Enzymatic and Histological Evaluations of Gut and Liver in Rainbow
Trout (Oncorhynchus mykiss) fed with Rice Protein Concentrate based
diets
Francesco Gai1*, Laura Gasco2, Franco Daprà3, Giovanni B. Palmegiano1 & Benedetto Sicuro3
1CNR Institute of Science of Food Production, Turin Division, Via L. da Vinci 44,
Grugliasco (TO), Italy
2Department of Animal Science, Via L. da Vinci 44, Grugliasco (TO), Italy 3Department of Animal Production, Via L. da Vinci 44, Grugliasco (TO), Italy
*Corresponding Author: Francesco Gai;CNR Institute of Science of Food Production, Turin Division, Via L. da Vinci 44, 10095 Grugliasco (To), Italy, telephone: +39 0116709232; fax: +39 0116709297; e-mail address: francesco.gai@ispa.cnr.it
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Abstract
Rice protein concentrate (RPC) is a plant protein source with high nutritional value in terms of proteins and lipids. This study was carried out to evaluate the intestinal response of rainbow trout fed with RPC using biochemical and histological observations. A stock of 360 juvenile rainbow trout (62.4±0.3 g) were randomly distributed into 12 fibre-glass tanks and fed with four experimental diets obtained by including graded levels of RPC (RPC0%, 20%, 35% and 53%) replacing fish meal on
dry weight basis. The experimental plan, consisting of four treatments and three
replicates, lasted 94 days. At the end of the experiment, 15 fish each treatment were killed and different tissues were sampled for biochemical and histological analysis. Proteolytic enzyme results showed that total alkaline proteases were unaffected. Amylase and lipase activities showed slight differences between the groups for all examined tracts. Histologically, the intestine in all experimental groups showed no inflammatory process. Liver samples of fish fed with RPC based diets were characterized by a slight decrease in lipid vacuolizations and a decreasing trend in the Ceroid substances Presence Index (CPI). In conclusion, RPC did not show detrimental effects on the intestinal morphology and modification of tissue enzyme activity in rainbow trout.
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The use of plant protein sources in aquaculture as fishmeal substitute is a extensively studied subject (Krogdahl et al. 2003; Palmegiano et al. 2005; Gatlin et al. 2007), since fishmeal will be a limited resource for fish feedstuff production in the future. In the past decade, in fact, a great deal of research in aquaculture nutrition dealt with fishmeal and fish oil substitution with alternative sources. As far as alternative protein sources are concerned, the best growth performances were achieved through utilization of glutens and/or plant protein concentrates. The most studied sources have been: soybean meal (Tibaldi et al. 2006; Escaffre et al. 2007) and plant mixture meals based on corn gluten,
wheat gluten and extruded peas (De Francesco et al. 2004; De Francesco et al. 2007).
Among the plant protein sources, rice protein concentrate (RPC) is a potential good raw material for fish nutrition due to its high protein (75% crude protein) and lipid content (11% ether extract). RPC is the insoluble proteinic fraction separated from rice starch and obtained from a rice wet milling process. The RPC showed good productive traits in rainbow trout and blackspot sea bream diets at the inclusion level of 20%, as reported by Palmegiano et al. (2006) and Palmegiano et al. (2007), respectively.
Although the digestive process in fish is not studied as much as in mammals, the enzyme profile seems to be a good physiological indicator of feed utilisation. The digestive enzyme content in the digestive processes is crucial to allow the ingested macronutrients (proteins, carbohydrates and fats) to be broken down into simple molecules, which can then be absorbed and used in metabolic pathways (Guillaume and Choubert, 2001). The digestive enzyme activity quantification is a useful method in order to provide clear information about the nutritional value and possible interactions between anti-nutritional factors and fish digestive enzymes of a new formulated fish feed (Refstie et al. 2006; Corrêa et al. 2007). A panel of different assays, such as digestive enzymes and histological stains were selected and tested on rainbow trout (Oncorhynchus mykiss, Walbaum) fed RPC graded 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63
inclusion level diets. Determination of digestive enzyme activity was carried out to evaluate the possible interaction between RPC and digestive processes in rainbow trout. Moreover, in order to evaluate the possible detrimental effects of RPC on the intestinal tracts and liver, several histological stains were performed on these tissues. This study is a follow-on from a previous nutritional trial, carried out by Palmegiano et al. (2006), focused on fish growth, feed conversion efficiency and digestibility of the RPC diets. Instead the present work aimed to examine, using biochemical and histological observation, the effects of the alternative protein-rich ingredient RPC on rainbow trout gastrointestinal digestive function in order to assess whether this ingredient may be satisfactorily and safe used in a trout feedstuff.
