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Research article
Dietary Exogenous α-Amylase Modulates the Nutrient Digestibility, Digestive Enzyme Activity, Growth-Related Gene Expression, and Diet Degradation Rate of Olive Flounder (Paralichthys olivaceus)
1Core-Facility Center for Tissue Regeneration, Dong-Eui University, Busan 47340, Republic of Korea
2Department of Aquaculture, Sylhet Agricultural University, Sylhet-3100, Bangladesh
3Aquafeed Research Center, National Institute of Fisheries Science, Pohang 37517, Republic of Korea
J. Microbiol. Biotechnol. 2023; 33(10): 1390-1401
Published October 28, 2023 https://doi.org/10.4014/jmb.2303.03033
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Graphical Abstract

Introduction
Olive flounder (
Similar to terrestrial animals, such as pigs and poultry [12], dietary exogenous enzymes can be used to reduce the impact of anti-nutritional factors in carnivorous fish diets containing plant-based feedstuffs [13]. Dietary supplementation with these enzymes increases the levels of substrates available to the intestinal microbial community, thereby improving nutrient digestion, synthesis of bioactive molecules, intestinal integrity, and fish growth [14]. Dietary administration of exogenous enzymes has been extensively studied in poultry and swine and applied in aquaculture feed to reduce both phytic acid levels and the anti-nutritional effects of non-starch polysaccharides to enhance the utilization of phosphorus and carbohydrates, respectively [15].
Several alternative animal- and plant-based sources have been proposed to satisfy the lipid and protein requirements of specific aquaculture species. However, the nutritional compositions of these alternative sources are often quite different from those of fish meal (FM) and fish oil (FO) [16]. Enhancing feed digestion and nutrient assimilation not only improves the well-being of fish but also increases their aquaculture profitability. The global carbohydrase market is dominated by xylanase, glucanase, and other commercially available carbohydrases, such as α-amylase, β-mannanase, α-galactosidase, and pectinase, which can hydrolyze carbohydrate polymers to produce low-molecular-weight oligosaccharides or polysaccharides [15, 17].
Amylase, an important endogenous digestive enzyme that degrades starch [18], is present in various fish species. Therefore, addition of exogenous amylase enzyme to diet formulations may facilitate the breakdown of complex carbohydrate polymers to produce glucose as an energy source in these fish [19]. Stone was the first to report that exogenous α-amylase supplementation in aquaculture feed increases the starch digestibility of silver perch (
In this study, we aimed to identify and quantify the effects of different doses of exogenous α-amylase-inoculated diet (soybean meal: 10.3%, tapioca starch: 10%, and wheat flour: 11.5%) on the growth, feed utilization, and apparent digestibility coefficient (ADC) of
Materials and Methods
All experiments were approved by and conducted at the Aquafeed Research Center (Pohang), National Institute of Fisheries Science (NIFS), Republic of Korea, following the NIFS regulations on the Care and Use of Laboratory Animals (approval no. 2021-NIFSIACUC-07).
Experimental Diet Formulation
Compositions of the experimental diets and proximate analyses results are shown in Table 1. Basal diets were prepared by thoroughly mixing the dry ingredients in an electric mixer, followed by extrusion in a twin-screw extruder (ATX-II; Fesco Precision Co., Korea) under the following conditions: feeder supply speed, 70 kg/h; conditioner temperature, 80°C; barrel temperature, 120–130°C; main screw speed, 650 rpm. The key ingredients for proteins (FM and soybean meal), carbohydrates (tapioca starch and wheat flour), and lipids (FO) in the experimental diets were purchased from Suhyup Feed Co. (Uiryeong, Korea). The required amount of α-amylase from
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Table 1 . Ingredient composition of the experimental diets fed to olive flounder over a 12-week growth trial (% of DM basis).
Ingredients Diet no. (AA supplementation level, U/kg diet) AA0 AA100 AA200 AA400 Fishmeal 60.0 Soybean meal 10.3 Tapioca starch 10.0 Wheat flour 11.5 Fish oil 5.0 Mineral mixture1 1.0 Vitamin mixture2 1.0 Choline 0.5 Cr2O3 0.5 α-amylase3 0 0.01 0.02 0.04 NaCl 0.2 0.19 0.18 0.16 Total 100 100 100 100 Analyzed nutrients compositions (as-is basis) Moisture (%) 4.63 5.02 4.73 4.86 Crude protein (%) 51.1 51.2 50.9 51.0 Crude lipid (%) 10.2 9.8 10.4 10.0 Crude ash (%) 13.8 13.8 13.9 13.6 Gross energy (cal/g) 4932 4925 4942 4926 1Mineral premix contained the following amount which were diluted in cellulose (g/kg premix): NaCl, 30.3; MgSO4-7H2O, 95.6; NaH2PO4-2H2O, 60.8; KH2PO4, 167.3; CaH4(PO4)2-H2O, 94.7; Ferric citrate, 20.7; ZnSO4-7H2O, 15.3; Ca-lactate, 212.8; CuCl, 0.14; AlCl3-6H2O, 0.105; KI, 0.105; Na2Se2O3, 0.01; MnSO4-H2O, 1.4; CoCl2-6H2O, 0.7.
2Vitamin premix contained the following amount which were diluted in cellulose (g/kg premix): L-ascorbic acid, 171.1; myoinositol, 181.8; DL-a-tocopheryl acetate, 18.9; niacin, 36.4; p-aminobenzoic acid, 18.2; Ca-D-pantothenate, 12.7; riboflavin, 9.1; thiamin hydrochloride, 2.7; pyridoxine hydrochloride, 1.8; menadione, 1.8; retinyl acetate, 0.73; folic acid, 0.68; D-biotin, 0.27; cholecalciferol, 0.003.
3α-amylase from
Aspergillus oryzae (30 units/mg; Sigma Aldrich, St. Louis, USA
Fish Rearing and Feeding Trial
Juvenile
Sample Collection
At the end of the feeding trial, fish were starved for 24 h, and all surviving fish in the tanks were caught and weighed. Subsequently, three fish from each tank (nine fish/diet group) were anesthetized with tricaine methanesulfonate (MS-222; 100 mg/l, buffered to pH 7.4) for whole-body composition analysis. An equal number of fish was sampled for blood collection using heparinized syringes to quantify the plasma amylase activity.
Variables Measured for Growth and Feed Utilization Parameters
At the start of the experiment, fish body weight was measured to calculate the initial body weight (IBW). During the feeding trials, the amount of supplemented feed in each tank was monitored to calculate the feed utilization parameters. At the end of the trial, the final body weight (FBW), weight gain (WG, %), specific growth rate (SGR), feed conversion ratio (FCR), protein efficiency ratio (PER), and survival rate (%) were calculated using the following equations:
IBW (g) = Initial weight of total fish in tank/Fish number
FBW (g)= Final weight of total fish in tank/Fish number
WG (%) = ([FBW – IBW]/IBW) × 100
SGR (%/day) = ([ln FBW – ln IBW]/day) × 100
FCR = Dry feed intake/Wet body weight gain
PER =Wet weight gain/Protein fed
Survival (%) = (number of fish at the end of the trial/number of fish at the beginning of the trial) × 100.
