Effects of emodin and vitamin C on growth performance, biochemical parameters and two HSP70s mRNA expression of Wuchang bream (Megalobrama amblycephala Yih) under high temperature stress

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Abstract

In order to study the effects of dietary emodin, high-dose vitamin C (Vc) and their combination on growth of Wuchang bream (Megalobrama amblycephala Y.) and its resistance to high temperature stress, 1200 healthy Wuchang bream with initial body weight of 133.44 ± 2.11 g were randomly divided into four groups: a control group fed with basal diet (containing 50.3 mg/kg Vc) and three treated groups fed with basal diets supplemented with 60 mg/kg emodin, 700 mg/kg Vc, and the combination of 60 mg/kg emodin + 700 mg/kg Vc, respectively. After feeding for 60 days, the growth performance of Wuchang bream was measured. Then 25 fish per tank were exposed to heat stress of 34 °C. The biochemical parameters of blood and liver, and expression levels of liver two HSP70s mRNA before and after heat stress were determined and the cumulative mortality of each group under heat stress was counted. The results showed that before stress, compared with the control, the weight gain (WG) and specific growth rate (SGR), serum total protein (TP), lysozyme (LSZ), and alkaline phosphatase (ALP) levels, liver superoxide dismutase (SOD) activity and expression level of HSP70 mRNA significantly increased in emodin and Vc groups while feed conversion rate (FCR), serum cortisol (COR), triglyceride (TG) and liver malondialdehyde (MDA) contents decreased (P < 0.05); liver catalase (CAT) activity also significantly increased in emodin group (P < 0.05). Although serum TP, LSZ, and liver HSP70 mRNA levels significantly increased and liver MDA level decreased in combination group (P < 0.05), no synergism was observed.

After heat stress, compared with the control, the serum TP, LSZ, ALP levels, liver SOD, CAT activities, and expression levels of HSC70 and HSP70 mRNAs increased in emodin and Vc groups in varying degrees and serum COR, glucose, glutamic-pyruvic transaminase (GPT), glutamic-oxaloacetic transaminase (GOT), TG and liver MDA levels decreased to some extent. Although these parameters had similar changing trend as above ones in combination group, it did not show any synergism either. Statistics showed that under heat stress, the cumulative mortalities of emodin and Vc groups, except at 6 h in emodin group, were significantly lower than that of the control (P < 0.05) while the difference between the combination and control groups was not significant (P > 0.05). Thus, the basal diet supplemented with 60 mg/kg emodin or 700 mg/kg Vc could promote the growth of Wuchang bream, reduce FCR, increase non-specific immunity of fish, antioxidant capacity, and two HSP70s mRNA expression levels, and enhance resistance to heat stress in fish. However, the combination of emodin and high-dose Vc showed no better effect.

Highlights

► Fish fed with 60 mg/kg emodin, 700 mg/kg vitamin C, and their combination for 60 days. ► Then 25 fish per tank were exposed to heat stress of 34 °C. ► Either emodin or high-dose vitamin C could promote the growth of fish, lower FCR. ► Increasing non-specific immunity of fish, antioxidant capacity and resistance to heat stress. ► The combination of emodin and high-dose Vc did not result in better effect.

Introduction

In aquaculture, fish often encounter high temperature, crowding, water quality deterioration and the invasion of bacteria and viruses. All these adverse environmental factors might disturb the balance and harmony between fish and the environment, causing stress response in fish [1]. Although it is a protective response, if the stress intensity is too high or the stress lasts too long, it may cause immunosuppression in fish, resulting in increased susceptibility to various pathogens [2], [3], and impaired growth [4]. In addition, a large number of antibiotics and hormones are added in feed to control fish diseases, resulting in drug resistance and residues and other issues, detrimental to the sustainable development of aquaculture, seafood safety, and human health. Therefore, more attention is being paid to improving fish immunity and anti-stress through nutrition ways.

