PARENT SESSION
IMPLANTATION AND PREGNANCY - B

Tuesday, August 3, 2004
10:30 AM–12:30 PM
Buchanan Courtyard

(520) COMPARISON OF IN VIVO DEVELOPMENTAL COMPETENCY OF CLONED PIG EMBRYOS DERIVED FROM THREE DIFFERENT ACTIVATION STRATEGIES.

Lai, Liangxue¹, Im, Gi-Sun ¹, Liu, Zhonghua¹, Hao, Yanhong¹, Samuel, Melissa¹, Wax, David¹, Rieke, August¹, Murphy, Clifton¹, Prather, Randall, ¹ Animal Science Research Center, University of Missouri-Columbia, Columbia, Missouri

ABSTRACT- In this report, we compared the long term in vivo developmental competency of cloned pig embryos derived from three different activation strategies including electrical pulse, electrical pulse plus chemical agent and chemical agents alone. The aim of this experiment is to provide some information to evaluate efficiency of the present activation methods for long term development of swine nuclear transfer (NT) embryos. Primary fetal fibroblasts were isolated from a day 35 porcine fetus. In vitro matured oocytes were enucleated by aspirating the first polar body and adjacent cytoplasm. The donor cells were injected into the perivitelline space of the oocyte. In the first group, fusion and activation of reconstructed oocytes was induced simultaneously with 2 DC pulses of 1.2 kV/cm for 30 sec in a medium with high calcium concentration (1.0 mM). In the second group, reconstructed oocytes obtained from the same fusion and activation condition as the first group were further activated with demethylaminopurine (DMAP) for 4 h. In the third group, reconstructed oocytes were fused in a medium with low calcium concentration (0.1 mM) and then were activated by thimerosal (200 M), followed by dithiothreitol (DTT) (8mM). After cultured for 18-22 h, 113-120 embryos were transferred into each recipient. The pregnancy rates of recipients among the three groups were almost same, group I (5/7), group II (3/6), group III (5/7).The rates of development to term of recipients between group I (2/7) and group II ( 2/6) were not different , but were lower than in group III (5/7).The rate of development to term of NT embryos in group I (4/840, 0.5%) was lower than those in group II (8/713, 1.2%) and group III (13/828, 1.6%). Our results indicate that, all the three activation methods could give rise to NT embryos with capacity to develop to term. Further, treatment with the chemical agent such as DMAP may be helpful to improve the rate of development to term of NT embryos. Thimerosal + DTT activation appears to improve the rate of development to term of both recipients and embryos.

KEY WORDS: Activation, pig, Nuclear transfer, in vivo development