199 Strategies to improve canine oocyte in vitro maturation

Abstract

Canine oocyte in vitro 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 48 h 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 48 h 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 72 h 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 10 μL mL⁻¹ ITS). Supplementation with 1 μL mL⁻¹ ITS demonstrated a beneficial effect by improving maturation rates up to 20%, compared to control and 10 μL mL⁻¹ supplemented group (4% and 6% MII, respectively) after 72 h of IVM. For experiment 3, oocytes were cultured in SOF medium with or without ITS (0 and 1 μL mL⁻¹ ITS) under two oxygen tensions (5% and 20% O₂) for 48 h. Results from this experiment demonstrated that the combination of low oxygen tension and ITS (5% O₂ and 1 μL mL⁻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 1 μL mL⁻¹ ITS) and low oxygen without ITS (5% O₂ and 0 μL mL⁻¹ ITS) groups. These treatments were able to increase MII rates compared with the control group (20% O₂ and 0 μL mL⁻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.