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Regular Article |
a Department of Veterinary Physiology and Pharmacology,
b Large Animal Medicine and Surgery,
c Anatomy and Public Health, College of Veterinary Medicine, Texas A&M University, College Station, Texas 77843
d The Fels Institute of Molecular Biology and Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140
We investigated the use of direct nuclear injection using the Piezo drill and activation by injection of stallion sperm cytosolic extract for production of cloned equine embryos. When metaphase II horse oocytes were injected with either of two dosages of sperm extract and cultured 20 h, similar activation rates (88% vs. 90%) and cleavage rates (49% vs. 46%) were obtained. The successful reconstruction rate of horse oocytes with horse somatic cell donor nuclei after direct injection using the Piezo drill was 82%. Four dosages of sperm extract (containing 59, 176, 293, or 1375 µg/ml protein) and two activation times (1.5–2 vs. 8–10 h after nuclear transfer) were examined. Cleavage and activation (pseudopronucleus formation) rates of oocytes injected with sperm extract containing 59 µg/ml protein were significantly (P < 0.05) lower than any other dosage. The percentage of embryos cleaving with normal nuclei in oocytes injected with the 1375 µg/ml preparation 1.5–2 h after donor injection was significantly (P < 0.05) higher than that of the 293 µg/ml preparation 8–10 h after donor injection (22 vs. 6%). Embryos developed to a maximum of 10 nuclei. Interspecies nuclear transfer was performed by direct injection of horse nuclei into enucleated bovine oocytes, followed by chemical activation. This resulted in 81% reconstruction (successful injection of the donor cell), 88% cleavage, and 73% cleavage with normal nuclei. These results indicate that direct nuclear injection using the Piezo drill is an efficient method for nuclear transfer in horse and cattle oocytes and that sperm extract can efficiently activate horse oocytes both parthenogenetically and after nuclear transfer
1 Supported by the Bio-Arts Research Corporation and the Link Equine Research Endowment Fund (Texas A&M University). Y.G.C. was supported by grant HD38381 from the National Institutes of Health.
2 Correspondence. FAX: 979 845 6544; khinrichs{at}cvm.tamu.edu
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