Effect of dietary protein levels on growth performance, hematological parameters and digestive enzymes in juveniles of Brycon amazonicus (Spix & Agassiz, 1829)

: A 52-day experiment was conducted to determine the crude protein (CP) requirements of juvenile matrinxã Brycon amazonicus, and to evaluate their resulting growth performance, hematological parameters and enzymatic activities. Sixty fish (29.03g ± 1.16g) were distributed in 12 tanks (310 L) with a completely randomized design, and maintained at four dietary crude protein levels (270, 320, 350, 390 g.kg -1 ) for 52 days. The results revealed that the fish fed diet 390 g.kg -1 CP had the best final weight, weight gain, feed conversion ratio, specific growth rate, protein efficiency ratio and lipid retention rate. The same could be stated for hematocrit, number of circulating erythrocytes, triglycerides and total proteins of the hematological profile (p<0.05). In the whole body composition, dry matter content was lower in the fish fed 390 g.kg -1 CP, while lipid content was higher in the fish fed 350-390 g.kg -1 CP (p<0.05). No differences were observed in CP and ash (p>0.05), or in the activities of digestive enzymes (p>0.05). In short, our findings suggest benefits of the 390 g.kg -1 CP feed for being the most adequate for this species’ juvenile stage. hydroxy


Introduction
In animal production segments, aquaculture is the fastest growing activity in the world because the world's population is eating more fish, which is a driver of higher production [1]. During the fish breeding process, supplying diets that meet the nutritional requirements of a given species and breeding stage is one of the determining factors for productive efficiency, and is related to zootechnical performance [2][3]. Understanding how nutrition influences fish metabolism and health allows us to formulate adequate diets that meet species requirements, and are viable from the zootechnical and financial points of view [4]. Of all nutrients, protein is the most important because it makes up about 65-75% of total fish weight (based on dry matter) [5]. Therefore, adequate protein supply is necessary to not only allow proper fish development, but to also preserve water quality in breeding tanks [6]. Of all the nutrients used in diet formulation, protein is one of the main obstacles for fish farming to develop in the Amazon [7].
The activities of digestive enzymes influence how fish use food. Understanding these activities is important because it allows us to optimize fish diet formulation [8]. As blood is one of the most dynamic tissues of the organism, and changes in it occur according to the type of diet consumed, it is possible to evaluate fish nutritional status by analyzing hematological characteristics [9,10]. Knowledge of the mean values of hematological parameters in the natural fish environment and captivity in the most diverse commercial breeding systems is important for identifying physiological changes due to nutrition and environmental factors, such as homeostasis conditions and stress [11].
Of the native fish species farmed in the State of Amazonas, tambaqui (Colossoma macropomum), matrinxã (Brycon amazonicus), and pirarucu (Arapaima gigas) are the most noteworthy [12]. Matrinxã is the second most cultivated species in the State of Amazonas, and the most cultivated species in the northern and central mesoregions of this state for its superior zootechnical performance, quality and good consumer acceptance [13]. It also adapts easily to the efficient use of protein sources of either animal or vegetable origin [14]. However, information on crude protein (CP) levels in matrinxã diets is still divergent, which evidences the need for research to generate knowledge about the protein needs of this species' juveniles [15].
In this context, the present study evaluated increasing CP levels in the diet of juvenile matrinxã (270, 320, 350, 390 g/kg -1 CP), and correlated them with zootechnical performance, hematological parameters and the determination of digestive enzyme activity. This study sought to establish the ideal protein level for this species' juvenile phase to achieve food efficiency and to optimize production.

Experimental diets
Four experimental isoenergetic diets (16.74 MJ crude energy/kg) with increasing protein levels (270, 320, 350, 390 g/kg -1 CP) were used (Table 1). Diets were formulated based on nutritional recommendations for omnivorous fish species [16]. Formulations were prepared in the Aquatic Organism Nutrition Laboratory at the Nilton Lins University. Ingredients were milled in a 0.5 mm mesh knife mill (Wiley Mill, TE-650/1), homogenized in a humidified "Y" automatic mixer (20% water) and processed in a single thread extruder through a 4 mm dye. Then they were dried in a forced ventilation oven at 55ºC for 24 h and kept in a freezer until they were used at -18°C.

