Bovine Seminal Plasma Research Articles

Protein profile and functionality of spermatozoa from two semen collection methods in Bali bulls

The aim of this study was to evaluate the effect of two semen collection methods (electro-ejaculation [EE] and transrectal massage [RM]) on in vitro sperm functionality and protein composition of seminal plasma in Bali bulls. Ten untrained Bali bulls were selected for semen collection by EE and RM. Parameters analysed were acrosome and plasma membrane integrity, sperm motility (by CASA), normal morphology, functionality (sperm penetration assay), acrosome reaction, total protein content and protein profiles (by 2D-PAGE). Bulls collected by RM had a higher (p<0.05) percentage of spermatozoa with intact acrosome and plasma membrane, functionality and individual motility, and a lower proportion of seminal plasma, total protein content and lower ratio of low molecular weight proteins than those collected by EE. Analysis of 2D-PAGE gel detected about 116 spots in the range of 10–250kDa and isoelectric points (pI) ranging from 3 to 10. Approximately 52% of seminal plasma protein spots were represented by four major protein fractions with molecular weights around 37–45kDa (15.66%), 25–30kDa (12.46%), 14–16kDa (11.73%) and 12–15kDa (11.52%). Ten of the seminal plasma proteins identified by mass spectrometry belonged to major bovine seminal plasma proteins. A very significant finding in this study was related to the two proteins identified, PGK and PLA2, with MW of approximately 37–40 and 50–55kDa and pI of 8.5–8.8 and 5.2–6.0, respectively. These two protein spots can only be detected in the seminal plasma of ejaculates obtained through RM. In conclusion, semen quality as examined by in vitro sperm functionality was found to be better in RM than EE samples after treatment with heparin and calcium ionophore A-23187. In addition some low molecular weight proteins were up-regulated in the seminal plasma obtained from the EE method.

Bioactivity of ovulation inducing factor (or nerve growth factor) in bovine seminal plasma and its effects on ovarian function in cattle

To understand the role of ovulation-inducing factor (or nerve growth factor) (OIF [NGF]) in bovine seminal plasma, we (1) used an in vivo llama bioassay to test the hypothesis that bovine seminal plasma induces ovulation and CL development in llamas similar to that of llama seminal plasma when the dose of seminal plasma is adjusted to ovulation-inducing factor content (experiment 1) and (2) determined the effect of bovine seminal plasma on the interval to ovulation and luteal development in heifers (experiment 2). Within species, seminal plasma was pooled (n = 160 bulls, n = 4 llamas), and the volume of seminal plasma used for treatment was adjusted to a total dose of 250 μg of ovulation-inducing factor. In experiment 1, mature female llamas were assigned randomly to four groups and treated intramuscularly with either 10 mL of PBS (negative control, n = 5), 50-μg GnRH (positive control, n = 5), 6-mL of llama seminal plasma (n = 6), or 12 mL of bull seminal plasma (n = 6). Ovulation and CL development were monitored by transrectal ultrasonography. In experiment 2, beef heifers were given a luteolytic dose of prostaglandin followed by 25-mg porcine LH (pLH) 12 hours later to induce ovulation. Heifers were assigned randomly to three groups and given 12 mL bovine seminal plasma intramuscularly 12 hours after pLH treatment (n = 10), within 4 hours after ovulation (n = 9), or no treatment (control, n = 10). Ovulation was monitored by ultrasonography every 4 hours, and the CL development was monitored daily until the next ovulation. In experiment 1, ovulation was detected in 0/5, 4/5, 4/6, 4/6 llamas in the PBS, GnRH, llama seminal plasma, and bovine seminal plasma groups, respectively (P < 0.05). Luteal development was not different among groups. In experiment 2, the interval to ovulation was more synchronous (range: 4 vs. 22 hours; P < 0.0001) in heifers treated with seminal plasma before ovulation compared with the other groups. Luteal development was not different among groups; however, plasma progesterone concentrations tended to be greater in the postovulation treatment group compared with other groups. In summary, results confirmed the presence of bioactive ovulation-inducing factor in bull seminal plasma and supported the hypothesis that bovine and llama seminal plasma have similar ovulatory effects, using a llama bioassay. Treatment with bovine seminal plasma resulted in greater synchrony of ovulation in heifers pretreated with pLH. Plasma progesterone concentration tended to be higher in heifers given bovine seminal plasma within 4 hours after ovulation, suggesting that bovine ovulation-inducing factor is luteotrophic.

