Penetration of zona-free hamster ova as a test to assess fertilizing ability of bull sperm after frozen storage

Frozen stored sperm samples of two Holstein bulls (A and B) were compared for their abilities to interact with zona-free hamster ova. The percentage of hamster vitelli interacting with sperm from Bull A was significantly higher than that interacting with sperm from Bull B (94.5% vs. 68.2%, P<0.01), and these results were in good agreement with 60 day non-return data for the same months (68.2% for Bull A vs. 64.3% for Bull B). Sperm from Bull A also excelled in average numbers attached to vitelli, and in average numbers of sperm penetrated into the zona-free hamster ova. However, of the penetrated vitelli, sperm of Bull B resulted in more pronuclei. In these experiments the percentage of progressively motile sperm at insemination was highly correlated with the percentage of vitelli interacting with sperm. The percentages and numbers of sperm cells with intact acrosomes were significantly correlated with the average number of sperm attached per vitellus. These observations encourage further development of this test for assessing sperm fertilizing ability.     

Prediction of fertility of bovine semen: Preliminary studies with the hamster egg penetration test

The ability of liposome-treated fresh and frozen spermatozoa from two bulls to interact with zona-free hamster oocytes was examined to show whether the in vitro test results would correspond with in vivo fertility as indicated by the 60 to 90 d nonreturn to service rates which, using frozen semen, were 77 and 59%, respectively. The motility of spermatozoa in washed suspensions was also rated. Hamster test results were obtained using three ejaculates from each bull both as fresh and frozen semen. The results with frozen semen corresponded with fertility. The averages of three hamster tests for oocyte penetration rates and mean number of spermatozoa per penetrated oocyte comparing spermatozoa from the bull with the higher fertility with spermatozoa from the bull with the lower fertility were 91% and 2.7 versus 56% and 1.4, respectively. Spermatozoa washed from frozen semen from the bull with the higher fertility interacted with hamster oocytes at the higher rate even when sperm motility was rated the same for both bulls. By contrast, fresh spermatozoa from the lower fertility bull interacted with hamster oocytes at a higher rate than spermatozoa from the higher fertility bull in six tests, comparing six ejaculates of fresh semen from both bulls. Comparing the higher fertility bull with the lower fertility bull, the average of six tests for oocyte penetration rates and mean number of spermatozoa per penetrated oocyte were 60% and 1.6 versus 89% and 3.0, respectively. This suggests that this hamster test cannot be used with fresh semen to predict relative levels of fertility of frozen semen. Also, the subjective rating of sperm motility did not correspond with the in vitro oocyte penetrating ability of the spermatozoa.     

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

Fresh sperm from five bulls having nonreturn rates ranging from 48% to 77% were treated with 15.7, 21.0, 26.2, 31.5, 36.7, and 42.0 μM dilauroylphosphatidylcholine (PC12) to induce the sperm acrosome reaction (AR). Treated sperm were incubated 3 hr with zona-free hamster eggs at 39°C prior to fixation. The eggs were then stained and examined for sperm penetration. Differences in the percentages of motile sperm and of sperm exhibiting an AR among bulls were small when compared on a within-liposome-concentration basis. Increasing the PC12 concentration from 15.7 μM to 42.0 μM increased the percentage of sperm exhibiting an AR for all bulls. At the lowest lipid concentration (15.7 μM), the percentage of eggs penetrated by sperm from the five bulls was 6% to 36%, with 0% in controls. When sperm were incubated with increasing lipid concentrations, the egg penetration rate increased to over 80%, and the total number of sperm increased to over 100 per 36 eggs in each treatment for every bull. These penetration rates decreased at the highest lipid concentration. A correlation between the PC12 concentration maximizing egg penetration and the nonreturn rate of ?.63 was found. The correlation between the PC12 concentration maximizing the total number of penetrated sperm per treatment and the bull nonreturn rate was ?.96. It was concluded that PC 12 liposomes induce the AR in bull spermatozoa, which enables them to penetrate zona-free hamster eggs. High fertility bulls required less lipid to induce the AR than did lower fertility bulls. Consequently, this assay of fresh semen could provide a laboratory method to estimate the fertility of a bull.     

