Abstract:Circadian rhythms are a prevalent physiological phenomenon in organisms, referring to the adaptation and regulation of the internal clock to a 24-h cycle. This biological clock governs various physiological processes, such as the sleep-wake cycle, hormone secretion, and metabolic rate, enabling organisms to synchronize with external environmental changes between day and night. Although circadian rhythms have been thoroughly researched in terrestrial animals, their importance in aquatic animals has gradually gained attention in recent years. Fish and other aquatic animals rely on circadian rhythms to regulate their daily physiological and behavioral activities, such as feeding, swimming, and reproduction. The stability of circadian rhythms is essential for fish health. If the rhythm is imbalanced, the physiological activities of fish are disrupted, potentially leading to decreased digestive and absorptive capacity, weakened immune responses, and increased infection risk. Therefore, maintaining stable circadian rhythms indicates healthy aquaculture practices that promote overall fish health and aquaculture efficiency. The stable operation of circadian rhythms is influenced by various environmental factors, including light, temperature, salinity, dissolved oxygen, and density. These factors not only influence the formation of circadian rhythms but can also alter the metabolism and behavior of fish by disrupting physiological homeostasis. Despite environmental stress causing temporary imbalances in circadian rhythms, fish generally possess the capacity to readjust their physiological rhythms through adaptive changes and establish a new steady state suited to the new environment, thereby allowing their physiological rhythms to resynchronize with the external cycle and potentially approximate the rhythmic state of standard aquaculture environments. This phenomenon can be considered an expression of adaptability in fish. Therefore, stress resistance can be effectively assessed by monitoring changes in the stability of circadian rhythms in groups or families, thereby establishing a scientific basis for the genetic selection of stress-resistant traits in aquaculture. Circadian rhythms encompass various aspects, such as digestive and metabolic, immune, and endocrine rhythms. Among these, digestive and metabolic rhythms, fundamental to life activities, are particularly important for fish and other aquatic animals because they directly affect the efficiency of energy utilization by the body and subsequently influence the growth rate and health status. Digestive and metabolic rhythms are primarily monitored by detecting a series of key physiological and biochemical indicators, including digestive enzyme activity, glucose levels, cortisol concentrations, and triglyceride and cholesterol contents. These indicators can reflect the digestive function and stress status of fish at various time points. Despite applying these biochemical indicators in the health assessment of farmed fish, research remains limited regarding the impact of various aquaculture environments on the circadian rhythms of digestion and metabolism. Furthermore, there is even less investigation into how these rhythms are altered by different environmental pressures and their correlation with the stress resistance of fish, which requires further study. In this study, we aimed to investigate the circadian rhythm of digestive metabolism in turbot (Scophthalmus maximus) juveniles under various culture conditions. The experiment had three experimental groups, each representing distinct aquaculture environments: high-temperature group (23 °C, salinity 30), control group (16 °C, salinity 30), and low-salinity group (16 °C, salinity 10). The water temperature was controlled using a chiller, and salinity was regulated by adjusting seawater and freshwater flow rates. The light cycle was regulated by an automatic timer, with a light period occurring from 6:00 to 20:00 and a dark period occurring from 20:00 to 6:00 the next day. Feeding was conducted daily at 6:00 and 18:00, with each feeding session amounting to 1% of the body weight of the fish. Residual feed and feces were expeditiously cleaned to maintain water quality. The experiment lasted 30 days. After the experiment was concluded, sampling was performed every 4 h for 72 h (at 08:00, 12:00, 16:00, 20:00, 24:00, and 04:00) to analyze the weight gain of juvenile turbot in different aquaculture environments and changes in key digestive enzymes (trypsin, lipase, and amylase) and serum metabolites (cortisol, glucose, cholesterol, and triglycerides). The results showed that the average weight of the high-temperature group was significantly lower than that of the control and low-salinity groups (P ＜ 0.05), whereas no significant difference was observed between the low-salinity and control groups. The rhythmicity of trypsin, lipase, and amylase did not reach significant levels in any of the groups. However, the digestive enzyme activity in the control and low-salinity groups demonstrated regular fluctuations during the first 48 h, whereas the high-temperature group lacked clear regularity, resulting in earlier peaks and more drastic variations. The cortisol analysis indicated that the 72-h and 48-h rhythms in the control group were significant (P ＜ 0.05), whereas the low-salinity and high-temperature groups did not show significant rhythmicity. Cortisol levels in the high-temperature group were significantly higher than those in the other two groups at multiple measurement points (P ＜ 0.05), whereas no significant difference was detected in cortisol levels between the low-salinity and control groups. Glucose concentrations did not show significant rhythmicity in any group. However, the control and low-salinity groups demonstrated marked periodic changes in glucose levels during the first 48 h, whereas the high-temperature group showed irregular fluctuations. The analysis of triglyceride and cholesterol levels revealed significant rhythmicity in both the control and low-salinity groups over 48 h (P ＜ 0.05), whereas the high-temperature group did not show significant rhythmicity. The triglyceride levels in the low-salinity group were slightly lower than those in the control group at most measurement points, whereas the triglyceride and cholesterol levels in the high-temperature group were significantly higher than those in the control group (P ＜ 0.05). Our findings indicate that high temperature has a greater impact on the metabolism and digestive functions of juvenile turbot than low salinity, manifesting as significant stress responses and abnormal digestive enzyme activities. Serum cortisol and triglyceride levels can accurately reflect the health status and stress response of turbot under different environmental stress conditions and may serve as indicators for evaluating resilience breeding. This study elucidates the dynamic changes in the digestive metabolic rhythms of turbot across different aquaculture environments, providing novel insights into the metabolic regulation mechanisms affected by environmental stress. The results not only enhance the health management of turbot but also provide potential indicators and practical references for resilience breeding.
