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 associations among semen quality,oxidative DNA damage in human spermatozoa and concentrations of cadmium,lead and selenium in seminal plasma

To explore the associations among semen quality, oxidative DNA damage in human spermatozoa and concentrations of cadmium, lead and selenium in seminal plasma, 56 non-smoking subjects were asked to collect semen by masturbation into a sterile wide-mouth metal-free plastic container after 3 days of abstinence. The conventional semen parameters were analysed. The concentrations of Cd, Pb and Se in seminal plasma were detected using atomic absorption spectrophotometer. 8-OHdG levels in sperm DNA were measured using HPLC-EC. The results showed that the geometric mean concentrations of Cd, Pb and Se were 0.78, 7.8 and 51.4 microg/l, respectively. The geometric mean of 8-OHdG/10(6) dG was 51.4 (95% CI: 21.5-123.0). A significant inverse correlation exists between Cd and sperm density (r=-0.28, P<0.05), and between Cd and sperm number per ejaculum (r=-0.27, P<0.05). In contrast, there was a significantly positive correlation between Se and sperm density (r=0.50, P<0.01), between Se and sperm number (r=0.49, P<0.01), between Se and sperm motility (r=0.40, P<0.01), and between Se and sperm viability (r=0.38, P<0.01). No statistically significant correlation was observed between Pb and semen quality. A significant inverse correlation was observed between 8-OHdG and sperm density (r=-0.34, P<0.01), between 8-OHdG and sperm number per ejaculum (r=-0.30, P<0.01), and 8-OHdG and sperm viability (r=-0.24, P<0.05). 8-OHdG was significantly correlated with Cd in seminal plasma (r=0.55, P<0.01). A significant but weak positive correlation was found between 8-OHdG and Pb concentration in seminal plasma (r=0.28, P<0.05). In contract, a significant inverse correlation was observed between 8-OHdG and Se concentration in seminal plasma (r=-0.40, P<0.01). The results indicate that Cd in seminal plasma could affect semen quality and oxidative DNA damage in human spermatozoa. Se could protect against oxidative DNA damage in human sperm cells. Pb did not appear to have any association with the semen quality when concentration of Pb in seminal plasma was below 10 microg/l.     

Bull sperm surface "craters" and other aspects of semen quality

Semen from 200 Holstein bulls in an artificial insemination center was examined for the frequency of craters on the surface of sperm heads, as visualized with the aid of differential interference contrast microscopy. Semen from 100 of these bulls was examined in more detail in 2 experiments by staining with eosin-aniline blue to determine the relationship of unstained spermatozoa, and spermatozoa with normal acrosomes with apical ridges to the incidence of craters and fertility. Only 3 of 100 bulls had a substantial incidence of craters (15 to 23%), whereas the average of the other 97 bulls in 2 experiments was 1 to 3%. The percentage of sperm cells with craters was correlated (P < 0.05) with the percentage of unstained spermatozoa (r = -0.29 and sperm cells with normal acrosomes (r = -0.52) but was not significantly correlated (r = -0.24) with the nonreturn rate. One bull with many sperm cells with craters was slaughtered, and the epididymal spermatozoa were examined. The high incidence of sperm cells with craters was limited to one side, with the testis on that side having 2 Sertoli cell tumors. The remaining 2 bulls as well as one other that produced 16% of sperm cells with craters did so only temporarily. Within a few months crater sperm production had decreased and semen quality increased. The condition usually appears to be transitory, presumably due to temporary stress.     

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.     

Interrelationships among fluorometric analyses of spermatozoal function, classical semen quality parameters and the fertility of frozen-thawed bovine spermatozoa

