Curcumin Supplementation Enhances the Feeding and Growth of Largemouth Bass ( Micropterus salmoides ) Fed the Diet Containing 80 g/kg Fish Meal

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Introduction
Te contribution of aquaculture to food security and human health has been continuously growing with expansion of the aquaculture industry worldwide [1,2].Dietary protein sources and environmental impact are the main challenges threatening sustainability of aquaculture industry since the proportion of fed aquaculture in global aquaculture production increased, and more fed aquaculture production resulted in more dietary protein consumption and waste outputs [3].Among the protein ingredients used for formulating aquafeed, fsh meal is recognized as ideal because of its advantages in digestible protein content, amino acid balance, and growth-promoting factors and is widely used at high levels in carnivorous fsh feed.However, fsh meal is a limited ocean resource, and replacement of fsh meal with economic terrestrial protein ingredients, including animal or plant ingredients, is necessary for sustainable development of aquaculture industry.Based on intensive studies over decades, fsh meal content in aquafeed signifcantly declined, e.g., the average dietary fsh meal level for marine fnfshes was reduced from 43% in 1995 to 14% in 2017 [4].Several approaches, such as using previously blended protein ingredients (such as blends of PBM, meat and bone meal, feather meal, and blood meal) or gamma-ray irradiated protein ingredients (such as irradiated soybean meal and feather meal) as fsh meal alternate, supplementation of functional amino acids (such as taurine and glycine) and minerals (selenium) and elevating dietary protein levels, have been demonstrated as efcient in increasing fsh meal amount replaced from fsh diets [5][6][7][8][9][10][11][12].De Cruz et al. [13] reported that nucleotide supplementation could enhance growth of hybrid striped bass Morone chrysops × M. saxatilis fed with low fsh meal diets.Terefore, identifying the constituents with the function to improve feeding and growth of fsh fed low fsh meal diets should be a focus in future studies of fsh nutrition and feed technology.
Curcumin (CU) is a polyphenolic and hydrophobic phytochemical component of the turmeric herb Curcuma longa and has been recognized as an immunomodulator of human and domestic animals [14].Previous studies reported that CU supplementation could improve growth of crucian carp Carassius auratus, Nile tilapia Oreochromis niloticus, and large yellow croaker Larimichthys crocea [15][16][17], and positively afect hematological parameters, immunity, and disease resistance of Nile tilapia, carp Cyprinus carpio, and rainbow trout Oncorhynchus mykiss [18][19][20].However, it is uncertain if CU could be used as a functional constituent in low fsh meal diets for carnivorous fshes.
Largemouth bass Micropterus salmoides is a carnivorous fsh species with commercial importance to freshwater aquaculture.In 2020, largemouth bass aquaculture production achieved 621,300 tons, which ranked 15 th in inland fnfsh aquaculture production and 2 nd in inland carnivorous fsh aquaculture production in the world [3].Fish meal content in commercial largemouth bass feed was generally more than 400 g/kg, which could be reduced to 160 g/kg by soybean meal, soy protein concentrate, or cottonseed protein concentrate as an alternative ingredient [21][22][23] or 80 g/kg by using poultry byproduct meal as an alternative ingredient [24].To our knowledge, the formulated diet containing 160 g/kg fsh meal has been successfully used in largemouth bass farming, and 80 g/kg is the minimum fsh meal level that could satisfy fast growth of the fsh.In the present study, we evaluated if CU could be used as a functional constituent to improve feeding, growth, and feed utilization efciency of largemouth bass fed a diet containing 80 g/kg fsh meal, to explore the potential to further reduce fsh meal level in commercial fsh feed.

Curcumin, Feed Ingredients, and Test Diets.
Curcumin (purity >98%) was purchased from Dulai Biotechnology Co., Ltd.(Shanghai, China).Feed ingredients, including animal and plant protein ingredients, were purchased from Hongli Feed Company (Huzhou, China).Te proximate composition of the feed ingredients is shown in Table 1.
Two single factor trials (I and II) were designed.In trial I, poultry by-product meal (PBM) replaced 60% (P16) and 80% (P8) of the fsh meal in a reference diet (R) that was formulated to contain 400 g/kg fsh meal.In trial II, the diet containing 80 g/kg fsh meal (P8) served as control (C), and curcumin (CU) was added at 5000 (C5) and 10000 (C10) mg/kg in diet C, respectively.Protein and lipid levels of the test diets were 520 and 120 g/kg, respectively [25].Formulation and proximate compositions of the test diets are shown in Te feed ingredients were ground with a hammer mill into particles that can pass through a mesh (pore size 500 μm).To prepare the test diets, the feed ingredients of each diet were weighed and mixed as described in Wang et al. [24], and pellets were extruded with an SLP-450 single screw machine (Fishery Machinery and Instrument Research Institute, Chinese Academy of Fishery Sciences, Shanghai, China).Te extruding temperature was controlled at 80-100 °C.After drying under 25 °C in an air-conditioned room, the test diets were collected and preserved in plastic bags in a refrigerator (−20 °C).

