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Replacement of fish meal by a novel non-GM variety of soybean meal in cobia, Rachycentron canadum: Ingredient nutrient digestibility and growth performance
Aquaculture 416–417 (2013) 328–333
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Replacement of fish meal by a novel non-GM variety of soybean meal in cobia, Rachycentron canadum: Ingredient nutrient digestibility and growth performance Jorge A. Suarez a,⁎, Carlos Tudela a, Drew Davis a, Zachary Daugherty a, Matthew Taynor a, Lindsay Glass a, Ronald Hoenig a, Alejandro Buentello b, Daniel D. Benetti a a b
University of Miami, Rosenstiel School of Marine and Atmospheric Science, 4600 Rickenbacker Causeway, Miami, FL 33149, USA Schillinger Genetics and Navita Premium Feed Ingredients, 4401 Westown Parkway, Suite 225, West Des Moines, IA 50266, USA
a r t i c l e
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Article history: Received 12 August 2013 Received in revised form 24 September 2013 Accepted 26 September 2013 Available online 2 October 2013 Keywords: Cobia Digestibility Non-GM soy Fish meal replacement Aquafeeds
a b s t r a c t A constraint for the expansion of cobia aquaculture is the availability of high quality formulated diets which reduce or eliminate fish meal (FM) protein. Therefore, the nutritive value of a novel soybean cultivar, Navita™ (Navita, non-genetically modified and selectively bred soy), and regular, commodity soybean meal (SBM, de-hulled, defatted, roasted and solvent-extracted) was evaluated for cobia, Rachycentron canadum via separate digestibility and growth trials. In the first experiment Navita's apparent digestibility coefficients (ADC) were higher than those of SBM for nearly every nutrient evaluated. Crude protein ADCs were 82 and 69% for Navita and SBM, respectively. Apparent DC for amino acids ranged from 68 to 109% for Navita whereas, amino acid ADCs for SBM varied from 42 to 98%. The feeding trial utilized fish of a size that more closely resembles commercial cobia stocking (1.8 kg), and was conducted over a 91-day period. Experimental diets (iso-nitrogenous and iso-energetic) were formulated such that 67% of the FM protein in the reference diet was replaced by either a combination of SBM + soy protein concentrate (SPC, Solae Profine®) labeled MXSB-diet, or by a combination of SPC + Navita; Navita-diet, hereafter. A fourth experimental diet had 80% of the FM protein replaced by a combination of Navita + SPC and was identified as Navita-high. No significant differences (P N 0.05) were observed in fish fed the experimental diets for feed conversion ratio, protein efficiency ratio, feed efficiency, mean daily intake, gross protein intake, gross energy intake, visceral somatic index, muscle ratio, and hepatosomatic index. Fish fed the Navita-high diet had the lowest fish in:fish out ratio (FIFO) at 0.9 ± 0.16. These results indicate that Navita meal can be incorporated at very high levels in the diet of marine carnivorous fish such as cobia with no detriment to performance, making it a prime candidate for FM replacement in aquafeeds. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Aquaculture is a rapidly growing food-producing industry; however, there is still considerable potential for increased efficiency and efficacy of aquaculture through development of nutritious and cost-effective alternatives to traditional marine protein feedstuffs such as fish meal (FM). For cobia aquaculture, this is particularly relevant as suitable diets which reduce or eliminate FM protein are not yet commercially available. This species is the only member of the family Rachycentridae which is distributed worldwide in tropical and subtropical waters (Benetti et al., 2008; Ditty and Shaw, 1992; Shaffer and Nakamura, 1989). Cobia is well recognized for its fast growth and excellent meat quality and has been intensively farmed since the 1990s (Liao et al., 2004). The technology for reliable broodstock spawning and mass production of cobia fingerlings has been long established at the University ⁎ Corresponding author. Tel.: +1 305 968 5849; fax: +1 305 421 4889. E-mail addresses: [email protected] (J.A. Suarez), [email protected] (A. Buentello). 0044-8486/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquaculture.2013.09.049
of Miami Experimental Hatchery (UMEH). However, while the fundamental technology for cobia production is in place, more research is needed to resolve bottle-neck issues of cobia culture, particularly at the grow-out stage. One such limitation is the formulation of commercial diets with reduced FM levels that are both economical and environmentally friendly, while also maintaining optimal growth performance and disease resistance. The global FM supply remains relatively static but demand and price continue to increase (Naylor et al., 2009). Therefore, FM is a raw material that cannot be relied upon for aquaculture expansion. In contrast, soybean meal (SBM) has long been recognized as an excellent source of protein for animals and humans (Baker, 2000). However, SBM inclusion levels as replacement of FM in aquafeeds are limited by species-specific digestive physiology and by the presence of both heat-resistant and thermo-labile anti-nutritional factors (ANFs). While solvent-extracting and cooking may significantly reduce the biological activity of temperature-sensitive protease inhibitors, this processing also renders protein less available for absorption at the gastrointestinal level. Therefore, the present study was conducted to evaluate the apparent digestibility coefficients (ADCs) of protein and amino acids of a novel
J.A. Suarez et al. / Aquaculture 416–417 (2013) 328–333
variety of non-genetically modified (GM) soybean product (Navita™) and contrast these coefficients against those obtained from fish fed conventional SBM. In addition, a study oriented at maximizing FM replacement in cobia diets, using fish close to marketable size was performed. The use of protein sources such as standard SBM, soy protein concentrates (SPC) and non-GM soy from selective breeding programs could greatly improve the profitability and ecologic sustainability of the industry. The overarching objective was to improve both the ecological and economic efficiencies of formulated feeds for cobia, Rachycentron canadum. 2. Materials and methods Care and handling of the fish as well as procedures used in this study were reviewed and approved by the University of Miami Animal Care and Use Committee. Navita™ meal was donated by Navita Premium Feed Ingredients (NPFI), West Des Moines, IA. This is a genetically unique, patented non-GM soy cultivar, which contains increased levels of protein and amino acids for animal feed and reduced ANF levels. Beans are made into a defatted meal by conventional methods but this particular variety is selectively bred to have up to 20% higher protein density than the commodity SBM and ultra-low levels of oligosaccharides (0.6%, raffinose + stachyose). In contrast, conventional SBM has 6% of oligosaccharides. 2.1. Digestibility trial — experimental diets Digestibility diets (Table 1) were manufactured at the Oceanic Institute, Waimanalo, HI and were comprised of a 70:30 mixture of the reference diet and the Navita™ meal. By including the reference diet at 70%, and with the crude protein contents inherent to the test ingredient, the resulting experimental diet had an analyzed crude protein and lipid values of 45 and 12%, respectively, which satisfies the requirements defined for cobia (Fraser and Davies, 2009). Yttrium oxide (Y2O3) was included at 0.5% of the reference diet as an inert, indigestible marker. The dietary ingredients were first ground to a uniform particle size (b 1mm) in a hammer mill, then batch-mixed in a commercial ribbon blender
Table 1 Reference and test diets for determination of apparent digestibility coefficients in cobia. Ingredient
Reference diet
Navitaa
Regular soybean mealb
g/kg of diet Pollock fish mealc Dextrin Menhaden oil Mineral premixd Vitamin premixe Carboxylmethyl cellulosef Cellufilf Yttrium oxideg Test ingredient Total
612.6 120.0 69.4 50.0 40.0 30.0 73.0 5.0 – 1000
g
700.0
700.0
300.0 1000
300.0 1000
a Navita Premium Feed Ingredients, West Des Moines, IA, (g/kg) 558.8 crude protein, 893.3 dry matter, 5.8 crude lipid, 31.0 crude fiber, 58.1 ash, non-genetically modified soy. b Archer Daniels Midland, Decatur, IL, 480.0 crude protein, 933.8 dry matter, 18.4 crude lipid, 38.0 crude fiber, 61.9 ash, commodity roasted/cooked and hexane extracted, genetically modified soy. c Alaska Pollock (Theragra clalcogramma) meal Genuine Alaska Pollock Producers, Seattle, WA, (g/kg) 698.8 crude protein, 734.5 dry matter, 7.9 crude lipid. d Composition (g/kg): Ca(H2PO4)2∙H2O, 136.00; Ca(C6H10O6)∙5H2O, 348.553; FeSO4∙7H2O, 5.00; MgSO4∙7H2O, 132.00; K2HPO4, 240.00; NaH2PO4∙H2O, 88.00; NaCl, 45.00; AlCl3∙6H2O, 0.084; KI, 0.15; CuSO4∙5H2O, 0.50; MnSO4∙H2O, 0.70; CoCl2∙6H2O, 1.00; ZnSO4∙7H2O, 3.00; NaSeO3, 0.0127. e Composition (g/kg): ascorbic acid, 50; DL-calcium pantothenate, 5.0; choline chloride, 36.2; inositol, 5.0; menadione sodium bisulfite, 2.0; niacin, 5.0; pyridoxine HCl, 1.0; riboflavin, 3.0; thiamine mononitrate, 0.5; DL-α-tocopherol acetate (250 IU/g), 8.0; vitamin A palmitate (500,000 IU/g), 0.2; micro-mix, 10.0; cellulose, 874.1. Micro-mix composition (g/100 g): biotin, 0.50; folic acid, 1.8; vitamin B12, 0.02; cholecalciferol (40 IU/μg), 0.02; cellulose, 97.66. f USB-Affymetrix, Cleveland, OH. g Sigma-Aldrich Company, St. Louis, MO.
