Almost 40 y ago the scientific community was taking actions to control environmental factors that contribute to variation in the responses of laboratory animals to scientific manipulation. Laboratory animal diet was recognized as an important variable. During the 1970s, the American Institute of Nutrition, National Academy of Science, Institute of Laboratory Animal Resources, and Laboratory Animals Centre Diets Advisory Committee supported the use of 'standard reference diets' in biomedical research as a means to improve the ability to replicate research. As a result the AIN76 purified diet was formulated. During this same time, the laboratory animal nutritionist at the NIH was formulating open-formula, natural-ingredient diets to meet the need for standardized laboratory animal diets. Since the development of open-formula diets, fixed-formula and constant-nutrient–concentration closed-formula laboratory animal natural ingredient diets have been introduced to help reduce the potential variation diet can cause in research.

As an approach to investigating mechanisms of anesthetic action, studies using selective breeding of animal stocks with different anesthetic sensitivity have increased during recent years. Mice are an ideal model for such studies due to their small size, short reproductive cycle, well-established behavioral endpoints of anesthesia, and well-known genetic background. Because single litters are not large enough for simultaneous selective breeding and conservation of the stock, mice must be used from successive litters. However, the stability of isoflurane anesthetic potency across successive litters has not been reported. In the present study, 24 (12 male and 12 female) outbred ICR mice were mated. Each pair was allowed to produce 2 successive litters. Offspring were separated by sex after weaning at 21 d of age. Reproductive characteristics were documented, including litter size at birth, sex ratio at weaning, and neonatal mortality. At 65 to 75 d of age, the median effective dose (ED₅₀) of inhaled isoflurane was measured in mice from the 2 parities by using a bracketing design. Loss of righting reflex was chosen as the criterion for successful anesthesia. The 2 parities did not differ significantly with regard to reproductive parameters and isoflurane ED₅₀ of offspring. These results indicate that offspring in the second litter from the same parents can be used for stock conservation and anesthesia research.

Ketamine–acepromazine–xylazine (KAX) has long been a popular combination of injectable anesthetics for use in laboratory rodents. These drugs are compounded extemporaneously at research facilities because a commercial mixture is not available. This study was designed to determine an appropriate period of use for this mixture by examining its safety, stability, and efficacy at 30-d intervals over an aging period of 270 d. For as long as 270 d after compounding, most of the data collected (chemical stability, sterility, pH, particulate formation, times to loss of righting reflex in injected mice and rats, and histopathology from these animals) supported the finding that the component drugs do not change or degrade. However, mice and rats did show significant differences in anesthetic responses after injection with KAX mixtures of different ages. In light of these findings, we suggest that KAX remains safe, stable, and efficacious for at least 180 d after mixing, and that 180 d constitutes an appropriate period of use for this drug combination when stored in a dark, room-temperature environment.

Cynomolgus (or longtailed) macaques (Macaca fascicularis) are used extensively as laboratory animals in biomedical research. Their use in Singapore, an emerging biomedical hub in Southeast Asia, is now increasing widely, with research subjects currently originating from Singapore, Vietnam, and Pulau Bintan, Indonesia. Limited data exist on the genetic and phenotypic polymorphisms and phylogenetic relationships of these groups, and the animals are used as research subjects without regard to potential differences or homogeneity. Here we characterize their phenotypes by using established primatology tools to detail morphometrics and pelage erythrism and saturation. Pelage analyses supported the Gloger rule, in which heavily pigmented forms predominate near the equator, with Singaporean and Bintan macaques having darker pelage than Vietnamese macaques. Morphometric variation patterns suggest a tendency toward insular dwarfism and correlate generally with the Bergmann rule, in which body mass increases with latitude and colder climate. Although the 3 populations all belong to the nominotypical subspecies M. f. fascicularis, phenotypic differences are evident and are valuable tools to analyze their phylogeographic history and phylogenetic relationships.