Feeding trial
This study was carried out following the recommendations on Ethical Principles in Animal Experimentation adopted by the Brazilian National Council for Animal Experimentation Control (CONCEA). It was approved by the Ethical Animal Use Commission (CEUA) at the Nilton Lins University with approval No. 017/2016 and at the Federal University of Amazonas with approval N°. 001/2017. All the experimental procedures were followed according to local guidelines [17]. The trial was conducted in Manaus, Amazonas (Brazil). The experimental system consisted of 12 tanks (310 L) in a system with partial water renewal and constant artificial aeration. During the trial, fish were fed 3 times (8 and 12 am, and 5 pm) a day until apparent satiety. Water parameters, such as temperature, pH, oxygen and ammonia, were measured daily, with respective values of 29±0.80°C, 6.25±0.50, 7.80±0.60 mg/L and 0.25±0.10 mg/l, following Resolution N°. 357 of 2005, of the National Council of the Environment (CONAMA) for freshwater. The photoperiod was maintained at 12h:12h light: dark.
Juvenile matrinxã Brycon amazonicus (N=60) were acquired from the Center for Technological Training and Production in Aquaculture (CTTPA) in the municipality of Presidente Figueiredo, State of Amazonas. They were allowed 10 days to adapt to laboratory conditions. Juvenile matrinxã, with a 29.03 g±1.1 mean initial weight, were randomly distributed to tanks at a density of five fish per tank, and each experimental diet was randomly assigned to three replications.
At the end of the 52-day feeding trial, fish were counted and weighed after not being fed for 24 h and four fish per tank were randomly sampled. Two of these fish were immediately euthanized by a 50 mg/L clove oil overdose [18] and frozen for the body composition analysis. Two other sampled fish were used for blood and tissue collections. Blood was obtained from the caudal vein and stored separately in tubes with or without 10% EDTA. Blood samples without anticoagulants were centrifuged at 3000 rpm for 15 minutes 3h after blood clotting to obtain plasma [19]. Plasma was recovered after centrifugation at 3000 rpm for 15 minutes and stored at −80°C until the analyses. Therefore, fish were killed by clove oil overdose (50 mg/L) [18]. Fish length and weight, and viscera and liver weights, were recorded to determine the body index. The stomachs, intestines and livers of these fish were collected and stored at −80°C until the analyses.

Growth parameters
The effects of the experimental diets on zootechnical performance, feed utilization and the hepato-somatic index were determined by calculating the parameters set out below.

Body composition
Proximate fish compositions were collected from two fish in each experimental unit (6 fish per treatment). Body composition was determined by the standard methods of the Association of Official Analytical Chemists [20]. The following were determined: content moisture by drying for 24 h at 110ºC to constant weight; protein by the Kjeldahl method (N × 6.25); crude fat by diethyl ether extraction; ash by heating at 450ºC for 24 h; crude fiber by the Weende method [21]. The nitrogen-free extract (NFE) for diets was determined as the remainder of CP, crude fat, ash and crude fiber.

Hematological parameters
Whole blood was used to evaluate the hematological variables. The determination of the number of circulating erythrocytes (RBC, x10 6 /µL) was made in a Neubauer chamber according to the method of Collier [22]; Hematocrit (Htc, %) was determined by the microhematocrit method, following the methodology of Goldenfarb et al. [23]; hemoglobin concentration (Hb, g/dL) was assayed by the cyanometahemoglobin method and spectrophotometer readings [24]. The hematimetric indices were also calculated as established by Wintrobe [25]: mean corpuscular volume (MCV, fL), mean corpuscular hemoglobin (MCH, pg), and mean corpuscular hemoglobin concentration (MCHC, g/dL).
The analyses of glucose (enzyme glucose GOD-PAP) and triglycerides (Triglycerides Liquicolor GPO-PAP) were performed from plasma. The analyses of total protein (Total Protein) and cholesterol (Cholesterol Liquicolor) were varied out from serum. All the analyses were run using specific commercial kits (In Vitro Diagnostica Ltda, Itabira/MG) for each parameter, with subsequent readings by a spectrophotometer (Multiskan ™, Thermo Fisher Scientific, Brazil).