Materials and Methods
Commercial rice protein concentrate (RPC) was purchased from the CBH Qingdao Co. LTD (Quindao, China). Three experimental diets were obtained by including graded levels of RPC (RPC 20%, 35% and 53%) and tested against a fish meal based diet (RPC0).
The feeds were manufactured in the laboratory at the Experimental Station of the Department of Animal Science of the University of Torino by means of a pelleting process using a 3.5 mm diameter. Pellets were dried in a stove overnight at 50 °C and then refrigerated at 6 °C until utilization. The total amino acid composition of RPC and the diets was determined as shown by Palmegiano et al. (2006). Experimental feeds met the nutritional requirements for rainbow trout as reported by NRC (1993), except diet RPC53 where the lysine level was marginally limiting for rainbow trout requirements. No free amino acids were supplemented to the diets.
Formulation and proximate composition of the diets are reported in Table 1. Diets were analysed to be isoproteic (CP 47%) and isoenergetic (22 MJ kg−1 DM), has been reported
in a previous study (Palmegiano et al. 2006). In order to evaluate the possible RPC detrimental effects on the liver no hepatoprotector additives were added to the diets. A 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89
selection of 360 juvenile rainbow trout (initial mean body weight 62.4±0.3 g) were individually weighed to obtain a homogeneous stock of fish and randomly distributed into 12 fibre-glass tanks (W 1.00 m, L 3.00 m, H 0.35 m) supplied by an open water circuit 8 L/min. The adopted experimental design was balanced, monofactorial with four treatments per three replicates (4x3). The feeding trial lasted 94 days, after a 2-week period of acclimatisation to the tanks and diets. Daily feeding rate was 1.5% of the wet biomass and the feedstuff was distributed by hand, twice a day. Feed intake was checked each time even all the supplied feed was consumed, no feed reject events were recorded during the trial, as reported in the previous study, Palmegiano et al. (2006).
At the end of the experiment, the fish were killed six hours after the last meal, 5 fish each tank (15 fish for treatment) were sampled and gut repletion was checked. The pyloric caeca (PC), proximal (PI) and distal intestine (DI) were separated and all visible fat removed. All the tissues with their digestive contents were immediately frozen in liquid nitrogen and stored at –80°C before analysis. Tissues were homogenized using a polytron (Kinematica, Lucerne, Switzerland) in a cold Tris-HCl 50mM (pH 7.0) buffer to a final concentration of 50 mg mL−1. The homogenate was then centrifuged at 4° C at 7500 (xg)
for 10 min. The supernatant containing the crude extracts was picked up and stored at –20° C before analysis as proposed by Santigosa et al. (2008). Enzyme activity was expressed as total enzyme activity, as reported by Krogdahl and Bakke-McKellep (2005), and calculated as follows:
Total activity = [Enzyme activity (U/mL) x volume (mL) ]/body weight (kg).
Results are reported as enzyme Units released per Kg of body weight (BW) (Units Kg-1
BW). One enzyme unit was defined as the amount of enzyme that catalysed the release of 1 μg of product min-1.
Total alkaline protease activity (TPA) 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114
The effect of different pH incubations on proteolytic activity of the crude enzyme extract was determined on the base of the casein hydrolysis assay by Kunitz (1947). The pH values of different buffers were previously optimised for alkaline proteases: 0.2 M phosphate buffer (pH 8.0) and 0.1 M glycine–NaOH (pH 9.0–10.0) were chosen. The enzyme-substrate mixture consisted of 0.75 mL 2% (w/v) casein in water, 0.75 mL selected buffer and 0.1 mL crude enzyme extract incubated in a water bath for 1 h at 37° C. A total of 2.25 mL trichloroacetic acid (TCA), 5% (w/v) was then added to the reaction mixture to stop the reaction. This mixture was incubated for 30 minutes at 4° C before centrifuging at 3500 rpm for 15 min. Absorbance of the supernatant was recorded at 280 nm to measure the amount of released tyrosine. The blank used for this assay was prepared by incubating a mixture of the casein, water and buffer for 1 h at 37° C, followed by the addition of a crude extract and TCA. One unit of enzyme activity was defined as 1 μg of tyrosine released per min-1.
