Seminal ribonuclease synthesis in bull reproductive organs during ontogenesis

Seminal ribonuclease (AS RNase) is synthesized in the epididymis, ampullary glands and seminal vesicles of sexually mature bulls. During sexual maturation of Czech red-spotted bulls it first begins to be synthesized in the seminal vesicles and ampullary glands, after the age of 20 weeks. At this age practically none is synthesized in the epididymis. As soon as the epithelial cells of the seminal vesicles start to synthesize the enzyme and secretion of the fluids of this organ begins, synthesis per ml fluid is almost the same as in sexually mature bulls. AS RNase synthesis in the cauda epididymidis begins after the age of 27 weeks and is individually variable. AS RNase synthesis in the reproductive organs depends on the testosterone concentration in the blood plasma of the bull.     

Qualitative and quantitative analyses of seminal ribonuclease in reproductive tract fluids of bulls

Bovine seminal ribonuclease (BS RNase) is synthetized in the ampulary gland and seminal vesicles of the bull as shown by indirect immunofluorescence and by using a sensitive sandwich-linked immunosorbent assay (ELISA). The ampullary and seminal vesicle BS RNase concentrations in samples from 15 bulls were 656 μg/ml and 1285 μg/ml, respectively. Seminal plasma BS RNase levels in 22 breeding bulls were slightly lower than in the seminal vesicles—1132 μg/ml. There were no significant differences in the concentration of this enzyme in seminal plasma of 16 bulls ranked as having high and low fertility. Even with its immunosuppressive activity this enzyme does not seem important for the protection of sperm cells in the female reproductive tract.     

The effect of bull seminal ribonuclease on reproductive organs and sexual behaviour in male rats

Wistar male rats received an intratesticular injection (at 114 and 265 days of age) of 3 mg of partially purified bull seminal ribonuclease (AS RNase) or saline. It was found that sexual behaviour (initiation of copulation as well as copulatory behavioural pattern) of experimental males was not changed, but the ability of these males to fertilize females was evidently suppressed. In addition to significantly lower weights of testes and epididymis, inhibition of seminiferous epithelium development (aspermatogenesis) associated with the absence of spermatozoa was determined in cauda epididymidis in experimental animals. However, Leydig cells remained without changes. Plasma testosterone levels of AS RNase treated males were not altered in comparison with the controls. Thus AS RNase specifically impaired spermatogenesis but did not influence androgen action and sexual behaviour.     

Seminal selenium concentrations and spermatozoal abnormalities in beef bulls

The relationship between seminal selenium (Se) concentration and spermatozoal abnormalities in 24 Angus and 12 Simmental bulls maintained on a Se adequate diet was studied. Two semen samples were collected by electroejaculation 50 days apart from each bull. Measurements of primary and secondary spermatozoal abnormalities, seminal Se concentration, and blood plasma Se concentration were determined at each semen collection. The mean (chi +/- SD ) Se concentration of semen (0.535 +/- 0.267) was approximately 8 fold greater than the Se concentration of blood plasma (0.069 +/- 0.066) and the values were similar for both collections. Spermatozoa concentration was correlated (r = 0.50; P<.01) with seminal Se concentration; however, seminal Se concentration was not highly correlated (P<.01) with primary spermatozoal abnormalities (r = -0.29) and secondary spermatozoal abnormalities (r = 0.16). This study indicates that the Se concentration of semen is high relative to blood plasma in bulls maintained on a Se adequate diet; however, the seminal Se concentration is not highly correlated with spermatozoal abnormalities.     

Effect of age and environmental factors on semen quality, glutathione peroxidase activity and oxidative parameters in simmental bulls

