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Comparing the effects of different dietary organic acids on the growth, intestinal
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short chain fatty acids, and liver histopathology of red hybrid tilapia
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(Oreochromis sp.) and potential use of these as preservatives
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Mahdi Ebrahimi 1, Nor Hafizah Daeman 2, Chou Min Chong 2,, Ali Karami 3, Vikas
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Kumar 4, Seyed Hossein Hoseinifar 5, Nicholas Romano 2*
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Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia
Department of Veterinary Preclinical Sciences, Faculty of Veterinary Medicine,
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43400 Serdang, Selangor, Malaysia
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3
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Universiti Putra Malaysia, 43400 Selangor, Malaysia
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Systems, Kentucky State University, Frankfort, KY, USA
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5
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University of Agricultural Sciences and Natural Resources, Gorgan, Iran
Department of Aquaculture, Faculty of Agriculture, Universiti Putra Malaysia,
Laboratory of Aquatic Toxicology, Faculty of Medicine and Health Sciences,
Division of Aquaculture, College of Agriculture, Food Science and Sustainable
Department of Fisheries, Faculty of Fisheries and Environmental Sciences, Gorgan
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* Corresponding author: Nicholas Romano
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Department of Aquaculture, Faculty of Agriculture, Universiti Putra Malaysia, 43400
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Serdang, Selangor, Malaysia
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Email address: [email protected]
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Abstract
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Dietary organic acids are increasingly being investigated as a potential means
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of improving growth and nutrient utilization in aquatic animals. A 9-week study was
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performed to compare equal amounts (2%) of different organic acids (sodium
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butyrate, acetate, propionate or formate) on the growth, muscle proximate
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composition, fatty acid composition, cholesterol and lipid peroxidation, differential
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cell counts, plasma biochemistry, intestinal short chain fatty acids (SCFA) level and
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liver histopathology to red hybrid tilapia (Oreochromis sp.) (Initial mean weight of
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2.87 g). A second experiment was performed to determine their effects on lipid
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peroxidation and trimethylamine (TMA) when added at 1% to tilapia meat and left
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out for 24 hours. The results of the first experiment showed no treatment effect to
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growth, feeding efficiencies, or muscle fatty acid composition but all dietary organic
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acids significantly decreased intestinal SCFA. Dietary butyrate and propionate
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significantly decreased muscle lipid peroxidation compared to the control group, but
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the dietary formate treatment had the lowest lipid peroxidation compared to all
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treatments. Muscle crude protein and lipid in tilapia fed the formate diet was
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significantly lower and higher, respectively, and showed evidence of stress based on
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the differential cell counts, significantly higher plasma glucose and liver glycogen, as
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well as inflammatory responses in the liver. Although a potential benefit of dietary
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organic acids was a reduction to lipid peroxidation, this could be accomplished post-
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harvest by direct additions to the meat. In addition, inclusions of butyrate and
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propionate to tilapia meat significantly decreased TMA, which might be a more cost-
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effective option to improve the shelf-life of tilapia products.
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Keywords: organic acids; liver histology; glycogen; short chain fatty acids;
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inflammation; lipid peroxidation; trimethylamine
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53
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Abbreviations
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56
FCR feed conversion ratio
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HSI hepatosomatic index
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MDA malondialdehyde
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SCFA
60
SGR specific growth rate
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TMA
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VSI viscerosomatic index
short chain fatty acids
trimethylamine
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64
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66
67
68
69
70
71
72
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3
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Introduction
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Organic acids are compounds with slightly acidic properties with less than
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seven carbon lengths that terminate with one or more carboxyl groups (Lückstädt
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2008). These are “generally regarded as safe” for animal and human consumption and
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have been used for many years as anti-microbials and preservatives in feeds. The
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most common include citric, propionic, formic, butyric and acetic acid or their salts
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(Ng and Koh 2016; Hoseinifar et al. 2016a). These are increasingly being investigated
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as dietary additives to various fish and crustacean species as a potential means of
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improving their growth, nutrient utilization and/or resistance to pathogenic bacteria
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(Ng and Koh 2016; Hoseinifar et al. 2016a). It is believed this is partly due to their
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slight acidifying properties that can inhibit pathogenic bacteria and/or enhance
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digestive enzyme activities (Castillo et al. 2014; Silva et al. 2016). In addition, some
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organic acids can act as readily available energy sources and/or feed attractants in
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tilapia or shrimp (Xie et al. 2003; Silva et al. 2013) as well as a protective agent
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against the damaging effects of oxidized oils in diets (Liu et al. 2014). There may also
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be potential implications to post-harvest quality, such as reducing lipid peroxidation
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in fish when included in the diets (Liu et al. 2015; Romano et al. 2016) and/or
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inhibiting bacterial spoilage bacteria in terrestrial meat (Ouattara et al. 1997). The
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implications of these to the production of trimethylamine (TMA), which is often used
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an indicator of bacterial spoilage such as from Shewanella putrefaciens and
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Photobacterium phosphoreum in fish (Dalgaard 1995), has received limited attention.
