Journal of the World Aquaculture Society
Demonstr ation That Feeds Containing <1% Fish Meal Can Support Growout Of Large Juvenile Red Drum, Sciaenops ocellatus, And Reduce Nutrient Waste
For Peer Review Journal: Journal of the World Aquaculture Society
Manuscript ID JWAS-16-114.R2
Manuscript Type: Applied Studies
Date Submitted by the Author: n/a
Complete List of Authors: Denson, Michael; Marine Resources Research Institute, Department of Natural Resources Sandifer, Paul; College of Charleston, School of Sciences and Mathematics; Hollings Marine Laboratory, College of Charleston Leffler, John; South Carolina Department of Natural Resources, Marine Resources Research Institute Yost, Justin; Marine Resources Research Institute, Department of Natural Resources Bearden, Daniel; National Institute of Standards and Technology Zeigler, Tom ; Zeigler Bros. Inc.
fish meal replacement, alternative protein sources, reduced pollution, Keywords: enzyme additives, red drum
Journal of the World Aquaculture Society Page 1 of 38 Journal of the World Aquaculture Society
1 1 2 3 1 Demonstration That Feeds Containing <1% Fish Meal Can Support Growout Of Large Juvenile 4 5 6 2 Red Drum, Sciaenops ocellatus, And Reduce Nutrient Waste 7 8 9 3 10 11 12 4 Michael R. Denson, Marine Resources Research Institute, South Carolina Department of Natural 13 14 15 5 Resources, 217 Fort Johnson Road, Charleston, SC 29412 USA 16 17 18 6 Paul A. Sandifer, CollegeFor of Charleston, Peer Hollings Review Marine Laboratory, 331 Fort Johnson Road, 19 20 7 Charleston, SC 29412 USA 21 22 23 8 John W. Leffler, Marine Resources Research Institute, South Carolina Department of Natural 24 25 26 9 Resources, 217 Fort Johnson Road, Charleston, SC 29412 USA 27 28 29 10 Justin Yost, Marine Resources Research Institute, South Carolina Department of Natural 30 31 11 Resources, 217 Fort Johnson Road, Charleston, SC 29412 USA 32 33 34 35 12 Daniel W. Bearden, National Institute of Standards and Technology, Hollings Marine 36 37 13 Laboratory, 331 Fort Johnson Road, Charleston, SC 29412 USA 38 39 40 14 Thomas R. Zeigler, Zeigler Bros., Inc., 400 Gardners Station Road, Gardners, PA 17324. 41 42 43 15 Corresponding Author: Paul A. Sandifer, College of Charleston, Hollings Marine Laboratory, 44 45 46 16 331 Fort Johnson Road, Charleston, SC 29412 USA. [email protected] 47 48 17 49 50 51 18 52 53 54 55 56 57 58 59 60 Journal of the World Aquaculture Society Journal of the World Aquaculture Society Page 2 of 38
2 1 2 3 19 Abstract 4 5 6 7 20 We conducted a 16-week feeding trial with large juvenile red drum, Sciaenops 8 9 21 ocellatus. Four diets were randomly assigned to six replicate tanks per treatment. Three 10 11 22 isonitrogenous (~44.5% protein) and isolipidic (~14.1% lipid) extruded diets were 12 13 14 23 formulated to compare a fish meal-based diet with diets using alternative protein sources. 15 16 24 Diet 1 contained 19.60% fish meal and 21.42% poultry meals as primary protein sources. 17 18 Two alternative dietsFor were formulated Peer reducing Review the fish meal to 0.61% by substituting 19 25 20 21 26 poultry meals (33.85%) and soybean protein concentrate (11.55% in Diet 2 and 11.70% in 22 23 27 Diet 3. Diet 3 also included Allzyme Vegpro ® and Allzyme ® SSF at 0.04%. Diet 4, a 24 25 26 28 “natural” diet consisting of chopped cigar minnows, squid, and shrimp, was used as a 27 28 29 positive control to compare growth rates of formulated feeds to near maximum growth 29 30 30 under these culture conditions. We found that reducing the amount of fish meal to <1% by 31 32 33 31 using alternative protein sources did not affect the growth rate, survival, or health of red 34 35 32 drum but improved assimilation of Phosphorus, reduced potential release of P to the 36 37 33 environment, and significantly lowered the amount of feeder fish needed in feed. The 38 39 40 34 control diet identified performance benchmarks for future feeds development work . 41 42 43 35 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Journal of the World Aquaculture Society Page 3 of 38 Journal of the World Aquaculture Society
3 1 2 3 36 4 5 37 The necessity for aquaculture to expand to provide animal protein and other products for 6 7 38 humans and to help spare fishery stocks from overexploitation has been emphasized many times 8 9 39 (e.g., Bardach et al. 1974; Pillay 1978; Sandifer 1979). Aquaculture has been growing at 10 11 12 40 sustained rapid rates for many years and now surpasses capture fisheries in the amount of 13 14 41 seafood produced for human consumption (FAO 2016). Concomitant with rapid growth has 15 16 42 come recognition of the need to eliminate or at least substantially reduce aquaculture’s reliance 17 18 For Peer Review 19 43 on fish meal and oil as feed ingredients and to decrease aquaculture’s contribution to nutrient 20 21 44 pollution of receiving waters (Naylor et al. 2009; Tacon and Metian 2008; Browdy and 22 23 24 45 Hargreaves 2009; Price and Morris 2013) 25 26 27 46 Red drum, Sciaenops ocellatus, is an important marine aquaculture species because of its 28 29 47 fast growth rate, wide environmental tolerances and market appeal (Sandifer et al. 1993; Faulk 30 31 48 2005). The cost of feed remains the single greatest cost for production of finfish, and fish meal is 32 33 34 49 often the most expensive component of fish feeds (Rana et al. 2009). Over the last decade, 35 36 50 numerous studies have been conducted to identify and evaluate alternative protein sources for 37 38 39 51 aquafeeds for a variety of freshwater and marine fish and crustaceans, including soybean and 40 41 52 other plant meals, blood meal, poultry meal and other animal processing by products (Hardy 42 43 53 2000; Rawles and Gatlin 2000; Emre et al. 2003; Erturk and Sevgili 2003; Hernandez et al. 2004; 44 45 46 54 Davis et al. 2005; Gaylord and Rawles 2005; Brinker and Reiter 2011). While a number of 47 48 55 studies have examined the potential to utilize soybean meal and oil and other feed ingredients to 49 50 56 partially replace fish meal in practical feeds for red drum (e.g., Reigh and Ellis 1992, Davis et al. 51 52 53 57 1995; Gaylord and Gatlin 1996; Meilahn et al. 1996; McGoogan and Gatlin 1997, Kureshy et al. 54 55 58 2000, Rossi et al, 2013, 2015, Minjarez Osorio et al. 2016), complete elimination of fish meal in 56 57 58 59 commercial diets has not yet been achieved. 59 60 Journal of the World Aquaculture Society Journal of the World Aquaculture Society Page 4 of 38
4 1 2 3 60 Two main end products of intensive modern finfish aquaculture are nitrogen (N) wastes 4 5 6 61 from deamination of proteins and indigestible phytate bound phosphorus (P) (USEPA 2005; 7 8 62 Debnath et al. 2008). These by products can have negative effects on fish health and growth 9 10 11 63 within production facilities as well as ecosystem impacts such as eutrophication (Goldburg and 12 13 64 Naylor 2005; Hall et al. 2011) that may lead to hypoxia and/or harmful algal blooms (Anderson 14 15 65 et al. 2008; Heisler et al. 2008). Improvements in prepared diets to mitigate these negative 16 17 18 66 impacts will be a leadingFor factor in Peer the long term sReviewustainability of aquaculture (USCOP 2004; 19 20 67 Rust et al. 2011). 21 22 23 68 Numerous products have been tested to improve P utilization, feed conversion ratios 24 25 26 69 (FCR), and protein uptake and retention efficiency (Jackson et al. 1996; Li and Robinson 1997; 27 28 70 Van Weerd et al. 1999; Hardy 2000; Cao et al. 2007; Buentello et al. 2010, Refstie et al. 2010). 29 30 71 Compounds that may reduce the pollution stream include new forms of phytase (i.e. enzymes 31 32 33 72 that release P from phytates in plants and make it available to consuming animals), acidifiers, 34 35 73 pre and probiotics, and essential oils (Debnath et al. 2005; Fox et al. 2006). While some of these 36 37 74 components have been adopted by the aquaculture feed industry, additional research is needed, 38 39 40 75 especially for carnivorous marine species (Buentello et al. 2010). 41 42 43 76 The present study was undertaken to: (1) determine the efficacy of using soybean and 44 45 77 poultry by product meals to replace nearly all of the fish meal in extruded prepared diets for red 46 47 48 78 drum under grow out conditions (~250 g to ~500 g); (2) evaluate the potential for reducing N 49 50 79 and P waste loading from red drum aquaculture via such substitutions, with and without enzyme 51 52 80 additives; and (3) to use Nuclear Magnetic Resonance (NMR) based metabolomics to measure 53 54 55 81 metabolic changes in various fish tissues in response to the diets presented. This paper focuses 56 57 82 on results from objectives 1 and 2; results from the NMR studies will be the subject of a separate 58 59 60 Journal of the World Aquaculture Society Page 5 of 38 Journal of the World Aquaculture Society