Fish and Feeding
Trials.Trials I and II were conducted in the feld station of the Zhejiang Institute of Freshwater Fisheries (Huzhou, China).Largemouth bass fngerlings were purchased from a commercial fsh farm located in Deqing (Huzhou, China).Te fsh were transported to the feld station and acclimated in two indoor recirculating aquaculture systems (RAS).Each RAS comprises 24 experimental tanks (water volume, 350 L).Prior to the start of the feeding trials, 720 fsh with similar body sizes were selected and reared in a RAS at 30 fsh/tank for one week.During the acclimation period, the fsh were fed with diet R twice daily.
Trials I and II were initiated and ceased simultaneously.At the start of the trials, the acclimatized fsh were pooled in four tanks after 24 h of feed deprivation.Twenty fsh were captured from the pooled tanks, weighed in bulk, and randomly released into one of 15 experimental tanks.Each diet treatment had three replicators, and the fsh were assigned to diet R, P16, and P8 in trial I and C, C5, and C10 in trial II (diets P8 and C were the same diet but was assigned as diferent diets in diferent trial).Initial body weight was 37.8 ± 0.2 g (mean ± SD, n = 15).Tereafter, three groups of 15 fsh each were collected from the pooled tanks.After the measurement of body weight, body length, liver weight, and viscera weight, the sampled fsh were preserved in a refrigerator (−20 °C) for analysis of body composition.
Te duration of trials I and II was eight weeks.Fish were fed to satiation at 8 : 00 and 16 : 00 every day.To ensure fsh were fed adequately and efciently, several pellets of a test diet were dropped in each tank, and feeding behavior of fsh in the tank was observed.Fish in the tanks were fed alternately until no fsh swam to the water surface to accept the dropped diet.Dead fsh were recorded and weighed.Water in the RAS was continuously aerated, recirculated, and treated with a bioflter, and recirculating rate of water in each tank was 3.0 L/min.Everyday 20% of water in the RAS was renewed with aerated tap water.Water temperature was monitored in the morning and afternoon daily and fuctuated from 25.8 to 30.8 °C.Dissolved oxygen was measured weekly and was always higher than 5.0 mg/L.
At the end of trials I and II, fsh were deprived of diet for 24 h.Tree fsh were captured from each tank, anaesthetized with clove oil (100 mg/L), and individually weighed.Te blood sample was collected with a 1 ml syringe rinsed with heparin sodium solution (1%, v/v) from the caudal vein of each fsh and transferred into a 2 ml Eppendorf tube.After centrifugation (15 min at 3000 rpm), the supernatant (plasma) was isolated and transferred into a 1.5 ml cryogenic vial and preserved in liquid nitrogen.Te liver was collected from each fsh following plasma sample collection and was preserved in liquid nitrogen.Tereafter, fsh were captured from each tank and weighed in bulk.Tree fsh were sampled from each tank, and the body weight, body length, viscera weight, and liver weight of the fsh were measured.Te samples of plasma and liver were preserved at −80 °C, and the samples of whole fsh were preserved at −20 °C, until physiological and chemical analyses.