Feed and Whole-Body Proximate Composition Analyses
Proximate composition analyses of the experimental feeds and whole fish bodies were conducted according to the standard methods of the Association of Official Analytical Chemists [26]. The collected fish samples were homogenized using an industrial food processor. Samples were dried in a convection oven at 105°C for 24 h to determine the moisture content. Crude protein content was determined using the Kjeldahl method (N × 6.25) after acid digestion with an auto Kjeldahl system (VAP50OT/TT125; Gerhardt GmbH & Co., Germany). Crude lipids were measured using the Soxhlet extraction method with the Tecator Soxtec System HT 1046 (Tecator AB, Sweden) after freeze-drying the samples for 20 h. Ash was analyzed by incineration at 550°C in a muffle furnace for 5 h. Feed energy content was determined using an isoperibol bomb calorimeter (Parr 6300; Parr Instrument Company Inc., USA).
Apparent Digestibility and Fecal Particle Size Test
After sample collection, ADC of the dry matter, proteins, lipids, carbohydrates, and energy was determined for the remaining fish. Each experimental diet was fed to the olive flounder (18 fish/ 1,000-L tank) at apparent satiation twice daily for two weeks. Then, fecal samples were collected by siphoning onto a mesh two hours after feeding to avoid leaching of the nutrients and stored in tubes at −20°C until required for analysis. The fecal samples collected daily were pooled per tank (three experimental units/treatment) for ADC and fecal particle size analyses. ADC of the dry matter, protein, lipids, carbohydrates, and energy of the diets was calculated using the following equation given by Bureau
ADC diet = 1 − ([F/D] × [Di/Fi]),
where D = % nutrient of diet, F = % nutrient of feces, Di = % digestion indicator of diet, and Fi = % digestion indicator of feces.
Fecal particle size was measured using a Mastersizer 3000 (Malvern, USA) fitted with a Hydro LV wet sample measurement accessory that has detectors ranging from 0.01 to 10.000 μM. Feces were suspended in water in the Hydro LV and circulated through the Mastersizer 3000 for 25 consecutive measurements, each of which was 5 s in duration. The first 25 measurements represent the particle size distribution of the fecal casts. The particle size distributions with 10, 50, and 90% of the total volume were calculated using the Mastersizer software.
Feed Degradation Rate
Feed degradation rate was determined using a modified version of the method described by Azarfar
Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)
To quantify the effects of α-amylase administration on the expression levels of immune- and growth-related genes in olive flounder, qRT-PCR was conducted as described by Hasan
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Table 2 . Gene specific primers used to quantify relative gene expression.
Gene Sense Oligonucleotide sequence (5` to 3`) Access No. Elongation factor-1-α (Reference) F GAGGTCAAGTCTGTGGAGAT AB915949 R GGTGGTTCAGGATGATGAC Tumor necrosis factor F CCCTATGAACTGTAACAGTTTG AB040448 R GTCAGGTACTTAACCCTCAT Interleukin 1 β F TGCTACCAGACCTTCAACAT AB070835 R TCTTTCCAGCAGACAGTGGT Interleukin 2 F ACATACGTACTTCAAGCTATCG KY307833 R GTAAAGATTCCACTTGGTCCA Immunoglobulin M F GCCTCCTTCTTCTGCTCTG AB109029 R CCTCAGTGGATGTTGTGATT Heat shock protein 70 F CAATGATTCTCAGAGGCAAG DQ662230 R TATCTAAGCCGTAGGCAATC Insulin-like growth factor 1 F ATGTCTAGCGCTCTTTCCTT AF061278 R CTTCTTGTTTTTTGTCTTGTCTG Insulin-like growth factor 2 F AGAACCGTGGGATCGTAGA AF091454 R TGCCACACCTCGTATTTG Transforming growth factor β 3 F TCCAAGGTATTCCGCTTCAA XM_020085122 R TTTGGCTTTGGGGTCATCT Growth hormone F TCCTCTCAGCCAATCACAGA M23439 R TACGTCTCCACCTTGTGCAT Growth hormone receptor F CCACAAACTGGAAATCATTGG AB058418 R CGAAAACAAGAACAACTGTGAG
Plasma Amylase Activity
Collected blood was centrifuged at 5,000 rpm (rcf: 7,168 ×
Statistical Analysis
Homogeneity of error variance was determined using Levene’s test, and the dependent variables were subjected to one-way analysis of variance using SAS Version 9.3 (SAS Institute, USA). When a significant (
Results
Growth, Feed Utilization, and Whole-Body Proximate Composition
After 12 weeks of feeding trial, no significant differences (
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Table 3 . Growth and feed utilization parameters of olive flounder fed with experimental diets for 12 weeks1.
AA0 AA100 AA200 AA400 P valueIBW2 273.2±3.9 273.5±3.4 272.2±1.8 273.3±0.6 0.939 FBW3 555.3±4.5 564.5±8.4 563.8±2.3 566.1±7.1 0.203 FI4 331.4±4.3 323.7±7.1 329.3±3.3 326.1±4.6 0.312 WG (%)5 103.3±2.1 106.4±1.4 107.1±2.2 107.1±2.3 0.126 SGR6 1.01±0.01 1.04±0.01 1.04±0.02 1.04±0.02 0.123 FCR7 1.16±0.02 1.11±0.04 1.10±0.02 1.11±0.02 0.054 PER8 1.66±0.06 1.76±0.06 1.73±0.04 1.76±0.03 0.117 Survival (%) 98.7±2.3 100 97.3±2.3 100 0.219 1Values are mean ± SD of three replicates (3 tank/group). Values without superscript letters within the same row in the table are not significantly (
p ≥ 0.05) different.2IBW: Initial body weight (g); 3FBW: Final body weight (g); 4FI: Feed intake (g/fish); 5WG: Weight gain (%); 6SGR: Specific growth rate (%/d); 7FCR: Feed conversion ratio; 8PER: Protein efficiency ratio
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Table 4 . Whole-body proximate composition (%, as-is basis) of juvenile rainbow trout fed experimental diets for 12 weeks1.
AA0 AA100 AA200 AA400 P valueMoisture 71.2±0.3 71.1±0.2 71.3±0.2 71.2±0.2 0.761 Crude protein 19.2±0.2 19.2±0.1 19.4±0.2 19.2±0.2 0.518 Crude lipid 5.02±0.08 5.12±0.09 4.92±0.11 5.07±0.13 0.326 Crude ash 3.43±0.18 3.36±0.06 3.52±0.28 3.51±0.19 0.834 1Values are mean ± SD of three replicates (3 tank/group). Values without superscript letters within the same row in the table are not significantly (
p ≥ 0.05) different.
