Cross comparison of seminal plasma proteins from cattle and buffalo (Bubalus bubalis)

The objective of this study was to evaluate seminal plasma proteins from cattle and buffalo (Bubalus bubalis), to identify differences between related species. Sixteen buffaloes and 16 cattle between 30 and 60 months of age were used. Semen collection was performed by electroejaculation, followed by macroscopic and microscopic subjective analyses. After analysis, the samples were centrifuged at 800 g for 10 min, and the supernatant (seminal plasma) was recentrifuged at 10,000 g for 30 min at 4°C. The total protein concentration was determined by the Bradford method, and the proteins were digested in solution for mass spectrometry (nLC-MS/MS). Multivariate statistical analysis was used to evaluate the proteomics results by non-hierarchical clustering the considering exponentially modified protein abundance index (emPAI). Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were used for clustering. Proteomics identified 78 proteins, and multivariate analysis showed 4 that were over-expressed in buffaloes (cystatin C, prosaposin, peptide YY and keratin type II cytoskeletal 5) and 9 in cattle (spermadhesin-1, seminal plasma protein PDC-109, ribonuclease 4, metalloproteinase inhibitor 2, acrosin inhibitor 1, seminal ribonuclease, C-type natriuretic peptide, angiogenin-1 and osteopontin). Among the proteins identified in seminal plasma, the C-type natriuretic peptide and metalloproteinase inhibitors were described for the first time in buffaloes. Some protease inhibitors were found over-expressed in buffaloes, and important proteins in seminal plasma of cattle were not identified or were found at lower expression levels in buffaloes, which can contribute to reproductive performance in this species.     

Label‐free proteome of water buffalo (Bubalus bubalis) seminal plasma

The study aimed to describe the Bubalus bubalis seminal plasma proteome using a label‐free shotgun UDMS^E approach. A total of 859 nonredundant proteins were identified across five biological replicates with stringent identification. Proteins specifically related to sperm maturation and protection, capacitation, fertilization and metabolic activity were detected in the buffalo seminal fluid. In conclusion, we provide a comprehensive proteomic profile of buffalo seminal plasma, which establishes a foundation for further studies designed to understand regulation of sperm function and discovery of novel biomarkers for fertility. MS data are available in the ProteomeXchange with identifier PXD003728.     

Comparison of two methods of seminal plasma removal on buffalo (Bubalus bubalis) sperm cryopreservation

Cryopreservation causes damage to spermatozoa, and methods minimizing this damage are therefore needed. Although much discussed, seminal plasma removal has become an alternative to improve sperm quality and viability after freezing and has been applied to different species in attempt to obtain good results. The objective of this study was to evaluate semen quality in buffaloes submitted to two methods for seminal plasma removal (filtration and centrifugation). Semen samples were collected from seven Murrah buffalo bulls (Bubalus bubalis) once a week for 8 weeks. Each ejaculate was divided into three groups: control (presence of seminal plasma), centrifugation and filtration. Sperm kinetics was evaluated with the computer‐assisted sperm analysis (CASA) system. Plasmalemma and acrosomal membrane integrity, mitochondrial membrane potential and reactive oxygen species (ROS) were measured by flow cytometry, and lipid peroxidation was evaluated by the thiobarbituric acid reactive substances (TBARS) assay. Seminal plasma removal did not improve sperm kinetics compared to the control group. Centrifugation increased the number of cells with damaged acrosomal membranes (0.77 ± 0.05) and filtration caused greater plasmalemma and acrosomal membrane damage (22.18 ± 1.07). No difference in the mitochondrial membrane potential was observed between groups. In contrast, ROS production was higher in the centrifugation group compared to the control and filtration groups, although no differences in TBARS formation were detected. In conclusion, seminal plasma removal did not improve the quality of thawed buffalo semen compared to control in terms of sperm kinetics, membrane integrity, mitochondrial membrane potential or lipid peroxidation.     

Paraoxonase 1 (PON1) activity in serum,follicular fluid and seminal plasma of sheep

This work aimed to describe the activity of paraoxonase 1 (PON1) in serum, follicular fluid and seminal plasma of sheep. Average serum PON1 activity was 286.8 ± 96.2 U/ml in females and 237.6 ± 18.9 U/ml in males. There was a positive correlation between PON1 activity in serum and follicular fluid in females, being twice higher in serum than in follicular fluid (148.8 ± 15.7 U/ml). PON1 activity in males’ serum was 10‐fold higher than in seminal plasma (21.18 ± 14.2 U/ml), and there was no correlation between PON1 activity in both compartments. Finally, this work suggests that PON1 activity of in sheep is higher compared to other mammalian species, and there is an association between PON1 in serum and follicular fluid only.     

