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Establishing normal plasma and 24-hour urinary biochemistry ranges in C3H, BALB/c and C57BL/6J mice following acclimatization in metabolic cages Michael J Stechman, Bushra N Ahmad, Nellie Y Loh, Anita A C Reed, Michelle Stewart, Sara Wells, Tertius Hough, Liz Bentley, Roger D Cox, Steve D M Brown and Rajesh V Thakker Lab Anim 2010 44: 218 DOI: 10.1258/la.2010.009128 The online version of this article can be found at: http://lan.sagepub.com/content/44/3/218

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Original Article Establishing normal plasma and 24-hour urinary biochemistry ranges in C3H, BALB/c and C57BL/6J mice following acclimatization in metabolic cages Michael J Stechman1, Bushra N Ahmad1, Nellie Y Loh1, Anita A C Reed1, Michelle Stewart2, Sara Wells2, Tertius Hough2, Liz Bentley3, Roger D Cox3, Steve D M Brown3 and Rajesh V Thakker1 1

Academic Endocrine Unit, Nuffield Department of Clinical Medicine, University of Oxford, Oxford Centre for Diabetes, Endocrinology and Metabolism (OCDEM), Churchill Hospital, Oxford OX3 7LJ; 2Mary Lyon Centre; 3Mammalian Genetics Unit, Medical Research Council, Harwell, Oxfordshire, UK Corresponding author: Professor Rajesh V Thakker. Email: [email protected]

Abstract Physiological studies of mice are facilitated by normal plasma and 24-hour urinary reference ranges, but variability of these parameters may increase due to stress that is induced by housing in metabolic cages. We assessed daily weight, food and water intake, urine volume and final day measurements of the following: plasma sodium, potassium, chloride, urea, creatinine, calcium, phosphate, alkaline phosphatase, albumin, cholesterol and glucose; and urinary sodium, potassium, calcium, phosphate, glucose and protein in 24- to 30-week-old C3H/HeH, BALB/cAnNCrl and C57BL/6J mice. Between 15 and 20 mice of each sex from all three strains were individually housed in metabolic cages with ad libitum feeding for up to seven days. Acclimatization was evaluated using general linear modelling for repeated measures and comparison of biochemical data was by unpaired t-test and analysis of variance (SPSS version 12.0.1). Following an initial 5– 10% fall in body weight, daily dietary intake, urinary output and weight in all three strains reached stable values after 3 –4 days of confinement. Significant differences in plasma glucose, cholesterol, urea, chloride, calcium and albumin, and urinary glucose, sodium, phosphate, calcium and protein were observed between strains and genders. Thus, these results provide normal reference values for plasma and urinary biochemistry in three strains housed in metabolic cages and demonstrate that 3– 4 days are required to reach equilibrium in metabolic cage studies. These variations due to strain and gender have significant implications for selecting the appropriate strain upon which to breed genetically-altered models of metabolic and renal disease.

Keywords: Metabolic cages, inbred laboratory mice, adaptation, refinement, renal function Laboratory Animals 2010; 44: 218– 225. DOI: 10.1258/la.2010.009128

Genetically-altered inbred laboratory mice are commonly used to investigate models of human disease and may be derived by either homologous recombination, transgenic or chemical mutagenesis routes.1 Investigation of metabolic and endocrine disorders such as bone disease, renal stones, renal failure, diabetes and hyperlipidaemia, which increase in frequency with age, may require specific measurement of plasma and urinary electrolytes by use of metabolic cages. Variability is an unavoidable problem when measuring these parameters in experimental animals and this may be due to fixed effects such as sex, age, strain, diet and environment, or random effects such as intra- and interindividual variation, social hierarchy, aggression and contamination of specimens.2 Controlling variability is therefore an Laboratory Animals 2010; 44: 218 –225

important element in the design and execution of metabolic cage experiments, particularly if Type II statistical errors are to be avoided. Use of metabolic cages also relies upon the incorrect assumption that mouse behaviour and physiology are not altered by solitary housing in a new environment. Measures that might reduce such variability, and hence optimize results, would therefore represent an important refinement to these experiments and potentially reduce the number of animals needed for such studies. Fixed effects can be managed by use of control groups matched for sex, age, strain and for diet and cage conditions. The inbred mouse has also permitted some of the variation between individuals to be diminished due to the genetic homogeneity of littermates of a particular age, sex and strain.

