Use of endogenous lipids and amino acids as unique oxidative substrates for porcine oocyte nuclear and cytoplasmic maturation

SERGIO MORADO; FLORENCIA IRIARTE; CAROLINA LETO; FRANCO PORTILLO; GIULIANA TRICERRI; STEPHANIA MADRID GAVIRIA; ELIZABETH BREININGER; PABLO CETICA

Bolonia

Congreso; International Congress on Animal Reproduction; 2022

It has been postulated that amino acids (Aa) and endogenous lipids can be used as energy substrates during porcine cumulus-oocyte complexes (COCs) in vitro maturation (IVM). However, those studies were conducted in complex media, so the use of other oxidative substrates could not be excluded. Our aim was to study if Aa and endogenous lipids may be used as unique oxidative substrates for the nuclear and cytoplasmic maturation of porcine oocytes. Immature COCs were obtained by aspiration of antral follicles from ovaries of slaughtered gilts and oocytes completely surrounded by a dense integer cumulus were selected. COCs were matured in: NCSU-37 (without pyruvate and glucose), NCSU-37+Glucose (negative and positive controls, respectively), NCSU-37+Aa, NCSU-37+Aa+Glucose, NCSU-37+Aa+Salicylate (Aa catabolism inhibitor); NCSU-37+L-carnitine (fatty acid oxidation stimulator), NCSU-37+Carnitine+Glucose and NCSU-37+Etomoxir (lipid catabolism inhibitor). Meiotic maturation (MII) rates were analyzed using Hoescht 33342 stain. To determine cytoplasmic maturation, COCs were co-incubated with 1x106 motile sperm/ml for 3h in modified Tris Buffer medium with 0.4% serum bovine albumin. Then zygotes were transferred to NCSU-23 medium under mineral oil at 39°C, 5% CO2 and 100% humidity and blastocyst rates were evaluated at day 7. The use of endogenous lipids was evaluated determining lipid levels in oocytes after IVM using Nile Red stain, while the use of Aa was evaluated by spectrophotometry, based on NADPH oxidation by glutamate dehydrogenase during the consumption of ammonia. Lipid levels and NADPH oxidation were analyzed by one way ANOVA, while MII and blastocyst rates were compared using a chi-square analysis for non-parametric data. P