Ram Seminal Plasma Research Articles

Various Saccharides Addition of Extender Ram Efficiency to Improve Cryopreservation in Semen Etawah Crossbreed Bucks with Seminal Plasma Replacement

Phospholipase in buck seminal plasma will trigger a coagulation incident resulting in the demise of the spermatozoa during liquid semen processing using milk or egg yolk substrates diluent. A plasma replacement process is performed to avoid this possibility. The cryopreservation process leads to sperm cell damage due to the freezing process, therefore cryoprotectant agents such as saccharides are required as protective agents. This study aimed to investigate the effect of various types of saccharides on the quality of frozen semen of etawah crossbreed (PE) bucks during cryopreservation and thawing with plasma replacement using Priangan ram semen plasma. Semen was collected using an artificial vagina once a week. Fresh PE bucks semen centrifuged at 3,000 RPM for 30 min. The supernatant (seminal plasma) was removed and replaced in equal volume with Priangan ram seminal plasma. Semen was divided into four treatments: goat semen with sheep semen plasma in Tris diluent (control); control with 0.6% dextrose added (monosaccharides); control with 0.6% sucrose added (disaccharides); control with 0.6% raffinose added (trisaccharides). Semen quality including percentages of motile spermatozoa, live spermatozoa, and intact plasma membrane (IPM) were evaluated after diluted, equilibrated, and thawing, respectively. Results of this study showed that viability and IPM of monosaccharides, disaccharides, and trisaccharides were significantly (P<0.05) higher than control (54.40 and 51.40; 55.00 and 53.60; 55.60 and 52.20 vs 48.40 and 52.20, respectively). The motility of disaccharides was significantly (P<0.05) higher compared to control and other diluent (47.00 vs 41.00, respectively). In conclusion, different types of saccharides were effective in maintaining the quality of etawah crossbreed buck frozen semen. The addition of disaccharides seems more effective compared to monosaccharides and trisaccharides in tris extender on the quality of etawah crossbreed buck frozen semen.

172 Untargeted metabolomic analysis of Zulu ram seminal plasma metabolome following selenium supplementation and their possibility as fertility biomarkers

172 Untargeted metabolomic analysis of Zulu ram seminal plasma metabolome following selenium supplementation and their possibility as fertility biomarkers

Metabolomic and Transcriptomic Changes Underlying the Effects of L-Citrulline Supplementation on Ram Semen Quality.

This study examined the effects of L-Cit supplementation on ram semen quality through metabolomics and transcriptomics. A total of 16 rams were randomly categorized into two groups. The control group was fed a basic diet, whereas the experimental group received feed supplemented with 12 g/d of L-Cit. Semen and blood were collected from the rams on days 0 and 72 to measure sugar, pyruvate, amino acid, and nontargeted metabolite contents. Additionally, hypothalamic and testicular tissues were collected for a transcriptomic analysis. We found 27 differential metabolites between the control and experimental groups, of which 21 were downregulated (p < 0.05) and 6 were upregulated (p < 0.05). Compared with the control group, xylose and pyruvate contents in seminal plasma increased by 43.86% and 162.71%, respectively (p < 0.01). Additionally, the levels of 11 amino acids showed a significant increase in seminal plasma (p < 0.01). Furthermore, 961 and 715 differentially expressed genes were detected in the hypothalamic and testicular tissues, respectively. The pathways of significant enrichment in the hypothalamus and testes were protein digestion, absorption, glycolysis/gluconeogenesis, and amino as well as nucleotide sugar metabolisms. In the present study, L-Cit improved protein synthesis and blood metabolism, consequently increasing the contents of most amino acids in ram seminal plasma. Specifically, the hypothalamus controlled the expression of glycolysis/gluconeogenesis-related genes in the testes through its metabolites released into the serum, thereby providing energy for sperm production, which led to a decrease in the sugar content of seminal plasma.