Physiological and Chemical Assay.
Prior to physiological analysis, the samples of plasma and liver were thawed at 4 °C.Te activity of antioxidant enzymes [superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and catalase (CAT)] and malondialdehyde (MDA) content in plasma were assayed with the kits purchased from the Nanjing Jiancheng  Bioengineering Institute (Nanjing, China).Te liver was homogenized with 0.86% physiological saline (1 : 4, w/v) in an ice bath.After centrifugation (5 min at 12000 rpm and 4 °C), supernatant of the liver sample was isolated, and concentration of the soluble protein was determined with the method described in Bradford [26], using bovine serum albumin as standard.
Activities of SOD, GSH-PX, and CAT and MDA content in the liver were assayed with the kits used for plasma assay.
Prior to chemical analysis, the sampled fsh were thawed, weighed, autoclaved (20 min at 120 °C), homogenized, and dried (6 h at 120 °C).Contents of moisture, crude protein, crude lipid, ash and phosphorus of the feed ingredients, test diets, and fsh were analyzed with the methods described in AOAC [27].Contents of crude protein and amino acids were analyzed with an 8400 auto-Kjeldahl-nitrogen analyzer (Foss, Sweden) and 433 amino acid analyzer (Sykam Company with Limited Liability, Germany), respectively.Gross energy content was analyzed with Parr 6200 calorimeter (Parr, USA).Carbon content was analyzed with an EA3000 element analyzer (Euro Vector, Italy).

Calculation and Statistics.
Calculation of feed intake, growth, feed conversion ratio (FCR), nutrient and energy retention efciencies (carbon: CRE; nitrogen: NRE; phosphorus: PRE; energy: ERE), morphological indexes [condition factor, hepatosomatic index (HSI), and viscerosomatic index (VSI)], waste outputs (carbon, nitrogen, and phosphorus), and fsh meal reliance [ratio of dietary consumption to fsh production (RCP)] were performed with the equations described by Wang et al. [24].Te retention efciency of amino acids (ARE) was calculated as follows: where I (g) is the feed consumption of fsh in each tank during the trials; W 0 (g) is the initial body weight of fsh in each tank; and W t (g) is the fnal body weight; N t and N 0 are the number of fsh alive in each tank at the end and start of the trials; C At (%) is the content of amino acids in the fsh body at the end of the trials and C A0 (%) at the start; C Af (%) is the content of amino acids in the test diets.Te infuences of fsh meal replacement level (among fsh fed diets R, P16, and P8) or CU supplementation (among fsh fed diets C, C5, and C10) on survival, growth, feed intake, feed utilization efciency, morphological indexes, and body composition were examined with one-way ANOVA.Te diferences in the above variables between the diet treatments in trials I and II were further examined with Duncan's test.Te diferences in ARE, activity of antioxidant enzymes (SOD, GSH-PX, and CAT) in plasma or liver, and MDA content in plasma or liver between diets R and P8 (trial I) or between diets C and C10 (trial II) were examined with independent t-test.Te diference between ARE and NRE for the same diet treatment in trials I and II was examined with paired t-tests.
Te statistical analysis, including one-way ANOVA, Duncan's test, independent t-test, and paired t-test, were performed with SPSS (version 24.0, SPSS, USA).Te signifcant level was set at P < 0.05.

Results
3.1.Survival, Feed Intake, Growth, and Feed Utilization Efciency.Survival was over 98% throughout the feeding trials.In trial I, feed intake and PRE were dependent (P < 0.05, Table 3), while fnal body weight, weight gain, FCR CRE, NRE and ERE were independent (P < 0.05), on fsh meal replacement level.Fish fed diet R exhibited higher feed intake relative to fsh fed diet P8 (P < 0.05), and exhibited lower PRE relative to fsh fed diet P16 (P < 0.05).In trial II, fnal body weight, weight gain, and feed intake were dependent (P < 0.05, Table 3), while FCR, CRE, NRE, PRE, and ERE were independent (P > 0.05), on CU supplementation level.Te weight gain and feed intake of fsh fed diet C10 were higher than that of fsh fed diet C (P < 0.05) but did not signifcantly difer from those of fsh fed diets C5 (P > 0.05).
In trial I, amino acid retention efciencies except proline were higher in fsh fed P8 than in fsh fed R (P < 0.05, Table 4).In trial II, no signifcant diferences were found in retention efciencies of Asp, Ser, Pro, Gly, Ala, His, and Lys between fsh fed diets C and C10 (P > 0.05).In trials I and II, the retention efciencies of Gly, Met, and Ala were higher, while the retention efciencies of Arg, Glu, His, Ile, Leu, Lys, Phe, Pro, Ser, Tyr, and Val were lower than NRE in fsh fed diets R, P8, C and C10 (P < 0.05).

