78 CHRONOLOGICAL EVENTS OF IN VITRO MATURATION IN CAMEL (CAMELUS DROMEDARIES) OOCYTES

Abstract

Experiments were conducted to investigate the chronological events and optimum time for in vitro oocyte maturation in the dromedary camel. Follicles measuring 3–12 mm were isolated from ovaries obtained from an abattoir and the oocytes harvested by teasing apart these follicles under a stereo microscope. Pooled oocytes were randomly distributed to 4-well dishes (20–25 per well) (Nunc, Denmark) containing 400 μL of the maturation medium (TCM-199 supplemented with 0.6 mg mL⁻¹ calcium lactate, 0.1 mg mL⁻¹ L-glutamine, 0.8 mg mL⁻¹ sodium bicarbonate, 1.4 mg mL⁻¹ HEPES, 0.25 mg mL⁻¹ pyruvate, 50 μg mL⁻¹ gentamicine, 10 μg mL⁻¹ FSH, 10 μg mL⁻¹ LH, 1 μg mL⁻¹ estradiol and 10% heat-inactivated estrous camel serum) and incubated at 38.5°C under 5% CO₂ for 4 to 48 h. After every 4 h (starting from 0 to 48 h), oocytes were denuded by treating them with hyaluronidase (1 mg mL⁻¹) followed by repeated pipetting. Denuded oocytes were mounted on glass slides and fixed in 3 : 1 ethanol : acetic acid for 24 h. Oocytes were stained with 1% aceto-orcein and examined under a phase contrast microscope at 400times. for each experimental group, 3 to 7 replications were made. Based on the visualization of the chromatin, oocytes were categorized as at germinal vesicle (GV), diakinesis (DK), metaphase-I (M-I), anaphase (Ana), metaphase-II (M-II) stage and those with no visible chromatin as NVC. At the start of maturation, 75.4% (43/57) of oocytes were at GV stage; however, none of the oocytes revealed a GV at 28 h of maturation (0/97). At 8 h of maturation 49.3% (34/69) of oocytes were at DK stage, and after 16 h of maturation 50% (49/98) of oocytes were at M-I stage. At 24 h of maturation the maximum number of oocytes were in Ana (24.7%, 21/85) stage. At 44 h the maximum number of oocytes had reached M-II stage (52%, 103/198) whereas, 10.6% (21/198) of the oocytes were at Ana stage. After 48 h the proportion of oocytes with NCV increased to 52.9% (45/85) and the proportion of M-II stage oocytes decreased to 37.6% (32/85). It may be concluded that 40–44 h of in vitro maturation yields the highest proportion of matured (M-II stage) oocytes suitable for further use in assisted reproductive technologies in camel.