Songklanakarin Journal of Science and Technology SJST-2019-0404.

R1 Pongjanyakul

Dietary Protein Requirement for Growth Performance and

Effects on Carcass Composition of Young Siamese spiny eel,
Macrognathus siamensis (GÜnther, 1861)

Journal: Songklanakarin Journal of Science and Technology

Manuscript ID SJST-2019-0404.R1
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Manuscript Type: Original Article

Date Submitted by the

30-Jan-2020
Author:
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Complete List of Authors: Pongjanyakul, Theerachai; Inland Fisheries Research and Development
Center Regional 4 (Ubon Ratchathani) , Department of Fisheries;
Ngamsnae, Praneet ; Ubon Ratchathani University
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Payooha, Kanjana; Ubon Ratchathani University

Grudpan, Jarungjit; Ubon Ratchathani University
BOONNGARM, Jongkon; Department of Fisheries
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Siamese spiny eel (Macrognathus siamensis), Dietary protein

Keyword:
requirement, Growth performances
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Dietary Protein Requirement for Growth Performance and Effects on Carcass
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7 Composition of Young Siamese spiny eel, Macrognathus siamensis (GÜnther, 1861)
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9 Theerachai Pongjanyakul1,*, Jongkon Boonngarm2, Praneet Ngamsnae3, Kanjana
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Payooha3 and Jarungjit Grudpan3
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1Inland Fisheries Research and Development Center Regional 4 (Ubon Ratchathani),
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16 Department of Fisheries, Department of Fisheries, Thailand
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18 3Rayong Inland Fisheries Research and Development Center, Rayong, Department of
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21 Fisheries, Thailand
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3Department of Fisheries, Faculty of Agriculture, Ubon Ratchathani University
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25 Warinchamrab, Ubon Ratchathani, Thailand

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* Corresponding author Email address: ptheerachai@gmail.com
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32 Abstract
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The study on protein requirement of young Siamese spiny eel, Macrognathus
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siamensis (GÜnther, 1861) was conducted using six different protein level (35, 40, 45, 50,
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39 55 and 60 % protein) with average gross energy of 450 kcal·100/g. The results
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41 demonstrated the maximum specific growth rate (SGR), % weight gain and daily weight
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44 gain were achieved at 55% protein while the fishes fed with 35% protein was the lowest.
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46 It was estimated by broken line regression that dietary protein level producing maximum
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48 growth was 46.50% protein. There was no significant difference (P>0.05) on survival rate
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51 amongst treatments. Protein efficiency ratio (PER) and apparent net protein retention
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53 (ANPR) were not significantly (P>0.05) affected by diet protein levels. No significant
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55 (P>0.05) effect of dietary protein levels was found on carcass moisture, crude protein,
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crude lipid and ash. However, carcass moisture, protein and ash were apparently increased
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7 in all fish groups after feeding trial, comparing to fish before the experiment.
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9 Keywords: Siamese spiny eel (Macrognathus siamensis), Dietary protein requirement,
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Growth performances
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14 1. Introduction
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16 The Siamese spiny eel (Macrognathus siamensis) naturally lives in canal, streams,
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18 swamp, ponds, paddy field and the mud, throughout Thailand, Lao, Cambodia and
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21 Vietnam (Khachonpisitsak, 2007; Rainboth, 1996). In Thailand, the species is an
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23 economically important native freshwater food fish and has very high demand of
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25 consumers who are willing to pay 150-200 baht/kg live fish and processed fish are usually
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traded in the market. During rainy and rice harvesting season, fish could be drawn out
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30 from trap ponds into the rice field areas to be caught. Although Siamese spiny eel is a
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32 small fish, this fish is a rich source of animal protein, essential fatty acids, vitamin and
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minerals. As well as many small fish species, Siamese spiny eel are eaten whole,
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including head, visceral and bones, they are particularly rich in bioavailable calcium and
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39 also rich in vitamin A, iron and zinc (Thilsted, 2010). However, the population of this
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41 species in natural water bodies becomes fast decreasing, due to degradation of

