341 IN VITRO MATURATION AND IN VITRO FERTILIZATION OF ALPACA (VICUGNA PACOS) OOCYTES: EFFECT OF TIME OF INCUBATION ON NUCLEAR MATURATION AND CLEAVAGE

Abstract

Experiments were carried out to evaluate the effect of incubation time on nuclear maturation (Experiment 1) and determine the cleavage rate of alpaca oocytes after of IVF time (Experiment 2) In Experiment 1, CCOs were collected from slaughterhouse ovaries and transported to the laboratory in a thermos flask containing a saline solution 0.9% with antibiotic antimycotic at 35°C. CCOs were aspirated from follicles >2 mm and pooled in a conical tube to sedimentation previous to evaluation under stereomicroscope and CCOs with a cytoplasm homogeneous and 2 or more layers of cumulus cells were transferred to plates with a 40-μL drop of maturation medium TCM-199 supplemented with 10% FCS (v : v) plus 0.5 μg mL-1 FSH, 10 μg mL-1 hCG, 0.2 mM sodium pyruvate, 50 μg mL-1 gentamicine, and 1 μg mL-1 Estradiol under mineral oil with 10-12 oocytes/drop. Oocytes were incubated under the following maturation times: 30, 34, and 38 h at 39°C in an atmosphere of 5% CO2 and high humidity. After each maturation time, CCOs were removed from maturation medium and washed with PBS supplemented with 10% FCS and 1 mgmL-1 of hyaluronidase and fixed in ethanol: acetic acid (3 : 1). Oocytes were placed on the slide with minimum medium and stained with 1% orcein for 5 min The slides were examined under a phase contrast microscope at × 400 to evaluate status of nuclear maturation and classified as germinal vesicle (GV); metaphase I (M-I), anaphase-telophase; metaphase II (M-II) and degenerated. Experiment 2: The same maturation method as Experiment 1 was used. Testes were collected of mature males from slaughterhouse and transported to the laboratory. Caudal epididymide was isolated. A prick was made on the convoluted tubules with a sterile hypodermic needle and the fluid, rich in spermatozoa, was aspirated in syringes containing 2 mL of Tris-fructose egg yolk extender. Motile spermatozoa were obtained by centrifugation: 700 g on a Percoll discontinuous gradient (22.5 :45.0%) for 25 min. The supernatant was removed by aspiration and pellet (containing viable spermatozoa) was resuspended in TL stock. Spermatozoa and oocytes were co-incubated for 18-20 h at 39°C with 5% CO2 and then cultivated in TCM-199 supplemented with 10% FCS (v: v), 0.2 mM sodium pyruvate, and 50 μg mL-1 gentamicine and evaluated at 48 h. Data were subjected to ANOVA. For Experiment 1, the proportions of oocytes reaching M-II stage was 18.9 ± 15.7, 42.9 ± 16.2, and 65.8 ± 8.1% for the 30, 34, and 38 h of culture, respectively, with difference to maturation time (P < 0.05). For Experiment 2, the cleavage rate was 9.5, 7.7, and 15.4% to 30, 34, and 38 h after of fertilization time 48 h culture. These results indicate that 38 or more h is required for the maturation and fertilization of alpaca oocytes. Grant 064 FINCyT-PIBAP 2008.