title: Effects of different activation treatments on fertilization of horse oocytes by intracytoplasmic sperm injection.
abstract: The effects of four reagents on the activation and subsequent fertilization of equine oocytes, and the development of these after intracytoplasmic sperm injection, were investigated. Cumulus-oocyte complexes collected from equine ovaries obtained from an abattoir were matured in vitro for 40-44 h in TCM199 medium before being injected, when in metaphase II, with an immobilized stallion spermatozoon. The cumulus-oocyte complexes were then subjected to one of five activation treatments: (a) 10 micromol ionomycin l(-1) for 10 min; (b) 7% (v/v) ethanol for 10 min; (c) 100 micromol thimerosal l(-1) for 10 min; (d) 250 micromol inositol 1,4, 5-triphosphate l(-1) injection; and (e) no treatment (control). After 18-20 h further culture, the cumulus-oocyte complexes were assessed for activation by observing whether they had progressed through second anaphase-telophase and had formed a female pronucleus. The proportions of oocytes activated after each treatment were: 16/27 (59%) for ionomycin; 14/25 (56%) for ethanol; 22/28 (79%) for thimerosal; 15/27 (56%) for inositol 1,4,5-triphosphate; and 0/20 (0%) for the untreated controls. Thus, significantly more oocytes (P < 0.05) were activated by treatment with thimerosal than by the other four treatments. The proportions of oocytes that cleaved to the two-cell stage at 24-30 h after sperm injection in the groups treated with ionomycin, ethanol and thimerosal were 7/20 (35%), 5/19 (26%) and 11/23 (48%), respectively. No cleavage was observed in any of the control oocytes or those treated with inositol 1,4, 5-triphosphate. Furthermore, evidence of normal fertilization was observed in 2/7 (29%), 2/5 (40%) and 7/11 (64%) of the oocytes treated with ionomycin, ethanol and thimerosal, respectively. These results demonstrated that: (a) it is possible to activate equine oocytes with the chemical stimulants, ionomycin, ethanol, thimerosal and inositol 1,4,5-triphosphate; (b) thimerosal is more effective than the other three reagents in facilitating both meiotic activation and normal fertilization of equine oocytes; and (c) chemical activation may also stimulate parthenogenetic cleavage of oocytes without concurrent changes in the head of the spermatozoon.
authors.affiliation: University of Cambridge, Department of Clinical Veterinary Medicine Equine Fertility Unit, Mertoun Paddocks, Woodditton Road, Newmarket, Suffolk CB8 9BH, UK.
authors.forename: X
authors.initials: X
authors.lastname: Li
authors.forename: L H
authors.initials: LH
authors.lastname: Morris
authors.forename: W R
authors.initials: WR
authors.lastname: Allen
mesh_terms: D000818:Animals; D016009:Chi-Square Distribution; D002970:Cleavage Stage, Ovum; D000431:Ethanol / Q000494:pharmacology; D005260:Female; D005307:Fertilization in Vitro / Q000379:methods / Q000662:veterinary*; D006736:Horses; D007267:Injections; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D008297:Male; D011310:Preservatives, Pharmaceutical / Q000494:pharmacology; D013268:Stimulation, Chemical; D013849:Thimerosal / Q000494:pharmacology
chemical_list: D007476:Ionophores; D011310:Preservatives, Pharmaceutical; D013849:Thimerosal; D000431:Ethanol; D015759:Ionomycin; D015544:Inositol 1,4,5-Trisphosphate