Presentation on theme: "Periparturient manipulation of mice is a valuable tool for the modern research facility. Techniques such as in vitro fertilization, embryo transfer, caesarian."— Presentation transcript:
Periparturient manipulation of mice is a valuable tool for the modern research facility. Techniques such as in vitro fertilization, embryo transfer, caesarian rederivation, and fostering are commonly used to create new animal models, rescue poorly breeding strains of mice, rescue valuable litters of mice when dams neglect their litters or die, and eradicate diseases. 1, 9, 10, 12, 16, 17 Fostering is the movement of a litter of mice from the birth (donor) dam to a recipient dam. Fostering has been used alone or as part of caesarian rederivation procedures to eradicate diseases such as mouse coronavirus 8, 13 and Helicobacter sp. 3, 20 It has also been used to save litters of rare strains of mice that are at risk due to neglect by the dam, agalactia, or the death of the dam 19 (personal communications). Current veterinary practice recommends the use of fostering pups within 48 hours of birth to recipient dams with age- matched litters. 5, 20, 21 Some sources advocate for complete replacement of the recipient dam’s litter with the fostered neonates. 8, 21 Others recommend inclusion of the recipient dam’s neonates to facilitate acceptance of the fostered neonates. 3, 11, 20. However, a review of these publications and others show that there is no clear scientific basis for the current recommended veterinary practice. It is likely that the current recommended veterinary practice of transfer prior to 48 hours of age has evolved from the use of caesarian rederivation, where the recommendations are easily met. However, global application of this recommendation can result in the euthanasia of valuable litters if an “appropriate” foster dam is unavailable when required. The hypothesis that female mice will readily accept litters that are not their own is supported by the observations of allonursing exhibited by females in polygamous mating systems. 18 Each female can be observed nursing the litters of her cagemates, and it can often be difficult to determine which female is the birth dam. However, there is also evidence that females will aggressively reject their own litters of mice under certain circumstances. 4 The majority of pup loss is described as due to stressors (e.g. noise, vibration) in the environment, injury by or competition with older pups in polygamous breeding systems, aggressive males, strain and genetically engineered mouse characteristics, and primiparous females. 14, 22 This study was performed to characterize standardized procedures to maximize the success of fostering techniques. Mice: In the interest of reducing the overall number of animals used in research, project mice were experimentally naïve rotationally outbred breeders that had successfully reared at least one litter and were no longer needed by our facility investigators. The strains that were utilized originated from a heterogeneous stock derived from 8 inbred strains and have been selected to exhibit physiologic or behavioral responses to ethanol administration - specifically seizures, hypo- or hyperthermia, and increased or decreased activity as assessed in an infrared activity monitor. 2, 15 These rotational outbred stocks were at least G75. The literature suggests that outbred strains of mice have an advantage as foster dams due to their ability to care for large litters of pups. 18, 21 We allowed natural mating without synchronization. When the females were identified as pregnant by observation or palpation after gestational day 17, they were moved to a maternity cage so that potential male aggression and pregnancy concurrent with lactation would not be confounding factors in the experiments. 22 Husbandry: The animal care facilities meet the requirements of all applicable federal regulations and is AAALAC accredited. All mice in this facility were housed in polycarbonate shoebox cages with corn cob bedding (Bed o- Cobs, Maumee, OH) and nestlets (Ancare, Belmore, NY). The cages were topped with filter tops and kept on ventilated racks (Thoren Caging Systems, Hazelton, PA). Cages were changed at least once weekly in a laminar flow changing station (Lab Products, Seaford, DE). Gloves were disinfected with a 10% bleach solution between cages. Soiled cages were sanitized in a mechanical cage washer with a finall rinse temperature of 180F (82C) and autoclaved prior to reuse. The rooms were kept on a 12:12-h light:dark cycle, and animals were provided rodent chow (LabDiet, St. Louis, MO) and tap water ad libitum. Temperature and humidity were maintained at 72F (22C) and at least 30%, respectively. Indirect exposure sentinel mice were used to screen the colony for pathogens on a quarterly basis. Five- week old female ICR (Taconic, Germantown, NY) mice that had been exposed to pooled dirty bedding from colony cages for a minimum of 28 days. Serum samples were analyzed for the presence of mouse rotavirus, mouse coronavirus, mouse parvovirus, minute virus of mice, pneumonia virus of mice, reovirus type 3, Sendai virus, mouse encephalomyelitis virus, and Mycoplasma pulmonis in house using the Smart Spot kit (Encinitas, CA). Internal and external parasite examinations were also