Indian Journal of Animal Research

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Indian Journal of Animal Research, volume 54 issue 7 (july 2020) : 835-840

Comparative Study on Phenotypic Differences in Eothenomys Miletus Under Food Restriction and Refeeding Between Xianggelila and Jianchuan from Hengduan Mountain Regions

Dong-min Hou1, Xiao-ying Ren1, Wan-long Zhu1,*, Hao Zhang1,*
1Key Laboratory of Ecological Adaptive Evolution and Conservation on Animals-Plants in Southwest Mountain Ecosystem of Yunnan Province Higher Institutes College, School of Life Sciences, Yunnan Normal University.Engineering Research Center of Sustainable Development and Utilization of Biomass Energy Ministry of Education. Key Laboratory of Yunnan Province for Biomass Energy and Environment Biotechnology, Kunming, 650500, China.
Cite article:- Hou Dong-min, Ren Xiao-ying, Zhu Wan-long, Zhang Hao (2019). Comparative Study on Phenotypic Differences in Eothenomys Miletus Under Food Restriction and Refeeding Between Xianggelila and Jianchuan from Hengduan Mountain Regions . Indian Journal of Animal Research. 54(7): 835-840. doi: 10.18805/ijar.B-1151.

ABSTRACT

Body mass regulation may be appeared regional differences, in order to investigate the physiological and behavioral changes in Eothenomys miletus from Shanggelila (XGLL) and Jianchuan (JC) under food restriction (FR) and refeeding (Re), body mass, food intake, resting metabolic rate (RMR), serum leptin levels, hypothalamic neuropeptides expression and activity behavior were measured. The results showed that areas and FR had significant effects on body mass, food intake, RMR, activity behavior, serum leptin levels, hypothalamic neuropeptides expression, masses of liver and small intestine in E. miletus. Body mass and serum leptin levels in XGLL were lower than that of JC, food intake, activity behavior, liver mass and RMR in XGLL were higher relative to in JC. All the indexes of the two areas of E. miletus can be restored to control levels after refeeding, showing phenotypic plasticity. In conclusion, physiological and behavioral characteristics illustrated that the influences of food and different regions on phenotypic plasticity, which had important significance for in-depth understanding of the survival and adaptation strategies of E. miletus in Hengduan mountain regions.

INTRODUCTION

Maintenance of energy homeostasis of animals were mainly affected by body mass, food intake and thermogenesis capacity, many characteristics of mammals showed obvious phenotypic plasticity changes (Visser et al., 2010; Gupta et al., 2017; Vinus et al., 2018). Food restriction reduced body mass of small mammals, and their body mass returned to the control levels after refeeding (Zhao et al., 2011). However, the results of energy expenditure and coping with food restriction and refeeding seem to be inconsistent (Cameron et al., 2011), which may reflect different energy strategies and behavior patterns of wild animals. Leptin is a protein hormone secreted by adipose tissue, which plays an important regulatory role in the hypothalamus on energy expenditure and energy intake (Friedman and Halaas, 1998). Previously studies on energy homeostasis of small mammals were mainly focused on leptin resistance (Tups, 2009), seasonal changes in food intake (McCue, 2010) and adaptive thermogenesis on energy balance (Manini, 2010). Hypothalamic neuropeptides were divided into appetite neuropeptide: NPY and AgRP, an appetite suppressant neuropeptide: POMC and CART, the former can stimulate feeding and inhibit energy consumption, while the latter can inhibit food intake and stimulate energy consumption (Morton et al., 2006). Leptin mainly by acting on NPY/AgRP and POMC/CART to regulate food intake, so as to maintain the relative balance of energy (Larsenal and Hunter, 2006). Studies had shown that food restriction can reduce serum leptin levels in animals and change hypothalamic neuropeptides expressions (Rousseas et al., 2003).
        
Plasticity of animal behavior characteristics were of great significance to adapt to environmental changes and improve their survival ability, uncertainty of food resource was an important problem faced by animals in the natural environment, and many animals adjust themselves to the changes of environmental conditions through active or passive behavior (Zhao et al., 2013; Dinani et al., 2019). For example, food restriction significantly increased the activity of Mus musculus (Sherwin, 1998), but significantly reduced the energy expenditure of Eutamias minimus (Hambly and Speakman, 2005). In addition, exogenous leptin injection can reduce the activity behavior of Cricetulus barabensis under food restriction (Zhao et al., 2013). It can be seen that the effect of food restriction on animals’ behaviors were not similar completely and there may be species differences.
        
Eothenomys miletus is an endemic species in China, which distributed mainly in Hengduan mountain region (Zheng, 1993). Different areas of E. miletus in terms of quantity and quality of food may face great seasonal fluctuations. Previous studies of our research group showed that food restriction significantly reduced body mass of E. miletus, and body mass returned to the control level after Refeeding (Zhu et al., 2014). In the present study, body mass, food intake, RMR, serum leptin levels, hypothalamus neuropeptide expression, body composition and activity behavior in E. miletus were measured under continuous food restriction for 4 weeks and refeeding for 4 weeks.