The purpose of the present study was to describe the technique for and findings of ultrasonographic examination of the rat uterus for diagnosis of early and midterm pregnancy. The uterus of anesthetized Wistar rats was examined between days 9 and 16 post coitum by transabdominal real-time ultrasonography by using a 12-MHz linear transducer. Pulsed-waved color Doppler sonography was used to measure the embryonic heart rate. The embryonic vesicles were detected with 25% false-negative diagnosis on day 9, 8% on day 10, and 0% thereafter. By day 12, the embryos were detected with measurable crown-rump length and heart rate. Ultrasonographic evidence of pregnancy in the rat was present by day 9 post coitum. Diagnosis of pregnancy was confirmed by detection of the embryo heart beat by day 12. Embryo characteristics were ultrasonographically measurable between days 9 and 16.

Increased numbers of mice housed per cage (that is, increased housing density) is seen as 1 way to reduce the costs of conducting biomedical research. Current empirically derived guidelines are based on the area provided per mouse depending on body weight as documented in the Guide for the Care and Use of Laboratory Animals. The current study aimed to provide a more scientific basis for housing density by examining the response of C57BL/6J and BALB/cJ mice to increased housing density from weaning to 5 mo of age, to determine those parameters most useful for future larger-scale studies. A wide range of phenotypic characteristics—including growth rate, body composition, hematology, serum biochemistry, hormone and metabolite measurements, in-cage telemetry, behavior, and cage microenvironment—was examined at various time points. The parameters showing greatest changes were: growth rate, which was significantly reduced in animals at the highest density; adrenal gland size, the proportion of adrenal cortex, and concentration of fecal corticosterone metabolites, all of which were increased at higher densities; and anxiety and barbering, which were more pronounced at higher densities. Cage microenvironment deteriorated with increasing density, but the increases in measured parameters were small, and their biologic impact, if any, was not apparent. The current findings indicate that mouse housing density can be increased 50% to 100% above the current recommendations (as floor area per mouse) with no or few apparent affects on mouse overall wellbeing. However, weight gain, fecal corticosterone metabolite levels, and barbering differed significantly with housing density and therefore are suggested as good measures of the response to alterations in housing.

Wild rodents are a potential source for pathogen introduction into laboratory animal research facilities. A study was designed to assess wild mice found at our institution by infectious disease surveillance. Wild white-footed mice (Peromyscus leucopus) were captured with live capture traps placed in areas in which wild mice had been reported in several animal facilities. Captured animals were euthanized by inhalation of CO₂, blood was collected by cardiocentesis (n = 10), and necropsy was performed (n = 8). Serum samples were negative for antibodies to mouse parvovirus (types 1 and 2), mouse minute virus, Sendai virus, pneumonia virus of mice, mouse hepatitis virus, Theiler murine encephalomyelitis virus, reovirus, rotavirus, lymphocytic choriomeningitis virus, mouse adenovirus, ectromelia virus, K virus, cilia-associated respiratory bacillus, and Mycoplasma pulmonis. Of the 8 animals that were necropsied, pelt and cecal examinations were negative for ectoparasites and pinworms, respectively. Histopathologic examination of brain, heart, lungs, liver, kidney, spleen, stomach, and small intestine revealed bacteria morphologically compatible with Helicobacter spp. in the cecal and colonic glands and occasionally in the gastric lumen and pits. Mesenteric lymph nodes and feces from 8 of the animals were submitted for PCR analysis for the detection of mouse parvovirus, mouse minute virus, mouse hepatitis virus, and Helicobacter spp.; 7 of the samples were PCR-positive for Helicobacter spp. At this time, wild mice found in our animal facilities do not appear to be a significant source of common laboratory mouse viral pathogens. However, they are a potential source of Helicobacter infections.

