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Semen cooling and cryopreservation have revolutionized the field of reproductive biotechnologies. However, challenges persist in maintaining sperm quality and viability during these processes. The unsatisfactory results and the main problems are associated with low quality, viability, morphological, structural, and DNA integrity, changes in plasma membrane, ability to interact with female tract and decrease in sperm fertilization potential. The objective of this study was to determine the effect of seminal plasma proteins with different molecular weights on the kinetic parameters of dog spermatozoa during cold storage at 4 °C. The proteins were isolated from the dogs’ seminal plasma ranging between 6-200 kDa, separated in four fractions. The ejaculates (n=15) were collected from 6 heathy dogs which were pooled. Spermatozoa were extended with Tris medium alone (control) or with addition of one of the isolated protein fractions, and were then incubated at 4 °C for 2 hours. Sperm incubated with seminal plasma proteins ranging between 10-15 kDa had significantly higher total motility (37.01±2.98%), sperm progressiveness (15.97±1.91%), curvilinear line velocity (37.46±3.75 μm/s), linearity (26.18±1.00%), and straightness (45.94±2.03%) compared to the other groups incubated with higher molecular weight proteins and the control group. The findings of this study indicated that the use of certain proteins in seminal plasma can be beneficial for reducing the detrimental effect of cooling at 4 °C and preserving the viability of dog spermatozoa. It seemed that the presence of 10-15 kDa proteins from canine seminal plasma rendered the spermatozoa less amenable to the negative influence during cooling.

Semen cooling and cryopreservation have revolutionized the field of reproductive biotechnologies. However, challenges persist in maintaining sperm quality and viability during these processes. The unsatisfactory results and the main problems are associated with low quality, viability, morphological, structural, and DNA integrity, changes in plasma membrane, ability to interact with female tract and decrease in sperm fertilization potential. The objective of this study was to determine the effect of seminal plasma proteins with different molecular weights on the kinetic parameters of dog spermatozoa during cold storage at 4 °C. The proteins were isolated from the dogs' seminal plasma ranging between 6-200 kDa, separated in four fractions. The ejaculates (n=15) were collected from 6 heathy dogs which were pooled. Spermatozoa were extended with Tris medium alone (control) or with addition of one of the isolated protein fractions, and were then incubated at 4 °C for 2 hours. Sperm incubated with seminal plasma proteins ranging between 10-15 kDa had significantly higher total motility (37.01±2.98%), sperm progressiveness (15.97±1.91%), curvilinear line velocity (37.46±3.75 μm/s), linearity (26.18±1.00%), and straightness (45.94±2.03%) compared to the other groups incubated with higher molecular weight proteins and the control group. The findings of this study indicated that the use of certain proteins in seminal plasma can be beneficial for reducing the detrimental effect of cooling at 4 °C and preserving the viability of dog spermatozoa. It seemed that the presence of 10-15 kDa proteins from canine seminal plasma rendered the spermatozoa less amenable to the negative influence during cooling. [ABSTRACT FROM AUTHOR]

In this study the individual profiles of seminal plasma proteins (SPP) in rams of three breeds – Me-rino, Pleven Blackhead and Ile de France  were analysed. The study was carried out with three rams at 3, 6 and 10 years of age, grown and fed under similar conditions. Eighteen ejaculates (6 ejaculates from each ram) were evaluated by Sperm Class Analyzer. The total SPP concentration was measured spectrophotometrically. The separation and characterisation of SPP was performed by HPLC and one dimensional polyacrylamide gel electrophoresis (SDS-PAGE). There were no significant differences between the characteristics of ejaculates and the main kinematic parameters of the sperm in the breeds studied. Chromatograms showed specific profiles with 9, 10 and 11 protein peaks for Merino, Pleven Blackhead and Ile de France breeds, respectively. The total SPP concentration was the highest in the Pleven Blackhead breed and the lowest in Ile de France breed. The major parts of SPP in the three breeds were identical. The seminal plasma of Merino breed contained proteins with molecular mass of 30.3 kDa, 15.7 kDa and 15.2 kDa that were not present in the other two breeds. In the Ile de France and Pleven Blackhead samples only, two proteins with molecular masses of 39.7 kDa and 21.1 kDa, were observed. In conclusion, the detection of specific proteins can be used as a biological marker for sheep breed identification.