Taking into account that semen quality depends on animal age and climate conditions and that oxidative stress has been reported to be a common cause of infertility, the objective of this study was to monitor indicators of oxidative stress and antioxidant protection during four seasonal periods in service bulls of various age to get better insight into the significance of these factors upon evaluating service bull semen. The research was conducted over a year on 19 Simmental service bulls. Animals were divided into two groups according to age; Group I consisted of younger bulls aged two to four yrs (n = 9), and Group II was comprised of older bulls aged five to ten yrs (n = 10). Semen samples were obtained once in the middle of every seasonal period and blood samples for biochemical analysis were collected by jugular venipuncture immediately after ejaculate collection. The activity of total glutathione peroxidase (T-GSH-Px), selenium-dependent glutathione peroxidase (Se-GSH-Px) and selenium-independent glutathione peroxidase (non-Se-GSH-Px), together with the intensity of lipid peroxidation (thiobarbituric acid reactive substances; TBARS) and oxidative protein damage (protein carbonyl content (PCC)) were measured in seminal plasma. In samples of spermatozoa and blood serum, the activity of Se-GSH-Px and TBARS and PCC concentrations were determined. Older service bulls had significantly higher ejaculate volume in summer in comparison with younger bulls, whereas the number of spermatozoa and progressive motility percentage did not significantly vary with age. Younger animals had lower progressive motility percentage during summer than in spring, with more intensive oxidative processes observed in seminal plasma (TBARS) and spermatozoa (TBARS and PCC). Based on the results presented here, it can be concluded that younger bulls are more sensitive to elevated ambient temperatures during the summer, when intensified prooxidative processes in semen plasma and spermatozoa eventually led to decreased sperm progressive motility with consequential semen quality deterioration.     

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.     

Active immunization of prepubertal bulls against testosterone: Seminal and testicular characteristics after puberty

Twenty-four Holstein bull calves were immunized at 1.0 month of age against either testosterone-17-hemisuccinate-human serum albumin (treated bulls) or against human serum albumin alone (control bulls). Booster injections were given monthly through five months of age. Bulls were reimmunized at six months of age with testosterone-17-hemisuccinate-equine serum albumin (treated bulls) or equine serum albumin alone (control bulls). At 12 months of age, eight treated and eight control bulls were electroejaculated twice daily for two days and then castrated. The remaining four bulls in each group were electroejaculated and castrated at 18 months of age. Active immunization against testosterone significantly elevated the binding of (3)H-testosterone in plasma within four weeks. Body weights of bulls were not affected by treatment. Concentrations of luteinizing hormone (LH) and follicle stimulating hormone (FSH) in plasma were generally not altered by treatment. At castration at 12 months of age, testosterone-immunized bulls tended to have greater (P < 0.15) parenchymal weights and had 30% greater (P < 0.07) daily sperm production (DSP) rates than control bulls; seminal characteristics (motility and intact acrosomes) were not affected. At 18 months of age, testosterone-immunized bulls had 21% greater (P < 0.07) parenchymal weights and 35% greater (P < 0.04) DSP rates than control bulls; again, seminal characteristics were not affected. It appears that prepubertal active immunization against testosterone is a potential means of increasing testicular size and sperm production rates in postpubertal bulls.     

Beta-galactosidase in the seminal plasma and reproductive organs of the bull

The distribution of beta-galactosidase activity was studied in different reproductive organs, seminal plasma and spermatozoa of the bull. The highest specific activity of beta-galactosidase was found in testis and in different parts of the epididymis, where the activity seemed to be partly in secretory (cauda secretion) and partly in non-secretory, bound form (caput to cauda epididymidis). Gel filtration on Sepharose 6B at pH 7.0 revealed two beta-galactosidase forms (GF-1, Mr approximately 500,000-600,000 and GF-2, Mr approximately 190,000-220,000) in reproductive organs and seminal plasma. The pH-optimum of both beta-galactosidase forms was about 3.75-4.75. Hg2+ and p-chloromercuribenzoate inhibited strongly these activities. Further, form GF-2 seemed to be slightly more sensitive to the thermal inactivation at 50-70 degrees C than form GF-1. In chromatofocusing beta-galactosidase activities in bull seminal plasma coeluted with those of the cauda epididymidis (pI-values 7.5-6.4). On the contrary, prostate, Cowper's gland, testis, ampulla and seminal vesicles had enzyme activities eluting at lower pI-values (6.3-4.2). Thus, the seminal plasma activity is mainly an indicator for the function of the epididymal cauda.     

