Transport, meiotic arrest, and biphasic maturation of canine oocytes

Abstract

Assisted reproduction is undeveloped in the canine compared to most domestic mammalian species. One of the contributing reasons is the inefficiency of in vitro maturation (IVM) of the canine oocyte. This project was designed to examine several factors regarding canine IVM, including in vitro oocyte transport conditions and oocyte arrest before IVM. Nine transport conditions were assessed for the ability to maintain oocyte viability during transport and subsequent in vitro culture. Whole reproductive tracts and excised ovaries were transported at either 20°C or 37°C in PBS. Collected oocytes were transported in either TCM-199 with Hank's salts or TL Hepes at 20°C or 37°C, or in a TCM-199 with Earl's salts based CO₂ buffered maturation medium at 37°C. Transporting collected canine oocytes in TL Hepes at 37°C appeared to be the most beneficial condition for maintaining oocyte viability and supporting subsequent IVM. Dibutyryl cyclic AMP (dbcAMP) was evaluated as a meiotic inhibitor for canine oocytes to maintain them in the germinal vesicle stage (GV) allowing time to gain cytoplasmic competence. Oocytes were cultured in 0, 0.5, 1.0, or 1.5 mM dbcAMP for 20, 31 or 42 hours. Success was determined by retention of the GV. Results indicate that every level of dbcAMP tested had a significant increase in GV retention over the control. The ability of the canine oocyte to resume meiosis after exposure to dbcAMP was evaluated using biphasic culture. For the first part of the experiment, oocytes were cultured for 20 hours in 0, 0.5, or 1.5 mM dbcAMP and then cultured for 48 hours in maturation medium with 0 or 0.01 [mu]g/ml of invasive adenylate cyclase (iAC). Results indicate that culturing the oocytes in 0.5 mM dbcAMP for 20 hours produced the highest rates of meiotic resumption and maturation to metaphase II (MII). The second part of this experiment evaluated culturing oocytes for 0, 24, or 48 hours in 0.5 mM dbcAMP and then transferring them to a maturation medium for 72 hours with or without iAC. Results indicate that there were no significant differences between the control and the treatment groups.