# Nutritional Performance of Juvenile Red Drum (Sciaenops ocellatus) Fed Various Fish, Shrimp, and Squid Diets

Authors: David A. Klett; Aaron M. Watson
Journal: Aquaculture Nutrition
 (2022)

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## Abstract

Aquaculture is the fastest-growing sector of protein production in the world. Due to the rising costs of fishmeal used as a critical ingredient to make pelleted fish feed, the industry is moving to replace fishmeal as a primary protein source without reducing the growth rate of aquacultured fish. A 12-week feeding trial utilizing juvenile red drum (Sciaenops ocellatus) was conducted to determine the performance of various combinations of natural diet components including fish (Decapterus punctatus), shrimp (Litopenaeus vannamei), and squid (Loligo opalescens and Illex), in addition to a commercial fishmeal-based pelleted feed, fed isocalorically to identify if there is an optimal combination for red drum growth and health. These results can provide information to develop fishmeal replacement diets (FMRDs) that can more closely mimic the performance of natural diets. Traditional aquaculture metrics showed that fish fed the diet comprised only of fish had the highest specific growth rate, condition factor, and protein conversion efficiency, with the lowest feed conversion ratio, indicating the fish component was the highest performing component for red drum growth on a calorically fixed ration. There were significant differences among eight groups found for traditional aquaculture metrics (p<0.05). The commercial pelleted feed performed better than all but the fish only natural diet treatments in terms of growth on a fixed ration, which indicated that it is nutritionally balanced. The results of this study show that there were performance differences between juvenile red drum fed various natural diets. There has been an investigation into the metabolome of these fish to identify potential metabolites for supplementation into FMRDs, which is not addressed in this paper.


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## Body

## 1. Introduction

The global aquaculture industry provides increasing production of seafood for human consumption as worldwide demand for fish grows, while aiming to put less direct harvesting stress on wild fish populations. As aquaculture production increases, it is becoming critical to identify and produce the most effective diets for grow-out. Fishmeal is currently one of the most used protein sources in fish feed due to its accessibility and nutritional value [1]. The expansion of aquaculture has caused a higher demand for fishmeal, which is a product of wild-caught fisheries. Fish oil, another product of wild-caught fisheries, is also used widely in feed for farm-raised animals. It is rich in omega-3 fatty acids, which are beneficial to humans and other animals. It takes approximately 10 to 20 kilograms of live fish to produce one kilogram of fish oil and 4.39 kilograms of fish to produce one kilogram of fishmeal [2]. Global wild-caught fishery yields have plateaued since ~1980, thus, making it important to find alternative ingredients to fishmeal for aquaculture feeds [1]. Aquaculture was a $250-billion-dollar global industry in 2018 that accounted for 82 million tons of fish produced (46% of world fish production) [1]. By 2030, it is projected that aquaculture production of fish will reach 109 million tons (54% of world fish production) [1]. To optimize feed costs, alternative ingredients to fishmeal, with supplements to minimize nutritional differences between the alternative ingredients and wild caught fishmeal-based feeds, need to be identified, evaluated, and validated. Soybean, corn, insect, and algae are some of the potential replacements for fishmeal that have been proven to be successful in some species [3–7]. However, it has been shown that these are not typically as effective for growing fish as fishmeal. These fishmeal replacement protein sources may lack essential nutrients that fishmeal contain that are important to fish health and growth. Taurine, lysine, and methionine are just some examples of nutrients necessary for growth that have been found to be present in fishmeal but are lacking in plant-based ingredients [8–10]. Not all nutrients missing from fish-meal replacement diets (FMRDs) that may help fish grow as well as natural diets (food eaten by fish in the wild) have been identified. Understanding nutritional requirements for fish and the metabolic and physiological responses to the diets can provide aquaculture feed producers with the information necessary to ensure they develop feeds that are nutritionally balanced and optimized for species-specific growth.One of the challenges facing feed development is determining imbalances or absence of important nutrients between FMRDs and fishmeal-based diets [11, 12]. FMRD ingredients provide protein or fat to fish but may not provide all essential nutrients that come from natural feed items. Examples of this imbalance are omega-3 long-chain polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DPA). It is important that alternative ingredients supply these fatty acids to aquaculture-raised fish, as they will be consumed by humans. These fatty acids are important to a number of human health processes, such as inflammation reduction, reproductive health, and increasing immune system strength [13]. It is critical to fully characterize the performance and nutritional gaps between natural feed items and FMRDs.Historically, aquaculture production and nutrition studies have relied on measuring lengths and weights, body composition, and feed utilization of fish fed different diets over time [12, 14, 15]. While these measurements do show how the diets affect fish production, they often do not answer questions about the physiological and metabolic responses to the different feeds that cause performance differences. Recently, analytical techniques such as nuclear magnetic resonance (NMR) spectroscopy have been used to investigate the metabolome of fish to better understand their nutrition, and how novel FMRDs affect production [16]. Sheedy et al. [17] identified N,N-dimethylglycine as a marker for starvation in abalone using NMR spectroscopy. More recently, Casu et al. [18] identified a potential marker for a specific dietary stress in red drum, N-formimino-L-glutamate (FIGLU).Profiling the composition of natural diets is critical in optimizing production of pelleted FMRDs for use in aquaculture. Different natural diet components are composed of varying levels of protein, ash, and fats, and therefore have different caloric content. Aquaculture feed studies tend to control for protein to determine the optimal amount needed in pelleted FMRDs [19–21]. Caloric intake is another important factor in these studies. To ensure fish consuming different diets are not benefitting from one certain feed over another if caloric content is significantly different, caloric intake needs to be controlled for. Caloric content is calculated using four calories per gram of proteins or carbohydrates and nine calories per gram of fats [22]. By controlling caloric intake, it is possible to identify which component of a natural diet most accurately meets fish requirements, by being the most balanced and having higher digestible protein. Controlling for caloric intake should also force nutritional deficiencies to appear more readily than when feeding to satiation, as it is more challenging for a limited amount of feed to overcome a deficiency.Carbohydrates, fats, and proteins are digested and metabolized differently, which can alter consumption rates, and therefore fish growth [23]. Using different levels of these nutrients in FMRDs can alter their performance. By looking at the composition of the feed items and differences in fish growth when fed these components in varying combinations, observed differences can help close the production gap between FMRDs and natural diets.Red drum,Sciaenops ocellatus, exhibits a high growth rate, high environmental tolerance ranges, and marketability, which makes it a quality species for aquaculture, and a candidate for offshore cage aquaculture in the Gulf of Mexico [4]. The species is recreationally and commercially important in the Southeastern United States and Gulf of Mexico. They have been reared in captivity for decades and much is known about their nutritional requirements. To date, nutritional requirements for the successful grow-out of juvenile red drum fed FMRDs have been determined [11, 15, 23]. However, there still exists a performance gap between these diets and the growth observed of red drum fed natural diets. Despite decades of nutritional studies of pelleted diets, survival and growth rates are still highest using cut fish, shrimp, and squid diets in recirculating aquaculture systems [20, 24]. This study explored the effects of feeding red drum calorically rationed diets of fish, shrimp, and squid separately and in various combinations to better understand nutritional requirements to gain information in the development of novel FMRDs that perform as well as natural diets.


## 2. Materials and Methods

### 2.1. Animal Rearing and Captivity

Broodstock red drum at the South Carolina Department of Natural Resources’ Marine Resources Research Institute (SCDNR) (Charleston, SC) underwent volitional spawning. Once the eggs hatched, resulting larvae were transferred to the Waddell Mariculture Center (WMC) (Bluffton, SC) and grown out to fingerlings in large in-ground ponds. They were then transferred to the Hollings Marine Laboratory (HML) (Charleston, SC) once they reached an average length between 30 and 40 mm. They were held at HML until a target average weight of 25 grams was reached, then the 12-week feeding trial began. The fingerlings were kept in a recirculating aquaculture system at HML for the duration of this study.The system used in this study consists of three groups of eight circular tanks (5’ diameter by 3’ tall, 1112 Liter volume) each with their own biological and mechanical filtration as well as UV sterilization, protein skimmer, and temperature control. For this study, all three systems were connected and on the same water supply.