A shipment of New Zealand white rabbits was infested with Leporacarus gibbus, a rabbit fur mite. This study compared the effectiveness of selamectin with that of imidocloprid plus permethrin in eliminating the mite infestation. Rabbits were divided into 2 groups, and either selamectin or imidocloprid plus permethrin was applied topically. Visual and microscopic examinations were performed on days 0, 1, 2, 3, 4, 5, 6, 13, and 27 for 5 sites (the left and right gluteal areas, neck, ventral tail, and abdomen). Mean percentage effectiveness for each treatment was calculated for each time point. Positive and negative predictive value, sensitivity, and specificity of visual examination were determined relative to microscopic assessment. In addition, location prevalence for the mites was determined. Both treatments were 100% effective by day 13, but selamectin was 100% effective by day 3. The positive predictive value of visual examination was 96%, its negative predictive value was 86%, sensitivity was 75%, and specificity was 98%. Parasite burden was most prevalent on the right and left gluteal areas. We conclude that although both imidocloprid plus permethrin and selamectin were effective against L. gibbus, treatment with selamectin more rapidly eliminated the infestation.

The frequency at which mouse cages are changed has important implications for the animals, animal care personnel, and facility managers. The objective of this study was to determine how bedding volume and the interval between changes affect microenvironmental conditions, health, and behavior of mice housed in individually ventilated cages (IVC). A total of 15 cages (n = 5 cages per bedding volume) housing ICR female mice (n = 5 animals per cage) were monitored for 17 d. Parameters monitored included clinical evaluation of each animal, appearance of the cage, fecal corticosterone levels, bedding weight, and mouse mass. Atmospheric analysis was performed daily to determine intracage ammonia cage humidity and temperature on a daily basis. Mice were videotaped for 10 min on days 1, 8, and 15, and videos were analyzed for abnormal behaviors. On day 17, 1 mouse from each cage was euthanized, and bronchoalveolar lavage was performed. Statistical differences in parameters were most often noted between low- and high-volume bedding groups. Correlation between visual appearance and actual intracage environmental conditions and mouse health and behavior at specific time points indicated cages that appear dirty to most observers did not have measurably adverse effects on the animals for any of the many parameters evaluated in this study. This study demonstrated that a 2-wk interval between cage changes for ICR female mice housed in IVC caging (with approximately 90 air changes per hour) and aspen chip bedding did not significantly affect measures of animal well-being in this study. This lack of effect occurred despite the appearance of excessive soiling by the 2-wk time point.

Contemporary rodent caging and equipment often are sterilized by steam autoclaves prior to use in facilities. This work assessed the microenvironment of unoccupied static mouse cages after steam sterilization to determine when internal temperatures had cooled to levels appropriate for rodent housing. Polycarbonate static cages containing food and corncob bedding were stacked (10 rows × 7 columns) in duplicate (front and back; n = 140 cages) on a storage truck and autoclaved to 249 °F (121 °C). Cages (n = 6) were assessed to represent top, middle, and bottom rows and edges of columns. After cage sterilization, hygrothermometers were placed in cages to measure internal cage temperature (IT), bedding temperature (BT), and cage humidity (CH) every 10 min for 150 min. At time 0, there were no significant differences in averaged temperatures or humidity across cage locations: IT, 95.9 °F; BT, 109.8 °F; and CH, 84.1%. Over time, significant positional effects occurred. Whereas IT and BT for cages in the center row cooled more slowly than those on the bottom row, CH in top row cages decreased more quickly compared with other cages. After 150 min, the average measures overall were IT, 75.8 °F; BT, 77.9 °F; and CH, 82.4%. Comparison of the overall measures at 150 min with those of cages cooled overnight (IT, 72.4 °F; BT, 71.0 °F; and CH, 49%) and cages housing mice (IT, 72.2 °F; BT, 70.7 °F; and CH, 82%) indicated that a poststerilization cooling period of greater than 2.5 h was necessary to achieve permissible rodent housing conditions at our institution, particularly with corncob bedding autoclaved within the cage.

