Mango pulp meal in diets for Nile tilapia (Oreochromis niloticus): Digestibility, Growth performance, Biochemical analysis, Digestive enzyme activity, hematological variables

ABSTRACT The objective of this study was to evaluate the potential of mango pulp meal (MPM) as an ingredient for extruded diets for Nile tilapia juveniles. In the first trial, the digestibility of energy, dry matter, crude protein, and amino acids was determined, using 80 juveniles (average weight 25.00 ± 0.50 g), fed with a reference diet and a test diet containing the proportion of 70% of the reference diet and 30% of the MPM. Chromium oxide (Cr2O6) was used as an digestibility indicator and feces were collected using the Adapted Guelph System. The second assay assessed the growth performance; biochemical analysis; digestive enzyme activity; hematological variables for 300 juveniles fish (average weight 26.66 ± 0.17 g), fed for 45 days with balanced diets containing 0 (control), 40, 80, 120, and 160 g of MPM per kg-1. The dry matter, crude protein, and crude energy apparent digestibility coefficients of MPM were 39.26, 50.18, and 44.5%, respectively. The growth performance of the animals was negatively impacted by the MPM, however the hematological parameters and plasma concentrations of albumin, total proteins and cholesterol were not altered. However, the increase on hepatic glycogen reserves, glycemia, and alanine aminotransferase plasmatic enzyme activity demonstrated that MPM diet inclusion promoted oxidative metabolism conditions that could cause hepatocellular damage and affect fish health, if fed for long periods or at higher levels of MPM than analyzed in this study.


INTRODUCTION
In aquaculture, the possibility of totally or partially replacing traditional ingredients used in diet formulations with alternatives is of great interest. In the evaluation process, the nutritional value of the ingredients must be considered, in addition to the possibility of replacing totally or partially the ingredients of traditional crops (GLENCROSS et al., 2007). Alternative ingredients include non-traditional crop products (DE SOUZA et al., 2018) or even residues from the agribusiness (FELIX E SILVA et al., 2020), enabling the reduction of the impact of activities with large disposal of by-products . The fruit agribusiness is an important economic activity in tropical and subtropical countries, and generates large amounts of waste during production and after harvest (MARTINS AND FARIAS, 1994;DURIGAN et al., 2005). Fruits with mechanical or aesthetic damage are often discarded during these phases, and can become a waste product (DE CAMPOS AND DE LIMA, 2014). On the other hand, fruits, as well as their parts are rich in carbohydrates such as reductors sugar, and pectical substances, which can make them potential substitutes for traditional energy ingredients (KROGDAHL et al., 2005). In this scenario, mango fruit (Mangifera indica L.) has the characteristics to be a possible substitute for traditional energetic ingredients (LAZZARI et al., 2015), with studies carried out with fish and other nonruminant organisms (VIEIRA et al., 2008;MELO et al., 2012;BEZERRA et al., 2014). In addition to the growth parameters of fish, the use of alternative ingredients can cause changes in animal's health and metabolism. When considering alternative energetic ingredients, due to the change in the carbohydrate matrix, we can observe differences in carbohydrate metabolism, consequently, energy and protein metabolism (DE SOUZA et al., 2018;FELIX E SILVA et al., 2020). In this context, the purpose of the present study was to determine the apparent digestibility (energy, dry matter, crude protein, carbohydrates and amino acids), growth performance; biochemical analysis; digestive enzyme activity; hematological variables of juvenile Nile tilapia (Oreochromis niloticus), by feeding them diets containing variable levels of mango pulp meal. Thus, to determine the potential use of this residue from the fruit agroindustry in the feeding of Nile tilapia.