ADC
Fish fed the experimental diets (AA100, AA200, and AA400) exhibited significant differences in carbohydrate ADC compared with the control (
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Table 5 . Apparent digestibility coefficients (%) of dry matter, crude protein, crude lipid, carbohydrate, and energy for experimental diets fed to olive flounder for 12 weeks1.
AA0 AA100 AA200 AA400 P valueDry matter 69.3±0.9 72.6±0.5 71.8±0.3 72.6±1.1 0.103 Crude protein 87.4±0.4 87.1±0.2 86.8±0.2 87.5±0.5 0.600 Crude lipid 89.5±0.3 89.6±0.2 90.0±0.1 90.4±0.4 0.217 Carbohydrate 83.2±0.5b 88.7±0.2a 87.7±0.1a 88.6±0.5a 0.001 Gross energy 86.6±0.4 86.3±0.3 86.0±0.2 86.2±0.6 0.680 1Values are mean ± SD of three replicated tanks. Values without/similar and different superscript letters within the same row in the table are not significantly (
p ≥ 0.05) and are significantly (p < 0.05) different, respectively.
Feed Degradation Rate (%)
AA0 and AA400 groups exhibited the lowest and highest feed degradation rates (%), respectively. Compared to the control, α-amylase significantly and dose-dependently increased the feed degradation rates in AA100, AA200, and AA400 groups to 41.8 ± 2.5, 55.3 ± 1.2, and 81.7 ± 2.9%, respectively (Table 6).
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Table 6 . Feed degradation rate (%) in different experimental feeds1.
AA0 AA100 AA200 AA400 p -valuePrepared pellet (g) 6.00 6.00 6.00 6.00 1.000 Soaked pellet (g) 4.71±0.14a 3.49±0.15b 2.68±0.07c 1.10±0.17d <0.001 Feed degradation rate (%)2 21.5±2.3d 41.8±2.5c 55.3±1.2b 81.7±2.9a <0.001 1Values are mean ± SD (3 times/group). Values without and different superscript letters within the same row in the table are not significantly (
p ≥ 0.05) and are significantly (p < 0.05) different, respectively.2Feed degradation rate (%): (dry pellet before soaking - dry pellet after soaking) / dry pellet before soaking × 100.
Feed pellets collected from the fish stomachs were gradually degraded at 2, 4, and 8 h. Interestingly, this degradation was time-dependent and higher diet morphological alteration/degradation was observed in the group with the highest α-amylase concentration (Fig. 1).
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Fig. 1. Photograph of the feed collected from flounder stomach after 2, 4, and 8 h of feeding supplemented with 0 (A00), 100 (AA100), 200 (AA200), and 400 (AA400) mg/kg of α-amylase.
Plasma α-Amylase Concentration and Fecal Particle Size
After 12 weeks of feeding, plasma α-amylase concentration increased (
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Fig. 2. α-Amylase activity of plasma between the experimental groups. The data represent the mean ± standard deviation (6 fish/group); values with different letters indicate significant differences (
p < 0.05).
In the 90% fecal particle size class, the particle size of AA0 group was significantly larger than those of the AA200 and AA400 groups. Moreover, 90% fecal particle size in olive flounder fed with the control (AA0) diet was < 134.0 ± 8.0 μM, whereas the AA100, AA200, and AA400 groups exhibited particle sizes of < 116.5 ± 2.5, < 95.8 ± 3.3, and < 93.5 ± 4.5 μM, respectively (Fig. 3; Table 7). In contrast, no significant variations were observed in the 50 and 10% size classes. Additionally, 90% particle sizes of the AA0 and AA100 groups were similar, whereas those of AA200 and AA400 were significantly different (
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Table 7 . Fecal particle size of olive flounder fed with four types of experimental diets for 12 weeks1.
Diet group Particle size (µM) 10% size class 50% size class 90% size class AA0 < 4.4±0.4a < 50.7±4.6a < 134.0±8.8a AA100 < 3.2±1.6a < 40.3±3.2a < 116.5±2.5ab AA200 < 3.8±0.10a < 41.8±0.9a < 95.8±3.3b AA400 < 2.8±0.5a < 36.7±3.4a < 93.5±4.5b 1Values are mean ± SD (3 tanks/group). Values with same and different superscript letters within the same column in the table are not significantly (
p ≥ 0.05) and are significantly (p < 0.05) different, respectively.
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Fig. 3. Fecal particle size of olive flounder fed with four types of experimental diets (
n = 3 tanks per diet). Feed was supplemented with 0 (AA00), 100 (AA100), 200 (AA200), and 400 (AA400) mg/kg of α-amylase.
Effects of α-Amylase on the Transcription of Immune- and Growth-Related Genes
Compared with the control, transcription levels of seven immune-related genes (tumor necrosis factor [
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Fig. 4. Profiles of gene expression in the muscle (A) and intestine (B) of olive flounder. Expression of these genes in olive flounder was measured by RT-qPCR after 12 weeks of feeding for the Control (AA0), AA100, AA200, and AA400 groups. Levels of gene expression were quantified relative to
elongation factor -1α transcription. The data are represented as the means ± standard deviation (6 fish/group); means that do not share the same letter differ significantly (p < 0.05).
In contrast, expression levels of growth-related genes, such as insulin-like growth factor
Discussion
In this study, dietary supplementation with three concentrations of α-amylase had no significant effects on the growth, feed utilization, and whole-body composition of olive flounder. Previous studies reported that cellulase and an enzyme cocktail comprising xylanase, amylase, cellulase, protease, and β-glucanase have no effects on the growth performance of
Also in this study, α-amylase had no significant effect on the ADC of dry matter, crude protein, lipid, and gross energy contents. However, an improved carbohydrate ADC was observed in all treatment groups. ADC of Indian major carp catla (
The carbohydrates (especially starch) that bind to the active site of the enzyme for digestion are hydrophilic [39], and our in vitro experiments suggested that the improvement in the carbohydrate degradation rates of olive flounder was due to the presence of α-amylase. This enzyme is also likely involved in changes in the feed structure in the stomach. However, further experiments are required to confirm these findings. Dietary supplementation with 200 and 400 mg/kg of α-amylase increased plasma amylase concentration in the experimental olive flounder. No previous study has estimated the activity of digestive enzymes in the blood in response to the application of exogenous enzymes. However, α-amylase application elevates glucose-6-phosphate dehydrogenase levels in the liver and blood of
GH binds to GH receptors and stimulates the synthesis of IGF-1 in the liver [49]. Our findings demonstrated that the levels of
In this study, olive flounder fed without α-amylase produced larger (90% size class) fecal particles than the 200 and 400 mg/kg enzyme-supplemented groups. Fecal particle size is a key indicator of digestion and mechanical stability of feces [54]. Starch is positively correlated with the production of larger fecal particles [55-57], whereas oligosaccharides in feed have little to no effect on the particle size or structure. Therefore, the larger fecal particles observed in the control group could be due to a higher level of starch in the digested food caused by a lack of amylase enzymes. In contrast, the two groups with the highest amylase doses produced smaller fecal particles, presumably because the carbohydrates in the feed were properly digested by the exogenous amylase. This also explains the increase in carbohydrate ADC observed in our experiment. Very few studies have assessed the effects of supplementation with exogenous enzymes (amylase, xylanase, cellulase, and gluconase) on fecal particle size in aquaculture species. A recent study by Welker
Conclusion
In this study, dietary application of α-amylase at 200 and 400 mg/kg improved the carbohydrate ADC, blood amylase content, feed degradation rate, and fecal particle size in olive flounder. Interestingly, α-amylase had no significant effects on the growth, feed utilization, and whole-body composition of this fish species. However, the effects of exogenous enzyme supplementation on the immunology, intestinal microbiome, serum biochemistry, and transcription of growth and digestive genes in cultured olive flounder require further elucidation in future studies. Moreover, the involvement of external enzymes in the activation of different physio-immunological pathways needs to be investigated in the future.