Cryopreserved spermatozoa from 8 bulls were used to examine the interrelationships among flow cytometric spermatozoal quality assessments and classical semen quality parameters and nonreturn rate estimates of fertility. The integrity of the sperm cell membrane and the functional capacity of the mitochondria were quantified by flow cytometry after concurrent staining with carboxydimethylfluorescein diacetate (CDMFDA), propidium iodide (PI), and rhodamine 123 (R123). For each sample a total of 10,000 stained spermatozoa were simultaneously quantified for the intensity of their green and red fluorescence. Three straws from each bull were each examined initially and following incubation at 37 degrees C for 3 hours to assess the rate of senescence. The proportion of spermatozoa retaining membrane integrity and having functional mitochondria, as determined by CDMFDA and R123 staining, were compared with classical semen quality assessments (sperm motility, acrosomal status, cellular and head morphology, presence of vacuoles/craters and cytoplasmic droplets) and with fertility (nonreturn to estrus rates). For individual ejaculates nonreturn rates, the range was from 61.8 to 78.8%, whereas the cumulative rates of several ejaculates for each bull ranged from 71.3 to 83.5%. The proportion of spermatozoa with functional membranes and mitochondria were positively correlated with the percentage of spermatozoa with normal morphology (r=0.82; P=0.01) and motility after 4 hours of incubation (r=0.78; P=0.02), but not with the estimates of fertility. The actual number of spermatozoa per straw staining with CDMFDA and R123 after 4 hours of incubation at 37 degrees C was correlated with the percentage of spermatozoa with normal morphology (r=0.73; P=0.04). Multiple regression equations indicated that combinations of semen quality measurements could be useful in estimating fertilizing potential.     

Sperm chromatin structure assay of bulls qualified for artificial insemination

The goal of our study was to find the relationship between fertility of bulls qualified for AI and the percentage of spermatozoa with abnormal chromatin structure as an independent parameter. We used the frozen semen of 8 mature bulls from one AI center. Each bull was represented by 3 ejaculates collected with at least 2-week intervals. Bull fertility was calculated on the basis of non-return ratio and was expressed as a scale where 100 points represented the average fertility of all the AI center's bulls. Bulls with lower or higher fertility received a lower or higher score respectively. Fertility scores of bulls used in the study ranged from 83 to 104 . Semen was processed according to the SCSA (sperm chromatin structure assay) method and was analyzed by flow cytometry. "Artificial" alpha(t) (alpha(t)=red/green+red fluorescence) and red fluorescence histograms were used for calculation of COMPalpha(t), SDalpha(t), %Red, %PeakR and MeanR parameters. The percentage of spermatozoa with abnormal chromatin ranged from 1.2% to 23.8%. A large variation among ejaculates was found for bulls with lower fertility. Fertility correlated significantly with COMPalpha(t) (-0.50, P < 0.05), SDalpha(t) (-0.55, P < 0.01), %Red (-0.53, P < 0.01), %PeakR (-0.58, P < 0.01) and MeanR (-0.45, P < 0.05). The SCSA method has a practical application in analyzing spermatogenesis disorders in bulls. If regularly applied, it allows us to identify and eliminate ejaculates with a high level of sperm chromatin abnormalities.     

The importance of porcine sperm parameters on fertility in vivo

It would be desirable to use semen parameters to predict the in vivo fertilizing capacity of a particular ejaculate. In animal production, an ejaculate is divided into multiple doses for artificial insemination (AI); therefore, it would be economically beneficial to know the functional quality (i.e., fertility) of the semen before it is inseminated. To identify a predictive assay of the fertilizing capacity of a porcine ejaculate, we performed 4 rapid assays of sperm quality (motility, viability, physiological status as assessed by chlortetracycline fluorescence, and ATP content) on samples from 9 ejaculates, before and after a thermal stress test (42.5 degrees C, 45 min). These parameters were subsequently correlated with in vivo fertility resulting from AI with 2 sperm doses, 3 x 10(9) or 0.3 x 10(9) motile cells in 70 mL (optimal or suboptimal sperm number per insemination, respectively) from these same ejaculates. No parameter was correlated to the fertility rates obtained after inseminating with the optimal semen doses, either before or after the thermal stress test (P > 0.05). However, with respect to the animals inseminated with the suboptimal semen dose, sperm motility (the percentage of motile spermatozoa as assessed visually by microscopy) prior to thermal stress was well-correlated to fertility rates (r = 0.783, P = 0.01). The percentage of spermatozoa displaying the chlortetracycline Pattern AR (acrosome reaction) was also statistically related to fertility (r = 0.05, P = 0.04), but the biological importance of this relationship is questionable given the small variation among ejaculates (range: 0 to 2%). No other sperm parameter was significantly related to fertility rates in this group (P > 0.05). These data, therefore, indicate that sperm motility is a useful indicator of sperm fertilizing capacity in vivo. Moreover, to identify a predictor of semen fertility it is critical that the number of spermatozoa used during insemination is sufficiently low to detect differences in sperm fertilizing efficiency.     