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Determining the effects of dietary organic acids to farmed fish or shellfish can
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be particularly important since these can be advertised as effective additives in their
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commercial diets. This practice, however, is often done without prior testing and
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solely based on positive findings from terrestrial livestock (Ng and Koh 2016). Such
4
100
advertising can be misleading considering the efficacy of organic acids to aquatic
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animals is largely dependent on the type as well as species (Ng and Koh 2016;
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Hoseinifar et al. 2016a). For example, dietary sodium propionate reduced the growth
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in Artic char Salvelinus alpinus (Ringø 1991), but the same organic acid improved the
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growth performance in both zebrafish (Danio rerio) (Hoseinifar et al. 2016b), Caspian
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white fish (Rutilus kutum) (Hoseinifar et al. 2017) and common carp (Safari et al.
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2017). Furthermore, formic acid and their salt reportedly improved mineral
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digestibility in rainbow trout (Oncorhynchus mykiss) (Vielma and Lall 1997; Morken
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et al. 2011), but an organic acid blend consisting of sodium formate and butyrate (at a
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2:1 ratio) significantly decreased nutrient digestibility in the same species (Gao et al.
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2011). In a more extreme case, dietary sodium citrate (at 1 – 4%) was toxic to tilapia
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based on lower growth and health status as well as liver damage that included
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necrosis and hemorrhaging (Romano et al. 2016). In contrast, similar dietary levels of
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citric acid improved growth and/or nutrient utilization in various fish species (Hossain
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et al. 2007; Khajepour and Hosseini 2012; Sarker et al. 2012).
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Considering the increasing interest in dietary acidifiers, and popularity of
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tilapia as a food fish that is only behind carp in overall aquaculture production (FAO
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2015), evaluating different dietary organic acids to the growth and health status of this
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commercially important species would likely benefit the industry. Therefore, two
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experiments were performed in this study which included, 1) a comparison of
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different dietary organic acid effects on the growth performance, feeding efficiencies,
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plasma and muscle biochemistry, intestinal short chain fatty acids as well as liver
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histopathology and glycogen content in red hybrid tilapia (Oreochromis sp.) and 2)
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comparing the efficacy of different organic acids on lipid peroxidation and TMA from
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tilapia meat left for 24 h at room temperature.
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Materials and Methods
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Experiment 1
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Experimental diets
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A total of five isonitrogenous diets were formulated to contain an equal
129
amount (2%) of different organic acids that included sodium butyrate, acetate,
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propionate or formate, while cellulose was added in the control diet at 2%. The
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organic acid level was chosen based on positive findings with various aquatic species
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(Khajepour and Hosseini 2012; Silva et al. 2013; Safari et al. 2016; Sukor et al. 2016).
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Danish fishmeal and soybean meal were the main protein sources, which were
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finely ground and sieved, and soybean oil was the main lipid source. All dry
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ingredients were thoroughly mixed for 30 min, followed by adding the lipid and then
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distilled water, and mixed again for an additional 30 min. The diets were then
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extruded according to Romano et al. (2016) and stored in air-tight plastic bags until
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use at -20°C. The proximate composition of the experimental diets was measured
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according to AOAC (1997) standard methods and was shown to be similar among
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treatments. The pH of the diets was measured according to Romano et al. (2015).
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Source of experimental animals and experimental design
Red hybrid tilapia (Oreochromis sp.) fingerlings (2 – 4 cm) were obtained
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from the Puchong Aquaculture Experimental Station, Universiti Putra Malaysia
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(UPM), and brought to the Wet Laboratory, Department of Aquaculture, Faculty of
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Agriculture, UPM. They were fed a commercial diet designed for tilapia (Ding Ding,
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Malaysia). After an initial two-day acclimation period in a 1,000 L fiberglass tank, a
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total of 20 apparently healthy fish were placed in glass aquaria filled with 70 L of de-
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chlorinated freshwater and continued to be fed the commercial tilapia diet. The
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following day, the aquaria were randomly assigned one of the five treatments, which
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yielded triplicates in each treatment, and the fish fed their respective diets to satiation
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twice each day for 9 weeks.
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In each aquaria, gentle aeration and individual pre-conditioned biofilters were
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provided, however, each day and week approximately 20% and 90% of the water was
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exchanged, respectively. The ammonia and nitrite levels were tested once per week
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prior to the water exchanges from each aquarium using a commercial test kit
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(Aquarium Pharmaceuticals®). The mean (±SD) ammonia-N and nitrite-N was 0.30 ±
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0.01 and 0.40 ± 0.02 mg l-1, respectively, and both never exceeded 1.0 mg l-1. The
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mean (±SD) dissolved oxygen, pH and temperature were also measured once per
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week using a digital probe (YSI 556, MPS) and these ranged from 5.4 ± 0.5 ppm, 7.8
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± 0.3, and 27 ± 0.2°C, respectively. The water source was tap water and sodium
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thiosulphate was used to neutralize any residual chlorine.
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After 9 weeks, the fish were euthanized with and overdose of clove essential
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oil and the final length (0.1 cm) and weights (0.01 g) were measured for later
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calculations of specific growth rates (SGR) and weight gain (WG) using the following
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equations.
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WG = [(final body weight – initial body weight) / initial body weight] ×100
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SGR for weight = specific growth rates (% day-1) = [(lnW1 – lnW0) / T] × 100; where
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W1 = final weight, W0 = initial weight and T = time in days
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SGR for length = specific growth rates (% day-1) = [(lnL1 – lnL0 )/ T] × 100; where L1
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= final weight, L0 = initial weight and T = time in days
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The feed conversion ratio (FCR) was also determined using the following
equation,
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FCR = total dry weight of diet fed (g) / wet weight gain (g)
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The blood from each fish was obtained to determine the differential cell
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counts and plasma biochemistry and then dissected for further analysis (see sections
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below).