In vitro fertility evaluation of cryopreserved ram semen and its correlation with relative in vivo fertility

The present study was designed to evaluate zona-free hamster ova assay conditions for cryopreserved ram semen and to investigate the correlation between ability to penetrate zona-free hamster ova and in vivo fertility. In vivo fertility was estimated for cryopreserved semen from 5 Merino rams using heterospermic insemination. Equal numbers of postthaw motile spermatozoa from a Merino ejaculate and pooled Suffolk ejaculates were mixed prior to insemination. Each Merino ejaculate was paired with the same pool of cryopreserved Suffolk semen. Relative in vivo fertility for each Merino ram was calculated as the proportion of offspring that were sired by the Merino (range 42 to 100%). These ejaculates also differed in their ability to penetrate zona-free-hamster ova (3.6 to 9.0 penetrated spermatozoa per ovum). Differences in penetration rate were correlated with in vivo fertility (P < 0.002, R2 = 0.69). Results of these studies suggest that the zona-free hamster ova bioassay may be a useful test in the assessment of cryopreserved ram sperm fertility.     

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.     

Modification of the zona-free hamster ova bioassay of boar sperm fertility and correlation with in vivo fertility

These studies were designed to evaluate the ability of the zona-free hamster ova bioassay to detect differences in fertility of boar sperm. In the first study, sperm from two previously infertile boars were compared to sperm from seven previously fertile boars. The percentage of zona-free hamster ova penetrated by sperm from the previously infertile boars was significantly lower than the percentage of ova penetrated by sperm from previously fertile boars (18% of ova penetrated vs. 83%, P < .001). In the 14 ejaculates from the previously infertile boars that had ejaculate motilities of 50% or greater, the percentage of zona-free hamster ova penetrated continued to be lower than in ejaculates from the fertile boars. One of the two previously infertile boars consistently had a normal semen analysis. The only two observed manifestations of his reduced fertility were his zero conception rate and the limited ability of his sperm to penetrate zona-free hamster ova. In the second study, females were inseminated with equal numbers of sperm from two previously fertile males and the paternity of offspring determined at birth. The experiment was replicated with four combinations of six boars. A high correlation was observed between the percentage of offspring sired and the ability to penetrate zona-free hamster ova (R = .89). Neither morphology nor the ability of the sperm to undergo an acrosome reaction during in vitro incubation was correlated with fertility in the competitive mating situation. These results suggest the zona-free hamster ova bioassay can improve the in vitro fertility assessment of fresh boar semen.     

Use of zona-free hamster ova to assess sperm fertilizing ability of bull and stallion

Zona-free hamster ova interacted with bull and stallion spermatozoa after treatment of ejaculated semen to capacitate the sperm cells. Sperm conditioning by prolonged incubation in BWW medium (18–26 hr) prior to insemination was effective for capacitation of bull and stallion sperm. Preincubation of bull sperm for 70 or 105 min in defined medium (DM) with NaCl content elevated to result in 350 m0sM/kg also led to penetration of hamster vitelli. More rapid sperm conditioning was possible and higher proportions of interacting vitelli followed insemination with bull or stallion sperm exposed to high ionic strength DM (380–390 m0sM/kg) for 10 min before incubation in isotonic DM prior to insemination, the treatment adopted in subsequent work. Initial efforts to assess relative fertilizing ability of freshly ejaculated semen from two fertile bulls (A and B) in A1 usage resulted in uniformly high ( > 90%) levels of sperm-vitelli interaction (for both) when the hamster ova employed resulted from superovulation with PMSG and HCG. Following use of ova from untreated hamsters sperm samples of bull A and bull B interacted with 53.8% and 84.9% (P < 0.05) of zona-free hamster ova, respectively. Conception data (60–90 day nonreturn rates) resulting from A1 with semen collected during the same interval but processed and stored in liquid nitrogen prior to use revealed an inverse relationship to proportions of vitelli interacting with fresh sperm; nonreturn rates were 69.3% and 66.3% for bull A and bull B, respectively. A similar treatment effected capacitation of frozen-stored bull semen to enable sperm-vitelli interaction. These findings encourage additional efforts to correlate testing of processed semen with fertility.     

