AMINO ACIDS AS UNIQUE OXIDATIVE SUBSTRATES DURING PORCINE OOCYTE NUCLEAR AND CYTOPLASMIC MATURATION

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

Jornada; XXIII Jornadas Anuales de la Sociedad Argentina de Biología; 2021

The aim of this work was to study the implication of amino acids (Aa) as unique oxidative substrates during porcine oocyte nuclear and cytoplasmic maturation in vitro. Immature cumulus-oocyte complexes (COCs) were obtained by aspiration of antral follicles from slaughtered gilts and then selected under a stereomicroscope. Oocytes surrounded by a dense cumulus were randomly distributed into 5 groups: NCSU-37 without pyruvate or glucose, NCSU-37 + glucose, NCSU-37 + Aa, NCSU-37 + Aa + glucose and NCSU-37 + Aa + salicylate (Aa catabolism inhibitor). All the groups were matured for 44h at 39ºC, 5% CO2 and 100% humidity. To determine meiotic maturation percentages oocytes were denuded and then stained with Hoechst 33342. The nuclear status of each oocyte was analyzed using an epifluorescence microscope with 330-380 (excitation) and 420 (emission) filters at x400. To evaluate Aa catabolism we used a spectrophotometric assay based on NADPH oxidation by glutamate dehydrogenase, quantifying the residual ammonia in each maturation medium. Data was expressed as ammonia production/COC/minute. To determine cytoplasmic maturation, COCs were co-incubated with 1x106 motile sperm/ml for 3 hours in modified Tris Buffer medium with 0,4% serum bovine albumin. Then zygotes were transferred to NCSU-23 medium under mineral oil at 39ºC, 5% CO2 and 100% humidity and the blastocyst rates were evaluated at day 7. Maturation percentages and blastocyst rates were compared using a Chi-square analysis for non-parametric data. The levels of residual ammonia in the maturation media were expressed as mean ± standard error mean and their interactions were analyzed by two-way ANOVA, using post-hoc general contrasts for comparison among treatments. Values with a p