Somatic cell nuclear transfer using transported in vitro-matured oocytes in cynomolgus monkey.

Somatic cell nuclear transfer (SCNT) is not successful so far in non-human primates. The objective of this study was to investigate the effects of stimulation cycles (first and repeat) on oocyte retrieval and in vitro maturation (IVM) and to evaluate the effects of stimulation cycles and donor cell type (cumulus and fetal skin fibroblasts) on efficiency of SCNT with transported IVM oocytes. In this study, 369 immature oocytes were collected laparoscopically at 24 h following human chorionic gonadotrophin (hCG) treatment from 12 cynomolgus macaque (Macaca fascicularis) in 24 stimulation cycles, and shipped in pre-equilibrated IVM medium for a 5 h journey, placed in a dry portable incubator (37 degrees C) without CO(2) supplement. A total of 70.6% (247/350) of immature oocytes reached metaphase II (MII) stage at 36 h after hCG administration, MII spindle could be seen clearly in 80.6% (104/129) of matured IVM oocytes under polarized microscopy. A total of 50.0% (37/74) of reconstructive SCNT embryos cleaved after activation; after cleavage, 37.8% (14/37) developed to the 8-cell stage and 8.1% (3/37) developed to morula, but unfortunately none developed to the blastocyst stage. Many more oocytes could be retrieved per cycle from monkeys in the first cycle than in repeated cycles (19.1 vs. 11.7, p < 0.05). There were no significant differences in the maturation rate (70.0 vs. 71.4%, p > 0.05) and MII spindle rate under polarized microscopy (76.4 vs. 86.0%, p > 0.05) between the first and repeat cycles. There were also no significant differences in the cleavage rate, and the 4-cell, 8-cell and morula development rate of SCNT embryos between the first and repeat cycles. When fibroblast cells and cumulus cells were used as the donor cells for SCNT, first cleavage rate was not significantly different, but 4-cell (50.0 vs. 88.9%, p < 0.05) and 8-cell (0 vs. 51.9%, p < 0.01) development rate were significantly lower for the former. In conclusion, the number of stimulation cycles has a significant effect on oocyte retrieval, but has no effect on maturation and SCNT embryo development; however, different donor cell types (cumulus and fibroblast) resulted in different developmental potentials of SCNT embryos.

Effects of elevated temperature in vivo on the maturational and developmental competence of porcine germinal vesicle stage oocytes.

Postslaughter processing of sow carcasses results in the ovaries being exposed to temperatures of 41.3 to 42.1 degrees C within a 30-min time frame. This study investigated whether the maturational and developmental competence of the recovered germinal vesicle stage oocytes could be compromised by post-slaughter processing. The results showed that the in vitro maturation rates of GV stage oocytes exposed to elevated temperature did not significantly differ from the corresponding controls (74.1 vs. 75.8%). Immunocytochemical staining revealed that elevated temperature did not adversely affect metaphase II spindle formation but resulted in extensive disruption of oocyte cytoskeletal organization. This, in turn, had a detrimental effect on parthenogenetic development compared with the corresponding nonheat-treated controls (cleavage rate = 27.7 vs. 65.3%, P < 0.01; blastulation rate = 6.7 vs. 20.6%, P < 0.01). Hence, transient exposure to elevated temperature during slaughter did not have any detrimental effects on nuclear maturation per se, but it did result in extensive cytoskeletal damage, which in turn drastically decreased the developmental competence.