The intestinal health of silver catfish Rhamdia quelen can be changed by organic acid salts, independent of the chelating minerals

Accepted Manuscript The intestinal health of silver catfish Rhamdia quelen can be changed by organic acid salts, independent of the chelating minerals

S.A. Pereira, G.F.A. Jesus, L. Cardoso, B.C. Silva, J.V.S. Ferrarezi, T.H. Fereira, F.C. Sterzelecki, J.K. Sugai, M.L. Martins, J.L.P. Mouriño PII: DOI: Reference:

S0044-8486(18)32758-3 https://doi.org/10.1016/j.aquaculture.2019.02.049 AQUA 633931

To appear in:

aquaculture

Received date: Revised date: Accepted date:

19 December 2018 19 February 2019 19 February 2019

Please cite this article as: S.A. Pereira, G.F.A. Jesus, L. Cardoso, et al., The intestinal health of silver catfish Rhamdia quelen can be changed by organic acid salts, independent of the chelating minerals, aquaculture, https://doi.org/10.1016/j.aquaculture.2019.02.049

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ACCEPTED MANUSCRIPT The intestinal health of silver catfish Rhamdia quelen can be changed by organic acid salts, independent of the chelating minerals S.A. Pereiraa, G.F.A Jesusa, L. Cardosoa, B.C. Silvab, J.V.S. Ferrarezia, T.H. Fereiraa F.C. Sterzeleckic, J.K. Sugaid, M.L. Martinsa, J.L.P. Mouriñoa a

AQUOS – Aquatic Organisms Health Laboratory, Aquaculture Department, Federal

University of Santa Catarina (UFSC), Rod. Admar Gonzaga 1346, CEP 88040-900, b

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Florianópolis, SC, Brazil.

EPAGRI - Company of Agricultural Research and Rural Extension of Santa Catarina,

LAPMAR - Marine Fisheries Laboratory, Aquaculture Department, Federal University

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Rua Joaquim Garcia, s/n, CEP 88340-000, Camboriú, SC, Brazil.

of Santa Catarina (UFSC), Servidão Beco dos Coroas, 503, CEP 88061-600 d

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Florianópolis, SC, Brazil.

Applied Enzymology Laboratory, Department of Biochemistry, Center for Biological

Sciences, Federal University of Santa Catarina (UFSC), Campus Universitário Prof. Dr.

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João David Ferreira Lima - Trindade. CEP 88 010-970, Florianópolis, SC, Brazil.

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E-mail: [email protected] (S.A. Pereira)

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Running title: Propionate increase in health intestinal fish

ACCEPTED MANUSCRIPT ABSTRACT Food additives based on organic acids or their salts promote numerous improvements in zootechnical parameters, digestive enzymatic activities, resistance to diseases and intestinal health. This study aimed to evaluate the influence of calcium and sodium chelated to propionic acid on the digestive enzymes activities, intestinal microbial community and histological alterations in the liver and intestine of the silver catfish Rhamdia quelen. Fish with an initial mean weight of 8.43±0.18 g were divided into a

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control and four treatments, with 15 fish in each replicate, and fed the following supplemented diets for 60 days, four times per day: unsupplemented control, Ca-

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propionate 0.25% (Ca0.25%), Ca-propionate 1% (Ca1%), Na-propionate 0.25% (Na0.25%)

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and Na-propionate 1% (Na1%). At the end of the assay, digestive enzymes activities in the gastrointestinal tract (GIT) was not affected by the chelating mineral, nor by its

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concentration to propionic acid. On the other hand, the most important digestive enzymes for silver catfish were lipase and acid protease. Regarding the intestinal microbial community, fish fed Ca0.25% showed a lower concentration of total

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heterotrophic bacteria and a higher lactic acid bacteria count, compared to Na1%supplemented fish, in addition to the maintenance of cordonal features and liver

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cholestasis. Fish fed a Ca0.25% or Na0.25% supplemented diet presented the best histomorphometry parameters, such as the greatest width and number of villi, a lower

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number of eosinophilic infiltrates and the absence of lymphocytic infiltrates. The organic acid propionate chelated to calcium at 0.25% improved the microbial composition and intestinal health of silver catfish, with no effects on the liver, the most

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catfish.

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important organ for depuration, and could be indicated as a feed additive for silver

Key words: Silurid fish, dietary supplementation, organic acids, lipase, protease, intestinal health, bacteria.

ACCEPTED MANUSCRIPT 1.

Introduction Aquaculture is a promising agricultural activity for the production of protein of an

animal origin. Among the aquaculture sectors, freshwater fish farming accounted for 93.8% of the total world fish production in 2016 (FAO, 2018). In continental waters, the most produced fish are tilapia and cyprinids, with a production of more than 45 million tons in 2016 (FAO, 2018). To diversify production, new species like native fish have

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gained ground in continental fish farming. Among the most cultured fish in southern Brazil, the native species silver catfish Rhamdia quelen (Quoy and Gaimard, 1824),

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commonly known as “jundiá” or silver catfish, has good zootechnical characteristics and market acceptance (Gomes et al., 2000).

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Of the 39.86 thousand tons of fish produced via continental Brazilian fish farming in 2013, 743.9 tons were silver catfish. According to the last survey, this species is the

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third most important for the state of Santa Catarina, after the production of tilapia and carp (Silva et al., 2017); for this reason, silver catfish was used as a model in this study.

