不同发育时期小鼠代谢笼实验的最佳适应期探讨

doi:10.12300/j.issn.1674-5817.2024.034

摘要/Abstract

摘要：

目的探究小鼠代谢笼实验中不同发育时期小鼠的最佳适应时间，以期为利用小鼠开展代谢研究提供参考。方法使用3个发育阶段（离乳期M1、青春期M2和成年期M3）的雄性C57BL/6J小鼠共80只进行为期7 d的代谢笼实验，每5 min记录小鼠摄食、饮水、能量消耗、呼吸商、体重和活动水平等数据。通过时间序列分解和综合聚类分析对数据进行处理，采用重复测量方差分析法并结合t检验进行统计学差异比较，以推断最佳适应时间。结果不同发育阶段的小鼠代谢活动存在明显差异（P<0.01）。与M2和M3小鼠相比，M1小鼠的运动水平较低（P<0.01），并且昼夜节律不明显。M1小鼠具有更高的氧气消耗量、二氧化碳呼出量和能量消耗水平，以及较低的呼吸商（均P<0.001），表明其主要以脂肪作为能量来源。对摄食、饮水和能量消耗等因素进行分析后发现，各发育阶段的小鼠在第1个光周期（0～12 h）与第2个光周期之间（24～36 h）存在明显差异（均P<0.05），而24 h后每日摄食量、饮水量不再有明显差异（均P>0.05）。综合多指标进行聚类分析后发现，小鼠在进入代谢笼的前24 h综合指标与后续6 d的综合指标不成簇，出现离群，表现出明显差异。结论小鼠代谢笼实验可以用于检测小鼠连续的生理学指标变化，结果提示小鼠进入代谢笼24 h就可以适应新的代谢笼环境，这为小鼠代谢实验设计提供了理论依据。

关键词: 代谢笼, 适应期, 摄食, 饮水, 聚类分析, 小鼠

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

中图分类号:

Q4-33

谭贺,杨晓辉,张大秀,等. 不同发育时期小鼠代谢笼实验的最佳适应期探讨[J]. 实验动物与比较医学, 2024, 44(5): 502-510. DOI: 10.12300/j.issn.1674-5817.2024.034.

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.

图/表 6

注：A～C，小鼠在X轴、Y轴和Z轴上打断红外射线的频次，离乳期（M1，n=8）小鼠在X和Z轴上运动频次显著低于青春期（M2，n=16）和成年期（M3，n=24）小鼠（^*P<0.05，^**P<0.01，^***P<0.001）；D～F，M1、M2和M3小鼠每5 min在所有方向上的运动距离总和。
">

图1 不同发育阶段的C57BL/6小鼠在代谢笼内的活动水平比较
注：A～C，小鼠在X轴、Y轴和Z轴上打断红外射线的频次，离乳期（M1，n=8）小鼠在X和Z轴上运动频次显著低于青春期（M2，n=16）和成年期（M3，n=24）小鼠（^*P<0.05，^**P<0.01，^***P<0.001）；D～F，M1、M2和M3小鼠每5 min在所有方向上的运动距离总和。

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.

注：A，氧气消耗量的变化；B，二氧化碳呼出量的变化；C，能量消耗（EE）水平的变化；D，小鼠代谢笼中呼吸商（RQ）变化。离乳期（M1，n=8）小鼠的O₂消耗水平、CO₂呼出水平和能量消耗水平显著高于青春期（M2，n=16）小鼠和成年期（M3，n=24）小鼠（^***P<0.001），而M3小鼠的RQ显著高于M1和M2小鼠（^***P<0.001）。以上数据均以体重（g）为基准进行校正，结果以mean±SEM表示，统计学显著性差异通过重复测量数据方差分析给出。
">

图2 不同发育阶段的C57BL/6小鼠在代谢笼中的代谢活动变化
注：A，氧气消耗量的变化；B，二氧化碳呼出量的变化；C，能量消耗（EE）水平的变化；D，小鼠代谢笼中呼吸商（RQ）变化。离乳期（M1，n=8）小鼠的O₂消耗水平、CO₂呼出水平和能量消耗水平显著高于青春期（M2，n=16）小鼠和成年期（M3，n=24）小鼠（^***P<0.001），而M3小鼠的RQ显著高于M1和M2小鼠（^***P<0.001）。以上数据均以体重（g）为基准进行校正，结果以mean±SEM表示，统计学显著性差异通过重复测量数据方差分析给出。

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.

