Construction Of Sika Deer-Bovine Interspecies Cloned Embryos

In this study, we constructed sika deer-bovine interspecies cloned embryo with MⅡ bovineoocytes as recipients which enucleated by squeezing method and sika deer pedicle periosteum cells asdonors. This experiment we designed to investigate the effects of field intensity, pulse number, pulseduration and recovery time before fusion in fusion rate, mortality rate and cleavage rate of reconstructedeggs. The main results were as follows:1. Growth curves and chromosome analysis of the sika deer pedicle periosteum cells1) The9thgeneration sika deer pedicle periosteum cells showed typical S-shaped curves forMammalian cells in the cultural conditions of temperature38.5℃, carbon dioxide content5%, saturatedhumidity and10%FBS.2）The indication of the test of the chromosome: The karyotype of9thgeneration sika deer pedicleperiosteum cell is2n=66. The results showed that the genetic feature of pedicle periosteum cells didn’tchange with the increase of culture passages, which can be used as donor cells for nuclear transfer.2. The effects of fusion parameters in fusion performance and cleavage rate1) The fusion rate in2.4KV/cm of field intensity (48.6%) were significantly higher than that in2.0KV/cm,2.2KV/cm and2.6KV/cm(25.2%,41.0%,31.1%，P＜0.05). But there was no significantdifference in the mortality rate and cleavage rate between four groups (P>0.05).2) There were no significant difference in fusion rate, mortality rate and cleavage rate between thepulse number of1,2and3(P>0.05).3) The fusion rate and cleavage rate at pulse duration of10μs (68.4%,47.0%) were significantlyhigher than that in20μs,30μs and40μs (52.9%,26.0%vs53.8%,6.3%vs37.5%,0; P＜0.05), but therewere no obvious difference in mortality rate between all experimental groups (P>0.05).4) Cleavage rate of recovery time before fusion in2h compared with the group of1h and3h hadremarkable difference(P＜0.05). But there was no obvious difference among the three groups (P>0.05).The results showed: the pedicle periosteum cells not only can normal growth and proliferation, butalso can’t change the genetic feature, which can be used as donor cells for nuclear transfer; theappropriate electric fusion parameters for sika deer-bovine interspecies nuclear transfer are2.4KV/cmof field intensity,1of pulse number,10μs of pulse duration and2h of recovery time before fusion.