Optimal dietary lipid and protein level for growth and survival of catfish Clarias magur larvae

Journal Pre-proof Optimal dietary lipid and protein level for growth and survival of catfish Clarias magur larvae Ishfaq Nazir Mir, P.P. Srivastava, I.A. Bhat, Y.D. Jaffar, N. Sushila, P. Sardar, S. Kumar, A.P. Muralidhar, K.K. Jain PII:

S0044-8486(19)32393-2

DOI:

https://doi.org/10.1016/j.aquaculture.2019.734678

Reference:

AQUA 734678

To appear in:

Aquaculture

Received Date: 11 September 2019 Revised Date:

3 November 2019

Accepted Date: 4 November 2019

Please cite this article as: Mir, I.N., Srivastava, P.P., Bhat, I.A., Jaffar, Y.D., Sushila, N., Sardar, P., Kumar, S., Muralidhar, A.P., Jain, K.K., Optimal dietary lipid and protein level for growth and survival of catfish Clarias magur larvae, Aquaculture (2019), doi: https://doi.org/10.1016/j.aquaculture.2019.734678. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

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Optimal dietary lipid and protein level for growth

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and survival of catfish Clarias magur larvae

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Ishfaq Nazir Mira, P.P Srivastavab, I.A Bhatc, Jaffar Y.Dd, N. Sushilad, P. Sardarb,

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S. Kumarb, A.P Muralidhare and K.K Jainb⃰

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a

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Vaniyanchavadi, Chennai- 603 103, India

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b

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Education, Mumbai-400 061, India

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c

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d

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Education, Mumbai-400 061, India

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e

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Andhra Pradesh, India

Institute of Fisheries Post Graduate Studies, Tamil Nadu Dr. J. Jayalalithaa Fisheries University,

Division of Fish Nutrition, Biochemistry and Physiology, ICAR-Central Institute of Fisheries

College of Fisheries Science, Gumla, Birsa Agricultural University, Ranchi, Jharkhand-834006, India Division of Aquatic Environment and Health Management, ICAR-Central Institute of Fisheries

ICAR-Central Institute of Fisheries Education, Kakinada Centre, Old Bumrah Shell Road, Kakinada,

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*Corresponding author:

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Dr. Ishfaq Nazir Mir

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Institute of Fisheries Post Graduate Studies

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TNJFU, Chennai- 603 103, India

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Email: [email protected]

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Phone: 7889850147

Abstract

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Large scale farming of Clarias magur (Indian walking catfish) is limited due to

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the non-availability of seed because of high mortality rate occurring during its larval

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rearing. The aim of the present study was to develop an optimal strategy based on

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the dietary lipids and proteins for enhancing the larval survival. A 3-week study from

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14-35 day post hatching (dph) was conducted to elucidate the effects of dietary lipid

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and protein level on growth, survival and mRNA expression of growth related genes

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of C. magur larvae. Significantly (P < 0.05) high growth on average wet weight basis

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and survival rate was observed in 8% dietary lipid inclusion level followed by 10%

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and 12% lipid contents from 14-35 dph larval rearing phase. The effect of dietary

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lipid on the body composition was also reflected on the lipid content of larvae where

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a significant difference was observed. Similarly, growth-related genes like growth

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hormone (GH) and insulin-like growth factor I (IGF-I) exhibited a significantly high

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expression at 8% lipid level compared to 10% and 12% whereas, insulin-like growth

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factor binding protein3 (IGFBP3) showed a reverse trend. Based on the results, 8%

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dietary lipid along with the protein combination improved the performances

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significantly with regard to growth and survival. However, this experiment did not

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help to differentiate whether the differences observed at different time points are due

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to dietary protein level or the age of larvae. Hence, another experiment was

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conducted in which diets were formulated to contain different protein levels (55, 50

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and 45%) with a constant 8% level of lipid in all the treatments along with

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commercial diet as control. The inclusion of 8% lipid and 55% protein was observed

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to show excellent performance with respect to growth, survival and the mRNA

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expression of important growth-related genes. Hence, a formulated diet with crude

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protein of 55% and 8% lipid can be suggested as the optimal microdiet for the larval

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rearing of C. magur.

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Keywords: Dietary lipid; Growth; Survival; Larval; Clarias magur

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1. Introduction

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Clarias magur, is a neotype of Clarias batrachus also known as Indian origin

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walking catfish and name change of this fish has also been supported through

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mitochondrial analysis of species identification (Devassy et al., 2016). The fish has a

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wide distribution over river basins in India, Bhutan, Nepal and Bangladesh (Ng and

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Kottelat, 2008). It has a good growth potential and efficient food conversion rate

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along with excellent nutritional profile owing to its high protein (15%), low fat (1%)

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and high iron (710 mg/100g) content, which makes the species to act as a suitable

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candidate species for aquaculture (Hossain et al., 2006; Argungu et al., 2013).

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However, the large-scale culture of this species is seriously hampered by the low

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seed availability due to the high mortality rates encountered during its early larval

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rearing, which is the major constraint in the commercial production of C. magur.

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A low survival could be due to several reasons such as lack of brood care,

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lack of knowledge on the optimum stocking density and the stage specific optimum

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nutrient requirements of this fish (Sahoo et al., 2004). Besides these reasons, the

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successful larval rearing also depends on the food and nutritional factors, which

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have a great influence on the growth and survival of larvae of any fish (Ramesh et

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al., 2014). Hence, there must be a proper feeding of the right food with optimum level

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of nutrients as required by the larvae.

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The growth pattern of fish larvae is very fast and its regulation during this

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rearing period is little known for most of the species. The growth regulation at

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molecular level is manifested by the expression of growth-related genes such growth

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hormone (GH), insulin-like growth factors (IGFs) and insulin-like growth factor

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binding proteins (IGFBPs). GH is released by the pituitary gland and its regulation is

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governed by the hypothalamic neuropeptides (Bolander, 2004). Along with growth,

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development, reproduction and some other physiological processes, it also regulates

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nutrient metabolism (Perez-Sanchez et al., 1991; Abdolahnejad et al., 2015).

