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.     

Genital papillomatosis in sperm whale bulls

Examination of 31 male sperm whales (Physeter catodon) caught off the western coast of Iceland revealed three cases of genital papillomatosis involving the unsheathed penis. One subadult and two sexually mature bulls were affected. Gross lesions resembled papillomas common in terrestrial mammalian species. Transmission electron microscopy of these lesions revealed nonenveloped intranuclear virus particles 28-40 nm in diameter and round to hexagonal in shape. In two cases immunoperoxidase staining was negative for group-specific papillomavirus antigen. These findings indicate that the spectrum of animal species affected with virus-associated genital papillomatosis includes at least one globally distributed species of the order Cetacea (whales, dolpins, and porpoises).     

Morphological abnormalities in zebrafish cryopreserved sperm

The aim of the present study was to evaluate the effect of cryopreservation on the morphology of zebrafish sperm (Danio rerio). Sperm from 30 males were collected and divided in two treatments: fresh and cryopreserved semen. The following were measured sperm morphology, motility and membrane integrity. Cryopreservation reduced motility, the number of normal cells and the membrane integrity, as well as increased the percentage of sperm abnormalities. The most frequent types of morphological changes found in cryopreserved semen were macrocephaly, loose head, degenerated head, proximal gout, curled tail and short tail. This study opens the way for further investigations on morphological changes and for a new classification of these changes in fish semen due to cryopreservation.     

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.     

The integrity of sperm chromatin in young tropical composite bulls

Sperm chromatin fragmentation is associated with subfertility, but its relationship with age progression in young bulls is poorly understood. The objective was to assess sperm chromatin fragmentation during the early post-pubertal development of 20 tropical composite bulls, using a sperm chromatin structure assay (SCSA) and sperm-bos-halomax (SBH). Bulls were subjected to bull breeding soundness evaluation (BBSE) at mean ages of 13, 18, and 24 mo. Traits measured included liveweight (WT), body condition score (BCS) and scrotal circumference (SC). Semen samples were collected by electroejaculation and assessed for mass activity (MA), motility (Mot), concentration (conc), sperm morphology and chromatin fragmentation. Concentration (r = 0.34, P = 0.0076), Mot (r = 0.36, P = 0.0041) and percentage of morphologic normal sperm (percent normal sperm (PNS); r = 0.31, P = 0.0132) were positively correlated with age. The percentage of sperm with proximal droplets (PD) was negatively correlated with age (r = −0.28, P = 0.0348), whereas neither SCSA nor SBH results were significantly correlated with age. The percentage of sperm with chromatin fragmentation using SCSA was correlated with PNS (r = −0.53, P < 0.0001), the percentage of sperm with head abnormalities (r = 0.68, P < 0.0001) and the percentage of intact sperm (Int) with SBH (r = −0.26, P = 0.0456). In summary, for assessment of sperm chromatin fragmentation, samples could be equally collected at 13, 18 or 24 mo of age, as results did not vary with age.     

Chromosome abnormalities in sperm of individuals with constitutional sex chromosomal abnormalities

The most common type of karyotype abnormality detected in infertile subjects is represented by Klinefelter's syndrome, and the most frequent non-chromosomal alteration is represented by Y chromosome long arm microdeletions. Here we report our experience and a review of the literature on sperm sex chromosome aneuploidies in these two conditions. Non mosaic 47,XXY Klinefelter patients (12 subjects) show a significantly lower percentage of normal Y-bearing sperm and slightly higher percentage of normal X-bearing sperm. Consistent with the hypothesis that 47,XXY germ cells may undergo and complete meiosis, aneuploidy rate for XX- and XY-disomies is also increased with respect to controls, whereas the percentage of YY-disomies is normal. Aneuploidy rates in men with mosaic 47,XXY/46,XY (11 subjects) are lower than those observed in men with non-mosaic Klinefelter's syndrome, and only the frequency of XY-disomic sperm is significantly higher with respect to controls. Although the great majority of children born by intracytoplasmic sperm injection from Klinefelter subjects are chromosomally normal, the risk of producing offspring with chromosome aneuploidies is significant. Men with Y chromosome microdeletions (14 subjects) showed a reduction of normal Y-bearing sperm, and an increase in nullisomic and XY-disomic sperm, suggesting an instability of the deleted Y chromosome causing its loss in germ cells, and meiotic alterations leading to XY non-disjunction. Intracytoplasmic injection of sperm from Y-deleted men will therefore transmit the deletion to male children, and therefore the spermatogenic impairment, but raises also concerns of generating 45,X and 47,XXY embryos.     

