260 IN VITRO DEVELOPMENTAL COMPETENCE OF PREPUBERTAL GOAT OOCYTES CULTURED IN GROWTH MEDIUM

Abstract

The prepubertal goat ovary presents a large number of small oocytes with a compromised competence to develop up to blastocyst stage. In pigs (Wu et al. 2006), using growth medium (GM) composed by low hormone concentrations, ascorbic acid, and insulin transferrin selenium (ITS) during the first 24 h of in vitro maturation (IVM) improved embryo development of small oocytes. The aim of this study was to test the GM in small prepubertal goat oocytes in order to increase blastocyst yield. The cumulus–oocyte complexes (COC) were recovered from prepubertal (1–2 months old) goat ovaries by slicing. The COC with a compact cumulus and homogeneous cytoplasm were selected and classified into 2 categories based on oocyte diameter: <125 μm and ≥125 μm. The ≥125 μm oocytes were matured in groups of 25 to 30 COC/100 μL drops of conventional IVM medium covered with mineral oil for 24 h (Treatment A). This medium was TCM-199 supplemented with 10% donor bovine serum, 10 μg mL^–1 FSH, 10 μg mL^–1 LH, 1 μg mL^–1 17β-oestradiol, and 100 μM cysteamine. The <125 μm oocytes were distributed into 3 experimental groups: Treatment B, COC matured in the conventional IVM medium; Treatment C, COC cultured in GM (TCM-199, 10% donor bovine serum, 0.04 μg mL^–1 FSH, 0.04 μg mL^–1 LH, 0.004 μg mL^–1 17β-oestradiol, 100 μM cysteamine, 100 μg mL^–1 ascorbic acid, and 5 μL mL^–1 ITS) for 12 h before placement for other 12 h in the conventional IVM medium, all drops of growth or maturation medium were covered with mineral oil; Treatment D, COC cultured during the first 12 h in GM and other 12 h into the conventional medium supplemented with 100 μg mL^–1 ascorbic acid and 5 μL mL^–1 ITS. After IVM, oocytes were fertilized for 24 h with a sperm concentration of 4 × 10⁶ spz mL^–1. Presumptive zygotes were cultured in SOF for 9 days. The cleavage rate was evaluated at 48 h post-insemination and blastocyst percentages at the final in vitro embryo culture (treatments A, B, C: 5 replicates; treatment D: 4 replicates). The results are shown in the Table 1. Cleavage and embryo development did not show different results when we compared small oocytes matured in GM to those matured in conventional IVM medium. However, the biggest oocytes (≥125 μm) showed the highest percentage of blastocyst development. The current study shows that the culture of small prepubertal goat oocytes in GM does not improve blastocyst yield.

Table 1.  Effect of growth medium on embryo development of small oocytes (<125 μm) from prepubertal goats