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(Ross 42-A-10, Charles Ross and Son Co., Hauppauge, NY), preconditioned at 94–99 °C and extruded (Wenger X-20, Wenger Manufacturing, Sabetha, KS) at 99–138°C through a 5-mm die to produce buoyant pellets. 2.2. Digestibility trial — fish and experimental conditions Juvenile cobia (initially weighing 286 ± 10 g/fish), raised in tanks at UMEH (Miami, FL) were seined and graded. These fish were stocked into four, 4500-L fiberglass round tanks at a density of 80 fish/tank. Sand-filtered well water entered each tank at a rate of 50 L/min (1600% exchange per day) and exited via an internal standpipe. Supplemental aeration was provided to each tank to maintain dissolved oxygen levels near saturation. Tanks were covered by a shading mesh with natural illumination. Water temperature, salinity and dissolved oxygen concentration were monitored daily (YSI-57) and averaged 24.4 °C, 33.5 ppt and 6.3 ppm, respectively. Water quality parameters were monitored weekly for pH, ammonia, nitrite and nitrate using a Hach™ water quality test kit and remained within recommended levels for cobia (Benetti et al., 2007) throughout the experiment. After a 7-day conditioning period during which all fish were fed the reference diet once per day (900 h) to apparent satiation, feeding of the test diet was initiated. Fish in each tank received the experimental diet for 7 days prior to the collection of fecal samples. Feces were collected 3–4 h post feeding at each collection period by the fecal stripping technique described by Austreng (1978) and Hajen et al. (1993), with modifications. Fish were stripped one day per week, for three consecutive weeks. Briefly, at the appointed collection period fish were dip-netted from the tank and a solution of MS-222 (500 mg/L; Finiquel, Argent Chemical, Redmond WA) was sprayed onto the gill cavity and the opercula held closed until complete relaxation of abdominal muscles was attained. Immediately afterwards, the gills were completely rinsed with a gentle stream of seawater for 30 s, flushing any residual anesthetic. Gentle pressure was then applied to the lower abdomen of the fish, thus expressing feces from the distal intestine onto clean stainless steel bowls. Care was taken to exclude urine, mucus, and other contaminants from the collection vessels. After fecal collection, fish were introduced into a plastic tub with oxygenated seawater until complete recovery. Fecal samples were oven-dried (60 °C) overnight, stored frozen (−80 °C) and sent on dry ice to the Fish Nutrition Laboratory of Texas A&M University for analyses. Yttrium oxide was analyzed by inductivity coupled plasma mass spectrometry (ICP-AES analysis, Perkin Elmer Optia 3000DV; Perkin Elmer, Wellesley, MA, USA) at 371 nm. All yttrium analyses were conducted in duplicate. Proximate composition of diets and fecal samples was analyzed using established methodologies for dry matter, crude protein (AOAC, 2005), lipids (Folch et al., 1957) and ash (AOAC, 2005). Crude protein was estimated by measuring total nitrogen by the Dumas method (Ebeling, 1968) and multiplied by 6.25. Dry matter was determined by heating the samples at 125 °C for 3 h, and ash was quantified after heating at, 650 °C for 3 h (AOAC, 1990). Crude lipid was determined by chloroform and methanol extraction (Folch et al., 1957). Gross energy content was measured by combustion via bomb calorimeter (Parr Instrument Company, Moline, IL, USA) using benzoic acid as a standard. Amino acid content was quantified after acid hydrolysis with 6 N HCl according to procedures described by Pohlenz et al. (2012). Calcium and Phosphorus were quantified by Inductively Coupled Plasma Spectroscopy. Plasma amino acids were assayed via HPLC following a fluorometric technique (Buentello and Gatlin, 2000) using pre-column derivatization with o-phthaldialdehyde (Sigma, St. Louis, MO). Navita ADCs for crude protein and amino acids were calculated from a comparison of the apparent nutrient digestibility of the reference diet and experimental diets as described by Forster (1999). 2.3. Feeding trial — systems, experimental diets and feeding The feeding trial was conducted in 12 cylindrical, fiberglass tanks (4000-L each) operated as a flow-through system with filtered water
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(Triton II sand filter) flowing at approximately 44 L/min (1600% exchange/day). Water quality parameters were monitored daily with a YSI Professional Plus meter (YSI, Inc., Yellow Springs, OH) and temperature remained at 24.7 ± 0.01°C throughout the trial. Salinity was monitored at about 33.5‰, pH 7.25 ± 0.03 and dissolved oxygen was maintained above 8.6 ± 0.11 ppm using low pressure electrical blowers and air stones. A natural photoperiod defined the cycle of hours of light and dark at Miami's latitude. Purified and practical ingredients for the experimental diets were purchased and sent to the Food Protein R&D Center at Texas A&M University for diet manufacturing via extrusion. A FM-based control diet (diet 1) – similar to commercial cobia diets – was formulated to contain 46% crude protein from menhaden FM (Special Select, Omega Protein, Houston, TX), SPC, SBM and wheat flour which was also use to provide starch-related binding (Table 2). Menhaden oil (Virginia Prime, Omega Protein) was included as lipid source and provided in combination with FM a total of 11% lipid and an estimated digestible energy level of 4.9 kcal/kg. Three experimental diets were derived from the control diet by replacing FM protein with an increased level of SPC (diet 2) or two different levels of Navita (diets 3 and 4) while maintaining diets iso-nitrogenous and iso-energetic. As plant protein sources increased in the diet an increased supplementation of glycine (Gly, USB-Affymetrix, Cleveland, OH) was used as palatability enhancer. Similarly, as vegetable protein increased in the diet supplemental dietary taurine (Tau) was added. When necessary, crystalline lysine-HCl and L-methionine were used to balance dietary levels of lysine and sulfur-containing amino acids. Diets met all the currently established nutritional requirements for juvenile cobia (Chou et al., 2001, 2004; Craig et al., 2006; NRC, 2011; Romarheim et al., 2008; Salze et al., 2010, 2011; Sun et al., 2006; Zhou et al., 2004).
