Sperm and oocyte treatments to improve the formation of male and female pronuclei and subsequent development following intracytoplasmic sperm injection into bovine oocytes

• Published in: Biology of reproduction Vol. 59; no. 4; pp. 918 - 924
• Main Authors: Rho, G.J. (University of Guelph, Ontario, Canada.), Kawarsky, S, Johnson, W.H, Kochhar, K, Betteridge, K.J
• Format: Journal Article
• Language: English
• Published: Madison, WI Society for the Study of Reproduction 01.10.1998
• Subjects: 6-DIMETHYLAMINOPURINE; Animals; Biological and medical sciences; Birth control; CATTLE; Cell Nucleus - drug effects; Cell Nucleus - physiology; Cell Nucleus - ultrastructure; Culture Media; Cytoplasm - drug effects; Cytoplasm - physiology; DEVELOPMENTAL COMPETENCE; DITHIOTHREITOL; Dithiothreitol - pharmacology; EMBRYO CULTURE; EMBRYO TRANSFER; Embryo, Mammalian - drug effects; Embryo, Mammalian - physiology; EMBRYONIC DEVELOPMENT; ENZYME INHIBITORS; Female; Fertilization in Vitro; Gynecology. Andrology. Obstetrics; INJECTION; IONOMYCIN; Ionomycin - pharmacology; IONOPHORES; Ionophores - pharmacology; Male; Medical sciences; Microinjections; NUCLEUS; OOCYTE ACTIVATION; Oocytes - drug effects; Oocytes - physiology; Oocytes - ultrastructure; OVA; Ploidies; PRETREATMENT; PRONUCLEAR FORMATION; PRONUCLEUS; Sex Differentiation - drug effects; Sperm-Ovum Interactions - drug effects; Sperm-Ovum Interactions - physiology; SPERMATOZOA; Spermatozoa - drug effects; Spermatozoa - physiology; Spermatozoa - ultrastructure; Sterility. Assisted procreation; Sulfhydryl Reagents - pharmacology; THIOLS; Embryonic development; Spermatozoa; Intracytoplasmic sperm injection; Ox; Germinal cell; Fecundation; In vitro; Pronucleus; Result; Vertebrata; Mammalia; Chromosomic decondensation; Artiodactyla; Ungulata; Oocyte
• ISSN: 0006-3363; 1529-7268
• DOI: 10.1095/biolreprod59.4.918

This study assessed pronuclear formation, the chromosomal constitution, and the developmental capacity of bovine zygotes formed by intracytoplasmic injection of oocytes with sperm, treated or not with dithiothreitol (DTT). Oocytes were matured in vitro for 22–24 h and then centrifuged so that sperm, prepared by swim-up in the presence or absence of 5 mM DTT, could be injected into the cleared area of the ooplasm. Injected oocytes were activated by treatment with 5 μM ionomycin (5 min) and, after a 3-h interval, with 1.9 mM 6-dimethylaminopurine (DMAP) for 3 h. They were then cocultured with bovine oviductal epithelial cells in M199. Sperm treatment resulted in a significantly higher proportion of male pronucleus formation 16 h after injection (40% vs. 11%; p < 0.0001) and a significantly higher rate of blastocyst development (24% vs. 10%; p < 0.005). Sixty-one percent of blastocysts produced with treated sperm were diploid. Of 12 blastocysts produced with treated sperm and sexed by a polymerase chain reaction, 4 were male and 7 female, and in one a definite diagnosis could not be made. Embryo transfer (2 embryos per heifer) resulted in pregnancies in 6 of 16 recipients at Day 49, but none was carried to term. These results show that the efficiency of bovine intracytoplasmic sperm injection can be improved by sperm pretreatment with DTT and by oocyte activation with ionomycin plus DMAP, although the developmental capacity of the resulting embryos remains limited.