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Growth hormone binds to its receptor in the target organ and mediates its action by

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the release of IGF-I (Moriyama et al., 2000). Insulin-like growth factors are the

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polypeptides required in the differentiation and proliferation in several vertebrates

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including fish (Perrot et al., 1999) and ultimately promote the body growth. The

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IGFBPs bind tightly with either of the circulating IGF’s in plasma specifically and

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regulates the growth rate of an animal. However, IGFBP3 in particular has IGF-

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dependent or IGF-independent roles to regulate the cellular proliferation (Baxter,

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2013). IGFBP3 is not considered as the major circulating IGFBP in teleosts, but

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there are some reports in fish like Zebrafish, yellowtail and flounder where it has a

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significant influence on the binding of IGF (Chen et al., 2004; Pedroso et al., 2009;

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Safian et al., 2016; Shimizu and Dickhoff, 2017).

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Among the nutrients dietary lipids are considered as the important source of

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energy and essential fatty acids for the fish in addition to have a role as the carriers

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of fat soluble vitamins (Watanabe, 1982). Fish is having a limited ability to utilize

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carbohydrates as a source of energy where lipids are the major sources for optimum

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growth and survival (Krogdahl et al., 2005). Dietary lipids also possess the protein

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sparing action where the optimum levels of lipid incorporation in the diets results in

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the efficient utilization of dietary protein for maximum nitrogen retention and growth

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performance (Cho and Kaushik, 1990). The excess lipid levels in diets might reduce

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the feed intake capacity along with the fat deposition in the fish resulting in a fatty

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fish which affects its market value (Watanabe, 1982; Martino et al., 2002; Mohanta et

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al., 2008). Hence, the knowledge on the optimal dietary lipid provides beneficial

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effects in the fish so for as growth and survival is concerned.

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Protein is an important nutrient and a major organic material in fish tissue,

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comprising about 65-75% proportion of the total on dry-weight basis (Wilson, 2002).

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Protein is also the most expensive ingredient in fish feeds, necessary for growth,

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maintenance and repairing of broken tissues, as well as in the production of

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hormones, enzymes and antibodies required for many vital processes of animals

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including fish. However, excessive protein in diet usually gets converted to energy

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and results in increased excretions of nitrogenous wastes into water, which affects

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the water quality and the growth of fish (McGoogan and Gatlin, 1999). Hence, it is

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essential to estimate the optimum dietary protein level for any fish to achieve their

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best growth at the lowest possible cost.

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As per our previous study the C. magur larvae have the high ability to utilize

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dietary lipids and protein from 13 dph onwards (Mir et al., 2018a and 2019a). This

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outcome of our previous study was used to devise a strategy based on the optimum

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utilization of dietary lipid and protein that could improve the survival rate of C. magur

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which is the major bottleneck for its early larval rearing. Besides, the evaluation of

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growth regulation as manifested by the expression of growth-related genes is

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necessary due to the fast growth rate of fish larvae. Hence, the aim of the present

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study was to evaluate the optimal dietary lipid and protein level in the diets and their

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effect on growth and survival of this fish.

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2. Materials and methods

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2.1. First experimental design

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A dietary strategy was conducted with variable levels of lipids with constant

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protein contents in all the treatments to observe an effect on the growth and survival

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of C. magur from 14-35 dph for three weeks, because this is the phase where a high

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mortality of larvae occurs in this fish. Larvae were reared in larval rearing tanks

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(2.72m×0.62m×0.15m) till the end of experiment as per our previous study (Mir et al.,

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2018a). During the rearing period, the different water quality parameters like

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temperature, dissolved oxygen and pH were found to be at optimum ranges of 28-

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30°C, 6-8 mgL-1 and 7.8-8.2 respectively. Throughout the rearing period, the water

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quality was maintained in normal ranges. Nine experimental diets were prepared

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with 8, 10 and 12% lipid level and 55, 50 and 45% crude protein levels. The

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reduction in the protein content from 55% to 45% was made in all treatments by

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considering the fact that the requirement of dietary protein decreases as the age of

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fish increases. The dietary combinations and the schedule followed for the

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experiment is mentioned in table 1. The experiment was conducted to see the effect

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of experimental diets on the growth, survival and mRNA expression of growth-related

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genes viz. growth hormone (GH), insulin-like growth factor-I (IGF-I) and insulin-like

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growth factor binding protein (IGFBP3).

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2.2. Formulation and preparation of experimental diets

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Commercially available practical feed ingredients (Table 2) were used to

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prepare the diets. Fish protein hydrolysate (FPH) and Soya protein hydrolysate

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(SPH) was purchased from Jeevan chemicals Pvt. Ltd. (Mumbai, India) and New

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Alliance Dye Chem Pvt. Ltd (Mumbai, India) respectively. Nine diets of different lipid

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and protein levels were formulated and prepared in the Fish Nutrition Laboratory of

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ICAR-CIFE (Central Institute of Fisheries Education). The diets were of 45% crude

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protein (CP) with 8% lipid or ether extract (EE)- F1; 50% CP 8% EE (F2); 55% CP

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8% EE (F3); 45% CP 10% EE (F4); 50% CP 10% EE (F5); 55% CP 10% EE (F6);

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45% CP 12% EE (F7); 50% CP 12% EE (F8) and 55% CP with 12% EE (F9) as

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shown in table 2.

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All the ingredients were weighed accurately and mixed thoroughly by the

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addition of adequate quantity of water, except soyalecithin, Vitamin C, vitamin-

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mineral mix and oil. The dough was steam cooked for 15 min in autoclave and

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cooled to room temperature. Then, butylated hydroxyl toluene (BHT) dissolved in

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measured amount of oil and premix of vitamin C and vitamin-mineral mix along with

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the betaine and choline chloride were mixed thoroughly in the dough for uniform

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distribution of micronutrients and other additives. Pellets were prepared by pelletizer

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having 1 mm diameter die size and were spheronized in spheronizer. The pellets

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obtained were air dried for some time and kept in hot air oven at 40°C till complete

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drying to achieve the final moisture level of 10-12% and later, crumbled to small size

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pellets. The pellets were packed in airtight polythene bags for use in the experiment.