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44 environments. Not recently, this broodstock of this fish were successfully induced its
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46 ovulations by hormone LHRH-a (Luteinizing Hormone Releasing Hormone analog)
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48 injection followed by natural spawning or artificial fertilizations (Saowakoon &
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Saowakoon, 2007).
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53 Appropriate feeding process for nursing 1-14 days old Siamese spiny eel larvae was
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55 feeding with Moina that obtained the best growth and survival. The larvae were then fed
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with live food such as blood worm and earthworm. Nevertheless, the substitution of a
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compound diet for live foods is necessary for reducing production costs and for sustaining
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7 production of high and constant quality juveniles (Cahu & Zambonino Infant, 2001).
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9 It is well known that proteins play a part in both as a structural component and
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provides energy for maintenance in fish. Inevitably, the dietary protein requirements of
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14 these organisms tend to be higher than other animals (NRC, 1993). Protein requirements
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16 for fish are 2-3 times higher than that of mammals (Pandian, 1987). However,
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18 requirements for protein from plant protein and fishmeal in fish are quite variable, and an
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21 adequate supply of dietary protein is essential if fish are to thrive and grow well (Walker
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23 & Berlinsky, 2011). Protein is the most expensive and significant in the optimization of
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25 economic feed costs nutrient of fish diets (Yao-Ping et al., 2009). Therefore, research on
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potentially cultural fish species starts with the determination of their dietary protein
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30 requirements. However, there is very little known about protein requirement of Siamese
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32 spiny eel, despite its feeding habits and breeding method are well documented
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(Saowakoon & Saowakoon, 2007). Furthermore, we know that the digestive tract of
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Siamese spiny eel is a straight tube, and the morphology of the fish similar to the
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39 carnivorous species, such as rice field eel (Monopterus albus) and the optimal protein
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41 content was 45 % protein (Ma, Wang, He, Feng, & Lu, 2014). Thus, objectives of this
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44 study were to determine the quantitative protein requirement and the effects of dietary
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46 levels on carcass composition of young Siamese spiny eel (Macrognathus siamensis),
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48 maintained in aquarium.
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2. Materials and Methods
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53 2.1 Experimental fish design and water quality
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55 Siamese spiny eel juveniles were prepared by induced breeding at culture facility of
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Inland Fisheries Research and Development center Regional 4 (Ubon Ratchathani),
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thereafter fishes were fed with moina for 30 days followed by weaning to acclimate to
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7 artificial diet 30 days.
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9 The experimental fishes average weight (0.22±0.04 g, n=20) were randomly selected
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and grouped in triplicate and assigned to one of the experimental diets, at the stocking
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14 rate of 25 fishes/aquarium. Culture facilities consist of 18 glass aquaria (45x90x45 cm)
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16 in a recirculating culture system with 35 cm water depth (140-liter). Each aquarium was
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18 provided with aeration and two shelters of diameter 1.5 inches, 14 cm long
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21 polyvinylchloride (PVC) pipes. Fishes were fed to apparent satiation three times daily at
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23 08.00, 12.00 and 16.00 h for the experimental period of 12 weeks. Fish of each replicate
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25 were counted and weighed fortnightly in bulk. The amount of feed consumed by the fish
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in each treatment was recorded daily. Final weight (g), total length (cm) and survivals
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30 were recorded.
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32 Water temperature and dissolved oxygen was monitored daily using YSI 550 DO
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meter. Water quality analyses were conducted three times a week for concentrations of
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total ammonia–nitrogen using a DR/2500 spectrophotometer and pH measured with a
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39 YSI 60 electronic pH meter. Alkalinity, Free CO2 and hardness were measured once a
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41 week by digital titration (HACH Company). All aquaria, some water were siphoned off
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44 and filled in prior to the first daily feeding to remove accumulated maters and kept
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46 maintaining a water depth 35 cm.
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49 2.2 Experimental diets and feeding
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52 Six experimental diets were formulated to contain increasing protein level 35, 40, 45,
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54 50, 55 and 60 % protein with average gross energy of 450 Kcal•100/g. Diets were
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56 prepared using fish meal and krill meal as the main protein sources. Each diet was
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59 prepared by first mixing the macro ingredients; fish meal, krill meal, rice bran and wheat
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flour, in Hammer mill for 5 min. The dry ingredients were pulverized, sieved through 0.5
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7 mm. All ingredients were thoroughly mixed and followed by addition of squid oil, tuna
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9 oil, vegetable oil, α-starch, cellulose, vitamin and mineral premixes, choline chloride and
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ascorbic acid. The mixed product was extruded with a grinder-Hobart at room
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14 temperature with 1.6-mm die and the outcome pellets were air-dried at temperature of 28-
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16 40 ˚C for about 3 days. In order to match the fish mouth size, pellets were then grinded
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18 by Molinex grinder and passed through 800 and 1,000 µm mesh sieves. The diets were
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21 kept at -18 ˚C in a freezer until the time of feeding. Analysis of the proximate composition
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23 of the experimental diets was carried out in Chonburi Aquatic Animal Feed Technology
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25 Research and Development Center. Determination for percentage of dry matter of the
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experimental diet i.e crude protein (Nх6.25), ether extract, crude fiber, ash and nitrogen-
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30 free extract (NFE) was conducted by standard (AOAC, 1985) methods. The ingredient
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32 composition and proximate analysis of the experimental diets are given in Table 1.
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36 Table 1. Formulation and composition analysis of the experimental diets (% Dry wt.
basis).
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40 2.3 Growth performance parameters and carcass composition analyses
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42 Six fishes from each dietary treatment (2 per replicate) were sampled and pooled
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45 for their proximate carcass composition analysis, at the beginning and the end of the
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47 experiment. Dry matter analysis was performed by drying for 24h at 105C, crude
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49 protein by Kjeldahl method, and crude lipid using a Soxhlet extraction apparatus, ash by
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52 combustion in muffle furnace at 550 C for 7 h, and energy by burning in a Gallenkamp
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54 bomb calorimeter (Leicestershire, England). Determination of carbohydrate content
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(nitrogen-free extract; NFE = 100 - (% protein + % lipid + % fiber + % ash) was
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59 calculated by difference. Specific growth rate (SGR), feed conversion ratios (FCR),
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weight gain (DWG), feed efficiency ratio (FE), daily feeding rate (DFR), protein
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7 efficiency ratio (PER), apparent net protein retention (ANPR) and survival ratio (SUR),
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9 were calculated as follows:
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SGR (%) = 100 (ln final wt - ln initial wt )/ t (days)
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14 FCR = Food consumed in g (dry weight)/ Live weight gain in g
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16 DWG (g/day) = (Mean final weight–Mean initial weight) / Culture period (days)
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FER = Mean weight gain/ Mean feed intake
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21 DFR (%/day) = 100 x (Daily feed intake)/(Initial weight+ Final weight)/2
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24 PER = Body weight gain (wet)/Apparent protein intake (dry)
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ANPR (%) = 100 (Final body protein-Initial body protein)/Apparent protein intake
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29 SUR (%) = 100  (Final number of fish)/ Initial number of fish

