The Future Is Not Bright: Evaluation of Rat Preferences for Color and Intensity of Light.

Light is a key factor influencing the welfare of laboratory rodents, but little is known about their optimal lighting condition. It i common knowledge that rats prefer dim light, so bright light is mitigated with red-tinted shelters or cages, which alter both the color and intensity of light. Because both aspects are altered, the contribution of each feature to rodent preference is unknown. Further, it is unknown if this preference is influenced by previous experience. We hypothesized that rats would prefer lower light intensity and that their preferences would be influenced by their housing environment. Breeder pairs of rats were randomly separated into four treatments groups: red 200 lux, red 25 lux, clear 200 lux, and clear 25 lux. The breeders' offspring were tested three times in an apparatus that offered access to each environment, and their preferences were analyzed. Generally, the rats preferred the lower-lux environments and showed no color preference. However, the rats from the clear, 200 lux cages, preferred clear caging and only showed a preference for 25 lux conditions during the second and third preference tests. These results suggest that the light intensity, more than color, should be considered when designing rodent housing and testing facilities.

Assessing the effect of compounds from plantar foot sweat, nesting material, and urine on social behavior in male mice, Mus musculus.

Home cage aggression causes poor welfare in male laboratory mice and reduces data quality. One of the few proven strategies to reduce aggression involves preserving used nesting material at cage change. Volatile organic compounds from the nesting material and several body fluids not only correlate with less home cage aggression, but with more affiliative allo-grooming behavior. To date, these compounds have not been tested for a direct influence on male mouse social behavior. This study aimed to determine if 4 previously identified volatile compounds impact home cage interactions. A factorial design was used with cages equally split between C57BL/6N and SJL male mice (N = 40). Treatments were randomly assigned across cages and administered by spraying one compound solution on each cage's nesting material. Treatments were refreshed after day 3 and during cage change on day 7. Home cage social behavior was observed throughout the study week and immediately after cage change. Several hours after cage change, feces were collected from individual mice to measure corticosterone metabolites as an index of social stress. Wound severity was also assessed after euthanasia. Measures were analyzed with mixed models. Compound treatments did not impact most study measures. For behavior, SJL mice performed more aggression and submission, and C57BL/6N mice performed more allo-grooming. Wound severity was highest in the posterior region of both strains, and the middle back region of C57BL/6N mice. Posterior wounding also increased with more observed aggression. Corticosterone metabolites were higher in C57BL/6N mice and in mice treated with 3,4-dimethyl-1,2-cyclopentanedione with more wounding. These data confirm previous strain patterns in social behavior and further validates wound assessment as a measure of escalated aggression. The lack of observed treatment effects could be due to limitations in the compound administration procedure and/or the previous correlation study, which is further discussed.

Evaluation of Ambient Sound, Vibration, and Light in Rodent Housing Rooms.

Excessive sound, vibration, and light are detrimental to rodent welfare, yet these parameters are rarely recorded in vivaria. Whether housing environments exceed the suggested thresholds and which specific factors may alter these parameters is generally unknown. The goal of this study was to determine how environmental factors may alter sound, vibration, and light at the room and cage levels. Measurements were made using an ultrasonic microphone, accelerometer, and light sensor. Measurement sites were 1) in open air at a central location in 64 rooms located in 9 buildings, and 2) inside an empty mouse or rat cage containing chow, water, and bedding and located on an animal transfer station (n = 51) or housing rack (n = 102). Information collected for each transfer station and rack measurement included the year of manufacture, the species on the rack, and the number of cages on the rack. For each location, a baseline measurement was taken with the transfer station turned off, followed by another measurement after the transfer station was turned on. In general, many factors influenced ambient sound, vibration, and light, indicating that values are not uniform across rodent rooms in the same institution or across cages in a single room. Sound peaks capable of startling rodents were measured in association with hallway ultrasonic motion sensors and during cage change. Vibration and light intensity were generally low when cages were located on the rack. In contrast, active transfer stations had more vibration and light intensity, reaching levels that were potentially stressful for rodents. These data reflect the ambient sound, vibration, and light that rodents experience during normal facility operations. These patterns may extend to other locations, but given the variability in all parameters, the data highlight the need for institutions to conduct their own monitoring.

Protein profiles from used nesting material, saliva, and urine correspond with social behavior in group housed male mice, Mus musculus.

Current understanding of how odors impact intra-sex social behavior is based on those that increase intermale aggression. Yet, odors are often promoted to reduce fighting among male laboratory mice. It has been shown that a cage of male mice contains many proteins used for identification purposes. However, it is unknown if these proteins relate to social behavior or if they are uniformly produced across strains. This study aimed to compare proteomes from used nesting material and three sources (sweat, saliva, and urine) from three strains and compare levels of known protein odors with rates of social behavior. Used nesting material samples from each cage were analyzed using LC-MS/MS. Sweat, saliva, and urine samples from each cage's dominant and subordinate mouse were also analyzed. Proteomes were assessed using principal component analyses and compared to behavior by calculating correlation coefficients between PC scores and behavior proportions. Twenty-one proteins from nesting material either correlated with affiliative behavior or negatively correlated with aggression. Notably, proteins from the major urinary protein family, odorant binding protein family, and secretoglobin family displayed at least one of these patterns, making them candidates for future work. These findings provide preliminary information about how proteins can influence male mouse behavior. SIGNIFICANCE: Research on how olfactory signals influence same sex social behavior is primarily limited to those that promote intermale aggression. However, exploring how olfaction modulates a more diverse behavioral repertoire will improve our foundational understanding of this sensory modality. In this proteome analysis we identified a short list of protein signals that correspond to lower rates of aggression and higher rates of socio-positive behavior. While this study is only correlational, it sets a foundation for future work that can identify protein signals that directly influence social behavior and potentially identify new murine pheromones.