Determination of oxidative stress parameters and DNA fragmentation on post‐thawed buck semen in the presence of ram seminal plasma and fetal calf serum

The aim of the study was to investigate the efficiency of ram seminal plasma and fetal calf serum on freezing of buck semen. Twenty ejaculates were collected using an electro-ejaculator and split into six groups. While FCS additive was not used in A1, A2 and A3 groups, 10% FCS was added to B1, B2 and B3 groups. These groups were then edited according to whether the buck or ram SP was involved. The design of the groups was done as follows: Group A1 (control 1), group A2 without buck SP, group A3 containing ram SP instead of buck SP. Groups B1 (control 2), B2 and B3 were the FCS added forms of these groups. Progressive sperm motility percentages in Group A1 and Group B2 were found to be higher when compared to the lowest Group B3. There were no significant differences between the groups in neither the levels of reactive oxygen species nor the enzyme and glutathione activities. In conclusion, the lack of statistical difference between the groups suggested that despite the supplements used but only when the buck spermatozoa structure was healthy, the cell could preserve acrosome, DNA and the integrity of membrane.

Structural and biophysical characterization of the major proteins from the seminal plasma of Dorper rams

Pregnancy rates using frozen semen from rams are higher than for horses. One of the factors that positively influences this effect is the composition of low-molecular-weight proteins from seminal plasma, since the amounts of these proteins are much lower in horses. The aim of this work was to purify the major protein components from ram seminal plasma for structural and biophysical characterization. First, the ram semen was collected and the plasma separated by centrifugation. The protein fractions were isolated by gel filtration chromatography, analyzed by circular dichroism spectroscopy and the amino acid sequence identified by mass spectrometry (LC-MSE), the results of which were used to model the protein structure by bioinformatics techniques. This protein was identified by LC-MSE as a spermadhesin, being an unglycosylated monomer with Tm = 69.3 °C and ΔHm= 371 kJ mol−1 at pH 7.0. This work describes for the first time the structural characterization of a spermadhesin from seminal plasma of Dorper rams.

Еffect of different breeds on the protein profile in ram seminal plasma

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.

Vasectomy and Photoperiodic Regimen Modify the Protein Profile, Hormonal Content and Antioxidant Enzymes Activity of Ram Seminal Plasma.

This work aimed to determine the contribution of the testis and epididymis and the effect of the photoperiodic regimen on ram seminal plasma (SP). Semen was collected from 15 mature rams located in an equatorial (Colombian Creole and Romney Marsh, eight intact and two vasectomized) or a temperate climate (Rasa Aragonesa, three intact and two vasectomized). SP proteins were analyzed by Bradford, SDS-PAGE and difference gel electrophoresis (DIGE). Melatonin and testosterone concentrations were quantified by ELISA, and activity of glutathione peroxidase (GPx), glutathione reductase (GRD), and catalase by enzymatic assays. Vasectomy increased protein concentration and the intensity of high molecular weight bands (p < 0.001), with no differences between breeds. DIGE revealed the absence of six proteins in vasectomized rams: angiotensin-converting enzyme, lactotransferrin, phosphoglycerate kinase, sorbitol dehydrogenase, epididymal secretory glutathione peroxidase and epididymal secretory protein E1. Vasectomy also decreased melatonin concentrations in seasonal rams, and testosterone in all of them (p < 0.001), but did not affect antioxidant enzyme activity. Equatorial rams showed lower melatonin and testosterone concentration (p < 0.01) and catalase, but higher GPx activity (p < 0.05). In conclusion, vasectomy modifies the protein profile and hormonal content of ram seminal plasma, whereas the exposure to a constant photoperiod affects hormonal concentration and antioxidant enzymes activity.