Juvenile cobia (1.87 ± 0.35 kg) were obtained from the reproduction program at UMEH and stocked at a density of 5 g/L (20 kg biomass/ tank). Prior to the start of the experiment numeric T-bar tags were inserted into each fish's musculature, just below the dorsal fin with the tag barb anchored through the boney fin ray supports. These tags were used for fish identification to track individual growth performance at three different weighing periods (days 29, 60 and 91). After implant recovery fish were conditioned on a commercial diet (Zeigler Bros., Inc., East Berlin, PA) and acclimated to the experimental conditions for 2 weeks prior to the feeding trial. Each experimental diet was then randomly assigned to specific tanks and fish were fed once a day (900h) ad libitum (~3% of body weight per day) for 91days. There were three replicate tanks per dietary treatment. The total feed consumption per tank was recorded daily. An initial sample of 5 fish was collected at the onset of the experiment and 3 additional fish per tank were secured at each harvest period, euthanized using MS-222 (800 mg/L), measured for weight and total length, and stored frozen (−80°C) until analyzed for moisture, ash, protein and lipid according to established procedures (Webb and Gatlin, 2003). An additional 3 fish per tank were euthanized in the same manner, dissected and the following parameters were determined:
Viscerosomatic indexðVSIÞ ð% Þ ¼ viscera weightðgÞ=fish weightðgÞ 100
Hepatosomatic indexðHSIÞ ð% Þ ¼ liver weightðgÞ=fish weightðgÞ 100
Muscle ratioðMRÞ ð% Þ ¼ muscle weightðgÞ 100=body weightðgÞ: Table 2 Formulated and analyzed composition of experimental diets for the feeding trial. Ingredient
Experimental diets 1
2
3
4
g/kg (dry weight basis) Menhaden fish meal, Special Select™a Soy protein concentrateb Regular soybean mealc Navitad Wheat floure Menhaden oila Mineral–vitamin premixf Glycineg Taurineg Lysine HClg g DL-methionine Cellufilg Total Analyzed composition (n = 3) Crude protein Crude lipid Moisture Ash Calcium Phosphorus Energy (kcal digestible energy/kg) a
256.5 102.5 320.0 0.0 160.0 59.5 60.0 10.0 5.0 0.0 0.9 24.0 1000.0
84.0 300.0 280.0 0.0 160.0 81.2 60.0 16.4 12.0 2.0 2.1 30.0 1000.0
84.0 190.0 0.0 376.5 159.0 82.3 60.0 16.4 12.0 1.0 4.9 14.0 1000.0
60.0 150.0 0.0 453.6 150.0 86.2 60.0 18.4 15.0 1.0 5.6 2.0 1000.0
473.0 112.0 91.0 108.0 22.0 18.0 4.9
493.0 103.0 83.0 84.0 15.0 13.0 4.9
487.0 105.0 74.0 84.0 13.0 13.0 4.9
502.0 98.0 82.0 83.0 10.0 11.0 4.8
Omega Protein Corp. (Hammond, LA, USA). Solae Profine VP, food-grade (intended for human consumption) St. Louis, MO, 720 g/kg crude protein. c Archer Daniels Midland, Decatur, IL, 480.0 crude protein, 933.8 dry matter, 18.4 crude lipid, 38.0 crude fiber, 61.9 ash, commodity roasted/cooked and hexane extracted, genetically modified soy. d Navita Premium Feed Ingredients, West Des Moines, IA, (g/kg) 558.8 crude protein, 893.3 dry matter, 5.8 crude lipid, 31.0 crude fiber, 58.1 ash, non-genetically modified soy. e Dawn Food Products, Inc., FL, 600 g/kg crude protein. f Same as d + e in Table 1. g USB-Affymetrix, Cleveland, OH. b
In addition, growth performance and feed consumption were evaluated using established parameters (AOAC, 2005; Zhou et al., 2010) including percent weight gain (WG), average daily gain (ADG), specific growth rate (SRG), mean daily intake (MDI), gross energy intake (GEI), gross protein intake (GPI), feed efficiency (FE), feed conversion ratio (FCR), protein efficiency ratio, (PER) and fish in:fish out (FIFO). Data was evaluated for normality using the Shapiro–Wilk test and no transformations were necessary. Mean ADCs for protein, energy and amino acids were contrasted using Student's t-tests to determine if the two sets of data (Navita vs. SBM) were significantly different from each other. Results from the feeding trial were analyzed via oneway analysis of variance (ANOVA), using Duncan's multiple-range test for mean separation. All statistical analyses were performed using the Statistical Analysis System (SAS) version 9.2 (SAS Institute Inc., Cary, NC). A P value equal to or less than 0.05 was taken to indicate statistical significance. 3. Results 3.1. Digestibility Cobia readily consumed the test diets at approximately 3% body weight and the 30% inclusion of Navita or SBM did not result in reduced diet palatability. Feces were collected exclusively from the distal gastrointestinal tract (rectum). The ADCs for protein, energy and amino acids varied considerably (Table 4) with significant differences (P b 0.05) detected among the fish fed diets containing Navita or SBM. The ADC for crude protein was almost 20% higher in Navita than for SBM. In general, most amino acid ADCs had numerically higher values for Navita than for SBM. For instance, increases of 20 to 80% or more were observed in ADCs for isoleucine, threonine, valine, cysteine, and serine in fish fed the Navita diet.
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3.2. Amino acids, growth performance and feed utilization All experimental diets utilized in the feeding trial presented a balanced profile for essential or indispensable amino acids (Table 3). The effects of amino acid supplementation were also evident for lysine, methionine and glycine. Table 5 summarizes several performance indicators for cobia fed the various experimental diets during the 3 weighing periods. No significant differences in VSI, MR, or HSI were identified among fish fed the four diets, indicating that there was no physiological alteration due to the increased inclusion levels of soybased products. In the present study, although slight differences were detected in WG and SGR these were only transitional in nature (for the 1st sampling period only) and fish fed all diets exhibited excellent growth, comparable to commercial conditions (Benetti et al., 2010), at the end of the experiment. As it relates to dietary formulation and growth, FIFO ratios remained significantly different (P N 0.05) throughout the experiment. 4. Discussion The determined ADCs for protein and amino acids, presented in Table 4, are generally lower than those described for cobia in previous digestibility studies (Zhou et al., 2004). The use of a different fecal collection technique – settling columns vs. manual stripping – may explain in part these differences. As explained by Glencross et al. (2005), test diets containing high levels of vegetable ingredients including SBM may result in fecal pellets of lesser density and thus prone to increased leeching. Therefore, the use of manual stripping may have rendered more accurate digestibility estimations in the present experiment. As expected, the magnitude of the ADC differences between the two techniques is in line with the values ascribed to the leaching of soluble nutrients into culture water (NRC, 2011). Significantly, of all the analyzed nutrients (18) twelve exhibited significantly higher ADCs (P N 0.05) for Navita as compared to conventional SBM. These results are in agreement to those obtained for rainbow trout (Barrows, 2011, personal communication) and may be due in part to the reduced oligosaccharide content of the Navita meal (N 0.5%, raffinose + stachyose) as compared to that present in conventional SBM (~6%). It is well established that complex carbohydrates including oligosaccharides are indigestible to monogastric animals including carnivorous fish (Francis et al., 2001; Glencross et al., 2003). Although expensive, the removal of Table 3 Amino acid composition of experimental diets for the feeding trial. Amino acid
Experimental diets 1
2
3
4
g/kg (dry weight basis) Essential Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine
23.0 10.0 19.0 31.0 25.0 7.0 22.0 17.0 21.0
22.0 10.0 17.0 28.0 17.0 6.0 24.0 14.0 18.0
29.0 12.0 21.0 35.0 26.0 9.0 26.0 18.0 23.0
31.0 13.0 21.0 37.0 26.0 10.0 27.0 19.0 24.0
Non-essential Alanine Aspartic acida Cysteine Glutamic acidb Glycine Proline Serine
22.0 29.0 1.0 48.0 36.0 23.0 18.0
16.0 24.0 1.0 41.0 34.0 21.0 17.0
20.0 34.0 1.0 57.0 41.0 26.0 22.0
21.0 35.0 2.0 60.0 44.0 27.0 24.0
a b
Aspartic acid + asparagine. Glutamic acid + glutamine.
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Table 4 ADCs for crude protein and amino acids from regular soybean meal or Navita in extruded diets for cobia. Test ingredient Navita
SBM
Pr N F
81.8
68.5
0.0360
8.67
Indispensable amino acids ADC Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine
102.6 86.0 76.2 76.3 68.3 76.6 74.5 74.2 74.5
97.9 79.2 58.1 64.8 65.0 82.0 73.1 41.5 58.4
0.0023 0.0415 0.0038 0.0118 0.0416 0.0655 0.588 0.000 0.0047
2.71 4.52 10.5 6.92 2.21 3.75 2.83 18.0 9.36
Dispensable amino acids ADC Alanine Aspartic acida Cysteine Glutamic acidb Glycine Proline Serine
88.3 91.0 77.2 86.1 100.5 80.1 108.7
80.1 87.6 64.9 78.8 78.9 72.5 73.3
0.1041 0.217 0.004 0.0064 0.0060 0.1316 0.0000
6.20 3.08 7.13 4.24 12.6 6.06 19.5
Crude protein ADC
a b
P.S.E.