Stechman et al. Reference normal plasma and urinary ranges

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However, there are reports that solitary housing is stressful to rodents3,4 and that social contact is preferred when animals are given the choice.5 Furthermore, environment, particularly if lacking in cage bedding or enrichment, has also been shown to increase biochemical markers of stress6 and housing and handling of mice may affect plasma biochemical and haematological values.7 Physiological changes, in keeping with stress, have also been reported; mice placed in metabolic cages with continuous cardiovascular monitoring exhibit transient increases in blood pressure and persistent tachycardia when studied for 48 h despite previous habituation8 and rats placed in metabolic cages exhibit significant changes in renal function.9 Thus, solitary housing and change of environment exert important effects upon rodent behaviour and physiology that will affect cardiovascular and renal functions after placement in metabolic cages. We hypothesized that solitary housing in a metabolic cage would lead to changes in feeding habits and water conservation secondary to stress, which would change as the animal became acclimatized to the foreign environment. To assess acclimatization, we singly housed males and females from three of the most commonly used inbred mouse strains in metabolic cages for up to seven days. Weight, water and chow consumption and daily urine volume were chosen as markers to assess adjustment to re-housing. The aims of this study were to define the period of time required for stabilization of weight, feeding habits and urine output, and to obtain reference values for a range of parameters of metabolic functions that would facilitate studies of models of renal, bone, hyperlipidaemic and hyperglycaemic disorders, which are often associated with the ageing process.

Animals, materials and methods Animals

disorders that increase with ageing. The mice (between 15 and 20 in each group) were individually housed in metabolic cages (Cat No. 3600M021, Tecniplast, Kettering, UK) that had a floor area of 200 cm2 and the dimensions of length ¼ 32 cm, width ¼ 25 cm and height ¼ 36.5 cm. The mice were fed ad libitum on water (10–12 ppm chlorine) and powdered chow (RM3, Special Diet Services) for up to seven days, and were inspected twice daily. Autoclavable red transparent igloos (Datesand Ltd, Manchester, UK) were provided as environmental enrichment. On each day of the study, animal weight, 24 h water and chow consumption, and 24 h urine volume were recorded and urine specimens were taken for analysis. Blood samples were collected on the final day of the study. Prior to analysis, all measurements of food/water intake and urine volume were expressed as values per 100 grams of body weight. Blood sample collection Whole blood samples were obtained by internal jugular venous bleeding, which was performed immediately after a terminal intraperitoneal injection of Euthatal (200 mg/mL sodium pentobarbital, Larkmead Veterinary, Didcot, UK). Samples were collected by capillary action into lithium heparin-coated paediatric Microvettew tubes (Sarstedt Ltd, Leicester, UK) and placed on ice. Each whole blood sample tube was centrifuged at 800 g for 10 min at 48C to yield approximately 125 mL of plasma which was stored at 2208C. Samples were analysed for sodium, potassium, chloride, urea, creatinine, calcium, phosphate, alkaline phosphatase activity, albumin, cholesterol and glucose using an Olympus AU400w analyser as described previously.10 Plasma calcium was adjusted for albumin concentration using the formula: plasma Ca2þ (mmol/L) 2 [( plasma albumin [g/L] 2 30) 0.017], as described previously.11

Inbred laboratory mice of the C57BL/6J (B6J) strain were obtained from the Jackson Laboratory (Bar Harbor, ME, USA) and BALB/cAnNCrl (CCrl) mice from Charles River (Kent, UK), and colonies bred at the Mary Lyon Centre (Harwell, UK). C3H/HeH (C3H) mice were an established substrain bred at the Mary Lyon Centre (Harwell, UK). Ethical approval for this study was obtained as part of the application for the UK Home Office Project Licence and all mice were kept in accordance with UK Home Office welfare guidance and Home Office project licence restrictions. Mice were maintained in controlled light (12 h light and dark cycle), temperature (21 + 28C) and humidity (55 + 10%). Mice had free access to water (10– 12 ppm [ parts per million] chlorine) and were fed ad libitum on a commercial diet (5.3% fat [corn oil], 21.2% protein, 57.4% carbohydrate, 4.6% fibre; Rat and Mouse Diet No. 3 [RM3], Special Diet Services, Essex, UK). All mice were treatment naive and socially housed in groups of 2– 5 mice until the start of study.

