Moribund sperm in frozen-thawed semen, and sperm motion end points post-thaw and post-swim-up, are related to fertility in Holstein AI bulls

The objectives were to compare testicular physical characteristics and post-thaw sperm characteristics and their associations with fertility in Holstein bulls used for AI. Ten Holstein bulls (4-5 y old) were classified as either high-fertility (HF) or low-fertility (LF; n = 5 each), based on adjusted 56-d non-return rates [non-return rate (NRR); range (mean ± SD): 55.6 ± 4.6 to 71.8 ± 1.3%). Testicular physical characteristics were not significantly different between the two groups. Four ejaculates were collected from each bull and cryopreserved. Several indexes of sperm motion (based on computer-assisted sperm analysis) at post-thaw and post-swim-up were correlated with NRR. Sperm from HF bulls were in transition to a hyperactivated motility pattern, whereas those from LF bulls had only a forward progressive motility pattern. In HF vs LF bulls, there was a greater percentage of viable sperm after thawing (60.6 ± 9.7 vs 49.5 ± 8.0%, P < 0.05) and after swim-up (70.9 ± 11.0 vs 63.0 ± 8.8%, P < 0.01); these two end points were positively correlated with fertility (r = 0.45, P < 0.01 and r = 0.78; P < 0.01, respectively). Furthermore, in HF vs LF bulls, the ratio of sperm recovered after swim-up to viable sperm in post-thaw semen was higher (P < 0.001), and the proportion of moribund sperm expressed as a percentage of live sperm differed (12.6 ± 3.4 vs. 16.4 ± 3.1%, P < 0.001) and was negatively correlated (r = −0.33, P < 0.05) with fertility. In conclusion, fertility of Holstein bulls maintained in a commercial AI center was not predicted by testicular physical characteristics, but it was associated with differences in moribund sperm in the inseminate, as well as characteristics of sperm post-thaw and after swim-up.     

Breed differences in competitive indices of Holstein and Jersey bulls and their association with sperm DNA fragmentation index and plasma membrane integrity

The objective was to evaluate the relationship of a competitive index (CI) determined by heterospermic performance and post-thaw semen quality of the same stored ejaculates. Semen from multiple ejaculates collected in succession from each bull (four Holstein and four Jersey) was pooled. Heterospermic doses (20x10(6)/straw) were made to obtain all possible Holstein-Jersey combinations (16 two-bull combinations) and contained 20x10(6) sperm/mL/bull. Cows at two University dairy farms were inseminated on observed or synchronized estrus. The sire of calves (N=460) were determined and a CI was determined for each bull (based on the number of calves sired). Prior to preparation of the heterospermic doses, a sub-sample of semen from each bull was taken, processed, frozen, and stored concurrently with heterospermic samples. Post-thaw semen samples (homospermic) from each bull were assessed for: sperm morphology, acrosome integrity, sperm motility parameters assessed by computer assisted sperm analysis (CASA), flow cytometry analysis of DNA Fragmentation Index (DFI), and Plasma Membrane Integrity (PMI). Heterospermic performance of Holstein bulls was superior to that of Jersey bulls. The DFI was negatively correlated to CI (r=-0.87; P<0.001), whereas the PMI (r=0.87; P<0.001) and total progressive motility (r=0.74; P<0.05) assessed by CASA were positively correlated to CI. In multivariate regression models, the DFI and PMI accounted for 87% variance in competitive index. In conclusion, bulls with less DFI and higher PMI had higher probabilities of siring calves.     

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.     

Infertility in a beef bull due to a failure in the capacitation process

The objective of this case report was to identify the cause of apparent idiopathic infertility in a Red Angus (beef) bull. Semen was collected by electroejaculation and submitted to a series of assays, including evaluation of sperm motility by computer-assisted sperm analysis (CASA), sperm morphology and DNA integrity, semen cryopreservation, AI, IVF, induction of the acrosome reaction, and determination of the level of sperm proteins associated with bull fertility potential. Total (92 ± 2%) and progressive (79 ± 4%) sperm motility; sperm concentration (1647 ± 429 × 10⁶ sperm/mL); proportions of morphologically normal sperm (83 ± 6%) and DNA integrity (96 ± 2), and acrosome-intact sperm (64 ± 4%) exceeded minimum acceptable values. Frozen sperm had good total (58.7 ± 6.7%) and progressive (43.9 ± 9.2%) motility immediately after thawing. However, AI of 16 heifers resulted in no pregnancies and blastocyst production rate (following IVF using sperm from this infertile bull) was nearly identical to that produced using dead sperm (a control of parthenogenesis; 2 ± 2 and 2 ± 3%; respectively P < 0.05). Treatment with a calcium ionophore (A23187) failed to induce the acrosome reaction in sperm from the infertile bull (P < 0.05). Evaluation of several proteins associated with the fertility potential of bulls revealed that the level of Binder Sperm Protein-1 (BSP1), known to be associated with the capacitation process, was much greater on sperm from the infertile bull compared to that of his sire. In conclusion, we inferred that the idiopathic infertility in this bull was caused by a failure to complete the capacitation process.     