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2.3. Sampling

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Sample collections were carried out randomly before feeding in the morning

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when guts were as empty as possible. For this experiment, six different pools of

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larvae were sampled for molecular analysis from the three rearing tanks each

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stocked with 500 larvae. The sampling was done at 21 dph (35 larvae), 28 dph (30

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larvae) and 35 dph (19 larvae) in all the treatments for gene expression studies after

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anaesthetizing with ice-cold water. Samples were also collected for carcass analysis

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from each replicate of all the treatments for first experiment after every week of

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experimental duration (132 larvae at 21 dph, 100 larvae at 28 dph and 75 larvae at

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35 dph).

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2.4. Proximate composition of diets and carcass

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The proximate analyses of diets and carcass were performed by prescribed

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method (AOAC, 1995) in the Fish Nutrition Laboratory of ICAR-CIFE.

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2.5. Growth Parameter

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Larval growth was estimated in terms of total body wet weight on average

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basis by sampling 10-20 larvae in a pre-weighed beaker containing water at 21 dph

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and 28 dph of experiment. The average weight of individual larva was calculated by

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dividing the total biomass of sampled larvae with the total number of collected

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specimens at 14 dph and 35 dph as initial and final weight of larvae. Similarly 10

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larvae at each time point were taken for measuring the length of larvae. Growth was

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estimated on end point basis and at each time point as well.

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2.6. Survival rate

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At the end of experiments, all the experimental tanks were dewatered and the

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number of the experimental animals in each tank was counted and the survival rate

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(%) was calculated by the following formula: % =

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ℎ

× 100

2.7. RNA extraction and cDNA synthesis

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Total RNA was isolated from the whole larval samples collected at different

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time points under study, using Trizol™ reagent (Invitrogen, USA) following

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manufacturer's protocol. The extracted RNA was subjected to DNase I (Thermo

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Scientific, USA) treatment for removing the genomic DNA as carried out in our

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previous study (Mir et al., 2019b). The RNA concentration and purity was then

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measured by Nanodrop spectrophotometer (Thermo Scientific, USA) at OD of

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260/280. First strand cDNA synthesis was carried out from DNase-treated RNA

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using RevertAidTM First Strand cDNA Synthesis kit (Thermo Scientific, USA) using

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Oligo dT primer as per manufacturer’s instructions.

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2.8. Quantitative real-time PCR (qRT-PCR)

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Quantitative PCR primers were designed (Table 3) from the nucleotide

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sequences of C. magur growth-related genes (GH, IGF-I and IGFBP3) available in

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NCBI under accession numbers AF416486.2, KX192142.1 and KC958590.1. The

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PCR efficiency (E) was calculated using the formula: E (%) = (10-1/slope-1) x 100. The

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primers ranging between 95% and 105% efficiency were considered significant for

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their use in the expression analysis (Kubista et al., 2006). The mRNA expression

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level of the selected genes was performed in LightCycler® Real-time PCR detection

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system (Roche, USA). Beta actin (β-actin) was used as reference gene for the

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expression pattern of growth-related genes.

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housekeeping gene among the three previously studied reference genes in C. magur

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(Mir et al., 2018b).

β-actin was validated as a stable

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The 25 µL reaction volume containing 12.5 µL of Maxima™ SYBR Green

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qPCR master mix (Thermo Scientific, USA), 1 µl (10 pM) of each primer, 1 µl (100

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ng) of cDNA and 9.5 µl nuclease free water was used for amplification in real-time

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PCR. Then, each sample was distributed into two wells, having the final volume of

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10 µl. The real-time PCR programme was as follows: initial denaturation of 10 min at

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95°C, followed by 40 cycles of denaturation at 95°C for 20 s, annealing at 57°C (GH

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gene), 56°C (IGF-I gene) and 58°C (IGFBP3 gene) for 30s and final extension of 30s

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at 72°C. The mRNA expression levels were performed using the 2−∆Ct method (Livak

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and Schmittgen, 2001).

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2.9. Second Experimental design and sampling

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Based on the outcome of first experiment, second experiment was designed

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as shown in the table 4. Control group taken in the second experiment was a

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commercial diet (Charoen Pokphand, India).

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Samples for second experiment were taken for analysis after anaesthetizing

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with ice-cold water. The six pools of samples both for RNA were collected at 21 dph

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(35 larvae), 28 dph (30 larvae) and 35 dph (19 larvae) in all the treatments each

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comprising three replicate tanks stocked with 300 larvae per tank. Samples were

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also collected for carcass analysis at the end of second experiment. All the collected

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samples were analyzed for growth, body composition and molecular studies as per

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the above mentioned methods.

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2.10. Statistical analysis

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The results of the growth, survival and quantitative real-time PCR expression

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were analysed by using one-way and two-way ANOVA with a significance level at P

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< 0.05. Shapiro-wilk’s test was performed to carry out the normality and, equality of

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variances were assessed by Levene’s test before performing ANOVA. All the

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statistics were conducted using SPSS 22.0 software (SPSS Inc., USA).

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3. Results

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Experiment I

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3.1. Growth

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The growth on average wet weight basis was evaluated in different treatments

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and is shown in figure 1. and table 5. A significant difference was observed among

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the treatments and higher values of average weight of larvae was found in T1 and T2

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treatments compared to T3 group. Significantly higher values of weight gain and

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length gain were observed at 8% dietary lipid level. The effect of lipid level on the

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growth revealed that 12% lipid has a decreasing effect on the weight of larvae,

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whereas significantly (P < 0.05) highest average weight has been observed in 8%

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dietary lipid level. Also age or protein has also a significant on effect on growth of

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larvae and was found to increase with the increasing age or decreasing dietary

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protein level. However, whether the effect was due to age or protein was not clear in

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this experiment, as each time point also represents a particular dietary protein level.

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The interaction of lipid and age/protein was not observed on the average wet weight

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of larvae.