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For the expansion of fish farming, there is a need for high-quality feed additives that provide better productive performance and disease resistance since silver catfish suffer from several bacterial and parasitic diseases. For many years, agricultural

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activities used growth promoters based on antibiotics; however, the indiscriminate use

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of this chemotherapeutic agent led to the contamination of the meat being produced, environmental pollution, in addition to the transfer of resistance plasmids between microorganisms (Cabelo et al., 2006, 2013; CLSI, 2006; Ng et al., 2016). In addition,

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antibiotics can disrupt the microbial community and damage some tissues, causing oxidative stress and increasing the possibility of the entry of pathogens (Kalghatgi et al., 2013; Reese et al., 2018; Yoon and Yoon, 2018); for this reason, the European Union

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and many countries have abolished the use of antibiotics in animal production (Lückstädts, 2006; MAPA, 2016). As a consequence, there is a search for biosecure substances that are capable of inducing improvements to cultivated animals. As an example, organic acid salts are already widely used in swine and poultry farming (Fang et al., 2014; Partanen and Mroz, 1999; Viola and Vieira, 2007). Organic acid salts are short-chain fatty acids that become stable when they are chelated to minerals, for example, calcium (Ca+2), potassium (K+1) and sodium (Na+1); as a consequence, they become organic acid salts. The most sought-after organic acid salts are potassium or sodium formate, calcium or sodium propionate, and sodium butyrate (Ng et al., 2016). These different organic acid

ACCEPTED MANUSCRIPT salts and their respective chelating minerals have already proven their effectiveness for numerous aquaculture species (Lückstädts, 2006, 2008; Ng et al. 2016; Pereira et al., 2018; Romano et al. 2015; Silva et al., 2013, 2015, 2016). The different salts of organic acids can influence several parameters, such as the productivity, immune system, resistance to diseases, nutrition, activities of digestives enzymes, as well as the composition of the intestinal microbiota. According to Silva et al. (2013), 2% sodium propionate, used in shrimp Litopenaeus vannamei, showed

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inhibitory activity in vitro against vibrios and reduced its number in the intestinal microbiota. Years later, Silva et al. (2015) observed an increase in the in vitro activity of

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trypsin and chymotrypsin in the presence of different molar concentrations of sodium

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acetate and propionate in the reaction system. The addition of citrate acid, calcium lactate and potassium formate to the diet of red drum, Sciaenops ocellatus, improved

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both pancreatic (trypsin and lipase) and intestinal enzymes (leucine-aminopeptidase and phosphatases), compared to unsupplemented fish (Castillo et al., 2014). However, it is unknown whether the chelating mineral has a direct influence on

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the effects of organic acid on the general and intestinal health of farmed animals. In a recent study, the mineral that chelated to propionic acid influenced the productive

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performance and disease resistance in R. quelen (Pereira et al., 2018). Nonetheless, little information is known regarding the effect of different chelating ions of organic acid

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salts on intestinal health. Therefore, the aim of this study was to evaluate the influence of calcium and sodium chelated to propionic acid on the digestive enzymes activities, intestinal microbial composition and histological alterations in the liver and intestine of

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silver catfish R. quelen.

Material and Methods

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2.1 Diet preparation Isonitrogenate and isocaloric diets were formulated based on the nutritional requirements of silver catfish according to NRC (2011) based on the channel catfish Ictalurus punctatus requirements (Table 1). Organic propionic acid chelated with calcium or sodium was used at concentrations of 0%, 0.25%, and 1%. The salt was added to the extruded diet instead of the cellulose filler, where the control unsupplemented fish received the inclusion of 1% cellulose. Analysis of the diets was made at the Nutrition Laboratory, UFSC (LabNutri/UFSC). Carbohydrate and energy analysis was performed by the RDC 360

ACCEPTED MANUSCRIPT method (Aoyama et al., 2003) and fiber, mineral material, moisture, and volatiles were analyzed using a protocol n. 108 (MAPA 1991) (Table 1). 2.2 Experimental design A total of 225 fish, with initial mean weight of 8.43±0.18 g, from Experimental fish farming of Camboriú (CEPC/EPAGRI, SC) were divided into polyethylene tanks of 100 L capacity with a usable volume of 70 L, 15 fish per tank (Piaia and Baldisserotto, 2000) with three replicates in each experimental group. The tanks were supplied by a

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recirculating system that was provided by the mechanical and biological filters (anaerobic and aerobic), which included ultraviolet sterilization and a controlled

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photoperiod of 12 h. The water was kept at 3 ppm salinity. The fish were fed at 5% of

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the biomass in four feedings per day (Carneiro and Mikos, 2005) and every 15 days fish were weighed and feed was adjusted. When an excess of food was noted, the next day

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10% reduction was standardized. The same was made when no food was noted and 10% more was added in the next day. Excess of food was removed from the tanks two or three times a day. Water quality was measured daily with a multiparameter HANNA®

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HI 9828, USA the temperature 25.91±1.40ºC and dissolved oxygen 8.00±0.75 mg·L-1. Alkalinity 34.00±2.82 mg·L-1 CaCO3, pH 7.20±0.17, ammonia 0.38±0.28 mg·L-1, and nitrite was less than 0.1 mg·L-1. This parameters were measured by the colorimetric

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method (Kit Acqua Análises®). The water quality parameters were kept within the safe

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values (Boyd and Tucker 2012).