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Plasma biochemistry and differential cells counts
The differential cell counts were performed according to Noga (2010). Briefly,
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one drop of blood was taken from three fish in each replicate and smeared onto glass
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slides. These slides were air-dried and fixed in absolute methanol for 1 min and then 3
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min in a May-Grünwald solution (BD Chemicals Ltd, England). After incubating the
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slides for 1 min in a PBS solution, these were then stained for 10 min in a 5% Giesma
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solution in order to accurately determine the cell type. At least 10 images were taken
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from the slides under a microscrope and the differential cell counts were performed
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with the aid of ImageJ software (CellC ImageJ; version 1.46d) (National Institute of
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Health, Bethesda, MD). The percentage of leukoyctes was determined using the
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following formula,
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[Absolute # of specific leukocyte in the blood / Total leukocyte count] × 100
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To measure the plasma biochemistry, the blood was obtained from syringes
previously coated with a saturated EDTA solution. The blood was pooled from all
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remaining fish in the same tank, to ensure the minimum required volume for
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measurements. This yielded triplicate samples in each treatment. After centrifuging
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the blood at 603 g for 10 min., the plasma was removed, placed in a new vial and kept
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at -20°C. The plasma phosphate, triglycerides and alanine aminotransferase (ALT)
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activity was determined using a Hitachi 902 automatic analyzer (Boehringer
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Manheim Diagnostics, Indianapolis, IN). For phosphate, these ions reacted with
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ammonium molybdate under acidic conditions to produce a phosphomolybdate
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complex that was measured spectrophotometry at 340 nm. The ALT was measured
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using an α-ketoglutarate reaction using a L-glutamyl-3-carboxy-4-nitroanilide rate
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method. Triglycerides were measured enzymatically using a series of coupled
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reactions in which triglycerides were hydrolyzed to produce glycerol. Glycerol was
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then oxidized using glycerol oxidase, and the reaction product H2O2 one of the
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reaction products, was measured quantitatively in a peroxidase catalyzed reaction that
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produces a color, and the absorbance was measured at 500 nm.
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Body indices, muscle TBARS, proximate composition and fatty acid composition
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After blood collection, the tilapia were then dissected to remove the viscera
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and then liver to calculate the viscerosomatic index (VSI) and hepatosomatic index
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(HSI), respectively. The VSI and HSI were calculated by dividing the viscera and
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liver by the final body weight, respectively. The liver and intestine from six fish in
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each treatment was then used for histopathological examination (see section below),
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while the remaining intestine was pooled in each replicate for short chain fatty acid
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analysis (see section below). From the remaining carcass, the heads, skin and fins
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were removed and the muscle was filleted, finely minced and pooled in each replicate.
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The muscle samples were then stored at -20°C for later determination of the
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proximate and fatty acid composition as well as for TBARS analysis (see below).
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The TBARS assay was analyzed in duplicate in each replicate from one gram
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of muscle and the production of malondialdehyde (MDA) was measured according to
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Lynch and Frei (1993). To produce a standard curve, 1,1,3,3,-tetraethoxypropane
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(TEP) from a 100 μM stock solution was made and the TBARS values are expressed
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as MDA equivalents (μM g-1). The fatty acid composition was also analyzed in
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duplicate from each replicate from one gram of muscle according to Ebrahimi et al.
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(2014). Meanwhile, from the remaining muscle sample, the proximate composition
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was determined according to AOAC (1997) standard methods.
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Intestinal short chain fatty acids
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After storing the intestinal samples with digesta at -20°C, these samples were
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finely minced using scissors and 1 ml of 20% metaphosphoric acid was added to 2 g
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of the intestine. This was then thoroughly homogenized using a homogenizer and the
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supernatant was then stored at -20°C and short chain fatty acids (SCFA) were
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measured according to Romano et al. (2016). Briefly, SCFA was measured on a gas-
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liquid chromatograph Quadrex 007 Series (Quadrex Corporation, New Haven, CT
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06525 USA) with a bonded phase fused silica capillary column (15m, 0.32mm ID,
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0.25 µm film thickness) in an Agilent 7890A gas-liquid chromatography (Agilent
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Technologies, Palo Alto, CA, USA) equipped with a flame ionization detector (FID).
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The injector/detector temperature was programmed at 220/230°C. The column
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temperature was set in a range of 70°C - 150°C with temperature programming at a
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rate of 7°C min-1 increments to facilitate optimal separation. There was an initial
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holding time of 1.5 min. once 70°C was reached and a final holding time at 3.5 min.
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at the end of the program. Authentic standards of acetic, propionic, butyric,
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isobutyric, valeric, isovaleric and 4-methyl-n-valeric acids (Sigma, St. Louis, Mo.,
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USA) were used for comparison to identify the peaks.
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Liver histopathology and Periodic-acid Schiff (PAS)
A total of four fish in each treatment were used for the histopathological
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examination of the liver. After dissection, the livers were immediately fixed in 10%
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(v/v) phosphate buffered formalin for 24 hours, followed by 70% ethanol (v/v) until
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processing. After processing, the tissues were embedded in paraffin wax, sectioned (5
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μm), stained with haemotoxylin and eosin or Periodic-acid Schiff (PAS) according to
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Karami et al. (2016). PAS staining intensity was quantified using ImageJ software
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(National Institute of Mental Health, Bethesda, Maryland, USA), and the staining area
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values were produced after using the same lower and upper threshold of 0 and 145,
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respectively.