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3.2. Survival rate

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The mortality of the larvae in all the three treatments was recorded every day

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throughout the experiment. At the end of experiment the survival rate was calculated

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which is shown in figure 2.

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3.3. Proximate composition of feed

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The different experimental diets were prepared to contain variable levels of

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lipid and protein. After proximate analysis the moisture content was found to be in a

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range of 8.28-8.99% and there was no significant (P > 0.05) difference among the

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treatments (Table 6). Crude protein content varied from 45.27% in F1 to 55.73% in

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F9 with a significant (P < 0.05) difference reflecting variable levels of protein in the

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diets. The ether extract was estimated in the range of 8.86-11.82% and there was a

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significant difference between the ether extract of different diets. The crude fiber

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content was found to vary from 3.25-5.46%. Total ash was estimated to lie in a range

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of 12.20-13.27% without a significant difference between the treatments. The

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calculated nitrogen-free extract ranged from 8.25-19.49% with a significant

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difference among the treatments. Finally, gross energy was estimated in bomb

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calorimeter which was observed to be almost similar without any significant

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difference in all the treatments and it ranged from 406-429 KCal/100g.

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3.4. Carcass analysis

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Data pertaining to the carcass analysis of different treatments at initial day

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(14th day), 21th, 28th and 35th day are given in table 7. The moisture content (%) was

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found within a range of 78.78 to 80.19. The crude protein content varied from 11.83-

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13.56% and no significant (P > 0.05) difference was observed in the treatments at all

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the time points. Ether extract was estimated and found to lie in a range of 1.05-

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2.96% with significantly (P < 0.05) high value at T3 group at the end of every week.

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The total ash varied from 1.78-2.65% and a significant (P < 0.05) difference was

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observed among the treatments during first and third week of experiment. The total

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carbohydrate content was calculated and observed to be in a range of 2.37% to

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4.53%.

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3.5. mRNA expression of growth-related genes

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The relative mRNA expression of growth related genes in different treatments

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is shown in table 8. A significant difference was found among the treatments and the

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expression pattern of GH gene started increasing in treatment T1 from 21 dph to 28

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dph and then further declined during 35 dph. Similarly, the same trend was observed

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in T2 group (10% lipid level). However, in T3 treatment (12% lipid level) there was no

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significant difference among the age groups within the treatment and also the

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expression of GH gene was low compared to the other two treatments. The

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expression of growth hormone (GH) gene was significantly (P < 0.05) high at T1 28

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which represents the 8% lipid level and 50% crude protein level at 28 dph. The

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mRNA expression pattern of IGF-I showed a non-significant difference between the

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T1 21, T1 28, T2 21 and T2 28 and was high as shown in table 7. The IGF-I mRNA

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expression was found to be low in T1 35 and T3 treatment. Then, the relative

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expression of IGFBP3 was evaluated which showed an increasing trend within the

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three treatments. A significantly (P < 0.05) high mRNA expression level was

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observed in T1 35 followed by T3 treatment and T2 35 as shown in figure 29.

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The effect of lipid level and age or protein level of diet on the expression of

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growth related genes is given in table 8. The mRNA transcript levels of all the

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studied growth related genes varied significantly (P < 0.05) with lipid levels and age

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or protein levels. It can be seen from the table that the expression of GH gene is

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significantly high in 8% lipid and lowest in the 12% lipid level. The age or protein has

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also an effect on GH gene expression with a significantly high expression at 21 dph

315

and 28 dph age groups and lowest at 35 dph. Similarly, in IGF-I a high level of

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expression was observed in 8 and 10% lipid level at 21 and 28 dph among all the

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treatments. However, a reverse trend was observed in IGFBP3 gene expression in

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which 12% lipid level exhibited a significantly high expression and with regard to age

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35 dph time point showed a significantly high level of mRNA expression. The

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interaction of lipid and age was observed only in GH gene expression among the

321

growth related genes.

322

The 8% lipid level was found to be optimal in improving the performance with

323

respect to growth, survival, mRNA expression of growth-related genes. However,

324

whether the growth performance and enhanced mRNA expression is influenced by

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protein level or age or both was not clear in this experiment. To understand the most

326

influential factor (age/dietary protein), a second experiment was conducted.

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Experiment II

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3.6. Growth on wet weight basis

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The growth of C. magur larvae on average wet weight (g) basis per larva of

330

different treatments was measured at the end of second experiment. It was observed

331

that the larvae showed a significant difference between the treatments (Fig. 3). The

332

dietary treatment D3 displayed a significantly (P < 0.05) high average wet weight

333

followed by D2 and D1. A significantly (P < 0.05) low growth was observed in the

334

control group.

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3.7. Survival rate

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The survival rate was observed to be significantly high (P < 0.05) in the D3

337

treatment followed by D2 and D1 as shown in figure 4. The lowest percentage

338

survival was found in the control group.

339

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3.8. Carcass analysis

341

The carcass analysis of samples survived at the end of third experiment was

342

conducted as shown in table 9. The moisture content of different treatments

343

including control varied from 80.52-81.43% and there was no significant difference in

344

the moisture content of respective treatments. The crude protein content on dry

345

weight basis was observed to be in a range of 12.14-12.94% and there was no

346

significant difference among the treatments. Ether extract was found to lie in a range

347

of 1.21-1.85% on dry weight basis and there was no significant difference among the

348

treatments. Total ash was observed to vary from 2.92-2.99% whereas, total

349

carbohydrate was found to lie in a range of 1.66-2.30% and also there was no

350

significant difference among the control group with other treatments.

351

3.9. mRNA expression of growth-related genes

352

The level of mRNA expression of different growth related genes in different

353

dietary treatments is shown in table 10. The dietary treatments influence the mRNA

354

expression of GH gene and a significantly (P < 0.05) high fold change in mRNA

355

expression was observed in the D3 (8% lipid and 55% CP) and D2 (8% lipid and

356

50% CP) groups followed by D1. The 45% dietary protein level has little effect on the

357

GH gene expression. Similarly, IGF-I expression was significantly (P < 0.05) high in

358

D3 treatment followed by D2 and D1. Hence, almost same trend was observed in

359

GH and IGF-I gene expression profiles with regard to dietary treatments, although,

360

the fold change in mRNA expression was more in GH gene in comparison to IGF-I.

