Use of sodium butyrate in the feeding of Arapaima gigas (Schinz, 1822) juvenile

Aquaculture 510 (2019) 248–255

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Use of sodium butyrate in the feeding of Arapaima gigas (Schinz, 1822) juvenile

T

Joaldo Rocha Luza,b, Ana Paula Souza Ramosa,c, José Fernando Bibiano Melod, ⁎ Luís Gustavo Tavares Bragaa, a

Postgraduate Program in Animal Science, Laboratory of Nutrition and Fish Feeding (AQUANUT), State University of Santa Cruz (UESC), 45662-900 Ilhéus, BA, Brazil Federal Institute of Education, Science and Technology Baiano (IFBaiano), Campus Uruçuca, 45680-000 Uruçuca, BA, Brazil c State University of Southwest Bahia (UESB), Campus Jequie, 45206-190 Jequie, BA, Brazil d Federal University of the Valley of Sao Francisco, (UNIVASF), Petrolina, PE, Brazil b

A R T I C LE I N FO

A B S T R A C T

Keywords: Additive Blood parameters Enzymatic activity Intestinal villi Performance

The objective of this study was to evaluate the sodium butyrate in the feeding of pirarucu juveniles, Arapaima gigas. Seventy - five juveniles of A. gigas (132.07 ± 3.12 g) were distributed in fifteen 310 L- tanks with constant aeration, filtration system and closed water recirculation. A completely randomized design the experiment, with four treatments including sodium butyrate (98%) in the diets (BS0 = control diet without sodium butyrate, BS1 = 0.5 g kg−1, BS2 = 1.0 g kg−1, BS3 = 1.5 g kg−1, BS4 = 2.0 g kg−1) with three replicates, and five juvenile treatment−1. The fish were fed three times day−1 (8, 12 and 16 h), until apparent satiety, for 45 days. Water quality parameters such as temperature, pH, dissolved oxygen and ammonia were monitored three times in the week. The growth parameters as weight gain, mean feed consumption, feed conversion ratio, specific growth rate, and survival were evaluated. It was performed chemical analysis of the whole body to establish dry matter, crude protein, ethereal extract, ash and gross energy. The hepatosomatic index and viscerosomatic index of juveniles also were evaluated. Histological sections of the intestine showed greater villous height and enzymatic activity of amylase, lipase and nonspecific alkaline protease. The blood plasma of juveniles was analyzed, as the hematocrit, total plasma protein concentration, triglyceride, glucose, and cholesterol. The inclusion of 1.17 g kg−1 of sodium butyrate in diets for A. gigas juveniles improves the growth parameters. This level favors feed conversion ratio increased intestinal villous height, without interfering in the blood parameters, and promote the adaptation of the enzymatic activity of amylase, lipase and nonspecific alkaline protease. Furthermore, it reduces the activity of aspartate aminotransferase and increases hepatic glycogen levels, making the animals more resistant to potentially stressful conditions in the growing environment.

1. Introduction The pirarucu (Arapaima gigas) is an endemic fish of the Amazon basin, with interesting characteristics for the cultivation, as: growth (Imbiriba, 2001), easy adaptability to commercial feed consumption high fillet yield, meat without intramuscular spines, and market demand (Cavero et al., 2003a; Castello, 2004; Fogaça et al., 2011). However, it produces is still incipient in view of the advantages mentioned above, and although recent nutritional studies have provided information about the species (Cipriano et al., 2015, 2016; Magalhães Júnior et al., 2017; Ribeiro et al., 2017), and there are no studies of the use of additives in the growth parameters and health of A. gigas. Organic acids are additives that improve the fish growth and health

⁎

(Ng and Koh, 2016; Ali et al., 2017; Jesus et al., 2019) and are being studied in several areas of animal nutrition (Lückstädt et al., 2014). Its beneficial characteristics are related to the palatability of the diets (Ramírez et al., 2017), increased protein digestibility (Lückstädt, 2008), growth gain (Liu et al., 2014), inhibition of pathogenic bacteria action (Bolivar et al., 2018), and improvements in immune responses (Hossain et al., 2007). Sodium butyrate has positive effects on the growth of aquatic animals (Ahmed and Sadek, 2014; Liu et al., 2016; Hoseinifar et al., 2017). The performance of this organic salt in the intestine promotes the absorption of nutrients, and have antioxidant characteristics (Wu et al., 2018). It also enables the supply of energy to enteric cells (Hoseinifar et al., 2017), and improves intestinal villi growth (Liu et al., 2014), with

