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.     

Zona pellucida binding and zona-induced acrosome reactions in horse spermatozoa: comparisons between fertile and subfertile stallions

Diagnostic tests that probe sperm function are needed to determine the potential etiologies of subfertility and to explore treatments of subfertility in stallions. Using epifluorescence and phase contrast microscopy, a comparison was made between ejaculates from 3 fertile and 3 subfertile stallions in which sperm-zona pellucida binding and acrosomal status were measured. Motile spermatozoa were selected by Percoll gradient centrifugation and were capacitated in vitro using TEST:TALP capacitation medium at 39 degrees C under humidified air containing 5% CO2. Concentration of motile spermatozoa was held constant during co-incubation with oocytes for fertile and subfertile ejaculates. The total number of zona pellucida-bound spermatozoa was higher for fertile stallions than for subfertile stallions (P < 0.05). Similarly, the percentage of acrosome reactions in zona pellucida-bound spermatozoa was higher for the 3 fertile stallions than for the 3 subfertile stallions (P < 0.05). These results indicate that spermatozoa from fertile stallions may interact with female gametes differently from that of subfertile stallions and suggest that sperm functions are measurable and may vary with fertility.     

Effects of long-term chilled storage of red deer epididymides on DNA integrity and motility of thawed spermatozoa

We have carried out a study on the influence of prolonged cold storage (5 degrees C) of Iberian red deer epididymides on post-thaw sperm motility and DNA integrity. Twenty-nine pairs of testes, with attached epididymides, were collected during November and December. Spermatozoa from one of each of the pairs were immediately recovered, evaluated and frozen (control). The remaining epididymides were cooled to 5 degrees C and stored for 24, 96 and 192 h (experimental groups), after which spermatozoa were collected and frozen. Samples were evaluated before freezing, after thawing, and after a 2-h period of incubation at 37 degrees C. Motility was evaluated by means of a CASA system and chromatin stability was assessed following the Sperm Chromatin Structure Assay (SCSA). Our results showed that, during the first 96 h, the motility (total and progressive) did not significantly decline when assessed after cryopreservation, although there was a significant decline when epididymides had been stored for 192 h at 5 degrees C (P<0.001). The present study demonstrates that motility and DNA status of thawed spermatozoa collected from refrigerated epididymes, at least 96 h post-mortem, were good enough to consider their eventual use. Most importantly, sperm DNA integrity after thawing was apparently not affected by storage time, even after 192 h.     

Morphometric differences in sperm head dimensions of fertile and subfertile stallions

Gross morphological evaluation of stallion spermatozoa is of clinical value in assessing male fertility in the horse. While of value, methods of subjective sperm classification yield highly variable results. Recent development of computer-assisted sperm morphometry analysis (ASMA) technology has allowed for the objective analysis of sperm head morphometry. In the current study, ASMA was employed to determine morphometric differences in sperm head dimensions between fertile and subfertile stallions. At least 200 spermatozoa from each of 10 fertile and 10 subfertile stallions were analyzed by a commercial ASMA instrument. The mean measurements for length, width, area, perimeter, and width/length for each stallion were recorded and group means compared by a two-sample t-test. The mean measurements for length, area and perimeter were significantly larger in the subfertile than the fertile group (5.77 microm vs 5.33 microm, 12.66 microm vs 11.37 microm and 14.59 microm vs 13.64 microm, respectively). The width of sperm heads from stallions in the subfertile group also tended to be larger than those of fertile stallions. The data suggest that differences in the dimensions of sperm heads may exist between fertile and subfertile stallions.     

