Effect of trypsin in semen on in vivo fertilization and early embryonic development in superovulated heifers

The effect of trypsin on the fertilizing capacity of bull semen was investigated as part of the evaluation of the addition of trypsin to semen as a method for destroying or inactivating infectious agents. Parts of the ejaculates from four bulls were treated with 0.3% trypsin solution. Both the treated and untreated aliquots of semen were frozen, thawed and used for the artificial insemination of superovulated heifers. Two hundred and thirty ova and embryos were collected from 22 heifers on day 7 after oestrus (insemination). One hundred and ten out of 164 (67%) embryos and ova from 15 heifers inseminated with trypsin-treated semen were classified as of transferable quality compared to 46 out of 66 (70%) in the control group of 7 heifers (p>0.05). There was no difference in the proportion of fertilized ova or degenerated embryos resulting from the control or trypsin-treated samples of frozen-thawed semen, which is consistent with results obtained previously using fresh semen.     

Effect of narrow sperm head shape on fertility in cattle

Seven experiments were done in feedlot heifers to determine the importance of various degrees of narrowness of the sperm head on fertility in feedlot heifers. Frozen semen used in these experiments was selected to be normal in all respects except for very high numbers of a single specific type of sperm head aberration. Semen with the sperm aberration in question and control semen were selected to be as similar as possible in dose and postthaw viability so that differences in fertility would be attributable to the morphological variant under study. Fertilization rates were determined by collecting embryos from the reproductive tracts of superovulated heifers which had been slaughtered seven days after insemination. Pregnancy rates and rates of embryonic loss were studied in estrus-synchronized heifers by repeated transrectal ultrasound examinations from day 22 to day 55 after insemination. Reproductive tracts were collected and examined after slaughter at 60 days postinsemination.

The combined results of these experiments show that a moderate degree of sperm head narrowness, in the absence of other seminal signs of a disturbance of spermatogenesis, is not detrimental to fertility. However, extreme narrowness of the postacrosomal region of the sperm head of most spermatozoa, as was found in two bulls without other seminal signs of a disturbance of spermatogenesis, resulted in significantly reduced fertility. The data suggest that, although a decision between normal and abnormal sperm morphology may contain a degree of subjectivity, of the defects studied only sperm with extreme narrowness of the post-acrosomal region are likely to reduce fertility.

     

Abaxial tail attachment of bovine spermatozoa and its effect on fertility

The prevalence of abaxial sperm tails, accessory tails and double tails, was determined from semen evaluation records of 600 western Canadian range bulls and 449 bulls in Canadian artificial insemination centers. Spermatozoa with abaxial tails were produced by 10.5% of bulls, however, only 0.48% produced sperm with greater than 50% abaxial tails, and 0.86% of the 1049 bulls produced sperm with accessory and double tails. Three experiments were done to determine the importance of abaxial sperm tails to fertility. In experiments 1 and 2, frozen semen with 88%, 50% and 0% abaxial tails was used to inseminate synchronized heifers, which were allowed to develop pregnancy, and superovulated heifers, which were slaughtered prior to embryo recovery seven days after breeding. In experiment 3, a bull which produced 100% abaxial sperm was used in a competitive mating situation with three control bulls in a herd of 118 cows. The combined results of these experiments indicate that spermatozoa with abaxial tail attachment fertilize ova at a normal rate and are not associated with any increase in embryonic death.     

