Effect of ionomycin associated with roscovitine, dehydroleucodine, cycloheximide, or ethanol on haploid activation of bovine oocytes.

SUVA, C.; CANEL N; SALAMONE D.

Versalles

Congreso; Annual Conference of the International Embryo Transfer Society,Versalles.; 2015

International Embryo Transfer Society

Haploid activation of bovine oocytes after ICSI is a routine procedure. However, embryos frequently contain an abnormal chromosome set as a result of the drugs employed. We compared the efficiency of ionomycin (Io) followed by roscovitine (ROSC), cycloheximide (CHX), ethanol, and dehydroleucodine (DhL) to induce haploid parthenogenetic activation in bovine. Pronuclear (PN) formation, second polar body (2PB) extrusion, embryo development, and ploidy of blastocysts were evaluated. To this aim, COC were aspirated from slaughtered ovaries and IVM for 22h. Oocytes were activated with 5µM of Io for 4min and then randomly allocated into 1 of the following treatments: 25 or 50µM ROSC or 10µgmL(-1) of CHX for 5 h; 15 or 30µM DhL for 3 h; or 5min of exposure to 7% ethanol 4h post-Io. Controls were Io followed by (1) 3h in TCM-199 and 3h in 1.9mM 6-DMAP (Io-3h-DMAP) and (2) 3h of exposure to 1.9mM 6-DMAP (Io-DMAP). Oocytes were cultured in SOF medium. The PN formation and 2PB extrusion were assessed by 5µgmL(-1) of propidium iodide oocyte staining, 17h after Io. Cleavage, morulae, and blastocyst stages were evaluated at Days 2, 5, and 8 of in vitro development, respectively. Chromosome number of blastocysts was evaluated by Giemsa staining. Data were analysed by Fisher´s test (P