Fertility of cryopreserved sperm in three bulls with different Robertsonian translocations

The fertility of three bulls carrying different Robertsonian translocations (rob(1;29), rob(14;17) and rob(26;29)) was evaluated. Oocytes-cumulus complexes obtained from slaughterhouse-derived ovaries were matured and then fertilised in vitro with frozen/thawed seminal material from the above mentioned subjects, and from control bulls with normal karyotype. An assessment was first made of the concentration, vitality and acrosome integrity of the seminal material to be sure that possible differences in the results of the in vitro fertilisation experiments were not due to seminal material quality. The results of the experiments, evaluated by the percentage of cleaved embryos and blastocysts per cleaved embryo, indicated that the three bulls carrying Robertsonian translocations had similar fertilising power and semen qualitative parameters to the controls. These data suggest that neither gametogenesys impairment nor decreased spermatozoa fertilising capacity is responsible for the reduced fertility in bulls with Robertsonian translocations. What the data do confirm is that the observed in vivo hypofertility for karyologically abnormal bulls is mainly due to early embryonic mortality.     

The relationship between sperm chromosomal abnormalities and sperm morphology in humans

It has been suggested that an assay for sperm morphology might prove useful as an initial screen in evaluating men at risk for an increased frequency of sperm chromosomal abnormalities. In this study, the technique for analysis of human sperm chromosomes after penetration of hamster eggs was employed to determine whether there is an association between the frequency of chromosomally and morphologically abnormal sperm. 30 healthy men of proven fertility were studied. The ages of the donors ranged from 22 to 55 years. The analysis was performed "blindly" so that the technician analysing the chromosome spreads had no knowledge of the age of the donors or of the individual frequencies of morphologically abnormal sperm. There was no significant relationship between the proportion of morphologically abnormal sperm and the proportion of chromosomally abnormal sperm when controlled for age. This was true for the total frequency of chromosomal abnormalities and also for numerical and structural chromosomal abnormalities. These results suggest that an assay of morphology is not a good indication of chromosomal normality in human sperm.     

A sperm mid-piece defect of epididymal origin in two Hereford bulls

Two unrelated Hereford bulls with “dag” sperm-cell defects were subjected to spermatological studies. The defect which involves coiling, folding and splitting of sperm mid-pieces, was found to have an epididymal origin.     

Ribavirin-induced sperm shape abnormalities in Wistar rat

Ribavirin (1-β-d-ribofuranosyl-1,2,4, triazole-3 carboxamide) is a broad-spectrum antiviral drug. This study was aimed to investigate the mutagenicity of ribavirin on germ cells by employing sperm morphology assay. Male Wistar rats were treated with water, cyclophosphamide (CP) 40 mg/kg, and ribavirin 20, 100 and 200 mg/kg (i.p.) for 5 consecutive days at intervals of 24 h. Following the last exposure, at 14, 28, 35, 42 and 70 days, the epididymal sperm smears were obtained and stained according to the standard procedure. One thousand sperms per animal were classified into normal and different abnormal types. Both CP and ribavirin-induced anomalies of head and tail of sperm except at 70 days. In CP groups, maximum incidence was observed at 28, 35 and 42 days. Ribavirin 20 mg/kg induced maximum incidence at 14 and 42 days, 100 mg/kg at 28 and 42 days and 200 mg/kg at 28-42 days. These results show that ribavirin is mutagenic to rat germ cells in a transient fashion.     

Diet and breed influence the sperm membranes of beef bulls

Semen was collected from 12 Hereford and 10 Simmental bulls at the conclusion of a 119-day Record of Performance growth trial. Within each breed, the bulls were fed a standard test ration (Diet 1) or an experimental diet consisting entirely of a pelleted concentrate with ground corn cobs as the primary fibre source (Diet 2). Semen was analyzed for motility and morphology while testicular tissue obtained at slaughter the day after semen collection was assessed for seminiferous tubule integrity; none of these parameters varied significantly with breed or diet. The fluidity of head plasma membranes from the spermatozoa was assessed with fluorescence polarization using tPNA. Fluidity decreased over the 160 minute observation period, indicating molecular rearrangments within the head membranes which may reflect sperm changes preceding fertilization. The fluidization displayed a breed-by-diet interaction since membrane fluidity differed significantly between breeds on Diet 1 and between diets for Simmental bulls. Fluidities of some samples were also analyzed with cPNA, and these differed significantly from those obtained with tPNA, indicating the presence of domains in sperm head membranes. Neither diet nor breed affected traditionally measured semen characteristics of Hereford and Simmental bulls, but the membrane dynamics differed between the 2 breeds, and diet affected the sperm membrane dynamics of Simmental bulls.     

