Flow of blood to the ovaries of ewes throughout the estrous cycle: Niswender,G.D., R.T. Moore,A.M. Akbar,T.M. Nett and M.A. Diekman: Biol. Repr., 13: 381, 1975. Department of Physiology and Biophysics,Colorado State University,Fort Collins,Colorado 80523

This study was undertaken to determine whether a specific antimycoplasmal immune response could be detected in the male bovine genital tract and to better define mechanisms of immunity at that site. Specific $\text{Mycoplasma agalactiae}$ subsp. $\text{bovis}$ agglutinins were titrated in the serum, semen and preputial mucus extracts of two bulls with $\text{M. agalactiae}$ induced chronic seminal vesiculitis and of one normal control bull. Titers from infected bulls averaged 64 for serum, 1024 for semen and <8 for preputial mucus extracts whereas the control bull titers were 16 for serum, <8 for semen, and <8 for preputial mucus extracts. Because of the high semen agglutinin titers from infected bulls it was proposed that semen titers may be more useful diagnostically than serum titers.Studies of immunoglobulin levels in semen revealed that IgA, IgG₁ and IgG₂ levels were all much higher in infected bulls than in the control bull. These high semen IgA levels together with the high semen agglutinin titers indicated a local secretory immune response in genital tracts of infected bulls.     

Update on treatment of vesiculitis in bulls

Four experiments were done to determine: (1) the effectiveness of early detection and treatment of vesiculitis in bulls; (2) whether antibiotic treatment at recommended dosages will result in adequate vesicular gland tissue concentrations of antibiotics to prevent in vitro bacterial growth; (3) whether intraglandular injection of antibiotics can be a successful alternative to systemic antibiotic treatment; and (4) the effectiveness of tilmicosin versus tulathromycin for treatment of clinical vesiculitis. In Experiment 1, there was a high rate of spontaneous remission from vesiculitis detected at 9-12 mo of age. Furthermore, there was no advantage for early antibiotic treatment versus no treatment for bulls of this age. In Experiment 2, bacteria on agar plates were exposed to fluid extracted from vesicular gland biopsies after antibiotic treatment of normal, healthy bulls. Although inadequate concentrations of antibiotics were achieved to inhibit bacterial growth when recommended dosages of various antibiotics were administered, doubling the antibiotic dosage increased in vitro bacterial growth inhibition. In Experiment 3, relatively nonirritating antibiotics were injected directly into the glands of bulls with clinical vesiculitis, demonstrating that intraglandular injections of antibiotic could be used as a successful alternative to systemic antibiotic treatment. Experiment 4 was a clinical field trial to compare the efficacy of tilmicosin versus tulathromide at recommended dosages for the treatment of clinical vesiculitis. Although the results favored tulathromycin, both antibiotics resulted in clinical cures of vesiculitis.     

Early detection and treatment of vesicular adenitis in bulls

The objective of this study was to determine whether early detection and treatment with tilmicosin would reduce the number of yearling bulls culled due to vesicular gland disease at the time of their first semen test. Bulls (n=2207) of 15 breeds at 17 performance test stations were examined by transrectal palpation at 9-12 months of age and assigned to three treatment groups: (1) positive treated group, receiving subcutaneous injections of tilmicosin every second day for three treatments (2) positive untreated control group, (3) negative untreated control group. Transrectal palpation of the glands was done again at a pre-sale semen test 28-70 d (mean=42.8 d) after the first examination. Semen was evaluated for the presence of pus and/or leukocytes by light microscopy. The proportion of bulls with vesiculitis was 4.4% (97/2207). At the pre-sale semen test the number of bulls with vesiculitis had decreased to 1.3% (29/2207); however, seven of these were new cases that developed after the first examination. Therefore, there was a recovery rate of 75/97 (77.3%) in the original group of bulls positive for vesiculitis. At the pre-sale test, there was no difference in the proportion of bulls with enlarged glands in the positive treated group (15/66) versus the positive untreated group (7/31) and no difference in the proportion of bulls with leukocytes or pus in their semen samples. The results of this experiment did not indicate any advantage in detection and treatment of vesiculitis before a year on age due to a high rate of spontaneous recovery.     

Distribution of Corynebacterium renale among healthy bulls with special reference to inhabitation of type III in the prepuce.