Amylase [EC 3.2.1.1/2] activity was determined according to Bernfeld (1955) using soluble starch as the substrate and maltose as standard disaccharides. One unit of enzyme activity was defined as 1 μg of maltose released per min-1.
Lipase [E.C. 3.1.1.3] activity was determined spectrophotometrically by hydrolysis of ρ-nitrophenyl myristate according to Iijima et al. (1998) with a modified method of Albro et al. (1985). Each assay (0.5 mL) contained 0.53 mM ρ-nitrophenyl myristate, 0.25 mM 2-methoxyethanol, 5 mM sodium cholate and 0.25M Tris-HCl (pH 9.0). One unit of enzyme activity was defined as 1 μg of ρ-nitrophenol released per min-1.
Histological assays:
Intestinal sections were evaluated following the criteria reported by Baeverfjord and Krogdahl (1996). Evaluated parameters consisted of the widening and shortening of the intestinal folds, the enterocyte supranuclear vacuolization extent, the lamina propria widening status villi and lymphocyte infiltration in the lamina propria and submucosa. Fish 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140
were sampled at the same time and in the same condition as those utilized for digestive enzyme assays. Five trout from each tank were sampled and the gut and liver were isolated (15 trout per each treatment). The visceral pack was gently separated from the perivisceral fat. Samples of the liver, pyloric caeca, proximal and the distal intestine were sampled and fixed in 4% buffered (pH 7.2) and isotonic formalin cooled at 4° C. Sample vials were stored at 4° C before analyses. After one week, the fixed tissues were embedded in paraffin wax, following normal histological procedures. Five µm thick paraffin sections were cut and collected on microscope slides for the histological stains. All the tissue samples were stained with the normal Mayer hematoxylin-eosin (HE) stain while on the liver samples
were also performed the PAS, PAS diastase and Sudan Black stains. The PAS diastase
stain was necessary to discriminate the PAS positive reaction due to glycogen from other PAS positivity, such as mucopolysaccaridic substances and glycoproteins. The Sudan Black stain was performed to observe and confirm the possible presence of ceroid substances. The Ceroid substances Presence Index (CPI) was calculated as a new parameter as follows: CPI = (number of livers with ceroid substances / total sampled livers).
A one-way analysis of variance (ANOVA) was used on the enzyme activity values. Any significant differences were ranged using the Duncan test according to the significance p level <0.05 using the GLM Procedure, SPSS (1999). Variance homogeneity was assessed on statistically significant results using a KS goodness of fit test. On pepsin activity, regression model was used and Pearson correlation coefficient was calculated.
Results and Discussion
Digestive Enzyme Activity The digestive enzyme activity (DEA) is reported in Table 2.
Considering the alkaline TPA of the intestine, a significant difference appeared in the PI with the lower TPA value for fish fed RPC53, whereas the DI tract showed no 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166
significant differences between the diets. Intestinal DEA change along the intestinal tracts, with the highest values detected in the PC intestinal region.
Amylase activity showed statistical differences for the PC tract, the highest activity was recorded in fish fed RPC20 and the lowest in fish fed RPC53. PI and DI tracts showed no significant differences between the diets.
Considering the lipase activity, no statistical differences appeared between the groups for all examined tracts. PC showed the highest values of lipase activity compared to the next intestinal tracts for all groups. The lipase activities of DI tracts were lower than the assay minimum detectable level for all the treatments.
Histological Studies
No severe changes and inflammatory process were found on the intestinal tracts except one fish, fed with the RPC35 diet, which was affected by a slight PI inflammation. (Table 3, Fig. 1). The observed liver parameters (Table 4) were the presence or absence of lipid vacuoles, glycogen granules and ceroid substances. The HE was used to evaluate hepatocyte lipid vacuolizations (Fig. 2). The PAS and PAS-diastase stains were used as evaluation tools for the presence of hepatocyte glycogen granules (Fig. 3). Finally, the Sudan black stain allowed ceroid substance detection (Fig. 4). The fish fed experimental diets were characterized by a frequent liver lipid vacuolisations. No glycogenosis was observed among the fish groups, although one fish fed with the RPC0 diet showed intense PAS stains. High numbers of fish with ceroid substances droplets were recorded in the RPC0 and the RPC20 groups, and a decreasing trend was recorded for the other fish groups. The CPI showed a opposite trend at the dietary RPC content. Moreover, the trout group fed with RPC53 was characterized by the higher number of free ceroid substance livers.
