38 Novel method of vitrification and warming techniques for canine mature oocytes and early pregnancy confirmation

Somatic cell nuclear transfer (SCNT) is the primary method for producing canine disease models and conserving endangered species, necessitating a reliable supply of oocytes. However, owing to the unique physiological characteristics of canines that ovulate in an immature state and the lack of proper establishment of ART in an in vitro environment, the IVM of canine oocytes remains unestablished. Thus, SCNT relies on surgical retrieval of mature oocytes using oviductal flushing. Effective cryopreservation of mature oocytes is essential, but the unique reproductive physiology of canines, which includes ovulation in an immature state and high lipid content in oocyte cytoplasm, complicates this process. Previous studies on canine oocyte cryopreservation have focused mainly on immature oocytes, with limited success and practical application. This study aimed to establish an effective vitrification and warming procedure for canine mature oocytes using melatonin supplementation. This procedure was evaluated with regard to its effects on oocyte viability, intracellular ROS levels, developmental potential, and cloning efficiency. Mature oocytes were retrieved from mongrel female dogs in the estrus stage using a surgical oviduct flushing method. The oocytes were then subjected to vitrification warming with or without melatonin supplementation (vitrified+mel and vitrified groups, respectively). Survival rates, intracellular ROS levels, mitochondrial distribution, and developmental competence after parthenogenetic activation (PA) were evaluated. Additionally, SCNT was performed to assess cloning efficiency and pregnancy rates. The results indicated no significant difference in survival rates between the control, vitrified, and vitrified+mel groups. However, melatonin significantly reduced intracellular ROS levels in vitrified-warmed oocytes and embryos compared with the vitrified group. Developmental competence after PA showed that while vitrified group embryos did not progress beyond the 8-cell stage, the vitrified+mel group developed to the blastocyst stage, similar to the control group. The SCNT results demonstrated improved fusion rates in the vitrified+mel group compared with the vitrified group (52.63 ± 4.17% vs. 30.13 ± 6.71%). Reconstructed embryos from both groups were transferred to surrogate mothers (four in the vitrified group and six in the vitrified+mel group, respectively), and two of the surrogate mothers in the vitrified+mel group had a confirmed pregnancy and maintained it until mid-term. In conclusion, despite no full-term pregnancies, these findings suggest melatonin’s potential to enhance cloning efficiency and early development in canine oocytes. In addition, this study successfully establishes a vitrification-warming procedure for canine mature oocytes, demonstrating melatonin’s protective effects on oocyte viability and developmental potential. These findings offer a promising approach for canine oocyte cryopreservation, which is crucial for producing disease models and conserving endangered species.

This work was supported by Korea Drug Development Fund funded by Ministry of Science and ICT, Ministry of Trade, Industry, and Energy, and Ministry of Health and Welfare (HN22C057100) and BK21 FOUR Program by Chungnam National University Research Grant, 2024.