### 2.2. Husbandry

Dissolved oxygen and temperature levels were routinely monitored by SCDNR Mariculture Staff as the fish were reared in ponds at WMC until they were transferred back to Charleston. Upon transfer to the recirculating aquaculture system at HML, water quality measurements of dissolved oxygen, pH, salinity, and temperature (every other day) were measured using a YSI probe (Model Pro 10102030, Professional Series, YSI, Inc., Yellow Springs, Ohio), and ammonia, nitrite, and nitrate (weekly) were measured using a spectrophotometer (Hatch Model # DR2500) (Table1). The fish were fed a commercial pelleted diet to satiation twice per day until they reached the target weight of 25 grams to ensure an equivalent starting metabolic profile and body composition.Table 1
Daily water quality measurements (temperature, dissolved oxygen, salinity, and pH) and weekly water quality measurements (ammonia, nitrite, and nitrate) taken throughout the 12-week feeding trial.
Water quality parameterRangeMean±SDnTemperature °C24.40 – 24.8024.70±0.10246Dissolved oxygen mg L-15.74 – 7.026.47±0.22222pH7.47 – 8.107.87±0.13246Salinity ppt28.66 – 32.9931.04±1.21246Ammonia mg L-10.00 – 0.190.05±0.0472Nitrite mg L-10.01 – 0.040.01±0.0171Nitrate mg L-11.10 – 7.503.30±1.1071


### 2.3. Feeding Trial

An 86-day feeding trial preceded by a 68-day conditioning period was conducted once the fingerlings reached an average weight of 25 grams. Twenty-five fish (600 total, average weight of27.45±0.52 grams) were randomly placed into 24 tanks for the duration of the study, where they were fed different cut diets comprised of fish (Decapterus punctatus), shrimp (Litopenaeus vannamei), and squid (Loligo opalescens and Illex) or a fishmeal-based commercial pelleted feed. Frozen feed was purchased at Haddrell’s Point Tackle (Charleston, SC). Eight treatment diets with three replicates randomly assigned were fed to the fish two times per day. Fish were not fed on sampling days. The amount of feed given at each feeding was based on caloric intake on average for the 25 fish in each tank, to control for the number of calories each tank receives (Table 2). Caloric intake for each tank was standardized, based on number of calories fed to the fish, shrimp, and squid diet treatment group. Table 3 shows the composition of each diet component, which was used to calculate calories per gram of each component. In a previous SCDNR study, juvenile red drum ate, on average, 8.4% of their body weight when fed the fish, shrimp, and squid diet to satiation (unpublished data). The current study reduced the amount fed to 7.8% body weight to ensure all feed was consumed. Since this study controlled for caloric intake, the different treatments were fed different amounts (grams), so the 7.8% value for the fish, shrimp, and squid diet was designed to prevent any treatment from receiving more feed than could be consumed. The number of calories per gram of fish fed at 7.8% of their body weight in fish, shrimp, and squid was calculated to be the number of calories each group would receive in total from any of the combinations of feed items (0.07 cal per g fish per day). This feeding rate is close to what others have fed to red drum fingerlings in similar feeding trials [23, 25, 26]. Amounts of every other treatment diet were then calculated to feed the same number of calories to each tank.Table 2
Conversion factors of calories per gram of dry and wet weights of each feed and water percentage for each experimental diet component. These factors were used to calculate how much wet feed to feed to each tank isocalorically based on tank biomass.
Experimental diet componentCalories/g dry weightCalories/g wet weight% WaterD. punctatus fish3.880.9974.56L. vannamei shrimp3.680.9574.30L. opalescens squid4.090.8277.77Illex squid4.501.0080.00Table 3
Composition of the different components used in the experimental diets. Values are listed as proportions.
Experimental diet componentCrude proteinMoistureCrude fatCrude fiberAshD. punctatus fish0.740.030.100.000.14L. vannamei shrimp0.830.030.020.040.10L. opalescens squid0.770.020.070.010.07Illex squid0.680.050.200.120.07Conditioning feed0.40 - 0.550.10 – 0.150.04 – 0.015Pelleted trial feed0.400.100.04The diets used in this feeding trial (followed by their notation) were comprised of fish, shrimp, and squid (FSHSQ), fish and shrimp (FSH), fish and squid (FSQ), shrimp and squid (SHSQ), fish only (F), shrimp only (SH), squid only (SQ), and commercial fishmeal-based pellets (PELL).


### 2.4. Data Collection

At weeks 0, 3, 6, 9, and 12, fish were weighed by tank with total tank biomass recorded. At week 0, ANOVA testing was used to confirm each treatment was at a statistically similar starting biomass. At week 0 (before placing 25 fish per tank), 30 fish were randomly sacrificed to assess initial proximate compositions. At weeks 3, 6, 9, and 12, 72 fish were sacrificed (three from each tank, nine from each treatment). Fish were euthanized in a lethal solution of diluted tricaine methanesulfonate (0.5 g L-1 buffered with 1 g L-1 sodium bicarbonate). Immediately after specimens were euthanized, livers were excised, weighed, and snap-frozen in liquid nitrogen. Intestine and muscle samples were also taken and snap frozen in liquid nitrogen to be used in future studies at SCDNR. At the end of the feeding trial, 80 (10 from each treatment) fish were sacrificed. 40 of the fish were preserved as whole fish, while the other 40 had muscle samples collected. Combined with the 30 fish (15 whole fish and 15 muscle samples) sacrificed at the beginning of the trial, these samples (whole fish and muscle) were then dried in an oven at 80° C with percent water and dry matter calculated. Dried whole fish and muscle samples were homogenized using a Hamilton Beach Coffee Grinder and then sent to the Clemson Agricultural Service Laboratory for proximate analysis.


### 2.5. Statistical Analysis

Statistical analysis was completed using the software program R-Studio Version 1.1.456. All data were tested for normality. Data that was not normal was log transformed. Analysis of Variance and Tukey’s Post Hoc tests were used to determine statistical differences between the diet treatments for each index calculated, using a significance value ofp<0.05.


### 2.6. Equations

(1)SpecificGrowthRate:100∗lnfinalweightgrams–lninitialweightgramsdaysfed,HepatosomaticIndex:100∗liverweightgramstotalweightgrams,ConditionFactor:100∗weightgramstotallengthcm^3,FeedConversionRatio:wetordryfedgramsfinalbiomass–initialbiomass,DryMatterRatio:FCR∗%drymatterweightoffeedsupplied%dryweightofbiomassgained,Protein Intake (per fish): dry grams of protein fed to each fish(2)ProteinEfficiency:drybiomassgainedfinalbiomass–initialbiomassdrygramsproteinfed,FeedIntake:100∗meanindividualdrymatterfeedintake/initialindividualweight∗finalindividualweight∗0.586days,ProteinConversionEfficiency:dryproteingain∗100dryproteinoffered.


## 2.1. Animal Rearing and Captivity

Broodstock red drum at the South Carolina Department of Natural Resources’ Marine Resources Research Institute (SCDNR) (Charleston, SC) underwent volitional spawning. Once the eggs hatched, resulting larvae were transferred to the Waddell Mariculture Center (WMC) (Bluffton, SC) and grown out to fingerlings in large in-ground ponds. They were then transferred to the Hollings Marine Laboratory (HML) (Charleston, SC) once they reached an average length between 30 and 40 mm. They were held at HML until a target average weight of 25 grams was reached, then the 12-week feeding trial began. The fingerlings were kept in a recirculating aquaculture system at HML for the duration of this study.The system used in this study consists of three groups of eight circular tanks (5’ diameter by 3’ tall, 1112 Liter volume) each with their own biological and mechanical filtration as well as UV sterilization, protein skimmer, and temperature control. For this study, all three systems were connected and on the same water supply.


## 2.2. Husbandry

Dissolved oxygen and temperature levels were routinely monitored by SCDNR Mariculture Staff as the fish were reared in ponds at WMC until they were transferred back to Charleston. Upon transfer to the recirculating aquaculture system at HML, water quality measurements of dissolved oxygen, pH, salinity, and temperature (every other day) were measured using a YSI probe (Model Pro 10102030, Professional Series, YSI, Inc., Yellow Springs, Ohio), and ammonia, nitrite, and nitrate (weekly) were measured using a spectrophotometer (Hatch Model # DR2500) (Table1). The fish were fed a commercial pelleted diet to satiation twice per day until they reached the target weight of 25 grams to ensure an equivalent starting metabolic profile and body composition.Table 1
Daily water quality measurements (temperature, dissolved oxygen, salinity, and pH) and weekly water quality measurements (ammonia, nitrite, and nitrate) taken throughout the 12-week feeding trial.
Water quality parameterRangeMean±SDnTemperature °C24.40 – 24.8024.70±0.10246Dissolved oxygen mg L-15.74 – 7.026.47±0.22222pH7.47 – 8.107.87±0.13246Salinity ppt28.66 – 32.9931.04±1.21246Ammonia mg L-10.00 – 0.190.05±0.0472Nitrite mg L-10.01 – 0.040.01±0.0171Nitrate mg L-11.10 – 7.503.30±1.1071


## 2.3. Feeding Trial

An 86-day feeding trial preceded by a 68-day conditioning period was conducted once the fingerlings reached an average weight of 25 grams. Twenty-five fish (600 total, average weight of27.45±0.52 grams) were randomly placed into 24 tanks for the duration of the study, where they were fed different cut diets comprised of fish (Decapterus punctatus), shrimp (Litopenaeus vannamei), and squid (Loligo opalescens and Illex) or a fishmeal-based commercial pelleted feed. Frozen feed was purchased at Haddrell’s Point Tackle (Charleston, SC). Eight treatment diets with three replicates randomly assigned were fed to the fish two times per day. Fish were not fed on sampling days. The amount of feed given at each feeding was based on caloric intake on average for the 25 fish in each tank, to control for the number of calories each tank receives (Table 2). Caloric intake for each tank was standardized, based on number of calories fed to the fish, shrimp, and squid diet treatment group. Table 3 shows the composition of each diet component, which was used to calculate calories per gram of each component. In a previous SCDNR study, juvenile red drum ate, on average, 8.4% of their body weight when fed the fish, shrimp, and squid diet to satiation (unpublished data). The current study reduced the amount fed to 7.8% body weight to ensure all feed was consumed. Since this study controlled for caloric intake, the different treatments were fed different amounts (grams), so the 7.8% value for the fish, shrimp, and squid diet was designed to prevent any treatment from receiving more feed than could be consumed. The number of calories per gram of fish fed at 7.8% of their body weight in fish, shrimp, and squid was calculated to be the number of calories each group would receive in total from any of the combinations of feed items (0.07 cal per g fish per day). This feeding rate is close to what others have fed to red drum fingerlings in similar feeding trials [23, 25, 26]. Amounts of every other treatment diet were then calculated to feed the same number of calories to each tank.Table 2
Conversion factors of calories per gram of dry and wet weights of each feed and water percentage for each experimental diet component. These factors were used to calculate how much wet feed to feed to each tank isocalorically based on tank biomass.
Experimental diet componentCalories/g dry weightCalories/g wet weight% WaterD. punctatus fish3.880.9974.56L. vannamei shrimp3.680.9574.30L. opalescens squid4.090.8277.77Illex squid4.501.0080.00Table 3
Composition of the different components used in the experimental diets. Values are listed as proportions.
Experimental diet componentCrude proteinMoistureCrude fatCrude fiberAshD. punctatus fish0.740.030.100.000.14L. vannamei shrimp0.830.030.020.040.10L. opalescens squid0.770.020.070.010.07Illex squid0.680.050.200.120.07Conditioning feed0.40 - 0.550.10 – 0.150.04 – 0.015Pelleted trial feed0.400.100.04The diets used in this feeding trial (followed by their notation) were comprised of fish, shrimp, and squid (FSHSQ), fish and shrimp (FSH), fish and squid (FSQ), shrimp and squid (SHSQ), fish only (F), shrimp only (SH), squid only (SQ), and commercial fishmeal-based pellets (PELL).