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Experiment 2
Three live tilapia (25 – 32 g) were purchased from a local grocery store and
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brought to the lab. The fish were the euthanized with an overdose of MS-222 and
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then de-skinned and filleted to obtain the muscle. The muscle from three fish were
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then thoroughly homogenized in a food processor, and then five equal portions were
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separated, weighed and an equal level of different organic acids at 1% (w/w) that
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included sodium acetate, butyrate, propionate or formate was added. This level was
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chosen based on a previous pilot study showing that 2% led to excessive water loss
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and an unappealing color and texture. A control was used that received no organic
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acid additions. The organic acids were mixed with the fish meat by hand, using
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gloves, and between mixing gloves were changed to prevent contamination. From
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these, six separate samples (1 g) from each treatment were placed in aluminum foil
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trays, covered with a plastic tray and left at room temperature (28°C) for 24 hours.
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After 24 h, the samples showed obvious signs of deterioration that included an
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obvious “fishy” smell and a color change from white to a more brownish color. The
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samples were then wrapped in aluminum foil and stored at -20°C for measurements of
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malondialdehyde (MDA) and trimethylamine (TMA) in triplicate as indicators of fish
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spoilage within 2 days. Measurements of MDA were performed as previously stated
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while TMA was measured according to Hamid et al. (2013).
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Statistical analysis
Data was also subjected to a one-way ANOVA and Chi-square analysis for
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survival after testing for homogeneity of variance. If significant differences (P < 0.05)
287
were detected, a Dunnett post-hoc test was performed to identify differences among
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treatments. Meanwhile, to only compare the organic acid groups, data from the
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control was omitted and a one-way ANOVA was used. For all statistical analysis the
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SPSS statistical package version 22.0 was used.
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Results
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Experiment 1
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Survival, growth, feeding efficiencies and body indices
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The survival of tilapia was high and ranged from 95.0 – 98.3%, with no
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significant difference detected among treatments (P > 0.05). Similarly, no difference
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to growth performance, feeding efficiency or body indices were detected (P > 0.05)
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among the different treatments (Table 2).
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Muscle proximate composition, cholesterol, lipid peroxidation and fatty acid
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composition
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The proximate composition, cholesterol and malondialdehyde (MDA)
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equivalents of the tilapia muscle are shown in Table 3. There was no significant
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treatment effect on the muscle moisture or crude ash. However, the crude protein and
305
lipid were significantly lower and higher, respectively, for tilapia fed dietary formate
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compared to those fed the control or butyrate diets. Meanwhile, the muscle
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cholesterol was the highest in the control treatment, which was significantly higher
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compared to fish in propionate and formate treatments. Finally, lipid peroxidation was
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significantly lower in the organic acid fed fish, with the formate treatment being
310
significantly lower than all others.
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The dominant fatty acids in the muscle of tilapia were C18:1n-9 at 32.2%
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followed by C18:2n-6 at 27.4%. Meanwhile, among the major long chain
313
polyunsaturated fatty acids, C20:4n-6, C20:5n-3 and C22:6n-3 these were 2.1, 0.7 and
314
2.3%, respectively. The results showed that none of the individual fatty acids or
315
groups were significantly affected by dietary organic acids (data not shown).
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Plasma biochemical composition and differential cell counts
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The biochemical composition of the plasma from tilapia fed diets with
319
different organic acids additions are shown in Table 4. Plasma phosphate was
320
significantly higher in the propionate treatment compared to the butyrate treatment.
321
Plasma potassium was significantly higher for tilapia fed the propionate diet
322
compared to the acetate, butyrate and control treatments while plasma sodium was
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significantly lower for tilapia fed the propionate diet compared to the acetate and
324
butyrate treatments. Plasma glucose was significantly higher for tilapia in the dietary
13
325
formate treatment compared to those in the control, butyrate or acetate treatments. No
326
significant treatment effect was detected to plasma calcium, chloride or triglycerides.
327
Among the organic acid treated diets, formate significantly affected some of
328
the differential cells counts of tilapia (Table 5). The percentage of red blood cells and
329
granulocytes were significantly higher for tilapia fed the formate treated diets,
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compared to the control, while the percentage of lymphocytes were significantly
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lower. Meanwhile, the granulocyte to lymphocyte (G:L) ratio was significantly higher
332
for tilapia in the dietary formate treatment compared to those in the control.
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Intestinal short chain fatty acids
The measured short chain fatty acids (SCFAs) within the intestine (containing
336
digesta) of tilapia consisted of acetic acid, butyric acid and propionic acid, which are
337
shown in Fig. 1. The results showed that the dietary supplementation of organic acids
338
significantly decreased all the intestinal SCFAs compared to the control. For fish fed
339
the formate diet, acetic acid was the lowest and was significantly lower than the
340
butyrate treatment. On the other hand, propionic acid was significantly higher for fish
341
in the formate treatment compared to the other dietary organic acid treatments.
