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.     

Sperm binding, in vitro fertilization, and in vitro embryonic development of bovine oocytes fertilized with spermatozoa incubated with norepinephrine

The final stages of sperm maturation, fertilization, and early embryonic development occur within the oviduct and are essential for successful reproduction in mammals. Norepinephrine was previously identified in native bovine oviductal fluid and its in vitro effects on bull sperm capacitation and the acrosome reaction have been determined. It was unknown how physiological concentrations of norepinephrine influence sperm binding, fertilization, and embryo development. Therefore, the objective of this study was to determine if pre-incubating bovine spermatozoa with physiological concentrations of norepinephrine prior to insemination of bovine oocytes would improve sperm-oocyte binding, fertilization, and embryonic development in vitro. Norepinephrine, in concentrations representing those measured in bovine oviductal fluid, was used to treat bovine spermatozoa prior to insemination. Spermatozoa incubated in norepinephrine were used to inseminate bovine oocytes matured in vitro, and oocytes were evaluated for sperm binding and fertilization. Additional experiments were conducted to evaluate how early in the co-incubation period oocytes were fertilized by spermatozoa pre-incubated with norepinephrine, and to test the developmental competence of those oocytes fertilized with norepinephrine-treated sperm. Sperm binding to the zona pellucida was reduced by pre-incubation with norepinephrine. Rates of fertilization and embryo development did not increase as a result of pre-incubating spermatozoa with norepinephrine, but as early as 4h after insemination, spermatozoa treated with 20 ng/ml norepinephrine fertilized more oocytes than spermatozoa incubated in medium alone. Interestingly, this concentration of norepinephrine was found to capacitate spermatozoa in previous studies. These data suggest that oocytes fertilized by spermatozoa incubated in 20 ng/ml norepinephrine fertilize earlier in vitro than sperm pre-incubated in medium alone, and provide additional support for the role of norepinephrine in sperm capacitation and the acrosome reaction.     

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

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

Induction of the acrosome reaction in human spermatozoa by a fraction of human follicular fluid

Human ejaculated spermatozoa were washed through a Percoll gradient, preincubated for 10 hr in a defined medium containing serum albumin, and then induced to undergo rapid acrosome reactions by addition of human follicular fluid or a Sephadex G-75 column fraction of the fluid. Induction by follicular fluid did not occur when the spermatozoa were preincubated for only 0 or 5 hr. The reactions were detected by indirect immunofluorescence using a monoclonal antibody directed against the human sperm acrosomal region. The percentage of acrosomal loss counted by transmission electron microscopy agreed with that counted by immunofluorescence. The apparent molecular weight of the Sephadex G-75 fraction containing the peak of acrosome reaction-inducing activity was 45,000 ± 4,200 (SD). The occurrence of physiological acrosome reactions was supported by: assessing motility (no significant loss of motility occurred during the treatment period when sperm were preincubated with bovine serum albumin), transmission electron microscopy (the ultrastructural criteria for the acrosome reaction were met), and zona-free hamster oocyte binding and penetration (spermatozoa pretreated with the active fraction of follicular fluid, then washed and incubated with oocytes, showed significantly greater binding to and penetration of oocytes). The stimulation of the acrosome reaction by follicular fluid is apparently not due to blood serum contamination; treatment of preincubated spermatozoa with sera from the follicular fluid donors had no effect on the spermatozoa. The nature of the active component(s) in that fraction is currently being investigated.     

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

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

Effect of albumin on fertilization of mouse ova in vitro

Serum albumin is an obligatory component of the incubation medium for the fertilization of mouse ova. Normal, untreated bovine serum albumin supports high rates of fertilization of cumulus-free ova both with and without their zonae pellucidae. Heat-treated or trichloroacetic acid-extracted bovine serum albumin is unable to support the fertilization of a majority of zona-intact ova but fertilization of zona-free ova is unimpaired. Spermatozoa incubated in medium containing heated bovine serum albumin fertilize zona-intact ova when 2 mM caffeine is present but the progress of sperm head decondensation is delayed when compared to normal controls. Trichloroacetic acid extracted BSA preferentially and irreversibly inhibits zona penetration by spermatozoa, but this effect is not mediated by an inhibition of spermatozoal motility or zona-binding ability. This effect occurs after only a 10-min preincubation of the spermatozoa in the extracted BSA or when the medium contains only a 10% (v/v) proportion of this albumin. It is estimated that mouse spermatozoa under the conditions used take 2 hr to penetrate the zonae pellucidae of 50% of ova and effect fertilization.     

