148 Effect of concentration of methionine in maturation and culture medium on cleavage rate of oocytes from alpaca (Lama pacos)

Abstract

Maturation time of oocytes from alpacas is around 38 to 40 h (Huanca et al. 2009) that would induce an increase in reactive oxygen species during in vitro maturation and IVF and cause cytotoxic damage to gametes. The objective of this study was to determine the optimal concentration of methionine during in vitro maturation on cleavage rate of alpacas oocytes following IVF. Cumulus-oocyte complexes were collected from slaughterhouse ovaries and transported in a thermos flask containing a saline solution 0.9% and antibiotic, antimycotic at 35°C. Cumulus-oocyte complexes were aspirated from follicles >2 mm and evaluated with a stereomicroscope for selection. Only cumulus-oocyte complexes with a homogeneous cytoplasm and with 2 or more layers of cumulus cells were selected to be cultured in 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⁻¹gentamycin and 1 μg mL⁻¹ oestradiol under mineral oil by 38 h. Testes of mature males were collected from a slaughterhouse and transported to the laboratory. Caudal epididymide was isolated, and fluid, rich in spermatozoa, was aspirated in syringes containing 2 mL of Tris-fructose-egg yolk extender. Motile spermatozoa were obtained by centrifugation at 700 × g in a Percoll discontinuous gradient (22.5: 45.0%) for 10 min. The supernatant was removed by aspiration, and the pellet was resuspended in TL stock and centrifuged again at 700 × g for 5 min. Spermatozoa and oocytes were co-incubated by 18 h at 39°C with 5% CO₂. Presumptive zygotes were culture in KSOMaa medium and evaluated at 72 h. The treatments include 0, 14 and 21 μM of methionine in maturation and culture medium. Data were analysed by ANOVA, and results are presented in Table 1. The results suggest that addition of methionine in maturation and culture medium improve the cleavage rate in oocytes from alpacas.

Table 1.  Cleavage rate (%) following in vitro maturation at different concentrations of methionine

Proyect 405-PNICP-PIAP-2014, INNOVATE-PERU, is acknowledged.