Biophysical characterization of the interaction of O-acylcholines with the major bovine seminal plasma protein, PDC-109.

Thermodynamic parameters characterizing the interaction of O-lauroylcholine (OLC) and O-myristoylcholine (OMC) with PDC-109 have been determined by isothermal titration calorimetric studies. Titration of OLC or OMC with PDC-109 resulted in exothermic peaks, the magnitude of which decreased with subsequent injections, showing saturation of the binding sites at high protein concentration and indicating that the binding process is associated with negative enthalpy change (ΔH° < 0). Association constant (Ka), Stoichiometry (n), and thermodynamic parameters, ΔH°, ΔS° and ΔG° for the binding of OLC to PDC-109 have been obtained at different temperatures, with the values obtained at 25 °C being 5.08 × 105 M−1, 0.47, −4.46 kcal mol−1, 11.13 cal mol−1 K−1 and −7.78 kcal mol−1, respectively. Values of thermodynamic parameters obtained at different temperatures indicate that the binding of PDC-109 to O-lauroylcholine is governed by a negative enthalpy contribution. The interaction of O-lauroylcholine with PDC-109 was also studied by monitoring changes in the protein intrinsic fluorescence emission intensity. The association constant, Ka, for the interaction of PDC-109 with O-lauroylcholine from fluorescence studies at 25 °C was estimated to be 5.46 × 104 M−1, which is somewhat lower than the Ka value determined by ITC. Circular dichroism spectroscopic studies indicate that interaction of O-lauroylcholine with PDC-109 induced changes in the secondary and tertiary structure of PDC-109. Differential scanning calorimetric studies revealed that the thermal unfolding temperature of PDC-109 increases in the presence of OMC, indicating that binding of O-acylcholines stabilizes the protein structure.

Desalted and lyophilized bovine seminal plasma delays induction of the acrosome reaction in frozen-thawed bovine spermatozoa in response to calcium ionophore

Cryopreservation is partially damaging and induces capacitation-like changes in spermatozoa. Seminal plasma (SP) contains a variety of biochemical components, such as protein and lipids, which are specific for the regulation of sperm cell function including those effective for decapacitation of spermatozoa. Therefore, this study tested the hypothesis that desalted and lyophilized SP could prevent premature capacitation (cryocapacitation) of Japanese Black bull spermatozoa. Seminal plasma was desalted by using Sephadex G-25 desalting column and lyophilized before added to semen extender at final concentrations 0, 2.5, 12.5, and 25 mg/mL. Frozen-thawed sperm progressive motility, acrosomal integrity, abnormal morphology, and the calcium ionophore A23187–induced acrosome reaction were assessed. Protein and lipid compositions in SP were analyzed by SDS-PAGE and thin-layer chromatography, respectively. The results revealed that progressive motility, intact acrosome, and abnormal morphology were not substantially modified by addition of SP. Stimulation of spermatozoa with calcium ionophore A23187 resulted in a time-dependent induction of the acrosome reaction, which was delayed by the desalted and lyophilized SP. There was no difference in the protein profile of SP before and after gel filtration. In total, 19 protein bands with molecular masses ranging from 5.2 to 185.8 kDa were detected and those of 185.8, 80, 34, 20.8, 18.8, 17.5, and 10 kDa were considered as novel proteins. Neutral lipids and phospholipids before and after gel filtration were the same, and the detected neutral lipid spots were monoacylglycerol, cholesterol, 1,2- and 1,3-disaturated diacylglycerol, 1,2- and 1,3-saturated, unsaturated diacylglycerol, whereas the detected phospholipid spots were sphingomyelin, phosphatidylcholine, phosphatidylserine, and three species of phosphatidylinositol, phosphatidylethanolamine, cerebroside, and polyglycerol phosphatide. The results suggest that premature capacitation during freeze-thaw processes could be reduced by adding desalted and lyophilized SP.