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.     

Effect of platelet activating factor on the motility and acrosome reaction of buffalo (Bubalus bubalis ) spermatozoa

Platelet activating factor (PAF; 1-0-alkyl-2 acetyl-sn-glycerol-3 phosphocholine) has been shown to have a wide range of biological activities. In this study, PAF was used to induce acrosome reactions in fresh as well as frozen-thawed buffalo spermatozoa at different incubation periods and PAF levels. As the period of incubation increased, there was a gradual decrease in motility and increase in acrosome reaction in both fresh and frozen-thawed spermatozoa. With increasing PAF levels, the motility of fresh spermatozoa decreased and acrosome reaction increased whereas in frozen-thawed semen, motility remained almost constant, and the increase in acrosome reaction was not pronounced. Differences in motility and acrosome reaction among different bulls, types of semen, periods of incubation and PAF levels were significant (P < 0.01). A PAF level of 100 microM and an incubation period of 15 min were found to be optimum for inducing acrosome reaction in buffalo spermatozoa, since at this combination acrosome reaction increased significantly (P < 0.01) over that of the control without much loss of motility.     

Species-specific sperm-egg interaction affects the utility of a heterologous bovine in vitro fertilization system for evaluating antelope sperm

The purpose of this study was to evaluate cryopreserved fringe-eared (FE) oryx (Oryx gazella callotis) sperm function using a heterologous in vitro fertilization (IVF) system previously developed to study scimitar-horned (SH) oryx (Oryx dammah) spermatozoa. Semen was collected by electroejaculation from FE oryx (n = 2) and SH oryx (n = 2), evaluated immediately postcollection, and cryopreserved. Thawed spermatozoa were evaluated for motility, forward progression, and acrosomal status immediately post-thaw, after Percoll-separation, and 1, 2, 3, and 8 h after culture in IVF medium. In vitro-matured cow oocytes (n = 924) were inseminated with either domestic bull, FE, or SH oryx spermatozoa and after an 8-h coincubation period, half the oocytes were fixed and examined for sperm penetration, polyspermy, and male pronuclear formation. The remaining oocytes were placed into in vitro culture and evaluated for cleavage after 48 h. Overall, there were no between-species differences in sperm motility and acrosome integrity. However, an effect of time (P < 0.05) and a species-by-time interaction (P < 0.05) were detected for both parameters. Penetration, male pronuclear formation, and embryo cleavage were high (>90%, >85%, and >70%, respectively) for oocytes inseminated with domestic bull and SH oryx spermatozoa and did not differ (P > 0.05) between species. In contrast, very few oocytes (2.8%, 4 of 141) inseminated with FE oryx sperm were penetrated. Cleavage was rare (8.0%, 16 of 200) in oocytes inseminated with FE oryx spermatozoa and did not differ (P > 0.05) from that in parthenogenetic controls (4.2%, 3 of 72). Furthermore, FE oryx spermatozoa were incapable of penetrating zona-free cow oocytes. These results indicate that species-specific differences in gamete interaction may exist even between very closely related nondomestic bovids.     

Role of spermatozoal platelet-activating factor in fertilization

Platelet-activating factor (PAF), a potent lipid mediator of inflammation, has been shown to play a role in both the implantation and viability of mammalian embryos. We examined whether human and mouse spermatozoa release PAF during in vitro incubation and assessed the effect of exogenous PAF and the PAF receptor antagonist WEB 2086, a thieno-triazolodiazepine, on mouse in vitro fertilization (IVF) rate. PAF biological activity was detected in 11 samples of leukocyte-free, purified human spermatozoa (28 pg PAF/10(6) cells/24 hr) and 5 samples of epididymal mouse spermatozoa (7.8 pg PAF/10(6) cells/3 hr). Exogenous PAF (10(-8) and 10(-6) M) increased (p less than 0.01) the fertilization rate 2- and 3-fold, respectively of mouse oocytes by mouse epididymal spermatozoa. 10(-4) M PAF, however, reduced sperm motility and decreased (p less than 0.05) the fertilization rate. 10(-6) M WEB 2086, decreased IVF to approximately 50% of the control fertilization rate (42% vs. 89%). WEB 2086 treatment also promoted the attachment of supernumerary spermatozoa to both fertilized and unfertilized oocytes. The fertilization rate in the presence of WEB 2086 returned to control levels when zona-pellucida-free oocytes were employed, indicating that WEB 2086 did not interfere with the spermatozoal acrosome reaction. These data suggest that PAF, of spermatozoal origin, may be important in mammalian fertilization.     