5 1 2 3 83 manuscript. Although most nutritional studies of this type use very small fish (2 10 g in weight) 4 5 6 84 and trials of short duration (30 60 days), we chose to use larger red drum that more accurately 7 8 85 reflect dietary needs and fish metabolism during grow out to harvestable size. 9 10 11 86 12 13 87 Materials and Methods 14 15 88 Experimental Design 16 17 18 89 A 16 week feedingFor trial was Peer conducted using Review large (~260 g) juvenile red drum held in a 19 20 21 90 recirculating aquaculture system (RAS) housed in the National Oceanic and Atmospheric 22 23 91 Administration’s Hollings Marine Laboratory (Charleston, SC, USA). The RAS was originally 24 25 92 designed as three independent systems, but for this study they were integrated into a single 26 27 28 93 system of 24 (1400 L) tanks connected to three sand filters, three fluidized beds, three protein 29 30 94 fractionators, and three UV sterilizers, that shared water equally among all tanks. Integrating into 31 32 95 one system permitted uniformity of water conditions in all tanks and allowed us to remove 33 34 35 96 blocking effects and execute a completely random experimental design. 36 37 38 97 Juvenile red drum were provided by the South Carolina Department of Natural 39 40 98 Resources’ aquaculture program. Prior to the study, the fish were maintained in outdoor flow 41 42 43 99 through tanks using ambient Charleston Harbor, SC, USA seawater and were transported in 44 45 100 oxygenated hauling tanks to the laboratory where they were acclimated to laboratory temperature 46 47 101 and salinity conditions over a one week period. Fifteen fish were hand graded into each tank in 48 49 50 102 an attempt to achieve equal size distributions of fish in all tanks. All fish were hand fed to 51 52 103 satiation once daily using a standard commercial diet (Zeigler Bros. Inc., Gardners, PA, USA; 53 54 Silver ® diet; 40% protein, 10% fat) until they were placed on the test diets on Day 1 of the 55 104 56 57 105 experiment. On Day 0 three fish were removed from each tank for metabolomics analysis. The 58 59 60 Journal of the World Aquaculture Society Journal of the World Aquaculture Society Page 6 of 38
6 1 2 3 106 remaining 12 fish in each tank were weighed individually. Temperature, dissolved oxygen (DO), 4 5 6 107 salinity, and pH were monitored daily using a YSI Pro Plus Multi Parameter Water Quality 7 8 108 Meter (YSI Inc., Yellow Springs, OH, USA), and ammonia, nitrite, and nitrates were monitored 9 10 11 109 weekly using a DR3900 benchtop spectrophotometer (Hach, Loveland, CO, USA) (Table 1). 12 13 110 Water quality measurements in all tanks were within ~ + 5% of each other throughout the study. 14 15 16 111 Diet Formulation and Analysis 17 18 For Peer Review 19 112 Four diets were randomly assigned to six replicate tanks per treatment. Three 20 21 22 113 isonitrogenous (~44.5% protein) and isolipidic (~14.1% lipid) pelleted diets were formulated to 23 24 114 compare a fish meal based diet with diets using alternative protein sources (Table 2). A Basal 25 26 115 Diet (Diet 1, BD) for red drum, which was typical of commercial feeds sold to red drum growers, 27 28 29 116 contained 44% crude protein and 14.5% crude lipids (T. Zeigler, personal communication). The 30 31 117 diet contained 19.60% fish meal and 21.42% poultry meals as the primary protein sources. Two 32 33 34 118 alternative diets were formulated reducing the fish meal to 0.61% by increasing poultry meals 35 36 119 (33.85%) and adding soybean protein concentrate (11.55% in Diet 2 [Low Fish meal, LFM] and 37 38 120 11.70% in Diet 3 [Low Fish meal + Additives, LFM+A]). Additives in Diet 3 were Allzyme 39 40 ® ® 41 121 Vegpro , an enzyme complex reported to improve digestion of vegetable proteins, and Allzyme 42 43 122 SSF, a refined fungal product reported by the manufacturer to make amino acids, energy, Ca, and 44 45 123 P more digestible (Alltech, Inc., Nicholasville, KY, USA). The Alltech Enzymes were chosen by 46 47 48 124 the commercial feed manufacturer, Zeigler Bros. Inc., based on recent and effective use with 49 50 125 chickens and fish (Hooge et al. 2010, El Sayed et al, 2014, Zhao et al. 2015). The inclusion rate 51 52 126 (0.04%) was based on the supplier’s recommendation. The extruded diets were formulated and 53 54 55 127 manufactured by Zeigler Bros. Inc. using standard industry practices and ingredients in order to 56 57 128 simulate commercial feeds as closely as practical. Diet 4, a “natural” diet (ND) consisting of 58 59 60 Journal of the World Aquaculture Society Page 7 of 38 Journal of the World Aquaculture Society
7 1 2 3 129 equal portions by wet weight of chopped cigar minnows, Decapterus punctatus (purchased 4 5 6 130 locally), squid, Loligo sp., (purchased locally), and Pacific white shrimp, Litopenaeus 7 8 131 vannamei,(produced by the SCDNR’s Waddell Mariculture Center), was used as an internal 9 10 11 132 positive control, i.e., a benchmark of the red drum growth potential under the specific conditions 12 13 133 of this RAS. The Pacific white shrimp were used as a substitute for similar, local penaeid shrimp 14 15 134 because of their consistent availability during the study. The Natural Diet was also important in 16 17 18 135 the NMR studies to helpFor establish Peera baseline metabo Reviewlomics profile. Fish were fed by hand twice 19 20 136 daily to satiation and excess feed was siphoned from the tanks after 20 minutes and cessation of 21 22 137 feeding. Composite samples of each diet were taken periodically throughout the experiment and 23 24 25 138 sent to the Clemson University Agricultural Service Laboratory (Clemson, SC, USA) and the 26 27 139 New Jersey Feed Laboratory (Trenton, NJ, USA) for proximate, mineral, and amino acid 28 29 140 analyses by standard methods (http://www.clemson.edu/ public/regulatory/ ag_svc_lab/ 30 31 32 141 index.html; http://www.njfl.com/index.html) (Table 3). 33 34 142 35 36 37 143 Sample Collection 38 39 40 144 The diet performance study was conducted for 114 days (April 9 – August 1, 2013). All 41 42 145 fish from the 24 tanks were individually weighed and measured on Day 0, Day 51 and Day 114. 43 44 On Day 114 the complete liver and body cavity fat of three fish from each tank were also 45 146 46 47 147 collected to calculate the hepatosomatic index (HSI) and Intraperitoneal Fat Index (IPF). At each 48 49 148 sampling period, three fish were randomly removed from each tank, anesthetized with tricaine 50 51 1 52 149 methanesulfonate (MS 222) (100 mg L ) and 2 mL of blood were drawn from the caudal vein in 53 1 54 150 a heparinized tube. These fish were then exposed to a lethal dose of MS 222 (500 mg L ). 55 56 57 58 59 60 Journal of the World Aquaculture Society Journal of the World Aquaculture Society Page 8 of 38
8 1 2 3 151 Tissue samples of the right lobe of the liver, and muscle from the right dorsal musculature were 4 5 6 152 extracted immediately and frozen in liquid nitrogen for later metabolomic analyses. 7 8 9 153 Seventeen red drum that were not part of the experiment (wet weight 24 g to 1661 g), 10 11 154 were used to establish a regression from which the N and P content of the experimental fish prior 12 13 14 155 to final harvest could be estimated. These fish had been raised in the same facility and fed the 15 16 156 standard 40% protein commercial diet that was used for the experimental fish prior to study 17 18 o 157 commencement. They Forwere dried Peerto constant weight Review at 100 C and homogenized. To ensure that 19 20 21 158 all parts of the fish were well sampled, between three and ten replicates of each homogenized 22 23 159 fish, depending on size of fish, were submitted to the Clemson University Agricultural Service 24 25 26 160 Laboratory for analysis of total N and total P on a dry weight basis. There were no significant 27 2 2 28 161 relationships between wet weight of fish and N P content (adj r = 0.099, P = 0.118; adj. r = 29 30 162 0.000, P = 0.993, respectively), so based on beginning wet weight of fish at Day 1 the mean N 31 32 33 163 content of samples analyzed was 10.13% of total weight and P content was 2.37% of total 34 35 164 weight. 36 37 38 165 Because the experimental fish had been fed different diets, the N P content of fish at the 39 40 41 166 end of the study was measured directly. One fish was taken randomly from each of the 24 tanks 42 43 167 resulting in six replicate fish for each of the four diets. Fish were prepared as above and ten 44 45 168 replicate samples from each fish were sent to the Clemson Agricultural Service Laboratory for 46 47 48 169 analysis. The actual mean N P content values measured for each of the four treatments were used 49 50 170 in all calculations. 51 52 53 171 Samples of each experimental feed were collected every two weeks and frozen. These 54 55 56 172 samples were then combined and three to seven replicates were sent to the Clemson Agricultural 57 58 59 60 Journal of the World Aquaculture Society Page 9 of 38 Journal of the World Aquaculture Society
