population. The objective of this study was to analyze the differences between the five most common methodologies, to study their correlations and to establish their cut-off values, sensitivity and specificity in predicting male infertility. We found differences in SDF between fertile donors and infertile patients in TUNEL, SCSA, SCD and alkaline Comet assays, but none with the neutral Comet assay. The alkaline COMET assay was the best in predicting male infertility followed by TUNEL, SCD and SCSA, whereas the neutral COMET assay had no predictive power. For our patient population, threshold values for infertility were 20.05% for TUNEL assay, 18.90% for SCSA, 22.75% for the SCD test, 45.37% for alkaline Comet and 34.37% for neutral Comet. This work establishes in a comprehensive study that the all techniques except neutral Comet are useful to distinguish fertile and infertile men. © 2013 American Society of Andrology and European Academy of Andrology.

  2. Damage to Sperm DNA Mediated by Reactive Oxygen Species: Its Impact on Human Reproduction and the Health Trajectory of Offspring.

    Science.gov (United States)

    Gavriliouk, Dan; Aitken, Robert John

    2015-01-01

    Disruptions to the genetic integrity of the mammalian spermatozoon play a major role in determining the subsequent developmental trajectory of the embryo. This chapter examines the causative links that connect DNA damage in human spermatozoa and the appearance of mutations in the progeny responsible for a variety of clinical conditions from autism to cancer. Integral to this discussion is an abundance of evidence indicating that human spermatozoa are vulnerable to free radical attack and the generation of oxidative DNA damage. The resolution of this damage appears to be initiated by the spermatozoa but is driven to completion by the oocyte in a round of DNA repair that follows fertilization. The persistence of unresolved oxidative DNA damage following zygote formation has the potential to create mutations/epimutations in the offspring that may have a profound impact on the health of the progeny. It is proposed that the creation of oxidative stress in the male germ line is a consequence of a wide variety of environmental/lifestyle factors that influence the health and well-being of the offspring as a consequence of mutational change induced by the aberrant repair of oxidative DNA damage in the zygote. Factors such as paternal age, subfertility, smoking, obesity, and exposure to a range of environmental influences, including radio-frequency electromagnetic radiation and xenobiotics, have all been implicated in this process. Identifying the contributors to oxidative stress in the germ line and resolving the mechanisms by which such stressors influence the mutational load carried by the progeny will be an important task for the future. This task is particularly pressing, given the extensive use of assisted reproductive technologies to achieve pregnancies in vitro that would have been prevented in vivo by the complex array of mechanisms that nature has put in place to ensure that only the fittest gametes participate in the generative process.

  3. Sperm preparation for fertilization

    NARCIS (Netherlands)

    Gadella, B.M.

    2014-01-01

    Description This book contains 19 chapters that discuss theoretical and applied andrology for domestic, zoo and wild animals. Topics include semen and its constituents; sperm production and harvest; determinants of sperm morphology; sperm preparation for fertilization; practical aspects of semen

  4. Carrier molecules and extraction of circulating tumor DNA for next generation sequencing in colorectal cancer.

    Science.gov (United States)

    Beránek, Martin; Sirák, Igor; Vošmik, Milan; Petera, Jiří; Drastíková, Monika; Palička, Vladimír

    The aims of the study were: i) to compare circulating tumor DNA (ctDNA) yields obtained by different manual extraction procedures, ii) to evaluate the addition of various carrier molecules into the plasma to improve ctDNA extraction recovery, and iii) to use next generation sequencing (NGS) technology to analyze KRAS, BRAF, and NRAS somatic mutations in ctDNA from patients with metastatic colorectal cancer. Venous blood was obtained from patients who suffered from metastatic colorectal carcinoma. For plasma ctDNA extraction, the following carriers were tested: carrier RNA, polyadenylic acid, glycogen, linear acrylamide, yeast tRNA, salmon sperm DNA, and herring sperm DNA. Each extract was characterized by quantitative real-time PCR and next generation sequencing. The addition of polyadenylic acid had a significant positive effect on the amount of ctDNA eluted. The sequencing data revealed five cases of ctDNA mutated in KRAS and one patient with a BRAF mutation. An agreement of 86% was found between tumor tissues and ctDNA. Testing somatic mutations in ctDNA seems to be a promising tool to monitor dynamically changing genotypes of tumor cells circulating in the body. The optimized process of ctDNA extraction should help to obtain more reliable sequencing data in patients with metastatic colorectal cancer.

  5. Sperm function test

    Directory of Open Access Journals (Sweden)

    Pankaj Talwar

    2015-01-01

    Full Text Available With absolute normal semen analysis parameters it may not be necessary to shift to specialized tests early but in cases with borderline parameters or with history of fertilization failure in past it becomes necessary to do a battery of tests to evaluate different parameters of spermatozoa. Various sperm function tests are proposed and endorsed by different researchers in addition to the routine evaluation of fertility. These tests detect function of a certain part of spermatozoon and give insight on the events in fertilization of the oocyte. The sperms need to get nutrition from the seminal plasma in the form of fructose and citrate (this can be assessed by fructose qualitative and quantitative estimation, citrate estimation. They should be protected from the bad effects of pus cells and reactive oxygen species (ROS (leukocyte detection test, ROS estimation. Their number should be in sufficient in terms of (count, structure normal to be able to fertilize eggs (semen morphology. Sperms should have intact and functioning membrane to survive harsh environment of vagina and uterine fluids (vitality and hypo-osmotic swelling test, should have good mitochondrial function to be able to provide energy (mitochondrial activity index test. They should also have satisfactory acrosome function to be able to burrow a hole in zona pellucida (acrosome intactness test, zona penetration test. Finally, they should have properly packed DNA in the nucleus to be able to transfer the male genes (nuclear chromatic decondensation test to the oocyte during fertilization.

  6. Function of the sperm nuclear matrix.

    Science.gov (United States)

    Shaman, Jeffrey A; Yamauchi, Yasuhiro; Ward, W Steven

    2007-01-01

    Mammalian spermatozoa contain some of the most highly compact chromatin. This is due to the DNA binding proteins, the protamines, which replace most of the histones during spermiogenesis. This chromatin, however, shares some features with somatic cell chromatin. One of these is the organization of DNA into loop domains attached at their bases to a proteinaceous nuclear matrix. Several groups have shown that the sites at which DNA associates with the sperm nuclear matrix contain chromatin structures that are linked with specific functions. Recent data also suggest that the sperm nuclear matrix plays essential roles in the paternal pronucleus of the newly fertilized oocyte, suggesting that the sperm cell provides more information to the new embryo than solely the genetic material it delivers. Here, we will review these data which together give insight into the functional significance and requirements of sperm nuclear structure.

  7. Etiologies of sperm oxidative stress

    Directory of Open Access Journals (Sweden)

    Parvin Sabeti

    2016-04-01

    Full Text Available Sperm is particularly susceptible to reactive oxygen species (ROS during critical phases of spermiogenesis. However, the level of seminal ROS is restricted by seminal antioxidants which have beneficial effects on sperm parameters and developmental potentials. Mitochondria and sperm plasma membrane are two major sites of ROS generation in sperm cells. Besides, leukocytes including polymer phonuclear (PMN leukocytes and macrophages produce broad category of molecules including oxygen free radicals, non-radical species and reactive nitrogen species. Physiological role of ROS increase the intracellular cAMP which then activate protein kinase in male reproductive system. This indicates that spermatozoa need small amounts of ROS to acquire the ability of nuclear maturation regulation and condensation to fertilize the oocyte. There is a long list of intrinsic and extrinsic factors which can induce oxidative stress to interact with lipids, proteins and DNA molecules. As a result, we have lipid peroxidation, DNA fragmentation, axonemal damage, denaturation of the enzymes, over generation of superoxide in the mitochondria, lower antioxidant activity and finally abnormal spermatogenesis. If oxidative stress is considered as one of the main cause of DNA damage in the germ cells, then there should be good reason for antioxidant therapy in these conditions

  8. Role of surfactants in the interaction of dye molecules in natural DNA polymers.

    Science.gov (United States)

    You, H; Spaeth, H; Linhard, V N L; Steckl, A J

    2009-10-06

    Solutions and powders formed from salmon sperm deoxyribonucleic acid (DNA) reacted with the cationic surfactant cetyltrimethylammonium chloride (CTMA-Cl) incorporated fluorescent rhodamine molecules: anionic sulforhodamine 640 (SRh) or cationic/zwitterionic rhodamine 640 perchlorate (RhP). The role of the cationic surfactant in the interaction between rhodamine dye and DNA-surfactant molecules has been investigated in both solution and solid state using optical spectroscopy and electrophoresis. Unexpectedly, the dye molecules did not interact directly with DNA, rather the DNA double helix acted as a template for the interaction between dye molecules and CTMA in the DNA/CTMA complex. The SRh and RhP molecules yield different fluorescence characteristics with increasing DNA/CTMA amount, indicating different configurations between the CTMA ligands.

  9. The structural variety of DNA-DPPC-divalent metal cation aggregates: SAXD and SANS study

    Science.gov (United States)

    Uhríková, D.; Pullmannová, P.; Kučerka, N.; Funari, S. S.; Teixeira, J.; Balgavý, P.

    2009-02-01

    We examine the structure of aggregates formed due to DNA interaction with dipalmitoylphosphatidylcholine (DPPC) in presence of Ca2+ and Zn2+ using small-angle synchrotron X-ray diffraction (SAXD) and neutron scattering (SANS). SAXD shows structural heterogeneity as a function of the cation concentration and temperature: At low cation concentration (˜1 mM), aggregates show two DPPC phases, one with a lateral segregation of DNA and cation, while higher cation concentration improves the DNA packing and the condensed lamellar phase is observed in DNA+DPPC+20mMion2+ aggregates. The SANS detected the dissolution of the condensed lamellar phase into unilamellar DPPC+Zn2+ vesicles due to gel ↦ liquid-crystal phase transition in DNA+DPPC+20mM Zn2+ aggregates with the short fragmented salmon sperm DNA.

  10. Sperm viability - Determination of sperm viability using fluorescence microscopy

    Science.gov (United States)

    To determine the percentage of viable sperm in a semen sample using stains that differentiates viable (live) sperm from nonviable (dead) sperm. Viable sperm are detected by SYBR-14, which stains the sperm nuclei green. Nonviable sperm are detected by propidium iodide (PI), which stains the sperm red...

  11. Diagnostic Accuracies of the TUNEL, SCD, and Comet Based Sperm DNA Fragmentation Assays for Male Infertility: a Meta-analysis Study.

    Science.gov (United States)

    Cui, Zhao-Lei; Zheng, De-Zhu; Liu, Yao-Hua; Chen, Liang-Yuan; Lin, Dong-Hong; Feng-Hua Lan

    2015-01-01

    Recent studies have provided new insights into the diagnostic value of sperm DNA fragmentation (SDF) for male factor sterility. This study aimed to systematically evaluate the diagnostic accuracy of the SDF test for male infertility. Eligible studies were retrieved by searching electronic databases. The quality of the studies was assessed on the basis of quality assessment for studies of diagnostic accuracy (QUADAS) criteria tool. The bivariate metaanalysis model was employed to summarize the diagnostic indices and plot the summary receiver operator characteristic (SROC) curve by using Meta-disc 1.4 software. Influence analysis, meta-regression, and publication bias assay were all conducted through Stata 12.0 software. Our bivariate random effect meta-analysis yielded an AUC (area under curve) value of 0.9211 with a sensitivity (95% confidence interval) of 0.80 (0.78 - 0.82) and specificity of 0.83 (0.80 - 0.86) for the use of the SDF test in differentiating infertile males from normal fertile controls. Moreover, our subgroup analysis suggested that SDF analysis with a single TUNEL test resulted in an AUC value of 0.9506, with a pooled sensitivity of 0.77 (0.74 - 0.80) and specificity of 0.91 (0.87 - 0.94), while SCD and Comet assays displayed a combined sensitivity of 0.77 (0.67 - 0.81) or 0.91 (0.88 - 0.94), and specificity of 0.84 (0.75 - 0.91) or 0.63 (0.54 - 0.70), accompanied by an AUC value of 0.8408 or 0.9473. The SDF assay confers a relatively high diagnostic accuracy for infertility detection, among which the TUNEL based methodology seems to achieve higher accuracy than the SCD and Comet assays.

  12. Male and Couple Fertility Impairment due to HPV-DNA Sperm Infection: Update on Molecular Mechanism and Clinical Impact—Systematic Review

    OpenAIRE

    Salvatore Gizzo; Bruno Ferrari; Marco Noventa; Emanuele Ferrari; Tito Silvio Patrelli; Michele Gangemi; Giovanni Battista Nardelli

    2014-01-01

    Recent evidences identify Human Papillomavirus (HPV) sperm infection as a possible cause of male and couple infertility. It acts through different mechanisms at various steps of human conception and early gestational development. We performed a systematic review to assess the role of HPV semen infection on male and couple infertility. Analysis of available and eligible data does not permit us to fund clear evidences about clinical impact of HPV infection on fertility, although sperm parameter...

  13. Importance of sperm morphology during sperm transport and fertilization in mammals.

    Science.gov (United States)

    García-Vázquez, Francisco A; Gadea, Joaquín; Matás, Carmen; Holt, William V

    2016-01-01

    After natural or artificial insemination, the spermatozoon starts a journey from the site of deposition to the place of fertilization. However, only a small subset of the spermatozoa deposited achieves their goal: to reach and fertilize the egg. Factors involved in controlling sperm transport and fertilization include the female reproductive tract environment, cell-cell interactions, gene expression, and phenotypic sperm traits. Some of the significant determinants of fertilization are known (i.e., motility or DNA status), but many sperm traits are still indecipherable. One example is the influence of sperm dimensions and shape upon transport within the female genital tract towards the oocyte. Biophysical associations between sperm size and motility may influence the progression of spermatozoa through the female reproductive tract, but uncertainties remain concerning how sperm morphology influences the fertilization process, and whether only the sperm dimensions per se are involved. Moreover, such explanations do not allow the possibility that the female tract is capable of distinguishing fertile spermatozoa on the basis of their morphology, as seems to be the case with biochemical, molecular, and genetic properties. This review focuses on the influence of sperm size and shape in evolution and their putative role in sperm transport and selection within the uterus and the ability to fertilize the oocyte.

  14. Cryptic choice of conspecific sperm controlled by the impact of ovarian fluid on sperm swimming behavior.

    Science.gov (United States)

    Yeates, Sarah E; Diamond, Sian E; Einum, Sigurd; Emerson, Brent C; Holt, William V; Gage, Matthew J G

    2013-12-01

    Despite evidence that variation in male-female reproductive compatibility exists in many fertilization systems, identifying mechanisms of cryptic female choice at the gamete level has been a challenge. Here, under risks of genetic incompatibility through hybridization, we show how salmon and trout eggs promote fertilization by conspecific sperm. Using in vitro fertilization experiments that replicate the gametic microenvironment, we find complete interfertility between both species. However, if either species' ova were presented with equivalent numbers of both sperm types, conspecific sperm gained fertilization precedence. Surprisingly, the species' identity of the eggs did not explain this cryptic female choice, which instead was primarily controlled by conspecific ovarian fluid, a semiviscous, protein-rich solution that bathes the eggs and is released at spawning. Video analyses revealed that ovarian fluid doubled sperm motile life span and straightened swimming trajectory, behaviors allowing chemoattraction up a concentration gradient. To confirm chemoattraction, cell migration tests through membranes containing pores that approximated to the egg micropyle showed that conspecific ovarian fluid attracted many more spermatozoa through the membrane, compared with heterospecific fluid or water. These combined findings together identify how cryptic female choice can evolve at the gamete level and promote reproductive isolation, mediated by a specific chemoattractive influence of ovarian fluid on sperm swimming behavior. © 2013 The Authors. Evolution published by Wiley Periodicals, Inc. on behalf of The Society for the Study of Evolution.

  15. Solea senegalensis sperm cryopreservation: New insights on sperm quality.

    Directory of Open Access Journals (Sweden)

    Marta F Riesco

    Full Text Available Cryopreservation of Senegalese sole sperm can represent an alternative to overcome some reproductive problems of this species. However, it is important to guarantee the safe use of cryopreserved sperm by selecting an appropriate protocol according to a high demand quality need to be ensured. It has been demonstrated that traditional assays such as motility and viability do not provide enough information to identify specific damage caused by cryopreservation process (freezing and thawing. Specific tests, including lipid peroxidation and DNA damage, should be performed. In the present study, motility and lipid peroxidation were performed as specific tests allowing us to discard cryopreservation conditions such as methanol as internal cryoprotectant and bovine serum albumin as external cryoprotectant. In addition, a caspase 3/7 detection by flow cytometry was performed to analyze apoptosis activity in the best selected conditions. Moreover, new highly sensitive tests based on transcript number detection have recently been described in fish sperm cryopreservation. For this reason, a transcript level detection assay was performed on certain oxidative and chaperone genes related to fertilization ability and embryo development (hsp70, hsp90BB, hsp90AA, gpx to select the best cryopreservation conditions. DMSO+ egg yolk proved to be the best cryoprotectant combination in terms of transcript level. This study describes an optimized cryopreservation protocol for Solea senegalensis sperm demonstrating for the first time that transcript degradation is the most sensitive predictor of cell status in this species after cryopreservation.

  16. The sperm epigenome: implications for the embryo.

    Science.gov (United States)

    Gannon, John R; Emery, Benjamin R; Jenkins, Timothy G; Carrell, Douglas T

    2014-01-01

    Recent advances, including the human genome project and numerous studies of cancer and other diseases, have shown that the genetic code is not simply limited to the sequence of the four bases of DNA but also includes epigenetic programming, heritable changes that affect gene expression [Riggs A, Martinssen R, Russo V (2007) Introduction. In: Riggs A, Martinssen R, Russo V (eds) Epigenetics mechanisms of gene regulation. Cold Spring Harbor Press, New York]. The science of epigenetics is important in understanding many diseases and biological processes, including in identifying the causes of disease and better understanding the mechanisms by which the environment can affect gene expression [Carrell Fertil Steril 97 (2):267-274, 2012]. This chapter will focus on the epigenome of sperm and particularly highlight the potential role of the sperm epigenome in embryogenesis.The sperm epigenome is unique and highly specialized because of the unique nature and function of sperm and because of the diverse requirements for successful fertilization. Due to the need for motility, sperm chromatin must be compacted and highly organized. During spermiogenesis the chromatin is packaged tightly into the sperm head by the replacement of most histones with protamines. This allows for protection of the DNA from the hostile environment in the female reproductive tract. Remaining histones can have chemical modifications to the tails of the protein that either facilitate or repress gene transcription. Sperm, like embryonic stem cells, have a unique pattern of histone modifications that includes both activating and silencing marks in the promoters of genes associated with development. These bivalent marks, along with DNA hypomethylation, comprise a unique state in which the key genes are "poised" for possible activation in embryogenesis. Sperm epigenetic abnormalities have been linked with multiple diseases including male factor infertility and poor embryogenesis.

  17. Characterization of the interaction of a mono-6-thio-β-cyclodextrin-capped CdTe quantum dots-methylene blue/methylene green system with herring sperm DNA using a spectroscopic approach.

    Science.gov (United States)

    Shen, Yizhong; Liu, Shaopu; Wang, Lei; Yin, Pengfei; He, Youqiu

    2014-11-01

    Novel, water-soluble CdTe quantum dots (QDs) capped with β-cyclodextrin (β-CD) and ~ 4.0 nm in diameter were synthesized in aqueous solution, and characterized using transmission electron microscopy (TEM). A fluorescence-sensing system based on the photoinduced electron transfer (PET) of (mono-6-thio-β-CD)-CdTe QDs was then designed to measure the interaction of phenothiazine dyes [methylene blue (MB) and methylene green (MG)] with herring sperm DNA (hsDNA). This fluorescence-sensing system was based on a fluorescence "OFF-ON" mode. First, MB/MG adsorbed on the surface of (mono-6-thio-β-CD)-CdTe QDs effectively quenches the fluorescence of (mono-6-thio-β-CD)-CdTe QDs through PET. Then, addition of hsDNA restores the fluorescence intensity of (mono-6-thio-β-CD)-CdTe QDs, because hsDNA can bind with MB/MG and remove it from the as-prepared (mono-6-thio-β-CD)-CdTe QDs. In addition, detailed reaction mechanisms of the (mono-6-thio-β-CD)-CdTe QDs-MB/MG-hsDNA solution system were studied using optical methods, by comparison with the TGA-CdTe QDs-MB/MG-hsDNA solution system. Copyright © 2014 John Wiley & Sons, Ltd.

  18. The effect of human papillomavirus infection on sperm cell motility.

    Science.gov (United States)

    Lai, Y M; Lee, J F; Huang, H Y; Soong, Y K; Yang, F P; Pao, C C

    1997-06-01

    To investigate the presence of human papillomavirus (HPV) in human sperm cells and to evaluate potential effects of HPV on the sperm functions. A descriptive clinical study. Specimens of semen were collected from 24 randomly selected patients who attended the fertility clinics at Chang Gung Memorial Hospital. The presence of HPV DNA and RNA were examined by polymerase chain reaction. Semen quality and sperm cell function were analyzed by computer-aided autoanalyzer. Human papillomavirus type 16 DNA and RNA were found in 6 (25%) and 2 (8%) of the sperm cells specimens, respectively. Human papillomavirus type 18 DNA and RNA were present in 11 (46%) and 5 (21%) of the same sperm cells specimens, respectively. Incidence of asthenozoospermia among patients infected with either HPV was significantly higher than in those without HPV in their sperm cells (75% versus 8%). Although performance of curvilinear velocity, straight-line velocity, and mean amplitude of lateral head displacement was significantly lower in HPV-infected specimens, the differences of linearity, beat cross frequency, and straightness were not statistically significant. These results suggest that human papillomavirus can be found in human sperm cells and that certain HPV-specific genes are actively transcribed. Sperm motility parameters seem to be affected by the presence of HPV in the sperm cells, and also the incidence of asthenozoospermia may be associated with HPV infection.

