Abstract

L, Wilfredo Huanca et al. Effect of time of incubation on nuclear maturation and cleavage post in vitro fertilization of alpaca oocytes. Rev. investig. vet. Perú [online]. 2014, vol.25, n.4, pp.468-476. ISSN 1609-9117.  http://dx.doi.org/10.15381/rivep.v25i4.10782.

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. Gameteswereco-incubated for 18 h at 39 °C with 5% CO 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

Keywords : in vitro fertilization; oocytes; maturation; alpaca.

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