Corresponding author at: UESC, Jorge Amado Road, Km 16, 45654-245 Ilheus, Bahia, Brazil. E-mail addresses: [email protected] (J.R. Luz), [email protected] (L.G.T. Braga).

https://doi.org/10.1016/j.aquaculture.2019.05.065 Received 19 February 2019; Received in revised form 27 May 2019; Accepted 27 May 2019 Available online 28 May 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.

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2.4. Growth parameters

increased absorption and weight gain (Ahmed and Sadek, 2014). In fish blood parameters, the use of sodium butyrate can increase the defense blood cells such as lymphocytes and monocytes (Ali et al., 2017), and positively alter blood parameters in fish (Ahmed and Sadek, 2014). According to Omosowone et al. (2018), can favors increased deposition of crude protein in the fish whole body, and it still reduces the population of Gram-negative bacteria in the intestinal region (Rimoldi et al., 2018). The objective of this study was to evaluate the performance parameters, the chemical composition of the whole body, intestinal villi morphology, blood parameters and enzymatic activity of A. gigas juveniles fed with diets containing different levels of sodium butyrate.

The biometry of the animals was performed at the beginning and end of the experiment and the growth parameters analyzed were: weight gain (g), mean feed consumption (g), feed conversion ratio, and survival (%). Among the 75 animals used in the experiment, 15 specimens were randomly collected (one per replicate), (three per treatment), underwent numbing with a diluted benzocaine solution according to the protocol of Ranzani-Paiva et al. (2013), for intracardiac puncture for blood collecting samples (2–3 mL). Subsequently, these fish were submitted to euthanasia with the medullary section. The anatomical parts (intestine and liver) were removed and fixed in formalin (10%) for histological analysis, and frozen for enzyme activity study and carcass chemical analyses.

2. Material and methods 2.5. Body chemical composition 2.1. Location and biological material In the AQUANUT the fish carcasses were lyophilized for dry matter determination, ground and processed for gross energy determination using a calorimetric pump IKA C-200. Other chemical analyzes such as crude protein, ethereal extract and ash (Association of Official Analytical Chemistry (AOAC), 2000) were carried out at the Laboratory of Forage and Pasture of the State University of Southwest of Bahia (UESB).

The experiment was approved by the Ethics Committee on the Use of Animals of the State University of Santa Cruz (CEUA/UESC – Protocol N°. 001/2016). Seventy-five juveniles of Arapaima gigas with initial weight mean of 132.07 ± 3.12 g, and were acquired in partnership with the Marway farm (Canavieiras, Bahia, BR) and transferred to the Laboratory of Fish Feeding and Nutrition (AQUANUT) of the State University of Santa Cruz (UESC), Ilheus, Bahia, BR. The fish were acclimatized for 10 days in 15,310 L-tanks, with constant aeration, filtration system and closed water recirculation.

2.6. Histological analyses The sections of the anterior and middle intestine of A. gigas were submitted to the routine histology technique, obtaining cuts of 5 μm thickness and stained in Hematoxylin and Eosin (HE) in the Laboratory of Histopathology of the Veterinary Hospital of UESC. The histological slides were analyzed for measure the intestinal villi height and were photo-documented at the Center of Electron Microscopy of UESC, with the use of Photonic Microscope Leica DMI 300B.

2.2. Water quality parameters Water quality parameters were monitored three times week−1 with a digital multiparameter (YSI Professional ProPlus). Every 3 days, 30%renewal of the total volume of water was performed to maintain ammonia levels below 2 mg L−1. The water quality parameters measured were: temperature, 27.09 ± 0.41 °C; dissolved oxygen, 7.57 ± 0.77 mg L−1; pH, 6.54 ± 0.37; and ammonia 1.89 ± 0.75 mg L−1, and were within the acceptable values for tropical species (Núñez et al., 2011), including the concentration of ammonia for A. gigas (Cavero et al., 2003b; Cavero et al., 2004).

