The integrity of sperm chromatin in young tropical composite bulls

Sperm chromatin fragmentation is associated with subfertility, but its relationship with age progression in young bulls is poorly understood. The objective was to assess sperm chromatin fragmentation during the early post-pubertal development of 20 tropical composite bulls, using a sperm chromatin structure assay (SCSA) and sperm-bos-halomax (SBH). Bulls were subjected to bull breeding soundness evaluation (BBSE) at mean ages of 13, 18, and 24 mo. Traits measured included liveweight (WT), body condition score (BCS) and scrotal circumference (SC). Semen samples were collected by electroejaculation and assessed for mass activity (MA), motility (Mot), concentration (conc), sperm morphology and chromatin fragmentation. Concentration (r = 0.34, P = 0.0076), Mot (r = 0.36, P = 0.0041) and percentage of morphologic normal sperm (percent normal sperm (PNS); r = 0.31, P = 0.0132) were positively correlated with age. The percentage of sperm with proximal droplets (PD) was negatively correlated with age (r = −0.28, P = 0.0348), whereas neither SCSA nor SBH results were significantly correlated with age. The percentage of sperm with chromatin fragmentation using SCSA was correlated with PNS (r = −0.53, P < 0.0001), the percentage of sperm with head abnormalities (r = 0.68, P < 0.0001) and the percentage of intact sperm (Int) with SBH (r = −0.26, P = 0.0456). In summary, for assessment of sperm chromatin fragmentation, samples could be equally collected at 13, 18 or 24 mo of age, as results did not vary with age.     

Assessment of sperm quality through fluorometry and sperm chromatin structure assay in relation to field fertility of frozen-thawed semen from Swedish AI bulls

We investigated fluorometry to study sperm viability and flow cytometry to study sperm chromatin structure. We also assessed sperm quality after thawing relative to field fertility after AI as shown by 56-day non-return rates (56-d NRR) Frozen-thawed semen samples were obtained from 20 Swedish Red and White bulls (1 to 3 semen batches/bull) and the fertility data were based on 6,369 AIs. Fluorometry enabled simultaneous detection of sperm viability and concentration in Hoechst 33258-stained semen samples. Sperm chromatin structure assay (SCSA) evaluated denaturability of sperm nuclear DNA in situ after acid treatment. The intensity of fluorescence in non-permeabilized samples was negatively (r = -0.60, P < 0.001) correlated with microscopically-assessed sperm viability, and the fluorescence of permeabilized semen samples significantly (r = 0.67, P < 0.001) correlated with sperm concentration as assessed by hemocytometry. From the fluorescence output, the calculated percentage of damaged cells was negatively (r = -0.71, P < 0.001) correlated with the number of live cells derived from the microscopic assessment of sperm viability and concentration. This variable was significantly correlated with fertility results both at batch (r = -0.39, P < 0.05), and bull (r = -0.57, P < 0.01) levels. The SCSA variables SDalphat and COMPalphat were significantly (r = -0.59-0.64, P < 0.001) correlated with sperm viability variables after thawing but only the COMPalphat correlated significantly (r = -0.53, P < 0.05) with fertility results and solely at the bull level. The results indicate that fluorometric assessment is in good agreement with other practiced procedures and can be performed with sufficient accuracy. The SCSA may be a valuable complement for routinely practiced microscopic evaluation of sperm morphology of AI bull semen     

The relationship of increased susceptibility of sperm DNA to denaturation and fertility in the stallion

