Nutritional Enhancement of Long-Chain Omega-3 Fatty Acids in Tilapia (Oreochromis honorum)

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1 The Israeli Journal of Aquaculture - Bamidgeh, IJA_ , 7 pages The IJA appears exclusively as a peer-reviewed on-line open-access journal at To read papers free of charge, please register online at registration form. Sale of IJA papers is strictly forbidden. Nutritional Enhancement of Long-Chain Omega-3 Fatty Acids in Tilapia (Oreochromis honorum) Corilee A. Watters 1 *, Lee S. Rosner 1, Adrian A. Franke 2, Warren G. Dominy 3, RuthEllen Klinger-Bowen 4, Clyde S. Tamaru 4 1 Department of Human Nutrition, Food and Animal Sciences, University of Hawaii at Manoa, 1955 East West Road, AgSci 216, Honolulu, Hawaii, USA 2 University of Hawaii Cancer Center, 1236 Lauhala Street, Honolulu, Hawaii, USA 3 Oceanic Institute, Kalanianaole Highway, Waimanalo, Hawaii, USA 4 Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, 1955 East-West Road, AgSci 218, Honolulu, Hawaii, USA (Received , Accepted ) Key words: sustainability, aquaculture, fishmeal, EPA, DHA, algae Abstract The health benefits of the long-chain omega-3 eicosapentaenoic (EPA) and docosahexaenoic (DHA) fatty acids that are obtained primarily from fish consumption are numerous. However, supplies of the small pelagic fish that are high in EPA and DHA and used in aquaculture feeds are diminishing, causing greater use of plant-based feeds that are low in EPA and DHA. Farmraised tilapia can be grown on feeds that are low in fishmeal, EPA, and DHA, but low levels of these fatty acids in fillets undermine the health benefits of consuming fish. A six-month feeding study was conducted on tilapia (Oreochromis honorum) to test the effect of augmenting a plant-based feed with corn oil, dried Schizochytrium sp. algae that are rich in DHA, or fish oil. These three experimental feeds were compared with a commercial feed containing fishmeal. The average levels of EPA+DHA for all samples within a treatment were significantly higher in fish fed the feed supplemented with Schizochytrium sp. (185 mg/100 g) or the commercial feed (177 mg/100 g) than in fish fed the feed supplemented with fish oil (138 mg/100 g) or corn oil (120 mg/100 g). Thus, algae represent a sustainable source of long-chain omega-3 fatty acids that can raise levels of healthy fats in aquacultured tilapia fed plant-based diets. Growth was greatest in fish fed the commercial feed, likely due to the higher amount and better quality of protein. * Corresponding author. cwatters@hawaii.edu

2 2 Watters et al. Introduction The health benefits of long-chain omega-3 polyunsaturated fatty acids, specifically eicosapentaenoic (20:5 ω-3, EPA) and docosahexaenoic (22:6 ω-3, DHA) acids, include prevention of heart disease, heartbeat irregularity, and blood clot formation, and the inhibition of inflammation (Connor, 2000; Harris et al., 2008). Long-chain omega-3 have been suggested to be of benefit in metabolic syndromes, rheumatoid arthritis, pulmonary disorders, and psychiatric disorders (Herbaut, 2006; Das, 2008; Simopoulos, 2008), as well as support brain function and development (Bourre, 2006). Western diets are rich in omega-6, but deficient in omega-3 fatty acids. A high dietary omega-6 or omega- 6/omega-3 ratio may play a role in western chronic diseases (Yam et al., 1996; Dubnov and Berry, 2003; Simopoulos, 2008). Enrichment of EPA and DHA in food sources could increase their intake and improve human health (Li et al., 2009). Fish is currently the primary food source of EPA and DHA in the western diet (Weaver et al., 2008). The American Dietetic Association and Dietitians of Canada recommends consumption of two 4-ounce servings of fatty fish per week, which provides about 500 mg of EPA and DHA combined (Kris-Etherton et al., 2007). Surging aquaculture production and reliance on fishmeal for fish feeds are threatening wild ocean stocks (Naylor et al., 2000). The fishmeal industry relies on small pelagic species such as herrings, sardines, and anchovies, which represent more than 22% of the total worldwide catch (FAO, 2008). Total aquaculture production consumed more fish biomass than it produced, an unsustainable practice if the industry continues to expand (Naylor et al., 2000). The use of plant-based feed sources in aquaculture is increasing, primarily due