Mapping the major proteome of reproductive fluids and sperm membranes of rams: From the cauda epididymis to ejaculation

The present study evaluated the major proteome of ram seminal plasma and the main secretions that contribute to its formation, such as the cauda epididymal and accessory sex gland fluids. The study also investigated sperm membrane protein profiles before and after ejaculation. First, semen was collected from six rams (using artificial vagina) to obtain seminal plasma and ejaculated sperm. Then, rams were vasectomized for collection of accessory sex gland fluid (using artificial vagina). Next, rams were slaughtered and cauda epididymal fluid (CEF), seminal vesicle fluid, bulbourethral gland fluid and cauda epididymal sperm were properly collected. Proteins from reproductive fluids and sperm membranes were analyzed by 2-D SDS-PAGE, tandem mass spectrometry and bioinformatics. There we 386 proteins and 256 isoforms identified in all samples. The most abundant seminal plasma proteins were BSP1, BSP5 and spermadhesins (bodhesin-2 and spermadhesin Z13-like). These proteins were present in similar patterns in maps of accessory sexgland fluid, with very low quantities in the CEF and absent in the bulbourethral gland secretion. Thus, practically all BSPs and spermadhesins come from seminal vesicles. Bulbourethral gland fluid brought bactericidal/permeability-increasing protein-containing Family A member 1 isoforms, superoxide dismutase [Cu-Zn] and betamicroseminoprotein to seminal plasma. CEF was the major provider of clusterin, epididymal-specific lipocalin-5-like isoform, epididymal secretory gluthathione peroxidase, epididymal secretory protein E1 and prostaglandin-H2 D-isomerase to seminal plasma. Albumin came from all reproductive fluids. BSPs and spermadhesins were present in 2-D maps of ejaculated sperm but absent in cauda epididymal sperm. These proteins come from the seminal vesicles and bind to sperm at the moment of ejaculation. Other proteins of ejaculated and epididymal sperm membranes were mostly associated to energy production, cell adhesion and proteolytic activity (ATP synthases, disintegrin, metalloproteinase domain-containing protein 32, carboxypeptidase Q and cytosol aminopeptidase). In conclusion, there is a well-orchestrated sequence of events to form the major seminal plasma proteome, with specific contributions from cauda epididymis, seminal vesicles and bulbourethral glands. The present data contribute to a better understanding of male reproductive biology and how sperm functions are affected by the noncellularmicro environment of semen.

Quantitative Proteomic Analysis of Seminal Plasma, Sperm Membrane Proteins, and Seminal Extracellular Vesicles Suggests Vesicular Mechanisms Aid in the Removal and Addition of Proteins to the Ram Sperm Membrane.

Quantitative proteomic studies are contributing greatly to the understanding of the spermatozoon through the provision of detailed information on the proteins spermatozoa acquire and shed in the acquisition of fertility. Extracellular vesicles (EVs) are thought to aid in the delivery of proteins to spermatozoa in the male reproductive tract. The aim of this study is to isolate, identify and quantify EV proteins isolated from ram seminal plasma. Ram sperm plasma membrane proteins are also isolated using nitrogen cavitation and identified to better understand the interplay of proteins between the sperm membrane and extracellular environment. The categorization of proteins enriched in the EV population according to their function revealed three main groupings: vesicle biogenesis, metabolism, and membrane adhesion and remodeling. The latter group contains many reproduction-specific proteins that show demonstrable links to sperm fertility. Many of these membrane-bound proteins show testicular expression and are shed from the sperm surface during epididymal maturation (e.g., testis expressed 101; TEX101 and lymphocyte Antigen 6 Family Member K; LY6K). Their association with seminal EVs suggests that EVs may not only deliver protein cargo to spermatozoa but also assist in the removal of proteins from the sperm membrane.

Homologous Seminal Plasma and Glutathione Promote Pre-Capacitation Motility and Structural Stability of Cryopreserved Ram Spermatozoa