The relationship between fertility and susceptibility of sperm DNA to denaturation was determined in a group of 84 actively breeding, clinically fertile stallions. Susceptibility of DNA to denaturation was determined using the sperm chromatin structure assay (SCSA). The SCSA measures, mean of alpha-t (mean alpha t), standard deviation of alpha-t (SD alpha t), and the COMP of alpha-t (cells outside the main population)] were significantly correlated with the percentage seasonal pregnancy rate (SPR; mean alpha t, r = -0.24, P < or = 0.05; % COMP alpha t, r = -0.27, P < or = 0.05); percentage pregnant per first cycle (FCP; SD alpha t, r = -0.30, P < or = 0.01; % COMP alpha t, r = -0.42, P < or = 0.0001); and the percentage pregnant per cycle (PC; mean alpha t, r = -0.31, P < or = 0.01; SD alpha t, r = -0.32, P < or = 0.01; % COMP alpha t, r = -0.41, P < or = 0.0001). This study describes detectable intrinsic variation in sperm chromatin structure among fertile stallions (SPR, mean = 83%; FCP, mean = 58%; PC, mean = 57%) in an active breeding population (number of mares bred/stallion/year, mean = 37), in the absence of overt reproductive abnormalities and apparent diseases such that an increase in the susceptibility of sperm DNA to denaturation is associated with reduced fertility, both in terms of efficiency of reproduction (FCP and PC) and seasonal pregnancy rate (SPR). Both COMP alpha t and mean alpha t were useful indicators of fertility, with COMP alpha t being the only SCSA value able to identify mean differences between fertility groupings for SPR and FCP, and overall it was the most reliable indicator of fertility in this group of stallions. The SCSA is able to evaluate a compartment of the spermatozoa which is different from that of traditional tests for sperm quality such as motility and morphology.     

Evaluation of DNA fragmentation of freeze-dried mouse sperm using a modified sperm chromatin structure assay

Freeze-dried sperm is applicable to the storage and transport of genetic material. We recently reported that freeze-dried mouse sperm required temperatures lower than −80 °C for long-term preservation and concluded that it was necessary to explore freeze-drying conditions before long-term preservation of sperm becomes viable. In the current study, we determined the percentage of sperm with elevated levels of DNA fragmentation using a sperm chromatin structure assay (SCSA), a technique not previously reported for the evaluation of freeze-dried mouse sperm. We applied SCSA to mouse sperm freeze-dried under four conditions (various combinations of primary drying pressure of 0.04 and 0.37 hPa and storage temperatures of 4 and −80 °C) and compared the results with the embryonic developmental rates of freeze-dried sperm after intracytoplasmic sperm injection (ICSI) and with comet assay results. The DNA fragmentation index values under the four conditions determined by SCSA had good correlation with the developmental rate to the blastocyst stage of embryos from ICSI with freeze-dried mouse sperm. We concluded that the SCSA method applied to freeze-dried mouse sperm after storage will lead to not only clarification of the developmental rate derived from ICSI using freeze-dried sperm but also to improvements in the freeze-drying and storage processes.     

Subtle membrane changes in cryopreserved bull semen in relation with sperm viability,chromatin structure,and field fertility

This study investigated the use of annexin-V/PI assay to assess sub lethal changes in bull spermatozoa post-thawing, and to further relate these changes to results obtained by fluorometric assessment of sperm viability and sperm chromatin structure assay (SCSA), as well as field fertility (as 56-day non-return rates, 56-day NRR) after AI. Frozen-thawed semen samples were obtained from 18 Swedish Red and White bulls (one to three semen batches/bull) and fertility data was based on 6900 inseminations. The annexin-V/PI assay revealed that post-thaw semen samples contained on average 41.8+/-7.5% annexin-V-positive cells. Most of the annexin-V-positive cells were dying cells, i.e. also PI-positive. The incidence of annexin-V-positive cells was negatively related (r=-0.59, P<0.01) to the percentage of viable cells, as detected by fluorometry. The incidence of annexin-V-positive spermatozoa significantly correlated to the SCSA variable xalphat (r=0.53, P<0.05). The incidence of annexin-V-negative, dead cells was the only annexin-V/PI assay variable that correlated significantly with fertility both at batch (r=-0.40, P<0.05), and bull (r=-0.56, P<0.05) levels. Among sperm viability variables, subjectively assessed sperm motility (r=0.52-0.59, P<0.01), CASA-assessed sperm motility (r=0.43-0.61, P<0.05), and the incidence of live spermatozoa, expressed as total numbers (r=0.39-0.54, P<0.05), or percentage values (r=0.68-0.68, P<0.01), correlated significantly with field fertility both at batch, and bull levels. Among the SCSA variables, only the COMP alphat correlated significantly (r=0.33-0.51, P<0.05) with fertility results. The results indicate a certain proportion of bull spermatozoa express PS on their surface after thawing, e.g. they have altered membrane function, and that the incidence of such cells is inversely correlated to sperm viability, and positively correlated to abnormal sperm chromatin condensation since they eventually undergo necrosis.     