Morphological Indexes and Body Composition.
In trial I, body lipid content was dependent (P < 0.05, Table 5), while condition factors, HSI, VSI, and body contents of moisture, crude protein, ash, carbon, phosphorus, and gross energy were independent (P > 0.05) on fsh meal replacement level.fsh fed diets P16 and P8 exhibited lower body lipid content relative to fsh fed diet R (P < 0.05).In trial II, dietary CU level did not result in signifcant alternation in condition factors, HSI, VSI, and body composition (P > 0.05).

Fish Meal Reliance and Waste Outputs.
In trial I, RCP, carbon waste, and phosphorus waste were dependent (P < 0.05, Table 6), while nitrogen waste was independent (P > 0.05), on fsh meal replacement level.fsh fed diet P8 exhibited lower RCP, carbon waste, and phosphorus waste relative to fsh fed diet R (P < 0.05).In trial II, CU supplementation level infuenced RCP and carbon waste (P < 0.05) but did not infuence wastes of nitrogen and phosphorus (P > 0.05).fsh fed diet C exhibited lower RCP relative to fsh fed diet C5 (P < 0.05) and lower carbon waste relative to fsh fed diets C5 and C10 (P < 0.05).

Activity of Antioxidant Enzymes and MDA Content.
No signifcant diference was found in SOD activity in plasma either between fsh fed diets R and P8 (P > 0.05, trial I) or between fsh fed diets C and C10 (P > 0.05, trial II).In trial I, the activities of GSH-PX and CAT as well as MDA content in plasma were higher in fsh fed diet R than in fsh fed diet P8 (P < 0.05, Figure 1).In trial II, the activities of GSH-PX and CAT as well as MDA content in plasma were higher in fsh fed diet C10 than in fsh fed diet C (P < 0.05).

