161 IN VITRO FERTILIZATION OF DROMEDARY CAMEL (CAMELUS DROMEDARIUS) OOCYTES WITH EPIDIDYMAL SPERMATOZOA

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.