In vitro maturation, fertilization and embryo culture of porcine gametes in a microfluidic environment.

摘要

In traditional porcine IVF systems, there is a high incidence of polyspermy, a pathological condition which results in aberrant embryonic development. Efforts to improve the in vitro embryo production process in pigs have included modifying the culture medium, the concentration of spermatozoa and the medium volume. The development of microfluidic devices (microchannels) has opened new avenues for manipulation of the IVF system to improve the efficiency and overall production of porcine embryos by more closely mimicking the function of the oviduct.; Results from IVF experiments revealed that oocyte number (10 or 15) did not have an effect on fertilization parameters in either IVF system. Irrelevant of oocyte number, IVF of porcine oocytes in the microchannels produced a higher incidence of monospermic penetration as compared to oocytes fertilized in the microdrop system with comparable penetration and male pronucleus formation rates.; When culturing the IVF embryos, it was found that the cleavage percentages were similar between both IVF systems. Significant differences between blastocyst production in both systems resulted when embryos were cultured in NCSU-23 supplemented with 0.4% BSA. Upon addition of 0.8% BSA to NCSU-23, the overall mean cleavage rates at 48 h were similar for the both IVF systems. The blastocyst development from embryos produced after IVF in the microchannels was not different (p > 0.05) from those produced from the microdrop fertilization system.; The initial experiments on the integration of IVM, IVF, and embryo culture within the same microchannel (integrated microchannel system; IMC) resulted in similar cleavage rates at 48 h of culture. Comparisons of static and dynamic (addition/removal of 20 μL twice daily) embryo culture of IVM-IVF oocytes within the IMC and the microdrop system revealed similar 2- and 4-cell development at 48 h and supported progression to the morula stage comparable to the static microdrop culture system.; These experiments were designed to examine the effects of in vitro fertilization within the microchannel environment and to compare the results to the traditional microdrop culture system. Additionally, the initial experiments of integrating the steps of the IVP process within a single microchannel were investigated in order to determine some baseline parameters when using this novel IVP system.