Soy is one of the most common sources of protein in many commercial formulas for laboratory rodent diets. Soy contains isoflavones, which are estrogenic. Therefore, soy-containing animal diets might influence estrogen-regulated systems, including basal behavioral domains, as well as affective behavior and cognition. Furthermore, the isoflavone content of soy varies, potentially unpredictably confounding behavioral results. Therefore researchers are increasingly considering completely avoiding dietary soy to circumvent this problem. Several animal studies have investigated the effects of soy free diets but produced inconsistent results. In addition, most of these previous studies were performed in outbred rat or mouse strains. In the current study, we assessed whether a soy-free diet altered locomotion, exploration, nesting, anxiety-related behaviors, learning, and memory in C57BL/6 mice, the most common inbred strain used in biomedical research. The parameters evaluated address measures of basic health, natural behavior, and affective state that also are landmarks for animal welfare. We found minor differences between feeding groups but no indications of altered welfare. We therefore suggest that a soy-free diet can be used as a standard diet to prevent undesirable side effects of isoflavones and to further optimize diet standardization, quality assurance, and ultimately increase the reproducibility of experiments.

Murine norovirus (MNV) and mouse parvovirus (MPV) are among the most common adventitial viruses seen in laboratory mice, and infections arise in barrier facilities despite rigorous biosecurity programs. Some authors have implicated nonsterilized feed as a source of MPV in rodent facilities, but none have conclusively documented viral particles in the feed. In this study, we hypothesized that both viruses can resist the pelleting process but not subsequent irradiation or autoclaving, thus revealing a potential source of outbreaks in rodent facilities. To test this hypothesis, we contaminated powdered feed with 10-fold concentrations of MNV and MPV and fed it to both Swiss Webster (SW) and C57BL/6NTac (B6) mice to determine a 'powdered ID₅₀' according to seroconversion over a 28-d period. We repeated the experiment by using powdered feed that we contaminated with increasing viral doses (as no. of powdered ID₅₀) and subsequently pelleted; from these results, we determined a 'pelleted ID₅₀.' Finally we assessed the effect of irradiation and autoclaving on contaminated pellets by using the same experimental design. The powdered ID₅₀ was relatively low and identical in both mouse strains (2.51 × 10² pfu) for MNV but higher in B6 (copy number, 3.20 × 10⁶) than SW (3.98 × 10⁴ copies) for MPV. As hypothesized, mice were infected by contaminated rodent feed despite the pelleting process. Indeed, pelleting resulted in a 1- to 2-log increase in ID₅₀ in both strains for MNV and MPV. Irradiation and autoclaving of infected pellets effectively prevented seroconversion of mice exposed to all doses of MNV, whereas a single mouse seroconverted at the highest dose of MPV (1.35 × 10⁷ copies). These data suggest that both MNV and MPV remain infectious after conditions reproducing the rodent chow pelleting process and that nonsterilized rodent chow might be a source of viral outbreaks.

Although efficient sanitization is a high priority for Animal Care and Use Programs (ACUP), the cage sanitation process for animals used in biomedical research can be labor-intensive. To increase the efficiency of sanitation services provided by ACUP, the current project used the Lean Six Sigma methodology to reduce the time to sanitize mouse cages by implementing countermeasures to reduce causes of waste within this process for the Animal Care Operations (ACO) group at a leading public research university. Lean Six Sigma was selected for use in this project given its goal to make organizations' routine operations flow as smoothly and efficiently as possible and its applicability to biomedical and research settings as discussed in the literature. Through observation of the ACO's existing mouse cage sanitation process and brainstorming sessions with the ACO staff, the existing process was mapped, a baseline measurement of the current process performance was established, the process was analyzed to identify potential causes of waste, and appropriate countermeasures were implemented to improve the process. These countermeasures involved actions specifically targeted at reducing process delays, which included implementing new procedures, clarified schedules, and visual controls. As a result of this project, ACO's average mouse cage sanitation process cycle time was reduced by 35 min, and the time saved was reinvested within the ACO to provide technicians additional time to complete other valuable tasks and thus support animal research endeavors at this university.

Gnotobiotic animal research has expanded markedly over the past decade. Although germ-free animals were first described more than 100 y ago, little evidence-based guidance is available on best operational procedures. A key aspect of gnotobiotic technology is the sterilization of animal enclosures, most commonly flexible vinyl film isolators. The objective of this study was to determine the most effective methods for chemical sterilization of gnotobiotic isolators and associated equipment. As test microbes, we used bacteria from 4 different accidental isolator contaminations that occurred in a gnotobiotic core facility. Identification by 16S ribotyping revealed facultative anaerobic firmicutes, including several Paenibacillus and Bacillus species, and obligate aerobic actinobacteria, namely Micrococcus luteus, among the contaminants. We selected 6 products commonly used for disinfecting hospital rooms, kitchens, and veterinary facilities to represent chlorine-oxide– and peroxide-based disinfectants and tested the hypothesis that these 2 classes are equally effective. However, evaluation of bactericidal and sporicidal activity in liquid cultures revealed that chlorine oxide-based disinfectants were more effective than peroxide-based disinfectants. In both groups, various products effectively sterilized gnotobiotic isolators by fogging in field tests, although bactericidal concentrations were markedly higher than those in suspension cultures, and effectiveness was contact-time–dependent. In addition, in both groups, some disinfectants were excessively corrosive to ferrous metals and acrylic. These results demonstrate that no single disinfectant has all desirable properties and that the different characteristics of disinfectants must be balanced during their selection. However, chlorine oxide-based disinfectants were generally more effective and less corrosive than peroxide-based products.

