Journal of Animal Reproduction and Biotechnology (한국동물생명공학회지)

The Korean Society of Animal Reproduction and Biotechnology (한국동물생명공학회)
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Optimization of the in vitro fertilization system in pigs

  • Song-Hee Lee (Department of Animal Science, Chungbuk National University) ;
  • Xiang-Shun Cui (Department of Animal Science, Chungbuk National University)
  • Received : 2023.05.23
  • Accepted : 2023.06.09
  • Published : 2023.06.30
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Abstract

Background: Despite considerable technological advancements, polyspermy remains a significant challenge in in vitro fertilization (IVF) procedures in pigs, disrupting normal embryonic development. Here, we aimed to determine whether optimal fertilization conditions reduce the polyspermy incidence in pigs. Methods: In vitro-matured oocytes were co-incubated with sperm according to a modified two-step culture system. Results: In the first experiment, oocytes were briefly co-incubated with sperm, washed in IVF medium, and then moved to fresh IVF medium for 5 or 6 h. Although the 6 h sperm-free cultured group had a higher penetration rate than the 5 h cultured group, the polyspermy rate significantly increased in the 6 h sperm-free cultured group. The gamete co-incubation period was either 20 or 40 min. The 40 min cultured group had a higher rate of blastocyst formation and number of total cells in blastocysts than the 20 min cultured group. In experiment 2, oocytes were inseminated with sperm separated by Pecroll treatment. Percoll treatment increased the rate of oocyte penetration and blastocyst formation compared to the control. In experiment 3, fertilized oocytes were cultured in 25 µL microdroplets (10 gametes/drop) or 500 µL (100 gametes/well) of culture medium in 4-well plates. The large volume of medium significantly reduced the number of dead oocytes and increased the rate of blastocyst formation compared to the small volume. Conclusions: Collectively, these results demonstrate that various fertilization conditions, including modified co-culture period, active sperm separation, and culture medium volume, enhance fertilization efficiency and subsequent embryonic development by decreasing polyspermy occurrence.

Keywords

Acknowledgement

This work was supported by the National Research Foundation (NRF) of Korea grant funded by the Korea government (MSIT) (No. 2022R1A2C300769), Republic of Korea.

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