Aspartic acid + asparagine. Glutamic acid + glutamine.
ethanol-soluble components of plant-based meals has significant influence on their apparent digestibility, with clearly identifiable effects improving the digestibility of the nitrogen, organic matter and nitrogenfree extractive components. In addition, conventional SBM is known to cause serious enteropathies in the intestine of salmonids, commonly referred to as SBM-induced enteritis (Krogdahl et al., 2003), and these affections of the intestinal tract are closely correlated with raffinose and stachyose, saponins, lectins and other ANFs present in soy products (Refstie et al., 1998, 2005). As in dogs and chickens, it is possible that excessive dietary raffinose and stachyose in fish may not only disrupt nutrient digestibility (Yamka et al., 2004), but also alter microbial populations in the gastrointestinal tract (GIT) and also reduce the apparent metabolizable energy (Perryman and Dozier, 2012) as well as modify certain indices of the immune system (Grieshop et al., 2004). Overall, results indicate that the use of Navita meal in cobia diets results in an improved digestibility of key nutrients. The present experiment pioneers in documenting growth performance of cobia of a size similar to that utilized in commercial operations from initial stocking to market size and thus should provide valuable information to fish farmers and aquafeed manufacturers as it relates to the developmental stage during which maximum feed consumption occurs. The similar growth performance attained as vegetable protein sources replaced dietary FM was expected and is in line with previous reports of juvenile cobia exhibiting similar production characteristics when fed plant-based diets (Lunger et al., 2006, 2007; Salze et al., 2010; Zhou et al., 2006). What is perhaps more relevant is that this was accomplished with minimum amino acid supplementation (Table 2) and relaying only on SBM, SPC and Navita meal as protein sources. Further, the perfect survival (100%) of all experimental organisms during the entire trial serves as clear evidence of the nutritional sufficiency of all experimental diets. Data from Table 5 indicates that while all diets supported optimal growth and no significant differences (P N 0.05) were found for any of the evaluated performance parameters, FIFO ratios were always best for the high Navita inclusion diet (diet 4). This is one of the most contentious issues in aquaculture so it deserves special consideration. The debate has centered primarily around the use of fish oil (FO) and FM in salmon diets, and a wide range of different values have been quoted for the number of tons of wild fish it takes to produce a ton of farmed salmon (Jackson, 2010). Presently, aquaculture converts 65% of the wild fish reduced into FM at a FIFO ratio of about 0.7 (Jackson,
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Table 5 Performance indicators for commercial-size cobia fed the experimental diets at different sampling periods. Final weight
WG1
ADG2
SGR3
MDI4
GEI5
GPI6
FE7
PER8
FCR9
kg
%
g/d
%/d
g/d
kcal/d
g/d
g gain/g fed
%
g fed/g gain
Sample period 1–29 d 1 2 3 4 Pr N F P.S.E.
2.38 2.58 2.50 2.38 0.24 0.46
35.5a 24.4b 35.6a 33.1a 0.014 15.04
20.5 17.6 22.1 19.9 0.62 3.9
1.02a 0.74b 1.02a 0.97a 0.008 0.37
35.7 34.3 39.5 34.1 0.4 4.3
174.8 168.1 192.9 165.0 0.4 21.3
16.9 16.9 19.2 17.1 0.5 2.1
0.56 0.51 0.57 0.57 0.82 0.08
1.20 1.00 1.17 1.14 0.7 0.17
1.80 1.95 1.81 1.74 0.87 0.28
2.08c 1.17b 1.10b 0.91a 0.003 0.52
Sample period 2–60 d12 1 2 3 4 Pr N F P.S.E.
2.80 3.00 2.84 2.81 0.43 0.60
56.2 49.9 52.3 54.5 0.1 17.8
16.9 15.8 16.4 16.4 0.94 1.91
0.76a 0.61b 0.69a 0.71a 0.04 0.21
34.3 33.5 35.3 33.2 0.81 2.60
167.7 164.5 172.2 160.7 0.78 12.70
16.2 16.5 17.2 16.6 0.86 1.27
0.49 0.47 0.46 0.49 0.60 0.03
1.00 0.95 0.95 0.98 0.45 0.06
2.0 2.1 2.1 2.0 0.69 0.13
2.30c 1.27b 1.31b 1.00a 0.000 0.54
Sample period 1–90 d13 1 2 3 4 Pr N F P.S.E.
3.15 3.3 3.27 3.1 0.60 0.75
78.5 64.1 75.3 73.1 0.24 28.9
15.0 13.9 15.4 14.2 0.72 1.64
0.62 0.53 0.59 0.58 0.25 0.18
35.9 34.4 36.1 35.9 0.90 2.75
175.6 168.7 176 173.9 0.93 13.20
16.9 17.0 17.5 18.0 0.80 1.40
0.41 0.40 0.43 0.39 0.30 0.02
0.88 0.82 0.88 0.78 0.07 0.05
2.4 2.5 2.3 2.5 0.31 0.13
2.8b 1.5a 1.4a 1.3a 0.000 0.63
Dietary treatment
FIFO10
11
Values in a column that do not have the same superscript letters are significantly different according to Duncan's multiple range test (P b 0.05). 1 Weight gain = ((final weight − initial weight) × 100) / initial weight. 2 ADG = average daily gain. 3 SGR = 100 × (ln average final weight − ln average initial weight) / numbers of days. 4 MDI = mean daily intake. 5 GEI = gross energy intake. 6 GPI = gross protein intake. 7 FE = feed efficiency. 8 PER = protein efficiency ratio. 9 FCR = feed conversion ratio. 10 FIFO = fish in:fish out ratio = (g fish meal in the diet + g fish oil in the diet) / (g fish meal from wild fish + g fish oil from wild fish) × FCR. 11 Initial weight: 1.86 ± 0.3 kg; final biomass density: 6.5 ± 0.3 kg of fish/m3. 12 Final biomass: density 7.6 ± 0.4 kg of fish/m3. 13 Final biomass: density 8.7 ± 0.5 kg of fish/m3.
2010; Kaushik and Troell, 2010; Tacon and Metian, 2008). Reported FIFO ratios for salmon aquaculture and marine fish in general are 2.2 and 1.9, respectively (Jackson, 2010). In the current study with cobia, FIFO ratios of 0.9, 1.0 and 1.3 were obtained for fish fed diet 4 with the highest concentration of Navita meal, and only 5.0% inclusion of FM and 8.9% of FO, for the 3 sampling periods (Table 5). These ratios were significantly lower than those obtained for fish fed all other diets and point to the fact that during the grow out when the bulk of the feed is consumed – the most important operation cost in aquaculture – diets can be formulated such that a high FM replacement is attained with no detriment to growth performance, feed efficiency or survival. Interestingly, a public analysis recently conducted by Agralytica (Young, 2013, personal communication) indicates that, on average, percent costs of SPC (Brazilian, feed grade, ~60% crude protein) and Navita are 63 and 50% that of Peruvian anchovy meal, respectively. It thus follows that, while providing equivalent growth, Navita may provide a more economical alternative to FM. Although not reflected in growth (Table 5) or biological indices (Table 6), digestibility data from the present experiment is particularly important from both commercial and environmental perspectives. Not only the inclusion of Navita meal improved the FIFO ratios but the documented higher ADCs indicate that more nutrients are being deposited into tissue, thus lessening the environmental footprint of cobia aquaculture. A complete characterization of the effects of incorporating Navita in fish diets, assessing generated waste and water quality, is necessary to confirm this notion. Also, the physiological effects involved in metabolizing plant-based proteins for carnivorous marine fish are largely unknown and should be further investigated. A relevant finding of the present experiments is the fact that Navita can be incorporated at very high levels (45%) in the diet of
marine carnivorous fish with no detriment to performance. These results have been validated in successive feeding trials with these and other fish species.
Acknowledgments We wish to express our gratitude to the Illinois Soybean Association (Mr. Mark Albertson) for funding this research. Our sincere thanks to Mr. Francisco de la Torre and Dr. Michael Cremer for their support. Also the assistance of the Oceanic Institute (Dr. Warren Domini), the Fish Nutrition Laboratory at Texas A&M University (Dr. Delbert Gatlin) and the Food Protein Research & Development Center (Dr. Mian Riaz) also at Texas A&M University in diet manufacturing and sample analysis
Table 6 Biological indices of commercial-size cobia at the end of the feeding trial. Dietary treatment
MR1
VSI2
HSI3
1 2 3 4 Pr N F P.S.E.
48.3 46.2 46.4 43.4 0.459 3.49
9.2 10.7 9.5 9.0 0.179 1.00
2.1 1.8 2.0 2.0 0.720 0.33
Values in a column that do not have the same superscript letters are significantly different according to Duncan's multiple range test (P b 0.05). 1 MR: muscle ratio = muscle weight ∗ 100 / body weight. 2 VSI: viscero-somatic index = VSI weight ∗ 100 / body weight. 3 HSI: hepato-somatic index = HSI weight ∗ 100 / body weight.
