The objective of the present study was to describe the proteins from the seminal plasma of buffalo and correlate these proteins with sperm motility. Ejaculates from sixteen Murrah buffalo were used. Semen collection was performed by electroejaculation, and the ejaculate was evaluated by macroscopic (volume) and microscopic analysis (subjective motility and vigor, as well as sperm concentration). After the analysis, the samples were centrifuged (800g for 10 min and 10,000 for 30 min at 4 °C), and the supernatant (seminal plasma) was used to determine total protein concentration by the Bradford method. Based on total protein concentration, an aliquot (50 μg) was taken to conduct protein in-solution digestion for nano-LC-ESI-Q-TOF mass spectrometry analysis. Samples were divided into two groups, minimal (little sperm motility) and greater (typical sperm motility), based on non-hierarchical clustering considering motility and emPAI protein value. The data were analyzed by multivariate statistical analysis using principal component analysis (PCA) and partial analysis of minimum squares discrimination (PLS-DA). Forty-eight proteins were detected in the seminal plasma, and fifteen were common to two groups. There were six proteins that were significantly different between the groups. The main functions of proteins in seminal plasma were catalytic and binding activity. Spermadhesin protein, ribonuclease, 14-3-3 protein zeta/delta and acrosin inhibitor were in greater amounts in seminal plasma from the group with greater sperm motility; prosaposin and peptide YY were in greater amounts in the group with little sperm motility. The proteins detected in the greater motility group were correlated with sperm protection, including protection against oxidative stress, lipid peroxidation, protease inhibition and prevention of premature capacitation and acrosome reaction. In the group with little sperm motility, one of the identified proteins is considered to be an antifertility factor, whereas the function of other identified protein is not definitive. Results from the present study add to the knowledge base about the molecular processes related with sperm motility, and these findings can be used for determining potential markers of semen quality.

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Sperm cryopreservation has become an indispensable tool in reproductive biology. However, frozen/thawed semen has a short lifespan due to loss of sperm cell integrity. To better understand which sperm cell structures are compromised by the cryopreservation process and apoptosis markers, the sperm of five healthy mature dogs was analyzed in this study. Analysis was performed after collection, cooling, and thawing via computer assisted sperm analyzer (CASA) and evaluation of membrane fluidity and permeability, phosphatidylserine translocation (Annexin V), membrane integrity, mitochondrial membrane potential, membrane lipid peroxidation (LPO) and activity of the apoptotic markers caspases 3 and 7 by flow cytometry. Cryopreservation decreased total and progressive motility and the percentage of rapid sperm (P < 0.01). Damage to sperm cells was confirmed by Annexin V (P < 0.01), indicating that capacitation-like changes were induced by the cryopreservation procedures. An increase in sperm membrane fluidity was also noted in frozen/thawed samples (P < 0.01). Plasma and acrosomal cell membranes were affected (P < 0.01), with decreases in the subpopulation displaying high membrane potential (P < 0.01). Membrane LPO was increased in thawed sperm compared to cooled sperm (P < 0.05) but was not different from that in fresh sperm. No differences were observed in caspase 3 and 7 activity after cooling, freezing, or thawing. In conclusion, total and progressive motility, plasma membrane integrity and mitochondrial membrane potential suffered from the deleterious effects caused by cryopreservation, unlike the activity of caspases that remained stable during the freezing process.

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Evaluation of the damage caused by the sperm preservation process is crucial to improving fertilization rates. The objective of this study was to evaluate the effects of refrigeration temperature (5 C and 15 C) and storage time (0, 12, 24, 48, and 72 hours) on apoptotic markers in equine semen. Membrane phosphatidylserine translocation index, caspase activation index, and DNA fragmentation index were analyzed using epifluorescence microscopy. Analysis of variance was used for statistical analysis, and Tukey test was used to compare means. The significance level was set at P < .05. The results demonstrated that for transport duration shorter than 24 hours, semen quality was maintained when stored at either 5 C or 15 C. A storage temperature of 5 C should be used when it is necessary to transport semen for longer than 24 hours. There was a significant decrease in semen quality after 48 hours of refrigeration.

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. K E Y W O R D Smale, proteomics, semen, spectrometry-mass | INTRODUC TI ONBuffaloes are marked by rusticity and were the first species inserted in regions in which cattle farming was infeasible due to climatic and/ or geographical conditions. Posteriorly, with the improvement in productive techniques associated with species characteristics such as high adaptability and fertility, potentiality and productive longevity, feed conversion efficiency, meat and milk production capacity, traction strength, resistance to parasitism and development of infectious diseases, they were reintroduced into areas already devoted to cattle All authors have contributed equally to this work.

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This study investigated the binding capacity of equine spermatozoa (both ejaculated and from different epididymal regions) to the oviductal epithelial cells (OECs) culture, before and after cryopreservation, using an oviduct explant assay. Ejaculated and epididymal sperm from caput, corpus, and cauda of 10 stallions were diluted and submitted to freezing process. Fresh and frozen-thawed sperm were evaluated for sperm kinematics, plasma membrane integrity (PMI) and incubated with oviduct explants. The cryopreservation process decreased significantly the sperm motility parameters of ejaculated sperm, and corpus and cauda epididymal sperm (P < .05). The percentage of PMI was significant higher in fresh samples versus frozen-thawed samples, in all analyzed groups (P < .05). Binding of ejaculated spermatozoa to oviduct epithelium was significantly higher than caput, corpus, or cauda epididymal sperm (P < .05). The caput epididymal sperm showed no binding capacity to oviduct explants; thus, significantly more sperm recovered from the corpus and cauda epididymis were bound to OEC compared to caput epididymal sperm (P <.05). No differences were observed in ejaculated and epididymal sperm before and after cryopreservation (P > .05). In conclusion, the ejaculated sperm has higher binding capacity than epididymal sperm, suggesting that the seminal plasma plays an important role in the establishment of the oviductal sperm reservoir. The cryopreservation process did not affect the binding capacity of ejaculated or epididymal spermatozoa to oviductal epithelium.