Each sample was centrifuged at 800 g for 10 min at 48C and 160 mL aliquots stored at 2208C. Each was diluted twofold with distilled water and analysed for sodium, potassium, calcium, phosphate, glucose and total protein using an Olympus AU400w analyser as described previously.11 The resultant values were doubled to provide the actual value. In order to control for animal size and creatinine clearance, the concentrations of urinary sodium, potassium, calcium and phosphate in mmol/L were expressed as a ratio to the concentration of urinary creatinine in mmol/L. Creatinine clearance in mL/min/kg was calculated using the formula UV/P 1/1440, where U is the urinary concentration of creatinine, V is the 24 h urinary volume in mL, P is the plasma concentration of creatinine in mmol and 1440 is the number of minutes in a 24 h period. Values were then adjusted for the weight of each mouse in kg as described previously.12

Metabolic cage studies

Statistical analysis

Twenty-four- to 30-week-old mice were selected for study to facilitate the establishment of reference values for metabolic

The method of general linear models for repeated measures (SPSS version 12.0.1) was used to examine for changes in

Analysis of urine specimens

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body weight, dietary intake and urine output over time. If overall within-subjects variation was significant on univariate analysis, pair-wise comparisons were performed to determine the point at which each parameter reached steady state. Linear mixed models (SPSS version 12.0.1) were used to test for differences due to strain and sex over the time of each experiment. Unpaired Student’s t-test with a Bonferroni correction for multiple testing was used to compare parametric variables between sexes, derived by the formula a/n (where a ¼ 0.05, and n ¼ the total number of variables tested) and P , 0.0026 was considered statistically significant. Overall analysis of variance (ANOVA) was used to compare parametric variables between strains and post hoc comparisons by t-test were subject to an in-built Bonferroni correction set at 0.05 significance level (SPSS version 12.0.1).

Results Body weight, food and water intake and urinary output Following placement in metabolic cages, mice of both sexes from the three strains were observed to use the environmental enrichment by either sitting inside or on top of the autoclavable igloos. However, mice of both sexes from the three strains had a 5 –10% decrease in body weight over the first two days, after which weight stabilized but the mice did not regain weight to their prestudy values (Figure 1 and Table 1). This initial weight loss coincided with a gradual increase in chow and water intake (Figure 1). In a pilot study of five male and five female C3H and CCrl mice that were studied for seven days, mean chow and water intake per 100 g body weight, and 24 h urine volume per 100 g body weight increased after

Figure 1 Changes in weight, water and food intake and urinary output over a 5- or 7-day period in metabolic cages. Body weight on day 0 is the immediate prestudy weight and the values for each of the other measurements are those at the end of that 24 h period. Initial metabolic cage studies in two of the mouse strains (C3H/HeH – graphs [a]—[d] and BALB/cAnNCrl – graphs [e] –[h]) indicated that acclimatization with stable values for weight, water and food intake and urinary output had occurred by day 5, and studies in mice from the C57BL/6J strain (graphs [i]—[l]) were therefore performed for a 5-day period only. Data were collected from male and female C3H/HeH (a–d) and BALB/cAnNCrl (e–h) mice (n ¼ 5 for each group) and C57BL/6J mice (i –l) (n ¼ 20 for each group). Mean 24 h per 100 g body weight values for water and chow intake and mean 24 h urine volume per 100 g body weight increased from the starting value until the 3rd to 4th day (error bars indicate 95% confidence intervals)


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Table 1 Mean weights for mice placed in metabolic cages Strain Day

Daily measurement Weight†

0 1 2 3 4 5

% Weight loss by day 3 P valuea

BALB/cAnNCrld

C3H/HeH

C57BL/6J

Male

Female

Male

Female

Male

Female

41.5 39.4b 38.9 38.0 38.0 37.3 8.4 (n ¼ 15) ,0.0005

40.2 37.9b 37.8 37.2 37.5 36.9 7.5 (n ¼ 15) ,0.0005

33.5 31.9b 31.0c 30.7 30.1 29.9 8.4 (n ¼ 15) ,0.0005

24.8 24.1b 23.7c 23.6 23.7 23.4 4.8 (n ¼ 15) ,0.0005

35.1 33.1b 32.7 32.1 31.9 31.1 8.5 (n ¼ 19) ,0.0005

29.1 27.6b 27.6 27.4 27.1 26.7 5.8 (n ¼ 20) ,0.0005

Day 0 is the immediate prestudy weight, and the subsequent weights on each day are at the end of each 24 h period Mean weight in g a Overall P value obtained on univariate analysis for each group; bP , 0.0005 and cP , 0.005 on pair-wise comparison with previous day; dBALB/cAnNCrl mice weighed significantly less than both C3H/HeH and C57BL/6J mice, P ¼ 0.0014 †

