A comprehensive proteomic analysis of the accessory sex gland fluid from mature Holstein bulls

The expression of proteins in accessory sex gland fluid (AGF) of proven, high use mature Holstein bulls was evaluated. Thirty-seven bulls with documented fertility based on their non-return rates were studied. AGF was obtained by artificial vagina after bulls were surgically equipped with cannulae in the vasa deferentia. Samples of AGF were evaluated by two-dimensional SDS-PAGE, gels stained with Coomassie blue and polypeptide maps analyzed by PDQuest software. A master gel generated by the software representing the best pattern of spots in the AGF polypeptide maps was used as a reference for protein identification. Proteins were identified by Western blots and capillary liquid chromatography-nanoelectrospray ionization tandem-mass spectrometry (CapLC-MS/MS). The product ion spectra were processed using Protein Lynx Global Server 2.1 prior to database search with both PLGS and MASCOT (Matrix Science) software. The entire NCBI database was considered for mass fingerprint matching. An average of 52+/-5 spots was detected in the AGF 2D gels, which corresponded to proteins potentially involved in capacitation (bovine seminal plasma protein-BSP-A1/A2 and A3, BSP 30 kDa, albumin); sperm membrane protection, prevention of oxidative stress, complement-mediated sperm destruction and anti-microbial activity (albumin, clusterin, acidic seminal fluid protein--aSFP, 5'-nucleotidase--5'-NT, phospholipase A2--PLA2); acrosome reaction and sperm-oocyte interaction (PLA2, osteopontin); interaction with the extracellular matrix (tissue inhibitor of metalloproteinase 2, clusterin) and sperm motility (aSFP, spermadhesin Z13, 5'-NT). The 20 spots distinguished in all gels were matched to proteins associated with these functions. Proteins identified by tandem mass spectrometry as ecto-ADP-ribosyltransferase 5 and nucleobindin, never described before in the accessory sex gland secretions, were also detected. In summary, we identified a diverse range of components in the accessory sex gland fluid of a select group of Holstein bulls with documented fertility. Known characteristics of these proteins suggest that they play important roles in sperm physiology after ejaculation.     

Proteomic markers of low and high fertility bovine spermatozoa separated by Percoll gradient

In the context of artificial insemination, male fertility is defined as the ability to produce functional spermatozoa able to withstand cryopreservation. We hypothesized that interindividual variations in fertility depend on the proportion of the fully functional sperm population contained in the insemination dose. The objective of this study was to identify protein markers of the fully functional sperm subpopulation. Insemination doses from four high‐fertility (HF) and four low‐fertility (LF) bulls with comparable post‐thaw quality parameters were selected for proteomic analysis using iTRAQ technology. Thawed semen was centrifuged through a Percoll gradient to segregate the motile (high density [HD]) from the immotile (low density [LD]) sperm populations. Sperm proteins were extracted with sodium deoxycholate and four groups were compared: LD and HD spermatozoa from LF and HF bulls. A total of 498 unique proteins were identified and quantified. Comparison of HD spermatozoa from HF and LF bulls revealed that five proteins were significantly more abundant in the HF group (AK8, TPI1, TSPAN8, OAT, and DBIL5) whereas five proteins were more abundant in the LF group (RGS22, ATP5J, CLU, LOC616319, and CCT5). Comparison of LD spermatozoa from HF and LF bulls revealed that four proteins were significantly more abundant in the HF group (IL4I1, CYLC2, OAT, and ARMC3) whereas 15 proteins were significantly more abundant in the LF group (HADHA, HSP90AA1, DNASE1L3, SLC25A20, GPX5, TCP1, HIP1, CLU, G5E622, LOC616319, HSPA2, NUP155, DPY19L2, SPERT, and SERPINE2). DBIL5, TSPAN8, and TPI1 showed potential as putative markers of the fully functional sperm subpopulation.     