MATERIAL AND METHODS
This experiment was performed at the Aquaculture Laboratory of Universidade Federal do Vale do São Francisco -UNIVASF -, Campus Ciências Agrárias, in Petrolina city Pernambuco state. This study was approved by the ethics committee of Universidade Federal da Bahia, process number 54/2015 of CEUA/EMVZ/UFBA. The experiment had two phases. The purpose of the first phase was to evaluate the chemical composition and amino acids profile of mango pulp meal (MPM), and to determine its apparent digestibility coefficients (ADCs) of dry matter (ADCDM), crude protein (ADCCP) and gross energy (ADCGE), and availability of essential amino acids (AEAAs) and non-essential amino acids (ANEAAs) for juvenile Nile tilapia. The purpose of the second phase was to determine the growth performance of juvenile tilapia that received diets containing levels of MPM, and the effects of MPM levels on metabolism, intestinal enzymatic activity, and metabolic plasma concentrations.

Manufacture and chemical composition of MPM
Multiple cultivars mangoes fruits, such as Tommy Atkins, unsuitable for human consumption, were collected at the "Mercado do Produtor" in Juazeiro/BA. The fruits were processed in a Max Machine pulper, model MDP-150, to separate the epicarp, mesocarp (pulp) and endocarp. Thus, the pulp, the part with the highest concentration of soluble carbohydrates, was dried in a forced ventilation oven (55 ° C) for 48 hours. The dry pulp was ground in a knife mill, using a 1 mm sieve. At the end of the processing, mango pulp meal (MPM) was obtained (Table 1).  a Mineral and vitamin mixture: Cobalt (minimum) 80 mg kg -1 ; Copper (minimum) 3.500 mg kg -1 ; Iron (minimum) 20 g kg -1 ; Iodine (minimum) 160 mg kg -1 ; Manganese (minimum) 10.000 mg kg -1 ; Selenium (minimum) 100 mg kg -1 ; (minimum) 24 mg kg -1 ; Folic acid (minimum) 1200 mg kg -1 ; Nicotinic acid (minimum) 20 g kg -1 ; Uric acid (minimum) 10.000 mg kg -1 ; Biotin (minimum) 200 mg kg -1 ; Choline (minimum) 100 g kg -1 ; Inositol (minimum) 25 g kg -1 ; Vitamin A (minimum) 2.400.000 UI kg -1 ; Vitamin B1 (minimum) 4.000 mg kg -1 ; Vitamin B2 (minimum) 4.000 mg kg -1 ; Vitamin B12 (minimum) 8.000 mg kg -1 ; Vitamin C (minimum) 60 g kg -1 ; Vitamin B2 (minimum) 4.000 mg kg -1 ; Vitamin B6 (minimum) 3.500 mg kg -1 ; Vitamin D3 (minimum) 600.000 The experimental fish were fed five times a day between 07:00 and 17:00 , until they were visibly satiated. One hour after the last feeding of the day, water circulation was closed, each tank was cleaned, and the water was partially changed, while the central blower remained active. Fecal collection tubes, which were individually attached to the bottom each tank, were collected, and stored in coolers with ice. At 0,600 hr the morning after collections, fecal collection tubes were centrifuged. Material in the sediment was saved and other material was discarded. The material was then fine-strained and deposited in plastic bottles labeled for each tank. Feces were pooled between collections into the same bottle for each replicate. These samples were kept frozen at -21°C until the end of the collection period.