day 1 until day 5, after which these values stabilized (Figures 1a –h). Further studies were therefore continued only up to day 5 in 10– 20 mice of each sex, from the C3H, CCrl and B6J strains (Figures 1i – l). The mean 24 h volume of water and grams of chow consumed per 100 g of body weight, and volume of urine output per 100 g of body weight for days 1 – 5 varied significantly within each group over the five days of the study (see Table 2). The water and chow intake and urinary output reached steady state by days 3 – 4, indicating that this was the period that the mice required for acclimatization. Male mice from all three strains required a longer period to acclimatize than the female mice, as was reflected by urinary output that reached a steady state in female mice at least one day earlier than in male mice for all three strains (Table 2). Following the period of acclimatization, the dietary intake per 100 g body weight, and 24 h urine volume per 100 g body weight varied significantly between the three strains

for all variables, except water intake in male mice (Table 3). Per gram of body weight, the food and water intake observed in C3H mice was significantly lower than mice of the CCrl and B6J strains; this was probably a reflection of the lower body weight of these strains (Table 1). Significant gender differences within strains were noted when the water and dietary intake of males and females of the CCrl and B6J strains were compared and also in the urinary output of C3H and B6J males and females (Table 3). Plasma biochemistry Significant differences in eight of the 12 measurements undertaken for plasma concentrations measured on day 5 of metabolic cage studies were observed between the strains and genders (Table 4). Thus, plasma urea concentrations in the C3H and the CCrl strains were significantly higher in males than females (P , 0.0005 for both) and

Table 2 Mean daily dietary intake and urinary volume in mice of each sex from three inbred strains Mean 24 h measurement Water intake

Overall P valuea Chow intake†

Overall P valuea Urinary output

Overall P valuea

Strain Day 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5

C3H/HeH Male 7.8 11.3b 12.0 12.2 15.2 ,0.0005 6.2 8.1b 9.1c 8.7 11.4 ,0.0005 3.1 4.4b 4.5 5.2 6.7 ,0.0005 (n ¼ 15)

BALB/cAnNCrl Female a

8.5 9.7c 10.5 11.5 12.2 ,0.0005 7.6 9.0b 10.1 9.4 10.9 ,0.0005 4.0 4.2 4.0 4.1 4.7 0.12 (n ¼ 15)

Male 8.4 13.0b 13.9 13.7 15.3 ,0.0005 7.6b 10.9b 12.5c 13.0 14.8 ,0.0005 2.0d 2.8b 3.8b 4.6b 4.9 ,0.0005 (n ¼ 15)

C57BL/6J Female a

15.2 16.3 16.5 17.5 14.9 0.089 12.3b 15.8b 16.8 17.4 16.5 ,0.0005 3.2 4.7b 5.8 5.7 5.2 ,0.0005 (n ¼ 15)

Male

Female

10.2 12.9b 14.6 13.1 13.3 0.008 6.2 10.9b 11.0 11.1 11.2 ,0.0005 5.3 6.3 7.5b 7.1 7.3 0.001 (n ¼ 19)

17.9a 20.5c 21.6 22.9 23.5 ,0.0005 11.0 14.6b 15.2 15.6 16.5 ,0.0005 6.3 7.5b 8.3 9.9 10.4 ,0.0005 (n ¼ 20)

mL/day/100 g body weight g/day/100 g body weight a Overall P value obtained on univariate analysis; bP , 0.001 and cP , 0.05 on pair-wise comparison with previous day a Water intake varied significantly between males and females of the three strains, P ¼ 0.0025; bchow intake varied significantly between BALB/cAnNCrl mice and mice of C3H/HeH and C57BL/6J strains (P ¼ 0.0023); durinary output in male BALB/c mice was significantly lower than males of the C3H/HeH and C57BL/6J strains (P , 0.0005) †

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Table 3 Normative values, mean (SD), for 24 h water and food intake, and urinary output in three inbred mouse strains after three days acclimatization in metabolic cages Strain

C3H

Sex

Male

Water intake (mL/100 g body wt/day) Chow intake (g/100 g body wt/day) Urinary output (mL/100 g body wt/day)

12.2 (1.4)

BALB/c Female

Male a

11.4 (3.1)

Female

Male a

a

13.7 (2.4)

17.5 (3.4)

13.1 (4.6)

Female b

8.7 (1.3)b

9.4 (2.2)c

13.0 (1.5)bb

17.4 (2.8)

11.1 (3.0)bb

5.2 (1.3)d

4.1 (1.1)

4.6 (1.0)

5.7 (1.9)

7.1 (2.3)da

(n ¼ 15)

(n ¼ 15)

ANOVA P value

C57BL/6J

(n ¼ 15)

(n ¼ 15)

(n ¼ 19)