361

On the other hand, there was no significant (P > 0.05) effect of the dietary lipid and

362

protein combinations on the IGFBP3 mRNA expression.

363

The dietary effect of protein and age along with their interaction on mRNA

364

expression of growth related genes is shown in table 10. Dietary protein level has a

365

significant effect on the mRNA expression of growth related genes. A significantly (P

366

< 0.05) high level of expression of GH and IGF-I were observed at 55% followed by

367

50% and 45% level of dietary proteins. While, a reverse trend has been observed in

368

IGFBP where a significantly (P < 0.05) high mRNA expression was showed by the

369

45% dietary protein level. It was observed that age or the different time points have

370

no significant (P > 0.05) effect on the fold change expression of growth related

371

genes. Interaction effect of protein and age were not observed in GH, IGF-I and

372

IGFBP3 genes expression.

373

Hence from the second experiment 55% dietary protein level was observed to

374

be optimal for promoting the growth and survival of C. magur larvae.

375

4. Discussion

376

Dietary lipid has a great influence on growth and survival of fish and its

377

deficiency causes the poor performance with regard to growth and survival in fish

378

larvae (Rainuzzo et al., 1997). Like other animals, fish larvae also require an optimal

379

dietary lipid level to maintain high growth and survival. In the present study, effect of

380

varying level of lipid on larval growth measured as average wet weight along with

381

survival percentage was estimated. The different dietary lipid levels of treatments

382

have a significant effect on growth and survival rate. It was observed that dietary

383

treatment of 8% followed by 10% lipid inclusion resulted in a significantly higher

384

growth and survival of C. magur larvae. 12% dietary lipid level resulted in decreased

385

growth which might be due to its accumulation on the walls of intestine to hamper the

386

absorption of essential nutrients for growth and survival. The high lipid level might

387

also result in decreased accessibility of digestive enzymes for their respective

388

substrates which could also result in poor growth and survival. The 8% lipid level

389

seems to be optimal as high growth and survival was observed. Previously the

390

optimum dietary lipid requirement of 7-9% for a diet containing 40% crude protein in

391

young C. batrachus was reported where, weight gain and specific growth rate were

392

significantly higher (P < 0.05) in fish fed diets containing 7% dietary lipid (Anwar and

393

Jafri, 1995). Chou et al. (2001) found a higher weight gain at 9% lipid level in juvenile

394

cobia fish, which supports our study. On the contrary, the study by Zheng et al.

395

(2010) on Pelteobagrus vachelli revealed a higher survival at 11.1% and 15.1%. This

396

difference may be due to the carnivorous nature of the species in the above study,

397

which advocates lage absorption efficiency for lipids. In our study, poor performance

398

with regard to growth and survival was observed in 12% lipid level. This poor

399

performance could be due to the accumulation of large lipid droplets in the

400

enterocytes of intestine, which might reduce the absorption efficiency of essential

401

nutrients utilized for growth and survival (Morais et al., 2005). High dietary lipid levels

402

were also observed to lead a growth reduction in fish larvae (Izquierdo et al., 2000;

403

Gawlicka et al., 2002; Ai et al., 2008). Similar results were found in juvenile cobia

404

(Chou et al., 2001), on-growing turbot (Regost et al., 2001) and yellow croaker larvae

405

(Ai et al., 2008) after fed with high dietary lipids.

406

Proximate composition of different diets implied the difference in crude protein

407

level and crude lipid level as assigned for the diets. The difference in the composition

408

is discernable as the diets were formulated with variable level of lipids and protein to

409

see their effect on different parameters of magur larvae. Body composition of whole

410

larvae was evaluated during initial and end of every week of the experiment. The

411

samples for body composition collected at 14th, 21st, 28th and 35th day showed a

412

significant difference with regard to the ether extract and ash content. The body lipid

413

content increased significantly when the dietary lipid level increased from 8% to

414

12%. Hence, lipid has a significant effect on body composition of fish. The results

415

corroborate with the Gomez-Requeni et al. (2013), Han et al. (2014) and Saez-

416

Royuela et al. (2015), where the lipid content of whole body increased as the lipid

417

level of diets were raised.

418

The nutritional impact on the growth at transcriptional level has been receiving

419

much attention in aquaculture. In the present study, the effect of different dietary

420

treatments vary in lipid inclusion levels on the expression of genes related to growth

421

were studied. It was observed that the GH gene expression was decreased with

422

increase in dietary lipids. Similarly, IGF-I mRNA transcript level was significantly

423

higher in 8% and lowest in 12% lipid diets. High dietary lipid level results in the

424

differences in growth in Senegalese sole (Dias et al., 2004). Campos et al. (2010)

425

also concluded a negative correlation of growth with dietary lipid levels in Solea

426

senegalensis when molecular expression of growth related genes were studied.

427

Kumar et al. (2018) observed the highest mRNA expression of IGF-I at 7% lipid level

428

in Labeo rohita fingerlings, which is in agreement with our study. However, IGFBP3

429

displayed the opposite results with that of GH and IGF-I. The growth inhibiting effect

430

of IGFBP might be due to the tight binding of this protein with IGF-I which makes the

431

later unavailable for binding with IGF-I receptor for growth promotion. The results are

432

justified because of the well known fact of IGFBP3 as negative regulator of cell

433

growth, differentiation and development (Kim et al., 1997; Lee et al., 1997; wood et

434

al., 2005). The growth related genes were also observed to vary in the different age

435

groups selected in the present study. GH and IGF-I expressions were found to

436

decrease from 21 dph to 35 dph. This might be due to the effect of dietary protein, as

437

the level decreases after every week from 55% at 45% during the period of

438

experiment. Opposite patterns were seen in IGFBP3 expression level whose high

439

mRNA expression at 12% lipid based diets reflects the growth reduction due to the

440

growth inhibiting property of this protein (IGFBP3). Although IGFBP3 plays different

441

roles in different species of fish, but it was reported that IGFBP3 has anti-proliferative

442

effects by preventing the interaction of IFG with its receptor (Yamada et al., 2009).

