APPARENT DIGESTIBILITY COEFFICIENTS OF FEEDSTUFF USED IN TAMBAQUI DIETS

Quality of fish diet can be evaluated by quantifying the ability of fish to digest food. Therefore, this study aimed to determine the apparent digestibility coefficients (ADC) for crude protein (CP), ether extract (EE) and gross energy (GE) in nine protein ingredients and seven energy ingredients most commonly used in commercial diets for tambaqui. In total, 510 tambaqui juveniles were fed test diets containing a reference diet supplemented with an inert digestibility marker (Cr2O3) and each ingredient tested. Digestibility analyses showed that corn gluten had the highest ADCCP (98.09%) and ADCGE (96.91%), whereas alcohol yeast had the lowest ADCCP (63.17%) and ADCGE (46.24%). Protein ingredients were revealed to be excellent sources of lipids (ADCEE > 85.0%), with the exception of wheat gluten (ADCEE: 63.73%). Corn (94.50%) and wheat bran (86.08%) showed the highest ADCCP of all energy ingredients. No significant differences in ADCEE were observed across the energy ingredients tested. Of the energy ingredients tested, corn oil (95.70%), fish oil (93.61%), soybean oil (93.31%) and corn (88.70%) had the highest ADCGE. The use of protein ingredients and energy ingredients with high digestibility coefficients, such as the ones reported, can guide the formulation of top quality diets for tambaqui.


INTRODUCTION
In recent years, the preference for healthier foods has gained increasing attention.Fish is one of the most-traded food commodities worldwide.It is rich in protein, minerals, and vitamins, is low in fat content, and contains omega-3 fatty acids (FAO, 2014).Aquaculture has been responsible for the impressive growth in the supply of fish for human consumption (FAO, 2016) and studies on fish feeding and nutrition are Hellen BUZOLLO 1Thiago Matias Torres do NASCIMENTO 1   Lidiane Cristina Gonçalves de SANDRE 1 Ligia Maria NEIRA 1 Rosângela Kiyoko JOMORI 2 Dalton José CARNEIRO 1 crucial because feeding accounts for up to 70% of costs in the production cycle (MORO and RODRIGUES, 2015).
The determination of apparent digestibility coefficients (ADC) is the first step in evaluating the nutritional quality and utilization efficiency of an ingredient in complete diets for animals.These coefficients provide an indication of the nutrient or energy fraction of the ingested feedstuff that is not excreted in the feces, but is used by the animal metabolism (NRC, 2011), thereby positively affecting production rates.Moreover, information on ADC can be useful to reduce excretion of nutrients in the production environment, contributing to sustainable fisheries and providing the basis for further studies on the nutritional needs of fish.
Brazil is known for its high freshwater species diversity.The tambaqui (Colossoma macropomum) is the most produced native species in continental aquaculture, with great growth in 2016 with 136.99 thousand tons (IBGE, 2016), due to its easy juvenile availability, fast growth, and excellent taste.Tambaqui is an omnivorous fish that adapts to a wide variety of sources of protein and energy in your diet.In fact, it can be fed commercial diets or take advantage of the primary productivity of the pond by utilizing plankton as a source of protein (GOMES and SILVA, 2009).Brazil produce a large amount of diversified feedstuff and the digestibility studies for this species are rare, and most studies have focused on the use of local alternative sources (SILVA et al., 2003(SILVA et al., , 2007;;GUIMARAES et al., 2014), limiting the availability of data for the formulation of balanced diets that maximize production.Thus, this study aimed to determine the apparent digestibility coefficients (ADC) for crude protein, ether extract, and gross energy in nine protein ingredients and seven energy ingredients most commonly used in commercial tambaqui diets.