Fertilizing characteristics of bovine sperm with flattened or indented acrosomes

Frozen semen from a control bull (C: 89% morphologically normal sperm) and two bulls with acrosomal defects (K1: 92% flattened acrosomes; K2: 82% indented acrosomes) were used to investigate the fertilizing ability of bull sperm with flattened or indented acrosomes. In experiment 1, frozen-thawed sperm were evaluated for acrosomal integrity with fluorescent microscopy. In experiment 2, proteolytic activity of the acrosomal contents of sperm was evaluated through a gelatin digestion assay. In experiment 3, an IVF test system was used to determine the ability of sperm with flattened or indented acrosomes to bind to bovine oocytes and penetrate the zona pellucida. In experiment 4, IVM zona-free bovine oocytes (ZFO) were fertilized and examined to evaluate sperm chromatin decondensation. In experiment 1, bulls K1 and K2 had a lower proportion of sperm with intact acrosomes (0 and 13.6 +/- 4.5%, respectively) than bull C (30.2 +/- 5.6%) after 2h of incubation. In experiment 2, the proportion of sperm with proteolytic activity, as indicated by gelatin digestion around sperm heads, did not differ among bulls (C: 55%, n=410; K1: 43%, n=426; K2: 48%, n=324). In experiment 3, a lower proportion of sperm with flattened (K1) or indented acrosomes (K2) bound to oocytes than sperm from the control bull, C. The percentage of zona penetrated (55%, n=20; 13%, n=23; 4%, n=25) and the mean (+/- S.E.M.) number of sperm penetrating these zona pellucida (19.7 +/- 2.5; 6.9 +/- 1.0; and 2.6 +/- 0.5) was higher (P<0.05) for bull C than for bulls K1 or K2, respectively. In experiment 4, the percentage of ZFO penetrated (95%, n=20; 52%, n=30; 30%, n=33) and the mean (+/- S.E.M.) number of sperm with chromatin decondensation (7.8 +/- 1.6; 0.8 +/- 0.2; and 0.3 +/- 0.1) were also higher (P<0.05) for the control bull, C than for bulls K1 or K2, respectively. Results suggest that although sperm with the flattened or indented acrosomes had a tendency to undergo spontaneous acrosome reaction on incubation after thawing, the proteolytic activity of the acrosomal contents appeared to be normal. Sperm with the flattened or indented acrosomes also appeared to have a reduced ability to fuse with oolemma as demonstrated by confocal microscopy. This would impair the ability to penetrate ooplasm and undergo sperm chromatin decondensation.     

Relative efficacy of conventional sperm parameters and sperm penetration bioassay to assess bull fertility in vitro

Frozen semen samples from 10 bulls were thawed and actively motile sperm recovered using a swim-up technique. Calcium ionophore A23187 at 0.5 microM concentration was used for 1 min to induce the acrosome reaction in the sperm. Mature female golden hamsters were superovulated with 50 IU of equine chorionic gonadotrophin followed 56 h later with 75 IU of human chorionic gonadotrophin. The cumulus mass was recovered 17 h after hCG treatment by puncturing the oviducts in the infundibulum region. Subsequently, cumulus cell mass and zona pellucida were digested by 0.1% hyaluronidase and 0.1% trypsin, respectively, to yield zona-free hamster eggs (ZFE). A sperm penetration bioassay was performed by coincubating capacitated sperm at 5 X 10(6) concentration and ZFE for 3 h at 38 degrees C in an air incubator. The conception rate of the bulls was based of an average of 82.6 cows per bull with pregnancy status confirmed by rectal palpation. It was found to be strongly correlated (p < 0.01, r = 0.723) with fertilization percentage, whereas percent motile sperm, percent viable sperm and percent sperm with intact acrosomes were not significantly correlated with the conception rate (r = 0.210, -0.021 and -0.468, respectively). Results of the present study suggest that the sperm penetration bioassay can be reliably used to test the fertilizing potential of bull sperm in vitro.     

Evaluation of relative fertility of cryopreserved goat sperm

This study was designed to compare differences in the in vivo fertility of cryopreserved goat semen assessed by heterospermic insemination with differences in in vitro analyses. Five groups of does were inseminated with mixed frozen-thawed semen from different pairs of bucks. The percentage of offspring sired by each buck in the pair was compared with the relative ability of spermatozoa from that frozen-thawed ejaculate to penetrate zona-free hamster ova, relative post-thaw acrosomal integrity, ability to undergo an acrosome reaction during in vitro capacitation, and assessments of sperm motility. In 4 of the 5 different insemination pairs, the ratio of offspring born was other than 1:1. Acrosomal integrity, ability of spermatozoa to undergo an acrosome reaction, and parameters of sperm motility were not correlated with differences in relative fertility in this experiment using ejaculates from fertile bucks. The ability of spermatozoa to fuse with the oocyte plasma membrane was highly correlated with relative in vivo fertility (R(2) = 0.78, P = 0.04). This suggests that fusion with the oocyte plasma membrane is an event in the fertilization process in which significant variation exists among fertile bucks. Assessment of ability of spermatozoa to fuse with zona-free hamster ova may contribute to analysis of post-thaw fertility of frozen-thawed buck semen.     