## 2.4. Data Collection

At weeks 0, 3, 6, 9, and 12, fish were weighed by tank with total tank biomass recorded. At week 0, ANOVA testing was used to confirm each treatment was at a statistically similar starting biomass. At week 0 (before placing 25 fish per tank), 30 fish were randomly sacrificed to assess initial proximate compositions. At weeks 3, 6, 9, and 12, 72 fish were sacrificed (three from each tank, nine from each treatment). Fish were euthanized in a lethal solution of diluted tricaine methanesulfonate (0.5 g L-1 buffered with 1 g L-1 sodium bicarbonate). Immediately after specimens were euthanized, livers were excised, weighed, and snap-frozen in liquid nitrogen. Intestine and muscle samples were also taken and snap frozen in liquid nitrogen to be used in future studies at SCDNR. At the end of the feeding trial, 80 (10 from each treatment) fish were sacrificed. 40 of the fish were preserved as whole fish, while the other 40 had muscle samples collected. Combined with the 30 fish (15 whole fish and 15 muscle samples) sacrificed at the beginning of the trial, these samples (whole fish and muscle) were then dried in an oven at 80° C with percent water and dry matter calculated. Dried whole fish and muscle samples were homogenized using a Hamilton Beach Coffee Grinder and then sent to the Clemson Agricultural Service Laboratory for proximate analysis.


## 2.5. Statistical Analysis

Statistical analysis was completed using the software program R-Studio Version 1.1.456. All data were tested for normality. Data that was not normal was log transformed. Analysis of Variance and Tukey’s Post Hoc tests were used to determine statistical differences between the diet treatments for each index calculated, using a significance value ofp<0.05.


## 2.6. Equations

(1)SpecificGrowthRate:100∗lnfinalweightgrams–lninitialweightgramsdaysfed,HepatosomaticIndex:100∗liverweightgramstotalweightgrams,ConditionFactor:100∗weightgramstotallengthcm^3,FeedConversionRatio:wetordryfedgramsfinalbiomass–initialbiomass,DryMatterRatio:FCR∗%drymatterweightoffeedsupplied%dryweightofbiomassgained,Protein Intake (per fish): dry grams of protein fed to each fish(2)ProteinEfficiency:drybiomassgainedfinalbiomass–initialbiomassdrygramsproteinfed,FeedIntake:100∗meanindividualdrymatterfeedintake/initialindividualweight∗finalindividualweight∗0.586days,ProteinConversionEfficiency:dryproteingain∗100dryproteinoffered.


## 3. Results

### 3.1. Feeding Trial

In total, nine mortalities occurred throughout the feeding trial, none of which were considered related to the dietary treatments. Three mortalities occurred in tanks fed shrimp only; two occurred in tanks fed fish, shrimp, and squid; and two occurred in a tank fed shrimp and squid. Two mortalities (one from a tank fed pellets, and one from a tank fed shrimp and squid) were due to handling incidents. No statistical differences were detected for mortalities between diet treatments (p>0.05). Water quality stayed within acceptable conditions for red drum growth and survival (Table 1) [27].Table4 summarizes the results from the 12-week feeding trial that shows how the fish responded to the different diets. There were significant differences (ANOVA; p<0.05) in specific growth rate (SGR), hepatosomatic index (HSI), final average fish weight (FW), final average total length (TL), feed conversion ratio (FCR), percent viscera (PV), condition factor (CF), protein efficiency (PE), dry matter ratio (DMR), and protein conversion efficiency (PCE) among the treatment groups (Table 4). Protein intake (PI) and feed intake (FI) showed expected significant differences among the treatment groups, due to the different compositions of the feed components. The data recorded in this study are presented as dry weight and per fish, unless otherwise noted.Table 4
Traditional Aquaculture Production Characteristics from the 12-week feeding trial.Mean±standarddeviation values are listed. Significance was tested with ANOVA using p<0.05. Means that were not statistically different are denoted with similar superscripts based on Tukey’s post hoc testing using p<0.05. ∗Indicates data was log transformed to fit the assumptions of ANOVA before testing.
DietFSHSQFSHFSQSHSQFSHSQPELLp valueSGR1.61±0.07a1.76±0.01a1.66±0.07a1.16±0.05bde2.18±0.04c1.30±0.08d1.02±0.06e1.83±0.01ap<0.05Final Wt (g)109.90±19.10a122.90±15.60a113.10±17.80a74.20±16.20b180.80±18.30c85.40±15.20ab66.50±11.60b133.40±24.70ap<0.05Final TL (mm)217.67±15.06a234.33±8.90ac219.11±13.46a189.44±12.02bd249.33±11.49c198.22±13.07abd186.78±11.65d226.89±14.22acp<0.05CF1.03±0.04ab0.99±0.02a1.01±0.04a1.00±0.02a1.10±0.07ab1.03±0.03ab0.99±0.03a1.06±0.07abp<0.05HSI∗0.79±0.20a0.73±0.20a0.76±0.21a0.65±0.18a1.70±0.67b0.66±0.16a0.81±0.20a3.11±1.06cp<0.05FCR dry: wet0.90±0.02a0.84±0.00a0.85±0.05a1.32±0.05b0.63±0.01c1.26±0.04b1.44±0.08bd1.15±0.09bep<0.05FCR wet: wet3.70±0.08a3.23±0.01ab3.66±0.20abc5.64±0.22d2.48±0.05e4.92±0.14f6.77±0.37gn/ap<0.05% viscera6.56±1.25ab4.83±0.81a6.44±3.69ab5.53±0.94a6.71±0.98ab5.98±1.25a7.32±1.25ab9.34±1.53bp<0.05PI57.11±3.10a64.19±1.37b52.11±2.51ac45.25±0.78d72.23±1.74e57.64±2.16abc38.02±0.90f49.86±1.82bcdp<0.05PE1.48±0.07a1.49±0.02a1.65±0.08a1.13±0.07b2.12±0.04c1.26±0.26b1.02±0.07b2.12±0.20cp<0.05FI1.57±0.03abc1.65±0.04ab1.51±0.01ac1.52±0.03ac1.61±0.01abc1.64±0.02ab1.44±0.01c2.40±0.06dp<0.05DMR3.59±0.17a3.17±0.03a3.17±0.33a7.41±0.81b1.80±0.06a6.78±0.62bc8.76±1.05bc6.06±1.78bp<0.05PCE26.69±1.11abd26.64±0.33abd28.56±1.27abc21.10±1.44abd36.27±0.71bc23.74±5.10abd19.00±1.18ad33.30±3.05abcp<0.05SpecificGrowthRateSGR:100∗lnFinalWeightgrams–lnInitialWeightgrams/DaysFed. ConditionFactorCF:100∗Weightgrams/TotalLengthcm^3. HepatosomaticIndexHSI:100∗LiverWeightgrams/TotalWeightgrams. FeedConversionRatioFCR:WetorDryFedgrams/FinalBiomass–InitialBiomass. ProteinIntakePIperfish:Drygramsfedtoeachfish. ProteinEfficiencyPE:Drybiomassgained/dryproteinfed. FeedIntakeFI:100∗meanindividualdrymatterfeedintake/initialindividualweight∗finalindividualweight∗0.5/86days. DryMatterRatioDMR:FeedConversionRatio∗%drymatterweightoffeedsupplied/%dryweightofbiomassgained. ProteinConversionEfficiencyPCE:Dryproteingain∗100/dryproteinoffered.Three groupings appeared based on final weight, with the group fed fish only finishing with the highest weight gain (p<0.05) (Table 4). The groupings were as follows: group 1: F; group 2: PELL, FSH, FSQ, and FSHSQ; and group 3: SHSQ, SH, and SQ.The group fed fish only had the highest average FW, longest average TL, had the highest CF, and the lowest FCR. The fish groups fed the pelleted diet had the highest HSI and the heaviest livers. The groups fed the diets of SHSQ, SH, and SQ had the lowest final average FW and the highest FCR. One interesting finding was that the FCR for amount of dried feed fed for the pellet diet was higher (lower performing) than the FSHSQ, FSH, FSQ, and F groups, while outperforming the FSHSQ, FSH, and FSQ groups in growth.PI was measured to compare the differences between the diet treatments since dietary treatments were isocaloric but not isonitrogenous. Compared to the growth results, the natural diets compare very similarly based on the order of amount of PI. As fish grew larger, the calories needed to be fed to these tanks increased, therefore, increasing the amount of dry protein being fed to each fish. The pelleted feed differed from the natural feed items because it contained 40% protein composition, while the natural diet components each contained at least 70% protein composition (Table3).There were three significantly different groups among the treatments in PE values (Table4). Lower performing groups, in terms of growth, SHSQ, SH, and SQ, had approximately a 1 : 1 ratio of dry weight gain to dry protein fed. The combination diets that included fish (FSHSQ, FSH, and FSQ) had approximately a 1.5 : 1 ratio of dry weight gain to dry protein fed. The F and PELL groups had approximately a 2.2 : 1 ratio of dry weight gain to dry protein fed.The DMR produced significantly different groupings similar to the FCR. Diets that included the fish component had lower DMR values than the other natural diets and the pelleted diet. The F group showed the lowest DMR, followed by the FSHSQ, FSH, and FSQ groups, then the SHSQ, SH, SQ, and PELL with the highest DMR values.There were significant differences in FI across the natural diets (p<0.05) simply due to the setup of feeding based on calories. Each component had different crude proximate compositions, which caused the weights of dry feed fed to each tank differ to make sure each tank was fed the same calories per gram of biomass in the tank. The PELL was fed significantly more dry feed (g) per day than the natural diets, due to the composition of the dry pellets (Table 3) making the pellets less calorically dense when compared to the dried natural feeds.