Moribund sperm in frozen-thawed semen, and sperm motion end points post-thaw and post-swim-up, are related to fertility in Holstein AI bulls

The objectives were to compare testicular physical characteristics and post-thaw sperm characteristics and their associations with fertility in Holstein bulls used for AI. Ten Holstein bulls (4-5 y old) were classified as either high-fertility (HF) or low-fertility (LF; n = 5 each), based on adjusted 56-d non-return rates [non-return rate (NRR); range (mean ± SD): 55.6 ± 4.6 to 71.8 ± 1.3%). Testicular physical characteristics were not significantly different between the two groups. Four ejaculates were collected from each bull and cryopreserved. Several indexes of sperm motion (based on computer-assisted sperm analysis) at post-thaw and post-swim-up were correlated with NRR. Sperm from HF bulls were in transition to a hyperactivated motility pattern, whereas those from LF bulls had only a forward progressive motility pattern. In HF vs LF bulls, there was a greater percentage of viable sperm after thawing (60.6 ± 9.7 vs 49.5 ± 8.0%, P < 0.05) and after swim-up (70.9 ± 11.0 vs 63.0 ± 8.8%, P < 0.01); these two end points were positively correlated with fertility (r = 0.45, P < 0.01 and r = 0.78; P < 0.01, respectively). Furthermore, in HF vs LF bulls, the ratio of sperm recovered after swim-up to viable sperm in post-thaw semen was higher (P < 0.001), and the proportion of moribund sperm expressed as a percentage of live sperm differed (12.6 ± 3.4 vs. 16.4 ± 3.1%, P < 0.001) and was negatively correlated (r = −0.33, P < 0.05) with fertility. In conclusion, fertility of Holstein bulls maintained in a commercial AI center was not predicted by testicular physical characteristics, but it was associated with differences in moribund sperm in the inseminate, as well as characteristics of sperm post-thaw and after swim-up.     

Associations of scrotal circumference with semen traits in young beef bulls

Associations of percent live sperm, sperm number, sperm concentration, sperm motility, and potential breeding efficiency score with scrotal circumference were studied using data from the records of 465 Polled Hereford and 264 Simmental bulls. Data were subdivided by breed and within breed into three groups according to scrotal circumference (SC) size. Group I bulls had SC measurements <32 cm. Group II included bulls with SC measurements between 32 cm and the mean measurement of the breed (i.e., 35 cm for Polled Hereford and 38 cm for Simmentals). Group III was comprised of bulls with SC values above their respective breed averages. Age and body weight differences were also evaluated in the three groups. Average age differences among bulls in the three groups differed by not more than nine days in the two breeds. Average body weight differences among the three groups were statistically significant (P<0.05) with the maximum difference between any two groups within breed being 80 pounds. Average differences in percent live sperm and sperm number were greater (P<0.05) in Groups II and III than in Group I. For sperm concentration ratings, sperm motility ratings and potential breeding efficiency score, and for both breeds, Group I had a significantly lower (P<0.05) number of bulls classified satisfactory and a significantly greater (P<0.05) number of bulls classified unsatisfactory than did groups II and III.     

Infertility in a beef bull due to a failure in the capacitation process

The objective of this case report was to identify the cause of apparent idiopathic infertility in a Red Angus (beef) bull. Semen was collected by electroejaculation and submitted to a series of assays, including evaluation of sperm motility by computer-assisted sperm analysis (CASA), sperm morphology and DNA integrity, semen cryopreservation, AI, IVF, induction of the acrosome reaction, and determination of the level of sperm proteins associated with bull fertility potential. Total (92 ± 2%) and progressive (79 ± 4%) sperm motility; sperm concentration (1647 ± 429 × 10⁶ sperm/mL); proportions of morphologically normal sperm (83 ± 6%) and DNA integrity (96 ± 2), and acrosome-intact sperm (64 ± 4%) exceeded minimum acceptable values. Frozen sperm had good total (58.7 ± 6.7%) and progressive (43.9 ± 9.2%) motility immediately after thawing. However, AI of 16 heifers resulted in no pregnancies and blastocyst production rate (following IVF using sperm from this infertile bull) was nearly identical to that produced using dead sperm (a control of parthenogenesis; 2 ± 2 and 2 ± 3%; respectively P < 0.05). Treatment with a calcium ionophore (A23187) failed to induce the acrosome reaction in sperm from the infertile bull (P < 0.05). Evaluation of several proteins associated with the fertility potential of bulls revealed that the level of Binder Sperm Protein-1 (BSP1), known to be associated with the capacitation process, was much greater on sperm from the infertile bull compared to that of his sire. In conclusion, we inferred that the idiopathic infertility in this bull was caused by a failure to complete the capacitation process.     