Alterations in primate sperm motility with maturation and during exposure to theophylline

Micropuncture was used to collect pure suspensions of sperm from the caput and cauda regions of chimpanzee epididymides, which were analyzed with a Motion Analysis VP-110. Sperm recovered from the caput region showed no forward motility. Incubation of these sperm with cauda epididymal fluid affected motility in 62%–90% of the sperm. Dilution of cauda sperm into buffer containing >50 mM theophylline resulted in immediate initiation of progressive forward motility. Although this motility was maintained by at least 50% of the sperm for over 5 hr, these “activated” caput sperm did not penetrate zona-free hamster ova. These data show that sperm from the caput epididymis of the chimpanzee have the capacity for normal motility but do not have the capacity to bind to and penetrate an ovum. Cauda epididymal chimpanzee sperm were motile at the time of recovery and this motility was maintained for over 5 hr. These sperm penetrated both hamster zona-free ova and intact chimpanzee ova. These data show that sperm from the cauda epididymis of the chimpanzee have the capacity for normal motility and also have the capacity to bind to and penetrate an ovum. This is the first use of computer assisted analysis to quantify motility in maturing nonhuman primate sperm.     

Evaluation of assay conditions for the zona-free hamster ova bioassay of boar sperm fertility

The ability to penetrate zona-free hamster ova may be a very useful test of fresh and frozen boar sperm fertility. These studies were designed to optimize assay conditions prior to evaluation of the accuracy of the bioassay in predicting boar sperm fertility. The ability to penetrate zona-free hamster ova was greater in sperm washed on a Percoll gradient than in sperm washed by dilution and centrifugation. Penetrating ability was greater in sperm from the sperm-rich fraction than from the whole ejaculate but did not differ among different aliquots of the sperm-rich fraction and did not decrease when the prewashing interval was increased from 15 to 85 min. Frequency of collection of ejaculates (1, 3, or 5 times per week) did not affect the penetrating ability of the sperm. Penetration rate was greater when sperm were coincubated with zona-free hamster ova at 39°C compared to 37°C. Sperm from an infertile boar had reduced penetrating ability compared to sperm from fertile boars (11% vs 93%, P < .001). These studies suggest that the zona-free hamster ova bioassay may be a useful assessment of fresh boar sperm fertility.     

Embryo quality and andrological study of two subfertile bulls versus five control bulls with normal fertility

Andrological studies and embryo morphology evaluation of superovulated cows were performed on 2 randomly selected subfertile dairy bulls whose semen was used for artificial insemination and on 5 control bulls with normal fertility. Neither sperm motility studies, nor sperm morphology or testicular measurements differed between the subfertile and the control bulls. Altogether 315 ova were recovered from 41 superovulated cows inseminated with semen collected from either the subfertile or the normal control bulls. The spermatozoa of one of the 2 subfertile bulls was shown to have a decreased ability to fertilize superovulated ova, while the other subfertile animal, the bull with the lowest noreturn rate, was found by chromosome analysis to have a reciprocal translocation (60, XY, rcp 20:24), causing embryonic death. We suggest that subfertile bulls should not be used in commercial embryo transfer programs nor in artificial insemination and that andrological studies on subfertile bulls with good sperm motility should include evaluation of 6- to 7-day-old ova from superovulated cows to determine if the fertilization rate is normal or impaired. A chromosome analysis should also be performed when a subjertile bull has a normal fertilization rate of ova.     

A chromosomal method to distinguish between X- and Y-bearing spermatozoa of the bull in zona-free hamster ova

The interspecific in-vitro fertilization system using zona-free hamster ova was adopted to distinguish chromosomally between X- and Y-bearing bull spermatozoa. Frozen semen samples were thawed and washed with modified BWW medium including 0.3% BSA (pH 7.2) to remove cryoprotective medium. Motile spermatozoa were recovered from the semen by the 'swim-up' method. These spermatozoa were treated with ionophore A23187 to facilitate capacitation. Adequate capacitation of spermatozoa was found by their movement patterns and the insemination was performed at the optimum time. The fertilized ova were cultured in Medium TC199 with 10% FBS including podophyllotoxin and vinblastine until they reached first cleavage metaphase. Chromosome slides were prepared by our gradual fixation-air drying method. The success rate of the method was 56% of the number corresponding to the zona-free ova used. Altogether 1116 spermatozoa from 4 different bulls were successfully analysed and the ratio of X- and Y-bearing spermatozoa was 542:574 (P greater than 0.3).     

