Amino acid supplementation of a simple inorganic salt solution supports efficient in vitro maturation (IVM) of bovine oocytes

Abstract

Defining oocyte in vitro maturation (IVM) conditions allows for improved reproducibility and efficiency of bovine embryo production. IVM conditions for bovine oocytes have been extensively studied, but beneficial effects of individual supplements remain controversial. This study compared methods of cumulus oocyte complex (COC) isolation, and culture medium requirements, for IVM in order to define optimal conditions. Antral follicles in ovaries were sliced or aspirated to isolate COCs. Brilliant cresyl blue staining of COCs was used to determine the most effective collection technique and the effect of hormones and groups of amino acids in the culture medium was investigated. Our results showed COCs isolated through aspiration had greater meiotic competency to reach MII. Oocyte maturation was achieved with the addition of 1 µg/mL FSH, while estrogen and human chorionic gonadotrophin did not increase the number of MII oocytes. We also provide novel data, that supplementation of a simple inorganic salt solution with L-proline, L-glutamine and essential amino acids in combination, but not individually, resulted in nuclear maturation comparable to TCM199, a more complex medium containing all 20 common amino acids, vitamins, inorganic salts and FBS. Replacement of FBS with BSA in this simplified medium creates a defined medium which provides conditions for IVM that enable reproducible maturation rates.