Characterization of recombinant murine binder of sperm protein homolog 1 and its role in capacitation

Sperm capacitation is a maturation step that is deemed to be essential for sperm to fertilize an oocyte. A family of proteins, the binder of sperm (BSP), are known to bind choline phospholipids on sperm membranes and promote capacitation in bulls and boars. Recently, BSP-homologous genes have been identified in the epididymal tissues of human (BSPH1) and mouse (Bsph1, Bsph2). The aim of this study was to determine the binding characteristics of the murine binder of sperm protein homolog 1 (BSPH1) and evaluate its effects on sperm capacitation. Since it is not possible to purify the native BSP proteins from human and mouse in sufficient quantity, a murine recombinant BSPH1 (rec-BSPH1) was produced and used for the functional studies. Similarly to BSP proteins from other species, rec-BSPH1 bound to gelatin, heparin, phosphatidylcholine liposomes, and sperm. Both native BSPH1 and rec-BSPH1 were detected on the head and the midpiece region of sperm, although a stronger signal was detected on the midpiece region when sperm were incubated in a capacitating media containing bovine serum albumin. More importantly, murine rec-BSPH1 was able to capacitate sperm, but was unable to induce the acrosome reaction. These results show that murine epididymal BSPH1 shares many biochemical and functional characteristics with BSP proteins secreted by seminal vesicles of ungulates, and suggest that it might play a similar role in sperm functions.     

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.     

Bovine seminal plasma phospholipid-binding proteins stimulate phospholipid efflux from epididymal sperm.

Several studies have shown that sperm capacitation was accompanied by a change in the lipid composition of the sperm membrane. In cattle, the major proteins of (bovine)seminal plasma (BSP proteins: BSP-A1/A2, BSP-A3, and BSP-30-kDa) potentiate sperm capacitation induced by high-density lipoprotein (HDL). Our recent studies indicate that these proteins and HDL stimulate sperm cholesterol efflux during capacitation. In order to gain more insight into the mechanisms of BSP-mediated sperm capacitation, we studied whether or not BSP proteins induce phospholipid efflux from epididymal sperm membrane. By direct determination of choline phospholipids on unlabeled epididymal sperm, the results show that sperm incubated in the presence of BSP-A1/A2 protein lost 34.4% of their choline phospholipids compared with the control (11.5%). Similar results were obtained using labeled epididymal sperm. Labeling was carried out by incubating washed epididymal sperm for 1 h with medium containing [(3)H]palmitic acid. The majority of the label was incorporated into sperm phosphatidylcholine. Studies of sperm phospholipid efflux were done by incubating the labeled sperm with purified BSP proteins, delipidated BSA, or bovine seminal ribonuclease (RNase, control protein). When labeled ([(3)H]phospholipid) epididymal sperm were incubated with BSP proteins (20-120 microg/ml) for 8 h, the sperm lost [(3)H]phospholipid in a dose-dependent manner (maximum efflux of approximately 30%). After the incubation with BSP proteins, the efflux particles were fractionated by size-exclusion chromatography. Analysis of the fractions obtained showed that the [(3)H]phospholipid was associated with BSP proteins. BSA (6 mg/ml) stimulated a specific phospholipid efflux of approximately 22%. In contrast, bovine RNase (120 microg/ml) did not stimulate phospholipid efflux. These results indicate that BSP proteins participate in the sperm cholesterol and phospholipid efflux that occurs during capacitation.     

