Amending Reduced Fish Meal Feeds with Phospholipids to Improve Performance of Hybrid Striped Bass

Objective: In carnivorous fishes like hybrid striped bass Morone chrysops female x M. saxatilis male, sparing dietary fish meal with soybean meal may lead to reduced feed intake and growth performance. Dietary phospholipid supplements may help improve feed intake and growth. The present study was designed to test the effects of dietary supplementation with marineor soy-origin lecithins (sources of phospholipids) on the growth performance of hybrid striped bass fed reduced fish meal, soybean meal-based feeds. Methods: We evaluated the growth performance of hybrid striped bass fed diets containing 30% soybean meal or 10% fish meal/65% soybean meal with and without supplementation of soyor marine-origin lecithin at 2 or 4% of the diet. Juvenile fish (33 ± 0.6 g, mean ± SEM) were stocked in a recirculation aquaculture system (8 fish per tank), and diets were randomly assigned to tanks in quadruplicate (n=4). Fish were fed assigned diets daily to apparent satiation for 9 weeks, after which standard metrics of growth performance were assessed. Results: Growth performance was generally superior among fish fed the lecithinsupplemented diets. This effect was particularly evident among fish fed the 10% fish meal diet supplemented with 2 or 4% marine lecithin, which exhibited significantly greater feed intake, weight gain, and specific growth rate than those fed the 10% fish meal or 30% fish meal feed. Feed conversion ratio was not statistically different among the dietary treatments. Conclusion: Our results suggest amending reduced fish meal feeds with phospholipids can improve feed intake and growth in hybrid striped bass, and supplementing the diet with marine lecithin in particular may increase hybrid striped bass growth beyond that typically achieved with fish meal-based formulations.


Introduction
The increased utilization of soybean meal in commercial aquafeeds has helped reduce fish meal dependence and made a significant contribution towards the development of costeffective and sustainable feeds for the fish farming sector [1]. Nonetheless, the extent to which fish meal can be spared by alternatives like soybean meal is still widely debated, and varies among taxa. In omnivorous fish diets, soybean meal typically allows for total replacement of fish meal without impacting the growth of the fish, whereas issues of palatability, digestibility or essential amino acid composition typically limit the performance of carnivorous fish when all or most of the fish meal fraction of the diet is replaced [1]. Although soy-derived proteins are welldigested by hybrid striped bass Morone chrysops female x M. saxatilis male [2,3], various experiments have confirmed that soybean meal (amended with methionine supplements) could not fully replace fish meal without impairing specific growth rate

Journal of Animal Research and Nutrition ISSN 2572-5459
and feed conversion ratio [4,5,6]. Often, impaired performance is linked to reduced intake of soybean meal-based feeds. The organoleptic profile of soybean meal is distinct from that of fish meal, and palatability factors (or the lack thereof) are commonly invoked to explain reduced acceptance of reduced or fish mealfree feeds by carnivorous fish. However, performance may also be influenced by the loss of nutrients present in fish meal but not soybean meal. Notably, fish meal is a rich source of phospholipids, with the lipid fraction (5-13%, by weight) typically containing twice as much polar lipid as neutral lipid [7]. It is possible that supplementation with potentially beneficial, though perhaps not strictly essential phospholipids may improve intake and performance of fish fed reduced fish meal feeds.
The addition of phospholipids, in the form of lecithins, to aquafeeds has been shown to increase growth by increasing total feed intake in some fish. Feed intake and growth of rainbow trout Oncorhynchus mykiss [8,9], Japanese flounder Paralichthys olivaceus [10], amberjack Seriola dumerili [11] and Atlantic salmon Salmo salar [12,13] were improved by dietary supplementation with soy lecithin. Although soy lecithin is more commonly used as a feed supplement, marine lecithin is also commercially available and may prove useful as a feed supplement. Marine lecithin, derived from fish, is high in 22:6n-3 (docosahexaenoic acid), an essential fatty acid for normal growth and development. While supplementation with lecithin may increase feed intake and growth performance, supplementation with essential fatty acids in the context of more 'potent' phospholipids (compared to neutral lipids) may further promote growth [7,14,15]. Little research has been undertaken to compare supplementation of marine lecithin with the more widely used soy lecithin. Accordingly, the present study was designed to test dietary supplementation with marine-or soy-origin phospholipids on the growth performance of hybrid striped bass fed reduced fish meal, soybean meal-based feeds.

