A comparative study of parthenogenic activation and in vitro fertilization of bubaline oocytes.
The effect of chemical activation protocols on in vitro-matured oocytes were compared to results with IVF (natural activation). Buffalo ovaries were collected in normal saline and transported to the laboratory within 2 h of slaughter. Good quality oocytes, collected by aspiration from 3 to 10 mm follicles, were matured for 22-24 h. Matured oocytes were subjected to either IVF (control) or chemical activation (treatment). For IVF, in vitro-matured oocytes were co-incubated with in vitro-capacitated approximately 1x10(6) frozen/thawed sperm of a Murrah bull and fertilized in modified synthetic oviductal fluid (mSOF) medium. Chemicals for oocytes activation comprised (a) 7% ethanol (ET) for 7 min+2.5 mM 6-dimethyl amino purine (6-DMAP) for 4h, (b) 7% ET for 7 min+10 microg/ml cycloheximide (CHX) for 6h and (c) 7% ET for 7 min+2.5 mM 6-DMAP+10 microg/ml CHX for 6 h. To study embryo development, fertilized and chemically activated oocytes were cultured in mSOF medium for up to 8 days. In this study, a mean of 1.9+/-0.02 maturable oocytes/ovary were recovered and 90.4% matured. Cleavage rate was significantly higher following ET+DMAP, ET+CHX and ET+CHX+DMAP activation (52.5%, 52.5% and 44.4%, respectively) compared to IVF (36.5%, 23.4% and 26.8%, respectively). Blastocyst development (30.9% versus 15.2%) was also significantly higher following ET+CHX+DMAP activation than IVF. The results of parthenogenesis reveal that buffalo oocytes had better inherent developmental competence and that the poor cleavage and embryo development following IVF may be due partly to the poor quality of frozen/thawed sperm, improper sperm capacitation and/or fertilization.
Mishra
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Department of Molecular Biology and Genetic Engineering, College of Basic Science and Humanities, GB Pant University of Agriculture and Technology, Udham Singh Nagar, Uttaranchal, India. mishrav@30gigs.com
Misra
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Sharma
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D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D002020:Buffaloes / Q000196:embryology / Q000502:physiology*; D018929:Cell Culture Techniques; D003513:Cycloheximide / Q000494:pharmacology*; D004622:Embryo, Mammalian / Q000502:physiology; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary*; D008297:Male; D009865:Oocytes / Q000502:physiology*; D010312:Parthenogenesis / Q000187:drug effects* / Q000502:physiology*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology
D011500:Protein Synthesis Inhibitors; C001020:N(6),N(6)-dimethyladenine; D003513:Cycloheximide; D000225:Adenine