Isaria cicadae Miquel Improves the Growth Performance, Physiological Response and Meat Quality of Giant Freshwater Prawn, Macrobrachium rosenbergii

Wenjuan Yan1, Qunlan Zhou2, Bo Liu2,3*, Cunxin Sun2, Huimin Zhang3 and Changyou Song2 1Zhejiang BioAsia Pharmaceutical Company, Co., Ltd., China 2Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture and Rural Affairs, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, China 3Wuxi Fisheries College, Nanjing Agriculture University, Wuxi 214081, China Article Information Received 15 January 2019 Revised 19 July 2019 Accepted 20 September 2019 Available online 06 November 2020


INTRODUCTION
A quaculture has progressed as one of the fastest food producing segments subsequently to agriculture, contributing significantly to global food security. Crustacean is one of the major parts of the global aquaculture production (Asaikkutti et al., 2018). The giant freshwater prawn, Macrobrachium rosenbergii, is a famous economically species among crustaceans, which distributed from the south of China to the East of China, due to its faster growth, better tolerance to the environment and delicious meat quality (Mohan et al., 2016). There were lots of reports on the nutrient requirement of giant freshwater prawn, such as protein level (Teshima et al., 2006), amino acid (Dong et al., 2018), lipid (Muralisankar et al., 2014), vitamins (Asaikkutti et al., 2016, etc. There were also some reports related the use of plant minerals (Tavabe et al., 2013;Muralisankar et al., 2015), extraction in the diet of giant freshwater prawn (Liu et al., 2010a(Liu et al., , 2010bSriket et al., 2011;Chang et al., 2013;Rattanavichai and Cheng, 2015). However, most of them were limited on the improvement of immunity. A little information is available on the role of the meat quality of giant freshwater prawn.
Isaria cicadae Miquel is a traditional Chinese medicine, which is widely used as a tonic for nourishment as well as a functional food, for it possesses remarkable biological activity including immunomodulatory activity, antioxidation, anti-aging, antitumor, anti-inflammation and amelioration in renal function . It has been found that the main active components are adenosine and polysaccharide. Polysaccharides are prebiotic substance which is widely accepted as a dietary ingredient for regulating growth and health status of organisms including humans (Bohn and BeMiller, 1995;Zekovic et al., 2005). In the aquaculture industry, dietary supplementation of polysaccharides (k-carrageenan, laminaran, β-glucan, chitosan, chitin, fucoidan, mannan oligosaccharide) can O n l i n e

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regulate the survival, growth and immune performance in fish and crustaceans (Chang et al., 2000;Murthy et al., 2009;Rodregeuz et al., 2007;Meshram et al., 2014;Sivagnanavelmurugan et al., 2014;Kumar et al., 2006). It was also reported that the culture medium after culturing I. cicadae could improve the growth, decrease the feed conversion ratio, and increase the immunity of chicken (Liu et al., 2012). However, no reports could find about the effects of I. cicadae on the crustaceans and fish. Hence, the current study was performed to evaluate the effect of dietary I. cicadae on the growth performance and feed utilization of giant freshwater prawn, to investigate the plasma biochemistry factors related the protein metabolism and to find the changes on the muscle quality of prawn so as to provide the theoretical basis for prophylaxis of shrimp diseases.

Ethics statement
The experiments were performed in accordance with the guidelines on the care and use of animals for scientific purpose set by the Institutional Animal Care and Use Committee (IACUC) of the Chinese Academy of Fishery Science (CAFS). This study was specifically approved by the Committee on the Ethics of Animal Experiments of Freshwater Fisheries Research Center at Chinese Academy of Fishery Science. All efforts were made to minimize the suffering of the animals.

Diet preparation
According to Wang et al. (2004), the basal diet was formulated with 40.54% crude protein and 7.16% crude lipid (Table I). The protein ingredients of basal diet included fishmeal, shrimp meal, soybean meal, and the lipid was fish oil and soybean oil. The fruiting bodies of I. cicadae was added into the basal diet with the levels as 0 (D1, control diet), 100 mg Kg -1 (D2), 200 mg Kg -1 (D3), 400 mg Kg -1 (D4) and 800 mg Kg -1 (D5). All ingredients except fish oil and soybean oil were ground into powder through 60 mesh sieve, mixed with fish oil, soybean oil and water, then forced through a pelletizer (die diameter 1.0 and 2.0 mm, South China University of Technology, Guangzhou China) and dried in a ventilated oven at 45°C. The diameter of diet was 1mm. After drying, all diets were sealed in bags and stored at -15°C until used.

