Effects of experimentally generated bull antisperm antibodies on in vitro fertilization.

To test the hypothesis that bull antisperm antibodies have the capacity to interfere with fertilization, antisperm antibodies were generated in three 13-mo-old Holstein bulls by auto-immunizing each bull with sperm three times. All bulls produced serum antisperm IgG1 and IgG2 antibodies. No serum antisperm IgA nor seminal plasma antisperm antibodies of any isotype could be detected by ELISA. Western blots were performed with immunopurified IgG1 and IgG2 from pre- and post-immunization sera from one test bull. Both post-immunization IgG1 and IgG2 recognized a 45-kDa sperm antigen. Serum samples from a normal bull stud population tested by ELISA had significantly higher levels of antisperm antibodies than did heifers. The bull stud serum samples giving the highest ELISA values differed from those of the immunized bulls in that their antisperm antibodies were of the IgM isotype only. Bull sperm were incubated with serum from the immunized and control bulls, then added to bovine oocytes in vitro. Incubation of sperm with post-immunization serum reduced in vitro fertilization rates (p < 0.01). This study demonstrated that antisperm IgG1 and IgG2 generated by sperm auto-immunizations reduced fertility in vitro, and therefore naturally occurring antisperm antibodies may affect fertility in bulls.     

Presence of antisperm antibodies in the serum of Sardinian adult people. II. Study on a selected infertile population in north Sardinia.

Among many possible causes of infertility an immunological factor must be considered, in particular the presence of antibodies to spermatozoa in the serum. In previous researches we confirmed the presence of antisperm antibodies in the serum of healthy men of North Sardinia in percentage equal to 2.4% of the considered population. The aim of the present study is to evaluate the correlation between the presence of antisperm antibodies and infertility. The presence of antispermatozoal antibodies has been evaluated in the serum of 124 men (aged 25-50) affected by unexplained infertility utilizing the indirect immunofluorescence and ELISA methods. The presence of antisperm antibodies has been found equal to 19.3% of the tested sera. These results show that the incidence of antisperm antibodies is higher in the serum of male partners from infertile couples than in healthy subjects (P < 0.01), also in a relatively segregated and pure population like the one studied.     

Effects of active immunization of the ram and bull against luteinizing hormone

Active immunization of ram lambs and bull calves against highly purified LH results in the production of antibodies which bind radiolabeled ovine LH. Antibody titers were not dependent upon the dose (0.1 or 1.0 mg) of antigen given but were found to be greater when the antigen consisted of LH conjugated to a carrier protein; in this case human serum globulin (hSG). The presence of LH antibodies in ram sera seemed of little consequence. The presence of antibodies in sera of immunized bulls, however, appeared to neutralize endogenous LH sufficiently to suppress serum testosterone and reduce testicular size. Androgen deficiency, produced by immunization against oLH-hSG, resulted in atrophy of the testes, seminal vesicles and epithelial cells lining both epididymides and seminal vesicles. Reduced weight gain and lack of development of the secondary sexual characteristics were at levels expected of surgical castrates (steers). In summary, active immunization of young beef bulls against LH and in particular, oLH-hSG, results in a castration-like response which has been compared with that achieved in other species by luteinizing hormone releasing hormone (LHRH) immunocastration. The success of LH immunocastration in field trials with young bulls and investigations aimed toward reversibility are yet to be determined.     

Identification of bovine sperm specific polypeptides reactive with antisperm antibodies.

The present study was taken to characterize molecular weights of sperm specific polypeptides antigenic to rabbits and calf with the aim to assess their immunoreactivity with IgG antibodies in sera from immuno-infertile cows. Seropositivity for antisperm IgG antibodies in 75 repeat breeder and 15 pregnant control cattle was tested by cellular ELISA using washed spermatozoa antigen from 4 bulls. Molecular weights of bovine sperm polypeptides antigenic to rabbit and calf were determined by 10% SDS-PAGE and Western blotting. Molecular weights of sperm peptides reactive with sera from immuno-infertile cows were also determined. Seropositivity of antisperm IgG antibodies for bull I, II, III and IV was 23.6, 14.6, 26.6 and 20%, respectively. A total of 16 polypeptides were discernible on gel. Out of these, 7 polypeptides were immunoreactive with sera from hyperimmunized rabbits as compared to 3 poly-peptides which reacted with sera from hyper-immunized calf. Only two polypeptides were reactive with sera from immuno-infertile cows. Variable number of sperm polypeptides and their immunoreactivity have been reported in different species. Antigenicity of different polypeptides in sperm needs further investigations.     

