Evaluation of EnzoMeal (EZM) in Commercial Production Diets for Rainbow Trout (Oncorhynchus mykiss)

Transcription

1 Evaluation of EnzoMeal (EZM) in Commercial Production Diets for Rainbow Trout (Oncorhynchus mykiss) Final Report Steven R. Craig. Ph.D H. William Harris, MD, Ph.D Marlies Betka, Ph.D Submitted December 1, 2015

2 Executive Summary A ten-week feeding trial was conducted to investigate the potential use of EnzoMeal (EZM) from Battelle in commercial diets formulated for rainbow trout (Oncorhynchus mykiss). Two diets were formulated to be identical except for the inclusion rate of the EZM (18 and 33%). Additionally, two more diets were formulated to contain a fermented soy protein concentrate, Hamlet Protein s HP- 300, at the same levels of inclusion (18 and 33%). These diets were compared to a control commercial diet which is in use at Homestead Springs Trout Farm in Fredericktown, OH as its standard grow-out production feed. All diets were formulated to provide 45% crude protein and 16% total lipid. After ten weeks, there were no significant differences with respect to growth, feed conversion ratios, specific growth rates and survival. Histological evaluation of specific important metabolic tissues, such as the liver, kidney, and spleen indicated no morphological abnormalities related to dietary treatment. Additionally, histological examination of the gastrointestinal tract provided no evidence of detrimental impacts of high soy inclusion, specifically as related to distal enteritis, a common malady in salmonids fed high levels of soy protein. While there were trends indicating a decrease in growth performance at the higher inclusion levels of both soy products, the data generated from the present trial indicates that EZM can be effectively incorporated into commercial diets for rainbow trout at relatively high inclusion levels without detrimental impacts to production performance. Higher inclusion levels are most likely possible with additional dietary considerations.

3 Introduction Research dealing with alternative proteins suitable for fish meal replacement in aquafeeds has been conducted for the past thirty years with mixed results (Drew et al, 2007; Sullivan, 2008; Naylor et al, 2009; Rust et al, 2010; Li et al, 2011; Suresh, et al, 2011, Ayadi et al 2012). Fish meal inclusion rates have dropped over the past decade, and the use of alternate proteins, particularly defatted soybean meal, has increased rather dramatically during this time frame. Nevertheless, the aquaculture industry utilized over 70% of the global fish meal supply and over 80% of the fish oil in More work obviously needs to be done to provide economically viable alternatives. The use of plant-based alternative protein sources in fish meal replacement studies has been well documented and works very well in a wide variety of species. The continued use of fish meal in aquafeed formulations is generally considered unsustainable for the long-term growth of the aquaculture industry. Over the past decades, tremendous advances have been made with respect to the reduction/elimination of fish meal from the formulations of many important commercially cultured species. Rainbow trout have been traditionally farmed and presently represent the second largest species in terms of production in the United States. However, traditional rainbow trout feeds have relied upon high levels of fish meal and fish oil, and this practice is simply not sustainable in the long term. The biological effects of reduction or elimination of fish meal from rainbow trout diets have been studied extensively (see Gatlin et al. 2007). In general, inclusion rates of commodity soybean meal higher than 15% of the diet in commercially formulated feeds has resulted in reductions in performance in terms of growth and overall health status in rainbow trout (Sealey et al., 2009). Lower concentrations of commodity soy protein are better tolerated by the fish but can produce changes in the distal intestinal architecture and diarrhea in trout (Rumsey, 1994). These impacts on the distal intestine have been described as sub-acute enteritis (Baeverfjord and Krogdahl, 1996; Bakke-McKellep et al., 2000). EnzoMeal, with the elimination of oligosaccharides and subsequent increase in crude protein represents a product that deserves further evaluation in a commercially-derived feed in a high value, well-recognized aquaculture species such as rainbow trout. This report details the findings of a tenweek feeding trial with this species which investigated the impacts of a low and high inclusion level of EZM in commercially formulated and manufactured extruded aquafeeds. Methods and Materials A ten-week feeding trial was conducted with rainbow trout at Homestead Springs in Fredericktown, OH following established, standardized protocols developed over the last 10 years and validated through numerous peer-reviewed publications. A sub-population of approximately 3,000 rainbow trout, initial weight of g per fish, was separated from the main production population at Homestead Farms and conditioned to the experimental system for a period of 2-3 weeks to ensure complete acclimatization to the experimental conditions. After the conditioning period, the fish were re-grouped together for restocking into the experimental tanks. At this point, average fish weight was approximately 25 g each. Each tank contained 40 fish, which were weighed as a group with each group being within + 5% of each other in terms of initial tank weight. Initial tanks weights were g, which corresponds to an average fish weigh of 25.7 g each. Each tank of fish was randomly assigned to a dietary treatment (n=3 tanks per treatment; n=120 fish per dietary treatment). The diets were formulated to provide 45% crude protein and 16% total lipid based upon current commercial production diets utilized at Homestead Springs (Control). The experimental diets compared EZM with a perceived competitor in the marketplace Hamlet Protein (HP-300) at two levels low and high. The LOW treatments included each soy product at equal protein contributions,