  19. Semen analysis and sperm function testing.

    Science.gov (United States)

    Franken, Daniel R; Oehninger, Sergio

    2012-01-01

    Despite controversy regarding the clinical value of semen analysis, male fertility investigation still relies on a standardized analysis of the semen parameters. This is especially true for infertility clinics in both developing and developed countries. Other optional tests or sophisticated technologies have not been widely applied. The current review addresses important changes in the analysis of semen as described in the new World Health Organization (WHO) manual for semen analysis. The most important change in the manual is the use of evidence-based publications as references to determine cutoff values for normality. Apart from the above mentioned changes, the initial evaluation and handling methods remain, in most instances, the same as in previous editions. Furthermore, the review evaluates the importance of quality control in andrology with emphasis on the evaluation of sperm morphology. WHO sperm morphology training programmes for Sub-Saharan countries were initiated at Tygerberg Hospital in 1995. The external quality control programme has ensured that the majority of participants have maintained their morphological reading skills acquired during initial training. This review reports on current sperm functional tests, such as the induced acrosome reaction, and sperm-zona pellucida binding assays, as well as the impact of sperm quality in terms of DNA integrity, and the relationship of sperm function tests to sperm morphology.

  20. Effect of semen preparation technique and its incubation on sperm quality in the Moroccan population.

    Science.gov (United States)

    Aboulmaouahib, S; Madkour, A; Kaarouch, I; Saadani, B; Sefrioui, O; Louanjli, N; Copin, H; Cadi, R; Benkhalifa, M

    2017-08-01

    In in vitro fertilisation (IVF), sperm preparation as critical part and influencing the sperm quality is especially dependent on the chosen technique itself and incubation parameters including temperature and CO2. In this study, we compared firstly density-gradient centrifugation technique (DGC) to the adapted DGC using the sperm pellet of 80% fraction (DGC/80P) in order to improve the sperm yield. Secondly, this study led to evaluate different sperm incubation conditions based on temperature effect (room temperature (RT = 23°C) versus 35°C) and in the other hand, with or without 5% CO2 during 24 hrs. Based on evaluating sperm conventional parameters and the DNA damage using TUNEL assay, our result showed that DGC/80P increased sperm quality compared to DGC with 25% of improvement. For temperature incubation effect after 24 hrs, 35°C increased the DNA damage and decreased the sperm quality while RT could improve sperm motility by 38%. Moreover, the sperm incubation with 5% CO2 after 24 hrs realised a negative impact on sperm parameters and its DNA damage. Indeed, for current IVF practice, a good sperm quality can be maintained for several hours at room temperature, while the sperm preparation is processed using the DGC/80P without CO2. © 2016 Blackwell Verlag GmbH.

  1. Sperm competition in birds.

    Science.gov (United States)

    Birkhead, T R

    1998-05-01

    Sperm competition in birds occurs when a female is inseminated by more than one male during a single breeding cycle. Despite most birds being socially monogamous, sperm competition is widespread and results in frequent extra-pair paternity. Sperm competition is a fundamental part of sexual selection since it results in differential reproductive success among males. Male adaptations to sperm competition include relatively large testes, large sperm stores and long spermatozoa, mate guarding and frequent pair copulations. Females show no obvious morphological adaptations to sperm competition but, by controlling whether copulations are successful, they probably determine its frequency and extent. Despite this, the evolutionary benefits females acquire from extra-pair fertilizations are poorly understood. Experiments in which females are inseminated with equal numbers of spermatozoa from two males usually show last male sperm precedence. Understanding the mechanism of sperm competition requires understanding of why the last male to inseminate a female fertilizes a disproportionate number of eggs. The data from sperm competition studies on the domestic fowl, turkeys and zebra finches are consistent only with a passive sperm loss model of sperm competition. The mechanism is as follows: after insemination, spermatozoa enter the sperm storage tubules located in the oviduct, from which they are lost at a constant rate over days or weeks. All else being equal, the interval between two inseminations determines the probability of fertilization: the second of two inseminations fertilizes most eggs simply because, by the time fertilization occurs, fewer of these spermatozoa have been lost. Other factors also affect the outcome of sperm competition: the timing of insemination relative to oviposition, the differential fertilizing capacity of males and differences in the numbers of spermatozoa inseminated; as a consequence, last male sperm precedence is not automatic. On the basis

  2. Diagnostic accuracy of sperm chromatin dispersion test to evaluate sperm deoxyribonucleic acid damage in men with unexplained infertility.

    Science.gov (United States)

    Feijó, Cinthia M; Esteves, Sandro C

    2014-01-01

    To compare the sperm chromatin dispersion (SCD) test and the terminal uridine nick-end labeling (TUNEL) assay for assessment of sperm DNA damage. Prospective comparative experimental study. Andrology laboratory. Twenty subfertile men with unexplained infertility. Sperm DNA damage was determined in the same semen samples using the TUNEL assay with fluorescence microscopy and the SCD test with bright-field microscopy. Correlation coefficient and receiver operating characteristic analysis outcomes. The TUNEL assay was used as the reference standard to identify optimal cutoff points for assessing DNA damage by SCD. The SCD test detected a significantly higher proportion of sperm with damaged DNA (20.6% ± 14.0%) than the TUNEL assay (11.5% ± 7.3%). Spearman's rank correlation showed that the methods were not comparable (r = 0.29). Receiver operating characteristic analysis revealed that 15% was the best SCD cutoff point to classify patients within the same levels of DNA fragmentation, normal or abnormal, as determined by the TUNEL assay, with an accuracy of 69%. The SCD test is more sensitive than the TUNEL assay for the assessment of DNA damage in men with unexplained infertility. Although the methods are poorly correlated, SCD may discriminate men with normal and abnormal sperm DNA damage with moderate accuracy when compared with TUNEL. It is important to distinguish between the methods because they differently evaluate sperm DNA damage. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  3. Ovarian fluid impacts flagellar beating and biomechanical metrics of sperm between alternative reproductive tactics

    Czech Academy of Sciences Publication Activity Database

    Butts, I. A. E.; Prokopchuk, Galina; Kašpar, V.; Cosson, J.; Pitcher, T. E.

    2017-01-01

    Roč. 220, č. 12 (2017), s. 2210-2217 ISSN 0022-0949 Institutional support: RVO:60077344 Keywords : competition * salmon * Oncorhynchus tshawytscha * spawning * reproductive strategy * sperm competition * cryptic female choice Subject RIV: EG - Zoology Impact factor: 3.320, year: 2016

  4. Effect of saffron on rat sperm chromatin integrity.

    Science.gov (United States)

    Mardani, Mohammad; Vaez, Ahmad; Razavi, Shahnaz

    2014-05-01

    Currently, relation between reactive oxygen species (ROS) ROS concentration and semen quality was indicated. Saffron has traditionally been not only considered as a food additive but also as a medicinal herb, which has a good antioxidant properties. The aim of this study was to evaluate the protection potency of saffron and vitamin E on sperm chromatin integrity. Thirty adult male Wistar rats divided equally into saffron (100 mg/kg), vitamin E (100 mg/kg) and control (0.5cc distilled water /day) groups. After 60 days, cauda epididymis dissected and sperm cells were used for analysis of sperm chromatin packaging by chromomycin A3 (CMA3) staining, and sperm chromatin susceptibility to acid denaturation by acridine orange (AO) staining. The mean percentage of CMA3 positive sperm was significantly decreased in saffron and vitamin E groups relative to control group (psaffron and vitamin E groups as compared with control group (psaffron can protect sperm against DNA damage and chromatin anomalies.

  5. Correlation between Different Patterns of Hypo-Osmotic Swelling and Sperm Functional Tests

    Directory of Open Access Journals (Sweden)

    Farzaneh Bassiri

    2013-01-01

    Full Text Available Background: Sperm membrane integrity is not only important as a barrier between intraandextra-cellular spaces, but also it can be considered as a sign of DNA integrity. Hypoosmoticswelling test reflects membrane integrity and has been used to evaluate spermquality. Intracytoplasmic sperm injection (ICSI in adjunct with hypo-osmotic swellingtest (HOST has been used for treatment of males with asthenozoospermia. Therefore,this study aims to evaluate correlation of different pattern of HOST with sperm parameters,protamine deficiency and apoptosis.Materials and Methods: In this case-control study, sixteen semen samples were randomlycollected from infertile normozospermic men. Semen samples were divided intotwo portions as follows: one portion was assessed for sperm parameters according toWorldHealth Organization (WHO-2010, while the other portion, after applying HOSTprocedure, was used for assessment of sperm morphology, protamine deficiency and lateor early apoptosis. Statistical analysis was carried out using the Statistical Package forthe Social Studies (SPSS 11.5.Results: Our results showed that, the lowest odds ratio (OR of abnormal sperm headmorphology and abnormal acrosome was in d-sperm as compared to a-pattern or nonviablespermatozoa (p=0.00, p=0.01. In addition, a significant correlation was observedbetween d-sperm with sperm concentration and percentage of DNA damage (p=0.03and p=0.04, respectively. A significant correlation was observed between percentageof sperm motility and DNA fragmentation (r=-0.56; p=0.01. Furthermore, significantcorrelations were observed between percentages of early apoptotic sperm with protaminedeficiency and sperm concentration (p=0.009 and p=0.01, respectively.Conclusion: Significant correlations exist between d-pattern and sperm DNA integrity.Semen samples with low sperm concentration have low percentage of d-sperm which aremature and intact sperms.

  6. Desvio da proporção de sexo e da integridade do DNA dos espermatozóides bovinos centrifugados em gradientes de densidade contínuos Alteration of sex ratio and DNA integrity of bovine sperm centrifuged in continuous density gradients

    Directory of Open Access Journals (Sweden)

    Alberto Lopes Gusmão

    2010-03-01

    Full Text Available O objetivo, neste trabalho, foi verificar o desvio da proporção de sexo e a presença de fragmentação do DNA, pela técnica de TUNEL (“In situ terminal deoxinucleotidyl transferase mediated dUTP nick end labeling assay”, em espermatozoides bovinos centrifugados em gradientes de densidade de Percoll ou OptiPrep durante a separação espermática. Doses de sêmen de touros foram descongeladas, e cerca de 40 milhões de espermatozoides foram depositados sobre cada gradiente de densidade compostos por Percoll ou OptiPrep com três camadas entre 1.110g/mL e 1.123g/mL, em tubos de 15mL, em que permaneceram por 24h a 4°C antes da deposição dos espermatozoides. Os tubos foram centrifugados a 500xg por 15min a 22°C. Os sobrenadantes foram aspirados, e os sedimentos, recuperados para verificação da fragmentação do DNA pela técnica de TUNEL. Obteve-se um desvio dos embriões produzidos in vitro para fêmeas no gradiente de Percoll (62% de fêmeas, em relação aos grupos OptiPrep e Controle (47,1 e 48,7% de fêmeas, respectivamente. Não foi detectada fragmentação do DNA dos espermatozoides nas amostras centrifugadas, tanto no gradiente de Percoll quanto de OptiPrep. Dessa forma, foi possível realizar a sexagem espermática, com uma maior porcentagem de espermatozoides X do que o grupo controle, por meio de metodologia mais simples e sem provocar danos ao DNA dos espermatozoides.The objective of the present study was to verify the sex ratio and presence of DNA fragmentation by TUNEL technique (In situ terminal deoxinucleotidyl transferase mediated dUTP nick end labeling assay in bovine spermatozoa centrifuged in density gradients of Percoll or OptiPrep during the sperm separation. Approximately 40 million of frozen/thawed bovine spermatozoa were deposited on each density gradient composed of Percoll or OptiPrep with three layers ranging from 1.110g/mL to 1.123g/mL in polystyrene tubes of 15mL. The tubes were kept at 4°C for 24h before

  7. Advanced sperm selection techniques for assisted reproduction.

    Science.gov (United States)

    McDowell, Simon; Kroon, Ben; Ford, Emily; Hook, Ysanne; Glujovsky, Demián; Yazdani, Anusch

    2014-10-28

    Assisted reproductive technologies (ART) such as in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) bring together gametes outside of the body to enhance the probability of fertilisation and pregnancy. Advanced sperm selection techniques are increasingly being employed in ART, most commonly in cycles utilising ICSI. Advanced sperm selection techniques are thought to improve the chance that structurally intact and mature sperm with high DNA integrity are selected for fertilisation. Advanced sperm selection strategies include selection according to surface charge; sperm apoptosis; sperm birefringence; ability to bind to hyaluronic acid; and sperm morphology under ultra-high magnification. These techniques theoretically improve ART outcomes. To evaluate the impact of advanced sperm selection techniques on ART outcomes. Systematic search of electronic databases (Cochrane Menstrual Disorders and Subfertility Group Specialised Register, the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE, PsycINFO, Cumulative Index to Nursing and Allied Health Literature (CINAHL), Latin American and Caribbean Health Science Information Database (LILACS)), trials registers (ClinicalTrials.gov, Current Controlled Trials, World Health Organization International Clinical Trials Registry Platform), conference abstracts (Web of Knowledge) and grey literature (OpenGrey) for relevant randomised controlled trials. We handsearched the reference lists of included studies and similar reviews. The search was conducted in May 2014. We included randomised controlled trials (RCTs) comparing an advanced sperm selection technique versus standard IVF or ICSI or versus another advanced sperm selection technique. We excluded studies of sperm selection using ultra-high magnification (intracytoplasmic morphologically selected sperm injection, or IMSI), as they are the subject of a separate Cochrane review. Quasi-randomised and pseudo-randomised trials were

  8. An immunological approach of sperm sexing and different methods for identification of X- and Y-chromosome bearing sperm

    Directory of Open Access Journals (Sweden)

    Shiv Kumar Yadav

    2017-05-01

    Full Text Available Separation of X- and Y-chromosome bearing sperm has been practiced for selection of desired sex of offspring to increase the profit in livestock industries. At present, fluorescence-activated cell sorter is the only successful method for separation of X- and Y-chromosome bearing sperm. This technology is based on the differences in DNA content between these two types of sperm and has been commercialized for bovine sperm. However, this technology still has problems in terms of high economic cost, sperm damage, and lower pregnancy rates compared to unsorted semen. Therefore, an inexpensive, convenient, and non-invasive approach for sperm sexing would be of benefit to agricultural sector. Within this perspective, immunological sperm sexing method is one of the attractive choices to separate X- and Y-chromosome bearing sperm. This article reviews the current knowledge about immunological approaches, viz., H-Y antigen, sex-specific antigens, and differentially expressed proteins for sperm sexing. Moreover, this review also highlighted the different methods for identification of X- and Y-sperm.

  9. Sperm antigens in fertilization.

    Science.gov (United States)

    Saling, P M

    1990-02-01

    A review of sperm antigens involved in fertilization includes a description of sperm differentiation, seminal fluid components that coat sperm, sperm antigens involved in binding to the zona pellucida (ZP), antigens involved in the acrosome reaction, in zona pellucida penetration, and those active in fusion with the ova membrane. Sperm antigens are located in certain domains of the cell, and they are altered during capacitation and passage through the female tract. Caltrin and acrosome-stabilizing factor are applied by seminal fluid. At least 2 antigens have been studied that occur in sterile women, although one cross reacts with milk proteins. Some antigens active in ZP binding are trypsin, proacrosin, acrosin, PH-20 from guinea pigs, and rabbit sperm autoantigen I. Antigens involved in the acrosome reaction, such as M42, are likely to cross react with other body proteins that also entail exocytosis. A mouse antigen involved in ZP penetration, MS 207 is well characterized. PH-30 from guinea pigs and M29 from mouse participate in sperm-egg membrane fusion, as does fertilization antigen I from human and mouse sperm which is know to cause infertility. Oddly, patients' sera react with polymers but not monomers of this antigen. Studies with antisperm antibodies suggest that it will not be necessary to agglutinate all sperm to block fertility, only to inhibit a single sperm epitope and function. It will probably be feasible to inhibit multiple successive events, and possibly to induce temporary immunity.

  10. Sperm competition and the evolution of sperm design in mammals

    National Research Council Canada - National Science Library

    Tourmente, Maximiliano; Gomendio, Montserrat; Roldan, Eduardo R S

    2011-01-01

    .... The hypothesis that, when ejaculates compete with rival males, an increase in sperm size would make sperm more competitive because it would increase sperm swimming speed, has generated contradictory...

  11. Do "sperm trading" simultaneous hermaphrodites always trade sperm?

    OpenAIRE

    Nils Anthes; Michiels, Nico K.

    2005-01-01

    Sperm trading can be a mechanism to solve the conflict over sex roles in hermaphrodites with copulation, sperm competition, and sperm digestion. If present, sperm donation depends on sperm receipt, resulting in conditional reciprocal inseminations. Conditional reciprocity can involve three traded commodities: penis intromissions on a yes-or-no basis, intromission durations (indicating ejaculate size), or sperm transfer. If present, animals that refuse to donate (cheaters) should be deserted b...

  12. Evaluation of male germ cell toxicity in rats: correlation between sperm head morphology and sperm comet assay.

    Science.gov (United States)

    Trivedi, P P; Kushwaha, S; Tripathi, D N; Jena, G B

    2010-12-21

    The present study was aimed to investigate the germ cell toxicity of doxorubicin and find out the possible correlation between sperm head morphological evaluation and sperm comet assay, which are used to assess male germ cell toxicity. The correlation between these two assays was validated using a potent germ cell toxicant, doxorubicin, in male Sprague-Dawley rats. Doxorubicin was administered intra-peritionally at the doses of 1.25, 2.5 and 5mg/kg weekly once for a period of 5 weeks and all the animals were sacrificed after 1 week of receiving the last dose. The germ cell toxicity of doxorubicin was assessed using oxidative stress parameters, sperm head morphology, sperm comet assay, halo assay and histology in testes as the end point of evaluation. A significant increase in the % abnormality in sperm head was found in the animals treated with 2.5 and 5mg/kg/week doxorubicin. Doxorubicin treatment significantly increased the DNA damage of sperm in a dose-dependent manner as observed by sperm comet assay parameters. A strong positive correlation was observed between the sperm head morphological evaluation and the sperm comet assay. Therefore, it can be concluded that the damage in genetic material of sperm may result into abnormalities in the sperm head morphology. The sperm head morphological evaluation is considered to be essential for the assessment of male germ cell toxicity by several regulatory bodies like the Organization for Economic Cooperation and Development (OECD) and the International Conference on Harmonization (ICH). However, acceptance of the sperm comet assay by regulatory authorities as a standard genotoxicity test for assessing male germ cell toxicity still requires further validation of the assay. 2010 Elsevier B.V. All rights reserved.

  13. Sperm preparation for fertilization

    OpenAIRE

    Gadella, B.M.

    2014-01-01

    Description This book contains 19 chapters that discuss theoretical and applied andrology for domestic, zoo and wild animals. Topics include semen and its constituents; sperm production and harvest; determinants of sperm morphology; sperm preparation for fertilization; practical aspects of semen cryopreservation; evaluation of semen in the andrology laboratory; genetic aspects of male reproduction; emerging techniques and future development of semen evaluation and handling and applied androlo...

  14. Maternal inheritance of deltamethrin resistance in the salmon louse Lepeophtheirus salmonis (Krøyer is associated with unique mtDNA haplotypes.