Assessment of chromatin status (SCSA) in epididymal and ejaculated sperm in Iberian red deer, ram and domestic dog

Abnormal chromatin condensation is not detected using classical techniques for sperm analysis. SCSA has demonstrated its usefulness in sperm chromatin analysis in several species (human, bull, stallion and boar). In this work, we studied sperm samples from red deer, ram and dog to analyze the differentiation of chromatin structure applying SCSA in epididymal and ejaculated spermatozoa. Epididymal samples were obtained from the caput, corpus and cauda by means of cuts, and ejaculated ones were obtained by electroejaculation (deer), artificial vagina (ram) and digital manipulation (dog). SCSA results suggested different critical points in sperm maturation (spermatozoa with loose chromatin to more condensed chromatin) among species: from corpus to cauda in ram and from caput to corpus in deer and dog. Moreover, we also detected differences in ruminants and dog, reflected in the appearance of SCSA plots. Indeed, ram and deer samples rendered two peaks within the sperm main population (sperm with condensed chromatin), whereas only one was detected in dog. Although some differences were observed between cauda and ejaculated samples, SCSA parameters indicated good chromatin condensation, making these samples suitable for germplasm banking. Some species-dependent modifications in the analysis of the results may be necessary to take full advantage of its analytical power.     

Scrotal heat stress induces altered sperm chromatin structure associated with a decrease in protamine disulfide bonding in the stallion

A variety of testicular insults can induce changes in the structure of spermatozoal chromatin, resulting in spermatozoal DNA that is more susceptible to acid-induced denaturation. The degree of change in the DNA can be measured using the sperm chromatin structure assay (SCSA). The SCSA measures the relative amounts of single- and double-stranded DNA after staining with the metachromatic dye, acridine orange. Here we used a stallion model (n = 4) to study the effects of scrotal heat stress on spermatozoal DNA. This model was created by insulating stallion testes for 48 h and collecting sperm daily thereafter for 60 days. Changes in the SCSA were then correlated with protamine disulfide content and protamine types and levels. Results of the SCSA indicated that the susceptibility of spermatozoal DNA to denaturation was dependent on the spermatogenic cell stage that the ejaculated sperm was in at the time of the heat stress. Spermatozoa with altered DNA had a decrease in the extent of disulfide bonding that was associated with an increase in the susceptibility of DNA to denaturation. However, there were no detectable changes in either the protamine type or level. Thus, in this model, decreased disulfide bonding is associated with an increased susceptibility of spermatozoal DNA to denaturation in the absence of protamine changes.     

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

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 < 0.017) than the sorted samples and that there was no difference between the samples in tail length (TL) (P = 0.36). The SCSA showed that the DNA fragmentation index (DFI) was higher for conventional than the sorted samples (P = 0.011), but the standard deviation of DFI (SD-DFI) was higher for the sorted samples (P < 0.001). We conclude that the NCA and SCSA can be used in assessing DNA integrity in bovine 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.     