Dynamics of sperm DNA fragmentation in domestic animals II. The stallion

The mixed success of equine artificial insemination programs using chilled and frozen-thawed semen is most likely associated with the variable response of the sperm cell to the preservation process and the fact that stallions are not selected on the basis of reproductive performance. We propose that the traditional indicators of sperm viability do not fully account for male factor infertility in the stallion and that knowledge of sperm DNA damage in the original semen sample and during semen processing may provide a more informed explanation of an individual stallion's reproductive potential. This study reports on the validation of a sperm DNA fragmentation test based on the sperm chromatin dispersion test (SCD) for stallion spermatozoa and on its application to semen that was chilled (4 degrees C; n=10) or frozen-thawed (n=13). Semen samples were collected by artificial vagina and the proportion of sperm with fragmented DNA determined. Seminal plasma was then removed by centrifugation and the sperm pellet re-suspended in commercial extenders prior to being chilled or cryopreserved using standard industry protocols. Chilled semen was cooled slowly to 4 degrees C and stored for 1h before commencing the analysis; cryopreserved semen was thawed and immediately analyzed. Following chilling or cryopreservation, the semen samples were incubated at 37 degrees C and analyzed for SCD after 0, 4, 6, 24 and 48 h storage. The results of this investigation revealed that there was no significant difference in the sperm DNA fragmentation index (sDFI) of sperm evaluated initially after collection compared to those tested immediately after chilling or cryopreservation. However, within 1h of incubation at 37 degrees C, both chilled and frozen-thawed spermatozoa showed a significant increase in the proportion of sDFI; after 6h the sDFI had increased to over 50% and by 48 h, almost 100% of the sperm showed DNA damage. While the sDFI of individual stallions at equivalent times of incubation was variable, an analysis of the rate of change of sDFI revealed no difference between stallions or the way in which the semen was preserved. In terms of sperm DNA fragmentation dynamics, the highest intensity of sperm DNA damage occurred in the first 6h of incubation. We suggest that the SCD test can be used as a routine assessment tool for the development and refinement of preservation protocols designed to reduce stallion sperm DNA damage.     

The quantification of lipid and protein oxidation in stallion spermatozoa and seminal plasma: seasonal distinctions and correlations with DNA strand breaks, classical seminal parameters and stallion fertility

The goal of this work was to correlate oxidative stress caused by reactive oxygen species (ROS) and DNA damage with classic semen parameters in spermatozoa and seminal plasma of fertile and subfertile stallions. Oxidation was measured in both lipids and proteins, using the thiobarbituric acid reactive species (TBARS) assay and the DNPH carbonyl groups assay, respectively. Sperm DNA damage was monitored using the TUNEL assay. These parameters were monitored in samples obtained during the breeding and the non-breeding seasons. In general, fertile stallions showed better classical semen parameters, and those parameters improved from the non-breeding to the breeding season, although an increase in sperm production was accompanied by a decrease in the semen quality from subfertile stallions in the breeding season. In terms of oxidation levels we found that there were clear differences whether lipids or proteins were considered. In the breeding season there seemed to be a tendency towards normalizing lipid oxidation in spermatozoa and seminal plasma, and protein oxidation in the seminal plasma, of both fertile and subfertile animals. Thus, differences monitored in the non-breeding season were no longer visible. Interestingly, a higher level of protein oxidation was found in the sperm of fertile animals in the breeding season. Considering that there were positive correlations between sperm protein oxidation and sperm motility and vitality, these results suggests that the oxidation of semen proteins may be important for sperm function. On the other hand, lipid oxidation in the seminal plasma seemed to be a general indicator for sperm damage. In the non-breeding season positive correlations between lipid and protein oxidation levels in both sperm and seminal plasma and several defects in sperm function were found, but only for subfertile animals, thus suggesting that lipid and protein oxidation may aid in the identification of subfertile stallions during the non-breeding season. Levels of ROS production never seemed to result in compromised sperm DNA integrity, indicating that measurements were within physiological levels and/or that there is an efficient antioxidant activity in stallion sperm cells.     

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.     

Heterogeneity of sperm nuclear chromatin structure and its relationship to bull fertility

The relationship between sperm nuclear chromatin structure and fertility was evaluated in two groups of Holstein bulls: Group 1, 49 mature bulls, and Group 2, 18 young bulls. Fertility ratings had been estimated for Group 1 and nonreturn rates were known for Group 2. Semen samples were measured by the sperm chromatin structure assay (SCSA): sperm were treated to induce partial in situ DNA denaturation, stained with acridine orange, and evaluated by flow cytometry. Acridine orange intercalated into double-stranded DNA emits green fluorescence upon excitation with 488 nm light, and red fluorescence when associated with single-stranded DNA. An index of DNA denaturation per cell is provided by alpha-t [alpha t = red/(red + green) fluorescence]. The standard deviation (SD alpha t), coefficient of variation (CV alpha t) and proportion of cells outside the main population (COMP alpha t) of the alpha t distribution quantify the extent of denaturation for a sample. Intraclass correlations of the alpha t values were high (greater than or equal to 0.70), based on four collections obtained over several years from Group 1 bulls. Negative correlations were obtained between fertility ratings and both SD alpha t (-0.58, p less than 0.01) and COMP alpha t (-0.40, p less than 0.01) in Group 1, and between nonreturn rates and both SD alpha t (-0.65, p less than 0.01) and COMP alpha t (-0.53, p less than 0.05) in Group 2. These data suggest that the SCSA will be of value for identification of low fertility sires and poor quality semen samples.     