The fish were divided into five groups, with three replicates, to evaluate the effects of supplemented diet during 60 days:

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1. Unsupplemented (Control); 2. Fish fed supplemented diet with 0.25% calcium propionate (Ca 0.25%);

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3. Fish fed supplemented diet with 1% calcium propionate (Ca 1%); 4. Fish fed supplemented diet with 0.25% sodium propionate (Na 0.25%); 5. Fish fed supplemented diet with 1% sodium propionate (Na 1%).

2.3 Enzymatic analysis Sixty days after supplementation, four fish per tank were frozen at -20 ºC for posterior enzymatic analysis. Firstly, the gastrointestinal tract (GIT) of two fish per tank was carefully dissected on ice, weighed (w) and homogenized (van Potter®) in chilled distilled water in a 1:8 (w:v) proportion for 2.5 minutes (5 shakes of 30 seconds with

ACCEPTED MANUSCRIPT approximately 5 minutes for cooling) and centrifuged at 20,817 g (Eppendorf 5804 R) for 15 min at 4ºC to obtain the enzymatic crude extract. All enzymatic assays were incubated at 25°C and absorbance was read with a microplate reader (Spectramax, Plus-384, Molecular Devices, Sunnyvale, CA), by transferring an aliquot of 300 μl of the hydrolysis products to the well, except for the lipase activity, whose reaction was carried out directly in the microplate wells. a) Soluble protein concentration

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The soluble protein concentration present in the enzymatic crude extract (EB) was determined according to the method of Bradford (1976), using bovine serum albumin

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(BSA) as a standard.

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b) Lipase activity

Lipase activity was determined from the hydrolysis of the synthetic substrate p-

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nitrophenyl myristate, according to Saele et al. (2010). Activity was monitored each minute for 30 min and read at 404 ɳm. Lipase activity was expressed in specific activity (μmol of p-nitrofenol produced min-1 mL-1 mg-1 of protein).

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c) Amylase activity

Amylase activity was determined by hydrolysis of the starch (E. Merck,

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Darmstadt, Germany) based on the Rick and Stegbauer (1974) method according to Baloi et al. (2017). The quantification of amylase product (reducing sugar) was

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estimated by 3,5-dinitrosalicylic acid (DNS), having maltose as the standard curve. Amylase activity was also expressed in specific activity (μmol of maltose min-1 mL-1 mg-1 of protein).

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d) Acid protease activity

To determine the acid protease activity, the hydrolysis of the bovine hemoglobin

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(Sigma, St. Louis, USA; Anson, 1938) was used, slightly modified according to VegaOrellana et al. (2006). The products of the acid hydrolysis were determined using a tyrosine (E. Merck S.A., Darmstadt, Germany) as standard curve, read at 280 ɳm and expressed in specific activity (μmol of tyrosine min-1 mL-1 mg-1 of protein). e) Alkaline protease activity The alkaline protease activity was determined by the hydrolysis of azocasein (Sigma, St. Louis, USA) following the Garcia-Carreño et al. (1997) method. Briefly, after stopping the reaction, the incubation system was maintained at 4 °C for 15 min and centrifuged at 11,000 g for 5 min. The absorbance of the supernatant was read at 366

ACCEPTED MANUSCRIPT ɳm and the total alkaline protease was expressed in specific activity (Δ A366 ɳm min-1 mL-1 mg-1 of protein). 2.4 Microbiological analysis of the intestine Intestines of four fish per tank were aseptically collected, weighed, macerated and serially diluted 1:10 to obtain 10-4 to 10-9 concentration for seeding in Man Rogosa Sharpe medium (MRS, Himedia® for lactic acid bacteria) with aniline blue (10 g L-1) and tryptone soy agar (TSA, Himedia® for heterotrophic bacteria). Dilution 10-1 to 10-4

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were seeded in thiosulphate-citrate-bile salts sucrose agar (TCBS, Himedia® for vibrionaceae) and Cetrimide agar (Himedia® for Pseudomonas sp.). All culture

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mediums were incubated at 30ºC for 24 h except for MRS which was incubated at 35ºC

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for 48 h (Jatobá et al., 2008). 2.5 Histological analysis

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The anterior medial portion of the intestine and liver of three fish per tank were collected and fixed in 10% buffered formalin, processed by routine methods, embedded in paraffin and sectioned into 3-µm thick cross-sections (Humason, 1979). The slides

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were stained with haematoxylin eosin (HE) to observe the intestine tissue and liver in an Axio Imager A.2 phase interference contrast microscope (DIC; Zeiss, Gottingen,

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Germany).

In the intestinal tissue, the length, area, width and perimeter of the villi, the

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number of villi, and the number of goblet cells per villus were assessed using photomicrographs made using the DIC, equipped with an image capture system with Zen Pro software (Zeiss, Germany).