Chemical analysis of ingredients, diets, and faeces
After each experimental period, feces were dehydrated in a forced ventilation oven at 55°C for 48 h; the same procedure was applied to the ingredients and the experimental diets, but only for 24 h. All sample materials were then ground in a ball mill. Dry matter and ash content were measured according to standard methods (AOAC, 2005). Moisture content was determined by drying the samples to a constant weight at 105°C in a furnace (TE-391-1, Tecnal). Nitrogen content was determined using the Micro-Kjeldahl (Tecnal) method, and the crude protein content was estimated by multiplying the nitrogen content by 6.25 (FURUKAWA AND TSUKAHARA, 1966). Lipid content was determined by ether extraction in a multi-unit Soxhlet apparatus (TE-188/6, Tecnal) for 16 h. Ash content was measured by combustion. Samples were dried in a muffle furnace (TE-1100-1P, Tecnal) at 550°C for 6 h, and crude energy content was assessed using an adiabatic bomb calorimeter (Parr 1266, Parr Instruments Co., Moline, Illinois, USA). Amino acids were measured commercially by CBO Analysis Laboratory Ltda., Valinhos, São Paulo, Brazil by hydrolyzing a 0.3-mg sample in 1 mL of 6 N HCl for 22 h. The obtained sample was diluted in 0.02 N HCl and injected into an automatic AA analyzer (Hitachi L-888, Tokyo, Japan). Hydrolysis was performed to recover the amino acids using 4-N methanesulfonic acid for tryptophan analysis, and sulfuric acid for amino acid analysis. Chromium (III) oxide (Cr2O3) levels were measured using inductively coupled plasma atomic emission spectrophotometry (ICP-AES, Vista-MPX, Varian, Palo Alto, California, USA) after digestion with perchloric acid, following a technique modified from Bezerra Neto and Barreto (2011). Concentrations of total phenols and tannins in MPM and in experimental diets were determined according to the (AOAC, 2005) acetone extraction method (70%), and colorimetric titration with the Folin-Ciocalteau reagent, sodium carbonate (20%), and insolubilized polyvinylpyrrolidone.
Standardized readings were derived from a tannic acid curve (0.1 mg mL -1 ) and the results were expressed as mg equivalents of tannic acid per kg of dry matter (DM).

Calculation of apparent digestibility coefficients and availability of amino acids
Nutrient and energy ADCs, in addition to AEAAs and ANEAAs, were calculated based on chromium levels in food and feces according equations 1 and 2, following (NRC, 2011): where ADC (n) = apparent digestibility coefficients, Equation 2: , where Dreference and Dingredient are the percentages of the nutrients or kcal g-1 gross energy in the reference diet and test ingredients, respectively.

MPM inclusion trial
Fish, experimental conditions, facilities, and diets manufacture A 45 day trial was conducted to evaluate performance of 300 male, sexually reversed, juvenile Nile tilapia (average weight 26.66 ± 0.17 g) given diets with different concentrations of MPM. The fish were obtained from AAT International Ltda. (Paulo Afonso-BA) and randomly distributed into 20 plastic circular tanks (500 L each). Each tank had a recirculation system with a physical central active carbon filter and a perlon-type acrylic cover. Aeration was supplied to tanks with a central blower connected to plastic hoses leading to porous stones. Five diet types were tested, containing 0, 40, 80, 120, or 160 g of MPM per kg of diet (Table 3). Each diet type was randomly assigned to each of four tanks. Fish were fed four times a day until visible satiation, at 08,00 hr, 11,00 hr, 14,00 hr, and 17,00 hr.  Furuya et al. (2010) Water quality parameters (temperature, pH, and dissolved oxygen) were measured daily in the morning and in the afternoon throughout the entire experimental period using a multiparametric digital Alfakit ® (AT1100) rig.

Growth performance
After the 45 day feeding experiment period, all fish were starved for a 24 hours, and then anesthetized in order to collect blood and biometric data. After this, fish were euthanized and their livers were dissected for further analysis. Data collected on fish included: final weight (FW); final biomass (FB); weight ).