The usefulness of combining traditional sperm assessments with in vitro heterospermic insemination to identify bulls of low fertility as estimated in vivo

To date, no single in vitro assessment can estimate bull fertility. This research was aimed at evaluating the ability of a series of laboratory assessments to assign 50 Holstein Friesian bulls grouped as low (ER-NRR<-1.5), medium (-0.5相似文献   

Sperm morphology and fertility of progeny-tested AI dairy bulls in Sweden

Use of bull semen with high levels of sperm abnormalities, reflecting genital dysfunction, is not recommended for artificial insemination (AI) since it would most likely lead to subfertility. Sperm quality, including sperm morphology, may deteriorate with increasing age of the bull thus becoming a source of concern when using older, progeny-tested AI bull sires. Although a relationship between sperm morphology and fertility after AI in progeny-tested bull sires has been reported, it is yet unclear which sperm abnormalities are most critical. This constituted the core aim of a 22-month long retrospective study in proven (aged 60-84 months at the start of the study) AI sires of the Swedish Red (SR, n=8) and Swedish Holstein (SLB, n=4) breeds where their semen (107 freezing batches in total, built by a single ejaculate (n=3) or pooling two consecutive ejaculates (n=104) collected at 1-3 months interval), were subjected to detailed morphological examinations on wet- and dry, stained smears. Attention was paid to between- and within-bull variations with regard to presence and level of sperm abnormalities. Sperm morphology differed significantly between sires and ejaculates, with 6/12 sires having ejaculates containing >10% of morphologically deviating sperm head shapes, a commonly used threshold for young AI bulls in Sweden. However, with the exception of pear-shaped or narrow-at-the-base anomalies, the mean values for individual defects were always within the limits expected for a normal bull sire, and were therefore considered acceptable. The percentage of morphologically normal spermatozoa was positively related to fertility, whose output differed significantly among bulls. Among sperm abnormalities, the proportion of morphologically deviating sperm head shapes were negatively correlated with fertility, pear-shaped sperm heads in particular. In conclusion, the relationship between sperm morphology and fertility after AI calls for frequent (2-3 months interval) detailed assessments of sperm morphology in AI stud bull sires.     

Effect of reducing sperm concentration during IVF on the ability to distinguish between bulls of high and low field fertility: work in progress

The objectives of this study were to evaluate the effect of sperm dose and sire on the fertilization rate, cleavage rate and blastocyst yield following insemination in vitro, to examine the relationship between these parameters and field fertility in cattle, and to examine the relationship between blastocyst quality and sire used in IVF. Frozen semen from four bulls with 150-day nonreturn rates ranging from 57 to 78% was used. In Experiment 1, oocytes were inseminated with sperm from one of the four bulls at concentrations ranging from 0.016 to 0.5 x 10(6)sperm/ml. A proportion of presumptive zygotes were fixed at 17 h post-insemination (hpi), while the remainder was transferred to in vitro culture (IVC) in droplets of synthetic oviduct fluid (SOF). Cleavage at 48 hpi and the percentage of oocytes reaching the blastocyst stage by Day 8 were recorded. In Experiment 2, to assess blastocyst quality, after insemination with semen from one of the four bulls, presumptive zygotes were cultured in SOF until Day 7. Blastocysts for each bull were removed and vitrified/warmed and survival was recorded at 24, 48 and 72 h after warming. Regardless of bull used, a concentration of 0.125 x 10(6)sperm/ml or above resulted in higher blastocyst yields than any lower concentration used. Fertilization and cleavage rates were also higher at higher sperm concentrations. The best predictor of field fertility was fertilization rate at a concentration of 0.5 x 10(6)sperm/ml (r=0.94, P<0.0001). There was also a significant correlation between cleavage rate at a concentration of 0.5 x 10(6)sperm/ml and nonreturn rate (r=0.90, P<0.0001). In Experiment 2, blastocysts derived from one bull, HTA, were of superior quality as measured by survival 24h after thawing, although these differences were less significant at the subsequent time points measured. In conclusion, these data show that differences between the field fertility of bulls can be determined at sperm concentrations routinely used in IVF. Lowering the sperm concentration does not increase the likelihood of optimizing the differences in fertility or cleavage rate between bulls of different field fertility. We have also demonstrated that the bull can have a significant effect on the quality of blastocysts produced using IVF techniques.     