Proteomics of cauda epididymal fluid from mature Holstein bulls

The proteome of cauda epididymal fluid (CEF) from Holstein bulls was defined. Fluid was collected from the vas deferens, subjected to 2-D SDS-PAGE and spots identified by CapLC-MS/MS and MALDI-ToF/ToF. Because albumin accounted for 21.1% of all spot intensities in the gels examined by PDQuest, samples were subjected to albumin depletion and then analyzed again as before. Original CEF gels had 114 ± 3 spots, including as the most abundant: albumin, epididymal secretory protein E1, prostaglandin d-synthase and gelsolin. Epididymal fluid also expressed: clusterin, transferrin, N-acetyl-β-glucosaminidase, cauxin, glutathione peroxidase, acidic seminal fluid protein (aSFP), aldehyde reductase, α-l-fucosidase, α-1-β-glycoprotein, apolipoprotein A-1, β actin, calmodulin, cathepsin D, cystatin E/M, enolase, galectin 3-binding protein, leucine amino-peptidase and nucleobindin. Albumin depletion decreased that very spot to 10% of its original intensity and the resulting gels had, on average, 137 ± 4 spots. Spots identified as dipeptidyl-peptidase 7, angiotensin-converting enzyme, arylsulfatase A, aspartylglucosaminidase, serine protease inhibitors, new isoforms of calmodulin, cystatin E/M and a 17-kDa nucleobindin appeared only in depleted maps. This study is the first to report nucleobindin and aSFP as epididymal components. We suggest that CEF proteins act to facilitate membrane remodeling, transport of lipophilic substances, protect sperm and prevent premature acrosome reaction.     

Effect of reducing sperm concentration during IVF on the ability to distinguish between bulls of high and low field fertility: work in progress

The objectives of this study were to evaluate the effect of sperm dose and sire on the fertilization rate, cleavage rate and blastocyst yield following insemination in vitro, to examine the relationship between these parameters and field fertility in cattle, and to examine the relationship between blastocyst quality and sire used in IVF. Frozen semen from four bulls with 150-day nonreturn rates ranging from 57 to 78% was used. In Experiment 1, oocytes were inseminated with sperm from one of the four bulls at concentrations ranging from 0.016 to 0.5 x 10(6)sperm/ml. A proportion of presumptive zygotes were fixed at 17 h post-insemination (hpi), while the remainder was transferred to in vitro culture (IVC) in droplets of synthetic oviduct fluid (SOF). Cleavage at 48 hpi and the percentage of oocytes reaching the blastocyst stage by Day 8 were recorded. In Experiment 2, to assess blastocyst quality, after insemination with semen from one of the four bulls, presumptive zygotes were cultured in SOF until Day 7. Blastocysts for each bull were removed and vitrified/warmed and survival was recorded at 24, 48 and 72 h after warming. Regardless of bull used, a concentration of 0.125 x 10(6)sperm/ml or above resulted in higher blastocyst yields than any lower concentration used. Fertilization and cleavage rates were also higher at higher sperm concentrations. The best predictor of field fertility was fertilization rate at a concentration of 0.5 x 10(6)sperm/ml (r=0.94, P<0.0001). There was also a significant correlation between cleavage rate at a concentration of 0.5 x 10(6)sperm/ml and nonreturn rate (r=0.90, P<0.0001). In Experiment 2, blastocysts derived from one bull, HTA, were of superior quality as measured by survival 24h after thawing, although these differences were less significant at the subsequent time points measured. In conclusion, these data show that differences between the field fertility of bulls can be determined at sperm concentrations routinely used in IVF. Lowering the sperm concentration does not increase the likelihood of optimizing the differences in fertility or cleavage rate between bulls of different field fertility. We have also demonstrated that the bull can have a significant effect on the quality of blastocysts produced using IVF techniques.     