Development of a zona-free hamster ova bioassay for goat sperm

In vitro conditions for a zona-free hamster ova bioassay of caprine sperm fertility were assessed. Washing the cryopreserved sperm by dilution and centrifugation resulted in greater ability to penetrate zona-free hamster ova than allowing the sperm to swim-up into medium. A 12-h preincubation of sperm prior to insemination of the zona-free hamster ova was optimal. Sperm had greater ability to penetrate the zona-free hamster ova when incubated in a Tris-buffered medium than when incubated in Ham's F-10 (95 vs 2%, P<0.001), although motility was not well maintained in the Tris-buffered medium. Ten million sperm/ml was sufficient for maximum penetration. The ability to penetrate zona-free hamster ova was positively but not significantly correlated with the sperm head ultrastructure, suggesting the two techniques may assess different aspects of sperm fertility.     

Platelet activating factor improves the in vitro penetration of zona free hamster eggs by buffalo (Bubalus bubalis) spermatozoa

Twelve buffalo bulls of Murrah breed, selected on the basis of their conception rates, were classified into low-, moderate- and high-fertility groups. Frozen semen was thawed and treated with 200 microM platelet activating factor (PAF) for 15 min at 37 degrees C and 5% CO2. In both treated and control (no PAF) semen samples (five replicates per bull), the following were assessed: motility, acrosome reaction (AR) evaluation (for 10 replicates of each bull), and zona-free hamster oocyte penetration test--to determine aspects of fertilization in vitro, viz., sperm attached per ovum (SA/O), fertilization percent (FP), fertilization index (FI), and polyspermic ova (PO). There was an effect of group (P < 0.01) on all parameters; all except motility were increased by PAF treatment. However, the group X treatment interaction was not significant for any parameter. The overall mean values of motility, AR, SA/O, FP, FI, and PO, for controls, treated spermatozoa and (net change) were: 42.89 +/- 0.85, 36.65 +/- 0.85, (-6.24); 28.94 +/- 0.46, 61.44 +/- 0.58, (32.50); 126 +/- 2, 145 +/- 2, (19); 74.21 +/- 1.59, 89.11 +/- 1.18, (14.90); 0.79 +/- 0.02, 1.10 +/- 0.03, (0.31) and 5.22 +/- 1.22, 21.69 +/- 1.88, (16.47)%, respectively. In conclusion, PAF significantly increased the AR and other aspects of fertilization, despite a small reduction in motility.     

In vitro assessment of sperm from bulls of high and low field fertility

The aim of this study was to investigate the reasons for differences in field fertility of bulls following insemination with frozen-thawed semen. The study was carried out in two separate parts over two years and comparisons were made between 5 high and 4 low fertility Holstein Friesian bulls as determined by their either 90 day non-return rate (Year 1) or calving rate (Year 2). Two high fertility Limousin bulls were included in Year 1 for comparative purposes. The ability of sperm from each bull to penetrate artificial mucus was assessed (Year 1 = 7 replicates; Year 2 = 5 replicates). Glass capillary tubes (2 per bull per replicate) were filled with artificial mucus and incubated with sperm stained in 1% Hoechst 33342 for 30 min at 37 °C. The number of sperm were subsequently counted at 10 mm intervals along the tube between 40 and 80 mm markers. Sperm mitochondrial activity of each bull was assessed by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay (4 replicates in each year). Sperm were incubated with MTT for 1 h at 37 °C following which the absorbance of formazan was read using a spectrophotometer. Sperm viability after thawing was assessed for each bull using a live/dead sperm viability kit (Year 1 = 3 replicates; Year 2 = 4 replicates). A minimum of 250 cells were assessed per bull in each replicate and classified as either live or dead. Finally, the ability of sperm to fertilize oocytes in vitro and their ability to develop to blastocyst stage embryos were assessed (5 replicates in each year involving 220 to 306 oocytes per bull). Data transformation to normalize residuals was required for mucus sperm penetration (square root) and IVF (cleavage and blastocyst rate) results (arcsin). The mean number of sperm counted at each 10 mm mark between 40 and 80 mm was higher in the high fertility (56.0; 95% CI 39.5 to 75.3) compared to the low fertility (42.9; 95% CI 29.3 to 59.1) Holstein Friesian bulls but the difference did not reach formal significance (P = 0.09). Fertility status had no effect on the ability of sperm to reduce MTT to formazan (mean absorbance 0.34 ± 0.051 and 0.30 ± 0.044) or on the percentage of live sperm per straw (mean 47.3 ± 5.47 and 32.4 ± 4.66) for high and low fertility Holstein Friesian bulls respectively. Oocyte cleavage rate following insemination with sperm from high fertility Holstein Friesian bulls was significantly higher than with sperm from low fertility Holstein Friesian bulls [76.7% (95% CI 60.9 to 89.4) and 55.3 (95% CI 40.4 to 69.7) respectively, P = 0.04]. There was no significant effect of bull fertility on blastocyst rate [34.7% (95% CI 21.1 to 49.6) and 24.2 % (95% CI 14.1 to 36.0) for the high and low fertility Holstein Friesian bulls, respectively; P = 0.2]. In conclusion, sperm from high fertility bulls tended to be more effective in penetrating artificial mucus and to have an increased ability to fertilize oocytes in vitro; however, once fertilization occurred subsequent embryo development was not significantly affected by fertility status.     