### 3.2. Results over Time

Total weight gained, CF, HSI, PV, and TL gained were tracked at each sampling time point. At week 0, all groups were statistically similar (p>0.05) for each index.For CF, a slight increase in value was apparent for each diet over the course of the feeding trial. The order of values showed that the F and PELL diets finished with a higher CF than the group of other natural components. The FSQ, SHSQ, SQ, and PELL groups saw a decrease in CF from week 0 to week 3, then an increase in CF until the end of the feeding trial.For HSI at week 3 through week 12, the PELL group was statistically different (p<0.05) than the other seven diet treatments, which were not statistically different from each other (p>0.05). All the natural diets saw a decrease in HSI values at week 3. At week 6, no diets showed significant differences to their week 3 values, with the F treatment showing a slight increase. At week 9, the F treatment was statistically different (p<0.05) than the other natural diets with another increase in HSI. The final sampling at week 12 showed the PELL treatment with the highest HSI value, followed by the F group, both statistically different (p<0.05) from each other and from the other six natural diet treatments, which were not statistically different from one another (p>0.05).At week 3, the natural diets start showing lower percent viscera values than they began with. The values of the pelleted diet treatment group stay significantly above the natural diets. As the feeding trial went longer, this value started to fall for the pelleted diet treatment group. The natural diets formed two significantly different groups, but no grouping based on particular components was observed.


### 3.3. Compositional Analysis

Table5 shows the results of the standard mineral and compositional analysis for the whole-body samples for each diet. Percent ash, fat, and protein were statistically different among the diets (p<0.05). The mineral content between whole fish sampled from each diet was also statistically different (p<0.05), except for iron (p>0.05). The fish sampled from the commercial pellet diet showed higher percent body fat than the fish sampled from the natural diets, while they also showed lower percent body protein composition.Table 5
Compositional and mineral analysis of the red drum whole fish samples in the feeding trial. Initial and final whole fish samples were reported.Mean±standarddeviation values are listed. Significance was tested with ANOVA using p<0.05.
DietConditionFSHSQFSHFSQSHSQFSHSQPELLpAsh %15.32±0.1118.40±1.7018.14±0.8517.66±0.5019.12±1.0516.94±1.0518.98±0.3918.00±1.3215.30±1.90p<0.05TotalN %10.12±0.1912.13±0.2712.13±0.3011.77±0.6312.11±0.3211.83±0.3912.28±0.1211.90±0.0810.92±0.34p<0.05Crude protein %63.27±1.1975.82±1.6975.83±1.8573.57±3.9775.68±2.0173.95±2.4276.77±0.7774.42±0.5168.28±2.15p<0.05Crude fat %17.88±0.364.73±1.774.77±1.425.49±2.962.93±1.676.32±4.472.78±1.324.52±2.3512.73±1.59p<0.05P ppm24349.30±501.2629560.34±3480.5630778.78±1165.7228219.84±2387.2431724.34±2261.3928697.16±898.2831021.58±2596.9926772.46±3013.8825772.64±4249.52p<0.05K ppm12226.60±304.3513532.64±996.4814292.44±758.9012955.48±602.8314451.48±555.4513116.40±431.8613833.40±1041.2412555.64±885.9612296.88±492.68p<0.05Ca ppm40167.86±1107.1046666.98±6843.4147075.90±1317.9043916.02±3291.3650366.54±3146.1444435.06±897.3248822.76±3726.1544728.18±4643.7542568.36±6526.68p<0.05Mg ppm1665.12±171.721724.04±188.641822.68±64.061672.14±90.291864.26±86.581594.02±57.601811.80±136.111566.72±152.351445.10±139.20p<0.05Zn ppm67.91±1.0848.90±1.6548.94±1.3147.80±2.8158.39±1.2748.70±2.0051.81±3.0352.72±6.9549.45±4.61p<0.05Cu ppm3.46±0.131.73±0.151.80±0.481.73±0.211.47±0.742.42±0.441.16±0.341.04±0.271.85±0.49p<0.05Mn ppm35.45±1.5811.11±3.379.44±1.227.10±1.5016.08±2.895.78±1.5615.22±2.5311.50±4.2032.68±6.18p<0.05Fe ppm48.23±2.6251.00±20.6847.30±4.2739.86±7.3640.77±4.8540.05±1.6239.96±7.3135.06±7.7230.15±5.90p>0.05S ppm7013.28±178.9810108.12±780.879061.44±465.819768.80±510.9710935.46±263.008891.86±307.068207.76±488.5310180.38±804.796398.76±259.40p<0.05Na ppm6611.42±1567.275876.17±861.645993.77±372.205535.06±447.886770.50±526.835412.16±481.796141.56±513.135975.73±658.645267.26±461.42p<0.05Table6 shows the results of the standard mineral and compositional analysis for the muscle fillet samples for each diet. Once again, the muscle fat content of the fillet samples of the fish fed the conditioning diet and pelleted diet were statistically higher than those fed the natural diets. However, protein percent differences were not observed between the fillet samples fed the pelleted and natural diets. The fillet samples from the fish fed pelleted diet showed significantly higher fat content than the natural diets (p<0.05). The mineral content found in the muscle samples of each diet was shown to be statistically different (p<0.05) for calcium, sodium, zinc, and sulfur. Phosphorus, potassium, magnesium, copper, manganese, and iron did not show statistical differences (p>0.05).Table 6
Compositional and mineral analysis of the red drum muscle samples in the feeding trial. Initial and final muscle fillet samples were reported.Mean±standarddeviation values are listed. Significance was tested with ANOVA using p<0.05.
DietConditionFSHSQFSHFSQSHSQFSHSQPELLpAsh %6.70±0.786.64±0.256.60±0.256.66±0.216.70±0.336.24±0.236.42±0.196.36±0.466.08±0.22p>0.05TotalN %13.99±0.1913.96±0.3214.30±0.2013.90±0.1213.77±0.3214.31±0.1313.97±0.4513.85±0.2114.04±0.20p<0.05Crude protein %87.43±1.1987.28±2.0089.42±1.2286.91±0.7486.07±2.0289.45±0.8387.32±2.8486.59±1.2887.78±1.23p<0.05Crude fat %2.63±0.360.88±0.711.03±0.941.06±0.551.17±0.361.28±0.631.56±0.191.09±0.243.08±1.07p<0.05P ppm9928.76±501.2610395.00±903.9610708.40±709.5110488.46±360.859942.80±895.1810480.24±302.4210592.02±574.4810195.24±917.4010044.52±596.97p>0.05K ppm17911±304.3520038.08±1479.8720870.78±1346.5920427.88±661.5319380.24±2075.3920442.96±466.8020273.68±1191.5419760.10±1831.7618549.84±1158.90p>0.05Ca ppm3085.26±1107.102242.74±536.22193.86±398.851959.88±324.861645.66±372.852176.64±874.191889.72±326.061417.46±683.101793.56±437.96p<0.05Mg ppm1460.96±171.721439.80±105.081431.26±63.041410.20±59.741384.52±120.361404.40±41.491478.80±66.231370.94±118.201339.24±103.30p>0.05Zn ppm26.90±1.0818.46±1.7719.36±1.1119.45±1.4819.12±1.6324.30±8.7021.06±1.2319.37±2.0723.44±2.47p<0.05Cu ppm1.81±0.131.24±0.181.45±0.201.26±0.101.41±0.171.74±1.131.70±0.521.65±0.351.53±0.13p>0.05Mn ppm4.59±1.580.88±0.170.86±0.120.72±0.080.86±0.074.422±8.601.54±0.351.11±0.432.57±0.54p>0.05Fe ppm22.14±2.6215.70±5.6416.11±7.3111.66±1.4911.42±2.7324.56±28.8516.08±4.2011.13±1.869.66±1.85p>0.05S ppm8426.33±178.9812058.08±782.1210107.62±429.9212626.64±710.3612316.80±931.5411233.94±410.519643.58±528.5613247.56±1214.338388.02±559.76p<0.05Na ppm2804.40±1567.275284.91±841.084497.20±981.334926.20±956.295713.72±1205.844571.54±1050.064826.34±459.404864.27±820.654607.09±1189.70p<0.05


