title: Effect of sperm extract injection volume, injection of PLCzeta cRNA, and tissue cell line on efficiency of equine nuclear transfer.
abstract: We evaluated the effect of different activation methods on blastocyst development after equine nuclear transfer. All activation treatments were followed by incubation in 2 mM 6-dimethylaminopurine for 4 h. In Experiment 1, reconstructed oocytes were injected with sperm extract for 0.1, 0.2, 0.4, 0.8, or 1.6 sec using a FemtoJet injection device, then treated with ionomycin. The blastocyst rate (9.8%) for 0.1-sec injection was significantly higher than that for 0.2 sec (0%) or 0.8 sec (1.4%). In Experiment 2, injection of murine PLCzeta cRNA before or after ionomycin treatment did not increase blastocyst development (0 and 4.5%) over a control treatment (injection of sperm extract after ionomycin exposure; 5.6%). Transfer of 10 blastocysts produced in Experiments 1 and 2 resulted in five pregnancies, all lost before 70 days of gestation. In Experiment 3, cells from a second biopsy sample from the same horse produced significantly more blastocysts than did the original sample (4/44 vs. 0/58; p < 0.05). Transfer of these four blastocysts produced two viable foals. In Experiment 4, blastocyst development rates did not differ between oocytes in metaphase I or II at the time of nuclear transfer (16.7 and 3.0%, respectively). A healthy foal was produced from a blastocyst originating from a metaphase I oocyte.
authors.affiliation: College of Veterinary Medicine and Biomedical Sciences, Texas A&M University , College Station, TX 77843-4466, USA.
authors.forename: Young-Ho
authors.initials: YH
authors.lastname: Choi
authors.forename: David L
authors.initials: DL
authors.lastname: Hartman
authors.forename: Rafael A
authors.initials: RA
authors.lastname: Fissore
authors.forename: Sylvia J
authors.initials: SJ
authors.lastname: Bedford-Guaus
authors.forename: Katrin
authors.initials: K
authors.lastname: Hinrichs
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000166:cytology / Q000502:physiology; D002460:Cell Line; D004622:Embryo, Mammalian / Q000502:physiology; D006736:Horses; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000502:physiology; D054801:Phosphoinositide Phospholipase C / Q000235:genetics* / Q000378:metabolism; D018075:RNA, Complementary / Q000235:genetics / Q000378:metabolism*; D013094:Spermatozoa / Q000737:chemistry*
chemical_list: D007527:Isoenzymes; D018075:RNA, Complementary; D054801:Phosphoinositide Phospholipase C; C499234:Plcz1 protein, mouse