Influence of Amino Acid Supplementations in Juvenile Yellow Perch Fed Plant Protein Combinations

Distillers dried grains with solubles (DDGS) in combination with soy protein concentrate (SPC) with and without an essential amino acid (EAA) complex were assessed as protein alternatives in juvenile Yellow Perch Perca flavescens diets. Diets contained 5% FM, 40% SPC, and 20% or 40% DDGS each with or without EAA. No mortalities or health assessment differences were observed during the trial and all fish readily accepted the experimental diets. Diets supplemented with EAA produced greater weight gain, improved feed conversion, and apparent protein digestibility. Performance was consistently improved for fish fed diets containing amino acid supplements. Based on these results, Yellow Perch are able to utilize high levels of the plant proteins, accompanied with EAAs, as a FM replacer.


Introduction
A primary concern for aquaculture production is the development of cost-effective feeds, and is a constraining factor that hampers the rapidly growing global aquaculture industry [1] [2].Feed ingredient prices, such as fishmeal (FM) are following an increasing cost trend; however, FM production will likely remain static [3].Fish meal is the traditional animal protein used in fish feeds [4] and generally accounts for the most expensive ingredient in fish diets, translating into one of the largest expenditures in aquaculture production [2] [5] [6].Prices for Open Journal of Animal Sciences FM have risen sharply since 2008 and since 2014 have fluctuated around $1500 per metric ton (Index Mundi.com2018).As a result, less costly plant and animal alternatives are included to reduce costs [7].However, plant-based ingredients often contain anti-nutritional factors (ANF), are less digestible, and do not offer the same balance of amino acids that FM provides [2].With the increased demand for feed supplies to support culture of existing and new species for the food fish market, it is vital that feeds research continues to evolve with a focus on sustainable, less costly energy-protein and lipid-ingredients.Substitution of FM with plant products will become more common as these plant products become available in large quantities and processing methods improve nutrient composition, palatability and digestibility, and decrease ANFs.
Corn ethanol production has increased over 160 percent since 2000 from 1.6 billion to 15.8 billion gallons in 2017 [8].With the increase in ethanol production, the availability of distillers dried grains with solubles (DDGS) has increased.An expected annual production of 40 million metric tons will eventually be produced by the U.S. fuel ethanol industry providing a readily available feed source to developing markets, especially in the Midwest Corn Belt where most ethanol plants are located [9] DDGS are a corn fermentation coproduct derived from ethanol production that typically yields protein ranging from 27% to 31% [10] [11] [12] [13], and lipid ranges from 8% to12% [12].Several studies have tested DDGS in aquaculture feeds [3] [14]- [20].Only one study to date [21] has investigated the use of DDGS in Yellow Perch diets, and some success was found.
Similarly, numerous soybean feedstuffs have been evaluated and are available for aquaculture and considered important ingredients in many freshwater fish diets [22] [23].Generally, defatted SBM has been the most commonly tested soybean feedstuff, however, some studies have tested SPC supplementation in fish diets [24] [35].Kasper et al. [36] and Schaeffer et al. [21] are the only known published studies that have evaluated soybean ingredients in Yellow Perch diets.
Reducing the cost of fish feeds by replacing FM with plant-based proteins is anticipated to reduce production costs, given the lower commodity pricing.Such replacements is not direct, because plant-based proteins often have an inferior essential amino acid (EAA) composition when compared to FM [37] [38] and may have several ANFs [22] [23] [36] [38] [39].Thus, mixed plant-based proteins along with EAA supplements may be required to achieve a balance in compound feeds that more closely mimics the nutritional profile of FM to meet production objectives and control operating costs.The objective of this study was to evaluate Yellow Perch performance fed diets that included mixed blends of DDGS and SPC in Yellow Perch diets, with and without amino acid supplements.