The present research was focused on the differentiation of specific proteins in the seminal plasma (SP) of two horse breeds  Holsteiner (n=4) and East Bulgarian (n=4) and their relation with indivi¬dual or breed characteristics, kinematic parameters of spermatozoa and the sperm head area. After CASA analysis of 8 ejaculates, no statistical differences in the kinematic parameters of the sperms between the two horse breeds were found out with the exception of the sperm head area (P

The objective of this study was to identify seminal plasma proteins associated with semen traits (mass motility (MM; %), live sperm (LS; %), abnormal sperm (AS; %), hypo-osmotic swelling test (Host; %), pH, colour, viscosity (Visco), ejaculate volume (VOL; ml), normal chromatin (NC; %), live sperm with reacted acrosome (LR; %), live sperm with unreacted acrosome (LU; %), dead sperm with reacted acrosome (DR; %), dead sperm with unreacted acrosome (DU; %)) in Egyptian Barki rams. Depending on MM%, the animals were divided into three different groups (high (≥ 80% MM), medium (50-80% MM) and low-fertility (≤50% MM) group). The seminal plasma proteins were separated using one-dimensional sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoresis technique based on their molecular weight. The actual means for MM, LS, AS, Host, pH, colour, Visco, VOL, NC, LR, LU, DR and DU were 69.93%, 94.40%, 6.76%, 20.14%, 6.94, 3.30, 3.04, 0.75 mL, 53.52%, 44%, 15.78%, 19.36% and 20.86%, respectively. The correlation coefficients between MM and each of LS (0.61) and NC (0.89) were highly positive and significant (P<0.05). Semen samples displayed a total of 8 distinct protein bands in the SDS-PAGE of seminal plasma proteins. The identified protein bands ranged in molecular weight from 14 to 248 kDa. The high and medium fertility ram groups showed six of the bands. However, all eight protein bands were seen in the group of low-fertility rams. It is notable that the two bands solely identified in this group are those with molecular weights of 29 and 35 kDa. Negative and high correlations (P<0.05) were observed between these two bands and each of MM and LS, these correlations were -0.87 and -0.36 for the 29 kDa band and were -0.62 and -0.56 for the 35 kDa band, respectively. The reduced motility and low fertility of the rams in this group may be caused by these two bands of protein. In contrast, there were positive and significant correlation between the bands of 14 kDa, 25 kDa, and 248 kDa and MM, LS, NC, and LR (P<0.05). Relevant correlations between mass motility and other seminal characteristics were found in Barki rams. Potential seminal plasma proteins have identified as biomarkers for sperm mass motility and other related seminal and fertility associated traits. [ABSTRACT FROM AUTHOR]

In this study the individual profiles of seminal plasma proteins (SPP) in rams of three breeds - Merino, Pleven Blackhead and Ile de France - were analysed. The study was carried out with three rams at 3, 6 and 10 years of age, grown and fed under similar conditions. Eighteen ejaculates (6 ejaculates from each ram) were evaluated by Sperm Class Analyzer. The total SPP concentration was measured spectrophotometrically. The separation and characterisation of SPP was performed by HPLC and one dimensional polyacrylamide gel electrophoresis (SDS-PAGE). There were no significant differences between the characteristics of ejaculates and the main kinematic parameters of the sperm in the breeds studied. Chromatograms showed specific profiles with 9, 10 and 11 protein peaks for Merino, Pleven Blackhead and Ile de France breeds, respectively. The total SPP concentration was the highest in the Pleven Blackhead breed and the lowest in Ile de France breed. The major parts of SPP in the three breeds were identical. The seminal plasma of Merino breed contained proteins with molecular mass of 30.3 kDa, 15.7 kDa and 15.2 kDa that were not present in the other two breeds. In the Ile de France and Pleven Blackhead samples only, two proteins with molecular masses of 39.7 kDa and 21.1 kDa, were observed. In conclusion, the detection of specific proteins can be used as a biological marker for sheep breed identification. [ABSTRACT FROM AUTHOR]

