Exp, Anim, 48(1), 59-62, 1999 —Note— Production of Germfree Mice by Embryo Transfer Masanori OKAMOTO and Tsuneya MATSUMOTO Laboratory Animal and Plant Sciences, National Institute of Radiological Sciences, ‘Anagawa 4-9-1, Inage-ku, Chiba-shi 263-8555, Japan Abstract: We applied the embryo transfer technique to germfree (GF) mouse production. Embryos harvested from superovulated mice were transferred aseptically, in a sterile environment, to the uterus of GF recipient females which had been mated with vasectomized GF males. One of the recipients became pregnant and delivered offspring. Sterilty tests confirmed that the vasectomized males, newborns, recipient female mice, ‘embryo-containing culture media, and the inside of the vinyl film isolator were germtree. These results suggest that the embryo transfer technique can be successfully applied to the production of GF mice. Key words: Aseptic technique, embryo transfer, germfree mice Conventionally, germfree (GF) mice are produced by hysterectomy. ‘The uterus is removed from the dam immediately before delivery and the fetus is taken out and given to a GF foster mother for nursing (4, 5, 7] But, this technique has some limitations. For one thing, determining the optimal time to remove the uterus is difficult, The survival rate of the offspring may be reduced if delivery occurs earlier than expected or if surgical delivery is performed too early. Furthermore, foster nursing requires skill, so that obtaining GF pups with a high probability of survival is not always pos- sible with this technique, 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 the embryo transfer technique [6]. Other researchers have reported that pathogenic microorgan- isms can be eliminated by transferring embryos harvested from infected animals to clean recipient mice [I-3, 10, 11). 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. 1 Five mature female JckMCH (ICR) mice (CLEA, Ja: pan) served as donors for embryo collection after they ‘were mated with mature males of the same strain. The embryo donors and males were specific pathogen free (SPF) mice purchased at 5 to 6 weeks of age and reared in our conventional animal facilities until 8 to 10 weeks Animals: of age, when they were used for the experiment. The animal rooms were maintained at 22 + 2°C and were lit (Received 3 October 1997 | Accepted 7 September 1998) ‘Address corresponding: M. Okamoto, Laboratory Animal and Plant Sciences, National Institute of Radiological Sciences, Anagawa 4-91 Inage-ku, Chibashi 263-8535, Japan Part ofthis study was presented atthe twenty-seventh Annual Meeting ofthe Japanese Association of Germpree Life and Gnotobiotogy Nagoya, Japan, January, [994 and XUth International Symposiam on Grotobiology. Honolulu, USA, June, 1996, and published as respective proceedings© M. OKAMOTO Al from 6:00 to 20:00, Nine 12- to 18-week-old GF fe- male C3H/HeMS mice, which had been maintained at our laboratory animal facility at the National Institute of Radiological Sciences (NIRS), served as recipients after having been mated with nine vasectomized GF male C3H/HeMs mice bred at NIRS. The animals used in the present study were treated and/or handled ac- cording to the “Recommendations for Handling of Laboratory Animals for Biomedical Research”, com- piled by the Committee on Safety and Handling Regulations for Laboratory Animal Experiments, NI All instruments and devices for surgery and embryo ‘manipulation were sterilized in either an autoclave or a gas sterilizer, or by filtration or with peracetic acid, and placed on a clean bench. Preparation of vasectomized sterile males: ‘To pre- pare the vasectomized males, a flexible vinyl film isolator containing 6- to 7-week-old GF males was con- rected to the clean bench to be used for surgery. The sleeve between the flexible vinyl film isolator and the clean bench, and the clean bench itself, were sterilized with peracetic acid, The GF males were placed on the \ND T. MATSUMOTO clean bench and anesthetized, The spermatic duct of the males was cauterized with a soldering iron. Aseptic collection and transfer of embryos: A trans- parent vinyl film curtain covered the anterior plane of the clean bench. A stereoscopic microscope was steri- lized by gas in a sterilization cylinder. The cylinder was then connected to the clean bench. Ten donor mice were superovulated with 5 IU of PMSG (Serotropin; Teikokuzoki Co. Ltd., Japan) and 5 1U of CG Gonatropin; Teikokuzoki