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New Zealand White (NZW) laboratory rabbits (Oryctolagus cuniculus), as well as their ancestors the European Rabbit, are a social species that exhibit numerous benefits to being housed accordingly. Although these rabbits are innately gregarious, certain behaviors can still arise when kept in captivity, which if left unchecked, can confound research results or lead to wounding, which in extreme cases can be severe. To prevent these issues, there must be a well-structured plan for the monitoring and maintenance of paired laboratory rabbits. The purpose of this protocol is to present effective procedures for establishing newly paired NZW rabbits as well as methods for successful maintenance. Multiple methods have been tested for the creation of newly paired female rabbits from the vendor, but the most efficacious technique emphasizes capitalizing on the stress bonding from transport, urine marking, pairing in a neutral cage with no forced sharing of resources and a system of monitoring and intervention. To determine the best method of housing paired rabbits in a standard caging environment, data were collected to generate a behavioral ethogram. Behaviors were then quantified as positive, neutral or negative and were tracked across the lifespan of the pair to determine which behaviors indicated pair success or failure. With the newfound knowledge of socially housed laboratory NZW rabbit behavior, enrichment intervention was applied to alleviate aggression and prevent wounding, thus resulting in a higher percentage of successful pairs. Through several years of trialing different pairing methods, the development of the ethogram and the resulting enrichment interventions, understanding of the highly complex social constructs that dominate pair housed rabbit behavior has dramatically increased and allowed for the provision of more species-specific care and increased standards of welfare.

Recent studies have revealed some of the most frequently used analgesics in mice are not effectively treating postoperative pain. Our laboratory sought to compare and assess the validity and reliability of 2 cageside pain assessments that we recently developed for use in mice-nesting consolidation and grooming transfer tests. We then applied these tests to compare the efficacy of commonly used analgesics-buprenorphine (0.1 mg/kg SC every 12 h for 48 h) and carprofen (30 mg/kg in drinking water for 72 h)-alone and in multimodal combination as a refinement for treating postoperative pain in mice. Briefly, C57BL/6 and CD1 male and female mice underwent assessment under conditions of baseline, anesthesia-analgesia, and laparotomy. Results showed that multimodal analgesia displayed the greatest analgesic coverage over the postoperative period, whereas buprenorphine showed slightly less coverage, and carprofen and saline groups displayed signs of pain at most postoperative time points. After anesthesia-analgesia, buprenorphine and multimodal mice lost significant body weight in the absence of a painful stimulus and displayed other significant drug-related changes. Animals treated with carprofen showed few drug-related changes after anesthesia-analgesia but also demonstrated minimal benefit from postsurgical analgesia. Overall, multimodal analgesia was more effective for treating postsurgical pain in mice than the single-analgesic protocols we tested; however, effects on weight loss need to be considered during analgesic selection. Nesting consolidation and grooming transfer tests were valid and highly reliable over time, in inbred and outbred mice, in male and female mice, under different housing conditions. In addition, the nesting consolidation test had excellent reliability between observers. These findings can be used in refining the detection and treatment of postoperative pain in mice.

Guinea pigs (Cavia porcellus) are a frequently used species in research, often involving potentially painful procedures. Therefore, evidence-based recommendations regarding analgesia are critically needed to optimize their wellbeing. Our laboratory examined the efficacy of carprofen and extended-release (ER) buprenorphine, alone and as a multimodal combination, for relieving postsurgical pain in guinea pigs. Animals were assessed by using evoked (mechanical hypersensitivity), nonevoked (video ethogram, cageside ethogram, time-to-consumption test), and clinical (weight loss) measurements for 96 h during baseline, anesthesia-analgesia, and hysterectomy conditions. In addition, ER buprenorphine was evaluated pharmacologically. Guinea pigs treated with a single analgesic showed increased mechanical sensitivity for at least 96 h and indices of pain according to the video ethogram for as long as 8 h, compared with levels recorded during anesthesia-analgesia. In contrast, animals given both analgesics demonstrated increased mechanical sensitivity and behavioral evidence of pain for only 2 h after surgery compared with anesthesia-analgesia. The cageside ethogram and time-to-consumption tests failed to identify differences between conditions or treatment groups, highlighting the difficulty of identifying pain in guinea pigs without remote observation. Guinea pigs treated with multimodal analgesia or ER buprenorphine lost at least 10% of their baseline weights, whereas weight loss in carprofen animals was significantly lower (3%). Plasma levels for ER buprenorphine exceeded 0.9 ng/mL from 8 to 96 h after injection. Of the 3 analgesia regimens evaluated, multimodal analgesia provided the most effective pain control in guinea pigs. However the weight loss in the ER buprenorphine-treated animals may need to be considered during analgesia selection.