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Replacement of fish meal by a novel non-GM variety of soybean meal in cobia, Rachycentron canadum: Ingredient nutrient digestibility and growth performance Jorge A. Suarez a,⁎, Carlos Tudela a, Drew Davis a, Zachary Daugherty a, Matthew Taynor a, Lindsay Glass a, Ronald Hoenig a, Alejandro Buentello b, Daniel D. Benetti a a b
University of Miami, Rosenstiel School of Marine and Atmospheric Science, 4600 Rickenbacker Causeway, Miami, FL 33149, USA Schillinger Genetics and Navita Premium Feed Ingredients, 4401 Westown Parkway, Suite 225, West Des Moines, IA 50266, USA
a r t i c l e
i n f o
Article history: Received 12 August 2013 Received in revised form 24 September 2013 Accepted 26 September 2013 Available online 2 October 2013 Keywords: Cobia Digestibility Non-GM soy Fish meal replacement Aquafeeds
a b s t r a c t A constraint for the expansion of cobia aquaculture is the availability of high quality formulated diets which reduce or eliminate fish meal (FM) protein. Therefore, the nutritive value of a novel soybean cultivar, Navita™ (Navita, non-genetically modified and selectively bred soy), and regular, commodity soybean meal (SBM, de-hulled, defatted, roasted and solvent-extracted) was evaluated for cobia, Rachycentron canadum via separate digestibility and growth trials. In the first experiment Navita's apparent digestibility coefficients (ADC) were higher than those of SBM for nearly every nutrient evaluated. Crude protein ADCs were 82 and 69% for Navita and SBM, respectively. Apparent DC for amino acids ranged from 68 to 109% for Navita whereas, amino acid ADCs for SBM varied from 42 to 98%. The feeding trial utilized fish of a size that more closely resembles commercial cobia stocking (1.8 kg), and was conducted over a 91-day period. Experimental diets (iso-nitrogenous and iso-energetic) were formulated such that 67% of the FM protein in the reference diet was replaced by either a combination of SBM + soy protein concentrate (SPC, Solae Profine®) labeled MXSB-diet, or by a combination of SPC + Navita; Navita-diet, hereafter. A fourth experimental diet had 80% of the FM protein replaced by a combination of Navita + SPC and was identified as Navita-high. No significant differences (P N 0.05) were observed in fish fed the experimental diets for feed conversion ratio, protein efficiency ratio, feed efficiency, mean daily intake, gross protein intake, gross energy intake, visceral somatic index, muscle ratio, and hepatosomatic index. Fish fed the Navita-high diet had the lowest fish in:fish out ratio (FIFO) at 0.9 ± 0.16. These results indicate that Navita meal can be incorporated at very high levels in the diet of marine carnivorous fish such as cobia with no detriment to performance, making it a prime candidate for FM replacement in aquafeeds. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Aquaculture is a rapidly growing food-producing industry; however, there is still considerable potential for increased efficiency and efficacy of aquaculture through development of nutritious and cost-effective alternatives to traditional marine protein feedstuffs such as fish meal (FM). For cobia aquaculture, this is particularly relevant as suitable diets which reduce or eliminate FM protein are not yet commercially available. This species is the only member of the family Rachycentridae which is distributed worldwide in tropical and subtropical waters (Benetti et al., 2008; Ditty and Shaw, 1992; Shaffer and Nakamura, 1989). Cobia is well recognized for its fast growth and excellent meat quality and has been intensively farmed since the 1990s (Liao et al., 2004). The technology for reliable broodstock spawning and mass production of cobia fingerlings has been long established at the University ⁎ Corresponding author. Tel.: +1 305 968 5849; fax: +1 305 421 4889. E-mail addresses: [email protected] (J.A. Suarez), [email protected] (A. Buentello). 0044-8486/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquaculture.2013.09.049
of Miami Experimental Hatchery (UMEH). However, while the fundamental technology for cobia production is in place, more research is needed to resolve bottle-neck issues of cobia culture, particularly at the grow-out stage. One such limitation is the formulation of commercial diets with reduced FM levels that are both economical and environmentally friendly, while also maintaining optimal growth performance and disease resistance. The global FM supply remains relatively static but demand and price continue to increase (Naylor et al., 2009). Therefore, FM is a raw material that cannot be relied upon for aquaculture expansion. In contrast, soybean meal (SBM) has long been recognized as an excellent source of protein for animals and humans (Baker, 2000). However, SBM inclusion levels as replacement of FM in aquafeeds are limited by species-specific digestive physiology and by the presence of both heat-resistant and thermo-labile anti-nutritional factors (ANFs). While solvent-extracting and cooking may significantly reduce the biological activity of temperature-sensitive protease inhibitors, this processing also renders protein less available for absorption at the gastrointestinal level. Therefore, the present study was conducted to evaluate the apparent digestibility coefficients (ADCs) of protein and amino acids of a novel
J.A. Suarez et al. / Aquaculture 416–417 (2013) 328–333
variety of non-genetically modified (GM) soybean product (Navita™) and contrast these coefficients against those obtained from fish fed conventional SBM. In addition, a study oriented at maximizing FM replacement in cobia diets, using fish close to marketable size was performed. The use of protein sources such as standard SBM, soy protein concentrates (SPC) and non-GM soy from selective breeding programs could greatly improve the profitability and ecologic sustainability of the industry. The overarching objective was to improve both the ecological and economic efficiencies of formulated feeds for cobia, Rachycentron canadum. 2. Materials and methods Care and handling of the fish as well as procedures used in this study were reviewed and approved by the University of Miami Animal Care and Use Committee. Navita™ meal was donated by Navita Premium Feed Ingredients (NPFI), West Des Moines, IA. This is a genetically unique, patented non-GM soy cultivar, which contains increased levels of protein and amino acids for animal feed and reduced ANF levels. Beans are made into a defatted meal by conventional methods but this particular variety is selectively bred to have up to 20% higher protein density than the commodity SBM and ultra-low levels of oligosaccharides (0.6%, raffinose + stachyose). In contrast, conventional SBM has 6% of oligosaccharides. 2.1. Digestibility trial — experimental diets Digestibility diets (Table 1) were manufactured at the Oceanic Institute, Waimanalo, HI and were comprised of a 70:30 mixture of the reference diet and the Navita™ meal. By including the reference diet at 70%, and with the crude protein contents inherent to the test ingredient, the resulting experimental diet had an analyzed crude protein and lipid values of 45 and 12%, respectively, which satisfies the requirements defined for cobia (Fraser and Davies, 2009). Yttrium oxide (Y2O3) was included at 0.5% of the reference diet as an inert, indigestible marker. The dietary ingredients were first ground to a uniform particle size (b 1mm) in a hammer mill, then batch-mixed in a commercial ribbon blender
Table 1 Reference and test diets for determination of apparent digestibility coefficients in cobia. Ingredient
Reference diet
Navitaa
Regular soybean mealb
g/kg of diet Pollock fish mealc Dextrin Menhaden oil Mineral premixd Vitamin premixe Carboxylmethyl cellulosef Cellufilf Yttrium oxideg Test ingredient Total
612.6 120.0 69.4 50.0 40.0 30.0 73.0 5.0 – 1000
g
700.0
700.0
300.0 1000
300.0 1000
a Navita Premium Feed Ingredients, West Des Moines, IA, (g/kg) 558.8 crude protein, 893.3 dry matter, 5.8 crude lipid, 31.0 crude fiber, 58.1 ash, non-genetically modified soy. b Archer Daniels Midland, Decatur, IL, 480.0 crude protein, 933.8 dry matter, 18.4 crude lipid, 38.0 crude fiber, 61.9 ash, commodity roasted/cooked and hexane extracted, genetically modified soy. c Alaska Pollock (Theragra clalcogramma) meal Genuine Alaska Pollock Producers, Seattle, WA, (g/kg) 698.8 crude protein, 734.5 dry matter, 7.9 crude lipid. d Composition (g/kg): Ca(H2PO4)2∙H2O, 136.00; Ca(C6H10O6)∙5H2O, 348.553; FeSO4∙7H2O, 5.00; MgSO4∙7H2O, 132.00; K2HPO4, 240.00; NaH2PO4∙H2O, 88.00; NaCl, 45.00; AlCl3∙6H2O, 0.084; KI, 0.15; CuSO4∙5H2O, 0.50; MnSO4∙H2O, 0.70; CoCl2∙6H2O, 1.00; ZnSO4∙7H2O, 3.00; NaSeO3, 0.0127. e Composition (g/kg): ascorbic acid, 50; DL-calcium pantothenate, 5.0; choline chloride, 36.2; inositol, 5.0; menadione sodium bisulfite, 2.0; niacin, 5.0; pyridoxine HCl, 1.0; riboflavin, 3.0; thiamine mononitrate, 0.5; DL-α-tocopherol acetate (250 IU/g), 8.0; vitamin A palmitate (500,000 IU/g), 0.2; micro-mix, 10.0; cellulose, 874.1. Micro-mix composition (g/100 g): biotin, 0.50; folic acid, 1.8; vitamin B12, 0.02; cholecalciferol (40 IU/μg), 0.02; cellulose, 97.66. f USB-Affymetrix, Cleveland, OH. g Sigma-Aldrich Company, St. Louis, MO.
