Evaluation of Potential Low-stress Handling Methods in Crl:CDSD Rats (Rattus norvegicus).

Low-stress handling methods have been studied in detail in mice, but relatively little research exists concerning preferred handling methods in rats. Most recommendations for low-stress handling of rats have been extrapolated from the mouse literature, despite known differences in handler interaction between the 2 species. The goal of the current study was to evaluate common methods of handling in rats, including application of recognized, low-stress handling methods from other species to rats, in order to determine relative stress levels associated with the handling methods. Seventy male and 70 female, 8-wk-old, Crl:CDSD rats, were housed either individually or in pairs, and were handled weekly or daily using one of the following methods: encircling of the torso (standard thoracic hold), handled using a tunnel, handled using a protective bite glove, handled using a soft paper towel, or tickled prior to being handled by the torso ( n = 10 per sex per treatment group). Body weight and clinical observations were scored at each handling session, abbreviated functional observation batteries were performed every other week, and an interaction test and hematology were conducted prior to study and on the day of study termination. Rats that were socially housed and handled weekly using the standard thoracic hold showed the least evidence of stress, while those that were singly housed and handled weekly using a protective bite glove or tunnel showed the highest level of stress. These effects were predominantly seen in males. This study suggests that standard low-stress handling methods used for other species may not be optimal for rats, and that additional research is needed to identify alternative methods to the standard thoracic hold that would further reduce stress during handling in rats.

The potential for nutritional components of food items used for enrichment of research animals to act as confounding variables in toxicology studies.

Produce and other non-certified foods may be provided to laboratory animals for enrichment, but this practice can generate scientific concerns, particularly if these food items contain nutrients that are pharmacologically active or affect animals' consumption of the basal diet. The author reviews information on potential for a number of nutritional components of food items to affect study data. On the basis of published effect levels, he proposes an upper limit for the consumption of each component in enrichment items relative to the amount present in a standard basal diet. He then assesses the amounts of these nutritional components in a broad range of food enrichment items and proposes a maximum serving size for each item for several common laboratory animals. Total caloric content and sugar content are the limiting components for many enrichment food items, but most items may be used as enrichment for laboratory animals without affecting study results, as long as the amounts of the items provided are managed.

The presence and management of contaminants in non-certified, agriculturally sourced food items used as enrichment for laboratory animals.

One enrichment strategy for laboratory animals is the provision of food variety and foraging opportunities. Fresh agricultural items, including produce or packaged human food items, provide variation in palatability, texture and complexity and can therefore be used as enrichment for lab animals. But concerns are often raised that these food items might sometimes carry contaminants that could affect research subjects and confound experimental results. The author discusses the potential for agriculturally sourced foods used as enrichment for lab animals to be contaminated with mycotoxins, microorganisms and pesticide residues and the effects these contaminants might have on lab animals. He also suggests strategies for reducing the risk of contamination.

Auditing laboratory rodent biosecurity programs.

A rodent biosecurity program that includes periodic evaluation of procedures used in an institution's vivarium can be used to ensure that best practices are in place to prevent a microbial pathogen outbreak. As a result of an ongoing comprehensive biosecurity review within their North American and European production facilities, the authors developed a novel biosecurity auditing process and worksheet that could be useful in other animal care and use operations. The authors encourage other institutions to consider initiating similar audits of their biosecurity programs to protect the health of their laboratory animals.

A novel rat model of hereditary hemochromatosis due to a mutation in transferrin receptor 2.

Sporadic iron overload in rats has been reported, but whether it is due to genetic or environmental causes is unknown. In the current study, phenotypic analysis of Hsd:HHCL Wistar rats revealed a low incidence of histologically detected liver iron overload. Here we characterized the pathophysiology of the iron overload and showed that the phenotype is heritable and due to a mutation in a single gene. We identified a single male rat among the 132 screened animals that exhibited predominantly periportal, hepatocellular iron accumulation. This rat expressed low RNA levels of the iron regulatory hormone hepcidin and low protein levels of transferrin receptor 2 (Tfr2), a membrane protein essential for hepcidin expression in humans and mice and mutated in forms of hereditary hemochromatosis. Sequencing of Tfr2 in the iron-overloaded rat revealed a novel Ala679Gly polymorphism in a highly conserved residue. Quantitative trait locus mapping indicated that this polymorphism correlated strongly with serum iron and transferrin saturations in male rats. Expression of the Gly679 variant in tissue culture cell lines revealed decreased steady-state levels of Tfr2. Characterization of iron metabolism in the progeny of polymorphic rats suggested that homozygosity for the Ala679Gly allele leads to a hemochromatosis phenotype. However, we currently cannot exclude the possibility that a polymorphism or mutation in the noncoding region of Tfr2 contributes to the iron-overload phenotype. Hsd:HHCL rats are the first genetic rat model of hereditary hemochromatosis and may prove useful for understanding the molecular mechanisms underlying the regulation of iron metabolism.