Scanning electron-microscopical and other observations of sperm fertilization reactions in Limulus polyphemus L. (Merostomata: Xiphosura).

Sperm fertilization reactions of Limulus polyphemus were examined by scanning electron and/or light microscopy. The following were considered: sperm motility, attachment of sperm to egg, acrosome reaction, and penetration of the acrosomal filament. The spermatozoa after semination are non-motile and become active only in close proximity to a defined region surrounding the egg. Egg materials diffusing into this region induce sperm motility and stimulate large numbers of spermatozoa to move towards the egg surface. Each sperm initially attaches by the apical tip and undergoes the acrosome reaction which causes a more permanent secondary attachment by the adhesion of acrosomal contents to the egg surface. The acrosome reaction also initiates the penetration of the acrosomal filament through the egg envelope, an event occurring in 70-80% of the attached spermatozoa (about 10(6). Shortly after this penetration, a secondary reaction occurs which involves a spiralling of the flagellum and an incorporation into the sperm body of the flagellar fibrous components, which then become closely apposed to the sperm nucleus. These sperm fertilization reactions were performed or initiated with 0-34 M CaCl2 in whole eggs, egg sections, excised egg envelopes and/or the outer basement lamina of the egg envelope. The Limulus fertilization system is very valuable since sperm reactions can be examined biochemically, which may lead to a better understanding of the chemical mechanisms involved in sperm-egg interactions in all animal species.     

Hamster zonae pellucidae cannot induce physiological acrosome reactions in chemically capacitated hamster spermatozoa in the absence of albumin

Cauda epididymal hamster spermatozoa were capacitated with D-penicillamine in a chemically defined (protein-free) medium (= "chemical" capacitation). Hamster zonae pellucidae were incapable of inducing functional acrosome reactions in chemically capacitated hamster sperm in a protein-free medium during sperm-egg coincubation. The culture medium used throughout incubation was a modified Tyrode's solution containing 10 mM sodium lactate, 100 microM sodium pyruvate and 1.0 mg/ml polyvinylalcohol (TLP-PVA). Sperm motility was maintained in all media with PHE (20 microM penicillamine, 100 microM hypotaurine, and 1.0 microM epinephrine). Additional D-penicillamine (125 or 500 microM) or 3 mg/ml bovine serum albumin (high control: TALP-PVA) was used to capacitate sperm during preincubation at 1-2 X 10(6) sperm/ml for 4.0 h at 37 degrees C in 5% CO2 in air. Sperm were then coincubated (2 X 10(4) sperm/ml) with cumulus-free hamster eggs in TALP-PVA or TLP-PVA +/- additional D-penicillamine (total: 500 or 125 microM) for 1.5 or 6.0 h. Percent egg penetration was used as the definitive index of sperm capacitation and functional acrosome reactions. Chemically capacitated sperm did not penetrate eggs (0.0 +/- 0.0%) in the absence of albumin during 1.5 h of sperm-egg coincubation. When sperm were chemically capacitated with 125 microM or 500 microM D-penicillamine, then coincubated with eggs for 6.0 h in the absence of albumin, only 18.8 +/- 28.6% and 23.7 +/- 29.7%, respectively, of eggs were penetrated. Significantly (p less than 0.05) more eggs (67.7 +/- 22.4%) were penetrated when coincubated with chemically capacitated sperm for 1.5 h in medium containing albumin. These results demonstrate that zonae pellucidae of hamster eggs require the presence of albumin to efficiently induce functional acrosome reactions in sperm that are chemically capacitated with D-penicillamine.     