The present research was focused on the differentiation of specific proteins in the seminal plasma (SP) of two horse breeds - Holsteiner (n=4) and East Bulgarian (n=4) and their relation with individual or breed characteristics, kinematic parameters of spermatozoa and the sperm head area. After CASA analysis of 8 ejaculates, no statistical differences in the kinematic parameters of the sperms between the two horse breeds were found out with the exception of the sperm head area (P<0.05), which can be considered as a morphometric marker of breed affiliation. The values for rapid sperm in East Bulgarian and Holsteiners were 28.1±0.2 µm2 and 19.9±0.3 µm2 respectively. The chromatographic analysis demonstrated specific quantitative and qualitative protein content of the individual chromatographic peaks (11 for Holsteiner and 15 for the Eastern Bulgarian breed), with similarity to the basic proteins. Three specific proteins with a molecular mass of 76 kDa, 21.6 kDa and 24.3 kDa, were differentiated by SDS PAGE in the Holsteiner breed, whereas in the Eastern Bulgarian horse breed they had a lower protein mass - 30.1 kDa and 14.2 kDa and 12.6 kDa. In conclusion, differences in the specific protein profile of Holsteiner and Eastern Bulgarian horse breeds are individually and naturally determined without significant effect on sperm kinematics. The sperm head area was a breed-specific difference. [ABSTRACT FROM AUTHOR]

Researchers have been done to identify а proteins contains in canine seminal plasma and relate them to quality, sperm motility, vigor and their role on capacitating, acrosome reaction, hyperactivation and etc. In addition, determination of specific canine seminal plasma proteins serving as biomarkers of some reproductive diseases. It is known that proteins, like: spermadhesin protein family, osteiopontin, kallikrein, some heparin-binding proteins and etc. are associated with sperm quality and play an important role during fertilization. Also, there is a data that specific proteins have an effect on hyperactiovation on canine spermatozoa, gamete interaction and etc. Specific seminal plasma proteins have been identified as markers for some diseases. Thus the present literature review aims to append to more growing studies demonstrates that the canine seminal plasma proteins have a different impact and also to give new insight of aspects related to the wide roles of seminal plasma proteins and their effect and influence on fertilization process. [ABSTRACT FROM AUTHOR]

Seminal plasma proteins already demonstrated to reflect the testicular environment function and important regulatory mechanisms. However, it is crucial to understand which of these proteins participate in probable altered pathways in testicular germ cell tumours and after unilateral orchiectomy. In this study, we proposed to verify, by a multiplex approach, the levels of DNA damage and apoptosis pathways' proteins, in seminal plasma of men before and after unilateral orchiectomy, and also in control men. Comparing pre‐ and post‐orchiectomy groups, just the apoptosis pathways' proteins presented different levels, in which Bad was lower and Bcl2, Akt, caspase‐9, p53 and caspase‐8 were higher after orchiectomy. When comparing pre‐ and post‐orchiectomy groups with control, both presented lower levels of ChK1, Chk2, H2AX, p53 and p21, for DNA damage pathway. Regarding the apoptosis pathway, lower levels of JNK, Bcl2, Akt, caspase‐9, p53 and caspase‐8 and higher levels of Bad were observed before orchiectomy. The post‐orchiectomy group did not differ from controls, demonstrating a probable restoration on its proteins levels. We can conclude that testicular tumours can alter both of the assessed pathways, and its removal is associated with a probable restoration of the apoptosis pathway. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the effect of different percentage of seminal plasma proteins with different molecular weight on sperm motility and fertility parameters (farrowing rate (FR), number of live-born pigs (PBA) per litter and percentage of unsuccessful insemination). A total of 50 sperm-rich ejaculate fractions were collected (one per boar) using the gloved hand method. The quality parameters of the semen samples were first evaluated at the farm. Further assessment of sperm quality was performed on a CASA - computer assisted semen analysis by two competent operators. Seminal plasma protein fractions were obtained by AOAC - Association of Official Analytical Chemists as a chemical method. The assessment of reproductive performance was carried out based on collected data of three parameters in selected 9696 sows: FR, PBA per litter and percentage of unsuccessful insemination. Protein fractions were divided in to three groups (10 - 20kDa, 21 - 30kDa and 31 - 40kDa) Proteins with 10 - 20kDa did not have significant effect and correlation with analyzed parameters. Significant differences were recorded in farrowing rate between samples with up to 80 % compared to samples with 10% of proteins with 21 - 30kDa. Significant differences were recorded in unsuccessful insemination between samples with different percentage of proteins with 31 - 40kDa. Results of this study have shown the effect of different percentage of certain fraction of seminal plasma proteins on boar ejaculates fertility potential. [ABSTRACT FROM AUTHOR]