Low molecular weight factor in bovine caudal epididymal fluid that stimulates calcium uptake in caput spermatozoa

Secretions from the mammalian epididymis contain proteins that bind to developing sperm and are presumed to play a role in sperm maturation. The biochemical functions in sperm of most of these proteins are not known. In this report we describe the presence of a low molecular weight compound in bovine caudal epididymal luminal fluid (CF) that has a potent stimulatory effect on calcium (⁴⁵Ca²⁺) uptake in immature caput epididymal spermatozoa. The studies were initially undertaken to characterize the effect of the protein caltrin, present in bovine seminal plasma (BSP), on calcium uptake into caput spermatozoa. Caltrin is known to block calcium influx into mature bovine sperm. Unexpectedly, the kinetics of calcium uptake into caput sperm showed a biphasic response when treated with BSP, namely, a stimulation of uptake at 1 to 5 min and inhibition of uptake after this time. Since caudal sperm do not show this biphasic response, we reasoned that BSP contained a factor derived from CF that must interact with developing sperm before the binding of caltrin to sperm can prevent further calcium uptake. We first demonstrated that preincubation of caput sperm with CF eliminated the biphasic calcium uptake effect induced in caput sperm by BSP and that caudal fluid alone had a potent stimulatory effect on calcium uptake in caput sperm. Half-maximal stimulation (fivefold over control) occurred at a caudal fluid protein concentration of 0.27 mg/ml. Partial purification of the factor indicates that it is of low molecular weight (MW ～ 1,000), but further chemical characterization has not been carried out and its epididymal site of origin is not known. The results indicate that the regulation of intracellular calcium levels in sperm differs in immature and mature bovine sperm in that an epididymal factor promotes calcium uptake during epididymal maturation, and the seminal fluid protein caltrin prevents it at ejaculation.     

Induction of epididymal boar sperm capacitation by pB1 and BSP-A1/-A2 proteins, members of the BSP protein family

A family of proteins designated BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa, collectively called BSP (bovine seminal plasma) proteins, constitute the major protein fraction of bull seminal plasma. BSP proteins can stimulate sperm capacitation by inducing cholesterol and phospholipid efflux from sperm. Boar seminal plasma contains one homologous protein of the BSP family, named pB1; however, its physiological role is still unknown. In the current study, we report a novel method to purify pB1 from boar seminal plasma by chondroitin sulfate B-affinity chromatography and reverse-phase-high performance liquid chromatography. We also studied the effect of pB1, BSP-A1/-A2, and whole boar seminal plasma on boar sperm capacitation. Boar epididymal sperm were washed, preincubated in noncapacitating medium containing pB1 (0, 2.5, 5, 10 or 20 microg/ml), BSP-A1/-A2 (0 or 20 microg/ml) proteins, or whole seminal plasma (0, 250, 500, or 1000 microg/ml), then washed and incubated in capacitating medium. Acrosomal integrity was assessed by chlortetracycline staining. The status of sperm capacitation was evaluated by the capacity of sperm to undergo the acrosome reaction initiated by the addition of the calcium ionophore, A23187. The pB1 and BSP-A1/-A2 proteins increased epididymal sperm capacitation as compared with control (sperm preincubated without proteins). This effect reached a maximum level at 10 microg/ml pB1 and at 20 microg/ml BSP-A1/-A2 (2.3- and 2.2-fold higher than control, respectively). Whole boar seminal plasma did not induce sperm capacitation. In addition, pB1 bound to boar epididymal sperm and was lost during capacitation. These results indicate that BSP proteins and their homologs in other species induce sperm capacitation in a similar way.     