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 simplified fertility test in the dairy bull utilizing superovulated dairy cows

Dairy bull fertility level has received less attention than production transmitting ability. A simplified fertility test may be beneficial. A study was designed to test the use of tris-(1-aziridinyl)-phosphine oxide (TEPA) treated sperm, which arrests early cell division of the fertilized egg, in heterospermic insemination of superovulated cows. Semen samples were collected and pooled from University of Illinois dairy bulls. Semen samples were washed once, suspended in Illini Variable Temperature diluent (IVT) and incubated with or without TEPA (1.0 to 5.0 mg/ml) for 15 min. Samples were then washed again to remove excess TEPA. Additions of 1.0 to 5.0 mg/ml TEPA to sperm concentrations of 8 x 10(8) sperm/ml had no adverse effect on motility or morphology. The first part of the study utilized superovulated cows inseminated with treated (six cows) or untreated (six cows) sperm in different samples from the same bulls. Secondly, superovulated cows (eight cows) were artificially inseminated with treated and untreated split ejaculates from the same bulls. Lastly, superovulated cows (five cows) were heterospermically inseminated with treated (bull No. 1) and untreated (bull No. 2) spermatozoa. Out of 54 and 39 ova recovered in control and test cows, 40 blastocysts and 31 embryos arrested at the one- to five-cell stage resulted, respectively. Out of a predicted 123 ovulations, 78 fertilized ova were recovered; 40 of these were fertilized by control spermatozoa and 36 by TEPA-treated spermatozoa for parts one and two of the study respectively. These results indicated no significant difference in fertilizability of ova between control and TEPA-treated spermatozoa. Of 41 fertilized ova recovered (part 3), bull No. 1 fertilized significantly more ova (mean +/- standard deviation 5.0 +/- 2.3) than bull No. 2 (2.6 +/- 1.8). Results indicate a difference in fertility between bulls.     

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.     

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.     

Short communication: Extraction of sp18 family membrane proteins on posterior head of bull sperm and the effect of anti‐sp18 IgG on sperm motility and murine in vitro fertilization

Abstract

Bovine sperm heads were separated via ultrasonic treatment and centrifugation. Anti‐bull sperm IgG was produced by immunizing rabbits with acrosome‐reacted bull sperm heads. SDS PAGE patterns revealed that the main membrane proteins on acrosome‐reacted bull sperm head were sp18 family, including 18, 16, and 14 kD, which represented about 64% of the total membrane proteins in bull sperm. Indirect immunofluorescence shown sp18 antigens primarily distributed in postacrosomal and proximal tail regions. Western blot analysis revealed that the anti‐bull sperm IgG reacted with sp18 antigens in acrosome‐reacted bull sperm head and bull seminal plasma. Anti‐bull sperm IgG also reacted with 14, 16, 18, 42, 57 and 60 kD proteins in fresh bull, mouse and rabbit sperm. Anti‐sp18 IgG caused agglutination of bull and rabbit sperm, but had no effect on murine sperm. In murine in vitro fertilization trials, preincubating capacitated sperm with 0.364 mg/ml of anti‐sp18 IgG resulted in a decrease in the fertilization rate from 75.6% in the controls to 50.8% in the experimental groups (p<0.001).     

Characterization and activity of angiotensin-converting enzyme in Holstein semen

The study was designed to perform immunodetection in spermatozoa and seminal plasma, immunolocalization in spermatozoa, and evaluation of the enzymatic activity of angiotensin-converting enzyme (ACE) in the semen of Holstein bulls. We used ejaculates from five bulls as part of a regular collection of semen. The monoclonal anti-ACE antibody recognized a single protein band with 100 kDa in detergent extract prepared from sperm and in seminal plasma. ACE enzymatic activity in sperm was 43.7, 21.3, 45.6, 60.0, and 57.7 mU/mL in bulls 1, 2, 3, 4, and 5, respectively, and 0.3, 2.3, 3.0, 2.3, and 2.6 mU/mL in seminal plasma of the same bulls, respectively. The average percentages of sperm with acrosome reactions after treatment with heparin were 28.3%, 28.6%, 35.2%, 25.0%, and 32.3%, respectively. These values were higher than the percentages of acrosome reactions in controls and the captopril group (P<0.05), although no difference was seen between the captopril and control groups (P>0.05). After 4h of incubation, motility in the control group (32.9%) was significantly higher than that in the heparin (15.7%) and captopril (12.1%) groups. No difference was found in motility after the capacitation assay in the heparin and captopril groups (P>0.05). In conclusion, ACE was immunologically localized in the acrosome of the spermatozoa of Holstein bull, the specific enzymatic activity of ACE in detergent-extracted spermatozoa and seminal plasma was inhibited by captopril, and this ACE inhibitor reduced the percentage of sperm with progressive motility and acrosome reactions after capacitation in vitro.     

