Castration Eliminates Conspecific Aggression in

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Sep 5, 2012 - that early castration could eliminate fighting in the male CD1 sentinels and ... normal eating, drinking, nesting, and grooming. No twitching,.
Journal of the American Association for Laboratory Animal Science Copyright 2012 by the American Association for Laboratory Animal Science

Vol 51, No 5 September 2012 Pages 594–599

Castration Eliminates Conspecific Aggression in Group-Housed CD1 Male Surveillance Mice (Mus musculus) Jennifer LS Lofgren*, Susan E Erdman, Christine Hewes, Catrina Wong, Rebecca King, Tony E Chavarria, Allan R Discua, James G Fox, and Kirk J Maurer† Microbiologic surveillance is essential for murine health maintenance. At our institution, female progeny of inhouse-bred CD1 mice are used in both the transgenic facility and health-surveillance program. To reduce overall animal use, the male progeny, otherwise slated for euthanasia due to a lack of utility, also were enrolled as sentinels. However, veterinary technicians noted excessive fighting among cohoused male surveillance mice that was not resolved by environmental enrichment. After review of factors known to influence aggression in male mice, early castration was selected as the most likely approach to eliminate aggressive behavior among cohoused male mice. Male mice were castrated before 1 mo of age and then placed into the surveillance program. Each week, veterinary technicians recorded all incidences of fighting in cages of castrated and noncastrated male surveillance mice to determine differences between groups. Over a 3-mo period, the overall prevalence of fighting in cages of intact male mice was 64% (14 of 22 cages); although all intact male mice were used preferentially for complete necropsy surveillance time points, one of these cages required separation and 4 cages housed mice that incurred severe fight wounds requiring both separation and euthanasia. In comparison, a 0% (0 of 16 cages) prevalence of fighting was observed among castrated male mice. Castration eradicated pain and distress associated with fighting, thereby constituting a refinement, and allowed the use of male mice from the breeding colony for surveillance, thereby reducing the total number of mice bred for surveillance. In conclusion, castration is a minimally invasive, safe, humane, rapid method to eliminate conspecific aggression among male CD1 surveillance mice.

Our institution maintains SPF rodent facilities, and maintaining this status is critical, given that most infectious agents are known to affect research results and reliability. In addition, pathogen elimination efforts are difficult, expensive, and laborious. Because many murine pathogens cause no gross clinical signs in infected animals, ‘sentinel’ animals are used to detect the presence of these microbial agents. To ensure that colonies remain free of specified pathogens, all of the rodent facilities at our institution support an active surveillance program. In each mouse holding room, 6 sentinel CD1 mice are housed in a rat cage and, in an organized rotation, are exposed to dirty bedding from colony mice in that room. The sentinels are sampled for endo- and ectoparasites and undergo serologic analysis for viral agents and mycoplasma every 2 mo. In addition, bacteriology and histology of tissue samples are performed every 6 mo. Concurrently, CD1 mice are bred inhouse for the transgenic core facility and to supply mice for this sentinel program. Female pups are used as recipients for embryo transfer, are cycled back into the breeding program, or are introduced into the surveillance program. Traditionally, additional female CD1 mice were ordered from vendors to meet the numbers required for the surveillance program. As a refinement initiative, the male CD1 mice bred inhouse, which would otherwise be euthanized due to their lack of prescribed use, were integrated into the surveillance

Received: 19 Jan 2012. Revision requested: 17 Feb 2012. Accepted: 19 Mar 2012. Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts. *Corresponding author. Email: [email protected] †Current affiliation: Center for Animal Resources and Education, Department of Biomedical Sciences, Cornell University, Ithaca, New York.

program. However, this practice had an unfortunate sequela: the male surveillance mice had a high prevalence of intracage aggression, often necessitating separation or euthanasia of affected mice. In addition to being a significant animal welfare concern, chronic social stress has known immunosuppressant effects that may be counterproductive to specific and sensitive serologic screening.5,11,12 In addressing this welfare concern, we reviewed the numerous factors reported to influence aggression in adult male mice. These factors include stocking density, cage space, enrichment, cagemate familiarity, olfactory cues, and the influence of sex hormones.9,20,22,25,31 After carefully weighing each of these factors, we determined that stocking density, cage space, and enrichment had been optimized for both animal welfare and achieving the goals of the surveillance program. Littermates designated for the surveillance program were maintained as a group, therefore cagemates were familiar. However, sentinel mice were unavoidably exposed to exogenous olfactory cues from dirty bedding from multiple cages.20,22 We hypothesized that early castration could eliminate fighting in the male CD1 sentinels and concurrently support the needs and goals of the health monitoring program.

