Efforts to correlate laboratory with field observations on bull sperm fertility

This work represents efforts towards development of the zona-free hamster ovum sperm penetration assay for predicting relative levels of fertility for semen from individual bulls. Results reported here followed insemination of hamster vitelli with bull sperm, after frozen storage, with observations of sperm acrosomes and parallel inseminations of more than 1000 cows with semen from each bull. The average 75-day non-return rate for the four bulls was 74.0% (range 71.6 to 75.6). Laboratory studies indicated the following: the percentage of sperm with intact acrosomes varied from 55 to 73 between bulls, the percentage of motile sperm varied from 41 to 64, the percentage of sperm with progressive motility ranged from 24 to 40, the number of sperm interacting per (zona-free hamster) ovum ranged from 1.6 to 3.8, the number of sperm attached per ovum ranged from 1.4 to 2.9, the number of sperm within each penetrated ovum ranged from 1.5 to 1.8, the percentage of ova interacting with sperm ranged from 76 to 92, the percentage of ova penetrated ranged from 62 to 85, and the percentage of ova with male pronuclei ranged from 33 to 49. Although predictive ranking in the laboratory of these bulls with less than 4% variation in fertility levels was not possible, the zone-free hamster ovum test could be useful in identifying potentially subfertile bulls before they enter a young sire-sampling program.     

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.     

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.     

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.     

Use of salt-stored zonae pellucidae for assessing rabbit sperm capacitation for in vitro fertilization

Zonae pellucidae of tubal and follicular oocytes were collected and prepared for salt storage. Cumulus and corona radiata cells were removed from oocytes with hyaluronidase and a small bore pipette. The oocytes (referred to as zonae since vitelli were rendered nonfunctional) were stored in a salt solution at 4°C. Using in utero capacitated sperm, the penetrability of zonae from tubal and follicular oocytes stored immediately after collection was compared to controls, i.e., in vitro development of tubal ova to the 4-cell stage within 24 hr. The penetration rates were 100% (8 penetrated/ 8 inseminated), 77.8% (7 penetrated/ 9 inseminated), and 100% (10 fertilized/10 inseminated), respectively, and these were not statistically different. The mean (x ) numbers of sperm able to penetrate the zonae, into the perivitelline space (PVS) for tubal (34.0) and follicular (1.1) oocytes were significantly different (P < 0.01). However, following maturational incubation before salt storage, zonae of tubal and follicular origin showed no significant differences in penetrability of in utero capacitated sperm when assessed by percent penetration, or mean numbers of sperm cells reaching the PVS: tubal zonae, 100% (15/15), and follicular zonae, 100% (18/18), and mean number of sperm in the PVS (x [tubal zonae] = 12.4, and x [follicular zonae] = 11.8). The penetrability of tubal zonae with and without maturational incubation was compared, and no significant differences in penetrability by in utero capacitated sperm were present when assessed by percent penetration nonmatured 92.6% (25/27) and matured 93.3% (28/30) and mean number of sperm in the PVS (x [nonmatured] = 3.33 and x [matured] = 2.41). In vitro capacitation of ejaculated rabbit sperm by serum treatment was assessed by the penetration of salt-stored zonae, zonae-free hamster oocytes (ZFHO), and in vitro fertilization of freshly collected tubal oocytes. None of 60 salt-stored zonae and none of 31 tubal oocytes were penetrated, and these values were significantly (P < 0.005) smaller than the 9 of 78 (12%) zona-free hamster ova that were penetrated by sperm cells from the same sample. In vitro capacitation of ejaculated rabbit sperm by washing and preincubation was assessed by the penetration of salt-stored zonae, zona-free hamster oocytes (ZFHO), and fertilization of freshly collected tubal oocytes. Seventy-six of 80 salt-stored zonae were penetrated, and this was significantly (P < 0.005) greater than the 67 of 87 tubal oocytes fertilized and 30 of 35 ZFHO penetrated, which were not significantly different. The salt-stored zonae were more readily penetrated by capacitated sperm when compared to tubal oocytes. However, the ZFHO are more penetrable than salt-stored zonae and tubal oocytes when incompletely capacitated sperm is used. A useful role for this approach in studies dealing with sperm fertilizing ability is anticipated.     

