Inclusion of soybean meal into summer flounder (Paralichthys dentatus) feeds: An environmentally-friendly protein alternative to fish meal and a potential immunostimulant

Final Report for GNE10-013

Project Type: Graduate Student
Funds awarded in 2010: $14,748.00
Projected End Date: 12/31/2011
Grant Recipient: University of Rhode Island
Region: Northeast
State: Rhode Island
Graduate Student:
Faculty Advisor:
Dr. David Bengtson
University of Rhode Island
Faculty Advisor:
Dr. Marta Gomez-Chiarri
University of Rhode Island
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Project Information

Summary:

There has been increasing emphasis on the replacement of fish meal (FM) with protein from alternative sources, particularly soybean meal (SBM). Although inclusion of SBM into diets for summer flounder at a level of more than 60% replacement leads to decreased growth, replacement of at least 40% results in a reduction in mortality following bacterial challenge. This project was designed to determine if products in soybean meal (as opposed to the more purified soy protein concentrate, SPC) may be responsible for protection of summer flounder against bacterial challenge through immunostimulation. Summer flounder (Paralichthys dentatus) were fed one of three diets utilizing SBM or SPC: control diet (100% of protein from FM), SBM diet (40:60 ratio of FM:SBM as protein source), or SBM:SPC (40:30:30 ratio of FM:SBM:SPC as a protein source). The fish fed the SBM:SPC diet grew significantly more (final weight 36.40±6.16g) than either the control or SBM (24.40±2.26g, 22.54±6.12 final weight, respectively) fed fish (ANOVA, df=2, F-value=15.36, p=0.0044, ?=0.05). Feed conversion ratios (FCRs) for the SBM/SPC fed group (1.01±0.02) was significantly lower than the SBM fed group (1.33±0.07), however it was not significantly lower than the control group (1.21±0.07) (ANOVA, df=2, F-value=7.77, p=0.021, ?=0.05, with Tukey’s Post-Test). Survival following challenge was higher for the fish fed diets which included soy, though not significantly higher. Immune parameters tested (hematocrit, plasma lysozyme activity, bactericidal activity, plasma protein content, and respiratory burst activity) 7 days following bacterial challenge indicated no immunostimulation at this point. Replacement of FM with soy products can result in equal, if not better growth rates at the 60% level. Further experiments need to be done to evaluate the mechanisms of protection against bacterial challenge provided by soybean meal.

Introduction:

World capture fisheries have been in a stable state throughout much of the past decade, and it has been estimated that the maximum capture fisheries potential from global waters has been reached (in 2007 as many as 50 percent of stocks were labeled as fully exploited) (FAO 2008). With US wild groundfish stocks in decline, the US must increase its aquaculture production, or risk increasing the already $8 billion annual trade deficit in seafood (FAO 2008). Aquaculture is currently growing faster than any other animal-producing sector (8.9% annually), currently most of the growth (89%) is found mainly in Asian countries, especially China (FAO 2008).

The continued growth of sustainable aquaculture production depends upon the development of reliable, ecologically sound protein sources to replace fish meal in aquaculture feeds. Fish meal is currently the main source of protein in carnivorous fish feeds (Murray et al. 2010), and the demand for fish meal available for aquafeeds is expected to exceed supply within the next decade (Gatlin et al. 2007). Fish meal is the protein source of choice for many reasons: high protein content, good amino acid profile, high nutritional digestibility, lack of antinutrients, and wide availability (Gatlin et al. 2007).

Fish meal has become extremely expensive as aquaculture production has grown worldwide. As of July 2011 the price was $1,469 per metric ton, which is over 3.7 times higher than the bulk price of soybean meal of $389 per metric ton (www.indexmundi.com). Due to the growing demand of fish meal and the limited supply, it is now apparent that the aquaculture industry must look to alternative, and in many instances terrestrial, protein sources to keep expanding. The soybean (Glycine max), when processed, produces a high quality source of protein (Gatlin et al. 2007) that can replace fish meal as an economically and nutritious alternative.Replacing fish meal with soy protein has shown promising results for many species of flatfish, such as Japanese flounder (Paralichthys olivaceus) (Kikuchi, 1999; Sun et al. 2007), Egyptian sole (Solea aegyptiaca) (Bonaldo et al. 2006), and Atlantic halibut (Hippoglossus hippoglossus) (Murray et al. 2010). The presence of several antinutritional factors in soybean meal, however, prevents full growth performance on fish fed high levels of replacement of fish meal with soybean meal, so the level and mode of incorporation of soybean meal into fish diets needs to be carefully evaluated (Sitja-Bobadilla et al. 2005; Sun et al. 2007; Salah Azaza et al. 2009). Soy protein concentrate does not appear to have the same growth-limiting drawbacks. Widespread incorporation of soybean meal or soy protein concentrate into diets for marine aquaculture not only opens up a new market for soy products, it is also one way to make carnivorous aquaculture much more sustainable.