342
343
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Liver histopathology and Periodic-acid Schiff (PAS)
The liver of fish from the control, butyrate, acetate and propionate treatments
345
appeared to have normal sinusoid structure with uncongested veins and intact
346
hepatocytes (Fig. 2a). However, for fish in the formate treatments, there were white
347
blood cell infiltrations and what appeared to be lipofuscin that were distributed among
348
the hepatocytes and pancreas (Fig. 2b). In addition, the liver was stained significantly
349
more for PAS-positive material in the formate treatment compared to all other
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350
treatments (Fig. 3a,b; 4).
351
352
Experiment 2
353
Malondialdehyde (MDA) and trimethylamine (TMA) in fish meat
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The amounts of MDA and TMA in tilapia meat after being left at room
355
temperature for 24 hours are shown in Figs. 5 and 6, respectively. All organic acids
356
significantly reduced MDA compared to the control, and no significant different was
357
detected among the organic acid treatments. Meanwhile, TMA was significantly
358
reduced in tilapia meat with sodium butyrate or propionate additions compared to the
359
control, while no significant difference in the TMA was detected between the sodium
360
formate treatment with the other treatment groups.
361
362
Discussion
363
The use of butyric, acetic, and propionic acid salts have not yet been
364
investigated as dietary additives to tilapia, and were shown to have no effect on their
365
growth or feeding efficiencies. These did, however, decrease muscle lipid
366
peroxidation and significantly reduced intestinal short chain fatty acids (SCFA) when
367
included in the diets of tilapia. Similarly, dietary sodium formate also led to
368
significantly lower intestinal SCFA and the lowest lipid peroxidation among the
369
treatments, however this also appeared to induced stress and compromised health
370
based on the differential cell count, altered plasma and muscle biochemistry as well as
371
liver histopathology.
372
Much of the research on dietary organic acids to tilapia have focused on
373
sodium formate or potassium diformate (KDF) at levels ranging from 0.2 – 1.2%,
374
which were generally found to impart certain benefits including better growth and/or
15
375
nutrient utilization (Ng et al. 2009; Zhou et al. 2009; Liebert et al. 2010; Elala and
376
Ragaa 2015). For instance, Liebert et al. (2010) found that dietary sodium formate at
377
0.3% significantly improved feeding efficiency in tilapia but the same amount of
378
dietary KDF was not as effective. On the other hand, a dietary inclusion of KDF at
379
0.3% was reported to significantly improve the growth, feeding efficiency and protein
380
digestibility in tilapia (Elala and Ragaa 2015). Higher levels of dietary KDF from 0.3
381
to 1.2% were found to slightly improve tilapia growth and feeding efficiencies,
382
although this was not significant (Zhou et al. 2009). In the current study, the dietary
383
inclusion of all the tested organic acids at 2% was chosen based on positive findings
384
when using similar levels to various aquatic species (e.g. Khajepour and Hosseini
385
2012; Silva et al. 2013; Safari et al. 2016; 2017; Sukor et al. 2016). Nevertheless, in
386
the case of sodium formate, this was likely excessive leading to several adverse
387
changes in tilapia.
388
Although dietary sodium butryrate, acetate or propionate had no effect on the
389
muscle proximate composition, dietary sodium formate significantly decreased and
390
increased muscle crude protein and crude lipid, respectively, which suggests impaired
391
nutrient utilization. This may have been caused, in part, to stress based on the various
392
measured parameters in the current study. The liver histopathology from only the
393
sodium formate treatment revealed hydropic vacuolation, an inflammatory response,
394
and lipofuscin-like material, which were predominately distributed around the
395
pancreatic tubules. White blood cell infiltrations are often associated with the
396
presence of lipofuscin, which is a yellow-brown pigment produced from the oxidation
397
of polyunsaturated fatty acids that may accumulate within the liver of fish due to
398
toxins or nutritional deficiencies (Agius and Roberts 2003). This finding is indicative
399
of impaired liver function and appears to be further supported by the elevated plasma
16
400
ALT, which sometimes used as an overall health status indicator of the liver during
401
nutritional studies in fish (Zhang et al. 2008; Kumar et al. 2010; Liu et al. 2015). This
402
is because when the liver is damaged leading to hepatocyte lysis, more enzymes are
403
released into the plasma. Moreover, the significantly higher and patchier distribution
404
of liver glycogen as well as the elevated plasma glucose, red blood cells (and
405
therefore lower white blood cells), granulocytes and graulocyte:lymphocyte (G:L)
406
ratio are all consistent with stress in fish (Blaxhall and Daisley 1973; Davis et al.
407
2008; Van Rijn and Reina 2010; Karami et al. 2016).