Circadian variations of plasma LH and testosterone in adult swamp buffalo bulls

Three swamp buffalo bulls aged 1.5, 1.10 and 2 years were submitted to frequent blood sampling every 15 m during a period of 25 h using an indwelling infusion set. Plasma LH and testosterone were quantified by radioimmunoassay technique. The levels of the two hormones in each individual exhibited episodic and nonrhythmic patterns. The number of LH peaks varied according to individval, ranging from no peak in one bull to 2 in the other two bulls. The mean LH concentrations during the period of study for each bull were 0.74, 0.33 and 1.17 ng/ml. Whereas the number of testosterone peaks varied between 1-10 and the average testosterone concentrations were 0.1, 0.33 and 0.55 ng/ml for the younger to the older bulls respectively. The testosterone peaks related to the LH peaks in each individual bull.     

Effects of pretreatment with adrenocorticotropin on endocrine and behavioral responses of bulls to sexual activity

Peripheral concentrations of cortisol, growth hormone and testosterone were determined in two experiments which examined the endocrine and behavioral responses of sexually mature Angus bulls to an estrous female (Experiment 1) and to female exposure 5 hours following an adrenocorticotropin (ACTH) injection (Experiment 2). Sexual activity of bulls in Experiment 1 significantly increased levels of cortisol when compared with concentrations before exposure to a female. Administration of ACTH in Experiment 2 consistently elevated levels of cortisol by 30-fold (P<0.01) when compared with pre-ACTH concentrations. This heightened level of cortisol persisted throughout the period of exposure to an estrous cow, although a gradual decline in cortisol concentrations occurred over time (P<0.05). In Experiment 1, growth hormone profiles tended to increase in response to sexual activity (P<0.10), whereas in Experiment 2, growth hormone increased in response to ACTH administration (P<0.01) and to female exposure (P<0.01). Concentrations of testosterone were unaffected (P>0.10) by mating activity in Experiment 1. In Experiment 2, acute suppression (P<0.01) in testosterone concentrations 5 hours after ACTH administration coincided with the exposure period to the estrous female. Frequencies of mounting behavious (penis extension, mounting, intromission and ejaculation) exhibited by ACTH-treated bulls were significantly lower compared with the frequencies two days earlier. Exogenous ACTH administration suppressed reproductive behaviors of bulls and altered secretion of cortisol, growth hormone and testosterone. Furthermore, these data provide evidence that specific mating behaviors of the bull can be influenced by circulating steroids.     

Male accessory sex glands produce heparin-binding proteins that bind to cauda epididymal spermatozoa and are testosterone dependent

Heparin binds to bovine sperm and stimulates capacitation in vitro. Seminal plasma alters the ability of epididymal sperm to bind heparin, and several heparin-binding proteins (HBPs) have been identified in bull seminal plasma. This study had three objectives: 1) to identify production sites of seminal plasma HBPs, 2) to determine which HBPs bound to cauda epididymal sperm, and 3) to determine whether presence of HBPs was testosterone dependent. Proteins from bull or rat seminal vesicles, prostates, and bulbourethral glands were separated by heparin affinity high-performance liquid chromatography. HBPs were found in all accessory glands of rats and bulls, but the major source of bovine seminal plasma HBPs appeared to be seminal vesicles. Between 25% and 50% of the protein from each gland bound to the heparin column, and NaCl concentrations required to elute proteins ranged from 0.15 to 1.4 M. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that major HBPs were relatively small, with molecular weights between 13 and 31 kDa, but some HBPs also exhibited higher molecular weights, between 40 and 100 kDa. Radioiodinated HBPs from each bovine gland were incubated with epididymal sperm. Labeled HBPs binding to sperm exhibited molecular weights of 14, 16, 24, and 30 kDa as determined by SDS-PAGE and autoradiography. The HBP content of the accessory sex glands decreased significantly in castrated rats and was restored to levels of sham-operated controls by testosterone replacement. Heparin-binding proteins may play a role in fertilization by attaching to sperm surfaces, enabling heparin-like glycosaminoglycans in the female reproductive tract to induce capacitation.     

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.     