to the scarcity and cost of wild fish for feed (Delgado et al., 2003) Freshwater farm-raised fish such as tilapia provide alternatives to wild-caught ocean fish and can be grown without fishmeal-containing feed. However, replacing fishmeal or fish oil in fish feeds with vegetable oils reduces the long-chain omega-3 content in the fish fillet (Olsen et al., 1990; Tocher et al., 2002; Karapanagiotidis et al., 2007). Tilapia has one of the highest omega-6 contents among farmed fish and up to 40-fold higher omega-6/omega-3 ratios than coldwater fish (Weaver et al., 2008). Continued consumption of farmed tilapia fed diets containing vegetable oil could undermine reliance on fish as a major source of EPA and DHA. Single-cell marine algae such as Schizochytrium sp. are promising alternative sources of long-chain omega-3. These microalgae can be grown heterotrophically and are commercially available as dried products. Schizochytrium sp. are effective in enriching Artemia nauplii and rotifers (Barclay and Zeller, 1996), Atlantic salmon (Miller et al., 2007), and seabream larvae (Ganuza et al., 2008). Channel catfish fed an all-plant diet supplemented with 0-2% dried Schizochytrium sp. for 9 weeks had linear increases in EPA and DHA as well as increased growth and feed efficiency (Li et al., 2009). Our current study tested if a plant-based feed with added Schizochytrium sp. could be used to improve the EPA+DHA content of tilapia relative to the same feed supplemented with fish or corn oil, or a commercial fishmeal-based feed. Materials and Methods The feed trial was conducted at the University of Hawaii at Manoa College of Tropical Agriculture and Human Resources, Windward Community College Aquaculture Complex, Kaneohe, Hawaii. The study was initiated on April 1, 2011, and carried out for six months. Four feed treatments were replicated with three tanks of Oreochromis honorum, each. Three treatments were based on Cargill Sportsman Choice Catfish Feed TM, chosen because it is almost completely lacking in EPA and DHA. Three sources of fat were added to create isocaloric and isonitrogenous feeds: corn oil (Mazola TM, Best Foods, Englewood Cliffs, New Jersey), Schizochytrium sp. flakes (Alga-Mac 3050 TM, Aquafauna Bio-Marine, Inc., Hawthorne, CA), or menhaden fish oil (Omega Protein Inc., Reedville, Virginia). The fat-coating procedure was carried out at the Oceanic Institute, Waimanalo, Hawaii, and involved slowly drizzling the corn oil, Schizochytrium sp. flakes blended in water, or fish oil over the feed pellets while they rotated in a 1.5 HP, 71-l paddle mixer. Ethoxyquin (0.0145% of the final feed weight) was blended with the corn oil,

3 Enhancement of omega-3 fatty acids in tilapia 3 Schizochytrium sp. emulsion, or fish oil prior to the fat-coating procedure. The mixtures were blended 15 min. After blending, the Schizochytrium feed was dried overnight to below 10% moisture content in a forced-air drying cabinet. The fourth treatment was Silver Cup Trout Feed TM (Skretting USA, Tooele, Utah), a commercial feed produced with fishmeal and containing high levels of EPA and DHA. Feeds were held in air-conditioned storage until use. Total EPA+DHA contents of the corn oil, Schizochytrium sp., menhaden oil, and Silver Cup feeds were 4.0, , 734.9, and mg/100 g, respectively (Table 1). Twenty-five 3-4 month-old fish, fed Silver Cup prior to initiation of the study, were added to each of twelve 341-l flow-through tanks, randomized to the feed treatments. Fish were weighed and counted at the onset of the study and every two months. Each week, feed for each tank was transferred to a labeled container, then provided a little at a time to prevent the accumulation of uneaten food in the tank. Therefore, consumed feed was assumed to equal given feed and was determined by subtracting the weight of the feed in the container at the end of the week from the weight at the beginning. Water temperature, dissolved oxygen, ph, and total ammonia nitrogen (TAN) were measured weekly. Water temperature ranged C (mean 22.7±1.3), dissolved oxygen ppm (mean 8.0±0.8), ph (mean 7.7±0.3), and TAN ppm (mean 0.18±0.11), within optimal limits for tilapia growth (Balarin and Haller, 1982). Three fish were sampled immediately prior to randomizing the treatments. Four samples were removed from each tank after two months. Due to mortality and to