4 approximately 18% of the total diet. The HIGH treatments compared EZM with HP-300 at levels of 33% of the total diet (See Table 1). The fish were fed to apparent satiation four times daily and weighed every two weeks as a group to monitor performance and to determine feed conversion ratio values. Feeding rates followed those developed for commercial trout production at Homestead Springs and feed consumption was closely monitored so that accurate feed conversion ratio (FCR) values could be calculated. All data were analyzed utilizing appropriate statistical procedures with differences in means considered significant at α < Table 1: Diet formulations for the control and experimental production feeds. See text for details. Control Low HP300 Low EZM High HP300 High EZM Ingredient Fish meal Poultry By-Product meal Corn protein concentrate EnzoMeal (EZM) HP Feather meal Blood meal Wheat Menhaden oil Soybean oil Vitamin premix Mineral Premix Taurine Choline CL Vitamin C Other ingredients (macrominerals etc) Astaxanthin Calculated Composition, as-is basis Crude Protein, % Lipid, %

5 Growth and FCR While growth was monitored throughout the feeding trial through the biweekly weight samples of each individual tank, final weights were taken at the end of the trial for the determination of weight gain (as measured by percent increase from initial weight) and specific growth rates (SGR). These data will be utilized to determine the overall feed conversion ratios for fish fed the various experimental diets. Additionally, all fish in each experimental tank for each respective diet (n=120) were individually weighed and measured to determine a distribution curve of weights and lengths for fish fed their respective diets. Biological and Physiological Sampling At the beginning of the feeding trial, 10 rainbow trout were euthanized and sampled for physiological and histological examination. For these procedures, fish were euthanized by an overdose of MS222, weighed and measured, photographed and blood samples taken via heparinized capillary tubes. Blood samples were spun in a hematocrit centrifuge (5 min) and the hematocrit measured. The plasma from several capillaries per fish was pooled and analyzed in a blood gas analyzer (Radiometer ABL 77) for sodium, chloride, calcium and potassium. Liver and spleen tissues were then removed via dissection and weighed immediately (live wet weight) in order to calculate hepatosomatic (HSI) and spleen-somatic indices (SSI). Aliquots of these tissues, as well as kidney, proximal and distal intestine tissue aliquots were then fixed in 10% buffered formalin for >24 h. Tissues were cut to smaller sizes, dehydrated in ethanol and paraclear and embedded in paraffin according to standard histological techniques. Sections of approximately 7 μm were cut and affixed to slides. A total of 4-12 sections per sample were then mounted per slide. These sections were utilized to determine if the feeding of these experimental diets resulted in any indication of distal enteritis, as well as any other pathology in terms of targeted organs (ie spleen, liver, kidney). These same procedures and analyses were conducted on 10 fish per dietary treatment at the end of the feeding trial in order to determine the biological and physiological impacts of the EZM and HP-300 at the two dietary inclusion levels investigated relative to fish fed the control diet Results Growth Parameters There were no significant differences related to dietary treatment in any of the growth parameters measured during the ten week feeding trial (See Table 2 for complete results).

