| The cryopreservation of mouse sperm provides an economical option for preserving a large number of mouse strains generated by transgenic and targeted mutation methodologies. However, for some reasons, it is difficult to cryopreservate mouse sperm. During our study, sperm from different mouse strains were frozen and we use the IVF rate of frozen-thawed sperm as the criterion to decide whether the cryopreservation is success or not. Our researches supply a method of IVF using frozen-thawed sperm and provide a foundation to use mouse sperm cryopreservation in the mouse model archiving.1 Influence factors to mouse sperm cryopreservation1.1 Genetic backgroundWe applied the mouse sperm freezing technique and in vitro fertilization of frozen-thawed sperm in our laboratory. Sperm from B6D2F1, KM, ICR, DBA/2J, BALB/cA and C57BL/6J mouse were frozen in 18% raffinose/3% skim milk cryoprotective solution. The viability and fertility of the post-thawed sperm from all strains were evaluated comparing against their fresh sperm used as the control.1.2 AgeSperm from 10, 15, 20 weeks old KM mice were frozen and superovulation oocytes were inseminated with frozen-thawed spermatozoa. The donator mice of different age were divided into two groups, one group mated 5 days before sperm cryopreservation and the other did not.In conclusion, the progressive motility and fertility of sperm in each strain were affected after cryopreservation. The fertilization rates of the same strains oocytes inseminated with frozen-thawed spermatozoa were 68.4%, 33.7%, 47.2%, 27.8% and 9.0% respectively in KM, ICR, DBA/2J, BALB/c and C57BL/6J mouse. These results demonstrated that successful cryopreservation is related to genetic background of mouse strains and that the viability and fertility of frozen-thawed sperm depended on mice ages and whether they had mated before the sperm cryopreservation or not. 10-week old mouse are too young to freeze their sperm, and 15-20 weeks old mice can be used in cryopreservation, but older mouse should mate before the sperm cryopreservation.2 Difference of the IVF rate between frozen-thawed sperm and fresh spermFrozen-thawed mouse sperm were fertilized with nude oocytes or oocytes wrapped with cumulus oophorus and matrix (COM). The difference of the IVF rate of the frozen sperm in the same fertilization time and different capacitation time was compared. And the difference in the same capacitation time and different fertilization time was compared too. The fresh sperm were used as the control.This study demonstrated that COM is necessary to IVF, especially when the sperm are low ability. 1 to 1.5 hours for capacitation in vitro is necessary for fresh sperm IVF, but the capacitation of thawed sperm changes during the frozen and thawed process. The IVF speed of the thawed sperm is faster than it of the fresh sperm.3 Mouse sperm cryopreservation technique in embryo engineeringEmbryos obtained from in vitro fertilization (IVF) of the frozen sperm were cultured, cryopreserved or transplanted to the pseudopregnant recipients. 47%~56% of 2-cell embryos inseminated with thawed sperm developed into live offspring by transplantation and 57% progressed to the expanded blastocysts under in vitro culture condition; 80% of 2-cell stage embryos retained alive after cryopreservation, and 50% were able to develop into pups. The difference was no significant between embryos inseminated with the fresh sperm and the frozen ones. So our studies illustrated that the mouse sperm cryopreservation could be applied to many fields of embryo engineering.4 Application of sperm cryopreservation to KM mouse breedingSperm of KM mouse from Wu Han Institute of Biologic Products and Shanghai Biochemistry Pharmaceuticals Factory were frozen and transported. Frozen-thawed sperm were inseminated with superovulation oocytes of Shanghai Laboratory Animal Center, Chinese Academy of Sciences. The 2-cell embryos were transplanted to pseudopregnant recipients. The activity rate of frozen-thawed sperm, the IVF rate and th...
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