Abstract Reduced glutathione (GSH) and homologous ram seminal plasma (HSP), used as additives in cryopreserving (CP) media prior to freezing, showed conflicting results in retaining structural integrity and progressive motility in post-thawed ram spermatozoa. The aims of this research were: (1) to assess the effect of GSH and/or HSP supplementation via soybean-lecithin CP extender on cryopreserved ram spermatozoa viability, morphology and motility pattern; and (2) to assess the effect of incubation in the context of the previous aim. Quantitatively and qualitatively, homogenized and pooled ram ejaculates (N=10) were extended with one of the following extenders: control (C) – tris-based, GSH and HSP-free, experimental-1 (E1) – C + GSH 5 mM, experimental-2 (E2) – C + HSP 20 % and experimental-3 (E3) - GSH 5 mM + HSP 20 %. Following thawing, samples were taken at 0- and 3-hours from each group (n=10) and were assessed for spermatozoa viability, morphology, and motility pattern. C-0h samples yielded a spermatozoa population with low viability, altered head morphology and highly deviated motility pattern. E3-3h samples yielded spermatozoa with unaffected viability, head morphology and high progressive motility. In conclusion, E3 extender added to cryopreserved-thawed ram spermatozoa is most efficient in obtaining high viability, unaltered head morphology, and progressive motility.

Ram seminal plasma and its functional proteomic assessment

Ejaculation results in the confluence of epididymal spermatozoa with secretions of the accessory sex glands. This interaction is not a prerequisite for fertilisation success, but seminal factors do play a crucial role in prolonging the survival of spermatozoa both in vitro and in vivo by affording protection from handling induced stress and some selective mechanisms of the female reproductive tract. Reproductive biologists have long sought to identify specific factors in seminal plasma that influence sperm function and fertility in these contexts. Many seminal plasma proteins have been identified as diagnostic predictors of sperm function and have been isolated and applied in vitro to prevent sperm damage associated with the application of artificial reproductive technologies. Proteomic assessment of the spermatozoon, and its surroundings, has provided considerable advances towards these goals and allowed for greater understanding of their physiological function. In this review, the importance of seminal plasma will be examined through a proteomic lens to provide comprehensive analysis of the ram seminal proteome and detail the use of proteomic studies that correlate seminal plasma proteins with ram sperm function and preservation ability.

Seminal plasma effects on sperm handling and female fertility

The components of ruminant seminal plasma and their influence on the fertility of spermatozoa are reviewed. Seminal plasma can both inhibit and stimulate sperm function and fertility through the multifunctional actions of organic and inorganic components. These effects are now better understood because the composition of the seminal plasma, including its protein content and that of other structures, specifically membrane vesicles, has been clarified. Spermatozoa gain motility and fertilizing capacity as they transit the epididymis under the influence of factors produced by that organ. At ejaculation, inhibitory (termed "decapacitation") factors, sourced from the accessory sex glands, bind to the sperm surface. The major proteins isolated and characterised in ram seminal plasma, whose specific functions are yet to be determined, originate from the vesicular gland and comprise a spermadhesin together with proteins with fibronectin-II domains. In vitro handling of spermatozoa in preparation for artificial insemination (AI), involving processes such as dilution, cooling, freezing, re-warming and sperm sexing by flow cytometric sorting, can remove seminal plasma and may modify the proteins bound to the sperm surface. This destabilises the membranes and may pre-capacitate the spermatozoa, shortening their fertilizing lifespan. These changes may be reversible by seminal plasma fractions but responses differ depending on the type of sperm pre-treatment. Fertility after AI of ruminant semen may be improved if the role of seminal plasma proteins and their effect, if added individually or in combination to spermatozoa at different stages of preservation, or other manipulations such as flow cytometric sorting, can be determined.

Changes in melatonin concentrations in seminal plasma are not correlated with testosterone or antioxidant enzyme activity when rams are located in areas with an equatorial photoperiod