A significant concern in laboratory animal medicine is contamination due to pathogen outbreaks and how to adequately decontaminate small equipment. Many factors play a role in the selection of the decontamination method including cost, efficacy, personnel time and safety. Chlorine dioxide (ClO₂) gas is an effective method, but decontamination often requires a ClO₂ gas generator with a specialized air-tight exposure chamber. Although this method works well for large-scale decon- tamination, the use of a gas generator may be impractical and too costly for smaller-scale decontamination. The goal of this study was to create and validate an effective, small-scale decontamination method that uses ClO₂ gas and which is an affordable, efficient, safe, and reproducible. First, we identified a product that generates ClO₂ gas after the combination of 2 dry reagents. To find an affordable exposure chamber, we evaluated the ability of 4 household totes with gasket-seal lid systems to retain ClO₂ gas and relative humidity (RH). The efficacy of decontamination was validated by concurrently using 2 different biologic indicators (BI), Bacillus atrophaeus (B.a.) and Geobacillus stearothermophilus (G.s.). All household totes evaluated held sufficient gas and RH for a 15-h cycle, providing adequate contact time to inactivate both BI evaluated. Our results suggest that a total exposure dose of 71 ± 42 ppm-h of ClO₂ gas over 15 h at 90% or greater RH is adequate to inactivate both B.a. and G.s. There was no statistical significance between the 2 BI as indicators for decontamination; 65 of 230 (28.3%) B.a. and 75 of 230 (32.6%) G.s spore strips were positive for growth (P = 0.36). In conclusion, we successfully combined a variety of low-cost materials to establish an effective, small-scale method to decontaminate laboratory equipment. Depending on the size of the tote and whether BI are used, the cost of our method is roughly 1% that of large-scale ClO₂ gas generators used with specialized air-tight exposure chambers.

Opioid analgesics have immunomodulatory properties, which often result in immunosuppression. Sustained-release buprenorphine (SR-Bup) has recently become available as an analgesic for pain management in mice, and little is known regarding potential effects of SR-Bup on the murine immune response. To this end, we immunized female CD1 mice with ovalbumin in complete Freud adjuvant and then treated them with either saline, SR-Bup, Bup-HCl, or SR-vehicle (SR-Veh) for 18 d. Splenocytes were isolated for culture and stimulation to assess cytokine responses, and blood was collected to determine serum antibody responses to ovalbumin. In all treatment groups, levels of IL10, TNFα, and IFNγ increased in ovalbumin-stimulated splenocytes compared with unstimulated splenocytes. Cytokine responses after stimulation did not differ between treatment groups except for IL10, which was significantly higher in SR-Bup–treated mice compared with those given saline or Bup-HCl. The antibody response was significantly increased after immunization but did not differ across treatment groups, except that the response to SR-Veh was lower. These results suggest that the immunomodulatory effects of prolonged treatment with SR-Bup on innate and adaptive immunity are negligible.

In the development of cancer therapeutics, no suitable replacements for the use of animals that are capable of modeling such complex disease processes are currently available. In orthotopic models, surgery is often required to access the target organ for tumor cell inoculation. Historically analgesics have been withheld in such models in light of potential effects on tumor development. The current study evaluated the effect of the opioid buprenorphine on tumor growth of a human ovarian cancer cell line (OVCAR5 OT luc2 mCherry). Female CB17 SCID mice (n = 150) underwent surgery for orthotopic inoculation and were assigned to 1 of 3 treatment groups: vehicle control, 1 dose of buprenorphine, or 2 doses of buprenorphine administered perioperatively. Bioluminescence imaging revealed no significant difference on tumor engraftment rate or growth between control and analgesia-treated groups. These data demonstrate that acute, perioperative analgesia with buprenorphine did not alter tumor growth. Although further research is needed to evaluate potential effects of buprenorphine in other cell lines and mouse strains, the justification for withholding analgesia and the potential influence of pain and stress due to insufficient analgesia in these models should be considered thoroughly.