Biochemical analysis
Plasmatic evaluations of metabolic intermediates were made using LABTEST ® industrial kits for colorimetric determination of glucose (GLUCOSE LIQUIFORM ® ), total proteins (TOTAL PROTEINS ® , BIURETO method) and total cholesterol (CHOLESTEROL LIQUIFORM ® ). Both analyses were conducted with a biochemical semiautomatic veterinary device (Doles ® , model D-250).
Free plasmatic amino acid dosage was measured using the Ninhidrine 0,10% method in propanol, with a standard curve based on a glycine solution of 1.00 millimolar (mM), and read via spectrophotometer wavelength of 570 nanometers (nm). The plasmatic activity of the enzyme alanine aminotransferase (ALT) was measured with an ALT/GPT Liquiform ® kit and read with a spectrophotometer with a wavelength of 340 nm at one and three minutes into the reaction. The activity was considered the difference between the two measurements. Glycogen content of liver tissue was determined following Bidinotto et al. (1997). Liver fragments were weighed and then put in potassium hydroxide (KOH) with 6.00 normal (N). Glycogen contents were extracted with ethanol, centrifuged, and reacted with potassium sulphate (K2SO4), water, and phenol. Reaction readings were taken using a spectrophotometer with a wavelength of 480 nm, and compared to the standard curve for glucose, with values in expressed in micromoles per milligram of hepatic tissue (µmol mg -1 ).

Digestive enzyme activity
For intestinal amylase evaluation, six fish were randomly selected from each treatment and a 3.00 cm portion of intestinal segment was collected from each (before final gastric expansion). Each adipose tissue segment and the rest of the mesentery were taken. Each intestinal fragment was weighed, put in a sample tube with a 2.00 mL buffer solution (tris-phosphate-acid phosphoric-glycerin, pH = 7.00), and macerated using a mechanical tissue refrigerated homogenizer. After tissue maceration, tubes were centrifuged at 12,000 rpm for three minutes in a refrigerated centrifuge. Then, intestinal amylase enzyme activity was measured using an Amylases Bioclin ® commercial test kit and a semiautomatic evaluator (Trademark Doles ® , model D-250). Values were expressed as international units of activity in grams of tissue (U g -1 ).

Hematological variables
Hematological variables were determined for fresh blood taken from fish tail veins using heparinized syringes. Hematocrits (HT) were measured by a centrifugation method with micro capillary tubes (heparinized in vitro), and compared with a percentage table of micro hematocrits (GOLDENFARB et al., 1971). To measure hemoglobin values (HB), grams per each deciliter of blood (g dL -1 ) (COLLIER, 1944). Corpuscular hemoglobin concentration index (CHCM) was calculated as CHCM = 100 (HB HT -1 ), with HT expressed in g dL -1 .

Statistical analysis
Data were tested for normality using Shapiro-Wilk tests, and were analyzed by polynomial regression and linear response plateau (LRP) analysis with the minimums squared method, according to the model Y = L + U * (RX), where Y = value of the variable analyzed, X = percentage of MPM inclusion, L = plateau response of the studied variable; U = slope of the line, and R = inclusion of MPM estimated by the intercept point (ROBBINS et al., 1979). The Dunnett test was performed to compare the control treatment with the other treatments. Models with the best fit for each variable were chosen. All procedures were conducted the SAS ® University Edition statistical package.

RESULTS
In both trials, there was no mortality of experimental fish. During the trials, average water temperatures were 26.00 ± 0.50ºC, pH was 6.80 ± 0.40, and dissolved oxygen was 4.80 ± 0.30 mg L -1 .

Digestibility Trial
Apparent digestibility of crude energy and crude protein was low, 44.55% and 50.18%, respectively. The average digestibility of amino acids was 56.81%, with a value of 56.28% for the average digestibility of essential amino acids (Table  4). Apparent digestibility coefficients of nutrients; 2 Availability of essential amino acids; 3 Availability nonessential amino acids.

MPM inclusion trial
All growth performance variables showed differences between treatments with the inclusion of MPM and the control treatment (p < 0.05), except for FI and FCR (Table 5). None of the variables fit a model with a biological explanation to the experimental design of the study. No differences were observed between hematological variables. Among the plasma variables, difference was observed in glucose, AST, ALT and TPAA, as well in hepatic glycogen (p< 0.05) ( Table 6). Only plasma glucose adjusted to a model with biological explanation (Figure 1). 1 ALT = alanine amino transferase; 2 TPAA = total plasmatic amino acids; 3 HT = hematocrit; 4 HB = hemoglobin; 4 CHCM = corpuscular hemoglobin concentration index; 5 Glycogen expressed in micro moles of glycosil-glucose for each milligram of hepatic tissue.

