Till now, the exact role of melatonin in male infertility hasn't been fully discovered. Moreover, the intracellular signaling pathways activated by melatonin in buffalo spermatozoa especially on the level of cryopreservation process haven't been reported. The current study aimed simply to clarify the precise role of this hormone during buffalo semen cryopreservation. Semen samples were obtained randomly from 6 fertile buffalo bulls (aged 3 to 5 years). Weekly, two consecutive ejaculates were collected from each bull for successive six weeks duration using an artificial vagina. The ejaculates were pooled to eliminate variability between the evaluated samples. After that, semen samples were subjected to extension with Tris-based extender supplemented with different concentrations of melatonin (0.1, 0.25, 0.5, 0.75 and 1 mM) Vs. Trisbased extender only (control). Then they were processed to cryopreservation and thawing to assess the different semen characteristics. It had been found that 0.1 mM of melatonin significantly (p<0.05) improved post thawing motility, viability index, acrosomal integrity, total antioxidant capacity, superoxide dismutase and glutathione reductase activity. On the other hand, it significantly (p<0.05) decreased the rate of lipid peroxidation, aspartate amino transferase, alanine aminotransferase, alkaline phosphatase and DNA fragmentation. All of these previously enhanced semen characteristics were reflected positively on its in vitro fertilizing capacity, as well as the percent of harvested embryos. In conclusion melatonin supplementation to the extension media of buffalo semen during processing significantly enhanced its characteristics. Moreover, the preceding results focusing more light on the potential roles of melatonin in regulating male reproduction.

Reproductive diseases may have destructive effects on the fertility of cattle. Bovine viral diarrhoea virus (BVDV) and bovine herpes virus-1 (BoHV-1) are potent viral pathogens linked to reproduction. Thus, the aim of this study was to utilize raw semen samples for conventional and molecular detection of BVDV and BoHV-1, simultaneously. Additionally, the effect of virus infection on the semen quality of naturally infected bulls has been investigated. Therefore, 40 bulls were employed for semen collection, evaluation and testing for both viruses by virus isolation, direct fluorescent antibody technique (FAT) and SYBR Green real-time PCR assay. In virus isolation results, no cytopathic effect (CPE) was observed for BVDV on cell culture whereas, eight (20%) samples displayed characteristic grape-like clusters of cells for BoHV-1. By direct FAT, 12 (30%) positive BVDV and 8 (20%) positive BoHV-1 samples were confirmed. SYBR Green real-time PCR analysis using 48 h inoculated semen samples revealed 14 (35%) and 8 (20%) positive samples for BVDV and BoHV-1, respectively. Statistical analysis of semen evaluation parameters showed a significant difference between viral-infected and free groups represented by increased sperm abnormalities and decreased sperm motility, liveability and concentration. However, there was no significant difference among BVDV, BoHV-1 and mixedinfected groups. The study concluded that BVDV and/or BoHV-1 infected bulls expressed low semen quality. Real-time PCR was confirmed to be the ideal laboratory assay for detection of both viruses in semen.

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Objectives of current study were to identify single nucleotide polymorphisms (SNP) in heat shock protein70 (HSP70) promoter gene of bulls (cattle and buffalo) and its association with semen quality traits and some enzymatic, hormonal and lipid parameters during winter and summer seasons. Semen samples of 37 animals (Cattle n=21 and buffalo n=16), were used in the study. Biochemical Parameter and hormonal profile were estimated. Qualities of some of fresh and frozen-thaw semen were evaluated. PCR-SSCP was used to detect SNPs and different patterns were sequenced. The study demonstrate all the measured biochemical parameters and hormonal profile of cattle and buffalo were significantly different (P<0.05) in winter as compared to summer. Results of PCR-SSCP and sequencing detected a new synonymous SNP 812 T>C transition was the same in both species and could clear two genotypes TT and TC. Association analysis revealed significance difference (P<0.001) with all the fresh and some of frozen-thawed semen traits in cattle population and T allele seemed to be the favorable allele. Regarding to buffalo population, different genotypes has significant difference in term of fresh motility, frozen motility at 1h and viability index. The results of association between biochemical and hormonal parameters there were no significance except for Aspartate-aminotransferase (AST) in cattle population and T 3 level in buffalo population and C allele appeared to be the favorable allele. This the first study concerned with the association of HSP70 polymorphisms and semen quality, in winter and summer seasons in buffalo population. Conclusion: High temperature has negative impact on semen quality. Biochemical and hormonal profile can be used as good indicator for semen value. Polymorphisms in HSP70 gene can affect semen quality. We suggest to be used in artificial insemination programs as marker assisted selection for anti-heat stress and good reproductive bulls.