Sperm transport and survival in the mare

Following the deposition of semen in the mares uterus, spermatozoa must be transported to the site of fertilization, be maintained in the female tract until ovulation occurs, and be prepared to fertilize the released ovum. Sperm motility, myometrial contractions, and a spontaneous post-mating uterine inflammation are important factors for the transport and survival of spermatozoa in the mares reproductive tract. Fertilizable sperm are present in the oviduct within 4 hours after insemination. At this time, the uterus is the site of a hostile inflammatory environment. Our data suggest that spermatozoa trigger an influx of polymorphonuclear neutrophils (PMNs) into the uterine lumen via activation of complement. Furthermore, seminal plasma appears to have a modulatory effect on the post-mating inflammation through its suppressive effect on PMN chemotaxis and migration. Spermatozoa that safely have reached the oviduct can be stored in a functional state for several days, but prolonged sperm storage in the female tract is not required for capacitation and fertilization in the horse. The caudal isthmus has been proposed as a sperm reservoir in the mare. The pattern of sperm transport and survival of spermatozoa in the mares reproductive tract are different between fertile and subfertile stallions, between fertile and some infertile mares, and between fresh and frozen-thawed semen. Possible explanations for these differences include a selective phagocytosis of damaged or dead spermatozoa, impaired myometrial activity in subfertile mares, bio-physiological changes of spermatozoa during cryopreservation, and the removal of seminal plasma during cryopreservation of equine semen.     

Motility and fertility of equine spermatozoa in a milk extender after 12 or 24 hours at 20 degrees C

The effects of extender and storage at 20 degrees C on equine spermatozoa were evaluated in two experiments using embryo recovery as the end point. In both experiments, inseminations were every other day, starting on Day 2 or 3 of estrus or after a 35-mm follicle was detected, with 250 x 10(6) progressively motile cells (based on initial evaluation). In Experiment 1, semen from two stallions was used to compare the motility and fertility of spermatozoa maintained in a) heated skim milk extender at 37 degrees C with insemination in <1 h; b) E-Z Mixin extender at 37 degrees C with insemination in <1 h; and c) E-Z Mixin extender at 37 degrees C with cooling to 20 degrees C and insemination after storage for 12 h at 20 degrees C. The percentage of motile spermatozoa was 34% after 12 h compared to 55% at 0 h (P < 0.05). However, the percentage of mares from which an embryo was recovered 6.5 d after ovulation was 62, 56, and 50% for Treatments A, B, and C (P > 0.05). In Experiment 2, semen from three stallions was used to compare the motility and fertility of spermatozoa in a) E-Z Mixin extender at 37 degrees C with insemination in <1 h or b) E-Z Mixin extender at 37 degrees C with cooling to 20 degrees C and insemination after storage for 24 h at 20 degrees C. The percentage of motile spermatozoa was 17% after 24 h compared to 54% at 0 h (P < 0.05). There was no difference between treatments (P > 0.05) in the percentage of mares from which an embryo was recovered 6.0 d after ovulation (68 vs 62%) or among stallions. Thus, stallion semen extended in E-Z Mixin was held at 20 degrees C for 24 h without a marked decline in fertility.     