6 Table 2. Growth parameters measured during the ten week feeding trial. See text for details. Diet Initial Weight Final Weight % Increase from Initial Weight FCR SGR Survival Control EZM-Low EZM-High HP-300-Low HP-300-High Survival in all experimental groups was excellent, ranging from a low of 98.0% in fish fed the EZM High diet to a high of 99.3% in rainbow trout fed the Control diet. Fish fed the remaining diets returned intermediate survival rates, all above 98%. The only mortalities were due to jumpers during the first two weeks of the trial. The fish were healthy and robust throughout the feeding trial period. Growth over the ten week feeding trial was excellent, with fish fed all diets performing in line with previous growth rates at Homestead Springs. Due to the artesian nature of the spring water at Homestead Springs, water temperatures run an average of C, which does result in slightly lower growth rates for rainbow trout when compared to higher culture temperatures. Growth, as measured as percent increase from initial weight ranged from a low of 582% in trout fed the EZM High diet to a high of 627% in fish fed the Control diet. Fish fed the remaining diets returned intermediate values of 584, 606 and 616% in fish fed the EZM Low, HP-300 High and HP-300 Low diets, respectively (Figure 1). Figure 1. Percent increase from initial weight for rainbow trout fed the experimental diets for 10 weeks. See text for details. Feed conversion ratio values (FCR; g feed fed/g gained) were outstanding, with fish fed all diets returning very favorable FCR values ranging from a high of 1.04 in fish fed the EnzoMeal High diet

7 to a low of 0.94 in fish fed the Control diet. Fish fed the remaining diets returned intermediate FCR values of 0.96, 0.97 and 0.97 in fish fed the HP-300 Low, HP-300 High and ENZ Low diets, respectively. Specific growth rates (SGR; percent of total body weight gain/day) ranged from a low of 2.74% in rainbow trout fed the EZM High diet to a high of 2.83% in fish fed the Control diet. Fish fed the remaining diets returned intermediate SGR values of 2.75, 2.79 and 2.81% in fish fed the ENZ Low, HP-High and HP-Low diets, respectively. Distributions of Fish Weights In an effort to determine the potential impacts of dietary treatment on the distribution of fish weights within each tank, every fish was weighed individually at the end of the trial and a distribution curve generated showing the number of fish within each weight category, with each category being 24 g (Figure 2). Figure 2. Compilation of body weight distributions for trout fed one of five different diets. Individual fish from each of 5 groups fed different diets were weighed upon completion of the trial and their weights grouped into categories of 24gm each. Fish from individual groups are denoted either by solid columns (production or lower concentration feed ingredient feeds) or crosshatched column (experimental feeds containing higher concentrations of test feed ingredients). See text for details. This distribution graph shows that fish fed the Control diet were more tightly grouped around the weight category of g. The two experimental diets at low soy concentrations (HP-300 Low and EZM Low) had very similar fish numbers in this weight category as observed in fish fed the

8 Control diet. However, fish fed these two diets also had larger numbers of fish in the weight category just below ( g), which illustrates the numerical, but non-significant, difference observed in growth rates between fish fed the Control diet and these two soy-based diets. Fish fed the two diets containing higher levels of soy, EZM High and HP-300 High had larger numbers of fish in this smaller size category ( g), and less fish in the larger weight category ( g), again indicative of the results discussed related to growth in this ten week feeding trial. It is interesting to note that some of the largest fish grown in the trial, in the weight categories above 251 g, although representing a low percentage of the total, were fed the EnzoMeal diets, indicating the biological potential of this feed ingredient. Figure 3 shows the distribution of fish fed the Control diets in comparison to the EZM Low and High diets. Again, one can see more clearly the distribution trends related to the soy inclusion levels, EZM Low has more, larger fish than EZM High, as compared to fish fed the Control diet. Figure 3: Comparison of the distribution of body weights of rainbow trout fed either production feed (solid black columns) or feed containing EnzoMeal (red columns) at inclusion rates of either 18% (solid red columns) or 33% (crosshatched red columns). See text for details. Figure 4 shows the distribution of fish fed the HP-300 Low and High diets. Again, one can see more clearly the distribution trends related to the soy inclusion levels as observed in fish fed the diets in that the group of fish fed the HP-300 Low had more, larger fish than those fed the EZM High diet.

