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54. Production of Germfree Mice by Embryo Transfer
Masanori Okamoto and Tsuneya Mastumoto
Keywords: aseptic technique, embryo transfer, germfree mice
We have been investigating the application of reproductive biotechnology to the strain maintenance of laboratory animals. We have reported that cleaning of Sendai virus-infected mice is possible with an embryo transfer technique. Other researchers have reported that pathogenic microorganisms can be eliminated by transferring embryos harvested from infected animals to clean recipient mice. In the present study, we investigated the applicability of the embryo transfer technique to GF mouse production.
The procedure for producing GF mice by embryo transfer is outlined in the flow chart in Fig. 18. Mature female Jcl:MCH (ICR) mice served as donors for embryo collection after they were mated with mature males of the same strain. The embryo donors and males were mice purchased at 5 to 6 weeks of age and reared in our conventional animal facilities until 8 to 10 weeks of age, when they were used for the experiment. GF female C3H/HeMS mice, which had been maintained at our laboratory animal facility, served as recipients after having been mated with vasectomized GF male of the same strain. To prepare the vasectomized males, a flexible vinyl film isolator containing GF males was connected to the clean bench to be used for aseptic surgery. The spermatic duct of the males was cauterized with a soldering iron. Donor mice were superovulated with 5 IU of PMSG and hCG injected 48 h apart. After mating, the uterus of females with a copulatory plug (plug discovery: day 1) was perfused by modified Whitten's medium for embryo collection on day 4. Embryos of morphologically normal morula and blastocyst stages were stored in a dish containing medium. These embryos were immediately transferred to the uterus horn of recipients on day 3 with aseptic techniques. All sterility tests were performed, in accordance with the methods recommended by the Japan Experimental Animal Research Association with Thioglycollate (TGC), Cooked Meat Medium (CM), Potato Dextrose Broth and GAM semisolid (GAM) as clinical test media. To confirm that the embryos had been collected aseptically, the embryos collected from the donor mice were placed in embryo culture medium. Half of the embryo-containing culture medium was then combined with one of the following, TGC, CM or GAM, and the other half was combined with one of the other clinical test media. The test media were then incubated and observed at 20 or 37°C for two weeks.
The results of the sterility tests on the samples from the fresh feces of the nine vasectomized males, the water bottle rubber stopper, the inside floor of the vinyl film isolator and the gloves were all negative. Microscopic examination of the gram-stained and unstained fecal smears revealed no evidence of microorganisms or parasites in the vasectomized males. The results of the sterility tests of all culture media containing embryos showed they were sterile. The sterility tests of the fresh feces collected from the recipient mice and the inside of the isolator after embryo transfer and delivery were negative. The same test was also carried out on one of the mice cared for in the same isolator, and the result was negative. The autopsy and microscopy of the caecum and duodenum contents of one of the female mice from the same vinyl film isolator revealed no microorganisms or parasites. From the remaining two donor mice, we collected 17 and 12 morphologically normal morulae and blastocysts, respectively. These embryos were transferred to two recipients. One of the recipients, which had a copulatory plug, delivered a litter of six pups after the transfer of 12 embryos. When these pups were weaned, sterility tests on their fresh feces were negative.
The production of GF mice is necessary for the preparation of specific pathogen-free animals and the cleaning of infected animals. Hysterectomy has conventionally been used at our laboratory animal facility to produce GF mice. The average rate of weanlings to newborns produced using the hysterectomy technique over the past three years at our animal facility is 40.0% (156/390). The results of the present study suggest that GF mice can be successfully produced by embryo transfer in addition to conventional hysterectomy. This study shows it is necessary to simplify the experimental procedure from embryo collection to embryo transfer in order to efficiently produce GF mice. Embryo transfer to the GF recipient mouse using frozen-thawed embryo should be examined. It is desirable to establish a procedure for producing GF mice, combined with such reproductive biotechnology techniques as cryopreservation of embryos and spermatozoa; such a goal is useful for strain maintenance and transportation of GF mice, and in vitro fertilization.
Publications:
Okamoto, M. and Matsumoto, T.: Exp. Anim., 48, 59-62, 1999.