Assessment of in vitro sperm characteristics in relation to fertility in dairy bulls

The performance of frozen-thawed spermatozoa from 10 Holstein bulls in a range of in vitro diagnostic tests and the relationship with adjusted in vivo fertility data was determined. The tests included an assessment of motility (subjective and computer-assisted), morphology, concentration, viability, acrosomal and chromatin integrity conducted immediately post-thaw and after swim-up, in conjunction with membrane status (CTC staining) and migration in an artificial cervical mucus. Adjusted in vivo fertility correlated with subjectively assessed post-thaw motility (r=0.672, p=0.033), post-thaw straight-line velocity (r=0.636, p=0.048), post-thaw sperm morphology (r=-0.762, p=0.010), post-thaw sperm viability (r=0.635, p=0.048), the concentration of spermatozoa after swim-up (r=0.649, p=0.042), sperm morphology after swim-up (r=-0.687, p=0.028), the number of spermatozoa migrating 10mm into artificial cervical mucus (r=0.632, p=0.050) and the distance migrated by the vanguard spermatozoon in artificial mucus (r=0.701, p=0.024). A stepwise regression analysis identified tests which, when combined, produced models with a strong correlation (R(2)>0.9) to fertility.     

Immunoneutralization of inhibin modifies hormone secretion and sperm production in bulls.

The objective of this study was to examine the role of endogenous inhibin in the regulation of FSH, LH, and testosterone secretion and sperm production in bulls. Bulls were actively immunized against bovine inhibin alpha 1-26 gly-tyr (bINH) conjugated to human alpha globulin (HAG) or HAG alone (controls) and emulsified in Freund's complete adjuvant. Primary immunization was at 14 wk of age, followed by booster immunizations in Freund's incomplete adjuvant at 28, 30, and 34 wk of age. Ten days after each booster immunization, scrotal circumferences and body weights were measured, and blood was sampled for determination of bINH antibody titer. Ten days after the third booster, blood was sampled at 1-h intervals for 8 h to quantify serum concentrations of FSH, LH, and testosterone. After this blood sampling period, bulls were castrated and testicular sperm production was determined. Serum diluted 1:4,000 from bINH-immunized bulls bound 36%, 52%, and 53% of radioiodinated bINH after the first, second, and third boosters, respectively. Serum from controls bound less than 1% radioiodinated bINH. After the third booster, serum concentrations of FSH and testosterone were increased (p less than 0.05) and LH concentrations were decreased (p less than 0.001) in bINH-immunized bulls compared with controls. After the third booster, daily sperm production per gram of testicular parenchyma was increased (p less than 0.05) in bINH-immunized bulls compared with controls. Scrotal circumferences and body weights were similar between treatment groups throughout the experiment. We concluded that inhibin has a role in regulation of secretion of gonadotrophins and testosterone and testicular sperm production, but not testicular growth, in bulls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Elevated testicular temperature modulates expression patterns of sperm proteins in Holstein bulls

The objective of this study was to determine the effects of elevated testicular temperature on the expression patterns of sperm proteins in bulls. Ejaculates were collected from sexually mature Holstein bulls (n = 6) twice weekly for 10 weeks. Testicular temperature was elevated in all bulls by scrotal insulation for 72 consecutive hours during Week 2. Triton X-100 extracts of sperm proteins were prepared and subjected to SDS-PAGE. Sperm proteins in the 110 kDa range decreased from pre-thermal insult to Day 28 (start of thermal insult = Day 0), concurrent with decreases in sperm concentration, motility, and morphology, and subsequently increased, approaching pre-thermal insult values by Day 40. Based on mass spectrometry, this band was comprised of angiotensin converting enzyme (ACE), Hexokinase-1, and the alpha-4 subunit of Na(+)/K(+)ATPase. Changes in the expression patterns of these proteins were confirmed by immunoblotting, including the use of a custom antibody against the alpha-4 subunit of Na(+)/K(+)ATPase (testis-specific isoform). Furthermore, a 25 kDa sperm protein (identified as tissue inhibitor of metalloproteinase-2; TIMP-2), had a low expression level in pre-thermal insult samples, increased to Day 28 of post-thermal insult, and subsequently decreased to the pre-thermal insult level by the end of the experimental period. In conclusion, we identified proteins that may serve as molecular markers of impaired sperm function due to elevated testicular temperature, with important implications for fertility predictions. To our knowledge, this is the first report of the identification of a testis-specific isoform of Na(+)/K(+)ATPase in bovine sperm.     

Effect of long-term immunization against inhibin on sperm output in bulls.

