Production of cloned horse foals using roscovitine-treated donor cells and activation with sperm extract and/or ionomycin.
We evaluated the effect of different activation treatments on the production of blastocysts and foals by nuclear transfer. Donor cells were prepared using roscovitine treatment, which has previously been associated with increased production of viable offspring. All activation treatments were followed by culture in 6-dimethylaminopurine (6-DMAP) for 4 h. In experiment 1, blastocyst production after activation by injection of sperm extract followed by treatment with ionomycin was significantly higher than that for activation with a serial treatment of ionomycin, 6-DMAP, and ionomycin (12.5 vs 2.8%; P < 0.05) and tended to be higher than that for injection of sperm extract alone (3.4%; P = 0.07). In experiment 2, there were no significant differences in blastocyst development among treatments with ionomycin once or twice, sperm extract then ionomycin, or ionomycin then sperm extract (range 4.6-7.3%). Overall, transfer of 26 blastocysts resulted in 16 pregnancies (62%) and 9 live foals (35% of transferred embryos). Treatment with sperm extract followed by ionomycin produced a live foal rate per embryo transferred of 5/10 (50%). One foal died of pneumonia 48 h post partum and one foal died at 1 week of age after complications during induction of anesthesia; the remaining seven foals are currently 10-14 months of age.
Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas 77843, USA. khinrichs@cvm.tamu.edu
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Hinrichs
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Choi
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Varner
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Hartman
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001755:Blastocyst / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D018929:Cell Culture Techniques; D002970:Cleavage Stage, Ovum; D019976:Cloning, Organism / Q000379:methods*; D004624:Embryo Transfer; D047108:Embryonic Development; D005260:Female; D006736:Horses; D015759:Ionomycin / Q000494:pharmacology*; D008297:Male; D008845:Microinjections; D053652:Nuclear Transfer Techniques; D018587:Oocyte Donation / Q000662:veterinary; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D011247:Pregnancy; D011256:Pregnancy Outcome; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011687:Purines / Q000494:pharmacology*; D000077546:Roscovitine; D020554:Sperm Injections, Intracytoplasmic
D047428:Protein Kinase Inhibitors; D011687:Purines; D000077546:Roscovitine; D015759:Ionomycin; C001020:N(6),N(6)-dimethyladenine; D000225:Adenine; D002118:Calcium