    Directory of Open Access Journals (Sweden)

    Greta Carmona-Antoñanzas

    Full Text Available Parasitic infections by the salmon louse, Lepeophtheirus salmonis (Krøyer, cause huge economic damage in salmon farming in the northern hemisphere, with combined treatment costs and production losses in 2014 having been estimated at US$ 350 million for Norway (annual production 1.25 million tonnes. The control of L. salmonis relies significantly on medicinal treatments, supplemented by non-pharmacological approaches. However, efficacy losses have been reported for several delousing agents, including the pyrethroid deltamethrin. The aim of the present study was to analyse the genetic basis of deltamethrin resistance in L. salmonis. Deltamethrin median effective concentrations (EC50 were 0.28 μg L-1 in the drug susceptible L. salmonis strain IoA-00 and 40.1 μg L-1 in the pyrethroid resistant strain IoA-02. IoA-00 and IoA-02 were crossed to produce families spanning one parental and three filial generations (P0, F1-F3. In three families derived from P0 crosses between an IoA-00 sire and an IoA-02 dam, 98.8% of F2 parasites (n = 173 were resistant, i.e. remained unaffected after exposure to 2.0 μg L-1 deltamethrin. F3 parasites from these crosses showed a deltamethrin EC50 of 9.66 μg L-1. In two families of the inverse orientation at P0 (IoA-02 sire x IoA-00 dam, 16.7% of F2 parasites were resistant (n = 84, while the deltamethrin EC50 in F3 animals was 0.26 μg L-1. The results revealed a predominantly maternal inheritance of deltamethrin resistance. The 15,947-nt mitochondrial genome was sequenced and compared among six unrelated L. salmonis strains and parasites sampled from wild salmon in 2010. IoA-02 and three further deltamethrin resistant strains, established from isolates originating from different regions of Scotland, showed almost identical mitochondrial haplotypes. In contrast, the mitochondrial genome was variable among susceptible strains and L. salmonis from wild hosts. Deltamethrin caused toxicity and depletion of whole body ATP

  15. Sperm preparation: state-of-the-art--physiological aspects and application of advanced sperm preparation methods.

    Science.gov (United States)

    Henkel, Ralf

    2012-03-01

    For assisted reproduction technologies (ART), numerous techniques were developed to isolate spermatozoa capable of fertilizing oocytes. While early methodologies only focused on isolating viable, motile spermatozoa, with progress of ART, particularly intracytoplasmic sperm injection (ICSI), it became clear that these parameters are insufficient for the identification of the most suitable spermatozoon for fertilization. Conventional sperm preparation techniques, namely, swim-up, density gradient centrifugation and glass wool filtration, are not efficient enough to produce sperm populations free of DNA damage, because these techniques are not physiological and not modeled on the stringent sperm selection processes taking place in the female genital tract. These processes only allow one male germ cell out of tens of millions to fuse with the oocyte. Sites of sperm selection in the female genital tract are the cervix, uterus, uterotubal junction, oviduct, cumulus oophorus and the zona pellucida. Newer strategies of sperm preparation are founded on: (i) morphological assessment by means of 'motile sperm organelle morphological examination (MSOME)'; (ii) electrical charge; and (iii) molecular binding characteristics of the sperm cell. Whereas separation methods based on electrical charge take advantage of the sperm's adherence to a test tube surface or separate in an electrophoresis, molecular binding techniques use Annexin V or hyaluronic acid (HA) as substrates. Techniques in this category are magnet-activated cell sorting, Annexin V-activated glass wool filtration, flow cytometry and picked spermatozoa for ICSI (PICSI) from HA-coated dishes and HA-containing media. Future developments may include Raman microspectrometry, confocal light absorption and scattering spectroscopic microscopy and polarization microscopy.

  16. Atlantic Salmon Smolt Monitoring

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Annual data are collected as part of smolt trapping operations using fish trapping methods. Traps collect emigrating salmon smolts to identify cohort...

  17. Atlantic Salmon Telemetry Monitoring

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Annual telemetry data are collected as part of specific projects (assessments within watersheds) or as opportunistic efforts to characterize Atlantic salmon smolt...

  18. Calcitonin Salmon Nasal Spray

    Science.gov (United States)

    ... bottle and turn to tighten. Then take the plastic cover off of the top of the spray unit. ... room temperature in an upright position. Replace the plastic cover to keep the nozzle clean. Opened calcitonin salmon ...

  19. Zinc protects sperm from being damaged by reactive oxygen species in assisted reproduction techniques.

    Science.gov (United States)

    Wu, Jinxiang; Wu, Shiqiang; Xie, Yuanzhi; Wang, Zhengyao; Wu, Ruiyun; Cai, Junfeng; Luo, Xiangmin; Huang, Suzhen; You, Liuxia

    2015-04-01

    The aim of this study was to explore the effect of zinc on hydrogen peroxide-induced sperm damage in assisted reproduction techniques. First, sperms were selected from semen samples of 20 healthy men prepared by density gradient centrifugation. Selected sperm were treated with either 0.001% H(2)O(2), 12.5 nM ZnCL(2), 0.001% H(2)O(2) + 12.5 nM ZnCL(2) or 0.9% NaCl(2) (control). After this treatment, the motility, viability, membrane integrity and DNA fragmentation of sperms in each group were analysed by Goodline sperm detection system, optical microscopy and sperm DNA fragmentation assay. Poorer motility, vitality, membrane integrity and more DNA damage were found in sperms treated by H(2)O(2), compared with control. When sperms were treated with both H(2)O(2) and zinc, however, all indicators were improved compared with H(2)O(2) alone. There was a close association between oxidative stimulation and sperm injury; zinc could inhibit hydrogen peroxide-induced damage of sperm in assisted reproductive technology. However, the presence of zinc in culture medium can decrease the sperm quality without addition of peroxide. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  20. Proteomic analysis of chinook salmon (Oncorhynchus tshawytscha ovarian fluid.

    Directory of Open Access Journals (Sweden)

    Sheri L Johnson

    Full Text Available The ovarian, or coelomic, fluid that is released with the egg mass of many fishes is increasingly found to play an important role in several biological processes crucial for reproductive success. These include maintenance of oocyte fertility and developmental competence, prolonging of sperm motility, and enhancing sperm swimming speed. Here we examined if and how the proteome of chinook salmon (Oncorhynchus tshawytscha ovarian fluid varied among females and then sought to examine the composition of this fluid. Ovarian fluid in chinook salmon was analyzed using 1D SDS PAGE and LC-MS/MS tryptic digest screened against Mascot and Sequest databases. We found marked differences in the number and concentrations of proteins in salmon ovarian fluid across different females. A total of 174 proteins were identified in ovarian fluid, 47 of which were represented by six or more peptides, belonging to one of six Gene Ontology pathways. The response to chemical stimulus and response to hypoxia pathways were best represented, accounting for 26 of the 174 proteins. The current data set provides a resource that furthers our understanding of those factors that influence successful egg production and fertilisation in salmonids and other species.

  1. Melatonin ameliorates the adverse effects of leptin on sperm

    Directory of Open Access Journals (Sweden)

    Fayez A Almabhouh

    2017-01-01

    Full Text Available This study examined the effects of melatonin on leptin-induced changes in sperm parameters in adult rats. Five groups of Sprague-Dawley rats were treated with either leptin or leptin and melatonin or melatonin for 6 weeks. Leptin was given daily via the intraperitoneal route (60 μg kg−1 body weight and melatonin was given in drinking water (10 mg kg−1 or 20 mg kg−1 body weight per day. Upon completion, sperm count, sperm morphology, 8-hydroxy-2-deoxyguanosine, Comet assay, TUNEL assay, gene expression profiles of antioxidant enzymes, respiratory chain reaction enzymes, DNA damage, and apoptosis genes were estimated. Data were analyzed using ANOVA. Sperm count was significantly lower whereas the fraction of sperm with abnormal morphology, the level of 8-hydroxy-2-deoxyguanosine, and sperm DNA fragmentation were significantly higher in rats treated with leptin only. Microarray analysis revealed significant upregulation of apoptosis-inducing factor, histone acetyl transferase, respiratory chain reaction enzyme, cell necrosis and DNA repair genes, and downregulation of antioxidant enzyme genes in leptin-treated rats. Real-time polymerase chain reaction showed significant decreases in glutathione peroxidase 1 expression with increases in the expression of apoptosis-inducing factor and histone acetyl transferase in leptin-treated rats. There was no change in the gene expression of caspase-3 (CASP-3. In conclusion, the adverse effects of leptin on sperm can be prevented by concurrent melatonin administration.

  2. Microscale integrated sperm sorter.

    Science.gov (United States)

    Chung, Yaokuang; Zhu, Xiaoyue; Gu, Wei; Smith, Gary D; Takayama, Shuichi

    2006-01-01

    This chapter describes the design and fabrication of a passively driven microfluidic sperm sorter using soft lithographic microfabrication techniques. This self-contained device can separate motile sperm from nonmotile sperm and other cellular debris. The sorting system is small (coin sized) and structurally simple. It comprises two inlets; two outlets; a sorting channel; and arrays of horizontally oriented reservoirs that function as passively driven, constant-flow-rate pumps. Sperm with higher motility are sorted out from the rest of the semen samples based on their ability to swim through interfaces between adjacent laminar streams into separate streamlines, whereas the nonmotile sperm and debris remain in their initial streamlines. The device, which we call a microscale integrated sperm sorter, does not rely on any external power sources or controllers and incorporates all sample loading and sorting functions necessary to prepare high-quality sperm for in vitro fertilization. This self-contained, inexpensive, and portable device may also be useful for developing convenient sperm motility assays that can be used at the point of care or at home.

  3. INFECTIOUS SALMON ANEMIA

    Directory of Open Access Journals (Sweden)

    M. Yu. Shchelkanov

    2017-01-01

    Full Text Available The aim of this work consists in the analysis of modern scientific conceptions about infectious salmon anemia (ISA etiologically linked with ISAV (infectious salmon anemia virus (Orthomyxoviridae, Isavirus. ISA is deadly disease of Salmonidae fishes.Discussion. ISA began to extend actively among salmon breeding farms since the extremity of the XX century and poses nowadays serious threat of fishing industry as there are no not only anti-ISAV chemopreparates and effective vaccines, but also scientifically based ideas of ISAV ecology. In the offered review data on the discovery history, taxonomical status, virion morphology and genome structure as well as ecology of ISAV, clinical features, pathogenesis and laboratory diagnostics, actions in the epizootic foci for the prevention of further distribution and prophylaxis of ISA, arrangement for protection against salmon louses and utilized approaches to anti-ISAV vaccines development are discussed. There is very important that ISAV is capable to be transferred by salmon louses – pelagic crustaceans (Copepoda: Caligidae that allows to classify ISAV as arbovirus ecological group which are transferred due to biological transmission by arthropods (copepods to vertebrate animals (salmons. It is the only example known so far when representatives of Crustacea act as a vector for arboviruses.Conclusion. Investigation of ISAV ecology turns into one of "touchstones" allowing to judge technological readiness of mankind to master resources of the World Ocean. 

  4. Salmon 2100: the future of wild Pacific salmon

    National Research Council Canada - National Science Library

    Lackey, R.T; Lach, D.H; Duncan, S.L

    2006-01-01

    Realistic options to restore and sustain wild salmon runs in California, Oregon, Washington, Idaho and southern British Columbia are identified by 36 salmon scientists, resource managers, and policy experts...

  5. Benthic monitoring of salmon farms in Norway using foraminiferal metabarcoding

    DEFF Research Database (Denmark)

    Pawlowski, Jan; Esling, Philippe; Lejzerowicz, Franck

    2016-01-01

    The rapid growth of the salmon industry necessitates the development of fast and accurate tools to assess its environmental impact. Macrobenthic monitoring is commonly used to measure the impact of organic enrichment associated with salmon farm activities. However, classical benthic monitoring can...... of macrofauna-based benthic monitoring. Here, we tested the application of foraminiferal metabarcoding to benthic monitoring of salmon farms in Norway. We analysed 140 samples of eDNA and environmental RNA (eRNA) extracted from surface sediment samples collected at 4 salmon farming sites in Norway. We sequenced...... appears to be a promising alternative to classical benthic monitoring, providing a solution to the morpho-taxonomic bottleneck of macrofaunal surveys....

  6. Sperm storage potential and daily sperm production of brown male ...

    African Journals Online (AJOL)

    Testes and epididymis were homogenised separately in 0.154M NaCl. Sperm reserves in the homogenates were determined. Sperm production efficiency and daily sperm production were also determined from testicular homogenates and epididymal sperm reserves from epididymal homogenate. The results showed that ...

  7. [Lipids in the sperm plasma membrane and their role in fertilization].

    Science.gov (United States)

    Niu, Dong-Mei; Wang, Jun-Jun

    2009-07-01

    Sexual reproduction is marked by the fusion of the sperm cell with the oocyte during fertilization to produce the diploid zygote, in which the lipids in the sperm plasma membrane play an important role. Due to the loss of most cell organelles and DNA transcription, spermatozoa lack protein expression and vesicular transport. However, the lipids of the sperm plasma membrane undergo complicated dynamic changes, which may facilitate the capacitation, binding with zona pellucida, acrosome reaction and fusion of the sperm cell with the oocyte. This paper summarizes the progress in the studies of the lipids in the sperm plasma membrane, their composition, structure, peroxidation, metabolism and role in fertilization.

  8. Salmon lice – impact on wild salmonids and salmon aquaculture

    Science.gov (United States)

    Torrissen, O; Jones, S; Asche, F; Guttormsen, A; Skilbrei, O T; Nilsen, F; Horsberg, T E; Jackson, D

    2013-01-01

    Salmon lice, Lepeophtheirus salmonis, are naturally occurring parasites of salmon in sea water. Intensive salmon farming provides better conditions for parasite growth and transmission compared with natural conditions, creating problems for both the salmon farming industry and, under certain conditions, wild salmonids. Salmon lice originating from farms negatively impact wild stocks of salmonids, although the extent of the impact is a matter of debate. Estimates from Ireland and Norway indicate an odds ratio of 1.1:1-1.2:1 for sea lice treated Atlantic salmon smolt to survive sea migration compared to untreated smolts. This is considered to have a moderate population regulatory effect. The development of resistance against drugs most commonly used to treat salmon lice is a serious concern for both wild and farmed fish. Several large initiatives have been taken to encourage the development of new strategies, such as vaccines and novel drugs, for the treatment or removal of salmon lice from farmed fish. The newly sequenced salmon louse genome will be an important tool in this work. The use of cleaner fish has emerged as a robust method for controlling salmon lice, and aquaculture production of wrasse is important towards this aim. Salmon lice have large economic consequences for the salmon industry, both as direct costs for the prevention and treatment, but also indirectly through negative public opinion. PMID:23311858

  9. Pacific Coastal Salmon Recovery Fund

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Congress established the Pacific Coastal Salmon Recovery Fund (PCSRF) to monitor the restoration and conservation of Pacific salmon and steelhead populations and...

  10. Pacific salmon (Oncorhynchus spp.) runs and consumer fitness: growth and energy storage in stream-dwelling salmonids increase with salmon spawner density

    Science.gov (United States)

    Rinella, Daniel J.; Wipfli, Mark S.; Stricker, Craig A.; Heintz, Ron A.; Rinella, Matthew J.

    2012-01-01

    We examined how marine-derived nutrients (MDN), in the form of spawning Pacific salmon, influenced the nutritional status and δ15N of stream-dwelling fishes. We sampled juvenile coho salmon (Oncorhynchus kisutch) and Dolly Varden (Salvelinus malma) during spring and fall from 11 south-central Alaskan streams that ranged widely in spawning salmon biomass (0.1–4.7 kg·m–2). Growth rate (as indexed by RNA–DNA ratios), energy density, and δ15N enrichment in spring-sampled fishes increased with spawner biomass, indicating the persistence of spawner effects more than 6 months after salmon spawning. Point estimates suggest that spawner effects on nutrition were substantially greater for coho salmon than Dolly Varden (268% and 175% greater for growth and energy, respectively), indicating that both species benefitted physiologically, but that juvenile coho salmon accrued more benefits than Dolly Varden. Although the data were less conclusive for fall- than spring-sampled fish, they do suggest spawner effects were also generally positive during fall, soon after salmon spawned. In a follow-up analysis where growth rate and energy density were modeled as a function of δ15N enrichment, results suggested that both increased with MDN assimilation, especially in juvenile coho salmon. Our results support the importance of salmon runs to the nutritional ecology of stream-dwelling fishes.

  11. Enantiopure copper(II) complex of natural product rosin derivative: DNA binding, DNA cleavage and cytotoxicity.

    Science.gov (United States)

    Fei, Bao-Li; Yin, Bin; Li, Dong-Dong; Xu, Wu-Shuang; Lu, Yang

    2016-12-01

    To develop chiral anticancer drug candidates for molecular target DNA, the synthesis and characterization of a novel enantiomerically pure copper(II) complex [Cu 1 Cl 2 ] (2) of an optically pure ligand N-(pyridin-2-ylmethylene) dehydroabietylamine (1) was carried out. The coordination geometry of the copper center is a distorted square-planar arrangement. The interactions of 1 and 2 with salmon sperm DNA were investigated by viscosity measurements, UV, fluorescence and circular dichroism (CD) spectroscopic techniques. All the results reveal that 1 and 2 interacted with DNA through intercalation and 2 exhibited a higher DNA binding ability. Further, 1 and 2 could cleave supercoiled pBR322 DNA by single strand and 2 displayed stronger cleavage ability in the presence of ascorbic acid. In vitro cytotoxicity of 1 and 2 against HeLa, SiHa, HepG-2 and A431 cancer cell lines was studied using CCK-8 assay. The results indicate that 2 had a superior cytotoxicity than 1 and the widely used drug cisplatin under identical conditions. Flow cytometry analysis demonstrates 2 produced death of HeLa cancer cells through an apoptotic pathway. Cell cycle analysis shows that 2 mainly arrested HeLa cells at the S phase. A novel enantiomerically pure copper(II) complex [Cu 1 Cl 2 ] (2) of an optically pure ligand N-(pyridin-2-ylmethylene) dehydroabietylamine (1), based on natural product rosin has been synthesized. 2 has the potential to act as effective anticancer drug.

  12. Identifying salmon lice transmission characteristics between Faroese salmon farms

    DEFF Research Database (Denmark)

    Kragesteen, Trondur J.; Simonsen, Knud; Visser, AW

    2017-01-01

    Sea lice infestations are an increasing challenge in the ever-growing salmon aquaculture sector and cause large economic losses. The high salmon production in a small area creates a perfect habitat for parasites. Knowledge of how salmon lice planktonic larvae disperse and spread the infection...... between farms is of vital importance in developing treatment management plans to combat salmon lice infestations. Using a particle tracking model forced by tidal currents, we show that Faroese aquaculture farms form a complex network. In some cases as high as 10% of infectious salmon lice released at one...

  13. Concurrent injection of a rhabdovirus-specific DNA vaccine with a polyvalent, oil-adjuvanted vaccine delays the specific anti-viral immune response in Atlantic salmon, Salmo salar L.

    Science.gov (United States)

    Skinner, Lisa A; LaPatra, S E; Adams, A; Thompson, K D; Balfry, S K; McKinley, R S; Schulte, P M

    2010-04-01

    Vaccines are commonly used in salmonid aquaculture as a method of disease prevention. Although there is a substantial amount of published research regarding the immunological and physiological effects following the injection of different polyvalent vaccines and DNA vaccines, there are no published reports examining the physiological and immunological effects of concurrent vaccine injection, which is the situation encountered in aquaculture. Using key immunological parameters such as lysozyme activity and specific antibody titres we examined the short-term activation of the immune response of cultured Atlantic salmon (Salmo salar L.) following concurrent injection with a traditional, polyvalent, oil-adjuvanted vaccine (AV) and an IHNV-specific DNA vaccine (DV). Our results indicate that different aspects of the innate and adaptive immune responses are influenced in either a positive or negative manner. While concurrent vaccine injection elicited an increase in lysozyme activity, changes in antibody titre (Ab) were antigen specific. The production of anti-Aeromonas salmonicida Abs was significantly greater in the combined vaccine group at 296 degree days post-vaccine injection (dd pvi), while the production of anti-Listonella anguillarum Abs was significantly greater at 106 dd pvi in the combined vaccine group. Of even greater interest was the apparent delay in production of IHNV-specific neutralizing antibodies (NAb) when the DV was injected concurrently with the polyvalent AV. The results indicated that concurrent injection of a polyvalent oil-AV and a DV can be beneficial to the production of antibodies; however, the specific anti-viral response may be delayed. Copyright 2010 Elsevier Ltd. All rights reserved.

  14. Mutagenic effect of radionuclides incorporated into DNA of Drosophila melanogaster. Progress report, 1977--1978. [Effects of tritium decay in labelled sperm

    Energy Technology Data Exchange (ETDEWEB)

    Lee, W.R.

    1978-01-01

    Three experiments were conducted in which Drosophila melanogaster males were labeled with (5-/sup 3/H)-deoxycytidine, (8-/sup 3/H)-deoxyguanosine, or L-(3-/sup 3/H)-arginine in the larval stage. In the first two experiments with 5-/sup 3/H-dC and 8-/sup 3/H-dG, the labeled males upon eclosion were divided into two groups: 1) those mated to females that from emergence as adults were sustained on a media deficient in protein, called sugar agar media; and 2) those mated to females that were on a standard nutritious media. All males from the arginine experiment were mated to females stored on the standard media. The labeled males were allowed to mate for two days during which time the females stored on standard media deposited fertile eggs. Those F/sub 1/ progenies developing from the brood of eggs laid during the first 48 hrs following mating were called the non-stored group, as the spermatozoa were not stored in the female for more than 48 hrs. Progenies of males that were mated to females sustained on the sugar agar media were referred to as the stored group because the females on the sugar agar did not lay eggs. After 14 days the females were transferred to regular corn meal agar to induce egg laying. The average time of storage of sperm in the females' seminal receptacles for the stored group was 18 days. The progenies from the non-stored and stored broods were analyzed for mutations thus giving a comparison of the mutation frequency for each class of mutants detected before and after storage of the tritium-labeled sperm. The difference between the mutation frequency following storage and that before storage is the increase in mutations attributed to tritium disintegrations occurring during the storage period. One determination for each experiment, 5-/sup 3/H-dC, 8-/sup 3/H-dG, or /sup 3/H-Arg. has been made.