Enzyme activities
Stomach and intestines were homogenized in TRIS HCL buffer 50 mM with CaCl2 at pH 8.0, and the stomach extract in saline containing CaCl2. After homogenization, an aliquot of 2.0 mL of each sample was centrifuged and cooled to 4ºC for 4 minutes at 10 rpm. The supernatant was stored in tubes and kept frozen at -80ºC, until the analysis to determine enzyme activity. All enzyme activities were measured at 37 °C in a microplate reader (Multiskan GO, ThermoScientific, USA), by monitoring changes in absorbance.
Protein concentration was determined according to Bradford [26] using bovine serum albumin as standard.
To establish acid protease activity, a version of the hemoglobin method adapted by Khaled et al. [27] was followed. A buffer of 100 mM of sodium citrate with 20 mM of CaCl2 was used and as substrate hemoglobin with pH 2.0. The reaction was incubated for 60 minutes at 25 ºC and was interrupted by the addition of 50 µL of 10% trichloroacetic acid. The precipitate was removed by centrifugation at 10000 rpm/5min for the supernatant reading at 275 nm, expressed as 1 µmol tyrosine/minute/mg protein.
Tyrosine was used as a standard.
To determine alkaline protease activity, a version of the azocasein method adapted by Sarath et al. [28] was employed. As a buffer, 100 mM Tris-HCl, pH 7.6, 20 mM CaCL2, and 20 mM NaCl with 0.25% azocasein were used as a substrate. The reaction was incubated for 60 minutes at 25 °C and was interrupted by adding 50 µL of TCA (10%). The precipitate was removed by centrifugation at 10000 rpm/5 min. for the supernatant reading at 440 nm, expressed as 1 µmol of hydrolyzed substrate/mg protein/min.
The non-specific lipase activity in the homogenized stomach and intestine was determined by the methodology adapted from Albro et al. [29] using a mixture of 200 mM Tris-HCl, 10 mM CaCl2, 10 mm NaCl, 1 mM Tamodeoxycholate, 0.05% Triton X-100 and 0.05% gum Arabic as a buffer at pH 8.0, and 2 mM P-nitrophenyl-myristate as a substrate. The microplate reading was taken at 405 nm after hydrolysis and expressed as 1 µmol of hydrolyzed substrate/mg of protein/minute.
Amylase was determined by the adapted method Bernfeld [30], by which the reaction was performed using a 100 mM na+ acetate buffer at pH 7.6, with 20 mM CaCl2 and 10 mM NaCl and the homogenized product was added and then incubated for 60 minutes at room temperature. The reaction was interrupted by adding 50 µL of 1% starch. The reading was taken at 554 nm and expressed as 1 µmol of glucose/mg of protein/minute. To establish the maltose calibration curve, 50 µL lugol diluted to 0.1% I2 and 0.3% I was used.

Data analysis
The results were expressed as mean±SEM. Data normality was previously evaluated by the Shapiro-Wilk test and homogeneity of variance was verified by the Levene test. Data were analyzed by a one-way analysis of variance (ANOVA), followed by Tukey's post hoc test, to check for significant differences between treatments. Statistical analyses were conducted using the SPSS software (version 20.0, IBM, Armonk, NY, USA). Differences were considered statistically significant when p<0.05.