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In the liver, the following alterations were considered: maintenance of the cordonal aspect, congestion in the large vessels and in the sinusoids, sinusoidal

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dilatation, necrosis, eosinophilic and lymphocytic infiltrate, melanomacrophage centres and loss of glycogen. For hepatocytes, we observed uniformity in the size of the cells and nuclei, ballooning aspect, hepatocyte and nucleus hypertrophy, displacement of the nucleus, pyknosis, karyolysis and karyorrhexis. For this organ, values were assigned for the histological changes, according to the degree of intensity: 0 (absence of alteration), 1 (mild alteration, corresponding to < 25% of the tissue area), 2 (moderate alteration, 25 to 50% of the tissue area) and 3 (severe alteration, > 50% of the tissue area), according to the method described by Schwaiger et al. (1997), with slight modifications (Brum et al., 2018). a) Transmission electron microscopy (TEM)

ACCEPTED MANUSCRIPT To verify the integrity of the intestinal cells and the microvilli, as well as the presence of bacteria, samples of the intestinal tract were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate (pH 7.2) containing 0.2 M sucrose (Schmidt et al., 2010), post-fixed in 1% osmium tetroxide for 4 h, dehydrated in graded acetone series and then embedded in Spurr’s resin. Ultrathin sections were made in an ultramicrotome (Leica, Reicheit Ultracut S, Vienna, Austria) and contrasted using uranyl acetate and lead citrate. The

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photomicrographs were made in a TEM JEM 1011 (JEOL, Tokyo, Japan) at 80 kV. b) Scanning electron microscopy (SEM)

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Samples were fixed and processed using the same methods used for TEM up to

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the dehydration stage, in which an ethanolic serial solution was used and dried at the critical point EM-CPD-030 (Leica, Heidelberg, Germany). Subsequently, the samples

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were adhered to metal holders and sputter-coated (Bal-Tec Sputter Coater, CED 030) with gold. The photomicrographs were made in a SEM JSM 6390 LV (JEOL Ltd., Tokyo, Japan) at 20 kV. Statistical analysis

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3.

All data were submitted to Shapiro-Wilk and Levene test to verify the normality

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and variance homoscedasticity, respectively. Data that did not present homogeneity of variance were transformed in Log2 (x+1) for microbial counting and square root for

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digestive enzymes. After that, the data were submitted to unifactorial variance analysis and the averages separated by Tukey test. All tests considered a significance of 5% with

4. 4.1

Results

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the software Statistica 10.0.

Enzymatic analysis

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After 60 days of supplementation, it was not possible to observe a clear influence (without significant difference) of the addition of propionates, regardless of the concentration or chelating mineral, on the digestive enzymes activities. The activities of the enzymes quantified in the GIT of silver catfish, in descending order, were lipase, acid protease, amylase and alkaline protease (Figure 1). 4.2

Microbiological analysis of the intestine At the end of the experimental period, the intestine of the fish supplemented with

propionate chelated to calcium in both concentrations (Ca0.25% and Ca1%) presented lower counts of total heterotrophic bacteria (p = 0.001) in comparison to the control group and that supplemented with Na1% (Figure 2). The highest counts (p = 0.003) of

ACCEPTED MANUSCRIPT total lactic acid bacteria were in fish supplemented with calcium and sodium propionate in the lowest concentrations (Ca0.25%, Na0.25%) in comparison to fish supplemented with the highest concentrations (Figure 2). 4.3

Histological analysis Regarding the histological alterations in the liver, fish supplemented Ca0.25%

showed a greater (p = 0.014) maintenance of the cordonal aspect in comparison with the

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control group. The cholestase was significantly higher (p = 0.026) in the liver of fish supplemented with Na1% compared to those fed with Ca0.25% and control. The

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hepatocyte core displacement indexes were lower (p = 0.037) in fish supplemented with Na0.25% compared to the Ca0.25%, Ca1% and Na1% fed animals, but did not differ from the

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control group (Table 2).

After 60 days of culture, it was observed that the intestine of the fish

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supplemented with propionate independent of the chelating mineral at the lowest concentrations (Ca0.25% and Na0.25%) had the best values of villi width (p = 0.001),

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number of villi (p = 0.001), and lower concentrations of eosinophilic (p = 0.019) and lymphocytic infiltrates (p = 0.001) compared to animals supplemented with propionates at the highest concentrations independent of the chelator (Ca1% and Na1%) (Table 3).

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a) Transmission electron microscopy (TEM)

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Using transmission electron microscopy (TEM), it was not possible to observe significant differences in the density and length of microvilli. However, TEM confirmed that, regardless of the treatment, the intestines of the silver catfish presented total

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membrane integrity of the intestinal mucosa. Moreover, the intestinal epithelium was intact, with well-defined enterocytes, integrity of the occlusion and adhesion zones, and

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without observable vacuoles or intercellular spaces (Figure 3). Photomicrographs of the transmission electron microscopy of the unsupplemented group (Figure 3A) demonstrated the abundance of microvilli (MV), and well-defined cell nucleus (noted by the asterisk, *), secretion granules (SG) and the occlusion zone (OZ). Photomicrographs of the group supplemented with Ca0.25% (Figure 3B) clearly showed the numbers of microvilli (MV), well-juxtaposed, and secretion granules (SG). For the group supplemented with Ca1% (Figure 3C), the photomicrographs showed the numbers of microvilli (MV) and a well-defined occlusion zone (OZ). Photomicrographs of the group supplemented diet Na0.25% (Figure 3D) revealed more spaced microvilli (MV) and secretion granules (SG). For the group supplemented with Na1% (Figure 3D), the

ACCEPTED MANUSCRIPT photomicrographs clearly showed the numbers of microvilli (MV), well-juxtaposed and apical to the occlusion zone (OZ) and adhesion zone (AZ), in the base direction. b) Scanning electron microscopy (SEM) In this study, the SEM of the intestinal tract of silverfish, regardless of the treatment, presented the following, in the broad cross-section: well-formed serosa (S), longitudinal muscle (ML), well-preserved villi (V) and lumen (Figure 4A1, B1, C1, D1 and E1).

