Cloning in cattle and goats : the first DNA replication and the development of nuclear transfer embryos

Abstract

In cattle, the objectives of the study were to investigate the onset and the length of the first DNA replication in the 1-cell stage of bovine somatic nuclear transfer embryos produced from the different state of recipient cytoplasts and to evaluate its influence on the development of the embryos. Cumulus-oocyte complexes obtained from slaughterhouse ovaries were matured in vitro then enucleated. In the non-activated group (NT-MII), the enucleated oocytes were fused with starved ear skin fibroblasts and activated immediately with cycloheximide (CHX) and cytochalasin B for 5 h. In the activated group (NT-ACT), the enucleated oocytes were activated with 7% ethanol for 5 min and cultured in CHX for 2 h, fused with donor cells and incubated in CHX for an additional 3 h. Nuclear transfer (NT) embryos were then cultured in vitro. DNA synthesis determination was performed using immunocytochemistry. At 5-hour post fusion (hpf), DNA synthesis started in NT-ACT embryos (10 of 27 embryos) whereas it was not observed in NT-MII group (0 of 25 embryos, p< 0.001). The DNA replication ended at 18 hpf in NT-MII group, however 9 of 25 embryos (36%) in NT-ACT group still synthesized DNA at this time (P<0.001). Development rates to the blastocyst stage were significantly higher in NT-MII group than in NT-ACT group (51.1% versus 22.8% of cultured embryos, p< 0.001). These data demonstrate that the DNA synthesis is longer and starts earlier in somatic nuclei transferred into activated cytoplasts than in those transferred into non-activated cytoplasts. In goats, the effect of activation protocols on the development of cloned goat embryos was investigated. Immature oocytes obtained from FSH-stimulated goats were matured and enucleated before NT. Donor cells were prepared from a locally bred goat, After fusion, the reconstructed embryos were activated with either ionomycin or ethanol followed by culturing in 6-dimethylaminopurine (6- DMAP) and cytochalasin B (CB). There were no significant differences between the NT embryos derived from the ionomycin and ethanol groups, in fusion (86.3% versus 82.9%, p>0.05), cleavage (90.5% versus 82.4%, p>0.05) and for morula/blastocyst rates (9.5% versus 5.9%). Pregnancy rates at Day 30 to 45 were also not significantly different in the recipients receiving NT embryos from ionomycin group (3/5, 60%) and those from ethanol group (1/5, 20%, p>0.05). Another study was conducted to determine the effect of repeated surgical oocyte collection by laparotomy on the numbers of follicles aspirated and oocytes recovered, as well as its effect on pregnancy in the donors. Retrospective data obtained during the past two years (2002-2004) were analyzed. Immature and matured oocytes were collected from FSH-stimulated donor goats. For immature oocyte collection, a total of 1,476 follicles were aspirated from 50 does in 69 sessions and 1,242 oocytes were recovered (21.4 ± 1.5, mean ± SEM, oocytes/donor/session), for a collection rate of 84.1%. For matured oocyte collection, a total of 398 corpora lutea were observed in 19 donors subjected to 20 collections by means of flushing the oviducts, and 333 oocytes recovered (16.7 ±2.1, mean ± SEM, oocytes/donor/session), for a collection rate of 83.7%. No significant differences in the number of follicles aspirated and oocytes obtained were found between the collections (P>0.05). In conclusion, the results indicate that stages of recipient cytoplasts affect the kinetics DNA replication during the first cell cycle as well as the development NT embryos in bovine. Somatic nuclei obtained from locally bred goats, could be activated by ionomycin or ethanol treatment in combination with 6-DMAP plus CB. Repeated OPU in the two consecutive sessions, in the same donors, had no significant influence on the numbers of follicles, oocytes with good quality as well as the collection efficiencies (P>0.05). Therefore, the oocyte collection in goats, at laparotomy, can be performed two to three times without any detrimental effects on pregnancy of the oocyte donors.