Methods
Experimental Diets.-Fourexperimental feeds were formulated to incorporate FM (70% crude protein; 9.7% lipid), SPC (73% crude protein; 0% lipid), and Open Journal of Animal Sciences 20% or 40% DDGS (29% crude protein; 11.9% lipid) (Table 1).Each DDGS level was formulated for testing with and without an essential amino acid (EAA) complex (arginine, lysine, isoleucine, histidine, glycine, methionine, and a sulfonic acid-taurine).EAA deficiencies of experimental diets were determined a priori from feedstuff analyses and minimum EAA concentrations for inclusion were conformed to recommended levels suggested for Yellow Perch by Hart et al. [41].Diets were formulated to contain similar crude protein levels (46.7 ± 0.84 [mean ± SE]) and gross energy (13.75 ± 0.32 MJ/kg) (Table 2).Gross energy   2) [2].Feed were processed using a single screw, autogenous extruder (Bepex International LLC, Minneapolis, Minnesota), with a barrel length of 600 mm, a barrel length to diameter ratio of four, and barrel speed of 228 rpm.Feeds were extruded into 2-mm diameter pellets, dried at room temperature, coated with fish oil, crumbled and sieved to achieve uniform pellet size, and stored at −20˚C.Diets were analyzed for crude protein (AOAC 2006, method  Fish and Culture System.-Age-0Yellow Perch were held in a 340-L flow-through system and feed trained to accept a commercial pelleted diet (Bio-Diet Grower, Bio-Oregon, Longview, Washington) for a period of 60 days.Following the feed training interval, 288 (10.8 ± 0.6 g) fish were randomly stocked into 16, 110-L tanks to provide four replicate experimental units per diet.Total tank weights (±0.5 g) were measured and a random subsample of 25 fish was selected for initial individual length (±1 mm) and weight (±0.1 g) measurements.Fish were fed the commercial diet for an additional two-week acclimation period Open Journal of Animal Sciences then diet treatments were randomly assigned to replicate tanks.
The Water quality remained stable throughout the duration of the trial.Water pH levels varied from 8.0 -8.6 during the trial.Un-ionized free ammonia (calculated from total ammonia nitrogen) ranged from 0.003 -0.01 mg/L.Nitrate-nitrogen ranged from 2.6 -6.2 mg/L.Nitrite-nitrogen ranged from 0.02 -0.05 mg/L.Water temperatures were held constant at 23˚C and photoperiod was maintained at 15 h light: 9 h dark for the duration of the feeding trial.
Fish were hand-fed rations of 2.0% -2.75% of tank biomass, split into two feedings daily.Consumption was monitored daily to ensure satiation feeding, estimate feed intake, and to minimize feed waste.Total tank weights were measured every 21 days and feed rations were adjusted according to tank weight and observed consumption.Upon completion of the feeding trial, total tank weights were measured as well as individual lengths and weights.Five randomly selected fish from each treatment were euthanized with MS-222, pending necropsy.Fish Analyses.-Performanceindices were used to determine responses to treatment diets.Percent weight gain was calculated as WG = 100 × (final weight (g) -initial weight (g))/initial weight (g)) [2].Feed conversion ratio was calculated as FCR = (weight of diet fed (g)/total wet weight gain (g)) [2].Protein efficiency ratio was calculated as PER = (weight gain (g)/protein fed (g)) [2].Consumption was measured as total feed fed minus feed remaining during the feeding trial.
Proximate analysis was determined on the fillets including crude protein Statistical Analysis.-Allresponse variables were analyzed using analysis of variance (ANOVA) to assess DDGS inclusion levels and amino acid supplementation treatment effects.Significant ANOVA results (p ≤ 0.05) were further analyzed with Tukey's range tests to determine mean differences [42].SYSTAT (version 11) software (SPSS Inc.Chicago, Illinois) and Microsoft Excel (Microsoft, Redmond, Washington) were used to perform all statistical analyses.

