170 POST-THAW VIABILITY OF IN VITRO-PRODUCED BOVINE EMBRYOS CULTURED EITHER IN HOST CAPRINE REPRODUCTIVE TRACTS OR IN VITRO
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In vitro culture of bovine embryos is usually associated with poor pregnancy rate following cryopreservation. The objective of this study was to compare the post-thaw viability of in vitro-produced bovine zygotes, cultured in vitro or in the reproductive tract of a host goat. Cumulus-oocyte complexes were matured in vitro, and in vitro fertilization was carried out with frozen-thawed semen as per standard laboratory procedures. At 18-20 h post-fertilization, zygotes were stripped of remaining cumulus cells and randomly separated into culture treatments. In three replicates, a total of 606 embryos were surgically transferred 12 to 24 h post-ovulation to the oviducts of an estrous-synchronized goat (VIVO) and 550 embryos were cultured in G1.3 for 72 h and then moved to G2.3 medium for 96 h and in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 (IVC). On Day 7, embryos were flushed from the excised tract with a 69.5% recovery rate or removed from culture. Embryos were classified according to IETS criteria with grades and stages recorded. All data were analyzed using the one-way analysis of variance and means were compared using Student's t-test. No differences were seen in the percentage of freezable quality embryos per total recovered between the two groups (34.3% vs. 32.3% for IVC and VIVO, respectively). However, there was a significant difference in the pre-freezing stage between the two culture groups (Stage 5.5 0.22 vs. Stage 4.8 0.26 for IVC and VIVO, respectively; P < 0.05), but no difference in the quality grade. All embryos greater than Stage 4, Grade 2 were frozen in groups of 5-10 in ethylene glycol with sucrose (Vigro Ethylene Glycol Freeze Plus; Bioniche Animal Health, Belleville, Ontario, Canada) in 0.25-mL straws. After thawing, embryo groups were washed, rehydrated, and incubated in G2.3 as above. Morphology was assessed by assigning grade and stage objectively at 24 h and 48 h post-thaw. Post-thaw viability in vitro was not different between groups (73.4% vs. 72.7% for IVC and VIVO, respectively). The average changes in morphology post-thaw from pre-freezing to 24 h and from 24 h to 48 h within each freezing group were determined. There was no significant difference in the mean change in stage (0.67 0.15 vs. 0.82 0.17 at 24 h and 0.31 0.09 vs. 0.37 0.10 at 48 h for IVC and VIVO, respectively) or grade (0.60 0.15 vs. 0.41 0.17 at 24 h and 0.03 0.06 vs. 0.14 0.07 at 48 h for IVC and VIVO, respectively) at either observation point. These results suggest that culture of in vitro-fertilized bovine embryos in the caprine reproductive tract did not alter post-thaw development or improve post thaw viability compared to in vitro cultured controls. However, morphological evaluation is too subjective to successfully predict pregnancy rate after transfer; therefore, further study is needed to determine if there are differences in pregnancy rates between these culture methods.