performed. Currently this facility is free of all pathogens described above, including rodent pinworms and mites. Fostering Methods: We followed a standard fostering protocol to ensure uniform handling of all pups. 8, 11 First, the donor and recipient dams were removed from their cages and placed in separate clean cages. The litter to be fostered was gently picked up and mixed with dirty bedding, nestlet, and (if indicated by the assigned treatment group) other pups from the recipient dam's cage. When mixing the pups, they were gently arranged in the palm of the hand, in contact with nestlet and bedding from the recipient dam’s cage to transfer the recipient dam’s scent. All pups were placed back in the nest and the recipient dam was returned to the cage. The cage were placed on a flat rack (with filter top) to allow visualization without disruption of the cage. The cage was monitored visually every 15 minutes for the first 60 minutes; if there was evidence of rejection by the dam (agitation, carrying the pups around), the pups were removed from the cage and humanely euthanized by administration of a barbiturate overdose (Sleepaway, Fort Dodge Laboratories, Inc., Fort Dodge, IA; 260 mg/mL; 0.1mL/pup IP). The cages were visually assessed at least twice daily and not disturbed for the first 72 hours after fostering in order to decrease potential cannibalism. 11 After this period, we returned the cages to the ventilated rack and performed daily observations for the remainder of the experiment. Any pups found dead were necropsied, if possible, to determine cause of death. Each recipient dam raised a litter of at least 5 and no greater than 10 pups (at least 3 will be fostered pups). Donor and recipient strains were of different coat colors, e.g. black or gray, to facilitate pup identification at weaning (19-21 days of age). To minimize overproduction of animals, breeding pairs were established to ensure appropriate age intervals to allow each female to potentially serve as both a donor and a recipient. Any pups unable to be fostered to another dam as part of this project were euthanized by injectable barbiturate overdose as described above. Experimental Protocols: Experiment 1: As described in the Introduction, some authors recommend the addition of fostered neonates to a recipient dam’s litter to increase the success of the fostering manipulation. 3, 11 The first experiment was designed to examine the validity of this recommendation. As it has been suggested that older pups may reduce the successful foster rate of young pups through injury to the younger pups or interference with their ability to nurse, we selected three age groups, detailed below (Table 1), to characterize this potential confounding factor. By spacing the groups by approximately 3 days of age, the pups developed significantly different abilities to ambulate and compete between groups. Experiment 2: As described in the Introduction, some authors recommend the complete replacement of the recipient dam’s pups with the fostered litter to increase the success of the manipulation. 8, 21 This experiment (Table 2) was designed to examine the validity of this recommendation, by exchanging litters between two dams. Table 1: Treatment groups, rationale, and results for Experiment 1. 1.Carthew, P., M. J. Wood, and C. Kirby Pathogenicity of mouse hepatitis virus for preimplantation mouse embryos. J. Reprod. Fertil. 73: Crabbe, J. C., T. J. Phillips, A. Kosobud, and J. K. Belknap Estimation of genetic correlation: interpretation of experiments using selectively bred and inbred animals. Alcohol Clin. Exp. Res. 14: Crisler-Roberts, R., Z. Ge, M. T. Kearney, K. B. Singletary, et al Evaluation of Helicobacter hepaticus bacterial shedding in fostered and sex-segregated C57BL/6 mice. Comp. Med. 55: Curtin, L Choosing the Right Foster Dam. Tech Talk 11(2): Hansen, A. K Health status and health monitoring. In Handbook of Laboratory Animal Science, 2nd ed. CRC Press, Boca Raton, FL. 6.Hickman, D. L. 2004a. Persistent shedding of mouse hepatitis virus in mouse lines selected for genetic differences in alcohol sensitivity. Contemp. Top. Lab. Anim. Sci. 43(5): Hickman, D. L., D. J. Beebe, S. L. Rodriguez-Zas, and M. B. Wheeler Comparison of static and dynamic medium environments for the culture of preimplantation mouse embryos. Comp. Med. 52:122 8.Hickman D. and K. Thompson Multi-phase approach to eradicate enzootic mouse coronavirus infection. Contemp. Top. Lab. Anim. Sci. 43(5): Hogan, B Manipulating the mouse embryo: a laboratory manual, 2nd. Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. 10.Homberger, F. R Enterotropic mouse hepatitis virus. Lab Anim. 31: Jackson Laboratories The postnatal period. On-line reference at [Accessed 4/19/06.]http://www.informatics.jax.org/silver/chapters/4-4.shtml 12.Kong, F. Y., G. Zhang, Z. S. Zhong, Y. L. Li, Q. Y. Sun, and D. Y. Chen Tranplantation of male pronucleus derived from in vitro fertilization of enucleated oocyte into parthenogentically activated oocyte results in live offspring in mouse. Zygote. 