Effect of Cytochalasin B pretreatment on developmental potential of ovine oocytes vitrified at the germinal vesicle stage.
Oocyte cryopreservation remains a challenge in most mammalian species because of their sensitivities to chilling injuries. Relaxation of the cytoskeleton during vitrification may improve post-thaw viability and pre-implantation embryo development. The aim of this study was to investigate the effect of cytochalasin B (CB) pre-treatment before vitrification on viability, frequencies of in vitro fertilisation (IVF) and subsequent development of ovine cumulus-oocyte complexes (COCs) vitrified at the germinal vesicle (GV) stage using cryoloop. COCs obtained at slaughter were randomly divided into two groups and incubated with or without 7.5µg/mL CB for 60 min. Oocytes from each group were then vitrified using a cryoloop or used as toxicity and controls. Oocytes were then matured, fertilised, and cultured in vitro for 7 days. Viability following vitrifiaction and warming, fertilisation events following IVF and subsequent pre-implantation embryo development were evaluated. No significant differences were observed in survival rates between CB treated and non-treated oocytes in both vitrified and toxicity groups. Frequencies of fertilisation were increased in CB-vitrified group (oocytes pre-treated with CB before vitrification) than those vitrified without CB pre-treatment (57.0% vs 40.7%). Cleavage was significantly lower (P < 0.05) in vitrified and CB-vitrified oocytes at both 24 hpi (12.5% vs 9.1%) and 48 hpi (25.0% vs 16.2%) than in other groups. Based on the numbers of cleaved oocytes, (48 hpi), 16.1% and 18.8% of the cleaved embryos developed to blastocysts in both vitrified and CB-vitrified groups. These values did not differ significantly from those obtained in CB-control group (37.8%). No significant differences were observed in mean cell numbers per blastocyst between all groups. In conclusion, pre-treatment of ovine GV oocytes with cytochalasin B as cytoskeleton stabilizer before vitrification increased frequencies of in vitro fertilisation and subsequently resulted in production of good quality late stage pre-implantation embryos following IVF.
Division of Animal Sciences, School of Biosciences, The University of Nottingham, Sutton Bonington Campus, Loughborough, UK. Department of Theriogenology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt. adelreda902@hotmail.com.
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Moawad
Division of Animal Sciences, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, UK.
J
J
Zhu
Division of Animal Sciences, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, UK.
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I
Choi
Division of Animal Sciences, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, UK.
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D
Amarnath
Division of Animal Sciences, School of Biosciences, The University of Nottingham, Sutton Bonington Campus, Loughborough, UK.
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KH
Campbell
D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary*; D003571:Cytochalasin B / Q000008:administration & dosage / Q000378:metabolism*; D047108:Embryonic Development / Q000187:drug effects; D005260:Female; D005307:Fertilization in Vitro / Q000379:methods / Q000662:veterinary*; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000254:growth & development*; D012756:Sheep; D058989:Vitrification
D003571:Cytochalasin B