Relationship of seminal plasma level and extender type to sperm motility and DNA integrity

The relationship between seminal plasma level (0, 10, or 20%) and extender type [Kenney type (EZ-Mixin-CST) or Kenney-modified Tyrodes-KMT] to the susceptibility of sperm DNA to denaturation and sperm motility measures were investigated in cooled (5 degrees C) stallion sperm. Three ejaculates from each of three fertile stallions were collected in an artificial vagina and processed as follows: diluted one part uncentrifuged semen with four parts of extender to a final concentration of 20% seminal plasma in either CST or KMT (20% CST; 20% KMT); diluted to a final concentration of 25 million sperm/mL in either CST or KMT (10% CST; 10% KMT); centrifuged to remove virtually all seminal plasma and resuspended in either CST or KMT (0% CST-Cent; 0% KMT-Cent); centrifuged semen to remove virtually all seminal plasma and resuspended with previously filtered seminal plasma from the same stallion in either CST or KMT to a final concentration of 20% seminal plasma (20% CST-Cent; 20% KMT-Cent). Sperm motion characteristics were determined by CASA and DNA integrity (%COMP, percent of cells outside the main population) evaluated by the Sperm Chromatin Structure Assay prior to cooling, and after 24 and 48 h cooled-storage at 5 degrees C. After 48 h of storage at 5 degrees C, extenders with 0% seminal plasma (0% CST-Cent, 0% KMT-Cent) maintained highest quality DNA (P < 0.05), but 0% KMT-Cent maintained higher velocity measures (P < 0.05) than 0% CST-Cent. Total sperm motility was highest (P < 0.05) in 0% CST-Cent, 0% KMT-Cent, 10% CST, 20% CST-Cent, and 20% CST compared to the other treatment groups. Progressive sperm motility was highest (P < 0.05) after 48 h of storage in the treatment with 10% seminal plasma in Kenney extender (10% CST), despite a reduction in DNA integrity. Regardless of extender type, addition of 20% seminal plasma following centrifugation resulted in almost a two-fold increase in %COMP(alpha t), even though one of the treatments (20% CST-Cent) maintained total and progressive motility similar to treatments with no seminal plasma, suggesting that sperm motility and DNA integrity may respond independently to environmental conditions. Overall, better quality sperm features (motility and DNA) were maintained in sperm from which seminal plasma was removed followed by resuspension in either Kenney extender or modified Kenney Tyrodes-type extender.     

Differential ubiquitination of stallion sperm proteins: possible implications for infertility and reproductive seasonality

Antibodies against ubiquitin, a universal proteolytic marker, show increased cross-reactivity with defective spermatozoa in men and bulls. We investigated sperm ubiquitination in the stallion, a seasonally polyestrous mammal. Immunofluorescence and immunoelectron microscopy demonstrated that anti-ubiquitin antibodies bind to the surface of both membrane-intact and aldehyde-fixed spermatozoa. Cross-reactivity to the ubiquitin-conjugating enzyme E2 was also detected in sperm. Immunohistochemistry showed that ubiquitinated spermatozoa were first detected in the caput epididymis, coincident with a strong accumulation of ubiquitin and ubiquitin C-terminal hydrolase, protein gene product 9.5, in the apical stereocilia of the epididymal epithelium. Testicular spermatozoa did not display significant ubiquitin cross-reactivity. Similarly, lesser accumulation of ubiquitin cross-reactive substrates was identified in the accessory sex glands. Semen samples were collected from three fertile stallions and one subfertile stallion between December and February and probed for ubiquitin by flow cytometry and immunoblotting. Flow cytometric analysis showed that sperm from the subfertile stallion had higher ubiquitin levels than sperm from the other three stallions. In addition, immunoblot analysis of sperm proteins from the subfertile stallion showed two unique ubiquitin cross-reactive bands that were not present in sperm extracts from the three fertile stallions. To screen for a possible role for ubiquitin in seasonal changes in sperm production, semen samples from two fertile stallions were collected in March, June, September, and December and subjected to a flow cytometric ubiquitin assay. The lowest levels of ubiquitin-labeled sperm were found in March, approximately coincident with the onset of the natural horse breeding season. A progressive increase in sperm ubiquitin levels was found during summer and fall, with a peak in December. These data suggest that stallion sperm are differentially ubiquitinated during epididymal maturation and that this ubiquitination may reflect changes in sperm numbers and semen quality. The association between changes in sperm ubiquitination and seasonal changes in sperm production will be subjected to further studies in a larger cohort of animals.     

