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Effects of disulfide bond reducing agents on sperm chromatin structural integrity and developmental competence of in vitro matured oocytes after intracytoplasmic sperm injection in pigs

¹ Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, College of Animal Sciences and Technology, China Agricultural University, Beijing 100094, People's Republic of China² College of Animal Sciences and Technology, Yunnan Agricultural University, Kunming 650201, People's Republic of China³ College of Veterinary Medicine, China Agricultural University, Beijing 100094, People's Republic of China⁴ Department of Obstetrics and Gynecology, University of California Irvine, 101 Theory, Suite 200, Irvine, California 92697, USA

Correspondence should be addressed to J-H Tian; Email: tianjh{at}cau.edu.cn

W-M Cheng and L An contributed equally to this work

We recently reported that electrical activation followed by secondary chemical activation greatly enhanced the developmental competence of in vitro matured porcine oocytes fertilized by intracytoplasmic sperm injection (ICSI). We hypothesized that sperm treatment with disulfide bond reducing agents will enhance the development competence of porcine embryos produced by this ICSI procedure. We examined the effects of glutathione (GSH), dithiothreitol (DTT), GSH or DTT in combination with heparin on sperm DNA structure, paternal chromosomal integrity, pronuclear formation, and developmental competence of in vitro matured porcine oocytes after ICSI. Acridine orange staining and flow cytometry based sperm chromatin structure assay were used to determine sperm DNA integrity by calculating the cells outside the main population (COMP T). No differences were observed in COMP T values among GSH-treated and control groups. COMP T values in GSH-treated groups were significantly lower than that in DTT-treated groups. Following ICSI, GSH treatments did not significantly alter paternal chromosomal integrity. Paternal chromosomal integrity in sperm treated with DTT plus or minus heparin was also the lowest among all groups. GSH-treated sperm yielded the highest rates of normal fertilization and blastocyst formation, which were significantly higher than that of control and DTT-treated groups. The majority of blastocysts derived from control and GSH-treated spermatozoa were diploid, whereas blastocysts derived from DTT-treated spermatozoa were haploid. In conclusion, sperm treatment with GSH enhanced the developmental capacity of porcine embryos produced by our optimized ICSI procedure.