  15. Effect of centrifugal fractionation protocols on quality and recovery rate of equine sperm.

    Science.gov (United States)

    Edmond, A J; Brinsko, S P; Love, C C; Blanchard, T L; Teague, S R; Varner, D D

    2012-03-15

    Centrifugal fractionation of semen is commonly done to improve quality of human semen in assisted-reproduction laboratories, allowing sperm separation based on their isopycnic points. Sperm with morphologic abnormalities are often more buoyant, promoting their retention above defined density media, with structurally normal sperm passing through the media following centrifugation. Three experiments were conducted to evaluate the effects of density-medium type, centrifuge-tube size, sperm number, and density-medium volume (column height) on stallion sperm quality and recovery rate in sperm pellets following centrifugation. In all three experiments, equine semen was initially centrifuged to increase sperm concentration. In Experiment 1, semen was layered over continuous or discontinuous gradients. For Experiment 2, semen was layered over three column heights of continuous gradients in 15- or 50-ml conical-bottom tubes. For Experiment 3, increasing sperm numbers were layered over continuous gradient in 15- or 50-ml conical-bottom tubes. Following centrifugation, sperm pellets were evaluated for sperm morphologic quality, motility, DNA integrity, and recovery rate. Centrifugal fractionation improved (P continuous gradient increased (P 0.05) by column height of gradient. Increasing sperm number subjected to gradient centrifugation decreased (P < 0.05) sperm recovery rate when 15-ml tubes were used. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. Low Sperm Count

    Science.gov (United States)

    ... lump or swelling in the testicle area A history of testicle, prostate or sexual problems Groin, testicle, penis or scrotum surgery Request an Appointment at Mayo Clinic Causes The production of sperm is a complex ...

  17. Physiological consequences of the salmon louse (Lepeophtheirus salmonis) on juvenile pink salmon (Oncorhynchus gorbuscha): implications for wild salmon ecology and management, and for salmon aquaculture.

    Science.gov (United States)

    Brauner, C J; Sackville, M; Gallagher, Z; Tang, S; Nendick, L; Farrell, A P

    2012-06-19

    Pink salmon, Oncorhynchus gorbuscha, are the most abundant wild salmon species and are thought of as an indicator of ecosystem health. The salmon louse, Lepeophtheirus salmonis, is endemic to pink salmon habitat but these ectoparasites have been implicated in reducing local pink salmon populations in the Broughton Archipelago, British Columbia. This allegation arose largely because juvenile pink salmon migrate past commercial open net salmon farms, which are known to incubate the salmon louse. Juvenile pink salmon are thought to be especially sensitive to this ectoparasite because they enter the sea at such a small size (approx. 0.2 g). Here, we describe how 'no effect' thresholds for salmon louse sublethal impacts on juvenile pink salmon were determined using physiological principles. These data were accepted by environmental managers and are being used to minimize the impact of salmon aquaculture on wild pink salmon populations.

  18. First study of sperm mediated gene transfer in Egyptian river buffalo

    Directory of Open Access Journals (Sweden)

    Mohamed S. Hassanane

    2017-12-01

    Full Text Available The present study was carried out to find the best treatments for enhancing the ration of insertion of a desired gene construct (pEGFP-N1 onto the sperm of buffalo as the first step for the production of transgenic buffalo using sperm mediated gene transfer (SMGT. The tested conditions were plasmid DNA concentration, sperm concentration, transfecting agent concentration: Dimethyle sulphoxide (DMSO and time of transfection. The study proved that the best conditions for producing transgenic embryos were incubation sperm solution its concentration is 107/ml sperm with 3% DMSO: with 20 µg/ml from the linarized DNA, for 15 min at 4 °C are the best conditions to produce transgenic buffalo embryo using sperm mediated gene transfer.

  19. Sperm as a noninvasive gene delivery system for preimplantation embryos.

    Science.gov (United States)

    Chan, P J; Kalugdan, T; Su, B C; Whitney, E A; Perrott, W; Tredway, D R; King, A

    1995-05-01

    To determine if sperm could be manipulated to be a noninvasive transport carrier for the delivery of gene fragments to the blastocyst. Sperm cells carrying foreign DNA fragments from human papillomavirus (HPV) types 16, 18, 31, and 33 were allowed to migrate from one end of an artificial reproductive tube and to come in contact with hatching mouse blastocysts at the other end of the tube. The blastocysts were then washed and analyzed for the presence of the foreign DNA fragments. Clinical and academic research environment. Detection of amplified products from transferred foreign DNA using the polymerase chain reaction and primers targeted at the E6-E7 region for different HPV types. Polymerase chain reaction analyses showed transference of DNA HPV type 18 to the blastocysts. Not all types of DNA fragments were transferred equally. The results suggested the possibility of using sperm as a noninvasive gene delivery system for passing on gene fragments to preimplantation embryos. It was demonstrated that certain DNA fragments were easier to deliver than others, indicating the necessity for exploring all the factors involved in the mechanism of the transference process. The study also serves to highlight the possibility of unintentional transmission of viral or bacterial DNA to the developing embryo via the sperm.

  20. Sperm length, sperm storage and mating system characteristics in bumblebees

    DEFF Research Database (Denmark)

    Baer, Boris; Schmid-Hempel, Paul; Høeg, Jens Thorvald

    2003-01-01

    Multiple insemination induces sperm competition and may select for longer, faster moving sperm in species where sperm is short-lived and egg fertilization takes place almost immediately after ejaculation. Here we report the first detailed analysis of sperm length in social insects with long......-term storage of sperm, using three bumblebee species with different mating systems as models. We show that individual males produce only one size-class of sperm, but that sperm length is highly variable among brothers, among unrelated conspecific males, and among males of different species. Males of Bombus...... hypnorum, a species with multiple-mating queens, have longer sperm than males of B. terrestris and B. lucorum whose queens are single mated. Although the sample size on the species level was too small to perform a phylogenetic analysis, this finding supports the hypothesis that, all other things being...

  1. The effects of male age on sperm analysis by motile sperm organelle morphology examination (MSOME

    Directory of Open Access Journals (Sweden)

    Silva Liliane FI

    2012-03-01

    Full Text Available Abstract Background This study aimed to investigate the influence of age on sperm quality, as analysed by motile sperm organelle morphology examination (MSOME. Methods Semen samples were collected from 975 men undergoing evaluation or treatment for infertility. Sperm cells were evaluated at 8400× magnification using an inverted microscope equipped with Nomarski (differential interference contrast optics. Two forms of spermatozoa were considered: normal spermatozoa and spermatozoa with large nuclear vacuoles (LNV, defined as vacuoles occupying > 50% of the sperm nuclear area. At least 200 spermatozoa per sample were evaluated, and the percentages of normal and LNV spermatozoa were determined. The subjects were divided into three groups according to age: Group I, less than or equal to 35 years; Group II, 36-40 years; and Group III, greater than or equal to 41 years. Results There was no difference in the percentages of normal sperm between the two younger (I and II groups (P >0.05. The percentage of normal sperm in the older group (III was significantly lower than that in the younger (I and II groups (P P >0.05. The percentage of LNV spermatozoa was significantly higher in the older group (III than in the younger (I and II groups (P P P Conclusion The results demonstrated a consistent decline in semen quality, as reflected by morphological evaluation by MSOME, with increased age. Considering the relationship between nuclear vacuoles and DNA damage, these age-related changes predict that increased paternal age should be associated with unsuccessful or abnormal pregnancy as a consequence of fertilisation with damaged spermatozoa. Given that sperm nuclear vacuoles can be evaluated more precisely at high magnification, these results support the routine use of MSOME for ICSI as a criterion for semen analysis.

  2. Study of the effect of varicocelectomy on sperm proteins expression in patients with varicocele and poor sperm quality by using two-dimensional gel electrophoresis.

    Science.gov (United States)

    Hosseinifar, Hani; Sabbaghian, Marjan; Nasrabadi, Davood; Modarresi, Tahereh; Dizaj, Ahmad Vosough Taqi; Gourabi, Hamid; Gilani, Mohmmad Ali Sadighi

    2014-06-01

    To compare proteomic profiles of spermatozoa from patients with varicocele and poor sperm quality before and after varicocelectomy. This work was designed as a prospective and observational study. The study was based on 20 men with varicocele grade 3 and poor sperm quality undergoing varicocelectomy at the Fertility Unit of Royan institute in 2009. Two semen samples were collected, one before varicocelectomy and the other after surgery. Protein separation was done by two-dimensional protein electrophoresis, and analyzed by gel densitometry and mass spectrometry. Differential sperm protein expression levels were measured by gel densitometry. Comparison of the sperm parameters showed that sperm motility and concentration were increased after varicocelectomy. At the level of protein, a total of 3 protein spots were identified whose expression was significantly lower in sperm samples before varicocelectomy compared with after surgery including heat shock protein A5 (HSPA5), superoxide dismutase 1 (SOD1) and δ-subunit of the catalytic core of mitochondrial adenosine triphosphate synthase (ATP5D). High grade varicocoele affects sperm protein expression presumably because of increasing testicular temperature. These proteins play essential roles in sperm production, DNA integrity protection, and sperm motility. This novel study demonstrates that varicocelectomy can improve both sperm quality and proteins expression.

  3. Using Phylogenetic Analysis to Detect Market Substitution of Atlantic Salmon for Pacific Salmon: An Introductory Biology Laboratory Experiment

    Science.gov (United States)

    Cline, Erica; Gogarten, Jennifer

    2012-01-01

    We describe a laboratory exercise developed for the cell and molecular biology quarter of a year-long majors' undergraduate introductory biology sequence. In an analysis of salmon samples collected by students in their local stores and restaurants, DNA sequencing and phylogenetic analysis were used to detect market substitution of Atlantic salmon…

  4. Healthy Sperm: Improving Your Fertility

    Science.gov (United States)

    ... and what you can do to improve your fertility. By Mayo Clinic Staff Do your sperm pass ... understanding the various factors that can affect male fertility — then consider steps to help your sperm become ...

  5. Cryoprotectant-free freezing of the goat epididymal sperm.

    Science.gov (United States)

    Katanbafzadeh, H; Barati, F; Tabandeh, M

    2014-01-01

    Cryoprotectant free approach successfully removed the impact of physical and chemical damages in preserving human sperm in a vitrification protocol. There is no any report on this technology in farm animal sperm freezing. The aim of the present study was to find the efficacy of cholesterol-loaded cyclodextrin (CLC; 1 mg per 60 million) and sucrose (0.1 and 0.2 M) on freezing of the goat epididymal sperm. Caudal epididymides (n=5 pairs) were dissected, incised and incubated in the Tris-BSA solution for 15 min, followed by swim-up at room temperature. Sperm was loaded in 0.25 mL French straws and cooled on nitrogen vapor for 3 min then immersed in liquid nitrogen and remained for 48 h. Then the straws thawed by immersing in 37 degree C waterbath for 30 sec and analyzed. The results showed the impact of freezing on the goat epididymal sperm motility, viability and DNA fragmentation that were improved by incorporation of CLC and sucrose (0.2 M). In conclusion, the goat epididymal sperm was frozen in a cryoprotectant-free freezing model. CLC and 0.4 M sucrose protected the goat epididymal sperm against freezing-induced damages.

  6. DNA fragmentation in spermatozoa

    DEFF Research Database (Denmark)

    Rex, A S; Aagaard, J.; Fedder, J

    2017-01-01

    Sperm DNA Fragmentation has been extensively studied for more than a decade. In the 1940s the uniqueness of the spermatozoa protein complex which stabilizes the DNA was discovered. In the fifties and sixties, the association between unstable chromatin structure and subfertility was investigated....... In the seventies, the impact of induced DNA damage was investigated. In the 1980s the concept of sperm DNA fragmentation as related to infertility was introduced as well as the first DNA fragmentation test: the Sperm Chromatin Structure Assay (SCSA). The terminal deoxynucleotidyl transferase nick end labelling...... (TUNEL) test followed by others was introduced in the nineties. The association between DNA fragmentation in spermatozoa and pregnancy loss has been extensively investigated spurring the need for a therapeutic tool for these patients. This gave rise to an increased interest in the aetiology of DNA damage...

  7. Does CO2 enhance short-term storage success of Chinook salmon (Oncorhynchus tshawytscha) milt?

    Science.gov (United States)

    Bencic, D C; Ingermann, R L; Cloud, J G

    2001-07-01

    Successful short-term storage of salmonid milt depends on numerous factors, including temperature, fluid volume, and gaseous environment, with storage at low temperatures under an atmosphere of 100% O2 being the most common method. Salmonid sperm maintained in a storage environment with elevated carbon dioxide (CO2) levels, such as the approximately 4% CO2 in exhaled air, are not motile when activated. While these modest levels of CO2 inhibit sperm motility, the effect is reversible within hours after exposure to a CO2-free oxygenated environment. Therefore, the effect of CO2 (as a component gas in the storage environment) on chinook salmon (Oncorhynchus tshawytscha) sperm motility and viability was examined. The hypothesis of the current investigation was that CO2-exposure with subsequent CO2 removal would be beneficial during short-term chinook salmon milt storage. Milt samples were collected from mature (adult) and precocious (jack) male chinook salmon and stored under various CO2 and O2 levels at 3 to 4 degrees C for up to 14 days. Milt samples were then removed from the incubation environments and maintained under CO2-free humidified air with continuous mixing for 4 h at 10 degrees C before analysis of motility. The resultant motility of samples incubated under 3.5% or less CO2 was not different than controls during the 14 d incubation period; motility of samples stored under higher CO2 tensions were significantly lower. The motility of samples incubated under 3.5% CO2 reached the maximum recovered motility after 2 h exposure to CO2-free humidified air, while the motility of sperm incubated under 13.4% CO2 levels recovered no motility even after 6 h exposure to CO2-free humidified air. The motility of samples incubated under normoxia was significantly greater than that of samples incubated under hyperoxia (approximately 90% O2) at both 7 and 14 d, regardless of the CO2 level. Sperm viability was relatively unaltered by any of the incubation conditions examined

  8. Reduction of DNA contamination in RNA samples for reverse transcription-polymerase chain reaction using selective precipitation by compaction agents.

    Science.gov (United States)

    Añez-Lingerfelt, Mariaclara; Fox, George E; Willson, Richard C

    2009-01-01

    An important problem in measurement of messenger RNA (mRNA) levels by reverse transcription-polymerase chain reaction (RT-PCR) is DNA contamination, which can produce artifactually increased mRNA concentration. Current methods to eliminate contaminating DNA can compromise the integrity of the RNA, are time-consuming, and/or are hazardous. We present a rapid, nuclease-free, and cost-effective method of eliminating contaminating DNA in RNA samples using selective precipitation by compaction agents. Compaction agents are cationic molecules that bind to double-stranded nucleic acids, driven by electrostatic interactions and steric complementarity. The effectiveness and DNA selectivity of six compaction agents were investigated: trivalent spermidine, Triquat A, and Triquat 7; tetravalent spermine and Quatro-quat; and hexavalent Quatro-diquat. Effectiveness was measured initially by supernatant UV absorbance after precipitation of salmon sperm DNA. Effectiveness and selectivity were then investigated using differences in RT-PCR C(t) values with synthetic mixtures of human genomic DNA and total RNA and with total RNA isolated from cells. With 500 microM spermidine or Triquat A, the supernatant DNA could not be detected up to 40 cycles of PCR (C(t)12.6), whereas the C(t) for the mRNA was increased by only five cycles. Therefore, spermidine and Triquat A each show strong DNA selectivity and could be used to eliminate contaminating DNA in measurements of mRNA.

  9. Reduction of DNA Contamination in RNA Samples for RT-PCR using Selective Precipitation by Compaction Agents

    Science.gov (United States)

    Añez-Lingerfelt, Mariaclara; Fox, George E.; Willson, Richard C.

    2017-01-01

    An important problem in measurement of mRNA levels by RT-PCR is DNA contamination, which can produce artifactually increased mRNA concentration. Current methods to eliminate contaminating DNA can compromise the integrity of the RNA, are time-consuming, or are hazardous. We present a rapid, nuclease-free, and cost-effective method of eliminating contaminating DNA in RNA samples using selective precipitation by compaction agents. Compaction agents are cationic molecules which bind to double-stranded nucleic acids, driven by electrostatic interactions and steric complimentarity. The effectiveness and DNA-selectivity of six compaction agents were investigated: trivalent spermidine, Triquat A, and Triquat 7; tetravalent spermine and Quatro-quat; and hexavalent Quatro-diquat. Effectiveness was measured initially by supernatant UV absorbance after precipitation of salmon sperm DNA. Effectiveness and selectivity were then investigated using differences in RT-PCR Ct values with synthetic mixtures of human genomic DNA and total RNA, and with total RNA isolated from cells. With 500 μM of spermidine or Triquat A, the supernatant DNA could not be detected up to 40 cycles of PCR (Ct ≥12.6) while the Ct for the mRNA was increased by only 5 cycles. Therefore, spermidine and Triquat A each show strong DNA-selectivity and could be used to eliminate contaminating DNA in measurements of mRNA. PMID:18831957

  10. Low diversity in the mitogenome of sperm whales revealed by next-generation sequencing

    Science.gov (United States)

    Alana Alexander; Debbie Steel; Beth Slikas; Kendra Hoekzema; Colm Carraher; Matthew Parks; Richard Cronn; C. Scott Baker

    2012-01-01

    Large population sizes and global distributions generally associate with high mitochondrial DNA control region (CR) diversity. The sperm whale (Physeter macrocephalus) is an exception, showing low CR diversity relative to other cetaceans; however, diversity levels throughout the remainder of the sperm whale mitogenome are unknown. We sequenced 20...

  11. Chelating agents in combination with rosmarinic acid for boar sperm freeze-drying.

    Science.gov (United States)

    Olaciregui, Maite; Luño, Victoria; González, Noelia; Domingo, Paula; de Blas, Ignacio; Gil, Lydia

    2017-09-01

    The presence of DNA protective agents in the medium is necessary to maintain sperm functionality after freeze-drying procedure. The objective of this study was to investigate the effect of chelating agents, ethylene diaminetetraacetic acid (EDTA) and ethylene glycoltetraacetic acid (EGTA), in combination with rosmarinic acid (RA) on DNA integrity of freeze-dried boar sperm. We also examined the effect of these agents on the in vitro developmental ability of porcine oocytes following sperm injection (ICSI). Heterospermic mix, obtained from ejaculated sperm of three boars, was freeze-dried in two different chelating agents' media: 50mM EDTA or 50mM EGTA, and in these media supplemented with 105μM of rosmarinic acid. Frozen-thawed sperm was used as control. After rehydration, samples were subjected to DNA damage detection using Sperm Chromatin Dispersion test. ICSI was performed to verify the ability of freeze-dried sperm to participate in embryonic development. Five replicated trials were carried out for each group. In the presence of rosmarinic acid, the percentage of spermatozoa with DNA damage decreased significantly (p=0.010), without differences between the two chelating agents combination. EDTA solution preserves more efficiently DNA integrity of boar sperm than EGTA solution (p=0.002). There were no significant differences among the studied groups related to the blastocyst formation rate. Results suggested that the addition of rosmarinic acid to the medium improves sperm DNA integrity after freeze-drying, but does not promote fertilization and blastocyst development. We also observed a similar percentage of embryos production with freeze-dried and with frozen-thawed sperm. Copyright © 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  12. On mammalian sperm dimensions.

    Science.gov (United States)

    Cummins, J M; Woodall, P F

    1985-09-01

    Data on linear sperm dimensions in mammals are presented. There is information on a total of 284 species, representing 6.2% of all species; 17.2% of all genera and 49.2% of all families have some representation, with quantitative information missing only from the orders Dermoptera, Pholidota, Sirenia and Tubulidentata. In general, sperm size is inverse to body mass (except for the Chiroptera), so that the smallest known spermatozoa are amongst those of artiodactyls and the largest are amongst those of marsupials. Most variations are due to differences in the lengths of midpiece and principal piece, with head lengths relatively uniform throughout the mammals.

  13. Nickel (II) Ions Interaction with Polynucleotides and DNA of Different GC Composition

    CERN Document Server

    Bregadze, Vasil G; Melikishvili, Sophie Z; Melikishvili, Zaza G

    2009-01-01

    The goal of the work was to study the role of GC alternative dimmers in the binding of DNA with Ni (II) ions. The method of ultraviolet difference spectroscopy has been applied to investigate Ni (II) ions interactions with DNA extracted from Clostridium perfringens, Mice liver (C3HA line), Calf thymus, Salmon sperm, Herring sperm, E.coli, Micrococcus luteus and polynucleotides Poly (dA-dT)xPoly (dA-dT), Poly (dG)x Poly (dC), Poly (dG-dC)xPoly (dG-dC). It is shown that Ni (II) ions at outer-spherical binding with DNA double helix from the side of the major groove choose more stable dimmers 3^'-C-G-5^' . . 5^'-G-C-3^' and get bound with N7 atoms of both guanines in dimmer forming G-G interstrand crosslink. It directly correlates to the process of forming point defects of Watson-Crick wrong pair type (creation of rare keto-enolic and amino-imino tautomeric forms) and depurinization.