Bovine embryo development after IVF with spermatozoa having abnormal morphology

The study was conducted to evaluate the effects of scrotal insulation on semen samples collected from bulls on embryonic development after IVF. Semen samples were obtained and cryopreserved from four Holstein bulls before and after a scrotal insulation period of 48 h (Day 0). Three types of samples were used for IVF: (1) semen from the test bulls collected 5 d prior to scrotal insulation (pre-insult); (2) semen from Day 13 (2-week post-insult; 2-week PI); and (3) semen from Day 20 (3-week PI). After 18 h of sperm-oocyte co-incubation, the zygotes were cultured for 8 d when a developmental score (0=degenerate, 1=2-cell embryo through 5=blastocyst) was assigned to each embryo. The post-thaw morphological evaluation of sperm samples revealed a decrease (P<0.01) in the percentages of normal spermatozoa in the 3-week PI samples in comparison with the pre-insult samples for Bulls I and III (74-22.3% and 67.7-0.5 %, respectively). The percentage of vacuolated spermatozoa increased significantly for Bull II. The cleavage and blastocyst formation rates and embryo development scores were affected (P<0.01) by the interaction of bull by sample collection time. For Bulls I and III (severe responders) the scrotal insulation effects persisted from the time of cleavage through blastocyst formation. In contrast, the cleavage and blastocyst formation rates for Bulls II and IV were unaffected, despite high percentages of vacuolated spermatozoa present in the post-insult samples for Bull II. In conclusion, the use of scrotal insulation to elevate scrotal temperature was an effective method to obtain semen samples with high percentages of abnormal spermatozoa. The decrease in embryonic development after IVF when using spermatozoa with morphological abnormalities seemed to be multifaceted and related to changes in head morphology.     

Fertility-related proteomic profiling bull spermatozoa separated by percoll

Infertility or subfertility of bovine spermatozoa may lead to disintegration of the breeding system and large economic losses. Recently, proteomics have identified candidates for the sperm fertility biomarkers, but no definite studies have clearly identified the relationship between the proteome and sperm fertility after proteomic study. Therefore, to determine the clinical value of the protein markers identified by proteomic study, we first compared the protein expression profiles of spermatozoa from high and low fertility bulls using 2-dimensional electrophoresis. We then investigated the relationship between protein expression and the fertility of individual bulls as assessed by Western blot analysis. Five proteins, enolase 1 (ENO1), ATP synthase H(+) transporting mitochondrial F1 complex beta subunit, apoptosis-stimulating of p53 protein 2, alpha-2-HS-glycoprotein, and phospholipid hydroperoxide glutathione peroxide, were more highly represented in high fertility bulls, whereas three proteins, voltage dependent anion channel 2 (VDAC2), ropporin-1, and ubiquinol-cytochrome-c reductase complex core protein 2 (UQCRC2), were more highly represented in low fertility bulls. Among those proteins, ENO1, VDAC2, and UQCRC2 were significantly correlated with individual fertility. Therefore, these results suggest that concurrent comparisons between protein expression and other fertility assays may represent a good in vitro assay to determine sperm fertility.     

Effect of seminal phospholipid-binding proteins and follicular fluid on bovine sperm capacitation.

Bovine seminal plasma (BSP) contains a family of novel phospholipid-binding proteins (BSP-A1/-A2, BSP-A3, and BSP-30-kDa; collectively called BSP proteins) that potentiate sperm capacitation induced by heparin or by serum high-density lipoprotein (HDL). BSP proteins stimulate lipid efflux from sperm that may occur during the early events of capacitation. Here, we investigated the role of BSP proteins, bovine follicular fluid (FF), and bovine follicular fluid HDL (FF-HDL) in sperm capacitation. FF and FF-HDL alone stimulated epididymal sperm capacitation (19.5% +/- 0.8% and 18.2% +/- 2.8%, respectively, control, 9.0% +/- 1.9%) that was increased by preincubation with BSP-A1/-A2 proteins (30.2% +/- 0.4% and 30.9% +/- 1.5%, respectively). In contrast, lipoprotein-depleted follicular fluid (LD-FF) alone was ineffective, and a preincubation with BSP-A1/-A2 proteins was necessary before sperm capacitation was stimulated (up to 22.8% +/- 1.4%). The interaction of BSP proteins with FF components was analyzed using ultracentrifugation, Lipo-Gel electrophoresis, SDS-PAGE, and gel filtration. We established that the BSP proteins interact with factors present in FF including FF-HDL. Additionally, we obtained evidence that BSP proteins, found associated with FF-HDL, were released from the sperm membrane during capacitation. These results confirm that the BSP proteins and the FF-HDL play a role in sperm capacitation.     