In temperate climates, photoperiod and melatonin regulate ram reproduction, modulating hormonal secretions, sperm quality, and seminal plasma composition. Information on the effect of an equatorial photoperiod (12L:12D) on ram reproduction, however, is scarce, and no data on hormonal concentrations and antioxidant enzyme activity in seminal plasma have been reported. Thus, the variation was investigated of melatonin and its relationship with testosterone and antioxidant enzyme activity in the seminal plasma of three sheep breeds in Colombia, when there was a consistent photoperiod during two dry and two rainy seasons per year. Semen was collected once a week from 12 mature rams (four of each breed: Colombian Creole, Hampshire, and Romney Marsh). Seminal plasma was obtained by centrifugation. The concentration of melatonin and testosterone were quantified along with the enzymatic activity of glutathione peroxidase (GPx), glutathione reductase (GRD), and catalase (CAT). Correlation analyses between melatonin and testosterone concentrations or enzymatic activity were also performed. Melatonin concentration was affected by season (P < 0.05) but not breed, with lesser concentrations in the first rainy season. Testosterone concentration, however, was affected by breed and season, with greater concentrations (P < 0.01) in the Hampshire and Romney Marsh rams during the second dry season. Regarding antioxidant enzyme activity, there was only seasonal variation in GPx activity (P < 0.05). When correlation analyses were used for data assessments, there was a negative correlation between melatonin and testosterone concentrations in Hampshire rams. In conclusion, melatonin concentrations in seminal plasma of rams that were located in an area with an equatorial photoperiod was affected by the climatological season but there was no positive correlation with testosterone concentration or antioxidant enzyme activity.

Use of anti-phosphotyrosine and PDK1 monoclonal antibodies enables observation of the effects of media on bull sperm second messenger signaling during capacitation

In temperate climates, photoperiod and melatonin regulate ram reproduction, modulating hormonal secretions, sperm quality, and seminal plasma composition. Information on the effect of an equatorial photoperiod (12L:12D) on ram reproduction, however, is scarce, and no data on hormonal concentrations and antioxidant enzyme activity in seminal plasma have been reported. Thus, the variation was investigated of melatonin and its relationship with testosterone and antioxidant enzyme activity in the seminal plasma of three sheep breeds in Colombia, when there was a consistent photoperiod during two dry and two rainy seasons per year. Semen was collected once a week from 12 mature rams (four of each breed: Colombian Creole, Hampshire, and Romney Marsh). Seminal plasma was obtained by centrifugation. The concentration of melatonin and testosterone were quantified along with the enzymatic activity of glutathione peroxidase (GPx), glutathione reductase (GRD), and catalase (CAT). Correlation analyses between melatonin and testosterone concentrations or enzymatic activity were also performed. Melatonin concentration was affected by season (P < 0.05) but not breed, with lesser concentrations in the first rainy season. Testosterone concentration, however, was affected by breed and season, with greater concentrations (P < 0.01) in the Hampshire and Romney Marsh rams during the second dry season. Regarding antioxidant enzyme activity, there was only seasonal variation in GPx activity (P < 0.05). When correlation analyses were used for data assessments, there was a negative correlation between melatonin and testosterone concentrations in Hampshire rams. In conclusion, melatonin concentrations in seminal plasma of rams that were located in an area with an equatorial photoperiod was affected by the climatological season but there was no positive correlation with testosterone concentration or antioxidant enzyme activity.

Characterization of interleukin-6 in the caprine testis

In temperate climates, photoperiod and melatonin regulate ram reproduction, modulating hormonal secretions, sperm quality, and seminal plasma composition. Information on the effect of an equatorial photoperiod (12L:12D) on ram reproduction, however, is scarce, and no data on hormonal concentrations and antioxidant enzyme activity in seminal plasma have been reported. Thus, the variation was investigated of melatonin and its relationship with testosterone and antioxidant enzyme activity in the seminal plasma of three sheep breeds in Colombia, when there was a consistent photoperiod during two dry and two rainy seasons per year. Semen was collected once a week from 12 mature rams (four of each breed: Colombian Creole, Hampshire, and Romney Marsh). Seminal plasma was obtained by centrifugation. The concentration of melatonin and testosterone were quantified along with the enzymatic activity of glutathione peroxidase (GPx), glutathione reductase (GRD), and catalase (CAT). Correlation analyses between melatonin and testosterone concentrations or enzymatic activity were also performed. Melatonin concentration was affected by season (P < 0.05) but not breed, with lesser concentrations in the first rainy season. Testosterone concentration, however, was affected by breed and season, with greater concentrations (P < 0.01) in the Hampshire and Romney Marsh rams during the second dry season. Regarding antioxidant enzyme activity, there was only seasonal variation in GPx activity (P < 0.05). When correlation analyses were used for data assessments, there was a negative correlation between melatonin and testosterone concentrations in Hampshire rams. In conclusion, melatonin concentrations in seminal plasma of rams that were located in an area with an equatorial photoperiod was affected by the climatological season but there was no positive correlation with testosterone concentration or antioxidant enzyme activity.