443

The same effect was found in our study where a down regulation of IGFBP3 has

444

been observed at higher dietary lipid levels.

445

The studied parameters were also influenced simultaneously by protein level

446

as well as the age of the larvae. However, it was not clear from the first experiment

447

that whether the effect on the studied parameters was due to protein or age of larvae

448

because every week of this experiment represents a particular level of protein as

449

mentioned in the material and methods section. Thus, to understand the most

450

influential factor (age/dietary protein), a second experiment was conducted.

451

In this experiment, three experimental diets and one control (commercial) diet

452

were fed to four different groups of fish. The diets vary in the levels of proteins with a

453

constant level of 8% lipid in all the treatments except control. After the end of

454

experiment at 35 dph, the growth was estimated on average wet weight basis. The

455

significant difference in the average wet weight of larvae was observed at the end of

456

experiment II. The treatment D3 (8% lipid and 55% protein) showed a highest value

457

of average wet weight compared to the rest of treatments including control. Survival

458

percentage was also found to be significantly high in D3 followed by D2, D1 and

459

control. In the control and treatment containing 45% crude protein level low average

460

wet weight and survival percentage was observed. These results indicated that low

461

protein inclusion in the diets of C. magur larvae have suppressing effects on the

462

growth and survival rate. The suppressing effect in the control group might be due to

463

the insufficient lipid and protein required for optimum growth and survival of magur

464

larvae, as after the proximate composition of commercial diet i.e, control, crude

465

protein and ether extract were found to be 39.5% and 7.1% respectively. The dietary

466

lipid and protein requirement for optimum growth and survival varies from species to

467

species. In earlier studies the efficient growth was obtained at 36.5% and 33.2%

468

dietary protein in hybrid Clarias catfish post-larvae and butter catfish fry (Giri et al.,

469

2003; Paul et al., 2012).

470

It is well accepted that the growth hormone (GH)-insulin-like growth factor-I

471

(IGF-I) axis plays a crucial role in the neuroendocrine regulation of vertebrate

472

growth and their expression is affected by nutritional status directly or indirectly

473

(Wood et al., 2005). Dietary protein level has a close correlation with GH and

474

IGF-I mRNAs, as reported previously in Sparus aurata (Perez-Sanchez et al.,

475

1995), Lates calcarifer (Dyer et al., 2004) and Oreochromis niloticus (Qiang et al.,

476

2012). However, the mechanism is not very clear and may be partially attributed

477

due to its ability of modulating transcriptional activities of the GH-IGF-I genes and

478

serum binding proteins such as IGFBP (Wood et al., 2005).

479

In experiment II, the mRNA expression levels of genes related to the

480

growth have been seen to be influenced by varying levels of dietary proteins. GH

481

transcript levels were found to be influenced by lipid and protein, however, no

482

such effect was seen due to age. The 8% lipid and 55% protein inclusion resulted

483

in significantly high expression level of GH gene among all the treatment groups.

484

Optimum protein level favours the molecular expression of growth related genes

485

which can be reflected from the increased average wet weight in the present study at

486

high dietary protein. IGF-I gene showed the same trend as that of GH gene with

487

better performance in D3 treatment among all the groups. The appropriate protein

488

and lipid level efficient for the promotion of growth varies with species. Tu et al.

489

(2015) also observed an up-regulation in the expression of IGF-I in Carassius

490

auratus gibelio with the increasing dietary protein level till it reaches the optimum

491

level. Kumar et al. (2018) observed a high expression of IGF-I at 26% and 7%

492

protein and lipid level respectively in the diets of L. rohita. However, age did not

493

influence the mRNA levels of the GH gene in the present study. Dietary protein

494

content exhibited a significant effect on IGFBP expression with a decreasing trend as

495

the protein level was increased, indicating the favourable endocrine modulation for

496

better growth. As the low protein content of diets has less growth promoting effects

497

as seen with the low expression of IGF-I. The high IGFBP expression might be a

498

conserved physiological mechanism to hamper the IGF-stimulated growth and

499

developmental process under unfavourable situations and with inappropriate diets

500

(Kajimura et al., 2005). There are many reports on growth suppressing effects of

501

IGFBP but the exact mechanism of how dietary inclusion of lipid and proteins leads

502

to its up-regulation is still unclear.

503

5. Conclusion

504

The dietary combination of 8% lipid and 55% protein showed excellent

505

performance with regard to growth, survival and also the mRNA expression of all the

506

selected genes. Hence, the study can be concluded that composition of dietary

507

treatment containing 8% lipid and 55% protein could be suggested as the microdiet

508

for the larval rearing of C. magur in order to alleviate the huge mortality and losses

509

encountered in the culture of this potential catfish species. Furthermore, as dietary

510

lipids and proteins could influence the lipid and protein metabolic pathway, hence a

511

regulatory mechanism should be elucidated in the future studies.

512

Conflict of interest

513

The authors report no conflict of interest.

514

Acknowledgements

515

The authors are grateful to Director and Vice-Chancellor, ICAR-Central Institute of

516

Fisheries Education, Mumbai, India for providing support and necessary facilities for

517

carrying out this experiment.

518

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Table 1: First experimental design to evaluate the effect of graded dietary lipid levels

Age in day post

T1 (8% lipid)

T2 (10% lipid)

T3 (12% lipid)

14-21

55% CP

55% CP

55% CP

21-28

50% CP

50% CP

50% CP

28-35

45% CP

45% CP

45% CP

hatch (dph)

T1- Treatment 1; T2- Treatment 2; T3- Treatment 3.

