Attempted sexing of bovine spermatozoa by fractionation on a Percoll density gradient

Bovine spermatozoa were fractionated on Percoll density gradients into two major subpopulations of motile spermatozoa and a minor fraction containing mostly nonmotile spermatozoa with abnormal morphology. Fractionation required the addition of bovine serum albumin and a continuous Percoll gradient buffered with sodium bicarbonate. It is postulated that, under suitable ionic conditions, the binding of bovine serum albumin to spermatozoa amplifies subtle differences between subpopulations. These studies were directed toward separating Y- and X-bearing spermatozoa. However, when the subpopulations were evaluated by flow cytometry, their Y:X ratios were similar to that of an unfractionated control.     

Absence of BoLA class I antigens on bovine spermatozoa

Summary. Forty AI bulls were tested for BoLA class I antigens by means of eight specific polyclonal reagents. By means of immobilization and sperm penetration tests these antigens were not detected on sperm cells. Isoimmunization studies with the use of sperm as antigenic stimuli and insemination of frozen spermatozoa diluted in specific reagents did not prove the presence of BoLA class I antigens on bovine spermatozoa. The cytotoxic tests used in this investigation were not reliable.     

Sperm volumetric dynamics during in vitro capacitation process in bovine spermatozoa

Previous studies have demonstrated that sperm head morphometry can be used as a potential diagnostic tool for detecting biophysical changes associated with sperm viability in bovine spermatozoa. In this study, sperm head morphometry was used to investigate its value as a biophysical marker for detecting volumetric changes in bovine spermatozoa under in vitro capacitating and non-capacitating incubation conditions. To further test this hypotesis, aliquots of pooled, washed bovine sperm were incubated in either Tyrode’s complete medium with heparin (TCMH; a capacitating medium containing Ca²⁺, NaHCO₃ and heparin), Tyrode’s complete medium heparin-free (TCM; a medium containing just Ca²⁺ and NaHCO₃) or Tyrode’s basal medium (TBM; a non-capacitating medium free of Ca²⁺, NaHCO₃ and heparin, used as control). Aliquots of sperm were processed for morphometric analysis at different incubation-time intervals (0, 3 and 6 h at 38°C), and the chlortetracycline assay was used simultaneously to confirm the ability of the sperm to undergo capacitation (B pattern) and the acrosome reaction (AR pattern) status in each medium. After 3 h of incubation under TCMH conditions, a significant increase was observed in the percentage of B and AR patterns and a significant decrease was found in all sperm morphometric parameters (P<0.01). Interestingly, after 6 h of incubation in TCMH, the percentage of B and AR patterns increased drastically over time and marked differences were found in the dimensional and shape parameters, which were significantly smaller compared with TBM or TCM media (P<0.001). Significant correlations were observed between sperm size and AR pattern (r=−0.875, P<0.01). In conclusion, sperm head morphometry can be used as a potential biophysical marker for detecting volumetric changes during capacitation process in bovine spermatozoa.     

Effects of elevated testicular temperature on morphology characteristics of ejaculated spermatozoa in the bovine

A mild thermal insult to the testes was studied with respect to ejaculated sperm motility and morphology. A 48-h scrotal insulation was administered to 6 young Holstein bulls whose semen was collected by artificial vagina twice in succession at 3-d intervals for 7 wk. For assessment of results, collection days were grouped in the follwing way: Period 1 (control) = Days -6, -3 and 0, where Day 0 = initiation of scrotal insulation after semen collection; Period 2 = Days 3, 6 and 9 (spermatozoa presumed to be in the epididymis or rete testes during scrotal insulation); and Period 3 = Days 12 and 15 through 39 (spermatozoa presumed to be in spermatogenesis during scrotal insulation). Daily sperm output per collection day was 5.3±0.7 × 10⁹ and did not differ across the experimental periods. Moreover, Periods 1 and 2 did not differ in the mean percentage of motility or in the mean percentage of abnormal spermatozoa (69.1±2.5 and 19.6±5.7%, respectively, for Period 1). Morphological change was first noted on Day 12 (47.5±27.4% abnormal) and peaked on Day 18 (86.3±24.3%). Motility depression began on Day 12 and reached a nadir on Day 15 (42.0±9.8%). Bulls varied in the type of abnormal spermatozoa produced and in magnitude of response; however, specific abnormalities appeared in ejaculates in a predictable chronological sequence following scrotal insulation (Day 0). The sequence was: tailless, (Days12 to 15); diadem, (Day 18); pyriform and nuclear vacuoles, (Day 21); knobbed acrosome, (Day 27); and Dag defect, (Day 30).     

