(56) Cryoprotective Treatment and Vitrification Stimulate the Oocyte Activation Cascade in Swine

Jada Nix^1,2; Katelin Huang¹; Emily Kiesewetter¹; Cari Green¹; Ty Montgomery¹; Melissa Samuel¹; Kiho Lee^1,2

1. Division of Animal Sciences, University of Missouri, Columbia, Missouri USA

2. National Swine Resource and Research Center, University of Missouri, Columbia, Missouri USA

Abstract Text:

Pigs are important biomedical models and agricultural commodities, making the long-term cryogenic storage of their gametes crucial for advancing valuable genetics. However, vitrification and retainment of developmental competency in pig oocytes has proven to be difficult. This study aimed to understand how the vitrification process affects swine oocyte biology to improve the technology. For vitrification, oocytes were placed in propylene and ethylene glycol (17.5% w/v each in modified MU4 embryo culture medium) on Cryolocks in groups of 20-50 (5-8 droplets at a total volume of 3µL), stored in liquid nitrogen, then thawed and rehydrated in modified MU4 medium containing reducing concentrations of sucrose. First, we investigated if vitrification differently affects the viability of oocytes before or after in vitro maturation. Staining the oocytes with fluorescein diacetate revealed that MII oocytes survived vitrification at a significantly higher rate than cumulus-enclosed GV oocytes (83% vs. 0% respectively, P< 0.01). Next, we investigated the developmental competence of vitrified versus fresh MII oocytes. Vitrification and thawing of MII oocytes significantly decreased embryo development at cleavage and blastocyst stages when fertilized and cultured in vitro (77% and 39% control vs. 61% and 6% vitrified for cleavage and blastocyst rates, respectively, P< 0.01). Finally, to identify mechanistic actions leading to the low developmental potential, we decided to investigate biomarkers of oocyte activation following cryoprotective agent (CPA) treatment, vitrification, and thawing. We used immunofluorescent stain (Lectin PNA From Arachis hypogaea) and microscopy to determine differences in cortical granule distribution and quantity. Images were randomized and three researchers (blinded to image group) sorted each image based on fluorescence distribution. The images were classified into four grades based on a guideline published by R. Jochems et, al. in 2021 that was modified for this experiment (1. thick band of staining around the cytoplasm periphery, 2. thin band of staining, 3. some staining but no band, or 4. no staining). CPA treated and vitrified oocytes have significantly different cortical granule distribution than control MII oocytes (P< 0.001). Comparison of total fluorescence intensity shows that CPA treated and vitrified oocytes also have significantly less cortical granules than MII controls (P< 0.0001). These data point to cryoprotective treatment inducing cortical granule exocytosis and this change being conserved through vitrification and thawing. Additionally, we assessed intracellular zinc dynamics (staining with FluoZin-3 AM), since zinc release is associated with oocyte activation and cortical granule exocytosis. We detected no significant differences in mean fluorescence intensity, but the CPA treatment group did show a trend toward greater zinc content (P=0.059) when compared to controls. Collectively, our data support the hypothesis that cryoprotective treatment and vitrification may prematurely initiate the oocyte activation cascade, thereby disrupting normal fertilization processes and embryonic development. Understanding the mechanisms of vitrification induced changes to normal oocyte biology is crucial for protocol optimization, and this will ultimately enable the effective preservation of valuable genetics and better utilization of pigs for agricultural and biomedical purposes.