Yellow semen syndrome (YSS) is the most widely recognized problem among male turkeys. Yellow semen is of low quality and, when used for insemination, results in reduction of fertility and hatchability. Elevated level of serum albumin-like protein accession no. XP_003205725 is a characteristic feature of yellow seminal plasma suggesting albumin role in YSS pathology. However, knowledge regarding the expression of albumin in the reproductive tract in relation to YSS is very limited. The aim of this study was to identify albumin secretion and localization sites in the turkey reproductive tract in relation to YSS. Reproductive tract tissues and liver originating from turkeys producing white semen (WS) and YSS were used for analysis of albumin mRNA expression and its localization using immunohistochemistry. Moreover, albumin abundance in tissues, blood and seminal plasma was analyzed using two dimensional electrophoresis and western blot analysis. Albumin mRNA expression was found in all parts of the reproductive tract. Apart from the liver, the highest expression of albumin was found in the ductus deferens in YSS turkeys. The testicular spermatids, Leydig, and myoid cells and the epithelium of the epididymis and ductus deferens were the main secretion sites of albumin in the reproductive tract in turkeys. Higher albumin abundance was found in the reproductive tract and seminal plasma of YSS toms compared to WS toms. Our results demonstrated that germ cells from spermatocytes to spermatids, Leydig cells, and myoid cells synthesized and secreted albumin in turkey testis, and epithelial cells are the main secretion sites in epididymis and ductus deferens. Ductus deferens secretion of albumin seems to be mostly responsible for YSS. Over-secretion by the ductus deferens may be the main origin of albumin abundance in YSS semen. Knowledge regarding disturbances of albumin secretion in relation to YSS may be useful for future work on studies related to better understanding the molecular basis of YSS. [ABSTRACT FROM AUTHOR]

To date there is still no optimal biotechnology which ensures maximum preservation of the functional parameters of the spermatozoa from buffaloes, boars and dogs. The aim of this research is to study the biological potential of seminal plasma proteins that are specific only to ejaculates with high cryotolerance and good quality parameters of the spermatozoa. The motility and velocity parameters of the spermatozoa were assessed by computer-assisted sperm analysis. Seminal plasma proteins were separated by size-exclusion liquid chromatography and characterized by polyacrylamide gel electrophoresis and mass spectrometry. Based on the results obtained, sperm diluents and methods for biological evaluation of the fertilization potential of the spermatozoa from buffalo bulls, boars and dogs were created and proposed for practical application.

Semen is a heterogeneous and complex cell suspension in a protein rich fluid with various functions, some of them well known, other still unexplained. The aim of the current research is focused on canine seminal plasma proteins and their functional relationship with capacitation of spermatozoa. Attempts were made to identify canine seminal plasma proteins with effect on sperm capacitation and motility. High- Performance Liquid Chromatography was done and 4 seminal plasma protein fractions were obtained and further characterized by electrophoresis (7.6 kDa - 200 kDa). Computer-assisted sperm analysis after incubation of spermatozoa with separated seminal plasma proteins revealed that Fraction 1, consisting of high molecular weight proteins (70 - 200 kDa), could increase the percentage of spermatozoa with progressive and rapid motility. Incubation with seminal plasma proteins from Fraction 1 leads to a significant increase of VCL (Curvilinear velocity) and decrease of LIN (Linearity) of spermatozoa, when compared to the other test samples and the control sample. Proteins with high molecular weight probably have the most significant influence on the process of hyperactivation and capacitation of canine spermatozoa. [ABSTRACT FROM AUTHOR]