Autoproteolytic activity in the seminal plasma of the domestic fowl

Acid and neutral proteolytic activity was found in chicken seminal plasma. This proteolytic activity can markedly change the plasma polypeptide pattern due to partial protein degradation.     

Immunoreactive prolactin, progesterone and luteinizing hormone in the seminal plasma of buffalo (Bubalus bubalis).

The presence of immunoreactive prolactin, luteinizing hormone (LH) and progesterone in buffalo seminal plasma is reported for the first time. Correlations were obtained between various semen attributes and the levels of the above immunoreactive hormones. Statistically significant (P < 0.01) negative correlations were found between the levels of immunoreactive prolactin in semen and sperm motility and viability. The coefficient of multiple linear correlation (R2) between the levels of immunoreactive progesterone, prolactin and LH in the seminal plasma and the various attributes of semen revealed that immunoreactive progesterone and prolactin showed stronger interactions than did LH. The biological significance of these immunoreactive hormones in semen is discussed.     

补肾生精散对种公牛精液品质和血浆生殖激素浓度的影响

选用10头么拉水牛种公牛,探讨添加补肾生精散对种公牛血浆促黄体素LH、促卵泡素FSH、睾酮T浓度和精液品质的影响.用药后血浆LH浓度上升,FSH和T浓度下降,种公牛鲜精量、鲜精活力、冻精活力有所改善,说明补肾生精散可改善水牛种公牛精液品质.     

Changes of fructose concentrations in seminal plasma and glucose and testosterone concentrations in blood plasma in rams over the course of a year

The present study was performed to examine seasonal changes in the fructose concentrations of seminal plasma and glucose and testosterone concentrations of blood plasma over the course of a year (from November 2004 to November 2005) using 5 Suffolk rams. Osmolality of the seminal plasma was also measured. The fructose concentrations in the seminal plasma increased as the breeding season approached, with the maximum in October (179.8 mg/dl) and the minimum in May (6.9 mg/dl), although there were no significant differences during the year. Osmolality of the seminal plasma in February (304 mOsm) was significantly (P<0.05) lower than in January (325 mOsm), July (327 mOsm), and August (325 mOsm). It was also significantly (P<0.05) lower in November (308 mOsm) than in January and August. The blood plasma glucose concentration in October (79.3 mg/dl) was significantly (P<0.05) higher than in January and February (43.2 and 43.7 mg/dl, respectively). The blood plasma testosterone (T) concentrations were significantly (P<0.05) higher in September (8.5 ng/ml) and October (10.2 ng/ml) than in other months. The fructose concentrations in the seminal plasma appeared to be related to the glucose and T concentrations in the blood plasma. These results show that fructose concentrations in the seminal plasma and blood plasma glucose and T concentrations tended to increase during the breeding season, with the highest concentrations in October.     

Estimation of endogenous levels of osteopontin,total antioxidant capacity and malondialdehyde in seminal plasma: Application for fertility assessment in buffalo (Bubalus bubalis) bulls

This study was attempted to identify subfertile bulls by quantifying the endogenous levels of osteopontin (OPN), total antioxidant capacity (TAC) and malondialdehyde (MDA) in seminal plasma of buffalo bulls. On the basis of conception rate, buffalo bulls were classified into two groups: high‐fertile (conception rate >50%) and subfertile bulls (conception rate <40%). A total of 100 ejaculates (10 ejaculates from each bull) were collected through artificial vagina method. The concentration of OPN, TAC and catalase (CAT) of high‐fertile bulls was found to be higher (p < .05) than that of subfertile bulls. Further, MDA level in seminal plasma was found to be lower (p < .05) in high‐fertile bulls compared with subfertile bulls. The fertility status had no effect on the superoxide dismutase (SOD) concentration in seminal plasma of both the groups. The levels of OPN (r = .678, p = 0.013) and TAC (r = .648, p = .042) were found to be positively correlated with bull fertility and the level of MDA (r = ?.718, p = .019) was found to be negatively correlated with bull fertility. However, the fertility of bulls was not found to be significantly correlated with SOD, CAT and sperm motility. In conclusion, seminal OPN, TAC and MDA tended to be more realistic in identification of subfertile bulls from breeding herds.     

Testosterone concentration in the seminal plasma of cocks

Testosterone concentrations in the seminal plasma of cocks ranged from 0.46 to 5.05 ng/ml and were substantially lower than in blood plasma. No significant variation was noted in seminal plasma testosterone concentrations during the light phase of the day, whereas the concentration in blood declined over this period. Spermatozoal concentration and seminal testosterone decreased in the third sample of the semen collected sequentially at 3 h intervals. Testosterone concentrations in seminal plasma (1.57 +/- 0.17 ng/ml) and in the semen from the ductus deferens (1.34 +/- 0.24 ng/ml) were not significantly different.     

