252 EFFECT OF FOLLICLE-STIMULATING HORMONE ADDITION ON IN VITRO MATURATION AND CLEAVAGE OF ALPACA (VICUGNA PACOS) EMBRYOS

Abstract

We have previously reported that alpaca oocytes require between 38 and 42 h of maturation time (Huanca et al. 2010 Reprod. Fertil Dev. 22(1), 327). The objective of this study was to evaluate the effect of the addition of FSH in the maturation medium on nuclear maturation and cleavage rate. Ovaries were collected from a slaughterhouse and transported to the laboratory in a thermos flask containing a saline solution 0.9% with antibiotic antimycotic at 35°C. Cumulus–oocyte complexes (COC) were obtained by slicing of ovaries with a scalpel and were pooled in a conical tube for sedimentation before evaluation. The 476 COC with homogeneous cytoplasm and 2 or more layers of cumulus cells were transferred to plates with a 40-μmL drop of maturation medium TCM-199 supplemented with 10% FCS (vol:vol), 10 μg mL^–1 of hCG, 0.2 mM sodium pyruvate, 50 μg mL^–1 of gentamicine, and 1 μg mL^–1 of oestradiol plus 0.5 μg mL^–1 of FSH (Folltropin, Bioniche Animal Health, Belleville, Ontario, Canada) according the following treatments: T1 (Control): FSH by 42 h, T2: 21 h with FSH + 21 h without FSH, T3: 21 h without FSH + 21 h with FSH. The COC were maintenance under mineral oil with 10 to 12 oocytes per drop and maturated 42 h at 39°C in an atmosphere of 5% CO₂ and high humidity. After the maturation time, part of the COC were removed from maturation medium and washed with PBS supplemented with 10% FCS and 1 mg/mL 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 the status of nuclear maturation and classify as germinal vesicle (GV), metaphase I (MI), anaphase–telophase, metaphase II (MII), and degenerated. The other part of oocytes was fertilized in vitro using epididymal sperm. Motile spermatozoa were obtained by centrifugation at 600 × g on a percoll discontinuous gradient (22.5:45.0%) for 10 min. After the supernatant was removed by aspiration, the pellet was resuspended in TL HEPES and centrifuged again at 300 × g by 5 m. The pellet was resuspended in TL Stock. Gametes were coincubated for 18 h at 39°C with 5% CO₂ and high humidity. Presumptive zygotes were culture in KSOM supplemented with 1 mM of glutamine, 0.3 mM of sodium pyruvate, 50 μg mL^–1 of gentamicine, ethylenediaminetetraacetic acid, amino acids essentials and nonessentials, and BSA by 3 days and cultured in SOF medium for 7 days. Cleavage rate was evaluated at 72 h. Proportional data were compared by chi-square test. The proportions of oocytes reaching MII stage were 64.9 ± 8.1, 49.2 ± 9.4, and 53.8 ± 7.5% for T1, T2, and T3, respectively. Cleavage rates were 39.1, 36.3, and 33.1% and blastocysts rates were 13.6, 16.1, and 14.5% for T1, T2, and T3, respectively. The results suggest that the moment of addition of FSH does not have an effect on the maturation and cleavage rate of alpaca oocytes.