289 EFFECTS OF THE STORAGE OF BOVINE OVARIES ON THE NUCLEAR MATURATION AND DEVELOPMENT OF IN VITRO PRODUCED EMBRYOS

Abstract

The objectives of this study were to investigate effects of storage of bovine ovaries on the maturation of oocytes and to determine the optimal maturation time for oocytes obtained from the stored ovaries. Ovaries were obtained at a local abattoir and transported in physiological saline to the laboratory (18°C, 3 h; storage group). As a control, oocytes were collected from ovaries without storage. Other ovaries were kept in a plastic bag without solution (Bag-group) or with saline (Saline-group). These ovaries were preserved at 20°C for 18 h. Then cumulus-oocyte complexes were collected and maturated in TCM-199 + 5% CS. In Experiment 1, to investigate effects of the storage methods of bovine ovaries on the timing of germinal vesicle breakdown (GVBD) and the progression to MII in oocytes obtained from ovaries, oocytes were fixed every 2 h (0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 h) from the start of in vitro maturation, and then stained for examination of their nuclear stage. In Experiment 2, to investigate effects of length of in vitro maturation (18, 20, 22, 24 h) of oocytes (18-h, 20-h, 22-h and 24-h group, respectively) obtained from the ovaries stored in a saline for 18 h at 20°C on the subsequent in vitro development after IVF and IVC. Following insemination, the presumptive zygotes were cultured in CR1aa + 5% CS for 6 days to assess the development of embryos on Day 2 (Day 0 = the day of IVF) for rates of cleavage and on Day 6 for rates of embryo development to morulae (M), compacted morulae (CM), and blastocyst (BL) stages. The data of nuclear stage were analyzed by ANOVA after transformation to arcsine, and the rates of embryo development were analyzed by chi-square. There were two peaks of GVBD in the storage group, one occurred at 2 h of maturation culture, the other at 4–8 h of culture as control. There were between-treatment differences in the timing of increase in the rates of oocytes to reach MII. After 12 h of culture 21.2 ± 1.1% of oocytes in the Saline-group and 11.6 ± 4.6% of oocytes in the Bag-group reached MII, but no oocytes in the control group reached MII (P < 0.05). Furthermore, the rate of oocytes in the Saline-group matured to MII at 20 h of culture was lower than that of the control group (Bag-group: 67.9 ± 7.3%; Saline-group: 61.2 ± 14.5%; control: 82.9 ± 5.3%) (P < 0.05). The rates of embryos that cleaved after IVF of IVM oocytes in the 18-h group (90.2 ± 7.0%) was higher than those of the other groups (20-h group: 81.3 ± 8.2%, 22-h group: 80.5 ± 13.2%, 24-h group: 75.8 ± 6.0%) (P < 0.05). The rate of embryos developed to M, CM, and BL stages in the 18-h group (48.4 ± 6.7%) was the highest among the treatments, and significantly higher than that of the 24-h group (36.2 ± 6.7%) (P < 0.05). These results indicated that the timing of undergoing GVBD and reaching MII of oocytes obtained from the stored ovaries was earlier than that of oocytes obtained from the non-preserved ovaries, and the optimal maturation time for oocytes obtained from stored ovaries was 18 h.

This work was supported by The Ito Foundation, Tokyo, Japan.