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(Ross 42-A-10, Charles Ross and Son Co., Hauppauge, NY), preconditioned at 94–99 °C and extruded (Wenger X-20, Wenger Manufacturing, Sabetha, KS) at 99–138°C through a 5-mm die to produce buoyant pellets. 2.2. Digestibility trial — fish and experimental conditions Juvenile cobia (initially weighing 286 ± 10 g/fish), raised in tanks at UMEH (Miami, FL) were seined and graded. These fish were stocked into four, 4500-L fiberglass round tanks at a density of 80 fish/tank. Sand-filtered well water entered each tank at a rate of 50 L/min (1600% exchange per day) and exited via an internal standpipe. Supplemental aeration was provided to each tank to maintain dissolved oxygen levels near saturation. Tanks were covered by a shading mesh with natural illumination. Water temperature, salinity and dissolved oxygen concentration were monitored daily (YSI-57) and averaged 24.4 °C, 33.5 ppt and 6.3 ppm, respectively. Water quality parameters were monitored weekly for pH, ammonia, nitrite and nitrate using a Hach™ water quality test kit and remained within recommended levels for cobia (Benetti et al., 2007) throughout the experiment. After a 7-day conditioning period during which all fish were fed the reference diet once per day (900 h) to apparent satiation, feeding of the test diet was initiated. Fish in each tank received the experimental diet for 7 days prior to the collection of fecal samples. Feces were collected 3–4 h post feeding at each collection period by the fecal stripping technique described by Austreng (1978) and Hajen et al. (1993), with modifications. Fish were stripped one day per week, for three consecutive weeks. Briefly, at the appointed collection period fish were dip-netted from the tank and a solution of MS-222 (500 mg/L; Finiquel, Argent Chemical, Redmond WA) was sprayed onto the gill cavity and the opercula held closed until complete relaxation of abdominal muscles was attained. Immediately afterwards, the gills were completely rinsed with a gentle stream of seawater for 30 s, flushing any residual anesthetic. Gentle pressure was then applied to the lower abdomen of the fish, thus expressing feces from the distal intestine onto clean stainless steel bowls. Care was taken to exclude urine, mucus, and other contaminants from the collection vessels. After fecal collection, fish were introduced into a plastic tub with oxygenated seawater until complete recovery. Fecal samples were oven-dried (60 °C) overnight, stored frozen (−80 °C) and sent on dry ice to the Fish Nutrition Laboratory of Texas A&M University for analyses. Yttrium oxide was analyzed by inductivity coupled plasma mass spectrometry (ICP-AES analysis, Perkin Elmer Optia 3000DV; Perkin Elmer, Wellesley, MA, USA) at 371 nm. All yttrium analyses were conducted in duplicate. Proximate composition of diets and fecal samples was analyzed using established methodologies for dry matter, crude protein (AOAC, 2005), lipids (Folch et al., 1957) and ash (AOAC, 2005). Crude protein was estimated by measuring total nitrogen by the Dumas method (Ebeling, 1968) and multiplied by 6.25. Dry matter was determined by heating the samples at 125 °C for 3 h, and ash was quantified after heating at, 650 °C for 3 h (AOAC, 1990). Crude lipid was determined by chloroform and methanol extraction (Folch et al., 1957). Gross energy content was measured by combustion via bomb calorimeter (Parr Instrument Company, Moline, IL, USA) using benzoic acid as a standard. Amino acid content was quantified after acid hydrolysis with 6 N HCl according to procedures described by Pohlenz et al. (2012). Calcium and Phosphorus were quantified by Inductively Coupled Plasma Spectroscopy. Plasma amino acids were assayed via HPLC following a fluorometric technique (Buentello and Gatlin, 2000) using pre-column derivatization with o-phthaldialdehyde (Sigma, St. Louis, MO). Navita ADCs for crude protein and amino acids were calculated from a comparison of the apparent nutrient digestibility of the reference diet and experimental diets as described by Forster (1999). 2.3. Feeding trial — systems, experimental diets and feeding The feeding trial was conducted in 12 cylindrical, fiberglass tanks (4000-L each) operated as a flow-through system with filtered water
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(Triton II sand filter) flowing at approximately 44 L/min (1600% exchange/day). Water quality parameters were monitored daily with a YSI Professional Plus meter (YSI, Inc., Yellow Springs, OH) and temperature remained at 24.7 ± 0.01°C throughout the trial. Salinity was monitored at about 33.5‰, pH 7.25 ± 0.03 and dissolved oxygen was maintained above 8.6 ± 0.11 ppm using low pressure electrical blowers and air stones. A natural photoperiod defined the cycle of hours of light and dark at Miami's latitude. Purified and practical ingredients for the experimental diets were purchased and sent to the Food Protein R&D Center at Texas A&M University for diet manufacturing via extrusion. A FM-based control diet (diet 1) – similar to commercial cobia diets – was formulated to contain 46% crude protein from menhaden FM (Special Select, Omega Protein, Houston, TX), SPC, SBM and wheat flour which was also use to provide starch-related binding (Table 2). Menhaden oil (Virginia Prime, Omega Protein) was included as lipid source and provided in combination with FM a total of 11% lipid and an estimated digestible energy level of 4.9 kcal/kg. Three experimental diets were derived from the control diet by replacing FM protein with an increased level of SPC (diet 2) or two different levels of Navita (diets 3 and 4) while maintaining diets iso-nitrogenous and iso-energetic. As plant protein sources increased in the diet an increased supplementation of glycine (Gly, USB-Affymetrix, Cleveland, OH) was used as palatability enhancer. Similarly, as vegetable protein increased in the diet supplemental dietary taurine (Tau) was added. When necessary, crystalline lysine-HCl and L-methionine were used to balance dietary levels of lysine and sulfur-containing amino acids. Diets met all the currently established nutritional requirements for juvenile cobia (Chou et al., 2001, 2004; Craig et al., 2006; NRC, 2011; Romarheim et al., 2008; Salze et al., 2010, 2011; Sun et al., 2006; Zhou et al., 2004).