Reproductive status of cows and incidence of antisperm antibodies

Serum and mucus samples from three groups of cows, i.e., repeat breeder cows, fertile cows, and virgin heifers were tested for anti-spermatozoal antibodies. Agglutinating (Aggl) and immunofluorescent (IF) antibodies were found in serum from all cows. Positive agglutination occurred in 26%, 32%, and 0% of mucus samples tested from the repeat breeder cows, fertile cows, and virgin heifers, respectively. Positive results on IF tests on mucus samples from the same cows were 37%, 28%, and 13%, respectively. In contrast, no sperm-immobilizing antibodies were found in any samples. There was no apparent correlation between the incidence and/or titer of the Aggl or IF antibodies and fertility status of the animal. These antibodies were assumed to be produced naturally (natural antibodies) with no need of female exposure to sperm antigens, since all virgin heifers also demonstrated Aggl and IF antibodies in their sera.The results from this study indicate that antibodies against sperm apparently were not responsible for reduced fertility in this particular group of repeat breeder cows.     

Immunoelectrophoretic study of esterase in blood serum, spermatozoa and genital tract fluids of bulls

Immunoelectrophoretic methods were used to observe the formation of precipitates with esterase activity in the blood serum, spermatozoa and the genital tract fluid of bulls, and also in the follicular fluid of cows. Fractions with esterase activity were found in the seminal vesicle fluid and the ampullar fluid of all investigated animals. Considerable individual differences in the formation of precipitates with esterase activity were found in the mentioned fluids. Ejaculated and ampullar spermatozoa, spermatozoa from the tail of the epididymis and the testicle fluid formed fractions with esterase activity only in some individuals. Esterase activity was not detected in the fluid of the tail of the epididymis of bulls nor in the follicular fluid of cows.     

The prevalence of chlamydiae of bulls from six bull studs in Germany

Although there are indications for venereal transmission of chlamydiae in cattle, epidemiological data on the presence of these bacteria in bulls and bull semen in particular is still incomplete. We investigated semen (n=120), preputial washing samples (n=121) and faeces (n=122) of bulls from six bull studs located within five Federal States of Germany for the presence of chlamydiae using omp1-PCR and partial omp1 sequencing. Blood serum was examined for chlamydial antibodies using an indirect ELISA (n=122). Chlamydiae were found in 11 (9.2%), 13 (10.7%) and 22 (18.0%) of the semen, preputial washing and faecal samples, respectively. Among individual chlamydial species identified, Chlamydophila (Cp.) psittaci predominated in semen and preputial washing samples, and Cp. pecorum in faeces. Cp. abortus was the third frequently observed species. Chlamydial antibodies were detected in a total of 62 (50.8%) bulls. Bull studs differed in regard to the number of bulls found chlamydia-positive in faeces and serologically positive. No correlation was observed between serological data and PCR of semen, preputial washing samples or faeces. Standard ejaculate parameters did not differ between bulls that were chlamydia-positive and -negative in semen. In conclusion, detection of chlamydiae in semen of bulls suggests a potential for venereal transmission. Chlamydiae appear to be widespread within the bull population in Germany. Serological testing failed to identify bulls shedding chlamydiae in their semen.     

Relationships among seminal culture, seminal white blood cells, and the percentage of primary sperm abnormalities in bulls evaluated prior to the breeding season

Semen samples from 100 beef breed bulls were evaluated for sperm morphology (phased contrast microscopy), seminal white blood cells, and the presence of potential reproductive pathogens. Eligibility required visualization of the glans penis throughout semen collection. Based on clinical spermiograms, bulls were grouped into normal, marginal, or unsatisfactory morphology classifications. The 3 experimental groups were similar in age and scrotal circumference and differed significantly in the percentage of primary sperm abnormalities. Most semen samples (94%) contained one or more potential reproductive pathogens (Hemophilus somnus. Mycoplasma bovigenitalium, Arcanobacterium pyogenes and Ureaplasma diversum). No significant relationship could be demonstrated between primary abnormalities and the assigned culture score. Our experimental results suggest that clinicians should interpret clinical semen culture results with great care. No significant relationship could be demonstrated between primary abnormalities and assigned white blood cell (WBC) score, although, only 1% of the samples was scored >5 WBC per high power field. The use of seminal WBC score may be valid adjunct to routine semen evaluation when that threshold is the basis for clinical decisions.     