2.7. Blood parameters The hematocrit values of A. gigas was determined by globular volume using the microhematocrit technique. Blood aliquots were centrifuged at 12,000 rpm for 10 min for serum separation (15 μL of potassium fluoride + EDTA), and plasma. With these components, the concentrations of glucose and cholesterol were analyzed by means of the reflectance technique and total plasma proteins with a manual clinical refractometer according to the protocol of Ranzani-Paiva et al. (2013). This procedure was performed at the Laboratory of Veterinary Clinical Analyzes of UESC.

2.3. Experimental design and treatment The experimental design was defined as completely randomized with five treatments. After the adaptation period (10 days), the fish were individually weighed and were randomly redistributed in 15 tanks, five fish tank−1, and with three replicates. The experimental diets were formulated using the program SuperCrac® with nutritional values based on previous work (Cipriano et al., 2015, 2016; Magalhães Júnior et al., 2017). The preparation and production of the diets occur in the animal's food factory Pratigi Alimentos, with the extrusion of pellets with a particle size of 4–6 mm. The diets received the addition of the different levels of sodium butyrate (98% - Adimix Pure, Nutriad Nutrição Animal Ltda, SP, BR) applied “on top”, replacing the wheat bran, and manually spread 1% soybean oil and subsequently kept in a stove with ventilation, at 42 °C for 12 h. The formulated treatments were: BS0 - control group, without addition of sodium butyrate in the diet; BS1–0.5 g kg−1 of sodium butyrate in the diet; BS2–1.0 g kg−1 of sodium butyrate in the diet; BS3–1.5 g kg−1 of sodium butyrate in the diet; BS4–2.0 g kg−1 of sodium butyrate in the diet (Table 1). The fish were fed three times a day (8, 12 and 16 h), until apparent satiety during 45 days, between the months of September and November of 2018. The pellets of the diets were consumed by the fish immediately after the contact with the water.

2.8. Enzymatic assays Enzymatic analyses were performed to determine the activity of the intestinal enzymes: amylase (Park and Johnson, 1949), lipase (Gawlicka et al., 2000) and nonspecific alkaline protease using commercial kit of Labtest ref.99 (Sarath et al., 1989); for the liver: aspartate aminotransferase (commercial kit Labtest AST/GOT liquiform Ref. 109), and glycogen concentrations (Bidinotto et al., 1998), held in the Laboratory of Aquaculture of the University of the Valley of São Francisco (UNIVASF). 2.9. Statistical analysis The data obtained at the end of the experiment were submitted to the normality and homoscedasticity test. The data were submitted to analysis of variance at 5% of probability, except for food conversion, and was applied a quadratic broken-line regression, using the computational program “R” (R Core Team, 2015). 249

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Table 1 Dietary composition for Arapaima gigas juveniles with different levels of sodium butyrate. Ingredients (%)

Levels of inclusion of sodium butyrate in diet (g kg−1) BS0

BS1

BS2

BS3

BS4

Soybean meal (45%) Wheat bran (16%) Corn gluten meal (60%) Fish-meal (55%) Fish hydrolyzate (15%) Meat and bone meal (45%) Corn bran Flour of poultry viscera (58%) Soy oil Sodium butyratea Premix vit-minb Common salt Antifungal – BHTc Antioxidant Ascorbic acid vit C mon 35 Total

27.40 16.10 14.90 5.00 1.00 11.00 7.30 11.90 4.50 0.00 0.50 0.28 0.08 0.01 0.02 100.00

27.40 15.60 14.90 5.00 1.00 11.00 7.30 11.90 4.50 0.50 0.50 0.28 0.08 0.01 0.02 100.00

27.40 15.10 14.90 5.00 1.00 11.00 7.30 11.90 4.50 1.00 0.50 0.28 0.08 0.01 0.02 100.00

