174 THE EFFECT OF DIFFERENT OVARY TRANSPORT TEMPERATURES ON IN VITRO DEVELOPMENT AND POST-THAW SURVIVAL OF BOVINE EMBRYOS

Abstract

The present study was carried out to investigate the effect of different ovary transport temperatures on in vitro development and post-thaw survivability of bovine embryos. Bovine ovaries were collected at a local slaughterhouse and transported at 4 different temperature categories to the laboratory: 7–10°C (T1), 11–17°C (T2), 18–25°C (T3), and above 26°C (control group). The cumulus–oocyte complexes (COCs) were aspirated from 2–8 mm antral follicles using a syringe with an 18 gauge needle. Selected COCs were washed in HEPES-buffered tissue culture medium (TCM-199) supplemented with 5% FBS. Sets of 50 COCs were matured for 22 h in 4-well dishes of TCM-199 supplemented with 5% FBS, 10 µg mL^-1 LH, and 10 µg mL^-1 FSH, that had been previously covered with mineral oil and equilibrated in an atmosphere of 5% CO₂ in air at 39°C. Mature COCs were fertilized with frozen–thawed semen treated with BO medium. To evaluate nuclear maturation to the metaphase II stage, the matured COCs were fixed in 1 : 3 acetic acid–ethanol for 30 s and stained with 3% basic Fuchsin. For embryo freezing, Day 7 and 8 blastocysts were equilibrated for 15 min in 1.8 M ethylene glycol as a cryoprotectant. Embryos were loaded into 0.25-mL straws at room temperature, plunged directly into a cooling chamber, kept at -7°C for 10 min, including time for seeding, and further cooled to -35°C at -0.3°C min^-1; after 2 min at this temperature, they were plunged into liquid nitrogen. Thawing was performed by keeping straws at room temperature for 10 s, followed by immersion in a water bath at 37°C. The appearance of the embryos was evaluated immediately after warming and again at 24-h intervals for at least 3 days. The development rate was assessed by the re-expansion of the blastocoel and the hatching of blastocysts. Results were compared by ANOVA. The rates of maturation (to metaphase II), cleavage, and development to blastocysts were compared among treatment groups. Furthermore, frozen–thawed blastocysts were in vitro cultured to compare the survivability among groups. The maturation rates in the T1, T2, and T3 groups (24/40, 60.0%; 25/41, 61.0%; and 30/44, 68.2%, respectively) were significantly lower than that in the control group (36/44, 81.8%; P < 0.05). The cleavage rates in the T1 and T2 groups (61/116, 52.6% and 66/121, 54.5%) were significantly lower than that in the control group (112/134, 83.6%; P < 0.05). However, there was no difference in the development rate to blastocysts among all groups (27.9–33.0%; P > 0.05). The survivability of frozen–thawed embryos was significantly lower in the T1 group (6/13, 46.2%) than in the T2 (11/16, 68.8), T3 (13/18, 72.2%), and control groups (19/26, 73.1%; P < 0.05). In conclusion, the results suggest that ovary transport at 26°C may be optimal for better in vitro development and survival of frozen–thawed embryos produced in vitro. Furthermore, exposure of ovaries to temperatures below 10°C during transport may significantly decrease both in vitro development and survivability of frozen-thawed blastocysts.