Comparison between the toluidine blue stain and the Feulgen reaction for evaluation of rabbit sperm chromatin condensation and their relationship with sperm morphology

Sperm chromatin alteration is an important feature that can affect fertility of the male rabbit. This study compared toluidine blue staining with Feulgen reaction (as methods for evaluating chromatin alteration) and investigated the relationship between sperm morphology and chromatin alteration. Seven hundred rabbit ejaculates of animals with unknown fertility were used. Primary and secondary morphological sperm abnormalities were evaluated in semen smears with phase-contrast microscopy. Chromatin alterations were evaluated in semen smears stained with toluidine blue (pH 4.0 and 5.0) and with the Feulgen reaction. While the three methods were equally efficacious for identification of chromatin alterations, toluidine blue staining was more appropriate to characterize the intensity of chromatin alterations. The correlation between primary sperm defects and chromatin alteration was high and positive, suggesting that sperm chromatin structure affected sperm head morphology. The correlation between secondary sperm defects and chromatin alteration was also positive, but lower. The final chromatin compaction occurs in the epididymus, where secondary sperm defects originate. Therefore, the causes of secondary sperm defects could also intervene with final chromatin compaction. In summary, the toluidine blue stain was an effective means of evaluating the sperm chromatin alteration in rabbit spermatozoa.     

Normospermic versus teratospermic domestic cat sperm chromatin integrity evaluated by flow cytometry and intracytoplasmic sperm injection

Teratospermia (>60% of morphologically abnormal spermatozoa) is well documented in felids. Even morphologically normal spermatozoa from teratospermic ejaculates have reduced ability to undergo tyrosine phosphorylation, acrosome react, and bind and penetrate oocytes compared with normospermic (<40% abnormal spermatozoa) counterparts. However, it is unknown whether fertilization deficiencies originate at a nuclear level. This study examined whether fertilization failure also was attributable to abnormal sperm chromatin, using the sperm chromatin structure assay (SCSA), in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI). Aliquots of unprocessed and swim-up-processed (to isolate morphologically normal spermatozoa) spermatozoa from teratospermic and normospermic domestic cats were analyzed by the flow cytometric SCSA. Swim-up-processed sperm were incubated with in vivo-matured oocytes or used for ICSI. Teratospermic ejaculates expressed more (P < 0.05) chromatin heterogeneity (abnormal chromatin structure) than their normospermic counterparts, both in unprocessed and swim-up-processed samples. Fertilization success in vitro was higher (P < 0.05) from normo- compared with teratospermic inseminates. Similar (P > 0.05) proportions of oocytes fertilized after ICSI using spermatozoa from normo- and teratospermic cats. Results reveal that teratospermia in the cat is expressed at the nuclear level as increased sperm chromatin heterogeneity, but ICSI showed that this does not apparently affect fertilization rates if the zona pellucida and oolemma can be bypassed.     

Evaluating human sperm DNA integrity: relationship between 8-hydroxydeoxyguanosine quantification and the sperm chromatin structure assay

In our work, we have used 8-hydroxy-deoxyguanosine (8-OH-dG), one of the major oxidative products of sperm DNA, in a population of patients consulting for infertility. We found an inverse relationship between sperm concentration and the log of the ratio of 8-OH-dG to dG (P < 0.01). On the same patients' sperm samples, the sperm chromatin structure assay (SCSA) was performed. An inverse relationship was observed between the DNA fragmentation index and sperm concentration (P < 0.001). There was also a positive relationship between SCSA and log 8-OH-dG/dG. This indicates that DNA fragmentation measured by the SCSA originates in part from oxidative products. In a few patients, antioxidant treatment decreased the DNA fragmentation index below the threshold of 30% that is crucial for subfertility.     

The sperm chromatin structure assay: a review of clinical applications

The sperm chromatin structure assay (SCSA) was introduced by as a method to determine the susceptibility of sperm DNA to denaturation and how those results related to fertility. This initial study used human sperm and was followed by studies in bulls and boars . This assay was one of the first to introduce the technique of flow cytometry, which has the ability to evaluate specific sperm compartments of large numbers of sperm in a short time, as a methodology to evaluate sperm quality and further define the relationship of sperm quality to fertility. For any assay to be of use clinically, it must not only be validated and adapted for the species of interest, but guidelines that associate specific levels of fertility with assay results must be defined. This review will describe how our laboratory uses the SCSA for clinical diagnosis of reduced fertility in the stallion.     