Materials and Methods

Animals. Adult Crl:CD1(ICR) female and male surveillance mice were bred inhouse as part of the transgenic core program and maintained under strict barrier conditions. Male CD1 mice (age 3 to 4 wk) from the same breeding colony were selected for castration surgeries. Mice in this barrier were SPF for ecto- and endoparasites and antibody-negative for all known viral and bacterial mouse pathogens except murine norovirus, which was

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not monitored routinely. Once released from the barrier, surveillance mice were placed in 71 rooms located in 6 facilities. All of the animal facilities surveyed were AAALAC-accredited and maintained under the consistent environmental conditions of 22 ± 1 °C, 30% to 70% humidity, 10 to 15 air changes hourly, and a 12:12-h light:dark cycle. All surveillance mice were maintained in static caging and housed in polycarbonate microisolation cages with filter tops (Allentown, Allentown, NJ) that were commercially designed as rat caging (48.3 cm × 26.6 cm × 15.24 cm, equivalent to 214 cm2 per mouse). Heat-treated hardwood bedding (Beta Chip, NEPCO, Warrensburg, NY) and nesting pads (Ancare, Bellmore, NY) were provided, in addition to ad libidum pelleted diet (RMH 3000, Purina Mills, Richmond, IN) and filtered tap or reverse-osmosis–purified water. In addition, surveillance mice were provided polyvinyl chloride tubes (generic) or red plastic igloos (Bio-Serv, Frenchtown, NJ) or both. The Massachusetts Institute of Technology Committee on Animal Care approved all activities. Exposure of sentinel mice to dirty bedding. Each mouse room had a dedicated cage of surveillance mice. Each surveillance cage was stocked initially with 6 mice; every 2 mo, 2 mice were removed without replacement for evaluation until all mice in that cage had been used, after which a new group of 6 mice were placed. Before dirty cages were brought to the cagewash, a 40cm3 plastic scoop was used to transfer 1 scoop of dirty bedding from each of 30 dirty cages into the sentinel cage. This volume of dirty bedding transfer ensured a ratio of 1 part clean to 2 parts dirty bedding in the sentinel cage. The dirty cages designated for sampling were selected on a weekly rotating basis, by rack. The cage change frequency was once weekly for static caging and every other week for ventilated caging, with the exception of as-needed spot cleaning. Sentinels were sampled for serologic and parasitologic testing every 2 mo, with complete necropsies performed at 6-mo intervals on mice euthanized via CO2. Measuring incidence of fighting. Animal care technicians that noted fight behavior or fight wounds during normal husbandry procedures placed a health-check card to alert veterinary staff. Veterinary technicians daily monitored all animal rooms for new health-check cards and performed physical exams and behavior assessments, as well as any necessary interventions. Veterinary technicians assessed all ongoing clinical cases weekly during grand rounds. Veterinary technicians were trained to recognize fight wounds and instructed to take the appropriate veterinary intervention where required. A spreadsheet form (Excel 2004, Microsoft, Redmond, WA) was used by veterinary technicians weekly to document information regarding all surveillance cages, including those identified by health-check card as housing mice with evidence of new or ongoing fighting, such as observed fight behavior or fight wounds. This spreadsheet included the date, building, room, sex of mice, presence of fighting, and any necessary course of action taken, including separation of the aggressor and euthanasia when fight wounds were severe. Mice from a total of 71 animal rooms from 6 animal facilities were monitored for 3 mo prior to implementing the castration program and again for 3 mo after integration of castrated male mice. Surgical castration. During the initial 3-mo monitoring period, castrations were performed in preparation for the introduction of castrated male mice into the surveillance program. All instruments were sterilized (Germinator 500 Glass Bead Dry Sterilizer, Roboz, Gaithersburg, MD) before use and between animals. Intact CD1 male mice (age 3 to 4 wk) from our inhouse breeding colony were anesthetized with tribromoethanol (250 to 500 mg/kg IP; Avertin, Sigma Pharmaceuticals, Monticello,