Juvenile cobia (1.87 ± 0.35 kg) were obtained from the reproduction program at UMEH and stocked at a density of 5 g/L (20 kg biomass/ tank). Prior to the start of the experiment numeric T-bar tags were inserted into each fish's musculature, just below the dorsal fin with the tag barb anchored through the boney fin ray supports. These tags were used for fish identification to track individual growth performance at three different weighing periods (days 29, 60 and 91). After implant recovery fish were conditioned on a commercial diet (Zeigler Bros., Inc., East Berlin, PA) and acclimated to the experimental conditions for 2 weeks prior to the feeding trial. Each experimental diet was then randomly assigned to specific tanks and fish were fed once a day (900h) ad libitum (~3% of body weight per day) for 91days. There were three replicate tanks per dietary treatment. The total feed consumption per tank was recorded daily. An initial sample of 5 fish was collected at the onset of the experiment and 3 additional fish per tank were secured at each harvest period, euthanized using MS-222 (800 mg/L), measured for weight and total length, and stored frozen (−80°C) until analyzed for moisture, ash, protein and lipid according to established procedures (Webb and Gatlin, 2003). An additional 3 fish per tank were euthanized in the same manner, dissected and the following parameters were determined:
Viscerosomatic indexðVSIÞ ð% Þ ¼ viscera weightðgÞ=fish weightðgÞ 100
Hepatosomatic indexðHSIÞ ð% Þ ¼ liver weightðgÞ=fish weightðgÞ 100
Muscle ratioðMRÞ ð% Þ ¼ muscle weightðgÞ 100=body weightðgÞ: Table 2 Formulated and analyzed composition of experimental diets for the feeding trial. Ingredient
Experimental diets 1
2
3
4
g/kg (dry weight basis) Menhaden fish meal, Special Select™a Soy protein concentrateb Regular soybean mealc Navitad Wheat floure Menhaden oila Mineral–vitamin premixf Glycineg Taurineg Lysine HClg g DL-methionine Cellufilg Total Analyzed composition (n = 3) Crude protein Crude lipid Moisture Ash Calcium Phosphorus Energy (kcal digestible energy/kg) a
256.5 102.5 320.0 0.0 160.0 59.5 60.0 10.0 5.0 0.0 0.9 24.0 1000.0
84.0 300.0 280.0 0.0 160.0 81.2 60.0 16.4 12.0 2.0 2.1 30.0 1000.0
84.0 190.0 0.0 376.5 159.0 82.3 60.0 16.4 12.0 1.0 4.9 14.0 1000.0
60.0 150.0 0.0 453.6 150.0 86.2 60.0 18.4 15.0 1.0 5.6 2.0 1000.0
473.0 112.0 91.0 108.0 22.0 18.0 4.9
493.0 103.0 83.0 84.0 15.0 13.0 4.9
487.0 105.0 74.0 84.0 13.0 13.0 4.9
502.0 98.0 82.0 83.0 10.0 11.0 4.8
Omega Protein Corp. (Hammond, LA, USA). Solae Profine VP, food-grade (intended for human consumption) St. Louis, MO, 720 g/kg crude protein. c Archer Daniels Midland, Decatur, IL, 480.0 crude protein, 933.8 dry matter, 18.4 crude lipid, 38.0 crude fiber, 61.9 ash, commodity roasted/cooked and hexane extracted, genetically modified soy. d Navita Premium Feed Ingredients, West Des Moines, IA, (g/kg) 558.8 crude protein, 893.3 dry matter, 5.8 crude lipid, 31.0 crude fiber, 58.1 ash, non-genetically modified soy. e Dawn Food Products, Inc., FL, 600 g/kg crude protein. f Same as d + e in Table 1. g USB-Affymetrix, Cleveland, OH. b
In addition, growth performance and feed consumption were evaluated using established parameters (AOAC, 2005; Zhou et al., 2010) including percent weight gain (WG), average daily gain (ADG), specific growth rate (SRG), mean daily intake (MDI), gross energy intake (GEI), gross protein intake (GPI), feed efficiency (FE), feed conversion ratio (FCR), protein efficiency ratio, (PER) and fish in:fish out (FIFO). Data was evaluated for normality using the Shapiro–Wilk test and no transformations were necessary. Mean ADCs for protein, energy and amino acids were contrasted using Student's t-tests to determine if the two sets of data (Navita vs. SBM) were significantly different from each other. Results from the feeding trial were analyzed via oneway analysis of variance (ANOVA), using Duncan's multiple-range test for mean separation. All statistical analyses were performed using the Statistical Analysis System (SAS) version 9.2 (SAS Institute Inc., Cary, NC). A P value equal to or less than 0.05 was taken to indicate statistical significance. 3. Results 3.1. Digestibility Cobia readily consumed the test diets at approximately 3% body weight and the 30% inclusion of Navita or SBM did not result in reduced diet palatability. Feces were collected exclusively from the distal gastrointestinal tract (rectum). The ADCs for protein, energy and amino acids varied considerably (Table 4) with significant differences (P b 0.05) detected among the fish fed diets containing Navita or SBM. The ADC for crude protein was almost 20% higher in Navita than for SBM. In general, most amino acid ADCs had numerically higher values for Navita than for SBM. For instance, increases of 20 to 80% or more were observed in ADCs for isoleucine, threonine, valine, cysteine, and serine in fish fed the Navita diet.
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3.2. Amino acids, growth performance and feed utilization All experimental diets utilized in the feeding trial presented a balanced profile for essential or indispensable amino acids (Table 3). The effects of amino acid supplementation were also evident for lysine, methionine and glycine. Table 5 summarizes several performance indicators for cobia fed the various experimental diets during the 3 weighing periods. No significant differences in VSI, MR, or HSI were identified among fish fed the four diets, indicating that there was no physiological alteration due to the increased inclusion levels of soybased products. In the present study, although slight differences were detected in WG and SGR these were only transitional in nature (for the 1st sampling period only) and fish fed all diets exhibited excellent growth, comparable to commercial conditions (Benetti et al., 2010), at the end of the experiment. As it relates to dietary formulation and growth, FIFO ratios remained significantly different (P N 0.05) throughout the experiment. 4. Discussion The determined ADCs for protein and amino acids, presented in Table 4, are generally lower than those described for cobia in previous digestibility studies (Zhou et al., 2004). The use of a different fecal collection technique – settling columns vs. manual stripping – may explain in part these differences. As explained by Glencross et al. (2005), test diets containing high levels of vegetable ingredients including SBM may result in fecal pellets of lesser density and thus prone to increased leeching. Therefore, the use of manual stripping may have rendered more accurate digestibility estimations in the present experiment. As expected, the magnitude of the ADC differences between the two techniques is in line with the values ascribed to the leaching of soluble nutrients into culture water (NRC, 2011). Significantly, of all the analyzed nutrients (18) twelve exhibited significantly higher ADCs (P N 0.05) for Navita as compared to conventional SBM. These results are in agreement to those obtained for rainbow trout (Barrows, 2011, personal communication) and may be due in part to the reduced oligosaccharide content of the Navita meal (N 0.5%, raffinose + stachyose) as compared to that present in conventional SBM (~6%). It is well established that complex carbohydrates including oligosaccharides are indigestible to monogastric animals including carnivorous fish (Francis et al., 2001; Glencross et al., 2003). Although expensive, the removal of Table 3 Amino acid composition of experimental diets for the feeding trial. Amino acid
Experimental diets 1
2
3
4
g/kg (dry weight basis) Essential Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine
23.0 10.0 19.0 31.0 25.0 7.0 22.0 17.0 21.0
22.0 10.0 17.0 28.0 17.0 6.0 24.0 14.0 18.0
29.0 12.0 21.0 35.0 26.0 9.0 26.0 18.0 23.0
31.0 13.0 21.0 37.0 26.0 10.0 27.0 19.0 24.0
Non-essential Alanine Aspartic acida Cysteine Glutamic acidb Glycine Proline Serine
22.0 29.0 1.0 48.0 36.0 23.0 18.0
16.0 24.0 1.0 41.0 34.0 21.0 17.0
20.0 34.0 1.0 57.0 41.0 26.0 22.0
21.0 35.0 2.0 60.0 44.0 27.0 24.0
a b
Aspartic acid + asparagine. Glutamic acid + glutamine.
331
Table 4 ADCs for crude protein and amino acids from regular soybean meal or Navita in extruded diets for cobia. Test ingredient Navita
SBM
Pr N F
81.8
68.5
0.0360
8.67
Indispensable amino acids ADC Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine
102.6 86.0 76.2 76.3 68.3 76.6 74.5 74.2 74.5
97.9 79.2 58.1 64.8 65.0 82.0 73.1 41.5 58.4
0.0023 0.0415 0.0038 0.0118 0.0416 0.0655 0.588 0.000 0.0047
2.71 4.52 10.5 6.92 2.21 3.75 2.83 18.0 9.36
Dispensable amino acids ADC Alanine Aspartic acida Cysteine Glutamic acidb Glycine Proline Serine
88.3 91.0 77.2 86.1 100.5 80.1 108.7
80.1 87.6 64.9 78.8 78.9 72.5 73.3
0.1041 0.217 0.004 0.0064 0.0060 0.1316 0.0000
6.20 3.08 7.13 4.24 12.6 6.06 19.5
Crude protein ADC
a b
P.S.E.