表1 小鼠进入代谢笼内前3 d的各指标适应效果评价 (

x ˉ

±se)

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

xˉ±se)
">

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^***

表1 小鼠进入代谢笼内前3 d的各指标适应效果评价 ( $x ˉ$ ±se)

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^***

表2 小鼠进入代谢笼内第1个与第2个光周期的各指标适应效果评价 (

x ˉ

±se)

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

xˉ±se)
">

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

表2 小鼠进入代谢笼内第1个与第2个光周期的各指标适应效果评价 ( $x ˉ$ ±se)

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

注：A，小鼠在代谢笼中每5 min内所有方向上的运动总和；B，小鼠每5 min在X轴上的运动频次；C，小鼠每5 min在Y轴上的运动频次；D，小鼠每5 min在Z轴上的运动频次；E，小鼠在代谢笼中消耗氧气的变化；F，小鼠在代谢笼中呼出二氧化碳的变化；G，小鼠在代谢笼中每5 min内摄取食物的质量；H，小鼠在代谢笼中每5 min内饮水量。M1为离乳期小鼠；M2为青春期小鼠；M3为成年期小鼠。
">

图3 不同发育阶段的小鼠进入代谢笼后的适应指标数据可视化分析
注：A，小鼠在代谢笼中每5 min内所有方向上的运动总和；B，小鼠每5 min在X轴上的运动频次；C，小鼠每5 min在Y轴上的运动频次；D，小鼠每5 min在Z轴上的运动频次；E，小鼠在代谢笼中消耗氧气的变化；F，小鼠在代谢笼中呼出二氧化碳的变化；G，小鼠在代谢笼中每5 min内摄取食物的质量；H，小鼠在代谢笼中每5 min内饮水量。M1为离乳期小鼠；M2为青春期小鼠；M3为成年期小鼠。

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.

注：离乳期小鼠进入代谢笼的第1天（M1_1）、青春期小鼠进入代谢笼的第1天（M2_1）和成年期小鼠进入代谢笼的第1天（M3_1）数据均出现离群，不与同组内数据成簇。 Kcal_hr，小鼠每小时能量消耗/mL；VCO₂，小鼠二氧化碳呼出量；VO₂，小鼠氧气消耗量/mL；VH₂O，小鼠饮水量mL；Allmeters.5m，小鼠每5 min总运动距离/m；Pedmeters.5m，小鼠每5 min内定向运动总距离/m；Foodupa.5m，小鼠每5 min摄食量/g；Waterupa.5m，小鼠每5 min饮水量/mL。
">

图4 不同发育阶段的小鼠进入代谢笼后各适应指标数据的多因素聚类分析
注：离乳期小鼠进入代谢笼的第1天（M1_1）、青春期小鼠进入代谢笼的第1天（M2_1）和成年期小鼠进入代谢笼的第1天（M3_1）数据均出现离群，不与同组内数据成簇。 Kcal_hr，小鼠每小时能量消耗/mL；VCO₂，小鼠二氧化碳呼出量；VO₂，小鼠氧气消耗量/mL；VH₂O，小鼠饮水量mL；Allmeters.5m，小鼠每5 min总运动距离/m；Pedmeters.5m，小鼠每5 min内定向运动总距离/m；Foodupa.5m，小鼠每5 min摄食量/g；Waterupa.5m，小鼠每5 min饮水量/mL。

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。

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