408
It could be speculated that a reduction to the white blood cell ratio in tilapia
409
fed sodium formate could make fish more susceptible to bacterial challenge. It has
410
been demonstrated, however, that dietary KDF at 0.2% or 0.3% significantly
411
improved the resistance of tilapia to the bacterial pathogens Streptococcus agalactiae
412
(Ng et al. 2009) and Aeromonas hydrophila (Elala and Ragaa 2015). Ng et al. (2009)
413
attributed these findings to decreased pathogenic bacteria within the intestine while
414
Elala and Ragaa (2015) found increased innate immunological responses as well as
415
proliferation of lactic acid bacteria. An altered bacterial composition was
416
demonstrated in tilapia fed dietary KDF from 0.3 to 1.2% (Zhou et al. 2009). It is
417
unclear whether the higher dietary levels used in the current study, compared to the
418
studies by Ng et al. (2009) and Elala and Ragaa (2015), would offer similar
419
protection. There appears to be an indication that the intestinal bacterial composition
420
or amount in the tilapia were altered in all the dietary organic acid treatments since
421
there was a significant decrease to intestinal short chain fatty acids (SCFA) which are
422
only produced by bacterial fermentation (Clements 1991). This finding in the current
423
study is in agreement with significantly lower SCFA within the intestine of tilapia
424
after being fed diets with sodium citrate (Romano et al. 2016). It cannot be totally
17
425
ruled out, however, that the higher intestinal SCFA within the control group in this
426
study as well as those of Romano et al. (2016) could be related to the higher cellulose
427
content of 2% and 1 – 4%, respectively. This is because, despite cellulose being added
428
as an inert filler in experimental diets (Nates 2015), tilapia have been shown to
429
ferment this ingredient based on elevated intestinal SCFA, albeit at slightly lower
430
amounts than α-starch (Kihara and Sakata 1997). Formulating the diets differently,
431
such as lower organic acid inclusions to reduce dietary cellulose inclusions and/or
432
increasing the amount of cellulose in all diets, might better elucidate the cause for this
433
response. A somewhat unexpected finding was the significant reduction to intestinal
434
acetic, propionic and butyric acids when these were supplied in salt form and more
435
research is required to determine the cause. It is important to note that the digesta was
436
still present within the intestine at the time of sampling and if the gut microbes were
437
reduced, as observed in tilapia fed a combination of various organic acid types (Ng et
438
al. 2015; Koh et al. 2016), the dietary SCFA might have been absorbed by the time
439
sampling was performed. A time series experiment to investigate any correlation
440
between SCFA and total colony forming units in the intestine could be worthwhile.
441
Despite the use of organic acids as preservatives in livestock feeds for many
442
decades (Lückstädt 2008), there is little information regarding their applications to
443
fish post-harvest. Recently, however, it was demonstrated that dietary sodium citrate
444
significantly reduced lipid peroxidation in rainbow trout (Li et al. 2015) and tilapia
445
(Romano et al. 2016). In the study by Li et al. (2015) dietary sodium citrate depressed
446
antioxidant enzyme activity, while in the case tilapia, dietary sodium citrate was toxic
447
(Romano et al. 2016). Therefore, both authors stated this finding could be related to
448
the chelating or anti-oxidant properties of sodium citrate, as opposed to an improved
449
physiological condition. The results of the in vitro experiment also showed an
18
450
improvement to lipid oxidative stability indicating the potential to act as a
451
preservative. It has been similarly shown that sodium acetate significantly reduced
452
peroxide value and thiobarbituric acid in salmon fillets over 15 days of storage in a
453
refrigerator (Sallam 2007).
454
In addition to improving oxidative stability in vitro, it was also shown that
455
sodium butyrate and propionate significantly reduced the production of TMA, which
456
is a by-product of spoilage bacteria such as Shewanella putrefaciens and
457
Photobacterium phosphoreum in fish (Dalgaard 1995). The anti-microbial properties
458
of organic acids are well known (Ng and Koh 2016), and both butryric and propionic
459
acid were shown to be toxic to P. phosphoreum and at lower concentrations than
460
acetic acid (Zeb et al. 2014). On the other hand, a sodium acetate solution was
461
reportedly more effective than sodium citrate or sodium lactate at decreasing various
462
spoilage bacteria in salmon fillets over 15 days of storage in a refrigerator (Sallam
463
2007). Considering each organic acid has their own unique Pka value, which affects
464
the amount dissociated organic acids and thus their anti-microbial properties (Ng and
465
Koh, 2016), further research should consider the pH and potential changes during the
466
deterioration of fish meat.
467
In conclusion, the use of dietary organic acids reduced lipid peroxidation but
468
the unaffected growth, and in the case of sodium formate inducing stress, might be
469
related to the organic acid levels being excessive to tilapia and requires further
470
investigations. Such research may potentially improve productivity but also cost-
471
effectiveness if lower supplementations are beneficial. On the other hand, the direct
472
inclusion of the organic acids into tilapia meat reduced lipid peroxidation while
473
sodium butyrate and propionate appeared to reduce bacterial spoilage. These findings
474
may have important post-harvest implications and should prompt further
19
475
investigations to optimal inclusion levels and any effect to various organoleptic
476
qualities.
477
478
479
Acknowledgements
This study was funded by a grant from Universiti Putra Malaysia (UPM);
480
project no. GP-IPB/2014/9440403. We would like to sincerely thank the two
481
anonymous reviewers for their constructive comments to improve this manuscript.
482
483
484
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671
672
673
674
25
675
Figure legends:
676
677
Figure 1 Mean (±SE) intestinal short chain fatty acids (SCFA) from red hybrid
678
tilapia after being fed diets with different types of organic acid additions. Different
679
letters among each SCFA indicates significant differences.
680
681
Figure 2 Histological sections of the liver from red hybrid tilapia in the control
682
treatment (a) and in the dietary formate treatment (b). For fish in the control,
683
butyrate, acetate, and propionate treatments, the livers showed normal sinusoid
684
structure and uncongested veins and pancreas (P). However, for fish in the formate
685
treatment, there were signs of lipofuscin (Lip) and white blood cell (WBC)
686
infiltrations that were often observed around the pancreas (P). Magnification × 40; H
687
& E staining.
