310 ENRICHING A DEFINED MATURATION MEDIUM IMPROVES SUBSEQUENT EMBRYONIC DEVELOPMENT OF BOVINE OOCYTES CULTURED IN SMALL AND LARGE GROUPS

Abstract

When performing Ovum Pick Up (OPU) in unstimulated animals, the number of oocytes collected per donor is often reduced. Culturing oocytes and embryos in small groups impairs embryonic development by comparison with embryos cultured in large groups: less blastocysts are obtained and their appearance is delayed. The aim of the study was to evaluate the effect of an enriched define maturation medium on further embryonic development of oocytes and embryos cultured in large and small groups during IVM, IVF and IVC. Bovine cumulus-oocyte complexes (COC) were collected from abattoir ovaries, selected on morphological criteria, and then allocated to two maturation media: TCM 199 + 10 ng mL⁻¹ mEGF or the same medium enriched with 5 μg mL⁻¹ insulin, 5 μg mL⁻¹ transferrin, 5 ng mL⁻¹ selenium, 19 ng mL⁻¹ IGF-1, 2.2 ng mL⁻¹ FGF, 90 μg mL⁻¹ L-cystein, 28 μM myo-inositol, 100 μM β-mercaptoethanol, 75 μg mL⁻¹ ascorbic acid, 720 μg mL⁻¹ glycine, 0.1 mg mL⁻¹ glutamine, 5 UI mL⁻¹ hCG and 10 UI mL⁻¹ eCG. For both media, COC were cultured either in groups of 18 to 20 (large) or in groups of 4 to 5 (small) in 500 μL of medium in 4-well plates. After 24 h maturation at 39°C and in 5% CO₂ in air, the COC were fertilized and then cultured in modified SOF medium with 5% FCS at 39°C and in 5% CO₂, 5% O₂ and 90% N₂. No selection was performed after the maturation step and the oocytes and embryos were kept in small or large groups throughout the experiment. Blastocyst development was evaluated at Day 7 and 8 post insemination. Results are shown in Table 1. As expected, a significant decrease was observed in blastocyst rates when oocytes and embryos were cultured in small groups. Enriching the maturation medium led to an important increase in blastocyst rates regardless of the number of oocytes cultured together, but the increase was greater when culture was performed in small groups from the maturation step (56% increase at Day 8 v. 31% increase for embryos cultured in large groups). The enriched medium also accelerated the appearance of the blastocysts in embryos cultured in small groups (74% of the blastocysts appeared on Day 7 instead of 50% in the control medium) which could indicate an improvement in blastocyst quality. The rate of hatching was not significantly increased. In conclusion, enriching the maturation allowed an increase in the developmental competence of abattoir oocytes matured in small and large groups. Although further experiments are needed, this could be of particular interest to improve embryonic development from OPU oocytes.

Table 1 
Effect of an enriched defined maturation medium on blastocyst development from oocytes cultured in small or large groups