Meloxicam is the most frequently used NSAID in birds; however, its elimination t_1/2 is highly variable among species. Because zebra finches that require analgesia could benefit from receiving meloxicam, we performed a pharmacokinetic study involving a single intramuscular dose of 1 or 2 mg/kg. Data analysis showed that C_max, t_1/2, and elimination rate constants were not significantly different between the 2 doses. In contrast, C_max for 1- and 2-mg/kg doses of meloxicam approached a significant difference, and those for AUC_0-∞ were significantly different. Importantly, a plasma concentration of 3500 ng/mL, considered a target level for meloxicam in other avian species, was maintained for approximately 9.5 h in finches that received 2 mg/kg, which was 4 h longer than in birds given 1 mg/kg. Both doses reached low plasma concentrations by 12 h after administration. Subsequently, 8 total doses of 1 or 2 mg/kg were administered to birds at 12-h intervals; these regimens caused no significant changes in select biochemical analytes or the Hct of meloxicam-treated birds. In addition, histopathologic changes for injection sites, kidney, liver, proventriculus, and ventriculus were minimal and similar between control and experimental groups after the multiple doses. These results suggest a 12-h or more frequent dosing interval is likely needed in zebra finches and that meloxicam at 1 or 2 mg/kg IM twice daily for 4 d is safe. The higher dose might provide longer analgesia compared with the lower dose, but a pharmacodynamics evaluation of meloxicam in zebra finches is needed to confirm analgesic efficacy.

Meloxicam is a common analgesic for rodents. Because meloxicam is only formulated commercially for companion animals, it requires dilution to achieve doses appropriate for small, laboratory species. Compounded multidose vial (cMDV) are often created to dilute and store a diluted drug. However, chronic cMDV use runs the risk of contamination and becoming a potential source of nosocomial infection. In this study, we created 15 cMDV by diluting meloxicam with sterile water (dilution, 1:10). cMDV were punctured once daily for 30 d. To determine the sterility of the diluted meloxicam, we assessed 8 cMDV for bacterial growth on days 0, 10, 20, 30, and 365 and tested them for endotoxin on days 0, 30, and 365. In addition, the stability of the remaining 7 cMDV was assessed on days 0, 10, 20, 30, and 365, by using liquid chromatography–diode assays. No bacterial growth or endotoxin was detected at any time point, and the drug concentrations remained stable over 365 d. Given the results this study, we believe that cMDV of diluted meloxicam can remain sterile and stable for 365 d.

Female athymic nude rats (Rattus norvegicus; n = 45; age, 6 wk) were used in an IACUC-approved protocol to investigate mechanisms and potential treatments associated with brain, spine, and spinal cord metastases from triple negative breast cancer. The analgesic plan included the use of buprenorphine SR LAB (0.6 mg/kg; 0.11 mL/rat) subcutaneously and an oral NSAID delivered via the water. Thirty-seven rats reached the experimental end point at 3 mo after xenotransplantation and were euthanized for tissue harvest. Grossly, all 37 rats had nodules in the subcutis over the shoulders; these were identified as small, cystic structures (diameter, approximately 0.25 cm). The cysts and haired skin were submitted for LC-MS/MS (liquid chromatography-tandem mass spectrometry) and histopathology. Histologically, the cysts were lined by fibrous connective tissue mildly infiltrated by macrophages, lymphocytes, and plasma cells. Adjacent blood vessels were rimmed by a mild infiltrate of lymphocytes and plasma cells. The cysts contained variable accumulations of a light pink, proteinaceous fluid. The cause for the cysts could not be determined histologically; there was no evidence of neoplasia. LC-MS/MS analysis revealed that the cysts contained buprenorphine. We hypothesize that the lack of T cells and a cell-mediated immune response in these rats prevented the dissolution of the vehicle and absorption of the buprenorphine. The manufacturer provides a cautionary statement regarding the use of this formulation in nude mice due to skin reactions, but to our knowledge, this report is the first description of an apparent lack of absorption of the drug in immunodeficient animals.

Opiates play an important role in the control of pain associated with thoracotomy in both people and animals. However, key side effects, including sedation and respiratory depression, could limit the use of opiates in animals that are lethargic due to cardiac disease. In addition, a rare side effect—neuroexcitation resulting in pathologic behavioral changes (seizures, mania, muscle fasciculation)—after the administration of morphine or hydromorphone is well-documented in many species. In pigs, however, these drugs have been shown to stimulate an increase in normal activity. In the case presented, we describe a Yorkshire-cross pig which, after myocardial infarction surgery, went from nonresponsive to alert, responsive, and eating within 30 min of an injection of hydromorphone. This pig was not demonstrating any signs associated with pain at this time, suggesting that the positive response was due to neural stimulation. This case report is the first to describe the use of hydromorphone—a potent, pure μ opiate agonist—for its neurostimulatory effect in pigs with experimentally-induced cardiac disease.

This retracts the article entitled, "Effects of Sodium Lighting on Circadian Rhythms in Rats" by Xian Chen, Chang-Ning Liu, and Judith E Fenyk-Melody published in the May issue vol 58, issue 3, p 311–320.¹ This article is being retracted with the support of all 3 coauthors due to methodological and authorship issues.