Preliminary results at the University of Rhode Island suggest that soybean meal can replace at least 40% of fish meal in a diet for summer flounder without a significant decrease in growth (Enterria 2006). Further research needed to be done to optimize the level to which fish meal can be replaced by soybean meals in summer flounder diets. Another main issue facing commercial grow-out of flatfish is disease outbreaks when fish are raised at a high density. At the only marine finfish hatchery in the northeast, GreatBay Aquaculture (Portsmouth, NH), there have been outbreaks of vibriosis which has caused widespread mortality of summer flounder (George Nardi per. comm.) and recent outbreaks of disease at the only summer flounder grow-out site in the northeast, Local Oceans (Hudson, NY). In light of these issues, commercial aquaculture companies are particularly interested in any opportunities to strengthen immune function and prevent disease outbreaks. A recent study at the University of Rhode Island demonstrated possible immunostimulatory effects of soybean meal replacement of fish meal in diets for summer flounder (Lightbourne 2010), suggesting that partial replacement of fish meal with soybean meal in marine finfish diets may provide several economical benefits to farmers, including lower cost and protection against disease outbreaks. The goal of this research was to determine if soybean meal (as opposed to the more purified soy protein concentrate) may be responsible for protection of summer flounder against bacterial challenge through immunostimulation. Increased knowledge on the mechanisms of protection against disease challenge by soybean meal in summer flounder will benefit the aquaculture industry and open a new market to soybean farmers.

Bonaldo A., Roem A.J., Pecchini A., Grilli E., Gatta P.P. 2006. Influence of dietary soybean meal levels on growth, feed utilization, and gut histology of Egyptian sole (Solea aqgyptiaca) juveniles. Aquaculture. 261: 580-586.

Enterria A. 2006. Partial replacement of fish meal with plant protein sources in diets for
summer flounder (Paralichthys dentatus). Masters Thesis. University of Rhode Island, Kingston.

FAO 2008. The State of World Fisheries and Aquaculture 2008. in: Department, F.a.A. (Ed.). Food and Agriculture Organization of the United Nations, Rome.

Gatlin D.M.I., Barrows F.T., Brown P., Dabrowski K., Gaylord T.J., Hardy R.W., Herman E., Hu G., Krogdahl A., Nelson R., Overturf K., Rust M., Sealey W., Skonberg D., Souza E.J., Stone D., Wilson R., Wurtele E. 2007. Expanding the utilization of sustainable plant products in aquafeeds: a review. Aquaculture Research. 38: 551-579.

Indexmundi. 2011. Internet, online. Soybean Meal Monthly Price. http://www.indexmundi.com/commodities/?commodity=soybean–?meal. Site visited 8/25/2011.

Kikuchi K. 1999. The use of defatted soybean meal as a substitute for fish meal in diets of Japanese flounder (Paralichthys olivaceus). Aquaculture. 179: 3-11.

Lightborne C. 2010 effects of soybean meal replacement with added taurine in fish meal diets for summer flounder (Paralichthys dentatus). Masters Thesis. University of Rhode Island, Kingston RI.

Murray H.M., Lall S.P., Rajaselvam R., Boutilier L.A., Blanchard B., Flight R.M., Colombo S., Mohindra V., Douglas S.E. 2010. A nutrigenomic analysis of intestinal response to partial soybean meal replacement in diets for juvenile Atlantic halibut, Hippoglossus hippoglossus, L. Aquaculture. 298: 282-293.

Salah Azaza M., Kammoun W., Abdelmouleh A., Kraiem M.M., 2009. Growth performance, feed utilization, and body composition of Nile tilapia (Oreochromis niloticus L.) fed with differently heated soybean-meal-based diets. Aquaculture International. 17: 507-521.

Sitja-Bobadilla A., Pena-Llopis S., Gomez-Requeni P., Medale F., Kaushik S., Perez-Sanchez J., 2005. Effect of fish meal replacement by plat protein sources on non-specific defense mechanisms and oxidative stress in gilthead sea bream (Sparus aurata). Aquaculture. 249: 387-400.

Sun M., Kim Y.C., Okorie O.E., Lee S., Devnath S., Yoo G., Bai S.C., 2007. Evaluation of fermented soybean curd residues as an energy source in diets for juvenile olive flounder, Paralichthys olivaceus. Journal of the World Aquaculture Society. 38: 536-542.

Project Objectives:
  • Objective 1: Create feeds with fish meal replaced by SBM or SPC to various levels, with balanced amino acid profiles, including additions of taurine and phytase. Objective completed February 2011. The three experimental diets were designed in collaboration with Dr. Lee at the University of Rhode Island. All three diets were designed to be isonitrogenous and isocaloric.

    Objective 2. Feed the diets with SBM or SPC inclusion to summer flounder. Evaluate growth, feed conversion, hematological profiles, immune function, and histological changes to the intestines.Objective was completed by June 2011. A 12 week feed trial was completed at the Marine Biological Laboratory in Woods Hole, MA. Wet weight and length measurements we taken every 2 weeks.

    Objective 3. Evaluate effect of SBM on mortality when challenged with Vibrio harveyi. Objective completed June 2011. Following the completion of the feed trial, the fish were transported to the University of Rhode Island Pathobiology Laboratory, at the Blount Aquaculture Research Facility. Here the bacterial challenge was performed, and mortality monitored, and subsequent immune function analyzed.

    Objective 4. Disseminate results to commercial partners, and present findings at sustainable aquaculture venues in the Northeast. Since the project has ended there have not been any conferences in the Northeast in which this particular project would be appropriate. I have submitted an abstract to present orally at the World Aquaculture Society meeting in February in Las Vegas, and I will submit an abstract to the Northeast Aquaculture Conference and Expo when it becomes available in the fall of 2012. I also have been in touch with both Great Bay Aquaculture and Ziegler to coordinate a trip to present my findings to them this fall.