Binder of Sperm Proteins protect ram spermatozoa from freeze-thaw damage

Cryopreservation causes sub-lethal damage which limits the fertility of frozen thawed spermatozoa. Seminal plasma has been investigated as a cryoprotectant, but has yielded inconsistent results due to considerable variation in its constituents. Individual seminal plasma proteins offer an ideal alternative to whole seminal plasma, and several have been correlated with freezing success. Binder of Sperm Proteins (BSPs) are abundant ram seminal plasma proteins which have been suggested to have significant protective effects on ram spermatozoa during cold shock. This is in direct opposition to bull spermatozoa, where BSPs cause sperm deterioration during in vitro handling. We investigated the potential of BSP1 and BSP5 to prevent freezing associated damage to important functional parameters of ram spermatozoa. BSPs purified by size exclusion chromatography improved post thaw motility and penetration through artificial mucus. Highly purified BSP1 and BSP5, isolated by gelatin affinity and RP-HPLC, improved motility and membrane integrity, and reduced post thaw protein tyrosine phosphorylation. Exposure to BSP5 before freezing increased the amount of phosphatidylethanolamine on the sperm surface after thawing. Neither BSP1 nor BSP5 prevented freezing associated changes in membrane lipid disorder. These results suggest that BSPs may significantly improve freezing outcomes of ram spermatozoa.

Melatonin receptors MT1 and MT2 are expressed in spermatozoa from several seasonal and nonseasonal breeder species

Melatonin is a ubiquitous and multipurpose molecule, and one of its roles is to regulate reproduction in some seasonal mammals. Our group has previously reported the variation in the melatonin levels in ram seminal plasma along the year and identified MT1 and MT2 receptors in ram spermatozoa. The objective of this study was to elucidate whether the presence of melatonin receptors (MT1 and MT2) in the sperm plasma membrane, and melatonin in the seminal plasma is related to seasonal breeding. For this purpose, the presence of melatonin receptors and the levels of melatonin in seminal plasma have been examined in several species: donkey and stallion as long-day breeders; red deer as a wild, short-day, highly seasonal breeder (epididymal spermatozoa); bull as a conventional nonseasonal breeder; boar as a seasonal breeder under management techniques; and dog as possible a seasonal breeder not regulated by melatonin. We have detected measurable levels of melatonin in the seminal plasma of all ejaculated semen samples (from donkey, stallion, boar, bull, and dog). Also, and for the first time, we have demonstrated the presence of MT1 and MT2 melatonin receptors in the spermatozoa of all these species, regardless their type of reproduction or sperm source (ejaculated or epididymal), using indirect immunofluorescence techniques and Western blotting. Our findings suggest that melatonin and melatonin receptors may be universally distributed in the reproductive system of mammals and that the sperm melatonin receptors cells may not be necessarily related with seasonal reproduction. Furthermore, the presence of MT1 at the cytoplasmic droplet in immature ejaculated stallion spermatozoa found in one sample and epididymal red deer spermatozoa suggests that melatonin may be involved in specific functions during spermatogenesis and sperm maturation, like protecting spermatozoa from oxidative damage, this activity being mediated through these receptors.