Magneto‐optical characteristics of human sperms: normal and deformed

In this study we report on magnetic orientation of human sperms. Samples were taken from 17 donors. Normal human sperms became oriented with their long axis perpendicular to the magnetic field (1 T maximum). Total orientation was achieved with magnetic field of about 1 T, while for abnormal sperms the magnetic behavior was different. The dependence of the measured degree of orientation on the intensity of the magnetic field was in good agreement with the theoretical equation for the magnetic orientation of diamagnetic substances. As a result of a numerical analysis based on the equation, the anisotropic diamagnetic susceptibility of normal sperm was found to be Δ_χ = 8 × 10^–20 J/T². The degree of orientation was influenced by the alterations in the shape of the head, body or the tail. It has been suggested that the DNA in the sperm head retain the strong magnetic anisotropy to counterbalance the magnetic anisotropy retained by flagellum microtubules. Recent studies demonstrated a well‐defined nuclear architecture in human sperm nucleus, where the head morphology has significant correlation with sperm chromatin structure assay SCSA. Then, as the methods to evaluate SCSA can be difficult and expensive our simple magnetic orientation technique can be an alternative to diagnose alteration in DNA. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Assessment of in vitro sperm characteristics in relation to fertility in dairy bulls

The performance of frozen-thawed spermatozoa from 10 Holstein bulls in a range of in vitro diagnostic tests and the relationship with adjusted in vivo fertility data was determined. The tests included an assessment of motility (subjective and computer-assisted), morphology, concentration, viability, acrosomal and chromatin integrity conducted immediately post-thaw and after swim-up, in conjunction with membrane status (CTC staining) and migration in an artificial cervical mucus. Adjusted in vivo fertility correlated with subjectively assessed post-thaw motility (r=0.672, p=0.033), post-thaw straight-line velocity (r=0.636, p=0.048), post-thaw sperm morphology (r=-0.762, p=0.010), post-thaw sperm viability (r=0.635, p=0.048), the concentration of spermatozoa after swim-up (r=0.649, p=0.042), sperm morphology after swim-up (r=-0.687, p=0.028), the number of spermatozoa migrating 10mm into artificial cervical mucus (r=0.632, p=0.050) and the distance migrated by the vanguard spermatozoon in artificial mucus (r=0.701, p=0.024). A stepwise regression analysis identified tests which, when combined, produced models with a strong correlation (R(2)>0.9) to fertility.     

Biology of sperm chromatin structure and relationship to male fertility and embryonic survival

Embryonic mortality in mammals is typically thought to result from 'female factor' infertility. There is growing evidence, however, that the status of sperm chromatin (DNA) at the time of fertilisation can also influence embryonic survival. During the final stages of spermatogenesis (spermiogenesis) a number of unique biochemical, morphological and physiological processes take place that are associated with marked changes in the structure of sperm chromatin. In early stages of spermatogenesis, sperm DNA is associated with histone nucleoproteins and structured into classical nucleosome core particles similar to other somatic cells. As spermiogenesis proceeds, the histone nucleoproteins are replaced by transition proteins which are subsequently replaced by protamines. At the completion of spermiogenesis the chromatin of mature sperm has a toroidal structure that is tightly compacted and resistant to denaturation. The compaction is necessary to protect sperm chromatin during transit through the epididymis and female reproductive tract. Disruption to chromatin remodelling during spermiogenesis results in chromatin that is susceptible to denaturation. Inappropriate chromatin structure has been shown in a number of mammalian species to be related to male infertility, and specifically the failure of embryonic development. A range of techniques are available to assess chromatin status in sperm but arguably the most informative is the sperm chromatin structure assay (SCSA). The SCSA is a flow cytometric assay that uses the metachromatic properties of acridine orange to measure the susceptibility of sperm chromatin to acid-induced denaturation. A relationship has been demonstrated, primarily in men, between the SCSA outcome and the probability of continued embryonic development and the establishment of pregnancy after fertilisation. The contribution of sperm chromatin instability to reproductive wastage in both natural mating and assisted reproduction warrants further investigation as it may prove valuable as a means of decreasing the incidence of embryonic mortality. In this regard, it is possible that 'male factor' infertility may emerge as an even more important component in embryonic development.     