Cooperators

Click linked name(s) to expand
  • Dr. David Bengtson
  • Dr. Marta Gomez-Chiarri

Research

Materials and methods:

Diet Preparation: Three diets were manufactured at the Food Science and Nutrition Center, at the University of Rhode Island (West Kingston, RI). The three diets were formulated as follows (Table 1): Control, no soy protein, all protein from fish meal; Partial replacement of 60% FM with SBM (prior unpublished research has indicated that 40% SBM replacement is tolerated without reduction in growth rate, though 70% inclusion will result in a loss of growth (Dave Bengtson, per. comm.)); Partial replacement of 60% FM with a 50:50 mix of SBM and SPC(potential immunostimulatory effects have been hypothesized to be attributed to non-starch polysaccharides in soybean meal, but are removed from soy protein concentrate). The diets were formulated to be isoenergetic and isonitrogenous and were evaluated through proximate analysis prior to the start of feeding trials. The three experimental diets contained 50% total crude protein (CP) and were fortified with taurine (all chemicals purchased from Sigma-Aldrich Co, St. Louis, MO, unless otherwise noted) (1%) and phytase (for diets containing soy products) (0.2-0.3%). Crude protein (CP), crude lipid (CL), moisture, fiber, and ash of all diets were analyzed using AOAC (1995) methods. All soy products were donated to the project by ADM (Arthur Daniels Midland Company, Decatur, IL), fish meal was donated to the project by the USDA fish laboratory, Idaho, USA (International Protein Corp. (anchovy, 69.6% protein, 9% oil)).

Fish, rearing conditions, and sampling protocol: The feed trial was carried out at the Marine Biological Laboratory (Woods Hole, MA), on a temperature and lighting controlled recirculating system. Following the feed trial, the fish were transported to the Blount Aquaculture Research Facility (Narragansett, RI), where the bacterial challenge was performed, and subsequent immunological analysis. Two hundred juvenile Summer flounder (Paralichthys dentatus) (length 7.1 ± 0.1cm, weight 3.6 ± 0.1g; mean ± SEM here and throughout text, unless otherwise noted) were obtained from GreatBay Aquaculture (Portsmouth, NH), and were transported to the Marine Biological Laboratory (MBL, Woods Hole, MA) prior to the start of the feed trials. All fish were fed a commercial diet (Skretting Gemma Diamond 0.8mm, Stavanger, Norway) twice daily by hand during the 2 week acclimation period. Twenty fish were randomly distributed to each of 9 50L aquaria, comprising triplicate aquaria for each of the three dietary treatments. Fish were fed twice daily at the rate of 5% wet body weight per day (recalculated every 2 weeks), for a period of 12 weeks. All uneaten feed was siphoned every day. The fish were held in a recirculating system at the MBL under natural lighting, and filtration and temperature (17.6 ± 0.1ºC) were controlled throughout the feeding trial. Individual fish wet weight and total length was determined every two weeks and feed levels adjusted accordingly. Weight gain, length gain, feed conversion ratio, specific growth rate, condition factor, and survival were calculated as follows:

Weight gain: W=final weight – initial weight
Length gain: L=final length – initial length
Feed conversion ratio: FCR= dry weight feed/ wet weight fish
Specific growth rate: C = [(Ln Wtf – Ln Wti)/d x 100]; where Wtf= final wet weight, Wti = initial wet weight, and d = number of feeding days.
Fulton’s Condition factor (K): K = 100 x fish weight (g)/fish length (cm)3
Survival: S = (final number of fish/ starting number of fish) x 100

Bacterial challenge: Following the conclusion of the feeding trial, all fish were transferred to the Blount Aquaculture Research Facility at URI. All fish were grouped within each treatment, and randomly redistributed into 20L aquaria as follows: each dietary treatment (three total) comprised four tanks, of which two tanks had 10 fish each (challenged, for bacterial injection), and two tanks had seven fish each (control, filtered artificial seawater injected). The fish were fed twice daily (5% body weight), and left for one week to acclimate to the new tanks. There were no statistical length or weight differences between fish in the different tanks or within groups.The optimum bacterial cell concentration to use in the challenge (LC50, concentration lethal to 50% of the fish) was determined prior to the challenge by the Karber method (Barros et al., 2002). Fish were placed in 20L tanks and injected intraperitoneally with 100 µlof solution containing the desired concentration of Vibio harveyi (a known pathogen of summer flounder, Soffientino et al. 1999), or filtered artificial sterile seawater. Mortality was monitored and recorded twice daily for seven days following injection. Post-challenge, bacteria were isolated from the peritoneal fluid, and confirmed to be V. harveyi by amplification and sequencing of a portion of the 16S rDNA gene using polymerase chain reaction. At the conclusion of the challenge (day 7) all remaining fish were anesthetized with tricaine methane sulphonate (MS-222 (100mg/L for 5 min). Blood was drawn from the caudal vein (less than 0.5ml per fish) using 27gauge needles attached to 1ml syringes rinsed with 0.2mM EDTA and was placed in 1.5ml eppendorf tubes with 2-3 drops 0.2mM EDTA as an anticoagulant. Since blood volumes from each individual fish were so small, samples were pooled within each tank. Following blood drawing, all of the fish were euthanized with an overdose of MS-222 (250mg/L for 10 min).

Hematocrit determination: Hematocrit was immediately measured by filling a hematocrit microcapillary tube two-thirds with whole blood, and then centrifuged for 5 minutes at 12000g. Percentage of packed cell volume (PCV) (hematocrit) was determined using the hematocrit reader (Ibraham et al. 2010).