An investigation of the fertilizing characteristics of pyriform-shaped bovine spermatozoa

Two breeding trials were done to determine the effect of pyriform-shaped bovine spermatozoa on fertility. In breeding trial (1), heifers were superstimulated by injecting follicle-stimulating hormone (FSH) and randomly allotted to an affected Bull A (n = 21) or a Control Bull, C (n = 18). Semen from Bull A contained 85% pyriform spermatozoa while that from the Control Bull C contained 91% normal sperm and 2% pyriform spermatozoa. Fertilization rate was lower (P = 0.01) for Bull A (total of 63 ova/embryos; 68.5%) than for the Control Bull, C (total of 81 ova/embryos; 84.4%). In breeding trial (2), 37 oestrus-synchronized heifers were randomly allotted to Bull A (n = 19) or Bull C (n = 18). Pregnancy rates at Day 60 (37% and 61% for Bulls A and C, respectively; P = 0.22) and rate of embryo/fetal loss between Days 22 and 60 of pregnancy (23% and 8% for Bulls A and C, respectively; P = 0.55) were not different. In vitro experiments involving the same Bull A and another Control Bull, B, were designed to explain the mechanism of infertility caused by pyriform sperm. The mean (+/- SEM) number of sperm bound to the zona pellucida was lower (P < 0.05) for Bull A (24.6+/-1.2) than for the Control Bull, B (46.6+/-1.9) and the percentage of zonae penetrated by sperm from Bull A (56.0%) and Bull B (82.8%) was also different (P < 0.05). The percentage of pyriform sperm from Bull A bound to (53%), and penetrating (49%) the zona pellucida was lower (P < 0.05) than that in the inseminate (85%). Although fertilization rates (64.1% and 72.8%) were not different (P = 0.5), cleavage rates (48.2% vs. 74.1%) and morula production rates (24.8% vs. 37.7%) were less (P < 0.05) for Bull A than for Bull B, respectively. In summary, pyriform sperm had reduced capability to bind to and penetrate the zona pellucida, and zygotes (resulting from the fertilization of oocytes by pyriform sperm) appeared to have a reduced ability to initiate cleavage.     

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.     

Stimulation of rhesus monkey sperm capacitation by cyclic nucleotide mediators

Capacitation of rhesus monkey spermatozoa was assessed by monitoring sperm flagellar beat and trajectory changes during incubation in vitro and by determining sperm penetration into rhesus oocytes and hamster zona-free ova. Rhesus sperm capacitation in vitro depended on the addition to the culture medium of the cyclic nucleotide mediators, caffeine and dibutyryl cyclic AMP. Capacitation was correlated with the development of hyperactivated motility. Spermatozoa treated with the cyclic nucleotide mediators, and showing hyperactivated motility, penetrated 57.4% of all rhesus oocytes and fertilized 88.9% of mature rhesus oocytes that were morphologically normal. Control spermatozoa did not penetrate any of the eggs. Some sperm penetration into hamster ova occurred but was not statistically significant. These data provide a basis for achieving in-vitro fertilization in the rhesus monkey and information on specific sperm motility characteristics associated with fertilizing ability.     

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.     

Changes in exposed membrane proteins during in vitro capacitation of boar sperm

Exposed plasma membrane proteins were labeled with 125I before and after incubation of boar sperm under capacitating conditions. Labeled protein profiles were compared to the ability of the sperm to penetrate zona-free hamster ova. Quantitatively, the labeled sperm membrane proteins were primarily low Mr prior to capacitation. The majority of the labeled seminal plasma protein was also low Mr. After capacitation, two new proteins (64,000 Mr and 78,000 Mr) were labeled. Sperm did not exhibit these exposed membrane proteins when incubated under noncapacitating conditions. Appearance of these proteins was not correlated to the percentage of acrosome-reacted sperm. Although the 64,000 Mr protein was not consistently observed, the relative labeling of the 78,000 Mr protein was highly correlated with the ability of sperm to fuse with zona-free hamster ova. The 78,000 Mr protein may be a sperm protein involved in fusion with the egg plasma membrane.     