Lipid Selectivity in Lipid Efflux Induced by Proteins and Peptides

Some proteins and peptides have the ability to extract lipids from membranes. In some systems, such as some toxins, it leads to cell death. In other systems, this process is vital. For example, bovine seminal plasma contains phosphocholine-binding proteins that associate with sperm membranes upon ejaculation and cause lipid efflux; this effect modifies the membrane lipid composition, an essential step in the maturation process of fertile sperm. In general, the affinity of these peptides/proteins for membranes and their impact on membrane permeability are well characterized. However the induced lipid efflux and particularly its lipid specificity are much less investigated. Using model membranes with different compositions, we identified the lipid selectivity of the membrane lipid efflux of two systems. First, we characterized the lipid efflux induced by BSP1, the most abundant Binder-of-Sperm protein in bull seminal plasma. The protein binding displays a distinct affinity for phosphatidylcholine. However, even though the protein anchoring on membranes is PC-specific, we demonstrated that the lipid extraction is practically without any lipid specificity; BSP1 molecules solubilise a lipid patch to form small complexes with a stoichiometry of 10-15 lipids per protein. Second, we characterized the lipid specificity of lipid efflux induced by melittin, a helical peptide with a secondary amphipathic character. Phosphatidylethanolamine, and cholesterol, two molecular species ordering bilayers, reduce the membrane affinity of melittin. We determined that, in parallel, the lipid fraction extracted by melittin is depleted of these lipids. It is proposed that the peptide accumulates in regions of bilayers where the bilayers are less ordered and, when its concentration is sufficient, detaches this patch from the membrane. These findings illustrate the need to investigate globally lipid efflux processes, from the peptide/protein binding to the mixed lipid-peptide/protein self-assembly formation.

An Overview on Binder of Sperm Proteins of Cattle

Bovine seminal plasma is a protein rich fluid, which plays major role in the fertility of sire. Proteins are derived from blood as well as accessory sex glands. Binder of sperm proteins are major proteins in seminal plasma characterized by their binding ability to heparin like glycosaminoglycans. These proteins destabilize the membrane of spermatozoa by interacting with glycosaminoglycans of female reproductive tract. Relatively much less proteomic has been done in seminal fluid as compare to other protein rich fluids. The functional and expression pattern studies of bulls with different fertility abilities can provide insights into the selection of sires.

Impact of Seminal Chemical Elements on the Oxidative Balance in Bovine Seminal Plasma and Spermatozoa.

Mutual relationships between selected chemical elements (Na, K, Fe, Cu, Mg, and Zn), basic motility characteristics (motility and progressive motility), and markers of the oxidative balance (superoxide dismutase, catalase, glutathione, albumin, and malondialdehyde) were investigated in bovine seminal plasma and spermatozoa. Computer assisted sperm analysis was used to assess the motility parameters; mineral concentrations were determined by the voltammetric method and flame absorption spectrophotometry; antioxidants and malondialdehyde were evaluated by UV/VIS spectrophotometry. Concentrations of chemical elements in both seminal fractions were in the following descending order: Na > K > Zn > Mg > Fe > Cu. Higher amounts of all minerals and nonenzymatic antioxidants were detected in the seminal plasma (P < 0.01; P < 0.001), while higher MDA concentration and activity of enzymatic antioxidants were recorded in the cell lysates (P < 0.01; P < 0.001). Na, Fe, Cu, Mg, and Zn were positively correlated with the motility and antioxidant parameters (P < 0.05; P < 0.01; P < 0.001). Inversely, K exhibited the positive associations with malondialdehyde (P < 0.05). This study demonstrates that most chemical elements are integral components of bovine semen and are needed for the protection against oxidative stress development.