Respiratory burst activity: The remaining blood was stored on ice, and transported to the Center for Biotechnology and Life Sciences, at the University of Rhode Island campus (Kingston, RI) for further analysis. Respiratory burst activity (RBA) was measured using a modified protocol based on Brubacher and Bols (2001) and Cathcart et al. (1983), which measures fluorescence of DCF when converted from H2DCFDA through radical oxygen species.The substrate 2’,7’-dihydrodichlorofluorescin-di-acetate (H2DCFDA, Invitrogen, USA) was converted to dichlorofluorescin (DCF) by incubating H2DCFDA (0.5ml of 1mM in ethanol)with 2.0ml 0.01N NaOH for 30 min at room temperature in the dark. The hydrolysate was then neutralized with 10ml of 25mM sodium phosphate buffer, diluted by 100x with sodium phosphate buffer, and stored on ice. Whole blood (100µl) was placed into the wells of flat bottomed 96-well microtiter plates in triplicate. The plate was then incubated for 1hr at 16ºC to allow cells to adhere to the sides of the microplate. The supernatant was removed and the wells were rinsed 3x with phosphate buffered saline (PBS). The activated substrate DCF (50 µl) was then added, as well as 50 µl of the blood cell activator phorbolmyristate acetate (PMA; Sigma, St. Louis, MO, USA, final concentration of 1 µg/ml). Fluorescence was measured immediately using a spectrofluorometer (excitation at 480nm and emission at 530nm) and then every 5 minutes after for 75 min, at which point the values began to plateau. Values are represented as relative fluorescence units, after the value of the blank has been removed.

Plasma protein: Blood samples were centrifuged at 3500 rpm for 30 minutes, and the plasma was removed and transferred to separate eppendorf tubes by pipette. The plasma samples were stored at 4°C for 24hrs until further analysis. Plasma protein was measured following the Coomassie (Bradford) Protein Assay (Bradford, 1976) using bovine serum albumin as standards.

Plasma lysozyme content: Plasma lysozyme content (per mg of protein) was measured following the standard hen egg-white lysozyme (HEWL) turbidimetric assay (Litwack, 1955). Briefly, 50µl plasma or lysozyme (HEWL) standard dilution (2.0-0.625 mg/ml) was added to individual wells of s 96-well microplate in triplicate. 150µl lysophilized Micrococcus lysodeikticus (0.75 mg/ml) was added to each well, and the absorbance was read at 450nm immediately. The absorbance was read again after 5 minutes, and a standard curve was plotted. The standard curve yielded a R2=0.982, therefore the trend line equation was used to determine plasma lysozyme content (each decrease in absorbance of 0.001/min is 1 international unit (IU) lysozyme).

Plasma bactericidal activity: Plasma bactericidal activity was determined following a modified procedure of El-Boshy et al. (2010). Fresh plasma (40 µl) or Hank’s Balanced Salt Solution (HBSS; positive control) were added in triplicate to wells of 96 round bottom well microtiter plate and incubated for 2.5 h with 20µl aliquots of an overnight culture of V. harveyi. Following incubation, 20µl MTT (2.5 mg/ml; Sigma) was added, and the plate was incubated at room temp for 10 min to allow the formation of formazan. The plates were then centrifuged for 10 min at 3200 rpm, the supernatant discarded, and the precipitate was dissolved in 150µl of DMSO. The absorbance of the dissolved formazan was read at 580 nm, and the bactericidal activity was calculated as the decrease in the number of viable V. harveyi cells by subtracting the absorbance of samples from that of controls and reported as absorbance units.

Statistics: All data were analyzed using the General Linear Model procedure using SAS computer software (SAS 9.2 TS Level 2M2, Cary, NC, USA). Mean results were subjected to one-way analysis of variance (ANOVA), and significant results were further analyzed using Tukey’s Post-Tests. The significance level of 0.05 was chosen, and all data are presented as mean ± s.e.m. (standard error of the mean).

Barros, M.M., Lim, C., Klesius, P.H., 2002. Effect of soybean meal replacement by cottonseed meal and iron supplementation on growth, immune response, and resistance of Channel Catfish (Ictalurus puctatus) to Edwardsiella ictaluri challenge. Aquaculture. 207: 263-279.

Bradford M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry. 72, 1-2: 248-254.

Brubacher J.L., and Bols N.C. 2001. Chemically de-acetylated 2?,7?-dichlorodihydrofluorescein diacetate as a probe of respiratory burst activity in mononuclear phagocytes. Journal of Immunological Methods. 251, 1-2: 81-91.

Cathcart R., Schwiers E., Ames B.N. 1983. Detection of picomole levels of hydroperoxides using a fluorescent dichlorofluorescein assay. Analytical Biochemistry. 134, 1: 111-116.

El-Boshy M.E., El-Ashram A.M., Abdel Hamid F.M., Gadalla H.A. 2010. Immunomodulatory effect of dietary Saccharomyces cerevisiae, ?-glucan and laminaran in mercuric chloride treated Nile tilapia (Oreochromis niloticus) and experimentally infected with Aeromonas hydrophila. Fish & Shellfish Immunology. 28, 5-6: 802-808.

Ibrahem M.D., Fathi M., Mesalhy S., Abd El-Aty A.M. 2010. Effect of dietary supplementation of inulin and vitamin C on the growth, hematology, innate immunity, and resistance of Nile tilapia (Oreochromis niloticus). Fish and Shellfish Immunology. 29, 2: 241-246.

