have been described for the pig (Prather et al., 1991, 1997a, b; Kim et al., 1998; Wang et al., 1998a, b), limited information is available for directly comparing the activation efficiency of various agents. Donor nuclei can be fused with the recipient cyto- plasm of matured oocytes by means of an electrical pulse, and simultaneous activation can be prevented by using a calcium-free pulse medium (Sun et al., 1992). Exposure of reconstructed embryos to calcium-free medium for a period of time after fusion is thought to be necessary to prevent activation, which might pro- mote complete exchange of nuclear and cytoplasmic proteins and subsequent reprogramming of the trans- planted nuclei (Terlouw, 1993). However, the effect of time in calcium-free medium on activation of oocytes is not known. Another technique that is frequently used during nuclear transfer is the staining of DNA with bisbenz- imide (Hoechst 33342). Following staining the cells are exposed to ultraviolet (UV) light which allows Introduction According to recent reports (Tao et al., 1991a, b), devel- opment of porcine nuclear transfer embryos derived from differentiated cells is less efficient compared with that of other species such as sheep (Wells et al., 1997; Wilmut et al., 1997), cattle (Cibelli et al., 1998a, b; Kato et al., 1998) and mouse (Wakayama et al., 1998). One rea- son for this low effectiveness is the lack of efficient oocyte activation methods. Many procedures that work well in other species are often ineffective for the activation of porcine oocytes (for review see Prather, 1997a). Although many methods of oocyte activation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 Zygote 8 (February), pp 69–77. © 2000 Cambridge University Press Printed in the United Kingdom All correspondence to: Randall S. Prather, 162 Animal Sci- ence Research Center, University of Missouri-Columbia, Columbia, MO 65211, USA. Tel: +1 (573) 882 6414. Fax: +1 (573) 882 6827. e-mail: pratherr@missouri.edu *Current address: Nexia Biotechnologies, Inc., Ste-Anne-de- Bellevue, Quebec H9X 3R2, Canada. Optimisation of porcine oocyte activation following nuclear transfer Tao Tao*, Zoltán Macháty, Lalantha R. Abeydeera, Billy N. Day and Randall S. Prather Department of Animal Sciences, University of Missouri-Columbia, Columbia, Missouri, USA Date submitted: 26.8.99. Date accepted: 7.11.99 Summary Experiments were conducted to examine the effects of (a) different activation methods, (b) incubation time in calcium-free medium and (c) bisbenzimide staining on the activation and subsequent develop- ment of pig oocytes. Oocytes were matured in vitro and activated by one of the following methods: com- bined thimerosal/dithiothreitol (DTT) treatment, calcium ionophore A23187 treatment followed by incu- bation in the presence of 6-dimethylaminopurine (6-DMAP), electroporation, and electroporation followed by incubation with cytochalasin B. There were no significant differences in the activation rate (ranging from 70.0% to 88.3%) and the percentage of cleaved embryos after activation (ranging between 48.8% and 58.8%) among the four treatment groups (p < 0.05). The rate of development to the blastocyst stage in oocytes activated by thimerosal/DTT (10.0%) or electroporation followed by cytochalasin B treatment (12.3%) was significantly higher (p < 0.05) than in the group activated with A23187/6-DMAP (2.5%). Both the activation rate and the rate of blastocyst formation in oocytes that were incubated in Ca 2+ -free medium for 8 h before thimerosal/DTT activation were significantly lower (p < 0.05) than in those incubated for 0, 1 or 4 h. Intracellular Ca 2+ measurements revealed that the Ca 2+ homeostasis in these oocytes were severely altered. Staining of oocytes with 5 μg/ml bisbenzimide for 2 h decreased the quality of blastocysts and increased the rate of degenerated embryos at day 6. Two activation protocols (thimerosal/DTT and electroproation) were used for activation after nuclear transfer; the rate of nuclear formation did not differ in the oocytes activated by the two different methods. Keywords: Activation, Nuclear transfer, Oocyte, Porcine