Seasonal variations in serum levels of thyroid hormones and their relation with seminal quality and libido in buffalo bulls

Blood samples from 15 Murrah buffalo bulls, (10- to 15- years-old) were collected during the summer, monsoon and winter seasons. The serum samples were analysed for thyroxine (T4) and triiodothyronine (T3) by RIA. Semen samples from these bulls were evaluated for various attributes. Sexual behaviour of these bulls was also recorded during the different seasons and was expressed as reaction time and refusal response. T4, T3, (T4 + T3) level and T4:T3 ratio did not reveal significant difference between seasons. Similarly, seminal characteristics did not exhibit any seasonal variation except for the percentage of live spermatozoa. However, the refusal response (no interest in mounting and ejaculating) was highest during summer months. T4 was significantly correlated to T(3) (r=0.53). Overall, T4 showed a positive correlation with seminal volume and initial motility while T3 exhibited a positive correlation with total sperm concentration and percentage of live spermatozoa. T3 was negatively correlated with refusal response only during the monsoon season. Correlation with other seminal and behavioural characteristics were not significant. The results indicated that although the sexual interest of buffalo bulls is reduced during the summer, the bulls can produce semen throughout the year under appropriate feeding and management conditions.     

Obstructive lesions of the genital tract in men

Obstructive lesions of the efferent and epididymal ducts in man were a common finding at necropsy. Although they demonstrated evidence of resorption of spermatozoa, they did not appear to be associated with the development of spermagglutinating antibodies in most cases. It is concluded that local spermiophagy within the genital tract in men is insufficient to lead to the development of antisperm autoallergy as detected by the tray agglutination test.     

Two-dimensional polyacrylamide gel electrophoresis of bovine seminal plasma proteins and their relation with semen freezability

The objective of this study was to evaluate the low weight (10-30 kDa) protein profile of bovine seminal plasma using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and to determine if any of these proteins was associated with semen freezability. Seminal plasma was collected from 16 bulls of high or low semen freezability. Twelve protein spots were identified from the 2D gel (15%); six of these were present in all samples. Of the 12 proteins found, three spots, present in all samples, 3 (15-16 kDa), 5 (16-17 kDa), and 7 (10-12 kDa) had nonsignificant variation among bulls, regardless of their freezability classification. Four proteins were more abundant (P<0.05) in seminal plasma samples collected from bulls with high semen freezability than in samples of bulls with low semen freezability: the spots 3 (15-16 kDa, pI 4.7-5.2), 7 (11-12 kDa, pI 4.8-4.9), 11 (13-14 kDa, pI 4.0-4.5), and 23 (20-22 kDa, pI 4.8-5.2). On the other hand, spot 25 (25-26 kDa, pI 6.0-6.5) was more abundant (P<0.05) on seminal plasma samples from bulls with low semen freezability. The N-terminus sequence of protein 7 was identical to the acidic seminal fluid protein (aSFP). Protein 23 (after trypsin digestion) had structural similarity to bovine clusterin. We concluded that there were differences in the seminal plasma protein profile from bulls with low and high semen freezability; aSFP, clusterin, proteins 3 and 11 may be used as semen freezability markers; and protein 25 was related to low semen freezability.     