## 3.1. Feeding Trial

In total, nine mortalities occurred throughout the feeding trial, none of which were considered related to the dietary treatments. Three mortalities occurred in tanks fed shrimp only; two occurred in tanks fed fish, shrimp, and squid; and two occurred in a tank fed shrimp and squid. Two mortalities (one from a tank fed pellets, and one from a tank fed shrimp and squid) were due to handling incidents. No statistical differences were detected for mortalities between diet treatments (p>0.05). Water quality stayed within acceptable conditions for red drum growth and survival (Table 1) [27].Table4 summarizes the results from the 12-week feeding trial that shows how the fish responded to the different diets. There were significant differences (ANOVA; p<0.05) in specific growth rate (SGR), hepatosomatic index (HSI), final average fish weight (FW), final average total length (TL), feed conversion ratio (FCR), percent viscera (PV), condition factor (CF), protein efficiency (PE), dry matter ratio (DMR), and protein conversion efficiency (PCE) among the treatment groups (Table 4). Protein intake (PI) and feed intake (FI) showed expected significant differences among the treatment groups, due to the different compositions of the feed components. The data recorded in this study are presented as dry weight and per fish, unless otherwise noted.Table 4
Traditional Aquaculture Production Characteristics from the 12-week feeding trial.Mean±standarddeviation values are listed. Significance was tested with ANOVA using p<0.05. Means that were not statistically different are denoted with similar superscripts based on Tukey’s post hoc testing using p<0.05. ∗Indicates data was log transformed to fit the assumptions of ANOVA before testing.
DietFSHSQFSHFSQSHSQFSHSQPELLp valueSGR1.61±0.07a1.76±0.01a1.66±0.07a1.16±0.05bde2.18±0.04c1.30±0.08d1.02±0.06e1.83±0.01ap<0.05Final Wt (g)109.90±19.10a122.90±15.60a113.10±17.80a74.20±16.20b180.80±18.30c85.40±15.20ab66.50±11.60b133.40±24.70ap<0.05Final TL (mm)217.67±15.06a234.33±8.90ac219.11±13.46a189.44±12.02bd249.33±11.49c198.22±13.07abd186.78±11.65d226.89±14.22acp<0.05CF1.03±0.04ab0.99±0.02a1.01±0.04a1.00±0.02a1.10±0.07ab1.03±0.03ab0.99±0.03a1.06±0.07abp<0.05HSI∗0.79±0.20a0.73±0.20a0.76±0.21a0.65±0.18a1.70±0.67b0.66±0.16a0.81±0.20a3.11±1.06cp<0.05FCR dry: wet0.90±0.02a0.84±0.00a0.85±0.05a1.32±0.05b0.63±0.01c1.26±0.04b1.44±0.08bd1.15±0.09bep<0.05FCR wet: wet3.70±0.08a3.23±0.01ab3.66±0.20abc5.64±0.22d2.48±0.05e4.92±0.14f6.77±0.37gn/ap<0.05% viscera6.56±1.25ab4.83±0.81a6.44±3.69ab5.53±0.94a6.71±0.98ab5.98±1.25a7.32±1.25ab9.34±1.53bp<0.05PI57.11±3.10a64.19±1.37b52.11±2.51ac45.25±0.78d72.23±1.74e57.64±2.16abc38.02±0.90f49.86±1.82bcdp<0.05PE1.48±0.07a1.49±0.02a1.65±0.08a1.13±0.07b2.12±0.04c1.26±0.26b1.02±0.07b2.12±0.20cp<0.05FI1.57±0.03abc1.65±0.04ab1.51±0.01ac1.52±0.03ac1.61±0.01abc1.64±0.02ab1.44±0.01c2.40±0.06dp<0.05DMR3.59±0.17a3.17±0.03a3.17±0.33a7.41±0.81b1.80±0.06a6.78±0.62bc8.76±1.05bc6.06±1.78bp<0.05PCE26.69±1.11abd26.64±0.33abd28.56±1.27abc21.10±1.44abd36.27±0.71bc23.74±5.10abd19.00±1.18ad33.30±3.05abcp<0.05SpecificGrowthRateSGR:100∗lnFinalWeightgrams–lnInitialWeightgrams/DaysFed. ConditionFactorCF:100∗Weightgrams/TotalLengthcm^3. HepatosomaticIndexHSI:100∗LiverWeightgrams/TotalWeightgrams. FeedConversionRatioFCR:WetorDryFedgrams/FinalBiomass–InitialBiomass. ProteinIntakePIperfish:Drygramsfedtoeachfish. ProteinEfficiencyPE:Drybiomassgained/dryproteinfed. FeedIntakeFI:100∗meanindividualdrymatterfeedintake/initialindividualweight∗finalindividualweight∗0.5/86days. DryMatterRatioDMR:FeedConversionRatio∗%drymatterweightoffeedsupplied/%dryweightofbiomassgained. ProteinConversionEfficiencyPCE:Dryproteingain∗100/dryproteinoffered.Three groupings appeared based on final weight, with the group fed fish only finishing with the highest weight gain (p<0.05) (Table 4). The groupings were as follows: group 1: F; group 2: PELL, FSH, FSQ, and FSHSQ; and group 3: SHSQ, SH, and SQ.The group fed fish only had the highest average FW, longest average TL, had the highest CF, and the lowest FCR. The fish groups fed the pelleted diet had the highest HSI and the heaviest livers. The groups fed the diets of SHSQ, SH, and SQ had the lowest final average FW and the highest FCR. One interesting finding was that the FCR for amount of dried feed fed for the pellet diet was higher (lower performing) than the FSHSQ, FSH, FSQ, and F groups, while outperforming the FSHSQ, FSH, and FSQ groups in growth.PI was measured to compare the differences between the diet treatments since dietary treatments were isocaloric but not isonitrogenous. Compared to the growth results, the natural diets compare very similarly based on the order of amount of PI. As fish grew larger, the calories needed to be fed to these tanks increased, therefore, increasing the amount of dry protein being fed to each fish. The pelleted feed differed from the natural feed items because it contained 40% protein composition, while the natural diet components each contained at least 70% protein composition (Table3).There were three significantly different groups among the treatments in PE values (Table4). Lower performing groups, in terms of growth, SHSQ, SH, and SQ, had approximately a 1 : 1 ratio of dry weight gain to dry protein fed. The combination diets that included fish (FSHSQ, FSH, and FSQ) had approximately a 1.5 : 1 ratio of dry weight gain to dry protein fed. The F and PELL groups had approximately a 2.2 : 1 ratio of dry weight gain to dry protein fed.The DMR produced significantly different groupings similar to the FCR. Diets that included the fish component had lower DMR values than the other natural diets and the pelleted diet. The F group showed the lowest DMR, followed by the FSHSQ, FSH, and FSQ groups, then the SHSQ, SH, SQ, and PELL with the highest DMR values.There were significant differences in FI across the natural diets (p<0.05) simply due to the setup of feeding based on calories. Each component had different crude proximate compositions, which caused the weights of dry feed fed to each tank differ to make sure each tank was fed the same calories per gram of biomass in the tank. The PELL was fed significantly more dry feed (g) per day than the natural diets, due to the composition of the dry pellets (Table 3) making the pellets less calorically dense when compared to the dried natural feeds.


## 3.2. Results over Time

Total weight gained, CF, HSI, PV, and TL gained were tracked at each sampling time point. At week 0, all groups were statistically similar (p>0.05) for each index.For CF, a slight increase in value was apparent for each diet over the course of the feeding trial. The order of values showed that the F and PELL diets finished with a higher CF than the group of other natural components. The FSQ, SHSQ, SQ, and PELL groups saw a decrease in CF from week 0 to week 3, then an increase in CF until the end of the feeding trial.For HSI at week 3 through week 12, the PELL group was statistically different (p<0.05) than the other seven diet treatments, which were not statistically different from each other (p>0.05). All the natural diets saw a decrease in HSI values at week 3. At week 6, no diets showed significant differences to their week 3 values, with the F treatment showing a slight increase. At week 9, the F treatment was statistically different (p<0.05) than the other natural diets with another increase in HSI. The final sampling at week 12 showed the PELL treatment with the highest HSI value, followed by the F group, both statistically different (p<0.05) from each other and from the other six natural diet treatments, which were not statistically different from one another (p>0.05).At week 3, the natural diets start showing lower percent viscera values than they began with. The values of the pelleted diet treatment group stay significantly above the natural diets. As the feeding trial went longer, this value started to fall for the pelleted diet treatment group. The natural diets formed two significantly different groups, but no grouping based on particular components was observed.