Distribution of Corynebacterium renale among apparently healthy bulls reared in Hokkaido was investigated. The organism was detected from 46 (39.3%) of 117 specimens of preputial cavity washing and from 60 (51.7%) of 116 specimens of semen. The isolates studied in this survey belonged to type III, except a few which belonged to type II. No type I strain was isolated from any bull. C. renale type III was isolated from the prepuce in six of seven bulls slaughtered and from urethra in three, but not at all from any other organ. In the seven bulls, no macroscopic changes were seen, but a slight infiltration of lymphocytes and formation of lymph nodules were noticed in the prepuce. No other microscopical changes could be demonstrated in any other organ. No serum antibody response was detected. To ascertain the virulence of C. renale isolated from the bulls, a strain of type II was inoculated into the urinary bladder of a healthy cow. The cow exhibited fever and hematuria on and after the 10th day. Typical cystitis was proved when the cow was necropsied on the 14th day after inoculation. From these result it is conceived that C. renale type II organisms inhabit the prepuce of apparently healthy bulls at a high rate, without inducing any disturbance.     

Intraglandular injection of antibiotics for the treatment of vesicular adenitis in bulls

Two experiments were designed to determine the efficacy of intraglandular antibiotic treatment in beef bulls. Experiment 1 was designed to evaluate the glandular tissue reaction to intraglandular antibiotic treatment. Experiment 2 was conducted to determine the efficacy intraglandular injection of antibiotics for the treatment of naturally occurring cases of vesicular adenitis. Healthy beef bulls (n=15), 2 and 3 years of age, were randomly allocated to three equal treatment groups to receive 10% of the daily recommended parenteral dose of penicillin, ceftiofur, or oxytetracycline in a volume of 6 mL injected directly into one of the vesicular glands. Ultrasonography was performed before, immediately after, and at 24, 48 and 168 h after intraglandular injection. The size and hardness of vesicular glands injected with oxytetracycline was greater (P<0.01) than those injected with ceftiofur. Ultrasonographic pixel intensity increased (P<0.01) after treatment with antibiotics, especially after treatment with oxytetracycline or penicillin. In Experiment 2, yearling beef bulls with clinical vesicular adenitis (n=14) were referred to the Western College of Veterinary Medicine for treatment. Eight bulls had unilateral and six had bilateral vesicular adenitis. The most common isolate was Arcanobacterium pyogenes. Corynebacterium pseudotuberculosis was isolated from one bull. Bulls were subjected to rectal palpation and ultrasonography of the vesicular glands, semen collection by electroejaculation, and intraglandular treatment with ceftiofur (n=13) and if necessary, a second intraglandular treatment of penicillin (n=6). One bull was treated only with an initial intraglandular injection of penicillin. Bulls were evaluated once a week over 6 weeks by palpation of the glands, and evaluation of semen. All bulls recovered from vesicular adenitis after 3-6 weeks. There was a difference in the amount of pus (P=0.042), leukocytes (P<0.001) and blood (P=0.003) present in ejaculates from before treatment to 3 weeks after treatment. Pixel intensities in ultrasonographic images of healthy or affected vesicular glands, whether treated or untreated, did not change over time. Intraglandular injection of ceftiofur in yearling bulls via the ischiorectal fossa was effective for treating vesicular adenitis.     

Amplification and Characterization of Bull Semen Infected Naturally with Foot-and-mouth Disease Virus Type Asial by RT-PCR

To investigate the security of semen biologically, 15 bull semen samples were collected (of which 5 exhibited clinical signs of Foot-and-mouth disease) and identified by RT-PCR and virus isolation. The results indicated that the semen of the infected bulls were contaminated by Foot-and-mouth disease virus (FMDV), but FMDV was not detected in semen samples from those bulls not showing clinical signs of Foot-and-mouth disease (FMD). This is the first report of the presence of FMDV in bull semen due to natural infection in China. The analysis of the partial sequence of the VP1 gene showed that the virus strain isolated from semen has 97.9% identity with the virus isolated from vesicular liquid of infected bulls showing typical signs of FMD and belonged to the same gene sub-group.     

