137 EFFECT OF FLUNIXIN MEGLUMINE IN CO-CULTURE MEDIUM ON THE DEVELOPMENT OF IN VITRO MATURED AND FERTILIZED BOVINE EMBRYOS

Abstract

PG concentration is often increased during uterine manipulation with embryo transfer. Embryo viability is affected by the increase in the PGF_2α concentration accompanying manipulation of the uterus during embryo transfer. Schrick et al. (2001 Theriogenology 55, 370 abst) observed that treatment with flunixin meglumine, an inhibitor of prostaglandin, increased pregnancy rates depending on the stage and quality of embryos transferred. On the other hand, prostaglandin was secreted by a cumulus cell monolayer in an in vitro culture of bovine oocytes. The present study aimed to assess the effects of flunixin meglumine in culture medium on the development of in vitro-matured and fertilized bovine embryos. COCs were collected from ovaries of slaughtered cows by aspiration. The COCs were matured for 20 h in TCM-199 supplemented with 5% fetal bovine serum (FBS) and antibiotics at 38.5°C under an atmosphere of 2% CO₂ in air. Matured COCs were inseminated with 1.0 × 10⁷ sperm mL⁻¹ in BO medium (Brackett and Oliphant 1975 Biol. Reprod. 12, 260–274) containing 5 mM theophillin and 5 μg mL⁻¹ heparin for 5 h. All of the inseminated oocytes were introduced into the maturation medium that had been kept with the cumulus cells in the CO₂ incubator. At 48 h after insemination, all embryos over the 4-cell stage were cultured in TCM-199 plus 5% FBS supplemented with each of five concentrations of flunixin meglumine (0, 0.0025, 0.005, 0.01, and 0.025%) with a cumulus cell monolayer. Development to the blastocyst stage and quality were examined at Days 7 to 8 (Day 0 = day of insemination) using a microscope. The experiment was replicated four times. Data were analyzed by the chi-square test. The total blastocyst rates from the over-4-cell embryos were 61.2 (52/89), 53.7 (44/89), 65.6 (59/90), 57.3 (51/89), and 33.7% (31/92) for 0, 0.0025, 0.005, 0.01, and 0.025%, flunixin meglumine, respectively. The total blastocyst rate with the flunixin meglumine concentration of 0.025% was significantly lower than those with the other concentrations (P < 0.05). The proportion of grade 1 blastocysts with the flunixin meglumine concentration of 0.005% was significantly higher than that with the 0, 0.0025, and 0.025% concentrations (27.8 vs 11.2, 14.6, and 5.4%; P < 0.05). Our present results show that the addition of 0.005% flunixin meglumine to the co-culture medium is positively associated with blastocyst quality in bovine embryos.