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Experimental system and animals Healthy prawn fry were obtained from South Taihu Freshwater Hatchery Company, Huzhou, China. Prior to the feeding trial, prawn were fed with control diet (D1) for 2 weeks to acclimate to the experimental diet and conditions. After fasting for 24 h, prawn fry (initial weight 0.243 ± 0.003 g, n = 50) were randomly distributed into 30 concrete tanks (2.0 m × 1.50 m × 0.70 m) with 50 prawn in each tanks. Each diet was randomly assigned to six tanks. Some meshes were put into the tank to help the prawn avoid the attack from others. Water from an underground source was used. Daily silt was siphoned before feeding with an exchange of one-third of water in each tank once every two weeks.
Prawns were hand-fed with the experimental diets three times daily at 7:00, 12:00 and 18:00. The amount of feeding diet was 5% to 10% of the prawn weight based on the feed intake every day. The residue of the feed was collected half hour after feeding. During the 9 week feeding trial, the number and weight of dead prawn and feed consumption were recorded every day. Photoperiod was set at natural conditions. Water temperature fluctuated from 25 to 30 °C, pH was ranged 7.8-8.5 and ammonia nitrogen was less than 0.5 mg L -1 . Dissolved oxygen was proved by aeration round the clock.

Sample collection and analysis
At the end of the feeding trial, prawns were starved for 24 h to evacuate the alimentary tract contents prior to sampling period. All prawns in each tank were counted and weighed for calculating the relative indexes. 18 prawns per group (three prawns per tank) were taken, and then blood samples were collected from the cardiocoelom using syringes. Alsever's solution was used as the anticoagulant, and the proportion of haemolymph to the anticoagulant was 1:1 (Rodriguez et al., 1995). The blood was centrifuged (3500×g, 10 min, 4°C) to obtain plasma. Plasma was separated and stored at -80°C until use. At last twelve prawns from the same tank were dissected and got the muscle to detect the meat quality.

Biochemical measurements related to protein metabolism
Twelve prawns of each group (2 prawns/each tank, 6 tanks per group) were selected randomly for haemolymph biochemical parameters. The aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were determined by the colorimetric method using a Mindray Auto Bio-chemical Analyzer (BS-400, Mindray, P.R. China) described as Zhou et al. (2018). The total protein (TP), albumin (ALB) and urea nitrogen (UN) contents were detected by the colorimetric method using a Mindray Auto Bio-chemical Analyzer (BS-400, Mindray, P.R.

Meat quality of prawn Compression loss and cooking loss
The water holding capacity of the meat was evaluated by the compression loss according to the method of Zhou et al. (2018) with some modification. 12 prawns of each group (2 prawns/each tank, 6 tanks per group) were prepared to detect compression loss. Before packaging with layers of filter paper, the samples were weighed in advance. Then, they were pressured with the persistent power of 35 kg for 3 min using confining pressure-free strain control instrument (Nanjing Soil Instrument Factory, Nanjing, P.R. China). 12 prawns of each group (2 prawns/ each tank, 6 tanks per group) were used to determine cooking loss. Cooking loss was performed according to Qi et al. (2018), with some modifications.

Texture profile analysis
Twelve prawns of each group (2 prawns/each tank, 6 tanks per group) were selected randomly for texture profile analysis (TPA) with XT plus texture analyser (Stable Micro Systems Ltd., Godalming, UK). Hardness, springiness, cohesiveness, gumminess, chewiness and resilience were determined according to De Huidobro et al. (2005) and analyzed with software Texture Expert Exceed (Stable Micro Systems Ltd).

Sensory evaluation
Sensory evaluation was carried out according to Yang et al. (2015) with some modifications. A ten-member sensory panel was trained before evaluation. According to the Chinese standard GB/T 22210-2008, each panellist was experienced in assessing sensory evaluation, including odour, whiteness, brightness, color, flavor, delicacy, succulency, fishiness and tenderness. All attributes were scored on five-point scales with small modifications (Kang et al., 2011). Each evaluation session was conducted at the same time of all samples. A total of ninety prawn samples were evaluated, and each sample was evaluated twice.