Table 2: Composition of the Experimental Diets (% DM)

Ingredients

45/8

50/8

55/8 45/10 50/10 55/10 45/12 50/12 55/12

F1

F2

F3

F4

F5

F6

F7

F8

F9

Level

level

level

level

level

level

level

level

level

Fish meal

32

32

32

32

32

32

32

32

32

Soybean meal

5.5

5.5

5.5

5.5

5.5

5.5

5.5

5.5

5.5

Shrimp meal

8

8

8

8

8

8

8

8

8

Soya protein

10.6

14.5

18.5

10.6

14.5

18.5

10.6

14.5

18.5

10.6

14.5

18.5

10.6

14.5

18.5

10.6

14.5

18.5

Wheat flour

21.48

13.18

5.18

19.48

11.18

3.18

17.48

9.18

1.18

Sunflower: Fish oil

3.5

4

4

5.5

6

6

7.5

8

8

Soyalecithin

3

3

3

3

3

3

3

3

3

Vitamin C

0.3

0.3

0.3

0.3

0.3

0.3

0.3

0.3

0.3

Betaine

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

Vit-min mix

3

3

3

3

3

3

3

3

3

BHT

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.2

0.2

0.2

0.2

0.2

0.2

0.2

0.2

0.2

CMC

1.7

1.7

1.7

1.7

1.7

1.7

1.7

1.7

1.7

Total

100

100

100

100

100

100

100

100

100

hydrolysate Fish protein hydrolysate

mix (1:1)

Choline chloride

Table 3: Primers used for the qRT-PCR analysis of growth related genes Primer name

Primer sequence (5´-3´)

Purpose

GH F

CGAGGGCAACCTGAGGAAGAGC

GH R

CTCGCTCTCACACGCCCCCT

IGF-I F

CGGCATCGTGGACGAATGCT

IGF-I R

TGGTGTTTTGGGCGGTGTCTG

IGFBP F

CTGACCGTGCGGCAACATGAAC

IGFBP R

TCGTCAGGGCAGTCCACAAACG

Beta Actin F

TGCCCCAGAGGAGCACCCTG

Beta Actin R

GACCAGAGGCGTACAGGGACAGC

Quantitative PCR

Quantitative PCR

Quantitative PCR

Quantitative PCR

Table 4: Second experimental design to evaluate the effect of graded dietary protein levels Treatments

Diet

Control (C)

Commercial diet

D1

45% CP and 8% lipid

D2

50% CP and 8% lipid

D3

55% CP and 8% lipid

C- Control; D1-Dietary combination 1; D2-Dietary combination 2; D3-Dietary combination 3.

Table 5: Effects of dietary lipid and age/protein levels on the average wet weight of C. magur Treatments

Average wet wt. (g)

T1 8×55×21 T1 8×50×28 T1 8×45×35 T2 10×55×21 T2 10×50×28 T2 10×45×35 T3 12×55×21 T3 12×50×28 T3 12×45×35 P value Effect of lipid 8 10 12 SEM P value Effect of age/protein 21/55 28/50 35/45 SEM P value Lipid×Age/protein

0.14bc±0.02 0.25bc±0.03 0.65a±0.17 0.12bc±0.01 0.17bc±0.02 0.54a±0.02 0.10c±0.02 0.12bc±0.01 0.32b±0.07 0.002 0.34a 0.27ab 0.18b 0.04 0.001 0.12b 0.18b 0.50a 0.04 0.02 NS

Mean values in the same column with different superscript differ significantly (P < 0.05). Data expressed as mean ± SEM, n=10.

Table 6: Proximate composition (% dry matter basis) of the different experimental diets Treatment

Moisture

Crude protein

Ether extract

Crude fiber

Total ash

NFE

Gross energy (Kcal/100g)

C

8.46±0.22

39.50±0.27

7.14±0.55

4.89±0.05

11.63±0.23

28.38±0.69

406±3.03

F1

8.96±0.53

45.27±1.96

8.86±0.30

5.22±0.05

12.20±0.22

19.49±0.68

427±7.00

F2

8.99±0.52

50.82±0.75

8.96±0.78

5.46±0.06

12.32±0.15

13.43±1.18

410±4.01

F3

8.64±0.93

54.86±0.18

8.13±0.68

4.19±0.22

12.87±0.27

11.31±1.11

404±3.03

F4

8.63±0.27

45.36±0.78

10.69±1.02

4.33±0.15

13.17±0.17

17.80±0.89

429±2.01

F5

8.33±0.07

49.77±0.34

10.40±0.25

4.16±0.09

13.27±0.34

14.07±0.60

422±5.02

F6

8.82±0.35

55.72±0.23

10.17±0.20

3.32±0.28

12.91±0.33

9.06±0.49

423±13.00

F7

8.28±0.11

44.85±0.15

11.82±0.51

4.59±0.09

12.51±0.54

17.95±1.06

423±7.07

F8

8.42±1.09

49.78±1.51

11.56±0.63

4.34±0.10

12.91±0.17

12.99±3.11

420±5.02

F9

8.33±0.10

55.73±0.19

11.67±0.50

3.25±0.19

12.77±0.12

8.25±0.32

415±7.07

Data expressed as mean ± SEM, n=3.

Table 7: Carcass analysis (% wet weight basis) of whole body of C. magur of different experimental groups at different stages

Treatment

Moisture

Dry matter

Crude protein

Ether Extract

Total Ash

Total carbohydrate

14th day Initial

79.82±0.31

20.18±0.31

12.52±0.09

1.05±0.11

2.08±0.16

4.53±0.15

1.36b±0.10

1.80b±0.07

3.86±0.33

T1

79.67±0.77

20.33±0.77

21th day 13.31±0.49

T2

80.11±0.45

19.89±0.45

12.08±0.56

1.53b±0.09

2.51a±0.11

3.76±0.93

T3

79.31±0.64

20.67±0.64

13.42±0.14

1.93a±0.10

1.89b±0.13

3.44±0.32

1.43c±0.12

2.58±0.28

3.55a±0.72

T1

79.85±0.37

20.15±0.37

28th day 12.58±0.52

T2

79.69±0.69

20.31±0.69

12.99±0.38

1.30b±0.15

2.62±0.09

3.41a±0.24

T3

79.80±0.36

20.20±0.36

13.42±0.40

1.63a±0.10

2.65±0.16

2.49b±0.12

35th day T1

78.78±0.83

21.22±0.83

13.56±0.28

1.71b±0.18

2.13a±0.11

3.81a±0.62

T2 T3

79.71±0.75 80.19±0.27

20.29±0.75 19.80±0.27

12.77±0.72 11.83±0.34

2.67a±0.25 2.96a±0.27

2.48a±0.23 1.78b±0.05

2.37b±0.47 3.23a±0.39

Mean values in the same column at different time points with different superscript differ significantly (P < 0.05). Data expressed as mean ± SEM, n=3.