Comparison of the BullMate sperm quality analyzer with conventional means of assessing the semen quality and breeding soundness of beef bulls

An instrument called the Optibreed, BullMate sperm quality analyzer (SQA) contains a densitometer for determining sperm cell concentration and an optical sensor to evaluate light deflections caused by sperm movement. Analysis of light deflections enables the generation of a value called the sperm quality index (SQI). The SQI represents the quality of a semen sample defined by sperm motility, concentration, viability and morphology. The SQA was compared to conventional, microscopic techniques for determining percent motile sperm and sperm concentration in bull semen samples and evaluated for its ability to classify bulls as satisfactory or unsatisfactory potential breeders. Semen samples were collected from 105 mature beef bulls by electroejaculation (day 1) and from 51 of the same bulls by internal artificial vagina (IAV) on day 2. SQI values were arranged into 20 categories in increments of 50units from 0 to 1000units. Percent motile sperm and sperm concentration values from both methods were significantly positively correlated (P<0.000) with respective r values of 0.82 and 0.80. A calculation of kappa to evaluate the differences in percent motile sperm generated by each system yielded a value of 0.20 and 0.54 for unweighted and weighted determinations, respectively. The intraclass correlation coefficient used to evaluate the reliability of sperm cell concentration determinations was 0.62 (P<0.05). SQI values generated on days 1 and 2 ranged from 0-994 to 0-906, respectively. Bulls were categorized as satisfactory or unsatisfactory potential breeders in all categories. The most appropriate SQI for determining whether a bull was a satisfactory or unsatisfactory potential breeder was 500 with respective sensitivity, specificity, positive predictive values (PPV) and negative predictive values (NPV) of 81, 65, 90.79 and 44.83%. In this experiment the BullMate SQA was not a reliable substitute for conventional semen analysis and was not useful for determining bull breeding soundness.     

Effect of hCG injection on prostaglandin E concentrations in ram seminal plasma

Ram and bull seminal plasma, respectively, contain 0.5-20 microg PGE/ml and 5-10 ng PGE/ml. To demonstrate that PGE concentrations in the seminal plasma are related to sperm quality and could be affected by hormonal stimulation in vivo, four rams were injected with 500 IU hCG, in and out of season. The rams responded 1 week after hCG with a 1.5- to 4-fold increase in seminal plasma PGE. The PGE peak was temporally separate from the hCG-induced rise in seminal plasma testosterone which was observed after 1 day. Using a simulated cryptochid ram, peaks in seminal fluid PGE were found to be associated with increased sperm velocity and sperm counts. In bulls, PGE concentrations in the seminal plasma of good bulls were significantly higher than that found in poor and cryptorchid bulls.     

Immunohistochemical localization of phospholipase A2 in the bovine seminal vesicle and on the surface of the ejaculated spermatozoa.

1. Approximately 150-fold purified phospholipase A2 (PLA2) from bovine seminal vesicle fluid was injected into rabbit to prepare antibodies. 2. Produced antisera blocked PLA2 activity in bovine seminal plasma, seminal vesicles and its fluid and it gave single precipitation lines with the same samples. No cross-reactivity was detected with other reproductive tissues of bull as well as human seminal plasma. 3. Using indirect peroxidase technique PLA2 was localized in the apical part of epithelia cells of the bull seminal vesicle and also some minor immunohistochemical reactions were observed in the tubular lumen. Indirect peroxidase staining gave weak or no reaction at all to seminal vesicles of immature bulls. This suggests that the enzyme may be under hormonal control. 4. By indirect immunofluorescence method ejaculated spermatozoa of bull revealed immunoreaction which was not uniform and it was restricted to the middle piece, acrosome as well as postacrosomal region, but no specific immunostaining could be found on the surface of the epididymal spermatozoa. 5. Enzyme visualization by immunoelectron microscopic labelling showed a predominant localization in membrane particles inside the lumen of bovine seminal vesicle but some gold particles were also seen in granules, larger vacuoles and in cytoplasm of epithelia cells.     

Effects of cold shock treatment on angiotensin-converting enzyme activity and on semen characteristics in roosters and bulls

Angiotensin-converting enzyme (ACE) activity has been determined in the semen of certain avian and mammalian species as well as its release during cold shock. The maximum and minimum levels of this enzyme were found in mammalian spermatozoa and in seminal plasma, respectively. It was found that ACE activity in mammalian spermatozoa was more pronounced than in the seminal plasma, whereas in the avian species a revers pattern was observed. However, there were no significant differences in ACE activity in spermatozoa and seminal plasma between layer and broiler strains of avian species. By contrast, ACE activity in the spermatozoa and seminal plasma of buffalo bulls was significantly higher (P/ 0.01) than in cattle bulls. Cold shock did not significantly alter semen characteristics in avian species, while a significant (P/ 0.01) decrease in sperm live counts and motility as well as a corresponding increase in morphological abnormalities were observed in the spermatozoa of cattle and buffalo bulls due to cold shock.     