In vitro evaluation of cryopreserved bovine sperm and its relation to field fertility in fixed‐time artificial insemination

This study aimed to assess characteristics of bovine cryopreserved sperm and evaluate its relation to field fertility in fixed‐time artificial insemination (FTAI). Semen samples of 16 bulls were used to inseminate 811 Nellore cows, and four of these bulls were also used to inseminate 101 Nellore heifers. Samples of the same ejaculate used for FTAI from each bull were analysed in the laboratory after thawing. Sperm motility and vigour were subjectively assessed by light microscope, and integrity of the plasma and acrosome membranes, and H₂O₂ production were evaluated by flow cytometer. Relation among sperm characteristics and pregnancy rate of cows and heifers were evaluated by univariate and multivariate logistic regression. Subjective sperm motility and vigour did not affect the probability of pregnancy in cows or heifers. In univariate analysis for pregnancy in cows, sperm traits related to acrosome injury positively affected probability of pregnancy mainly when associated with plasma membrane integrity; H₂O₂ production seems to be less important than plasma membrane integrity in affecting probability of pregnancy. In multivariate analysis, sperm traits related to injured acrosome positively affected probability of cow and heifer pregnancies while intact acrosome was negatively related to cow pregnancy. Intact plasma membrane and high H₂O₂ production were positively related to cow pregnancy but negatively related to heifer pregnancy. Results suggest that a capacitation‐like status of the acrosome may benefit probability of pregnancy in cows.     

Fertility in Single‐ovulating and Superovulated Dairy Heifers after Insemination with Low Dose Sex‐sorted Sperm

The present study was performed to test fertility in single‐ovulating and superovulated dairy heifers after insemination with low dose sex‐sorted sperm under field conditions. Some parameters, including the dosage, deposition site and timing, were assessed with the pregnancy rates after artificial insemination (AI). Moreover, the use of oestrus synchronization in combination with sorted sperm was evaluated. Besides that, we also improved the embryo production efficiency in superovulated dairy heifers by optimizing the timing of inseminations and repartitioning the sexed sperm dosage among multiple inseminations. The conception rate (52.8%) in heifers after low dose (2 × 10⁶) insemination with sorted sperm deep into the uterine horn did not differ (p > 0.05) from that (59.6%) of conventional AI (1 × 10⁷ non‐sorted sperm) and that of deep insemination with low dose non‐sorted sperm (57.7%). There was also no difference (p > 0.05) between conception rates after single (51.7%) and double (53.8%) deep insemination with sorted semen. Heifers inseminated with sorted sperm at synchronous oestrus had a lower pregnancy rate (48.1%) than heifers at spontaneous oestrus (53.6%), but this did not reach statistical difference (p > 0.05). The average number of transferable embryos collected in vivo from heifers inseminated with sorted sperm (4.81 ± 2.04) did not differ (p > 0.05) from that obtained from heifers after insemination with non‐sorted sperm (5.36 ± 2.74). Thus, we concluded that the pregnancy rate after deep intra‐uterine insemination with low dose sorted sperm was similar to that of non‐sorted sperm, which was either also deposited at a low dose deep intra‐uterine or into the uterine body. Sychronization of oestrus can be beneficial in combination with sorted sperm to optimize the organization and management of dairy herds. The results from superovulated heifers demonstrated that our insemination regime can be used to obtain a comparable embryo production efficiency with sorted sperm than with non‐sorted sperm.     

Artificial insemination outcomes in beef females using bovine sperm with a detectable fertility-associated antigen

In this study, semen samples from 25 bulls that had passed a breeding soundness evaluation were analyzed for the presence or absence of a 31-kDa protein, known as fertility-associated antigen (FAA), on spermatozoal membranes. Eighteen bulls had FAA on sperm (FAA-positive) and seven were devoid of FAA on sperm (FAA-negative). A single ejaculate from each bull was extended and frozen with 25 to 30 x 10(6) sperm in .5-mL straws. Crossbred replacement heifers (n = 865) were estrus-synchronized and artificially inseminated either at timed AI or 12 h after they were detected in estrus. Mature cows (n = 285) were inseminated 12 h after they were detected in estrus during a 45-d AI period. Pregnancy rates (pooled) to first AI service for females (n = 764) inseminated with FAA-positive sperm were 65.6% and were 49.7% for females (n = 386) inseminated with FAA-negative sperm (P < .005). Among the estrus-synchronized replacement heifers, pregnancy rates to synchronized AI service for heifers (n = 550) inseminated with FAA-positive sperm were 62% and were 45.7% for heifers (n = 315) inseminated with FAA-negative sperm (P < .005). These data indicate that pregnancy rates to first AI service at spontaneous and synchronized estrus are higher when using semen from bulls with detectable FAA on spermatozoal membranes compared to semen from bulls devoid of FAA on membranes. Fertility-associated antigen is an important determinant for fertility potential of sperm from bulls to be used in AI breeding programs.     