Selection of a high quality subpopulation of frozen thawed bovine spermatozoa by filtration in a Sephadex column. An assessment of functional effects of progesterone

It is accepted that cryopreservation exerts deleterious effects on functional characteristics of mammalian spermatozoa. Conventional procedures for processing frozen-thawed gametes, such as centrifugation, produce additional damage. In the present work, we investigated the efficacy of processing bovine cryopreserved semen by filtration in a Sephadex column (SF group) or by washing by centrifugation (100 g, 10 min, twice) (W group); the results obtained from both procedures were compared to untreated samples (C group). The effects of in vitro addition of progesterone (10 μM, 20 min) upon sperm functional activity were studied also. The evaluated sperm parameters were concentration, motility (progressive or non progressive cells), viability and acrosome reaction. They were measured at time 0 (immediately after processing) or after 4 h incubation in capacitating conditions. Sperm concentration was (× 10⁻⁶): 37.5 ± 5.4 in C, 8.3 ± 2.1 in W and 12.5 ± 2.9 in SF. The percentages of motile, progressive, viable or acrosome intact gametes were significantly higher in SF than in W or in C. in SF group, after 4 h incubation in capacitating conditions, progesterone increased significantly the population of acrosome reacted cells whereas this parameter was not modified when the cells were incubated in absence of heparin. Motility and viability were not modified by the hormone. We conclude that Sephadex filtration method is an adequate tool to obtain a subpopulation of spermatozoa with superior quality, as assessed by motility, viability and acrosomal integrity; besides, our results strongly support that, as in other species, progesterone would be a physiological inductor of acrosome reaction in bovine.     

Morphological characteristics of stallion spermatozoa

A study of the morphological characteristics of stallion spermatozoa was conducted at the semen laboratory of the Department of Animal Production during four breeding seasons. A total of 590 ejaculates collected from 216 stallions aged from 2 to 26 years and including 13 breeds or colour types was examined. Overall means for the spermatozoal characteristics of these stallions and the classes of head and tail abnormalities are presented and compared with results of other works. Scanning electron micrographs are included to illustrate recognised abnormalities.     

In vitro fertility and motility characteristics of frozen-thawed boar epididymal spermatozoa separated by Percoll

Frozen-thawed epididymal spermatozoa from four boars were separated through a Percoll gradient, and motility characteristics and in vitro fertility were assessed. Percoll-separated spermatozoa had a significantly higher percentage of motile and progressively motile spermatozoa than those that were not separated (P < 0.0001). However, there were no clear differences in other motility parameters between Percoll-separated and un-separated spermatozoa. Furthermore, sperm agglutination was decreased by Percoll separation (P < 0.05). The effects of Percoll separation on in vitro fertility of spermatozoa differed among boars. In addition, there were large differences in fertility between sperm samples in vitro. Sperm samples, which indicate highly motile and progressively motile, did not always show high in vitro fertility. Furthermore, there was no distinct pattern between fertility in vitro and motility parameters. There was no difference in fertility in vitro between Percoll-separated and un-separated spermatozoa from two of the four boars. However, in vitro fertility of Percoll-separated spermatozoa was higher than that of un-separated spermatozoa from the other two boars.     

