Short-term Preservation of Porcine Oocytes in Ambient Temperature: Novel Approaches

Cai-Rong Yang,Heide Schatten,Qing-Hua Zhang,Lei Guo,Xin Huang,Qing-Yuan Sun,Yi Hou,Yanchang Wei,De-Qiang Miao,Zhonghua Liu,Jing-Shan Tong,Austin Cooney

doi:10.1371/journal.pone.0014242

Abstract

The objective of this study was to evaluate the feasibility of preserving porcine oocytes without freezing. To optimize preservation conditions, porcine cumulus-oocyte complexes (COCs) were preserved in TCM-199, porcine follicular fluid (pFF) and FCS at different temperatures (4°C, 20°C, 25°C, 27.5°C, 30°C and 38.5°C) for 1 day, 2 days or 3 days. After preservation, oocyte morphology, germinal vesicle (GV) rate, actin cytoskeleton organization, cortical granule distribution, mitochondrial translocation and intracellular glutathione level were evaluated. Oocyte maturation was indicated by first polar body emission and spindle morphology after in vitro culture. Strikingly, when COCs were stored at 27.5°C for 3 days in pFF or FCS, more than 60% oocytes were still arrested at the GV stage and more than 50% oocytes matured into MII stages after culture. Almost 80% oocytes showed normal actin organization and cortical granule relocation to the cortex, and approximately 50% oocytes showed diffused mitochondria distribution patterns and normal spindle configurations. While stored in TCM-199, all these criteria decreased significantly. Glutathione (GSH) level in the pFF or FCS group was higher than in the TCM-199 group, but lower than in the non-preserved control group. The preserved oocytes could be fertilized and developed to blastocysts (about 10%) with normal cell number, which is clear evidence for their retaining the developmental potentiality after 3d preservation. Thus, we have developed a simple method for preserving immature pig oocytes at an ambient temperature for several days without evident damage of cytoplasm and keeping oocyte developmental competence.

Highlights

Oocyte preservation and transport of oocytes are important aspects of research and experimentation which oftentimes needs to be performed at a convenient time or place that is different from the site of oocyte collection
When porcine oocytes were preserved in porcine follicular fluid (pFF) and Fetal calf serum (FCS) for 3 d, 65% oocytes in pFF and 64% oocytes in FCS were arrested at the germinal vesicle (GV) stage at 27.5uC
Only 4% was arrested at the GV stage in TCM-199

Summary

Introduction

Oocyte preservation and transport of oocytes are important aspects of research and experimentation which oftentimes needs to be performed at a convenient time or place that is different from the site of oocyte collection. Low temperature freezing and vitrification are the only practical methods for long-term preservation of oocytes and embryos. Conventional cryopreservation methods (slow-freezing) proved that porcine oocytes were highly sensitive to temperature below 15uC [2], and the formation of ice crystals from the lipid content of the cytoplasm was recognized as the main reason for the high sensitivity to low temperature [3]. Cryoprotectant (CPA) toxicity and osmotic injury to the oocytes often occur during the thawing/warming phase [1]. These problems hampered the application of oocyte cryopreservation. It is essential to have a new method available for oocyte preservation based on physiological conditions without adding drugs or thawing/warming while not damaging developmental competence of porcine oocytes

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Conclusion