Optimal Adaptation Period for Metabolic Cage Experiments in Mice at Different Developmental Stages

doi:10.12300/j.issn.1674-5817.2024.034

Abstract

Abstract:

Objective To investigate the optimal adaptation period for mice at different developmental stages during metabolic cage experiments, aiming to provide a reference for conducting metabolic research using mice. Methods A total of 80 male C57BL/6J mice at three developmental stages (weaning period M1, adolescent M2, and adulthood M3) were subjected to a 7-day metabolic cage experiment. Data on food intake, water intake, energy expenditure, respiratory quotient, body weight, and activity levels were recorded every five minutes. The collected data were processed using time series decomposition and comprehensive cluster analysis. Statistical differences were compared using repeated measures ANOVA combined with t-test to determine the optimal adaptation period. Results Significant differences in metabolism were observed among mice in different developmental stages (P<0.01). Compared with adolescent (M2) and adult (M3) mice, weaned mice (M1) exhibited lower activity level (P<0.01) and less distinct circadian rhythm. M1 mice had higher oxygen consumption, carbon dioxide production, and energy expenditure, as well as a lower respiratory quotient (all P<0.001), indicating that they mainly relied on fat as an energy source. Analysis of food intake, water intake, and energy expenditure revealed significant differences between the first light cycle (0-12 h) and the second light cycle (24-36 h) across all developmental stages (all P<0.05) . However, there was no significant difference in daily food intake or water intake after 24 hours (both P>0.05). Comprehensive cluster analysis of multiple indicators showed that the overall indicators of mice during the first 24 hours in the metabolic cages did not cluster with those of the subsequent 6 days, demonstrating significant differences. Conclusion Metabolic cage experiment can be used to detect continuous physiological changes in mice. The results suggest that mice can adapt to new metabolic cages environment within 24 hours, providing a theoretical basis for the design of metabolic experiments using mice.

Key words: Metabolic cage, Adaptation period, Food intake, Water intake, Cluster analysis, Mice

CLC Number:

Q4-33

TAN He,YANG Xiaohui,ZHANG Daxiu,et al. Optimal Adaptation Period for Metabolic Cage Experiments in Mice at Different Developmental Stages[J]. Laboratory Animal and Comparative Medicine, 2024, 44(5): 502-510. DOI: 10.12300/j.issn.1674-5817.2024.034.

Figures/Tables 6

Figure 1 Comparison of activity levels of C57BL/6 mice at different developmental stages in metabolism cages
Note：A-C, Frequency of infrared beam interruptions on X, Y, Z axes, the movement frequencies of weaned mice (M1, n=8) on X and Z axes was significantly lower than that of adolescent (M2, n=16) and adult (M3, n=24) mice (^*P<0.05，^**P<0.01, ^***P<0.001); D-F, Total movement distance of M1, M2, M3 mice in all directions every 5 minutes in metabolism cage.

Figure 2 Metabolic activity changes of mice in metabolism cages across 3 age groups
Note：A, Changes in oxygen consumption; B, Changes in carbon dioxide exhalation; C, Changes in energy expenditure (EE) levels; D, Changes in metabolic cage respiratory quotient (RQ) in mice. O₂ consumption, CO₂ exhalation, and EE of weaned mice (M1, n=8) were significantly higher than those of adolescent (M2, n=16) and adult (M3, n=24) mice (^***P<0.001). RQ of M3 mice was significantly higher than that of M1 and M2 mice (^***P<0.001). Data were calibrated on body mass (g), and results were expressed as mean±SEM, statistically significant differences were shown by repeated measures ANOVA.

Table 1 Evaluation of adaptation effects on various indicators in metabolic cages during the first three days

适应时间 Adaptation time	指标 Indicators	离乳期(M1) Weaned (M1)	青春期(M2) Adolescent (M2)	成年期(M3) Adult (M3)
第1天 0-24 h	摄食量/g	4.43±1.12	4.27±1.02	5.02±1.51
	饮水量/mL	3.38±0.62	2.98±0.59	3.50±1.01
	XYZ轴移动频次/次	222.21±23.20	289.41±40.00	368.90±39.49
第2天 24-48 h	摄食量/g	4.69±0.80	4.25±0.99	4.50±1.21
	饮水量/mL	3.57±0.73	3.15±0.75	3.44±0.77
	XYZ 轴移动频次/次	186.22±32.15	240.27±32.29^***	264.98±36.63^***
第3天 48-72 h	摄食量/g	4.78±1.00	3.82±0.67	4.78±1.14
	饮水量/mL	4.78±1.00	3.05±0.66	3.43±0.82
	XYZ 轴移动频次/次	168.21±54.76	213.24±24.71^***	250.81±25.62^***