IA). We selected tribromoethanol for this study because the surveillance mice used were generated in our transgenic core, where this agent has had a long and established history for this use.21 Although tribromoethanol does not provide pain relief, it reliably induces narcosis and, because mice are given analgesics before recovery, postoperative pain is minimized.16 Once anesthetized, mice were prepared aseptically for surgery (Figure 1 A through C). An open castration was performed, followed by separate closure of the body wall and skin with cold-sterilized 5-0 silk (Figure 1 D through J). With experience, the surgical steps typically can be completed in about 5 min. Immediately after surgical closure, the mice were given buprenorphine (0.1 mg/kg SC; Hanna Pharmaceutical Supply, Wilmington, DE) and warmed sterile saline (1 mL SC). Mice then were put in clean recovery cages located on circulating warm-water blankets. When fully ambulatory, mice were placed in clean, home cages with nesting pads and several pellets of food on the floor. Mice were assessed for signs of unalleviated postoperative pain, with the option of additional analgesia; however, all mice appeared to be pain-free and quickly resumed normal eating, drinking, nesting, and grooming. No twitching, flinching, or belly pressing behavior indicative of abdominal pain was observed. Mice were monitored daily by surgical staff for approximately 7 to 10 d, at which time any remaining sutures were removed, and the castrated male mice were placed into the surveillance program. Statistics. Data recorded by veterinary technicians in intact and castrated male mice over their respective 3-mo observation periods were compared by using the Student t test. Statistical analysis was performed by using GraphPad Prism 4.0 (GraphPad Software, La Jolla, CA), with a P value of less than 0.05 considered significant.

Results

Prevalence of fighting in intact male surveillance mice. During the first week of monitoring, 47 rooms contained female surveillance mice, none of which were fighting. In contrast, during that same week 22 rooms contained male surveillance mice, 50% of which were fighting; 9% of the cages with male mice that fought had to be separated, and 27% required both separation and euthanasia of mice due to the severity of their wounds. Over the following 3-mo period, the overall prevalence of fighting in cages housing intact male mice was 64% (14 of 22 cages); one of these cages required separation only, whereas 4 cages required both separation and euthanasia of mice due to fighting (Figure 2). No fighting among female surveillance mice was reported at any time during the study. Effect of castration on fighting in male surveillance mice. No postoperative complications from the castration surgeries were reported. In the 3-mo after the addition of castrated surveillance male mice, the prevalence of fighting among castrated mice was 0% (0 of 16 cages; Figure 3). Castration significantly (P < 0.0001) decreased fighting among male mice.

Discussion

Euthanizing male mice to spare them the pain and distress resulting from fighting behavior is a common practice in the laboratory animal field. On average, 65% of male mice, possibly as high as 80% when mice are from aggressive strain backgrounds, are euthanized prior to weaning.14,31 This frequency highlights an important target for refinement. In the present study, more than half of the intact male surveillance mice monitored over the 3-mo observation period were noted to be 595

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Figure 1. Castration surgery. The inguinal–perineal region was (A) clipped and scrubbed with alternating swabs of (B) povidone–iodine and (C) alcohol. (D) The skin and body wall caudal to the penis were incised with sterile scissors, and the individual testicles were identified and retracted through the incision. (E through G) The exposed testicular artery and vein were double-ligated and (H) cut. The (I) body wall and (J) skin were closed in 2 layers.

fighting or have fight-associated wounds. The wounds were often considerable: 36% of cages in which fighting was recorded necessitated separation of mice, their euthanasia, or both. On

identifying this important animal welfare concern, intact male mice were used preferentially for complete necropsy surveillance time points. In an effort to diagnose the source of stress

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Figure 2. Prevalence of fighting, separation and both separation and euthanasia during the 3 mo prior to the introduction of castrated male mice. Among the 64% of cages of intact male mice identified as fighting, 7% required separation alone, 29% required both separation and euthanasia, and 64% did not require intervention. All intact male mice were used preferentially for complete necropsy surveillance time points.