Methods
Two practical feeds (40% protein, 14% lipid) previously evaluated in the Center for Fisheries, Aquaculture, and Aquatic Sciences (CFAAS, Carbondale, Illinois) [6] and formulated to be isolipidic/ isonitrogenous and to meet all known nutritional requirements of juvenile hybrid striped bass were slightly modified for use in the present work (  [16]. Fatty acid methyl esters (FAME) were obtained via acid-catalyzed transmethylation and identified/quantified via GC-FID (GC-17A, Shimadzu Corporation, Kyoto, Japan) as described by [17] ( Table 2). Dietary lipid class composition was determined using procedures adapted from [18]. Briefly, crude lipid was extracted from feeds as before and resuspended in chloroform. Lipid classes were separated using 3-mL aminopropyl solid phase extraction columns (Supelclean™ LC-NH2 SPE Tubes, Supelco, Bellefonte, Pennsylvania) fitted to a Supelco VISIPREP™ (Supelco, Bellfonte, Pennsylvania) vacuum separation unit. Neutral lipids were eluted using a 2:1 chloroform:2-proponal solution, and polar lipids were subsequently eluted using methanol. Fatty acid methyl esters were prepared, identified, and quantified in the same manner as those derived from total lipid samples ( Tables 3, 4).
Juvenile hybrid striped bass (33 ± 0.6 g, mean ± SEM) were obtained from Keo Fish Farm (Keo, Arkansas) and stocked at 8 fish/tank into 24 tanks of a recirculation aquaculture system consisting of 150-L tanks with associated mechanical (bead filter) and biological (submerged media biofilter) filtration and supplemental aeration. Photoperiod was maintained at a 12:12 light/dark cycle, and temperature was maintained via ambient air temperature. Temperature and dissolved oxygen were monitored daily (YSI 550A, Yellow Springs Instruments, Yellow 56 ± 4 54 ± 3 a Formulated to contain the following per 100 g of premix: 8.955 g cellulose, 8.3 g all-rac-alpha tocopheryl acetate, 12.5 g inositol, 25.0 g L-ascorbyl-2-polyphosphate, 12.5 g nicotinic acid, 6.25 g calcium pantothenate, 13.16 g menadione sodium bisulfate complex, 1.25 g thiamine mononitrate, 2.5 g pyridoxine hydrochloride, 7.5 g riboflavin, 1.0 g vitamin A palmitate, 0.45 g folic acid, 0.5 g cyanocobalamin, 0.01 g cholecalciferol, and 0.125 g biotin. b Formulated to contain the following per 100 g of premix: 55.381 g cellulose, 24.897 g zinc oxide, 14.933 g ferrous sulfate, 3.47 g manganese oxide, 0.967 g cupric carbonate, 0.262 g potassium iodide, 0.06 g sodium selenate, and 0.03 g cobalt carbonate. c Yelkin ® TS soy-derived lecithin, Archer Daniels Midland, Decatur, Illinois. d Marine Natural Lecithin LC 60, Phosphotech Laboratories, Saint-Herblain Cedex, France. Springs, Ohio), whereas alkalinity, total ammonia-, nitrite-, and nitrate-nitrogen were quantified every 7-10 days. Water quality parameters were maintained within ranges suitable for hybrid striped bass culture [19] throughout the trial (temperature -24.3 ± 0.0 °C; dissolved oxygen -5.5 ± 0.0 mg/L; total ammonia -0.1 ± 0.1 mg/L; nitrite-nitrogen -0.0 ± 0.0 mg/L; nitrate-nitrogen -9.2 ± 0.9 mg/L; alkalinity -306.0 ± 6.7 mg/L of CaCO 3 ; mean ± SE). Dietary treatments were randomly assigned to quadruplicate tanks (4 tanks/treatment, n = 4) and all fish were fed assigned diets once daily to apparent satiation. After 9 weeks of culture, fish were counted and group weighed by tank. Three fish were then randomly selected from each tank, sedated (bath immersion in a solution of tricaine methanesulfonate), euthanized by single cranial pithing, weighed, and dissected to remove and weigh liver and intraperitoneal fat tissues. Group and individual-based data were used, along with daily feeding records, to calculate production performance metrics as follows:

Results
Amending reduced fish meal hybrid striped bass feeds with marine or soy-derived phospholipids altered the composition of the feeds and the growth performance they yielded. Although proximate composition was relatively consistent among the feeds (Table 1), the diets were distinctly different with respect to total lipid FA composition ( Table 2). These differences in fatty acid composition were particularly evident within the neutral 2015 Vol.