The objective of this study was to evaluate relationships between common semen quality estimates including sperm motility, sperm morphology, spontaneous capacitation status and seminal plasma proteins and boar fertility using heterospermic inseminations and subsequent paternity testing. All boars (n = 12) used in the study had excellent semen quality (≥70% normal sperm) that resulted in average farrowing rates and litter sizes of 88.9 ± 0.7% and 11.7 ± 0.1 pigs, respectively. Their ejaculates were combined to make heterospermic insemination doses in such a way that each boar was tested against all of his contemporaries. The proportion of piglets sired by each individual was used to separate boars into three fertility groups: High (71.6 ± 4.8%; n = 3); Medium (51.6 ± 3.8%; n = 6); and Low (25.2% ± 5.3%; n = 3). Ejaculates from High fertility boars had more motile sperm with normal acrosomes that moved faster in a straight-line and were more likely to undergo an acrosome reaction (p ≤ 0.05) compared with their counterparts in the Low fertility group. Ejaculates from High fertility boars contained the greatest concentrations of three seminal plasma proteins (25.9 kD/5.9 pI; 55.1 kD/4.8 pI; and 70.1 kD/5.2 pI; p ≤ 0.05), whereas concentrations of a 19.1 kD/6.8 pI were highest in semen from Low fertility boars (p ≤ 0.05). Multiple regression analyses indicated that concentrations of the 25.9 kD/5.9 pI seminal plasma protein explained 66% of the variation observed in the proportion of pigs sired within a litter among boars (p ≤ 0.00001). These results demonstrate that heterospermic inseminations and subsequent paternity testing is an effective technique for defining relationships between common semen quality tests and fertility, especially in situations where reproductive performance of all the boars is high. Motility, normal acrosome morphology, average linear velocity of motile sperm, and the proportion of sperm capable of an acrosome reaction were all positively associated with boar fertility. However, concentrations of a 25.9 kD/5.9 pI seminal plasma protein were the best single semen characteristic for ranking boars in terms of their fertility. [ABSTRACT FROM AUTHOR]

Background: Seminal plasma is a set of secretions from accessory male reproduction organs which influence spermatic functions. Climate changes that affect gametogenesis may produce low reproduction efficiency in bulls. Electrophoresis is a great help in the diagnosis of reproduction pathologies and in animal differentiation with regard to fertility within the context of climate changes. Current research aims at studying the influence of the rainy and dry periods in testicle and epididymis morphometry, semen characteristics and protein of seminal plasma by SDS-PAGE electrophoresis in extensively bred Nellore and Simental bulls. Materials, Methods & Results: Five Nellore and two Simental bulls, at mean age of four years, were employed. Semen collection was undertaken during the rainy (spring and summer) and dry (autumn and winter) at 15-day intervals. One hundred and fifty-four ejaculations were analyzed and scrotal, testicle and epididymis measurement was provided. Samples of semen plasma were centrifuged and conditioned at -196°C till electrophoresis processing. Proteins were extracted from 200 µL from each sample in an extraction buffer composed of 0.625 M Tris - HCl, pH 6.8; 2% SDS, 5% β - mercaptoethanol and 20% glycerol. Proteins were quantified and electrophoreses processed. Data were evaluated by analysis of variance and means compared by Tukey's test at 5%. The expression TV = 0.0396 x (average testicle length) x (scrotal perimeter)² was used for testicle volume (TV), with TV = 460.14 cm³ for the Simental breed during the rainy season and TV = 571.26 cm³ during the dry season. Nellore bulls showed TV = 524.75 cm³ during the rainy season and 515.13 cm³ during the dry season. TV increased in Simental bulls during the dry period, whereas Nellore breed increased during the rainy one. There was an increase (P < 0.05) in big and total defects during the rainy season for both breeds. Further, 30 kDa in the seminal plasma occurred in all samples for both breeds and seasons under analysis. Band 179 kDa occurred in 43% and 14% of samples from Nellore and Simental breeds respectively. Molecular weights of identified bands in the gels varied between 4 and 205 kDa. Discussion: Testicle length axis varied significantly between breeds at specific seasons. Variation may be due to adaptation and anatomic plasticity of the testicles within specific seasons of the year under analysis. TV rise in Simental bulls during the dry period may have contributed towards the decrease of minor and major spermatic defects with an improvement of semen quality. Spermatic motility during the dry period and total spermatic defects during the rainy season for Simental breed provided fall in semen quality. Morphological abnormalities of spermatozoa are lower in the dry season. Season affects testicle morphometry and protein composition of the bulls' seminal plasma. Protein band 179 kDa occurred in Nellore bulls during the two analyzed seasons, a fact that has not been described in the literature. [ABSTRACT FROM AUTHOR]

