160 In vitro maturation of pre-pubertal goat oocytes and their development after chemical activation

Abstract

Experiments were conducted to study in vitro maturation of pre-pubertal goat oocytes and their developmental potential after chemical activation. Cumulus-oocyte complexes (n = 1170) collected from the ovaries of pre-pubertal goats slaughtered at a local abattoir were matured in TCM-199 supplemented with 0.15 mg mL⁻¹ l-glutamine, 0.25 mM sodium pyruvate, 0.1 mM l-cysteine, 20 ng mL⁻¹ epidermal growth factor, 10 mg mL⁻¹ FSH, 10 mg mL⁻¹ LH, 1 μg mL⁻¹ oestradiol and 10% FCS for 24 h at 39°C under 5% CO₂ in humidified air. In Experiment 1, matured oocytes were activated (r = 6) with either 5 mM ionomycin (n = 85) or 7% ethanol (n = 91) followed by culture in 6-DMAP for 4 h. All the activated oocytes were then cultured in KSOM supplemented with 3 mg mL⁻¹ BSA and were fixed and stained with Hoechst 33342 after 18 h of culture to evaluate their activation status. In Experiment 2, oocytes activated with 5 mM ionomycin and 6-DMAP were cultured for 7 days (r = 6) in 1 of the 4 different culture media [Charles Rosenkrans medium (CR-1), modified TCM-199, KSOM and SOF] to study their developmental potential. All media were supplemented with 3.0 mg mL⁻¹ BSA for the first 3 days and 10% FCS for the subsequent 4 days. Of these pre-pubertal oocytes, 59% reached metaphase II stage, and 83% of these oocytes were classified as activated in the group using ionomycin in comparison with 69% in the group using ethanol as an activating agent (P < 0.05). No difference was observed in the cleavage rate of activated oocytes cultured in any of the 4 culture media (65.7 v. 55.0 v. 61.0 v. 56.2%, respectively). However, the development to blastocyst stage was observed in only KSOM (16%) and SOF (5%) media. In conclusion, the present study demonstrates that pre-pubertal goat oocytes can mature in vitro and can be activated with 5 mM ionomycin, and KSOM, and to a lesser extent SOF, supports development to the blastocyst stage. We plan to use these oocytes as a cytoplast source for interspecies somatic cell NT; however, before that, more studies are needed to evaluate their requirements in culture media to enhance their development to the blastocyst stage.