  14. Effects of sera taken from women with recurrent spontaneous abortion on sperm motility and apoptosis

    Directory of Open Access Journals (Sweden)

    Tahereh Talaei-khozani

    2011-01-01

    Full Text Available Background: Recurrent spontaneous abortion impacts almost 1% of couples. The sera from women with unexplained recurrent spontaneous abortion (URSA have toxic effects on embryos that grow in the uterus. Therefore, the abnormal condition of the uterus may also affect sperm qualities.Objective: The objectives of this study were to search if these sera could induce DNA denaturation in sperm nuclei and also it could reduce sperm motility.Materials and Methods: Sera of 20 women with URSA history and sera from 20 women with at least two healthy children were added to the sperms samples from 20 healthy men for 2 hours. The sperm motility was assessed after incubation with sera. The samples were stained with Tdt mediated dUTP nick end labeling (TUNEL assay for DNA fragmentation. The samples were analyzed with flow cytometry and the percentage of the TUNEL positive sperms were calculated. The data were analyzed by t-test.Results: The incubation of the sperm samples in sera with URSA lead to a decrease in the percentage of the motile sperm from 55% in control to 41% in the treated group, significantly (p=0.038. The percentage of the sperm with abnormal fragmented DNA increased after incubation with URSA (26.6% compare to the control (21.2%; however, it was not significant.Conclusion: It seems that sera from URSA patients could not induce a significant increase in the percentage of the sperms with nuclei contain DNA fragmentation. However, the sera of women with URSA could affect the fertility rate by reduction of the sperm motility.

  15. Validation of the FACSCount AF system for determination of sperm concentration in boar semen

    DEFF Research Database (Denmark)

    Hansen, C.; Christensen, P.; Stryhn, H.

    2002-01-01

    A flow cytometric method has been developed for rapid determination of sperm concentration in semen from various mammalian species.* All cells containing DNA are stained with SYBR-14 or propidium iodide (PI) and sperm concentration is determined in relation to an internal standard of fluorescent...... with the spectrophotometric method ( CV = 6.3%). These results indicate that the FACSCount AF System is a valuable tool for precise and accurate assessment of sperm concentration in boar semen and that use of this system may lead to production of more uniform insemination doses containing a specific number of sperm per dose....

  16. Identification of multiple HPV types on spermatozoa from human sperm donors

    DEFF Research Database (Denmark)

    Kaspersen, Maja D; Larsen, Peter B; Ingerslev, Hans Jakob

    2011-01-01

    Human papillomaviruses (HPV) may cause sexually transmitted disease. High-risk types of HPV are involved in the development of cervical cell dysplasia, whereas low-risk types may cause genital condyloma. Despite the association between HPV and cancer, donor sperm need not be tested for HPV...... according to European regulations. Consequently, the potential health risk of HPV transmission by donor bank sperm has not been elucidated, nor is it known how HPV is associated with sperm. The presence of 35 types of HPV was examined on DNA from semen samples of 188 Danish sperm donors using a sensitive...

  17. Effects of copper ions on DNA binding and cytotoxic activity of a chiral salicylidene Schiff base.

    Science.gov (United States)

    Fei, Bao-Li; Xu, Wu-Shuang; Tao, Hui-Wen; Li, Wen; Zhang, Yu; Long, Jian-Ying; Liu, Qing-Bo; Xia, Bing; Sun, Wei-Yin

    2014-03-05

    A chiral Schiff base HL N-(5-bromo-salicylaldehyde)dehydroabietylamine (1) and its chiral dinuclear copper complex [Cu2L4]·4DMF (2) have been synthesized and fully characterized. The interactions of 1 and 2 with salmon sperm DNA have been investigated by viscosity measurements, UV, fluorescence and circular dichroism (CD) spectroscopic techniques. Absorption spectral (Kb=3.30 × 10(5)M(-)(1) (1), 6.63 × 10(5)M(-)(1)(2)), emission spectral (Ksv=7.58 × 10(3)M(-)(1) (1), 1.52 × 10(4)M(-)(1) (2)), and viscosity measurements reveal that 1 and 2 interact with DNA through intercalation and 2 exhibits a higher DNA binding ability. In addition, CD study indicates 2 cause a more evident perturbation on the base stacking and helicity of B-DNA upon binding to it. In fluorimetric studies, the enthalpy (ΔH>0) and entropy (ΔS>0) changes of the reactions between the compounds with DNA demonstrate hydrophobic interactions. 1 and 2 were also screened for their cytotoxic ability and 2 demonstrates higher growth inhibition of the selected cancer cells at concentration of 50 μM, this result is identical with their DNA binding ability order. All the experimental results show that the involvement of Cu (II) centers has some interesting effect on DNA binding ability and cytotoxicity of the chiral Schiff base. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Relative resistance of Pacific salmon to infectious salmon anaemia virus.

    Science.gov (United States)

    Rolland, J B; Winton, J R

    2003-09-01

    Infectious salmon anaemia (ISA) is a major disease of Atlantic salmon, Salmo salar, caused by an orthomyxovirus (ISAV). Increases in global aquaculture and the international movement of fish made it important to determine if Pacific salmon are at risk. Steelhead trout, Oncorhynchus mykiss, and chum, O. keta, Chinook, O. tshawytscha, coho, O. kisutch, and Atlantic salmon were injected intraperitoneally with a high, medium, or low dose of a Norwegian strain of ISAV. In a second challenge, the same species, except chum salmon, were injected with a high dose of either a Canadian or the Norwegian strain. Average cumulative mortality of Atlantic salmon in trial 1 was 12% in the high dose group, 20% in the medium dose group and 16% in the low dose group. The average cumulative mortality of Atlantic salmon in trial 2 was 98%. No signs typical of ISA and no ISAV-related mortality occurred among any of the groups of Oncorhynchus spp. in either experiment, although ISAV was reisolated from some fish sampled at intervals post-challenge. The results indicate that while Oncorhynchus spp. are quite resistant to ISAV relative to Atlantic salmon, the potential for ISAV to adapt to Oncorhynchus spp. should not be ignored.

  19. Reproductive success in wild and hatchery male coho salmon.

    Science.gov (United States)

    Neff, Bryan D; Garner, Shawn R; Fleming, Ian A; Gross, Mart R

    2015-08-01

    Salmon produced by hatcheries have lower fitness in the wild than naturally produced salmon, but the factors underlying this difference remain an active area of research. We used genetic parentage analysis of alevins produced by experimentally mixed groups of wild and hatchery coho salmon (Oncorhynchus kisutch) to quantify male paternity in spawning hierarchies. We identify factors influencing paternity and revise previously published behavioural estimates of reproductive success for wild and hatchery males. We observed a strong effect of hierarchy size and hierarchy position on paternity: in two-male hierarchies, the first male sired 63% (±29%; s.d.) of the alevins and the second male 37% (±29%); in three-male hierarchies, the first male sired 64% (±26%), the second male 24% (±20%) and the third male 12% (±10%). As previously documented, hatchery males hold inferior positions in spawning hierarchies, but we also discovered that hatchery males had only 55-84% the paternity of wild males when occupying the same position within a spawning hierarchy. This paternity difference may result from inferior performance of hatchery males during sperm competition, female mate choice for wild males, or differential offspring survival. Regardless of its cause, the combination of inferior hierarchical position and inferior success at a position resulted in hatchery males having only half (51%) the reproductive success of wild males.

  20. Determination of atropine sulfate using a novel sensitive DNA-biosensor based on its interaction on a modified pencil graphite electrode.

    Science.gov (United States)

    Ensafi, Ali A; Nasr-Esfahani, Parisa; Heydari-Bafrooei, Esmaeil; Rezaei, B

    2015-01-01

    A novel, selective, rapid and simple electrochemical method is developed for the determination of atropine sulfate. UV-Vis and differential pulse voltammetry are used to study the interaction of atropine sulfate with salmon sperm ds-DNA on the surface of salmon sperm ds-DNA modified-pencil graphite electrode (PGE). For this purpose, a pencil graphite electrode (PGE) modified with multiwall carbon nanotubes (MWCNTs), titanium dioxide nanoparticles (TiO2NPs), and poly-dialyldimethylammonium chloride (PDDA) decorated with ds-DNA is tested for the determination of atropine sulfate. The electrochemical oxidation peak current of adenine and guanine bonded on the surface of ds-DNA/PDDA-TiO2NPs-MWCNTs/PGE is used to obtain the analytical signal. Decreases in the intensities of guanine and adenine oxidation signals after their interaction with atropine sulfate are used as indicator signals for the sensitive determination of atropine sulfate. Using ds-DNA/PDDA-TiO2NPs-MWCNTs/PGE and based on the guanine signal, linear calibration curves were obtained in the range of 0.6 to 30.0 μmol L(-1) and 30.0 to 600.0 μmol L(-1) atropine sulfate with low detection limits of 30.0 nmol L(-1). The biosensor shows a good selectivity for the determination of atropine sulfate. Finally, the applicability of the biosensor is evaluated by measuring atropine sulfate in real samples with good accuracy. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Running title: Sperm cytogenetic alterations and male infertility

    Directory of Open Access Journals (Sweden)

    Hossein Mozdarani

    2016-11-01

    Full Text Available Male infertility is caused by many factors including genetics. Although part of genetic damages are inherited and could be traced in blood leukocytes, but those de novo alterations induced in spermatogenesis are not part of diagnostic work up. De novo alterations might be the cause of many idiopathic conditions of male infertility. The aim of this study was to evaluate DNA damage, sex chromosomal aneuploidy and DAZ microdeletion in sperms of subfertile males in comparison with normal healthy individuals. Whole blood and semen samples were obtained from 75 subfertile and 45 normal men. Semen samples from karyotypically normal subfertile and normal individuals were used for DNA fragmentation, sex chromosome aneuploidy and DAZ microdeletion analysis. Sperm DNA damage was assessed by alkaline comet assay, chromosome aneuploidy and DAZ microdeletion was assessed using a combined primed in situ labeling and fluorescent in situ hybridization (PRINS-FISH method. A significantly high percentage of DNA fragmentation was observed in subfertile patients compared to control. Similar observation was observed for sex chromosome aneuploidy and DAZ microdeletion (p < 0.01. A relatively small interindividual difference was seen in all three assays performed. However DAZ microdeletion was observed as mosaic form in Y bearing sperms. Results indicate that subfertile males experience higher genome instability in spermatogenesis expressed as DNA damage and consequently sperm chromosomal aneuploidy or microdeletions. Occurrence of de novo genetic alterations caused by environmental chemico-physical genotoxic agents during spermatogenesis might be one of the causes of idiopathic male infertility.

  2. Advancing age increases sperm chromatin damage and impairs fertility in peroxiredoxin 6 null mice

    Directory of Open Access Journals (Sweden)

    Burak Ozkosem

    2015-08-01

    Full Text Available Due to socioeconomic factors, more couples are choosing to delay conception than ever. Increasing average maternal and paternal age in developed countries over the past 40 years has raised the question of how aging affects reproductive success of males and females. Since oxidative stress in the male reproductive tract increases with age, we investigated the impact of advanced paternal age on the integrity of sperm nucleus and reproductive success of males by using a Prdx6−/− mouse model. We compared sperm motility, cytoplasmic droplet retention sperm chromatin quality and reproductive outcomes of young (2-month-old, adult (8-month-old, and old (20-month-old Prdx6−/− males with their age-matched wild type (WT controls. Absence of PRDX6 caused age-dependent impairment of sperm motility and sperm maturation and increased sperm DNA fragmentation and oxidation as well as decreased sperm DNA compaction and protamination. Litter size, total number of litters and total number of pups per male were significantly lower in Prdx6−/− males compared to WT controls. These abnormal reproductive outcomes were severely affected by age in Prdx6−/− males. In conclusion, the advanced paternal age affects sperm chromatin integrity and fertility more severely in the absence of PRDX6, suggesting a protective role of PRDX6 in age-associated decline in the sperm quality and fertility in mice.

  3. Equine sperm-neutrophil binding.

    Science.gov (United States)

    Alghamdi, Abdorrahman S; Madill, Scott; Foster, Douglas N; Troedsson, Mats H T

    2015-04-01

    When mares are inseminated repeatedly, protein molecules from the seminal plasma (SP) prevent sperm-neutrophil binding and reduced fertility. The molecule(s) responsible for sperm-neutrophil binding is not known and the identification of beneficial SP proteins is complicated by their large numbers and abundant variation. We examined several important aspects of sperm-neutrophil binding to ultimately facilitate the identification and isolation of the molecule(s) responsible. First, we raised anti-equine P-selectin antibodies to determine the involvement of this adhesion molecule in sperm-neutrophil binding. While these antibodies identified equine P-selectin, they did not inhibit sperm-neutrophil binding. However, acrosome-reacted equine sperm expressed a molecule similar to the ligand recognition unit of P-selectin. Second, we attempted to characterize SP protein binding to equine sperm and gauge their affinity. Biotinylated SP proteins were incubated with fresh sperm, washed over a viscous medium, electrophoresed, and probed with avidin. Several SP proteins bound to sperm with a strong affinity to withstand these treatments. This finding may prove valuable for future attempts to identify and characterize specific SP molecules. Lastly, we compared the secretions from male sex organs/glands on sperm motility, sperm-neutrophil binding, and their protein profile. We expected fewer proteins from individual organs/glands, which would facilitate isolation and identification of target molecules. While each secretion had a varying effect on motility and sperm-neutrophil binding, the protein profile was as complex as that seen in whole SP, indicating that collection of proteins from individual sources will not facilitate this work. Together, these experiments answer several important questions related to sperm-neutrophil binding, sperm-SP proteins interaction, and the complexity of the SP proteome. © 2015 by the Society for the Study of Reproduction, Inc.

  4. Development of the NBT assay as a marker of sperm oxidative stress.

    Science.gov (United States)

    Tunc, Ozlem; Thompson, Jeremy; Tremellen, Kelton

    2010-02-01

    Oxidative stress is a well-established cause of male infertility, with reactive oxygen species (ROS) causing infertility principally by impairing sperm motility and DNA integrity. Currently, most clinics do not test their infertile patients for the presence of oxidative stress because the available tests are expensive or difficult to perform. As antioxidant therapy may improve sperm DNA integrity and pregnancy outcomes, it has become apparent that there is an unmet clinical need for an inexpensive and easy-to-perform assay to identify sperm oxidative stress. The aim of this study was to develop a standardized protocol for performing a photometric nitro blue tetrazolium (NBT) assay for the measurement of seminal ROS production via production of coloured formazan, whilst correlating these results with impaired sperm function (motility and DNA integrity). Semen samples from 21 fertile and 36 male aetiology infertile men were assessed for ROS production (NBT assay), sperm DNA integrity (TUNEL), apoptosis (Annexin V) and sperm motility. Infertile men's semen contained on average fourfold higher levels of ROS than fertile men. The production of ROS by sperm was positively correlated with sperm DNA fragmentation and apoptosis, whilst being negatively correlated with sperm motility. Receiver-operating characteristic plot analysis established a cut-off point of 24 microg formazan/10(7) sperm having a sensitivity of 91.7% and a specificity of 81% for determining the fertility status of an individual. This study has been successful in establishing a standardized protocol for performing a photometric seminal NBT assay that has significant clinical utility in identifying men with impaired fertility because of oxidative stress.

  5. Human papillomavirus found in sperm head of young adult males affects the progressive motility.

    Science.gov (United States)

    Foresta, Carlo; Garolla, Andrea; Zuccarello, Daniela; Pizzol, Damiano; Moretti, Afra; Barzon, Luisa; Palù, Giorgio

    2010-02-01

    To evaluate the prevalence of human papillomavirus (HPV) sperm infection and its correlation with sperm parameters in a cohort of young adult males. Cross-sectional clinical study. Andrology and Microbiology sections at a university hospital. A cohort of 200 young adult male volunteers (18 years old), 100 with previous sexual intercourse and 100 without previous sexual intercourse. Seminal parameters, sperm culture for HPV and fluorescence in situ hybridization (FISH) analysis for HPV detection in the sperm head. Statistical analysis was performed with a two-tailed Student's t-test. Results of HPV investigation were compared to sperm parameters and results of FISH analysis. HPV infection was present in sperm cells of 10 subjects among those 100 young adults who already had unprotected intercourse and its presence was associated with reduced sperm motility. Furthermore, infected samples showed that about 25% of sperm had an HPV DNA positivity at the head site, but it is unclear whether it was integrated in the nucleus or not. This is the first report estimating the percentage of HPV-positive sperm in infected subjects and the association between HPV infection and sperm motility. Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  6. Infectious diseases of Pacific salmon

    Science.gov (United States)

    1954-01-01

    Investigations on infectious diseases of Pacific salmon due to micro-organisms other than viruses are reviewed. The etiological agents include trematodes, fungi, protozoa and bacteria. Bacteria have been found to be the most important agents of disease in the several species of Pacific salmon. Kidney disease, due to a small, unnamed Gram-positive diplobacillus, causes serious mortalities in young salmon reared in hatcheries. The disease has also been found in wild fish. Aquatic myxobacteria are important agents of disease both in the hatchery and in the natural habitat. One of the myxobacteria, Chondrococcus columnaris, causes disease at relatively high water temperatures. The problem of the taxonomy of this organism is discussed. Another myxobacterium, Cytophaga psychrophila, has been found responsible for epizootics in coho salmon at lower water temperatures, i.e., in the range of 40° to 55° F. In outbreaks of gill disease in young salmon, myxobacteria of several kinds have been implicated.

  7. Human papillomavirus sperm infection and assisted reproduction: a dangerous hazard with a possible safe solution.

    Science.gov (United States)

    Garolla, Andrea; Lenzi, Andrea; Palù, Giorgio; Pizzol, Damiano; Bertoldo, Alessandro; De Toni, Luca; Foresta, Carlo

    2012-04-01

    Human papillomavirus (HPV) infection has been demonstrated in the sperm of a large percentage of sexually active males and is associated with an impairment of sperm parameters, with a particular negative impact on sperm motility, suggesting a possible role in male infertility. Conventional sperm selection techniques have a low efficiency in removing HPV. Evaluation of sperm parameters, terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling test to evaluate DNA fragmentation and fluorescence in situ hybridization or immunohistochemistry for HPV were performed on semen samples from infected patients (n= 22), control subjects (n= 13) and on pooled control sperm samples incubated with HPV16-L1 (HPV capsid), before and after direct swim-up and modified swim-up (with added Heparinase-III). Moreover, cytofluorimetry for HPV detection was performed in pooled sperm pre- and post-incubation with HPV 16-L1 before and after direct and modified swim-up. Statistical analysis was performed with a two-tailed Student's t-test. Direct swim-up reduces the number of HPV-infected sperm by ~24% (PHPV DNA both from naturally and artificially infected sperm. Enzymatic treatment with Heparinase-III tended to decrease sperm motility, viability and DNA integrity but the effects were not significant. This study shows that Heparinase-III treatment seems not to affect spermatozoa in vitro and suggests that this treatment should be investigated further as a means of preparing sperm from patients who are infected with HPV in order to reduce the risk of HPV infection when using assisted reproduction techniques.

  8. Mitochondria functionality and sperm quality.

    Science.gov (United States)

    Amaral, Alexandra; Lourenço, Bárbara; Marques, Mónica; Ramalho-Santos, João

    2013-01-01

    Although mitochondria are best known for being the eukaryotic cell powerhouses, these organelles participate in various cellular functions besides ATP production, such as calcium homoeostasis, generation of reactive oxygen species (ROS), the intrinsic apoptotic pathway and steroid hormone biosynthesis. The aim of this review was to discuss the putative roles of mitochondria in mammalian sperm function and how they may relate to sperm quality and fertilisation ability, particularly in humans. Although paternal mitochondria are degraded inside the zygote, sperm mitochondrial functionality seems to be critical for fertilisation. Indeed, changes in mitochondrial integrity/functionality, namely defects in mitochondrial ultrastructure or in the mitochondrial genome, transcriptome or proteome, as well as low mitochondrial membrane potential or altered oxygen consumption, have been correlated with loss of sperm function (particularly with decreased motility). Results from genetically engineered mouse models also confirmed this trend. On the other hand, increasing evidence suggests that mitochondria derived ATP is not crucial for sperm motility and that glycolysis may be the main ATP supplier for this particular aspect of sperm function. However, there are contradictory data in the literature regarding sperm bioenergetics. The relevance of sperm mitochondria may thus be associated with their role in other physiological features, particularly with the production of ROS, which in controlled levels are needed for proper sperm function. Sperm mitochondria may also serve as intracellular Ca²⁺ stores, although their role in signalling is still unclear.

  9. The predictive value of various indicators of sperm for male fertility

    Directory of Open Access Journals (Sweden)

    A. Yu. Metelev

    2015-01-01

    Full Text Available Introduction. DNA fragmentation of sperm is one of the possible causes of reduced fertility potential of men. However, a significant correlation between conventional semen parameters and sperm DNA fragmentation was not found. This fact determines the relevance of the study of the influence of various parameters of sperm on male fertility.Materials and methods. The study included 60 men, aged 26–36 years (median – 30 years with idiopathic infertility and the level of DNA fragmentation of sperm is higher than 15 %. These men were treated with hyperbaric oxygen therapy, after 3 months in vitro fertilization performed partners of these men. DNA fragmentation of sperm cells was determined by TUNEL (upper limit of normal – 15 %. The level of reactive oxygen species (ROS of the ejaculate were determined by chemiluminescence (upper limit of normal – 0.64 mV/s.Results. The frequency of pregnancy in vitro fertilization was following: 62.8 and 64.7 % (p > 0.05 for the total number sperm of spermatozoa < 38 × 106 /ejaculate and ≥ 39 × 106 /ejaculate, respectively; 63.3 and 63.6 % (p > 0.05 for mobility (a + b of spermatozoa < 40 and ≥ 40 %, respectively; 58.3 and 64.6 % (p > 0.05 for normal forms of spermatozoa < 4 and ≥ 4 %, respectively; 67.3 and 20.0 % (p < 0.05 for the level of DNA fragmentation of sperm ≤ 15 and > 15 %, respectively; 64.9 and 33.3 % (p < 0.05 for the level of ROS in semen ≤ 0.64 and > 0.64 mV/s, respectively.Conclusion. The probability of pregnancy after in vitro fertilization significantly depends on the levels of sperm DNA fragmentation in the sperm and level of ROS in semen.

