Laboratory mouse housing conditions can be improved using common environmental enrichment without compromising data

Abstract

Animal welfare requires the adequate housing of animals to ensure health and well-being. The application of environmental enrichment is a way to improve the well-being of laboratory animals. However, it is important to know whether these enrichment items can be incorporated in experimental mouse husbandry without creating a divide between past and future experimental results. Previous small-scale studies have been inconsistent throughout the literature, and it is not yet completely understood whether and how enrichment might endanger comparability of results of scientific experiments. Here, we measured the effect on means and variability of 164 physiological parameters in 3 conditions: with nesting material with or without a shelter, comparing these 2 conditions to a "barren" regime without any enrichments. We studied a total of 360 mice from each of 2 mouse strains (C57BL/6NTac and DBA/2NCrl) and both sexes for each of the 3 conditions. Our study indicates that enrichment affects the mean values of some of the 164 parameters with no consistent effects on variability. However, the influence of enrichment appears negligible compared to the effects of other influencing factors. Therefore, nesting material and shelters may be used to improve animal welfare without impairment of experimental outcome or loss of comparability to previous data collected under barren housing conditions.

Fig 1. Workflow for the screens of this study.

DEXA, dual-energy x-ray absorptiometry; IpGTT, intraperitoneal glucose tolerance test; NMR, nuclear magnetic resonance; PPI, pre-pulse inhibition; SHIRPA, Smithkline Beecham, MRC Harwell, Imperial College, the Royal London Hospital Phenotype Assessment.

Fig 2. Influence of main factors (enrichment, sex, cohort) on mean of parameters in 2 mouse strains.

(A) The heatmap shows the results for all metric parameters (rows) and influencing factors and their double interactions (columns). The B6 and D2 data are on the left and right of each column, respectively. The influence of main factors was evaluated using linear models. A variable selection using the BIC was performed. Reference categories are enrichment none, sex female, and the first cohort. The differences between means of parameters are expressed as color-coded estimators β of the respective model in percent of the intercept of the model. The estimator β describes the change induced by the respective factors. The intercept is the mean of the parameter of the reference group and is set as 100%. Grey shade: variable selection using the BIC yielded nonrelevant influencing factor, thus no effect can be assumed; violet shade: factor is relevant for the model, i.e., mean is increased compared to reference group; yellow shade: factor is relevant for the model, i.e., mean is decreased compared to reference group; strength of color reflects size of difference. “X” marks parameters that were not analyzed. (B) Color grading scheme: the color grading illustrates the change in percent of the intercept; the strength of color reflects the size of difference. ABR, auditory brain stem response; ALAT/GPT, Alanine aminotransferase/Glutamat pyruvat transaminase; ALP, Alkaline phosphatase; ASAT/GOT, Aspartate aminotransferase/Glutamat oxalacetat transaminase; ASR, acoustic startle response; AU400, name of clinical chemistry analyzer; AUC, area under the curve; BIC, Bayesian Information Criterion; BN, background noise; CNPG3, substrate of test reaction (2-Chloro-4-Nitrophenyl-α-D-Maltotriosid); DEXA, dual-energy x-ray absorptiometry; HR, heart rate; HRV, heart rate variability; IgE, immunoglobulin E; IpGTT, intraperitoneal glucose tolerance test; LDH, Lactat-dehydrogenase; LVIDd, left ventricular end-diastolic internal diameter; LVIDs, left ventricular end-systolic internal diameter; LVPW, left ventricular posterior wall; P1-P4, startle amplitude following pulse of 67 (P1), 69 (P2), 73 (P3), 81 (P4) dB; PP (67, 69, 73, 81), startle amplitude and PPI following startle pulse (110 dB) with preceding pre-pulse of 67, 69, 73, 81 dB; PPI, pre-pulse inhibition; qNMR, quantitative nuclear magnetic resonance; QTc, corrected QT; R1, region analyzed (whole body excluding the skull); SHIRPA, Smithkline Beecham, MRC Harwell, Imperial College, the Royal London Hospital Phenotype Assessment; ST 110, acoustic startle response at 110 dB.