## 3.3. Compositional Analysis

Table5 shows the results of the standard mineral and compositional analysis for the whole-body samples for each diet. Percent ash, fat, and protein were statistically different among the diets (p<0.05). The mineral content between whole fish sampled from each diet was also statistically different (p<0.05), except for iron (p>0.05). The fish sampled from the commercial pellet diet showed higher percent body fat than the fish sampled from the natural diets, while they also showed lower percent body protein composition.Table 5
Compositional and mineral analysis of the red drum whole fish samples in the feeding trial. Initial and final whole fish samples were reported.Mean±standarddeviation values are listed. Significance was tested with ANOVA using p<0.05.
DietConditionFSHSQFSHFSQSHSQFSHSQPELLpAsh %15.32±0.1118.40±1.7018.14±0.8517.66±0.5019.12±1.0516.94±1.0518.98±0.3918.00±1.3215.30±1.90p<0.05TotalN %10.12±0.1912.13±0.2712.13±0.3011.77±0.6312.11±0.3211.83±0.3912.28±0.1211.90±0.0810.92±0.34p<0.05Crude protein %63.27±1.1975.82±1.6975.83±1.8573.57±3.9775.68±2.0173.95±2.4276.77±0.7774.42±0.5168.28±2.15p<0.05Crude fat %17.88±0.364.73±1.774.77±1.425.49±2.962.93±1.676.32±4.472.78±1.324.52±2.3512.73±1.59p<0.05P ppm24349.30±501.2629560.34±3480.5630778.78±1165.7228219.84±2387.2431724.34±2261.3928697.16±898.2831021.58±2596.9926772.46±3013.8825772.64±4249.52p<0.05K ppm12226.60±304.3513532.64±996.4814292.44±758.9012955.48±602.8314451.48±555.4513116.40±431.8613833.40±1041.2412555.64±885.9612296.88±492.68p<0.05Ca ppm40167.86±1107.1046666.98±6843.4147075.90±1317.9043916.02±3291.3650366.54±3146.1444435.06±897.3248822.76±3726.1544728.18±4643.7542568.36±6526.68p<0.05Mg ppm1665.12±171.721724.04±188.641822.68±64.061672.14±90.291864.26±86.581594.02±57.601811.80±136.111566.72±152.351445.10±139.20p<0.05Zn ppm67.91±1.0848.90±1.6548.94±1.3147.80±2.8158.39±1.2748.70±2.0051.81±3.0352.72±6.9549.45±4.61p<0.05Cu ppm3.46±0.131.73±0.151.80±0.481.73±0.211.47±0.742.42±0.441.16±0.341.04±0.271.85±0.49p<0.05Mn ppm35.45±1.5811.11±3.379.44±1.227.10±1.5016.08±2.895.78±1.5615.22±2.5311.50±4.2032.68±6.18p<0.05Fe ppm48.23±2.6251.00±20.6847.30±4.2739.86±7.3640.77±4.8540.05±1.6239.96±7.3135.06±7.7230.15±5.90p>0.05S ppm7013.28±178.9810108.12±780.879061.44±465.819768.80±510.9710935.46±263.008891.86±307.068207.76±488.5310180.38±804.796398.76±259.40p<0.05Na ppm6611.42±1567.275876.17±861.645993.77±372.205535.06±447.886770.50±526.835412.16±481.796141.56±513.135975.73±658.645267.26±461.42p<0.05Table6 shows the results of the standard mineral and compositional analysis for the muscle fillet samples for each diet. Once again, the muscle fat content of the fillet samples of the fish fed the conditioning diet and pelleted diet were statistically higher than those fed the natural diets. However, protein percent differences were not observed between the fillet samples fed the pelleted and natural diets. The fillet samples from the fish fed pelleted diet showed significantly higher fat content than the natural diets (p<0.05). The mineral content found in the muscle samples of each diet was shown to be statistically different (p<0.05) for calcium, sodium, zinc, and sulfur. Phosphorus, potassium, magnesium, copper, manganese, and iron did not show statistical differences (p>0.05).Table 6
Compositional and mineral analysis of the red drum muscle samples in the feeding trial. Initial and final muscle fillet samples were reported.Mean±standarddeviation values are listed. Significance was tested with ANOVA using p<0.05.
DietConditionFSHSQFSHFSQSHSQFSHSQPELLpAsh %6.70±0.786.64±0.256.60±0.256.66±0.216.70±0.336.24±0.236.42±0.196.36±0.466.08±0.22p>0.05TotalN %13.99±0.1913.96±0.3214.30±0.2013.90±0.1213.77±0.3214.31±0.1313.97±0.4513.85±0.2114.04±0.20p<0.05Crude protein %87.43±1.1987.28±2.0089.42±1.2286.91±0.7486.07±2.0289.45±0.8387.32±2.8486.59±1.2887.78±1.23p<0.05Crude fat %2.63±0.360.88±0.711.03±0.941.06±0.551.17±0.361.28±0.631.56±0.191.09±0.243.08±1.07p<0.05P ppm9928.76±501.2610395.00±903.9610708.40±709.5110488.46±360.859942.80±895.1810480.24±302.4210592.02±574.4810195.24±917.4010044.52±596.97p>0.05K ppm17911±304.3520038.08±1479.8720870.78±1346.5920427.88±661.5319380.24±2075.3920442.96±466.8020273.68±1191.5419760.10±1831.7618549.84±1158.90p>0.05Ca ppm3085.26±1107.102242.74±536.22193.86±398.851959.88±324.861645.66±372.852176.64±874.191889.72±326.061417.46±683.101793.56±437.96p<0.05Mg ppm1460.96±171.721439.80±105.081431.26±63.041410.20±59.741384.52±120.361404.40±41.491478.80±66.231370.94±118.201339.24±103.30p>0.05Zn ppm26.90±1.0818.46±1.7719.36±1.1119.45±1.4819.12±1.6324.30±8.7021.06±1.2319.37±2.0723.44±2.47p<0.05Cu ppm1.81±0.131.24±0.181.45±0.201.26±0.101.41±0.171.74±1.131.70±0.521.65±0.351.53±0.13p>0.05Mn ppm4.59±1.580.88±0.170.86±0.120.72±0.080.86±0.074.422±8.601.54±0.351.11±0.432.57±0.54p>0.05Fe ppm22.14±2.6215.70±5.6416.11±7.3111.66±1.4911.42±2.7324.56±28.8516.08±4.2011.13±1.869.66±1.85p>0.05S ppm8426.33±178.9812058.08±782.1210107.62±429.9212626.64±710.3612316.80±931.5411233.94±410.519643.58±528.5613247.56±1214.338388.02±559.76p<0.05Na ppm2804.40±1567.275284.91±841.084497.20±981.334926.20±956.295713.72±1205.844571.54±1050.064826.34±459.404864.27±820.654607.09±1189.70p<0.05


## 4. Discussion

### 4.1. End of Feeding Trial Results

This study used caloric intake as the control for rationing, instead of protein, which others have used in similar studies [9, 19, 28]. This was beneficial because each diet component had different crude compositions (Table 3), which allowed the differences of the overall diet compositions to affect production, rather than one part, such as protein. There were differences observed in traditional aquaculture metrics that may have been masked if fed to satiation. The “Law of the Minimum” states that the nutrient that is important for growth, or other body function, that is in the lowest amount for what is needed, will control the yield, or in this case, growth [29]. There may be a nutrient that is lacking that is important for growth found in the shrimp and squid components that are found in a higher amount in the fish component and pellet diet. This may explain the significant differences in growth between the groups fed the fish component compared to the other groups.One noted observation concerning the pelleted feed was that the fish ate considerably slower compared to the fish that ate the natural diets. It is encouraging, however, to see how well the pelleted feed performed when fed in equal rations, based on caloric intake, with the natural diets. Improvements must be made to close the performance gap that exists [1, 30] when feeding to satiation, as previous research has shown juvenile red drum will consume significantly higher volumes of natural feed items [24].The fish fed the pelleted diet showed significantly larger livers, and thus, higher HSI than the natural diet treatments. A larger liver may mean more energy storage and less conversion of this energy to be used in cellular processes, which is potentially indicative of a deficiency. HSI can be an indicator for fat accumulation and energy storage in the body of fish [31, 32]. The pelleted diet had significantly less protein in the formulation, but fat levels were not significantly different than the natural diet components. No noticeable differences in health were observed along with the increased livers other than significantly higher percent viscera of the whole-body weight. Further analysis to determine the cause of the larger livers is needed. HSI is a parameter that can be used to estimate the energy status, or energy reserves, in fish since the liver is an important energy storage organ [31]. The PELL group had the highest HSI values, followed by the F group, then the natural diet groups. The other natural diets had lower values not statistically different from each other (p>0.05). This could indicate that these six groups had low energy storage, while the F and PELL groups had more energy storage. This could also indicate that the F and PELL groups were using the energy from their food differently that led to a higher energy storage as opposed to directly for growth, potentially due to a bottleneck of resources at a metabolic checkpoint. This will be further analyzed in a future metabolomics study using the liver samples collected from this study to understand what the mechanisms are for these results. Furthermore, research into fat content of the muscle may help explain what was observed. Lack of fat content or poor muscle quality could indicate if there is a metabolic pathway inhibited, leading to changes in liver sizes. The PELL group may have had an overabundance of a specific nutrient or biomolecule that caused extra storage that may not have been usable elsewhere [31]. However, fish fed the pelleted diet still grew well, so this is more likely an indication that the squid and shrimp components have some imbalances and limitations compared to the fish and pellet components. The HSI values reported in this study are similar to other HSI values for juvenile red drum in feeding trials [21, 33, 34].CF is a parameter that can be used to estimate energy status, specifically lipid levels in fish [31], which can be an indicator of health. Across the treatment diets in this study, there was statistical significance (p<0.05) between the CF of each treatment diet. The F group had the highest CF, again suggesting that there are potential nutrients found in the fish component that provides the red drum higher quality or more balanced nutritional inputs. A grow-out study of red drum for stock enhancement completed by Deloach [35] reported CFs that were similar to the CF reported in this study. The CF reported in this study could be lower than wild or captive red drum fed to satiation, since these fish were fed on a caloric ration. With this said, the CF reported in this study are representative of healthy fish and show the fish were eating enough when compared to other studies with juvenile red drum [35, 36].The SH group performed second worst in terms of growth. A previous study using shrimp waste meal in a pelleted form fed to red drum showed poor growth compared to fish bycatch meal and other fishmeal [37]. Li et al. [37] hypothesized this was due the high chitin content present in the shrimp. The Li et al. [37] study reported shrimp seemed to limit growth in juvenile red drum, similar to the current study. Further research into the digestibility and nutritional profile of shrimp could help better explain any limitations it may have as a dietary component for red drum.The proximate composition and standard mineral analyses were performed for whole fish and muscle samples. Tables5 and 6 show these results, respectively. There were more differences observed in the whole fish samples than in muscle samples, most likely due to muscle representing only a single type of tissue, mainly composed of protein, and not typically involved in intermediate physiology. For the minerals in the whole fish samples, phosphorus, potassium, magnesium, copper, manganese, and iron showed no differences among the treatments (p>0.05). Sodium, zinc, calcium, and sulfur were found to be statistically different among the treatments (p<0.05). A comparison of the natural diets and the pelleted diet showed no deficit or surplus minerals [38] in the pelleted feed muscle samples. This supports that the pelleted feed used in this study helps produce quality fillets for human consumption.The compositional analysis showed that for the whole-body samples, there were significant differences in protein, ash, and fat percentages (p<0.05). The composition of the whole fish samples showed the fish fed the pelleted diet had higher fat with lower protein and ash percentages than the fish fed the natural components. This indicates that there is more fat being stored and less protein and ash production occurring in fish fed the fishmeal-based pelleted feed. There seems to be a lack of a specific nutrient or imbalance with the pelleted feeds compared to the natural diets, which is important to know because the development of FMRDs needs to have balanced nutritional makeup so aquaculture-raised fish can perform as well, or better than, wild fish. The composition of the muscle samples showed significant differences in protein and fat (p<0.05) but did not show differences in ash (p>0.05). One positive note to report from this analysis is that the muscle samples from the fish fed the pelleted diet did not have lower levels of protein than those fed the natural diets. There were elevated fat levels in the muscle samples, however, indicating that the quality of the meat from the fish fed the pelleted diet were different than those fed the natural diets.The results of the feeding trial and traditional aquaculture metrics showed that researchers should look closely at why the fish-only diet, and other diets containing fish, resulted in the highest levels of production. From there, the causes behind the production gap between this natural component and FMRDs can be better identified. A strategy for supplementation in FMRDs to close the production gap can then be developed.