  10. Comparative evidence for the evolution of sperm swimming speed by sperm competition and female sperm storage duration in passerine birds.

    Science.gov (United States)

    Kleven, Oddmund; Fossøy, Frode; Laskemoen, Terje; Robertson, Raleigh J; Rudolfsen, Geir; Lifjeld, Jan T

    2009-09-01

    Sperm swimming speed is an important determinant of male fertility and sperm competitiveness. Despite its fundamental biological importance, the underlying evolutionary processes affecting this male reproductive trait are poorly understood. Using a comparative approach in a phylogenetic framework, we tested the predictions that sperm swim faster with (1) increased risk of sperm competition, (2) shorter duration of female sperm storage, and (3) increased sperm length. We recorded sperm swimming speed in 42 North American and European free-living passerine bird species, representing 35 genera and 16 families. We found that sperm swimming speed was positively related to the frequency of extrapair paternity (a proxy for the risk of sperm competition) and negatively associated with clutch size (a proxy for the duration of female sperm storage). Sperm swimming speed was unrelated to sperm length, although sperm length also increased with the frequency of extrapair paternity. These results suggest that sperm swimming speed and sperm length are not closely associated traits and evolve independently in response to sperm competition in passerine birds. Our findings emphasize the significance of both sperm competition and female sperm storage duration as evolutionary forces driving sperm swimming speed.

  11. Short-Term Storage of Human Spermatozoa in Electrolyte-Free Medium Without Freezing Maintains Sperm Chromatin Integrity Better Than Cryopreservation1

    Science.gov (United States)

    Riel, Jonathan M.; Yamauchi, Yasuhiro; Huang, Thomas T.F.; Grove, John; Ward, Monika A.

    2011-01-01

    Previous attempts to maintain human spermatozoa without freezing were based on short-term storage in component-rich medium and led to fast decline in motility and increased incidence of chromosome breaks. Here we report a new method in which sperm are maintained without freezing in an electrolyte-free medium (EFM) composed of glucose and bovine serum albumin. Human sperm were stored in EFM or human tubal fluid medium (HTFM) or were cryopreserved, and their motility, viability, and DNA integrity were examined at different intervals. Cryopreservation led to significant decline in sperm motility and viability and induced DNA fragmentation. Sperm stored in EFM maintained motility and viability for up to 4 and 7 wk, respectively, much longer than sperm stored in HTFM (comet assay, was also maintained significantly better in EFM than in HTFM. One-week storage in EFM yielded motility and viability similar to that of cryopreserved sperm, but DNA integrity was significantly higher, resembling that of fresh sperm. After several weeks of storage in EFM, sperm were able to activate oocytes, undergo chromatin remodeling, and form normal zygotic chromosomes after intracytoplasmic sperm injection. This study demonstrated that human spermatozoa can be stored in EFM without freezing for several weeks while maintaining motility, viability, and chromatin integrity and that 1-wk storage in EFM offers better protection of sperm DNA integrity than cryopreservation. Sperm storage in EFM may become a viable option for the physicians working in assisted reproduction technology clinics, which would avoid cryodamage. PMID:21593474

  12. Coupling sperm mediated gene transfer and sperm sorting techniques: a new perspective for swine transgenesis.

    Science.gov (United States)

    De Cecco, Marco; Spinaci, Marcella; Zannoni, Augusta; Bernardini, Chiara; Seren, Eraldo; Forni, Monica; Bacci, Maria Laura

    2010-09-15

    Flow cytometric separation of X and Y chromosome-bearing spermatozoa has been demonstrated to be effective in pigs, allowing the use of boar sexed semen in in vitro trials. Sperm Mediated Gene Transfer (SMGT) is a widely used and efficient technique for the creation of transgenic animals. The present research intended to prove that it is possible to associate sperm sexing with the SMGT technique in order to speed up the assessment of homozygous lines of transgenic pigs. In the first experiment, the sorting protocol was modified in order to obtain the highest DNA uptake by sorted spermatozoa. In the second experiment, spermatozoa that had undergone only sperm sorting, only SMGT, or both procedures (Sorted-SMGT) were used for in in vitro fertilization of in vitro matured oocytes. In the third experiment, transformed blastocysts of the desired gender (male) were obtained with Sorted-SMGT in an in vitro fertilization trial. The method we developed here allowed us to produce transgenic swine blastocysts of pre-determined gender, giving a positive answer at the aim to couple SMGT and sperm sorting in swine, obtaining fertile spermatozoa able to produce transgenic embryos of pre-determined gender. Copyright 2010 Elsevier Inc. All rights reserved.

  13. DNA binding of sunitinib: Spectroscopic evidence via circular dichroism and nuclear magnetic resonance.

    Science.gov (United States)

    Kiss, Eszter; Mirzahosseini, Arash; Hubert, Ágnes; Ambrus, Attila; Őrfi, László; Horváth, Péter

    2018-02-20

    Sunitinib is a non-selective tyrosine kinase inhibitor, but in its chemical structure there can be discovered certain features, which suggest the ability to bind to DNA. These elements are the planar aromatic system and the tertiary amine function, which is protonated at the pH of the organism. In this study, the binding of the drug sunitinib to DNA was investigated using circular dichroism (CD), 1 H NMR and UV spectroscopies, along with CD melting. For these studies DNA was isolated from calf thymus (CT), salmon fish sperm (SS), and chicken erythrocyte (CE), however for our purposes an artificially constructed and highly purified plasmid DNA (pUC18) preparation proved to be the most suitable. DNA binding of the drug was confirmed by shifts in the characteristic CD bands of the DNA, the appearance of an induced CD (ICD) signal in the upper absorption region of sunitinib (300 nm-500 nm), and the evidence from CD melting studies and the NMR. Based on the CD and NMR measurements, it can be assumed that sunitinib has a multiple-step binding mechanism. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Transgene transmission in chickens by sperm-mediated gene ...

    Indian Academy of Sciences (India)

    Transgene transmission in newborn chicks was evaluated by in vivo enhanced green fluorescent protein (EGFP) expression, RT-PCR and PCR analysis. DNA internalization was limited to sperm samples washed twice. The presence of DMSO or DMAc during transfection had no effect on fertilization or hatching rates.

  15. Morphology, Structure of Dimorphic Sperm, and Reproduction in the Hermaphroditic Commensal Bivalve Pseudopythina tsurumaru (Galeommatoidea: Kellidae)

    DEFF Research Database (Denmark)

    Lützen, Jørgen; Jespersen, Åse; Takahashi, Tohru

    2004-01-01

    Galeommatoide, commensal bivalve, reproduction, dimorphic sperm, sperm ultrastructure, spermatozeugma......Galeommatoide, commensal bivalve, reproduction, dimorphic sperm, sperm ultrastructure, spermatozeugma...

  16. The role of human papillomavirus on sperm function.

    Science.gov (United States)

    Garolla, Andrea; Pizzol, Damiano; Foresta, Carlo

    2011-08-01

    To review the role of human papillomavirus (HPV) on sperm parameters, fertility and implication of the use of infected sperm cells in assisted reproduction. HPVs are agents of the most common sexually transmitted disease and can lead to warts and cancers both in men and women.A high incidence of HPV infection has been demonstrated in sperm from sexually active men with and without risk factors for HPV and from infertile patients. Semen infection is associated to an impairment of sperm parameters suggesting a possible role in male infertility. Interestingly, it has been demonstrated that when HPV is present in semen only a percentage of total cells are infected and the virus can be localized in sperm or in exfoliated cells with different impact on sperm motility. Moreover, infected sperm are able to penetrate the oocyte, to deliver HPV genome in the oocyte and HPV genes can be actively transcribed by the fertilized oocyte. Recently an increased risk of pregnancy loss has been demonstrated in couples undergoing in-vitro fertilization and particularly when HPV DNA was present in semen samples of male partners. To date, no effective treatment, control strategy and prevention is provided for men despite the reported high incidence of HPV semen infection. Because this infection in men is also a problem for partners, and because growing evidence suggests that semen infection may cause infertility and early miscarriage, more attention should be paid to male HPV infection. This study reviews the more recent literature about the role of HPV infection on sperm function and human reproduction.

  17. Sperm whale clicks

    DEFF Research Database (Denmark)

    Møhl, Bertel; Wahlberg, Magnus; Madsen, Peter T.

    2000-01-01

    . A sound generator weighing upward of 10 tons and with a cross-section of 1 m is expected to generate high-intensity, directional sounds. This prediction from the Norris and Harvey theory is not supported by published data for sperm whale clicks ~source levels of 180 dB re 1 mPa and little, if any...... of the continental shelf off Andenes, Norway, in the summers of 1997 and 1998. With this system, source levels up to 223 dB re 1 mPa peRMS were recorded. Also, source level differences of 35 dB for the same click at different directions were seen, which are interpreted as evidence for high directionality....... This implicates sonar as a possible function of the clicks. Thus, previously published properties of sperm whale clicks underestimate the capabilities of the sound generator and therefore cannot falsify the Norris and Harvey theory....

  18. Cryopreservation of Fish Sperm

    Science.gov (United States)

    Kurokura, Hisashi

    Present status of research activities in cryopreservation of fish gamete in aquaculture field was introduced. More than 59 fish species have been reported in the research histories and nearly half of them were studied during recent 10 years. This means that the research activities are increasing, though commercial profit have not obtained yet. Fish species of which sperm can successfully cryopreserved is still limited comparing to numerous species in telost. One of the major obstacle for improvement of the technique is existence of wide specie specific variance in the freezing tolerance of fish sperm. The varianc can possibly be explaind thorugh the informations obtained by the studies in comparative spermatology, which is recently activated field in fish biology.

  19. Identifying salmon lice transmission characteristics between Faroese salmon farms

    DEFF Research Database (Denmark)

    Kragesteen, Trondur J.; Simonsen, Knud; Visser, AW

    2017-01-01

    between farms is of vital importance in developing treatment management plans to combat salmon lice infestations. Using a particle tracking model forced by tidal currents, we show that Faroese aquaculture farms form a complex network. In some cases as high as 10% of infectious salmon lice released at one...... farm site enter a neighboring fjord containing another farm site. Farms were characterized as emitters, receivers or isolated, and we could identify two clusters of farms that were largely isolated from each other. The farm characteristics are a valuable input for the development of management plans...... for the entire Faroese salmon industry...

  20. Quality decline and oxidative damage in sperm of freshwater crab Sinopotamon henanense exposed to lead.

    Science.gov (United States)

    Li, Na; Hou, Yu-Hua; Jing, Wei-Xin; Dahms, Hans-Uwe; Wang, Lan

    2016-08-01

    Lead (Pb) induces male infertility in vertebrates. Whether lead is related to reproductive abnormalities in aquatic invertebrates remains uncertain. In this work, effects of Pb on the sperm quality and oxidative stress of the freshwater crab Sinopotamon henanense were investigated after 3, 5 and 7d exposure to different Pb concentrations (0, 3.675, 7.35, 14.7, 29.4 and 58.8mg/L). Sperm quality indices including sperm plasma-membrane integrity and acrosomal-membrane integrity were measured by flow cytometry. DNA integrity was measured by fluorescence microscopy. The results showed that Pb levels in sperm increased significantly upon Pb exposure in most treated groups, sperm plasma-membrane integrity, acrosomal-membrane integrity, and DNA integrity were reduced at higher concentrations after 5 d and 7d. Oxidative stress of sperm induced by Pb was reflected in significant up-regulation of reactive oxygen species (ROS) levels after 3, 5 and 7d. A significant reduction of the total antioxidant capacity levels occurred after exposure to 14.7mg/L Pb and above at 7d compared to the control. The results of oxidative damage to lipids, proteins and DNA of sperm showed that malondialdehyde, protein carbonylation and DNA-protein crosslinks were increased in a concentration- and time-dependent manner. Our findings document that Pb can induce harmful effects on several reproductive endpoints in a freshwater crab. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Paternal aging and associated intraindividual alterations of global sperm 5-methylcytosine and 5-hydroxymethylcytosine levels.

    Science.gov (United States)

    Jenkins, Timothy G; Aston, Kenneth I; Cairns, Bradley R; Carrell, Douglas T

    2013-10-01

    To evaluate the relative intraindividual changes in sperm 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) levels associated with age and to compare the levels of 5-hmC in mature human sperm to blood DNA. Prospective research study. University-based andrology and in vitro fertilization (IVF) laboratory. Fifteen known fertile sperm donors, 22 other known fertile controls, and 41 male blood donors from a general population tissue bank. None. Measurements of global 5-mC and 5-hmC levels via an enzyme-linked immunosorbent assay (ELISA)-based assay. Global sperm 5-mC levels exhibit a statistically significant increase with age, and a similar trend was seen in 5-hmC levels. On average, in the age ranges we analyzed, 5-mC increased by 1.76% per year, and 5-hmC, though more variable, increased by approximately 5% per year. Additionally, we found that 5-hmC levels in sperm are 32.59% of that found in blood DNA. Global sperm DNA methylation patterns are stable over short periods of time but increase with age, which raises important questions regarding the risks of advanced paternal age. Additionally, as we would predict for a transcriptionally quiescent cell type, 5-hmC levels are statistically significantly lower in human sperm than in blood DNA. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  2. Reliability of the comet assay in cryopreserved human sperm.

    Science.gov (United States)

    Duty, S M; Singh, N P; Ryan, L; Chen, Z; Lewis, C; Huang, T; Hauser, R

    2002-05-01

    Although the comet assay has potential value for measuring DNA damage in large epidemiological human sperm studies, it is impractical to perform the assay daily on fresh semen samples. Therefore, before its use in epidemiological studies, the reliability of the comet assay in measuring DNA damage in cryopreserved sperm should be compared with that in fresh human sperm. Semen samples from 16 men were cryopreserved in liquid nitrogen (LN) using four methods: flash freezing with and without cryopreservative, and programmable freezing with and without cryopreservative. Neutral microgel electrophoresis was performed and comets were stained with YOYO-1. Comet length was measured using an eyepiece micrometer at x400 magnification. The highest correlation was between comet assay results obtained from fresh human semen compared with semen flash frozen without cryopreservative (R = 0.88). However, the method of cryopreservation, as compared with other sources of variability, accounted for only 6% of the variability. Inter-individual variability accounted for 20%, and individual sperm-to-sperm variability within an ejaculate accounted for 65%. Flash-freezing in LN without cryopreservative most closely reproduced the results obtained using fresh human semen samples, and thereby represents the most appropriate cryopreservation method for human semen in epidemiological studies utilizing the neutral comet assay.

  3. Cryopreservation of microencapsulated canine sperm.

    Science.gov (United States)

    Shah, Shambhu; Otsuki, Tsubasa; Fujimura, Chika; Yamamoto, Naoki; Yamashita, Yasuhisa; Higaki, Shogo; Hishinuma, Mitsugu

    2011-03-01

    The objective was to develop a method for cryopreserving microencapsulated canine sperm. Pooled ejaculates from three beagle dogs were extended in egg yolk tris extender and encapsulated using alginate and poly-L-lysine at room temperature. The microcapsules were cooled at 4 °C, immersed in pre-cooled extender (equivalent in volume to the microcapsules) to reach final concentration of 7% (v/v) glycerol and 0.75% (v/v) Equex STM paste, and equilibrated for 5, 30 and 60 min at 4 °C. Thereafter, microcapsules were loaded into 0.5 mL plastic straws and frozen in liquid nitrogen. In Experiment 1, characteristics of microencapsulated canine sperm were evaluated after glycerol addition at 4 °C. Glycerol exposure for 5, 30 and 60 min did not significantly affect progressive motility, viability, or acrosomal integrity of microencapsulated sperm compared with pre-cooled unencapsulated sperm (control). In Experiment 2, characteristics of frozen-thawed canine microencapsulated sperm were evaluated at 0, 3, 6, and 9 h of culture at 38.5 °C. Pre-freeze glycerol exposure for 5, 30, and 60 min at 4 °C did not influence post-thaw quality in unencapsulated sperm. Post-thaw motility and acrosomal integrity of microencapsulated sperm decreased more than those of unencapsulated sperm (P < 0.05) following glycerol exposure for 5 min. However, motility, viability and acrosomal integrity of microencapsulated sperm after 30 and 60 min glycerol exposure were higher than unencapsulated sperm cultured for 6 or 9 h (P < 0.05). In conclusion, since microencapsulated canine sperm were successfully cryopreserved, this could be a viable alternative to convention sperm cryopreservation in this species. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. Genetic variation in time and space : Microsatellite analysis of extinct and extant populations of Atlantic salmon

    DEFF Research Database (Denmark)

    Eg Nielsen, Einar; Hansen, Michael Møller; Loeschcke, V.

    1999-01-01

    Information on genetic composition of past and present populations may be obtained by analyzing DNA from archival samples. A study is presented on the genetic population structure of extant and extinct local populations of Atlantic salmon from 1913 to 1989 using dried scales as a source of DNA...

  5. Cryopreservation of epididymal stallion sperm.

    Science.gov (United States)

    Olaciregui, M; Gil, L; Montón, A; Luño, V; Jerez, R A; Martí, J I

    2014-02-01

    Any event that makes semen collection or mating impossible, such as death, castration, or injury, may terminate a stallion's breeding career. Fortunately, stallion sperm which are capable of fertilization can be harvested from the epididymis, and frozen for future use. However, the fertility of frozen-thawed epididymal sperm has been found to be lower than that of ejaculated sperm. Therefore, this study aimed to optimize the fertility of frozen epididymal stallion sperm by investigating the effects of different cryoprotectants and freezing protocols on sperm quality. Dimethylformamide was tested alone or combination with pasteurized egg yolk as substitute of fresh egg yolk. In addition, the effect of the pre-freeze stabilization on sperm quality was analyzed. Heterospermic samples obtained from stallion epididymis were collected and cryopreserved in lactose-egg-yolk extender or in the same extender with varying content of cryoprotectant and content of egg yolk, stabilized and no-stabilized. Sperm motility, viability, hypoosmotic swelling test (HOST) and acrosome integrity were evaluated post-thawing. No improvement was observed on the replacement of fresh yolk by pasteurized egg yolk, whereas the results suggest that dimethylformamide is a cryoprotectant suitable for cryopreservation of equine epididymal semen, even better than glycerol. In addition, we found that the stabilization before freezing on epididymal stallion sperm, can improve sperm quality parameters. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Salmon Population Summary - Impacts of climate change on Pacific salmon

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — This work involves 1) synthesizing information from the literature and 2) modeling impacts of climate change on specific aspects of salmon life history and...

  7. SALMON 2100: THE FUTURE OF WILD PACIFIC SALMON

    Science.gov (United States)

    Many experts have concluded that wild salmon recovery efforts in western North America (especially California, Oregon, Washington, Idaho, and southern British Columbia), as earnest, expensive, and socially disruptive as they currently are, do not appear likely to sustain biologic...

  8. Luminal fluid of epididymis and vas deferens contributes to sperm chromatin fragmentation.

    Science.gov (United States)

    Gawecka, Joanna E; Boaz, Segal; Kasperson, Kay; Nguyen, Hieu; Evenson, Donald P; Ward, W Steven

    2015-12-01

    Do the luminal fluids of the epididymis and the vas deferens contribute to sperm chromatin fragmentation (SCF) in mice? The luminal fluids of both organs are required for activating SCF in mice, but the vas deferens luminal fluid does this more efficiently than that of the epididymis. Mice sperm have the ability to degrade their DNA in an apoptotic-like fashion when treated with divalent cations in a process termed SCF. SCF has two steps: the induction of reversible double-strand DNA breaks at the nuclear matrix attachment sites, followed by the irreversible degradation of DNA by nuclease. Single stranded DNA breaks accompany SCF. Luminal fluids from two reproductive organs of the mouse (B6D2F1 strain), the epididymis and vas deferens, were extracted and tested for SCF activation with divalent cations using four different combinations of the sperm and the surrounding luminal fluids: (i) in situ--sperm were kept in their luminal fluid and activated directly; (ii) reconstituted--sperm were centrifuged and resuspended in their luminal fluid before SCF activation; (iii) mixed--sperm were centrifuged and resuspended in the luminal fluid of the other organ; (iv) no luminal fluid--sperm were centrifuged and reconstituted in buffer. All four experiments were performed without (controls) and with divalent cations (resulting in SCF). For each experimental condition, two different mice were used and the analyses averaged. DNA damage by SCF was analyzed by three different methods, the sperm chromatin structure assay (SCSA), terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) analysis and field inversion gel electrophoresis. In all three assays that we used, the vas deferens luminal fluid was much more efficient in stimulating SCF in the sperm from either source than that of the epididymis (P sperm were capable of initiating lower levels of SCF in the absence of luminal fluid (P sperm during their transit providing a mechanism to degrade the DNA. We

  9. Altered sperm chromatin structure in mice exposed to sodium fluoride through drinking water.

    Science.gov (United States)

    Sun, Zilong; Niu, Ruiyan; Wang, Bin; Wang, Jundong

    2014-06-01

    This study investigated the effects of sodium fluoride (NaF) on sperm abnormality, sperm chromatin structure, protamine 1 and protamine 2 (P1 and P2) mRNA expression, and histones expression in sperm in male mice. NaF was orally administrated to male mice at 30, 70, and 150 mg/l for 49 days (more than one spermatogenic cycle). Sperm head and tail abnormalities were significantly enhanced at middle and high doses. Similarly, sperm chromatin structure was also adversely affected by NaF exposure, indicating DNA integrity damage. Furthermore, middle and high NaF significantly reduced the mRNA expressions of P1 and P2, and P1/P2 ratio, whereas the sperm histones level was increased, suggesting the abnormal histone-protamine replacement. Therefore, we concluded that the mechanism by which F induced mice sperm abnormality and DNA integrity damage may involved in the alterations in P1, P2, and histones expression in sperm of mice. Copyright © 2012 Wiley Periodicals, Inc.