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.

Most academic research colonies of mice are endemically infected with enterohepatic Helicobacter spp. (EHS). We evaluated EHS prevalence in surveillance mice before and after a 10-y period of requiring that imported mice be free of EHS by embryo transfer rederivation or purchase from approved vendors. In 2009, composite fecal samples from CD1 surveillance mice representing colony health in 57 rooms located in 6 facilities were evaluated for EHS infection by using PCR assays. Fecal samples were screened with primers designed to detect all known EHS, and positive samples were further assayed by using primers specific for H. hepaticus, H. bilis, H. rodentium, and H. typhlonicus. Most EHS were detected in surveillance mice within the first month of dirty bedding exposure, with prevalence ranging from 0% to 64% as monoinfections or, more commonly, infections with multiple EHS. Compared with 1999 prevalence data, EHS remained endemic in colonies importing the lowest number of EHS-free mice. EHS were absent or the prevalence was greatly reduced in colonies receiving the highest percentage of EHS-free mice. This study demonstrates that the management decision to require exclusive importation of EHS-free mice reduced EHS prevalence on an institutional scale without intensive labor and expense associated with other techniques or interference with research objectives.

The objective of this study was to determine the effect of body position, chest wrap, and sedation on functional residual capacity (FRC) in 6 healthy dogs. Functional residual capacity was determined by helium dilution (re-breathing) whilst in different clinically relevant conditions. These conditions included the standing (sternal) and lateral positions in unsedated dogs and then again both standing and lateral following chest bandaging, and sedation with acepromazine, IV and butorphanol, IV. The mean FRC at each measurement point was determined, as was the change in FRC (delta FRC) from one measurement point to another. Analysis of variance (ANOVA) with repeated measures with Fisher’s LSD post hoc test was used to evaluate the effect of interventions. The differences in delta FRC were evaluated using a t-test or Wilcoxon rank-sum test. P < 0.05 was considered significant. The mean FRC at baseline, defined as standing, unsedated and unwrapped, was 75.3 ± 23.8 mL/kg. Body position or sedation had the most profound effect on FRC with right lateral recumbency lowering FRC by a median of 20.4 mL/kg and sedation lowering FRC by a median of 19.8 mL/kg. Common clinical procedures and positioning result in lowered FRC in healthy deep-chested dogs. In critically ill or injured dogs, the iatrogenic loss of FRC through chest bandaging, sedation, or body position may be clinically relevant.

Higher prevalence of helminth infections in Helicobacter pylori infected children was suggested to potentially lower the life-time risk for gastric adenocarcinoma. In rodent models, helminth co-infection does not reduce Helicobacter-induced inflammation but delays progression of pre-malignant gastric lesions. Because gastric cancer in INS-GAS mice is promoted by intestinal microflora, the impact of Heligmosomoides polygyrus co-infection on H. pylori-associated gastric lesions and microflora were evaluated. Male INS-GAS mice co-infected with H. pylori and H. polygyrus for 5 months were assessed for gastrointestinal lesions, inflammation-related mRNA expression, FoxP3[superscript +] cells, epithelial proliferation, and gastric colonization with H. pylori and Altered Schaedler Flora. Despite similar gastric inflammation and high levels of proinflammatory mRNA, helminth co-infection increased FoxP3[superscript +] cells in the corpus and reduced H. pylori-associated gastric atrophy (p < 0.04), dysplasia (p < 0.02) and prevented H. pylori-induced changes in the gastric flora (p < 0.05). This is the first evidence of helminth infection reducing H. pylori-induced gastric lesions while inhibiting changes in gastric flora, consistent with prior observations that gastric colonization with enteric microbiota accelerated gastric lesions in INS-GAS mice. Identifying how helminths reduce gastric premalignant lesions and impact bacterial colonization of the H. pylori infected stomach could lead to new treatment strategies to inhibit progression from chronic gastritis to cancer in humans., RO1-CA67529, R01DK052413, PO1CA26731, P01 CA028842, P30ESO2109, R01DK065075