9 1 2 3 173 Services Laboratory or the New Jersey Feed Laboratory for proximate and element analyses 4 5 6 174 (Table 3). N and P values were used to calculate the loadings to each tank in the experiment. 7 8 9 175 Calculations 10 11 12 176 Fish production performance was characterized by calculating several parameters as 13 14 15 177 follows: 16 17 18 178 Weight gain (%) = 100For x ((mean weightPeer @ Day 51Review – mean initial weight) + (mean final 19 20 179 weight – mean weight @ Day 52)) / mean initial weight 21 22 23 180 Weight gain per day (g/d) = ((mean weight @ Day 51 – mean initial weight) + (mean 24 25 26 181 final weight – mean weight @ Day 52)) / 114 days 27 28 29 182 Specific Growth Rate (SGR) (% body weight/day) = 100 x (natural log mean final weight – 30 31 183 natural log mean initial weight) / 114 days (Trushenski et al. 2011) 32 33 34 184 Hepatosomatic Index (HSI) = 100 x (total liver weight / total body weight) 35 36 37 185 Intraperitoneal Fat Index (IPF) = 100 x (body cavity fat weight / total body weight) 38 39 40 186 Feed Intake (% body weight/day) = 100 x mean individual dry matter feed intake / 41 42 43 187 ((initial individual weight x final individual weight) ^ 0.5) / 114 days) (Trushenski et al. 44 45 188 2011) 46 47 48 189 FCR (feed conversion ratio) = weight of dry or wet feed supplied / wet weight of biomass 49 50 51 190 gained. 52 53 54 191 DMR (dry matter ratio) = FCR x % dry matter weight of feed supplied / % dry weight of 55 56 192 biomass gained; i.e. dry weight of feed / dry weight of biomass gained (Boyd et al. 2007). 57 58 59 60 Journal of the World Aquaculture Society Journal of the World Aquaculture Society Page 10 of 38
10 1 2 3 193 FMR (fish meal ratio) = FCR x % fish meal in feed supplied /100 (Boyd et al. 2007) 4 5 6 7 194 FFE (feed fish equivalence) = FMR x 4.5 (approximately 4.5 kg of live feeder fish are 8 9 195 required to produce 1 kg of fish meal; Boyd et al. 2007) 10 11 12 196 The efficiencies of N and P assimilation were calculated by the following equations: 13 14 15 197 NitCR (nitrogen conversion ratio) = DMR x % N in dry weight feed; i.e. dry 16 17 18 198 weight of N suppliedFor in feed Peer / dry weight Reviewof biomass gained 19 20 21 199 PhosCR (phosphorus conversion ratio) = DMR x % P in dry weight feed; i.e. 22 23 200 dry weight of P supplied in feed / dry weight of biomass gained 24 25 26 27 201 The amount of pollution resulting from each diet was described by the waste production 28 29 202 ratio (WPR), which indicates the weight of waste produced kg 1 wet weight gain of the culture 30 31 203 species (Boyd, et al 2007). The total P and total N in feeds not utilized by fish and released into 32 33 34 204 the system as waste were calculated by mass balance. 35 36 37 205 (Total Nutrient Lost from System as Pollution) = (Total Nutrient in Fish at Start) + (Total 38 39 206 Nutrient added through Feed) – (Total Nutrient removed in Fish on Day 51) – (Total 40 41 42 207 Nutrient in Fish at End) 43 44 45 208 Waste Production Ratio (WPR) = (DMR – 1) x % dry matter in cultured species /100 (Boyd et 46 47 209 al. 2007) 48 49 50 Nitrogen Loss Ratio (g/kg) = grams of N lost as waste / kilogram of wet weight 51 210 52 53 211 biomass of the cultured species gained 54 55 56 212 Phosphorus Loss Ratio (g/kg) = grams of P lost as waste / kilogram of wet 57 58 59 60 Journal of the World Aquaculture Society Page 11 of 38 Journal of the World Aquaculture Society
11 1 2 3 213 weight biomass of the culture species gained 4 5 6 7 214 Statistical Analyses 8 9 10 215 All statistical tests were run using SigmaPlot 12.5 (Systat Software Inc., San Jose, CA, 11 12 216 USA). Data were analyzed by One Way ANOVA. All data were subjected to Shapiro Wilk and 13 14 15 217 the Equal Variance tests provided in SigmaPlot to determine that the data passed normality and 16 17 218 homoscedasticity assumptions, respectively. Data that failed either assumption were analyzed by 18 For Peer Review 19 219 a Kruskal Wallis ANOVA test on ranks, a non parametric equivalent. The Holm Sidak multi 20 21 22 220 comparison method was applied to data analyzed by parametric one way ANOVAs, while either 23 24 221 the Tukey or Student Neuman Keuls multi comparison method was used with Kruskal Wallis 25 26 222 nonparametric ANOVAs. The multi comparison tests used P = 0.05 as the criterion for defining 27 28 29 223 significant differences among treatments. 30 31 32 224 Results 33 34 35 225 Fish Production and Health 36 37 38 226 Fish survival was 100% throughout the experiment for all treatments; the only reduction 39 40 41 227 in fish per treatment were animals randomly selected for sacrifice at Day 51. Final mean 42 43 228 individual weight, mean individual weight gain as a percent of initial mean weight, mean weight 44 45 46 229 gain/day, and SGR (Table 4) were statistically higher for the natural, positive control diet (Diet 47 48 230 4), indicating that fish on this diet grew significantly faster and larger than those on the three 49 50 231 extruded diets, among which multi comparison tests could detect no differences. Similarly, there 51 52 53 232 were no differences in HSI and IPF of fish raised on the three extruded diets, but those fed Diet 4 54 55 233 had significantly lower values. A slight increase in growth rates was noted among fish fed all 56 57 234 three test diets following the removal of three fish from each tank on day 51 (Fig. 1). A similar 58 59 60 Journal of the World Aquaculture Society Journal of the World Aquaculture Society Page 12 of 38
12 1 2 3 235 and more pronounced growth rate increase was observed for fish fed the natural diet (Diet 4). It 4 5 6 236 is possible that the decrease in fish density per tank after day 51 contributed to the increased 7 8 237 growth rates recorded. However, fish densities were relatively low in all tanks throughout the 9 10 11 238 study, particularly in terms of biomass of fish to water volume. This was higher in the Diet 4 12 13 239 tanks, and these fish grew the most rapidly. 14 15 16 240 Nutritional Performance 17 18 For Peer Review 19 241 Fish in all four diet treatments consumed approximately the same dry weight of feed/day 20 21 22 242 as a percentage of their body weight. However, fish fed Diet 4 grew approximately twice as 23 24 243 rapidly as those on the extruded diets and consumed considerably more feed. The FCR for Diet 25 26 244 4, calculated on a wet weight basis, was twice that of the prepared diets. This diet consisted of 27 28 29 245 fresh cut shrimp, squid, and fish with very high water content compared to the dry extruded 30 31 246 feeds. The DMR provides a more informative comparison, and that of Diet 4 was significantly 32 33 34 247 less than those of the prepared diets. 35 36 37 248 Calculation of FMR for each extruded diet showed that Diet 1 incorporated 38 39 249 approximately 32 times the amount of fish meal as the two diets formulated to reduce fish meal, 40 41 250 although all three prepared diets supported comparable growth characteristics (Fig. 1). Similarly, 42 43 44 251 FFE was significantly higher for the fish meal based diet. Approximately 1.7 kg of feeder fish 45 46 252 were required for Diet 1 to produce 1 kg of cultured fish, while the two LFM diets (Diets 2 and 47 48 1 49 253 3) required only about 0.05 kg kg of cultured fish produced. Diet 4, which consisted entirely of 50 51 254 unprocessed marine protein, required significantly more, 3.6 kg of FFE, to produce a kg of 52 53 255 cultured fish biomass. 54 55 56 57 58 59 60 Journal of the World Aquaculture Society Page 13 of 38 Journal of the World Aquaculture Society
13 1 2 3 256 Fish fed Diets 1 and 3 required statistically more N/kg of fish biomass produced than did 4 5 6 257 fish fed Diets 2 and 4. Diet 4 had the lowest ratio of feed supplied N to biomass produced, 7 8 258 although based on the Holm Sidak multiple comparison test (P = 0.05) it was not statistically 9 10 11 259 different from Diet 2. 12 13 14 260 One study hypothesis was that by reducing fish meal in Diet 2 (LFM), and by including 15 16 261 enzyme additives in Diet 3 (LFM+A), P uptake efficiency might increase. However, we found no 17 18 262 significant difference betweenFor these Peer diets, both suReviewpplying approximately 0.055 kg of P in the 19 20 21 263 feed to produce a kg of fish biomass (Table 4). Fish fed Diet 1 required significantly more P to 22 23 264 produce a kg of fish biomass, while Diet 4 produced a kg of fish biomass with significantly less 24 25 26 265 P supplied by the feed. 27 28 29 266 Pollution Potential 30 31 32 267 N, P, and solids are the most important water pollutants resulting from intensive 33 34 268 aquaculture (USEPA 2005). Calculation of the WPR demonstrated that the three extruded diets 35 36 37 269 were not significantly different from each other and produced between 1.34 kg – 1.51 kg of 38 39 270 waste (dry matter basis) kg 1 of live fish product. The ND diet produced significantly less waste 40 41 271 than did the three pelleted diets, approximately 0.57 kg kg 1 of live fish product (Table 4). 42 43 44 272 Similarly, the N loss ratio indicated that there were no significant differences among the three 45 46 273 extruded diets in amounts of N waste produced. The extruded diets ranged between 90.7 g – 99.7 47 48 1 49 274 g of N waste produced kg of live fish product. By contrast, Diet 4 resulted in only 70.2 g of N 50 1 51 275 waste kg of live fish biomass produced. Note that these numbers represent only the weight of N 52 53 276 itself, not the mass of N compounds in which it is contained, such as ammonia, nitrate, or 54 55 1 56 277 organic N. Diets 2 and 3 created significantly less P waste (8.39– 9.82 g kg of wet weight 57 58 59 60 Journal of the World Aquaculture Society Journal of the World Aquaculture Society Page 14 of 38