METHODS
The experiment was carried out in the Laboratory of Aquatic Organism Nutrition of the Aquaculture Center of São Paulo State University (Jaboticabal, São Paulo, Brazil).This study was conducted in accordance with the ethical principles for animal experimentation adopted by the Brazilian College of Animal Experimentation (COBEA) and was approved by the Ethics Committee on Animal Use (CEUA) at São Paulo State University, Faculty of Agricultural and Veterinarian Sciences, SP, Brazil, under protocol n. 015810/11.
A reference diet was formulated (Table 1) to determine the ADCs of each ingredient.In total, 16 ingredients were analyzed and divided into two groups: a) energy ingredient: broken rice, wheat bran, corn, sorghum, corn oil, soybean oil, and fish oil; b) protein ingredient: soybean meal, cottonseed meal, corn gluten meal, wheat gluten meal, alcohol yeast (spray-dried), salmon meal, tilapia processing residue meal, poultry by-product meal and feather meal.Ingredients were ground and analyzed (Table 2).16 test diets containing 695 g kg -1 of the reference diet mixture, 5 g kg -1 of chromium-III oxide (Cr 2 O 3 ) (used as the inert digestibility marker) and 300 g kg -1 of each ingredient tested (except for the oils and wheat gluten, 100 g kg -1 ) were prepared.The inclusion of only 100 g kg -1 for wheat gluten meal was due to gluten's cohesive and visco-elastic properties that provide the binding needed for the pellet, but that may also result in a rubbery, dry pellet (DAY et al., 2006) and for the oils the inclusion of the 300 g kg -1 would cause an interference in the starch expansion (MOREIRA et al., 2015) and in the acceptability of the diets by the fish.For the preparation of diets, the ingredients were ground, manually mixed, and the diets were then extruded (Extecc  Maquinas, Ribeirão Preto, Brazil, model Ex Micro) into granules with diameter of approximately 5mm.The granules were dried in an oven with forced-air ventilation at 55 °C for 24 h.
The digestibility trial consisted of 17 treatments (16 test diets and one reference diet) and four replications arranged in a randomized entirely design.In total, 510 tambaqui (Colossoma macropomum) juveniles (mean initial weight: 70.0 ± 8.58 g) were used in the study.The animals were kept in 34 experimental tanks (430-L) provided with continuous aeration and water circulation.The physicochemical parameters of the water (mean ± standard deviation, pH: 7.85 ± 0.17; temperature: 29.72 ± 0.34 °C; dissolved oxygen: 5.71 ± 0.34 mg L -1 ; and total ammonia: 189.17 ± 59.29 µg L -1 ) in the tanks were within the acceptable range for the species (ARAUJO-LIMA and GOMES, 2010).
according to the modified Guelph system described by ABIMORAD and CARNEIRO (2004).Fecal collection from the four replicates of the 17 treatments (16 test diets and 1 diet reference) was divided into two periods: 1st period -distribution of replicates 1 and 2 in 34 feeding tanks.The adaptation to the diets was carried out for seven days.On day 8, feces were collected from replicate 1 and on day 9 from replicate 2. 2nd period -redistribution of the diets of replicates 3 and 4 in 34 feed tanks.The adaptation to the diets was carried out for seven days.On day 8 feces were collected from replicate 3 and on day 9 from replicate 4. In both periods the fish from each replicate were fed to apparent satiation and transferred after the last feeding of the day (18:00 h) to the conical tanks and therefore, the collections were performed during the night.The feces were collected into Falcon conical tubes (kept on ice to reduce feces degradation), every three hours, for ease management of the animals, according to previous project, until 6:00 h of the next day.All feces collected from each replicate were lyophilized using a Thermo Electron Corporation Fisher  freeze dryer and analyzed.
Chromium-III oxide concentrations in diets and feces were determined by spectrophotometry following nitric-perchloric digestion according to the methodology described by FURUKAWA and TSUKAHARA (1966).Ether extract (EE) was determined by acid hydrolysis (AOAC, 2000).Crude protein (CP) was determined by automated combustion using a LECO FP 528 nitrogen analyzer (LECO Instruments, St Joseph, Michigan, USA).Gross energy (GE) was determined by bomb calorimetry (AOAC, 2000) using a C2000 basic IKA calorimeter.Apparent digestibility coefficients (ADC) of the nutrients in the diets were calculated according to the equation of NOSE (1960): The ADC for a nutrient in a test ingredient was calculated from the digestibility coefficient for the reference diet and the test diets according to the following equation of FORSTER (1999): where: a = CP, EE, or GE contribution of the reference diet to the CP, EE, or GE content of the test dietm multiplied by 69.5%; b = CP, EE, or GE contribution of test ingredient to CP, EE, or GE content of the test diet, multiplied by 30%.For wheat oils and gluten: a = multiplied by 90% and b = multiplied by 10%.
ADCs of ingredients were analyzed separately according to the classification of ingredients (protein or energy).The data were analyzed using a parametric analysis of variance (ANOVA) and tested for error normality and homoscedasticity of variances.When significant differences were detected, the means of the variables were compared using the Tukey's test at the 5% significance level.All analyses were performed using SAS 9.1 software (SAS Inst.Inc., Cary, North Carolina, USA).
The ingredients used in the experimental diets were obtained from four brazilian industries Guabi  , Coplana  , Agromix  and Grupo Ambar Amaral  , with exception of salmon meal that was imported from Chile.