Selenium and glutathione peroxidase in seminal plasma of men and bulls

High levels of selenium and glutathione peroxidase (GSH-Px) were found in bull seminal plasma but low concentrations in human seminal plasma. In man the seminal plasma selenium was associated with two macromolecules separable by gel filtration, but no GSH-Px was found in the same fractions. Selenium in bull seminal plasma was associated with two proteins, which could be separated by gel filtration and anion exchange chromatography. Both macromolecules coeluted with GSH-Px activity and had identical optima at pH 7.0. Their responses to thermal treatment, however, differed. Seminal vesicle secretory fluid in the bull contained both these proteins, while the larger molecule was also found in fractionations of ampulla, prostate and Cowper's glands. The larger enzyme form is evidently a tetramer of the smaller one. Both enzyme forms were extremely sensitive to heavy metals and some divalent metal ions. GSH caused an activation while other reducing agents were suppressive. Triton X-100 had no effect, while sodium deoxycholate was inhibitory. These properties are typical for a phospholipid hydroperoxide GSH-Px. It is concluded that this selenium-dependent enzyme may be important in the protection of bovine spermatozoa against damage caused by oxygen radicals, while in man such a mechanism is not functional.     

Electroejaculation increased vocalization and plasma concentrations of cortisol and progesterone, but not substance P, in beef bulls

Electroejaculation is a reliable method of obtaining a semen sample for a bull breeding soundness examination, but is sometimes regarded as painful. Substance P is a neuropeptide involved in the integration of pain, stress, and anxiety. We hypothesized that substance P is a measure of pain in bulls following electroejaculation. The specific objective was to compare vocalization and plasma concentrations of cortisol, progesterone, and substance P immunoreactivity in bulls following electroejaculation. Nine Angus bulls (501.9 ± 14.3 kg) were used. Blood samples were collected at −60, −30, 0, 2, 10, 20, 30, 45, 60, 75, 90, 120 min relative to treatment. At Time 0, bulls were subject to electroejaculation, rectal probe insertion without electroejaculation, or no manipulation. Treatments were administered contemporaneously to three bulls. Treatments were repeated weekly until each bull had received each treatment in a 3 × 3 Latin square design. More bulls (P = 0.0147) in the electroejaculation group vocalized (5 of 9 bulls; 55.6%) when compared to controls (0 of 9 bulls; 0%). Mean plasma cortisol and progesterone concentration following electroejaculation in bulls were higher (P < 0.05) than concentrations in probed and control bulls through the 45 min sample. However, mean plasma substance P concentration following electroejaculation in bulls (77.2 ± 17.2 pg/mL) was not different (P = 0.6264) from probed (79.1 ± 17.2 pg/mL) or control bulls (93.4 ± 17.2 pg/mL). A significant increase in vocalization and plasma cortisol and progesterone concentrations in bulls following electroejaculation was likely owing to acute stress. However, the lack of a difference in plasma concentrations of substance P after electroejaculation was interpreted as a lack of pain associated with nociception.     

beta-N-acetylglucosaminidase in the reproductive organs and seminal plasma of the bull

The highest specific activity of beta-N-acetylglucosaminidase (beta-NAG) was found in the different parts of the epididymis, where the activity seemed to be partly in secretory and partly in non-secretory, tissue-bound form. Epididymal spermatozoa also contained moderate beta-NAG activity. The beta-NAG was separated by chromatofocussing and anion exchange chromatography with HPLC into multiple forms with distinct pI values (8.0-4.0). The cauda epididymidis, ampulla and the seminal vesicles formed the major secretory sources of the high beta-NAG activity in bull seminal plasma. The major secretory forms of beta-NAG in caput and cauda epididymidis showed distinct elution profiles. In the fractionation with gel filtration on Sepharose 6B, the beta-NAG activities derived from bull testis and caput epididymidis had smaller molecular weights than did the secretory enzymes in seminal plasma, seminal vesicle secretion and cauda epididymidis. Maximum activity of all beta-NAG isoenzymes was observed at pH 5.0. They were almost totally inactivated at 60 degrees C and about 75-80% of the activity was lost at 55 degrees C. All the isoenzymes were strongly inhibited by thiol reagents but not with other metal ions and chelating agents. Histochemical studies showed a strong granular (lysosomal) reaction for beta-NAG in basal cells and basal parts of the principal cells in all but the initial segment of the epididymis. An apical (secretory) reaction was prominent in the distal caput and corpus as well as in distal cauda. After the distal caput the luminal sperm mass became increasingly mixed with a beta-NAG-positive material. The epithelial cells of the ampulla and seminal vesicle displayed a moderate apical (secretory) reaction.     

