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 cagesNote：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 groupsNote：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 stagesNote：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 cagesNote：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。

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