Metabolomic fingerprinting of bull spermatozoa for identification of fertility signature metabolites

The objective of the study was to identify the fertility‐associated metabolites in bovine spermatozoa using liquid chromatography‐mass spectrometry (LC‐MS). Six Holstein Friesian crossbred bulls (three high‐fertile and three low‐fertile bulls) were the experimental animals. Sperm proteins were isolated and protein‐normalized samples were processed for metabolite extraction and subjected to LC‐MS/MS analysis. Mass spectrometry data were processed using iMETQ software and metabolites were identified using Human Metabolome DataBase while, Metaboanalyst 4.0 tool was used for statistical and pathway analysis. A total of 3,704 metabolites belonging to various chemical classes were identified in bull spermatozoa. After sorting out exogenous metabolites, 56 metabolites were observed common to both the groups while 44 and 35 metabolites were found unique to high‐ and low‐fertile spermatozoa, respectively. Among the common metabolites, concentrations of 19 metabolites were higher in high‐fertile compared to low‐fertile spermatozoa (fold change > 1.00). Spermatozoa metabolites with variable importance in projections score of more than 1.5 included hypotaurine, d ‐cysteine, selenocystine. In addition, metabolites such as spermine and l ‐cysteine were identified exclusively in high‐fertile spermatozoa. Collectively, the present study established the metabolic profile of bovine spermatozoa and identified the metabolomic differences between spermatozoa from high‐ and low‐fertile bulls. Among the sperm metabolites, hypotaurine, selenocysteine, l ‐malic acid, d ‐cysteine, and chondroitin 4‐sulfate hold the potential to be recognized as fertility‐associated metabolites.     

Use of chromomycin A3 staining in bovine sperm cells for detection of protamine deficiency

Sperm chromatin integrity is essential for accurate transmission of male genetic information, and normal sperm chromatin structure is important for fertilization. Protamine is a nuclear protein that plays a key role in sperm DNA integrity, because it is responsible for sperm DNA stability and packing until the paternal genome is delivered into the oocyte during fertilization. Our aim was to investigate protamine deficiency in sperm cells of Bos indicus bulls (Nelore) using chromomycin A₃ (CMA₃) staining. Frozen semen from 14 bulls were thawed, then fixed in Carnoy's solution. Smears were prepared and analyzed by microscopy. As a positive control of CMA₃ staining, sperm from one bull was subjected to deprotamination of nuclei. The percentage of CMA₃-positive bovine sperm did not vary among batches. Only two bulls showed a higher percentage of CMA₃-positive sperm cells compared to the others. CMA₃ is a simple and useful tool for detecting sperm protamine deficiency in bulls.     

Phospholipid hydroperoxide glutathione peroxidase: expression pattern during testicular development in mouse and evolutionary conservation in spermatozoa

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a selenoprotein belonging to the family of glutathione peroxidases and has been implicated in antioxidative defense and spermatogenesis. PHGPx accounts for almost the entire selenium content of mammalian testis. In an attempt to verify the expression pattern of PHGPx, testes of mouse mutants with arrest at different stages of germ cell development and testes of mice at different ages were subjected to immunostaining with a monoclonal anti-PHGPx antibody. PHGPx was detected in Leydig cells of testes in all developmental stages. In the seminiferous tubuli, the PHGPx staining was first observed in testes of 21-day-old mice which is correlated with the appearance of the first spermatids. This result was confirmed when the testes of mutant mice with defined arrest of germ cell development were used. An immunostaining was observed in the seminiferous tubuli of olt/olt and qk/qk mice which show an arrest at spermatid differentiation. In Western blot analysis of proteins extracted from testes of mutant mice and from developing testes, two signals at 19- and 22-kDa were observed which confirm the existence of two PHGPx forms in testicular cells. In mouse spermatozoa, a subcellular localization of PHGPx and sperm mitochondria-associated cysteine-rich protein (SMCP) was demonstrated, indicating the localization of PHGPx in mitochondria of spermatozoa midpiece. For verifying the midpiece localization of PHGPx in other species, spermatozoa of Drosophila melanogaster, frog, fish, cock, mouse, rat, pig, bull, and human were used in immunostaining using anti-PHGPx antibody. A localization of PHGPx was found in the midpiece of spermatozoa in all species examined. In electronmicroscopical analysis, PHGPx signals were found in the mitochondria of midpiece. These results indicate a conserved crucial role of PHGPx during sperm function and male fertility.     

