Optimization Of Porcine In Vitro Fertilization System Based On Microfluidic Chip

Domestic porcine is the highest proportion of livestock industry in China.At the same time,porcine plays an important role in the field of human medical biotechnology.Porcine play an important role not only in the application of gene editing to improve the productivity and safety of livestock,but also in the research of making animal models of specific diseases.At present,although the technology of porcine embryo in vitro production has been established,large-scale production and application still can not be realized.This is mainly due to the low efficiency of in vitro fertilization(IVF)compared with other species.The main reason is that the phenomenon of polyspermy into the egg easily occurs in the process of in vitro fertilization.There are several reasons for polyspermy in oocytes: the nuclear and cytoplasmic maturation of oocytes is inconsistent;Sperm capacitation in vitro is insufficient;The environment of fertilization and early embryo development in vitro is far less than that in vivo.In order to strengthen the research and utilization of domestic porcine in breeding and biomedicine,it is necessary to develop a more efficient porcine IVF system.In the traditional in vitro fertilization system,all operations are completed in four well plate culture dish and micro drop culture dish,including a large number of fluid exchange procedures and tedious manual operations,which will cause mechanical and chemical damage to the embryo.As a new biotechnology,microfluidic chip can alleviate this problem.According to the bionics principle,microfluidic chip simulates the physiological process of sperm sorting,sperm egg combination and embryo culture in the natural mating process of vagina and uterus.The application of microfluidic technology makes the process of in vitro fertilization and the culture environment of early embryo closer to the natural process in vivo,so as to realize the improvement and optimization of in vitro fertilization system.In this experiment,a kind of polydimethylsiloxane(PDMS)microfluidic chip with sperm sorting function was designed,and four kinds of microchannels including short straight microchannel,long straight microchannel,short curved microchannel and long curved microchannel were constructed for sperm sorting experiment.The results showed that the long curved microchannel had the best sorting effect,and the sperm motility could reach 85.28% ± 3.24%;The integrity rate of sperm plasma membrane was 85.77% ± 2.65%;The number of sperm zona pellucida binding was 163.30 ± 2.93;The cleavage rate of IVF was 72.50% ± 2.78%,significantly better than the control group 52.50 ± 4.79%(P<0.01).This indicates that the application of microfluidic technology has optimized the in vitro fertilization system of porcine.Based on the above results,an integrated microfluidic chip integrating sperm screening,fertilization and embryo development was designed and fabricated,and the chip was used for in vitro fertilization.Compared with the traditional IVF method,integrated microfluidic chip significantly improves the simultaneous interpreting of IVF results.The blastocyst rate increased from 5.40% ± 0.70% to 10.30% ± 1.20%(P < 0.05),and improved the quality of embryo development(the number of blastocyst cells increased from 60.40 ± 2.79 to 91.00 ±3.82)(P<0.01).At the same time,the integrated microfluidic chip can reduce the in vitro operation time and steps,and save reagent consumables.The experiments in this study proved the effectiveness of microfluidic chip in optimizing IVF system,and this technology has a good application prospect in IVF production.