title: Development of a pronuclear DNA microinjection technique for production of green fluorescent protein-expressing bubaline (Bubalus bubalis) embryos.
abstract: Oocytes from abattoir-derived bubaline (Bubalus bubalis) ovaries were subjected to IVM and IVF; the objective was to develop a pronuclear DNA microinjection technique to produce embryos expressing green fluorescent protein (GFP). The largest proportion (61.2%) of zygotes in which one (1 PN) or two pronuclei (2 PN) were visible was when centrifugation (14,000 x g for 15 min) was done 16 h after insemination. Centrifugation had no adverse effects on cleavage rate, development to morulae/blastocysts, and total cell number of embryos. Piercing the pronuclear but not the plasma membrane reduced (P<0.05) cleavage rate (44.0% vs. 51.0%), without affecting subsequent development. Following microinjection of a GFP-DNA construct, cleavage rate (55.9, 38.9, and 30.9%) and proportion of cleaved embryos that developed to morulae (39.9, 25.6, and 15.5%) and blastocyst stages (22.4, 13.4, and 2.8%) were higher (P<0.05) for non-injected controls than for those injected with buffer alone, which, in turn, were higher (P<0.05) than for those injected with buffer containing 5 microg/mL DNA. The cleavage rate (39.2% vs. 34.8%) and proportion of cleaved embryos that developed to morulae/blastocysts (37.5% vs. 10.9%) were higher (P<0.05) for microinjected zygotes with 2 PN than for those with 1 PN. The cleavage rate and the proportion of cleaved embryos that developed to morulae and blastocysts were higher (P<0.05) following culture of microinjected zygotes in mCR2aa medium (40.7, 32.7, and 9.1%, respectively) compared to those for mSOFaa (33.3, 26.0, and 6.5%, respectively) or after culture in TCM-199+co-culture with buffalo oviductal epithelial cells (31.2, 25.0, and 4.5%, respectively). The proportion of embryos expressing GFP was higher (P<0.01) for 2 PN than for 1 PN zygotes (15.9% vs. 13.7%). Thirty-five embryos expressed GFP; the proportion of mosaic embryos (62.8%) was higher (P<0.01) than of embryos in which all blastomeres expressed GFP (37.2%); eight and two of those embryos developed to the morula and blastocyst stages, respectively.
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute, Karnal, Haryana 132001, India.
authors.forename: V
authors.initials: V
authors.lastname: Verma
authors.forename: S K
authors.initials: SK
authors.lastname: Gautam
authors.forename: P
authors.initials: P
authors.lastname: Palta
authors.forename: R S
authors.initials: RS
authors.lastname: Manik
authors.forename: S K
authors.initials: SK
authors.lastname: Singla
authors.forename: M S
authors.initials: MS
authors.lastname: Chauhan
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000502:physiology; D002020:Buffaloes / Q000196:embryology*; D002467:Cell Nucleus / Q000648:ultrastructure; D004247:DNA / Q000008:administration & dosage*; D046149:Embryo Culture Techniques / Q000662:veterinary; D047108:Embryonic Development; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary*; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics*; D008297:Male; D008845:Microinjections; D009028:Morula / Q000502:physiology; D015053:Zygote / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D049452:Green Fluorescent Proteins; D004247:DNA