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34 2.4 Statistical analysis
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To determine the significant differences amongst treatments at the 0.05
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40 significance level, all data on growth, feed conversion and survival rates were subjected
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42 to one-way analysis of variance and Tukey’s test. The equation broken-line regression
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model is Y = L+U(R-X), which Y is the growth parameter (% weight gain) chosen to
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47 predict the protein requirement, L is the ordinate and R is the x-axis of the breakpoint R
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49 is taken as the predicted requirement and U is the slope of the line for X, which
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51 represented the optimum dietary protein level for fish. (Robbins, Norton, & Baker, 1979:
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54 Robbins, Saxton, & Southern,2006)
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6 3. Results
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8 Overall water quality values ranged during the study period, water temperature,
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10 25.7-28.7 ºC; air temperature, 27.50-30.50 ºC; dissolved oxygen, 4.30-5.80 mg/l; free
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13 CO2, 1.35-2.80 mg/l; pH, 5.0-7.9; hardness, 35.60-52.20 mg/l as CaCO3; alkalinity,
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15 15.20-18.75 mg/l as CaCO3; and total ammonia–nitrogen, 0.029-0.403 mg/l (Table 2).
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17 These major water quality parameters ranges were within suitable levels for living and
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growth of tropical freshwater fishes (Boyd, 1979), and similar reported water quality
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22 results of juvenile rice field eel (Ma et al., 2014)

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25 Table 2. Water quality parameters of all treatments during the study period.
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29 3.1 Growth performance