MATERIALS AND METHODS

Animals and experimental design
 
Adult E. miletus used in this study were captured from farmland near the city of Shangri-La (XGLL) (27°90’ N, 99°83’ E; altitude 3,321m; mean temperature in winter 2.1°C) and the country of Jianchuan (JC) (26°43’ N, 99°75’ E; altitude 2,590 m; mean temperature in winter 8.4°C) from the Hengduan mountain region in winter (December 2017). Then transported to School of Life Sciences of Yunnan Normal University, which were housed individually (260×160×150 mm) and were maintained at a room temperature of 25 ± 1°C, under a photoperiod of 12 h light: 12 h dark (lights on at 08:00 h); food (standard rabbit pellet chow; produced by Kunming Medical University, Kunming) and water were provided ad libitum for 1 week. All animal procedures were compliant with the Animal Care and Use Committee of the School of Life Science, Yunnan Normal University. This study was approved by the Committee (13-0901-011).
 
Experiment 1
 
Effects of food restriction and refeeding on body mass, food intake, RMR and activity behavior in E. miletus. 16 adult weight-matched E. miletus of XGLL and JC were housed individually (were maintained at 12L: 12D (light on at 08:00am), 25±1°C, respectively) and kept for 1 weeks to familiarize with the environment. After the acclimatizing period, the animals of XGLL and JC were acclimated to food restriction (80% of ad libitum food intake) for 28 days and then refeeding for another 28days, animals were acclimated for 8 weeks. Food intake was calculated as the mass of food missing from the hopper, subtracting orts mixed in the bedding. Body mass, food intake, RMR and activity behavior were measured every day.
 
Experiment 2
 
Effects of food restriction and refeeding on body mass, serum leptin levels, hypothalamic neuropeptide genes expression, body compositions and gastrointestinal tract in E. miletus. 41 adult weight-matched E. miletus from two regions (XGLL, N=22 and JC, N=19) were selected, which were maintained at 12L: 12D (light on at 08:00am), 25±1°C, respectively. Animals of one region were randomly assigned to a control group or a food restriction and refeeding group (FR-Re). Controls were fed ad libitum during 8 weeks, while FD-Re were fed 80% of ad libitum food intake for 4 weeks, then fed ad libitum for a further 4 weeks. On day 28, animals were randomly selected from FD-Re group for the measurement of body mass, serum leptin levels, body composition, gastrointestinal tract mass and length, and hypothalamic neuropeptide genes expression. These measurements were taken again from the remaining animals of each group (8W Control group and 8W FD-Re group) at 8 weeks. All animals were sacrificed between 09:00 h and 11:00 h by decapitation and animals were dissected to evaluate organ morphology. Blood was centrifuged at 4,000 rpm for 30 min after a 30 min interval. Blood serum was collected and stored at -75°C prior to hormone measurement.

Measurement of metabolic rates, food intake and activity behavior
 
Body mass, food intake, RMR and behavioral activity were measured using the metabolic system (BXY-R, Sable Systems). E. miletus were acclimated to calorimetry cages prior to 30 min the study and data collection (Weir 1949). 
 
Measurement of morphology, serum leptin levels and hypothalamic neuropeptide gene expression
 
Measurements of Morphology was details in Zhu et al., (2015). Serum leptin levels were determined by radioimmunoassay (RIA) with the 125I Multi-species Kit (Millipore) and leptin values were determined in a single RIA; the lowest level of leptin that can be detected by this assay is 1.0 ng/mL when using a 100-μL sample size (instructions for Multi-species Kit). The inter- and intra-assay variabilities for leptin RIA were 3.6% and 8.7%, respectively. Measurements of hypothalamic neuropeptide gene expression was detailed in Zhu et al., (2015).
 
Statistical analysis
 
Data were analyzed using the software package SPSS 15.0. Prior to all statistical analyses, data were examined for assumptions of normality and homogeneity of variance using Kolmogorov–Smirnov and Levene tests, respectively. Differences in body mass, food intake, RMR, activity behavior, serum leptin levels and hypothalamic neuropeptide genes expression were analyzed by two-way ANOVA, and differences in body compositions and gastrointestinal tract were analyzed by two-way ANCOVA with body mass as a covariate, followed by Tukey’s post hoc test. Results are presented as means ± SE and P<0.05 was considered to be statistically significant.

RESULTS AND DISCUSSION

Body mass, food intake, RMR and behavior
 
Areas had significant effect on body mass of E. miletus (F=213.87, P<0.01), the influence of FR and Re on body mass was different significantly (F=2.48, P<0.01, Fig 1). The influence of areas on food intake of E. miletus was significantly different (F=287.72, P<0.01), FR and Re had significant effects on food intake (F=14.64, P<0.01, Fig 2). Areas affects RMR of E. miletus significantly (F=1250.77, P<0.01), FR-Re had a significant effect on RMR of E. miletus (F=10.03, P<0.01, Fig 3). Areas and FR-Re had significant effects on activity behaviors of E. miletus (areas: F=134.61, P<0.01; FR-Re: F = 18.85, P<0.01, Fig 4).
 

Fig 1: Effects of food restriction and refeeding on body mass in Eothenomys miletus between. XGLL and JC.