精子生育相关性抗原对奶牛妊娠率影响研究

为研究精子生育相关性抗原对奶牛妊娠率的影响,测试了16头种公牛精子的生育相关性抗原,进行人工授精,比较奶牛的妊娠情况.结果显示:测试的16头种公牛精子生育相关性抗原,其中13头呈阳性,3头阴性,阴性率为18.75%.在用FAA阳性精液进行人工授精的520头奶牛中,有433头奶牛怀孕,妊娠率为83.26%.而FAA阴性授精的80头奶牛中,只有52头奶牛怀孕,妊娠率为65.0%(P<0.01).因此,奶牛人工授精FAA阳性精子妊娠率比FAA阴性精子高18个百分点,大大提高了奶牛的妊娠率.     

黑白花公牛冻精受精力的检测—穿透仓鼠卵的试验

本试验用SPA法(Sperm Penetration Assay)即精子穿透分析法对8头黑白花公牛冻精的受精力进行检测,以预测各公牛精液受精力的水平。结果:每卵表面平均精子数为3.2～9.5个,每卵内精子数为1.6～3.4个,被穿透的卵子比例即穿透率为34.9～65.7％,具雄原核的卵子比例即原核卵率为16.0～32.4％。这些数值与各公牛冻精授精500多头母牛的效果作对照,8头公牛平均80(70～90)天的不返情率为56％(45.8～71.1％),不返情率与雄原核卵率,穿透率呈高度正相关,相关系数分别为 0.82和 0.92。 另一组试验结果表明:授精时间对穿透率特别是对雄原核形成有很大影响。授精2小时后观察,没有雄原核形成,只有附着于卵子表面的精子;授精4小时后,精子已通过卵黄膜进入卵内,并开始形成雄原核;授精6小时后形成发育良好的雄原核。 这些观察结果有助于利用这项技术在生产上对公牛精液的受精力进行客观评定,为选择后备公牛提供重要的生理指标,特别是对评定冷冻精液的品质具有一定的参考价值。并为精子顶体反应、受精机理、体外受精、遗传学和胚胎学等领域的研究提供新的手段。     

Development of Procedures for Sex-sorting Frozen–Thawed Bovine Spermatozoa

Dairy bull sperm may be sex‐sorted, frozen and used to artificially inseminate heifers with acceptable fertility if the herd is well‐managed. One drawback to the technology is that donor bulls must be located within a short distance of the sorting facility in order to collect semen, which limits the number of bulls from which sorted sperm are available. A successful method used to overcome this limitation in sheep is sex‐sorting from frozen–thawed semen and refreezing for artificial insemination. This technique is attractive to the dairy industry, and therefore a series of three experiments was designed to investigate the optimal methods to prepare, sex‐sort and re‐freeze frozen–thawed bovine sperm. Sperm were prepared for sorting by density gradient separation in either PureSperm^® or BoviPure?, followed by staining in one of three diluents (Androhep^®, Bovine Sheath Fluid + 0.3% BSA or TALP buffer). Sperm were sorted and collected into Test yolk buffer, and frozen in an extender containing 0, 0.25, 0.375 or 0.5% Equex STM Paste. Frozen–thawed sperm were better orientated (p = 0.006) and had fewer damaged membranes (8.7 ± 0.6% vs 19.5 ± 2.4%; p = 0.003) after centrifugation in PureSperm^® rather than BoviPure? gradients. Sperm orientation (p < 0.05) and motility (69.9 ± 3.0 vs 55.6 ± 4.0; p < 0.001) were highest after staining in Androhep^® rather than in TALP buffer. Sperm were more motile (58.2 ± 4.7 vs 38.7 ± 3.5; p < 0.001) and had better acrosome integrity (74.3 ± 2.9 vs 66.8 ± 2.0; p < 0.001) after freezing in an extender containing 0.375% Equex STM Paste than in extender without Equex. Hence, a protocol has been developed to allow frozen–thawed bull sperm to be sex‐sorted with high resolution between the sexes, then re‐frozen and thawed with retention of motility and acrosome integrity.     