Effectiveness of two systems for transporting equine semen

The storage and transport of cooled, liquid semen is an effective way of facilitating the use of desirable stallions for breeding mares located on distant farms. The Equitainer System is the most widely used transport container and it has been shown that it is possible to ship semen in this container and obtain good conception rates. However, the cost of Equitainers is high, and stud-farms that ship large quantities of semen have tended to rely on cheaper alternatives, even though little documentation exists concerning their reliability, especially under extreme temperature conditions. Two different containers for transporting equine semen (the Equitainer and a styrofoam box) were compared in their effectiveness at maintaining semen quality (i.e. sperm motility and plasma membrane integrity) during 24 h of storage. The transport containers were stored at 2 different environmental temperatures, i.e., room temperature (20 degrees C) and 37 degrees C. Thirty-seven ejaculates from 10 Standardbred stallions (3 to 6 samples per stallion) were examined. Sperm function and plasma membrane integrity were assessed using a Mika Motion Analyzer and a fluorescein stain (Calcein AM/Ethidium homodimer) in fresh diluted semen that had been stored for 24 h at room temperature (20 degrees C). Another 18 ejaculates from 5 stallions were examined using methods described above, but the transport boxes were kept at a high environmental temperature (37 degrees C). After storage at room temperature, there was no significant difference in total sperm motility and frequency of spermatozoa with an intact plasma membrane between the 2 types of transport boxes. A significant difference was seen in linear sperm motility, with the Equitainer being the better container. However, a significant difference was also seen in average path velocity, with the styrofoam box being the better container. After storage at 37 degrees C, the Equitaner maintained semen quality better. A significant difference was seen in total sperm motility, average path velocity, lateral head displacement and frequency of spermatozoa with an intact plasma membrane between the 2 types of transport boxes. Although, both transport containers were satisfactory when used under normal conditions. The Equitainer seemed superior under more extreme temperatures and during longer transport periods (> 24 to 30 h).     

Administration of oxytocin immediately after insemination does not improve pregnancy rates in mares bred by fertile or subfertile stallions

It is probable that reduced pregnancy rates in mares bred to subfertile stallions is attributable, in part, to the reduced number of normal spermatozoa that colonize the oviduct. Administration of oxytocin stimulates both uterine and oviductal contractility. The hypothesis that oxytocin may enhance sperm transport to/into the oviducts, and thereby increase pregnancy rates, was tested in 2 trials. For both trials, fertile estrous mares with follicles > or = 35 mm in diameter were inseminated once at 24 h after administration of 1500 to 2000 U hCG. The inseminate dose was limited to 100 million spermatozoa in order to lower pregnancy rates and thus increase the chance of detecting a treatment effect. Pregnancy status was determined by transrectal ultrasound examination 14 to 16 d after insemination. In Trial 1, 49 mares were inseminated with 4 mL extended semen from 1 of 3 stallions (1 fertile and 2 subfertile males). Immediately after insemination, the mares were administered either 20 U oxytocin or 1 mL saline intravenously. In Trial 2, 51 mares were inseminated with 4 mL extended semen from 1 of 4 stallions (1 fertile and 1 subfertile male used in Trial 1, and 2 additional fertile males). Immediately after insemination, and again 30 min later, mares were administered either 5 U oxytocin or 0.25 mL saline intramuscularly. To test for effects of treatment with oxytocin and for the interaction between semen quality and treatment, a generalized linear mixed regression model was used that accounted for the split-plot design (treatment within stallions), the random effect of stallion, the fixed effect of semen quality, the binary outcome of a single breeding trial, and the varying number of trials per stallion/treatment groups. Three treatment protocols or regimens were used: placebo, 5 U oxytocin injected twice intramuscularly, and 20 units oxytocin injected twice intravenously. Semen was classified as high (fertile stallions) or low (subfertile stallions) quality. No interaction between semen quality and treatment was detected (P > 0.10). The pregnancy rate of mares treated with oxytocin immediately after insemination was 30% (15/50) compared with 50% (25/50) for mares treated with saline immediately after breeding. Administration of oxytocin did not affect pregnancy rates (P > 0.10).     