Effects of cage density, sanitation frequency, and bedding type on animal wellbeing and health and cage environment in mice and rats.

The objective of the current study was to evaluate the effects of cage density, sanitation frequency, and bedding type on animal growth and welfare. At weaning, Sprague-Dawley rats and C57BL/6 mice were allocated to treatment groups according to sex, bedding type (shredded aspen, cellulose, or a 50:50 mixture), and cage density and sanitation frequency (inhouse cage density standards and sanitation procedures measured against Guide recommendations) for an 8-wk period. Body weight, feed disappearance, cage ammonia, ATP concentrations, behavior, morbidity, and mortality were assessed weekly; fecal corticosterone, microbiology, and lung histopathology (rats only) were evaluated at the culmination of the trial. In both rats and mice, parameters indicative of animal health and welfare were not significantly affected by cage density and sanitation frequency or bedding type. Occasional effects of feed disappearance and cage ammonia concentrations due to density and sanitation guidelines were noted in rat cages, and bedding type affected cage ammonia and ATP concentrations. Periodic spikes of cage ammonia and ATP concentrations were recorded in mouse cages maintained according to inhouse compared with Guide standards and in cages containing aspen compared with cellulose or aspen-cellulose mixed bedding. Ongoing studies and historical data support the finding that deviations or exceptions from the cage density and sanitation frequency standards set forth in the Guide do not negatively affect animal health, welfare, or production parameters at our institution. These parameters appear to be credible measures of animal health and wellbeing and may be useful for evaluating performance standards for animal husbandry.

A simple method for assessing analgesic requirements and efficacy in rodents.

Evaluation of pain in the clinical setting is an ongoing challenge for veterinarians, researchers and IACUCs. Behavioral assessment, a common technique for evaluating pain, is subjective and difficult to translate into quantifiable data. The authors propose measuring changes in body weight, food consumption and water consumption as a simple and objective method for evaluating postsurgical pain and analgesic efficacy in rodents.

Refinement of the dosage and dosing schedule of ketoprofen for postoperative analgesia in Sprague-Dawley rats.

Though ketoprofen is commonly used in rodent surgical procedures, an optimal dosing regimen has not yet been established. The authors sought to refine ketoprofen dosage requirements in rats and to determine whether one or two doses were needed. In one experiment they compared the effects of one preoperative dose of ketoprofen with those of two perioperative doses (3 mg per kg body weight). In a second experiment they compared the effects of two different dosages of ketoprofen (3 or 5 mg per kg body weight). Results show that all regimens tested were similarly effective in curbing post-surgical weight loss and reduction in food and water consumption; therefore, a single dose of 3 mg per kg body weight was the most efficient.

Development of a percutaneous cerebrospinal fluid collection technique in F-344 rats and evaluation of cell counts and total protein concentrations.

A simple technique for cerebrospinal fluid (CSF) collection was developed in F-344 rats. Cell counts and total protein concentrations were evaluated to assess sample quality. While the 50 to 70 mu L samples of CSF collected on three different days showed a progressive decrease in the total erythrocyte and nucleated cell counts, no significant changes were observed in the total protein concentrations. Progressive decreases in the total erythrocyte count correlated positively with the decreases in volume of CSF collected. Our data suggest that collection of less than 50 mu L of CSF will give a better quality of CSF in F-344 rats. This is the first report of cellular and protein parameters in the CSF of F-344 rats.

Duration of clinical effects and absence of referred hyperalgesia after femoral vein cannulation in rats.