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31 Weekly increments in mean body weight of young Siamese spiny eels are shown in
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33 Figure 1 overall growth performances and other related parameters are given in Table 3.
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36 The weights of fish in all treatments were increased and varied with the dietary protein
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38 content (Figure 1). However, mean survival rates did not significantly different (P>0.05)
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40 among treatments and mortalities were not affected by treatments (Table 3). The highest
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final weight 0.85g occurred at 55 % dietary protein level while the lowest final weight
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45 gain of 0.50 g observed at the lowest dietary protein level of 35%. Similarly, the highest
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47 final length 6.42 cm was obtained from fish fed 55% protein diet, which was significantly
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(P0.05) different from 35% protein. Percentage weight gain was significantly different
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52 (P0.05) between 35 % protein and the rest dietary protein levels. Although fish fed the
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54 55% protein diets showed the highest specific growth rate, but did not significantly (P>
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57 0.05) differ from those fed 45, 55 and 60% protein diets.
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The fish fed with the higher protein levels were increased in % SGR (1.11-1.84) and
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7 feed efficiency ratio (FER) (0.36-0.63). Daily feeding rate (DFR), Protein efficiency ratio
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9 (PER) tended to increase as the dietary protein level increasing from 35 to 50% but were
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decreased at the protein levels of 55 to 60%. The highest feed conversion ratio (FCR) was
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14 observed at the protein level of 35%, while the lowest was at 60% but was not
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16 significantly (P>0.05) different when fish were fed diets with protein levels from 40, 45,
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18 55 and 60%. Apparent net protein retentions (ANPR) were not significantly different (P
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21 >0.05) in all dietary protein levels. The overall survival rate was 72-84%, with no
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23 significant differences (P>0.05) amongst groups.
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26 Table 3. Growth performance, feed utilization efficiency and protein utilization efficiency
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of fish fed experimental diets for 12 weeks.
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30 Figure 1. Changes in average weight of young Siamese spiny eel (Macrognathus
31 siamensis), fed with different protein content diets.
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34 3.2 Optimum dietary protein level
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36 Based on percentage weight gain, daily weight gain, and specific growth rate, the
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38 dietary protein requirement of young Siamese spiny eel was apparently observed at 55%
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40 protein. However, in accordance with the growth response to the feeding experimental,
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body weight gain as an index (y) and dietary protein level (x) were fitted using the broken-
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45 line model and a regression analysis map constructed (Figure 2). The appropriate level of
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47 dietary protein was calculated to be 46.50 % protein of the dry feed that produced
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maximum growth for young Siamese spiny eel in this study.
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53 Figure 2. Optimum dietary Protein level for young Siamese spiny eel (Macrognathus
54 siamensis), determined by Breakpoint regression analysis.
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56 3.3 Fish carcass Composition
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The effects of dietary protein levels on carcass composition of young Siamese spiny
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7 eel are presented in Table 3. Fish body moisture content (15.76-18.17), crude protein
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9 (68.86-70.68), crude lipid (6.54-8.30) and ash (16.40-18.44) did not differ significantly
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(P>0.05) among all fish groups after the experiment.
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15 Table 4. Proximate carcass composition of young Siamese spiny eel (Macrognathus
16 siamensis), fed diets containing graded levels of protein over 12 weeks.
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Fish body moisture, protein, lipid and ash were not clearly related to dietary protein
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21 level. Fishes in this study were young and sexually immature; however, carcass moisture,
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23 protein and ash increased in all fish groups after feeding trial comparing to fish before the
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experiment, and there was no significant (P>0.05) effect of dietary protein on carcass
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28 lipid.
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30 4. Discussion
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32 4.1 Growth
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35 From this study results, the dietary protein level producing maximum growth of this
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37 experimental fish, calculated by broken line regression was 46.50% protein. This
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39 optimum protein level for young Macrognathus siamensis (GÜnther, 1861) is close to the
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demand for protein of other eel-like appearence species such as swamp eel: Monopterus
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44 albus 45% protein (Ma et al., 2014) and Japanese eel; Anguilla japonica 45% protein
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46 (Okorie et al., 2007). When compare to the protein demand (35-45% protein) of Anguilla
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australis (Engin & Carter, 2006), this study result is slightly higher, and probably due to
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51 the relatively small size of the fish that were used in this study. However, the present
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53 result is quite close to the dietary protein requirement of juvenile American eel; Anguilla
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55 rostrata 47% protein (Tibbetts, Lall, & Anderson, 2000). It was found that at an excessive
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dietary protein levels, the growth performances responding to dietary protein levels in
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7 Siamese spiny eel exhibited similar trend in decreasing growth rate to all previous studies.
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10 Generally, the optimum dietary protein requirement is known to differ between
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13 different fish species. These may be due to differences in strains (NRC, 1993), and
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15 environmental factors and size or stages of their growth development. In the present
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17 study, with 35 and 40% protein diets, fish feed lower protein diets showed significantly
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lower growth than the higher protein groups. On the other hand, with diets containing 45-
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22 50 % protein, fish had sufficient protein for their maximum growth. Further, increases in
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24 dietary proteins did not result in significantly increase in growths. Excessive dietary
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26 protein levels (55-60%) in this study trended to decrease in growth rates. This similar
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29 apparent growth-depressing effect of fish fed high protein diets has also been reported for
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31 other fish species, in carnivorous fish such as bagrid catfish; Mystus nenurus (Ng, Soon,
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33 & Hashim, 2001), snakehead; Channa striata (Mohanty & Samantaray, 1996; Wee &
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36 Tacon, 1982) and striped catfish; Pangasianodon hypopthalmus (Jayant et al., 2018),
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38 herbivorous fish such as tawes, Puntius gonionotus (Wee & Ngamsnae, 1987) and
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40 omnivorous freshwater fish such as rohu; Labeo rohita (Satpathy, Mukherjee, & Ray,
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2003) and Nile tilapia; Oreochromis niloticus (Siddiqui, Howlader, & Adam, 1988).
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46 It was advised that the content of dietary protein should be maintained at a suitable
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48 level to minimize feed costs. Otherwise, when dietary protein level the requirement, the
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fish excretes more ammonia nitrogen into the surrounding environment, thus reducing the
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53 quality of the culture water. Growth of the fish also might be affected since extra energy
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55 is required to delaminate the excess amino acids absorbed (Jauncey, 1982). This
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conclusion have been confirmed by Lim, Sukhawongs, & Pascaual (1979) who concluded
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that the lower weight gain of milkfish Chanos chanos, fed diets with protein levels above
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7 the optimum could be due to insufficient non-protein energy in the high protein diets
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9 which caused part of the dietary protein to be metabolized and use for energy.
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13 Feed conversion (FCR) values ranged from 1.62 to 2.27, with significant differences
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15 between the lowest protein diet (35% protein) and the two high groups (55 and 60 %
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17 protein) of experimental diets. However, similar distinct trend for FCR to decrease with
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increasing dietary protein levels was observed in Rohu Labeo rohita (Nandeesha et al.,
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22 1994) and Zacco barbata (Shyong, Huang, & Chen, 1998). Protein efficiency ratio (PER)
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24 ranged from 0.98 to 1.05, with could not be easily categorized into groups. However,
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26 apparently PER decreased when dietary protein level increased. This trend was also
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29 reported in bighead carp (Santiago & Reyes, 1991) and Zacco barbata (Shyong et al.,
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31 1998). This occurrence could explained that fish often show the greatest protein
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33 conversion efficiency when fed a dietary protein less than that yielding maximum growth
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36 and feed efficiency (Jobling, 1994).
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39 Apparent net protein retention (ANPR) in this study for Siamese spiny eel ranged from
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41 15.12% to 17.81%, with not significantly different amongst all treatments but apparently
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44 higher in the lower diets. Previous studies reported that the protein retention for different
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46 dietary protein levels depends on the type of energy provided by the ingredients (Ogino
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48 & Saito, 1970) and different between species (Boyd & McNevin, 2015). For example,
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while for striped catfish, Pangasianodon hypopthalmus and Nile tilapia, Oeochromis
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53 niloticus whole- body composition in retrieval is between 20.9-22.4% (Abdel-Tawwab,
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55 2012; Jayant et al., 2018), and for atlantic salmon, Salmo salar is up to 52.9% (Grisdale-
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Helland & Helland, 1997). Cowey, Blair, Adron, & Pope (1971) reported that ANPR and
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PER values for plaice were higher with diets containing carbohydrate than with diets
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7 without carbohydrate, even though those diets were isocaloric. In this study, the highest
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9 ANPR and PER of Siamese spiny eel were found with the 40% protein diet, nearly the
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lowest dietary protein concentration. This result empirically confirms the conclusion that
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14 the fish fed diets containing higher carbohydrate exhibit higher PER and ANPR values.
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16 4.2 Fish carcass composition
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18 The protein level in diets was not significantly affected on carcass composition of
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21 Siamese spiny at this stage of development. Although body moisture content tended to
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23 increase with the dietary protein level, changes in protein, fat and ash contents were not
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25 clearly related to the dietary treatment. Gunasekera, Silva, Collins, Gooley, & Ingram
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(2000) and Yao-Ping et al. (2009) found that gross body composition in Murray cod
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30 Maccullochella peelii peelii juvenile and Barbodes caldwelli juvenile were not affected
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32 by increasing dietary protein levels but the fish tended to have lower body protein and
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higher body lipid content when fed low protein diet. The carcass lipid levels generally
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increased with increasing dietary protein from 35% to 50% protein, then decreased after
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39 reached the optimum protein level for growth. A similar trend has been reported in other
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41 experiments with Nibea diacanthus (Li, Wen, Zhao, Li, & Zhu, 2016) and Japanese eel;
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44 Anguilla japonica (Nose & Arai, 1972).
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47 5. Conclusion
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49 The calculated dietary protein requirement for young Siamese spiny eel was 46.50%
50
51 when determined by the broken-line regression model using % final weight gain as the
52
53
54 indicator. However, the effectiveness of this recommended level practical diets, will
55
56 depend on the essential amino acid composition provided by suitable plant and animal
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feedstuffs. Future study on practical diets for Siamese spiny eel emphasized on essential
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7 amino acid requirements and their optimum levels needed be conducted.
8
9
10 Acknowledgements
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14 This research was sponsored by the Agriculture Research Development Agency
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16
(Public Organization), ARDA this support was appreciated. The authors would like to
17
18
19 thank Miss Laddaval Krongpong, Director of Chonburi Aquatic Animal Feed Technology
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21 Research and Development Center for providing research facilities for analysis of the
Fo