DISCUSSION
The levels of dry matter (DM) and crude protein (CP) of the mango pulp meal were similar to those describe for de Lima et al. (2011) for Nile tilapia and Vieira et al. (2008) for poultry, respectively, with DM = 941.00 g kg -1 and CP = 44.40 g kg -1 and DM = 922.30 g kg -1 and CP = 38.70 g kg -1 , by studying the composition of industrial solid residues from the fruit pulp industry, containing fruit peels and seeds. Concentrations of crude fiber (CF) observed were different from the actual level of CF = 149.90 g kg -1 and GE = 3.724 kcal kg -1 in the study de Lima et al. (2011), and 146.00 g kg -1 and 3.906 kcal kg -1 in the study by Vieira et al. (2008). Digestibility coefficients for MS, PB, and EB of MPM in our study were lower than those described by de Lima et al.  et al. (2015), mango pulp contains carbohydrates in the forms of fructose, cornstarch, and pectins. These molecules store high amounts of energy in chemical bonds (VERGARA-VALENCIA et al., 2007). However, pectin is a non-starchy carbohydrate and has low digestibility because it is resistant to breakdown by enzymatic hydrolysis or heat treatment (OLIVEIRA et al., 2007). Although Nile tilapia are omnivorous and capable of consuming pectins and other non-starchy carbohydrates normally included in their diet (KROGDAHL et al., 2005), such carbohydrates can adversely affect hydrolysis of proteins. This can occur even in fish species such as Nile tilapia (KROGDAHL et al., 2005), which have active β-galactosidase (type of pectin) in their anterior and middle intestines (TANIGUCHI AND TAKANO, 2004). Furthermore, Irvin et al. (2016), described negative effects of pectin on digestibility of energy and nutrients in barramundi fish (Lates calcarifer), and pectin could also affect the viscosity and transit velocity in intestines. These results are supported by those of Castillo and Gatlin (2015), who found that addition of exogenous carbohydrase to the diet of juvenile tilapia increased the digestibility of vegetable ingredients. Therefore, compared with pulp residues, we would expect that MPM has lower digestibility of nutrients and energy, and lower availability of amino acids, because of greater interference from soluble fibers that are indigestible, such as pectin. Increasing concentrations of MPM lead to a significant performance changes (p > 0.05) in the juvenile tilapia tested during our study, results are not consistent with those of de Lima et al. (2011) andMelo et al. (2012). Although both of those studies used juvenile tilapia, they were conducted using agroindustrial residues resulting from mango pulp meal processing. These two studies did not detect any performance decrease with an inclusion of a maximum 150 or 300 g MPM per kg of diet, respectively. In contrast, Souza et al. (2013), who tested MPM that included peels, observed a reduction in performance of Nile tilapia with increasing MPM concentrations. Therefore, we advise against inclusion of MPM levels over 100 g kg -1 because of the harmful effects of phenols, such as tannins contained in peels, even though this possibility was not directly assessed in our study. Melo et al. (2012) replaced corn bran in juvenile Nile tilapia diets with variable levels of mango pulp (0, 33, 66, or 100%) and did not observe any effect of mango pulp on feed intake or FCR, as we did in our study. However, Souza et al. (2013), observed a negative effect on feed intake and FCR of a MPM including peels, at concentrations of over 200 g of MPM per kg of diet. This negative effect may have been due to phenolic contents of fruit peels that were incorporated in the MPM. Pinto et al. (2001) reported negative effects of tannins on the digestion process of juvenile tilapia, for concentrations equal to or higher than 6 g kg -1 . These concentrations were much higher than the ones in our study (total phenols of 0.62 gAT kg -1 ). According to Rosset et al. (2016), ingestion of large quantities of fructose leads to an increase in the production of metabolic intermediates involved in fructolysis, the process of converting fructose to glucose, lactate, glycerol, and fatty acids, or for use in oxidative metabolism in