Litwack G. 1955. Photometric Determination of Lysozyme Activity. Journal of Experimental Biology and Medicine. 89, 3: 401-403.

Soffientino, B., Gwaltney, T., Nelson, D., Specker, J., Mauel, M., Gomez-Chiarri, M., 1999. Infectious necrotizing enteritis and mortality caused by Vibrio carchariae in summer flounder (Paralichthys dentatus) during intensive culture. Diseases of Aquatic Organisms. 38: 201-210.

Research results and discussion:

Results

Growth and feed conversion: All three diets were generally uniform as far as chemical composition and proximate analysis. The fish in the control group started at average weight of 7.06±0.06g, and the SBM and SBM/SPC groups started the trial at 7.06±0.07 and 7.04±0.08, which was not significantly different. Change in both length and weight was highest in the group fed the SBM/SPC diet (Fig. 1). At the conclusion of the trial, the SBM/SPC group increased the average weight per fish to 36.40±6.16g, significantly better than both the Control fed group (24.40±2.26g) and the group fed the SBM diet (22.54±6.12) (ANOVA, df=2, F-value=15.36, p=0.0044, ?=0.05).

The SBM/SPC fed group had a significantly higher specific growth rate (SGR, 2.80±0.07 g d-1) compared to the other two experimental groups (ANOVA, df=2, F-value=29.43, p=0.001, ?=0.05, with Tukey’s Post-Test; Table 2). Feed conversion ratio for the SBM/SPC fed group (1.01±0.02) was significantly lower than the SBM fed group (1.33±0.07), however it was not significantly lower than the Control fed group (1.21±0.07) (ANOVA, df=2, F-value=7.77, p=0.021, ?=0.05, with Tukey’s Post-Test). Condition factor (K) was only different between the Control and SBM fed groups (1.12±0.01, 1.24±0.02, respectively) (ANOVA, df=2, F-value=10.38, p=0.011, ?=0.05, with Tukey’s Post-Test). There were no differences in survival throughout the entire experiment (ANOVA, df=2, F-value=4.20, p=0.0723, ?=0.05; Table 2).

Survival to Bacterial Challenge and Immune Parameters: All of the fish which were injected with filtered artificial seawater (FASW) as a control survived the 7 day challenge period. Within the groups of fish that were experimentally injected with Vibrio harveyi, there was significantly reduced survival compared to the FASW group, however, no differences between groups fed different diets (Kruskal-Wallis Test Nonparametric ANOVA, df=2, p=0.080, ?=0.05; Table 3).

Hematocrit was approximately 29% for groups when injected with FASW. However, once experimentally infected with bacteria, the hematocrit for all 3 groups decreased to between 24-26%, though there were no statistically significant differences between groups. For the groups injected with the control (FASW), plasma protein was highest for the SBM group (138.0±21.3 mg/ml), and for the groups injected with bacteria, plasma protein was highest for the SPC group (142.4±8.3 mg/ml). Lack of significance may have been due to large variation between samples.

Respiratory burst activity (RBA) was measured using adherent blood cells. At no point throughout the 75 minute period in which fluorescence was measured was a statistically significant difference between the experimental groups. All groups regardless of diet resulted in higher RBA values after injection with bacteria as compared to FASW (Fig. 2). Of those injected with bacteria, the group fed the control diet also resulted in higher RBA after 75 minutes (91.5±14.7 rfu) as compared to the two experimental diets (78.3±7.4 rfu SBM, and 78.3±5.7 rfu SPC), however, again, this difference was not significantly higher.

The group fed the SBM diet and challenged with FASW (control) show the lowest level of lysozyme activity (24.8±6.0 units/ml) when saline injected. The group fed the control was slightly higher (36.8±5.1 units/ml), and the group fed the SBM/SPC diet (43.3.±3.3 units/ml) was significantly higher than the SBM fed group (p<0.05). When considering the groups injected with bacteria, there were no differences between groups, however the SBM/SPC fed group was significantly higher (p<0.05) following injection with FASW (43.3±3.3 units/ml) as compared to injection with bacteria (17.0±4.4 units/ml) (Fig. 2).

Regardless of diet following either FASW or bacteria injection, fish in the group fed the control diet showed higher bactericidal activity (lower levels of formazan indicate less bacteria in the sample). However due to high variability in the two groups fed the experimental diets, there were no statistically significant differences in plasma bactericidal activity between any of the groups.

Discussion

The main impediments to the expansion of summer flounder aquaculture, an important food fish in the Atlantic US, include feed costs and disease outbreaks. The current study examined the impact on growth performance of replacing 60% of the main protein source (fish meal or FM) in a summer flounder diet with either soybean meal (SBM) or a 50:50 ratio of SBM to soy protein concentrate (SPC) for 12 weeks. The diet containing the SBM:SPC mix proved to provide better growth than the fish meal control or the diet containing 60% SBM, indicating that soy-based products can replace most of the fish meal in carnivorous fish diets as a source of protein when most of the antinutritional factors present in soybean meal are removed by purification. We also searched to confirm previous results indicating that diets containing soybean meal provide protection against bacterial challenge (Lightbourne 2011) and determine if that protection is due to stimulation of the innate immune responses of the fish. Our results from the bacterial challenges were ambiguous and difficult to interpret due to large experimental variation, warranting further research on this subject.