9 Figure 4. Comparison of the distribution of body weights of rainbow trout provided feed containing either 18% Hamlet Protein HP-300 (solid blue columns) or 33% HP-300 (crosshatched blue columns). See text for details. Biological Indices There were no significant differences related to dietary treatment in any of the biological indices measured during the ten week feeding trial (See Table 3 for complete results). The following biological indices were measured: hematocrit; hepatosomatic index (HSI); spleensomatic index (SSI); and the length of distal intestine related to body weight (DI/BW). In the following graphs, the diet designations were changed as follows for ease of identification on the X- axis: HP-300=HP-H; HP-300 Low=HP-L; EnzoMeal High=EZM-H and EnzoMeal Low=EZM-L. Hematocrit Hematocrits ranged from 39.5% in rainbow trout fed the Control diet to 42.6% in fish fed the HP-H diet. Fish fed the remaining diets returned intermediate hematocrit values ranging from 40.7 to 42.2%. All observed hematocrit were well within accepted levels for healthy rainbow trout (Figure 5).

10 Figure 5. Hematocrit (packed cell volume) from rainbow trout fed the experimental diets for ten weeks. Hepatosomatic index The hepatosomatic index (HSI), which measures size of the liver relative to total body weight, was also not significantly impacted by dietary treatment. Values for HSI ranged from a high of 1.6 in fish fed both HP diets and the EZM-H to a low of 1.4% in fish fed the Control diet (Figure 6). Fish fed the EZM-L diet returned a HSI value of 1.53%. All values for HSI observed in the present study were well within accepted levels for healthy rainbow trout. Figure 6. Hepatosomatic index (HSI) from rainbow trout fed the experimental diets for ten weeks.

11 Spleen-somatic index The spleen-somatic index (SSI), which measures size of the spleen relative to total body weight, was also not significantly impacted by dietary treatment. Values for SSI ranged from a high of 1.25% in fish fed the Control diet to a low of 0.91% in fish fed the HP-H diet (Figure 7). All values for SSI observed in the present study were well within accepted levels for rainbow trout. Figure 7. Spleen-somatic index (SSI) from rainbow trout fed the experimental diets for ten weeks. Length of distal intestine related to body weight The length of the distal intestine relative to total body weight (DI/BW), is an indicator of GI tract modification in response to dietary treatments. There was no significant impact of dietary treatment on the DI/BW in the present trial. Values for DI/BW ranged from a high of 1.24% in fish fed the Control diet to a low of 0.94% in fish fed the HP-H diet (Figure 8). Interestingly, while there were no significant effects of diets, fish fed the soy-based diets had very similar DI/BW values, differing only by four hundredths of a percent. Values for DI/BW observed in the present study were well within accepted levels for rainbow trout feeding on complete diets. Figure 8. Length of distal intestine related to body weight (DI/BW) from rainbow trout fed the experimental diets for ten weeks.

12 Plasma Ion Levels After measuring hematocrit levels, the resulting plasma was analyzed for sodium (Na), chloride (Cl), potassium (K) and calcium (Ca) levels. There were no significant differences in any of these plasma ions due to dietary treatment. Figures 9 and 10 illustrate the plasma ion levels from rainbow trout in the present study. All values for all ions were well within acceptable ranges for healthy rainbow trout under culture conditions. Figure 9. Plasma sodium (Na; left panel) and chloride (Cl; right panel) from rainbow trout fed the experimental diets for ten weeks. Values are in mmolel -1. Figure 10. Plasma sodium (K; left panel) and chloride (Ca; right panel) from rainbow trout fed the experimental diets for ten weeks. Values are in mmolel -1. Control HP-H HP-L EZM-H EZM-L Control HP-H HP-L EZM-H EZM-L