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.     

Assessment of bull fertility using a mucus penetration test and a human chorionic gonadotrophin stimulation test

The relative predictive abilities of an in vitro mucus penetration test and a human chorionic gonadotrophin (hCG) stimulation test were compared for assessing bull fertility. Cervical mucus was collected from 22 Holstein cows and then stored in liquid nitrogen. Semen was collected from 13 Holstein bulls and evaluated for standard semen parameters. The mucus penetration test was performed with fresh ejaculates, and the results were expressed as the distance traveled by the vanguard spermatozoa during incubation at 38 degrees C for 10 min. Increases in plasma testosterone levels were determined by a ratio of testosterone concentration before and after hCG injection. Sperm motility and mucus penetration were significantly correlated with the conception rate. However, no significant correlation was confirmed between the increased plasma testosterone levels and conception rate. The results indicate that the mucus penetration test is an effective method for predicting the fertility of bulls.     

Dilauroylphosphatidylcholine liposome effects on the acrosome reaction and in vitro penetration of zona-free hamster eggs by bull sperm: II. A fertility assay for frozen-thawed semen

Frozen-thawed sperm from five bulls with fertility rates ranging from 48% to 77% were treated with seven concentrations of dilauroylphosphatidylcholine (PC12) liposomes to induce an acrosome reaction (AR) that enabled sperm to penetrate eggs. Treated sperm were incubated with liposomes for 7 min prior to insemination of zona-free hamster eggs in vitro. Sperm and eggs were incubated 3 hr at 39°C prior to fixation, staining, and examination for sperm penetration and nuclear decondensation. The percentage of motile sperm immediately after thawing as well as after treatment with liposomes had a low correlation with sire fertility (r = .39 and ?.63, respectively). The percentage of sperm exhibiting an AR was more highly correlated with fertility (r ? ?.85). Similar correlations were found between fertility and the penetration rates of zona-free hamster eggs or the total number of penetrating sperm. When data for two high and for two lower fertility buils were each grouped to increase information per data point the correlation between the PC12 concentration giving the maximum proportion of eggs penetrated and fertility was r = .92 (P ≤ .05). The correlation between the PC12 concentration producing the most total sperm penetrating the eggs and fertility r = .97 (P ≤ .05). It was concluded that PC12 liposomes induced an AR in bull sperm frozen-thawed in egg yolk extender. Frozen-thawed sperm from low fertility bulls require less PC12 to induce the AR and to penetrate zona-free hamster eggs than do sperm from higher fertility bulls. These differences in lipid requirements may help to provide a quick, direct laboratory assay method to estimate the fertility of frozen bull semen.     

Relationship between embryo development in vitro and 56-day nonreturn rates of cows inseminated with frozen-thawed semen from dairy bulls

Frozen-thawed bull semen with > 50% post-thaw motility from 40 batches (21 bulls, 2 consecutive ejaculates per batch) was used for fertilization (IVF) and embryo development in vitro to assess the relationship between field and laboratory fertility using a retrospective approach. Each frozen batch was tested in 3 or 4 replicates with 30 oocytes per replicate. Field fertility, quantified as the 56-d nonreturn rate and based on 89 to 441 artificial inseminations per frozen batch, ranged between 46.2 and 74.8%. The cleavage and blastocyst rates after IVF varied from 29.0 to 81.9% and from 1.8 to 32.0%, respectively, with significant differences among frozen batches. Rates of cleavage and blastocyst formation were significantly related to the nonreturn rate (r = 0.59, P < 0.001; r = 0.35, P < 0.05, respectively). The interaction between cleavage and blastocyst rate was 0.69 (P < 0.001). Significant variations (P < 0.05) among frozen semen batches within 15 bulls with >/= 2 different semen batches were found for the nonreturn rate (13.3%) of 2 bulls, for cleavage rates (26.7%) in 4 bulls and for blastocyst rates (20.0%) in 3 bulls. Significant differences (P < 0.05) among replicates within the 40 frozen semen batches were only found in 3 batches (7.5%) for the cleavage rate and in 7 batches (17.5%) for blastocyst rate. Overall, bull and frozen semen batch were the greatest sources of variation in the cleavage rate (30.6 and 29.4%, respectively), while testing date was the greatest source of variation in the blastocyst development rate (21.7%). The results indicated that in vitro fertilization and, to a lesser extent, culture to the blastocyst stage could be useful in estimating the potential fertilizing ability of frozen-thawed semen from dairy bulls.     