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23 proximate composition of the experimental diets and fish carcass, and Mr. Supat Sripat,
24
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26 Director of Inland Fisheries Research and Development Regional 4 (Ubon Ratchathani)
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28 for providing facilities in feeding trial of the experimental fish.
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30
31 References
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33
34
35 Abdel-Tawwab, M. (2012). Effects of dietary protein levels and rearing density on
36
On

37 growth performance and stress response of Nile tilapia, Oreochromis niloticus

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39
(L.). International Aquatic Research, 4(1), 3. https://doi.org/10.1186/2008-6970-
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42 4-3
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44 Association of Official Analytical Chemists. (1985). Official Methods of Analysis (16th
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46 ed.). Arlington, VA: Author. Auburn University, Agricultural Experiment
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49 Station.
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51 Boyd, C. E. (1979). Water quality in warmwater fish ponds. Auburn, Alabama, USA:
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53 Boyd, C., & McNevin, A. (2015). Aquaculture, Resource Use, and the Environment.
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Willey Blackwell, New Jersey.
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9 2), 161–180. https://doi.org/10.1016/S0044-8486(01)00699-8
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14 marine flatfish. Growth of the plaice Pleuronectes platessa on diets containing
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16 proteins derived from plants and other sources. Marine Biology, 10(2), 145–153.
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18 https://doi.org/10.1007/BF00354830
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21 Engin, K., & Carter, C. G. (2006). Growth and food utilization of the Australian short-
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23 finned eel, Anguilla australis australis (Richardson) given paired iso-energetic
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25 diets with increasing crude protein content. Animal Science, 82(2), 169–174.
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30 Grisdale-Helland, B., & Helland, S. J. (1997). Replacement of protein by fat and
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32 carbohydrate in diets for atlantic salmon (Salmo salar) at the end of the
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39 Gunasekera, R. M., Silva, S. S. D., Collins, R. A., Gooley, G., & Ingram, B. A. (2000).
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41 Effect of dietary protein level on growth and food utilization in juvenile Murray
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44 cod Maccullochella peelii peelii (Mitchell). Aquaculture Research, 31(2), 181–
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46 187. https://doi.org/10.1046/j.1365-2109.2000.00417.x
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48 Jauncey, K. (1982). The effects of varying dietary protein level on the growth, food
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conversion, protein utilization and body composition of juvenile tilapias
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53 (Sarotherodon mossambicus). Aquaculture, 27(1), 43–54.
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55 https://doi.org/10.1016/0044-8486(82)90108-9
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Jayant, M., Muralidhar, A. P., Sahu, N. P., Jain, K. K., Pal, A. K., & Srivastava, P. P.
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7 (2018). Protein requirement of juvenile striped catfish, Pangasianodon
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14 Jobling, M. (1994). Fish Bioenergetics. London, England: Chapman & Hall.
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16 Khachonpisitsak, S. (2007). Taxonomy of Spiny Eels (Symbranchiformes:
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18 Mastacembelidae) Thailand (Master’s Thesis, Chulalongkorn University,
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21 Bangkok, Thailand).
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23 Li, W., Wen, X., Zhao, J., Li, S., & Zhu, D. (2016). Effects of dietary protein levels on
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30 https://doi.org/10.1007/s12562-015-0945-9
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32 Lim, C., Sukhawongs, S., & Pascual, F. P. (1979). A preliminary study on the protein
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requirements of Chanos chanos (Forskal) fry in a controlled environment.
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Aquaculture, 17(3), 195–201. https://doi.org/10.1016/0044-8486(79)90123-6
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39 Ma, X., Hu, Y., Wang, X.-Q., Ai, Q.-H., He, Z.-G., Feng, F.-X., & Lu, X.-Y. (2014).
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41 Effects of practical dietary protein to lipid levels on growth, digestive enzyme