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In addition, scanning photomicrography showed a great diversity and bacterial concentration (Figure 4A2, B2, C2, D2 and E2). Most of the bacteria were

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morphologically characterized as coccus, as noted by the arrow, and were present in all

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experimental groups (Figure 4A, B, C, D and E2). However, some bacteria that were morphologically bacillus were only found in the group supplemented with Ca0.25%, as

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indicated by the asterisk (Figure B2). With SEM, we observed some bacteria that were morphologically similar to vibrionaceae in the groups supplemented with the highest concentration of propionate (1%), independent of the chelating mineral agent (Ca or

Discussion The benefits of using organic acid salts as a food supplement for aquatic animals

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Na), as noted by the triangle (Figure 2C2 and E2).

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are already well known (Lückstädts, 2006, 2008; Ng et al., 2016; Pereira et al., 2018; Romano et al., 2015; Silva et al., 2013, 2015, 2016). However, the interaction mechanism of mineral chelants in organic acid and its effects on intestinal health are

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still scarce. In the present study, we verified that the mineral chelator influences intestinal health. Calcium chelated to propionic acid at the lowest concentration (0.25%)

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presented a better composition of the cultivable microbiota (lower concentration of total heterotrophic bacteria and higher lactic acid count), compared to the group supplemented with Na1%, as well as better maintenance of the appearance cord and less observation of foci of cholestasis However, the chelating mineral is not the only aspect to act on intestinal health, but so does the concentration of the active principle, such as propionic acid. Fish fed a diet with a lower concentration of propionic acid (0.25%), independent of the chelating mineral (Ca+2 or Na+1), had better histomorphometric parameters, such as a greater width and number of villi, fewer eosinophilic infiltrates and the absence of lymphocytic infiltrate.

ACCEPTED MANUSCRIPT Digestive enzymatic activity of the gastrointestinal tract was not affected by the chelating mineral, nor its concentration to propionic acid. Changes in type, source, amount of nutrients and feed additives may alter the profile of the enzyme or the concentration of digestive enzymes (Ng et al., 2016). However, these changes were not evidenced in the present study. This is in contrast to Li et al. (2009), in which tilapia hybrids supplemented with 1% citric acid showed an increase in the activity of the digestive enzymes acid protease

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and amylase in comparison to the control group. As reported by Castillo et al. (2014) for red drum Sciaenops ocellatus supplemented with calcium lactate, citric acid and

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potassium diformate (KDF) increased the activities of pepsin, trypsin, lipase, leucine-

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aminopeptidase and phosphatases in comparison to the control group. In a study evaluating the mucosal protease and phosphatase activity of Caspian

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white fish Rutilus frisii kutum, Hoseinifar et al. (2016) observed that animals supplemented with 0.25 and/or 0.5% sodium propionate had elevations of these enzymes. However, in the present study, the Ca+2 and Na+1 minerals chelated to

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propionic acid had no influence on activity of the digestive enzymes. This could be associated with the type of organic salt, since the above-mentioned works are based on

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the use of acids and/or salts of smaller or equal-chain organic acids. It is also possible that different species respond differently to the supplementation diet.

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In the present study, the enzymes apparently most important for silver catfish were lipase and acid protease. The digestive enzymatic patterns largely reflect the dietary habits of fish (herbivore, detritus, omnivore and carnivore), as well as their

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digestive capacity (Smith, 1980). We observed that for R. quelen lipase and acid protease providing indications of their dietary habits. According to several studies,

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lipase activity is not closely related to the amount of dietary fat (Agrawal et al., 1975; Chesley, 1934; Nagase, 1964), but rather to a more efficient metabolic pathway in nutrient utilization. Acid proteases are produced and activated in the stomach at low pHs, and are of fundamental importance in the beginning of protein digestion for carnivorous fishes (Rotta, 2003). Such evidence supports the previous reports that R. quelen is a demanding omnivore, often being referred to as a carnivorous omnivore (Fracalossi and Cyrino, 2013; Rotta, 2003). In relation to the influence of the minerals chelating (sodium or calcium) to the propionic acid on the microbiota, animals supplemented with dose-independent calcium propionate (0.25 or 1%) had a lower concentration of total heterotrophic bacteria

ACCEPTED MANUSCRIPT compared to the control and the Na1% group, but propionate chelated to the minerals Ca+2 and Na+1 in a lower dose promoted greater proliferation of lactic acid bacteria. Some studies have shown the influence of the salts of organic acids on the intestinal microbiota of fish, but do not report the real effect of the mineral chelant. According to Katya et al. (2018), supplementation of a 0.4% blend of organic acids with OAA (formic acid, ammonium formate and propionic acid) and OAB (benzoic acid, fumaric acid and methionine hydroxy analogue) for olive sole,

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Paralichthys olivaceus, promoted a reduction of total heterotrophic bacteria, but did not influence vibrionaceae counts, as was observed in the present study. Thus, calcium

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chelated to propionic acid in the lowest concentration (0.25%) seems to decrease total

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intestinal microorganisms and increase beneficial bacteria (lactic acid). According to Michiels et al. (2002) and Dominguez (2004), in bacteria, Ca+2 is responsible for a wide

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variety of processes, from cycle and cell division to the maintenance of cell structure, mobility, transport and cellular differentiation processes, such as sporulation, and various protein functions, such as stability, enzymatic activity and signal transduction.

