273 EFFECT OF IONOMYCIN ASSOCIATED WITH ROSCOVITINE, DEHYDROLEUCODINE, CYCLOHEXIMIDE, OR ETHANOL ON HAPLOID ACTIVATION OF BOVINE OOCYTES

Abstract

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 22 h. Oocytes were activated with 5 µM of Io for 4 min and then randomly allocated into 1 of the following treatments: 25 or 50 µM ROSC or 10 µg mL^–1 of CHX for 5 h; 15 or 30 µM DhL for 3 h; or 5 min of exposure to 7% ethanol 4 h post-Io. Controls were Io followed by (1) 3 h in TCM-199 and 3 h in 1.9 mM 6-DMAP (Io-3h-DMAP) and (2) 3 h of exposure to 1.9 mM 6-DMAP (Io-DMAP). Oocytes were cultured in SOF medium. The PN formation and 2PB extrusion were assessed by 5 µg mL^–1 of propidium iodide oocyte staining, 17 h 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 < 0.05). Rates of 2PB extrusion were 75, 61.1, 60, 56.3, 54.6, and 42.9% for 15 µM DhL (n = 23), 50 µM ROSC (n = 22), Io-3h-DMAP (n = 9), CHX (n = 17), 25 µM ROSC (n = 22), and ethanol (n = 22), respectively, with no differences between groups. A PN was observed in over 81% of the oocytes activated with ethanol, 25 µM ROSC, CHX, 50 µM ROSC, and 15 µM DhL. Lower percentages of 2PB extrusion and PN formation were observed for 30 µM DhL (n = 22; 6.3 and 0%, respectively). The highest cleavage rates were 83.2% for 25 µM ROSC (n = 185), not differing from 78% in Io-DMAP (n = 159). Cleavage rates for 50 µM ROSC (n = 185), CHX (n = 143), and ethanol (n = 74; 80.5, 80.4 and 67.6%, respectively) were not different from Io-3h-DMAP (n = 78; 71.8%) and Io-DMAP. Cleavage rates for 15 µM DhL (n = 70) and 30 µM DhL (n = 93) were the lowest (48.6 and 25.8%). Blastocyst rates were the highest for CHX and 50 µM ROSC, not differing from Io-3h-DMAP (21.7 and 10.8 v. 18%). Very few or no blastocysts were obtained with ethanol, 25 µM ROSC, 30 µM DhL, and 15 µM DhL (4.1, 3.8, 1.1, and 0%, respectively), although ethanol was not different from Io-3h-DMAP. Chromosome number analysis showed that ethanol (n = 2) and CHX (n = 2) resulted in a higher percentage of haploid embryos (50% each), followed by 50 µM ROSC (n = 8), 25 µM ROSC (n = 3), and Io-3h-DMAP (n = 8; 37.5, 33.3% and 12.5%, respectively), although they were not different. Remaining embryos were diploid, aneuployd, or mixoployd. In conclusion, DhL and ROSC proved to be as effective as CHX or ethanol regarding 2PB extrusion and resulting ploidy, defining features when activating oocytes in ART, suggesting they could be efficiently used in bovine to assist ICSI.