avoid running out of fish in some tanks, only two fish were sampled from each tank after four and six months. Sampled fish were euthanized, weighed, and filleted. Samples that were not processed immediately after collection were held at -70 C. Pooled fillets Table 1. Fatty acid profiles and protein/fat contents of feeds for tilapia (Oreochromis honorum). from each tank were homogenized and two 10-g samples were freeze dried and immediately weighed to calculate dry mass and initial moisture content. One sample, each, of freeze-dried Schizochytrium and fish oil, and not freeze-dried catfish and Silver Cup feeds were analyzed at the University of Hawaii Cancer Research Center, Honolulu, Hawaii, according to the methods detailed by Li and Franke (2011). Data for corn oil was obtained from the USDA Nutrient Database. EPA+DHA and fish weight were analyzed by repeated measures ANOVA. Pairwise comparisons of means were carried out using Tukey s Multiple Range Test. Differences were considered significant if the significance level was less than Results The levels of fatty acids in the flesh of O. honorum at 2, 4, and 6 months are given in Table 2. EPA+DHA was significantly affected by the feed treatment (p = 0.006) and the interaction between feed and time (p = 0.04). Feeding duration had no significant main effect (p = 0.56). The average level of EPA+DHA of all samplings was highest in fish fed the diet containing Schizochytrium sp. (185 mg/100 g) but did not significantly differ Diet Supplemented catfish feed Schizochytrium Corn Menhaden oil oil sp. Fatty acid (mg/100 g) 1 Silver Cup Trout Feed 14: : :1 n : :1 n :2 n :3 n :2 n :4 n :5 n-3 (EPA) :5 n :6 n-3 (DHA) Macronutrient (%) 2 Total fat Total protein profiles were determined by analysis, except for the corn oil treatment that was determined by analysis of the catfish feed and the fatty acid profile of corn oil provided by the USDA Nutrient Database. 2 fat and protein contents of the trout feed were provided by manufacturer; contents of the experimental feeds were calculated using manufacturer analyses of the catfish feed and Schizochytrium flakes, with corn oil and menhaden oil included as 100% fat.

4 Mean fish wt (g) 4 Watters et al. Table 2. Fatty acids (mg/100 g wet wt) in fillets of tilapia (Oreochromis honorum) fed diets containing corn oil, Schizochytrium sp. (Schiz sp.), or fish oil, compared to those fed a commercial trout diet, Silver Cup. Initial 2 months 4 months 6 months Fatty acid Silver Corn Schiz Fish Silver Corn Schiz Fish Silver Corn Schiz Fish Silver Cup oil 1 sp. oil Cup oil 1 sp. oil Cup oil 1 sp. oil Cup 14: : :1 n : :1 n :2 n :3 n :2 n :4 n :5 n-3 (EPA) :5 n :6 n-3 (DHA) from the average level in fish fed Silver Cup (177 mg/100 g). These levels were significantly higher than in fish fed the diets containing fish oil (138 mg/100 g) or corn oil (120 mg/100 g). After six months, no EPA+DHA level significantly differed from the initial level (Fig. 1). Fish fed Silver Cup grew significantly faster and larger than fish fed the other feeds (p = 0.049; Fig. 2). Cold water temperatures limited growth in the first two months of the study but when the water temperature rose in June and July, growth improved noticeably. However, after 6 months, fish in all treatments were well below the minimum market size of 0.45 kg. Mortality occurred throughout the study. Fish were missing every month, likely the result of jumping out of the tank. Fish were missing from eight of the 12 tanks, including all treatments, with a mean loss of 3.6±4.3 fish/tank and a maximum loss of 13 fish/tank. An acute mortality event affected two tanks in July, with all fish in two of the fish oil replications dying within 24 days. At the same time, no mortality occurred in any other tank. Prior to this event, a wood preservative (Thompson s WaterSeal, Sherwin- Williams Co., Cleveland, Ohio) Fig. 1. EPA+DHA content in tilapia fed feeds containing different EPA+DHA sources: corn oil (CO), fish oil (FO), Schizochytrium sp. algae (SCH), or a commercial trout feed, Silver Cup (SC). P values indicate the significance of the final content of each treatment relative to the initial value in April. Corn oil Schizochytrium Menhaden oil Silver Cup Feed Fig. 2. Mean weight of tilapia fed feeds containing different EPA+DHA sources. Bars represent +/- one standard error of the mean.