In vitro assessment of sperm from bulls of high and low field fertility

The aim of this study was to investigate the reasons for differences in field fertility of bulls following insemination with frozen-thawed semen. The study was carried out in two separate parts over two years and comparisons were made between 5 high and 4 low fertility Holstein Friesian bulls as determined by their either 90 day non-return rate (Year 1) or calving rate (Year 2). Two high fertility Limousin bulls were included in Year 1 for comparative purposes. The ability of sperm from each bull to penetrate artificial mucus was assessed (Year 1 = 7 replicates; Year 2 = 5 replicates). Glass capillary tubes (2 per bull per replicate) were filled with artificial mucus and incubated with sperm stained in 1% Hoechst 33342 for 30 min at 37 °C. The number of sperm were subsequently counted at 10 mm intervals along the tube between 40 and 80 mm markers. Sperm mitochondrial activity of each bull was assessed by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay (4 replicates in each year). Sperm were incubated with MTT for 1 h at 37 °C following which the absorbance of formazan was read using a spectrophotometer. Sperm viability after thawing was assessed for each bull using a live/dead sperm viability kit (Year 1 = 3 replicates; Year 2 = 4 replicates). A minimum of 250 cells were assessed per bull in each replicate and classified as either live or dead. Finally, the ability of sperm to fertilize oocytes in vitro and their ability to develop to blastocyst stage embryos were assessed (5 replicates in each year involving 220 to 306 oocytes per bull). Data transformation to normalize residuals was required for mucus sperm penetration (square root) and IVF (cleavage and blastocyst rate) results (arcsin). The mean number of sperm counted at each 10 mm mark between 40 and 80 mm was higher in the high fertility (56.0; 95% CI 39.5 to 75.3) compared to the low fertility (42.9; 95% CI 29.3 to 59.1) Holstein Friesian bulls but the difference did not reach formal significance (P = 0.09). Fertility status had no effect on the ability of sperm to reduce MTT to formazan (mean absorbance 0.34 ± 0.051 and 0.30 ± 0.044) or on the percentage of live sperm per straw (mean 47.3 ± 5.47 and 32.4 ± 4.66) for high and low fertility Holstein Friesian bulls respectively. Oocyte cleavage rate following insemination with sperm from high fertility Holstein Friesian bulls was significantly higher than with sperm from low fertility Holstein Friesian bulls [76.7% (95% CI 60.9 to 89.4) and 55.3 (95% CI 40.4 to 69.7) respectively, P = 0.04]. There was no significant effect of bull fertility on blastocyst rate [34.7% (95% CI 21.1 to 49.6) and 24.2 % (95% CI 14.1 to 36.0) for the high and low fertility Holstein Friesian bulls, respectively; P = 0.2]. In conclusion, sperm from high fertility bulls tended to be more effective in penetrating artificial mucus and to have an increased ability to fertilize oocytes in vitro; however, once fertilization occurred subsequent embryo development was not significantly affected by fertility status.     

Sex ratio of early embryos fertilized in vitro with spermatozoa separated by percoll

Early bovine embryos were obtained by in vitro fertilization and sexing carried out by chromosome analysis. Separation of bovine X- and Y-bearing spermatozoa was performed using Percoll density gradient centrifugation and the enrichment of X-sperm proportion was investigated. Through treatment with vinblastin sulfate and podophyllotoxin, 880 (48.6%) of 1812 embryos at two- to seven-cell stages at 48 to 53 h after sperm-egg incubation produced metaphase spreads, and 399 (45.3%) of these were successfully sexed; the sexable rate reaching 53.4% for four-cell embryos. Sexing rates for embryos from the original sperm of two bulls were 69.6% (32/46) in Bull A and 54.2% (58/107) in Bull B. Embryos fertilized in vitro with sperm sedimented at the bottom of sperm centrifuged under conditions (I) 50 to 85% of Percoll, 15 °C; (II) 30 to 80%, 10 °C; (III) 30 to 80% 20 °C; (IV) 30 to 90%, 20 °C, gave rise to male sex ratios of (I) 58.3% in Bull A and 53.5% in Bull B, (II) 65.9% in Bull A, (III) 49.3% in Bull B and (IV)_66.7% in Bull B. In conclusion, Percoll density gradient centrifugation under these four conditions was unsuccessful in separating X- and Y-bearing bull spermatozoa.     