Calculation and statistical analysis
Data were the mean of the raw data from the same tank. Therefore, a total of six data were used to analyze every parameter. Then data were analyzed using SPSS version 13.0 for Windows (SPSS Inc., Chicago, USA).

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A one-way analysis of variance (ANOVA) was used to compare the means of the main effects and was followed by Duncan's test to detect significant differences between the means. All data are presented as mean ± standard error (SE). Significance was determined as a P value < 0.05.

Growth performance and feed utilization
Growth performance is shown in Table II. 75-84% survival rate was observed in for all treatments and no significant differences were found among the all groups (P > 0.05). All the treatment group of I. cicadae could significantly improve the specific growth rate (SGR) and protein efficiency ratio (PER) compared to the control group (P < 0.05). Meanwhile, the feed conversion rate of the prawn fed with100 mg Kg -1 and 800 mg Kg -1 I. cicadae was significantly lower than those fed with control diet (P < 0.05). The feed intake in all treatments was significantly lower than those in control group (P < 0.05).

Biochemical measurements related to protein metabolism
The blood biochemical parameters are shown in Table  III. The ALT activities of those fed with diet containing 100 mg Kg -1 and 800 mg Kg -1 I. cicadae were significantly higher than those fed with control diet (P < 0.05). While the AST activity of prawn fed with 400 mg Kg -1 I. cicadae were significantly lower than those fed with control diet (P < 0.05). The total protein, albumin and globulin contents in the plasma of all treatments were significantly higher than that of control group (P < 0.05). While the urea nitrogen of the treatment groups were keep similar with that of the control group except those fed with 800 mg Kg -1 I. cicadae, which was significantly higher than control group (P < 0.05).

Haemolymph non-specific immunity parameters
The haemolymph SOD, GSH-PX, MDA and lysozyme are shown in Table IV. The SOD activity of prawns in the group of 400 mg Kg -1 I. cicadae were significantly higher than that of the control group. The MDA contents of prawns in the group of 400 mg Kg -1 I. cicadae and 800 mg Kg -1 I. cicadae were significantly lower than those in the group of control and 100 mg Kg -1 I. cicadae. The GSH-PX activities of prawns in the group of 100 mg Kg -1 I. cicadae and 800 mg Kg -1 I. cicadae were significantly higher than those in the control group. There were no significant differences of the lysozyme activity among the groups.

Meat quality of the prawn
Compression loss and cooking loss of prawn are shown in Table V. The compression loss of the prawn fed with diets including I. cicadae was significantly higher than the control group except the group of 100 mg Kg -1 I. cicadae (P < 0.05). However, the cooking loss of the prawn in all treatment groups was similar with that of control group, while the cooking loss in the group of 800 mg Kg -1 I. cicadae was significantly lower than that in the group of 400 mg Kg -1 I. cicadae (P < 0.05).
The texture profile of giant freshwater prawn are shown in Table VI. The gumminess of the prawn fed diet with I. cicadae was significantly stronger than that of the control group (P < 0.05). The hardness and chewiness of the prawn of the treatment group were significantly stronger than those of the control group (P < 0.05), except of those fed with 400 mg Kg -1 I. cicadae. The springiness, cohesiveness and resilience of the prawn were similar in all groups (P > 0.05). Note: D1(control diet), D2(100 mg Kg -1 ), D3(200 mg Kg -1 ), D4(400 mg Kg -1 ) and D5(800 mg Kg -1 ). Five-point scales were used. Odour, 1: abnormal taste, 5: odourless; whiteness, 1: not white, 5: very white; brightness, 1: lacklustre, 5: bright; color, 1: light, 5: bright; flavor, 1: dislike, 5: like; delicacy, 1: tasteless, 5: delicious; succulency, 1: dry, 5: succulent; fishiness, 1: without fishy, 5: very fishy; and tenderness, 1: hard, 5: very tender. Sensory evaluations of the muscle, including odour, whiteness, brightness, color, flavor, delicacy, succulency, fishiness, and tenderness, of giant freshwater prawn were evaluated (Fig. 1). I. cicadae in diet did not significantly affect the brightness, color, flavor, delicacy, fishiness and tenderness of the giant freshwater prawns (P > 0.05), however it significantly changed the odour, whiteness and succulency of the muscle of the prawn (P < 0.05). The odour of the muscle of the prawn fed diet containing 100 mg Kg -1 Values are means ± SEM of six samples. Means in the same column with different superscripts are significantly different (P < 0.05). Survival rate (%) =100 × (final amount of prawn) / (initial amount of prawn); WGR (weight gain rate, %)= 100× (final prawn weight -initial prawn weight) / initial prawn weight; SGR (specific growth rate, % day -1 ) =100× (Ln final prawn weight −Ln initial prawn weight) / the experimental duration in days; FCR (feed conversion ratio) = dry diet fed / wet weight gain; PER (protein efficiency ratio) = wet weight gain/ dry protein intake; Feed intake (FI, g d -1 )= dry diet fed/ (amount of prawn × the experimental duration in days).   I. cicadae was significantly stronger than that of the control group (P < 0.05). The whiteness of the muscle of the prawn from the treatment group was significantly declined than that from the control group except the group of 800 mg Kg -1 I. cicadae (P < 0.05). The I. cicadae in the diet of giant freshwater prawn significantly decreased the succulency of the muscle compared with the control group (P < 0.05).