14 1 2 3 278 biomass produced) than did Diet 1 (13.50 g kg 1 of wet weight biomass produced), while Diet 4 4 5 1 6 279 fish produced significantly less ((5.11 g of P kg of wet weight biomass) than any of the 7 8 280 extruded diets (Table 4). Again, these values represent only the weight of P itself, not the mass of 9 10 11 281 those compounds containing the P. 12 13 14 282 Discussion 15 16 17 283 Fish Production 18 For Peer Review 19 20 284 No differences in growth or survival were observed between Diet 1 and the two reduced 21 22 23 285 fish meal diets (Diets 2 and 3), demonstrating that virtually all of the fish meal in a commercially 24 25 286 prepared feed for red drum can be replaced by a combination of soybean meal and other 26 27 287 alternative protein sources without impacting the production (growth, survival) and health (as 28 29 30 288 indicated by HSI and IPF) of red drum. Previous studies with red drum had shown that some fish 31 32 289 meal could be replaced by soybean meal but not to the near elimination level (0.6%) 33 34 290 demonstrated here (e.g., reduction to 15% fish meal, Davis et al., 1995; reduction to 20% fish 35 36 37 291 meal, Meilahn et al. 1996; some reduction in replacement of fish oil but not fish meal by soybean 38 39 292 oil, Tucker et al. 1997; reduction of fish meal to 10% of diet by McGoogan and Gatlin 1997; 40 41 293 reduction of fish meal from 30% of diet to 10%, Kureshy et al. 2000). More recently, Rossi et al. 42 43 44 294 (2013) successfully replaced 50% of fish meal with soy and barley protein concentrate in red 45 46 295 drum feeds but did not replicate the 90% replacement reported by McGoogan and Gatlin (1997). 47 48 49 296 However, Rossi et al. (2015) were able to replace ~86% of fish meal with soy protein, and 50 51 297 Minjarez Osorio et al. (2016) substituted amino acid supplemented soy protein and corn protein 52 53 298 concentrates for 75% and 50% of fish meal protein, respectively, in red drum diets. Also, Rossi 54 55 56 299 and Davis (2014) were able to remove all fish meal from soybean meal based diets for Florida 57 58 59 60 Journal of the World Aquaculture Society Page 15 of 38 Journal of the World Aquaculture Society
15 1 2 3 300 pompano through substitution with meat and bone meal, but only when the feed also included 4 5 6 301 supplemental taurine. Trushenski et al. (2011) observed no effect on growth by reducing the 7 8 302 amount of fish meal in diets for juvenile cobia by 50%, but fish final mean weight and percent 9 10 11 303 weight gain were significantly lower when fish meal was completely eliminated. Here we 12 13 304 achieved almost an order of magnitude reduction in actual dietary content of fish meal below the 14 15 305 lowest previously reported levels that did not also include supplemental amino acid (0.6% of diet 16 17 18 306 as fish meal here comparedFor to ~5% Peer reported by McGoogReviewan and Gatlin 1997 and Davis and 19 20 307 Arnold 2004, and 6% by Rossi et al. 2013). Davis and Arnold (2004) and Rossi et al. (2015) also 21 22 308 used relatively large fish which, while still smaller (179 and 68 g, respectively), were more 23 24 25 309 comparable to those used in the present study than the very small fish employed in most other 26 27 310 studies. Rossi et al. (2015) noted that “advanced juvenile red drum appear to exhibit a higher 28 29 311 tolerance to soybean based diets than younger ones.” The present study supports this idea and 30 31 32 312 their observation regarding the importance of conducting feed trials at different life stages and to 33 34 313 evaluate performance under simulated grow out conditions. 35 36 37 314 The additives in Diet 3 were intended to improve the ability of fish to extract nutrients, 38 39 315 particularly P, from the plant based ingredients in the diet and improve nutritional efficiency 40 41 316 over Diet 2 (Vielma et al. 1998; Storebakken et al. 1998). Previous work with juvenile red drum 42 43 44 317 has shown that incorporation of dietary prebiotics is often beneficial, but some may have neutral 45 46 318 or even minor negative effects (Buentello et al. 2010, Zhou et al. 2010, Rossi et al. 2015). The 47 48 319 compound chosen for inclusion in the present study, Allzyme ® SSF, has been reported to 49 50 51 320 improve production of broiler chickens (Hooge et al. 2010) and various species of fish and 52 53 321 crustaceans (Debnath et al. 2005; Fox et al. 2006; Moura et al. 2012) and to positively affect fish 54 55 322 immune status and resistance to bacterial disease (El Sayed et al. 2014; Zhao et al. 2015). 56 57 58 59 60 Journal of the World Aquaculture Society Journal of the World Aquaculture Society Page 16 of 38
16 1 2 3 323 However, in this experiment, no differences in fish production performance were observed 4 5 6 324 between the diets with and without additives, suggesting that the enzyme additives used were not 7 8 325 effective, at least at the very low level of inclusion and size of fish tested. Similarly, Rossi et al. 9 10 ®, 11 326 (2015) found no effect of GroBiotic a compound previously observed by Buentello et al. 12 13 327 (2010) to be beneficial for red drum. Rossi et al. (2015) pointed out that prebiotics often were 14 15 328 most effective when used in situations where rearing conditions were suboptimal or fish were 16 17 18 329 stressed by disease, situationsFor that Peer did not occur here.Review In the present case, both diets 2 and 3 were 19 20 330 supplemented with poultry by product meal as a substitute for fish meal protein as well as having 21 22 331 soybean protein concentrate. It is possible that the animal based meals were adequate 23 24 25 332 replacements for fish meal and contained a complement of amino acids appropriate for the level 26 27 333 of growth observed in this study. It is likely that if a higher proportion of replacement proteins 28 29 334 came from plant sources that we would have seen an effect from the added enzyme. It may also 30 31 32 335 be that the animal based proteins masked the negative effect of the plant based protein sources. 33 34 336 In the wild, red drum feed on a variety of fish, crustaceans, polychaetes, molluscs, and 35 36 37 337 echinoderms (Overstreet and Heard 1978; Robinson 1988). Fish fed a diet prepared to simulate 38 39 338 their natural foods (Diet 4) grew twice as fast and large as those on the three extruded diets. It is 40 41 339 likely that the natural foods were more digestible and contained important micronutrients and/or 42 43 44 340 amino acid and fatty acid profiles that provided better nutrition at the right time than the prepared 45 46 341 diets . The much higher protein level of the natural diet (~73% compared to ~45% in the 47 48 342 extruded diets) may also have played a significant role in its better performance (Webb and 49 50 51 343 Gatlin 2003). The resulting growth curve (Fig. 1) demonstrates the potential biological 52 53 344 possibilities for growing red drum, even without genetic selection applied to the population, and 54 55 56 345 the substantial potential for improvement in performance of formulated rations. 57 58 59 60 Journal of the World Aquaculture Society Page 17 of 38 Journal of the World Aquaculture Society