In vitro effect of gamma-aminobutyric acid on bovine spermatozoa capacitation

Sperm capacitation is defined as the maturational changes that render a sperm competent for fertilization and occurs in the female reproductive tract. Identification of the factor/s that regulate sperm capacitation would allow the understanding of these phenomena. Among these factors, gamma-aminobutyric acid (GABA) has recently become as a putative modulator of sperm function. The aim of this study was to explore the presence of a GABAergic regulation of bovine sperm capacitation as well as the possible intracellular mechanisms involved. GABA was detected in fresh semen by a sensitive radioreceptor assay (spermatozoa, 0.064 +/- 0.003 nmoles/10(6) cells; seminal plasma, 23.21 +/- 1.16 nmoles/ml). Scatchard analysis of [(3)H]-muscimol binding to sperm membranes yielded a linear plot consistent with a single population of binding sites (K(d) = 3.87 nM, B(max) = 417 fmol/mg prot.). [(3)H]-muscimol specific binding to sperm membranes was significantly inhibited by the GABA A receptor (GABA A-R) antagonist bicuculline and by the agonists muscimol and isoguvacine. Addition of GABA to the incubation medium resulted in a concentration-dependent increase in the percentage of capacitated spermatozoa (chlortetracycline assay). We observed a significant increment on intracellular calcium and cyclic 3',5' adenosine monophosphate (cAMP) concentrations induced by GABA, being the cation influx abolished when the cell suspensions were coincubated with the antagonists bicuculline or picrotoxin. It is concluded that GABA induces sperm capacitation through an intracellular mechanism dependent on calcium influx and cAMP accumulation mediated by a specific GABA A-R.     

Cooling rates,storage temperatures and fertility of extended equine spermatozoa

Two experiments were designed to evaluate cooling rates and storage temperatures for stallion spermatozoa extended in caprogen (CAP), Cornell University extender (CUE), heated skimmilk (SM) and a nonfat-dried milk solids glucose extender (NFDMS-glucose). In Experiment 1, each extender was evaluated in a separate but similar 4 × 4 × 6 factorial trial using two ejaculates from each of six stallions. Aliquots of 66 × 10⁶ spermatozoa were transferred to each of 16 coded tubes and extended to 6 ml with SM, CAP, CUE or NFDMS-glucose. Four tubes of extended semen were either plunged into 5C water or cooled at a rate of ?1.0, ?0.5, or ?0.2C/min. Within each treatment, one tube of extended semen was maintained at 20C, 15C, 10C or 5C. Progressive spermatozoal motility was estimated immediately after dilution (0 h) and at 4, 8, 12, 24 and 36 h. Regardless of extender, all three slower cooling rates were superior (P<0.05) to plunging to 5C; storage temperatures of 20C and 15C were superior (P<0.05) for maintaining spermatozoal motility. Experiment 2 was designed so that all extenders could be evaluated simultaneously. Since CUE resulted in an immediate depression of spermatozoal motility, it was not evaluated further. Semen was collected from 12 stallions and each ejaculate was extended in SM, CAP and NFDMS-glucose. Semen was cooled at ?1.0C/min and maintained at either 20C or 15C. Spermatozoal motility was assessed as in Experiment 1. Overall, the CAP and NFDMS-glucose extenders were superior (P<0.05) to SM for maintenance of spermatozoal motility. Storage at 20C or 15C resulted in similar (P>0.05) spermatozoal motility. Two fertility trials compared the use of SM and NFDMS-glucose extenders. Embryo recovery 6 days post-ovulation (Experiment 3) and pregnancy rate 50 days postestrus (Experiment 4) was similar (P>0.05) for mares inseminated with spermatozoa extended in SM or NFDMS-glucose.     