Relationships among frozen-thawed sperm characteristics assessed via the routine semen analysis, sperm functional tests and fertility of bulls in an artificial insemination program

Frozen semen specimens from 22 Holstein bulls representing a wide range of field fertility levels or nonreturn rates (NRR) were used in this study. Semen specimens were thawed at 37 degrees C for a minimum of 30 sec, followed by assessment via a routine semen analysis (RSA) and other sperm functional tests. The RSA was performed by assessing sperm count, motility and morphological characteristics. Other sperm functional tests were performed by assessing the acrosomal membrane integrity, sperm penetration into the cervical mucus and the sperm membrane functional integrity. Following assessment of sperm characteristics, the fertility data of the various bulls were compared to the RSA and the functional tests results. Bulls of high and low fertility were similar in terms of sperm count and progressive motility (P > 0.05). Other characteristics measured by the RSA and functional tests were significantly higher in high fertility bulls (P < 0.05). Correlation coefficients among the various sperm characteristics and fertility of bulls were highly significant (P < 0.01). The highest correlation coefficients between sperm characteristics and fertility were obtained for motility (r = 0.53; P < 0.01), normal morphology (r = 0.59; P < 0.01) and swollen spermatozoa (r = 0.57; P < 0.01). Analysis of specific sperm swelling patterns showed that those patterns considered to reflect maximal sperm swelling were indicative of high fertility.     

Breeding soundness and libido examination of Belgian Blue and Holstein Friesian artificial insemination bulls in Belgium and The Netherlands

Data on breeding soundness and libido evaluations in Belgian Blue (BB) bulls are scarce. The present study compared results of breeding soundness and libido evaluations of young BB bulls to young Holstein Friesian (HF) bulls prior to acceptance into an AI program. Breed differences for breeding soundness exist between BB and HF bulls, as 93.7% of the young BB bulls failed the breeding soundness examination (BSE) compared to 59.3% of the HF bulls (P=0.0005). Within the BB breed, differences were present between bulls of different ages, and bull selection for better fertility with increasing age apparently influenced the results. The number of reasons for which bulls failed the test differed between the age groups in the BB breed, whereas a tendency for more failure reasons in the BB breed was noticed in the breed comparison. The most important reasons for failure were sperm morphology and scrotal circumference (SC), but far more BB bulls failed for these traits compared to the HF breed (82.8% versus 56.0% and 43.8% versus 17.6% in the BB and the HF breed for sperm morphology (P=0.0005) and SC (P<0.0001), respectively). The high proportion of BB bulls with a substandard SC and poor sperm morphology might suggest an increased prevalence of testicular hypoplasia or degeneration within this breed. Concerning libido, the reaction time did not differ either between breeds or between age groups within the BB breed, whereas mounting enthusiasm, although not different between the two breeds, did decline with increasing age, probably due to the greater mating experience of the older bulls. All in all, libido did not seem to be different between the breeds.     

Metabolomic fingerprinting of bull spermatozoa for identification of fertility signature metabolites

The objective of the study was to identify the fertility‐associated metabolites in bovine spermatozoa using liquid chromatography‐mass spectrometry (LC‐MS). Six Holstein Friesian crossbred bulls (three high‐fertile and three low‐fertile bulls) were the experimental animals. Sperm proteins were isolated and protein‐normalized samples were processed for metabolite extraction and subjected to LC‐MS/MS analysis. Mass spectrometry data were processed using iMETQ software and metabolites were identified using Human Metabolome DataBase while, Metaboanalyst 4.0 tool was used for statistical and pathway analysis. A total of 3,704 metabolites belonging to various chemical classes were identified in bull spermatozoa. After sorting out exogenous metabolites, 56 metabolites were observed common to both the groups while 44 and 35 metabolites were found unique to high‐ and low‐fertile spermatozoa, respectively. Among the common metabolites, concentrations of 19 metabolites were higher in high‐fertile compared to low‐fertile spermatozoa (fold change > 1.00). Spermatozoa metabolites with variable importance in projections score of more than 1.5 included hypotaurine, d ‐cysteine, selenocystine. In addition, metabolites such as spermine and l ‐cysteine were identified exclusively in high‐fertile spermatozoa. Collectively, the present study established the metabolic profile of bovine spermatozoa and identified the metabolomic differences between spermatozoa from high‐ and low‐fertile bulls. Among the sperm metabolites, hypotaurine, selenocysteine, l ‐malic acid, d ‐cysteine, and chondroitin 4‐sulfate hold the potential to be recognized as fertility‐associated metabolites.     