Effect of acrosomal defects on fertility of bulls used in artificial insemination and natural breeding

Four bulls that produced spermatozoa with a high percentage of abnormal acrosomes were individually placed in pens with females for 21 days. Frozen semen from 2 of the bulls was used for artificial insemination. One of the bulls was placed in a competitive mating situation with normal bulls at pasture. First service pregnancy rates were determined by transrectal ultrasonography 28 days after bull removal from breeding pens, or after the last artificial insemination. The results of competitive mating at pasture were determined from breeding observations, the phenotypic characteristics of calves sired, and blood typing for parentage. The results of these studies suggest that bulls that produce a high percentage of spermatozoa with indented acrosomes may have normal fertility when used in artificial insemination or in single sire mating; however, their fertility may be low when breeding competitively with bulls with normal spermiograms.     

Influence of the Donor Male on the Fertility of Frozen-Thawed Rabbit Sperm after Artificial Insemination of Females of Different Genotypes

In this work, the influence of the donor male on the fertility of cryopreserved rabbit sperm after artificial insemination into females from different genotypes was evaluated. Females belonged to three lines selected for maternal characteristics (A, V and H) and all the possible crosses between them. Sperm from five males from the line selected for one of the maternal characteristics (line V) was frozen individually in a Tris-citric acid-glucose diluent with 1.75 m of DMSO and 0.05 m of sucrose (final concentrations). After artificial insemination of the cryopreserved sperm, fertility rates and prolificacy were similar for all groups of females (56% of fertility rate and 7.2 total born). Significant differences between males were observed for both fertility (p < 0.05) and kindling (p < 0.01) rate. These differences could be because of differences in the freezing resistance of sperm from the different males.     

The Effect of Sperm Morphology and Sire Fertility on Calving Rate of Finnish Ayrshire AI Bulls

Good‐quality semen is a prerequisite for successful and profitable artificial insemination (AI) of modern dairy cattle. Fertility of the bulls is evaluated with andrological examinations and semen analyses, such as morphology. However, little attention has been paid to the inheritance of bull fertility. In this study, we correlated sperm morphology, birth year and station of 695 AI bulls with calving rate (CR). Sperm morphology was clearly associated with CR underlining the usefulness of morphological examination in the assessment of fertility. The correlation between the proportion of normal spermatozoa and CR was significant (p < 0.001). No significant differences were detected between stations or birth years. We also compared the CR of 695 AI bulls with the CR of their 27 sires to study the inheritance of fertility. Sire's CR did not correlate with the CR of the sons (p = 0.218). This result indicates that at least when sires of acceptable CR are used to produce sons for use in AI the inheritance of CR is not significantly correlated.     

Is sperm DNA fragmentation a good marker for field AI bull fertility?