Results
The growth performance and feed efficiency data are presented in Table 2. Fish well accepted the experimental diets and no mortality was recorded. FW, WG, the SGR and the LRR were higher in the fish fed the diet with 390 g/kg -1 CP, while lower FCR and PER were also observed in this diet. The parameters FW, WG and the SGR showed a positive linear effect with increasing protein inclusion levels. In the whole body composition (Table 3), dry matter content was lower in the fish fed 390 g/kg -1 CP. Lipid content was higher in the fish fed 350 g/kg -1 CP. No differences were observed in the CP and ash of the whole body composition.
No effects by dietary protein were seen for MCH, MCV and MCHC (Table 4). However, hematocrit (Htc) and the number of erythrocytes (RBC) were higher in the fish fed 390 g/kg -1 CP, while the triglyceride and total plasma protein levels were higher in the fish fed 350 and 390 g/kg -1 CP. Regarding digestive enzymes, acid protease, alkaline protease, lipase and amylase enzyme activity were evaluated in stomachs, anterior intestines and posterior intestines, and were not affected by dietary treatments (Table 5).

Discussion
Matrinxã is a carnivorous freshwater fish (trophic level 3.0) with a high cannibalism index in its initial growth phase [31]. In our study, the fish fed the highest CP level in diet showed higher growth rates than those that received the lowest CP levels in diet. Similar results have been reported for other carnivorous freshwater fish [32].
The growth performance of the juvenile matrinxã in this study fell within the growth performance range observed in other studies for this species. Our fish with the most WG were those fed 390 g/kg -1 CP, while the lowest WG value went to the fish fed 270 g/kg -1 CP. In a study by Izel et al. [15], conducted with juvenile matrinxã submitted to diets with 160, 190, 220, 250, and 280 g/kg -1 CP, the highest WG value was obtained in the fish fed 280 g/kg -1 CP, with FCR values of 2.04 for the 250 g/kg -1 and 280 g/kg -1 CP levels.
In the present study, the FCR values for the fish that received 270 g/kg-1 and 390 g/kg-1 CP were 1.66 and 1.36, respectively. This difference could be related to the increase in protein level, which resulted in a better use of diets, and was observed in the FCR. Yang et al. [33] noted this same behavior when evaluating increasing protein levels for Lepomis macrochirus, as did Ullah et al. [34] when they assessed rising protein levels for Tor putitora and observed the same behavior.
The PER had a negative linear effect on the tested diets, which suggests that the highest-level protein diet should not be used for protein deposition and may have been used as an energy source. This statement can be supported by the results found for the PER, the LRR and ether extracts in carcasses because fish retained more body fat when the protein level in diet increased. These results corroborate the findings reported in similar studies [33; 35,36].
The highest ether extract content in carcasses was observed in the animals fed the diet with 350 g/kg -1 of CP. Deposition of lipids in carcasses due to increased dietary protein is not economically desirable [37,38] because the amino acids that derive from protein have to be oxidized to be stored as energy reserves instead of being used for growth.
Blood parameters can be used as biological indicators to monitor fish well-being, particularly as a tool to diagnose animal stress [7; 39-41]. The biochemical composition of blood plasma reflects the metabolic situation of tissues in animals. It is also possible to evaluate changes in animal's organ function and adaptation as regards not only nutritional and physiological challenges, but also specific metabolic imbalances or imbalances of a nutritional origin [42,43]. The present study revealed that protein levels do not interfere with the results of hemoglobin and hematimetric indices. The highest hematocrit value was observed in the animals fed a diet containing 390 g/kg -1 CP. However, this value is considered normal because it falls within the reference values (23-35%) for the species without causing any health problems [44]. Higuchi et al. [45] fed Rhandia quelen with increasing dietary protein levels and did not observe any differences in the evaluated biochemical and hematological parameters.
Ferreira et al. [46] tested the effects of 360 and 450 g/kg -1 CP on diets associated with physical training in physiological parameters of juvenile matrinxã. They concluded that there was no protein level-erythropoietic variation interaction. Puppo et al. [47] evaluated hematological responses of Nile tilapia in relation to increasing CP levels (270, 300, 330, 360 g/kg -1 CP) in diets. They found no changes in erythropoiesis, which indicates that erythrogram changes are not related to dietary CP levels.
