41 Evaluation of two-stage delipidation on bovine embryo development and cryotolerance

Cryopreservation of bovine embryos is a crucial aspect of reproductive technologies in the livestock industry. However, cryopreservation reduces viability and pregnancy rates due to cryodamage and the presence of intracellular lipids increases susceptibility to cryodamage. The objective of this research is to explore the feasibility of delipidation (DeL) as a strategy to enhance bovine embryo vitrification. We hypothesised that the DeL process does not affect developmental potency but mitigates cryoinjuries by down-regulation of lipid metabolism-related genes. Bovine embryos were produced by IVF from ovaries obtained from abattoir and commercial cryopreserved semen from 2 bulls of proven fertility. A total of 590 presumptive zygotes on Day 1 after fertilization were randomly subjected to one of two treatment groups, namely DeL or sham control (Ctrl) group. Zygotes in the DeL group were incubated with 7.5 μg/mL cytochalasin B followed by first centrifugation (13 000g) and then hyperosmotic media (0.15 M sucrose) was added to create more perivitelline space before the second centrifugation. Embryos in the Ctrl group were processed in the same manner but without centrifugation. Cleavage and blastocyst rates were assessed on Days 2 and 7 post-IVF. Expanded blastocysts on Day 7 were vitrified using standard Cryotop vitrification method and were imaged for 24 h under time-lapse microscopes after warming. Time to re-expansion and time to hatch were analysed by proportional hazards model and embryos were collected after then for further gene expression, oxidative stress (ROS), or apoptotic (TUNEL) assay. Statistics are performed using unpaired, two-tailed Student’s t-test. Preliminary results indicate that delipidated zygotes have similar developmental potency as controls but have lower ROS staining intensity. Cleavage rates of the DeL and Ctrl groups were 80.3% and 84.3%, blastocyst rates were 29.8% and 30.6% (n ≥ 4), and the postwarmed re-expansion rate was 100% for both groups (n ≥ 33, P > 0.05). Time to hatching for the DeL group was 801 min compared with 968 min, (n ≥ 14, P = 0.1058). The ROS staining intensity was significantly lower in the DeL group (0.49 vs 1.00, relative intensity, n ≥ 6, P = 0.0003); while the portion of apoptotic cell stained with TUNEL was similar between groups (n ≥ 10, DeL = 6.25 vs Ctrl = 6.90, P = 0.72). In conclusion, the DeL technique does not have significant effects on embryo development rates but does represent a valuable technique to increase our understanding of lipid-mediated cryoinjury and may pave the way for the development of more effective cryopreservation protocols in bovine reproductive technologies, with broader implications for other mammalian species including human as well. Further research into lipid metabolism and cryoinjury-related gene expression such as Fabp3, Plin2, Plin3, Cpt2, and Cirbp is warranted.