A Three-dimensional Culture Method For Porcine Oocyle Maturation In Vitro Was Established By Alginate

The maturation quality of oocytes is directly related to the developmental potential of embryos,therefore in vitro maturation(IVM)is the basis of all embryo production technologies,as Oocytes need the surrounding cumulus cells to provide nutritional support through intercellular association,but the currently conventional IVM methods can not effectively maintain the association between oocytes and cumulus cells,which directly affects the quality of oocyte maturation.Three-dimensional culture technology can simulate the microenvironment of signal transduction between cells and extracellular matrix(ECM),and maintain the three-dimensional spatial structure between cultured cells.Therefore,this study intends to explore the influence of three 3D culture methods(suspended drop culture method,Matrigel embedding method and alginate brine gel embedding method)on oocyte IVM,and select one method for optimization on this basis,in order to establish a three-dimensional culture technology system suitable for porcine oocyte IVM,and improve the efficience of oocyte IVM.The main results of this study are as follows:(1)Effects of different 3D culture methods on porcine oocyte IVMThe effects of alginate saline gel embedding method,suspension drop culture method,matrix gel embedding method and conventional culture method on the maturation of porcine oocytes in vitro were compared.The results showed that 3D culture could avoid the adhesion phenomenon during oocyte maturation,and the oocyte after substrate gel and alginate embedding did not fall off,and the oocyte expanded and structured well.In addition,the incidence of oocyte GVBD and the excretion rate of the first polar body in the alginate saline gel embedding group and the matrix gel embedding group were significantly higher than those of conventional culture group.The proportion of uniform distribution of mitochondria in the 3D group was significantly higher than that of conventional culture group,particularly,the content of mitochondria in the alginate saline gel embedding group and the matrix gel embedding group was higher than that of conventional culture group,and the rate of parthenogenesis activation of blastocysts after oocyte maturation was higher than that of conventional culture group.(2)Optimization of sodium alginate gel embedding and decrosslinking systemAlginate saline gel crosslinking time,decrosslinking time,gel medium and other parameters were optimized.The results showed that when the crosslinking time reached more than 5 min,the gel could remain stable within44 h.The degradation rate,porosity,water absorption and swelling rate of alginate gel at 0.25 %(W/V)concentration were significantly higher than those of other experimental groups.The cytotoxicity at 5 min was significantly lower than that in other experimental groups,and the blastocyst rate at 2 min and 5 min was significantly higher than that in 12 min group.The maturation rate and blastocyst rate of alginate gel mixed with PM solution and PBS were significantly higher than those in the three steaming water group.(3)The effect of the optimized alginate saline gel culture system on porcine oocytes IVMIn the mature oocytes cultured in the optimized alginate saline gel system,the first polar body expulsion rate increased(75.6% vs 65.2%),the mortality rate decreased(21.3% vs 26.7%),the proportion of cortical granules content to peripheral oocytes increased significantly,and the abnormal spindle distribution of oocytes in MⅡ stage decreased significantly.Mitochondrial content and mitochondrial membrane potential were significantly increased,while ROS levels were significantly decreased.In addition,the parthenogenetic activation cleavage rate(74.2% vs 67.3%)and blastocyst rate after oocyte maturation were also significantly increased(23.5% vs 16.5%).These results indicated that the 3D maturation culture method established based on alginate saline gel could improve the in vitro maturation rate of oocytes and the embryonic development rate after parthenogenetic activation,reduce oxidative stress,promote the synchronous maturation of oocyte nucleus and cytoplasm,and improve the nucleo-cytoplasmic normality rate of IVM oocytes.