The impact of lead and cadmium on selected motility, prooxidant and antioxidant parameters of bovine seminal plasma and spermatozoa

The aim of this study was to investigate the effects of lead (Pb) and cadmium (Cd) content on basic motility characteristics (motility-MOT, progressive motility-PROG) as well as selected markers of the prooxidant-antioxidant balance (catalase-CAT, glutathione-GSH, malondialdehyde-MDA) in bovine seminal plasma and spermatozoa. Twenty five semen samples were collected from breeding bulls and used in the study. Motility analysis was carried out using the Computer Assisted Sperm Analysis (CASA) system. The samples were centrifuged, fractions of seminal plasma and spermatozoa were separated, lysates were prepared from the sperm cell fractions. Pb and Cd concentrations were determined by the voltametric method (ASV), antioxidants and MDA were analyzed by UV/Vis spectrophotometry. The analysis showed that the average concentration of Pb in the seminal plasma was 0.23 ± 0.02 μg/mL, while its amount in the sperm cells was significantly higher (0.41 ± 0.07 μg/mL; P < 0.05). The average Cd content in bovine seminal fractions was similar and non-significant: 0.09 ± 0.01 μg/mL in the seminal plasma and 0.11 ± 0.01 μg/mL in spermatozoa (P > 0.05). The correlation analysis revealed that both heavy metals were significantly negatively correlated with MOT and PROG (P < 0.05; P < 0.01; P < 0.001), CAT (P < 0.05; P < 0.01) as well as GSH (P < 0.05; P < 0.01) but significantly positively associated with MDA as the marker of lipid peroxidation (P < 0.05; P < 0.01). Moreover the samples were categorized in three quality groups (Excellent, Good, Moderate) according to their motility values. The lowest Pb, Cd and MDA concentrations but the best antioxidant characteristics were found in samples of the best quality, moderate quality samples exhibited the highest Pb, Cd and MDA content together with the worst antioxidant capacity. This study demonstrates that Pb and Cd are serious toxic elements, which are able to increase the risk of seminal oxidative stress development and a subsequent decrease of male fertility.

Sequestration of PDC‐109 Protein by Specific Antibodies and Egg Yolk Cryoprotects Bull Spermatozoa

PDC-109, one of the most abundant proteins in bovine seminal plasma, has detrimental effect on spermatozoa in a time- and concentration-dependent manner. Therefore, we hypothesized that sequestration of detrimental protein from ejaculates would be beneficial following cryopreservation of sperm cells. To this aim, we evaluated the effect of sequestration of PDC-109 either by anti-PDC-109 antibodies (Ab) or egg yolk (EY) alone or by the synergistic action of EY+Ab in minimizing cryoinjury to bull spermatozoa. PDC-109 protein was purified by applying two-step chromatography procedures. The purified protein was injected in rabbits to raise antibodies which were isolated using ion-exchange chromatography. After checking the Ab cross-reactivity, they were quantitated and added to ejaculates, either alone or in addition to EY in Tris-glycerol (TG) extender. Thus, ejaculates were processed in extender containing EY+TG (group I), Ab+TG (group II) or EY+Ab+TG (group III). Semen quality parameters (SQPs) viz. viability and acrosome integrity (FITC-PSA), cryoinjury to spermatozoa (chlortetracycline, CTC assay) and in vitro fertility of protein-sequestered-semen (zona-penetration assay) were evaluated. A significant (p<0.05) improvement in post-thaw SQPs as well as in non-capacitated spermatozoa observed at pre-freeze and post-thaw stages of cryopreservation in group III compared with other groups indicated reduction in protein-mediated cryoinjury. From this study, it can be concluded that sequestration of PDC-109 by synergistic action of EY+Ab as compared to either of them alone significantly improve sperm quality and minimize cryoinjury to bull spermatozoa upon storage at ultra-low temperatures.

Characterization of the cDNA and in vitro expression of the ram seminal plasma protein RSVP14

In previous studies we have shown that seminal plasma (SP) proteins can prevent and repair cold-shock membrane damage to ram spermatozoa. Three proteins of approximately 14, 20 and 22kDa, mainly responsible for this protective ability, were identified in ram SP. They are exclusively synthesized in the seminal vesicles and, consequently, named RSVP14, RSVP20 and RSVP22. The aim of this study is to characterize and express the RSVP14 gene to provide new insights into the mechanisms through which SP proteins are able to protect spermatozoa. Additionally, a first approach has been made to the recombinant protein production. The cDNA sequence obtained encodes a 129 amino acid chain and presents a 25-amino acid signal peptide, one potential O-linked glycosylation site and seven phosphorylation sites on tyrosine, serine and threonine residues. The sequence contains two FN-2 domains, the signature characteristic of the bovine seminal plasma (BSP) protein family and related proteins of different species. More interestingly, it was shown that RSVP14 contains four disulphide bonds and a cholesterol recognition/interaction amino acid consensus (CRAC) domain, also found in BSP and similar proteins. Analysis of the relationships between RSVP14 and other mammalian SP proteins revealed a 76–85% identity, particularly with the BSP protein family. The recombinant protein was obtained in insect cell extracts and in Escherichia coli in which RSVP14 was detected in both the pellet and the supernatant. The results obtained corroborate the role of RSVP14 in capacitation and might explain its protective effect against cold-shock injury to the membranes of ram spermatozoa. Furthermore, the biochemical and functional similarities between RSVP14 and BSP proteins suggest that it might play a similar role in sperm functionality.