17 1 2 3 346 Fish Health 4 5 6 7 347 Fish health was good throughout the study, as indicated by feeding behavior, 100% 8 9 348 survival, and HSI ratios within ranges seen in other red drum feeding studies (e.g., see 10 11 349 McGoogan and Gatlin 1997; Rossi et al. 2013, 2015; Minjarez Osorio et al. 2016). IPF values in 12 13 14 350 all treatments were higher than those seen in most other feeding studies, reflecting the much 15 16 351 larger size of fish used in the present study. 17 18 For Peer Review 19 352 Nutritional Performance 20 21 22 23 353 Feed consumption was equivalent for all four feeds, indicating that feed acceptance was 24 25 354 equal across treatments. However, since the Diet 4 fed fish grew twice as fast as those on other 26 27 355 diets, they consumed considerably more total feed, although the amount was equivalent as a 28 29 30 356 percent of body weight. Also, there were no indications of acceptability or palatability issues 31 32 357 with any of the test rations. Thus, the observed poorer growth rates of fish fed the prepared feeds 33 34 358 did not appear to be due to reluctance of the fish to consume them. Similarly, there were no 35 36 37 359 significant differences in FCR among the three extruded diets, and the ratios were generally 38 39 360 similar to those reported previously (Sandifer et al. 1993; numerous references cited in Tucker et 40 41 361 al. 1997). In contrast, the FCR for Diet 4 was considerably higher, but this diet consisted entirely 42 43 44 362 of fresh cut fish, shrimp and squid with very high water content (~67%). After conversion of all 45 46 363 the feeds and the cultured fish to a common standard dry weight basis, the DMR indicates that 47 48 49 364 Diet 4 was approximately twice as efficient as the three extruded diets in conversion of feed to 50 51 365 fish biomass. The much higher protein level, the fact that all of the protein was derived from 52 53 366 aquatic animal sources, and the greater efficiency of use by the fish likely resulted in the more 54 55 56 367 rapid weight gain observed in animals fed Diet 4. Recently, Minjarez Osorio et al. (2016) used a 57 58 59 60 Journal of the World Aquaculture Society Journal of the World Aquaculture Society Page 18 of 38
18 1 2 3 368 partial substitution of fish meal by soybean protein supplemented with crystalline amino acids 4 5 6 369 which matched the amino acid profile of the fish meal control diet, further supporting the idea 7 8 370 that soybean meal may not meet all the fish’s nutritional requirements as adequately as natural 9 10 11 371 foods do. 12 13 372 14 15 373 Potential Environmental Effects 16 17 18 374 The ecologicalFor consequences Peer of removing Review so many feeder fish from the oceans to 19 20 375 produce fish meal is an important concern (e.g., see Naylor et al. 2000). Aquaculture today 21 22 376 consumes 60% to 70% of all fish meal produced (Rust et al. 2011). Our Diet 1, which contained 23 24 25 377 only ~20% fish meal, used 34 times the quantity of feeder fish as the alternative LFM rations 26 27 378 (Diets 2 and 3). Since Diet 4 was entirely raw marine protein, on a feeder fish equivalent basis it 28 29 379 required twice the amount of sea life as Diet 1 and 72 times that of Diets 2 and 3. The present 30 31 32 380 study demonstrated that fish meal could be excluded almost entirely from prepared diets for large 33 34 381 juvenile red drum without negative consequences to growth or survival and without other 35 36 37 382 additives. If these findings can be replicated broadly at commercial scale, the result could be a 38 39 383 substantial reduction in demand for fish meal to support aquaculture which, in turn, could lessen 40 41 384 impacts to wild populations of feeder fish. 42 43 44 385 The DMR provides a good indicator of feed utilization for the four diets, and indirectly 45 46 386 the ability to reduce solid and dissolved solid wastes. In general, N, P, and solids are considered 47 48 387 to be the most important water pollutants resulting from intensive aquaculture (USEPA 2005). 49 50 51 388 No differences were observed in DMR among the three pelleted formulations, but the DMR of 52 53 389 Diet 4 was about half that of the prepared feeds (Table 4). Interestingly, the NitCR of Diets 1 and 54 55 390 Diet 3 were similar and statistically different from Diets 2 and 4, which were not different. 56 57 58 59 60 Journal of the World Aquaculture Society Page 19 of 38 Journal of the World Aquaculture Society
19 1 2 3 391 Statistically, less N was utilized in the production of biomass by the two latter diets. The 4 5 6 392 grouping of these diets and the absolute values of the parameters suggest that these differences 7 8 393 may be a statistical artifact. On the other hand, the PhosCR demonstrates that the two LFM diets, 9 10 11 394 although not different from each other, reduced by about 20% the weight of P required to 12 13 395 produce a kg of cultured fish biomass compared to Diet 1. This indicates that the reduced fish 14 15 396 meal diets improved P conversion efficiency which should lead to less P pollution in waste 16 17 18 397 effluents. Again, DietFor 4 had a significantly Peer lower Review P conversion to biomass ratio than any of the 19 20 398 three pelleted diets, requiring about 38% less P than Diet 1 and 22% less than diets 2 and 3. 21 22 399 The N and P Loss Ratios (Table 4) are also good indicators of pollution potential. There 23 24 25 400 were no significant differences in the NLRs among the extruded diets, although that for Diet 4 26 27 401 was significantly lower (~27%). The PLR was highest for Diet 1, significantly lower for Diets 2 28 29 402 and 3, and significantly lower again for Diet 4. P loss in effluent was reduced by about 20% in 30 31 32 403 Diets 2 and 3 compared to Diet 1. This suggests that reducing fish meal in prepared feeds may 33 34 404 lower P pollution in aquaculture effluents. Again, however, the PLR for Diet 4 was 62% lower 35 36 37 405 than that for Diet 1 and about 44% lower than those for Diets 2 and 3. The lack of significant 38 39 406 difference between Diets 2 and 3 in NLR or PLR suggests that the added enzymes in Diet 3 did 40 41 407 not reduce N or P loss by improving their assimilation. In contrast, Ai et al. (2007) and Biswas et 42 43 44 408 al. (2007) reported reductions in P in effluents from fish fed diets including phytases. However, 45 46 409 Rossi et al. (2015) did not observe beneficial effects of including GroBiotic A® in soybean based 47 48 410 diets for large juvenile red drum, although Burr et al. (2008) reported that addition of the same 49 50 51 411 prebiotic to a compressed pellet diet for red drum in which about half the protein was provided 52 53 412 by soybean meal resulted in improved digestibility. Finally, there were no differences in WPRs 54 55 413 among the three prepared feeds, but Diet 4 generated significantly (~60%) less overall waste than 56 57 58 59 60 Journal of the World Aquaculture Society Journal of the World Aquaculture Society Page 20 of 38
20 1 2 3 414 the extruded diets. These results suggest that, while low fish meal diets may reduce P in 4 5 6 415 aquaculture effluents compared to high fish meal feeds, food that more closely simulates a 7 8 416 natural diet would provide better overall waste reduction. 9 10 11 417 12 13 418 Summary and Conclusions 14 15 419 We found that substituting 97% of the fish meal in a standard prepared diet with other 16 17 18 420 protein sources did notFor affect the growthPeer rate, sur vival,Review or health of red drum, but did improve 19 20 421 the assimilation of P and potentially reduced the amount of P released to the environment. 21 22 422 Enzyme additives, of the types and concentrations used in this study, in a low fish meal diet did 23 24 25 423 not significantly improve nutrient assimilation, growth rate, or loss of nutrients in effluent. We 26 27 424 also found that the use of a positive, internal control “natural” diet established the performance 28 29 425 potential of the specific environment and culture system, thereby eliminating possible extraneous 30 31 32 426 influences on the performance of the experimental formulated diets. The natural diet also 33 34 427 identified clear benchmarks for growth rate, fish health, nutrient assimilation, and effluent 35 36 37 428 pollution reduction against which future feeds development work should be evaluated. We 38 39 429 suggest that when possible this type of control be incorporated in all nutrition studies. In a 40 41 430 similar vein, McGoogan and Gatlin (1997) found that a diet that used red drum muscle as the 42 43 44 431 primary protein source provided the best results in feeding trials with red drum and suggested 45 46 432 that a more “natural” diet could serve as a target against which to evaluate future studies on 47 48 433 protein sources for this species. While it may not be possible to achieve growth rates observed 49 50 51 434 with natural diets due to cost and other practical constraints, this should be the long term goal of 52 53 435 ongoing nutrition research. 54 55 436 56 57 58 59 60 Journal of the World Aquaculture Society Page 21 of 38 Journal of the World Aquaculture Society