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.     

Evaluation of Spermatological Parameters Used to Predict the Fertility of Frozen Bull Semen

Post-thaw motility, velocity and acrosome integrity of frozen semen were determined in 18 bulls with varying fertility (average non-return rates: 71.3 (± 2.8) - range: 65.2-75.7). Five semen straws were investigated from each bull. The average values for sperm motility (percentage motile spermatozoa), sperm velocity (graded from 0-3) and acrosome integrity (proportion of spermatozoa with intact acrosome) were 67.5%, 2.5 and 79.3%, respectively. Significant correlations were found between sperm motility and velocity, but not between sperm motility and acrosome integrity. Both sperm motility and velocity were significantly related to bull fertility. It was concluded that of the post-thaw semen characteristics investigated in this study these 2 parameters provided a reliable basis for prediction of bull fertility.     

Ejaculate-hormonal traits in the leopard cat (Felis bengalensis) and sperm function as measured by in vitro penetration of zona-free hamster ova and zona-intact domestic cat oocytes

Electroejaculate traits and circulating follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone concentrations were analyzed in adult leopard cats (Felis bengalensis), a rare felid species indigenous to east Asia. The ability of leopard cat sperm to bind and penetrate zona-free hamster ova and zona-intact domestic cat oocytes in vitro was examined as a means of testing sperm function. The influence of culture media [Biggers, Whitten, Whittingham (BWW) vs. modified Krebs Ringer bicarbonate (mKRB)], seminal plasma removal, and swim-up separation on sperm motility, sperm morphology, and oocyte penetration also were assessed. Sperm treatments included dilution of raw semen (DR), ejaculate centrifugation, and either resuspension (NS) or swim-up processing (SU). The percentage of oocytes penetrated (penetration rate) and the number of penetrated sperm/oocyte (penetration index) were determined. Ejaculates from each male consisted of at least a 50% sperm motility rating, and hormone concentrations in individual males were unrelated to any ejaculate trait measured concurrently on the same day. The SU technique improved (P less than 0.05) percent sperm motility and the proportion of structurally normal sperm compared to DR and NS treatments. Leopard cat spermatozoa were capable of binding to and penetrating hamster ova and domestic cat oocytes; however, penetration was influenced by culture medium and seminal processing. In the hamster assay, a higher (P less than 0.05) penetration rate and penetration index were achieved when mKRB was used for gamete incubation instead of BWW. NS processing also increased (P less than 0.05) overall penetration compared to DR and SU. In the cat oocyte assay, zona penetration rate was similar (P greater than 0.05) in the DR, NS, and SU aliquots; however, the zona penetration index was increased (P less than 0.05) by the NS compared to the DR and SU treatments. This study 1) provides baseline ejaculate and endocrine norms for the leopard cat, 2) demonstrates that leopard cat sperm undergo nuclear decondensation in hamster ova and penetrate zona-intact domestic cat oocytes, 3) indicates that seminal plasma removal enhances leopard cat sperm fertilizing ability and ovum penetration, and 4) suggests that heterologous oocyte penetration is effective for assessing factors influencing fertilization and sperm function in this nondomestic felid.