HSP-1/2, a major protein of equine seminal plasma, exhibits chaperone-like activity

The major bovine seminal plasma protein, PDC-109 exhibits chaperone-like activity (CLA) against a variety of target proteins. The present studies show that the homologous protein from equine seminal plasma, HSP-1/2 also exhibits CLA and inhibits the thermal aggregation of target proteins such as lactate dehydrogenase, and DTT-induced aggregation of insulin in a concentration-dependent manner. Phosphorylcholine binding inhibited the CLA of HSP-1/2, suggesting that aggregation state of the protein is important for this activity. These results demonstrate that HSP-1/2 functions as a molecular chaperone invitro, and suggest that it may protect other proteins of equine seminal plasma from unfolding/misfolding or aggregation. These results suggest that homologous proteins from the seminal plasma of other mammals also exhibit CLA, which will be physiologically relevant.

Chemical and physical requirements for lipid extraction by bovine binder of sperm BSP1

The bovine seminal plasma contains phosphocholine-binding proteins, which associate to sperm membranes upon ejaculation. These binder-of-sperm (BSP) proteins then induce a phospholipid and cholesterol efflux from these membranes. In this work, we determined physical and chemical parameters controlling this efflux by characterizing the lipid extraction induced by BSP1, the most abundant of BSP protein in bull seminal plasma, from model membranes with different composition. The model membranes were formed from binary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (Lyso-PC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) or cholesterol. The modulation of BSP1-induced lipid extraction from membranes by their chemical composition and their physical properties brings us to propose a 3-step extraction mechanism. First, the protein associates with membranes via specific binding to phosphocholine groups. Second, BSP1 penetrates in the membrane, essentially in the external lipid leaflet. Third, BSP1 molecules solubilize a lipid patch coming essentially from the outer lipid leaflet, without any lipid specificity, to ultimately form small lipid/protein auto-assemblies. The stoichiometry of these complexes corresponds to 10–15 lipids per protein. It is also shown that fluid-phase membranes are more prone to BSP1-induced lipid extraction than gel-phase ones. The inhibition of the lipid extraction in this case appears to be related to the inhibition of the protein penetration in the membrane (step 2) and not to the protein association with PC head groups (step 1). These findings contribute to our understanding of the mechanism by which BSP1 modify the lipid composition of sperm membranes, a key event in sperm capacitation.

Influence of Calcium on Milk Protein Aggregation and Binding Dynamics Between Binder of Sperm (BSP) Proteins and Milk Proteins.