Several authors have investigated the impact of FM replacement with SBM at various levels, which almost without exception, resulted in a decrease in growth (Rumsey et al. 1994; Refstie et al. 1998; Refstie et al. 2005; Hart et al. 2010). Research in salmonids has determined that the replacement of fish meal by either SBM or SPC must be much lower than 100% or the chance of reduced growth and enteritis of the gut is almost guaranteed (Rumsey et al. 1994; Refstie et al. 1998). The decrease in growth from diets where soybean meal replaces fish meal at high levels in carnivorous fish feeds may have to do with the impact of many antinutritional factors present in soybean meal (Grisdale-Helland et al. 2008). These include protease inhibitors (trypsin inhibitors), oligosaccharides (stachyose, raffinose, etc.), saponins, isoflavones, antigens (glycinin, conglycinin, lectins), phytate, and tannins (Refstie et al. 2005; Knudsen et al. 2007; Iwashita et al. 2009). Heat processing or alcohol extraction of the soybean meal to produce soy protein concentrate removes many of these factors, and produces a product which, for several species, allows for good growth rates without morphological intestinal changes (e.g. enteritis; Francis et al. 2001; Knudsen et al. 2007).In diets for Atlantic cod (Gadus morhua) soy protein concentrate can replace 22% of FM with no decrease in growth (Refstie et al. 2006), and other studies have shown even higher levels (40-70%) of fish meal replacement with soy protein concentrate may be possible for other carnivorous fish species (Berge et al. 1999; Mambrini et al. 1999; Refstie et al. 2003). In juvenile cod, soy protein concentrate was able to replace 100% of the fish meal in diets, with no observed enteritis or reduction in growth (Walker et al. 2010).

Our research confirms that using soy protein concentrate as a replacement for fish meal in summer flounder diets can result in equal if not better growth than using fish meal as the sole protein source. Soy protein concentrate has many of the antinutritional factors present in soybean meal removed, including protease inhibitors (trypsin inhibitors), oligosaccharides (stachyose, raffinose, etc.), saponins, isoflavones, antigens (glycinin, B-conglycinin, lectins), phytate, and tannins (Refstie et al., 2005; Knudsen et al., 2007; Iwashita et al, 2009), which results in better growth. The feed conversion ratio for the group of fish fed the SBM:SPC diet was the lowest of the three tested diets (including the control fish meal diet), suggesting that a decrease in the antinutritional factors in soybean-based feeds leads to a more efficient use of the diet by the fish and, consequently, a higher specific growth rate. These results confirm past reports of soy protein concentrate replacement diets resulting in at least as good growth rates, if not better, as compared to the control fish meal diet (Bengtson and Lee, in preparation). Planned future research at the University of Rhode Island will investigate histological changes associated with diets including SBM, SPC, and a mix of antinutritional factors to determine the effect of SBM and SPC on the intestinal morphology and the connection to growth and survivability.

Ongoing research in our group shows that soybean meal may have immunostimulatory properties that lead to protection against bacterial challenge (Lightbourne 2010 and preliminary results from other experiments). These properties may be due to some of the antinutritional factors present in soybean meal, but absent in soy protein concentrate. There are many molecules known to initiate immunostimulation, such as B- glucans, bacterial products or plant components which may directly activate the immune system resulting in the production of anti-microbial molecules (Bricknell and Dalmo 2005). Immunostimulation from molecules derived from plant sources is not a new concept; however the use of soy in fish feeds has mainly focused on the negative aspects of reduced growth and enteritis, instead of potential beneficial immune applications. On the other hand, high levels of protein replacement with soybean meal have been shown to cause an inflammatory response in Atlantic salmon (Salmo salar), resulting in reduced capacity to fight off bacterial infection (Krogdahl et al. 2000) and an increase in enteritis (Knudsen et al. 2007). In rainbow trout (Oncorhynchus mykiss), high (60-70%) replacement of fish meal with soybean was also correlated with a decrease in immune function (macrophage activity and histological modulation), in conjunction with a decrease in growth. Studies on immune activity as a function of SBM inclusion in the diet have come to the same conclusions. The antinutritional factors causing the enteritis in SBM, were producing a sustained inflammatory or hypersensitivity response, increasing neutrophil, monocyte and macrophage activity (Rumsey et al. 1994) as well as increased lysozyme and antibody levels (IgM) (Krogdahl et al. 2000).

Although we have found in previous experiments that replacement of fish meal with soybean meal in summer flounder diets at levels of 40–70% leads to protection to bacterial challenge (Lightbourne 2010), there was no significant reduction in mortality after bacterial challenge in the current experiment. In the current work, fish were fed either 60% replacement of FM with SBM or 60% replacement of FM with a even ratio of SBM:SPC, in which they were fed for 12 weeks. Past work investigated 40-70% replacement with SBM alone, and fed for 8 weeks. Possibly the effect of feeding a high SBM diet for 12 weeks instead of 8 had a more pronounced impact on the intestinal morphology, reducing the ability to protect against bacterial challenge. Another variable which may have affected the challenge experiment is past exposure to the pathogen. Vibrio harveyi is ubiquitous throughout New England waters, and the fish used in this experiment may have been exposed prior to the start of the experiment.

Our results indicate that replacement of fish meal with either soybean meal (60%) or a mix of soybean meal (30%) and soy protein concentrate (30%) for 12 weeks did not lead to significant differences in hematocrit, plasma protein, or respiratory burst activity in adherent blood cells of summer flounder, when measured 7 days after challenge with control (filtered sterile seawater) or bacteria. Our failure to detect differences between the groups may be due to lack of power (variation in the parameters within experimental groups was high). Further research is needed to evaluate the impact of soybean meal components on the immune responses of summer flounder, including evaluation of the effect of independent components of soybean meal, and examination of the effect of dose and length of exposure on selected immune responses.