Effect of relaxin on cryopreserved beef bull semen characteristics

This study aimed to improve the quality of cryopreserved beef bull (Piedmontese) semen by incorporation of relaxin in diluted semen before cryopreservation procedures. Semen samples were collected from 4 proven fertile bulls, using artificial vagina, once per week for 8 consecutive weeks and pooled together then diluted with Bullxcell® extender, and supplemented with different concentrations of relaxin (0 (control), 25, 50 and 100 ng/ml) before cooling, equilibration and freezing procedures. Frozen semen was thawed and assessed for motility by Computer-Assisted Sperm Analysis and vitality parameters such as acrosome, plasma membrane and DNA integrities, apoptosis, mitochondrial membrane potential, mucus penetration and SOD activity. The developmental potential of bovine embryos produced in vitro by using relaxin-treated was also investigated. In the present study, 50 and 100 ng/ml relaxin incorporation in extended bull semen before cryopreservation induced a reduction of sperm motility immediately after thawing (0h), whereas, during long incubation periods (1–2 h), relaxin showed a significant positive effect on sperm quality by improving the sperm motility and velocity parameters. Interestingly, sperm vitality was improved by 25 and 100 ng/ml relaxin and the blastocyst developmental rate was significantly increased in the 25 ng/ml relaxin group compared with controls (52/118, 44.0% vs. 32/116, 27.6%, respectively). These findings suggest a potential use of relaxin at the doses tested in the present study as an additive in the cryopreservation media of bull semen to improve sperm quality.     

Development of embryos after in vitro fertilization of bovine oocytes with sperm from either yaks (Bos grunniens) or cattle (Bos taurus)

The objectives of this study were to investigate differences in fertilization and development of embryos after in vitro fertilization of Bos taurus (cow) oocytes with sperm from either yaks (Bos grunniens) or Holstein bulls. Frozen-thawed spermatozoa (Holstein n=5 sires; yak n=5 sires) were evaluated for motility (forward progression) and acrosomal status immediately post-thaw and then 1, 2, 3, and 8h later. In vitro-matured cow oocytes (n=1652) were inseminated with either Holstein bull or yak spermatozoa and after an 18-h co-incubation period, a proportion of the oocytes were fixed and examined for sperm penetration, polyspermy, and male pronuclear formation. The remaining oocytes were cultured in vitro and evaluated for cleavage and blastocyst production rates. Overall, there were species differences (P<0.05) and an effect of time (P<0.01) in sperm motility and acrosome integrity. An effect (P<0.01) of a species-by-time interaction was detected for motility, but not for acrosome integrity. The percentage of oocytes penetrated and the formation of two pronuclei when cow oocytes were inseminated with yak spermatozoa (97.4% and 81.6%, respectively) were greater (P<0.01) than that achieved with Holstein bull spermatozoa (77.8% and 65.9%, respectively), but the incidence of polyspermy (>2 pronuclei) was similar (P>0.05; 10.8% vs. 15.8%). The yak male symbolxcow combination gave a higher cleavage rate than the Holstein male symbolxcow combination (P<0.05; 76.3% vs. 63.3%), but there was no difference in the blastocyst rate (17.9% vs. 14.5%). It is concluded that yak spermatozoa could successfully fertilize cattle oocytes and their hybrid embryos had normal competence to develop to blastocysts.     

Effects of experimentally generated bull antisperm antibodies on in vitro fertilization.