  10. Subversive practices of sperm donation - globalizing Danish sperm

    DEFF Research Database (Denmark)

    Willum Adrian, Stine

    into how the bending of boundaries by “inappropriate parents”, fertility travellers, private sperm banks and fertility clinics have been part in negotiating the changes of the legislation in practice, and thus been part of developing a Danish industry of sperm banking. The presentation is based on a multi...

  11. Subversive practices of sperm donation - globalizing Danish sperm

    DEFF Research Database (Denmark)

    Willum Adrian, Stine

    as the use of donated sperm continuously has been debated as an ethical issue, and increasingly been regulated. In this presentation I will discuss how Denmark became a destination for fertility travelling (sperm donation) as a result of various subversive strategies of family making. The article inquires...

  12. Factors influencing boar sperm cryosurvival.

    Science.gov (United States)

    Roca, J; Hernández, M; Carvajal, G; Vázquez, J M; Martínez, E A

    2006-10-01

    Optimal sperm cryopreservation is a prerequisite for the sustainable commercial application of frozen-thawed boar semen for AI. Three experiments were performed to identify factors influencing variability of postthaw sperm survival among 464 boar ejaculates. Sperm-rich ejaculate fractions were cryopre-served using a standard freezing-thawing procedure for 0.5-mL plastic straws and computer-controlled freezing equipment. Postthaw sperm motility (assessed with a computer-assisted semen analysis system) and viability (simultaneously probed by flow cytometry analysis after triple-fluorescent stain), evaluated 30 and 150 min postthaw, were used to estimate the success of cryopreservation. In the first experiment, 168 unselected ejaculates (1 ejaculate/boar), from boars of 6 breeds with a wide age range (8 to 48 mo), were cryopreserved over a 12-mo period to evaluate the predictive value of boar (breed and age), semen collection, transport variables (season of ejaculate collection, interval between collections, and ejaculate temperature exposure), initial semen traits, and sperm quality before freezing on sperm survival after freezing-thawing. In Exp. 2, 4 ejaculates from each of 29 boars, preselected according to their initial semen traits and sperm quality before freezing, were collected and frozen over a 6-mo period to evaluate the influence of interboar and intraboar ejaculate variability in the survival of sperm after cryopreservation. In Exp. 3, 12 ejaculates preselected as for Exp. 2, from each of 15 boars with known good sperm cryosurvival, were collected and frozen over a 12-mo period to estimate the sustainability of sperm cryosurvival between ejaculates over time. Boar and semen collection and transport variables were not predictive of sperm cryosurvival among ejaculates. Initial semen traits and sperm quality variables observed before freezing explained 23.2 and 10.9%, respectively, of the variation in postthaw sperm motility and viability. However, more that

  13. Embryo quality and IVF treatment outcomes may correlate with different sperm comet assay parameters.

    Science.gov (United States)

    Tomsu, M; Sharma, V; Miller, D

    2002-07-01

    Standard semen parameters have proven poor at predicting the outcomes of IVF treatment cycles. As recent studies suggest that the male genome may play an important role in early embryogenesis, this study attempts to correlate the level of sperm DNA damage in fresh semen and prepared sperm with the outcomes of conventional IVF treatment cycles. Forty patients embarking on IVF treatment were recruited into this prospective observational study. Both fresh semen and PureSperm-prepared sperm were processed using a modified comet assay 3-6 months prior to the patients' IVF treatment cycles. Comet head DNA (mean and integrated head density) and tail DNA parameters (length and moment) were measured separately. Significant correlations between total sperm concentration and between comet length, moment, mean head density with embryo quality were detected in fresh semen and prepared sperm. Surprisingly, no significant correlations between head and tail parameters were detected. Comet head and tail DNA parameters appear to be potentially useful as predictors of embryo quality and IVF outcomes, especially in couples with unexplained subfertility. The lack of correlation between head and tail parameters may be due to a different mechanism of DNA damage within these two compartments.

  14. Expression of human protamine P1 in sperm of transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Wyrobek, A.J.; Keith, C.; Stilwell, J.; Lowe, X. [Lawrence Livermore National Laboratory, CA (United States); Anderson, G. [Univ. of California, Davis, CA (United States)

    1994-12-31

    Transgenic mice were produced by pronuclear injection with DNA constructs containing human protamine P1 cDNA recombined with a murine protamine P1 promoter, and were identified by PCR. Expression of human P1 was investigated using huplm, a monoclonal antibody specific for human P1, applied to murine testicular cells, smears of epididymal sperm, and smears of detergent-isolated sperm nuclei. Various antibodies and nontransgenic littermates were used as controls. Two male founders (T3 and T7) sired more than five generations of transgenic offspring each with continued expression of human P1 in their sperm. Transgenic animals appear of normal fertility with sperm of typical nuclear morphology. The human P1 transgene was expressed postmeioticly in both lines, as expected. Nearly 100% of sperm of T3 and T7 hemizygotes labeled with huplm, consistent with complete diffusion of human P1 protein through the intercellular bridge of spermatogenic cells. Human P1 labeling of sperm nuclei was not visibly affected by sonication or by treatment with the detergent MATAB or the reducing agent DTT. A third founder female (T5) showed a transmission pattern consistent with insertion of the transgene into an X chromosome; her transgenic offspring expressed human P1 in only a small fraction of sperm. Human P1 transgenes may serve as efficient targets for germinal mutations and transgenicmice may provide promising models for investigating the DNA complexes.

  15. Social imaginaries, sperm and whiteness

    DEFF Research Database (Denmark)

    Andreassen, Rikke

    2017-01-01

    This article analyses narratives about so-called Viking babies and Viking sperm. Over the last few years an increasing number of British single women and lesbian couples have been creating families by becoming pregnant with Danish donor sperm, termed ‘Viking sperm’. Through analyses of British...

  16. Sperm preparation for ART

    Directory of Open Access Journals (Sweden)

    Schill Wolf-Bernhard

    2003-11-01

    Full Text Available Abstract The onset of clinical assisted reproduction, a quarter of a century ago, required the isolation of motile spermatozoa. As the indication of assisted reproduction shifted from mere gynaecological indications to andrological indications during the years, this urged andrological research to understand the physiology of male germ cell better and develop more sophisticated techniques to separate functional spermatozoa from those that are immotile, have poor morphology or are not capable to fertilize oocytes. Initially, starting from simple washing of spermatozoa, separation techniques, based on different principles like migration, filtration or density gradient centrifugation evolved. The most simple and cheapest is the conventional swim-up procedure. A more sophisticated and most gentle migration method is migration-sedimentation. However, its yield is relatively small and the technique is therefore normally only limited to ejaculates with a high number of motile spermatozoa. Recently, however, the method was also successfully used to isolate spermatozoa for intracytoplasmic sperm injection (ICSI. Sperm separation methods that yield a higher number of motile spermatozoa are glass wool filtration or density gradient centrifugation with different media. Since Percoll® as a density medium was removed from the market in 1996 for clinical use in the human because of its risk of contamination with endotoxins, other media like IxaPrep®, Nycodenz, SilSelect®, PureSperm® or Isolate® were developed in order to replace Percoll®. Today, an array of different methods is available and the selection depends on the quality of the ejaculates, which also includes production of reactive oxygen species (ROS by spermatozoa and leukocytes. Ejaculates with ROS production should not be separated by means of conventional swim-up, as this can severely damage the spermatozoa. In order to protect the male germ cells from the influence of ROS and to stimulate

  17. Integridade de DNA e morfologia espermática de ratos submetidos a campos eletromagnéticos de baixa frequência durante diferentes períodos do desenvolvimento = DNA integrity and sperm morphology of Wistar rats exposed to low frequency electromagnetic fields during different periods of development

    Directory of Open Access Journals (Sweden)

    Bruno Mendes Tenorio

    2011-07-01

    Full Text Available A exposição da sociedade a Campos Eletromagnéticos (CEM vemaumentando vertiginosamente em virtude da ampla expansão tecnológica observada nos últimos anos. Tanto a geração, como a distribuição e a utilização de energia elétrica podem gerar Campos Eletromagnéticos de baixa freqüência (50 e 60 Hz. Pesquisas vêm demonstrando que a exposição a estes CEM podem proporcionar alterações fisiológicassignificativas, apesar disto, ainda não estão totalmente esclarecidos a extensão destes efeitos, nem os mecanismos de ação que envolve a interação dos CEM com os organismos biológicos. O presente trabalho teve como principal objetivo verificar os efeitos dos CEM (60 Hz e 1 mT sobre a integridade de DNA e morfologia espermática de ratos sexualmente maduros, que foram expostos ao CEM durante diferentes períodos do seu desenvolvimento. Os resultados obtidos neste trabalho não encontraram indícios de alterações no DNA dos espermatozóides, porém, foram observadas alterações significativas na morfologia dos espermatozoides após a exposição ao CEM. Estas alterações namorfologia espermática podem reduzir o potencial reprodutivo. Portanto, devemos considerar o CEM como um potencial risco a saúde pública, recomendando-se a realização de mais pesquisas buscando estabelecer níveis seguros de exposição aos CEM.The society’s exposure to electromagnetic fields (EMF has been growing considerable due to the great technological expansion observed in the last few years. Generation as well as distribution and use of electric energy can generate low frequency electromagnetic fields (50 and 60 Hz. Issues have been demonstrating that EMF exposure could provoke significantly physiological changes, however, the extension of EMF effects weren’t totally clarified. The major objective of this issue was to evaluate the EMF (60 Hz and 1 mTeffects on DNA integrity and sperm morphology in Wistar rats with mature sexuality that were

  18. Etiopathogenesis of autoimmune responses against sperm

    Directory of Open Access Journals (Sweden)

    V. A. Bozhedomov

    2012-01-01

    Full Text Available The autoimmune reactions against sperm, which are accompanied by the elaboration of antisperm antibodies (AsAb, are one of the causes of male infertility.Objective: to specify the role of different factors (other than obstruction in the etiology of male immune infertility.Subjects and methods. Clinical and laboratory studies were made in 536 males from infertile couples (their age 18–45 years; a control group comprised fertile men whose wives were 8–16 weeks pregnant (n = 82. Their sperms were examined in accordance with the WHO recommendations. AsAbs in the sperm were determined by a MAR test. Oxidative stress was estimated using luminol-dependent chemiluminescence. Chromosome damage was identified from DNA fragmentation by chromatin dispersion in an inert agarose gel, by making a microscopic examination of halo formation after acid DNA denaturation and nuclear protein lysis. The blood levels of interferons (IFN and their natural and in vitro induced production were determined by the Campbell method. Reproductive tract infections were diagnosed by polymerase chain reaction.Results. There is a high significant correlation between the quantity of AsAb, on the one hand, and previous orchitis, as well as subclinical testicular injury, on the other hand. There was no correlation between varicocele and AsAb, but in the former, the risk of immune infertility and orchitis increases after injury; varicocelectomy promotes a reduction in AsAb with the higher degree of varicocele and a less marked autoimmune process. AsAbs are observed during Chlamydia infection with the increased production of IFN-γ. Cryptorchidism and orchiopexy, parotitis, epididymitis, herniotomy, chronic bacterial prostatitis, and other potential risk factors for decreased male fertility had no significant impacts on an odds ratio for developing immune infertility. In the majority (41 % of cases, immune infertility seems to be idiopathic, but it is accompanied by the

  19. Etiopathogenesis of autoimmune responses against sperm

    Directory of Open Access Journals (Sweden)

    V. A. Bozhedomov

    2014-11-01

    Full Text Available The autoimmune reactions against sperm, which are accompanied by the elaboration of antisperm antibodies (AsAb, are one of the causes of male infertility.Objective: to specify the role of different factors (other than obstruction in the etiology of male immune infertility.Subjects and methods. Clinical and laboratory studies were made in 536 males from infertile couples (their age 18–45 years; a control group comprised fertile men whose wives were 8–16 weeks pregnant (n = 82. Their sperms were examined in accordance with the WHO recommendations. AsAbs in the sperm were determined by a MAR test. Oxidative stress was estimated using luminol-dependent chemiluminescence. Chromosome damage was identified from DNA fragmentation by chromatin dispersion in an inert agarose gel, by making a microscopic examination of halo formation after acid DNA denaturation and nuclear protein lysis. The blood levels of interferons (IFN and their natural and in vitro induced production were determined by the Campbell method. Reproductive tract infections were diagnosed by polymerase chain reaction.Results. There is a high significant correlation between the quantity of AsAb, on the one hand, and previous orchitis, as well as subclinical testicular injury, on the other hand. There was no correlation between varicocele and AsAb, but in the former, the risk of immune infertility and orchitis increases after injury; varicocelectomy promotes a reduction in AsAb with the higher degree of varicocele and a less marked autoimmune process. AsAbs are observed during Chlamydia infection with the increased production of IFN-γ. Cryptorchidism and orchiopexy, parotitis, epididymitis, herniotomy, chronic bacterial prostatitis, and other potential risk factors for decreased male fertility had no significant impacts on an odds ratio for developing immune infertility. In the majority (41 % of cases, immune infertility seems to be idiopathic, but it is accompanied by the

  20. Linking individual migratory behaviour of Atlantic salmon to their genetic origin

    DEFF Research Database (Denmark)

    Jepsen, Niels; Eg Nielsen, Einar; Deacon, M.

    2005-01-01

    by increased aquaculture activities. The interpretation of results from studies of survival and behaviour of fish from such “mixed stocks” require information of the genetic background of individual fish. We used genetic analysis combined with radiotelemetry to study upstream migration of Atlantic salmon....... The results indicate that stocked, foreign salmon had a slightly higher mortality and moved more up and down in the river than the native salmon did, but all salmon had problems passing the physical obstructions in the river. The DNA analyses enabled us to compare the behaviour of fish of different genetic...... origin, but the interpretation of the results was hampered by a high mortality of tagged fish. This study demonstrates that the combination of recent genetic methods and telemetry provides a potent tool for better management of mixed stock fisheries...

  1. Selection of Sperm Based on Hypo-Osmotic Swelling May Improve ICSI Outcome: A Preliminary Prospective Clinical Trial

    Directory of Open Access Journals (Sweden)

    Nasim Charehjooy

    2014-03-01

    Full Text Available Background: The intra-cytoplasmic sperm injection (ICSI technique selects sperm according to morphology and motility. However, these parameters cannot predict the chromatin integrity of sperm. Considering the detrimental effects of DNA-damaged sperm on reproductive outcomes, novel sperm selection procedures have been proposed to circumvent the possibility of inseminating DNA-damaged sperm. It has been shown that different potential hypo-osmotic swelling test (HOST patterns possess the potential to differentiate between sperm that have intact or damaged chromatin. Therefore, for the first time, this preliminary study evaluates the role of HOST as a sperm selection procedure in a clinical setting. Materials and Methods: In this preliminary prospective clinical trial study, we divided infertile couples diagnosed with male infertility into two groups. In the treatment group (n=39, half of the oocytes were inseminated by sperm selected following density gradient centrifugation (DGC group. The remaining oocytes from the treatment group were inseminated by sperm chosen according to HOST pattern (c, d or e following DGC processing (HOST group. In the control group (n=63, all oocytes were inseminated by sperm chosen after DGC. Results: There was a significantly higher percentage of embryos that had good quality, implantation, and chemical pregnancy rates in the HOST group compared to the DGC group (p≤0.05. Conclusion: This study has shown that selecting sperm according to membrane functionality (HOST pattern rather morphology and viability may open a new window in our approach for determining the appropriate sperm for ICSI, particularly in individuals with severe male infertility (Registration Number: IRCT201307087223N2.

  2. Sperm membrane modulation by Sapindus mukorossi during sperm maturation.

    Science.gov (United States)

    Nivsarkar, Manish; Shrivastava, Neeta; Patel, Manoj; Padh, Harish; Bapu, Cherian

    2002-09-01

    To observe the alterations in the biochemical and biophysical changes in the sperm membrane during sperm maturation in male rats treated with the water extract of the fruit pericarp of S. mukorossi. Adult male Sprague-Dawley rats were gavaged the aqueous extract of the fruit pericarp of S. mukorossi at a dose of 50 mg/kg/d for 45 days. On day 46, the sperm parameters were observed in different sections of the epididymis and the sperm superoxide dismutase and the lipid peroxidation was determined and compared with the controls. The testis and epididymis were routinely prepared for histological examination under the light microscope. No significant differences in the sperm number and morphology were observed between the control and treated groups. However, a significant inhibition (P<0.05-0.01) of sperm motility in the caput, corpus and cauda regions of the epididymis was seen in the treated group. No significant histopathological changes were found in the testis and epididymis. The important finding was that in the treated animals, the spermatozoa showed an abnormal distribution of the superoxide dismutase activity, being minimum in the caput and maximum in the corpus, which was just opposite to that of the controls. The study provides a unique observation where the plant extract alters the sperm membrane physiology without change the testicular and epididymal morphology.

  3. Relationship between sperm parameters and intracytoplasmic sperm injection outcome

    Directory of Open Access Journals (Sweden)

    Shahla Chaichian

    2015-12-01

    Full Text Available Objectives: With the adventure of intracytoplasmic sperm injection (ICSI technique, great progresses have developed in the treatment of infertility. Concentration on the properties of male’s gamete has been encouraged by the increasing concerns about the causes of ICSI failure. We hence conducted this study to investigate the probable association of sperm parameters with ISCI outcome. Methods: A total of 523 couples referred to Isfahan Fertility and Sterility Center from January 2007 to June 2008 for ICSI. Semen analysis was performed before ICSI procedure according to the WHO criteria. Patients were assigned into successful ICSI (case and failed ICSI (control groups. Sperm parameters were then compared between the 2 groups. Results: One hundred and six patients (20% had successful ICSI results (case group compared with 417 couples (80% with undesirable ICSI outcomes (control group. Among evaluated factors, sperm agglutination (p = 0.007, sperm concentration (p = 0.043, leukocytospermia (p = 0.026 and head abnormality of sperm (p = 0.019 showed statistically significant differences between two groups with differing ICSI results. None of the other semen parameters revealed significant differences between these two groups. Conclusion: Our study showed that some sperm parameters are associated with desirable ICSI outcome. However, it is unclear whether these associations are causal.

  4. Sperm production and sperm morphology of Swedish Warmblood stallions.

    Science.gov (United States)

    Einarsson, S; Dalin, A-M; Lundeheim, N

    2009-02-01

    The present study investigated daily sperm output and sperm morphology of fresh semen in eight Swedish Warmblood stallions aged 5-8 years. They were used for artificial insemination, and their fertility during the breeding season of semen collection exceeded 60% per cycle. One ejaculate of semen was collected daily for 10 consecutive days from each stallion. The gel-free volume was measured, and the sperm concentration was assessed with a Bürker chamber. The volume of gel-free fraction was multiplied by the sperm concentration to give the total number of spermatozoa (TSN). Sperm morphology was examined in ejaculates collected on days 2, 5 and 10. An aliquot from each ejaculate was fixed in 1 ml formol-saline immediately after collection and examined under a phase-contrast microscope (magnification 1000x) to assess morphological abnormalities. Furthermore smears were prepared and stained according to Williams (carbolfuchsin-eosin) for a more detailed examination of the sperm heads under a light microscope (magnification 1000x). Analysis of variance was applied to data. Total spermatozoa number decreased progressively during the first 8 days of collection, and daily sperm output (DSO) was calculated as mean TSN of collections on days 8-10, being 6.4 x 10(9) spermatozoa. The overall percentages of morphologically normal spermatozoa in ejaculates collected on days 2, 5 and 10 were above 70%, being significantly lower in ejaculate 2 (68.6%) compared with ejaculates 5 and 10 (72.9% respectively 75.3%).

  5. New potential antitumoral fluorescent tetracyclic thieno[3,2-b]pyridine derivatives: interaction with DNA and nanosized liposomes

    Directory of Open Access Journals (Sweden)

    Calhelha Ricardo

    2011-01-01

    Full Text Available Abstract Fluorescence properties of two new potential antitumoral tetracyclic thieno[3,2-b]pyridine derivatives were studied in solution and in liposomes of DPPC (dipalmitoyl phosphatidylcholine, egg lecithin (phosphatidylcholine from egg yolk; Egg-PC and DODAB (dioctadecyldimethylammonium bromide. Compound 1, pyrido[2',3':3,2]thieno[4,5-d]pyrido[1,2-a]pyrimidin-6-one, exhibits reasonably high fluorescence quantum yields in all solvents studied (0.20 ≤ ΦF ≤ 0.30, while for compound 2, 3-[(p-methoxyphenylethynyl]pyrido[2',3':3,2]thieno[4,5-d]pyrido[1,2-a]pyrimidin-6-one, the values are much lower (0.01 ≤ ΦF ≤ 0.05. The interaction of these compounds with salmon sperm DNA was studied using spectroscopic methods, allowing the determination of intrinsic binding constants, K i = (8.7 ± 0.9 × 103 M-1 for compound 1 and K i = (5.9 ± 0.6 × 103 M-1 for 2, and binding site sizes of n = 11 ± 3 and n = 7 ± 2 base pairs, respectively. Compound 2 is the most intercalative compound in salmon sperm DNA (35%, while for compound 1 only 11% of the molecules are intercalated. Studies of incorporation of both compounds in liposomes of DPPC, Egg-PC and DODAB revealed that compound 2 is mainly located in the hydrophobic region of the lipid bilayer, while compound 1 prefers a hydrated and fluid environment.