21 1 2 3 437 Disclaimer 4 5 6 7 438 Certain commercial equipment, instruments, or materials are identified in this paper to 8 9 439 specify adequately the experimental procedure. Such identification does not imply 10 11 440 recommendation or endorsement by the National Institute of Standards and Technology, the 12 13 14 441 National Oceanic and Atmospheric Administration, or the SC Department of Natural Resources 15 16 442 nor does it imply that the materials or equipment identified are necessarily the best available for 17 18 443 the purpose. ReferenceFor to the content Peer of this paper Review for commercial advertising purposes with 19 20 21 444 regard to commercial equipment, instruments, or materials is prohibited (15 CFR 200.113). 22 23 24 445 Acknowledgments 25 26 27 446 We thank Colden Battey (NOAA, Hollings Marine Laboratory), Tracy B. Schock and 28 29 30 447 Miki Watanabe (NIST, Hollings Marine Laboratory), J. M. Monserrat (Federal University of Rio 31 32 448 Grande, Brazil) who was on sabbatical leave at NIST; Andrew Grosse and Patrick Biondo 33 34 449 (SCDNR), and Scott Snyder (formerly of Zeigler Bros.) for their assistance during the study. 35 36 37 450 Aaron Watson (SCDNR) provided helpful review comments and assistance with formatting. The 38 39 451 research was supported by a NOAA Aquaculture Program internal competitive grant and 40 41 452 contributions from NIST. This is contribution number __ from the SCDNR’s Marine Resources 42 43 44 453 Research Institute. 45 46 47 454 48 49 50 455 51 52 53 54 55 56 57 58 59 60 Journal of the World Aquaculture Society Journal of the World Aquaculture Society Page 22 of 38
22 1 2 3 456 Literature Cited 4 5 6 7 457 Ai, Q., K. Mai, W. Zhang, W. Xu, B. Tan, C. Zhang, and H. Li. 2007. Effects of exogenous 8 9 458 enzymes (phytase, non starch polysaccharide enzyme) in diets on growth, feed 10 11 459 utilization, nitrogen and phosphorus excretion of Japanese seabass, Lateolabrax 12 13 14 460 japonica s. Comparative Biochemistry and Physiology Part A 147: 502 508. 15 16 17 461 Anderson, D.M., J. M. Burkholder, W. P. Cochlan, P. M. Gilbert, C. J. Gobler, C. A. Heil, 18 For Peer Review 19 462 R. M. Kudela, M. L. Parsons, J.E. Jack Rensel, D. W. Townsend, V. L. Trainer, and 20 21 22 463 G. A. Vargo . 2008. Harmful algal blooms and eutrophication: examining linkages from 23 24 464 selected coastal regions of the United States. Harmful Algae 8:39 53. 25 26 27 465 Bardach, J. E., J. H. Ryther, and W. O. McLarney. 1974. Aquaculture: the farming and 28 29 30 466 husbandry of freshwater and marine organisms. Wiley Interscience, New York. 31 32 33 467 Biswas, A. K., H. Kaku, S. C. Ji, M. Seoka, and K. Takii. 2007. Use of soybean meal and 34 35 468 phytase for partial replacement of fish meal in the diet of red sea bream, Pagrus major. 36 37 38 469 Aquaculture 267(1 4): 284 291. 39 40 41 470 Boyd, C.E., C. Tucker, A. McNevin, K. Bostick, and J. Clay . 2007. Indicators of resource use 42 43 471 efficiency and environmental performance in fish and crustacean aquaculture. Reviews in 44 45 46 472 Fisheries Science 15:327 360. 47 48 473 Brinker, A. and R. Reiter . 2011. Fish meal replacement by plant protein substitution and guar 49 50 474 gum addition in trout feed, Part I: effects on feed utilization and feed quality. Aquaculture 51 52 53 475 310:350 360. 54 55 56 476 Browdy, C.L. and J.A. Hargreaves , editors. 2009. Overcoming Technical Barriers to the 57 58 59 60 Journal of the World Aquaculture Society Page 23 of 38 Journal of the World Aquaculture Society
23 1 2 3 477 Sustainable Development of Competitive Marine Aquaculture in the United States. U.S. 4 5 6 478 Department of Commerce, Silver Spring, MD USA. NOAA Technical Memo NMFS 7 8 479 F/SPO 100. 114pp. 9 10 11 480 Buentello, J.A., W.H. Neill, and D.M. Gatlin, III . 2010. Effects of dietary prebiotics on the 12 13 481 growth, feed efficiency and non specific immunity of juvenile red drum fed soybean 14 15 482 based diets. Aquacul. Res. 41:411 418. 16 17 18 483 Burr, G., M. Hume, W.For H. Neill, Peer and D. M. Gatlin Review III . 2008. Effects of prebiotics on nutrient 19 20 21 484 digestibility of a soybean meal based diet by red drum Sciaenops ocellatus (Linnaeus). 22 23 485 Aquaculture Research 39:1680 1686. 24 25 26 486 Cao, L., W. Wang, C. Yang, Y. Yang, J. Diana, A. Yakupitiyage, Z. Luo, and D. Li . 2007. 27 28 29 487 Application of microbial phytase in fish feed. Enzyme and Microbial Technology 40:497 30 31 488 507. 32 33 34 489 Davis, D.A. and C. R. Arnold . 2004. Red drums, Sciaenops ocellatus, production diets: 35 36 37 490 replacement of fish meal with soybean meal. J. Applied Aquacult. 15(3/4):173 180. 38 39 40 491 Davis, D. A., D. Jirsa, and C. R. Arnold. 1995. Evaluation of soybean proteins as replacement 41 42 492 for menhaden fish meal in practical diets for the red drum Sciaenops ocellatus. Journal of 43 44 45 493 the World Aquaculture Society 26(1):48 58. 46 47 48 494 Davis, D. A., C. L. Miller, and R. P. Phelps . 2005. Replacement of fish meal with soybean 49 50 495 meal in the production diets of juvenile red snapper, Lutjanus campechanus. J. World 51 52 53 496 Aquacult. Soc. 36(1): 114 119. 54 55 56 57 58 59 60 Journal of the World Aquaculture Society Journal of the World Aquaculture Society Page 24 of 38
24 1 2 3 497 Debnath, D., N. P. Salu, A. K. Pal, K. Baruah, S. Yengkokpam, and S. C. Mukherjee . 2005. 4 5 6 498 Present scenario and future prospects of phytase in aquafeed – review. Asian Australian 7 8 499 Journal of Animal Science 12:1800 1812. 9 10 11 500 El-Sayed, B. M., Y. Hakim, J. M. El-Sayed, and H. A. Ali . 2014. Effect of partial replacement 12 13 14 501 of yellow corn by pomegranate peel with or without allozyme SSF on growth 15 16 502 performance and health status of Oreochromis niloticus. World Journal of Fish and 17 18 503 Marine SciencesFor 6(12): 182 189. Peer Review 19 20 21 22 504 Emre, Y., H. Sevgili, and I. Diller . 2003. Replacing fish meal with poultry by product meal in 23 24 505 practical diets for mirror carp (Cyprinus carpio) fingerlings. Turkish Journal of Fisheries 25 26 506 and Aquatic Science 3:81 85. 27 28 29 30 507 Erturk, M. M. and H. Sevgili . 2003. Effects of replacement of fish meal with poultry by 31 32 508 product meals on apparent digestibility, body composition and protein efficiency ratio in 33 34 509 a practical diet for rainbow trout, Onchorynchus mykiss. Asian Australian Journal of 35 36 37 510 Animal Science 16(9):1355 1359. 38 39 40 511 FAO . 2016. The state of world fisheries and aquaculture 2016. Contributing to food security and 41 42 512 nutrition for all. Rome, Italy. 200 pp. 43 44 45 46 513 Faulk, C. K . 2005. Cultured aquatic species information programme. Sciaenops ocellatus. In: 47 48 514 FAO Fisheries and Aquaculture Department (online). Updated 09 Feb 2005; accessed 28 49 50 515 Oct 2015. http://www.fao.org/fishery/culturedspecies/sciaenopsocellatus/en. 51 52 53 54 516 Fox, J.M., A. L. Lawrence, A. J. Siccardi III, D. A. Davis, D. Ricque-Marie, E. Cruz- 55 56 517 Suarez, and T. M. Samocha . 2006. Phytase supplementation in aquaculture diets 57 58 59 60 Journal of the World Aquaculture Society Page 25 of 38 Journal of the World Aquaculture Society
25 1 2 3 518 improves fish, shrimp growth performance. Global Aquaculture Advocate, Feb 2006: 64 4 5 6 519 66. 7 8 9 520 Gaylord, T.G. and D. M. Gatlin III . 1996. Determination of digestibility coefficients of various 10 11 521 feedstuffs for red drum (Sciaenops ocellatus). Aquaculture 139:303 314. 12 13 14 15 522 Gaylord, T. G. and S. D. Rawles . 2005. The modification of poultry by product meal for use in 16 17 523 hybrid striped bass Morone chrysops X M. saxatilis diets. Journal of the World 18 For Peer Review 19 524 Aquaculture Society 36(3):363 374. 20 21 22 23 525 Goldburg, R. and R. Naylor . 2005. Future seascapes, fishing, and fish farming. Front Ecol 24 25 526 Environ 3(1): 21 28. 26 27 28 527 Hall, S.J., A. Delaporte, M.J. Phillips, M. Beveridge, and M. O’Keefe . 2011. Blue frontiers: 29 30 31 528 managing the environmental costs of aquaculture. The Worldfish Center, Penang, 32 33 529 Malaysia. 103 pp. 34 35 36 530 Hardy, R. W. 2000. New developments in aquatic feed ingredients, and potential of enzyme 37 38 531 supplements. Pages 216 226 in Cruz Suarez, L.E., D. Ricque Marie, H. Tapia Salazar, 39 40 41 532 M. A. Olvera Novoa and R. Civera Cerecedo. R., editors. Avances en nutricion acuicola 42 43 533 V. Memorias del V Simposium Internacional de Nutricion Acuicola. 19 22 Noviembre 44 45 46 534 2000. Merida, Yucatan, Mexico. 47 48 49 535 Heisler, J., P. M. Gilbert, J. M. Burkholder, D. M. Anderson, W. Cochlan, W. C. Dennison, 50 51 536 Q. Dortch, C. J. Gobler, C. A. Heil, E. Humphries, A. Lewitus, R. Magnien, H. G. 52 53 54 537 Marshall, K. Sellner, D. A. Stockwell, D. K. Stoecker, and M. Suddelson . 2008. 55 56 538 Eutrophication and harmful algal blooms: a scientific consensus. Harmful Algae 8:3 13. 57 58 59 60 Journal of the World Aquaculture Society Journal of the World Aquaculture Society Page 26 of 38