As mentioned before this study is a follow-on from a previous nutritional trial carried out by Palmegiano et al. (2006). Briefly, these authors concluded that RPC can be used at an
167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192
inclusion level of up to 20% without a growth performance reduction in terms of weight gain, specific growth rate and protein efficiency ratio. Over this inclusion level, reduced growth performance was mostly a consequence of reduced nutrient digestibility and energy intakes as the RPC level was increased in the diet.
Enzymatic investigations are crucial in order to clarify the effect of vegetal protein substitution in farmed fish (Corrêa et al. 2007; Palmegiano et al. 2006), even if a bibliographic comparison of the TPA activity is difficult for the lack of uniformity in laboratory protocols used (Hidalgo et al. 1999).
Digestive enzymes of fish have been reported in many species and their patterns of distribution are different among them (Corrêa et al. 2007). Considering the productive results obtained by RPC digestibility (Palmegiano et al. 2006) that showed a decrease in protein digestibility related with an increase of RPC inclusion, digestive enzymes were studied in the main portions of digestive tract. Considering in particular the protease activity in the different intestine portions, the decrease of feed digestibility previously registered (Palmegiano et al. 2006), is explained by a reduction of protease activity in proximal intestine in fish fed the highest RPC inclusion level (RPC53 group). Santigosa et al. (2008) reported that the replacement of fish meal by plant protein (PP) mixtures depressed trypsin and chymotrypsin activity and chymotrypsin in rainbow trout and sea bream, respectively. However, both trout and sea bream showed compensation mechanisms, such as an increase in the relative intestinal length and an up-regulation of trypsine activity in sea bream to achieve a digestive balance. Lundstedt et al. (2004) observed an unresponsive protease activity in a carnivorous fish, pintado (Pseudoplatystoma corruscans) fed different dietary protein and starch levels, but the same authors reported that chymotrypsin and trypsin activities were lower in the anterior intestine than in the other portions. In Salmonids pyloric caeca constitute a greater portion of the intestine than the other regions and in the present work, as reported for Atlantic 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218
salmon, the maximum TPA activity is shown in the PC tract. In the present study, the RPC inclusion in diets did not induce significant modifications of the intestinal TPA activity even if, due to the adopted method, trypsin and chymotripsin, that are pancreatic enzymes secreted into the lumen, were not detected in this study. Amylase activity in fish is directly related to a dietary carbohydrate level, as demonstrated in European sea bass and rainbow trout (Corrêa et al. 2007; Cahu & Zambonino Infante, 1994). In the present study, the highest values of amylase activity were recorded in the PC as well as reported by Corrêa et al. (2007) in tambaqui. In our study, the lowest values were recorded in fish fed the RPC53 diet and this result is in disagreement with the observation of Pérez-Jiménez et al. (2009) that found enhanced amylase activity in the pyloric caeca of on-growing common dentex (Dentex dentex), fed on higher carbohydrate and lower lipid level diets. On the basis of the amylase results we suppose that RPC, at the highest inclusion level, could be exert an inhibition effect on the fish amylase activity probably due to the presence of an anti-nutritional factor. Lipase activity showed the highest value in the PC tracts of fish fed RPC35 and RPC53 diets, although no significant differences appeared among the different groups . The lipase activity is strictly correlated with dietary levels of triglycerides (TG) and phospholipids (PL), as demonstrated in sea bass larvae fed diets containing different levels of these lipid fractions (Cahu et al. 2003; Zambonino Infante & Cahu 2007). It is well known that fish meal contains lipids rich in long chain TG, whereas rice lipids are rich in PL (Choi et al. 2005). In the present study, the raw RPC was characterized by 11% lipids therefore in the RPC53 diet the 43% of dietary lipid was provided by rice fats rich in PL that may influence the lipase activity.
Formulation of modern fish feeds should considers new foodstuffs for different species and the future development of this sector is related to the researcher ability to look new methods to overpass some physiological features that negatively influence fish feed 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243
utilisation. First step in this direction is the clarification of these bottlenecks, considering the digestive enzyme activity.