688
689
Figure 3 Histological sections of the liver showing the overall distribution of
690
Periodic-acid Schiff positive material in red hybrid tilapia from the control treatment
691
(a) and in the dietary formate treatment (b).
692
693
Figure 4 Mean (±SE) Periodic acid-Schiff (PAS) stain intensity (% area) in the liver
694
of red hybrid tilapia after being fed diets with different types of organic acid
695
additions. Different letters indicate significant differences.
696
697
Figure 5 Mean (±SE) malondialdehyde (MDA) equivalents (μM g-1) when different
698
organic acids were added at 1% to tilapia meat and left at room temperature for 24
699
hours. Different letters indicate significant differences.
26
700
Figure 6 Mean (±SE) trimethylamine (TMA) (μg ml-1) when different organic acids
701
were added at 1% to tilapia meat and left at room temperature for 24 hours. Different
702
letters indicate significant differences.
703
704
705
706
707
708
709
710
711
712
713
714
715
716
717
718
719
720
721
722
723
724
27
725
Table 1 Ingredient formulation and proximate composition (% dry matter) of the
726
experimental diets with different types of organic acids additions.
727
Experimental diets
Ingredients
Control
Butyrate
Acetate
Propionate
Formate
Danish fishmeal
14.39
14.39
14.39
14.39
14.39
Soybean meal
56.11
56.11
56.11
56.11
56.11
Soybean oil
7.68
7.68
7.68
7.68
7.68
Others a
19.14
19.14
19.14
19.14
19.14
Sodium butyrate b1
0.00
2.00
0.00
0.00
0.00
Sodium acetate b2
0.00
0.00
2.00
0.00
0.00
Sodium propionate b3
0.00
0.00
0.00
2.00
0.00
Sodium formate b4
0.00
0.00
0.00
0.00
2.00
α-cellulose c
2.68
0.68
0.68
0.68
0.68
Proximate composition
Dry matter
95.43
94.11
96.52
97.04
96.54
Crude protein
40.14
40.26
40.43
39.94
40.03
Crude lipid
9.54
9.59
9.36
9.24
9.77
Crude ash
10.17
9.96
10.38
10.37
10.81
Crude fiber
5.53
5.21
5.63
5.80
5.37
pH
5.93
5.95
6.30
6.27
6.07
728
a
729
methionine (Sigma Aldrich Co. M9625) 0.45%
730
b
731
propionate P1880, 4 Sodium formate 71539
732
c
Others: tapioca starch 12.39%, vitamin mix 3.15%, mineral premix 3.15%, LSigma-Aldrich Co. 1 Sodium butyrate 303410, 2 Sodium acetate S2889, 3 Sodium
α-Cellulose (Sigma Aldrich Co. C8002).
733
28
734
Table 2 Mean (± SE) growth performance, feed efficiencies, body indices and
735
survival (%) of red hybrid tilapia when fed diets with different types of organic acids
736
additions.
Experimental diets
Control
Butyrate
Acetate
Propionate
Formate
Initial weight
2.87 ± 0.01
2.86 ± 0.01
2.85 ± 0.01
2.87 ± 0.00
2.88 ± 0.01
Final weight
10.01 ± 0.41
10.22 ± 0.89
9.98 ± 0.04
11.12 ± 0.50
10.67 ± 0.45
Initial length
5.31 ± 0.01
5.30 ± 0.01
5.31 ± 0.01
5.29 ± 0.01
5.31 ± 0.01
Final length
8.24 ± 0.14
8.22 ± 0.25
8.17 ± 0.09
8.37 ± 0.07
8.30 ± 0.13
SGR weight
1.97 ± 0.06
2.00 ± 0.13
1.96 ± 0.01
2.14 ± 0.07
2.08 ± 0.07
0.70 ± 0.03
0.69 ± 0.05
0.68 ± 0.02
0.72 ± 0.01
0.71 ± 0.03
HSI
3.53 ± 0.44
3.67 ± 0.39
3.60 ± 0.48
3.56 ± 0.23
3.41 ± 0.35
VSI
10.26 ± 1.17
10.14 ± 1.03
9.94 ± 1.25
10.29 ± 0.81
9.83 ± 1.34
FCR
1.46 ± 0.06
1.59 ± 0.14
1.62 ± 0.06
1.48 ± 0.09
1.49 ± 0.10
Feed intake
200.73 ± 6.43
223.96 ± 5.21
227.20 ± 2.56
230.94 ± 8.72
222.05 ± 3.94
Survival (%)
96.67 ± 1.67
98.33 ± 1.67
96.67 ± 3.33
95.00 ± 2.89
98.33 ± 1.67
(% day-1)
SGR length
(% day-1)
737
No significant differences were detected among these parameters (p > 0.05).
738
739
740
741
742
743
744
29
Table 3 Mean (± SE) muscle proximate composition (% wet weight), cholesterol (mg 100 g-1) and malonaldehyde (MDA) equivalents (μM g-1)
of red hybrid tilapia fed diets with different types of organic acids additions.