Abstract: Whole seminal plasma (SP) enhances the function and fertility of frozen/thawed ram sperm. The objective of the current study was to investigate whether SP proteins capable of binding to molecules from the sperm plasma membrane were conserved among ram breeds, and whether these proteins were sufficient to overcome cryopreservation-induced reductions in sperm quality. Whole ram SP, obtained from rams of various breeds, improved progressive motility of frozen/thawed sperm at all times evaluated (P < 0.05); however, it did not improve total motility (15 min, P = 0.480; 30 min, P = 0.764; and 45 min, P = 0.795). To identify SP proteins responsible for this effect, a new method was developed to retain SP proteins that bound specifically to the sperm membrane by immobilization of sperm membrane proteins. These proteins specifically bound to the sperm surface, especially the acrosomal region. Lactotransferrin, epididymal secretory protein E1, Synaptosomal-associated protein 29, and RSVP-20 were identified (mass spectrometry) in this fraction. The retained SP proteins fraction repaired ultrastructural damage of frozen/thawed sperm and, with the addition of fructose, significantly improved motility of frozen/thawed sperm. We concluded that SP proteins that bound to the sperm membrane were conserved among ram breeds, and that when added to frozen/thawed semen (along with an energy source), they repaired ram sperm damage and enhanced sperm motility. [Copyright &y& Elsevier]

The article addresses the allocation of seminal plasma (SP) proteins on cattle sperm that was unleashed through sexual intercourse. The study was performed by applying immunochemistry and confocal microscopy on semen of 5 adult bulls in terms of the binding patterns of the fluid. Results prove that substances including prostaglandin D-synthase, nucleobindin, and bovine SP protein BSP-A3 contributed in the proper distribution of bull semen.

Objective: To evaluate the effects of two sublethal doses of gossypol on biochemical, enzymatic, and electrolytic properties of male rabbit seminal plasma. Design: Retrospective study. Setting: A university-affiliated laboratory. Animal(s): Fifteen adult male New Zealand white rabbits. Intervention(s): Two sublethal doses of gossypol (4 and 20 mg per kilogram of body weight, every other day) were given orally. Main Outcome Measure(s): Biochemical, enzymatic, and electrolytic properties of male rabbit seminal plasma. Result(s): Seminal plasma total protein and albumin were statistically significantly increased by treatment with gossypol. Total lipids concentration was statistically significantly decreased by treatment with both doses of gossypol, whereas cholesterol was statistically significantly decreased by treatment with the low gossypol dose. Seminal plasma lactate dehydrogenase and alanine aminotransferase activities were statistically significantly decreased by treatment with the low dose of gossypol, whereas activities of these enzymes as well as of aspartate aminotransferase were statistically significantly increased by treatment with the high dose. Treatment with the high dose of gossypol statistically significantly reduced K+ concentration in the seminal plasma, whereas it statistically significantly increased Na+ and the Na+-K+ ratio. Conclusion(s): Seminal plasma values of total protein, total lipids, cholesterol, aspartate aminotransferase, and Na+ concentration returned to control levels after withdrawal of gossypol, whereas the effect of this drug on other parameters continued during the recovery period. [Copyright &y& Elsevier]