Journal of Animal Research and Nutrition ISSN 2572-5459
and polar lipid fractions (Tables 3, 4). Reducing fish meal from 30 to 10% of the formulation necessitated the inclusion of additional oil to maintain total dietary lipid content. Greater inclusion of neutral lipid-rich fish oil thus increased the level of 20:5n-3 (eicosapentaenoic acid), 22:6n-3, and total long-chain polyunsaturated fatty acids (LC-PUFA) within the neutral lipid fraction of the 10% FM feed compared to the 30% FM feed. By replacing fish oil, lecithin supplementation progressively reduced the contribution of fish oil to neutral lipid and LC-PUFA within this lipid fraction. Sparing fish meal with alternative feedstuffs had the opposite effect on the prevalence of marine-associated fatty acids within the polar lipid fraction. Although the 10% FM formulation was amended with fish oil to balance total lipid content, phospholipids were not supplemented and thus the polar lipid fraction reflected only the residual phospholipids contributed by the plant-origin feedstuffs and remaining fish meal. As a result, the LC-PUFA-rich polar fraction in the 30% FM feed was largely replaced by an 18:2n-6 (linoleic acid)-rich polar lipid fraction in the 10% FM feed. Supplementation with marine lecithin corrected for this effect by progressively increasing the amount polar lipid-bound LC-PUFA, whereas supplementation with soy lecithin maintained an 18:2n-6-rich polar lipid profile.
Fish fed the 30% FM control feed exhibited feed intake (2.51% body weight/day), growth (weight gain = 272%, SGR = 2.12% body weight/day), and a feed conversion ratio (1.10) consistent with juvenile hybrid striped bass cultured under near-optimal conditions in recirculation systems (

Discussion
Previous research evaluating alternative proteins in hybrid striped bass feeds has indicated that aggressive fish meal sparing (e.g., reducing fish meal inclusion below 10%) or complete replacement can result in reduced growth performance, often       Table 2. b Only a single replicate was successfully analyzed; remaining replicates were lost