Inhibition of bovine sperm-oocyte fusion by the p-aminophenyl derivative of D-mannose

Several steps in the process of mammalian fertilization are mediated by carbohydrates. This study investigated the role of the p-aminophenyl derivative of d-mannose (APMP) during bovine fertilization. Inseminating cumulus-oocyte complexes (COCs) in the presence of increasing APMP concentrations resulted in a significant dose-dependent decrease of the fertilization rate (P < 0.05). No negative effect of 50 mM APMP on total sperm motility and progressive motility was found. Subsequently, the fertilization steps at which this blocking effect could be exerted were investigated, i.e., sperm penetration of the cumulus oophorus, sperm-zona binding, acrosome reaction, sperm-oolemma binding, and/or sperm-oocyte fusion. Inseminating cumulus-enclosed and cumulus-denuded oocytes in the presence of 50 mM APMP significantly decreased the fertilization rate to a comparable minimum level (P < 0.05). There was no significant relationship between the number of spermatozoa bound to the zona pellucida and the APMP concentration, and APMP nor d-mannosylated bovine serum albumin (BSA) suppressed or stimulated sperm acrosomal status. Inseminating zona-free oocytes in the presence of 50 mM APMP did not influence sperm-oolemma binding, but significantly inhibited sperm-oocyte fusion (P < 0.05). Preincubating zona-free oocytes with 200 microg/ml Con A but not with 50 mM APMP inhibited the sperm-oocyte fusion rate to the same extent as when the gametes were simultaneously exposed to 50 mM APMP. These data indicate that the blocking effect of APMP on bovine fertilization is mainly due to an inhibition of sperm-oocyte fusion, probably by specific obstruction of the sperm receptor sites that are responsible for the fusion process.     

Dilution of spermatozoa results in improved viability following a 24 h storage period but decreased acrosome integrity following cryopreservation

This study was designed to investigate the physiological factors affecting the reduced viability of cryopreserved spermatozoa following dilution. Ninety-six ejaculates were collected from 13 bulls and diluted to 10 x 10(6) and 60 x 10(6) sperm/ml in a commercial long term extender (Eqcellsire; IMV) and in an egg yolk extender. Samples diluted in the egg yolk extender were frozen in 0.25 ml straws. Samples diluted in the Eqcellsire were stored at room temperature for 24 h and assessed for sperm cell viability using SYBR14 and PI (Molecular Probes) and osmotic resistance. Frozen samples were thawed and assessed for viability, osmotic resistance and acrosome intergrity. Acrosome integrity was measured using Mitotracker, PI and PNA-FITC (Molecular Probes). Spermatozoa diluted to 10 x 10(6) sperm/ml and stored at ambient temperature had a higher proportion of viable spermatozoa (P < 0.01) and were less susceptible to osmotic stress (P < 0.01) than sperm diluted to 60 x 10(6) sperm/ml. Following cryopreservation there was no concentration-related difference in the proportion of viable spermatozoa and their relative susceptibility to osmotic stress. Spermatozoa diluted to lower cell concentrations had a higher proportion of viable cells that were acrosome reacted (P < 0.001). It is suggested that the higher proportion of acrosome reacted cells may result from an increased proportion of cells in a capacitated-like state in the spermatozoa diluted to lower concentrations. A Spearmans ranked correlation demonstrates a relationship between individual bull spermatozoa following dilution or cryopreservation for viability (r2 = 0.98; P < 0.001) or osmotic resistance (r2 = 0.87; P < 0.001) suggesting a variation in these characteristics between bulls.