Acknowledgments
This study was supported by the National Institute of Fisheries Science, Ministry of Oceans and Fisheries, Republic of Korea (R2023036).
Author Contributions
MTH: Data Curation. Methodology, Software, Investigation, Writing – Original Draft. HJK: Conceptualization, Methodology, Software, Investigation. SWH, SMJ, and KKW: Investigation, Resources, Data Curation. SHL: Supervision, Project Administration.
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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Related articles in JMB

Article
Research article
J. Microbiol. Biotechnol. 2023; 33(10): 1390-1401
Published online October 28, 2023 https://doi.org/10.4014/jmb.2303.03033
Copyright © The Korean Society for Microbiology and Biotechnology.
Dietary Exogenous α-Amylase Modulates the Nutrient Digestibility, Digestive Enzyme Activity, Growth-Related Gene Expression, and Diet Degradation Rate of Olive Flounder (Paralichthys olivaceus)
Md. Tawheed Hasan1,2†, Hyeon Jong Kim3†, Sang-Woo Hur3, Seong-Mok Jeong3, Kang-Woong Kim3, and Seunghan Lee3*
1Core-Facility Center for Tissue Regeneration, Dong-Eui University, Busan 47340, Republic of Korea
2Department of Aquaculture, Sylhet Agricultural University, Sylhet-3100, Bangladesh
3Aquafeed Research Center, National Institute of Fisheries Science, Pohang 37517, Republic of Korea
Correspondence to:Seunghan Lee, shlee5863@naver.com
†These authors contributed equally to this work.
Abstract
In this study, a 12-week feeding experiment was conducted to characterize the effects of exogenous α-amylase on the growth, feed utilization, digestibility, plasma α-amylase activity, feed degradation rate, and fecal particle size of olive flounder (Paralichthys olivaceus). Diet was supplemented with 0 (AA0; control), 100 (AA100), 200 (AA200), or 400 (AA400) mg/kg of α-amylase, respectively. Fish (273.1 ± 2.3 g) were stocked into 12 tanks (25 fish/1,000-L tank) and 3 tanks were randomly selected for each diet group. As a result, α-amylase was found to have no significant effects (p ≥ 0.05) on the growth, feed utilization parameters, and whole-body proximate compositions. α-Amylase-treated fish exhibited only a significant increase in the apparent digestibility coefficient of carbohydrates compared to the controls. In addition, in vitro analyses revealed that α-amylase dose-dependently increased (p < 0.05) the feed degradation rate, while photographs of the intestinal content after 2, 4, and 8 h of feeding demonstrated an improved degradation rate in the α-amylase-treated groups. Plasma α-amylase content was higher in the AA200 and AA400 groups, whereas the control group produced significantly larger-sized fecal particles (90% size class) than these two groups. In the intestine, no changes were observed in the expression levels of the immune-related TNF-α, IL-1β, IL-2, immunoglobulin-M, HSP-70, lysozyme, and amylase alpha-2A. However, growth-related genes IGF-1, IGF-2, TGF-β3, and growth hormone genes were upregulated in muscle tissues. Collectively, exogenous α-amylase has positive roles in the modulation of the digestibility coefficient, blood α-amylase concentration, growth-related gene expression, and diet degradation for improved digestion in olive flounder.
Keywords: Aqua-feed, feed additive, olive flounder, digestive enzymes, alpha-amylase, digestibility
Introduction
Olive flounder (
Similar to terrestrial animals, such as pigs and poultry [12], dietary exogenous enzymes can be used to reduce the impact of anti-nutritional factors in carnivorous fish diets containing plant-based feedstuffs [13]. Dietary supplementation with these enzymes increases the levels of substrates available to the intestinal microbial community, thereby improving nutrient digestion, synthesis of bioactive molecules, intestinal integrity, and fish growth [14]. Dietary administration of exogenous enzymes has been extensively studied in poultry and swine and applied in aquaculture feed to reduce both phytic acid levels and the anti-nutritional effects of non-starch polysaccharides to enhance the utilization of phosphorus and carbohydrates, respectively [15].
Several alternative animal- and plant-based sources have been proposed to satisfy the lipid and protein requirements of specific aquaculture species. However, the nutritional compositions of these alternative sources are often quite different from those of fish meal (FM) and fish oil (FO) [16]. Enhancing feed digestion and nutrient assimilation not only improves the well-being of fish but also increases their aquaculture profitability. The global carbohydrase market is dominated by xylanase, glucanase, and other commercially available carbohydrases, such as α-amylase, β-mannanase, α-galactosidase, and pectinase, which can hydrolyze carbohydrate polymers to produce low-molecular-weight oligosaccharides or polysaccharides [15, 17].
Amylase, an important endogenous digestive enzyme that degrades starch [18], is present in various fish species. Therefore, addition of exogenous amylase enzyme to diet formulations may facilitate the breakdown of complex carbohydrate polymers to produce glucose as an energy source in these fish [19]. Stone was the first to report that exogenous α-amylase supplementation in aquaculture feed increases the starch digestibility of silver perch (
In this study, we aimed to identify and quantify the effects of different doses of exogenous α-amylase-inoculated diet (soybean meal: 10.3%, tapioca starch: 10%, and wheat flour: 11.5%) on the growth, feed utilization, and apparent digestibility coefficient (ADC) of
Materials and Methods
All experiments were approved by and conducted at the Aquafeed Research Center (Pohang), National Institute of Fisheries Science (NIFS), Republic of Korea, following the NIFS regulations on the Care and Use of Laboratory Animals (approval no. 2021-NIFSIACUC-07).