In summary, our results confirm that a mix of soybean meal and soy protein concentrate can be successfully used to replace 60% of the fish meal, providing improved growth. Future research will further evaluate the impact of replacement of fish meal with different levels of soybean meal and soy protein concentrate on growth performance, susceptibility to bacterial challenge, and immunological response.

Berge G.M., Grisdale-Helland B., Helland S.J. 1999. Soy protein concentrate in diets for Atlantic halibut (Hippoglossus hippoglossus). Aquaculture. 178, 1-2: 139-148.

Bricknell I. and Dalmo R.A. 2005. The use of immunostimulants in fish larval aquaculture. Fish and Shellfish Immunology. 19, 5: 457-472.

Francis G., Makkar H.P.S., Becker K. 2001. Antinutritional factors present in plant–?derived alternate fish feed ingredients and their effects in fish. Aquaculture. 199, 3-4: 197?227.

Grisdale-Helland B., Helland S.J., Gatlin D.M. 2008. The effects of dietary supplementation with mannanoligosaccharide, fructooligosaccharide or galactooligosaccharide on the growth and feed utilization of Atlantic salmon (Salmo salar). Aquaculture. 283, 1-4: 16-?167.

Hart S.D., Bharadwaj A.S., Brown P.B. 2010. Soybean lectins and trypsin inhibitors, but not oligosaccharides or the interactions of factors, impact weight gain of rainbow trout (Oncorhynchus mykiss). Aquaculture 306: 310–314.

Iwashita, Y.; Suzuki, N.; Matsunari, H.; Sugita, T.; Yamamoto, T. 2009. Influence of soya saponin, soya lectin, and cholyltaurine supplemented to a casein-based semipurified diet on intestinal morphology and biliary bile status in fingerling rainbow trout Oncorhynchus mykiss. Fisheries Science. 75, 5: 1307-1315.

Knudsen D., Uran P., Arnous A., Koppe W., Frokiaer H. 2007. Saponin-containing subfractions of soybean molasses induce enteritis in the distal intestine of Atlantic salmon. Journal of Agricultural and Food Chemistry 55, 6: 2261?2267.

Krogdahl A., Bakke-Mckellep A.M., Roed K.H., Baeverfjord G. 2000. Feeding Atlantic salmon Salmo salar L. soybean products: effects on disease resistance (furunculosis), and lysozyme and IgM levels in the intestinal mucosa. Aquaculture Nutrition. 6, 2: 77–84.

Lightborne C. 2010. Effects of soybean meal replacement with added taurine in fish meal diets for summer flounder (Paralichthys dentatus). Masters Thesis. University of Rhode Island, Kingston RI.

Mambrini, M., Roem, A.J., Cravèdi, J.P., Lallès, J.P. and Kaushik, S.J. (1999). Effects of replacing fishmeal with soy protein concentrate and of DL-Methionine supplementation in high-energy, extruded diets on the growth and nutrient utilization of rainbow trout, Oncorhynchus mykiss. Journal of Animal Science. 77: 2990-2999.

Refstie S., Storebakken T., Roem A.J. 1998. Feed consumption and conversion in Atlantic salmon (Salmo salar) fed diets with fish meal, extracted soybean meal or soybean meal with reduced content of oligosaccharides, trypsin inhibitors, lectins and soya antigens. Aquaculture. 162, 3-4: 301-312.

Refstie S., Teikstra H.A.J. 2003. Potato protein concentrate with low content of solanidine glycoalkaloids in diets for Atlantic salmon (Salmo salar). Aquaculture. 216: 283?298.

Refstie, S., Sahlstrom S., Brathen E., Baeverfjord G., Krogedal P. 2005. Lactic acid fermentation eliminates indigestible carbohydrates and antinutritional factors in soybean meal for Atlantic salmon (Salmo salar). Aquaculture. 246, 1-4: 331-345.

Rumsey, G.L., Siwicki, A.K., Anderson, D.P., Bowser, P.R., 1994. Effect of soybean protein on serological response non-specific defense mechanisms, growth, and protein utilization in rainbow trout. Veterinary Immunology and Immunopathology. 41: 323-339.
Walker A.B., Sidor I.F., O’Keefe T.O., Cremer M., Berlinksy D.L. 2010. Partial Replacement of Fish Meal with Soy Protein Concentrate in Diets of Atlantic Cod. North American Journal of Aquaculture. 72, 4: 343-353.

Research conclusions:

One of the main disease issues facing summer flounder culture is infectious necrotizing enteritis (FINE), caused by the pathogenic bacterium Vibrio harveyi (Gauger et al. 2006). Both reduction of disease and sustainable feed production are two of the main obstacles facing aquaculture expansion in the northeast. If a diet can be designed for summer flounder which will results in equal if not better growth, at a fraction of the cost of the current fish meal-based diets, it will contribute to the economic viability of aquaculture expansion in the Northeast. If this same diet could result in some measure of immunostimulation, and a reduction in mortality due to disease, it could drastically impact investment in sustainable practices in aquaculture farming.