Experimental diets
Control
Butyrate
Acetate
Propionate
Formate
Moisture
77.89 ± 0.21
77.87 ± 0.45
77.79 ± 0.37
78.09 ± 0.71
78.02 ± 0.49
Crude protein
17.13 ± 0.13 a
16.97 ± 0.16 a
16.74 ± 0.23 ab
16.91 ± 0.18 ab
16.46 ± 0.15 b
Crude lipid
1.58 ± 0.12 a
1.84 ± 0.14 a
1.99 ± 0.10 ab
1.94 ± 0.15 ab
2.11 ± 0.11 b
Crude ash
2.29 ± 0.15
2.41 ± 0.19
2.24 ± 0.13
2.15 ± 0.12
2.31 ± 0.09
MDA
7.19 ± 0.18 d
6.01 ± 0.42 bc
6.49 ± 0.18 cd
5.46 ± 0.17 b
4.16 ± 0.28 a
Different superscripted letters within each row indicate significant differences (p < 0.05).
30
Table 4 Mean (± SE) plasma mineral content (mmol L-1), cholesterol (mmol L-1), triglycerides (mmol L-1), glucose (mmol L-1) and alanine
aminotransferase (ALT) (U L-1) of red hybrid tilapia fed diets with different types of organic acids additions.
Experimental diets
Control
Butyrate
Acetate
Propionate
Formate
3.03 ± 0.41 ab
3.00 ± 0.15 b
3.23 ± 0.09 ab
3.87 ± 0.13 a
3.40 ± 0.17 ab
Calcium
3.52 ± 0.15
3.35 ± 0.06
3.88 ± 0.24
3.49 ± 0.11
3.42 ± 0.30
Sodium
155.0 ± 2.6 ab
157.4 ± 2.0 a
159.4 ± 0.2 a
149.5 ± 2.2 b
155.3 ± 1.2 ab
Potassium
6.03 ± 0.44 a
5.83 ± 0.49 a
6.23 ± 0.45 a
8.37 ± 0.67 b
7.03 ± 0.60 ab
Chloride
132.9 ± 2.6
134.8 ± 2.2
135.8 ± 0.9
127.5 ± 2.2
132.2 ± 1.0
Cholesterol
3.85 ± 0.19
3.73 ± 0.23
3.90 ± 0.09
3.85 ± 0.18
3.72 ± 0.11
Triglycerides
2.07 ± 0.45
1.38 ± 0.20
1.79 ± 0.35
1.94 ± 0.22
1.77 ± 0.37
Glucose
3.90 ± 0.12 a
4.27 ± 0.09 ab
4.07 ± 0.17 ab
4.63 ± 0.18 bc
5.13 ± 0.32 c
ALT
13.70 ± 5.82 a
15.63 ± 1.25 a
18.03 ± 1.43 ab
19.33 ± 6.04 ab
28.27 ± 5.77 b
Phosphate
Different superscripted letters within each row indicate significant differences (p < 0.05).
31
Table 5 Mean (± SE) red blood cell (RBC) and white blood cell (WBC) counts (%) and differential WBC counts (% of total WBC) in red
hybrid tilapia fed diets with different types of organic acids additions.
Control
Butyrate
Acetate
Propionate
Formate
RBC
94.07 ± 0.69 a
93.96 ± 0.29 a
96.30 ± 0.95 ab
94.22 ± 0.47 a
96.93 ± 0.87 b
WBC
5.93 ± 0.21 a
6.04 ± 0.15 a
3.70 ± 0.48 ab
5.78 ± 0.19 a
3.07 ± 0.42 b
Lymphocyte
67.26 ± 6.46 ab
73.78 ± 5.04 a
71.28 ± 4.15 ab
66.27 ± 3.99 ab
56.27 ± 5.10 b
Thrombocyte
28.35 ± 6.93
19.87 ± 4.21
22.70 ± 4.34
25.11 ± 2.72
34.34 ± 7.02
Granulocyte
3.41 ± 0.47 a
5.62 ± 0.79 ab
4.96 ± 0.51 ab
6.98 ± 2.55 ab
8.16 ± 1.33 b
Monocyte
0.98 ± 0.98
0.74 ± 0.44
1.06 ± 0.68
1.64 ± 1.21
1.22 ± 0.79
G:L ratio
0.05 ± 0.00 a
0.08 ± 0.01 ab
0.07 ± 0.00 ab
0.11 ± 0.05 ab
0.14 ± 0.01 b
Different superscripted letters within each row indicate significant differences (p < 0.05).
32
Intestinal short chain fatty acids (mM)
1.20
Control
a
Butyric
1.00
a
0.80
b
0.60
Acetate
Propionate
bc
Formate
a
bc
0.40
b
c
b b
b
b
c
0.20
c
c
0.00
Acetic acid
Butyric acid
Fig. 1
33
Propionic acid
34
Fig. 2
35
36
Fig. 3
37
PAS staining intensity (% area)
45
b
40
a
35
a
a
a
30
25
20
Control
Butyrate
Acetate
Fig. 4
38
Propionate
Formate
10.0
b
μM g-1 MDA equivalent
9.0
8.0
7.0
6.0
5.0
a
4.0
a
a
3.0
a
2.0
1.0
0.0
Control
Acetate
Butyrate
Fig. 5
39
Propinate
Formate
0.14
0.12
a
a
ab
μg ml-1 TMA
0.10
b
0.08
b
0.06
0.04
0.02
0.00
Control
Butyrate
Acetate
Fig. 6
40
Propionate
Formate