Experimental Diet Formulation
Compositions of the experimental diets and proximate analyses results are shown in Table 1. Basal diets were prepared by thoroughly mixing the dry ingredients in an electric mixer, followed by extrusion in a twin-screw extruder (ATX-II; Fesco Precision Co., Korea) under the following conditions: feeder supply speed, 70 kg/h; conditioner temperature, 80°C; barrel temperature, 120–130°C; main screw speed, 650 rpm. The key ingredients for proteins (FM and soybean meal), carbohydrates (tapioca starch and wheat flour), and lipids (FO) in the experimental diets were purchased from Suhyup Feed Co. (Uiryeong, Korea). The required amount of α-amylase from
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Table 1 . Ingredient composition of the experimental diets fed to olive flounder over a 12-week growth trial (% of DM basis)..
Ingredients Diet no. (AA supplementation level, U/kg diet) AA0 AA100 AA200 AA400 Fishmeal 60.0 Soybean meal 10.3 Tapioca starch 10.0 Wheat flour 11.5 Fish oil 5.0 Mineral mixture1 1.0 Vitamin mixture2 1.0 Choline 0.5 Cr2O3 0.5 α-amylase3 0 0.01 0.02 0.04 NaCl 0.2 0.19 0.18 0.16 Total 100 100 100 100 Analyzed nutrients compositions (as-is basis) Moisture (%) 4.63 5.02 4.73 4.86 Crude protein (%) 51.1 51.2 50.9 51.0 Crude lipid (%) 10.2 9.8 10.4 10.0 Crude ash (%) 13.8 13.8 13.9 13.6 Gross energy (cal/g) 4932 4925 4942 4926 1Mineral premix contained the following amount which were diluted in cellulose (g/kg premix): NaCl, 30.3; MgSO4-7H2O, 95.6; NaH2PO4-2H2O, 60.8; KH2PO4, 167.3; CaH4(PO4)2-H2O, 94.7; Ferric citrate, 20.7; ZnSO4-7H2O, 15.3; Ca-lactate, 212.8; CuCl, 0.14; AlCl3-6H2O, 0.105; KI, 0.105; Na2Se2O3, 0.01; MnSO4-H2O, 1.4; CoCl2-6H2O, 0.7..
2Vitamin premix contained the following amount which were diluted in cellulose (g/kg premix): L-ascorbic acid, 171.1; myoinositol, 181.8; DL-a-tocopheryl acetate, 18.9; niacin, 36.4; p-aminobenzoic acid, 18.2; Ca-D-pantothenate, 12.7; riboflavin, 9.1; thiamin hydrochloride, 2.7; pyridoxine hydrochloride, 1.8; menadione, 1.8; retinyl acetate, 0.73; folic acid, 0.68; D-biotin, 0.27; cholecalciferol, 0.003..
3α-amylase from
Aspergillus oryzae (30 units/mg; Sigma Aldrich, St. Louis, USA.
Fish Rearing and Feeding Trial
Juvenile
Sample Collection
At the end of the feeding trial, fish were starved for 24 h, and all surviving fish in the tanks were caught and weighed. Subsequently, three fish from each tank (nine fish/diet group) were anesthetized with tricaine methanesulfonate (MS-222; 100 mg/l, buffered to pH 7.4) for whole-body composition analysis. An equal number of fish was sampled for blood collection using heparinized syringes to quantify the plasma amylase activity.
Variables Measured for Growth and Feed Utilization Parameters
At the start of the experiment, fish body weight was measured to calculate the initial body weight (IBW). During the feeding trials, the amount of supplemented feed in each tank was monitored to calculate the feed utilization parameters. At the end of the trial, the final body weight (FBW), weight gain (WG, %), specific growth rate (SGR), feed conversion ratio (FCR), protein efficiency ratio (PER), and survival rate (%) were calculated using the following equations:
IBW (g) = Initial weight of total fish in tank/Fish number
FBW (g)= Final weight of total fish in tank/Fish number
WG (%) = ([FBW – IBW]/IBW) × 100
SGR (%/day) = ([ln FBW – ln IBW]/day) × 100
FCR = Dry feed intake/Wet body weight gain
PER =Wet weight gain/Protein fed
Survival (%) = (number of fish at the end of the trial/number of fish at the beginning of the trial) × 100.
Feed and Whole-Body Proximate Composition Analyses
Proximate composition analyses of the experimental feeds and whole fish bodies were conducted according to the standard methods of the Association of Official Analytical Chemists [26]. The collected fish samples were homogenized using an industrial food processor. Samples were dried in a convection oven at 105°C for 24 h to determine the moisture content. Crude protein content was determined using the Kjeldahl method (N × 6.25) after acid digestion with an auto Kjeldahl system (VAP50OT/TT125; Gerhardt GmbH & Co., Germany). Crude lipids were measured using the Soxhlet extraction method with the Tecator Soxtec System HT 1046 (Tecator AB, Sweden) after freeze-drying the samples for 20 h. Ash was analyzed by incineration at 550°C in a muffle furnace for 5 h. Feed energy content was determined using an isoperibol bomb calorimeter (Parr 6300; Parr Instrument Company Inc., USA).
Apparent Digestibility and Fecal Particle Size Test
After sample collection, ADC of the dry matter, proteins, lipids, carbohydrates, and energy was determined for the remaining fish. Each experimental diet was fed to the olive flounder (18 fish/ 1,000-L tank) at apparent satiation twice daily for two weeks. Then, fecal samples were collected by siphoning onto a mesh two hours after feeding to avoid leaching of the nutrients and stored in tubes at −20°C until required for analysis. The fecal samples collected daily were pooled per tank (three experimental units/treatment) for ADC and fecal particle size analyses. ADC of the dry matter, protein, lipids, carbohydrates, and energy of the diets was calculated using the following equation given by Bureau
ADC diet = 1 − ([F/D] × [Di/Fi]),
where D = % nutrient of diet, F = % nutrient of feces, Di = % digestion indicator of diet, and Fi = % digestion indicator of feces.
Fecal particle size was measured using a Mastersizer 3000 (Malvern, USA) fitted with a Hydro LV wet sample measurement accessory that has detectors ranging from 0.01 to 10.000 μM. Feces were suspended in water in the Hydro LV and circulated through the Mastersizer 3000 for 25 consecutive measurements, each of which was 5 s in duration. The first 25 measurements represent the particle size distribution of the fecal casts. The particle size distributions with 10, 50, and 90% of the total volume were calculated using the Mastersizer software.
Feed Degradation Rate
Feed degradation rate was determined using a modified version of the method described by Azarfar
Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)
To quantify the effects of α-amylase administration on the expression levels of immune- and growth-related genes in olive flounder, qRT-PCR was conducted as described by Hasan
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Table 2 . Gene specific primers used to quantify relative gene expression..
