214 EFFECT OF LONG-DISTANCE TRANSPORTATION OF OVARIES ON THE DEVELOPMENT OF IN VITRO-MATURED–IN VITRO-FERTILIZED–IN VITRO-CULTURED BOVINE EMBRYOS

Abstract

Keeping ovaries for a long time before oocyte recovery affects the maturation rate and blastocyst rate (BL rate), but a change in the temperature of the ovary at the time of transportation does not affect the BL rate (Ribeiro et al. 2008 Anim. Reprod. Sci. 108, 171–179). The objective of this study was to investigate the effect of long-distance transportation of ovaries on the in vitro development of bovine oocytes into blastocysts. Ovaries in the transportation group (Group T) were collected from a slaughterhouse, placed inside a Thermos flask, and transported to the laboratory within 17 to 21 h. The Thermos flask was covered with a freezer pack in a foam polystyrene box (Nakatate et al. 2006 Reprod. Fertil. Dev. 18, 193). The temperature of the Thermos flask was measured with a temperature recorder. Cumulus–oocyte complexes (COC) were collected by aspirating 2- to 6-mm follicles from the ovaries. The COC were matured in TCM-199 supplemented with 5% calf serum and 0.02 AU mL^–1 of FSH. Groups of 20 COC were incubated in 100-μL drops of IVM media at 38.5°C under an atmosphere of 5% CO₂ in air for 20 h. After 18 h of gamete co-culture (3 × 10⁶ sperm mL^–1), the presumptive zygotes were cultured in CR1aa medium supplemented with 5% calf serum for 9 days at 38.5°C under an atmosphere of 5% CO₂, 5% O₂, and 90% N₂. Embryonic development was evaluated at 48 h after IVF (total cleavage rates, CL rate) and on Days 7 to 9 (BL rate). The ovaries in the control group (Group C) were collected from a slaughterhouse different from that of the Group T ovaries and transported to the laboratory in saline (23°C) in a Thermos flask within 3 h. These ovaries were then used for the collection of COC in the same way as for Group T. Data were analysed by chi-square test. A total of 25 experiments were performed using the ovaries from Groups C and T (n = 3945 v. n = 7218). The CL rate and the BL rate for Group T were significantly lower than those for Group C (41.8 v. 62.0% and 16.1 v. 29.7%; P < 0.01). In Group T, the duration of ovary transportation did not affect the CL rate (within 19 h, between 19 and 20 h, and more than 20 h), but the BL rate was significantly decreased with an increase in transport time (18.7 v. 16.1 v. 14.8%). The temperature change in the Thermos flask during transportation ranged from 12.0 to 25.1°C; the temperature was maintained virtually constant or decreased. The CL rates of the groups that underwent a temperature (t) change (t ≤ 5.0 and 5.0 < t ≤ 10.0) were not significantly different, but the BL rate in the group with 5.0 < t ≤ 10.0 was significantly lower than that of the other group (16.7 v. 13.5%; P < 0.01). These results suggest that the long-distance transportation of ovaries affects in vitro embryo development. Moreover, a decrease in temperature during transportation may have an adverse effect on blastocyst formation.