In vitro production of bovine embryo in tubes without gaseous atmosphere control

Abstract

The increase in OPU utilization requires an alternative to start the early phases of PIV without a controlled atmosphere. The development of a practical and simple way to transport/mature/fertilize might enhance the efficiency of the laboratory and consequently decrease its production costs. The aim of this study was to develop a method for cumulus oocytes complexes (CCO) maturation and fertilization in polypropylene tubes without atmosphere control kept in water bath. The in vitro maturation performed with controlled atmosphere was in TCM-199 modified (control group). The water bath treatments in tubes the TCM-199 had a addition of 25 mM of N-2-hidroxyethylpiperazine-N'-2-ethanosulfonic acid (HEPES). The CCO in vitro fertilization was performed in Talp-Fert medium with 10mM a 28.7 mM of HEPES, under mineral oil in tubes kept in water bath at 39°C, during 10 to 18 hours. Zygotes were cultured 4 well dishes, in bag system, with SOFaa medium with 5,00% estrus cow serum, under mineral oil, in incubator with 5,00%CO2, 5,00%O2 and 90,00%N2, at 39°C for 9 days. The results showed that CCO can be matured for 24 hours in tubes kept in water bath and also fertilized during 10 hours in tubes with Talp-Fert medium added of 25mM HEPES. The cleavage, D7 and D9 blastocysts rates in tubes without controlled atmosphere were similar (P>;0.05) to those in the incubator procedures. However, the fertilization in Talp-Fert medium added of 10mM a 28.7 mM of HEPES in tubes kept in water bath for 18 hours had a detrimental effect on the embryonic development.