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44 activities and body composition of juvenile rice field eel (Monopterus albus).
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46 Aquaculture International, 22(2), 749–760. https://doi.org/10.1007/s10499-013-
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48 9703-0
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Mohanty, S. S., & Samantaray, K. (1996). Effect of varying levels of dietary protein on
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53 the growth performance and feed conversion efficiency of snakehead Channa
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55 striata fry. Aquaculture Nutrition, 2(2), 89–94. https://doi.org/10.1111/j.1365-
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2095.1996.tb00013.x
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Nandeesha, M.C., K. Dathathri, D. Krishnamurthy, T.J. Vargese, B. Gangadhar, & N.R.
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7 Umesh. (1994). Effect of varied levels of protein on the growth and tissue
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9 boichemistry of stunted yearlings of rohu, Labeo rohita, in the absence of
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natural food. In, Fish Nutrition Reasearch in Asia (pp.93-99). Manila,
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14 Philippines: Asian Fish.Soc.Spec.Publ.
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16 National Research Council. (1993). Nutrient Requirements of Fish. Washington, D.C:
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18 National Academy Press.
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21 Ng, W.-K., Soon, S.-C., & Hashim, R. (2001). The dietary protein requirement of a
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23 bagrid catfish, Mystus nemurus (Cuvier & Valenciennes), determined using
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25 semipurified diets of varying protein level. Aquaculture Nutrition, 7(1), 45–51.

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30 Nose, T., & Arai, S. (1972). Optimum level protein in a purified diet for eel, Anguilla
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32 japonica. Bull. Freshwater Fish. Res. Lab. 22, 145-155.

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Ogino, C., & Saito, K. (1970). Protein nutrition in fish. 1. The utilization of dietary
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protein by young carp. Bulletin of the Japanese Society of Scientific Fisheries,
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39 36(3), 250–254.
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41 Okorie, E. O., Kim, Y.-C., Lee, S., Bae, jun-young, H. Yoo, J., Han, K., Bai, S., Park,
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44 G.-J., & Choi, S.-M. (2007). Reevaluation of the Dietary Protein Requirements
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46 and Optimum Dietary Protein to Energy Ratios in Japanese Eel, Anguilla
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48 japonica. Journal of The World Aquaculture Society - J WORLD AQUACULT
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SOC, 38, 418–426. https://doi.org/10.1111/j.1749-7345.2007.00113.x
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53 Pandian, T.J. (1987). Fish energetics. In: Animal energetics. Vol. 2. New York:
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55 Academic Press.
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Rainboth, W. J. (1996). Fishes of the Cambodia Mekong. Rome: Food and Agriculture
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7 Organization of The United States.
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9 Robbins, K.R., Norton, H.W., & Baker, D.H. (1979). Estimation of nutrient
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requirements from growth data. Journal of Nutrition, 109, 1710-1714.
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14 Robbins, K.R., Saxton, A.M., & Southern, L.L. (2006). Estimation of nutrient
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16 requirements using broken-line regression analysis. Journal of Animal Science
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18 84 (Suppl. E): E155-E165.
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21 Santiago, C. B., & Reyes, O. S. (1991). Optimum dietary protein level for growth of
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23 bighead carp (Aristichthys nobilis) fry in a static water system. Aquaculture,
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30 (Macrognathus siamensis; Günther, 1861). Proceeding of 45th Kasetsart
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Satpathy, B. B., Mukherjee, D., & Ray, A. K. (2003). Effects of dietary protein and
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lipid levels on growth, feed conversion and body composition in rohu, Labeo
On

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39 rohita (Hamilton), fingerlings. Aquaculture Nutrition, 9(1), 17–24.
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41 https://doi.org/10.1046/j.1365-2095.2003.00223.x
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44 Shyong, W.-J., Huang, C.-H., & Chen, H.-C. (1998). Effects of dietary protein
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46 concentration on growth and muscle composition of juvenile Zacco barbata.
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48 Aquaculture, 167(1), 35–42. https://doi.org/10.1016/S0044-8486(98)00313-5
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Siddiqui, A. Q., Howlader, M. S., & Adam, A. A. (1988). Effects of dietary protein
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53 levels on growth, feed conversion and protein utilization in fry and young Nile
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55 tilapia, Oreochromis niloticus. Aquaculture, 70(1), 63–73.
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Thilsted, S. H. (2010). The potential of nutrition-rich small fish species in aquaculture
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7 to improve human nutrition and health. Proceeding of the Global Conference
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9 on Aquaculture, 57–73.
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Tibbetts, S. M., Lall, S. P., & Anderson, D. M. (2000). Dietary protein requirement of
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14 juvenile American eel (Anguilla rostrata) fed practical diets. Aquaculture,
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16 186(1–2), 145–155. https://doi.org/10.1016/S0044-8486(99)00363-4
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18 Walker, A. B., & Berlinsky, D. L. (2011). Effects of Partial Replacement of Fish Meal
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21 Protein by Microalgae on Growth, Feed Intake, and Body Composition of
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23 Atlantic Cod. North American Journal of Aquaculture, 73(1), 76–83.
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25 https://doi.org/10.1080/15222055.2010.549030
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Wee, K.L., & A.G.J. Tacon. (1982). A preliminary study on the dietary protein
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30 requirement of juvenile snakehead. Bulletin of the Japanese Society for the
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32 Science of Fish, 48, 1463-1468.