26 1 2 3 539 Hernandez, A., S. Satoh, V. Kiron, T. Watanabe . 2004. Phosphorus retention efficiency in 4 5 6 540 rainbow trout fed diets with low fish meal and alternative protein ingredients. Fisheries 7 8 541 Science 70:580 586. 9 10 11 542 Hooge, D. M., J. L. Pierce, K.W. McBride, and P. J. Rigolin . 2010. Meta analysis of broiler 12 13 543 chicken trials using diets with or without Allzyme SSF enzyme complex. International 14 15 544 Journal of Poultry Sciences 9(9): 819 823. 16 17 18 545 Jackson, L.S., M. H. ForLi, and E. H.Peer Robinson . 1996.Review Use of microbial phytase in channel 19 20 546 catfish Ictalurus punctatus diets to improve utilization of phytate phosphorus. Journal of 21 22 547 the World Aquaculture Society 27(3):309 313. 23 24 25 548 Kureshy, N., D. A. Davis, and C.R. Arnold . 2000. Partial replacement of fish meal with meat 26 27 549 and bone meal, flash dried poultry by product meal, and enzyme digested poultry by 28 29 550 product meal in practical diets for red drum. North American Journal of Aquaculture 30 31 32 551 62:266 272. 33 34 35 552 Li, M. and E. H. Robinson . 1997. Microbial phytase can replace inorganic phosphorus 36 37 553 supplements in channel catfish Ictalurus punctatus diets. Journal of the World 38 39 40 554 Aquaculture Society 28(4):402 406. 41 42 43 555 McGoogan, B.B. and D. M. Gatlin III . 1997. Effects of replacing fish meal with soybean meal 44 45 556 in diets for red drum Sciaenops ocellatus and potential for palatability enhancement. J. 46 47 48 557 World Aquacult. Soc. 28(4): 374 385. 49 50 51 558 Meilahn, C. W., D. A. Davis, and C. R. Arnold . 1996. Effects of commercial fish meal 52 53 559 analogue and menhaden fish meal on growth of red drum fed isonitrogenous diets. 54 55 56 560 Progressive Fish Culturist 58:111 116. 57 58 59 60 Journal of the World Aquaculture Society Page 27 of 38 Journal of the World Aquaculture Society
27 1 2 3 561 Minjarez-Osorio, C., S. Castillo-Alvarado, D. M. Gatlin III, M. L Gonzalez-Felix, M. 4 5 6 562 Perez-Velazquez, and W. Rossi, Jr. 2016. Plant protein sources in the diets of the 7 8 563 sciaenids red drum (Sciaenops ocellatus) and shortfin corvina (Cynoscion parvipinnis): a 9 10 11 564 comparative study. Aquaculture 453:122 129. 12 13 14 565 Moura, G. de S., E. A T. Lanna, K. Filer, D. L. Falkoski, J. L. Donzele, M. G. de A. Olivera, 15 16 566 and S. T. de Rezende . 2012. Effects of enzyme SSF (solid state fermentation) in pellet 17 18 567 diets for Nile tilapia.For Revista Peer Brasileira de Review Zootecnia 41(10). 19 20 21 568 http://dx.doi.org/10.1590/S1516 35982012001000001. 22 23 24 569 Naylor, R. L., R. J. Goldburg, J.H. Primavera, N. Katusky, M.C.M. Beveridge, J. Clay, C. 25 26 570 Folke, J. Lubchenco, H. Mooney, and M. Troell . 2000. Effects of aquaculture on world 27 28 29 571 fish supplies. Nature 405:1017 1024. 30 31 32 572 Naylor, R.L., R. W. Hardy, D. P. Bureau, A. Chiu, M. Elliott, A. P. Farrell, I. Forster. D.M. 33 34 573 Gatlin, R. J. Goldburg, K. Hua, and P.D. Nichols. 2009 . Feeding aquaculture in an era 35 36 37 574 of finite resources. Proceedings of the National Academy of Sciences 106 (36):15103 38 39 575 15110. 40 41 42 576 Overstreet, R.M. and R. W. Heard . 1978. Food of the red drum, Sciaenops ocellata, from 43 44 45 577 Mississippi Sound. Gulf Research Reports 6:131 135. 46 47 578 Pillay. T. V. R. 1978. Progress of aquaculture. Pages 84 101 in E. M. Borgese and N. Ginsburg, 48 49 579 editors. Ocean Yearbook 1. University of Chicago Press, Chicago, IL. 50 51 52 580 Price, C.S. and J. A. Morris, Jr. 2013. Marine cage culture and the environment: twenty first 53 54 581 century science informing a sustainable industry. NOAA Technical Memorandum NOS 55 56 57 582 NCCOS 164, 158 pp. 58 59 60 Journal of the World Aquaculture Society Journal of the World Aquaculture Society Page 28 of 38
28 1 2 3 583 Rana, K.J., S. Siriwardena, and M. R. Hasan . 2009. Impact of rising feed ingredient prices on 4 5 6 584 aquafeeds and aquaculture production. FAO Fisheries and Aquaculture Technical Paper 7 8 585 541. Rome, Italy. 78 pp. 9 10 11 586 Rawles, S.D. and D. M. Gatlin III . 2000. Nutrient digestibility of common feedstuffs in 12 13 587 extruded diets for sunshine bass Morone chrysops ♀ x M. saxatilis ♂. Journal of the 14 15 588 World Aquaculture Society 31:570 579. 16 17 18 589 Refstie, S., G. Baeverfjord,For R. R.Peer Seim, and O. ReviewElvebo . 2010. Effects of dietary yeast cell wall 19 20 21 590 B glucans and MOS on performance, gut health, and salmon lice resistance in Atlantic 22 23 591 salmon (Salmo salar) fed sunflower and soybean mean. Aquaculture 305:109 116. 24 25 26 592 Reigh, R.C. and S. C. Ellis. 1992. Effects of dietary soybean and fish protein ratios on growth 27 28 593 and body composition of red drum (Sciaenops ocellatus) fed isonitrogenous diets. 29 30 594 Aquaculture 104(3 4):279 292. 31 32 33 595 Robinson, E.H. 1988. Nutritional requirements of red drum: a review. Contributions in Marine 34 35 596 Science (Suppl.) 30: 11 20. 36 37 597 Rossi, W. Jr. and D. A. Davis . 2014. Meal and bone meal as an alternative for fish meal in 38 39 40 598 soybean meal based diets for Florida pompano, Trachinotus carolinus L. Journal of the 41 42 599 World Aquaculture Society 45(6): 613 624. 43 44 Rossi, W. Jr., D. Moxely, A. Buentello, C. Pohlenz, and D. M. Gatlin III . 2013. Replacement 45 600 46 47 601 of fish meal with novel feed plantstuffs in the diet of red drum Sciaenops ocellatus: an 48 49 602 assessment of nutritional value. Aquacult. Nutr. 19:72 81. 50 51 52 603 Rossi, W. Jr., J. R. Tomasso, and D. M. Gatlin III . 2015. Production performance and non 53 54 604 specific immunity of cage raised red drum, Sciaenops ocellatus, fed soybean based diets. 55 56 605 Aquaculture 443:84 89. 57 58 59 60 Journal of the World Aquaculture Society Page 29 of 38 Journal of the World Aquaculture Society
29 1 2 3 606 Rust, M.B., F.T. Barrows, R.W. Hardy, A. Lazur, K. Naughten, and J. Silverstein . 2011. 4 5 6 607 The Future of Aquafeeds. NOAA/USDA Alternative Feeds Initiative. NOAA Technical 7 8 608 Memorandum NMFS F/SPO 124, National Oceanic and Atmospheric Administration, 9 10 11 609 Washington, DC. 12 13 610 Sandifer, P.A. 1979. The necessity for aquaculture development. Marine Technology Society 14 15 611 Journal 13 (3):35 39. 16 17 18 612 Sandifer, P.A., J.S. Hopkins,For A.D. Peer Stokes, and ReviewR.A. Smiley . 1993. Experimental pond grow 19 20 21 613 out of the red drum, Sciaenops ocellatus, in South Carolina. Aquaculture 118 (3/4):217 228. 22 23 24 614 Storebakken, T., K.D. Shearer, and A. J. Roem . 1998. Availability of protein, phosphorus and 25 26 615 other elements in fish meal, soy protein concentrate and fish meal, soy protein concentrate 27 28 29 616 based diets to Atlantic salmon, Salmo salar . Aquaculture 161:365 379. 30 31 32 617 Tacon, A.G.J. and M. Metian . 2008. Global overview on the use of fish meal and fish oil in 33 34 618 industrially compounded aquafeeds: trends and future prospects. Aquaculture 285:146 158. 35 36 37 38 619 Trushenski, J., J. Laporte, H. Lewis, M. Schwarz, B. Delbos, R. Takeuchi, and L.A. 39 40 620 Sampaio . 2011. Fish meal replacement with soy derived protein in feeds for juvenile cobia: 41 42 621 influence of replacement level and attractant supplementation. Journal of the World 43 44 45 622 Aquaculture Society 42(3): 435 443. 46 47 48 623 Tucker, J. W., Jr., W. A. Lellis, G. K. Vermeer, D. E. Roberts, Jr., and P. N. Woodward. 49 50 624 1997. The effects of experimental starter diets with different levels of soybean or 51 52 53 625 menhaden oil on red drum (Sciaenops ocellatus). Aquaculture 149:323 339. 54 55 626 USCOP . 2004. An ocean blueprint for the 21 st century. Final report of the U.S. Commission on 56 57 627 Ocean Policy. Washington, DC, 455 pp + appendices 58 59 60 Journal of the World Aquaculture Society Journal of the World Aquaculture Society Page 30 of 38
30 1 2 3 628 USEPA . 2005. Chapter 6 Water use, wastewater characterization, and pollutants of 4 5 6 629 concern. In Technical Development Document for the Final Effluent Limitations 7 8 630 Guidelines and New Source Performance Standards for the Concentrated Aquatic 9 10 11 631 Animal Production Point Source Category (Revised August 2004). EPA 821 R 12 13 632 04 012, U.S. Environmental Protection Agency Washington, DC. 14 15 16 633 Van Weerd, J. H. K.H.A. Khalaf, F. J. Aartsen, and P.A.T. Tijssen . 1999. Balance trials with 17 18 634 African catfishFor Clarias gariepinus Peer fed phytase trea Reviewted soybean meal based diets. 19 20 21 635 Aquaculture Nutrition 5:135 142. 22 23 24 636 Vielma, J., S. P. Lall, J. Koskela, F. J. Schoner, and P. Mattila . 1998. Effects of dietary 25 26 637 phytase and cholecalciferol on phosphorus bioavailability in rainbow trout 27 28 29 638 (Oncorhynchus mykiss). Aquaculture 163 (3 4): 309 323. 30 31 32 639 Webb, K.A. and D. M. Gatlin III . 2003. Effects of dietary protein level and form on production 33 34 640 characteristics and ammonia excretion of red drum Sciaenops ocellatus. Aquaculture 35 36 37 641 225:17 26. 38 39 40 642 Zhao, H., C. Li, B. H. Beck, R. Zhang, W. Thongda, D. A. Davis, and E. Peatman. 2015. 41 42 643 Impacts of feed additives on surface mucosal health and columnaris susceptibility in 43 44 45 644 channel catfish fingerlings, Ictalurus punctatus. Fish and Shellfish Immunology 46(2): 46 47 645 624 637. 48 49 50 646 Zhou, Q.-C., J. A. Buentello, and D. M. Gatlin III. 2010. Effects of dietary prebiotics on 51 52 53 647 growth response and intestinal morphology of red drum (Sciaenops ocellatus). 54 55 648 Aquaculture 309 (1 4):253 257. 56 57 58 59 60 Journal of the World Aquaculture Society Page 31 of 38 Journal of the World Aquaculture Society