The goal of this study was to assess the duration of pain-related clinical effects and referred hyperalgesia after surgery in rats. Isoflurane anesthesia with or without femoral vein cannulation was performed (n = 6 per group). Body weight and food and water consumption were monitored daily for 48 h, and tail-flick latency was measured twice daily for 24 h after surgery. Water consumption at 24 h after surgery was significantly decreased in the surgical group compared with baseline values and those of the anesthesia group. Body weight change and food consumption showed nonsignificant decreases compared with baseline in both groups 24 h after the procedure. There was a trend toward decreased food consumption after surgery compared with that for the anesthesia-alone group. Tail-flick latency was nonsignificantly decreased the afternoon after surgery compared with baseline values or that after anesthesia alone. Tail-flick latency was similar to baseline and between groups 24 h after surgery. All parameters were similar between groups and compared with baseline by 48 h after surgery. Our results show some changes in postsurgical pain-related parameters only during the initial 24-h period after femoral cannulation surgery, but only the change in water consumption was significant. Although this study involved only a small number of animals, our findings suggest that femoral vein cannulation produces a less painful stimulus than that seen in studies assessing these parameters after abdominal surgery. Hyperalgesia from a distant painful stimulus could not be measured in this model by using the tail-flick assay.

Duration of effects on clinical parameters and referred hyperalgesia in rats after abdominal surgery and multiple doses of analgesic.

This study evaluated the duration of clinical effects and referred hyperalgesia in rats (n = 10 per group) undergo ing abdominal surgery with analgesics (ketoprofen at 3 mg/kg and buprenorphine at 0.01 or 0.1 mg/kg) administered intramuscularly twice daily for 72 h beginning prior to surgery; no-surgery and no-analgesia control groups were included. Food and water consumption and body weight were monitored daily. As a measure of referred hyperalgesia, tail-flick latency was measured daily, before and 4 h after analgesia administration. Compared with those of the no-surgery controls, significant decreases in food consumption and body weight occurred 24 h after surgery without analgesics. There were nonsignificant reductions in these effects by analgesics, but the benefits were not significantly different than those of saline. These parameters continued to be decreased with variable significance in the buprenorphine groups at 48 and 72 h after surgery. In both buprenorphine-treated groups, water consumption was significantly increased at 24 h after surgery but not at 48 or 72 h. Tail-flick latency was not significantly different between the no-surgery and no-analgesia groups but was significantly increased 4 h after high-dose buprenorphine administration and declined nonsignificantly over time in the other groups. We conclude that painful effects from surgery are present primarily during the first 24 h after surgery. The analgesic regimens tested did not completely reduce these effects. Buprenorphine was associated with adverse effects for as long as 72 h after surgery. Referred hyperalgesia from this abdominal surgery could not be measured using the tail-flick assay.

Assessment of personal protective equipment used for facial mucocutaneous exposure protection in nonhuman primate areas.

Animal caretakers working in NHP areas must wear facial PPE to protect themselves from the zoonotic hazards related to splash exposures, but PPE that is uncomfortable may present its own risks. The authors evaluated the level of protection offered by several types of facial PPE against a variety of simulated facial mucocutaneous exposures of the sort that could occur during typical procedures in Old World NHP facilities and determined that less restrictive PPE can be used without compromising safety.

Use of body condition scoring to manage body weight in dogs.

The purpose of this study was to evaluate the use of a body condition scoring system as a tool to manage body weight in our dog colony. We believe that body condition extremes have a pronounced potential impact on pharmaceutical assessment studies. We selected animals from our colony for participation in a dietary management program based on body condition scoring and body weight. Dietary amount was adjusted according to the current and desired conditions of the animal. Animals were re-evaluated monthly, and diet was adjusted based on the progress the animal was making toward its goal. Retrospective analysis of the data showed that approximately 3 months of enrollment in the dietary management program was required to optimize body condition in our dogs. Nearly twice as many female than male animals were enrolled in the program for weight loss, and they were enrolled at a significantly younger age (784.3 days versus 1063.9 days, respectively, P = 0.02). Nearly six times as many female animals were enrolled for weight loss as weight gain. Conversely, twice as many males than females were enrolled for weight gain, but differences in age for these animals were not significant. There was a significant negative correlation (-0.68, P < 0.01) between age and rate of loss of body condition for female dogs. Body condition scoring was a less sensitive indicator of progress on the program than was body weight, with a 6.5% to 7.2% change in body weight seen for each incremental change in score. It took between 2 and 4 months for the body condition score to change by one increment. Correlations between change in body condition and body weight were poor, except in the group of male animals enrolled for weight loss (0.84, P < 0.01). We conclude that body condition scoring is useful for dietary management of a dog colony within the time frame we have described, with the caveat that correlations between weight change and body condition score change is somewhat variable. Older female dogs offered particular challenges for management of obesity.