Protein ingredients
Mean ADC values of protein ingredients are presented in Table 3. Concerning crude protein digestibility, corn gluten meal had the highest ADC CP (98.1%), but it was not significantly different from that of either soybean meal or wheat gluten meal (P>0.05).Additionally, alcohol yeast was less digestible protein ingredient.No significant differences in ADC CP were found between salmon meal, poultry by-product meal, and feather meal (P>0.05).Of the protein ingredients analyzed, cottonseed meal (79.62%) and tilapia processing residue meal (78.46%) had intermediate ADC CP values.Concerning ether extract, all protein ingredients had high ADC values ranging from 87.44 to 96.6%, except for wheat gluten (63.7%).Finally, in the case of gross energy, all protein ingredients had high ADC GE values (>70%), except for cottonseed meal and alcohol yeast, which showed the lowest values, 50.15 and 46.24%, respectively.Corn gluten meal had the maximum value (96.9%).

Energy ingredients
Mean ADC values of energy ingredients are presented in Table 4. Regarding crude protein digestibility, wheat bran (86.1%) and corn (94.5%) had the highest ADC CP values (P>0.05),whereas mean ADC CP was around 71% for sorghum and broken rice and no significant difference was observed between the two ingredients.In the case of ether extracts, mean ADC EE values were high (> 80%) for all energy ingredients.Concerning gross energy, oil  sources (corn, soybean, and fish) and corn had the highest ADC GE values, but no significant differences were observed across the four ingredients (P>0.05).Additionally, no significant difference in mean ADC GE was observed between broken rice (85.5%) and sorghum (81.1%,P>0.05).Wheat bran was the least digestible ingredient (68.23%).

Protein ingredients
The high ADC values of corn gluten meal observed in this study can be attributed to its high protein percentage (63%) and the low amount of fiber (1.01%) of this ingredient.The high ADC CP and ADC GE values of corn gluten meal was observed in omnivorous fish, such as pacu (Piaractus mesopotamicus) (ABIMORAD et al., 2008) and Nile tilapia (Oreochromis niloticus) (PEZZATO et al., 2002).However, there are a few drawbacks in using corn gluten meal, because its high carotenoid content imparts a yellow color to fillets (HU et al., 2012).
Our results for soybean meal are in line with the ones presented by ABIMORAD et al. (2008) for pacu, who found ADC CP and ADC GE values of 90.6% and 78.1%, respectively.This type of meal can be used in quantitative meal replacement studies as an alternative protein source for tambaqui because it contains most essential amino acids for fish (NRC, 2011) and is an affordable product, lowering production costs of diets (HERNÁNDEZ et al., 2007).
We observed a high protein digestibility of wheat gluten, even though it does not show efficient digestibility for ether extract and energy.Wheat gluten meal has low levels of important amino acids such as lysine and methionine (GATLIN et al., 2007), which may hinder its use and high cost.Despite being a good protein source, digestibility studies of wheat gluten in omnivorous species are rare (APPER-BOSSARD et al., 2013), but it is highly digestible for carnivorous species such as turbot (Psetta maxima) (BONALDO et al., 2011) and salmon (Salmo salar) (PRATOOMYOT, et al., 2010).
Two fish meal sources were evaluated in this study: one made from Nile tilapia filleting residue produced in Brazil and one made from salmon residue imported from Chile.Mean ADC CP and ADC GE of salmon meal were higher than those of Nile tilapia residue meal showing differences in the quality of by-products, and probably in their processing.Mean ADC CP was similar to the value reported by PEZZATO et al. (2002) for Nile tilapia (78.55%) and lower than that obtained by ABIMORAD and CARNEIRO (2004) for juvenile pacu (88.4%).Mean digestibility coefficients of tilapia processing residue meal were slightly lower than those of the other protein ingredients tested, exception of alcohol yeast and cottonseed meal.The low ADC CP of cottonseed meal may be related to its high fiber content, which is approximately 13.97% (ROSTAGNO et al., 2011).Also, this low digestibility coefficient may be due to high levels of gossypol, which is an antinutritional factor in cottonseed meal.Gossypol can be toxic to fish and accumulate in the liver and, if present in large amounts, may affect growth (PASTORE et al., 2013).Regarding cottonseed meal, we observed similar ADC CP than reported by PEZZATO et al. (2002) for Nile tilapia (74.87%), and lower ADC CP and ADC GE than reported by ABIMORAD and CARNEIRO (2004) for pacu (86% and 59.55%, respectively).
Alcohol yeast did not classify as a good protein and energy source for juvenile tambaqui.The low ADC CP of alcohol yeast may be related to the low digestibility of certain amino acids in yeast (HUYBEN et al., 2017).Other studies also reported low ADC CP and ADC GE for yeast: 34.70% and 86.20%, respectively, for Nile tilapia (HISANO et al., 2008) and 68.86% and 45.77%, respectively, for pacu (ABIMORAD and CARNEIRO, 2004).The high non-protein nitrogen and cell wall content in yeast, approximately 14% and 30%, respectively, hinder its use in animal feed (HAUPTMAN et al., 2014).Conversely, ether extract digestibility of yeast by juvenile tambaqui was remarkably high (91.41%).