### 4.2. Results through Time

The design of this feeding trial allowed for the observation of what happened with specific data trends over the course of the feeding trial. Understanding the time at which a fish changes internal health metrics over time can inform growers and researchers of what nutrients to provide at certain life stages. Knowing at what time these differences start to show can also provide insight into what is needed to produce FMRDs that can close the gap in fish performance observed compared to natural diets.The pelleted diet did not have a higher fat content than the natural diet components, however, the PELL group had significantly higher HSI values. The PELL groups were fed more fat over the course of the feeding trial due to there being more dry material fed compared with the natural diets. The type of fat in the pelleted feed, and how it was digested could also explain why the HSI values were higher compared to the natural diet treatment groups. The HSI value did not change for the PELL group from the initial sampling, due to the pelleted feed being similar in composition to the conditioning feed. It is worth noting that the fingerlings were fed crumble followed by 1.5 mm pellets for the conditioning period, then 3 mm pellets for the feeding trial, each having slightly different compositions (Table3). The natural diets were associated with a substantial decrease in HSI values from week 0 to week 12, and only the F group was associated with an increase in HSI until the end of the feeding trial. Having fish as a component mixed with shrimp and squid in the combination diets did not raise the HSI values, however.Different species of fish have different ranges of optimal CF values based on their body shapes and diets. Also, the optimal CF values change over a fish’s life cycle, meaning a CF for a healthy adult will be different than that for a juvenile. The FSQ, SHSQ, SQ, and PELL groups saw a decrease in CF from week 0 to week 3, then an increase in CF over the course of the feeding trial. All diets showed a slight increase in CF from week 0 to week 12. The lower-performing diets according to weight showed lower CF values. There were no clear statistical trends that separated any of the treatment diets, but it does appear the higher-performing diets according to weight (F and PELL) led to healthier fish due to their higher CF values.The differences observed in PV between the natural diets and the pelleted diet were most likely caused by the different compositions of the pelleted feed (Table3). The fish fed the pelleted feed could have incorporated fat into storage, not muscle growth or similar body functions, while the fat from the natural feeds was likely incorporated differently [32]. Further analysis of the actual fatty acid and overall lipid profiles, including the amount of saturated and unsaturated fats in these feeds, may provide clearer insight into this observation. Other feedstuffs in the pelleted diet may have contributed to the higher PV in fish fed the pelleted diet.The results observed in this study showed that the PELL and F groups had the highest PE. The second-highest performing group (FSHSQ, FSH, and FSQ) outperformed the lowest-performing group of diets (SHSQ, SH, and SQ), which did not include the fish component. Erturk and Sevgili [39] reported PE values slightly higher than in this study. Erturk and Sevgili [39] fed rainbow trout (Onchorynchus mykiss) experimental diets with poultry by product meal fed to satiation, which likely explains why their values were higher. This study shows FMRDs can perform well when compared to the natural diets and fishmeal-based pelleted diet. The fish in the fishmeal and natural diet treatments seemed to boost PE. This is further evidence that fish performs better compared to squid and shrimp for juvenile red drum growth and health.Each natural diet was converted to dry mass to compare with the dry pelleted feed. The PELL group was fed significantly more dry feed per fish per day, due to the compositional differences between the natural diet components and the pellets. This shows that juvenile red drum needs to be fed almost twice as much dry feed mass than the natural diet counterparts. If the fish were fed to satiation, they would have eaten more of the natural feeds compared to the pelleted feed. The actual mass of the wet natural diets fed to the fish resulted in a higher feed intake, but the water mass was significant. When comparing dry matter, the pelleted feed is not as calorically dense compared with the natural diet components. Future research into similar pelleted fishmeal-based feeds and FMRDs should focus on formulating a more calorically dense pellet, as filling up on the dry pellets may be causing gaps in growth performance.The differences in DMR observed between the diets in this study showed further evidence that diet performance was better with the fish component. DMR shows the relationship between dry food fed and dry biomass gained. The fish component seemed more efficient, as evidenced by groups with more growth with less intake. Interestingly, the PELL group, which showed the second-highest performance with regards to growth, statistically grouped with the lower performing natural groups (SHSQ, SH, and SQ) with regards to DMR. This is most likely due to the less dense caloric composition of the pellets.Due to the very different compositions of the pelleted feed used in PELL diet and the natural components of the natural diets, it was observed that the PELL group performed well in growth but performed worse in metrics such as FCR and DMR. FCR and DMR show differences in the production of the fish fed the pelleted feed compared to the natural feed. The protein from the pelleted feed was efficiently used by the fish. The overall conversion of the pelleted feed was not as good, perhaps due to the higher density of the pellets than the natural components. Improving FCR values in fish farming can lead to lower costs for feed and faster turnover for new batches of fish. The FCR values reported in this study were similar to those reported by Tucker et al. [34] for red drum.As global need for protein continues to increase with a static global fishery yield, aquafeed producers are challenged to create high-quality FMRDs [40]. The results of this study provide useful information to show where current commercial fishmeal-based pelleted diets are compared to natural diets for fish when fed isocalorically and can lead to future research to improve FMRDs for juvenile red drum production.