Boar sperm plasma membrane protein profile: Correlation with the zona-free hamster ova bioassay

This study was designed to compare differences among porcine sperm plasma membrane proteins with the ability of spermatozoa to interact with zona-free hamster ova. Sperm plasma membrane vesicles were recovered from 24 ejaculates from 10 fertile boars, and from cauda epididymal spermatozoa from 3 fertile and 1 very subfertile boar. Solubilized sperm plasma membrane proteins were run on 1D SDS-PAGE gels, transferred to western blots, stained, and analyzed for quantity of protein per band by scanning laser densitometry. Variation in the quantities of individual sperm plasma membrane proteins in the 20 identified bands were statistically compared with the ability of spermatozoa from the same ejaculate to penetrate zona-free hamster ova. The percentages of plasma membrane protein present in 3 bands (90, 84 and 60 kD) were positively correlated with the ability of spermatozoa from the same ejaculate to fuse with zona-free hamster ova (P = 0.002, 0.01, 0.04; R = 0.53, 0.40, 0.38, respectively). The quantities of protein in 2 other bands (69 and 35 kD) were significantly but negatively correlated with the results of the zona-free hamster ova bioassay (P = 0.02, 0.01; R = -0.42, -0.37, respectively). The sperm plasma membrane profiles were quantitatively similar between the ejaculated samples and the fertile epididymal samples. Six epididymal sperm plasma membrane proteins were present in statistically different quantities in the subfertile boar sample and the 3 fertile controls. The 90 kD band positively correlated with the hamster ova bioassay in the ejaculated samples was not detected in the subfertile epididymal sperm plasma membrane sample. These results suggest that protein(s) in one or more of the 3 positively correlated ejaculated sperm plasma membrane protein bands may be involved in sperm-oocyte interaction.     

In vitro fertilizing characteristics of bovine spermatozoa with multiple nuclear vacuoles: A case study

A study was designed to determine the in vitro fertilizing characteristics of bovine semen with a high percentage of spermatozoa with multiple nuclear vacuoles. In Experiment 1, a total of 620 oocytes was divided into 2 groups and inseminated with spermatozoa from 1 of 2 different bulls at a concentration of 2 × 10⁵/ml. After Percoll washes, 73.5 ± 3.0% of spermatozoa from Bull A contained multiple nuclear vacuoles, while no sperm cells from Bull B contained vacuoles. After 19.5 ± 0.5 h of co-incubation of oocytes with spermatozoa, loosely attached sperm cells were removed by washing, and the oocytes were fixed between 2 poly-l-lysine coated glass slides. Mean (±SD) percentage of fertilization was significantly lower (P < 0.05) in Bull A (19.7 ± 7.0%) than in Bull B (67.6 ± 4.5%). In one-third of the oocytes fertilized by spermatozoa from Bull A, sperm head decondensation was incomplete and normal male pronucleus formation did not occur. All oocytes fertilized by Bull B had normally decondensed sperm heads. Although fewer (P < 0.05) spermatozoa from Bull A were bound to the zona pellucida than from Bull B, the percentage of vacuolated sperm cells bound to the zona pellucida (73.3 ± 7.8%) did not differ from that in the inseminate. The mean number of sperm cells binding to fertilized oocytes was higher than to unfertilized oocytes for both bulls (P < 0.05). In Experiment 2, 748 salt-stored oocytes (zonae) were inseminated with semen from the same 2 bulls to determine the ability of spermatozoa to penetrate the zona pellucida. The percentage of zonae penetrated by spermatozoa from Bull A (69.9 ± 3.5%; a mean of 2.4 ± 2.3 spermatozoa) was lower (P < 0.05) than from Bull B (96.5 ± 14.7%; a mean of 11.3 ± 9.9). Although the proportion of vacuolated sperm cells from Bull A that bound to the zona pellucida did not differ from that in the inseminate, the proportion of those penetrating the zona pellucida (52.7%) was lower (P < 0.05). In summary, vacuolated sperm cells apparently gained access to the oocyte and bound to the zona pellucida, but they penetrated the zona pellucida at a lower rate and apparently did not form normal male pronuclei.     

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.     