DISCUSSION
It was found that I. cicadae could improve the growth of giant freshwater prawn, increase the protein utilization in  Values are means ± SEM of twelve samples. Means in the same column with different superscripts are significantly different (P < 0.05).
the diet and reduce feed conversion ratio in this study. It was similar result of anthraquinones extracted from Rheum officinale Bail (Liu et al., 2010a(Liu et al., , 2010b and Ganoderma lucidum polysaccharides (Mohan et al., 2016), which suggested the suitable level of Chinese herbs in the diet could increase the growth performance of giant freshwater prawn. This may be related with the active components in the herb extraction (Liu et al., 2010a(Liu et al., , 2010bSriket et al., 2011;Chang et al., 2013;Rattanavichai and Cheng, 2015). Based on our detection, the concentration of polysaccharide in I. cicadae reaches 71.8 g Kg -1 , and the concentration of adenosine is 0.85 g Kg -1 . Dietary polysaccharides and adenosine can be digested and used as potential immune matters and energy sources in fish and prawn. It was reported that polysaccharides, such as β-glucan, chitin and fucoidan in the diet could significantly increase the survival, growth performance, and protein efficiency ratio on M. rosenbergii, Litopenaeus vannamei and Penaeus monodon (Sung et al., 1994;Shiau and Yu, 1998;Chang et al., 2000;Murthy et al., 2009;Rodregeuz et al., 2007;Meshram et al., 2014;Sivagnanavelmurugan et al., 2014;Kumar et al., 2006). It was proved that dietary fructooligosaccharide at 0.4% significantly improved growth of M. rosenbergii, while the higher concentration (above 1%) induced oxidative stress (Chen et al., 2017). In this study, the results showed that all the treatment groups of 100-800 g Kg -1 I. cicadae could significantly improve the specific growth rate and protein efficiency ratio. The group pf 100 mg Kg -1 and 800 mg Kg -1 I. cicadae reduced feed conversion rate of the prawn compared with the control diet. Based on the feed conversion ratio and specific growth rates, the concentration of 100 mg Kg -1 I. cicadae was suit for the growth performance of M. rosenbergii. The influence of I. cicadae on the growth of M. rosenbergii was further confirmed with some blood parameters. It could be found that 400 mg Kg -1 I. cicadae significantly reduced the AST activity compared to the control. Similarly, Liu et al. (2010b) found that the doses of 0.1% -0.2% anthraquinones could reduce ALT and AST activities compared to the control prawn (Liu et al., 2010b). Nakano et al. (1999) who reported that AST and ALT activities in rainbow trout fed a diet containing astaxanthin under the oxidative stress were significantly lower than those of the control fish (Nakano et al., 1999). Rao et al. (2006) also found the AST and ALT activities in Labeo rohita significantly decreased in the dose of 0.05% Achyranthes aspera compared with the control fish (Rao et al., 2006). AST is important indices for diagnosis of hepatopancreas function and damage for aquatic animals (Ozaki, 1978). So, 400 mg Kg -1 I. Miquel in diet fed to M. rosenbergii may contribute to protect the hepatopancreas function.
The haemolymph protein content is used as an immune parameter indicating whether the prawn is healthy or not (Bachère et al., 1997). Rainbow trout treated with some fructoses improved haemolymph total protein content (Jeney et al., 1997). M. rosenbergii fed with 0.1%-0.2% anthraquinone extract also improved haemolymph total protein content. This experiment showed that the haemolymph total protein content significantly increased in fish fed with 0.4% anthraquinone extract before the stress, in fish treated with 0.2% anthraquinone extract at 6h and 12h after stress, and in fish treated with 0.4% anthraquinone extract at 12h, 24h after the stress as well. So, it indicated that the supplement of 0.1%~0.2% anthraquinone extract may elevate the haemolymph protein concentration, prevent the harm of the stress, improve the health of freshwater prawns and keep them in good condition to some degree. In this experiment, the total protein, albumin and globulin contents in the plasma of all treatments were significantly higher than that of control group. So, it indicated that the supplement of 100-800 mg Kg-1 I. cicadae elevated the haemolymph protein concentration, improved the health of freshwater prawns and kept them in good condition.
Aquatic animals have a complex system of multiple types of antioxidants, such as glutathione, catalase, superoxide dismutase and various peroxidases to prevent the oxidation of non-self antigens, to protect the organism from oxidative damage (Holmblad et al., 1999;Munoz et al., 2000). Activation of non-specific defense mechanisms in prawn is evident by increasing SOD, GSH-PX, which O n l i n e