mitochondria. A similar effect was found in our study, and by Bezerra et al. (2014), who tested levels of 200, 300, 400, and 500 g kg -1 of MPM in the diet of the tambaqui fish (Colossoma macropomum). That study reported changes in glycogen hepatic reserves corresponding to the level of MPM inclusion in diets. However, results of de Lima et al. (2011), who incorporated levels of 0, 50, 100, and 150 g kg -1 of MPM in the tilapia diet, differed from results in our study in that they did not observe an effect of MPM on glycogen reserves. These contrasting results might have occurred because the type of mango residue used by de Lima et al. (2011) had lower amounts of fructose, which was removed by a fruit processing step. Amylase, a pancreatic enzyme secreted in the intestine, is responsible for hydrolysis of polysaccharides. Its activity is related to the utilization of different levels of starchy carbohydrates in food, and varies according to feeding routines of species (STECH et al., 2009). The activity of plasmatic ALT increased with the inclusion MPM. this enzyme is present in several tissues of the body, especially hepatocytes. Elevation in plasma ALT is associated with increased permeability of cytoplasmic membranes, oxidative stress, and metabolic imbalance (JONSSON et al., 2002;KOBAYASHI et al., 2009). According to Rosset et al. (2016), fructose absorption has no regulation system like that of glucose. Hence, ingestion of large quantities of fructose can lead to a cytoplasmic increase in fructose concentration. Fructose would then be phosphorylated, leading to depletion of hepatic adenosine triphosphate (ATP) reserves, and an increase in the turnover of proteins, especially those involved in deamination of amino acids for energy purposes. Higher fructose levels in diets would reduce transamination of amino acids for energy production, leading to what Boscolo et al. (2011) described as a protein-saving effect of carbohydrates. In a subsequent study, higher concentrations of fructose increased the activity of ALT, leading to a prooxidative effect when 100 g kg -1 lyophilized mango pulp meal was included in the diet of rats (TOLEDO et al., 2013). This may have been because the lyophilized mango pulp meal led to a mobilization of amino acids in order to keep appropriate glucose levels in blood and to preserve hepatic glycogen reserves through gluconeogenesis. This side effect was also noted by (BEZERRA et al., 2014), who tested mango pulp levels (ranging from 200 to 500 g kg -1 ) in the diet of tambaquis. High levels of fructose in diets containing MPM can cause oxidative damage to hematocrits and increase plasmatic activity of enzymes similar to ALT. However, MPM levels did not influence hematologic variables. Akrami et al. (2013), researched the prebiotic effect of polymeric carbohydrates of fructose (fruitoligosaccharides) in a sturgeon species (Acipenser stellatus), and only observed an increase in average levels of white blood cells. They did not report any significant differences in the hematimetric index. The effect of including MPM on digestive enzyme activity was similar to that observed when replacing corn with mesquite bran in diets for Nile tilapia. Some enzymes have their production related to the amount of the substrate that catalyzes, thus, the lower proportion of starch induces less amylase production and directs the body's efforts to produce other carbohydrases (DE SOUZA et al., 2018). In our study, we prepared isoproteic and isoenergetic diets in order to control for carbohydrate compositions of MPM. Consequently, there none of the risk factors described above could have affected the hematologic variables we analyzed. We found that the inclusion of MPM in diets for Nile tilapia reduced the parameters of fish growth, on the other hand, the hematological parameters do not indicate a significant impact on health in the evaluated period. Furthermore, our data concerning metabolism indicated that inclusion of 54.14 g kg -1 or greater concentrations resulted in metabolism changes in proteins. Therefore, future research to evaluate metabolic and cellular damage by long-term ingestion of MPM could be valuable.