27.40 14.60 14.90 5.00 1.00 11.00 7.30 11.90 4.50 1.50 0.50 0.28 0.08 0.01 0.02 100.00

27.40 14.10 14.90 5.00 1.00 11.00 7.30 11.90 4.50 2.00 0.50 0.28 0.08 0.01 0.02 100.00

Analyzed composition Gross energy (kcal kg−1) Crude protein (%) Ethereal extract (%) Dry matter (%) Ash (%)

4590 45.58 13.61 91.74 11.10

4550 45.07 13.60 91.08 10.95

4510 45.49 13.57 91.15 10.93

4550 45.47 13.66 91.95 10.99

4560 45.02 13.30 91.99 10.98

a

98% - Adimix Pure of Nutriad Nutrição Animal Ltda. Premix Vitamin Mineral (Composition/kg of product): vit. A = 6,000,000 IU; vit. D3 = 2,250,000 IU; vit. E = 75,000 mg; vit. K3 = 3000 mg; vit. Thiamine = 5000 mg; riboflavin = 10,000 mg; vitropyrdoxin = 8000 mg; biotin = 2000 mg; vit. C = 192,500 mg; niacin = 30,000 mg; folic acid = 3000 mg; Fe = 100,000 mg; Cu = 600 mg; Mn = 60,000 mg; Zn = 150,000 mg; I = 4500 mg; Cu = 15,000 mg; Co = 2000 mg; Se = 400 mg. c BHT - Butylhydroxy-toluene. b

crude protein, ethereal extract, ash, and crude energy after the diet of the experimental diets can be observed. There was a difference (P < 0.05) between treatments, except for the dry matter (P = 0.4813). The regression analysis showed a quadratic effect for crude protein (estimated value in 1.03 g kg−1 and absolute value of 68.18%), ash (0.98 g kg−1 and 22.15%) and gross energy (0.81 g kg−1 and 4078.58 kcal kg−1), but the increase in the addition of sodium butyrate promoted linear growth in the ethereal extract when analyzing the chemical composition of the carcass of juveniles.

2.10. Calculations To evaluate how sodium butyrate interferes with the growth and morphology of A. gigas, some formulas were calculated: weight gain [WG = (total final weight - initial total weight)/number of animals], mean feed consumption [FC = initial total feed - weight of the final feedstock)/number of animals], feed conversion ratio [FCR = mean feed intake/mean weight gain], specific growth rate [SGR = 100 (ln final weight - ln initial weight)/day)] and survival [SUR = (final number of fish/fish initial number) * 100]. With the data, the following nutritional efficiency variables were determined: Hepatosomatic index (%) [HSI = (hepatosomatic weight/ whole body weight) × 100]; Viscerosomatic index (%) [VSI = (viscera weight/whole body weight) × 100].

3.3. Histological analyses In the histological analysis, it was evidenced that the regions of the anterior intestine had greater villous height than the medium intestine. Both values of the villous height of the anterior and middle part of the intestine of A. gigas juveniles exhibit a quadratic effect (Table 4). The estimated optimal level was 0.99 g kg−1 of sodium butyrate to the anterior intestine (925.00 μm), and 1.20 g kg−1 for the middle intestine (704.03 μm) (Figs. 2 and 3).

3. Results 3.1. Growth parameters The performance parameters of Arapaima gigas juveniles were affected (p < 0.05) by the inclusion of sodium butyrate in diets. There was a significant increase in juveniles mean feed consumption, feed conversion ratio, hepatosomatic index, and viscerosomatic index. On the other hand, no differences in weight gain, specific growth rate and survival (100%) were observed (Table 2). The inclusion of 0.59 g kg−1 of sodium butyrate in feed increased the consumption (135.39 g) of fish, and the inclusion of 1.17 g kg−1 of SB reduced the feed conversion ratio (1.13) (Fig. 1). Juveniles fed with 1.19 g kg−1 and 0.92 g kg−1 of the additive, also influenced the hepatosomatic index (1.45%) and the viscerosomatic index (8,6%), respectively.