  6. Orientation of sea urchin sperms in static magnetic fields: Compared to human sperms

    Science.gov (United States)

    Sakhnini, Lama; Dairi, Maheen

    In this study we report on magnetic orientation of sea urchin and human sperms. The sea urchin and human sperms became oriented parallel to the magnetic field (1 T maximum). The human sperms were totally oriented with magnetic field at about 600 mT. However, the sea urchin sperms show different behavior due to morphological differences between them and the human sperms.

  7. Freezing-induced uptake of disaccharides for preservation of chromatin in freeze-dried stallion sperm during accelerated aging.

    Science.gov (United States)

    Oldenhof, Harriëtte; Zhang, Miao; Narten, Katharina; Bigalk, Judith; Sydykov, Bulat; Wolkers, Willem F; Sieme, Harald

    2017-11-07

    Non-viable freeze-dried sperm have intact chromatin and can be used for fertilization via intracytoplasmic sperm injection. Freeze-dried sperm preferably should be stored at 4°C or lower, because DNA damage accumulates during storage at room temperature. Disaccharides are known to protect biomolecules both during freezing and drying, by forming a highly viscous glassy state. Their use for intracellular protection is challenging because cellular membranes are normally impermeable for disaccharides. In the current study, we demonstrate that membrane impermeable compounds, including lucifer yellow and trehalose, are taken up by stallion sperm when exposed to freezing. Trehalose uptake likely occurs during freezing-induced membrane phase transitions, but does not allow sperm to survive drying. Stallion sperm was freeze-dried in various formulations consisting of reducing or non-reducing sugars combined with albumin as bulking agent. Chromatin stability was studied during storage at 37°C, using the flow cytometric sperm chromatin structure assay and microscopic assessment of chromatin dispersion and DNA fragmentation after electrophoresis. Freeze-drying did not affect sperm chromatin, irrespective of the formulation that was used. DNA fragmentation index (DFI) values ranged from 5 - 8%. If sperm was freeze-dried without protectants or in a combination of glucose and proteins, DNA damage rapidly accumulated during storage at 37°C, reaching DFI values of respectively 95 ± 4 and 64 ± 42% after 1 month. DFI values of sperm freeze-dried with sucrose or trehalose ranged between 9 - 11% and 33 - 52% after 1 and 3 months storage, respectively. In conclusion, freeze-drying sperm with disaccharides results in uptake during freezing, which greatly reduces chromatin degradation during dried storage. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e

  8. Sperm glucose transport and metabolism in diabetic individuals.

    Science.gov (United States)

    Dias, Tânia R; Alves, Marco G; Silva, Branca M; Oliveira, Pedro F

    2014-10-01

    Individuals with diabetes mellitus (DM) present marked reduction in sperm quality and higher DNA damage in spermatozoa, evidencing that this metabolic disorder impairs male fertility. These effects are related to defective testicular metabolic pathways and signaling, resulting in altered sperm metabolism. Spermatozoa metabolize several substrates to ensure energy supplies and any alteration in this feature compromise sperm quality. For ATP production, spermatozoa require substrate availability and the involvement of specific hexose membrane carriers. DM is known to modulate the spermatozoa substrate consumption and/or production due to altered glycolytic behavior. In fact, glucose uptake and metabolism is highly deregulated in diabetic individuals. Herein, we present an overview of the implications of DM in sperm glucose uptake and metabolism. The understanding of these processes is essential to identify key mechanisms associated with DM-related male (in)fertility. Moreover, it may contribute to the development of therapeutics to counteract the dysfunction induced by DM in sperm metabolism. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  9. Recombinant hamster oviductin is biologically active and exerts positive effects on sperm functions and sperm-oocyte binding.

    Directory of Open Access Journals (Sweden)

    Xiaojing Yang

    Full Text Available Studies carried out in several mammalian species suggest that oviductin, also known as oviduct-specific glycoprotein or OVGP1, plays a key role in sperm capacitation, fertilization, and development of early embryos. In the present study, we used recombinant DNA technology to produce, for the first time, recombinant hamster OVGP1 (rHamOVGP1 in human embryonic kidney 293 (HEK293 cells. rHamOVGP1 secreted in the culture medium was purified by affinity chromatography. The resulting protein migrated as a poly-dispersed band of 160-350 kDa on SDS-PAGE corresponding to the molecular mass of the native HamOVGP1. Subsequent mass spectrometric analysis of the purified rHamOVGP1 confirmed its identity as HamOVGP1. Immunocytochemistry demonstrated binding of rHamOVGP1 to the mid-piece and head of hamster sperm and to the zona pellucida (ZP of ovarian oocytes. In vitro functional experiments showed that addition of rHamOVGP1 in the capacitation medium further enhanced tyrosine phosphorylation of two sperm proteins of approximately 75 kDa and 83 kDa in a time-dependent manner. After 3 hours of incubation in the presence of rHamOVGP1, a significant increase in acrosome reaction was measured. Pretreatment of either sperm or oocyte with 20 μg/ml of rHamOVGP1 prior to sperm-egg binding assay significantly increased the number of sperm bound to the ZP. Addition of rHamOVGP1 in the medium during sperm-egg binding with either oocyte or sperm pretreated with rHamOVGP1 also saw an increase in the number of sperm bound to ZP. In all experimental conditions, the effect of rHamOVGP1 on sperm-oocyte binding was negated by the addition of monoclonal anti-HamOVGP1 antibody. The successful production and purification of a biologically active rHamOVGP1 will allow further exploration of the function of this glycoprotein in reproductive function.

  10. Modifications in sperm quality of Wister Albino Rats by Ethanol ...

    African Journals Online (AJOL)

    Epidydymal sperm was collected and analyzed using standard procedures. Sperm analyses involved sperm count, sperm morphology test and sperm motility test. At the doses administered, P. amarus extract affected the sperm number, morphology and motility of treated animals. Epididymal sperm count and motility were ...

  11. Identification of multiple HPV types on spermatozoa from human sperm donors.

    Science.gov (United States)

    Kaspersen, Maja D; Larsen, Peter B; Ingerslev, Hans Jakob; Fedder, Jens; Petersen, Gert Bruun; Bonde, Jesper; Höllsberg, Per

    2011-03-29

    Human papillomaviruses (HPV) may cause sexually transmitted disease. High-risk types of HPV are involved in the development of cervical cell dysplasia, whereas low-risk types may cause genital condyloma. Despite the association between HPV and cancer, donor sperm need not be tested for HPV according to European regulations. Consequently, the potential health risk of HPV transmission by donor bank sperm has not been elucidated, nor is it known how HPV is associated with sperm. The presence of 35 types of HPV was examined on DNA from semen samples of 188 Danish sperm donors using a sensitive HPV array. To examine whether HPV was associated with the sperm, in situ hybridization were performed with HPV-6, HPV-16 and -18, and HPV-31-specific probes. The prevalence of HPV-positive sperm donors was 16.0% and in 66.7% of these individuals high-risk types of HPV were detected. In 5.3% of sperm donors, two or more HPV types were detected. Among all identified HPV types, 61.9% were high-risk types. In situ hybridization experiments identified HPV genomes particularly protruding from the equatorial segment and the tail of the sperm. Semen samples from more than one in seven healthy Danish donors contain HPV, most of them of high-risk types binding to the equatorial segment of the sperm cell. Most HPV-positive sperm showed decreased staining with DAPI, indicative of reduced content of DNA. Our data demonstrate that oncogenic HPV types are frequent in men.

  12. Identification of multiple HPV types on spermatozoa from human sperm donors.

    Directory of Open Access Journals (Sweden)

    Maja D Kaspersen

    Full Text Available Human papillomaviruses (HPV may cause sexually transmitted disease. High-risk types of HPV are involved in the development of cervical cell dysplasia, whereas low-risk types may cause genital condyloma. Despite the association between HPV and cancer, donor sperm need not be tested for HPV according to European regulations. Consequently, the potential health risk of HPV transmission by donor bank sperm has not been elucidated, nor is it known how HPV is associated with sperm. The presence of 35 types of HPV was examined on DNA from semen samples of 188 Danish sperm donors using a sensitive HPV array. To examine whether HPV was associated with the sperm, in situ hybridization were performed with HPV-6, HPV-16 and -18, and HPV-31-specific probes. The prevalence of HPV-positive sperm donors was 16.0% and in 66.7% of these individuals high-risk types of HPV were detected. In 5.3% of sperm donors, two or more HPV types were detected. Among all identified HPV types, 61.9% were high-risk types. In situ hybridization experiments identified HPV genomes particularly protruding from the equatorial segment and the tail of the sperm. Semen samples from more than one in seven healthy Danish donors contain HPV, most of them of high-risk types binding to the equatorial segment of the sperm cell. Most HPV-positive sperm showed decreased staining with DAPI, indicative of reduced content of DNA. Our data demonstrate that oncogenic HPV types are frequent in men.

  13. Female sperm storage in amphibians.

    Science.gov (United States)

    Sever, David M

    2002-02-01

    The three orders of extant amphibians are Gymnophiona, Anura, and Urodela. Although all gymnophionans apparently have internal fertilization and many are viviparous, female sperm storage is unknown. Internal fertilization has convergently evolved in a few anurans, but females of just one species, Ascaphus truei, are known to possess oviductal sperm storage tubules (SSTs). The SSTs of A. truei are similar anatomically to such glands in squamate reptiles. This similarity is convergence due to similar functional adaptations and/or internal design constraints. In salamanders and newts (Urodela), absence of sperm storage in females is the ancestral condition (three families). In the derived condition, sperm storage occurs in cloacal glands called spermathecae, and their possession is a synapomorphy for females in the suborder Salamandroidea (seven families). Salamandroids are the only vertebrates with cloacal sperm storage glands. In this paper, a phenetic analysis of variation in spermathecal characters reveals patterns of convergence in certain spermathecal characters in unrelated taxa that breed in similar habitats. In the family Salamandridae, a role in sperm nutrition for the spermathecal epithelium is questioned, and the widespread occurrence of spermiophagy is related to other reproductive strategies. I propose how the packaging of sperm in structurally different types of spermathecae may influence male paternity. Copyright 2002 Wiley-Liss, Inc.

  14. Sperm quality evaluation in Solea senegalensis during the reproductive season at cellular level.

    Science.gov (United States)

    Beirão, J; Soares, F; Herráez, M P; Dinis, M T; Cabrita, E

    2009-12-01

    Sperm quality seems to be one of the reasons for the reproduction constraints faced by Senegalese sole (Solea senegalensis) aquaculturists. Previous studies in this species indicated that the sperm quality of individuals kept in culture varies throughout the year and that different sperm subpopulations can be identified in ejaculates according to the motility pattern of spermatozoa. Aiming to better understand factors affecting sole sperm quality in captivity, sperm of 11 males was assessed during the reproductive season using different parameters: motility characteristics using CASA analysis; cell plasma membrane resistance to seawater hyperosmolarity; DNA fragmentation with single-cell gel electrophoresis; and early apoptosis, labeled with Annexin-V FITC. Computer-assisted sperm analyses motility data were treated using multivariate analysis to identify the presence of different spermatozoa subpopulations according to their motility pattern. Four distinct sperm subpopulations were obtained: Subpop1, which includes fast linear spermatozoa; Subpop2, made up of fast nonlinear spermatozoa; Subpop3, which includes slow linear spermatozoa; and Subpop4, which contains slow nonlinear spermatozoa. The sperm subpopulation structure varied with time after activation and with male. Low cell resistance to the seawater hyperosmotic conditions was noticed. The Annexin-V assay allowed the identification of an apoptotic population ranging from 6% to 20%. A high percentage of cells (64.1%) showed a DNA fragmentation level below 30%, but these values varied significantly between males. DNA fragmentation appears to be related to cell membrane resistance to hyperosmotic conditions faced by the cells when in contact with seawater. This condition seems to modulate the composition of the motile sperm population and performance after activation. This phenomenon could be related to the spermatozoa maturation process.

  15. Exposure of rainbow trout milt to mercury and cadmium alters sperm motility parameters and reproductive success

    Energy Technology Data Exchange (ETDEWEB)

    Dietrich, Grzegorz J., E-mail: dietrich@pan.olsztyn.pl [Department of Gamete and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10-747 Olsztyn (Poland); Dietrich, Mariola; Kowalski, R.K. [Department of Gamete and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10-747 Olsztyn (Poland); Dobosz, Stefan [Department of Salmonid Research, Inland Fisheries Institute, Rutki 83-330 Zukowo (Poland); Karol, Halina; Demianowicz, Wieslaw; Glogowski, Jan [Department of Gamete and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10-747 Olsztyn (Poland)

    2010-05-10

    In the current work, seminal plasma was used for the first time as an incubation medium for monitoring short-time exposure effects of sublethal concentrations of mercury and cadmium ions on rainbow trout sperm. Sperm motility parameters (CASA) and hatching rates were used as gamete quality markers. Additionally live/dead sperm viability test and comet assay of DNA fragmentation were performed. We demonstrated that computer-assisted sperm motility analysis (CASA) may serve as a predictor of reproductive success, when milt contaminated with heavy metals is used. Results presented in this study demonstrate that mercury ions altered sperm motility characteristics at 1-10 mg Hg{sup 2+}/l and 10 mg Cd{sup 2+}/l and hatching rates at 10 mg Hg{sup 2+}/l and 10 mg Cd{sup 2+}/l after 4 h of exposure. Although mercury ions affected sperm motility parameters immediately after dilution with milt as well as at 4 h of exposure, no differences in sperm motility parameters were found between intact and mercury-treated milt after 24 h of exposure. Our results suggest that rainbow trout seminal plasma has a protective role against the toxic effects of mercury ions of rainbow trout sperm motility.

  16. Effect of seminal plasma and sperm of boars valued by freezability on seminal cryopreservation

    Directory of Open Access Journals (Sweden)

    Francisco Javier Henao Uribe

    2016-07-01

    Full Text Available The aim of this study was to determine the effect of sperm and seminal plasma (SP on the freezability of porcine semen. Semen of eight commercial males from two farms in the central-western region of Colombia (four boars in each farm was frozen and tested to select two males with high freezability (MHF and two with low freezability (MLF, according to the percentage of functionally competent sperm (FCS. Immediately after the collection was completed, the SP and sperm from the males selected were separated by centrifugation to combine the two types of plasma with the two types of sperm, incubate them for three hours and then freeze them. The variables evaluated were: sperm morphology, structural and functional integrity of plasmatic membrane, progressive and total motility, DNA fragmentation, acrosome integrity, capacitated sperm and FCS. The combination of sperm and plasma of MHF recorded the highest value (P<0.01 of acrosome integrity (24.3 ± 0.082 vs 6.076 ± 0.16 when compared to MLF plasma and cells. Membrane structural integrity was higher (P<0.01 with MHF (53.56 ± 0.0395 than with MLF plasma (47.49 ± 0.0419. The differences in porcine semen freezability depend on interactions between seminal plasma and sperm.

  17. A study of the antioxidant effect of alpha lipoic acids on sperm quality

    Directory of Open Access Journals (Sweden)

    Siti Fatimah Ibrahim

    2008-01-01

    Full Text Available OBJECTIVE: Assisted reproductive techniques are useful in helping infertile couples achieve successful conception. Initial studies have shown that sperm cryopreservation, one step in assisted reproduction, causes a dramatic reduction in sperm quality. This has been attributed to, among other things, free radical activities. The aim of the present study was to minimize this oxidative attack by adding an antioxidant into the sperm microenvironment. Alpha lipoic acids were selected for this purpose for their efficient free radical scavenging properties and solubility in lipid and aqueous phases. METHODS: For this investigation, semen from six Boer bucks was pooled. Seminal analysis of the baseline prior to incubation of samples with different concentrations of Alpha lipoic acids (0.00625, 0.0125, 0.025, 0.05, 0.1 mmol/ml was performed, and post-seminal analysis was conducted after a one-hour incubation. The comet assay was used to observe the effect of Alpha lipoic acids on sperm DNA integrity. Statistical analysis using an unpaired t-test with a significance level of p<0.05 was then performed. RESULTS: Our results indicate that the sperm motility rate was improved after incubation with Alpha lipoic acids at a concentration of 0.02 mmol/ml. This concentration was also capable of reducing DNA damage. CONCLUSION: In conclusion, Alpha lipoic acids renders cryoprotection to sperm, thereby improving sperm quality.

  18. Sperm structure and sperm transfer in Pseudopythina subsinuata (Bivalvia, Galeommatoidea)

    DEFF Research Database (Denmark)

    Jespersen, Åse

    2009-01-01

    to the elongate cells. Most females contain one to three "sperm trees", structures consisting of a short stem and numerous branches. They are firmly implanted in the abfrontal part of the gill filament and protrude into the posterior part of the suprabranchial (brooding) chamber. Implantation of the trees causes...... the receptacle and thereupon fuse. A similar process is known in the allied P. tsurumaru, but the resulting structure ("sperm-carrying body") is not attached to the gills....

  19. Sperm Proteome Maturation in the Mouse Epididymis.

    Science.gov (United States)

    Skerget, Sheri; Rosenow, Matthew A; Petritis, Konstantinos; Karr, Timothy L

    2015-01-01

    In mammals, transit through the epididymis, which involves the acquisition, loss and modification of proteins, is required to confer motility and fertilization competency to sperm. The overall dynamics of maturation is poorly understood, and a systems level understanding of the complex maturation process will provide valuable new information about changes occurring during epididymal transport. We report the proteomes of sperm collected from the caput, corpus and cauda segments of the mouse epididymis, identifying 1536, 1720 and 1234 proteins respectively. This study identified 765 proteins that are present in sperm obtained from all three segments. We identified 1766 proteins that are potentially added (732) or removed (1034) from sperm during epididymal transit. Phenotypic analyses of the caput, corpus and cauda sperm proteomes identified 60 proteins that have known sperm phenotypes when mutated, or absent from sperm. Our analysis indicates that as much as one-third of proteins with known sperm phenotypes are added to sperm during epididymal transit. GO analyses revealed that cauda sperm are enriched for specific functions including sperm-egg recognition and motility, consistent with the observation that sperm acquire motility and fertilization competency during transit through the epididymis. In addition, GO analyses revealed that the immunity protein profile of sperm changes during sperm maturation. Finally, we identified components of the 26S proteasome, the immunoproteasome, and a proteasome activator in mature sperm.

  20. The relationship between sperm morphology and chromatin integrity in the koala (Phascolarctos cinereus) as assessed by the Sperm Chromatin Dispersion test (SCDt).

    Science.gov (United States)

    Johnston, Stephen D; López-Fernández, Carmen; Gosálbez, Altea; Zee, Yengpeng; Holt, William V; Allen, Camryn; Gosálvez, Jaime

    2007-01-01

    Koala (Phascolarctos cinereus) sperm nuclei show a tendency to swell after cryopreservation, but it is uncertain whether this phenomenon is associated with DNA fragmentation. In this study, we validated a modified version of the sperm chromatin dispersion test (SCDt) for use with koala spermatozoa, which is the first use of the test for a marsupial. Cryopreserved spermatozoa (multiple straws) from a single koala were used to explore the relationship between sperm morphology, viability, chromatin dispersion, and DNA fragmentation. A SCDt prototype kit (Sperm Halomax) was specifically developed for koala spermatozoa with the use of a lysing solution that did not contain dithiothreitol. DNA fragmentation of lysed and nonlysed spermatozoa was examined in microgel slides and validated by means of in situ nick translation (ISNT). The SCDt was then applied to the analysis of extended and frozen-thawed semen samples of 3 different koalas. Spermatozoa were classified into 3 distinct koala sperm morphotypes (KSMs) after the SCDt: 1) KSM-1, rod-shaped cells with no halo of DNA; 2) KSM-2, rounded nuclei with various degrees of halo formation about a dense chromatin core; and 3) KSM-3, rod-shaped or rounded nuclei consisting of an inner chromatin core but with large dispersed halos of stellar chromatin. Although ISNT after the SCDt did not label KSM-1, both KSM-2 and KSM-3 stained positively for DNA fragmentation. ISNT was not able to differentiate between KSM-2 and KSM-3. Although application of the SCDt to the spermatozoa of another 3 koalas showed no difference in the percentage of the 3 sperm morphotypes found between extended and frozen-thawed semen, thawed spermatozoa incubated at 35 degrees C for 2 hours showed an increase in the incidence of KSM-3 and a corresponding decrease in KSM-2. We propose that KSM-1 and KSM-2 represent nuclei that show either no, or only limited, sperm DNA fragmentation, respectively. It is likely that the halos formed around KSM-2 are from DNA