Co. Lid., Japan) injected 48 hr apart, ‘These mice were mated with males of the ‘same strain, and 5 females with a copulatory plug (plug discovery: day 1) were cuthanatized on day 4 post- mating. ‘They were immersed in a disinfecting bath solution and placed on the clean bench. The uterus ‘was removed from each donor and perfused by modi- fied Whitten’s medium [12] for embryo collection. Embryos which demonstrated morphologically normal morulae and blastocysts were stored in a dish contain- ing medium and placed on a warming plate. The recipients were moved to the clean bench via a sterile lock on day 3 of pseudopregnancy. ‘The normal em FLEXIBLE VINYL FILM ISOLATOR ae Ten emibrye donor $ ia ‘Superovuation injected with PMSG-hCG Mating 8X2 {(Five # mica) bays (CLEAN BENCH Embryo collection res | Sleeve | LL (ouity te) Ff Vasectomy ? [Aseptic Procedure] Fig. nigue, SL: Stele lock Morulae and Blastocysts > Embryo transfer Nine gormtroe Nine germiree recipient $ mice 7 mice Vasectomy * Infertle copulation + j o Pseudopregnancy oi ne pregnancy Delvery Weaning (Stent test) [sterile Environment] Experimental flowchart of germfree mice production by an embryo transfer tech-PRODUCTION OF GERMFREE MICE 61 bryos were immediately transferred to the recipients with « micropipette and aseptic techniques [6]. One of the recipients became pregnant, delivered a litter of six pups and nursed them. Sterility tests: Sterility ests were performed, in ac cordance with the methods recommended by the Japan Experimental Animal Research Association [8, 9] with Thioglycollate (TGC, Eiken Chemical Co. Ltd, Japan), Cooked Meat Medium (CM, Nissui Pharmaceutical Co. Ltd., Japan), Potato Dextrose Broth (PD, Difeo Labora- tories, USA) and GAM semisolid (GAM, Nissui Pharmaceutical Co. Lid, Japan) as clinical test media. To sterility test the vasectomized males, we collected a fresh fecal sample from each of the nine vasectomized males 4-5 days after surgery, placed it in a mid-sized test tube in suspension with 20 parts bottled water, soaked four sterilized swabs in the fecal suspension and inoculated the TGC, CM, PD and GAM test media with one swab each. The test media inoculated with samples from four of the test mice were then cultured at 37°C, while those from the other five test mice were cultured at 20°C, for 14 days each. In addition, we moistened four sterilized swabs with bottled water and wiped the inside of the rubber stopper of the water bottle of the vasectomized male, the interior floor of the vinyl film isolator and the gloves of the vinyl film isolator thoroughly, inoculated ‘TGC and PD with two swabs each and cultured them under the same condi- tions as the fecal samples. Furthermore, gram-stained and unstained smears of the fecal suspension were ex- amined microscopically for the presence of microorganisms and parasites. ‘The above sterility tests were carried out once a month for each isolator. To confirm that the embryos had been collected asepti- Table 1, Results of st cally, the embryos collected from the superovulated donor mice were placed in embryo culture medium. We collected 16, 13 and 15 morphologically normal ‘morulae and blastocysts from three of the donors. Half of the embryo-containing culture medium was then com: bined with one of TGC, CM or GAM, and the other half was combined with one of the other clinical test media (Table 1). The test media were then incubated and observed at 20 or 37°C for two weeks. The steril- ity of the recipients and pups cared for in the same vinyl film isolator was confirmed by checking their fresh feces at 4-5 days after embryo transfer and after delivery, by the same method as that used for the vas- ectomized males. When examining the recipient mice, samples from inside the vinyl film isolator were also tested for sterility by the above methods. Fresh fecal samples from the pups were also examined for sterility ‘when the pups were weaned at the age of three weeks. Microscopic examination ofthe cecal and duodenal con- tents was performed both with and without gram staining, when an autopsy was performed on one of the female mice cared for in the same vinyl film isolator in order to check for the presence of microorganisms and parasites. “The samples from the fresh feces of the nine vasecto- mized 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. Table I shows the results of the sterility tests of the embryo-containing culture media. All culture media containing embryos were