Energy ingredients
Energy ingredients showed high mean ADC values for protein, energy, and ether extract fractions, and thus can be included in commercial diets as less expensive energy sources.Mean ADC CP of corn (94.5%) for juvenile tambaqui was very high in this study.GUIMARAES et al. (2014) obtained a lower ADC CP (87.5%) for tambaqui, whereas ABIMORAD and CARNEIRO (2004) reported a lower ADC CP (84.38%) for pacu, another omnivorous species with similar digestive tract morphology.Conversely, BOSCOLO et al. (2002) obtained a high ADC CP of corn (93.4%) for Nile tilapia.Additionally, similar ADC GE values of corn were reported by ABIMORAD and CARNEIRO (2004) for pacu (86.69%).Few studies have estimated ether extract digestibility in fish diets.PEZZATO et al. (2002) in a study with Nile tilapia found values of ADC EE for corn (69.02%) lower than that observed in this study.
Similarly, to the results obtained with corn, protein and ether extract digestibility of wheat bran by tambaqui were high.Mean ADC CP was similar to the values reported by GUIMARAES et al. (2014) andABIMORAD et al. (2008) for tambaqui (84.4%) and pacu (87.7%), respectively, and lower than the values obtained by PEZZATO et al. (2002) and BOSCOLO et al. (2002) for Nile tilapia (91.13% and 91%, respectively).Mean ADC EE in this study was higher than the value reported by PEZZATO et al. (2002) for Nile tilapia (67.37%).Conversely, energy digestibility of wheat bran by tambaqui was low (68.2%),similar to what was found by GUIMARAES et al. (2014).Wheat bran was the least digestible ingredient for ADC GE (68.23%).The difference in energy digestibility of wheat bran compared to corn, sorghum, and broken rice, may be due to the high crude fiber content in wheat bran.The crude fiber, a component of the main feed ingredients, is indigestible to most fish species and should be avoided.High dietary crude fiber content shortens food transit time in the gut, affecting nutrient absorption (ENES et al., 2011).Even though sorghum is a possible replacement for corn due to their similar chemical composition and nutritional value, mean ADC CP of sorghum was significantly lower than of corn.The quality of the corn protein was higher, probably showing a better amino acid balance.GUIMARAES et al. (2014) andPEZZATO et al. (2002) also reported lower ADC CP in sorghum than in corn for tambaqui (71.4%) and Nile tilapia (67.83%), respectively.
Mean ADC CP of broken rice was similar to the value reported by GUIMARAES et al. (2014) for the same species (72.8%) and mean ADC GE (85.52%) was higher than the value (80.7%) reported by GUIMARAES et al. (2014).Additionally, mean ADC EE of broken rice was not significantly different from the ADC EE of the other protein ingredients tested, evidencing that tambaqui can adjust its feeding behavior based on the availability of food during cultivation.
Oils are important energy sources used in fish diets.In this study, the three oil sources tested were well utilized by tambaqui.Fish oil was produced from Nile tilapia meal processing.Even though oils are commonly utilized in fish diets, few studies have evaluated the digestibility of oils by freshwater species.GUIMARAES et al. (2014) andBOSCOLO et al. (2002) observed ADC GE values for soybean oil for tambaqui (89.85%) and Nile tilapia (92.7%) lower than the value observed in this study.Studies on corn oil are rare, but like soybean oil, it is a rich source of omega-6 polyunsaturated fatty acids and also a source of monounsaturated fatty acid, may be used as a replacement for animal oils to reduce production costs (PIEDECAUSA et al., 2007).
To our knowledge, this is the first study to evaluate a large number of feed ingredients commonly used in commercial diets for tambaqui.The apparent digestibility coefficients of the ingredients tested in this study will aid nutritionists in decision making regarding feed utilization parameters, which also include the quality of other nutrients, their pricing, availability, and seasonality.

CONCLUSION
In general, protein and energy ingredients are well utilized by juvenile tambaqui with high ADC values, except for alcohol yeast.The protein ingredients tested with better results were corn gluten meal and soybean meal, and among energy ingredients the better results were found with corn and oil sources.If inclusion limits are observed, the ingredients tested in this study can be used in commercial diets for tambaqui.

Table 1 .
Formulation and chemical composition of the reference diet (as fed basis).
Mean ± standard error of the mean (n=4).Values followed by the same letter in a column are not significantly different (Tukey's test, P > 0.05).DP: digestible protein; DEE: digestible ether extract; and DE: digestible energy.
Mean ± standard error of the mean (n=4).Values followed by the same letter in a column are not significantly different (Tukey's test, P > 0.05).DP: digestible protein; DEE: digestible ether extract; and DE: digestible energy.