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.     

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.     

Flow cytometric evaluation of sperm parameters in relation to fertility potential

Most laboratory methods used to evaluate semen quality have not correlated highly with fertilizing capacity. The discovery of a variety of fluorochromes and compounds conjugated to fluorescent probes has enabled a more widespread analysis of sperm attributes, and in conjunction with the flow cytometer, permit the evaluation of a large number of spermatozoa. A number of characteristics of sperm integrity, viability and function can be assessed by flow cytometry. The DNA status of spermatozoa has been determined using the metachromatic properties of acridine orange (AO). AO staining, when used in the sperm chromatin structure assay (SCSA), correlates with fertility in a number of species. DNA fragmentation can also be assessed using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, which identifies DNA strand breaks by labeling free 3'-OH termini with modified nucleotides. The status of the sperm acrosome can be determined using fluorescently labeled lectins and LysoTracker Green DND-26, a fluorescent acidotropic probe. Capacitation status has been observed through calcium-mediated changes using chlortetracycline (CTC) or by changes in membrane fluidity monitored by the binding of the fluorescent amphiphilic probe, Merocyanine 540. Fluorescently labeled annexin-V, C6NBD and Ro-09-0198 can also be used to detect changes in membrane phospholipid distribution. Cell viability can be determined using the propensity of propidium iodide (PI), ethidium homodimer-1 (EthD-1) or Yo-Pro-1 to permeate damaged membranes. These are generally more adaptable to clinical flow cytometry than the bisbenzimide membrane impermeable stain, Hoechst 33258, which excites in the ultraviolet range and requires UV laser equipment. Mitochondrial function can be determined using rhodamine 123 (R123) and MitoTracker Green FM (MITO) and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1). Flow cytometry is a tool that may be used in the future to monitor many new potential markers of sperm function.     

Relationships among lipid peroxidation, glutathione peroxidase, superoxide dismutase, sperm parameters, and competitive index in dairy bulls

Sperm membranes contain high concentrations of polyunsaturated fatty acids that are highly susceptible to oxidative damage that interferes with fertilization ability. The objective of this study was to determine associations among lipid peroxidation (thiobarbituric-acid-reactive substance concentration), antioxidant enzymatic activities in frozen spermatozoa, and competitive indices. Semen from multiple ejaculates collected in succession from each bull (four Holstein and four Jersey) was pooled. Heterospermic doses (20x10(6)sperm/0.5mL straw) were made to obtain 16 Holstein/Jersey combinations (equal number of sperm from each bull). Cows were inseminated on observed or synchronized estrus. The sire of calves (N=460) was determined; based on the number of calves sired, a competitive index was obtained for each bull. Prior to preparation of the heterospermic doses, a sub-sample of semen from each bull was taken, processed, frozen, and stored concurrent with heterospermic samples. After thawing, these homospermic samples were assessed for lipid peroxidation, superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity, DNA fragmentation index (DFI), plasma membrane integrity (PMI), and total progressive motility (assessed by CASA). Sperm lipid peroxidation and the competitive index were negatively correlated (r=-0.78; P<0.05), the DFI and sperm lipid peroxidation were positively correlated (r=0.86; P<0.001), and there were negative correlations (P<0.05) for sperm lipid peroxidation and both PMI and total progressive motility (r=-0.78 and -0.83, respectively). There was neither significant association between SOD activity and competitive index, nor between GPx activity and competitive index. In conclusion, bulls with lower sperm lipid peroxidation had higher chances of siring calves; this was attributed to the deleterious effects of lipid peroxidation on sperm plasma membrane integrity and sperm DNA, which may reduce sperm fertilizing potential.     

Sperm apoptosis in fresh and cryopreserved bull semen detected by flow cytometry and its relationship with fertility.