Objectives: Gastric colonisation with intestinal flora (IF) has been shown to promote Helicobacter pylori (Hp)-associated gastric cancer. However, it is unknown if the mechanism involves colonisation with specific or diverse microbiota secondary to gastric atrophy. Design: Gastric colonisation with Altered Schaedler's flora (ASF) and Hp were correlated with pathology, immune responses and mRNA expression for proinflammatory and cancer-related genes in germ-free (GF), Hp monoassociated (mHp), restricted ASF (rASF; 3 species), and specific pathogen-free (complex IF), hypergastrinemic INS-GAS mice 7 months postinfection. Results: Male mice cocolonised with rASFHp or IFHp developed the most severe pathology. IFHp males had the highest inflammatory responses, and 40% developed invasive gastrointestinal intraepithelial neoplasia (GIN). Notably, rASFHp colonisation was highest in males and 23% developed invasive GIN with elevated expression of inflammatory biomarkers. Lesions were less severe in females and none developed GIN. Gastritis in male rASFHp mice was accompanied by decreased Clostridum species ASF356 and Bacteroides species ASF519 colonisation and an overgrowth of Lactobacillus murinus ASF361, supporting that inflammation-driven atrophy alters the gastric niche for GI commensals. Hp colonisation also elevated expression of IL-11 and cancer-related genes, Ptger4 and Tgf-β, further supporting that Hp infection accelerates gastric cancer development in INS-GAS mice. Conclusions: rASFHp colonisation was sufficient for GIN development in males, and lower GIN incidence in females was associated with lower inflammatory responses and gastric commensal and Hp colonisation. Colonisation efficiency of commensals appears more important than microbial diversity and lessens the probability that specific gastrointestinal pathogens are contributing to cancer risk., National Institutes of Health (U.S.) (grant R01 AI37750), National Institutes of Health (U.S.) (grant R01 CA093405), National Institutes of Health (U.S.) (grant P30-ES02109), National Institutes of Health (U.S.) (grant P01 CA028842), National Institutes of Health (U.S.) (grant T32 RR07036)

Background & Aims Transgenic FVB/N insulin-gastrin (INS-GAS) mice have high circulating gastrin levels, and develop spontaneous atrophic gastritis and gastrointestinal intraepithelial neoplasia (GIN) with 80% prevalence 6 months after Helicobacter pylori infection. GIN is associated with gastric atrophy and achlorhydria, predisposing mice to nonhelicobacter microbiota overgrowth. We determined if germfree INS-GAS mice spontaneously develop GIN and if H pylori accelerates GIN in gnotobiotic INS-GAS mice. Methods We compared gastric lesions, levels of messenger RNA, serum inflammatory mediators, antibodies, and gastrin among germfree and H pylori–monoinfected INS-GAS mice. Microbiota composition of specific pathogen-free (SPF) INS-GAS mice was quantified by pyrosequencing. Results Germfree INS-GAS mice had mild hypergastrinemia but did not develop significant gastric lesions until 9 months old and did not develop GIN through 13 months. H pylori monoassociation caused progressive gastritis, epithelial defects, oxyntic atrophy, marked foveolar hyperplasia, dysplasia, and robust serum and tissue proinflammatory immune responses (particularly males) between 5 and 11 months postinfection (P, National Institutes of Health (U.S.) (Grant R01 AI37750), National Institutes of Health (U.S.) (Grant R01 CA093405), National Institutes of Health (U.S.) (Grant P30 ES02109), National Institutes of Health (U.S.) (Grant P01 CA028842), National Institutes of Health (U.S.) (Grant T32 RR07036)