Effects Of Emodin On Growth, Blood Physiology And Resistance To Ammonia Nitrogen Stress In Wuchang Bream(Megalobrama Amblycephala)under High Temperature

Emodin (1,3,8-trihydroxy-6-methyl-anthraquinone) is an active anthraquinone constituent extracted from the rhizome of rhubarb Rheum officinale Baill and Reynoutria japonica Houtt. Many actions have been reported for emodin, such as anti-bacterial and anti-inflammatory, antioxidation and scavenging free radicals, reducing blood lipid, protecting the liver, and regulating immunity. In aquaculture, fish often encounter high temperature, high ammonia nitrogen stress. Wuchang bream (M. amblycephala) is a major freshwater species cultured in China. In recent year’s high temperature caused diseases outbreak of cultured Wuchang bream showed an increasing trend, especially during summer, resulting high mortality. We sought to study the effects of emodin on growth and high ammonia nitrogen stress resistance in Wuchang bream, and the result would suggest new avenues for the alleviation of stress and prevention of fish diseases. Three hundred healthy fish (average initial weight of7.29±0.01g) were selected and randomly divided into four groups rearing at25℃and30℃temperatures. At each temperature, the fish were fed with basal diets and supplemented with25mg/kg emodin, respectively, for77days. After77feeding trial the fishes were challenged with high ammonia nitrogen (10mg/L) for14days; then infected with Aeromonas hydrophila. This study includes two aspects, such as:1. Effects of emodin on growth performance and blood parameters of Wuchang bream (M. amblycephala) under high temperature:At the end of the rearing experiment, the growth performance and blood physiology were investigated. The results showed that the weight gain rate (WGR), specific growth rate (SGR), hematocrite (HCT) and plasma lactate dehydrogenase (LDH) of the fish reared at30℃ significantly (P<0.05) increased, whereas the feed conversion ratio (FCR) and viserosomatic index (VSI), cortisol, aspertate aminotransferase (AST), urea (UREA), triglycerides (TG), significantly (P<0.05) reduced, compared to those reared at25℃. The SGR, hepatosomatic index (HSI), HCT and plasma LDH of the emodin group significantly (P<0.05) increased, but the FCR and feeding rate (FR), crotisol significantly (P<0.05) reduced, compared to the control group. There was an interaction between temperature and emodin for the total cholesterol (TC), complement3(C3), and complement4(C4)(P<0.05). Besides, the TC, C3, and C4of the emodin group reared at30℃were significantly higher than that at25℃(P<0.05). However, there was no significant difference was observed on white blood cell (WBC), red blood cell (RBC), haemoglobin (HGB), triiodothyronine (T3), thyroxine (T4), ALT, glucose, high density lipoprotein cholesterol (HDL-C), and low density lipoprotein cholesterol (LDL-C) among the groups. The results indicated that the feed with25mg/kg emodin can help to alleviate the long-term high temperature effects on blood physiology, improve immunity, and promote the growth of Wuchang bream.2. Effects of emodin on resistance to ammonia nitrogen stress and Aeromonas hydrophila infection of Wuchang bream (M. amblycephala) under high temperature:At the end of the rearing experiment, each group were exposed to high ammonia nitrogen stress (10mg/L) for14days and after stress infected with A. hydrophila. The plasma and liver indices, and the heat shock protein levels (HSPs) mRNA levels were investigated at0,2,7,14d, respectively. The results showed that compared with before stress,2d after stress, the plasma AST, UREA, glucose, and liver heat shock protein60(HSP60) mRNA level of the control group reared at25℃were significantly increased, whereas TG was significantly reduced (P<0.05). Meanwhile, the plasma cortisol, T3, T4, AST, UREA, TG, TC, C3, C4, liver HSP60, and HSP70mRNA levels of the control group reared at30℃were significantly increased (P<0.05).7d after stress, the plasma cortisol, T4, C4, and liver HSP70mRNA level of the control group reared at25℃were significantly increased, whereas the TC and liver HSP90mRNA level were significantly reduced (P<0.05).14d after stress, the plasma glucose and HDL-C of the control group reared at30℃and the C3of the control group reared at25℃were significantly increased, while LDL-C and nitric oxide (NO) significantly reduced (P<0.05). This indicated that the fish is more sensitve to ammonia nitrogen stress at30℃than25℃. Compared with the control group, the dose of25mg/kg emodin significantly (P<0.05) increased; the liver HSP60, HSP70, and HSP90mRNA levels at2and7d after stress; liver HSP70and HSP90 mRNA levels at14d after stress, and reduced plasma cortisol, reactive oxygen species (ROS) contents at7d after stress. In addition, during the whole stress period, the T3, T4, AST and HSP60mRNA level increased at first and then decreased. Furthermore, the long-term high temperature and ammonia nitrogen stress led to damaging of the head kidney cell. Emodin could affect the ultrastructure of head kidney cell of Wuchang bream.After infection with A. hydrophila, the cumulative mortalities of the groups from high to low were25℃control,30℃control,30℃25mg/kg emodin and25℃emodin group, respectively. The results suggested that fish had low resistance to diseases at30℃than25℃and25mg/kg could enhance resistance of Wuchang bream against A. hydrophila infection after ammonia nitrogen stress.In conclusion, the rearing and stress experiment indicate that the addition of25mg/kg emodin in the feed could promote growth, improve immunity, antioxidant capacity, three HSPs mRNA levels, and increase ammonia nitrogen stress resistance, and reduce stress mortality in Wuchang bream.