## 4.1. End of Feeding Trial Results

This study used caloric intake as the control for rationing, instead of protein, which others have used in similar studies [9, 19, 28]. This was beneficial because each diet component had different crude compositions (Table 3), which allowed the differences of the overall diet compositions to affect production, rather than one part, such as protein. There were differences observed in traditional aquaculture metrics that may have been masked if fed to satiation. The “Law of the Minimum” states that the nutrient that is important for growth, or other body function, that is in the lowest amount for what is needed, will control the yield, or in this case, growth [29]. There may be a nutrient that is lacking that is important for growth found in the shrimp and squid components that are found in a higher amount in the fish component and pellet diet. This may explain the significant differences in growth between the groups fed the fish component compared to the other groups.One noted observation concerning the pelleted feed was that the fish ate considerably slower compared to the fish that ate the natural diets. It is encouraging, however, to see how well the pelleted feed performed when fed in equal rations, based on caloric intake, with the natural diets. Improvements must be made to close the performance gap that exists [1, 30] when feeding to satiation, as previous research has shown juvenile red drum will consume significantly higher volumes of natural feed items [24].The fish fed the pelleted diet showed significantly larger livers, and thus, higher HSI than the natural diet treatments. A larger liver may mean more energy storage and less conversion of this energy to be used in cellular processes, which is potentially indicative of a deficiency. HSI can be an indicator for fat accumulation and energy storage in the body of fish [31, 32]. The pelleted diet had significantly less protein in the formulation, but fat levels were not significantly different than the natural diet components. No noticeable differences in health were observed along with the increased livers other than significantly higher percent viscera of the whole-body weight. Further analysis to determine the cause of the larger livers is needed. HSI is a parameter that can be used to estimate the energy status, or energy reserves, in fish since the liver is an important energy storage organ [31]. The PELL group had the highest HSI values, followed by the F group, then the natural diet groups. The other natural diets had lower values not statistically different from each other (p>0.05). This could indicate that these six groups had low energy storage, while the F and PELL groups had more energy storage. This could also indicate that the F and PELL groups were using the energy from their food differently that led to a higher energy storage as opposed to directly for growth, potentially due to a bottleneck of resources at a metabolic checkpoint. This will be further analyzed in a future metabolomics study using the liver samples collected from this study to understand what the mechanisms are for these results. Furthermore, research into fat content of the muscle may help explain what was observed. Lack of fat content or poor muscle quality could indicate if there is a metabolic pathway inhibited, leading to changes in liver sizes. The PELL group may have had an overabundance of a specific nutrient or biomolecule that caused extra storage that may not have been usable elsewhere [31]. However, fish fed the pelleted diet still grew well, so this is more likely an indication that the squid and shrimp components have some imbalances and limitations compared to the fish and pellet components. The HSI values reported in this study are similar to other HSI values for juvenile red drum in feeding trials [21, 33, 34].CF is a parameter that can be used to estimate energy status, specifically lipid levels in fish [31], which can be an indicator of health. Across the treatment diets in this study, there was statistical significance (p<0.05) between the CF of each treatment diet. The F group had the highest CF, again suggesting that there are potential nutrients found in the fish component that provides the red drum higher quality or more balanced nutritional inputs. A grow-out study of red drum for stock enhancement completed by Deloach [35] reported CFs that were similar to the CF reported in this study. The CF reported in this study could be lower than wild or captive red drum fed to satiation, since these fish were fed on a caloric ration. With this said, the CF reported in this study are representative of healthy fish and show the fish were eating enough when compared to other studies with juvenile red drum [35, 36].The SH group performed second worst in terms of growth. A previous study using shrimp waste meal in a pelleted form fed to red drum showed poor growth compared to fish bycatch meal and other fishmeal [37]. Li et al. [37] hypothesized this was due the high chitin content present in the shrimp. The Li et al. [37] study reported shrimp seemed to limit growth in juvenile red drum, similar to the current study. Further research into the digestibility and nutritional profile of shrimp could help better explain any limitations it may have as a dietary component for red drum.The proximate composition and standard mineral analyses were performed for whole fish and muscle samples. Tables5 and 6 show these results, respectively. There were more differences observed in the whole fish samples than in muscle samples, most likely due to muscle representing only a single type of tissue, mainly composed of protein, and not typically involved in intermediate physiology. For the minerals in the whole fish samples, phosphorus, potassium, magnesium, copper, manganese, and iron showed no differences among the treatments (p>0.05). Sodium, zinc, calcium, and sulfur were found to be statistically different among the treatments (p<0.05). A comparison of the natural diets and the pelleted diet showed no deficit or surplus minerals [38] in the pelleted feed muscle samples. This supports that the pelleted feed used in this study helps produce quality fillets for human consumption.The compositional analysis showed that for the whole-body samples, there were significant differences in protein, ash, and fat percentages (p<0.05). The composition of the whole fish samples showed the fish fed the pelleted diet had higher fat with lower protein and ash percentages than the fish fed the natural components. This indicates that there is more fat being stored and less protein and ash production occurring in fish fed the fishmeal-based pelleted feed. There seems to be a lack of a specific nutrient or imbalance with the pelleted feeds compared to the natural diets, which is important to know because the development of FMRDs needs to have balanced nutritional makeup so aquaculture-raised fish can perform as well, or better than, wild fish. The composition of the muscle samples showed significant differences in protein and fat (p<0.05) but did not show differences in ash (p>0.05). One positive note to report from this analysis is that the muscle samples from the fish fed the pelleted diet did not have lower levels of protein than those fed the natural diets. There were elevated fat levels in the muscle samples, however, indicating that the quality of the meat from the fish fed the pelleted diet were different than those fed the natural diets.The results of the feeding trial and traditional aquaculture metrics showed that researchers should look closely at why the fish-only diet, and other diets containing fish, resulted in the highest levels of production. From there, the causes behind the production gap between this natural component and FMRDs can be better identified. A strategy for supplementation in FMRDs to close the production gap can then be developed.


## 4.2. Results through Time

The design of this feeding trial allowed for the observation of what happened with specific data trends over the course of the feeding trial. Understanding the time at which a fish changes internal health metrics over time can inform growers and researchers of what nutrients to provide at certain life stages. Knowing at what time these differences start to show can also provide insight into what is needed to produce FMRDs that can close the gap in fish performance observed compared to natural diets.The pelleted diet did not have a higher fat content than the natural diet components, however, the PELL group had significantly higher HSI values. The PELL groups were fed more fat over the course of the feeding trial due to there being more dry material fed compared with the natural diets. The type of fat in the pelleted feed, and how it was digested could also explain why the HSI values were higher compared to the natural diet treatment groups. The HSI value did not change for the PELL group from the initial sampling, due to the pelleted feed being similar in composition to the conditioning feed. It is worth noting that the fingerlings were fed crumble followed by 1.5 mm pellets for the conditioning period, then 3 mm pellets for the feeding trial, each having slightly different compositions (Table3). The natural diets were associated with a substantial decrease in HSI values from week 0 to week 12, and only the F group was associated with an increase in HSI until the end of the feeding trial. Having fish as a component mixed with shrimp and squid in the combination diets did not raise the HSI values, however.Different species of fish have different ranges of optimal CF values based on their body shapes and diets. Also, the optimal CF values change over a fish’s life cycle, meaning a CF for a healthy adult will be different than that for a juvenile. The FSQ, SHSQ, SQ, and PELL groups saw a decrease in CF from week 0 to week 3, then an increase in CF over the course of the feeding trial. All diets showed a slight increase in CF from week 0 to week 12. The lower-performing diets according to weight showed lower CF values. There were no clear statistical trends that separated any of the treatment diets, but it does appear the higher-performing diets according to weight (F and PELL) led to healthier fish due to their higher CF values.The differences observed in PV between the natural diets and the pelleted diet were most likely caused by the different compositions of the pelleted feed (Table3). The fish fed the pelleted feed could have incorporated fat into storage, not muscle growth or similar body functions, while the fat from the natural feeds was likely incorporated differently [32]. Further analysis of the actual fatty acid and overall lipid profiles, including the amount of saturated and unsaturated fats in these feeds, may provide clearer insight into this observation. Other feedstuffs in the pelleted diet may have contributed to the higher PV in fish fed the pelleted diet.The results observed in this study showed that the PELL and F groups had the highest PE. The second-highest performing group (FSHSQ, FSH, and FSQ) outperformed the lowest-performing group of diets (SHSQ, SH, and SQ), which did not include the fish component. Erturk and Sevgili [39] reported PE values slightly higher than in this study. Erturk and Sevgili [39] fed rainbow trout (Onchorynchus mykiss) experimental diets with poultry by product meal fed to satiation, which likely explains why their values were higher. This study shows FMRDs can perform well when compared to the natural diets and fishmeal-based pelleted diet. The fish in the fishmeal and natural diet treatments seemed to boost PE. This is further evidence that fish performs better compared to squid and shrimp for juvenile red drum growth and health.Each natural diet was converted to dry mass to compare with the dry pelleted feed. The PELL group was fed significantly more dry feed per fish per day, due to the compositional differences between the natural diet components and the pellets. This shows that juvenile red drum needs to be fed almost twice as much dry feed mass than the natural diet counterparts. If the fish were fed to satiation, they would have eaten more of the natural feeds compared to the pelleted feed. The actual mass of the wet natural diets fed to the fish resulted in a higher feed intake, but the water mass was significant. When comparing dry matter, the pelleted feed is not as calorically dense compared with the natural diet components. Future research into similar pelleted fishmeal-based feeds and FMRDs should focus on formulating a more calorically dense pellet, as filling up on the dry pellets may be causing gaps in growth performance.The differences in DMR observed between the diets in this study showed further evidence that diet performance was better with the fish component. DMR shows the relationship between dry food fed and dry biomass gained. The fish component seemed more efficient, as evidenced by groups with more growth with less intake. Interestingly, the PELL group, which showed the second-highest performance with regards to growth, statistically grouped with the lower performing natural groups (SHSQ, SH, and SQ) with regards to DMR. This is most likely due to the less dense caloric composition of the pellets.Due to the very different compositions of the pelleted feed used in PELL diet and the natural components of the natural diets, it was observed that the PELL group performed well in growth but performed worse in metrics such as FCR and DMR. FCR and DMR show differences in the production of the fish fed the pelleted feed compared to the natural feed. The protein from the pelleted feed was efficiently used by the fish. The overall conversion of the pelleted feed was not as good, perhaps due to the higher density of the pellets than the natural components. Improving FCR values in fish farming can lead to lower costs for feed and faster turnover for new batches of fish. The FCR values reported in this study were similar to those reported by Tucker et al. [34] for red drum.As global need for protein continues to increase with a static global fishery yield, aquafeed producers are challenged to create high-quality FMRDs [40]. The results of this study provide useful information to show where current commercial fishmeal-based pelleted diets are compared to natural diets for fish when fed isocalorically and can lead to future research to improve FMRDs for juvenile red drum production.


## 5. Conclusions

The results of this study determined there is an optimal diet combination among fish, shrimp, and squid components when caloric intake is restricted and equivalent. The current study found that red drum fed fish only had the highest production metrics, and diets containing fish in combination with shrimp and or squid (FSHSQ, FSH, and FSQ) outperformed those without fish in their diet (SHSQ, SH, and SQ). From this,D. punctatus showed that it was the most valuable component of the natural diet. The composition (Table 3) does not indicate an obvious reason for this to have happened, as there are not extreme differences in proximate composition of the natural diet components. The performance differences could be due to amino acid or fatty acid profile differences, which were not addressed in this study. Further study into the metabolomics, combined with the amino and fatty acid profiles of fish and the diet components, can reveal more information to explain why the fish seems to be a better diet component for juvenile red drum than shrimp and squid [18].Ultimately, this research will lead to knowledge that will help feed producers develop higher-performing pelleted diets for juvenile red drum and other aquaculture species. Further research into the metabolome of the fish in this study may identify potential supplements for FMRDs as replacement protein sources continue to be used more in the aquaculture industry.



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