Emodin (1, 3, 8-trihydroxy-6-methyl-anthraquinone) is an active anthraquinone constituent extracted from the rhizome of rhubarb Rheum officinale Baill. Many actions have been reported for emodin, such as anti-bacterial and anti-inflammatory [5], antioxidation and scavenging free radicals [6], reducing blood lipid [7], protecting the liver [8], and regulating immunity [9]. We previously reported that anthraquinone extract (the main components: emodin, chrysophanol and rhein) can promote the growth of freshwater prawn (Macrobrachium rosenbergii) and enhance its immunity and resistance to high temperature [10].

Vitamin C (Vc), also known as l-ascorbic acid, is an essential nutrient for normal physiological functions in fish, and most teleosts are unable to synthesize ascorbic acid due to lack of l-gulonolactone oxidase (EC 1.1.3.8) responsible for the de novo synthesis of Vc [11]. Therefore, exogenous Vc is required in fish diet. Vc is an important immunostimulant [12]. Fish fed with high dosage of Vc can improve the immunity and resistance to pathogens [13], [14], [15], [16], [17], and alleviate the negative effects of stress [18], [19], [20]. As an important antioxidant, Vc can reduce the increase of tissue lipid peroxide and inhibit the decreased antioxidant enzyme activity induced by high temperature [19], [20], [21].

The 70 kDa heat shock proteins (HSP70s) are primarily encoded by two kinds of genes, a constitutive type HSC70 and inducible type HSP70 [22]. These two HSP70s play key roles in the cells, including molecular chaperone, antioxidant, synergetic immunity and other biological functions [23], [24], [25]. HSC70 is mainly associated with physiological processes such as cell division, proliferation and development [26], [27] whereas HSP70 is mainly involved in immune protection, improving cell viability and strengthening stress tolerance to adverse environment or pathogens [28], [29].

Wuchang bream (Megalobrama amblycephala Yih) is a major freshwater species cultured in China. In recent years diseases of cultured Wuchang bream showed an increasing trend, especially in summer, high temperature causes disease outbreak, resulting in high mortality. We sought to study the effects of emodin and high-dose Vc on growth and heat stress resistance in Wuchang bream, and the result would suggest new avenues for the alleviation of stress and prevention of fish diseases.

Section snippets

Experimental design and diets

Wuchang bream were obtained from Yixing fish farm of Freshwater Fisheries Research Center, ChineseAcademy of Fishery Sciences. Twelve hundred healthy fish with initial body weight of 133.44 ± 2.11 g were selected and randomly divided into four groups: the control group fed with basal diet (Table 1) and three treated groups fed with the basal diets supplemented with 60 mg/kg emodin, 700 mg/kg Vc, and the combination of 60 mg/kg emodin + 700 mg/kg Vc, respectively. Each diet was tested in

Effects of emodin and Vc on growth performance of Wuchang bream

Table 2 shows that both WG and SGR of emodin and Vc groups significantly increased in comparison to the control (P < 0.05), in which the WG increasing by 9.93% and 13.38%, respectively. Both WG and SGR of the combination group were not significant difference compared with those of the control (P > 0.05). The FCRs of emodin and Vc groups were significantly lower than that of the control (P < 0.05) whereas the difference of FCR between the combination group and control was not statistically

The effects of emodin and Vc on growth performance

In this experiment the basal diet supplemented with 60 mg/kg emodin could increase the WG and SGR and lower FCR of Wuchang bream. This is consistent with our previous report that 0.1%–0.2% anthraquinone extract could promote the growth of M. rosenbergii [10]. Vc is an essential nutrient needed to maintain the normal physiological functions of fish. High-dose Vc could improve the WG of matrinxã (Brycon amazonicus) [17] or the SGR of Asian catfish (Clarias batrachus) [15]. But some studies

Acknowledgments

This work was supported by the funds of the National Technology System for Conventional Freshwater Fish Industries of China (nycytx-49-18) and Integration and Demonstration of Freshwater Fish Septicemia Prevention and Cure Techniques (No. 200803013) from Ministry of Agriculture of China. We thank Dr. Ya-Xiong Tao (Auburn University, Auburn, Alabama, USA) for his help in editing the manuscript.

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