Nuclear maturation of canine oocytes cultured in protein-free media.
The objective of this study was to determine the ability of canine oocytes to complete nuclear maturation in a protein-free medium. Oocytes obtained from ovaries of bitches aged 6 months to 2 years were cultured either in TCM199 or CMRL1066 medium without protein supplementation in 5% or 20% O(2). Sixteen of 121 (13%) oocytes cultured in TCM199 reached metaphase II, but only 1 of 135 oocytes cultured in CMRL1066 did so (P < 0.05). Oxygen concentration did not affect nuclear maturation. An additional 103 oocytes were cultured in TCM199 for 48 hr, inseminated with chilled ejaculated spermatozoa, fixed in 1:3 acetic acid-ethanol and then stained with aceto-orcein; 34% of these oocytes were penetrated by spermatozoa. To determine developmental competence of oocytes cultured in a protein-free medium, 85 oocytes were cultured in TCM 199 for 48 hr, inseminated and then cultured; 7 early stage embryos were produced. The effects of growth hormone, beta-mercaptoethanol (betaME), luteinizing hormone (LH) and energy substrates, alone or in combination, on nuclear maturation of oocytes cultured in a protein-free medium were also determined. Growth hormone enhanced cumulus expansion, but did not improve nuclear maturation. beta-mercaptoethanol had no effect on nuclear maturation. However, percentages of MII oocytes significantly decreased when the oocytes were cultured for 48 hr in the medium containing LH or a high concentration of glucose (P < 0.05). In conclusion, canine oocytes are able to complete nuclear maturation in a protein-free medium. The specific type of medium and other supplements significantly influence the meiotic maturation of canine oocytes.
Songsasen
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Department of Biological Sciences, University of New Orleans; Audubon Center for Research of Endangered Species, New Orleans, Louisiana 70131, USA. nsongsasen@acres.org
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Leibo
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D000818:Animals; D018929:Cell Culture Techniques; D002467:Cell Nucleus / Q000378:metabolism / Q000502:physiology*; D016895:Culture Media, Serum-Free; D004285:Dogs; D005260:Female; D005947:Glucose / Q000378:metabolism; D013006:Growth Hormone / Q000378:metabolism; D007986:Luteinizing Hormone / Q000378:metabolism; D008297:Male; D008540:Meiosis; D008623:Mercaptoethanol / Q000378:metabolism; D009865:Oocytes / Q000254:growth & development*; D009866:Oogenesis; D010100:Oxygen / Q000378:metabolism; D019289:Pyruvic Acid / Q000378:metabolism
D016895:Culture Media, Serum-Free; D008623:Mercaptoethanol; D019289:Pyruvic Acid; D007986:Luteinizing Hormone; D013006:Growth Hormone; D005947:Glucose; D010100:Oxygen