3.4. Blood parameters In Table 5 the mean values of the blood parameters of A. gigas were evaluated as the hematocrit (26.66%), total plasma protein (3.16 g dL−1), triglycerides (92.23 mg dL−1), glucose (59.90 mg dL−1), and cholesterol (91.12 mg dL−1) and no difference was observed among the treatments. 3.5. Enzymatic assays The grater enzymatic activity has a quadratic effect for amylase in A. gigas, and occurs in the calculated inclusion of 0.88 g kg−1 of sodium butyrate in the diet; lipase and alkaline protease with the inclusion of 1.90 g kg−1 and 0.71 g kg−1 of sodium butyrate in the diet, respectively (Table 6). Juvenile's aspartate aminotransferase has a linear decreasing

3.2. Body chemical composition In Table 3 the body chemical analyzes of A. gigas for dry matter, 250

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Table 2 Performance parameters (mean ± SEM) analyzed in Arapaima gigas juvenile fed with increasing levels of sodium butyrate. Levels of inclusion of sodium butyrate (g kg−1)

Initial body weight Weight gain (g) Mean feed consumption (g)a Feed conversion ratiob Hepatosomatic index (%)a Viscerosomatic index (%)a Specific growth rate (%) Survival (%)

BS0

BS1

BS2

BS3

BS4

P-value

134.20 ± 1.32 115.36 ± 2.79 133.92 ± 5.83 1.16 ± 0.05 1.82 ± 0.01 10.02 ± 0.45 1.46 ± 0.03 100 ± 0.00

134.90 ± 1.05 124.21 ± 2.33 139.10 ± 4.94 1.12 ± 0.01 1.77 ± 0.10 9.36 ± 0.00 1.54 ± 0.02 100 ± 0.00

129.55 ± 1.48 109.15 ± 6.37 123.87 ± 20.53 1.12 ± 0.02 1.10 ± 0.11 7.42 ± 0.61 1.40 ± 0.06 100 ± 0.00

128.00 ± 1.17 115.43 ± 0.70 148.76 ± 3.01 1.29 ± 0.02 1.78 ± 0.14 10.44 ± 1.24 1.46 ± 0.01 100 ± 0.00

133.70 ± 1.09 111.51 ± 4.38 161.85 ± 18.21 1.45 ± 0.08 1.55 ± 0.00 10.03 ± 0.04 1.42 ± 0.04 100 ± 0.00

0.1576 0.1383 0.0154 0.0001 0.0190 0.0490 0.1709 1.0000

CV = Coefficient of variation; P < 0.05; a Quadratic effect. b Broken-line regression.

which help to protect against water leaching (Silva et al., 2016), besides allowing protection and release of its active principle only in the intestinal region of animals (Jesus et al., 2019). The sodium butyrate is an efficient additive promoter in the productive performance of aquatic animals (Hoseinifar et al., 2017; Tian et al., 2017), because it provides the energy supply to the enteric cells (Hoseinifar et al., 2017). In these cells, this fatty acid short-chain enters to the mitochondria undergoes β-oxidation by passing acetyl-CoA, which enters the cycle of tricarboxylic acid resulting in the reduction of NAD + and culminating subsequently with the production of ATP (Donohoe et al., 2011). In addition, it provides essential amino acids and nucleotide derivatives (Robles et al., 2013). Fig. 1. Feed conversion ratio of Arapaima gigas juveniles fed with increasing levels of sodium butyrate.

4.2. Growth parameters The fish species and the sodium butyrate concentration added to the conditions of the culture can interfere in the results of fish growth and body composition (Ng and Koh, 2016). The increase in feed consumption of Arapaima gigas juveniles after fed with the sodium butyrate can positively alter the palatability of the diets, being attractive (Ramírez et al., 2017). Best values of feed conversion ratio in juveniles occur with the sodium butyrate level of 1.17 g kg−1. For other species (Ctenopharyngodon idellus) the inclusion values in the diet are greater than those found in this experiment (Liu et al., 2016; Tian et al., 2017). According to Bicudo et al. (2012), for carnivorous fish, feed conversion ratio of 2.0 is considered appropriate to the culture. The feed conversion ratio of A. gigas in this study (1.10) was lower than those reported by Ituassú et al. (2005) and Crescêncio et al. (2005), with the same species. Possibly the formulated diets based on the digestible energy and protein plus the action of the sodium butyrate, improves the absorption and utilization of the nutrients, favoring a better use of the food. Cavero et al. (2003b) reported a similar feed conversion ratio value to our study (1.2), but with live feed in the diets. Lower values of the hepatosomatic index of A. gigas juveniles

