282 EFFECT OF THE MATURATION TIME ON THE OUTPUT OF THE INTRACYTOPLASMIC SPERM INJECTION (ICSI) OF PIG OOCYTES PRECULTURED WITH ROSCOVITINE

Abstract

This study was conducted to evaluate the effect of different maturation times on the output of intracytoplasmic sperm injection (ICSI) with pig oocytes precultured with roscovitine, a specific inhibitor of MPF kinase activity (Meijer and Raymond 2003 Acc. Chem. Res. 36, 417–425). Cumulus-oocyte complexes (COCs) were collected from nonatretic follicles from slaughtered gilts. Just after recovery, oocytes (n = 456) were cultured in the presence of 50 μ­M roscovitine for 28–30 h, which maintains them at the GV stage until use without affecting further development (Coy et al. 2004 Human Reprod. 19, 41–42). Then, oocytes were matured in a conventional maturation system (Coy et al. 2002 Reproduction 124, 279–288) for 36, 40, or 44 h. Matured oocytes were fertilized by ICSI. Injection was performed as described by Martin (2000 Biol. Reprod. 63, 109–112) but in D-PBS medium with 10% FCS. Injected oocytes were transferred to TALP medium (Rath et al. 1999 J. Anim. Sci. 77, 3346–3352) for 24 h, after which they were fixed and Hoechst-stained to check for fertilization. Results (data were analyzed by ANOVA and Tukey test) showed similar activation rates (oocytes with female pronucleus) for 36, 40, and 44 h (83.6%, 81.7%, and 90.7%, respectively). Differences were observed in male pronuclear formation (80.2%, 67.6%, and 57.8%) and in putative embryos (zygotes showing two pronuclei or two cell stage 24 hpi; 74.3%, 62.9%, and 55.1%) for 36, 40, and 44 h, respectively (P < 0.05), where the 36-h group showed the best result. Data show that pre-treatment of pig oocytes with roscovitine and further maturation for 36 h can improve the output of ICSI.

This work was supported by Ministerio de Ciencia y Tecnología (AGL2003-03144).