199 Strategies to improve canine oocyte invitro maturation

Abstract

Canine oocyte invitro maturation (IVM) is one of the challenges of animal reproduction because of low maturation and high degeneration rates. In the bitch, after ovulation, oocytes remain in an immature stage and acquire their competence in the intra- and extrafollicular (oviductal) environments. Oxidative stress and reactive oxygen species affect canine oocytes, which can be related to the high amount of lipids they contain. Therefore, the use of antioxidants such as insulin-transferrin-selenium (ITS) and lower oxygen tension during IVM could be beneficial for oocyte maturation and survival. The purpose of this study was to determine an optimum IVM culture medium and to evaluate the effect of ITS and lower oxygen tension in canine IVM. In experiment 1, TCM-199 and synthetic oviductal fluid (SOF) media were evaluated for their ability to promote nuclear maturation at 72 and 48h of culture. Also, two protein sources were used: 8% bovine serum albumin (BSA) and 2.5% fetal bovine serum (FBS), and media were supplemented with hormones. The results revealed that SOF with FBS and BSA had similar results to TCM-199 supplemented with FBS after 72 and 48h of IVM (MII rates of 7% and 4% for the 72-h group, and 4% and 10% for the 48-h group). Synthetic oviductal fluid supplemented with BSA but without FBS produced significantly higher degeneration rates compared with SOF with FBS and BSA (44% and 23%, respectively). Forty-eight hours of IVM decreased degeneration rates, with higher MII rates compared with 72h of IVM. In experiment 2, SOF medium supplemented with FBS and BSA was chosen. Oocytes were cultured in SOF with FBS and BSA supplemented at two concentrations of ITS (1 and 10μLmL−1 ITS). Supplementation with 1μLmL−1 ITS demonstrated a beneficial effect by improving maturation rates up to 20%, compared to control and 10μLmL−1 supplemented group (4% and 6% MII, respectively) after 72h of IVM. For experiment 3, oocytes were cultured in SOF medium with or without ITS (0 and 1μLmL−1 ITS) under two oxygen tensions (5% and 20% O2) for 48h. Results from this experiment demonstrated that the combination of low oxygen tension and ITS (5% O2 and 1μLmL−1 ITS) improved maturation rates up to 26.2%, although there were no statistically significant differences compared with high oxygen and ITS (20% O2 and 1μLmL−1 ITS) and low oxygen without ITS (5% O2 and 0μLmL−1 ITS) groups. These treatments were able to increase MII rates compared with the control group (20% O2 and 0μLmL−1 ITS). Parthenogenetic activation was performed on the low oxygen with or without ITS supplemented groups. The untreated group generated higher degeneration rates after 7 days of culture, and cleavage rates were low for both groups. Nevertheless, an oocyte at the 8-cell stage was obtained in the ITS-supplemented group. Taken together, these results indicate that ITS supplementation and low oxygen tension during IVM improve canine oocyte maturation.