Our objective was to determine whether an adult ferret can be intubated as many as 10 times per training session without resulting in trauma to the upper airway. In this program, 8 male ferrets rotated through intubation laboratories, limiting the use of each animal to once every 3 mo. Animals were examined by the veterinary staff after intubations to assess for trauma to upper airway tissue. Each examination was given a trauma grade of 0 for no visible signs of trauma, 1 if erythema of the larynx was present, 2 if visible excoriation of the mucus membranes was present, and 3 if bleeding (frank hemorrhage) was observed. The number of intubation attempts was restricted to 10 per animal per training session. A total of 170 intubations were completed on the ferrets during a 12-mo period. The average number of intubations per laboratory was 8.1 intubations per ferret. In addition, 1.8% of the intubations resulted in erythema (score, 1) after training, and 0.6% of the intubations resulted in excoriation (score, 2). Frank hemorrhage (score, 3) was not noted. The overall percentage of intubations resulting in any trauma during a training session was 0.02%. None of the animals have experienced any major complications to date. This ongoing training program has been used to teach neonatal intubation skills to emergency medicine residents for the past 12 mo. Ensuring the health and safety of the ferrets was paramount. Our results suggest that as many as 10 intubation attempts per session can be performed safely on each ferret without causing excessive trauma.

Despite the progressively increasing use of zebrafish (Danio rerio) in research, the most humane method of euthanasia for these fish has not been determined. Contemporary guidance documents state that hypothermia is an unacceptable method of euthanasia. The goal of this study was to compare rapid cooling and tricaine methanesulfonate (MS222) for zebrafish euthanasia. Zebrafish (n = 46) were euthanized by immersion in either an ice-water (4 °C or less) bath or unbuffered MS222 solution (250 mg/L; 25 to 30 °C). Another cohort (n = 10) was exposed to buffered MS222 to determine whether the acidity of the water alone caused distress. The times from exposure until the animals became unable to swim, right themselves, and death were measured, and signs of distress were recorded. Fish then were placed in a 'recovery tank' of system water to verify that recovery did not occur. Tissues were examined histologically. The mean time for euthanasia was longer and the number of fish exhibiting signs of distress was greater for fish exposed to MS222 than those exposed to chilled water. In addition, 4 of the 23 fish exposed to MS222 regained consciousness in the recovery tank, whereas none of 23 fish exposed to chilled water recovered. No histopathologic changes or evidence of ice crystal formation were seen in either group. In light of the faster time to death and fewer signs of distress in zebrafish euthanized by rapid cooling, we advocate this method as a humane veterinary practice.

Female Sprague–Dawley rats (n = 100; age, 3 wk) were fed diets that included a vitamin premix and either albumin or milk powder. Rats fed the albumin diet gained weight more slowly than did the other group. Between 19 and 28 wk of being fed the albumin diet, 12 rats died of bacterial cystitis and pyelonephritis. In addition, 2 more rats from the same dietary group developed peritonitis after ovariohysterectomy. Examination of the 44 rats fed the albumin diet that completed the 34-wk experiment revealed pyelonephritis in 68%, cystitis in 66%, urolithiasis in 27%, and nephrolithiasis in 5%. Squamous metaplasia of the transitional epithelium was present in all 44 rats, although other epithelia were histologically normal. Vitamin A deficiency was diagnosed after analyses of blood and liver samples. Analysis of the vitamin premix revealed approximately 25% of the expected amount of vitamin A. Because the milk powder contained sufficient vitamin A, deficiency did not occur in rats fed the milk powder diet. The major consequences of vitamin A deficiency in the rats were squamous metaplasia, bacterial infection, and calculus formation within the urinary tract. This report illustrates the importance of careful formulation and storage of vitamin premixes used in experimental diets. Vitamin A deficiency should be considered in rats with decreased weight gain and urinary tract disease even if ocular lesions are not present.

An 8-y-old gilt was evaluated after the onset of hemorrhagic perineal discharge. Uterine adenocarcinoma with metastases to the lungs and regional lymph nodes was diagnosed at necropsy. Tumor cells lacked expression of estrogen receptor α and progesterone receptor. This case represents the first reported uterine adenocarcinoma in a research pig and the first swine uterine neoplasia in which steroid hormone receptor expression was evaluated.