Occasional detection of Neospora caninum DNA in frozen extended semen from naturally infected bulls

Recently, the presence of Neospora caninum DNA in semen from naturally infected bulls was reported. In the present work, the presence and quantification of N. caninum by PCR techniques in frozen extended semen straws from naturally infected bulls was investigated. A total of 20 seropositive and five seronegative bulls raised for reproductive purposes in an AI centre were used. Ten extended semen straws from each bull obtained at different time-points during the previous 2 years were selected for Neospora testing. Eight of the seropositive bulls (40%) studied showed at least one positive straw to N. caninum DNA and 14 of their 180 semen straws examined (7.8%) were found to be positive. In all positive samples, N. caninum DNA was consistently detected in the cell fraction and not in the seminal plasma. However, the parasite number in each positive straw was under the detection level of real-time PCR. In parallel, 10 semen straws from each of the five seronegative bulls were also analyzed by the nested-PCR and no N. caninum DNA products were obtained. In order to check the consistent presence of N. caninum in a positive semen batch, three additional semen straws from the same batch of each positive straw from three seropositive bulls were analyzed but N. caninum DNA was only detected in one straw from one bull. In conclusion, we report the sporadic detection of N. caninum DNA in semen straws of naturally infected bulls but the low frequency of contaminated semen straws and the low parasite load observed indicate a minor chance of bovine neosporosis transmission by AI.     

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.     

Hormonal control of serving capacity in bulls

The hormonal control of serving capacity in bulls was investigated in four sets of identical twin bulls averaging 9.9 ± 1.8 (SD) (H), 4.1 ± 1.0 (M1), 4.3 ± 1.0 (M2) and 1.9 ± 1.0 (L) services in four standardised tests. Within each pair, twin brothers had similar serving capacity. One bull from each twin set was castrated while its brother remained entire. When the castrates were maintaining their nadir in serving capacity (4, 0, 1 and 0) services for H, M1, M2 and L respectively) each was given testosterone propionate (TP) (1 mg/kg body weight) at 4 to 5 day intervals for 3 weeks, and along with its entire twin brother, tested for serving capacity 1 day after TP administration. Serving capacity of each castrate returned to its high, medium or low precastrational level and to the contemporary level of its entire twin brother. The plasma testosterone levels of the H, M1 + M2 and L entire bulls were similar (16.0 ± 3.0, 16.0 ± 3.0 and 22.4 ± 3.0 ng/ml respectively) despite large differences in serving capacity. It was concluded that (i) the presence of testosterone above a threshold level (<7 ng/ml in the assay used) is necessary for the maintenance of serving capacity and (ii) differences in serving capacity between bulls are not due to differences in plasma testosterone levels but to differences in their somatic responsiveness to threshold levels of testosterone.     

Characterization of seminal plasma proteins and sperm proteins in ejaculates from normospermic bulls and bulls with thermally-induced testicular degeneration

Semen was collected from 5 mature beef bulls by electroejaculation before, during, and after 20 days of scrotal insulation. Thermally-induced testicular degeneration was irreversible in 3 of the bulls. Analysis of sperm and seminal plasma polypeptides revealed that 15 to 30 sperm polypeptides and 25 to 30 seminal plasma polypeptides were indistinguishable between bulls prior to the insulation treatment. Changes in the sperm polypeptides pattern appeared as early as 2 days after the insulation treatment and persisted for at least 11 months in 2 of the bulls. In the spermatozoa, there was a detectable loss of 31, 34, 49 and 58 kDa polypeptides and an appearance of 6 to 8 new major polypeptides, ranging from 32 to 83 kDa. The 83 kDa polypeptide was most prominent in the 2 bulls that regained normal sperm motility and morphology following the insulation period. The post-insulation appearance of a seminal plasma polypeptide (circa 60 kDa) was also identified in these 2 bulls. Seminal plasma polypeptides remained qualitatively unaltered by the insulation treatment in the 3 bulls with irreversible testicular degeneration.