13 Histology In order to determine if the experimental diets tested produced microscopic or tissue specific changes in each of the experimental groups of trout, various organs including liver, kidney, spleen, distal intestine and pyloric caeca were examined after standard formalin fixation, tissue dehydration and paraffin embedding followed by the production of sections that were attached to glass slides and stained using either hematoxylin-eosin (H&E) or Perls Prussian blue. Random sections from each of these tissues were examined carefully in an effort to identify morphological changes that might be indicative of metabolic or biochemical abnormalities arising from a specific diet. Moreover, the presence of changes in tissues from trout receiving one experimental diet can be used as a foundation for the identification of feed formulation or ingredient problems that can be corrected in subsequent work. Liver: It has been well documented that vacuolization of liver hepatocytes in fish can be caused by a variety of abnormalities, particularly fatty acid deficiencies arising from either feed rancidity or reactive oxygen species exposure. In previous feeding trials, our research group has identified various dietary combinations when formulated together produce both vacuolization of liver parenchyma and, in selected cases, deposition of iron laden hemosiderin. By contrast, examination of liver tissue from all experimental groups of fish in this trial revealed no significant vacuolization or changes in the hepatic parenchyma (Figure 11). All experimental groups and a commercial control diet showed no significant impacts of dietary treatment on the architecture of these important metabolic tissues.

14 Figure 11. Comparison of liver histology from rainbow trout fed one of five different diets. Representative sections are included from trout fed diets containing: A) 18% Hamlet Protein SPC; B) 33% Hamlet Protein SPC; C) 18% EnzoMeal; D) 33% EnzoMeal or E) Production feed containing fishmeal. The difference in coloration in Panel B vs. other Panels is caused by differences in de-staining times and is not reflective of any alterations in tissue content or architecture. See text for details.

15 Kidney: Examination of sections from kidney tissue of fish fed one of 4 experimental diets or Control diet showed a normal appearance of both glomerular and tubular elements of the kidney. Figure 12 shows the comparison of kidney histology from fish fed the Control and high soy inclusion diets (HP-300 High and EnzoMeal High). Since there was no difference in the kidney histology from rainbow trout fed the corresponding low inclusion diets, those data are not presented. Even at the high inclusion levels of soy protein, from both sources, there is no apparent difference with respect to the architecture of the kidney tissue. Figure 12: Comparison of kidney histology from rainbow trout fed one of five different diets. After completion of the trial, kidney tissue was obtained from trout selected from each test group and examined by light microscopy after processing and staining with H&E (see Methods). Representative sections are included from trout fed diets containing: A) 33% Hamlet Protein SPC; B) 33% EnzoMeal or E) Production feed containing fishmeal. Sections from trout fed diets containing either 18% Hamlet Protein SPC or 18% EnzoMeal displayed no differences from sections shown in Panels A and B (data not shown). The difference in coloration in Panel A vs. other Panels is caused by differences in de-staining times and is not reflective of any alterations in tissue content or architecture. See text for details.

16 Gastrointestinal Tract: Previous work by multiple research groups has documented the histological changes that occur after feeding salmonids diets containing high concentrations of soy-based feed ingredients (Refstie et al., 2001). In particular, it is generally appreciated that there are reductions in height of villi of the distal intestine as well as evidence of inflammation in the underlying submucosa. However, examination of sections of the distal GI tract from all experimental diets as well as control revealed no such reductions in villi nor any evidence of inflammatory cell infiltration of the intestinal submucosa (Figure 13). Figure 13. Comparison of gastrointestinal tract histology from rainbow trout fed one of five different diets. After completion of the trial, intestinal tissue was obtained from trout selected from each test group and examined by light microscopy after processing and staining with H&E (see Methods). Representative sections are included from trout fed diets containing: A) 18% Hamlet Protein SPC; B) 33% Hamlet Protein SPC; C) 18% EnzoMeal; D) 33% EnzoMeal or E) Production feed containing fishmeal. See text for details.

17 However, changes were observed in the same distal intestinal tissues using the Perls Prussian Blue staining method to detect the deposition of hemosiderin within intestinal mucosa and submucosa. While both Control and both 18% and 33% EnzoMeal fish displayed no evidence of hemosiderin deposition, fish fed a diet containing 33% Hamlet Protein as a feed ingredient showed significant tissue hemosiderin as shown in Figure 14. The presence of hemosiderin within the distal intestinal mucosa is abnormal and its presence suggests abnormalities that result in tissue iron sequestration. It is interesting that hemosiderin deposition was not observed in any other tissues examined (liver, kidney, pyloric caecae and spleen) for any of the experimental or Control diets (data not shown). Figure 14: Deposition of iron rich hemosiderin in gastrointestinal tract mucosa of rainbow trout fed diet containing 33% Hamlet Protein SPC. Sections identical to those shown in Figure 13 were processed using Perls Prussian Blue Staining (see Methods) and examined under conditions identical to Figure 15. Significant staining was present in mucosa from trout fed 33% Hamlet Protein SPC (Panel A) while all other groups including fish fed production feed (Panel B) showed no hemosiderin deposition. See text for details.