Table 2 Evaluation of adaptation effects on various indicators in metabolic cages during the first and second light periods

适应时间 Adaptation time	指标 Indicators	离乳期(M1) Weaned (M1)	青春期(M2) Adolescent (M2)	成年期(M3) Adult (M3)
第1个光周期 0-12 h	摄食量/g	1.52±0.44	1.16±0.47	1.58±0.72
第1个光周期 0-12 h	饮水量/mL	0.44±0.29	0.37±0.25	0.49±0.34
第2个光周期 24-36 h	摄食量/g	2.03±0.45^*	1.05±0.47	0.95±0.42^***
第2个光周期 24-36 h	饮水量/mL	1.29±0.43^***	0.54±0.35	0.67±0.31

Figure 3 Visualization analysis of adaptation indicators for mice in metabolic cages at different developmental stages
Note：A, The total movements of the mice in all directions during 5 minutes in metabolic cages; B, The frequency of movement along the X axis; C, The frequency of movement along the Y axis; D, The frequency of movement along the Z axis; E, Changes in oxygen consumption in metabolic cages; F, Changes in carbon dioxide exhaled in metabolic cages; G, The amount of food intake every 5 minutes by mice in metabolic cages; H, The amount of water intake by mice every 5 minutes in metabolic cages. M1, weaned mice; M2, adolescent mice; M3, adult mice.

Figure 4 Multifactor clustering analysis of adaptation indicators for mice at different developmental stages in metabolic cages
Note：The data from day 1 in metabolic cages for weaned mice (M1_1), adolescent mice (M2_1), and adult mice (M3_1) were outliers and did not cluster with the data from the same group. Kcal_hr，EE per hour/mL；VCO₂，mice carbon dioxide exhalation；VO₂，oxygen consumption/mL；VH₂O，water intake/mL；Allmeters.5m，total movement distance in 5 min/m；Pedmeters.5m，total directional movement distance along X, Y, Z axes in 5 min/m；Foodupa.5m，food intake in 5 min/g；Waterupa.5m，water intake in 5 min/mL。