effect in the enzyme activity with the increasing levels of sodium butyrate. Fish hepatic glycogen was also altered using sodium butyrate and had its optimum level estimated in 1.44 g kg−1. 4. Discussion 4.1. Dosage form and function mechanism of sodium butyrate The organic acids and its salts have been indicated for addition in fish diets, but in the aquatic environment, organic salts such as propionate (3C), acetate (2C) and format (1C) have greater leaching, but the same does not occur with lactate (3C), butyrate (4C) and citrate (6C) (Silva et al., 2013). According to the authors, although sodium butyrate shows greater stability in the diets, it is known that the addition of this organic salt after the extrusion process, may favor the digestibility of amino acids and macronutrients (Gao et al., 2011). Therefore, in order to achieve greater efficiency of the organic acids and its salts, the products must be coated with vegetable oil derivatives,

Table 3 The whole body chemical composition (mean ± SEM) of Arapaima gigas juveniles fed with increasing levels of sodium butyrate (based on dry matter). Levels of inclusion of sodium butyrate (g kg−1)

Dry matter (%) Crude protein (%)a Ethereal extract (%)b Ash (%)a Gross energy (kcal kg−1)*

P-value

BS0

BS1

BS2

BS3

BS4

25.09 ± 1.58 61.99 ± 0.54 16.04 ± 0.61 23.55 ± 0.06 4174 ± 11.55

28.14 ± 0.21 68.41 ± 0.17 16.10 ± 0.60 22.71 ± 0.55 4079 ± 57.29

26.45 ± 1.20 67.60 ± 1.01 17.17 ± 0.66 22.28 ± 0.38 4075 ± 27.12

26.07 ± 1.03 65.42 ± 1.88 17.74 ± 0.46 22.21 ± 0.32 4164 ± 12.41

26.88 ± 1.28 64.41 ± 1.11 17.67 ± 0.25 23.94 ± 0.02 4251 ± 8,08

CV = Coefficient of variation; P < 0.05; a Quadratic effect. b Linear effect. 251

0.4813 0.0028 0.0160 0.0016 0.0020

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Table 4 The intestinal villi height (mean ± SEM) of Arapaima gigas juveniles fed with increasing levels of sodium butyrate. Levels of inclusion of sodium butyrate (g kg−1)

Anterior intestine (μm) Middle intestine (μm)a

a

P-value

BS0

BS1

BS2

BS3

BS4

632.01 ± 45.45 491.57 ± 22.64

822.01 ± 39.01 576.35 ± 3.31

913.54 ± 28.29 786.39 ± 16.67

886.39 ± 15.36 623.26 ± 29.38

586.88 ± 21.03 629.92 ± 71.36

0.0000 0.0032

CV = Coefficient of variation; P < 0.05; a Quadratic effect.

deposition, as reported for carp (C. idellus) (Liu et al., 2016). Also, there was no weight gain for A. gigas juvenile, a contrast when compared to omnivorous fish such as common carp (Liu et al., 2016), tilapia (Oreochromis niloticus) (Ahmed and Sadek, 2014), and grass carp (Liu et al., 2016). The results of our study are in agreement with the literature, with rainbow trout (Oncorhynchus mykiss) (Gao et al., 2011; Ebrahimi et al., 2017) and tilapia (Oreochromis sp.), and also reported no difference in weight gain in these fish fed with diets supplemented with sodium butyrate. 4.3. Body chemical composition The differences in the chemical composition of the whole body of A. gigas juveniles after feeding with sodium butyrate are in agreement with results of Omosowone et al. (2018). According to Reda et al. (2016), organic acids such as butyrate improved percentages of dry matter, digestibility of crude protein, and mineral absorption, consequently improving the feed conversion ratio. In the African catfish (Clarias gariepinus) and tilapia, these effects were also evidenced, since the researchers reported a greater deposition of protein in the fish carcass (Omosowone et al., 2018).

