Study Of Mouse On The In Vitro Fertilization And Cryopreservation Of Spermatozoa

To establish a suit of method on the in vitro fertilization (IVF) of mouse, the different concentration and swimming up of mouse spermatozoa and the different time of the oocytes collected were compared. Five strains of mouse (CD-I, B6DF1, FVB, C57BL/6 and DBA) oocytes were removed cumulus cells, dissected partial zona-pellucida, surrounded by cumulus cells. The techniques of IVF, culture and embryos transfer were used. Results showed that the feasible concentration of mouse spermatozoa was 5-7 X 105/ml. Swimming up of mouse spermatozoa in C57BL/6 can improve the fertilization rate. The best time of the oocytes collected was 15h after injected hCG The higher rates of IVF and pregnancy were CD-land B6DF1. There was no significantly difference of the pregnancy rate in three disposal ways of oocytes in each of five strains of mouse. The rates of IVF of the denuded and zona-intact oocytes (10-23%)were evidently lower than that of oocytes surrounded by cumulus cells (34-60%). We found the positive effect of partial zona-pellucida dissection(PZD) on the insemination rate (41-65%), above all the strains of C57BL/6 and DBA. So this is the best method of IVF of C57BL/6 and DBA while the oocytes surrounded by cumulus cells of CD-1,B6DF1,FVB are directly used to IVF in the tests of fresh spermatozoa.to explore and establish a suit of method of cryopreserved mouse spermatozoa, two kind of cryopreservation protection solutions (CPS and RS318) and two thawed methods and two methods to dispose RS318 were studied. Four strains (B6DF1,CD-1,DBA, C57BL/6) of mouse spermatozoa were cooled at a high rate and low rate when RS318 was used .Then frozen-thawed spermatozoa and oocytes surrounded by cumulus cells were used for IVF under the control of unfrozen sperm. Four strains of mouse oocytes removed cumulus cells and of PZD were inseminated with sperm cooled at a high rate. The techniques of culture and embryos transfer were used. The rate of IVF and pregnancy was used to select good method of cryopreservation of mousespermatozoa. Results, there was no remarkable difference in the rate of IVF of the test of CPS and RS318 or two thawed methods or two methods to dispose RS318. The rates of IVF(35%,34%,17%)of B6DFl,CD-l,C57BL/6 mouse of tests of spermatozoa cooled rapidjy were remarkably lower than that of fresh spermatozoa(60%,59%,34%)or that of spermatozoa cooled slowly(62%,58%,30%)and the rates of pregnancy in the spermatozoa cooled tests were remarkably lower than them in fresh spermatozoa except for DBA. The fertilization rates of PZD oocytes (43-49%) in the tests of spermatozoa cooled rapidly were significantly higher than that of the zona-intact oocytes(7-19%) and higher than that of oocytes surrounded by cumulus cells (17-34%). So mouse spermatozoa cooled slowly is a feasible method. And dry thawed and dilution methods are used. PZD oocytes can improve the rate of IVF with frozen-thawed spermatozoa.