5 Enhancement of omega-3 fatty acids in tilapia 5 was applied to boards 5-10 m from the aquaculture set-up. The two affected tanks were those closest to this application. Post mortem examination was inconclusive, but consistent with toxicity. Given the lack of mortality in the third fish oil tank, there is no evidence to indicate toxicity related to the fish oil or any other feed. Discussion The EPA+DHA levels in fish fed the algae or commercial diet were higher than those typically reported for tilapia (Karapanagio-tidis et al., 2006, 2007; Shapira et al., 2009; USDA-ARS, 2011) and reflect the level of long-chain omega-3 fatty acids in the feeds. Silver Cup was formulated for trout, which has a high dietary DHA requirement (Bell et al., 2001). 12. Although we were unable to obtain such information from the manufacturer, the fishmeal/fish oil content of Silver Cup is likely greater than in the experimental diets or the level that is typically included in commercial feeds. Likewise, the level of Schizochytrium in the algae diet (3.56% by weight) was almost double the highest level (2%) in a plant-based feed that increased EPA+DHA levels in channel catfish (Ictalurus punctatus) by 480% in 9 weeks (Li et al., 2009). The commercial diet, Silver Cup, resulted in the fastest growth. Most likely, the higher amount of protein, the fish source of protein, palatability, and/or the higher fat content, rather than the level of long-chain omega-3, influenced the growth rate. The optimum feed protein content for growth of young O. niloticus (45 g) is 40-45% (Al Hafedh, 1999); Silver Cup was the only feed in our experiment with this protein level. Growth was lower and FCR was higher in rainbow trout (Oncorhynchus mykiss) fed plantbased diets than in those fed a fishmeal-based diet with an equivalent amount of protein, possibly due to amino acid imbalances in the soy component of the feed (Barrows et al., 2001). Palatability is also an issue with plant-based diets. In the present study, the protein component of the corn oil, algae, and fish oil diets was entirely plant-based. The commercial diet provided 10% total fat, near the optimum 12% for growth of O. niloticus x O. aureus (Chou and Shiau, 1996), and more than the other experimental feeds. Currently the cost of Schizochytrium sp. limits its use as a feed ingredient in juvenile and adult finfish production (Li et al., 2009). However, as the scale of Schizochytrium production increases and costs fall, judicious use of the DHA-rich algae as a finishing feed supplement should become viable. It will be important to determine the optimum duration the finishing feed needs to be provided. Since the tilapia were fed Silver Cup, a diet rich in long-chain omega-3, prior to the start of this study, our results cannot be used to determine how long a finishing feed needs to be provided to fish raised on a 100% plant-based diet. In the present study the highest EPA+DHA level was obtained in fish fed the algae diet by the fourth month, suggesting the time required for a finishing feed may be relatively short. Taste is another variable that needs to be explored with the use of supplemental algae. In channel catfish the addition of 0.5-2% Schizochytrium sp. to a plant-based diet increased growth and improved feed efficiency (Li et al., 2009). The use of higher levels in different species requires evaluation. The purpose of this research was to determine if supplementation of Schizochytrium sp. increases the long-chain omega-3 content in tilapia. Although growth and feed efficiency in fish fed this diet were lower than in fish fed the commercial fishmeal-based feed, much of this effect is probably due to the fish base of the feed rather than the algae component. Work is still needed to produce 100% plant-based feeds that yield equivalent growth and feed efficiency as fishmeal-based feeds. Juvenile O. niloticus fed a soy-based feed supplemented with essential amino acids and dicalcium phosphate resulted in an equivalent weight gain and FCR relative to a 10% fishmeal-based diet (Furuya et al., 2004). Raising tilapia on such a feed, supplemented with an algae source of long-chain omega-3 at the end of the production cycle, would optimize their nutritional value and minimize environmental impact. Acknowledgements The authors would like to thank Tetsuzan (Benny) Ron, Ph.D., Aquaculture Program Coordinator, University of Hawaii Aquaculture Program, Office of the Vice Chancellor for

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