found to be sterile. The re- sults of the sterility tests of the fresh feces collected lity tests of embryo culture media containing normal ‘morulae and blastocysts collected after mating Donor No.ofembryos Sterility ‘Culture Rewits mouse collected per mouse test temperature °O) A 6 20 Negative 2 Negative B B Tec 37 Negative cM 20 Negative © 5 Tec u Negative Gam 37 Negative © TGC: thioglycollate medium, CME: cooked meat medium, GAM: GAM semisolid medium.2 M, OKAMOTO AND T. MATSUMOTO Table 2, Production of germfree mice after embryo transfer Donor No.of Recipient mouse _embryoscollected” No. D 2 » E "7 Ww * Morphologically normal morulae and 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. ‘Tests on the fresh feces collected from the six wean- lings were also 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 12 and 17 morphologi- cally normal morulae and blastocysts, respectively. These embryos were transferred to two recipients. One of the recipients, which had a copulatory plug, deliv- cred a litter of six pups after the transfer of 12 embryos (Table 2). ‘The production of GF mice is necessary for the prepa ration of SPF animals and the cleaning of infected animals. The results of the present study suggest that GF mice can be successfully produced by embryo trans- fer in addition to conventional hysterectomy. Hysterectomy has conventionally been used at our labo- ratory animal facility to produce GF mice. The average rate of weanl gs to newborns produced using the hys- terectomy technique over the past three years at our animal facility is 40.0% (156/390). ‘This low rate is attributable to difficulty in determining the optimal tim- ing for surgical delivery and in the nursing of pups by the foster mother. ‘These two factors in turn appear to be related to the human manipulation of the pups dur- ing delivery and postnatal development. The present ‘method allows the recipient mice to deliver and nurse the pups without human intervention, but some aspects of the sterilization and preparation of instruments and devices for aseptic procedures for this method require technical improvement. Future research will focus on using a larger number of animals in order to establish a more efficient, practical technique for producing GF animals. This method for producing GF mice, in com- No. of embryor™ Pregnancy of No. of weanlings! transferred recipient _no, of pups born 2 » 66 10 “ 00 tocysts, ® Recipient with copulatory plug, pregnant. © Recipient without copulatory plug, not pregnant. bination with such reproductive engineering techniques ‘as eryopreservation of embryos [13] and spermatozoa [14] and in vitro fertilization, is certain to be useful for strain maintenance and transportation of GF mice. In this study, it is required that the experimental proce- dure from the embryo collection to the embryo transfer be simplified in order to efficiently produce GF mouse. ‘Therefore, it seems to be effective that the embryo trans- ferred to the GF recipient mouse as a problem to be ‘examined uses the frozen-thawed embryo. References 1. Carthew, P., Maureen, J.W., Wood, J., and Kirby, C. 1983, J. Reprod. Fert 69: 253-251. 2. Carthew, P., Mauteen, .W., Wood, J. and Kirby, C. 1985, J. Reprod. Fert. 73: 207-213. 3. Eglesome, MD., Hare, W.CD, Ver. J. 21: 106-112. 4, Luckey, T.D. 1963. A Text Book of Germfiee Life and Gnotobiology, Academic Press, New York and London. 5. Miyakawa, M. 1963. A Text Book of Germfree Animal, Ishiyaku Shuppan, Tokyo (in Japanese). 6. Okamoto, M., MatsushitS., and Matsumoto, T. 1990. Exp. Anim. 39: 601-603. 7. Pleasants, LR, 1968, pp. 47-63, Jn: The Germ-tree Animal in Research (Goates, M.E. ed.), Academic Press, London and New York, 8. Recommended Requirement for Sterility Test of Germfree Animals by Japan Experimental Animal Research Association, 1972. Exp. Anim. 21: 35-38 (in Japanese). 9. Maejima, K. and Nomura, T. 1975. Exp. Anim. 24: 177 and Singh, EL, 1980, Can, 1a. 10, Reetz, 1.C., Wullenweber-Shmidt, M., Kraft, V., and Hedsich, H.-J. 1988. Lab, Anim. Sei. 38: 696-701 11, Suauki, H., Yorozu, K., Watanabe, H., Nakura, M., and ‘Adachi, J. 1996. Exp. Anim. 45: 33-38. 12, Whiten, W.K, 1971, Advan, Bioscl 6: 129-141 13. Yokoyama, M., Wakasugi, N., and Nomura, T- 1981. pp. 113-117. In: Frozen Storage of Laboratory Animals (Zeitmaker, G.H. ed.), Gustav Fischer Verlag, Stuy New York. 14 Okamoto, M., Nakagata, N., Ueda, O., Kamada, N., and Suzuki, H, 1998, J. Mamm. Ova Res. 15: 77-80