EFFECT OF TIME OF INCUBATION ON NUCLEAR MATURATION AND CLEAVAGE POST IN VITRO FERTILIZATION OF ALPACA OOCYTES

Abstract

The study was carried out to evaluate the effect of incubation time on nuclear maturation and cleavage rate of alpaca oocytes after 72 hours post-fertilization. Cumulusoocyte complexes (CCOs) were collected from ovaries collected at slaughterhouse and transported in saline solution 0.9% with antibiotic antimycotic at 35 ºC. CCOs were aspirated from 2-6 mm follicles. Experiment 1: 502 oocytes were distributed in four maturation times (30, 34, 38, 42 hours), matured in TCM-199 supplemented with 10% fetal calf serum (FCS) (v:v), 0.5 μg/mL FSH, 10 μg/mL hCG, 0.2 mM sodium pyruvate, 50 μg/mL gentamicine and 1 μg/mL oestradiol, and cultivated at 39 ºC in an atmosphere of 5% CO2 and high humidity. After maturation, CCOs were removed from maturation medium and washed with PBS supplemented with 10% FCS and 1 mg/ml of hyaluronidase, and fixed in ethanol and acetic acid (3:1). Oocytes were placed on a glass slide, stained with 1% orcein and examined under microscope at 400x to evaluate nuclear maturation status. Experiment 2: 533 CCOs were culture under similar maturation protocols than experiment 1 and fertilized with epididymal spermatozoa. These were obtained by centrifugation at 700 g on a Percoll discontinuous gradient (22.5:45%) for 25 min. The supernatant was removed by aspiration and the pellet (containing viable spermatozoa) was re-suspended in TL Stock. Gametes were co-incubated for 18 h at 39 °C with 5% CO2 and cultivated in KSOM supplemented with 10% FCS (v:v), 0.2 mM sodium pyruvate and 50 μg/ml gentamicine, and evaluated in 72 hours. In experiment 1, 26.3±5.4, 52.6±6.7, 68.5±10.6 and 75.3±11.9% of oocytes were in M-II stage for the 30, 34, 38, and 42 h of culture respectively, with significant difference between 30 and 34 with respect to 38 and 42 h (p<0.05). In experiment 2, the cleavage rate was 9.5±4.8, 8.1±5.8, 15.6±9.2 and 19.8±8.0% for 30, 34, 38, and 42 h after culture, and without statistical difference between groups. These results indicate that is required 38-42 h for the maturation of alpaca oocytes.