Purification and quantification of lactoferrin in equine seminal plasma

Lactoferrin with a molecular mass of 80 kDa was purified from equine seminal plasma by heparin-Agarose affinity chromatography and Sephacryl S-200 gel filtration. Purified lactoferrin was found to be highly homogeneous on the bases of its migration as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and of the monospecificity of rabbit antibodies to the purified protein in immunoblotting of seminal plasma proteins. A sandwich enzyme-linked immunosorbent assay was developed for quantifying lactoferrin in equine seminal plasma. Seminal plasma lactoferrin concentrations in 23 normal stallions ranged from 42 to 453 microg/ml, with a mean value of 157 +/- 118 microg/ml (S.D.).     

Ram seminal plasma and fertility: results from an ongoing field study

The effects of partial replacement of ram semen diluent with ram seminal plasma on the fertility of ewes were studied. Crossbred Chios ewes (n = 152) were assigned to six groups. The oestrous cycles of the ewes were synchronised at the peak (Groups A, B, C and D) and at the end (Groups E and F) of the breeding season by means of intravaginal sponges impregnated with fluorogestone acetate (FGA) for 14 days. Four hundred IU of PMSG were injected intramuscularly at the time of sponge removal. Ewes of Groups A, C and E were artificially inseminated with ram semen diluted with skim milk extender, while those of Groups B, D and F with ram semen diluted with 50% skim milk and 50% ram seminal plasma. The addition of ram seminal plasma induced a significant increase (P < 0.05) in litter size in Groups B and D when compared with that of Groups A and C (1.85 and 1.88 vs. 1.39 and 1.52, respectively). This increase was not significant when insemination was performed at the end of the breeding season (2.0 vs. 1.4). These results indicate that the addition of seminal plasma can influence the fertility of ewes or the fertilising capacity of extended ram semen to some extent.     

Modulatory effects of bovine seminal plasma on uterine inflammatory processes

In this study, a simple model to simulate a uterine environment affected by subclinical endometritis was established by culturing isolated primary bovine uterine epithelial cells (pbUEC). Co-incubation of pbUEC and polymorphonuclear (PMN) granulocytes derived from peripheral bovine blood samples, was performed before testing the cell culture supernatant for production of interleukin-8 (IL-8) via ELISA. Cytokine secretion was only detectable after co-incubation of pbUEC with PMN, whereas neither pbUEC nor PMN alone generated IL-8 in relevant chemo attractive doses. Another objective was to examine the influence of bovine seminal plasma (SP) and vesicular gland fluid (VGF) on various functional parameters of PMN including cell viability, production of reactive oxygen species and chemotaxis. Analysis of these effects was conducted by flow cytometry. Viability of PMN was determined by staining the cells with propidium iodide. Seminal plasma was added to suspensions of PMN in increasing increments and resulted in a significant increase of cell membrane damaged PMN when using SP concentrations above 0.2%. The reactive oxygen species production of PMN suspensions, stimulated with phorbol-12-myristate-13-acetate, was significantly decreased by 30% up to 90% when adding 0.06-30‰ of either SP or VGF. The PMN transmigration induced by IL-8 was diminished by 50% when 0.4‰ of either SP or VGF were added. The results of this study indicate a potential regulatory impact of SP and VGF on inflammatory processes in the bovine uterus.     

Concentrations of oestrogens in blood plasma and seminal plasma of boars during the postpuberal period

Concentrations of oestrogens in the blood plasma and seminal plasma of mature boars are high. However, little is known about their concentrations after reaching sexual maturity. The aim of this study was to determine the concentration of oestrogens in blood plasma and seminal plasma of boars during the postpuberal period. Free and conjugated oestrone and oestradiol-17beta were determined by radioimmunoassay in blood from the testicular vein and artery, and peripheral circulation as well as in seminal plasma collected from 18 Polish Landrace boars. The animals were divided into three groups (n = 6) according to age (8, 12 and 16 months, respectively). Oestrone was predominant free and conjugated oestrogen. The highest values of oestrogens were measured in the testicular vein (p < or = 0.05). The concentrations of oestrogens in seminal plasma did not differ from those found in the peripheral circulation. An age-dependent increase in levels of all four oestrogens (p < or = 0.05) was observed. This can be associated with biochemical maturation of the reproductive system during the postpuberal period.