This paper aimed at investigating the potential use of sperm DNA fragmentation (SDF) to improve the routine screening of infertility of Holstein bulls. Cryopreserved sperm samples from 201 Holstein bulls provided by an AI center were used in the analyses of SDF at 0 (SDF_0) and 6 (SDF_6) h of incubation at 37°C. A refinement of the sperm chromatin dispersion test implemented in the Sperm-Halomax kit was employed to measure SDF. Records on routinely collected semen traits (volume, concentration, mass and individual motility evaluated in the fresh ejaculate, and individual motility in post-thawed semen straws) were provided by the AI center. Artificial insemination bull fertility was obtained from official field recording as successful or failed insemination. The results show that the average SDF was low (around 3.5%) at 0 and 6 h of incubation. A moderate effect of inbreeding depression was found. Estimated heritability for SDF traits were moderately high (0.41 and 0.29 for SDF_0 and SDF_6, respectively) and estimated repeatability of SDF measures in the same animal were high (0.73 and 0.70 for SDF_0 and SDF_6, respectively). An overall estimated service bull value (ESBV) obtained through statistical modeling that allowed for adjustment of systematic environmental effects not specific to a bull and of the female contribution to fertility, and the estimated genetic values (EGV) were obtained from field-recorded AI information. The ESBV and EGV were also obtained for all semen traits. Moderately large and negative Pearson correlation coefficients were observed between SDF traits and male fertility ranging from (-0.43 to -0.50; P <0.001). Results of stepwise regression analyses showed that SDF_6 had the largest partial r(2) (0.15 to 0.26) among all semen characteristics. Overall, the selected semen traits explained 25% and 31% of the observed variability in bull fertility measured as EGV and ESBV, respectively. When looking at the predictive ability of bull fertility categories, the results of discriminant and logistic regression analyses showed that low-fertility bulls (those in the 10th or lower percentile in the fertility distribution) can be accurately identified by using measures of SDF alone or in combination with sperm motility. Values of SDF around 7% to 10% could be used as indicators of low AI success.     

Post-Death Cloning of Endangered Jeju Black Cattle (Korean Native Cattle): Fertility and Serum Chemistry in a Cloned Bull and Cow and Their Offspring

To preserve Jeju black cattle (JBC; endangered native Korean cattle), a pair of cattle, namely a post-death cloned JBC bull and cow, were produced by somatic cell nuclear transfer (SCNT) in a previous study. In the present study, we examined the in vitro fertilization and reproductive potentials of these post-death cloned animals. Sperm motility, in vitro fertilization and developmental capacity were examined in a post-death cloned bull (Heuk Oll Dolee) and an extinct nuclear donor bull (BK94-13). We assessed reproductive ability in another post-death cloned cow (Heuk Woo Sunee) using cloned sperm for artificial insemination (AI). There were no differences in sperm motility or developmental potential of in vitro fertilized embryos between the post-death cloned bull and its extinct nuclear donor bull; however, the embryo development ratio was slightly higher in the cloned sperm group than in the nuclear donor sperm group. After one attempt at AI, the post-death cloned JBC cow became pregnant, and gestation proceeded normally until day 287. From this post-death cloned sire and dam, a JBC male calf (Heuk Woo Dolee) was delivered naturally (weight, 25 kg). The genetic paternity/maternity of the cloned JBC bull and cow with regard to their offspring was confirmed using International Society for Animal Genetics standard microsatellite markers. Presently, Heuk Woo Dolee is 5 months of age and growing normally. In addition, there were no significant differences in blood chemistry among the post-death cloned JBC bull, the cow, their offspring and cattle bred by AI. This is the first report showing that a pair of cattle, namely, a post-death cloned JBC bull and cow, had normal fertility. Therefore, SCNT can be used effectively to increase the population of endangered JBC.     

Sperm penetration assay as an indicator of bull fertility

To predict the fertility of frozen-thawed bull spermatozoa, a sperm penetration assay (SPA) using zona-free hamster oocytes was optimized, and the assay results were compared with data from field fertility expressed as the non-return rate (NRR). To increase sperm penetration, the spermatozoa were pre-incubated and coincubated with oocytes in media containing various concentrations of heparin (0 to 50 μg/ml). Coincubation with 10 μg/ml heparin showed the highest sperm penetration (P<0.05); it is considered to be the optimized SPA method. Sperm fertility index values obtained from WSPA were significantly correlated with the historic average NRR of 46 bulls (P<0.01). To determine the normal range for SPA, we established the lower limits of the sperm fertility index and set the cut-off value at 2.55, at which point the NRR was more than 70%, using the receiver operating characteristic curve. The overall accuracy for the 46 bulls was 95.7% (44/46) for both the low and high NRR, with a sensitivity of 95.5% (21/22) and a specificity of 95.8%. This protocol would make it easier to discriminate bulls according to their sperm fertilizing ability.     