Gonadal and extragonadal sperm reserves in Boran and Boran x Friesian bulls raised on two planes of nutrition in the Highlands of Ethiopia

Gonadal and extragonadal sperm reserves were determined in testicular and epididymal tissues obtained from Boran (n=10) and Boran x Friesian (n=12) bulls fed either a high or low plane nutrition diet for a 1-year period. The bulls were 32 months of age at castration. Mean (+/-SEM) daily body weight gains over a 1-year period were 776+/-34 and 264+/-34 g/day (P<0.001) for bulls on high and low nutrition, and their respective body weights at castration were 458+/-17 and 276+/-17 kg (P<0.01). Mean body weights did not differ between Boran and Boran x Friesian bulls. Mean scrotal circumference measurements were 32.1+/-0.6 and 28.8+/-0.6 cm for the high and low planes of nutrition (P<0.01) and 31.8+/-0.7 and 29.2+/-0.6 cm for the Boran and the Boran x Friesian bulls (P<0.05). Paired testes and epididymal weights averaged 432+/-22 and 313+/-22 g (P<0.01) and 46.3+/-2.0 and 32.5+/-2.0 g (P<0.001) for bulls on the high and low planes of nutrition, respectively. Boran x Friesian bulls had significantly heavier (P<0.05) testes, but epididymal weithts did not differ between breeds. Although daily sperm production per gram was not influenced by either plane of nutrition or breed, daily sperm production differed between high (5.3 x 10(9)) and low nutrition (2.9 x 10(9)) bulls (P<0.001) and between Boran (4.6 x 10(9)) and Boran x Friesian (3.5 x 10(9)) bulls (P<0.01). Extragonadal sperm reserves were significantly higher in high than in low nutrition bulls (13.1 x 10(9) vs 6.9 x 10(9); P<0.001) and in Boran than Boran x Friesian bulls (12.9 x 10(9) vs 7.1 x 10(9); P<0.01). The cauda epididymis contributed 50 to 54% to the total epididymal sperm reserves. It was concluded that the plane of nutrition influenced growth rates and testes and epididymal weights, and it improved gonadal and extragonadal sperm reserves in young Boran and Boran x Friesian bulls.     

Genome-wide profiling of sperm DNA methylation in relation to buffalo (Bubalus bubalis) bull fertility

The DNA methylation pattern in spermatozoa of buffalo bulls of different fertility status was investigated. Spermatozoa isolated DNA from two groups of buffalo bulls (n = 5), selected based on their artificial insemination–generated conception rate data followed by IVF efficiency, were studied for global methylation changes using a custom-designed 180 K buffalo (Bubalus bubalis) CpG island/promoter microarray. A total of 96 individual genes with another 55 genes covered under CpG islands were found differentially methylated in sperm of high-fertile and subfertile buffalo bulls. Important genes associated with biological processes, cellular components, and functions were identified to be differentially methylated in buffalo bulls with differential fertility status. The identified differentially methylated genes were found to be involved in germ cell development, spermatogenesis, capacitation, and embryonic development. The observations hint that methylation defects of sperm DNA may play a crucial role in determining the fertility of breeding bulls. This growing field of sperm epigenetics will be of great benefit in understanding the graded fertility conditions of breeding bulls in commercial livestock production system.     

