Study On Mouse Sperm Cryopreservation And Establishment Of Mouse Intracytoplasmic Sperm Injection(ICSI)

With the development of life science, as one of basis of life science, the laboratory animal has gained a degree in our country as well. Whereas mouse have the widest idioplasm using in many laboratory animals. So establishing a effective mouse idioplasm resources keeping protocol seems especially important. Embryo engineering and cryobiology using together has made other resources of kinds of laboratory animals keeped successfully . Mouse sperm cryopreservation is as one of the valid skills of keeping embryos. To a considerable part of strains mouse, In vitro fertilization of thawed sperms is very mellow, but to a part of particular strains mouse, the rate of IVF of thawed sperm is still low. As a result, Intracytoplasmic sperm injection(ICSI) in mouse is applied to resolve this situation.R18S3 and FERTIUPTM-CPA were used as cryopreservation protection solutions to freeze DBA/2,C57BL/6J,KM and B6.129S7 -Ldlr^tm1Her/J four strains mouse sperm. Three thawed methods were betaken, rate of IVF(In Vitro Fertilization) assessed the effect of frozen-thawed sperm. Three methods were: (1) Frozen sperm kept water bath at 37℃for 15min to thaw, thawed solution is HTF, (2) Frozen sperm kept water bath at 60℃for 6s then 37℃for 15min to thaw, thawed solution is also HTF, (3) Frozen sperm kept water bath at 60℃for 6s then 37℃for 15min to thaw, thawed solution is FERTIUP TM-PM. When R18S3 was used to freeze sperm,there were distinct difference in the rate of IVF of DBA/2(73.3%,88.4%,55.6%)and KM(64.9%,60.2%,39.6%)among three thawed methods ( P<0.05 ) ,but the results of C57BL/6J(3.0%,10.3%,10.3%) and B6.129S7-Ldlr^tm1Her/J (0%,5.0%,0%) were not notably different(P>0.05); the ratio of IVF of DBA/2 ( 33.6%,14.1%,91.6% ),C57BL/6J ( 8.4%,21.0%,4.9% ) and B6.129S7-Ldlr^tm1Her/J(8.2%,10.0%,28.9%)was remarkably different(P<0.05) while using FERTIUPTM-CPA, the rate of IVF of KM(48.1%,48.0%,48.1%)was not different(P>0.05). For DBA/2 and KM mouse sperm ,frozen with either R18S3 or FERTIUPTM-CPA ,chosen a fit thawed method can both get a ideal rate of IVF. No matter what using any cryopreservation protection solution, choosing any thawed method,rate of IVF of C57BL/6J and B6.129S7-Ldlr^tm1Her/J thawed sperm were relatively low.In order to improve the rate of In Vitro fertilization of C57BL/6J and B6.129S7-Ldlr^tm1Her/J mice′s frozen sperm , Intracytoplasmic sperm injection(ICSI) was used in this experiment. Frozen sperm of C57BL/6J and B6.129S7-Ldlr^tm1Her/J mice thawed at 37℃water for 15min . The head of sperm was separated by Piezo-electric actuator,then injected into the cytoplasm of mouse oocytes. The rate of IVF of C57BL/6J and B6.129S7-Ldlr^tm1Her/J mice reached 44.7% and 50.0% respectively. Compared to the ordinary IVF of frozen-thawed sperm(3.5%,4.3%), the result was enhanced obviously.