Table 8: Effects of dietary lipid and age levels on the expression of growth related genes of C. magur Treatments T1 8×55×21 T1 8×50×28 T1 8×45×35 T2 10×55×21 T2 10×50×28 T2 10×45×35 T3 12×55×21 T3 12×50×28 T3 12×45×35 P value Effect of lipid 8 10 12 SEM P value Effect of age 21 28 35 SEM P value Lipid×Age

IGFBP

GH 2.03ab±0.35 2.41a±0.51 0.41c±0.15 1.30b±0.23 1.63ab±0.33 0.51c±0.16 0.34c±0.17 0.23c±0.05 0.25c±0.07 0.002

IGF1 2.06a±0.52 2.07a±0.10 0.60b±0.13 1.96a±0.36 1.78a±0.49 0.48b±0.06 0.55b±0.14 0.41b±0.07 0.36b±0.07 0.001

0.79c±0.17 1.03c±0.05 1.84a±0.21 1.01c±0.27 0.89c±0.24 1.21bc±0.12 1.66ab±0.04 1.64ab±0.27 1.74ab±0.13 0.003

1.62a 1.15b 0.27c 0.15 0.002

1.58a 1.41a 0.44b 0.16 0.001

1.22b 1.04b 1.69a 0.10 0.002

1.23a 1.42a 0.39b 0.15 0.001 S

1.53a 1.42a 0.48b 0.16 0.003 NS

1.16b 1.19b 1.60a 0.10 0.017 NS

Mean values in the same column with different superscript differ significantly (P < 0.05). Data expressed as mean ± SEM, n=6.

Table 9: Carcass analysis (% wet weight basis) of C. magur whole body of different treatments at the end of second experiment Treatment

Moisture

Dry

Crude

Ether

Total

Total

matter

protein

Extract

Ash

carbohydrate

C

80.62±0.54 19.38±0.54 12.94±0.75 1.21±0.09 2.93±0.05

2.30±0.78

D1

80.59±0.57 19.41±0.56 12.91±0.29 1.50±0.25 2.99±0.08

2.00±0.06

D2

80.52±0.81 19.48±0.81 12.86±0.49 1.80±0.02 2.95±0.04

1.87±0.29

D3

81.43±0.88 18.57±0.88 12.14±0.61 1.85±0.26 2.92±0.11

1.66±0.45

Data expressed as mean ± SEM, n=3.

Table 10: Effects of dietary protein levels and age on the expression of growth related genes of C. magur

Treatments

GH

IGF-I

IGFBP

D1 8×45×21

1.43b±0.11

1.17c±0.19

2.29±0.71

b

c

D1 8×45×28

1.65 ±0.24

1.58 ±0.12

1.99±0.20

D1 8×45×35

1.37b±0.15

1.89c±0.43

2.32±0.13

D2 8×50×21

3.41a±0.59

3.09b±0.44

1.17±0.30

D2 8×50×28

4.18a±0.63

3.68ab±0.13

1.46±0.01

D2 8×50×35

3.50a±0.17

3.34ab±0.34

1.89±0.33

D3 8×55×21

4.99a±0.89

4.31a±0.36

1.13±0.31

a

a

D3 8×55×28

4.84 ±0.77

4.39 ±0.22

1.46±0.09

D3 8×55×35

4.82a±0.49

4.44a±0.59

1.46±0.43

P value Effect of Protein 45 50 55 SEM P value Effect of Age 21 28 35 SEM P value Protein×Age

0.00

0.001

0.16

1.48c 3.70b 4.88a 0.30 0.00

1.55c 3.37b 4.38a 0.20 0.00

2.20a 1.51b 1.36b 0.19 0.01

3.27 3.56 3.23 0.30 0.71 NS

2.85 3.21 3.22 0.20 0.23 NS

1.54 1.64 1.89 0.19 0.85 NS

Mean values in the same column with different superscript differ significantly (P < 0.05). Data expressed as mean ± SEM, n=6.

4.0

a

Weight gain (g)

b b

3.0

0.6 a

2.0 ab

0.3 1.0

Length gain (cm)

0.9

b

0.0

0.0

Treatments Fig. 1. Growth performance of different treatments at the end of first experiment in C. magur. Different lower case letter superscripts indicate significant differences (P < 0.05). Data was expressed as mean ± SEM, n=10.

90

Survival (%)

85 80

a

b b

75 70 65

Treatments

Fig. 2. Survival percentage of different treatments at the end of first experiment in C. magur. Different lower case letter superscripts indicate significant differences (P < 0.05). Data was expressed as mean ± SEM, n=3.

1.2

Mean wet weight (g)/larva

a 1.0 0.8

b bc

0.6 0.4

c

0.2 0.0

Treatments Fig. 3. Growth on average wet weight (g) basis of the different dietary treatments at the end of second experiment in C. magur. Data expressed as mean ± SEM, n=10.

100

a

Survival (%)

60

b

b

80 c

40 20 0

Treatments

Fig. 4. Survival percentage of different treatments including control at the end of second experiment in C. magur. Different lower case letter superscripts indicate significant differences (P < 0.05). Data expressed as mean ± SEM, n=3.

Highlights • • • •

Effect of graded dietary lipid and protein content was investigated on Indian walking catfish to determine the optimal levels. Significantly higher performance of growth and survival was observed at 8% dietary lipid level. 55% protein level was found be optimal for promoting growth and survival along with the expression of growth-related genes of Clarias magur larvae. 8% lipid and 55% protein inclusion in the diets of C. magur is recommended in its larval rearing phase of life cycle.

Conflict of interest The authors report no conflict of interest.