Fig. 2. Quadratic regression of the villi height of the anterior intestine of Arapaima gigas juveniles fed with increasing levels of sodium butyrate.

4.4. Histological analyses The A. gigas juveniles that consumed diets with inclusion of 1 g kg−1 of sodium butyrate, had an increase in its villi height, in the anterior and middle intestine. Since this additive acts, directly, in the intestine of fish, and acting in these regions (Abdel-Mohsenl et al., 2018), it promotes the release of energy into enteric cells, provides cells proliferation, differentiation of mucosal cells (Gálfi and Neogrády, 2001; Terova et al., 2016), and leads to a higher production of mucins that benefit the intestinal function (Piazzon et al., 2017). Sodium butyrate when consumed orally, also inhibits apoptosis and improves the integrity of intestinal cells (Wu et al., 2018). These results are similar to those related by Liu et al. (2016), which the organic acids cause growth and greater health to intestinal villi. Thus, they allow greater absorption and promote better utilization of nutrients (Robles et al., 2013; Omosowone et al., 2018). In addition, for its antioxidant function (Liu et al., 2016), the sodium butyrate can be administered for the purpose of preventing intestinal damage (Liu et al., 2014), and promote the health of this organ, leading to greater resistance to diseases (Abdel-Mohsenl et al., 2017).

Fig. 3. Quadratic regression of the villi height of the middle intestine of Arapaima gigas juveniles fed with increasing levels of sodium butyrate.

indicate that diets containing sodium butyrate are nutritionally balanced, since changes and/or higher values of this index may be due to antinutritional factors in the diet (Francis et al., 2001). Additionally, the viscerosomatic index of the A. gigas has a lower visceral fat

Table 5 The blood parameters (mean ± SEM) in Arapaima gigas juveniles fed with different levels of sodium butyrate. Levels of sodium butyrate (g kg−1)

Hematocrit (%) Total plasma protein (g dL−1) Triglycerides (mg dL−1) Glucose (mg dL−1) Cholesterol (mg dL−1)

P-value

BS0

BS1

BS2

BS3

BS4

26.00 ± 0.58 3.47 ± 0.35 102.87 ± 14.59 57.33 ± 8.09 86.07 ± 14.44

27.30 ± 0.88 3.27 ± 0.07 88.57 ± 4.94 48.57 ± 0.30 90.17 ± 9.83

27.00 ± 0.88 2.80 ± 0.20 71.67 ± 1.67 55.02 ± 6.35 90.30 ± 9.70

26.00 ± 0.10 3.10 ± 0.15 91.93 ± 10.04 70.67 ± 6.64 91.97 ± 15.57

27.00 ± 2.91 3.17 ± 0.15 106.13 ± 17.50 67.93 ± 15.01 97.13 ± 15.83

CV = Coefficient of variation. 252

0.8954 0.3030 0.2923 0.3960 0.9836

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Table 6 The digestive enzymes activities (IU) and hepatic glycogen (μmol g−1) (mean ± SEM) in Arapaima gigas juveniles fed with different levels of sodium butyrate. Levels of sodium butyrate (g kg−1)

a

Amylase Lipasea Nonspecific alkaline proteinasea Aspartate aminotransferaseb Glycogena

P-value

BS0

BS1

BS2

BS3

BS4

0.95 ± 0.18 3.04 ± 0.11 1.61 ± 0.09 2.31 ± 0.18 87.34 ± 15.12

0.69 ± 0.13 5.29 ± 0.37 0.69 ± 0.23 2.38 ± 0.05 179.61 ± 8.23

0.59 ± 0.08 4.97 ± 0.10 1.42 ± 0.20 0.44 ± 0.06 114.67 ± 15.59

0.68 ± 0.01 4.70 ± 0.32 1.48 ± 0.23 1.04 ± 0.05 244.13 ± 13.87

1.26 ± 0.21 5.92 ± 0.06 2.63 ± 0.23 0.71 ± 0.01 158.10 ± 27.19

0.0052 0.0413 0.0011 0.0250 0.0300

CV = Coefficient of variation; P < 0.05; a Quadratic effect. b Linear effect.