Cholesterol-to-phospholipid ratio in whole sperm and seminal plasma from fertile stallions and stallions with unexplained subfertility

Semen samples were collected from six fertile stallions and seven stallions with unexplained infertility. Percentages of motile sperm (77.5 +/- 11.3 versus 67.5 +/- 12.2, P = 0.2), and progressively motile sperm (70.8 +/- 13.6 versus 60.7 +/- 14.0, P = 0.2) were similar between fertile and subfertile stallions, respectively. Morphologic characteristics in ejaculates of control and affected stallions (% normal: 60.2 +/- 18.2 versus 52.9 +/- 11.3, P = 0.4; % abnormal heads 7.3 +/- 4.8 versus 12.1 +/- 5.0, P = 0.11; and % abnormal acrosomes 1.6 +/- 2.1 versus 3.0 +/- 3.4, P = 0.4) did not differ. After incubation with the calcium ionophore A23187, acrosome reaction rate of sperm from fertile stallions was 96 +/- 2.8% whereas only 2.9 +/- 2.5% of sperm from stallions with unexplained subfertility had acrosome reacted (P < 0.001). Molar amounts of cholesterol and phospholipid in whole sperm and seminal plasma did not differ (P > 0.1) between fertile and subfertile stallions. However, the molar ratio of cholesterol-to-phospholipid was 2.5 times greater in the seminal plasma (P = 0.09) and 1.9 times greater (P = 0.009) in whole sperm of subfertile stallions compared to fertile stallions.     

Association of classical semen parameters, sperm DNA fragmentation index, lipid peroxidation and antioxidant enzymatic activity of semen in ram-lambs

The objective was to determine relationships among classical semen characteristics, sperm chromatin structure assay (SCSA), lipid peroxidation and antioxidant enzymatic activity in ram-lamb semen. Fifty-seven ram-lambs were electroejaculated, and routine semen evaluation was conducted (as part of a breeding soundness evaluation). The percentage of sperm DNA fragmentation index (%DFI) and the percentage of sperm with abnormally high DNA stainability (HDS; immature spermatozoa) were determined by SCSA using the metachromatic properties of acridine orange. Semen was centrifuged at 800 x g for 15 min to separate spermatozoa and seminal plasma and the aliquots were stored at -70 degrees C until analyzed. Lipid peroxidation, superoxide dismutase (SOD), and glutathione peroxidase (GPx) levels in seminal plasma and spermatozoa were measured by spectrophotometric assays. The classical semen parameters were negatively related to lipid peroxidation and GPx activity in spermatozoa; motility and morphology were negatively related to %DFI (P < 0.05). Based on Kruskal-Wallis pair-wise comparison of median values among breeding soundness outcome groups, %DFI was lower in the satisfactory group compared to other groups (P < 0.05) and the lipid peroxidation and GPx activity in seminal plasma and spermatozoa were lower in satisfactory and questionable groups (P < 0.05). However, the SOD was lower in the unsatisfactory group (P < 0.05). In summary, classical semen parameters were negatively related to % DFI, lipid peroxidation and GPx activity in ram-lamb spermatozoa and seminal plasma. There were indications that SOD and GPx have crucial protective roles against the toxic effect of reactive oxygen species (ROS) in ram-lamb semen.     

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.     

Sperm transport and survival in the mare: a review

After the deposition of semen in the mare's uterus, spermatozoa must be transported to the site of fertilization, be maintained in the female tract until ovulation occurs, and be prepared to fertilize the released ovum. Sperm motility, myometrial contractions, and a spontaneous post-mating uterine inflammation are important factors for the transport and survival of spermatozoa in the mare's reproductive tract. Fertilizable sperm are present in the oviduct within 4 h after insemination. At this time, the uterus is the site of a hostile inflammatory environment. Our data suggest that spermatozoa trigger an influx of polymorphonuclear neutrophils (PMNs) into the uterine lumen via activation of complement. Furthermore, semen plasma appears to have a modulatory effect on the post-mating inflammation through its suppressive effect on PMN chemotaxis and migration. Spermatozoa that safely have reached the oviduct can be stored in a functional state for several days, but prolonged sperm storage in the female tract is not required for capacitation and fertilization in the horse. The caudal isthmus has been proposed as a sperm reservoir in the mare. The pattern of sperm transport and survival of spermatozoa in the mare's reproductive tract are different between fertile and subfertile stallions, between fertile and some infertile mares, and between fresh and frozen/thawed semen. Possible explanations for these differences include a selective phagocytosis of damaged or dead spermatozoa, impaired myometrial activity in subfertile mares, bio-physiological changes in spermatozoa during cryopreservation, and the removal of semen plasma during cryopreservation of equine semen.     