Fig 3. Smoothed histogram showing the influence of main factors (enrichment, sex, cohort) on means in 2 strains.

The influence is expressed as differences in percent of intercept for B6 (a) and D2 (b) mice. The intercept is the mean of the parameter of the reference group. The area under the curve is proportional to number of parameters, for which the variable selection using the BIC yielded relevance (yellow and violet shading in Fig 2); nonselected parameters (grey in the heatmap of Fig 2) are not included (number of selected factors for B6 [a]: enrichment nest: 69; enrichment double: 69; sex male: 118; cohort 2: 152; cohort 3: 152; number of selected factors for D2 [b]: enrichment nest: 88; enrichment double: 88; sex male: 144; cohort 2: 135; cohort 3: 135). The height of the histogram is calculated as density multiplied by total number of parameters and is proportional to number of parameters that fall in the respective bin of the x-axis.

Fig 4. Raw data of selected parameters on the background of the biological range for B6.

Raw data of the 4 main parameters of the open field test: (A) “distance traveled total,” (B) “number of rears total,” (C) “percent center distance total,” and (D) “center permanence time” as box- and whisker-plots. The box represents 25th percentile, median, and 75th percentile; the length of whiskers is maximally the 1.5-fold interquartile range but is determined by the last value within this range. All individual values are shown for each experimental group (“con,” nest, double) and every cohort (1, 2, 3) for female and male mice in the upper and lower plot, respectively, for each selected parameter (A–D). The range in the background gives 1 SD (dark shading) and 2 SD (bright shading) of >200 reference B6 female (red) and male (blue) mice. The reference mice were same-aged wild-type control mice from other phenotyping projects of the GMC and were measured within the same timespan as the mice used in this project. “con”, control; GMC, German Mouse Clinic.

Fig 5. Influence of main factors (enrichment, sex, cohort) on bootstrapped CVs in 2 strains.

The heatmap shows the results for all metric parameters (rows) and influencing factors (columns) for B6 (left column) and D2 (right column). Reference categories are enrichment none, sex female, and the first cohort. Results of the bootstrap method are summarized by computed confidence intervals for estimators (β), and bootstrapped CVs are then classified into the following 3 categories: confidence interval for β includes 0, thus no effect is assumed (grey); confidence interval for β is greater than 0, i.e., bootstrapped CVs are increased compared to reference group (violet); confidence interval for β is below 0, i.e., bootstrapped CVs are decreased compared to reference group (yellow). “X” mark parameters that were not analyzed. ABR, auditory brain stem response; ALAT/GPT, Alanine aminotransferase/Glutamat pyruvat transaminase; ALP, Alkaline phosphatase; ASAT/GOT, Aspartate aminotransferase/Glutamat oxalacetat transaminase; ASR, acoustic startle response; AU400, name of clinical chemistry analyzer; AUC, area under the curve; BIC, Bayesian Information Criterion; BN, background noise; CNPG3, substrate of test reaction (2-Chloro-4-Nitrophenyl-α-D-Maltotriosid); CV, coefficient of variation; DEXA, dual-energy x-ray absorptiometry; HR, heart rate; HRV, heart rate variability; IgE, immunoglobulin E; IpGTT, intraperitoneal glucose tolerance test; LDH, Lactat-dehydrogenase; LVIDd, left ventricular end-diastolic internal diameter; LVIDs, left ventricular end-systolic internal diameter; LVPW, left ventricular posterior wall; P1-P4, startle amplitude following pulse of 67 (P1), 69 (P2), 73 (P3), 81 (P4) dB; PP (67, 69, 73, 81), startle amplitude and PPI following startle pulse (110 dB) with preceding pre-pulse of 67, 69, 73, 81 dB; PPI, pre-pulse inhibition; qNMR, quantitative nuclear magnetic resonance; QTc, corrected QT; R1, region analyzed (whole body excluding the skull); SHIRPA, Smithkline Beecham, MRC Harwell, Imperial College, the Royal London Hospital Phenotype Assessment; ST 110, acoustic startle response at 110 dB.