18 Pyloric Caeca: Pyloric caeca in trout function to provide trypsin for food digestion and their morphology can be altered by changes in dietary composition (Shaibani et al., 2013). Examination of pyloric caeca tissue from fish in all experimental or Control groups showed no abnormalities (Figure 15). Figure 15: Comparison of pyloric caeca histology from rainbow trout fed one of five different diets. After completion of the trial, intestinal pyloric caeca tissue was obtained from trout selected from each test group and examined by light microscopy after processing and staining with H&E (see Methods). Representative sections are included from trout fed diets containing: A) 18% Hamlet Protein SPC; B) 33% Hamlet Protein SPC; C) 18% EnzoMeal; D) 33% EnzoMeal or E) Production feed containing fishmeal. See text for details.

19 Spleen: Changes in stress, diet and infections produce alterations in the splenic parenchyma which reflect changes in red and white cell turnover (Peters and Schwarzer, 1985). Detailed examination of the spleen tissues of all experimental and the control group fish revealed no abnormalities as shown in Figure 16. Figure 16: Comparison of spleen histology from rainbow trout fed one of five different diets. After completion of the trial, spleen tissue was obtained from trout selected from each test group and examined by light microscopy after processing and staining with H&E (see Methods). Representative sections are included from trout fed diets containing: A) 18% Hamlet Protein SPC; B) 33% Hamlet Protein SPC; C) 18% EnzoMeal; D) 33% EnzoMeal or E) Production feed containing fishmeal. The difference in coloration in Panel A vs. other Panels is caused by differences in de-staining times and is not reflective of any alterations in tissue content or architecture. See text for details.

20 Discussion Traditionally, commodity soybean meal has been effectively utilized in aquafeed formulations under rather strict incorporation levels due to the presence of antinutritional factors (ANF) such as trypsin inhibitors, raffinose, stachyose and others that can detrimentally impact production in many high value species (Hardy, 2010). It is believed that ANF and/or antigens present in soybean products stimulate inflammatory processes in the GI tract of many fish, particularly the salmonids, which alter and decrease the absorptive surface area of the proximal and distal intestine. Inflammatory mediated reduction of salmonid intestinal mucosa surface area is believed to reduce utilization of nutrients in the feed and produce watery, diarrhea-like excrement that is difficult for water treatment (Kaushik et al., 1995; Tacchi et al., 2012). While both trout and salmon develop enteritis in response to ingestion of soy protein containing feeds, comparison of the growth performance and respective digestibilities and nutrient retention data suggest that trout perform better as compared to salmon when challenged with high soy inclusion diets (Refstie et al., 2000). To circumvent the presence of these ANF, many feed companies simply limit inclusion of commodity soybean meal to 10-12% of the diet, which is tolerable for most commercially cultured fish species. Lower concentrations of commodity soy protein are better tolerated by the fish but can produce changes in the distal intestinal architecture and diarrhea in trout (Rumsey, 1994) which is problematic to maintain water quality using standard RAS system methods that are designed for clearance of larger trout fecal pellets. These impacts on the distal intestine have been described as sub-acute enteritis (Baeverfjord and Krogdahl, 1996; Bakke-McKellep et al., 2000). As efforts to find suitable, more sustainable replacement proteins for fish meal intensify, the need to increase the inclusion levels of soybean meal in aquafeed formulations has escalated as well. There has been a dedicated drive to eliminate the ANF in traditional soybean meal, either through the non-gm route of genetic selection, such as the case with Schillinger Genetics, through the GM route of elimination of these ANF through gene deletion/manipulation, or by the process of the production of a soy protein concentrate, of which there are a wide variety of strategies. There is a dire need for more available, cost-effective protein concentrate feedstuffs, and as soybean meal is the most widely utilized plant protein source in aquafeeds, the development of soy protein concentrates for the aquafeed industry is becoming more and more competitive. This feeding trial investigated two inclusion levels for an improved soybean meal (EnzoMeal) manufactured by Battelle under a licensing agreement with the Ohio Soybean Council. Additionally, a comparison was made with a well-know, fermented soy protein concentrate manufactured by Hamlet Protein HP-300. This comparison was chosen due to the similarity of EnzoMeal with HP300 with respect to crude protein levels and amino acid concentrations. Although widely utilized in the weanling piglet industry, HP-300 has not effectively penetrated the aquafeed industry in the US at the present time. These two feed ingredients were tested at