Sperm chromatin stability in frozen-thawed semen is maintained over age in AI bulls

The aim of the present study was to investigate the effect of age of the sire on the in vitro quality of frozen-thawed (FT) bull spermatozoa, both when tested immediately postthaw (PT) and when assessed after cleansing and selection through a swim-up (SU) procedure. Semen samples from six Swedish Red and White Breed (SRB) artificial insemination (AI) bulls at age 1 and again, at 4 years were collected and frozen in 0.25 ml plastic straws. Also, semen was collected from six Estonian Holstein (EHF) bulls at the ages of 3, 5, and 7 years and likewise processed. The FT semen was tested for the susceptibility of sperm nuclear deoxyribonucleic acid (DNA) to undergo acid-induced denaturation in situ, as quantified by flow cytometry (FCM). The DNA denaturability was expressed as function alpha t, i.e., as the ratio of red (denaturated DNA) to red + green (total cellular DNA) fluorescence intensity. The results were expressed as the percentage of cells with high alpha t values, i.e., cells outside the main population (% COMP alpha t). Morphological evaluation of the same samples was performed to detect general and sperm head abnormalities and differences between ages. Fertility results were available as non-return rates (NRRs) for the semen of the sires when they were 1 year (SRB) and 3 years (EHF) old, varying from 62.2 to 70.7% in SRB and from 52.2 to 76.0% in EHF animals. The COMP alpha t values ranged from 0.5-3.6% (PT) to 0.2-1.7% (SU) for SRB bulls and from 0.4-1.8% (PT) to 0.2-1.5% (SU) for EHF bulls. Both breeds lacked differences between ages, either PT or after SU. However, the SU procedure yielded a significantly higher population of spermatozoa with stable DNA following acid-induced denaturation, than PT samples (p < 0.001). No correlation was detected between field fertility and chromatin stability. The results indicate that for these bull populations, the SU procedure was able to select spermatozoa with stable chromatin from the bulk samples. However, the use of DNA denaturation as a challenge to assess sperm chromatin stability did not offer a more accurate tool to evaluate sperm quality than the conventional, light microscopical evaluation of morphology.     

Dynamics of sperm DNA fragmentation in domestic animals III. Ram

From a biological viewpoint spermatozoa are ejaculated by the male and received into the female while maintaining roughly constant temperature, which in most mammals is below the temperature of the soma. When ejaculated spermatozoa are used for artificial reproductive purposes a temperature excursion episode is produced, because the spermatozoa are often stored as frozen or chilled samples and the biological temperature is only recovered after insemination. In this study we have analyzed the effects of cooling (to 15 degrees C) and freezing ram spermatozoa on the subsequent sperm DNA fragmentation index (sDFI) during a varying period of storage at 37 degrees C. The aim was to emulate in vivo processes that cooled or frozen-thawed spermatozoa experience after insemination. The study was performed using commercial semen samples derived from rams regularly used for reproductive purposes. Semen samples were studied after a cooling or cryopreservation episode followed by biological temperature recovery and incubation up to 48h. The results indicated that when spermatozoa experience a severe (frozen) or mild (cooled) temperature excursion episode, major effects on sperm viability and DNA fragmentation are induced and cause the subsequent rapid decline of ram sperm quality. This effect could be detected just at the onset of the biological temperature recovery. Sperm DNA damage in cooled samples was observed after 5h of incubation at 37 degrees C, while this time was reduced to less than 60min in frozen-thaw samples. The dynamics of sDFI in different animals, analyzed under the same experimental conditions, was different from one sample to another, regardless of the method used for storage. Sperm viability was better preserved in cooled rather than in frozen samples. While for the frozen-thawed samples sperm viability was almost abolished after 5h of incubation, a stable proportion of viable spermatozoa (ranging from 20% to 60%) was observed in the cooled samples at the corresponding time points. Finally, with respect to the prevalence of sDFI in ram, the level commonly found was lower than 5% at the onset of the experiment. However, sDFI was higher than 5% in 25% of the samples and in 15% of rams this index exceeded 10%.