Home cage measures of Alzheimer's disease in the rTg4510 mouse model.

Alzheimer's disease affects an array of activities in patients' daily lives but measures other than memory are rarely evaluated in animal models. Home cage behavior, however, may provide an opportunity to back translate a variety of measures seen in human disease progression to animal models, providing external and face validity. The aim of this study was to evaluate if home cage measures could indicate disease in the rTg4510 mouse model. We hypothesized that sleep, nesting, and smell discrimination would be altered in mutant mice. Thirty-two transgenic mice were used in a Latin square design of four genotypes x both sexes x two diets. Half the mice received a doxycycline diet to suppress tauopathy and evaluate tau severity on various measures. At 8-, 12-, and 16-weeks old, 24 h activity/sleep patterns, nest complexity, and odor discrimination were measured. After 16-weeks, tau concentration in the brain was quantified. Mutant mice had increased tau concentration in brain tissue, but it was reduced by the doxycycline diet. However, only nest complexity was different between mutant mice and controls. Overall, tauopathy in rTg4510 mice does seem to affect these commonly observed symptoms in human patients. However, while running this study, a report showed that the rTg4510 mutant phenotype is not caused by the mutation itself, but confounding factors from transgene insertion. Combined with report findings and our data, the rTg4510 model may not be an ideal model for all aspects of human Alzheimer's disease.

Who's the Boss? Assessing Convergent Validity of Aggression Based Dominance Measures in Male Laboratory Mice, Mus Musculus.

Aggression among group housed male mice continues to challenge laboratory animal researchers because mitigation strategies are generally applied at the cage level without a good understanding of how it affects the dominance hierarchy. Aggression within a group is typically displayed by the dominant mouse targeting lower ranking subordinates; thus, the strategies for preventing aggression may be more successful if applied specifically to the dominant mouse. Unfortunately, dominance rank is often not assessed because of time intensive observations or tests. Several correlates of dominance status have been identified, but none have been directly compared to home cage behavior in standard housing. This study assessed the convergent validity of three dominance correlates (urinary darcin, tube test score, preputial gland to body length ratio) with wound severity and rankings based on home cage behavior, using factor analysis. Discriminant validity with open field measures was assessed to determine if tube test scores are independent of anxiety. Cages were equally split between SJL and albino C57BL/6 strains and group sizes of 3 or 5 (N = 24). Home cage behavior was observed during the first week, and dominance measures were recorded over the second. After controlling for strain and group size, darcin and preputial ratio had strong loadings on the same factor, which was a significant predictor of home cage ranking showing strong convergent validity. Tube test scores were not significantly impacted by open field data, showing discriminant validity. Social network analysis revealed that despotic power structures were prevalent, aggressors were typically more active and rested away from cage mates, and the amount of social investigation and aggression performed by an individual were highly correlated. Data from this study show that darcin and preputial ratio are representative of home cage aggression and provide further insight into individual behavior patterns in group housed male mice.

Compounds from plantar foot sweat, nesting material, and urine show strain patterns associated with agonistic and affiliative behaviors in group housed male mice, Mus musculus.

Excessive home cage aggression often results in severe injury and subsequent premature euthanasia of male laboratory mice. Aggression can be reduced by transferring used nesting material during cage cleaning, which is thought to contain aggression appeasing odors from the plantar sweat glands. However, neither the composition of plantar sweat nor the deposits on used nesting material have been evaluated. The aims of this study were to (1) identify and quantify volatile compounds deposited in the nest site and (2) determine if nest and sweat compounds correlate with social behavior. Home cage aggression and affiliative behavior were evaluated in 3 strains: SJL, C57BL/6N, and A/J. Individual social rank was assessed via the tube test, because ranking may influence compound levels. Sweat and urine from the dominant and subordinate mouse in each cage, plus cage level nest samples were analyzed for volatile compound content using gas chromatography-mass spectrometry. Behavior data and odors from the nest, sweat, and urine were statistically analyzed with separate principal component analyses (PCA). Significant components, from each sample analysis, and strain were run in mixed models to test if odors were associated with behavior. Aggressive and affiliative behaviors were primarily impacted by strain. However, compound PCs were also impacted by strain, showing that strain accounts for any relationship between odors and behavior. C57BL/6N cages displayed the most allo-grooming behavior and had high scores on sweat PC1. SJL cages displayed the most aggression, with high scores on urine PC2 and low scores on nest PC1. These data show that certain compounds in nesting material, urine, and sweat display strain specific patterns which match strain specific behavior patterns. These results provide preliminary information about the connection between home cage compounds and behavior. Salient compounds will be candidates for future controlled studies to determine their direct effect on mouse social behavior.

Proteome characterization of used nesting material and potential protein sources from group housed male mice, Mus musculus.

Laboratory mice (Mus musculus) communicate a variety of social messages through olfactory cues and it is often speculated that these cues are preserved in nesting material. Based on these speculations, a growing number of husbandry recommendations support preserving used nests at cage cleaning to maintain familiar odors in the new cage. However, the content of used nesting material has never been chemically analyzed. Here we present the first comprehensive proteome profile of used nesting material. Nests from cages of group housed male mice contain a variety of proteins that primarily originate from saliva, plantar sweat, and urine sources. Most notably, a large proportion of proteins found in used nesting material belong to major urinary protein ("MUP") and odorant binding protein ("OBP") families. Both protein families send messages about individual identity and bind volatile compounds that further contribute to identity cues. Overall, this data supports current recommendations to preserve used nesting material at cage cleaning to maintain odor familiarity.