22.9 (6.3)

a

15.6 (3.7) 9.9 (3.5)e

Males

Females

0.5

,0.0005

,0.0005

,0.0005

,0.0005

,0.0005

(n ¼ 20)

C ¼ BALB/c; C3 ¼ C3H; B6 ¼ C57BL/6J; ANOVA ¼ analysis of variance; SD ¼ standard deviation Comparison of each variable (males versus females within each strain) by unpaired t-test: aP , 0.005, bP , 0.0005 and dP ¼ 0.02 Overall P value on comparison of males from each strain and females from each strain by ANOVA: aP , 0.004 C3 versus C versus B6 females; bP , 0.004 C3 versus C and B6 males; cP , 0.0005 C3 versus C and B6 females; dP , 0.015 B6 versus C3 and C males; eP , 0.0005 B6 versus C3 and C females

significantly greater in CCrl males than in C3H or B6J males (ANOVA, P ¼ 0.01); B6J mice had significantly higher plasma glucose concentrations than age- and gendermatched C3H and CCrl mice (ANOVA, P , 0.0005 for both); plasma cholesterol concentrations were highest in C3H mice and lowest in the B6J mice (ANOVA, P , 0.0005) and varied significantly within strain between C3H and CCrl males and females (t-test, P ¼ 0.0006); plasma chloride concentrations were significantly lower in B6J mice than in C3H and CCrl mice (ANOVA, P , 0.0005 for males and P ¼ 0.003 for females); plasma calcium and plasma calcium adjusted for albumin concentration were significantly lower in B6J females than C3H females (ANOVA, P ¼ 0.001 for plasma calcium and P ¼ 0.043 for adjusted plasma calcium); plasma albumin concentrations varied significantly between females of the three strains (ANOVA, P , 0.0005) and significantly between C3H and B6J male mice (ANOVA, P ¼ 0.005). Lastly, the plasma potassium and phosphate concentrations in B6J male and

female mice were higher than the other two strains (ANOVA, P ¼ 0.02 and P ¼ 0.002, respectively, for males, and P , 0.0005 and P , 0.0005, respectively for females). Urine biochemistry Significant differences were observed in all measurements undertaken for urinary biochemical analysis on day 5 of metabolic cage studies, except of creatinine clearance (Table 5). Thus, both urinary glucose and 24 h excretion of urinary protein varied significantly according to sex (t-test, P ¼ 0.0004 and P , 0.0005, respectively); urinary glucose excretion was highest in CCrl females (ANOVA, P , 0.0005) and urine protein excretion was higher in B6J than in C3H and CCrl females (ANOVA, P ¼ 0.006); urinary sodium and potassium excretion were significantly greater in C3H males than in CCrl and B6J males (ANOVA, P , 0.0005 and P ¼ 0.001, respectively); urinary phosphate excretion was significantly greater in B6J females than in C3H and

Table 4 Mean (SD) plasma biochemistry values obtained on day 5 of metabolic cage studies ANOVA P value#

Strain

C3H

Sex

Male

Female

Male

Female

Male

Female

Males

Females

Na K Chloride Urea Creatinine† Ca ACa PO4 ALP Albumin‡ Cholesterol Glucose

155 (4) 7.6 (1.1) 117 (2) 11.6 (1.7)a 36 (6) 2.39 (0.12) 2.38 (0.12) 2.59 (0.93) 55 (15) 30.5 (1.6) 3.66 (0.53)ai 7.17 (1.61) (n ¼ 10– 15)

153 (3) 6.6 (0.3) 115 (2) 7.2 (1.1) 32 (2) 2.53 (0.13) 2.47 (0.14) 2.67 (0.41) 75 (21) 33.4 (1.6) 2.97 (0.43)i 8.09 (2.19) (n ¼ 10–15)

155 (2) 7.5 (0.9) 118 (3) 14.0 (2.5)ad 35 (4) 2.23 (0.17) 2.31 (0.13) 2.58 (1.21) 55 (12) 29.1 (1.9) 2.63 (0.31)a 9.79 (2.11) (n ¼ 10 –15)

153 (3) 7.4 (1.0) 116 (4) 9.8 (2.0) 35 (7) 2.36 (0.11) 2.35 (0.14) 2.63 (1.16) 61 (8) 30.5 (2.9) 2.00 (0.18) 9.01 (3.89) (n ¼ 10 –15)

155 (5) 9.8 (3.5)a 112 (4)c 11.5 (2.8) 35 (6) 2.25 (0.33) 2.27 (0.34) 3.91 (1.13)g 57 (29) 28.2 (1.9)h 1.58 (0.22) 13.56 (5.93)j (n ¼ 19)