Over the years, skim milk has been used as extender for cryopreservation of semen from domestic animals due to its protective role against cold shock. The protective effect is attributed to its protein fraction and approximately 80% of the total milk proteins are casein micelles. In skim milk, calcium ions are associated with casein micelles and also responsible for their internal stability. Calcium forms linkages between proteins as colloidal calcium phosphate or it directly binds to caseins. Recent studies have shown that major proteins of the bovine seminal plasma, named Binder of Sperm (BSP) proteins as well as their homologues from stallion, boar and ram interact with the constituents of milk-based extenders. This interaction is implicated in sperm protection during cooling and storage of semen. Thus, the aim of this study was to investigate if calcium ions modulate the interaction between milk proteins and BSP proteins from animals of agricultural importance. In order to evaluate such interaction, heated skim milk was incubated with seminal plasma proteins from bulls, stallions, boars and rams. Then, these solutions were loaded onto Sepharose CL-4B column previously equilibrated with buffer-C (20 mM Tris, 50 mM NaCl, 10 mM CaCl2, 0.02% sodium azide, pH 7.4). The proteins were eluted with buffer-C and fractions were analysed by immunobloting. In parallel, seminal plasma proteins alone was used as controls and subjected to gel filtration chromatography in buffer-C. In the presence of calcium, casein micelles and whey protein aggregated and co-eluted. Bull, stallion and boar BSP proteins also co-eluted with milk protein aggregates in the presence of calcium. These results suggest that, in these species, calcium modulates protein-protein interaction as a result of an increase in milk protein aggregation. In contrast, ram BSP proteins did not bind to milk protein aggregates, suggesting that ram BSP proteins may differ in structural organization as compared to the bull, boar and stallion BSP homologues. In conclusion, our results indicate that calcium influences the formation of larger casein aggregates and the binding of bull, stallion and boar BSP proteins to milk proteins. These findings are crucial to understand the mechanisms of sperm protection by extender components and for the development of novel extenders. (Supported by CAPES - Brazil and NSERC of Canada)

Selected heavy metals versus antioxidant parameters in bull seminal plasma – A comparative study

To investigate the effects of lead (Pb) and cadmium (Cd) content on basic motility characteristics (motility, progressive motility) and selected antioxidant parameters (total antioxidant status - TAS, superoxide dismutase - SOD, albumin - ALB) in the bovine seminal plasma semen samples were collected from breeding bulls and used in the study. Motility analysis was carried out using the Computer Assisted Sperm Analysis (CASA) system. Subsequently, the samples were centrifuged and fractions of seminal plasma were collected. Pb and Cd concentrations were determined by the voltametric method (ASV), antioxidant parameters were analyzed by UV/VIS spectrophotometry using commercial kits. The analysis showed that the average concentrations of the trace elements were 0.57 ± 0.01μg/mL for Pb and 0.11±0.01 μg/mL for Cd. The correlation analysis revealed that both heavy metals were negatively correlated with motility (r = −0.777; P < 0.001 for Pb and r = −0.786; P < 0.001 for Cd), progressive motility (r = −0.763; P < 0.001 for Pb and r = −0.792; P < 0.001 for Cd), TAS (r = −0.375; p > 0.05 and r = −0.334; P > 0.05, respectively), SOD (r = −0.746; P < 0.001 and r = −0.537; P < 0.05, respectively) as well as with ALB (r = −0.609; P < 0.01 and r = −0.699; P < 0.001, respectively). Moreover the samples were categorized in three quality groups (Excellent, Good, Medium) according to their motility values. The lowest Pb and Cd concentrations but the best antioxidant characteristics were found in samples of excellent quality, medium quality samples were described by the highest Pb and Cd concentration and the worst antioxidant power. This study demonstrates that Pb and Cd are serious toxic elements, which are able to increase the risk of oxidative stress development and a subsequent decrease of semen quality.

Top–down mass spectrometry reveals new sequence variants of the major bovine seminal plasma protein PDC‐109

The major protein of bovine seminal plasma, PDC-109, is a 109-residue polypeptide that exists as a polydisperse aggregate under native conditions. The oligomeric state of this aggregate varies with ionic strength and the presence of lipids. Binding of PDC-109 to choline phospholipids on the sperm plasma membrane results in an efflux of cholesterol and choline phospholipids, which is an important step in sperm capacitation. In this study, Fourier transform ion cyclotron resonance mass spectrometry was used to analyze PDC-109 purified from bovine seminal plasma. In addition to the previously known PDC-109 variants, four new sequence variants were identified by top-down mass spectrometry. For example, a protein variant containing point mutations P10L and G14R was identified along with another form having a 14-residue truncation in the N-terminal region. Two other minor variants could also be identified from the affinity-purified PDC-109. These results demonstrate that PDC-109 is naturally produced as a mixture of several protein forms, most of which have not been detected in previous studies. Native mass spectrometry revealed that PDC-109 is exclusively monomeric at low protein concentrations, suggesting that the protein oligomers are weakly bound and can easily be disrupted. Ligand binding to PDC-109 was also investigated, and it was observed that two molecules of O-phosphorylcholine bind to each PDC-109 monomer, consistent with previous reports.