Discussion
Indeed, dietary fsh meal replacement level depends on fsh species, alternative ingredients, and basal diet formulation [9,24].To our knowledge, the dietary fsh meal level for largemouth bass varied from 240 to 80 g/kg [6,8,9,24], and 80 g/kg is the minimum fsh meal content for largemouth bass when PBM is used alone as a fsh meal alternate [24].In the present study, no signifcant diference was found in weight gain between fsh fed diets R, P16, and P8.Tis result is consistent with the conclusion that dietary fsh meal content in largemouth bass diet could be reduced to 80 g/kg.Te weight gain of fsh fed diet C was lower than that of fsh fed diet C10 but did not signifcantly difer from that of fsh fed diet C5.Tese results suggest that the infuence of CU supplementation on growth of largemouth bass was positive and dose-dependent, and 10000 mg/kg CU supplementation could improve growth of fsh fed the diet containing 80 g/kg fsh meal.Terefore, CU could be a functional constituent to improve growth of largemouth bass fed low fsh meal diets.Several studies reported that CU supplementation could promote fsh growth, however, the dietary CU level that can accelerate fsh growth varied in these studies [16,20].For instance, the dietary CU levels that are sufcient to promote growth of Nile tilapia were 50 mg/kg [16] and 200 mg/kg [28].Te dietary CU levels that could improve growth of rainbow trout were 200 mg/kg [29], 400 mg/kg [30], and 10000 mg/kg [20].Weight gain of tilapia Oreochromis mossambicus increased with increasing dietary CU level from 0 to 5000 mg/kg, and then declined with further increase of dietary CU level to 10000 mg/kg [31].Weight gain of large yellow croaker Larimichthys crocea increased with increasing dietary CU level from 0 to 400 mg/kg, and then declined with increasing dietary CU level to 600 mg/kg [17].Li et al. [32] reported that CU supplementation from 120 to 600 mg/kg could increase feed intake, weight gain, and feed efciency of grass carp Ctenopharyngodon idella, and the suitable dietary CU level was 310 mg/kg.In the present study, the weight gain of largemouth bass increased with increasing dietary CU level from 0 to 10000 mg/kg, suggesting that 10000 mg/kg CU supplementation benefted growth of fsh fed diet containing 80 g/kg fsh meal.Tis result is inconsistent with the conclusion that growth O. mossambicus declined with increasing dietary CU level from 5000 to 10000 mg/kg [31].Considering CU is an expensive constituent and the efcacious CU supplementation level (from 50 to 10000 mg/kg) varies greatly among fsh species, more studies warrant to determine the minimum dietary CU supplementation level for fshes to identify if CU could be used as an economic dietary functional constituent in the commercial manufacture of fsh feed.
Te mechanisms by which CU promotes growth of fshes remains unclear.Te bioavailability of CU is limited by its low solubility in water, low absorption, chemical instability, and rapid metabolism [33]; therefore, the biological activity of CU might be dependent on its content and retention time in blood and tissues.Mahmoud et al. [14] indicated that CU might increase feed intake by improving diet palatability due to its attractive favor.Ahmed et al. [34] reported that CU supplementation could enhance antibacterial capacity of gilthead seabream.In the present study, fsh fed diet C10 exhibited higher feed intake, but nonsignifcantly altered FCR, CRE, NRE, PRE, and ERE, relative to fsh fed diet C. Tese results illustrate that increasing feed intake, rather than improving feed utilization efciency, should be the mechanism that CU supplementation improved growth of largemouth bass.No signifcant diferences were found in condition factors, HSI, VSI, and body composition between fsh fed diets C, C5, and C10, suggesting that CU supplementation could not obviously alter morphological indexes and body composition of largemouth bass.On the other hand, it is noted that retention efciency of Met and Gly was high relative to that of the other dispensable and indispensable amino acids in fsh fed diets R, P8, C, and C10.Te retention efciency, of amino acids (ARE) varied from 20 to 46%, while NRE varied from 31% to 34%.Te average ARE (30%) was close to average of NRE (33%).Tese results are consistent with the conclusion that Gly retention efciency was high in AREs and average ARE was close to NRE, in Japanese seabass fed the diets with fsh meal replaced by PBM [35].According to the present and previous studies, the retention efciency of single amino acid could refect the suitability of the dietary amino acid profle, and Met and Gly might be the important amino acids that play a crucial role in 6 Aquaculture Research modulating growth of largemouth bass.Rossi et al. [12] reported that glycine supplementation could improve growth of largemouth bass fed a diet in which fsh meal is almost completely replaced by soybean meal.As polyphenols, CU is recognized as an antioxidant and may be involved in reactive oxygen species (ROS) generation and scavenging.Some ROS generated in the process of cellular metabolisms, such as hydrogen peroxide (H 2 O 2 ), are signal molecules responsible for growth regulation [36].Excessive ROS can induce oxidative stress that results in growth retard and diseases, and the situation of oxidative stress is generally evaluated with MDA accumulation [37].Several enzymatic antioxidants, including SOD, GSH-PX and CAT, and nonenzymatic antioxidants, including vitamin E, vitamin C, and polyphenols, are responsible for ROS scavenging [38].Previous studies reported that CU supplementation could protect a freshwater teleost Anabas testudineus from oxidative damage [39], inhibit lipid peroxidation, and reduce MDA accumulation in liver of crucian carp and Nile tilapia [15,16,40], suppress infammatory response of crucian carp [41], and enhance activity of antioxidant enzymes (GSH-PX and CAT in plasma and SOD in liver) of large yellow croaker [17].In the present study, activities of GSH-PX and CAT in plasma as well as MDA content in plasma and liver were higher in fsh fed diet diet C10 Aquaculture Research than in fsh fed diet C. Tese results suggest that 10000 mg/kg CU supplementation might increase metabolism intensity of largemouth bass and generate more H 2 O 2 , and the H 2 O 2 in return stimulated feeding and growth as a signal molecule.In fsh fed diets C10 and C, the higher activities of GSH-PX and CAT in plasma and MDA content in plasma and liver are consistent with the higher feed intake and weight gain.Terefore, it is assumed that modulating ROS generation and scavenging might be a mechanism by which dietary CU infuenced feeding and growth of largemouth bass.Tis hypothesis remains to be tested in the future studies.Defciency in dietary protein source (such as fsh meal) and environmental pollution are two obstacles limiting the sustainability of fsh aquaculture [8,35,42].In the present study, the RCP of fsh fed diets diet P8 was 0.26, which was obviously lower than that (1.38) of fsh fed diet R but did not signifcantly difer from that of fsh fed diets C (0.26), C5 (0.29), and C10 (0.28).Tese results indicate that CU supplementation could not further improve the situation of fsh meal reliance in largemouth bass farming despite reducing dietary fsh meal level from 400 to 80 g/kg considerably saving fsh meal in feed manufacture.Tere was no signifcant diference in nitrogen waste either between fsh fed diets R, P16, and P8 or between fsh fed diets C, C5, and C10 and wastes of carbon and phosphorus were lower in fsh fed diet P8 than in fsh fed diet R. Tese results are consistent with the report that replacing fsh meal with PBM did not increase waste outputs of carbon, nitrogen, and phosphorus in largemouth bass farming [24].Carbon waste of fsh fed diet C was lower than that of fsh fed diets C5 and C10, suggesting that CU supplementation at 5000 and 10000 mg/kg could not beneft to reduce waste outputs.In comparison with previous studies, the RCP (0.28), nitrogen waste [56 g•N/(kg fsh gain)], and phosphorus waste [11 g•P/(kg fsh gain)] of fsh fed diet C10 was similar to those [RCP was 0.29, nitrogen waste was 56 g N/(kg fsh gain), and phosphorus waste was 17 g P/(kg fsh gain)] of Japanese seabass fed a diet with fsh meal replaced by PBM [35] and those (RCP was 0.25, nitrogen waste was 49 g•N/(kg fsh gain), and phosphorus waste was 10 g•P/(kg fsh gain)) of largemouth bass fed a diet with fsh meal replaced by PBM [24], but was obivously lower than those (RCP was 0.72, nitrogen waste was 102 g N/(kg fsh gain), and phosphorus waste was 28 g•P/(kg fsh gain)) of golden pompano fed a diet with fsh meal replaced by PBM [9].Terefore, fsh meal reliance and waste outputs of largemouth bass farming are similar are to those of Japanese seabass farming, but lower than those of golden pompano farming.
In summary, the minimum fsh meal content could be declined to 80 g/kg in largemouth bass diet, and CU supplementation at 10000 mg/kg could enhance feed intake and growth of fsh fed the diet containing 80 g/kg fsh meal.Dietary CU might increase feed intake of largemouth bass by modulating the generation and scavenging of ROS.

