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Various techniques such as AI and ET have been reported to improve reproductive efficiency and genetic potential in camelids. In vitro fertilization and the development of IVP embryos are considered an alternative for genetic improvement in this species. This study investigated the effects of different sperm cell concentrations (1, 2, 3 and 4 × 106 sperm mL–1), different capacitating materials (5 mM caffeine, 10 μg mL–1 of heparin, 10 mg mL–1 of theophylline, 1 mM calcium ionophore A23178 and 10 μg of heparin + 5 mM caffeine), post-slaughter epididymal flushing time and fertilization media supplements (Fert-TALP + 6 mg mL–1 of BSA and Fert-TALP + 3 mg mL–1 of polyvinylpyrrolidone ) on fertilization rates and subsequent development of dromedary camel oocytes. Cumulus–oocyte complexes obtained at slaughter were matured in vitro in TCM-199 for 36 h at 39°C in a humidified atmosphere of 5% CO2. For IVF, spermatozoa were collected from epididymides of slaughtered male camels at 1 to 2 h post-slaughter or after 24 h of epididymal storage at 4°C. The spermatozoa were then prepared for IVF by the swim-up technique. Following sperm capacitation, oocytes and spermatozoa were co-incubated for 18 h. Oocytes were then stained using aceto-orcein for evaluation of fertilization events. Presumptive zygotes were cultured in vitro in TCM-199 medium supplemented with 5% FCS for 9 days at 39°C in a humidified atmosphere of 5% O2, 5% CO2 and 90% N2. At least 3 replicates were performed for each experimental group. Data were analysed by chi-square test. Fertilization rates were 55.5, 62.5, 62.7 and 47.2% in oocytes inseminated with 1, 2, 3, or 4 × 106 sperm mL–1, respectively. Normal fertilization rate (oocytes with 2 pronuclei) was higher (P =  0.06) in oocytes inseminated with 2 × 106 sperm mL–1 (29.7%) than in those inseminated by 4 × 106 sperm mL–1 (11.1%). Treatment of epididymal spermatozoa with 5 mM caffeine significantly increased (P ≤ 0.05) fertilization rate (61.9%) compared with calcium ionophore A23178 (32.4%). These values were not significantly different from other groups (38.5, 54.1 and 50.0% in heparin, theophylline and heparin + caffeine, respectively). Normal fertilization was highest (25.4%) in oocytes inseminated with caffeine-treated spermatozoa. Insemination of oocytes in Fert-TALP medium containing BSA resulted in a higher fertilization rate (21.4%) compared with oocytes in polyvinylpyrrolidone-supplemented medium (5.7%; P =  0.06). Storage of camel epididymides at 4°C for 24 h did not affect fertilization rates. Cleavage rate (48 h post-insemination) was higher in oocytes fertilized with caffeine-treated spermatozoa than in oocytes in the theophylline group (26.8 vs 10.5%; P =  0.08). No significant difference was observed in the frequency of blastocyst development (5 days post-insemination) between the 2 groups (5.4 vs 2.6%); based on the number of cleaved oocytes, the same proportions of blastocyst embryos were reported (20.0 and 25.0%). Taken together, these results suggest that dromedary camel oocytes can be matured, fertilized and subsequently developed in vitro with high developmental potential. Epididymal spermatozoa at a concentration of 2 × 106 sperm mL–1 prepared in a medium containing caffeine as a capacitating agent can be used effectively in IVF of camel oocytes.

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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.

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