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However, the requirements for Ca+2 among microorganisms are distinct or even nonexistent, as, for example, for some fungi and some varieties of Escherichia coli (Youat,

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1993). Perhaps, the small amount of calcium (0.25%) in the R. quelen intestinal tract provoked a reduction of total heterotrophic bacteria and increased lactic acid bacteria.

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Regarding the histological alterations of the liver, the general aspects of this organ, independent of the treatment, were well maintained, indicating that the food supplements were not toxic to the animals, since the liver is a detoxifying organ.

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However, fish fed Ca0.25% demonstrated an improvement in the cordonal aspect and cholestasis; this indicates that calcium chelated to propionic acid in the lower

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concentration (0.25%) was responsible for better bile flow, in comparison to fish fed Na1%. Cholestasis is the reduction of bile flow by compromised secretion in hepatocytes. For normal secretion in the bile ducts, Ca+2 signaling to the glycoprotein inositol trisphosphate receptor (InsP3Rs) is of great importance since the scarcity or excess of Ca+2 can culminate in the incorrect function of InsP3Rs and the development of cholestasis (Martins et al., 2004). According to human studies, calcium supplementation provokes an alteration in the bile acid profiles, making them healthier (Lupton et al., 1996) and probably reducing the formation of cholestasis. In addition, supplementation of propionic acid chelated to calcium or sodium in the lowest concentration (0.25%) promoted an increased width and number of villi, as

ACCEPTED MANUSCRIPT well as a reduction of eosinophilic infiltrates and an absence of lymphocytes for silver catfish (Table 3). These findings suggest that propionate in the lowest independent concentration of the chelating mineral promotes greater energy for the epithelial cells. Through this surplus energy, enterocytes can multiply, resulting in an increased width and number of villi. Starting propionate supplementation at a higher concentration (1%) seems to promote excessive energy and culminates in reducing the width and number of villi, in relation to Ca0.25% and Na0.25%. In contrast to the present study, Adil et al. (2010)

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observed an increase in the villi height of the cut chicken jejunum with the addition of 3% organic acids (butyric, fumaric and lactic acid)

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The increase in villus height with the addition of organic acids may be related to

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the reduction of pathogenic microorganisms (a fact observed in the present research), possible infections and inflammatory processes, which can increase villi height,

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secretion, digestion and the absorption functions of nutrients by the mucosa. These alterations in the gastrointestinal structure may facilitate the digestion and absorption of nutrients in the small intestine, improving the productive performance of these animals,

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as reported by Pereira et al. (2018), Adil et al. (2010) and Samanta et al. (2010). In addition, the reduction of eosinophilic infiltrates and the absence of

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lymphocytes in the animals supplemented with Ca0.25% or Na0.25% may demonstrate an improvement in the overall intestinal health; there was a reduction of the concentration

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of total microorganisms and an increase in beneficial bacteria in these treatments (Figure 2). This may have promoted the reduction of inflammatory processes and improved intestinal dynamics, without requiring the excessive and unnecessary

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production of defense cells (eosinophils and lymphocytes). The increase of eosinophilic infiltrates in the gut indicates the migration of these cells from the bloodstream to this

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tissue, due to the stimuli of inflammatory processes, possible infections and/or infestation (Martins et al., 2004; Menezes et al., 2011; Ranzai-Paiva, 2013); this corroborates the higher concentrations of total heterotrophic bacteria and lower concentration of beneficial bacteria (lactic) found in fish fed the diet supplemented with Na1%, compared to those supplemented with Ca0.25%. Fish that received Na1% also presented a higher concentration of eosinophilic and lymphocytic infiltrates, in relation to those fed Ca0.25%. With such evidence, we can conclude that the mineral chelating to organic acid tested influences fish intestinal health. The organic salt propionate chelated to the mineral calcium or sodium at a concentration of 0.25% improved the microbial

ACCEPTED MANUSCRIPT composition and intestinal health of silver catfish, without degrading the functions and morphology of the main purification organ, the liver. 6. Acknowledgements The authors thank National Council of Scientific and Technological Development (CNPq) for financial support to J.L.P. Mouriño (301524/2017-3) and M.L. Martins (CNPq 305869/2014-0), Coordination of Improvement of Higher Education Personnel,

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Brazil (CAPES) - Finance Code 001 for financed part of this study and for PhD Scholarship to S.A. Pereira and J.J. Mattos biologist at the Federal University of Santa

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Catarina.

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Figure legends Figure 1: Enzyme activities of gastrointestinal tract of silver catfish Rhamdia quelen fed supplemented diet with calcium or sodium propionate at concentrations of 0.25 and 1% or unsupplemented (control) for 60 days. The specific activity (U mg-1 protein) are shown: Lipase (μmol of p-nitrophenol min-1 mL-1 mg-1 protein), amylase (μmol of maltose min-1 mL-1 mg-1 protein), acid protease (μmol of tyrosine min-1 mL-1 mg-1

ACCEPTED MANUSCRIPT protein), alkaline protease (ΔA366

ɳm

min-1 mL-1 mg-1 protein). Data are presented as

mean ± error deviation. *Different letters indicate a significant difference between the enzymatic activities by the Tukey test. No significant difference between treatments.

Figure 2: Concentration of bacteria per gram of intestine, in Log 10 of the colony forming units (CFU), of silver catfish Rhamdia quelen fed supplemented diet with calcium or sodium propionate at concentrations of 0.25 and 1% or unsupplemented

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(control) for 60 days. The concentrations of bacteria are shown: total heterophosphoric, vibrionacea, Pseudomonas sp. And total lactic acid. Data are presented as mean ± error

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deviation. *Different letters indicate significant difference by Tukey test (p<0.05).