Post-thawing quality of ram spermatozoa is impaired by inclusion of boar seminal plasma in the freezing extender

Addition of seminal plasma (SP) to freezing extenders may help reducing post-thawing sperm cryoinjuries. This study evaluated the effects of adding heterologous (boar) SP to the freezing extender on ram sperm quality after thawing. Protein content was quantified in pooled SP samples from rams and boars. Five ejaculates from four rams were frozen in a Tris-egg yolk-glycerol extender including: no (control), ram and boar SP (both at 20% of the volume, with 48μg protein/mL). After thawing, total and progressive motility for sperm treated with SP were lower than that of the control (P<0.05). For spermatozoa with intact acrosome, those in contact with boar SP had the lowest membrane integrity (P<0.05), but for spermatozoa with damaged acrosome, the lowest membrane integrity occurred for those in contact with ram SP (P<0.05). Greater percent of high post-thawing mitochondrial membrane potential occurred in the control than in contact with both SP (P>0.05). Apoptotic-like changes were similar among treatments (P>0.05), but there were fewer intact spermatozoa extended with boar SP than in the control (P<0.01). Contact with extenders including boar SP harmed ram sperm quality after thawing. Post-thawing motility was decreased for ram sperm extended in contact with both homologous and heterologous SP.

Evidence of melatonin synthesis in the ram reproductive tract.

Melatonin is a ubiquitous molecule found in a wide range of fluids, one of them being ram seminal plasma, in which it can reach higher concentrations than those found in blood, suggesting an extrapineal secretion by the reproductive tract. In order to identify the source of the melatonin found in ram seminal plasma, we first tried to determine whether the melatonin levels were maintained during the day. For this purpose, melatonin concentrations were measured in seminal plasma obtained from first ejaculates of six rams at 6:00 a.m. in total darkness, at 10:00 a.m. and at 14:00 p.m. The melatonin concentration was higher (p < 0.05) in ejaculates collected at 6:00 a.m. than at 10:00 and 14:00. There was no statistical difference between the latter. To further corroborate an extrapineal secretion of melatonin, the presence of the two key enzymes involved in melatonin synthesis, arylalkylamine-N-acetyltransferase (AANAT) and N-acetylserotonin-O-methyltransferase (ASMT) was analyzed by RT-PCR, q-PCR and Western-blot in ram testes, epididymis, and accessory glands. The RT-PCR showed the presence of the m-RNA codifying both AANAT and ASTM in all the tissues under study, but the q-PCR and Western-blot revealed that gene expression of these enzymes was significantly higher in the testis (p < 0.05). Immunohistochemistry confirmed the presence of AANAT and ASMT in the testis and revealed that they were found in the Leydig cells, spermatocytes, and spermatids. Also, measurable levels of melatonin were found in testicular tissue and the tail of the epididymis. In conclusion, our study indicates that the testes are one of the likely sources of the high levels of melatonin found in ram seminal plasma, at least during the day.

Interaction of milk proteins and Binder of Sperm (BSP) proteins from boar, stallion and ram semen

BackgroundMammalian semen contains a family of closely related proteins known as Binder of SPerm (BSP proteins) that are added to sperm at ejaculation. BSP proteins extract lipids from the sperm membrane thereby extensively modifying its composition. These changes can ultimately be detrimental to sperm storage. We have demonstrated that bovine BSP proteins interact with major milk proteins and proposed that this interaction could be the basis of sperm protection by milk extenders. In the present study, we investigated if homologous BSP proteins present in boar, stallion and ram seminal plasma display a similar affinity for the milk proteins in order to assess whether the mechanism of sperm protection by milk for these species could be general.MethodsSkim milk was incubated with seminal plasma proteins (boar, stallion and ram), chromatographed on a Sepharose CL-4B column and protein fractions were analyzed by immunoblotting.ResultsBoar, stallion and ram BSP proteins displayed affinity for a milk protein fraction (F1) mainly composed of α-lactalbumin, β-lactoglobulin, and κ-casein. They also had affinity for another milk protein fraction (F2) composed mostly of casein micelles. However, stallion BSP showed higher affinity for the fraction (F1).ConclusionsThese results further extend our view that the association of BSP proteins with milk proteins could be a general feature of the mechanism of mammalian sperm protection by milk to prevent detrimental effect of prolonged exposure of sperm to seminal plasma.