21 two inclusion levels low (18%) and high (33%) in an effort to ascertain whether the products induced any detrimental impacts upon production characteristics, as well as biological, physiological and histological indicators in rainbow trout. After ten weeks on the experimental diets, there were no indications of any significant detrimental impacts on production performance or health of the animals, even at the higher inclusion levels of 33% of the diet. While there were no significant differences with respect to growth or FCR values between any of the experimental feeds, there was a trend of reduced growth at the higher inclusion levels for each respective product. This is particularly evident looking at the weight distribution curves, where the diets with the higher inclusion levels of both HP-300 and the EnzoMeal produced larger numbers of fish in smaller weight classes as compared to the Control and both low inclusion diets (Figure 2). Since this was only a ten-week trial, a virtual snap-shot of a commercial production run, this trend would most likely become more pronounced, and thus significant, during an actual commercial length feeding trial. Nevertheless, the growth data from the current trial is a notable finding for the EnzoMeal in a controlled laboratoryscale trial which provide an excellent foundation for future diets with minimal dietary manipulation to optimize these higher inclusion levels diets to potentially eliminate the detrimental trends to growth. Another noteworthy finding from this feeding trial was the lack of evidence of enteritis in rainbow trout fed EnzoMeal, even at the higher inclusion rates of 33% of the diet. This is an important finding since it opens up the potential for higher inclusion rates of this product in diets designed for rainbow trout. The distal section of the GI tracts in fish fed the EnzoMeal High diet showed excellent architecture of the villi and surrounding mucosal tissues. Additionally, the presence of hemosiderin deposits in the mucosa of the GI tract in fish fed the HP-300 High diet were not observed in fish fed any of the remaining diets. Iron is an essential trace element micronutrient where its uptake into fish is tightly regulated by various transport, metabolic and degradative mechanisms. Iron is not very soluble and is not easily excreted. Therefore, excess absorbed or metabolized iron can accumulate and when it is deposited in cells of the intestine these can be sloughed off with feces. Alternately iron can be stored intracellularly, possibly in lysosomes as hemosiderin which is similar to other breakdown products such as lipofuscin and ceroid. These iron containing degradative compounds represent the wear and tear products of iron metabolism and may influence the susceptibility of cells to oxidation stress over time. Since the HP-300 product contained higher iron levels than the EZM product (200 ppm vs 85 ppm, respectively; Specification sheet for HP-300 attached as Appendix A), this deposition could be the result of the higher inclusion level of 33% manifested itself into hemosiderin deposits in the GI tract mucosa in fish fed the HP-300 High diet. The lack of hemosiderin deposits in fish fed the EnzoMeal High diet is a positive finding, as well as the lower total iron content. Overall, the EnzoMeal product performed well at both inclusion levels. Based upon these data, the lower inclusion level of 18% of the diet appears to be acceptable for commercial aquafeed formulations for rainbow trout. Higher inclusion levels certainly are obtainable with particular