Figure 3. Prevalence of fighting among male mice before and after introduction (arrow) of castrated male mice. During the 15 wk prior to introduction, the overall prevalence of fighting was 64%. During the 15 wk after introduction of castrated male mice, the prevalence of fighting was 0%.

resulting in the increased prevalence of fighting, we considered the major factors that contribute to aggressive behavior in male mice. These factors include interaction with unfamiliar male mice, lack of appropriate enrichment, cage size and stocking density, exogenous olfactory cues, and the influence of sex hormones.9,20,22,27,31 Maintenance of littermates in groups and sufficient environmental enrichment were the first factors addressed in our attempt to decrease fighting among intact male surveillance mice. In natural and laboratory settings, mice display less aggressive behavior toward family members than unfamiliar mice.22 Therefore, in the surveillance program, littermates were maintained in a group whenever possible, such that surveillance mice were not exposed routinely to unfamiliar cagemates. A single shelter or igloo-running wheel has been reported to increase aggressive behavior in mice, whereas multiple structures can act as visual barriers for subordinate escape and prevent dominant mice from controlling a single shared resource.9,18,28,30,31 Therefore, multiple enrichment devices (tubes, huts, running wheels) were provided to our mice, but the fighting among the intact male surveillance mice continued. Furthermore, for both colony and surveillance mice, nesting material was transferred from

the dirty to clean cage at cage changing—a long-term provision known to reduce stress in CD1 mice.28,30 Some of the factors known to induce aggression in mice such as cage size, stocking density, and exposure to olfactory cues were not amenable to change because they were required for the integrity of the dirty-bedding surveillance program. For example, although a decrease in aggressive behavior has been observed in smaller cage sizes, the use of larger cages was required to provide the dirty-to-clean bedding ratio necessary for optimal exposure to major mouse pathogens.26,27,29 The stocking density used in the current study—2 to 6 surveillance mice per cage—was similar to the 3 to 5 mice per cage recommended by prior research to reduce aggression.29 These cited recommendations were based on a stable group composition, whereas the current study required removal of 2 mice without replacement every 2 mo to provide optimal dirty-bedding exposure times for sensitive and specific detection of infection with or seroconversion to monitored mouse pathogens. The use of rat caging at this stocking density prevented overcrowding, a factor known to inhibit proper neuroendocrine and immunologic function.3,13,23,25 In addition, central to their function, surveillance mice must be exposed constantly to dirty bedding 597

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and the associated olfactory cues from foreign mice; these cues can intensify aggression in mice.15,31 The final factor explored as a potential means of reducing aggression among our intact male surveillance mice was alteration of sex hormones. Whereas 64% of intact males were noted to exhibit fight behavior, castrated mice did not have a single documented incidence of fighting. The castration method used did not expose a body cavity, caused little physical impairment, and is performed routinely on an outpatient basis in veterinary clinical practice, thus fulfilling the 2011 Guide’s definition of minor surgery.10 All castrated surveillance mice received perioperative analgesia, recovered from surgery without incident, and remained clinically normal. In future studies we hope to refine our anesthesia regimen and use remote cameras to facilitate increased postoperative monitoring during the nocturnal period and without the presence of a human observer. Reduced aggression toward cagemates in castrated mice, particularly when castrated prior to puberty, is well documented in the literature.4,8,17,19 This behavioral effect is specific to castration, because exogenous androgen therapy restores aggressive behavior.2,4,7,17 In addition, a review of the literature did not identify any reports of adverse effects of castration on immune function in humans or mice that would be deleterious to the use of castrated surveillance mice.1,6,24 The current study demonstrated that, through the addition of a minor surgery, castration served as both a refinement, by eradicating pain and distress resulting from fighting, and a reduction in animal numbers by allowing male mice from the breeding colony to be used as surveillance mice. Programs of sufficient size to breed their own surveillance mice or vendors providing surveillance mice may benefit from this early castration strategy also. Although the surgeries themselves require a certain amount of labor, the time and effort in addressing the veterinary concerns arising from fighting and the expense of ordering replacement female mice for culled males is obviated. In addition, these refinements were successful and did not compromise the needs and purpose of the dirty-bedding surveillance program. In conclusion, castration is a minimally invasive, safe, humane, rapid method to eliminate conspecific aggression in male CD1 surveillance mice.

Acknowledgement

Grant support: T32 RR007036 (JGF).

References

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