Figure 3: Photomicrographs of the transmission electron microscopy (TEM) of the

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intestinal epithelium of silver catfish Rhamdia quelen fed supplemented diet with calcium or sodium propionate at concentrations of 0.25 and 1% or unsupplemented (control) for 60 days. A-Unsupplemented; B- Fish fed supplemented diet with 0.25%

propionate (Ca

D- Fish fed supplemented diet with 0.25% sodium propionate (Na

E- Fish fed supplemented diet with 1% sodium propionate (Na

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calcium propionate (Ca 0.25%); C- Fish fed supplemented diet with 1% calcium

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microvilli; *: cell nucleus; SG: secretion granules; OZ: occlusion zone; AZ: adhesion. Figure 4: Photomicrographs of the transmission electron microscopy (TEM) of the intestinal epithelium of silver catfish Rhamdia quelen fed supplemented diet with

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calcium or sodium propionate at concentrations of 0.25 and 1% or unsupplemented (control) for 60 days. A1-Unsupplemented, broad cross-sectional view; A2-

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Unsupplemented, detailed and approximate view; B1- Fish fed supplemented diet with 0.25% calcium propionate (Ca 0.25%), broad cross-sectional view; B2- Fish fed supplemented diet with 0.25% calcium propionate (Ca 0.25%), detailed and approximate view; C1- Fish fed supplemented diet with 1% calcium propionate (Ca 1%), broad cross-sectional view; C2- Fish fed supplemented diet with 1% calcium propionate (Ca

1%),

detailed and approximate view; D1- Fish fed supplemented diet with 0.25%

sodium propionate (Na

0.25%),

broad cross-sectional view; D2- Fish fed supplemented

diet with 0.25% sodium propionate (Na 0.25%), detailed and approximate view; E1- Fish fed supplemented diet with 1% sodium propionate (Na

1%),

broad cross-sectional view;

E2- Fish fed supplemented diet with 1% sodium propionate (Na

1%),

detailed and

ACCEPTED MANUSCRIPT approximate view. S: serosa; ML: longitudinal muscle; V: villi; L: lumen; →: the arrows show bacteria with the morphology of coccus; *: the asterisks show bacteria with bacillus morphology; ∆: the triangles show bacteria with morphology similar to

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vibrionaceae.

ACCEPTED MANUSCRIPT Table 1: Formulation and composition analysis (g kg-1 of dry matter) of the experimental diets. Ingredients (%)

Control

Ca+20.25% Ca+21% Na

+1 0.25%

0%

Na +1 1%

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Corn bran 20.5 20.5 20.5 20.5 20.5 Soybean meal (48% CP) 45.45 45.45 45.45 45.45 45.45 Salmon residue flour (71% CP) 29 29 29 29 29 Fish oil 1 1 1 1 1 Oil of the soya 1 1 1 1 1 Hydroxy-Butylated Toluene (HBT) 0.05 0.05 0.05 0.05 0.05 Premix vitamin and mineral 1 1 1 1 1 1 2 Dicalcium phosphate 1 1 1 1 1 Cellulose 1 0.75 0 0.75 0 Ca-propionate3 0 0.25 1 0 0 4 Na-propionate 0 0 0 0.25 1 -1 Crude Energy (CB) cal·kg 3615.2 3557.2 3568.7 3599.7 3537.1 Crude protein (PB) 461.9 470.8 471.2 471.7 465.8 Ethereal extract 82.8 81.6 81.9 80.1 81.9 Total Carbohydrate 255.6 234.9 236.7 248.0 234.2 Ashes 93.6 96.3 93.2 98.0 91.9 Moisture 893.9 883.6 883.0 897.8 873.8 1 Levels of guarantee per kilo of the product: vit. A - 1.250.000 UI; vit. D3 - 350.000 UI; vit. E 25.000 UI; vit. K3 - 500 mg; vit. B1 – 5.000 g; vit B2 4.000 g; vit. B6 – 5.000 g; B12 – 10 mg; nicotinic acid – 15.000 mg pantothenic acid – 10.000 mg; biotin - 150 mg; folic acid - 1,25 mg; vit. C – 25.000 mg; Hill – 50.000 mg; Inositol 30.000 mg; Iron – 2.000 mg; Copper – 3.500 mg; Copper-chelated – 1.500 mg; Zinc – 10.500 mg; Zinc- chelated – 4.500 mg; Manganese – 4.000 mg; Selenium - 15 mg; Selenium-chelated – 15 mg; Iodine – 150 mg; Chrome – 80 mg e Vehicle (q.s.p).2 Dicalcium phosphate PA. 3 Purity 98-100,5%, solubility 1 g in 3 ml of water and moisture maximum of 5%. 4 Purity 99-100%, solubility 1 g in 1 ml of water and moistures maximum of 0.6%.