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Wee, K.L., & Ngamsnae, P. (1987). Dietary protein requirement of fingerlings of the
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herbivorous carp tawes, Puntius gonionotus (Bleeker). Aquaculture and
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39 Fisheries Management, 18, 121-129.
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41 Yao-Ping, L., Jian-Ming, C., Jin-Yun, Y., Xu-Xiong, H., Shen-Ying, L., Bin-Qian, S.,
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44 Zi-Liang, Y., Jian-Lin, G., & Li-Ping, Y. (2009). The effects of diet protein
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46 levels on the growth, body composition and digestive enzyme activities of the
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48 Barbodes caldwelli juvenile. Chinese Journal of Agricultural Biotechnology,
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6(3), 199–205. https://doi.org/10.1017/S1479236209990222
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Table 1. Formulation and composition analysis of the experimental diets (% Dry wt.
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6 basis).
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8 Dietary Protein levels
9 Ingredients
10 35 % 40 % 45 % 50 % 55 % 60 %
11
12 Fish meal 34.00 39.00 43.00 43.80 47.10 46.57
13 Krill meal 12.00 16.00 20.00 21.00 25.80 33.10
14 Gluten 3.00 3.00 3.00 3.00 3.00 3.00
15 Wheat flour 14.00 9.50 5.00 3.75 0.50 1.00
16
Rice bran 13.50 9.50 5.00 3.75 0.50 0.50
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18
Squid oil 2.45 1.88 1.80 1.14 0.58 0.00
19 Vegetable oil 6.65 6.20 5.80 4.65 3.95 1.50
20 Tuna oil 2.45 1.88 1.80 1.14 0.58 0.00
21 Ascorbic acid 0.10 0.10 0.10 0.10 0.10 0.10
Fo

22 Choline Chloride 0.20 0.20 0.20 0.20 0.20 0.20

23 Vitamin premix1 1.00 1.00 1.00 1.00 1.00 1.00
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Mineral Premix2 1.00 1.00 1.00 1.00 1.00 1.00
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26 α-starch 7.00 7.00 7.00 7.00 7.00 7.00
27 Cellulose 2.65 3.74 5.30 8.47 8.69 5.03
28 Total 100 100 100 100 100 100
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29 Proximate analysis (% Dry wt. basis)

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31 Moisture (%) 8.41 7.56 8.01 8.69 7.45 9.14
iew

32 Crude protein 36.20 41.84 46.02 49.90 54.04 60.06

33 Ether extract 15.74 13.71 13.68 11.31 10.03 6.98
34 Ash 7.31 7.86 8.21 8.25 8.68 9.07
35 Crude fiber 5.88 5.82 7.15 10.26 10.43 7.15
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NFE 26.46 23.21 16.93 11.59 9.37 7.60
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38 GE (kcal·100/g) 463.08 462.27 459.54 436.91 438.99 436.84
39 GE:P 12.79 11.05 9.99 8.76 8.12 7.27
40 1Vitamin mix (g/kg premix): vitamin A retinol acetate, 0.80; vitamin D3 cholecalciferol, 0.06;
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41 vitamin E a-tocopherol acetate, 4.00; vitamin K3 menadione, 8.00; thiamin, 2.00; riboflavin,
42 2.00; pantothenic acid, 6.00; pyridoxine, 2.00; folic acid, 0.50; niacin, 15; vitamin B12
43 cyanocobalamin, 0.02; inositol, 40.00; corn starch, 920.62.
44 2Mineral premix (g/kg mix): FeSO .7H O, 15; CuSO .H O, 0.3; ZnSO .7H O, 10;
45 4 2 4 2 4 2