References 24

1	GRUNDY S M. Metabolic syndrome update[J]. Trends Cardiovasc Med, 2016, 26(4):364-373. DOI: 10.1016/j.tcm. 2015.10.004 .
2	GUPTA A, GUPTA V. Metabolic syndrome: what are the risks for humans?[J]. Biosci Trends, 2010, 4(5):204-212.
3	GRIFFIN J L. Understanding mouse models of disease through metabolomics[J]. Curr Opin Chem Biol, 2006, 10(4):309-315. DOI: 10.1016/j.cbpa.2006.06.027 .
4	KULKARNI N M, JAJI M S, SHETTY P, et al. A novel animal model of metabolic syndrome with non-alcoholic fatty liver disease and skin inflammation[J]. Pharm Biol, 2015, 53(8):1110-1117. DOI: 10.3109/13880209.2014.960944 .
5	LUO Y H, LU H C, PENG D Q, et al. Liver-humanized mice: a translational strategy to study metabolic disorders[J]. J Cell Physiol, 2022, 237(1):489-506. DOI: 10.1002/jcp.30610 .
6	EVANS C C, LEPARD K J, KWAK J W, et al. Exercise prevents weight gain and alters the gut microbiota in a mouse model of high fat diet-induced obesity[J]. PLoS One, 2014, 9(3): e92193. DOI: 10.1371/journal.pone.0092193 .
7	RIDAURA V K, FAITH J J, REY F E, et al. Gut microbiota from twins discordant for obesity modulate metabolism in mice[J]. Science, 2013, 341(6150):1241214. DOI: 10.1126/science.1241214 .
8	TETRI L H, BASARANOGLU M, BRUNT E M, et al. Severe NAFLD with hepatic necroinflammatory changes in mice fed trans fats and a high-fructose corn syrup equivalent[J]. Am J Physiol Gastrointest Liver Physiol, 2008, 295(5): G987-G995. DOI: 10.1152/ajpgi.90272.2008 .
9	LIU Z W, HUANG M L, WU X, et al. PER1 phosphorylation specifies feeding rhythm in mice[J]. Cell Rep, 2014, 7(5):1509-1520. DOI: 10.1016/j.celrep.2014.04.032 .
10	ZHU S W, YEE B K, NYFFELER M, et al. Influence of differential housing on emotional behaviour and neurotrophin levels in mice[J]. Behav Brain Res, 2006, 169(1):10-20. DOI: 10.1016/j.bbr.2005.11.024 .
11	ISHIDA H, MITSUI K, NUKAYA H, et al. Study of active substances involved in skin dysfunction induced by crowding stress. I. effect of crowding and isolation on some physiological variables, skin function and skin blood perfusion in hairless mice[J]. Biol Pharm Bull, 2003, 26(2):170-181. DOI: 10.1248/bpb.26.170 .
12	STEYERMARK A C, MUELLER P J. Cage size affects feeding and energetics of captive rodents[J]. Physiol Biochem Zool, 2002, 75(2):209-213. DOI: 10.1086/338689 .
13	BARTOLOMUCCI A, PALANZA P, SACERDOTE P, et al. Individual housing induces altered immuno-endocrine responses to psychological stress in male mice[J]. Psychoneuroendocrinology, 2003, 28(4):540-558. DOI: 10.1016/s0306-4530(02)00039-2 .
14	MANSER C E, ELLIOT H, MORRIS T H, et al. The use of a novel operant test to determine the strength of preference for flooring in laboratory rats[J]. Lab Anim, 1996, 30(1):1-6. DOI: 10.1258/002367796780744974 .
15	MANSER C E, MORRIS T H, BROOM D M. An investigation into the effects of solid or grid cage flooring on the welfare of laboratory rats[J]. Lab Anim, 1995, 29(4):353-363. DOI: 10.1258/002367795780740023 .
16	BRUNNER L J, DIPIRO J T, FELDMAN S. Metabolic cage isolation reduces antipyrine clearance in rats[J]. J Pharm Pharmacol, 1994, 46(7):581-584. DOI: 10.1111/j.2042-7158.1994.tb03861.x .
17	KALLIOKOSKI O, JACOBSEN K R, DARUSMAN H S, et al. Mice do not habituate to metabolism cage housing: a three week study of male BALB/c mice[J]. PLoS One, 2013, 8(3): e58460. DOI: 10.1371/journal.pone.0058460 .
18	DUTTA S, SENGUPTA P. Men and mice: relating their ages[J]. Life Sci, 2016, 152:244-248. DOI: 10.1016/j.lfs.2015.10.025 .
19	PALANZA P. Animal models of anxiety and depression: how are females different?[J]. Neurosci Biobehav Rev, 2001, 25(3):219-233. DOI: 10.1016/s0149-7634(01)00010-0 .
20	STECHMAN M J, AHMAD B N, LOH N Y, et al. Establishing normal plasma and 24-hour urinary biochemistry ranges in C3H, BALB/c and C57BL/6J mice following acclimatization in metabolic cages[J]. Lab Anim, 2010, 44(3):218-225. DOI: 10.1258/la.2010.009128 .
21	尹媛, 陆璐, 王蕴, 等. 小鼠生活笼实验引入适应期的必要性研究[J]. 实验动物与比较医学, 2021, 41(4):358-362. DOI: 10.12300/j.issn.1674-5817.2020.154 .
	YIN Y, LU L, WANG Y, et al. Necessity of an acclimatization period in a home cage experiment of mice[J]. Lab Anim Comp Med, 2021, 41(4):358-362. DOI: 10.12300/j.issn.1674-5817.2020.154 .
22	ERIKSSON E, ROYO F, LYBERG K, et al. Effect of metabolic cage housing on immunoglobulin A and corticosterone excretion in faeces and urine of young male rats[J]. Exp Physiol, 2004, 89(4):427-433. DOI: 10.1113/expphysiol. 2004. 027656 .
23	TULI J S, SMITH J A, MORTON D B. Stress measurements in mice after transportation[J]. Lab Anim, 1995, 29(2):132-138. DOI: 10.1258/002367795780740249 .
24	Parliament European. Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes [S/OL]. (2010-10-20) [2024-02-20]..

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