Abstract: The objective of this study was to evaluate the protein profiles of seminal plasma in buffalo bulls and to examine their correlation with semen characteristics. Semen of 10 buffalo bulls were collected by a bovine artificial vagina. Semen characteristics (motility, morphology, viability and concentration) were recorded. A part of the semen sample (1ml) was diluted by tris-egg yolk-glycerol extender, packed in French straws and was frozen in liquid nitrogen. The straws were later thawed and semen characteristics were compared with those of the fresh semen. Seminal plasma was harvested by centrifugation; treated with cold ethanol and then, underwent SDS-polyacrylamide gel electrophoresis (PAGE). Twenty five protein bands were identified on the gel, of which those of <35.5kDa were prominent (72% of the bands). Of these protein fractions, 24.5kDa was significantly correlated with sperm progressive motility in fresh and viability in frozen-thawed semen while 45kDa bands were correlated with abnormal morphology in frozen-thawed semen; 55kDa protein fractions were correlated with sperm viability of fresh semen. Progressive motility, viability and abnormal sperm morphology of frozen-thawed semen were highly correlated with these parameters in the fresh semen. In conclusion, seminal plasma protein fractions in buffalo bulls are similar to those reported in other animal species and have some correlations with semen characteristics before and after freezing. [Copyright &y& Elsevier]

Abstract: Chromogranin-A (CgA) is the most distributed member of the granin family. Chromogranins are soluble anionic glycoproteins, found in the majority of the neuroendocrine and neural cells, co-stored with other endocrine substances (like insulin, glucagon, FSH and LH or NPY) in secretory granules. Outside the cell, it has been suggested that this peptide or one of its fragments, obtained by proteolytic cleavage, could act in an autocrine or paracrine way, regulating either the cell function or the contractibility of vascular segments. The purpose of the present study is to determine the distribution of chromogranin-A in the structures of the bovine testis. Immunohistochemical analysis was performed employing the biotin–streptavidin-peroxidase immunostaining technique in tissue specimens obtained at a local abbatoir. A CgA expression was found in the germinal epithelium at several stages of differentiation. Generally the strongest positive reaction was consistently observed in the basal compartment of the seminiferous tubules, with spermatogonia presenting a dense granular immunostaining pattern; a less intense reaction was also consistently recorded in type II spermatocytes and in round spermatids, which showed a more scattered disposition of CgA-positive granules. Clusters of Leydig cells also displayed a faint and homogeneous cytoplasmatic immunoreactivity for chromogranin-A. These results demonstrate a widely distribution of CgA-positive cells in the organism, and its presence in the testis raises the possibility of its participation in the cohort of local factors involved in the regulation of spermatogenesis. [Copyright &y& Elsevier]

Rainbow trout (Oncorhynchus mykiss) spermatozoa were incubated in artificial sperm motility inhibiting saline solution (SMIS), in SMIS containing seminal plasma proteins or in pure seminal plasma. In SMIS containing the total seminal plasma protein fraction or the <50 kDa protein fraction or in pure seminal plasma, significantly higher motility rates and swimming velocities could be activated than in SMIS without seminal plasma proteins and in SMIS containing the >50 kDa protein fraction. These preliminary results indicated that seminal plasma proteins have physiological functions in prolongation and stabilization of sperm viability when using sperm motility as viability index. [Copyright &y& Elsevier]

This study aimed to characterize the protein composition of fractionated seminal plasma (SP) by liquid chromatography mass spectrometry (LC–MS/MS) analysis and investigate its effects on survival of frozen-thaw (FT) boar spermatozoa following storage. Seminal plasma (SP) was fractionated by gel filtration chromatography to give two fractions, SP1 with more than 40 kDa (>40 kDa) and SP2 with less than 40 kDa (<40 kDa). SP1 and SP2 were subjected to LC–MS/MS and bioinformatics analysis. Following cryopreservation, FT boar semen (n = 7) was thawed in Beltsville Thawing Solution (BTS), BTS + SP1 or BTS + SP2, stored at different periods and subjected to post-thaw (PT) quality assessment. A total of 52 and 22 abundant proteins were detected in SP1 and SP2, respectively. FN1, ANGPTL1, and KIF15 proteins were more abundance in SP1, whereas a high abundance of spermadhesins (PSP-I and PSP-II) was detected in SP2. Proteins of the fractionated SP were involved in various biological processes, such as cell motility and signal transduction. The dominant pathway of SP1 proteins was the apelin signaling pathway (GNA13, MEF2D, SPHK2, and MEF2C), whereas a pathway related to lysosome (CTSH, CTSB, and NPC2) was mainly represented by SP2 proteins. In most of the boars, significantly higher motility characteristics, membrane integrity, and viability were observed in FT spermatozoa exposed to SP1 or SP2 compared with BTS. The results of our study confirm that a combination of several proteins from the fractionated SP exerted beneficial effects on the sperm membrane, resulting in improved quality characteristics following PT storage. [ABSTRACT FROM AUTHOR]