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Effects of I. cicadae on Giant Freshwater Prawn can lead to the decrease of MDA content (Liu et al., 2010a(Liu et al., , 2010b. In fish, Chinese herbal extracts in the diet can improve the antioxidant ability and reduce the mortality of fish (Xie et al., 2008;Christybapita et al., 2007). In prawns, it has been proved the Chinese herb such as anthraquinones extracted from Rheum officinale Bail could increasing the non-specific immunity of giant freshwater prawn even under the high temperature stress (Liu et al., 2010a(Liu et al., , 2010b. Consistent with these studies, in the present study the haemolymph SOD activity was enhanced significantly after feeding with 400 mg Kg -1 I. cicadae, the haemolymph GSH-PX activity was increased significantly after feeding with 100, 800 mg Kg -1 I. cicadae compared to the control group, which led to the significantly decrease of MDA content in the group of 400, 800 mg Kg -1 I. cicadae. Thus, the fruit of I. cicadae could reduce the damage of oxidative stress. Giant freshwater prawn is famous on its delicious meat quality (Mohan et al., 2016), while no report concern on its meat quality. We evaluated the meat quality of giant freshwater prawn in this study for the first time based on the water holding capacity, texture profile and sensory evaluation. Cooking loss and compression loss are the primary indicators of water holding capacity of meat (Chen et al., 2015). At present study, we found that 100 mg Kg -1 I. cicadae did not affect the cooking loss and compression loss increase the water holding capacity of muscle with the decrease trend of cooking loss in the treatment group.
Water mainly exists between the myofibrils and most immobilized water is within the myofibrils (Ali et al., 2015). Thus I. cicadae may lead to the increase of the water between the myofibrils, which may relate to the texture profile of the muscle. It was found that 100, 200, 800 mg Kg -1 I. cicadae could increase the gumminess, hardness and chewiness of the muscle of M. rosenbergii compared with the control, which belong to the texture profile of muscle. The gumminess, hardness and chewiness of prawn are usually significantly lower than those of beef, pork and chicken. Thus, the increase of gumminess, hardness and chewiness may result in the harder of muscle than the normal one, which may lead to more delicious of prawn. According the data of the sensory evaluation, the odour of prawn fed diet with 100 mg Kg -1 was increased and the whiteness and the succulency were reduced. It was a direct sensory that I. cicadae could make the prawn more delicious.
Therefore, it can be concluded that diet with 100 mg Kg -1 I. cicadae could improve the growth performance by increasing the protein efficient ratio and by increasing protein metabolism, enhance haemolymph GSH-PX, TP, ALB, GLB and reduce feed conversion ratio and haemolymph AST, MDA. Meanwhile 100 mg Kg -1 I. cicadae in diet could improve the meat quality of giant freshwater prawn, especially the odour compression loss, gumminess, hardness and chewiness of the muscle. So, the supplement of 100 mg Kg -1 I. cicadae in the diet can increase immune and anti-oxidation capability of prawns, enhance the meat quality and promote the growth of prawns. This work provides the theoretical basis for I. cicadae of the prawns feed.

Statement of conflict of interest
Authors have declared no conflict of interest.  (95)