The present study was conducted to detect sperm apoptosis in fresh and frozen semen and to determine its relationship with bull fertility. Three ejaculates were collected from five breeding bulls with different fertility levels and were cryopreserved using standard methods. Two flow cytometric methods were employed to measure apoptosis: an assay for phosphatidylserine (PS) translocation across the plasma membranes using fluorescein-labeled Annexin V and propidium iodide (PI), and an assay for nicked DNA using bromodeoxyuridine (BrdU), terminal deoxynucleotidyl transferase, and fluorescein-labeled anti-BrdU monoclonal antibody. Both assays showed that fresh sperm contained 10%-20% apoptotic sperm. Significant differences in the percentage of apoptotic sperm were observed among the bulls. Cryopreservation induced translocation of PS to the outer leaflet of the plasma membrane and caused most of the necrotic cells in fresh sperm to disintegrate. Bull fertility was significantly related to the percentage of necrotic or viable sperm in fresh semen as detected by the Annexin V/PI assay, to the number of apoptotic sperm in fresh semen as detected by the TUNEL assay, and to the level of chromatin or DNA condensation as detected by PI staining. The present study suggests that the presence of apoptotic spermatozoa in fresh semen could be one of the reasons for poor fertility in breeding bulls.     

Assessment of fresh and stored duck spermatozoa quality via in vitro sperm-egg interaction assay

An in vitro sperm-egg interaction assay was used to measue the quality of duck spermatozoa in fresh and stored semen. The inner perivitelline layer (IPVL), which had been separated from laid duck eggs, was incubated with spermatozoa in vitro. The number of points of sperm hydrolysis in the IPVL in vitro was logarithmically correlated with the fertility of the eggs laid by inseminated females, for both fresh semen (r = 0.85, P < 0.001) and stored semen at 5 degrees C for 24 h (r = 0.84, P < 0.001). After semen storage, the ability of spermatozoa to hydrolyze the IPVL decreased by 67.4% compared with the values for fresh semen, whereas egg fertility and sperm motility decreased by 47.8% and 15.2%, respectively. These results suggest that the in vitro sperm-egg interaction assay accurately reflects the fertilizing ability of fresh and stored duck spermatozoa and detects spermatozoal damage due to semen storage more sensitively than motility or fertility tests.     

Comparison of seminal quality in Holstein bulls as yearlings and as mature sires

Semen quality was compared in 5 Holstein bulls from samples collected as young sires (yearlings) and again as mature bulls after a mean interval of 1,265 d. At both sampling periods, the semen was examined for ejaculate volume, sperm numbers, post-thaw progressive motility and sperm viability. Sperm viability was assessed on cryopreserved samples with fluorescent SYBR-14 to stain living spermatozoa and propidium iodide (PI) to identify dead spermatozoa. The fluorescent populations of stained spermatozoa were quantified by flow cytometry. The percentages of living spermatozoa for the individual bulls, as determined by green fluorescence of SYBR-14, ranged from 44 +/- 3.1 to 54 +/- 0.3 for yearlings, and from 38 +/- 1.5 to 55 +/- 1.0 for mature sires. No differences in sperm viability were found between samples taken from yearling bulls and those of mature bulls. The percentage of spermatozoa stained with SYBR-14 was negatively correlated (r = -0.97; P = 0.0001) with the percentage of dead spermatozoa as indicated by PI staining. Comparisons of identical samples run on 2 different flow cytometers indicated that either flow instrument could be used to assess sperm viability. Although the individual bulls differed (P < 0.05) in ejaculate volume and sperm numbers as yearlings, they did not differ in these parameters as mature bulls. The average number of spermatozoa per ejaculate changed as a result of maturation, increasing from 6.2 +/- 1.0 to 10.7 +/- 1.1 x 10(9). Aging was significantly correlated with ejaculate volume (r = 0.76; P = 0.01) but not with the total number of spermatozoa per ejaculate (r = 0.51; P = 0.13). The maturational changes that occurred in the 5 bulls were minimal with the exception of the increased volume of the ejaculate and the number of spermatozoa per ejaculate.     

Relationship between sperm-zona pellucida binding assays and the 56-day nonreturn rate of cattle inseminated with frozen-thawed bull semen