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Table 2: Histopathological analysis in the liver of silver catfish Rhamdia quelen fed supplemented diet with calcium or sodium propionate at concentrations of 0.25 and 1% or unsupplemented (control) for 60 days. Data are presented as mean and standard deviation.* MCA: maintenance of the cordonal aspect; C: cholestasis; CLV: congestion in the large vessels; CS: congestion in the sinusoids; SD: sinusoidal dilatation; N: necrosis; EI: eosinophilic infiltrate; LI: lymphocytic infiltrate; MC: melanomacrophage centers; LG: loss of the glycogen; UCN: uniformity in the size of cells and nuclei; BA: ballooning aspect; HNH: hepatocyte and nucleus hypertrophy; DN: displacement of the nucleus; P: pyknosis; Ka: karyolysis and Kx: karyorrhexis. Histologica Na+1 Na+1 Cont Ca+2 Ca+21 l 0.25% 1% rol 0% 0.25% % alterations MC 0.62 1.27 1.00 0.93 1.00± b a ab ab ab A ±0.50 ±0.47 ±0.00 ±0.62 0.00 C 0.16 0.09 0.33 0.29 0.88± ±0.37a 0.30a ±0.49ab ±0.47ab 099b CL 1.74 1.55 1.50 1.21 1.75± V ±1.05 ±0.93 ±0.90 ±0.80 1.04 CS 1.43 1.36 1.83 1.71 1.25± ±0.90 ±0.81 ±1.03 ±0.99 0.71 N 1.04 1.55 1.33 1.50 1.00± ±0.97 ±0.93 ±0.78 ±1.02 0.00 EI 0.35 0.55 0.58 0.50 0.25± ±0.48 ±0.52 ±0.51 ±0.52 0.46 LI 1.29 1.64 1.58 1.86 1.50± ±0.77 ±1.12 ±1.08 ±1.03 0.93 MC 0.27 0.36 0.08 0.21±0.43 0.13± ±0.44 ±0.50 ±0.29 0.35 LG 0.24 0.55 1.17 1.36±1.34 0.88± ±0.43 ±1.04 ±0.94 0.99 UC 0.10 0.45 0.25 0.29±0.47 0.13± N ±0.29 ±0.69 ±0.45 0.35 BA 0.03 0.09 0.08 0.07±0.27 0.25± ±0.12 ±0.30 ±0.29 0.46 MC 0.27 0.36 0.08 0.21±0.43 0.13± ±0.44 ±0.50 ±0.29 0.35 HN 1.04 1.73 1.67 1.50±1.02 2.00± H ±0.66 ±1.01 ±0.98 1.07 DN 0.92 1.00 1.00 0.71±0.47a 1.00± ±0.29ab ±0.00b ±0.00b 0.00b P 1.91 1.91 1.67 1.86±1.03 1.75± ±1.05 ±1.04 ±0.98 1.04 Ka 1.09 1.55 1.00 1.29±0.73 1.75± ±0.62 ±0.93 ±0.00 1.04 Kx 1.27 1.91 1.50 1.43±0.85 1.50± ±0.78 ±1.04 ±0.90 0.93 * Different letters indicate significant difference by Tukey test (p<0.05).

p value

0.01 4 0.02 6 0.57 3 0.59 3 0.49 1 0.60 5 0.70 6 0.55 7 0.10 0 0.35 4 0.42 6 0.55 7 0.47 4 0.03 7 0.88 8 0.14 3 0.55 7

ACCEPTED MANUSCRIPT Table 3: Histomorphometric alterations in the middle portion of gut in silver catfish Rhamdia quelen fed supplemented diet with calcium or sodium propionate at concentrations of 0.25 and 1% or unsupplemented (control) for 60 days. Data are presented as means+standard deviation.* Histomorphomet ric alterations

Ca+2

Cont rol 0%

0.25%

Ca+21%

Na+ 1

0.25%

Na+1 1%

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Perimet 1.95 1.54 1.42±0.26 1.89 1.86 er (104·μm) ±0.19 ±0.74 ±0.40 ±0.88 Area 0.96 1.26 0.79±0.42 0.84 1.26 ((106·μm) ±0.26 ±0.65 ±0.87 ±0.33 Length 6.19 4.67 5.47±2.18a 5.71 6.26 2 a b b (10 ·μm) ±2.49 ±2.35 ±2.84ab ±2.84a Width 1.71 2.11 1.63±0.66c 2.43 1.98 2 bc a (10 ·μm) ±0.57 ±0.97 ±1.30a ±0.96b Number 5.01 8.14 7.25 7.85 goblet cells 7.16±0.68 ±2.35 ±4.56 ±5.06 ±3.63 (102) Number 25.7 31.3 23.38±2.02 27.2 23.0 c of villi 0±1.73b 6±6.96a 4±3.77a 9±2.37c Eosinop 51.4 8.82 53.41±30.6 9.19±12.1 41.9 hilic infiltrate 2±25.20 b ±12.04 a 6b 4a 5±28.40 b (%) Lympho NF NF 4.55±9.72* NF 3.45 cyte infiltrate ±8.67* (%) NF- Not Found. * Different letters indicate significant difference by Tukey (p<0.05).

p valu e 0.56 2 0.40 6 0.00 4 0.00 1 0.80 3 0.00 1 0.01 9 0.00 1 test

ACCEPTED MANUSCRIPT Highlights 

First study on the use of organic acid-chelated mineral on the enzymatic production, intestinal microbial composition and histological alterations in the liver and intestine.



The lowest concentrations of propionic acid chelated to calcium and sodium increased the concentration of beneficial bacteria in the intestinal tract, villi

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width, number of villi and lower concentrations of eosinophilic and lymphocytic infiltrates.

Calcium chelated propionic acid at concentrations of 0.25% and 1% reduced the

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number of total heterotrophic bacteria

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Propionic acid chelated to calcium 0.25% showed greater maintenance of the

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cordonal aspect of the liver.

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Figure 1

Figure 2

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Figure 4