152 (5) 11.8 (2.9)b 112 (3)c 10.9 (2.6) 37 (6) 2.25 (0.25)e 2.30 (0.25)f 4.15 (0.96)g 64 (21) 27.3 (1.2)h 1.58 (0.25) 20.83 (10.76)j (n ¼ 20)

0.99 0.02 ,0.0005 0.01 0.73 0.1 0.4 0.002 0.96 0.005 ,0.0005 ,0.0005

0.62 ,0.0005 0.003 0.001 0.04 0.001 0.04 ,0.0005 0.1 ,0.0005 ,0.0005 ,0.0005

BALB/c

C57BL/6J

C ¼ BALB/c; C3 ¼ C3H; B6 ¼ C57BL/6J; ACa ¼ calcium adjusted for plasma albumin (mmol/L); ALP ¼ alkaline phosphatase (U/L); ANOVA ¼ analysis of variance; SD ¼ standard deviation mmol/L † mmol/L – plasma creatinine (mmol/L) ‡ g/L; comparison of each variable (males versus females within each strain) by unpaired t-test: aP , 0.001, all others P . 0.0026 # Overall P value on comparison of males from each strain and females from each strain by ANOVA: post hoc analysis by t-test, aP , 0.05 B6 males versus C males; b P , 0.0005 B6 females versus C and C3 females; cP , 0.03 B6 males and females versus C and C3 males and females; dP , 0.03 C males versus C3 and B6 males; eP , 0.03 B6 females versus C3 females; fP ¼ 0.04 B6 females versus C3 females; gP , 0.03 B6 males and females versus C3 and C males and females; h P , 0.03 B6 males and C3 males, and B6 females versus C3 and C females; iP , 0.01 C3 males and females versus C males and females versus B6 males and females; jP , 0.01 B6 males and females versus C3 and C males and females


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Table 5 Mean (SD) urine biochemistry values obtained on day 5 of metabolic cage studies ANOVA P value#

Strain

C3H

Sex

Male

Female

Male

Female

Male

Female

Males

Females

Glucose Na:Cr† K:Cr† Ca:Cr† Pi:Cr† 24 h protein‡ Creatinine clearance}

1.0 (0.7)b 41 (8)c 71 (12)d 0.11 (0.04) 8.5 (2.7) 16.8 (3.9)ah 6.2 (1.6) (n ¼ 15)

2.4 (0.4)a 39 (8) 66 (9) 0.14 (0.03) 7.2 (1.7)f 5.9 (2.5) 7.0 (1.1) (n ¼ 15)

2.5 (0.8) 31 (7) 62 (14) 0.12 (0.04) 7.9 (2.9) 16.2 (5.1)a 8.1 (1.7) (n ¼ 15)

4.6 (2.5)aa 35 (9) 67 (10) 0.13 (0.03) 5.5 (1.8)f 1.8 (0.9) 8.3 (2.3) (n ¼ 15)

2.1 (0.8) 30 (4) 55 (10) 0.27 (0.13)e 9.3 (2.1) 11.5 (8.7) 8.6 (4.5) (n ¼ 19)

2.0 (1.0) 35 (9) 69 (22) 0.25 (0.10)e 9.3 (2.0)f 9.7 (10.7)g 10.7 (8.3) (n ¼ 20)

,0.0005 ,0.0005 0.001 ,0.0005 0.3 0.038 0.5

,0.0005 0.9 0.9 ,0.0005 ,0.0005 0.006 0.2

BALB/c

C57BL/6J

C ¼ BALB/c; C3 ¼ C3H; B6 ¼ C57BL/6J; ANOVA ¼ analysis of variance; SD ¼ standard deviation mmol/L † Na:Cr ¼ urinary sodium:creatinine ratio; K:Cr ¼ urinary potassium:creatinine ratio; Ca:Cr ¼ urinary calcium:creatinine ratio; Pi:Cr ¼ urinary phosphate:creatinine ratio ‡ mg/24 h } mL/min/kg; comparison of each variable (males versus females within each strain) by unpaired t-test: aP , 0.0005, all others P . 0.0026 # Overall P value on comparison of males from each strain and females from each strain by ANOVA: post hoc analysis by t-test, aP , 0.003 C females versus C3 and B6 females; bP , 0.003 C3 males versus C and B6 males; cP , 0.0005 C3 males versus C and B6 males; dP ¼ 0.001 C3 males versus B6 males; eP , 0.0005 B6 males and females versus C3 and C males and females; fP , 0.05 B6 females versus C3 females versus C females; gP ¼ 0.005 B6 females vs C females; h no significant difference on multiple testing

CCrl females (ANOVA, P , 0.0005). The most significant difference based upon strain was the greater amount of urinary calcium excreted by B6J mice of both sexes, represented by a doubling of the urinary calcium to creatinine ratio compared with both C3H and CCrl mice (ANOVA, P , 0.0005 for both). Creatinine clearance values were similar between males and females and between the three strains studied.