Clinical aspects of sperm DNA fragmentation detection and male infertility

Over the past 25 years, various methods have been developed to measure sperm DNA strand breaks in situ. Currently, there are four major tests of sperm DNA fragmentation, including the Comet, Tunel, sperm chromatin structure assay (SCSA) and the acridine orange test (AOT). The Comet assay is a light microscope technique where the sperm cells are mixed with melted agarose and then placed on a glass slide. The cells are lysed and then subjected to horizontal electrophoresis. The Tunel assay, another light microscope technique, transfers labeled nucleotide to the 3'OH group of a broken DNA strand with the use of terminal deoxynucleotidyl transferase. The fluorescence intensity of each scored sperm is determined as a "yes" or "no" for sperm on a light microscope slide or by channels of fluorescent intensity in a flow cytometer. The light microscope-based AOT, uses the metachromatic properties of acridine orange to stain sperm cells. The SCSA treats sperm with low pH to denature DNA at the sites of DNA strand breaks, followed by acridine orange (AO) staining of green for native DNA and red for denatured DNA as measured by flow cytometry (FCM) as well as % sperm with high DNA stainability (HDS: immature sperm with intact DNA related to decreased fertilization rates). The SCSA method has defined a 27-30% DNA fragmentation index (DFI) as the point in which a man is placed into a statistical category of taking a longer time to in vivo pregnancy, intra uterine insemination (IUI) and more routine in vitro fertilization (IVF) cycles or no pregnancy. Current data suggest that intracytoplasmic sperm injection (ICSI) may help overcome the diminished pregnancy prognosis with high DFI over the other ART or natural methods.     

Flow cytometric evaluation of boar semen by the sperm chromatin structure assay as related to cryopreservation and fertility

Boar semen from a heterospermic mating trial and semen cryopreserved by various methods were evaluated by the flow cytometric sperm chromatin structure assay (SCSA), which measures the susceptibility of sperm nuclear DNA to acid-induced denaturation in situ. Spermatozoa were treated with a pH 1.4 buffer and then stained with the metachromatic dye acridine orange. Acridine orange intercalated into double-stranded DNA (native) fluoresces green while single-stranded DNA (denatured) fluoresces red when excited with 488 nm light. The ratio of red to total fluorescence provides an index of normality/abnormality. The SCSA data on neat boar semen or semen in either Kiev-Merck or Pursel-Johnson extender and frozen directly on dry ice blocks or plunged into LN(2) did not differ within individual boars. Therefore, chromatin structure, as measured by the SCSA, was not influenced differently by these 2 methods of semen cryopreservation. When semen from 6 boars was mixed in equal sperm numbers in six 3-way combinations and inseminated into at least 3 Duroc gilts per combination, 4 of the 6 combinations yielded 2 litters, while the remaining 2 combinations yielded 3 litters. The SCSA correctly predicted both the high and low fertility boars based on a ratio of offspring as deviated from the theoretical percentage. Thus, the SCSA was found to be a valuable adjunct method for evaluating boar cemen quality.     