Relationships among fertility, scrotal circumference, seminal quality, and libido in Santa Gertrudis bulls

Forty Santa Gertrudis bulls were used to examine relationships among scrotal circumference, seminal quality, libido, and fertility [assessed as the percent pregnant of estrous females (PE rate) and the percent pregnant of females mated (PM rate)]. These bulls were selected from 220 two year old bulls to represent variations in scrotal circumference and seminal quality. Each of the 40 bulls were exposed to 100 cyclic Santa Gertrudis heifers for a 4-day (96 hr) breeding period. The number of estrous females available to each bull varied from 12 to 27. A breeding soundness examination (BSE) was conducted on each bull approximately 45 days prior to the 4-day breeding period and immediately after the 4-day breeding period. The three components of the BSE scroe (scrotal circumference, spermatozoal abnormalities and spermatozoal motility) were not significantly correlated with PE rate or PM rate at either evaluation. There was no significant correlation between PM rate and scrotal circumference; however, each of the 4 bulls having a scrotal circumference less than 30 cm had a PM rate below 31%. Relationships between seminal quality and PM rate were unclear and differed between the two evaluations. There was a trend for bulls having poor seminal quality at the first evaluation to improve by the second evaluation. Consequently, fluctuations in seminal quality between evaluations is one possible explanation for low correlations between seminal parameters and PM rate. Libido (number mated/number in estrus x 100) was positively correlated (r = 0.44) with PE rate. Using a stepwise regression procedure, the independent variables accounting for the most variation in PE rate (dependent variable) included libido, secondary spermatozoal abnormalities, and BSE score (r(2) = 0.44). Results of this study indicate that current methods of fertility evaluation did not accurately predict the fertility of individual Santa Gertrudis bulls as measured by PE rate and PM rate during a 4 day breeding period.     

Scrotal circumference measurements in 764 beef bulls.

A total of 764 breeding soundness examinations was conducted on beef bulls utilizing the method of examination and criteria for classifying bulls of the Society for Theriogenology. In addition to this examination each bull was subjected to scrotal circumference measurement and to weighing. Classification of the bulls according to breeding soundness potentials was as follows: 88% of the bulls were satisfactory potential breeders, 8% of the bulls were questionable potential breeders and 4% of the bulls were unsatisfactory potential breeders. The proportions of bulls in each classification; satisfactory, questionable, or unsatisfactory; were not different among the four breeds evaluated. The majority of bulls evaluated in this study were between 14 and 36 months of age and weighed between 900 and 1500 pounds. For the ages and weights evaluated, scrotal circumference measurement variances were not closely related to age and weight differences. There was a tendency shown for “Questionable” and “Unsatisfactory Potential Breeders” to have smaller scrotal circumference measurements. The study indicates that Angus, Charolais, Horned Hereford and Polled Hereford bulls of breeding ages and weights should have scrotal circumference measurements of at least 32 centimeters in order to be classified as “Satisfactory Potential Breeders”.     

Age-related differences of semen quality,seminal plasma,and spermatozoa antioxidative and oxidative stress variables in bulls during cold and warm periods of the year