Results
Fish readily accepted the experimental diets and fed actively consuming all feed at 2% of tank weight per day.No mortalities or nutritional deficiencies were observed during the trial and no fish exhibited any disease symptoms.
Post-hoc amino acid analysis of the diets revealed that only diets containing EAA supplements completely met or exceeded the suggested EAA required for Yellow Perch reported by Hart et al. [41] (Table 2).Diets without EAA failed to Table 3. Mean tank weight gain (WG), feed conversion ratio (FCR), protein efficiency ratio (PER), viscerosomatic index (VSI), hepatosomatic index (HSI), Fulton-type condition factor (K), muscle ratio (MR) of Yellow Perch fed experimental diets.Values are treatment means (±SE) for response variables.Values not significantly different (P > 0.05) have the same letter within a given column.showed that diet 4 (40% DDGS + EAA) had the highest crude protein while diet 1 (20% DDGS) had the lowest (Table 2).Analysis of muscle tissue revealed no statistical differences in muscle EAA concentrations (Table 4).

Discussion
Rinchard et al. [43] [44] stated that Yellow Perch readily accepted and consumed prepared FM diets and juveniles could be successfully weaned with prepared diets.
However, their diets contained FM as the primary protein source.In this study, all fish readily accepted and actively consumed diets with largely plant-based proteins.
No mortalities or signs of nutritional deficiencies occurred during the feeding trial, indicating that the Yellow Perch are able to utilize these combinations of plant ingredients.Schaeffer et al. [21] had nearly 100% survival (two mortalities) when testing combinations of DDGS and SBM in experimental diets fed to Yellow Perch.Kasper et al. [36] tested soybean feedstuffs in two different forms of SBM (solvent extracted, dehulled SBM and expelled-extruded SBM) in the diets of Yellow Perch and although mortality rates increased as SBM increased survival remained above 90%.Those findings, along with current results suggest Yellow Perch will utilize diets containing high amounts of plant products in conjunction with FM, and performance improves when EAA are included as demonstrated in this study.DDGS.Fish fed either diet with EAA displayed at least 140% weight gain.Similarly, Kwasek et al. [45] found that Yellow Perch growth improved when feeding wheat gluten-based diets supplemented with lysine.
Schaeffer et al. [21] showed that Yellow Perch had greatest growth when fed diets containing 40% DDGS + 9.5 SBM + 24% FM, but noted that growth decreased significantly after DDGS was incorporated at 50% DDGS.Similar studies have had success in using combinations of DDGS and soybean feedstuffs in other species.Nile Tilapia Oreochromis niloticus were fed diets containing 35% DDGS + 58% soy flour producing growth most similar to that of a commercial diet [15].DDGS was used to replace up to 30% SBM, along with lysine supplements, without significantly affecting growth in Channel Catfish Ictalurus punctatus [44].The authors of that study also reported that weight gain was highest for diets containing DDGS + SBM as opposed to diets testing cottonseed meal (CSM) with either DDGS or SBM.Similar results was seen in a study by Gaylord and Barrows [46] who found that Rainbow Trout diets incorporating plant based proteins with EAA supplements at two different protein (35%, 45%) levels had increased performance over un-supplemented diets.The authors noted that greater improvements were observed in diets containing the lower protein level.
Furuya et al. [47] found that FM can be completely replaced by SBM when supplemented with EAA (Lys, Met, Thr, Trp, Arg, Phe, His, Ile, Leu, Val) in the diets of Nile Tilapia.
EAA supplements had a positive impact on FCR and PER values of diets in this study.Similar results have been reported for other cultured species fed DDGS and soybean feedstuffs with amino acid supplements.An increase in FCR was observed for Rainbow Trout when lysine and methionine were incorporated [7].Likewise, increased FCR and PER values were observed in both juvenile and yearling Red Sea Bream Pragus major fed diets containing SPC when lysine and methionine were incorporated [29].Davies and Morris [37] found Rainbow Trout PER improved when EAA supplements were added to soy-based diets, however multiple EAA (met, lys, try, thr, arg, his) supplements proved to provide higher PER than diets containing just methionine and lysine supplements.
In this study, PER was significantly lower in fish fed the diet containing 20% DDGS without EAA (Table 3), no difference was observed in fish fed diets containing EAA or 40% DDGS.
Condition (K values) was significantly higher in fish fed diet 4 (40% DDGS + EAA), however, fish fed diets with EAA had higher K values when compared to the paired EAA-restricted diets.A similar trend was observed with DDGS inclusion, when DDGS were incorporated at 40% the K values were higher than when compared with the paired diets.These results are similar to those observed by Open Journal of Animal Sciences Schaeffer et al. [21] where the authors determined that Yellow Perch K values remained high when DDGS were included in the diet up to 40%, however lower K values were observed when DDGS reached 50% inclusion.These results indicate that Yellow Perch condition is supported up to a 40% DDGS inclusion and that EAAs support an even more favorable condition level.
Based on the somatic tissue analysis, Yellow Perch incorporated dietary EAAs into muscle at similar concentrations regardless of whether or not the diets were supplemented with additional EAA (Table 4).Proximate analysis revealed fillet crude protein was lower for fish fed diets with EAA when compared to diets without additional EAAs.Crude lipid levels in the muscle tissue was found to be higher in diets supplemented with EAA and diets with higher DDGS (Table 4), but diets containing 40% DDGS had the highest crude protein levels.Even though no significant differences were apparent, diets supplemented with EAA or 40% DDGS had higher MR than diets without supplements (Table 3).Schaeffer et al. [21] reported similar results in which no difference was observed in MR when Yellow Perch were fed various combinations of DDGS and SBM.
No significant difference was observed in HSI values indicating that plant protein or EAA inclusion had no impact on relative liver size.Schaeffer et al. [21] noted that HSI values increased as SBM decreased and DDGS increased, HSI values in their study were overall higher than in this study.Takagi et al. [29] determined that HSI of Red Sea Breamfed SPC-based diets was most similar to those fed a FM-based diet when lysine and methionine were supplemented in the diet.VSI values differed significantly in diet 1 and diet 3, however did not appear to be affected by EAA supplements (Table 2).Schaeffer et al. [21] reported no differences in VSI values regardless of DDGS and SBM inclusion when fed to Yellow Perch.