Effect of storage time and temperature on stallion sperm DNA and fertility

We used the sperm chromatin structure assay (SCSA) to study the change in stallion sperm DNA susceptibility to denaturation after exposure of extended semen to three different storage temperatures (5, 20, or 37 degrees C) at 7, 20, 31, and 46 h. In addition, we compared the rates of sperm DNA denaturation in fertile and subfertile stallions. Among fertile stallions, spermatozoa stored at 20 and 37 degrees C showed a significant (P < 0.05) rise in the SCSA measures (Mean(alpha1), S.D.(alpha(t)), and percent cells outside the main population-COMP(alpha(t))) overtime, with the degree of rise being more dramatic at 37 degrees C. Over all stallions, samples stored at 5 degrees C showed no significant (P > 0.05) changes in the SCSA values measured over time, indicating maintenance of chromatin quality for up to 46 h. The COMP(alpha(t)) from stallions classified as subfertile showed an increased susceptibility to denaturation or decline in chromatin quality between 20 and 31 h when stored at 5 degrees C; however, spermatozoa from fertile stallions did not change during the time intervals analyzed. These data suggest that sperm DNA from some subfertile stallions may decline at a greater rate than spermatozoa from fertile stallions when exposed to similar storage conditions.     

Lead chloride affects sperm motility and acrosome reaction in mice

Lead is highly toxic and persistent in the environment and, thus, a major concern for public health. In this study, the effects of lead chloride (PbCl₂) on mouse epididymal sperm were evaluated. Male mice were subcutaneously injected with 74 and 100 mg PbCl₂/kg body weight for four consecutive days. Sperm was collected from the epididymis and several parameters of sperm function, such as sperm density, motility, viability, mitochondrial function, acrosome integrity and morphology, were evaluated. Furthermore, DNA fragmentation was assessed by the terminal deoxylnucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick-end labelling (TUNEL) assay and chromatin integrity was evaluated by sperm chromatin structure assay (SCSA). In order to assess direct effects on existing sperm population, we sacrificed one group for each condition at day 5. The effects of lead upon one entire spermatogenic cycle were evaluated on day 35. Both lead concentrations used in this work affected sperm motility, although no significant differences were observed in sperm viability, mitochondrial function and DNA/chromatin integrity. However, a decrease in the percentage of intact acrosomes was also observed, mirroring a lead-induced premature acrosome reaction. Thus, the results obtained indicate that, together with impaired motility, the effect of lead toxicity on acrosome integrity, leading to premature reaction, may compromise the ability of sperm to fertilize the oocyte.     

Impact of cryopreservation and reactive oxygen species on DNA integrity, lipid peroxidation, and functional parameters in ram sperm

Assisted reproduction using frozen-thawed semen has practical advantages, although cryopreservation is detrimental to sperm fertility in most mammals. We examined the influence of cryopreservation and reactive oxygen species (ROS) on ram sperm DNA stability (using SCSA), lipid peroxidation (LPO), chlortetracycline fluorescence (CTC) patterns, motility and viability. In Experiment 1, DNA integrity, LPO, CTC, motility and viability tests were performed on fresh and cryopreserved sperm after 0, 6, and 24 hr in synthetic oviductal fluid (SOF). In Experiment 2, fresh sperm were incubated in serum-free SOF (SOF-S; 1, 4, and 24 hr) with 0, 50, 150, or 300 microM H2O2 then assayed. Cryopreservation increased the percentage of sperm with a high DNA fragmentation index (%DFI), decreased the percentages of motile and viable sperm at thawing (0 hr), but did not affect LPO. H2O2 (150 or 300 microM) increased %DFI after 24 hr. LPO or sperm viability were not affected by H2O2, although most motility parameters decreased. H2O2 decreased the percentage of chlortetracycline pattern F sperm at 4 hr and increased the percentage of acrosome-reacted sperm (pattern AR) after 1 hr. Pooled data of Experiment 2 showed LPO was positively correlated with SCSA (r = 0.29 to r = 0.59; P < 0.05 to P < 0.01), while most motility parameters and the percentage of viable sperm were negatively correlated with LPO (r = -0.30 to r = -0.38; P < 0.05 to P < 0.01). LPO was positively correlated with the percentage of pattern AR sperm (r = 0.33; P < 0.01). Cryopreservation and H2O2 promote DNA instability in ram sperm, though motility is a more sensitive indicator of oxidative stress than the other parameters investigated.