The purpose of freezing sperm or pre-implantation stage embryos is to protect against the loss of valuable, unique mouse stocks through breeding failure or disease, prevention of genetic drift and to eliminate the cost of maintaining mouse lines not actively in use. Another advantage is that during the process of embryo collection, most pathogenic organisms are excluded as in other methods used to rederive lines of mice.
Although more expensive than the sperm cryopreservation, embryo cryopreservation is recommended when mouse lines are to be terminated and the P.I anticipates that there will be a significant demand for supplying these lines to other labs. The large number of embryos collected will afford the P.I to share samples with collaborators and still have enough stock to revive the line in the future. The sperm cryopreservation is preferred for a back-up strategy while the line is ongoing, although it can also be used to preserve lines that are being terminated.
Embryos can be cryopreserved at all stages of pre-implantation development. The most robust method of embryo cryopreservation uses 8-cell stage embryos that are infused with a cryoprotectant, slowly lowered to –35C, and then rapidly submerged in, and stored in, liquid nitrogen. Sperm cryopreservation involves harvesting the cauda epididymis from euthanized males, releasing the sperm into a cryoprotective solution, loading the sperm suspension into semen straws, incubating the straws in the vapor phase of liquid nitrogen, plunging them into the liquid phase, and storing them in liquid nitrogen.
The the investigators shall provide the TMII with stud males and egg donors from the line or stock which they desire to preserve. Typically, 15 stud males are mated weekly with egg donors per cryopreservation session to produce embryos for cryopreservation. On average, it takes 4 embryo collection sessions to freeze down enough embryos to guarantee recovery. If provided with homozygous males, fewer sessions will be needed. However, it may take more sessions if the mouse strain has a low superovulation rate, the stud males have low fertility, or males are too old. Quality control is assured by freezing and thawing a representative sample of wild-type frozen embryos along with each line frozen down. Frozen embryos should be retrieved by the investigator one-week post-freezing for long-term storage in liquid nitrogen in the investigator’s own lab.
The number of embryos that are cryopreserved depends on several factors, including the number of embryo donors supplied by the client, their age and response to superovulation, and the fertility of the stud males. Due to inherent variation, no guarantee can be provided as to how many embryos will be frozen in any given session.
The recovery of mice from cryopreserved sperm has suffered from large strain-to-strain variations in efficiency. Sperm from popular strains such as C57BL/6J produced very low yields of fertilized embryos after thawing and performing in vitro fertilization (IVF). Recent improvements from Kumamoto University (Takeo and Nakagata, Biology of Reproduction 85, 1066 (2011)) have been shown to make sperm cryopreservation as reliable as embryo cryopreservation. ICSI can also be performed with cryopreserved sperm.
For sperm cryopreservation, the investigators shall provide with 3 young (2 to 6-month-old) healthy males which have been previously proved to be fertile. We obtain the sperm (the procedure requires the sacrifice of the male) and we freeze down 10 sperm aliquots per male. The frozen specimens should be retrieved by the investigator one-week post-freezing for long-term storage in liquid nitrogen in the investigator’s own lab.
Yields from in vitro fertilization (IVF) with thawed sperm can exhibit large variations from strain to strain. Validation of cryopreserved sperm is based either on motility of recovered sperm (at no additional cost) or by in vitro fertilization of oocytes and progression to 2-cell stage embryos. It should be noted that motility does not guarantee viability.