Infection of SDAV-immune rats with SDAV and rat coronavirus

Infection of rats with sialodacryoadenitis virus (SDAV) or rat coronavirus (RCV) is acute and self-limiting, and elimination and control of either virus is based on the assumption that recovered rats are immune to reinfection. To test this hypothesis, we examined whether SDAV-immune rats could be infected with RCV or reinfected with SDAV. Sprague Dawley (SD) rats were inoculated intranasally with SDAV or with culture medium alone and serial SDAV antibody titers were obtained. Eleven months after inoculation, when antibody titers had stabilized, SDAV-immune and nonimmune rats were challenged with SDAV or RCV, and euthanatized 3 or 6 days later. SDAV-immune rats challenged with SDAV or RCV manifested acute rhinitis associated with virus antigen by 3 days after inoculation, but no lesions or antigen were subsequently found in the lower respiratory tract, salivary glands or lacrimal glands. There was also a marked anamnestic increase in antibody titer by 6 days after challenge. SDAV-immune rats challenged with SDAV or RCV also transmitted infection to nonimmune cage mates. This study indicates that 11 months after primary infection with SDAV, rats can be infected with SDAV or RCV, but that the severity of disease is significantly reduced.     

Purification and characterization of fertility‐associated antigen (FAA) in bovine seminal fluid

Heparin‐binding proteins (HBP) recognized by a monoclonal antibody (M1) are produced by male accessory sex glands and bind to distinct regions of ejaculated bull sperm. Immunoblots of sperm proteins probed with M1 identified HBP variants of approximately 31‐, 24‐, and 21.5‐kDa that were associated with increased fertility of bulls. The purpose of this study was to identify the 31‐kDa HBP known as fertility‐associated antigen (FAA). FAA was isolated by heparin‐affinity chromatography and reversed‐phase high performance liquid chromatography near homogeneity. Biochemical characterization indicated that FAA was an unglycosylated, basic protein. FAA protein was detected in seminal vesicle and prostate gland homogenates, and FAA extracted from sperm membranes by treatment with hypertonic media was identical biochemically to seminal fluid‐derived FAA. N‐terminal sequence analysis of purified FAA yielded a 26 amino acid sequence (L K I X S F N V R S F G E S K K A G F N A M R V I V) with 73% identity to a recently identified human deoxyribonuclease (DNase) I‐like protein. Two internal amino acid sequences generated from lys‐C digested FAA were 85% and 92% identical to the same DNase I‐like protein. In conclusion, we have identified a bovine seminal heparin‐binding protein that binds to sperm and is indicative of bull fertility as being similar to the family of DNase I‐like proteins. These data demonstrate the presence of a novel DNase I‐like protein in bull accessory sex glands and form the groundwork for the identification of a candidate genetic marker for fertility of bulls. Mol. Reprod. Dev. 54:145–153, 1999. © 1999 Wiley‐Liss, Inc.     

Amplification and characterization of bull semen infected naturally with foot-and-mouth disease virus type Asia1 by RT-PCR

To investigate the security of semen biologically, 15 bull semen samples were collected (of which 5 exhibited clinical signs of Foot-and-mouth disease) and identified by RT-PCR and virus isolation. The results indicated that the semen of the infected bulls were contaminated by Foot-and-mouth disease virus (FMDV), but FMDV was not detected in semen samples from those bulls not showing clinical signs of Foot-and-mouth disease (FMD). This is the first report of the presence of FMDV in bull semen due to natural infection in China. The analysis of the partial sequence of the VP1 gene showed that the virus strain isolated from semen has 97.9% identity with the virus isolated from vesicular liquid of infected bulls showing typical signs of FMD and belonged to the same gene sub-group. Foundation items: State Science and Technology Support Program (2006DAD06A03) and Hi-tech Research and Development Program of China 863 (2006AA10A204).     

Effect of the knobbed acrosome defect in bovine sperm on IVF and embryo production

An IVF and culture system was used to determine the effect of the knobbed acrosome defect in bovine spermatozoa on fertilization and early embryonic development. Three bulls affected with knobbed acrosomes were identified as K+ (flattened acrosome), K2+ (indented acrosome) or K3+ (deep indentation of the acrosome) based on the predominant type of acrosomal aberration present in sperm of the respective bulls. After swim-up, all semen traits, except for acrosome morphology, were similar between bulls with varying degrees of the knobbed acrosome defect and a control bull, C. The mean number of spermatozoa bound to the zona pellucida was lower (P< 0.05) for the bulls with the knobbed acrosome defects (40.3 +/- 2.3, 29.5 +/- 1.6, 14.6 +/- 1.3, respectively, for Bulls K+, K2+ and K3+) than for Bull C (52.3 +/- 2.3). The percentages of zonae pellucidae penetrated by spermatozoa from Bulls K+ (51.2%), K2+ (49.5%) and K3+ (37.1%) were lower than that of Bull C (84.5%). No sperm with knobbed acrosome defects were found to have penetrated the zona pellucida. Fertilization rates for bulls with the knobbed acrosome defect, K+ (63.0%), K2+ (62.7%) and K3+ (22.6%), were significantly lower than that of the control bull (82.8%). Percentages of cleaved embryos, morulae and blastocysts produced were also lower for the bulls with knobbed acrosomes than that of the control bull. Results indicate that sperm with the knobbed acrosome defect had a reduced ability to bind to the zona pellucida, depending upon the severity of the defect, and that these aberrant spermatozoa did not penetrate the zona pellucida. The apparently normal spermatozoa coexisting in the inseminate of bulls with a high percentage of knobbed spermatozoa were also functionally deficient; oocytes penetrated by these spermatozoa had a reduced potential for fertilization, and resulting zygotes had a reduced ability for cleavage and embryonic development to the blastocyst stage. The results of the present study do not support the hypotheses that the knobbed acrosome defect is compensable.     

