Rabbits are widely used in clinical trials, and are also an important source of high-quality protein for humans ( Bosze Zs and Houdebine, 2006 Cullere and Dalle Zotte, 2018). Hence, selecting growing animal models to study the interference of mistimed eating on intestinal microbiological diurnal rhythm is of great significance to fill knowledge gaps regarding the impact of irregular lifestyle on the health of children and growing animals. However, there are still many challenged associated with studying the diurnal rhythms of human gut microbes, such as continuous multi-temporal sampling, differences in environmental and dietary composition, and particularly the lack of studies on children and growing animals ( Frazier and Chang, 2020). Therefore, in recent years, unhealthy lifestyles causing disturbances in the diurnal rhythm of intestinal microorganisms have become the focus of research ( Asher and Sassone-Corsi, 2015 Murakami and Tognini, 2019 Bishehsari et al., 2020). However, the increasing academic burden on students has become a serious concern, as it has contributed toward the annual increase in insufficient sleep and nocturnal diets in children, which in turn are associated to higher rates of obesity, cardiovascular disease, and chronic intestinal inflammatory diseases during childhood ( Eastman et al., 2015). Epidemiological investigations have determined that unhealthy lifestyle-promoted metabolic syndromes and intestinal inflammatory diseases result in heavy global economic burden ( Bishehsari et al., 2020). Unhealthy adult lifestyles that include shift work, nighttime social activities and jet lag are becoming more prevalent, and consequently, associated health problems have also become more prominent ( Parsons et al., 2015 Koshy et al., 2019). Collectively, these results provide a new perspective for the healthy feeding and management of growing animals. This is the first study to reveal that NRF reprograms the diurnal rhythm of the gut microbiome, promotes the diurnal expression of clock genes and tight junction genes via synchronization of microbial-driven serotonin rhythm and eating activity-driven body temperature oscillations, thereby improving intestinal health and reducing the risk of diarrhea in growing rabbits. In vitro simulation experiments further revealed that synchronization of microbial-driven serotonin rhythm and eating activity-driven body temperature oscillations, which are important zeitgebers, could promote the diurnal expression of clock genes and CLAUDIN-1 in rabbit intestinal epithelial cells (RIEC), and enhance RIEC proliferation. Furthermore, NRF strengthened the diurnal amplitude of body core temperature, and promoted the diurnal expression of intestinal clock genes ( BMAL1, CLOCK, REV-ERBα, and PER1), and genes related to the regulation of the intestinal barrier ( CLAUDIN-1), and intestinal epithelial cell self-proliferation and renewal ( BMI1). Moreover, NRF improved diurnal rhythm of tryptophan hydroxylase isoform 1 and serotonin. Overall, NRF was found to reduce the risk of diarrhea in growing rabbits, improved the diurnal rhythm and abundance of beneficial microorganisms, along with the production of beneficial metabolites, whereas reduced the abundance of potential pathogens ( Synergistes, Desulfovibrio, and Alistipes). At age 12 weeks, six rabbits were selected from each group, and caecum and cecal contents, as well as serum samples were collected at 4-h intervals during 24 h. In this study, 108 5-week-old weaned rabbits (nocturnal animals) were randomly subjected to daytime feeding (DF) and night-restricted feeding (NRF). However, whether the composition and diurnal rhythm of gut microbiota can be optimized by synchronizing the window period of eating with natural eating habits to reduce the risk of diarrhea remains unclear, especially in growing animals. The circadian misalignment of the gut microbiota caused by unusual eating times in adult animals is related to disease development. 3Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture and Rural Affairs, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, China.2National Rabbit Industry Technology System Qingdao Comprehensive Experimental Station, Qingdao, China.1 State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, China.Qiang-Jun Wang 1† Yao Guo 1† Ke-Hao Zhang 1 Lei Zhang 1 Shi-Xia Geng 1 Chun-Hua Shan 1 Peng Liu 1 Meng-Qi Zhu 1 Qiong-Yu Jin 1 Zhong-Ying Liu 1 Mei-Zhi Wang 1 Ming-Yong Li 2 Man Liu 2 Lei An 3 Jian-Hui Tian 3 Zhong-Hong Wu 1*
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