The effect of adjusting settings within a Computer-Assisted Sperm Analysis (CASA) system on bovine sperm motility and morphology results

Semen motility is the most widely recognized semen quality parameter used by Artificial Insemination (AI) centers. With the increasing worldwide export of semen between AI centers there is an increasing need for standardized motility assessment methods. Computer-Assisted Sperm Analysis (CASA) technology is thought to provide an objective motility evaluation; however, results can still vary between laboratories. The aim of present study was to verify the impact of different setting values of the CASA IVOS II on motility, concentration, and morphology of bovine semen samples frozen in an extender with or without egg yolk and then decide on optimal settings for a further validation step across AI centers. Semen straws from 30 different bulls were analyzed using IVOS II with twelve modified settings. No significant changes were observed in semen concentration, percentage of motile sperm or kinetic results for either extender type. However, increasing settings for both STR and VAP progressive (%) from Low, Medium, and High cut-off values significantly (p<0.05) reduced the percentage of detected progressive spermatozoa, in egg yolk extender from 49.5±15.2, 37.2±11.9 to 11.9±5.3%, and in clear extender from 51.9±9.1, 35.8±7.3 to 10.0±2.4%, respectively. In clear extender only, the modification of droplet proximal head length significantly affected the detection of normal sperm percentages (88.0± 4.7 to 95.0±0.6 and 96.0±0.6%) and of the percentage of detected proximal droplets (12.2±4.7, 2.5±2.7 to 0.6±0.2%) for Low, Medium and High values respectively (p<0.05). The identification of sensitivity within the CASA system to changes in set parameters then led to the determination of an optimal IVOS II setting. The existing variability among centers for these phenotypes was reduced when the standardized settings were applied across different CASA units. The results clearly show the importance of applied settings for the final CASA results and emphasize the need for standardized settings to obtain comparable data.     

Characterization of subpopulated articular chondrocytes separated by Percoll density gradient

The aim of this study was to develop a method for fractionation of articular chondrocytes from the entire thickness of the tissue. Isolated chondrocytes from rabbit articular cartilage fractionated by centrifugation in a discontinuous Percoll gradient resulted in four cell fractions with two differing properties. The lowest-density fraction consisted mainly of large cells with small nuclei proliferated actively, maintained the chondrocytic phenotype, and secreted larger amounts of proteoglycan. In contrast, the highest-density fraction consisted of small cells with large nuclei proliferated slowly, did not express the chondrocytic phenotype, and produced larger amounts of interleukin 1-induced nitric oxide. Comparing our results with other previous reports, we find that fraction 1 cells are likely originated from the deep layer of the articular cartilage, whereas fraction 4 cells are tentatively categorized as chondrocytes from the superficial layer of cartilage. Centrifugal fractionation of articular chondrocytes via Percoll density gradient permits clear separation of these heterogeneous cells into different phenotypic populations and allows distinguishing of cells from the different layers of articular cartilage. This simple novel method will provide ready separation of articular chondrocytes for the investigation of the pathogenesis of articular cartilage.     

Functional changes and motility characteristics of Japanese Black bull spermatozoa separated by Percoll