Seminal Plasma Proteome: Promising Biomarkers for Bull Fertility

The seminal plasma is a mixture of secretions from the testis, epididymis and male accessory sex glands. A number of seminal plasma proteins are associated with male fertility but most of these have not been studied in detail till now. Recently, proteomics has been used to show the differences in proteins profile in seminal plasma from high and low fertility bulls. For example, osteopontin, phospholipase A2, P25b, acidic seminal fluid proteins, α-l-fucosidase and cathepsin D are positively correlated with fertility of bulls and may act as useful fertility marker (s), while lipocalin-type prostaglandin D synthase, spermadhesin Z13, clusterin and ubiquitin are negatively correlated with fertility in bulls. Bovine seminal plasma proteins in seminal plasma act like a double-edged sword and showed a quadratic association with bull fertility. The physiological roles of the metalloproteinase-2 (TIMP-2), ecto-ADP-ribosyltransferase 5, nuclobindin, Niemann-Pick C2 and epididymal sperm-binding protein 1 and their relationship to bull fertility need further studies. This review summarizes the physiological functions of proteins of seminal plasma and their relation to bull fertility.

Effect of glutathione and bovine seminal plasma in lactose extender on viability of swamp buffalo frozen semen.

The aim of the study was to investigate the effect on viability of frozen swamp buffalo semen of glutathione and bovine seminal plasma in lactose extender. Semen from two swamp buffalo bulls was collected twice weekly using an artificial vagina. Pooled, good-quality fresh semen was divided into three parts and centrifuged at 3,000 rpm for 15 minutes in preparation for three treatments-substitution of buffalo seminal plasma with zero, 50 or 100% bovine seminal plasma (BS0, BS50 and BS100, respectively). Each semen aliquot was then divided in two parts, on which was diluted with lactose extender containing 1 mM glutathione (GSH) and the other diluted with lactose extender without GSH (0 mM GSH). Extended semen from all six treatments was cooled to 5 o C and then frozen in 0.25 ml straws. Mean motility percentages 0 and 30 minutes post thaw (PTM 0′ and 30′) with GSH were 38.33 and 34.29%, significantly higher (P < 0.05) than treatments without GSH (31.67 and 25.95%). PTM 0′ and 30′ were also higher (P < 0.05) with no substitution of bovine seminal plasma (BS0) than when buffalo seminal plasma was replaced with bovine seminal plasma at either 50 or 100%. Averages were 40.00 vs 34.46 and 30.54% (BS0 vs BS50 and BS100) at thawing and 36.96 vs 28.36 and 25.36% 30 minutes post-thaw. Mean percentages of live sperm (LD), intact plasma membrane (MPU) and intact acrosomal membrane (TAU) at thawing were not significantly different with or without addition of GSH. However,at 30′ post thawing, TAU and MPU were significantly higher in GSH treatments than inthose without GSH: 61.50 vs. 58.19% (MPU) and 59.81 vs. 57.38% (TAU). Mean percentages of LD, TAU and MPU 30′post thawing were higher with no substitution ofbuffalo seminal plasma (BS0) (P < 0.05) than to BS50 and BS100 treatments. In conclusion, the addition of glutathione (GSH) improved the quality of frozen swamp buffalo semen, but the partial substitution of buffalo seminal plasma with bovine seminal plasmaprovided no beneficial effects. Key Words: Swamp buffalo, Semen, Antioxidant, Glutathione, Seminal plasma