The current project is another step in the process towards designing the “super diet” which has all of the above attributes. Following the conclusion of this project, the United Soybean Board (USB) decided to fund another feed trial throughout the summer of 2011, concluding in an identical bacterial challenge to the one performed as part of the SARE project. The USB followed on to the logical next step following the SARE project. This was to design 7 diets, which further investigate the exact ratio of SBM:SPC which will result in not only good growth, but also protection against bacterial challenge. This feed trial resulted in the determination of a balance between growth and disease susceptibility (the best protection was achieved by a diet tested in the USB trial which had protein sourced at 40%FM, 12%SBM, 48%SPC ), and also determined that SPC alone appears to confer no protection against bacterial challenge. The results from the SARE and USB projects also helped our team to secure funding from Rhode Island Sea Grant to continue the work towards the sustainable diet for summer flounder. Both the feed manufacturer Ziegler, as well as Local Oceans and Great Bay Aquaculture have written letters of support, and have shown great interest in the results of these projects. As noted above, FINE is one of the more prevalent and devastating diseases effecting summer flounder aquaculture, and the results thus far have created quite a bit of excitement with our partners that a reduction of mortality may be possible without needing additional antibiotics or vaccinations.

Gauger E., Smolowitz R., Uhlinger K., Casey J., Gomez-Chiarri M. 2006. Vibrio harveyi and other bacterial pathogens in cultured summer flounder. Aquaculture. 260: 10?20.

Participation Summary

Education & Outreach Activities and Participation Summary

Participation Summary

Education/outreach description:

The results of this work will be presented at the annual meeting of the World Aquaculture Society in February in Las Vegas, NV (February 29 – March 2, 2012). The results will also be presented at the biennial Northeast Aquaculture Conference and Expo, which will take place in December 2012. The cooperative partners in this project, Ziegler feed manufacturers, Great Bay Aquaculture, and Local Oceans, have all been contacted, and meetings have been set up to present the conclusions of this work at their facilities. Follow-up studies will continue throughout the next 2 years, at which time articles and further dissemination of the knowledge gained from this project will continue through RI Sea Grant, and other outlets within the University of Rhode Island.

Project Outcomes

Project outcomes:

The results of the current work will result in greater investment in aquaculture, greater economic viability for aquaculture farms, and lower feed costs for fish farmers. As the aquaculture in both the Northeast as well as the US in general develops, more sophisticated feeds will be increasingly important. Farmers will continue to require feeds which not only produce good growth rates, but that are also sustainably sourced , and can provide reduced susceptibility to outbreaks of disease as fish are raised at high densities. Designing a feed which does not rely on such an increasingly scarce and expensive commodity as fish meal will provide further incentive for investors to provide funding for aquaculture expansion. The reduction of disease outbreaks is mandatory for farmers to feel comfortable making the investment in expanding their facilities, and the results of this project allow the industry to get closer to that goal.

With fish meal currently trading at $1347 per ton, and soy protein concentrate currently selling at approximately $1300-$1400 per ton, the price difference is negligible. However, the results of a feed trial following the SARE work (subsequent USB funded trial) show that growth is 58% higher on a FM: SBM:SPC (FM:SBM:SPC ratio of 40:30:30) diet compared to a FM only-based control diet. Survival following bacterial challenge (simulating a disease outbreak) is 79% higher when the fish are fed a diet with FM as well as SBM and SPC (FM:SBM:SPC ratio of 40:24:36; 125% better survival on a diet with a 40:12:48), instead of only FM as the main protein source. This analysis does not take into account sustainability, as fish meal prices will continue to rise as demand increases due to a limited supply, while soy output can increase with demand. Even though price of raw feed components may not make the feed initially cheaper, better feed conversion ratios, growth rates, and survival following a bacterial outbreak, will make using diets comprising more soy products a better choice for an aquaculture farmer.

Farmer Adoption

The fish farmers at Great Bay Aquaculture have expressed great interest in the need for cheaper and sustainable feed components. They have also stressed the importance of the reduction of susceptibility to disease. Currently the farmers in the Northeast use dip-vaccinations to prevent bacterial infections from Vibrio-type strains. If immunostimulation can be facilitated through feed components, the farmers may not need to vaccinate fish against these particular pathogens. This will result in lower costs to the farmers, and less handling of the fish, resulting in better general animal welfare. The feed manufacturers at Ziegler have also expressed a great desire to increase soy inclusion in their feeds, and any research which will result in greater replacement of fish meal will allow them to be more competitive. Since soy also has the potential immunostimulatory functions, they have been very interested in how the results of this project can be implemented into their feed production.

Assessment of Project Approach and Areas of Further Study:

Areas needing additional study

The results of this research helped to confirm what was already suspected about utilizing soy products for fish meal replacement in summer flounder feeds. Replacing FM with SPC (at least up to 60%) will result in good, if not better, growth rates as compared to control. This is excellent from both a sustainability standpoint, as well as economically due to the drastically reduced cost of SPC compared to FM. What was not as clear is the potential mechanism of immunostimulation due to the replacement of FM with either SBM or SPC. Following the conclusion of this project, a subsequent feed trial examined the exact ratio of FM:SBM:SPC necessary to provide a measure of reduced mortality due to bacterial infection. This follow-up project further confirmed the good growth rates seen in the SARE project, and also confirmed better survival for diets with FM replacement with soy products as compared to control. The logical next step for this project is to separate out the fractions that are present in SBM though absent in SPC and individually analyze them for their immunostimulatory properties. Through funding secured through RI SeaGrant, the next project is slated to start in February 2012, breaking down the antinutritional properties present in SBM, and individually characterizing their immunostimulatory properties in order to further build upon the knowledge gained through the SARE project.

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.