FISH PROTEIN HYDROLYSATE AS AN INGREDIENT IN DIETS FOR Arapaima gigas JUVENILES*

This study evaluated the dietary inclusion of a fish protein hydrolysate (FPH) derived from from tilapia trimmings, on physiological and growth parameters of juveniles of A total of Arapaima gigas. 180 arapaima juveniles (91.4 ± 2.7 g) were used in a complete randomized design with six treatments (n = 3). Fish were fed to apparent satiation four times a day for eight weeks, with diets containing increasing inclusion levels of FPH (0, 4, 8, 12, 16 and 20%). FPH diets did not affect growth and hemato-biochemical parameters of arapaima juveniles. The FPH from tilapia trimmings seems to be a suitable ingredient for arapaima over 90 g feeds, at least up to 20% inclusion level. No bioactive effects of the FPH could be detected.


INTRODUCTION
Arapaima gigas is one of the most important native species for Brazilian aquaculture. It is a carnivorous species with big growth rates, high fillet yield and high market value, which makes it very attractive for fish farming (IMBIRIBA, 2001;OLIVEIRA , 2012;VALLADÃO , 2016). et al. et al. However, there are limitations in juveniles supply mainly due to absence of techniques for artificial reproduction and high mortality rates observed in arapaima early life (NÚÑEZ et al., 2011;LIMA et al., 2015). Diets with an optimal cost-effectiveness and reducing susceptability to oportunistic pathogens are also lacking for this species. The use of diets with high biological value and immunostimulant action ingredients could reduce growth losses and mortality rates due to parasites.
Fish protein hydrolysates (FPH) have been described to possess bioactive peptides and amino acids which can act as antibiotics, antibacterial agents, antioxidants, and regulators of the activity of certain digestive enzymes (GILL , 1996). et al. FPH are considered potential ingredients for the aquafeed industry, due to its high protein content, as well as having flavoring action (CONCEIÇÃO et al. et al. , 2012;HE , 2013). The inclusion up to 10% FPH in juvenile diets for croaker, Pseudosciaena crocea, provided improvements in growth and immunological parameters (TANG , 2008). et al. However, these benefits are not present for all species. The addition of 15% and 25% FPH in diets for juvenile turbot, Scophthalmus maximus, reduced protein and energy digestibility due to gastrointestinal disorders (OLIVA-TELES et al., 1999).
Fish were fed experimental diet four times a day (8:00, 11:00, 14:00 and 17:00 h) during eight weeks. At the end of growth assay, fish were fasted during 24 h. where: e = nepper number; g = (ln(final weight) ln(inicial weight))/(length of the assay period);  Hepatosomatic Index (HSI) = liver weight (g)/body weight (g) x 100. Blood samples (1.5 mL) were taken by caudal venipuncture (6 fish tank -1 ), with ethylenediamine tetraacetic acid (EDTA-10%) coated syringes for hematological and biochemical parameters evaluation. From these a subsample (3 fish tank -1 ) were euthanized with anesthetic overdose (0.4 mL eugenol L -1 water) to collect the liver and calculate the HSI. From these subsamples, fish muscle were collected and frozen to posterior chemical composition analysis.
p Data with nonparametric distribution (HSI, RBC, MCH, MCV) were analyzed by Kruskal-Wallis test ( <0.05). Data under the significance level p were submitted to regression analysis to find the best model fit (CurveExpert Professional v 2.6.2). Since no model tested showed a suitables fit, Tukey test ( <0.05) were used to narrow down p the dose interval in order to obtain de closest value (ZAR, 2013 ).

RESULTS
All diets were equally accepted by fish, and no mortalities were observed during the trial. Growth parameters and HSI were not affected by inclusion of FPH in the diets (Table 2). Arapaima juveniles show a RGR of 1.5% day -1 (coefficient of variation (CV) = 4.3%); growing 2.2 g day -1 (CV = 7.8%) and 70 g month -1 (CV = 7.1%) among treatments.
p The 12% FPH diet decreased Ht compared to the control (Table 3).

DISCUSSION
Fish protein hydrolysate inclusion did not influence feed palatability provided by its free amino acids content, which can act as feeding attractant (CHOTIKACHINDA , 2013). It has et al. also been reported that prolonged or uncontrolled hydrolysis of fish protein may result in bitterness (KRISTINSSON and RASCO, 2000), decreasing fish intake. Good acceptance of all experimental feeds and the similar feed intake suggest that diets showed no bitter taste.
Amino acids are considered at feed stimulants for most fish species (VELEZ , 2007;BARATA et al. et al., 2009). However, turbot juveniles, Scophthalmus maximus, fed diets up to 35% FPH in substitution of fish meal showed no feed intake increase, as inosins rather than free amino acids are probably associated with feeding stimulation in this species (MACKIE and ADRON, 1978 In the present study, the zootechnical parameters were similar to that observed for arapaima reared in experimental conditions according to ITUASSÚ et al. (2005) that reported the individual weight gain of 110.9 ± 56.0 g and 1.5 ± 0.5% of specific growth rate in arapaima juveniles (initial weight of 120.7 ± 3.5 g) fed a diet with 43.4% of crude protein for 45 days. However, our results for arapaima as well as the results for Steubacheridion melanodermatum fed diets containing 4% and 6% of tilapia and sardine protein hydrolysate, respectively (LEWANDOWSKI , 2013), and et al. for juvenile turbot, , with Scophthalmus maximus the addition of 15% and 25% FPH (OLIVA-TELES et al., 1999), show no benefits of FPH on growth performance. In the later study FPH even lead to reduced protein and energy digestibility due to gastrointestinal disorders. So there may be species specificity for tolerance to higher levels of FPH, eventually with faster growing species (e.g., croaker and arapaima) tolerating higher levels compared to slower growing fish (e.g., turbot). The explanation for these conflicting results can also be related with FPH quality which is influenced by raw material and the manufacturing process conditions (FURLAN and OETTERER, 2002).
Moreover, it would be of interest to test high FPH hydrolysates in larvae and post-larvae stages of arapaima, as positive effects of FPH inclusion levels up to 10% feed basis, on growth performance and intestinal maturation, have been demonstrated in early stages of marine fish (CAHU and ZAMBONINO INFANTE, 2001;CONCEIÇÃO , 2011 Fish protein hydrolysates could enhance several health aspects of fish due to putative bioactive compounds positively affecting fish immune system (MURRAY , 2003;KHOSRAVI et al. et al., 2015). Haemato-biochemical parameters are important tool for evaluating the stressing agents effects which may compromise animal health (BLAXHALL, 1972;WENDELAAR BONGA, 1997). From our results, it is possible to suggest that diets formulated with FPH did not impair the physiological homeostasis of fish, as observed in Pagrus major fed with marine protein hydrolysates (KHOSRAVI, 2015). In addition, the inclusion of these bioactive compounds did not compromise the lipid and protein metabolism of fish, which is desirable for the maintenance of their performance; this same situation was observed in innate immune parameters and performance of challenge trials with pathogens.

CONCLUSIONS
FPH from tilapia trimmings can be safely used as an ingredient for feeds of arapaima over 90 g, with an inclusion level up to 20%. No bioactive effects of the FPH could be detected.

ACKNOWLEDGMENTS
Falbom Agroindustrial Company for the fish protein hydrolysate donative.