Stem cell-like outgrowths from in vitro fertilized goat blastocysts.
With an aim to isolate, culture and characterize goat embryonic stem cell-like cells derived from in vitro fertilized goat blastocysts, slaughterhouse derived goat oocytes were in vitro matured in maturation medium in 5% CO2 air at 38.5 degrees C. Matured oocytes were fertilized in vitro with fresh capacitated spermatozoa. Total 636 (36.5%) cleaved embryos were obtained which were further co-cultured with goat oviductal epithelial cells (GOEC) for 7-10 days. GOEC culture system was better for formation of morula (150; 44.3%) and hatched blastocyst (13; 3.8%) than embryo development medium culture system, [morula (69; 23.1%) and hatched blastocyst (5; 1.6%)]. Out of total blastocysts (48) the primary colonies were formed in 23.3% (7/30) blastocysts, and 66.6% (12/18) of hatched blastocysts. The cells of the inner cell mass (ICM) derived primary colonies were small, aggregated and tightly packed in nature forming embryoid bodies on further subculture. The colonies were stained to see the expression of alkaline phosphatase and positive result was obtained. Goat embryonic stem cell like outgrowths were also characterized for Oct-4 expression and positive result was found. It could be concluded that ICM cells were isolated from in vitro fertilized goat blastocysts and cultured for embryonic stem cell-like cells and expression of alkaline phosphatase and Oct-4 in these cells were positive.
Pawar
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Animal Biotechnology Center, National Dairy Research Institute, Karnal 132 001, India.
Malakar
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Akshey
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D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001755:Blastocyst / Q000166:cytology*; D018929:Cell Culture Techniques / Q000379:methods; D049109:Cell Proliferation; D002478:Cells, Cultured; D018920:Coculture Techniques; D046149:Embryo Culture Techniques / Q000379:methods*; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D047108:Embryonic Development; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D005260:Female; D005307:Fertilization in Vitro; D018507:Gene Expression Regulation, Developmental; D006041:Goats; D008297:Male; D009028:Morula / Q000166:cytology* / Q000378:metabolism; D050814:Octamer Transcription Factor-3 / Q000235:genetics; D009865:Oocytes / Q000166:cytology; D010057:Oviducts / Q000166:cytology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism
D050814:Octamer Transcription Factor-3; D000469:Alkaline Phosphatase