According to Tavares-Dias et al. [44], the RBC reference values for Brycon amazonicus varied between 1.13-1.56 x106.µL -1 ), but the present study obtained higher RBC values (between 2.68-3.34 x106.µL -1 ). In studies with Cyprinus carpio, Ahmed and Maqbool [48] observed a linear increase in RBC with varying levels from 250 to 500 g/kg -1 CP. They attributed this increase to the early release of the storage pool in the spleen. Abdel-Tawwab [49] believed that splenic activity was influenced by protein levels after verifying a linear increase in RBC levels under the influence of protein levels ranging from 250 to 450 g/kg -1 CP in Oreochromis niloticus. Different stress factors ranging from handling or forced physical exercise at the time of capturing these animals to performing final biometrics can also influence RBC levels.
The reduction in plasma triglycerides due to the increased protein in experimental diets may be related to greater lipid retention and a higher ether extract concentration in carcasses, which might indicate lower circulating triglyceride levels. Glencross et al. [50] observed how lipid retention increased at higher protein levels in the diet of Lates calcarifer. Ahmed and Maqbool [48] reported how incremented protein in diet increased the body lipid concentration in Cyprinus Carpio.
A drop in the total plasma protein levels with increasing protein content in diet may indicate higher protein consumption, as evidenced by the lowering protein efficiency rate. These results agree with Ahmed and Ahmad [51] and Ahmed and Maqbool [48], who demonstrated worse protein efficiency and greater body protein retention in diets with high CP levels for Oncorhynchus mykiss and Cyprinus Carpio, respectively, which may represent lower plasma protein levels, as herein observed.
Regarding digestive enzymes, no differences in activity were observed in the fish fed the experimental diets. Lazzari et al. [8] observed variations in trypsin, chymotrypsin and amylase activity in the digestive tract segments of Rhamdia quelen, and were linked with the protein source composition, which varied between meat and bone meal with sugar cane yeast, soybean meal and fish meal. Non specific acid and alkaline proteases were detected in all the digestive tract segments, but displayed more marked activity in the anterior and posterior intestine segments due to their characteristic alkaline pH. Several studies have correlated proteolytic activity, which varies depending on whether different protein levels for fish are included [52][53][54]. However, this relation was not observed in the present study, but was ratified by Almeida et al. [55]. Lundsedt et al. [56] conducted a study with juvenile pintado (Pseudoplatystoma corruscans), which were fed 200, 300, 400, and 500 g/kg -1 CP. These authors observed that the pintado stomach showed marked proteolytic activity but, as this did not respond to diet protein content, they were unable to assume that both enzymes responded to diet.
Lipase activity was not detected in stomachs, and its greatest activity took place in anterior intestines. This result may be correlated with the presence of pyloric caeca, which were homogenized with the anterior intestine portion. Gisbert et al. [57] stated that lipase action was more effective in the proximal portion of intestines and pyloric caeca, whose action extends at a lower activity rate to other digestive tract portions. In the stomach, lipase is responsible for hydrolyzing emulsified and low-melting-point lipids, which demonstrates lower lipolytic performance in this segment. Tok et al.
[58] conducted a study on Pangasianodon hypophthalmus and observed only a minor variation in the intestinal lipase enzyme in different protein diets as the same level of lipids was included in all the experimental diets. This finding corroborates the data found in this study. Amylase activity showed no significant differences between treatments and can be localized throughout the fish digestive tract.

Conclusions
It can be concluded that 390 g/kg -1 CP in diet increases zootechnical performance indices in juvenile matrinxã. The inclusion of different protein levels neither leads to hematological alterations nor interferes with the activities of the evaluated digestive enzymes. Future studies should be carried out to determine the ideal digestible protein and amino acid profile and, thus, optimal nutritional requirements.