Effect of insulin-transferrin-selenium and metformin on the in vitrofertilization of pigs

Abstract

Brief introduction: The in vitro production of pig embryos is important for both commercial purposes and biomedical studies. It has a low efficiency mainly because in vitro maturation (IVM) systems are inefficient. Supplementation of the IVM medium with insulin plus metformin(M) improves pig embryonic development. We demonstrated that insulin-transferrin-selenium (ITS) plus M increases nuclear maturation, decreases oxidative stress, increases glucose consumption and the viability of cumulus cells. Objective: Our aim was to study the IVF efficiency using ITS plus M during IVM. Methods: Porcine COC obtained by follicular aspiration from slaughterhouse ovaries were in vitro matured for 44 h in modifed M199, supplemented with hMG and dAMPc during the first 22 h. Oocytes were randomly distributed in four experimental groups containing: ITS (ITS 1 µ g/mL), M (M 10-4M), ITS + M and C (no supplement). Then matured oocytes were denuded and subjected to IVF (co-incubated 4 h with refrigerated boar semen 1 × 106 sperm/mL in modified M199). The presumptive zygotes were cultured in NCSU at 39 °C with 5% CO2, 7% O2 and 100% humidity. After 20 h from the beginning of the IVF, zygotes were fixed in paraformaldehyde 4% for 30 min, washed and stored in PBS-PVA until use. To assess fertilization parameters, zygotes were stained with Hoëchst and mounted for evaluation: penetration (penetrated/inseminated), masculine pronuclear formation (MPN/penetrated), monospermy (only one sperm head or male pronucleus/penetrated), and efficiency of fertilization (monospermic/inseminated). Data were analyzed by Fisher's exact test (significant P < 0.05). Main Outcomes and Results: The group matured with ITS + M showed a significantly higher efficiency of fertilization compared to control (ITS + M 30%, n = 43 vs C 11%, n = 45). Conclusions: The supplementation of IVM media with ITS + M is a good tool to improve oocyte quality. This is reflected in a higher IVF efficiency.