To test the hypothesis that bull antisperm antibodies have the capacity to interfere with fertilization, antisperm antibodies were generated in three 13-mo-old Holstein bulls by auto-immunizing each bull with sperm three times. All bulls produced serum antisperm IgG1 and IgG2 antibodies. No serum antisperm IgA nor seminal plasma antisperm antibodies of any isotype could be detected by ELISA. Western blots were performed with immunopurified IgG1 and IgG2 from pre- and post-immunization sera from one test bull. Both post-immunization IgG1 and IgG2 recognized a 45-kDa sperm antigen. Serum samples from a normal bull stud population tested by ELISA had significantly higher levels of antisperm antibodies than did heifers. The bull stud serum samples giving the highest ELISA values differed from those of the immunized bulls in that their antisperm antibodies were of the IgM isotype only. Bull sperm were incubated with serum from the immunized and control bulls, then added to bovine oocytes in vitro. Incubation of sperm with post-immunization serum reduced in vitro fertilization rates (p < 0.01). This study demonstrated that antisperm IgG1 and IgG2 generated by sperm auto-immunizations reduced fertility in vitro, and therefore naturally occurring antisperm antibodies may affect fertility in bulls.     

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.     

Testicular dysfunction is responsible for low sperm quality in Belgian Blue bulls

In a previous study, we demonstrated that Belgian Blue (BB) bulls have a higher prevalence of small scrota and poorer semen morphology compared to the Holstein Friesian (HF) breed in Belgium. The present study tested the hypothesis that the underlying reason for these BB traits negative to fertility was testicular degeneration, associated with an eventual hypoplastic background. At culling, sperm quality and testicular histology of BB bulls were assessed and compared to that of HF bulls. Besides semen quality being generally poorer in the BB breed, significantly more degenerative changes were encountered in BB compared to HF testicles (degeneration index: 37.7+/-11.9 versus 29.3+/-9.9 for BB and HF bulls, respectively; P=0.053). These results correlated to the percentage of normal spermatozoa (r=-0.44; P=0.024) and primary abnormalities (r=0.38; P=0.053). Moreover, the relative amount of collagen fibers present in the testicular interstitial connective tissue was correlated with % normal sperm (r=-0.47; P=0.017), primary defects (0.48; P=0.014), and the degeneration results (r=0.63; P<0.001). The % testicular interstitial collagen fibers differed significantly between breeds (10.6+/-4.0% for the BB versus 7.6+/-1.9% for the HF bulls; P=0.016). This increased amount of connective tissue in BB testes might hypothetically be responsible for the poorer sperm quality. This condition can be defined as a mild form of testicular hypoplasia, and might, in turn, be responsible for the higher sensitivity to testicular degeneration, which is encountered in the BB breed.     

Effects of heparin and sperm concentration on cleavage and blastocyst development rates of bovine oocytes inseminated with flow cytometrically-sorted sperm

The objective was to determine the optimal concentration of heparin for sperm capacitation, as well as the optimal sperm concentration for in vitro fertilization using flow cytometrically-sorted sperm from individual bulls. A total of 5327 bovine oocytes and sperm from four bulls were examined. Oocytes from slaughterhouse ovaries were matured in TCM199 for 22-24 h. Flow cytometrically-sorted sperm as well as unsorted control sperm from the same bulls were cryopreserved. For sperm from each of the four bulls, oocytes were inseminated in a three-by-three factorial design plus one control group (three heparin concentrations: 0, 2, and 10 microg/ml and three sperm concentrations: 0.5 x 10(6), 1.5 x 10(6), and 4.5 x 10(6) ml(-1); 10 microg/ml of heparin and 1.5 x 10(6) ml(-1) of sperm were used for the unsorted control). Presumptive zygotes were cultured in chemically defined media, CDM-1 and CDM-2 for 52-54 h and 96 h, respectively. Samples of about 10 oocytes from each of the 10 treatment groups per replicate were fixed at 18-20 h after insemination to determine sperm pronuclei formation and polyspermy. Increased polyspermy resulted as heparin and sperm concentrations increased (P < 0.05). A higher rate of polyspermy was found in oocytes inseminated with unsorted control sperm compared with sorted sperm (P < 0.05). Sperm of one of four bulls tested required no heparin and lower concentration (0.5 x 10(6) ml(-1)) to obtain optimal cleavage and blastocyst rates while optimal parameters for another bull were higher heparin (10 microg/ml) and sperm concentrations (4.5 x 10(6) ml(-1)). Optimal parameters for the other two were intermediate levels of heparin and sperm. Sperm appeared to be partially capacitated during the flow cytometric-sorting process used for sex pre-determination. When heparin and sperm concentrations were optimized for individual bulls, blastocyst production per oocyte was similar for sorted and unsorted sperm for three of the four bulls studied.