The CUB domain is a widespread 110-amino-acid module found in functionally diverse, often developmentally regulated proteins, for which an antiparallel β-barrel topology similar to that in immunoglobulin V domains has been predicted. Spermadhesins have been proposed as a subgrou0 of this protein family built up by a single CUB domain architecture. To test the proposed structural model, we have analyzed the structural organization of two members of the spermadhesin protein family, porcine seminal plasma proteins I/II (PSP-I/PSP-II) heterodimer and bovine acidic seminal fluid protein (aSFP) homodimer, using differential scanning calorimetry, far-ultraviolet circular dichroism and Fourier-transform infrared spectroscopy. Thermal unfolding of PSP-I/PSP-II and aSFP were irreversible and followed a one-step process with transition temperatures (Tm) of 60.5°C and 78.6°C, respectively. The calorimetric enthalpy changes (ΔHcat) of thermal denaturation were 439 kJ/mol for PSP-I/PSP-II and 660 kJ/mol for aSFP dimer. Analysis of the calorimetric curves of PSP-I/PSP-II showed that the entire dimer constituted the cooperative unfolding unit. Fourier-transform infrared spectroscopy and deconvolution of circular dichroic spectra using a convex constraint analysis indicated that β-structure and turns are the major structural element of both PSP-I/PSP-II (53% of β-sheet, 21% of turns) and aSFP (44% of β-sheet, 36% of turns), and that the porcine and the bovine proteins contain little, if any, α-helical structure. Taken together, our results indicate that the porcine and the bovine spermadhesin molecules are probably all-β-structure folds, such as the Greek-key pattern characteristic of many cabohydrate-binding protein domains cannot be eliminated. Finally, the same combination of biophysical techniques was used to characterize the residual secondary structure of thermally denatured forms of PSP-I/PSP-II and aSFP, and to emphasize the aggregation tendency of these forms. [ABSTRACT FROM AUTHOR]

• Seminal plasma (SP) regulates immune responses in the reproductive tract via cytokines. • Cytokine production by endometrial cells may thus depend on bull fertility. • Endometrial cells in culture were challenged with SP from high and low fertility bulls. • TGF-β1, TGF-β2, TGF-β3, IL-6 and IL-8 were measured after challenge with SP. • More cytokine production was stimulated by SP from low fertility bulls than high. Seminal plasma (SP) regulates immune responses in the female reproductive tract through specific cytokines. It is not known whether SP from high fertility bulls (H) differs from SP from low fertility bulls (L). In this study, the cytokine response of bovine endometrial epithelial cells (bEEC) in culture was investigated after challenge with SP from two bulls of below average (L) or three bulls of above average fertility (H). The bEECs were challenged with 1% or 4% SP from l - or H-fertility bulls (L1, L4, H1, H4, respectively) or 1% or 4% PBS as control (C1, C4) for 72 h. The culture media were analysed for concentrations (pg/million cells) of transforming growth factor beta (TGF-β1, TGF-β2 and TGF-β3) by Luminex, and Interleukin 6 and 8 (IL-6, IL-8) by ELISA. Challenge significantly affected production of TGF-ß1, TGF-ß2 and IL-8 compared to controls and was affected by bull fertility (p < 0.0001), SP concentration (p < 0.0001) and their interaction (p < 0.0001). A higher production of TGF-β1, TGF-β2 and IL-8 (p < 0.0001), and also IL-6 (p < 0.01), resulted from challenge with high doses of SP, being higher for L than H (p < 0.05). For TGF-β3, fertility of bull (p < 0.05). For TGF-B3, fertility of bull (p < 0.05) and the interaction between fertility and concentration of SP were significant (p < 0.01). In conclusion, 4% SP from L bulls stimulated more TGF-β1, TGF-β2, TGF-β3, IL-6 and IL-8 production than SP from H bulls, indicating that stimulation of the endometrium is relevant for fertility. Seminal plasma from high fertility bulls seems to affect cytokine production in utero positively in inseminated cows. [ABSTRACT FROM AUTHOR]