 

Fig 2: Effects of food restriction and refeeding on food intake in Eothenomys miletus between XGLL and JC.


 

Fig 3: Effects of food restriction and refeeding on RMR in Eothenomys miletus between XGLL and JC.


 

Fig 4: Effects of food restriction and refeeding on activity behavior in Eothenomys miletus between. XGLL and JC.


 
Body mass was an important indicator of animal nutrition and its stability depended on the balance of energy budget (Kouda et al., 2004). The results showed that food restriction significantly decreased body mass of two regions, which could return to the control level after refeeding, showing phenotypic plasticity, which was similar to the results of other rodents (Zhao et al., 2013). Body mass in E. miletus in XGLL was lower than that in JC, and the fluctuation of body mass in JC was greater than that in XGLL, indicating that E. miletus in JC had a greater response to food restriction, which may be related to the relatively abundant food resource in this region. Moreover, body mass loss in JC was greater than that in XGLL under food restriction, which may indicate that food resources in XGLL were less in winter and E. miletus responded to food resources shortage more quickly, and thus had a process of adaptation, so body mass loss was smaller. Many animals tend to decrease their RMR when they were faced with poor food quality (Zhang and Wang, 2008). In the present study, RMR in XGLL was significantly lower than that in JC. RMR in XGLL decreased rapidly after food restriction, while that of E. miletus in JC decreased gradually after food restriction. RMR performance and activity behavior were similar. The results supported the metabolism switch hypothesis and E. miletus need to decrease body mass and RMR to cope with the environment of food shortage. Animal activity plays an important role in energy metabolism (Hambly and Speakman, 2005). In the present study, the activity in XGLL was higher than that in JC. In the initial period of food restriction in the two regions, the activity behaviors of the E. miletus were different, E. miletus in JC increased their activity activities to obtain more food, which may be related to their habitats with richer food resources. In contrast, E. miletus in XGLL rapidly decreased their activity to decrease energy consumption. However, there was no significant difference in the activity behaviors of the E. miletus in the two regions under food restriction on day 7. Once the food recovered, it quickly increased their activity behaviors to increase their food intake.
 
Body compositions, serum leptin levels and hypothalamus neuropeptide expressions
 
Areas and FR-Re had significant effects on serum leptin levels in E. miletus (areas: F=13.85, P<0.01; FR-Re: F=9.52, P<0.01, Table 1). Serum leptin levels of E. miletus in XGLL was lower than that in JC. Similar trends were also found in liver mass and small intestine mass. Areas and FR-Re had significant effects on NPY expression, AgRP expression, POMC expression in E. miletus. Areas had a significant effect on CART expression. However, FR-Re had on effects on CART expression (Table 1).
 

Table 1: Effects of food restriction and Refeeing on serum leptin levels, hypothalamic neuropeptides expression and body composition of E.miletus in different region.


 
Previous studies have shown that liver mass returned to the control level after 48 h of refeeding, which may be related to the recovery of liver glycogen (Ji and Friedman, 1999). In the present study, it showed that food restriction can significantly decrease liver mass of the two areas of E. miletus and it can be restored to the control level after refeeding, indicating that E. miletus may rapidly invoke liver glycogen, because starvation would cause a sharp decrease in plasma glucose level (Mustonen et al., 2008). Food restriction also affects the mass of small intestine, which may be related to the low metabolic rate of the E. miletus under food restriction. Masses of liver and small intestine in E. miletus in XGLL were higher than that in JC, which may be related to the lower environmental temperature in XGLL in winter. Leptin plays an important role in body mass regulation, which acting as a signaling molecule to regulate food intake and energy expenditure (Ablenda et al., 2003). The results of our study showed that serum leptin levels in XGLL was lower than that in JC, which was consistent with  the change trend of body mass. Food restriction could significantly decrease serum leptin levels of E. miletus in the two areas, indicating that lower leptin concentrations could promote food intake, and the serum leptin levels of E. miletus could be restored to the control level during refeeding. The current results showed that food restriction affects the expressions of NPY, AgRP and POMC, suggesting that food restriction can increase the expression of NPY and AgRP, decreased the expression of POMC, then increased food intake. Expressions of NPY, AgRP, POMC and CART in different regions in E. miletus were different, suggesting that regional differences may also lead to changes in expressions of hypothalamic neuropeptides, thereby regulating the changes of body mass.

CONCLUSION

In conclusion, energy metabolism regulation in E. miletus in different regions showed geographical differences. Food restriction would reduce body mass and activity behavior in E. miletus in the two regions and increase activity behavior after refeeding, showing phenotypic plasticity. In addition, serum leptin levels and hypothalamic neuropeptide might be involved in body mass regulation of E. miletus in the two regions.

ACKNOWLEDGEMENT

This research was financially supported by National Science Foundation of China (No. 31760118; 31560126) and Young and Middle-aged Academic and Technical Leaders Reserve Talents Project of Yunnan Province (2019HB013). We wish to thank Pro. Burkart Engesser at Historisches Museum Basel, Switzerland for correcting the English usage in the draft. Thank you for the anonymous reviewers and the editor of the journal for their valuable comments.

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