5. Conclusion

Other authors (Ahmed and Sadek, 2014; Tian et al., 2017), associated the use of sodium butyrate to the villi growth and intestinal health, with an increase in weight gain. As reported by Liu et al. (2016), in which the addition of 2 g kg-1 of sodium butyrate to diets for carp (C. idellus) increased intestinal absorption, aiding in health and improving growth.

The inclusion of 1.17 g kg−1 of sodium butyrate in diets for Arapaima gigas juveniles improves the growth parameters. This additive level favors the feed conversion ratio, increased intestinal villous height, without interfering in the blood parameters, promoting the adaptation of the enzymatic activity of amylase, lipase and nonspecific alkaline protease. Furthermore, it reduces the activity of aspartate aminotransferase, and increases hepatic glycogen levels, making the animals more resistant to potentially stressful conditions in the growing environment.

4.5. Blood parameters Diets with the inclusion of sodium butyrate, formulated to meet the nutritional needs of the fish, did not alter the blood parameters, being within the appropriate values for the well being and health of fish (Drumond et al., 2010; Hoshino et al., 2017). On the other hand, Ahmed and Sadek (2014), reported that dietary supplementation with 3 g kg−1 of the sodium butyrate tilapia provided an increase in glucose content. It is known that blood parameters are susceptible to the diets ingredients and provide relevant information on the health status and physiological conditions of the fish (Buscaino et al., 2010). Nutritional deficiencies and diets imbalances can be perceived in response to blood parameters changes (Fazio et al., 2013), or stress generated by factors arising from the metabolism or physiology of the animal (Labarrère et al., 2012).

Acknowledgements This study was funded by the Foundation for Research Support of the State of Bahia (FAPESB) - Notice PRONEM PNE - 013/2014, aimed at strengthening the Arapaima gigas production chain in the southern Bahia, Brazil. Thanks to the company PRATIGI ALIMENTOS, by the preparation of experimental diets. Thanks to the MARWAY FARM for supplying the fish. We acknowledge the great contribution of the members of the laboratories of Hematology and Animal Pathology, both of the State University of Santa Cruz, by contributions in hematological and histological analyzes, respectively. To the teachers: Marianne Schorer, Luiz Vítor Vidal, Camila Meneghetti and Cínthia Pereira for the contributions in this work. To the Aquanut team for their support in the realization of the experiment.

4.6. Enzymatic assays The regulation of the enzymatic profile and alteration in the level of these activities in fish depend on the feeding conditions, and type of food offered (Fracalossi and Cyrino, 2012). This occurred in relation to amylase, lipase and nonspecific alkaline protease, however, there was no direct relation in A. gigas growth. On the other hand, the decrease of the aspartate amino transferase in the hepatic tissue of the juveniles has already been reported in other species of fish fed with organic acid supplementation (Hoseinifar et al., 2017). The evidenced that the sodium butyrate has no hepatotoxic effect, being able to activate a further reduction in the process of transamination in the liver (Ali et al., 2017). This may lead to the use of other energy sources for metabolic activities, in addition to amino acids, since the activity of AST is related to the catabolism of amino acids to regulate glucose levels and hepatic reserves (Melo et al., 2006). Additionally, the inclusion of butyrate sodium provided greater accumulation of energy reserve with the increase of glycogen in the liver as reported for Sparus aurata (Estensoro et al., 2016), but with the maintenance of blood glucose level. Sodium butyrate as a dietary supplement can be used in diets for the carnivorous fish A. gigas (pirarucu) in the juvenile phase since this acid provides greater absorption of nutrients contributing in the body composition of fish and does not present hepatotoxic action. In addition, sodium butyrate can act as a growth promoter, perform weight gain, and improves in fish health status (Robles et al., 2013; Liu et al., 2014; Ahmed and Sadek, 2014; Liu et al., 2016; Tian et al., 2017; Omosowone et al., 2018).

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