Beneficial Effect of Young Oocytes for Rabbit Somatic Cell Nuclear Transfer

Abstract

This study was designed to examine the effect of the age of rabbit oocytes on the developmental potential of cloned embryos. The metaphase II oocytes used for nuclear transfer (NT) were collected at 10, 12, 14, and 16 h post-hCG injection (hpi). The total number of oocytes collected per donor (21.4-23.7) at 12 to 16 hpi was similar, but significantly higher than that collected at 10 hpi (16.2). Additionally, a significant improvement in blastocyst development was achieved with embryos generated by electrically mediated cell fusion (56.0%), compared to those from nuclear injection (13.1 %) (Experiment 1). Markedly higher blastocyst development (45.8-54.5%) was also achieved with oocytes collected at 10-12 hpi than from those collected 14-16 hpi (8.3-14.3%) (Experiment 2). In Experiment 3, the blastocyst rates of NT embryos derived from oocytes harvested 12 hpi (39.2-42.8 %) were significantly higher than from those collected at 16 hpi (6.8-8.4 %) (p < 0.05), regardless of the donor cell age. Kinase activity assays showed variable changes of activity in rabbit oocytes over the period of 10-16 hpi; however, there was no correlation with preimplantational development (blastocyst rate vs. MPF, R = 0.326; blastocyst rate vs. MAPK, R = -0.131). Embryo transfer of NT embryos utilizing 12 hpi oocytes resulted in one full-term but stillborn, and one live cloned rabbit; thus, an efficiency of 1.7 % (n = 117) (Experiment 4). These results demonstrated that NT utilizing relatively young rabbit oocytes, harvested at 10-12 h after hCG injection, was beneficial for the development of NT embryos.

Recombinant Rabbit Leukemia Inhibitory Factor and Rabbit Embryonic Fibroblasts Support the Derivation and Maintenance of Rabbit Embryonic Stem Cells

“…Sexually mature (6-18 months old) New Zealand White (NZW) female rabbits were superovulated using our routine regime (Du et al, 2009), consisting of two 0.3-mg, two 0.4-mg, and two 0.5-mg administrations of folliclestimulating hormone (FSH; Folltropin-V, Bioniche Animal Health Canada, Belleville, Ontario, Canada) at intervals of 12 h, followed by 200 IU of human choriogonadotropin (hCG; Chorulon, Intervet Inc, Millsboro, DE, USA). Superovulated does were mated with fertile males and served as embryo donors.…”

“…Nuclear transfer, in combination with gene targeting in somatic cells, has been used as an alternative to produce gene-targeted pigs, cattle, sheep, and goats. Unfortunately, the rabbit has been proven to be an extremely difficult species to clone (Du et al, 2009). Since the first report of successful rabbit cloning a decade ago (Chesne et al, 2002), no gene-targeted transgenic rabbits were produced via this approach.…”

Two-staged nuclear transfer can enhance the developmental ability of goat–sheep interspecies nuclear transfer embryos in vitro

“…This process is defined as oocyte aging. Failed and abnormal fertilization was observed in aged oocytes, and this always causes increased abnormal or poor embryo development and early pregnancy loss of aged embryos after transfer (Wilcox et al 1998;Liu and Keefe 2002;Hall et al 2007;Liu et al 2007;Du et al 2009;Miao et al 2009). As shown in this study, when the time of delay activation was over 6 h, the blastocyst rate of FSNT-derived reconstructed embryos began to decrease, which may be attributed to oocyte aging.…”

“…In previous studies, using in vitro-matured sheep oocytes as nuclear recipients and goat fetal fibroblasts as nuclear donors, we constructed goat-sheep interspecies cloned embryos, but the blastocyst rate of interspecies cloned embryos was only 7.4% (Ma et al 2008a). This led us to think whether donor nucleus could be better reprogrammed by longtime exposure to non-activated heterogenous ooplasm, but prolonging exposure time can result in the aging of oocytes, which also influence the developmental ability of cloned embryos (Hall et al 2007;Liu et al 2007;Du et al 2009). Therefore, maybe a new SCNT procedure can enhance the developmental ability of cloned embryos, in which the donor nucleus can be exposed to the ooplasm for a long time; on the other hand, the aging of oocytes cannot occur.…”

Derivation of Rabbit Embryonic Stem Cells from Vitrified–Thawed Embryos

“…Sexually mature (6-18 months old) New Zealand white (NZW) female rabbits were superovulated using our routine regime (Du et al, 2009), consisting of two 3-mg, two 4-mg, and two 5-mg administrations of follicle-stimulating hormone (FSH; Folltropin, Bioniche Animal Health Canada, Belleville, Ontario, Canada) at intervals of 12 h, followed by 200 IU of human choriogonadotropin (hCG; Chorulon, Intervet Inc, Millsboro, DE, USA). Superovulated does were mated with fertile males and served as embryo donors.…”