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In vitro maturation of ovine oocytes in the absence of continuous 5% CO2 in a portable incubator
Abstract
This work was aimed at developing a method for in vitro maturation of oocytes in a portable incubator lacking C02. Ovine ovaries were collected from an abbatoir and transported in physiological saline at 360C to the laboratory. Cumulus-oocyte complexes were aspirated with a negative pressure vacuum pump and assigned to one of three treatment groups for maturation. 1) oocytes were matured in a 5% C02 incubator in tissue culture wells containing bicarbonate buffered Tissue Culture Medium199 (TCM199) supplemented with fetal calf serum (FCS), follicle stimulating hormone (FSH), luteinizing hormone (LH), and penicillin/streptomycin (pen/strep) (T1). 2) oocytes were matured in the same medium as T1, equilibrated with 5% C02 and overlayed with oil, in tubes in the portable incubator (T2). 3) oocytes were matured in the portable incubator, without C02 equilibration in tubes containing Hepes buffered TCM199 supplemented as previously described. After 24 hours at 390C, T1, T2, and T3 yielded, respectively, 72.55%,68.14%, and 66.96% mature. No significant difference existed between these. Some oocytes were then placed in fertilization medium supplemented with heparin. They were inseminated with frozen-thawed ram sperm and placed in a conventional incubator for 20-24 hours at 390C. Percentage fertilization was 44.09%, 58.62%, and 55.69% for T1, T2, and T3, respectively. T1 and T2 were significantly different. Some oocytes were then placed in Modified Brinster's Mouse Ova Culture (MBMOC) drops, containing bovine oviductal cells. These were then cultured at 39'C in 5% C02 and air for 7 days. T1, T2, and T3 resulted in 20.26%,16.94%, and 21.43% development to morulae, respectively. Blastocyst development for T1, T2, and T3 was, respectively, 4.01%, 3.06%, and 1.85%. No significant difference existed between these. Results indicate that maturation, fertilization, and developmental rates between oocytes matured in the portable incubator and oocytes matured in the conventional incubator are comparable. Ovine oocytes can be matured in a portable incubator lacking a C02 source. This technique shows promise for transportation of oocytes to laboratories where abbatoirs are not in close proximity and holds promise for transportation of oocytes from exotic animals in the field or remote locations to facilities capable of preserving the gametes.
Description
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Citation
Byrd, Susan Rebekah Walker (1995). In vitro maturation of ovine oocytes in the absence of continuous 5% CO2 in a portable incubator. Master's thesis, Texas A&M University. Available electronically from https : / /hdl .handle .net /1969 .1 /ETD -TAMU -1995 -THESIS -B974.
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