Discussion Our study, which has examined the effects of housing in metabolic cages on the dietary intake, urine output and weights of inbred mice, demonstrates that confinement in metabolic cages induces significant changes in the feeding behaviour, weight and urine output (Figure 1 and Tables 1 and 2). However, following a 3 – 4 day period, the mice acclimatized to conditions in the metabolic cages and this was evidenced by an attainment of a steady state in the dietary intake, urine output and weight of the mice (Figure 1 and Tables 1 and 2). This acclimatization was found to be influenced by the strain and sex of the mouse. These results are consistent with those previously reported that have demonstrated that metabolic cage confinement is stressful to rodents,8,9 and that there are significant differences in the phenotypic responses, which are dependent on the strain and sex of the mouse.13,14 The stress of confinement, or some other potential stressors, in metabolic cages evokes an increase in the activity of adrenal glucocorticoids; for example, solitary housing of mice is associated with elevations in faecal corticosterone concentrations for up to two weeks.15 This period of stress and the duration for acclimatization can be reduced by environmental enrichment, which reduces the amount of weight loss and the urinary corticosterone to creatinine ratios in mice.6 Thus, it is possible that the environmental enrichment, in the form of igloos, provided in this study was helpful in reducing the time taken to achieve

acclimatization as weight, dietary intake and urinary output stabilized by day 3 of confinement (Table 2), but further studies with and without environmental enrichment would be required to confirm this. Acclimatization occurred earlier in female mice than male mice, and the basis for this gender difference remains to be elucidated, although one possibility is that the stress response is accentuated in males by increased circulating androgen and/or testosterone concentrations. Significant gender differences among the strains in plasma and urinary biochemical parameters and changes in relation to acclimatization were also observed (Figure 1 and Tables 2, 4 and 5). Thus, male mice from the CCrl and C3H strains were found to have higher plasma urea and cholesterol concentrations, whereas females from the B6J strain had much higher plasma glucose concentrations than males (Table 4). In addition male mice, from the CCrl and C3H strains, excreted greater amounts of urinary protein than female mice (Table 5). These results, which demonstrate gender differences in three strains, emphasize the importance of studying males and females in parallel experiments, and are consistent with a previous report that investigated for changes relevant to the metabolic syndrome.16 Our findings for the concentrations of plasma sodium, chloride, total calcium, albumin and cholesterol in the 24- to 30-week-old mice from the three strains housed in metabolic cages (Table 4) are comparable to those previously reported in six-month-old mice, from three similar strains (C3HeB/FeJ, BALB/cByJ and C57BL/6J) that were not placed in metabolic cages but were instead grouphoused and fasted overnight prior to obtaining samples by retro-orbital bleeding.13 The similarities between our study and the previous study, which confirm the effects of strain and gender, indicate that the stress of confinement, fasting and methods of bleeding do not have a significant effect on the plasma concentrations of sodium, chloride, total calcium, albumin or cholesterol. However, the plasma concentrations of urea, creatinine, potassium and phosphate were higher in the mice from the metabolic