Dynamics of exogenous kallikrein-stimulated bovine sperm motility in the presence of seminal plasma kallikrein

Sperm motility is known to be activated and maintained by kallikrein contained within the seminal plasma. We studied the relationship between the levels of seminal plasma kallikrein activity and in vitro exogenous kallikrein-induced sperm motility enhancement. Semen samples were collected from Holstein-Friesian bulls and grouped on the basis of the initial total sperm motility into Group I with > 60 % (mean 75.3 +/- 1.8 %, n = 25), and Group II with < 60 % (mean 51.2 +/- 1.7%, n = 25). Seminal plasma kallikrein activity was measured with the aid of the specific chromogenic substrate S-2266. In Group I the mean activity was 0.983 +/- 0.042 microkat/L, and in Group II it was 0.805 +/- 0.063 microkat/L (P < 0.05). Then each semen sample was divided into a control and an experimental subgroup treated with 16.7 microkat/L of hog pancreatic kallikrein. Total sperm motility was monitored at 1-h intervals. It was found that the addition of exogenous kallikrein stimulated the sperm motility in both groups but in the 4th h after treatment the difference in sperm motility between the experimental and control subgroups of Group I was 12.4 % whereas in Group II it was 21.7 %. We concluded that adding exogenous kallikrein in vitro to semen samples with lower kallikrein activity in the seminal plasma enhanced total sperm motility more than adding it to ejaculates with higher levels of endogenous kallikrein activity.     

Effects of pulsed electromagnetic field on intervertebral disc cell apoptosis in rats

Despite numerous studies on pulsed electromagnetic field (PEMF) application, its effects of PEMF on intervertebral disc (IVD) have not yet been investigated in vivo. Accordingly, the effects of PEMF upon IVD in rats were evaluated through molecular surveys. Rats were divided into six groups: Group I and II were exposed to low and high frequency of PEMF (LF and HF, respectively). Group III and IV underwent induced disc degeneration and were exposed to low and high frequency of PEMF (LF/IDD and HF/IDD, respectively). Group V underwent induced disc degeneration (IDD), and group VI was control. The values of caspase 3, Bax, Bcl-2 and β-actin band density, as cell apoptotic markers, were obtained from band densitometry. Our results showed that the value of cleaved caspase-3 of cells and Bax/Bcl-2 ratio in IDD group increased significantly compared to the control group (p?p?相似文献   

Bull effect on the reproductive performance of mature and first calf-suckled Zebu cows in the tropics

This experiment was designed to determine if continuous bull exposure of heifers 2 wk post partum to a first calf (30 to 36 m old) or mature (42 to 72 m old) Zebú Brahman suckled cows reduces the interval to first estrus and the number of days open. Post partum first calf cows were randomly assigned to be exposed continuously to epidectomized bulls with surgical ablation of the dorsal ligament of the penis (Group 35 FBE, n = 25) or not exposed to bulls (Group FNE, n = 22). Post partum mature cows were likewise assigned to be continuously exposed (Group MBE, n = 24) or not exposed to bulls (Group MNE, n = 25). Milk samples were collected twice a week for progesterone assays to determine resumption of ovarian cyclic activity. All the cows were artificially inseminated for 120 d while in their respective treatment groups. The first postpartum estrus percentages were 60 < 90 d, 24.4%; and > 90 d, 33.7%. The interval to first estrus f-or Groups FBE and FNE was 75.4 and 104.2 d, respectively, and differed significantly (P < 0.01). For Groups MBE and MNE the interval to first estrus was 76.3 and 95.0 d, respectively (P < 0.05). The number of open days for Groups FBE and FNE was 77.3 and 114.9, respectively (P < 0.01); while for Groups MBE and MNE it was 73.0 and 98.6 d, respectively (P < 0.01). The results of the present study indicate that introduction of bulls to first calving cows or mature Zebú Brahman cows after parturition provides a practical and economical option for reducing the extended post partum anestrus interval and days open under tropical conditions.