Influence of oocyte selection, activation with a zinc chelator and inhibition of histone deacetylases on cloned porcine embryo and chemically activated oocytes development.

The aim of this study was to evaluate the effects of alternative protocols to improve oocyte selection, embryo activation and genomic reprogramming on in vitro development of porcine embryos cloned by somatic cell nuclear transfer (SCNT). In Experiment 1, in vitro-matured oocytes were selected by exposure to a hyperosmotic sucrose solution prior to micromanipulation. In Experiment 2, an alternative chemical activation protocol using a zinc chelator as an adjuvant (ionomycin + N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) + N-6-dimethylaminopurine (6-DMAP)) was compared with a standard protocol (ionomycin + 6-DMAP) for the activation of porcine oocytes or SCNT embryos. In Experiment 3, presumptive cloned zygotes were incubated after chemical activation in a histone deacetylase inhibitor (Scriptaid) for 15 h, with the evaluation of embryo yield and total cell number in day 7 blastocysts. In Experiment 1, cleavage rates tended to be higher in sucrose-treated oocytes than controls (123/199, 61.8% vs. 119/222, 53.6%, respectively); however, blastocyst rates were similar between groups. In Experiment 2, cleavage rates were higher in zygotes treated with TPEN than controls but no difference in blastocyst rates between groups occurred. For Experiment 3, the exposure to Scriptaid did not improve embryo development after cloning. Nevertheless, the total number of cells was higher in cloned zygotes treated with Scriptaid than SCNT controls. In conclusion, oocyte selection by sucrose as well as treatments with zinc chelator and an inhibitor of histone deacetylases did not significantly improve blastocyst yield in cloned and parthenotes. However, the histone deacetylases inhibitor produced a significant improvement in the blastocyst quality.

Effect of FSH treatment on cumulus oocyte complex recovery by ovum pick up and in vitro embryo production in beef donor cows.

This study was conducted to evaluate oocyte recovery and in-vitro blastocyst production of donor cows superstimulated for ovarian follicular development with FSH administered as twice-daily injections in saline or a single injection diluted in 0.5 % hyaluronan before oocyte aspiration. In Experiment 1, cows were treated with 160 mg of Folltropin-Vdiluted in saline, administered in four twice-daily i.m. injections for 2 days (Multiple FSH group); 160 mg of Folltropin-V diluted in hyaluronan and administered in a single i.m. injection (Single FSH group); or no FSH treatment (Control). In Experiment 2, donor cows were treated with either a single FSH i.m. injection or there was no treatment (Control) before ovum pick up (OPU) was performed. In both experiments, COCs collected using OPU were classified, matured, fertilized and cultured at 38.8 °C in a humidified atmosphere for 7 days. In Experiment 1, the number of follicles aspirated and COCs recovered were greater (P < 0.05) in cows treated with multiple and single doses of FSH. Number of blastocysts produced, however, did not differ among groups. In Experiment 2, mean number of follicles aspirated and COCs recovered were also greater (P < 0.05) in FSH-treated cows. Nevertheless, number of blastocysts produced did not differ. In summary, single and multiple FSH administrations induced similar follicular stimulation for OPU. Furthermore, with both FSH treatments there was induction of development of a larger number of follicles to be aspirated and COCs recovered by OPU compared with these values for donor beef cows with no FSH treatment for follicular stimulation.