22 dietary modifications to accommodate such levels. Our past experience with all plant protein-based feeds (ie fish meal-free) have indicated the importance of specific micronutrients and minerals that must be supplemented to maintain optimal growth. It certainly appears by the results of this work that the EnzoMeal inclusion rates in rainbow trout could be improved by following a similar strategy. References Ayadi, F.Y, K.A. Rosentrater, and K. Muthukamarappan, Alternative protein sources for aquaculture feeds. J. Aquacult. Feed Science and Nutrition 4(1):1-26. Baeverfjord, G. and A. Krogdahl. (1996). Development and regression of soybean meal induced enteritis in Atlantic salmon, Salmo salar L., distal intestine: a comparison with the intestines of fasted fish. Journal of Fish Diseases 19: Bakke-McKellep, A.M., C.McL. Press, G. Baeverfjord, A. Krogdahl, and T. Landsverk. (2000). Changes in immune and enzyme histochemical phenotypes of cells in the intestinal mucosa of Atlantic salmon, Salmo salar L., with soybean meal-induced enteritis. Journal of Fish Diseases 23: Drew, M.D., Borgeson,T.L. & Thiessen, D.L. (2007) A review of processing feed ingredients to enhance diet digestibility in finfish. Animal Feed Science and Technology 138: Gatlin, D.M. III, F.T. Barrows, D. Bellis, P. Brown, J. Campen, K. Dabrowski, T.G. Gaylord, R.W. Hardy, E. Herman, G. Hu, G., Ǻ. Krogdahl, R.Nelson, K. Overturf, K., M. Rust, W. Sealey, D. Skonberg, E. Souza, D. Stone, R. Wilson, and E. Wurtele. (2007). Expanding the Utilization of Sustainable Plant Products in Aquafeeds A Review. Aquaculture Research 38: Hardy, RW (2010). Use of soybean meals in diets of salmon and trout. Technical Review Paper United Soybean Board and American Soybean Association. Pages Kaushik, SJ, JP Cravedi, JP Lalles, J Sumpter, B Fauconneau and M Laroche. (1995) Partial or total replacement of fish meal by soybean protein on growth, protein utilization, potential estrogenic or antigenic effects, cholesterolemia and flesh quality in rainbow trout (O. mykiss). Aquaculture 133: Li, X. Rezaei, R., Li, P., & Wu, G. (2011) Composition of amino acids in feed ingredients for animal diets. Amino Acids 40: Naylor et al., (2009) Feeding aquaculture in an era of finite resources. PNAS 106(36): Peters, G. and R. Schwarzer. (1985). Changes in hemopoietic tissue of rainbow trout under influence of stress. Diseases of Aquatic Organisms. 1: 1-10.

23 Refstie, S, O Korsoen, T Storebakken, G Baeverfjord, I Lein and AJ Roem. (2000) Differing nutritional responses to dietary soybean meal in rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). Aquaculture 190: Rust, M.B, Barrows, F.T., Hardy, R.W., Lazur, A., Naughten, K., & Silverstein, J. (2010). The Future of Aquafeeds. NOAA/USDA Alternative Feeds Initiative. Rumsey, G.L., A.K. Siwicki, D.P. Anderson, and P.R. Bowser. (1994). Effect of soybean protein on serological response, non-specific defense mechanisms, growth and protein utilization in rainbow trout. Veterinary Immunology and Immunopathology 41: Sealey, WM, FT Barrows, CE Smith, K Overturf and SE LaPatra (2009). Soybean meal leveland probiotics in first feeding fry diets alter the ability of rainbow trout to utilize high levels of soybean meal during grow out. Aquaculture 293: Shaibani, M.E.; B.M. Amin and S. Khodabandeh. (2013). Starvation and refeeding effects on pyloric caeca structure of Caspian salmon (Salmo trutta caspius Kessler 1877) juvenile. Tissue and Cell. 45: Sullivan, K.B. (2008). Replacement of fishmeal by alternative protein sources in diets for juvenile black sea bass. MS thesis UNC Wilmington 85pp. Suresh, A.V., K.P. Kumaraguru, and S. Nates. (2011). Attractability and palatability of protein ingredients of aquatic and terretrial animal origin, and their practical value for blue shrimp, Litopenaeus stylirostris fed diets formulated with high levels of poultry byproduct meal. Aquaculture 319: Tacchi, L, CJ Secombes, R Bickerdike, MA Adler, C Venegas, H Takle and SA Martin. (2012) Transcriptomic and physiological responses to fishmeal substitution with plant proteins in formulated feed in farmed Atlantic salmon (Salmo salar). BMC Genomics 13:363.

24 Appendix A: Specification Sheet for Hamlet HP-300