Inoculation of bulls with bovine virus diarrhea virus: Excretion of virus in semen and effects on semen quality

Bovine virus diarrhea (BVD) virus was inoculated into 9 bulls. Semen samples were collected to determine if BVD virus could be isolated from semen and to determine if the virus affected semen quality. The reproductive tracts of 6 bulls were recovered following slaughter and subjected to virus isolation procedures and histologic examination.BVD virus was detected in 4 of 98 semen samples following inoculation. The virus was also recovered from the testicle of one bull following slaughter. Inoculation of BVD virus into bulls did not affect semen quality and did not cause gross or microscopic lesions in the reproductive tract. This experiment indicates that following inoculation of BVD virus into bulls, the virus may be shed in the semen. However, the probability of this occurring is low.     

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.     

Spermagglutinins in serum and seminal fluid of bulls and their relationship to fertility classification

Twenty-five satisfactory and 25 unsatisfactory potential breeder bulls were examined with a modified Shulman spermagglutination test for the presence of spermagglutinating antibodies in their serum and seminal fluid. This was done to determine whether antibodies to spermatozoa were a contributing factor to classification of the bull as an unsatisfactory potential breeder. No correlation was found between classification as an unsatisfactory potential breeder and the presence of spermagglutinating antibodies. In addition, indirect fluorescent antibody tests were performed to detect antisperm antibodies in the serum and seminal fluid of the bulls. Again, no correlation was found between antisperm antibodies and breeding soundness classification. Finally, there was no correlation between the age of the bulls and occurrences of spermagglutination antibodies in serum or seminal fluid.     

OESTROUS BEHAVIOUR, OVULATION AND THE FORMATION OF THE CORPUS LUTEUM IN THE AFRICAN ELEPHANT,

A female elephant was observed throughout most of oestrus, and was then shot. The cow was mated by many different bulls during the course of oestrus. Initially, there seemed to be little competition between bulls for the cow, but as oestrus wore on, fighting broke out amongst the bulls and one eventually appeared to establish mastery, driving the other bulls away. It was interesting that this master bull showed no discharge from his temporal glands.     

Superinfection of a chimpanzee with a second strain of human immunodeficiency virus.

Two chimpanzees, one (C-499) infected with the acquired immunodeficiency syndrome-associated retrovirus type 2 (ARV-2) strain of human immunodeficiency virus (HIV) and one (C-560) infected with the lymphadenopathy-associated virus type 1 (LAV-1) strain of HIV, were inoculated with approximately 10(4) tissue culture infective doses of the reciprocal strain. At the time of the second inoculation, both chimpanzees had high titers of HIV-specific antibodies, including antibodies that neutralized both virus strains. After inoculation of the second strain of HIV, the antibody titers in both chimpanzees increased 4- to 10-fold, and in one chimpanzee (C-499), the numbers of infectious peripheral blood mononuclear cells (PBMC) increased 1,000-fold to levels that are comparable with those observed after primary HIV infections. By restriction enzyme analysis of virus recovered from PBMC, both ARV-2 and LAV-1 were identified in C-499, thus demonstrating that superinfection had occurred.     

Diagnosis of bovine respiratory syncytial virus infection by complement fixation test.