Effect of linoleic acid albumin in a dilution solution and long-term equilibration for freezing of bovine spermatozoa with poor freezability

Despite normal eucrasia, mating desire and semen quality, sire bulls sometimes have spermatozoa with poor freezing tolerance. This study assessed effects of the addition of linoleic acid albumin (LAA) and long-term (LT) equilibrium to frozen semen on their sperm freezing tolerance. Immediately after collection using an artificial vagina and a breeding mount, semen was diluted with yolk citrate buffer; then, it was cooled slowly to 4°C during more than 5 h. Equilibrium treatment at 4°C was applied using the same extender supplemented with glycerol. Semen of bull A, with low sperm freezing tolerance, was treated with 1 mg/ml of LAA added to the first extender. The equilibrium treatment at 4°C was prolonged to 30 h. Significantly higher motility rates were obtained for the LT + LAA-treated sperm before and after freezing-thawing. However, for semen of bulls B and C with normal sperm freezing tolerance, the LT + LAA treatment barely exhibited a small effect on the motility rate. Almost no difference was found among bulls A, B and C in the motility rates of LT + LAA-treated sperm after freezing-thawing. No difference of fertility was apparent on LT + LAA-treated frozen sperm in comparison with normal sperm in embryonic collection and in vitro fertilization. It was not an aberration of fertility in vivo or in vitro. In addition, the conception rate of artificial insemination did not have a difference, and a normal calf was obtained. Results show that addition of LAA to an extender for frozen bovine spermatozoa and 30 h of low-temperature equilibrium might improve the motility of freezing-thawing spermatozoa with poor freezability. Sperm exhibited normal fertilization capability and ontogenic capability.     

Production of sexed bovine embryos in vitro can be improved by selection of sperm treatment and co-culture system

The study investigated the effects of sperm sorting, capacitation treatment and co-cultivation on sexed bovine in vitro embryo production. The effect of treatment and co-culture on production of embryos of the preferred sex from unsorted sperm was also studied. Sperm from five breeding bulls was used for fertilization of mature oocytes as follows : Experiment 1, sorted and unsorted sperm (bulls A-E) treated only with heparin in standard co-cultures; Experiment 2, sorted sperm (bulls A-E) treated with heparin-PHE (penicillamine, hypotaurine, and epinephrine) or heparin-caffeine in drop co-cultures; and Experiment 3, unsorted sperm (bull E) treated with either heparin-PHE or heparin-caffeine in both standard and drop co-cultures. In all bulls, treatment with heparin resulted in significantly (p < .05) reduced cleavage and blastocyst rates from sorted sperm, as compared with those from unsorted sperm. In bulls A, B, D and E, treatment of sorted sperm with heparin-PHE in drops significantly increased the blastocyst rate (p < .05). In unsorted sperm of bull E, heparin-PHE treatment in drops resulted in the XX/XY sex ratio inverse to that obtained by heparin-caffeine treatment in standard co-cultures (32.3%/67.7% and 66.7%/33.3%, respectively). In conclusion, the treatment of sorted sperm with heparin-PHE in modified drop co-cultures can be recommended for production of in vitro sexed embryos. The use of unsorted sperm for production of embryos of the preferred sex by selected capacitation treatment and co-culture can be the method of choice in bulls with low IVF yields from sorted sperm.     