128 BOVINE SEMINAL PLASMA INDUCES OVULATION IN LLAMAS

Ovulation-inducing factor (OIF) is a protein present in the seminal plasma of several species, including llamas, alpacas, pigs, cattle, sheep, horses and rabbits. In an initial study (Ratto et al. 2006 Theriogenology 66, 1102–1106), bovine seminal plasma induced ovulations in 26% (5/19) of llamas compared with 0% (0/19) in the placebo group, but induced proportionately less than in llamas treated with alpaca or llama seminal plasma (100%). It is important to highlight that treatments were based on volume of seminal plasma; the actual dose of OIF was unknown. In a later study (Tanco et al. 2011 Biol. Reprod. doi:10.1095/biolreprod.111.091876), OIF from llama seminal plasma had a dose-dependent effect on ovulation rate, corpus luteum (CL) diameter and progesterone production in llamas. The present study was designed to test the hypothesis that bovine seminal plasma induces ovulation and CL development in llamas comparable with that of llama seminal plasma, based on total dose of OIF. Within species, seminal plasma was pooled from 1 to 4 ejaculates per male (n = 145 bulls, n = 4 llamas). The concentration of OIF in the pooled seminal plasma was measured by radioimmunoassay and the volume of seminal plasma used for treatment was adjusted to reach a total dose of 250 μg of OIF. Mature female llamas were assigned randomly to 4 groups and given a single intramuscular dose of 10 mL of PBS (negative control, n = 5), 50 μg of gonadotropin-releasing hormone (GnRH; positive control, n = 5), 6 mL of llama seminal plasma (n = 6), or 12 mL of bull seminal plasma (n = 6). Ovulation and CL development were monitored by transrectal ultrasonography. The incidence of ovulation was compared among groups by Fisher's exact test. Nonserial data (i.e. follicle size at treatment, maximum CL diameter, day of maximum CL diameter and first day of CL detection) were compared among groups by ANOVA. The diameter of the preovulatory follicle at treatment did not differ among groups (P = 0.10). The incidence of ovulation was 0/5, 4/5, 3/6 and 4/6 in the groups treated with PBS, GnRH, llama seminal plasma and bovine seminal plasma, respectively (P &lt; 0.05). The incidence of ovulation did not differ among llamas treated with GnRH, llama seminal plasma, or bovine seminal plasma. Among the treatments that elicited ovulation, neither the maximum CL diameter nor the day of maximum CL diameter differed (P = 0.30 and P = 0.24, respectively). In addition, no difference was detected in the day of first detection of the CL (P = 0.25). Results document the bioactivity of OIF in the bovine seminal plasma of Bos taurus. These findings further support the notion that OIF is highly conserved among mammals and that seminal plasma exerts its effect in an OIF dose-related manner. This research was supported by the Natural Sciences and Engineering Research Council of Canada.

Correlation between sperm characteristics and testosterone in bovine seminal plasma by direct radioimmunoassay

The objectives of this study were to validate a non-extractive RIA for seminal testosterone and quantify the hormone using a solid-phase commercial kit, and study the correlation between testosterone in seminal plasma and sperm characteristics. Parallelism showed a correlation index r = 0.992 (Y = -5.47 + 1.073X; R² = 0.985), indicating that the non-extractive method presented is indicated particularly for assessment of testosterone when establishing comparisons between samples. Overall mean (±SD) of testosterone level was 0.60±0.65 ng/mL. Correlation was only found between the seminal concentrations of testosterone and pH of the semen.

Isothermal Titration Calorimetric Studies on the Interaction of the Major Bovine Seminal Plasma Protein, PDC-109 with Phospholipid Membranes

The interaction of the major bovine seminal plasma protein, PDC-109 with lipid membranes was investigated by isothermal titration calorimetry. Binding of the protein to model membranes made up of diacyl phospholipids was found to be endothermic, with positive values of binding enthalpy and entropy, and could be analyzed in terms of a single type of binding sites on the protein. Enthalpies and entropies for binding to diacylphosphatidylcholine membranes increased with increase in temperature, although a clear-cut linear dependence was not observed. The entropically driven binding process indicates that hydrophobic interactions play a major role in the overall binding process. Binding of PDC-109 with dimyristoylphosphatidylcholine membranes containing 25 mol% cholesterol showed an initial increase in the association constant as well as enthalpy and entropy of binding with increase in temperature, whereas the values decreased with further increase in temperature. The affinity of PDC-109 for phosphatidylcholine increased at higher pH, which is physiologically relevant in view of the basic nature of the seminal plasma. Binding of PDC-109 to Lyso-PC could be best analysed in terms of two types of binding interactions, a high affinity interaction with Lyso-PC micelles and a low-affinity interaction with the monomeric lipid. Enthalpy-entropy compensation was observed for the interaction of PDC-109 with phospholipid membranes, suggesting that water structure plays an important role in the binding process.