31 1 2 3 TABLE 1. Ranges, means and standard deviations (SD), across all tanks and dates, of water 4 5 6 quality parameters measured during the course of a diet study where red drum, Sciaenops 7 8 ocellatus, were fed four diets to evaluate alternative protein sources. 9 10 11 12 13 14 15 Parameter Units Range Mean ± SD (n) 16 17 18 Temperature Foro CPeer Review 23.7 25.4 25.0 ± 0.2 (1134) 19 20 21 Dissolved oxygen mg L 1 4.5 7.1 5.8 ± 0.3 (1133) 22 23 24 pH 6.9 – 8.2 7.9 ± 0.1 (1049) 25 26 27 1 28 Salinity g kg 27.7 31.5 29.9 ± 0.9 (1133) 29 30 1 31 Ammonia N mg L 0.01 – 0.46 0.16 ± 0.08 (388) 32 33 34 Nitrite N mg L 1 0.00 – 0.08 0.04 ± 0.02 (408) 35 36 37 Nitrate N mg L 1 1.50 – 6.00 3.22 ± 0.75 (408) 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Journal of the World Aquaculture Society Journal of the World Aquaculture Society Page 32 of 38
32 1 2 3 TABLE 2. Composition of three isonitrogenous (~44.5% protein) diets formulated for red drum, 4 5 6 Sciaenops ocellatus, used in a recirculating aquaculture system for feeding study to evaluate protein 7 8 sources as an alternative to fish meal and reduce phosphorus in effluent. 9 10 11 Ingredients Diet 1 Diet 2 Diet 3 12 13 (%) (%) (%) 14 15 16 17 18 Wheat For Peer Review23.46 23.85 23.85 19 20 21 Menhaden Fish Meal (62% protein) 19.60 0.61 0.61 22 23 24 Poultry By product Meal 13.13 25.96 25.96 25 26 27 Hydrolyzed Feather Meal 8.29 7.89 7.89 28 29 30 Profine VF Soybean Protein Concentrate 0 11.55 11.70 31 32 33 Corn Distillers Grains and Solubles 7.42 7.4 7.27 34 35 36 Blood Meal (92% protein) 6.10 2.38 2.30 37 38 39 Menhaden Fish Oil 4.37 5.0 5.0 40 41 42 43 Soy Oil 4.96 5.17 5.1 44 45 46 Hydrolyzed Poultry Protein Concentrate 2.0 2.0 2.0 47 48 49 Dried Yeast 2.0 2.0 2.0 50 51 52 HP 300 Soy Protein 2.0 2.0 2.0 53 54 55 Hydrolyzed Fish Protein Concentrate 1.67 0 0 56 57 58 59 60 Journal of the World Aquaculture Society Page 33 of 38 Journal of the World Aquaculture Society
33 1 2 3 Wheat Middlings 1.62 0.81 0.81 4 5 6 Solvent Extracted Soybean Meal (48% protein) 1.62 0.81 0.81 7 8 9 10 11 ® ® 12 Allzyme Vegpro and Allzyme SSF 0 0 0.04 13 14 15 Total Active ingredients 98.24 97.43 97.34 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39
40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Journal of the World Aquaculture Society Journal of the World Aquaculture Society Page 34 of 38
34 1 2 3 TABLE 3. Chemical analyses of diets used for red drum, Sciaenops ocellatus, in a recirculating 4 5 6 aquaculture system for a feeding study to evaluate protein sources as an alternative to fishmeal and to 7 8 reduce phosphorus in effluent. Test Diets 1 3 all contained approximately 44.5% protein. Diet 1 was 9 10 similar to commercial feed and contained 19.6% fish meal; Diet 2 contained only 0.6% fish meal; Diet 3 11 12 differed from Diet 2 only in containing additives designed to improve digestion of alternative proteins 13 14 sources. Diet 4 was a 73% protein control ration composed solely of natural products (fish, mollusks, and 15 16 crustaceans). Multiple samples of each feed were taken at different times during the experiment and 17 18 For Peer Review 19 analyzed by the Clemson Agricultural Services Laboratory (Clemson, SC, USA) or the New Jersey Feed
20 1 21 Laboratory (Trenton, NJ, USA). All determinations are reported as percent or as mg kg of the dry 22 23 weight (ppm). Values in the table represent the mean ± standard error; number of samples is shown in 24 25 parentheses. 26 27 28 29 30 Diet 1 Diet 2 Diet 3 Diet 4 31 32 33 Protein % 44.4 ± 0.6 (6) 44.1 ± 0.6 (6) 45.0 ± 0.4 (6) 72.9 ± 2.0 (3) 34 35 36 Fat % 14.5 ± 0.6 (6) 13.6 ± 1.0 (6) 14.2 ± 0.9 (6) 10.2 ± 0.7 (3) 37 38 39 Fiber % 3.5 ± 0.0 (2) 3.2 ± 1.5 (2) 3.2 ± 1.1 (2) 2.2 ± 0.1 (2) 40 41 42 Ash % 8.0 ± 0.5 (2) 6.7 ± 0.5 (2) 6.5 ± 0.2, 2 (2) 13.3 ± 2.2 (2) 43 44 45 Moisture % 6.4 ± 0.2 (5) 8.2 ± 0.1 (5) 6.9 ± 0.1 (5) *5 .0 ± 1.0 (3) 46 47 48 Ca % 1.86 ± 0.04 (6) 1.55 ± 0.03 (6) 1.49 ± 0.03 (6) 2.32 ± 0.09 (3) 49 50 51 P % 1.23 ± 0.02 (6) 1.06 ± 0.02 (6) 1.03 ± 0.02 (6) 1.56 ± 0.03 (3) 52 53 54 Mg % 0.16 ± 0.00 (6) 0.15 ± 0.00 (6) 0.16 ± 0.00 (6) 0.24 ± 0.01 (3) 55 56 57 58 59 60 Journal of the World Aquaculture Society Page 35 of 38 Journal of the World Aquaculture Society
35 1 2 3 Na % 0.28 (1) 0.2 (1) 0.19 (1) 0.53 (1) 4 5 6 K % 0.64 ± 0.00 (6) 0.66 ± 0.01 (6) 0.71 ± 0.01 (6) 1.23 ± 0.01 (3) 7 8 9 Mn ppm 86 ± 2 (6) 84 ± 2 (6) 88 ± 2 (6) 4 ± 0 (3) 10 11 12 Fe ppm 421 ± 10 (5) 268 ± 8 (6) 239 ± 4 (6) 85 ± 7 (3) 13 14 15 Zn ppm 123 ± 1 (6) 119 ± 3 (6) 121 ± 3 (6) 86 ± 0 (3) 16 17 18 Cu ppm 54 ± 1For (6) 55Peer ± 1 (6) Review 59 ± 1 (6) 51 ± 1 (3) 19 20 21 22 23 24 *Note that Diet 4 was dried prior to chemical analyses. Moisture content of Diet 4 was 67.23 ±3.07, 25 before drying. 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Journal of the World Aquaculture Society Journal of the World Aquaculture Society Page 36 of 38
1 2 3 1400 4 5 6 1200 7 8 9 1000 10 11 12 800 13 14 15 600 16 17 18 400 For Peer Review 19 grams/treatment Average 20 200 21 22 23 0 24 9 April 2013 (T0) 30 May 2013 (T1) 1 August 2013 (T2) 25 26 Date 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Journal of the World Aquaculture Society Page 37 of 38 Journal of the World Aquaculture Society
1 2 3 TABLE 4. Final fish production performance and waste production by dietary treatment for red drum, Sciaenops ocellatus, fed 4 5 6 different diets. The initial and final mean weight per fish are presented as +SD to describe the variation among the fish. All other 7 8 parameters are expressed as + SEM to describe the precision with which the mean was determined, and “(n)” indicates the number of 9 10 11 replicates for each treatment. Means designated by the letter A are not significantly different; letters B and C indicate significant 12 13 differences. For Peer Review 14 15 16 17 18 19 20 Parameter Basal Low FM Low FM & Additives "Natural" P-values for 1-way ANOVA 21 22 23 Diet – 1 Diet - 2 Diet - 3 Diet – 4 or Kruskal-Wallis test 24 25 Fish Production 26 27 Initial individual weight (g) 258 ± 27 A (72) 263 ± 29 A (72) 262 ± 25 A (72) 261 ± 28 A (72) 0.521 28 29 Survival (%) 100 100 100 100 30 31 Final individual weight (g) 563 ± 128 A (54) 588 ± 120 A (54) 591 ± 129 A (54) 1185 ± 186 B (54) < 0.001 32 33 Weight gain (% initial) 132 ± 6 A (6) 138 ± 5 A (6) 139 ± 7 A (6) 389 ± 10 B (6) <0.001 34 35 -1 36 Weight gain per day (g d ) 2.7 ± 0.1A (6) 2.8 ± 0.1A (6) 2.9 ± 0.2A (6) 8.1 ± 0.3B (6) <0.001
37 -1 38 SGR (% wet BW day ) 0.68 ± 0.02 A (6) 0.70 ± 0.02 A (6) 0.71 ± 0.03 A (6) 1.33 ± 0.02 B (6) <0.001 39 40 Hepatosomatic Index (HSI) 1.62 ± 0.14A (18) 1.68 ± 0.09A (18) 1.60 ± 0.11A (18) 0.89 ± 0.04B (18) <0.001 41 42 43 44 45 46 Journal of the World Aquaculture Society 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Journal of the World Aquaculture Society Page 38 of 38
1 2 3 Intraperitoneal Fat Index (IPF) 2.94 ± 0.17A (18) 3.17 ± 0.20A (18) 2.81 ± 0.23A (18) 1.82 ± 0.13B (18) <0.001 4 5 6 7 Nutritional Performance 8 9 Feed Intake (%BW day -1) 1.27 ± 0.05 A (6) 1.20 ± 0.03 A (6) 1.27 ± 0.04 A (6) 1.18 ± 0.01 A (6) 0.316 10 11 FCR (dry or wet wt/wet wt) 1.94 ± 0.08 A (6) 1.80 ± 0.03 A (6) 1.89 ± 0.28 A (6) 3.61 ± 0.11 B (6) <0.001 12 13 DMR (dry wt/dry wt) 5.58For ± 0.24 A (6)Peer 5.07 ± 0.08 A (6)Review 5.38 ± 0.33 A (6) 2.73 ± 0.03 B (6) 0.002 14 15 FMR (wet wt/wet wt) 0.380 ± 0.016 A (6) 0.011 ± 0.000 B (6) 0.012 ± 0.001 B (6) NA 0.003 16 17 18 FFE (wet wt/wet wt) 1.708 ± 0.073 A (6) 0.050 ± 0.001B (6) 0.052 ± 0.003 B (6) 3.606 ± 0.044 C (6) <0.001 19 20 NitCR (dry wt/dry wt) 0.397 ± 0.017 A (6) 0.358 ± 0.006 B (6) 0.387 ± 0.023 A (6) 0.319 ± 0.004 B (6) 0.006 21 22 PhosCR (dry wt/dry wt) 0.069 ± 0.003 A (6) 0.054 ± 0.001 B (6) 0.055 ± 0.003 B (6) 0.043 ± 0.001 C (6) <0.001 23 24
25 Pollution Production 26
27 28 WPR (dry wt/wet wt) 1.51 ± 0.08 A (6) 1.34 ± 0.03 A (6) 1.44 ± 0.11 A (6) 0.57 ± 0.01 B (6) 0.002 29 -1 30 Nitrogen loss ratio (g kg ) 99.7 ± 5.5 A (6) 90.7 ± 1.9 A (6) 98.5 ± 7.7 A (6) 70.2 ± 1.3 B (6) 0.001 31 32 Phosphorus loss ratio (g kg -1 ) 13.50 ± 0.95 A (6) 8.39 ± 0.27 B (6) 9.82 ± 1.08 B (6) 5.11 ± 0.17 C (6) <0.001 33 34 35 36 37 38 39 40 41 42 43 44 45 46 Journal of the World Aquaculture Society 47 48 49 50 51 52 53 54 55 56 57 58 59 60