To determine the effect of neutralization of inhibin on sperm output, 12 Holstein bulls were paired by birth date and weight on Day 1 of age. Each bull was actively immunized against bovine inhibin alpha1-26 gly-tyr (bINH) conjugated to human alpha globulin (HAG, n = 6 bulls) or HAG alone (controls, n = 6) at 60 days of age; booster immunizations were administered at 90, 104, 124, 270, and 395 days of age. Body weights and scrotal circumferences were measured at the time of primary immunization and at 10 days after each booster. In addition, jugular blood was obtained at 60, 70, 100, 114, 134, 280, and 405 days of age, during the 3-wk sperm collection period, and during a 6-h blood-sampling period after sperm collection to determine bINH antibody titer and concentrations of FSH, LH, testosterone, and estradiol. Beginning at 405 days of age, sperm output was measured 3 days/wk for 3 wk with two successive ejaculates collected each day for a total of 18 ejaculates per bull. During Days 60-405 of age, the increase in titer of bINH antibodies, scrotal circumference, and serum concentration of FSH was greater (p < 0.01) for the bINH-immunized compared with control bulls. There were significant (p < 0.01) pair x treatment interactions for sperm output and serum FSH and LH concentrations. Specifically, bINH-immunized bulls for four of the six pairs had nearly 50% greater serum FSH concentrations and sperm output. For the remaining two pairs, sperm output was lower and FSH was either lower or only marginally higher in the bINH-immunized bulls compared with controls. Also, the control bulls for the two remaining pairs produced more sperm than all but one bINH-immunized bull, and had markedly higher serum LH concentrations than all other bulls. To summarize, enhancement of sperm output after immunization against inhibin depends on the subsequent increment in FSH concentrations. We conclude that inhibin suppresses spermatogenesis. Thus, methods to immunoneutralize inhibin may have merit as a therapeutic route to enhance sperm production in reproductively maturing bulls.     

Methyl parathion-induced sperm shape abnormalities in mouse.

Metacid 50, the commercial grade of methyl parathion (O,O-dimethyl-O-4-nitrophenyl phosphorothionate), a commonly used organophosphorus insecticide, was tested for its genotoxicity in Swiss albino mice using the sperm abnormality assay. Sperms of albino mice were examined at two time intervals, 1 week and 5 weeks after a single acute oral treatment with the pesticide at four dose levels, viz., 75.0, 37.5, 18.75 and 9.375 mg/kg body weight corresponding to 1/2 LD50, 1/4 LD50, 1/8 LD50 and 1/16 LD50 values respectively. A dose-related statistically significant increase in the percentage of abnormal sperm observed indicates the genotoxic potency of methyl parathion.     

Ribavirin-induced sperm shape abnormalities in Wistar rat.

Ribavirin (1-beta-D-ribofuranosyl-1,2,4, triazole-3 carboxamide) is a broad-spectrum antiviral drug. This study was aimed to investigate the mutagenicity of ribavirin on germ cells by employing sperm morphology assay. Male Wistar rats were treated with water, cyclophosphamide (CP) 40 mg/kg, and ribavirin 20, 100 and 200 mg/kg (i.p.) for 5 consecutive days at intervals of 24h. Following the last exposure, at 14, 28, 35, 42 and 70 days, the epididymal sperm smears were obtained and stained according to the standard procedure. One thousand sperms per animal were classified into normal and different abnormal types. Both CP and ribavirin-induced anomalies of head and tail of sperm except at 70 days. In CP groups, maximum incidence was observed at 28, 35 and 42 days. Ribavirin 20 mg/kg induced maximum incidence at 14 and 42 days, 100 mg/kg at 28 and 42 days and 200 mg/kg at 28-42 days. These results show that ribavirin is mutagenic to rat germ cells in a transient fashion.     

Comparison of sperm quality of Belgian Blue and Holstein Friesian bulls

Few data are currently available on sperm quality of Belgian Blue (BB) bulls. The present study compared sperm quality of BB to Holstein Friesian (HF) bulls of several age categories, by means of a classical semen evaluation. Volume and concentration, and consequently total sperm output depended largely on age. Gross, total, and progressive motility, % live and % normal spermatozoa were significantly lower in the BB breed. Primary sperm abnormalities, such as nuclear vacuoles, midpiece defects and cytoplasmic droplets which were noticed most frequently, occurred far more in the BB breed. Hence, disturbances in spermiogenesis are deemed to be the cause of the poorer BB sperm quality. Since these sperm abnormalities occur significantly more in the BB breed than in the HF breed, it seems as if the BB breed is genetically predisposed to a higher susceptibility to environmental stresses which are known to interfere with normal spermiogenesis. The small scrota typical of the inbred BB breed might in part be responsible for this, and therefore selection for larger scrota in the BB breed is advisable.