The complement fixation test by the microtiter method was applied to the serological diagnosis of bovine respiratory syncytial (RS) virus infection. When used as complement fixing antigens, untreated infected cell culture fluid, fluorocarbon-treated, and ether-treated materials showed no differences in antigenicity among them. The complement fixing antigenicity of bovine RS virus appeared in bovine kidney and Vero cell cultures for the first time 4 days after inoculation. Both the infectivity and complement fixing antigenicity reached a maximum 6 days after inoculation. In detecting complement fixing antibody from infected cattle, the most outstanding specific reaction was obtained when 5% fresh normal calf serum had been added to the diluent of complement. Neutralizing and complement fixing antibodies were examined in serum samples from two cattle in the course of experimental infection. It was found that both antibodies turned to be positive 2 weeks after inoculation. There was a linear correlation between neutralizing and complement fixing antibody titers, when serum samples from 40 natural cases were tested in the acute and convalescent stages. In addition, common antigenicity was demonstrated between the virus of bovine origin and the Long strain of human RS virus by complement fixation test.     

Safety of Brucella abortus strain RB51 in bull elk

Some of the elk (Cervus elaphus nelsoni) of the Greater Yellowstone Area (Wyoming, Idaho, Montana; USA) are infected with Brucella abortus, the bacterium that causes bovine brucellosis. Brucella abortus strain RB51 vaccine is being considered as a means to control B. abortus induced abortions in cow elk. However, the most probable vaccination strategies for use in free-ranging elk might also result in some bull elk being inoculated, thus, it is important to insure that the vaccine is safe in these animals. In the winter of 1995, 10 free-ranging bull elk calves were captured, tested for B. abortus antibodies, and intramuscularly inoculated with 1.0 x 10(9) colony forming units (CFU) of B. abortus strain RB51. Blood was collected for hemoculture and serology every 2 wk after inoculation for 14 wk. Beginning 4 mo postinoculation and continuing until 10 mo postinoculation elk were serially euthanized, necropsied, and tissues collected for culture and histopathology. These elk cleared the organism from the blood within 6 wk and from all tissues within 10 mo. No lesions attributable to B. abortus were found grossly and only minimal to mild lymphoplasmacytic epididymitis was found in a few elk on histologic examination. In a separate study, six adult bull elk from Wind Cave National Park (South Dakota, USA) were taken to a ranch near Carrington (North Dakota, USA). Three were orally inoculated with approximately 1.0 x 10(10) CFU of RB51 and three were inoculated with corn syrup and saline. Ninety days post-inoculation semen was examined and cultured from these bulls. Strain RB51 was not cultured from their semen at that time. There were no palpable abnormalities in the genital tract and all elk produced viable sperm. Although they contain small sample sizes, these studies suggest that B. abortus strain RB51 is safe in bull elk.     

Human toxoplasmosis acquired by ingestion of goat's milk

Acute toxoplasmosis in three members of the same family was related to drinking unpasteurized goat's milk. The goats were reared, for milk production in a peri-domestic area. Based on indirect immunofluorescent reactions antibody titers greater than 1: 1024 were recorded in eight out of fifteen animals examined and were highest in the five lactating adult females. Toxoplasma was isolated, by inoculation of mice, from the milk of one of these females. Dogs reared in the same house showed no symptoms of acute toxoplasmosis and low antibody titers were detected in these animals. It was also found that the human infections could not have been due to the ingestion of food contaminated with oocysts.     

Immunity patterns during acute infection by Eimeria bovis

Cellular and humoral responses were investigated following gavage inoculation of 6-wk-old bull calves with 35,000-40,000 oocysts of Eimeria bovis. At 3-4-day intervals for 40 days after inoculation (DAI), blood was taken and assessed for serum IgG against merozoites and sporozoites of E. bovis. Proliferative responses of peripheral blood lymphocytes were measured following stimulation with either concanavalin A (Con A) or a soluble antigen derived from E. bovis oocysts (EbAg). Serum IgG against merozoites and sporozoites reached a peak of activity between 10 and 20 DAI, coinciding with oocyst shedding on days 17 to 24. Serum antibody titers had dropped to base levels by 40 DAI, although anti-merozoite titers remained elevated for the duration of the study (i.e., from days 12 and 20 to day 40). Con A stimulation of lymphocytes was not affected by infection; there was no evidence of suppressed or augmented responsiveness. Lymphocyte responses to EbAg had reached a maximum by day 20 and remained elevated throughout the study. These results indicate (a) that sporozoites and merozoites share antigens recognized by serum IgG, (b) that there is no episode of marked immunosuppression during acute infection, and (c) that cellular immunity is probably more important in resistance against reinfection than humoral immunity.     

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.