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ISSN 2572-5459 associated with somewhat distinct functions: whereas gall powder (containing phospholipids, deoxycholyltaurine, and other proteins) and synthetic cholyltaurine act primarily via maintenance of gut integrity and stimulation of digestive and metabolic activities, lecithin acts primarily by increasing feed intake [9]. Growth of Japanese flounder [10] and amberjack [11] fed fish meal-free diets was progressively improved by amending the diets with 2-4% soy lecithin, and the authors also attributed these effects to greater acceptance and intake of the lecithinsupplemented feeds. In a series of studies evaluating dietary inclusion of lecithin and soy protein in salmonid feeds, Poston [8,12,13,27] reported similar increases in feed intake and growth performance using lecithin as a supplement in feeds for rainbow trout and Atlantic salmon. Our results are consistent with these accounts, suggesting improved performance among fish fed the lecithin-amended feeds is due, in part, to increased feed intake. Although Sealey et al. [28] reported hybrid striped bass growth was unaffected by dietary supplementation with soy lecithin, the feeds used in this study contained approximately 60% fish meal (containing 2.6% lipid) and approximately 7% Fish Oil and were likely to have contained sufficient amounts of polar lipid as to preclude performance enhancement through additional supplementation.
In their studies of phospholipid supplementation in yellowtail Seriola quinqueradiata and gilthead seabream Sparus aurata, respectively, Harada [29] and Koven et al. [30] suggested the trimethyl group associated with the nitrogen moiety of phosphatidylcholine (the primary phospholipid associated with lecithins) could act as an attractant in fish feeds. The proposed attractant properties of phosphatidylcholine do not explain the differential results achieved with marine vs. soy lecithin in the present study or our previous work with these supplements in cobia Rachycentron canadum feeds [24]. As in the present study, as a consequence of reduced feed palatability and intake [6,19,20,21,22]. In our previous research, we have determined that 10% fish meal is the approximate threshold at which fish meal sparing begins to impact palatability and growth performance in juvenile hybrid striped bass: although statistically significant impairment was not observed in each of these studies, 10% fish meal appears to be a relatively conservative estimate of the minimum appropriate inclusion level for this taxon [22,24,25].
Our present results are consistent with the concept of a 10% fish meal inclusion threshold: whereas statistically significant impairment was not observed, fish fed the 10% FM feed exhibited numerically lower weight gain and SGR compared to those fed the 30% FM feed despite numerically higher feed intake of the 10% FM feed. It is somewhat unsurprising that the addition of nutrients which may be limiting in the 10% FM feed, specifically phospholipids, was generally associated with improvements in growth performance. What is surprising, however, is that certain of these amendments, most obviously marine lecithin, resulted in significant improvement over the 30% FM feed as well as the 10% FM feed.
Previous research has indicated that supplementation with lecithin can improve intake, digestibility, and utilization of aquafeeds, particularly those containing little-to-no fish meal. Although a preliminary study failed to demonstrate growth promotion [26], Iwashita et al. [9] reported supplementing fish meal-free rainbow trout diets with soy lecithin improved fish growth and feed intake, and also corrected for some histological abnormalities observed within the gut of fish fed the un-supplemented, fish meal-free diet. Similar results were also achieved with bovine gall powder and cholyltaurine supplements, though these products appeared to be more effective in maintaining gastrointestinal integrity and promoting growth than soy lecithin [9]. These authors concluded that the growth promoting effects of these supplements were

Journal of Animal Research and Nutrition ISSN 2572-5459
supplementing reduced fish meal cobia feeds with soy lecithin did not elicit the same magnitude of growth promotion as did supplementation with marine lecithin [24]. Although the diets were formulated to be replete with all nutrients known to be required for optimal growth in both studies, marine lecithin supplementation conferred greater benefit to the reduced fish meal feeds than equivalent supplementation with soy lecithin. This would seem to suggest a distinct functional property, not the potential attractant characteristics of a trimethyl group common in both phosphatidylcholine-rich lecithin products, is responsible for the particularly beneficial effects of supplementing the diet with marine lecithin. The primary difference between the two lecithin products is the fatty acid composition of the phospholipids they contain: the soy lecithin is particularly rich in 18:2n-6, whereas 22:6n-3 is abundant in the marine lecithin product. Previous research has demonstrated that provision of essential and physiologically relevant fatty acids like 22:6n-3 is critical for normal growth and development of many fishes [31], including hybrid striped bass [32], and that these nutrients are more potent when provided in the context of polar rather than neutral lipid [7,14,15]. Thus, a growth-promoting effect of providing additional phospholipid-bound 22:6n-3 vs. 18:2n-6 seems a plausible explanation of the present results. However, it is not clear what the underlying mechanism responsible for these differential effects might be.
In conclusion, it appears that amending reduced fish meal feeds with phospholipids can improve feed intake and growth in hybrid striped bass, and supplementing the diet with marine lecithin in particular may increase hybrid striped bass growth beyond that typically achieved with fish meal-based formulations. Of course, the incorporation of marine lecithin as a feed ingredient is counter to the rationale for fish meal sparing, particularly the arguments related to economic and sustainability concerns regarding fish meal. Although sustainability may be less of a concern for marine lecithin than for other marine-derived ingredients (marine lecithin is derived from processing wastes generated by industrialized marine fisheries), it will almost certainly be more expensive that soy lecithin, now and in the future. Whether the added cost of marine lecithin supplementation is justified by the apparent benefits of this type of ingredient will depend upon the circumstances at hand, e.g., the feed formulation, intended fish taxon, feedstuff pricing. Further research is recommended to further elucidate the potentially interrelated functions of phospholipids and the fatty acids they carry in determining feed acceptance and growth performance in cultured fish.