Genomewide analysis of bull sperm quality and fertility traits

Because the priority of AI industry is to identify subfertile bulls, a predictive model that allowed for the prediction of 91% bulls of low fertility was implemented based on seminological (motility) parameters and DNA status assessed both as DNA fragmentation index (DFI) and by TUNEL assay using sperm of 105 Holstein–Friesian bulls (four batches per bull) selected based on in vivo estimated relative conception rates (ERCR). Thereafter, sperm quality and male fertility traits of bulls were explored by GWAS using a high‐density (777K) Illumina chip. After data editing, 85 bulls and 591,988 SNPs were retained for GWAS. Of 12 SNPs with false discovery rate <0.2, four SNPs located on BTA28 and BTA18 were significantly associated (LD‐adjusted Bonferroni <0.05) with the non‐compensatory sperm parameters DFI and TUNEL. Other SNPs of interest for potential association with TUNEL were found on BTA3, in the same chromosome where associations with non‐compensatory in vivo bull fertility were already reported. Further suggestive SNPs for sperm membrane integrity were located on BTA28, the chromosome where QTL studies previously reported associations with sperm quality traits. Suggestive SNPs for ERCR were found on BTA18 in the vicinity of a site already associated with in vivo bull fertility. Additional SNPs associated with ERCR and sperm kinetic parameters were also identified. In contrast to other, but very few GWAS on fertility traits in bovine spermatozoa, which reported significant SNPs located on BTX, we have not identified SNPs of interest in this sexual chromosome.     

Pregnancy rate of heifers bred by an immunotolerant bull persistently infected with bovine viral diarrhoea virus

Twenty four maiden heifers were bred by natural route by a specific immunotolerant bull, that was persistently infected with Bovine Viral Diarrhoea virus (BVD virus). The quality of the bull's semen was normal. Twelve heifers became pregnant in the first oestrus cycle and the remaining twelve in the second oestrus cycle. This leads to the conclusion that such persistently infected bulls may have good fertilisation results. Nevertheless, it is felt that bulls persistently infected with the BVD virus must be excluded from artificial insemination centres because of the risk of introducing BVD virus in a herd by the semen.     

Relationship between Sperm Response to Glycosaminoglycans in vitro and Non-return Rates of Swedish Dairy AI Bulls

In this study, the relations between fertility (56‐day non‐return rates, 56‐day NRR) after artificial insemination (AI) and bull sperm characteristics post‐thaw, after swim‐up and after co‐incubation with heparin (Hep) and hyaluronan (HA), respectively, were determined, attempting to determine if such a procedure could be of value to evaluate the potential fertilizing ability of frozen‐thawed AI bull spermatozoa. Spermatozoa from 20 semen batches derived from 20 Swedish Red and White AI bulls ranging widely in their field fertility after AI (55–79% 56‐day NRRs) were evaluated with regards to post‐thaw motility, membrane integrity, and migration through a simple swim‐up procedure. Sperm viability and capacitation status were evaluated by two different vital staining procedures and chlortetracycline hydrochloride staining. Sperm motility and membrane integrity post‐thaw (e.g. indicators of sperm viability) were significantly correlated (r = 0.53, p < 0.05 and r = 0.59, p < 0.01, respectively) with fertility. Heparin (5 µg/ml) significantly (p lt; 0.001) increased the frequencies of capacitation and acrosome‐reaction (AR) among swim‐up separated spermatozoa, whereas HA at a concentration of 50 ng/ml did not have any significant capacitating effect. The incidences of capacitated or AR‐spermatozoa following Hep‐treatment were not correlated with fertility. On the other hand, the percentage of viable spermatozoa was significantly (p < 0.001) lower in Hep‐treated samples than in control and HA‐treated samples and was significantly (r = 0.49, p < 0.05) correlated with fertility after AI (56‐day NRR). The results indicate that the percentage of viable spermatozoa after swim‐up separation and heparin‐exposure from a selected population of AI bulls were significantly and positively related to the AI fertility of the donors and thus could be used as a parameter to determine the fertilizing ability of frozen—thawed AI bull spermatozoa.