Conclusion
In summary, Yellow Perch were able to effectively utilize the DDGS and SPC combinations presented in this study, although when added EAA supplementation improved performance characteristics.In general, performance was better when DDGS were incorporated at 40%; however, concentration did not appear to be a factor when EAA was added to the diets.In the case of 20% DDGS with EAA supplements, results were nearly the same as those provided by diets including 40% DDGS with EAAs.SPC appears to be a suitable alternative to replacing FM when combined with corn-based proteins.These results along with those of Kasper et al. [36] and Schaeffer et al. [21] demonstrate that plant-based proteins can be used as the primary source of protein in Yellow Perch diets.
feeding trial was conducted in a closed loop recirculation system consisting of a solids separation tank, bio-reactor, and 100-µm bag, charcoal, and UV filtration.Water turnover rate was 25 times per 24 hour period.Water tempera- nois).Dissolved oxygen (DO) was measured twice weekly using an YSI Model 55 DO meter (Yellow Springs Instrument Corp., Yellow Springs, Ohio).

Table 4 .
Resulting amino acid and proximate composition (%, dry matter basis) of Yellow Perch fillets.Values not significantly different (p > 0.05) have the same letter within a given row.Open Journal of Animal SciencesResults from this study demonstrate that Yellow Perch are able to effectively use combinations of DDGS and SPC as FM protein replacers and perform better when supplemented with EAA.Weight gain was significantly higher when diets contained 40% DDGS and when EAA were supplemented at both 20% and 40%