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cage study (Table 4) than those from the group-housed study,13 and these differences may be due in part to a continuing stress response induced by the confinement or to haemolysis associated with the method of venesection. Moreover, the mean plasma glucose concentrations in both sexes of the overnight fasted BALB/cByJ and B6J mice13 were lower than those from the non-fasted study, as expected. However, both sexes of the overnight fasted C3HeB/FeJ mice had higher mean plasma glucose concentrations than those in the non-fasted C3H strain (Table 4), indicating that this difference is likely to be significantly influenced by the substrain of the mouse. The mean plasma alkaline phosphatase activities in the mice from both sexes of the three strains from the metabolic cage study were lower than that from the group-housed mice, and this is likely attributed to the different assays used to assess this biochemical parameter, as previously reported for the activity of this enzyme.17 A previous study of fasted, group-housed adult mice at the ages of 3, 6 and 12 months has reported that age does not have significant effects on the majority of electrolyte and metabolite concentrations,13 with the exception of plasma urea concentrations, which declined with age in B6J mice, and alkaline phosphatase activity, which was elevated in the youngest group in all three strains. Our findings in the 24- to 30-week-old mice housed in metabolic cages are similar to those of these mice at three different ages, and these combined results suggest that the age of the adult animal is not a significant contributor in determining the concentrations of plasma electrolytes and metabolites that were measured in these studies (Table 4). The use of mouse models for investigating plasma and urinary phenotypic traits has revealed that these are relatively homogeneous in a given strain. However, when compared across multiple strains, the distribution of these traits is unimodal and mimics the situation seen in a human population.16 Thus, in human populations, certain individuals exhibit genetic variation of traits such as hypercalciuria,18 and others may be more susceptible to environmental and dietary influences leading to traits such as obesity.19 Knowledge of this phenotypic information in mice is particularly important when choosing a host strain for a genetically-altered mouse model since phenotypes within certain inbred strains may mimic or interact with the intended disease phenotype.13,16 Thus, differential susceptibility on the basis of inbred strain has already been noted in mouse models of the metabolic syndrome,16 diabetic nephropathy20 and renal hypercalciuria.21 Our data reveal strain differences in plasma glucose concentrations which were higher in B6J mice than in C3H and CCrl mice (Table 4), plasma cholesterol concentrations which were highest in C3H mice (Table 4), and urinary calcium excretion which was highest in B6J mice (Table 5) and these strain differences are consistent with previous plasma values obtained from wild-type inbred mice that have not been placed in metabolic cages.13 Impaired glucose tolerance has previously been described in the B6J strain and subsequently mapped to three quantitative trait loci on chromosomes 9, 11 (Gluchos2, the glucokinase gene) and 13 (Nnt, the nicotinamide nucleotide

transhydrogenase gene).22 The hyperglycaemia observed particularly in B6J female mice in our study (Table 4) is likely to be due to a combination of stress response and impaired glucose tolerance. The results of our study will be of help to investigators using these mouse strains to investigate glucose homeostasis, lipid metabolism and calcium metabolism. In summary, our results provide normative reference data for plasma and urinary measurements in 24- to 30-week-old mice from three inbred mouse strains following placement in metabolic cages for five days, and demonstrate differences in these parameters due to sex and strain of the mouse. In addition, our findings show that confinement in metabolic cages leads to significant changes in mouse behaviour and physiology that are consistent with a stress response, and that mice appear to acclimatize to their new environment, which contained some environmental enrichment, by days 3– 4. Funding: Grants from: Kidney Research UK (MJS, RVT), The European Union, EuReGene FP6 (NYL, AACR, LB, RDC, RVT), The Medical Research Council (AACR, TH, LB, RDC, SDMB, RVT) and The Wellcome Trust (BNA, NYL, AACR, RVT) and the University of Oxford (NYL). Conflicting interests: None.

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13 Champy MF, Selloum M, Zeitler V, et al. Genetic background determines metabolic phenotypes in the mouse. Mamm Genome 2008;19:318 –31 14 Klempt M, Rathkolb B, Fuchs E, de Angelis MH, Wolf E, Aigner B. Genotype-specific environmental impact on the variance of blood values in inbred and F1 hybrid mice. Mamm Genome 2006;17:93 –102 15 Hunt C, Hambly C. Faecal corticosterone concentrations indicate that separately housed male mice are not more stressed than group housed males. Physiol Behav 2006;87:519 –26 16 Svenson KL, Von Smith R, Magnani PA, et al. Multiple trait measurements in 43 inbred mouse strains capture the phenotypic diversity characteristic of human populations. J Appl Physiol 2007;102:2369 – 78 17 Davidson DF. Measurement of serum alkaline phosphatase. Enzyme Microb Technol 1979;1:9 –14 18 Hunter D, De Lange M, Snieder H, et al. Genetic contribution to bone metabolism, calcium excretion, and vitamin D and parathyroid hormone regulation. J Bone Miner Res 2001;16:371 – 8

19 Frayling TM, Timpson NJ, Weedon MN, et al. A common variant in the FTO gene is associated with body mass index and predisposes to childhood and adult obesity. Science 2007;316:889 – 94 20 Qi Z, Fujita H, Jin J, et al. Characterization of susceptibility of inbred mouse strains to diabetic nephropathy. Diabetes 2005;54:2628 – 37 21 Picard N, Van Abel M, Campone C, et al. Tissue kallikrein-deficient mice display a defect in renal tubular calcium absorption. J Am Soc Nephrol 2005;16:3602 – 10 22 Toye AA, Lippiat JD, Proks P, et al. A genetic and physiological study of impaired glucose homeostasis control in C57BL/6J mice. Diabetologia 2005;48:675 –86

(Accepted 14 March 2010)

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