Gene Sense Oligonucleotide sequence (5` to 3`) Access No. Elongation factor-1-α (Reference) F GAGGTCAAGTCTGTGGAGAT AB915949 R GGTGGTTCAGGATGATGAC Tumor necrosis factor F CCCTATGAACTGTAACAGTTTG AB040448 R GTCAGGTACTTAACCCTCAT Interleukin 1 β F TGCTACCAGACCTTCAACAT AB070835 R TCTTTCCAGCAGACAGTGGT Interleukin 2 F ACATACGTACTTCAAGCTATCG KY307833 R GTAAAGATTCCACTTGGTCCA Immunoglobulin M F GCCTCCTTCTTCTGCTCTG AB109029 R CCTCAGTGGATGTTGTGATT Heat shock protein 70 F CAATGATTCTCAGAGGCAAG DQ662230 R TATCTAAGCCGTAGGCAATC Insulin-like growth factor 1 F ATGTCTAGCGCTCTTTCCTT AF061278 R CTTCTTGTTTTTTGTCTTGTCTG Insulin-like growth factor 2 F AGAACCGTGGGATCGTAGA AF091454 R TGCCACACCTCGTATTTG Transforming growth factor β 3 F TCCAAGGTATTCCGCTTCAA XM_020085122 R TTTGGCTTTGGGGTCATCT Growth hormone F TCCTCTCAGCCAATCACAGA M23439 R TACGTCTCCACCTTGTGCAT Growth hormone receptor F CCACAAACTGGAAATCATTGG AB058418 R CGAAAACAAGAACAACTGTGAG
Plasma Amylase Activity
Collected blood was centrifuged at 5,000 rpm (rcf: 7,168 ×
Statistical Analysis
Homogeneity of error variance was determined using Levene’s test, and the dependent variables were subjected to one-way analysis of variance using SAS Version 9.3 (SAS Institute, USA). When a significant (
Results
Growth, Feed Utilization, and Whole-Body Proximate Composition
After 12 weeks of feeding trial, no significant differences (
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Table 3 . Growth and feed utilization parameters of olive flounder fed with experimental diets for 12 weeks1..
AA0 AA100 AA200 AA400 P valueIBW2 273.2±3.9 273.5±3.4 272.2±1.8 273.3±0.6 0.939 FBW3 555.3±4.5 564.5±8.4 563.8±2.3 566.1±7.1 0.203 FI4 331.4±4.3 323.7±7.1 329.3±3.3 326.1±4.6 0.312 WG (%)5 103.3±2.1 106.4±1.4 107.1±2.2 107.1±2.3 0.126 SGR6 1.01±0.01 1.04±0.01 1.04±0.02 1.04±0.02 0.123 FCR7 1.16±0.02 1.11±0.04 1.10±0.02 1.11±0.02 0.054 PER8 1.66±0.06 1.76±0.06 1.73±0.04 1.76±0.03 0.117 Survival (%) 98.7±2.3 100 97.3±2.3 100 0.219 1Values are mean ± SD of three replicates (3 tank/group). Values without superscript letters within the same row in the table are not significantly (
p ≥ 0.05) different..2IBW: Initial body weight (g); 3FBW: Final body weight (g); 4FI: Feed intake (g/fish); 5WG: Weight gain (%); 6SGR: Specific growth rate (%/d); 7FCR: Feed conversion ratio; 8PER: Protein efficiency ratio.
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Table 4 . Whole-body proximate composition (%, as-is basis) of juvenile rainbow trout fed experimental diets for 12 weeks1..
AA0 AA100 AA200 AA400 P valueMoisture 71.2±0.3 71.1±0.2 71.3±0.2 71.2±0.2 0.761 Crude protein 19.2±0.2 19.2±0.1 19.4±0.2 19.2±0.2 0.518 Crude lipid 5.02±0.08 5.12±0.09 4.92±0.11 5.07±0.13 0.326 Crude ash 3.43±0.18 3.36±0.06 3.52±0.28 3.51±0.19 0.834 1Values are mean ± SD of three replicates (3 tank/group). Values without superscript letters within the same row in the table are not significantly (
p ≥ 0.05) different..
ADC
Fish fed the experimental diets (AA100, AA200, and AA400) exhibited significant differences in carbohydrate ADC compared with the control (
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Table 5 . Apparent digestibility coefficients (%) of dry matter, crude protein, crude lipid, carbohydrate, and energy for experimental diets fed to olive flounder for 12 weeks1..
AA0 AA100 AA200 AA400 P valueDry matter 69.3±0.9 72.6±0.5 71.8±0.3 72.6±1.1 0.103 Crude protein 87.4±0.4 87.1±0.2 86.8±0.2 87.5±0.5 0.600 Crude lipid 89.5±0.3 89.6±0.2 90.0±0.1 90.4±0.4 0.217 Carbohydrate 83.2±0.5b 88.7±0.2a 87.7±0.1a 88.6±0.5a 0.001 Gross energy 86.6±0.4 86.3±0.3 86.0±0.2 86.2±0.6 0.680 1Values are mean ± SD of three replicated tanks. Values without/similar and different superscript letters within the same row in the table are not significantly (
p ≥ 0.05) and are significantly (p < 0.05) different, respectively..
Feed Degradation Rate (%)
AA0 and AA400 groups exhibited the lowest and highest feed degradation rates (%), respectively. Compared to the control, α-amylase significantly and dose-dependently increased the feed degradation rates in AA100, AA200, and AA400 groups to 41.8 ± 2.5, 55.3 ± 1.2, and 81.7 ± 2.9%, respectively (Table 6).
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Table 6 . Feed degradation rate (%) in different experimental feeds1..
AA0 AA100 AA200 AA400 p -valuePrepared pellet (g) 6.00 6.00 6.00 6.00 1.000 Soaked pellet (g) 4.71±0.14a 3.49±0.15b 2.68±0.07c 1.10±0.17d <0.001 Feed degradation rate (%)2 21.5±2.3d 41.8±2.5c 55.3±1.2b 81.7±2.9a <0.001 1Values are mean ± SD (3 times/group). Values without and different superscript letters within the same row in the table are not significantly (
p ≥ 0.05) and are significantly (p < 0.05) different, respectively..2Feed degradation rate (%): (dry pellet before soaking - dry pellet after soaking) / dry pellet before soaking × 100..
Feed pellets collected from the fish stomachs were gradually degraded at 2, 4, and 8 h. Interestingly, this degradation was time-dependent and higher diet morphological alteration/degradation was observed in the group with the highest α-amylase concentration (Fig. 1).
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Figure 1. Photograph of the feed collected from flounder stomach after 2, 4, and 8 h of feeding supplemented with 0 (A00), 100 (AA100), 200 (AA200), and 400 (AA400) mg/kg of α-amylase.
Plasma α-Amylase Concentration and Fecal Particle Size
After 12 weeks of feeding, plasma α-amylase concentration increased (
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Figure 2. α-Amylase activity of plasma between the experimental groups. The data represent the mean ± standard deviation (6 fish/group); values with different letters indicate significant differences (
p < 0.05).