The aims of this study were to determine the presence and quantities of antioxidative status and oxidative stress (OS) variables in the seminal plasma and spermatozoa of bulls of varying age during cold and warm periods of the year, and to establish the correlation of these variables with semen quality parameters. The study was conducted on two groups each comprising nine Simmental bulls: one group contained younger animals (aged 2 to 4 years) and the second older animals (aged 5 to 10 years). Semen samples were collected using an artificial vagina for biochemical analysis. Seminal plasma and spermatozoa activities of total superoxide dismutase (TSOD), manganese superoxide dismutase (MnSOD), copper–zinc superoxide dismutase (CuZnSOD), catalase (CAT), selenium-dependent glutathione peroxidase, reduced glutathione and concentrations of total protein (TP), thiobarbituric acid reactive substances (TBARS) and protein carbonyl content (PCC) were determined. Several antioxidants in seminal plasma were also determined: total glutathione peroxidase (TGSH-Px), selenium-independent glutathione peroxidase (Non-SeGSH-Px), uric acid, albumins (ALB) and alkaline phosphatase (ALP). Significantly higher spermatozoa motility was observed during the cold v. warm period, and a significantly higher volume and total number of spermatozoa per ejaculate was observed in older than in younger bulls. Significantly higher values of ALP, TP and ALB were found in seminal plasma of older bulls than in younger bulls during the warm period. The seminal plasma of younger bulls showed significantly higher activities of TSOD, MnSOD, CuZnSOD, TGSH-Px and Non-SeGSH-Px. Younger bulls had significantly higher PCC concentration and activity of CAT in seminal plasma than older bulls during the cold period. Significantly higher concentrations of PCC and TBARS, and activities of TSOD, MnSOD and CuZnSOD were established in spermatozoa of the younger than in older bulls during the warm period. It could be concluded that antioxidative and OS variables differ significantly depending on bull age and time of year. Younger bulls were more sensitive to elevated ambient temperatures during the warm period, when the higher enzymatic antioxidative protection in seminal plasma and spermatozoa were insufficient to counteract the intensive oxidative processes in spermatozoa, which eventually resulted in decreased spermatozoa motility. The estimation of antioxidative and OS variables in seminal plasma and spermatozoa may have practical value for the assessment of bull semen quality.     

Relationships between seminal vesicle size and copulatory activity in bulls

Two experiments were designed to investigate the hypothesis that dimensions of the seminal vesicle are positively correlated with copulatory behavior in bulls. In Experiment 1, thirty Santa Gertrudis bulls (16.5 mo old) were used to study the relationship between copulatory activity and seminal vesicle length and width (palpation per rectum), seminal vesicle thickness (ultrasonography), and volume. The bulls showed a low level of copulatory activity (6.0+/-1.5, 0.8+/-0.3 and 0.3+/-0.1 for mean number of mounts, intromissions and ejaculations, respectively) in 2 tests. The mean number of mounts but not the mean number of intromissions or ejaculations differed (P<0.01) among bulls. No significant correlations were found between seminal vesicle size and any of the variables of sexual behavior. In Experiment 2, 16 beef bulls (18 mo old) were used to study the relationship between copulatory activity and seminal vesicle length (palpation) or dorso-ventral thickness (ultrasonography). Differences were detected (P<0.01) among bulls in mean number of mounts, intromissions and ejaculations achieved in 2 serving capacity tests. No significant correlations were found between copulatory activity and seminal vesicle length or thickness. These data do not support the hypothesis of a positive relationship between seminal vesicle length or thickness with copulatory activity, even for bulls with marked diffences in intensity of sexual behavior.     

Variation in lipid profiles within semen compartments—the bovine model of aging

Semen lipid composition was examined in young and mature bulls. Given the specific roles of various semen compartments (i.e., seminal fluid, sperm head, and sperm tail) during fertilization, we hypothesized that altered fatty acid and cholesterol composition of a specific compartment might impair semen quality and sperm function. Semen samples were collected from five mature and five young Holstein Friesian bulls during the winter (December–January). Semen was evaluated by computerized sperm-quality analyzer for bulls and was centrifuged to separate the sperm from the seminal fluid. The sperm fraction was sonicated to separate its head and tail compartments. Cold extraction of lipids was performed, and fatty acids and cholesterol were identified and quantified by gas chromatography. Semen physiological features (concentration, motility, and progressive motility) did not differ between mature and young bulls. However, lipid composition within fractions varied between groups, with prominent impairments in the head compartment. In particular, the proportions of polyunsaturated fatty acids, omega-3 fatty acids, and docosahexaenoic acid in the intact sperm; seminal fluid; and sperm head were lower in semen collected from mature bulls than in that from young bulls. The finding suggests an age-differential absorption and/or metabolism through spermatogenesis. Reduced proportions of major fatty acids in mature bulls might reduce membrane fluidity, which in turn might affect the ability to undergo cryopreservation and/or oocyte-sperm fusion through fertilization.