It is well known that too high inclusion levels of plant protein (in particular soy products) in fish feeds can affect palatability, feed conversion and intestinal integrity. Intestinal lesions are common in different fish species studied, but their extent varies among species (Refstie et al. 2000; Ostaszewska et al. 2005). Soybean meal detrimental effects were reported in Atlantic salmon (Krogdahl et al. 2003; Bakke-McKellep et al. 2007) and rainbow trout (Romarheim et al. 2006). On the contrary, a gut histological examination in Atlantic salmon fed diets with legumes, oilseeds, or cereals did not found intestinal pathologies except than enteritis in the distal intestine of fish fed soybean meal (Aslaksen et al. 2007). Similar results were reported in another study on the intestinal tract of Atlantic salmon parr fed different varieties of soy and maize (Sanden et al. 2005).
In trout, the soybean containing diets can cause disturbances in intracellular digestion in enterocytes, pathological changes in pancreatic secretory epithelium and metabolic disturbances in the liver (Ostaszewska et al. 2005). In this study RPC did not induce histomorphological changes in rainbow trout, not even when RPC provides about 80% of dietary protein as in the case of the RPC53 diet. Similar results were reported by Daprà et al. (2009) in blackspot seabream (Pagellus bogaraveo) fed with RPC diets. These authors recorded minor changes in the intestinal morphology suggesting that the RPC supplementation did not induce severe inflammatory process in this fish.
In the present study, an high liver lipid vacuolization was recorded in all the fish groups. Others investigations carried out on rainbow trout fed with yellow lupin meal showed a reduction in the number of lipid vacuolizations in their livers (Glencross et al. 2004). Brassica by-products and gelatinized starch can modify liver histology of rainbow trout and a relationship was found between gelatinized starch and glycogen granules, as vacuole PAS positive-like structures (Pereira et al. 2002). The corn gluten meal based diets induced 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269
a more pronounced PAS positive reaction in gilthead seabream livers (Robaina et al. 1997). The afore mentioned studies utilized only one or two histological stains, providing a limited pattern whereas there several stains are necessary to evaluate the whole complex of the liver pattern. The use of HE or HE combined with PAS stains cannot provide an adequate view of the liver. Furthermore, there is the possibility that PAS positive-like structures cannot be represented by glycogen granules. Lipid vacuolizations may not always have pathological signs, but the presence of ceroid substances is considered a sub-pathological problem. Moreover, some PAS positive-like structures can be recognised as ceroid substances and not due to glycogen content, in fact PAS positive granules in PAS-diastase stained sections are also positive to Sudan black stain, indicating the presence of ceroid substances. Noteworthy is the fact that the CPI shows an opposite trend to the dietary RPC content. These results lead the authors to suppose that RPC have a slight liver protector effect displayed by the reduced presence of ceroid substances. A possible explanation of this fact can be attributed to RPC tocotrienols content and their powerful antioxidant action (Xu et al. 2001). Taking into account the results of the present study RPC may be used as partial fishmeal substitute up to 53% inclusion level without any detrimental effects on the intestinal morphology and physiology, unlike those observed by other authors with soybean in several fish species.
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Acknowledgments
Financial support for this work was provided by the MIPAF (Ministero delle Politiche Agricole e Forestali, Italy) VI Programma Quadro 2003–2005 Grant. The authors thank Mrs Sara Antoniazzi (ISPA-CNR. Torino) and Mrs Cristina Vignolini (Dept. Veterinary Morphophysiology) for her technical assistance during the laboratory analysis and Dr. Ivan Ferro for his careful management of the fish. All the authors contributed equally to the work described in this paper.
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Figure Captions
FIGURE 1 Proximal intestinal sections of rainbow trout stained with HE, the black arrow heads indicate the villi lamina propria.
A) Normal pattern with thin lamina propria. B) Tract with moderate changes with thick lamina propria and mucosa indentations.
FIGURE 2 Liver sections of rainbow trout stained with HE, showing different patterns of lipid vacuolisation. A) Hepatocytes without lipid vacuolisations. B) Hepatocytes with moderate presence of lipid vacuoles. C) High lipid vacuolisations.
FIGURE 3 Liver sections stained with PAS (series 1) and PAS-diastase (series 2) stains. A1-A2) Hepatocytes lacking in lipid vacuolisations. B1-B2) Hepatocytes with lipids vacuolisations. In A1 and B1 photos it is possible distinguish the glycogen granules.
FIGURE 4 Liver sections of rainbow trout stained with Sudan black stain. A) Section with high presence of ceroid substances, visible as dark granules into the hepatocytes B) Section without ceroid substances presence.
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