In the 90% fecal particle size class, the particle size of AA0 group was significantly larger than those of the AA200 and AA400 groups. Moreover, 90% fecal particle size in olive flounder fed with the control (AA0) diet was < 134.0 ± 8.0 μM, whereas the AA100, AA200, and AA400 groups exhibited particle sizes of < 116.5 ± 2.5, < 95.8 ± 3.3, and < 93.5 ± 4.5 μM, respectively (Fig. 3; Table 7). In contrast, no significant variations were observed in the 50 and 10% size classes. Additionally, 90% particle sizes of the AA0 and AA100 groups were similar, whereas those of AA200 and AA400 were significantly different (
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Table 7 . Fecal particle size of olive flounder fed with four types of experimental diets for 12 weeks1..
Diet group Particle size (µM) 10% size class 50% size class 90% size class AA0 < 4.4±0.4a < 50.7±4.6a < 134.0±8.8a AA100 < 3.2±1.6a < 40.3±3.2a < 116.5±2.5ab AA200 < 3.8±0.10a < 41.8±0.9a < 95.8±3.3b AA400 < 2.8±0.5a < 36.7±3.4a < 93.5±4.5b 1Values are mean ± SD (3 tanks/group). Values with same and different superscript letters within the same column in the table are not significantly (
p ≥ 0.05) and are significantly (p < 0.05) different, respectively..
-
Figure 3. Fecal particle size of olive flounder fed with four types of experimental diets (
n = 3 tanks per diet). Feed was supplemented with 0 (AA00), 100 (AA100), 200 (AA200), and 400 (AA400) mg/kg of α-amylase.
Effects of α-Amylase on the Transcription of Immune- and Growth-Related Genes
Compared with the control, transcription levels of seven immune-related genes (tumor necrosis factor [
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Figure 4. Profiles of gene expression in the muscle (A) and intestine (B) of olive flounder. Expression of these genes in olive flounder was measured by RT-qPCR after 12 weeks of feeding for the Control (AA0), AA100, AA200, and AA400 groups. Levels of gene expression were quantified relative to
elongation factor -1α transcription. The data are represented as the means ± standard deviation (6 fish/group); means that do not share the same letter differ significantly (p < 0.05).
In contrast, expression levels of growth-related genes, such as insulin-like growth factor
Discussion
In this study, dietary supplementation with three concentrations of α-amylase had no significant effects on the growth, feed utilization, and whole-body composition of olive flounder. Previous studies reported that cellulase and an enzyme cocktail comprising xylanase, amylase, cellulase, protease, and β-glucanase have no effects on the growth performance of
Also in this study, α-amylase had no significant effect on the ADC of dry matter, crude protein, lipid, and gross energy contents. However, an improved carbohydrate ADC was observed in all treatment groups. ADC of Indian major carp catla (
The carbohydrates (especially starch) that bind to the active site of the enzyme for digestion are hydrophilic [39], and our in vitro experiments suggested that the improvement in the carbohydrate degradation rates of olive flounder was due to the presence of α-amylase. This enzyme is also likely involved in changes in the feed structure in the stomach. However, further experiments are required to confirm these findings. Dietary supplementation with 200 and 400 mg/kg of α-amylase increased plasma amylase concentration in the experimental olive flounder. No previous study has estimated the activity of digestive enzymes in the blood in response to the application of exogenous enzymes. However, α-amylase application elevates glucose-6-phosphate dehydrogenase levels in the liver and blood of
GH binds to GH receptors and stimulates the synthesis of IGF-1 in the liver [49]. Our findings demonstrated that the levels of
In this study, olive flounder fed without α-amylase produced larger (90% size class) fecal particles than the 200 and 400 mg/kg enzyme-supplemented groups. Fecal particle size is a key indicator of digestion and mechanical stability of feces [54]. Starch is positively correlated with the production of larger fecal particles [55-57], whereas oligosaccharides in feed have little to no effect on the particle size or structure. Therefore, the larger fecal particles observed in the control group could be due to a higher level of starch in the digested food caused by a lack of amylase enzymes. In contrast, the two groups with the highest amylase doses produced smaller fecal particles, presumably because the carbohydrates in the feed were properly digested by the exogenous amylase. This also explains the increase in carbohydrate ADC observed in our experiment. Very few studies have assessed the effects of supplementation with exogenous enzymes (amylase, xylanase, cellulase, and gluconase) on fecal particle size in aquaculture species. A recent study by Welker
Conclusion
In this study, dietary application of α-amylase at 200 and 400 mg/kg improved the carbohydrate ADC, blood amylase content, feed degradation rate, and fecal particle size in olive flounder. Interestingly, α-amylase had no significant effects on the growth, feed utilization, and whole-body composition of this fish species. However, the effects of exogenous enzyme supplementation on the immunology, intestinal microbiome, serum biochemistry, and transcription of growth and digestive genes in cultured olive flounder require further elucidation in future studies. Moreover, the involvement of external enzymes in the activation of different physio-immunological pathways needs to be investigated in the future.
Acknowledgments
This study was supported by the National Institute of Fisheries Science, Ministry of Oceans and Fisheries, Republic of Korea (R2023036).
Author Contributions
MTH: Data Curation. Methodology, Software, Investigation, Writing – Original Draft. HJK: Conceptualization, Methodology, Software, Investigation. SWH, SMJ, and KKW: Investigation, Resources, Data Curation. SHL: Supervision, Project Administration.
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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Fig 4.

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Table 1 . Ingredient composition of the experimental diets fed to olive flounder over a 12-week growth trial (% of DM basis)..
Ingredients Diet no. (AA supplementation level, U/kg diet) AA0 AA100 AA200 AA400 Fishmeal 60.0 Soybean meal 10.3 Tapioca starch 10.0 Wheat flour 11.5 Fish oil 5.0 Mineral mixture1 1.0 Vitamin mixture2 1.0 Choline 0.5 Cr2O3 0.5 α-amylase3 0 0.01 0.02 0.04 NaCl 0.2 0.19 0.18 0.16 Total 100 100 100 100 Analyzed nutrients compositions (as-is basis) Moisture (%) 4.63 5.02 4.73 4.86 Crude protein (%) 51.1 51.2 50.9 51.0 Crude lipid (%) 10.2 9.8 10.4 10.0 Crude ash (%) 13.8 13.8 13.9 13.6 Gross energy (cal/g) 4932 4925 4942 4926 1Mineral premix contained the following amount which were diluted in cellulose (g/kg premix): NaCl, 30.3; MgSO4-7H2O, 95.6; NaH2PO4-2H2O, 60.8; KH2PO4, 167.3; CaH4(PO4)2-H2O, 94.7; Ferric citrate, 20.7; ZnSO4-7H2O, 15.3; Ca-lactate, 212.8; CuCl, 0.14; AlCl3-6H2O, 0.105; KI, 0.105; Na2Se2O3, 0.01; MnSO4-H2O, 1.4; CoCl2-6H2O, 0.7..