13: Lipman, N. S., C. E. Newcomer, and J. G. Fox Rederivation of MHV and MEV antibody positive mice by cross-fostering and use of the microisolator caging system. Lab. Anim. Sci. 37: Marques-de-Araujo, S. and M. A. Cardoso A laboratory cage for foster nursing newborn mice. Braz. J Med. Biol. Res. 32: Meyer, P. J. and T. J. Phillips Sensitivity to ketamine, alone or in combination with ethanol, is altered in mice selectively bred for sensitivity to ethanol’s locomotor effects. Alcohol Clin. Exp. Res. 27: Mochida, K., M. Ohkawa, K. Inoue, D. M. Valdez, Jr., M. Kasai, and A. Ogura Birth of mice after in vitro fertilization using C57BL/6 sperm transported within epididymides at refrigerated temperatures. Theriogenology. 64: Morrell, J. M Techniques of embryo transfer and facility decontamination used to improve the health and welfare of transgenic mice. Lab Anim. 33: Roulin, A Why do lactating females nurse alien offspring? A review of hypotheses and empirical evidence. Anim. Behav. 63: Silver, L. M Reproduction and Breeding. In Mouse Genetics: Concepts and Applications, Oxford University Press, New York, NY. 20.Singletary, K. B., C. A. Kloster, and D. G. Baker Optimal age at fostering for derivation of Helicobacter hepaticus-free mice. Comp. Med. 53: Watson, J, K. N. Thompson, and S. H. Feldman Successful rederivation of contaminated immunocompetent mice using neonatal transfer with iodine immersion. Comp. Med. 55: Whittingham, D. G. and M. J. Wood Reproductive Biology. In The Mouse in Biomedical Research: Volume III. Academic Press, Inc., New York, NY. Introduction Materials & Methods Research Plan & Results Acknowledgements Discussion Optimizing Mouse Fostering Protocols to Maximize Pup Survival M. Swan,* D. Hickman, VA Medical Center, Portland, OR Optimizing Mouse Fostering Protocols to Maximize Pup Survival M. Swan,* D. Hickman, VA Medical Center, Portland, OR GroupAge of Litter to be Fostered (mean % weaned ± standard deviation) Age of Recipient Dam’s Litter (mean % weaned ± standard deviation) Rationale 1 ( n = 11) <48 hours (95% ± 12% ) <48 hours (98% ± 8% ) Control (typical and recommended method of fostering) 2 ( n = 10) 5-7 days (100% ± 0%) 5-7 days (100% ± 0%) Determine if female with young pups will accept foreign young pups 3 (n = 9) days (100% ± 0%) days (100% ± 0%) Determine if female with older pups will accept foreign older pups 4 (n = 8) 5-7 days (100% ± 0%) <48 hours (88% ± 27%) Determine if female with neonates will accept foreign young pups 5 (n = 10) days (100% ± 0%) <48 hours (93 ± 11%) Determine if female with neonates will accept foreign older pups 6 (n = 9) <48hours (76% ± 43%) 5-7 days (100% ± 0%) Determine if female with young pups will accept foreign neonates 7 (n = 9) <48 hours (78% ± 39%) days (100% ± 0%) Determine if female with older pups will accept foreign neonates 8 (n = 9) 5-7 days (89% ± 33%) days (97% ± 6%) Determine if female with young pups will accept foreign older pups 9 (n = 9) days (100% ± 0%) 5-7 days (100% ± 0%) Determine if female with older pups will accept foreign young pups 10 (n = 7) <48 hours (0% ± 0%) Prenatal (0% ± 0%) Determine if preparturient female will accept foreign neonates 11 (n = 7) 5-7 days (22% ± 44%) Prenatal (29% ± 49%) Determine if preparturient female will accept foreign young pups 12 (n = 7) days (14% ± 38%) Prenatal (32% ± 47%) Determine if preparturient female will accept foreign older pups References Table 2: Treatment groups, rationale, and results for Experiment 2. GroupAge of Fostered Litter A (mean % weaned ± standard deviation) Rationale 1<48 hours Control (typical and recommended method of fostering) 2,48 hours5-7 daysDetermine success of fostering if dam is presented with a litter older (5-7 days) or younger (<48 hours) than she had been nursing 35-7 days10-12 daysDetermine success of fostering if dam is presented with a litter older (10-12 days) or younger (5-7 days) than she had been nursing 4<48 hours10-12 daysDetermine success of fostering if dam is presented with a litter significantly older (10-12 days) or younger (<48 hours) than she had been nursing The authors wish to thank the Portland VA Medical Center for supporting this research project. Special thanks to William Beckman, John DenHerder, Courtney Sherwood, Randy Baldwin, Bryan Bustamante, and Paul Bui for their technical assistance with this project. With the exception of fostering pups to prenatal dams, these studies showed that there was at least 75% chance of successful fostering, regardless of age of fostered litter, birth litter and the addition of pups to an existing litter versus the full replacement of the litter. A slight advantage of age was shown. The older the manipulated litter (whether fostered or birth), the more likely it was to survive to weaning. However, an analysis of variance controlling for age versus treatment showed no statistical significance between ages (data not shown). Our study concludes that the conventional practice of limiting fostering of pups to litters that are less than 48 hours of age to dams with age matched litters is not necessary. One can successfully foster litters of a variety of ages to dams with litters of a variety of ages and expect to be successful at least 75% of the time. Additionally, the addition of pups to an existing litter is no better or worse than total replacement of a litter. This confirms that either of these methods can successfully be used to facilitate fostering, depending on the clinical goal (i.e. disease control would likely require full removal versus salvage of a valuable litter where either would be acceptable).