Effect of sperm extract injection volume, injection of PLCzeta cRNA, and tissue cell line on efficiency of equine nuclear transfer.

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.