Glutamine, proline, and isoleucine support maturation and fertilisation of bovine oocytes

Successful in-vitro production of bovine embryos relies on meiotic maturation of oocytes in vitro (IVM) before they can be fertilised. High levels of IVM are currently achieved using a complex medium that contains all 20 common amino acids, namely TCM199, but can also be achieved using a simple inorganic salt solution containing non-essential amino acids, proline, and glutamine. Further simplification of the amino acid content of medium used for IVM could lead to a more defined medium that provides reproducible IVM. The aim of this study was, therefore, to determine the minimal amino acid requirements for bovine oocyte nuclear maturation, as measured by progression to metaphase II (MII) of meiosis. Supplementation of a simple medium composed of inorganic salts (M1 medium) with multiple amino-acid combinations showed that M1 containing glutamine, proline, and isoleucine resulted in nuclear maturation comparable to that of TCM199 (57.4 ± 3.4% vs 67% ± 1.7%, respectively) but was reduced when cystine (Cys2) to that seen with M1 alone (38.0 ± 2.2%). Viability of oocytes matured in this simplified medium was equal to those matured in TCM199 since the same proportion of zygotes with 2 pronuclei were observed following fertilisation in medium containing no amino acids (33.9 ± 6.5% vs 33.3 ± 3.6%, respectively). Addition of glutamine, proline and isoleucine to fertilisation medium also increased the proportion of zygotes but did not increase blastocyst development rates. Thus, a defined medium containing only glutamine, proline and isoleucine is sufficient for oocyte maturation and successful fertilisation.