Aspartic acid + asparagine. Glutamic acid + glutamine.
ethanol-soluble components of plant-based meals has significant influence on their apparent digestibility, with clearly identifiable effects improving the digestibility of the nitrogen, organic matter and nitrogenfree extractive components. In addition, conventional SBM is known to cause serious enteropathies in the intestine of salmonids, commonly referred to as SBM-induced enteritis (Krogdahl et al., 2003), and these affections of the intestinal tract are closely correlated with raffinose and stachyose, saponins, lectins and other ANFs present in soy products (Refstie et al., 1998, 2005). As in dogs and chickens, it is possible that excessive dietary raffinose and stachyose in fish may not only disrupt nutrient digestibility (Yamka et al., 2004), but also alter microbial populations in the gastrointestinal tract (GIT) and also reduce the apparent metabolizable energy (Perryman and Dozier, 2012) as well as modify certain indices of the immune system (Grieshop et al., 2004). Overall, results indicate that the use of Navita meal in cobia diets results in an improved digestibility of key nutrients. The present experiment pioneers in documenting growth performance of cobia of a size similar to that utilized in commercial operations from initial stocking to market size and thus should provide valuable information to fish farmers and aquafeed manufacturers as it relates to the developmental stage during which maximum feed consumption occurs. The similar growth performance attained as vegetable protein sources replaced dietary FM was expected and is in line with previous reports of juvenile cobia exhibiting similar production characteristics when fed plant-based diets (Lunger et al., 2006, 2007; Salze et al., 2010; Zhou et al., 2006). What is perhaps more relevant is that this was accomplished with minimum amino acid supplementation (Table 2) and relaying only on SBM, SPC and Navita meal as protein sources. Further, the perfect survival (100%) of all experimental organisms during the entire trial serves as clear evidence of the nutritional sufficiency of all experimental diets. Data from Table 5 indicates that while all diets supported optimal growth and no significant differences (P N 0.05) were found for any of the evaluated performance parameters, FIFO ratios were always best for the high Navita inclusion diet (diet 4). This is one of the most contentious issues in aquaculture so it deserves special consideration. The debate has centered primarily around the use of fish oil (FO) and FM in salmon diets, and a wide range of different values have been quoted for the number of tons of wild fish it takes to produce a ton of farmed salmon (Jackson, 2010). Presently, aquaculture converts 65% of the wild fish reduced into FM at a FIFO ratio of about 0.7 (Jackson,
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Table 5 Performance indicators for commercial-size cobia fed the experimental diets at different sampling periods. Final weight
WG1
ADG2
SGR3
MDI4
GEI5
GPI6
FE7
PER8
FCR9
kg
%
g/d
%/d
g/d
kcal/d
g/d
g gain/g fed
%
g fed/g gain
Sample period 1–29 d 1 2 3 4 Pr N F P.S.E.
2.38 2.58 2.50 2.38 0.24 0.46
35.5a 24.4b 35.6a 33.1a 0.014 15.04
20.5 17.6 22.1 19.9 0.62 3.9
1.02a 0.74b 1.02a 0.97a 0.008 0.37
35.7 34.3 39.5 34.1 0.4 4.3
174.8 168.1 192.9 165.0 0.4 21.3
16.9 16.9 19.2 17.1 0.5 2.1
0.56 0.51 0.57 0.57 0.82 0.08
1.20 1.00 1.17 1.14 0.7 0.17
1.80 1.95 1.81 1.74 0.87 0.28
2.08c 1.17b 1.10b 0.91a 0.003 0.52
Sample period 2–60 d12 1 2 3 4 Pr N F P.S.E.
2.80 3.00 2.84 2.81 0.43 0.60
56.2 49.9 52.3 54.5 0.1 17.8
16.9 15.8 16.4 16.4 0.94 1.91
0.76a 0.61b 0.69a 0.71a 0.04 0.21
34.3 33.5 35.3 33.2 0.81 2.60
167.7 164.5 172.2 160.7 0.78 12.70
16.2 16.5 17.2 16.6 0.86 1.27
0.49 0.47 0.46 0.49 0.60 0.03
1.00 0.95 0.95 0.98 0.45 0.06
2.0 2.1 2.1 2.0 0.69 0.13
2.30c 1.27b 1.31b 1.00a 0.000 0.54
Sample period 1–90 d13 1 2 3 4 Pr N F P.S.E.
3.15 3.3 3.27 3.1 0.60 0.75
78.5 64.1 75.3 73.1 0.24 28.9
15.0 13.9 15.4 14.2 0.72 1.64
0.62 0.53 0.59 0.58 0.25 0.18
35.9 34.4 36.1 35.9 0.90 2.75
175.6 168.7 176 173.9 0.93 13.20
16.9 17.0 17.5 18.0 0.80 1.40
0.41 0.40 0.43 0.39 0.30 0.02
0.88 0.82 0.88 0.78 0.07 0.05
2.4 2.5 2.3 2.5 0.31 0.13
2.8b 1.5a 1.4a 1.3a 0.000 0.63
Dietary treatment
FIFO10
11
Values in a column that do not have the same superscript letters are significantly different according to Duncan's multiple range test (P b 0.05). 1 Weight gain = ((final weight − initial weight) × 100) / initial weight. 2 ADG = average daily gain. 3 SGR = 100 × (ln average final weight − ln average initial weight) / numbers of days. 4 MDI = mean daily intake. 5 GEI = gross energy intake. 6 GPI = gross protein intake. 7 FE = feed efficiency. 8 PER = protein efficiency ratio. 9 FCR = feed conversion ratio. 10 FIFO = fish in:fish out ratio = (g fish meal in the diet + g fish oil in the diet) / (g fish meal from wild fish + g fish oil from wild fish) × FCR. 11 Initial weight: 1.86 ± 0.3 kg; final biomass density: 6.5 ± 0.3 kg of fish/m3. 12 Final biomass: density 7.6 ± 0.4 kg of fish/m3. 13 Final biomass: density 8.7 ± 0.5 kg of fish/m3.
2010; Kaushik and Troell, 2010; Tacon and Metian, 2008). Reported FIFO ratios for salmon aquaculture and marine fish in general are 2.2 and 1.9, respectively (Jackson, 2010). In the current study with cobia, FIFO ratios of 0.9, 1.0 and 1.3 were obtained for fish fed diet 4 with the highest concentration of Navita meal, and only 5.0% inclusion of FM and 8.9% of FO, for the 3 sampling periods (Table 5). These ratios were significantly lower than those obtained for fish fed all other diets and point to the fact that during the grow out when the bulk of the feed is consumed – the most important operation cost in aquaculture – diets can be formulated such that a high FM replacement is attained with no detriment to growth performance, feed efficiency or survival. Interestingly, a public analysis recently conducted by Agralytica (Young, 2013, personal communication) indicates that, on average, percent costs of SPC (Brazilian, feed grade, ~60% crude protein) and Navita are 63 and 50% that of Peruvian anchovy meal, respectively. It thus follows that, while providing equivalent growth, Navita may provide a more economical alternative to FM. Although not reflected in growth (Table 5) or biological indices (Table 6), digestibility data from the present experiment is particularly important from both commercial and environmental perspectives. Not only the inclusion of Navita meal improved the FIFO ratios but the documented higher ADCs indicate that more nutrients are being deposited into tissue, thus lessening the environmental footprint of cobia aquaculture. A complete characterization of the effects of incorporating Navita in fish diets, assessing generated waste and water quality, is necessary to confirm this notion. Also, the physiological effects involved in metabolizing plant-based proteins for carnivorous marine fish are largely unknown and should be further investigated. A relevant finding of the present experiments is the fact that Navita can be incorporated at very high levels (45%) in the diet of
marine carnivorous fish with no detriment to performance. These results have been validated in successive feeding trials with these and other fish species.
Acknowledgments We wish to express our gratitude to the Illinois Soybean Association (Mr. Mark Albertson) for funding this research. Our sincere thanks to Mr. Francisco de la Torre and Dr. Michael Cremer for their support. Also the assistance of the Oceanic Institute (Dr. Warren Domini), the Fish Nutrition Laboratory at Texas A&M University (Dr. Delbert Gatlin) and the Food Protein Research & Development Center (Dr. Mian Riaz) also at Texas A&M University in diet manufacturing and sample analysis
Table 6 Biological indices of commercial-size cobia at the end of the feeding trial. Dietary treatment
MR1
VSI2
HSI3
1 2 3 4 Pr N F P.S.E.
48.3 46.2 46.4 43.4 0.459 3.49
9.2 10.7 9.5 9.0 0.179 1.00
2.1 1.8 2.0 2.0 0.720 0.33
Values in a column that do not have the same superscript letters are significantly different according to Duncan's multiple range test (P b 0.05). 1 MR: muscle ratio = muscle weight ∗ 100 / body weight. 2 VSI: viscero-somatic index = VSI weight ∗ 100 / body weight. 3 HSI: hepato-somatic index = HSI weight ∗ 100 / body weight.
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