2Vitamin premix contained the following amount which were diluted in cellulose (g/kg premix): L-ascorbic acid, 171.1; myoinositol, 181.8; DL-a-tocopheryl acetate, 18.9; niacin, 36.4; p-aminobenzoic acid, 18.2; Ca-D-pantothenate, 12.7; riboflavin, 9.1; thiamin hydrochloride, 2.7; pyridoxine hydrochloride, 1.8; menadione, 1.8; retinyl acetate, 0.73; folic acid, 0.68; D-biotin, 0.27; cholecalciferol, 0.003..
3α-amylase from
Aspergillus oryzae (30 units/mg; Sigma Aldrich, St. Louis, USA.
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Table 2 . Gene specific primers used to quantify relative gene expression..
Gene Sense Oligonucleotide sequence (5` to 3`) Access No. Elongation factor-1-α (Reference) F GAGGTCAAGTCTGTGGAGAT AB915949 R GGTGGTTCAGGATGATGAC Tumor necrosis factor F CCCTATGAACTGTAACAGTTTG AB040448 R GTCAGGTACTTAACCCTCAT Interleukin 1 β F TGCTACCAGACCTTCAACAT AB070835 R TCTTTCCAGCAGACAGTGGT Interleukin 2 F ACATACGTACTTCAAGCTATCG KY307833 R GTAAAGATTCCACTTGGTCCA Immunoglobulin M F GCCTCCTTCTTCTGCTCTG AB109029 R CCTCAGTGGATGTTGTGATT Heat shock protein 70 F CAATGATTCTCAGAGGCAAG DQ662230 R TATCTAAGCCGTAGGCAATC Insulin-like growth factor 1 F ATGTCTAGCGCTCTTTCCTT AF061278 R CTTCTTGTTTTTTGTCTTGTCTG Insulin-like growth factor 2 F AGAACCGTGGGATCGTAGA AF091454 R TGCCACACCTCGTATTTG Transforming growth factor β 3 F TCCAAGGTATTCCGCTTCAA XM_020085122 R TTTGGCTTTGGGGTCATCT Growth hormone F TCCTCTCAGCCAATCACAGA M23439 R TACGTCTCCACCTTGTGCAT Growth hormone receptor F CCACAAACTGGAAATCATTGG AB058418 R CGAAAACAAGAACAACTGTGAG
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Table 3 . Growth and feed utilization parameters of olive flounder fed with experimental diets for 12 weeks1..
AA0 AA100 AA200 AA400 P valueIBW2 273.2±3.9 273.5±3.4 272.2±1.8 273.3±0.6 0.939 FBW3 555.3±4.5 564.5±8.4 563.8±2.3 566.1±7.1 0.203 FI4 331.4±4.3 323.7±7.1 329.3±3.3 326.1±4.6 0.312 WG (%)5 103.3±2.1 106.4±1.4 107.1±2.2 107.1±2.3 0.126 SGR6 1.01±0.01 1.04±0.01 1.04±0.02 1.04±0.02 0.123 FCR7 1.16±0.02 1.11±0.04 1.10±0.02 1.11±0.02 0.054 PER8 1.66±0.06 1.76±0.06 1.73±0.04 1.76±0.03 0.117 Survival (%) 98.7±2.3 100 97.3±2.3 100 0.219 1Values are mean ± SD of three replicates (3 tank/group). Values without superscript letters within the same row in the table are not significantly (
p ≥ 0.05) different..2IBW: Initial body weight (g); 3FBW: Final body weight (g); 4FI: Feed intake (g/fish); 5WG: Weight gain (%); 6SGR: Specific growth rate (%/d); 7FCR: Feed conversion ratio; 8PER: Protein efficiency ratio.
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Table 4 . Whole-body proximate composition (%, as-is basis) of juvenile rainbow trout fed experimental diets for 12 weeks1..
AA0 AA100 AA200 AA400 P valueMoisture 71.2±0.3 71.1±0.2 71.3±0.2 71.2±0.2 0.761 Crude protein 19.2±0.2 19.2±0.1 19.4±0.2 19.2±0.2 0.518 Crude lipid 5.02±0.08 5.12±0.09 4.92±0.11 5.07±0.13 0.326 Crude ash 3.43±0.18 3.36±0.06 3.52±0.28 3.51±0.19 0.834 1Values are mean ± SD of three replicates (3 tank/group). Values without superscript letters within the same row in the table are not significantly (
p ≥ 0.05) different..
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Table 5 . Apparent digestibility coefficients (%) of dry matter, crude protein, crude lipid, carbohydrate, and energy for experimental diets fed to olive flounder for 12 weeks1..
AA0 AA100 AA200 AA400 P valueDry matter 69.3±0.9 72.6±0.5 71.8±0.3 72.6±1.1 0.103 Crude protein 87.4±0.4 87.1±0.2 86.8±0.2 87.5±0.5 0.600 Crude lipid 89.5±0.3 89.6±0.2 90.0±0.1 90.4±0.4 0.217 Carbohydrate 83.2±0.5b 88.7±0.2a 87.7±0.1a 88.6±0.5a 0.001 Gross energy 86.6±0.4 86.3±0.3 86.0±0.2 86.2±0.6 0.680 1Values are mean ± SD of three replicated tanks. Values without/similar and different superscript letters within the same row in the table are not significantly (
p ≥ 0.05) and are significantly (p < 0.05) different, respectively..
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Table 6 . Feed degradation rate (%) in different experimental feeds1..
AA0 AA100 AA200 AA400 p -valuePrepared pellet (g) 6.00 6.00 6.00 6.00 1.000 Soaked pellet (g) 4.71±0.14a 3.49±0.15b 2.68±0.07c 1.10±0.17d <0.001 Feed degradation rate (%)2 21.5±2.3d 41.8±2.5c 55.3±1.2b 81.7±2.9a <0.001 1Values are mean ± SD (3 times/group). Values without and different superscript letters within the same row in the table are not significantly (
p ≥ 0.05) and are significantly (p < 0.05) different, respectively..2Feed degradation rate (%): (dry pellet before soaking - dry pellet after soaking) / dry pellet before soaking × 100..
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Table 7 . Fecal particle size of olive flounder fed with four types of experimental diets for 12 weeks1..
Diet group Particle size (µM) 10% size class 50% size class 90% size class AA0 < 4.4±0.4a < 50.7±4.6a < 134.0±8.8a AA100 < 3.2±1.6a < 40.3±3.2a < 116.5±2.5ab AA200 < 3.8±0.10a < 41.8±0.9a < 95.8±3.3b AA400 < 2.8±0.5a < 36.7±3.4a < 93.5±4.5b 1Values are mean ± SD (3 tanks/group). Values with same and different superscript letters within the same column in the table are not significantly (
p ≥ 0.05) and are significantly (p < 0.05) different, respectively..
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