Assays based on sperm-zona pellucida binding have been developed as diagnostic tests to predict the fertilizing potential of mammalian spermatozoa. Recently, we reported on the development of a sperm-zona pellucida binding assay (SZBA) for bull spermatozoa. The aim of the present study was to develop a hemi-zona assay (HZA) for bull spermatozoa and to investigate the relationship between SZBA and HZA outcomes and in vivo fertility. Frozenthawed semen samples from 8 fertile Swedish Red and White bulls (one ejaculate per bull) designated as the test semen samples and a single ejaculate from a fertile Holstein-Friesian bull designated as the control semen sample were used in this study. In the SZBA, 2 groups of 20 oocytes per semen test sample and in the HZA a minimum of 6 matching pairs of hemizonae were used for comparison of sperm binding with control semen. Sperm binding to matching hemi-zonae of individual semen samples was equal, and clearly demonstrated the feasibility of the HZA for cattle. A significant correlation was found between the SZBA and the HZA indices obtained from the different semen test samples (r = 0.42, P < 0.001; n = 67). There was no significant relation between the SZBA indices and the 56-d nonreturn rate of the test samples. However, the HZA indices of the semen test samples and the 56-d nonreturn rate were significantly correlated (r = 0.46, P < 0.0001; n = 67). It is concluded that HZA can be regarded as a potential assay for predicting the fertilizing ability of bovine semen samples. However, further studies using more semen samples are necessary to confirm this view.     

Effects of age and environmental factors on semen production and semen quality of Austrian Simmental bulls

More than 90% of the breeding stock of Austrian dual purpose Simmental cows is artificially inseminated. Knowledge of factors affecting sperm production and semen quality is of importance with regard to reproductive efficiency and thus genetic improvement as well as for the productivity and profitability of AI centers. Hence, semen data from two Austrian AI centres collected in the years 2000 and 2001 were evaluated. In total, 3625 and 3654 ejaculates from 147 and 127 AI bulls, respectively, were analysed regarding ejaculate volume, sperm concentration, percentage of viable spermatozoa in the ejaculate, total spermatozoa per ejaculate and motility. Effects accounted for were the bull (random), age of bull, collection interval, number of collection on collection day, bull handler, semen collector, temperature on day of semen collection, in the course of epididymal maturation (average temperature of days 1-11 before collection) and during spermatogenesis (average temperature of days 12-65 before collection). Age of bull significantly affected all traits (P<0.01 to P<0.001) except motility score in center 2. Ejaculate volume and total number of spermatozoa increased with age of bull while sperm concentration was lower in higher age classes (center 1). The collection team was also found to significantly influence semen quality traits. With increasing collection interval ejaculate volume and total number of spermatozoa increased significantly (P<0.05 to P<0.001) while collection intervals between 4-9 days and 1-6 days were superior with regard to sperm concentration and percentage of viable spermatozoa, respectively (P<0.10 to P<0.001). First ejaculates were superior with respect to ejaculate volumes, sperm concentrations and total number of spermatozoa per ejaculate (P<0.001). Temperature, either on day of semen collection or during epididymal maturation or spermatogenesis, had important but inconsistent effects on semen production and sperm quality. Overall, however, ambient temperatures in the range of 5-15 degrees C were found to be optimal for semen production.     

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.     

Binding of the anti-human sperm monoclonal antibody HS-11 to bull spermatozoa is correlated with fertility in vitro

The present study aimed to determine if there is bull to bull variation in the binding of the anti-human sperm monoclonal antibody (MAb) HS-11 to bull spermatozoa, and to investigate if there is any correlation between HS-11 binding to spermatozoa and in vitro fertility of the bulls tested. Semen samples of a single collection (split frozen in 0.5-ml straws) from 8 dairy bulls were used. Swim-up separated motile spermatozoa were incubated in 90-microl drops of capacitation medium (TALP+10 microg/ml heparin) at 39 degrees C, 5% CO2, 95% air. At 0, 2, 4 and 6 h of incubation HS-11 was added (1:1000 final concentration), and the MAb binding was assessed by indirect immunofluorescence assay (IIFA). The HS-11 binding was indicated by a bright green fluorescence of the sperm acrosome region. In vitro-matured, good quality bovine oocytes were randomly allocated to spermatozoa of each bull for in vitro fertilization. Sperm samples of 2 to 3 bulls were used in each trial until 4 replicates per bull were attained for IVF (n approximately 100 oocytes/bull) and IIFA experiments. Sperm capacitation status was assessed simultaneously using an egg yolk lysophosphatidylcholine- (LC) induced acrosome reaction assay. The binding of HS-11 to spermatozoa was maximum at 4 h of incubation in most (6/8) of the bull semen samples. Significant (P < 0.01) differences were observed between bulls in the binding of HS-11 to their spermatozoa (range 22 +/- 8 to 52 +/- 5%) at 4 h, but not within replicates. Similarly, variations (P < 0.05) in the cleavage rate were also seen (range 22 +/- 9 to 58 +/- 7%) between bulls. The HS-11 binding and cleavage were significantly correlated (r = 0.43; n = 32; P < 0.05). The highest percentage of spermatozoa underwent acrosome reaction in response to LC treatment at the 4-h incubation period. This and the linear relationship between HS-11 binding and the cleavage rate observed in the present study together strengthen our earlier suggestion that the binding of the monoclonal antibody HS-11 to bull spermatozoa on a time-dependent manner, may indicate capacitation changes. We conclude that 1) between-bull differences exist in HS-11 binding to spermatozoa, and in the cleavage rate, and 2) HS-11 binding to spermatozoa is correlated with fertility, as determined by the cleavage of bovine oocytes matured and fertilized in vitro.     