Spermatozoa from two Japanese Black bulls (Bull-ATF and Bull-KTG) were separated by centrifugation at 700 x g for 15min in modified TALP with or without 45-90% Percoll. Control washed spermatozoa and those collected from the bottom of 45 and 90% Percoll fractions were examined for viability and membrane integrity (using Hoechst bis-benzimide 33258 or propidium iodide and 6-carboxyfluorescein diacetate (PI-CFDA)), acrosomal status (using fluorescence isothiocyanate (FITC) conjugated Pisum Sativum agglutinin (PSA) and Peanut agglutinin (PNA), Naphthol Yellow S and Erythrosin B (NE) or triple staining (TS)), capacitation status (using chlortetracycline (CTC)), motility characteristics (using a computer-assisted sperm motion analysis system (CASA)) and for in vitro fertility. Percoll-separated spermatozoa showed greater viability and membrane integrity than controls, as determined by supravital staining. Differences were observed in the results regarding viability and acrosomal status of spermatozoa among sperm staining methods. Bull-ATF, which showed significantly greater in vitro fertility than Bull-KTG (P<0.05), showed a significantly higher rate of CTC-B-pattern (capacitated) spermatozoa (P<0.01) than Bull-KTG. The motility characteristics of control washed spermatozoa and those separated by 45-90% Percoll were analyzed by CASA. More motile and progressively motile spermatozoa were observed in the fraction at the bottom of the 90% Percoll solution than in the 45% Percoll fraction or in controls (P<0.01). Moreover, the spermatozoa of Bull-KTG, which showed lower in vitro fertility than Bull-ATF, did not show significant differences in motility from those of Bull-ATF. These results provided basic information about Japanese Black bull spermatozoa, and suggested that spermatozoa with greater motility and viability can be obtained by Percoll separation than without separation. However, Percoll separation did not enhance their in vitro fertility.     

Bull subfertility is associated with low levels of a sperm membrane antigen

During epididymal transit, mammalian spermatozoa acquire new surface antigens that may participate in gamete interaction. We have previously described a 26 kDa (P26h) epididymal hamster sperm protein that is proposed to be involved in fertilization. We have also identified its human homolog, P34H. Variability in the amount of P34H on spermatozoa from fertile and idiopathic infertile men provides strong evidence that this protein is a potential marker of male fertility. Since these sperm antigens constitute a family of proteins with common antigenicity, we have investigated the presence of a related protein in bovine sperm. In the present study, a P26h antiserum recognized two bull sperm proteins of 21 kDa and 25 kDa (MW) on SDS‐PAGE. We showed that P25b could be extracted with detergent as a surface protein, whereas the P21b was associated with non‐soluble intracellular structures. Sonication of whole sperm cell suspensions and subsequent Percoll gradient centrifugation revealed that P21b may be a flagellar protein whereas the P25b may be located in the head region. Western blot analysis was used to determine the amount of P25b and P21b proteins present on spermatozoa obtained from fertile and subfertile bulls. P21b protein levels were similar in fertile and subfertile bulls, but P25b protein levels were variable. Thus, all bulls with high Non‐Return Rates (fertile bulls) demonstrated high amounts of P25b, whereas P25b levels were decreased in semen from subfertile bulls. We conclude that the protein P25b is a potential fertility marker in the bull and consequently may provide an invaluation tool for the evaluation of bull fertility. Mol. Reprod. Dev. 52:57–65, 1999. © 1999 Wiley‐Liss, Inc.     

Polymorphisms in candidate genes as markers for sperm quality and boar fertility

Alpha-actinins (ACTN1 and ACTN4) and gamma-actin (ACTG2), were investigated as candidate genes on the basis of their known functions, for their possible association with sperm concentration, motility, semen volume per ejaculate, plasma droplets rate, abnormal sperm rate and the fertility traits, non-return rate and number of piglets born alive. Polymorphisms were identified in intron 18 (G>A) of the porcine ACTN1 gene and in exon 22 (A>C) of the porcine ACTN4 gene by comparative sequencing of animals from the Pietrain (PI) and Hampshire (HA) breeds. Pietrain (n = 244) and crossbreed PI x HA (n = 112) boars from an artificial insemination boar station were genotyped for the single nucleotide polymorphisms (SNPs) within ACTN1 and ACTN4 as well as for a previously described microsatellite within ACTG2. The study provides evidence for effects of ACTN1 on fertility and of ACTG2 on sperm quality traits, while no indication of impact of ACTN4 on any of these traits was found.