46 MnSO4.H2O, 0.5; NaCl, 30;MgSO4, 40; Ca(H2PO4)2, 400; KI, 0.05; Na2SeO3, 0.005; CoCl3
47 .6H2O, 0.5; zeolite, 503.645.
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Table 2. Water quality parameters of all treatments during the study period.
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9 Dietary protein levels (%)
Water quality Parameters
10 35 40 45 50 55 60
11 Water temperature (ºC) 25.80-29.50 26.00-30.43 25.70-29.70 26-28.70 25.70-28.70 25.70-28.70
12 Air temperature (ºC) 27.50-30.50 27.50-30.50 27.50-30.50 27.50-30.50 27.50-30.50 27.50-30.50
13 Dissolved oxygen (mg/l) 4.30-5.73 4.30-5.70 4.30-5.80 4.30-5.20 4.30-5.30 4.40-5.80
14 Free CO2 (mg/l) 1.50-2.60 1.45-2.60 1.35-2.50 1.50-2.80 1.50-2.60 1.60-2.50
15 pH 6.5-7.5 6.5-7.0 6.9-7.5 6.8-7.9 6.6-7.5 6.5-7.0
Hardness (mg/l as CaCO3) 35.80-45.70 35.60-47 35.80-50 35.80-45.70 36.20-52.20 36.67-51.67
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Alkalinity (mg/l as CaCO3) 16.20-17.58 16.70-18.75 17.40-18.05 15.20-18.25 16.45-17.15 17.30-18.67
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Total ammonia-N (mg/l) 0.037-0.110 0.029-0.207 0.045-0.095 0.051-0.403 0.039-0.082 0.053-0.236
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20 Table 3. Growth performance, feed utilization efficiency and protein utilization efficiency
21 of fish fed experimental diets for 12 weeks.
Fo

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23 Growth Parameters
Dietary protein levels (%)
24 35 40 45 50 55 60
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Initial weight (g) 0.22±0.04 0.22±0.04 0.22±0.04 0.22±0.04 0.22±0.04 0.22±0.04

25 Final weight (g) 0.50±0.03a 0.69±0.04b 0.78±0.03c 0.84±0.03c 0.85±0.06c 0.84±0.07c
26 Initial length (cm) 4.69±3.34 4.69±3.34 4.69±3.34 4.69±3.34 4.69±3.34 4.69±3.34
27 Final length (cm) 5.32±0.69a 5.78±1.09ab 5.38±0.40ab 5.81±0.40ab 6.42±0.24b 6.34±0.37ab
Specific Growth Rate (SGR) 1.11±0.09a 1.56±0.78b 1.73±0.06c 1.83±0.06c 1.84±0.10c 1.82±0.13c
28
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Daily Weight Gain (DWG) 3.77±0.57a 6.33±0.57b 7.68±0.58c 8.67±0.58c 8.67±0.58c 8.33±0.12 c
29 % Weight gain 123.37±14.85a 208.42±18.56b 249.12±14.03c 274.18±15.31c 276.11±27.17c 273.15±34.22c
30 % Daily Feeding Rate (DFR) 2.39±0.26a 2.69±0.37ab 2.82±0.22ab 2.96±0.37b 2.56±0.19ab 2.31±0.15a
31 Average Daily Feed intake (g) 0.63±0.09a 0.89±0.09b 1.03±0.81bc 1.13±0.11c 0.99±0.11bc 0.89±0.12b
iew

Protein Efficiency Ratio (PER) 0.98±0.21a 1.04±0.27a 1.04±0.16a 1.02±0.15a 0.98±0.07a 1.05±0.08a
32 Feed Efficiency Ratio (FER) 0.36±0.08a 0.43±0.11ab 0.48±0.08ab 0.51±0.07bc 0.53±0.04bc 0.63±0.05c
33 App. Net Protein Retention (ANPR) 15.60±0.49a 17.81±3.55a 16.53±1.31a 15.12±2.39a 15.46±1.96a 15.57±0.50a
34 Average Protein Intake (g) 0.23±0.04a 0.37±0.04b 0.48±0.04c 0.56±0.05c 0.53±0.06c 0.53±0.07c
Feed Conversion Ratio (FCR) 2.27±0.36b 1.91±0.34ab 1.84±0.16ab 1.85±0.28ab 1.69±0.29a 1.62±0.29a
35 Survival Rate 82.67±14.05a 72±4.00a 76±8.00a 84±4.00a 72±10.58a 80±10.58a
36
Row means with the same letters are not significantly different (P>0.05).
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40 Table 4. Proximate carcass composition of young Siamese spiny eel fed diets containing
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41 graded levels of protein over 12 weeks.

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Dietary Protein Levels (%) As percentage of dry matter (after 12 weeks)
45 Moisture Protein Lipid Ash
46 Before the Expt. 14.39 62.43 6.94 11.75
47 35 15.76 70.68 6.54 17.84
48 40 17.54 70.21 7.25 17.58
49 45 18.17 68.86 8.12 16.96
50
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50 17.85 69.82 8.30 16.89
52 55 17.15 70.50 7.82 16.40
53 60 17.58 69.84 6.68 18.44
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0.9
7 35%Protein
8 0.8
40%Protein
Average Weight (g)
9 0.7
45%Protein
10 0.6
50%Protein
11 0.5
55%Protein
12 0.4
13 60%Protein
0.3
14
0.2
15
16 0.1
17 0
18 0 2 4 6 8 10 12
19 Time (Weeks)
Fo
20
Figure 1. Changes in average weight of young Siamese spiny eel fed (Macrognathus
21
22
siamensis), with different protein content diets.
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Y = 12.76X - 309.36 for X < 46.50

27 300 Y = 275.37 for X > 46.50

28 250
29
%Weight gain

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30 200
31 150
32
33 100
34 46.50
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36 0
37 30 35 40 45 50 55 60 65
38 Dietary Protein Level (%)
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40 Figure 2. Optimum dietary Protein level for young Siamese spiny eel (Macrognathus
41
siamensis), determined by Breakpoint regression analysis.
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