Viability and fertility of rabbit spermatozoa diluted in Tris-buffer extenders and stored at 15 degrees C

Artificial insemination (AI) in rabbits is not extensive in the breeding programs of the rabbit meat industry. A limiting factor is related to the semen preservation. In order to improve the use of AI, two experiments have been conducted to evaluate sperm viability and fertility of rabbit semen chilled and stored at 15 degrees C after dilution in Tris-based extenders. In Experiment 1, pooled semen samples were diluted 1:10 (semen/extender) in four different Tris-based extenders (Tris-citric-glucose (TCG), TES-Tris-glucose (TTG), Tris-citric-fructose (TCF) and TES-Tris-fructose (TTF)) and stored at 15 degrees C. Sperm viability was evaluated at 0, 24, 48, 72 and 96 h following dilution for total sperm motility (TSM), forward progressive motility (FPM), plasma membrane integrity (PMI) and acrosome integrity (NAR). Viability of spermatozoa declined with time of storage (P<0.05), irrespective of the extender used. There were interactions between extender and time of storage (P<0.05) in all viability parameters evaluated. After 96 h of storage, TCG provided the highest sperm viability (P<0.05) and TTG the lowest (P>0.05). In Experiment 2, a field trial was conducted at a commercial farm to evaluate the conception and farrowing rates of rabbit spermatozoa extended in TCG. After synchronization of oestrous and induction of ovulation, 3713 does with different physiological conditions (nulliparous, primiparous, lactating and re-breeding) were inseminated one time (15x10(6) sperm per doses) with semen stored at 0 (n: 1275), 24 (n: 1503) and 48 h (n: 935) at 15 degrees C. Overall conception and farrowing rates were 77.1+/-0.7 and 70.4+/-0.7, respectively, and the mean litter size was 7.6+/-0.1. Fertility results were unaffected by the time of semen storage (P>0.05). Regardless of time of semen storage, fertility results were affected by the physiological conditions of does (P<0.05). Nulliparous and lactating does showed the highest fertility and primiparous the lowest. In summary, these results indicate that Tris-buffer extenders are effective for preserving viability and fertilizing capability of rabbit spermatozoa stored at 15 degrees C.     

Evaluation of seasonal variations of semen freezability in Leccese ram

The experiment was carried out in Southern Italy (41 degrees N latitude) to examine the effects of seasonal variations of semen freezability in Leccese ram. Semen from five rams, collected every 2 weeks for a whole year, was frozen in straws, using a system based on Tris-fructose egg yolk as extender to constitute semen doses of 100x10(6) spermatozoa. Post-thaw survival and acrosomal status of cells were assessed by dual staining by Hoechst 33258 and FITC-PSA. Three different forms of fluorescence distribution were displayed indicating sperm without acrosome (unstained cells), sperm with damaged acrosome (cells with incomplete fluorescence over the head), sperm with widespread fluorescence (cells completely fluorescent). Motility and kinetic rating at thawing and after 1 and 3h incubation (37 degrees C) were also assessed.Semen frozen in summer and autumn, corresponding to the breeding season, showed the highest (P<0.01) post-thaw survival of spermatozoa (41.7%) and the lowest (P<0.01) incidence of spermatozoa with damaged acrosome. The positive influence of the summer-autumn period was expressed also on motility and kinetic rating of spermatozoa at thawing. The integrity of the acrosomal membrane was positively correlated (P<0.01) with sperm viability before processing (r=0.32) and after thawing (r=0.51).In conclusion, the results show that season exerts a significant influence on semen freezability in Leccese ram, with the best performance occurring the summer and autumn period, corresponding to the reproductive season in temperate zones.