Figure 1 :
Figure 1: Antioxidant enzyme activities and malondialdehyde content in plasma and liver of largemouth bass.(a) Superoxide dismutase (SOD); (b) glutathione peroxidase (GSH-PX); (c) catalase (CAT); (d) malondialdehyde (MDA).R: reference diet; P8 and C (the same treatment): poultry by-product meal (PBM) replaced 80% of the fsh meal in diet R; C10: PBM replaced 80% of the fsh meal in diet R with 10000 mg/kg curcumin supplementation.Data are presented as mean ± SD (n � 3).Te superscripts present the results of independent t-test between R and P8 (capital letters) or between C and C10 (small letters).Te data with diferent superscripts are signifcantly diferent (P < 0.05).

Table 1 :
Proximate composition (g/kg) of the feed ingredients.
1 crude protein, crude lipid, and ash are expressed as the situation in natural storage (n � 2).2protein premix is a commercial product made by the Hongli Feed Company of Deqing county (Huzhou, China).Te protein premix comprised blood cell meal, soybean meal, and cottonseed protein meal.

Table 3 :
Survival, growth, feed intake, and feed utilization of largemouth bass.PBM replaced 80% of the fsh meal in diet R with 5000 and 10000 mg/kg curcumin supplementation, respectively.Data are presented as mean ± SD (n � 3).Te superscripts present the results of Duncan's test between R, P16 and P8 (capital letters) or between C, C5, and C10 (small letters).Te data in the same column with diferent superscripts are signifcantly diferent (P < 0.05).
1 R: reference diet; P8 and C (the same treatment): PBM replaced 80% of the fsh meal in diet R; C10: PBM replaced 80% of the fsh meal in diet R with 10000 mg/kg curcumin supplementation.Data are presented as mean ± SD (n � 3).Te superscripts present the results of independent t-test between R and P8 (capital letters) or between C and C10 (small letters).Te data in the same column with diferent superscripts are signifcantly diferent (P < 0.05).

Table 5 :
Morphological indexes and body composition of largemouth bass.

Table 6 :
Fish meal reliance and waste outputs of largemouth bass.RCP, ratio of fsh meal consumption to fsh production. 1 R: reference diet; P16: poultry by-product meal (PBM) replaced 60% of the fsh meal in diet R; P8 and C (the same treatment): PBM replaced 80% of the fsh meal in diet R; C5 and C10: PBM replaced 80% of the fsh meal in diet R with 5000 and 10000 mg/kg curcumin supplementation, respectively.Data are presented as mean ± SD (n � 3).Te superscripts present the results of Duncan's test between R, P16, and P8 (capital letters) or between C, C5, and C10 (small letters).Te data in the same column with diferent superscripts are signifcantly diferent (P < 0.05).