Ethanol inhibits human and hamster sperm penetration of eggs

The effect of alcohol on the fertilizing ability of both human and hamster spermatozoa was examined by an in vitro fertilization assay using hamster ova. Spermatozoa were incubated in capacitating media for 3 hr (hamster sperm) and 4 hr (human sperm). Hamster ova were inseminated with preincubated sperm and were examined after 2 to 3 hr. Ethanol was added to the capacitating media at concentrations of 25, 50, 100, 200, and 400 mg%. Fertilization of zona-free hamster eggs by human spermatozoa was reduced from 49.6% in no alcohol to 16.7% in 400 mg% ethanol. Fertilization of hamster eggs by hamster sperm revealed a reduction from 63.6% to 33.7% in cumulus-intact eggs and from 65.8% to 10.8% in cumulus-free eggs in the presence of ethanol at 400 mg%. Hamster sperm acrosome reaction was reduced from 47% to 12%. When these hamster sperm with reduced acrosome reaction were placed with zona-free hamster eggs, the 100% fertilization rate was not reduced; however, the fertilization index, which reflects the number of swelling sperm heads per egg, was reduced from 8.5 to 1.8. This suggests that as little as 12% of the sperm with an acrosome reaction is sufficient to fertilize 100% of the zona-free eggs. If ethanol was added to the insemination media only, there was no inhibition of fertilization by human sperm or hamster sperm that had been previously capacitated in an ethanol-free media. Removal of the ethanol from the preincubated sperm produced fertilization at control levels; thus the inhibitory effect is reversible. These results indicate that ethanol may affect fertilization by an inhibition of the capacitation and/or acrosome reaction process.     

Zinc does not inhibit the capacitation of human spermatozoa in vitro

The effects of zinc on human sperm motility, fertilizing capacity (as assessed by penetration of human spermatozoa into the zona pellucida-free hamster oocyte), and nuclear chromatin decondensation were investigated using spermatozoa from four fertile donors. Both sperm motility and the penetration of sperm into zona-free hamster ova were consistently impaired in media containing 1,000 μM zinc. Spermatozoa from one man were similarly affected at a concentration of 500 μM zinc, but no adverse effects were noted at this zinc concentration in experiments with other donors. Since decreased fertilizing capacity in response to zinc was always accompanied by a significant decline in both the percentage of motile cells and mean swimming speeds, it appears that all of these results reflect a general toxic effect on the cells. At lower concentrations (125–250 μM), zinc had no effect on human sperm motility nor their ability to undergo capacitation and penetrate zona-free hamster ova in vitro. For some donors, zinc (125–500 μM) stimulated both the attachment of spermatozoa to the hamster vitellus and the incorporation of spermatozoa into the hamster ooplasm. The decondensation of human sperm nuclear chromatin in sodium dodecyl sulfate was largely inhibited when zinc was added to the medium, but no significant changes in nuclear stability were apparent after capacitation in zinc-free medium. We conclude that zinc, when present in subtoxic concentrations, does not adversely affect the ability of human spermatozoa to undergo capacitation and penetrate zona-free hamster ova in vitro.     

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.     

Macaque sperm fusion with zona-free hamster oocytes

Abstract: Semen from six cynomolgus macaques was washed twice by centrifugation in BWW media and resuspended in 1:2 (vol:vol) of Tes-Tris-buffered eggyolk extract (EXT) diluent and BWW (EXT-BWW). Caffeine and dbcAMP were added to induce capacitation. Sperm were treated with calcium ionophore, A23187, followed by the addition of EXT to recover motility, then washed into BWW. The percent motile sperm was similar in ionophore-treated and control sperm suspensions, but motility of treated sperm decreased significantly by 20 min after treatment. The percentage of viable, acrosome-reacted sperm was 56.3% following ionophore treatment versus 4.0% in control suspensions. When ionophore-treated sperm were coincubated with zona-free hamster oocytes, 51 % of oocytes had evidence of sperm fusion and no oocytes were penetrated after incubation with control sperm. These results are consistent with the hypothesis that macaque sperm are capable of fusion with zona-free hamster oocytes if sperm are viable and acrosome-reacted.     

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.     

Pisum sativum agglutinin used as an acrosomal stain of porcine and caprine sperm

Fluorescein isothiocyanate-labelled pisum sativum agglutinin (FITC-PSA) was evaluated as an acrosomal stain for porcine and caprine sperm that had previously been stained with Hoechst 33258 to assess cell viability. The FITC-PSA procedure was as accurate as other procedures in assessing acrosomal presence or absence on either fresh or liquid-stored porcine sperm. Approximately half of the incubated porcine sperm with acrosomal loss maintained membrane impermeability to the Hoechst 33258; these were potentially viable acrosome-reacted sperm. The FITC-PSA procedure was significantly correlated with the assessment of acrosomal status of cryopreserved caprine sperm by transmission electron microscopy (TEM). However, TEM results indicated a higher percentage of caprine sperm with acrosomal loss. Ability to penetrate zona-free hamster ova was not highly correlated with the percentage of viable acrosome-reacted porcine or caprine sperm. The FITC-PSA procedure provides an estimate of viable acrosome reactions and may be a useful tool in the evaluation of sperm fertility.