Nutritional assessment of linseed meal (Linum usitatissimum L.) protein concentrate in feed 1 of silver catfish* 2 3 4 5 6 7
D. Pianessoa**, T.J. Adoriana, P.I. Mombacha, M.O. Dalcina, L. Loebensb, Y.B. Tellesa, S.S. 8
Roballoa, N.M. Lovattoa, L.P. Silvaa 9
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aDepartment of Animal Science, Federal University of Santa Maria, Av. Roraima nº 1000, 11
Cidade Universitária, Bairro Camobi, Santa Maria – RS, Brazil. CEP: 97105-900. 12
13
bFederal Institute of Education, Science and Technology Farroupilha, Campus Frederico 14
Westphalen, Linha 7 de Setembro, BR 386, s/n, Zona Rural, Frederico Westphalen – RS, 15
Brazil. CEP: 98400000. 16
17 18
** Corresponding author. Tel: 55 (55) 3220-8365; Fax: 55 (55) 3220-8240; EM: 19
pianessodirleise@gmail.com 20
Abstract 21
Linseed protein concentrate (LPC) was produced in the laboratory and subsequently a 22
feed assay was performed to evaluate the replace of increasing levels of fish meal protein for 23
LPC on growth, nutrient utilization, metabolic responses and goblet cells of silver catfish 24
(Rhamdia quelen). Five isoproteic and isocaloric diets were formulated with 0, 100, 200, 300 25
or 400 g/kg replace of fish meal protein for LPC. Each diet was randomly distributed to 26
quadruplicate groups of 25 fish (initial average weight of 6.13 g) per tank, totaling 20 tanks. 27
The crude protein of LPC was lower (P<0.05) than that of fish meal. However, its in vitro 28
digestibility was higher (P<0.05). Fish fed with LPC presented the same growth and nutrient 29
utilization (P>0.05) than animals submitted to the 0 g/kg LPC diet. Diets of 300 and 400 g/kg 30
LPC replacing fish meal protein provided higher (P<0.05) free amino acid content in plasma. 31
Hepatic protein was higher (P<0.05) in the 300 g/kg LPC treatment, differing from the 0 g/kg 32
LPC diet. Hepatic ammonia was higher (P<0.05) in fish submitted to 0 g/kg LPC diet, differing 33
from 200 and 300 g/kg treatments. Fish fed with 300 g/kg LPC presented more (P<0.05) goblet 34
cells, differing from the 0 g/kg LPC group. LPC presents equivalent nutritional quality and can 35
replace fish meal protein by up to 400 g/kg without causing metabolic and histological injuries 36
that affect growth and nutrient utilization. 37
38
Keywords: replace fish meal, vegetable protein, goblet cells, metabolism, Rhamdia quelen 39
40
Abbreviations: FW, final weight; BPD, body protein deposition; BFD, body fat deposition; 41
PRC, protein retention coeficiente; Free AA, free amino acids; ALT, alanine aminotransferase; 42
ALF, alkaline phosphatase; HSI, Hepatosomatic index. 43
44 45
1. Introduction 46
The increasing expansion of aquaculture has raised the demands on the availability, 47
quality and accessibility of ingredients for formulating rations of different species. Historically, 48
fish meal is the main protein ingredient used in aquaculture diets. However, the increase in 49
demand has raised the price and caused instability of supply and product quality in the 50
international market. Therefore, aquatic animal nutrition research has directed its studies in the 51
search for alternative sources of proteins to reduce the dependence of fish meal on aquaculture 52
nutrition (Deng et al., 2017; Ju et al., 2013; Kumar et al., 2012). 53
Some plant sources appear to be viable alternatives for total or partial replacement of 54
fish meal in diets of aquatic organisms (Lovatto et al., 2016, 2015, 2014; Gaylord et al., 2006; 55
Kaushik et al., 1995). However, most of the sources tested so far (soybean meal, corn gluten, 56
cottonseed meal and rapeseed) (Hardy, 2010) are also intensively used in raising other animals 57
(pigs, birds and large ruminants), increasing the competitiveness of use and reducing the 58
availability in the market. For these reasons, research has sought to meet demands with 59
ingredients of regionalized production, encouraging local agriculture and economy, generating 60
productive identity and reducing international competitiveness (Deng et al., 2017; Lovatto et 61
al., 2016). 62
Linseed (Linum uistatissimum L.) is an annual plant of cold seasons, produced in 63
southern Brazil. After extraction of its oil, the meal is obtained as a by-product that presents 64
high protein content (average 300 g/kg) (Oomah and Mazza, 1993). The use of this ingredient 65
in fish nutrition is limited by its high fiber concentration, especially its water soluble fraction 66
(Goulart et al., 2015; Alzueta et al., 2003), which may impair the availability and utilization of 67
dietary nutrients (Francis et al., 2001). One way to potentiate the use of linseed meal as a 68
substitute for fish meal in aquaculture diets is by the application of the protein concentration 69
process through the isoelectric precipitation of proteins (Ferri, 2006). This process allows 70
obtaining a concentrate with higher protein content and reduced in antinutrients, increasing its 71
inclusion potential in the diets, without causing metabolic disturbances that affect the growth 72
of the animals (Lovatto et al., 2014, 2015, 2016; Thiessen et al., 2004; Kaushik et al., 1995; 73
Mambrini et al., 1999). 74
In this scenario, we produced a linseed protein concentrate on laboratory scale, which 75
was tested as a substitute for fish meal protein in diets for silver catfish (Rhamdia quelen), 76
evaluating the growth, deposition of nutrients in the carcass, metabolic and histological 77
responses of the animals. 78
79
2. Materials and methods 80
81
2.1 Preparation of the samples and protein concentrate obtaining 82
Linseed meal (Linum usitatissimum), courtesy of Giovelli & Cia Ltda (Guarani das 83
Missões, RS, Brazil), was used for extracting the mucilage (Goulart et al., 2015) and then dried 84
at 55°C in an air circulation oven and ground in refrigerated micro mill (MA -630, Marconi). 85
The oil was removed with hexane (P.A. Synth, Brazil) at the ratio 1:2 (weight / volume) in four 86
sequential washes, resulting in the demucilated and degreased linseed meal used to obtain 87
linseed protein concentrate (LPC). 88
The LPC extraction and precipitation was carried out at the Fish Farming Laboratory of 89
the Federal University of Santa Maria (Universidade Federal de Santa Maria - UFSM), RS, 90
Brazil, by protein isoelectric point, according to methodologies described by Smith et al. (1946) 91
and Lovatto et al. (2016). The analysis of the LPC protein content (factor N x 6.25) followed 92
AOAC 960.52 method (1995). 93
94 95
2.2 In vitro digestibility protein 96
The in vitro digestibility protein test was carried out in duplicate in the samples of fish 97
meal and linseed protein concentrate (LPC). We followed the methodology proposed by 98
Mauron (1973), with modifications made by Dias et al. (2010). The method is based on the 99
digestion of the sample by the pepsin (1: 10,000, Nuclear) and pancreatin enzyme (Sigma). 100
101
2.3 Experimental diet 102
Five experimental isoproteic (370 g/kg of crude protein) and isocaloric (13 MJ/kg) diets 103
were formulated (Table 1), to meet the nutritional and essential amino acid requirements 104
according to Meyer and Fracalossi (2004) and Montes-Girao and Fracalossi (2006). 105
The control diet contained fish meal (fish fillet residue flour - Oreochromis niloticus, 106
sieved at 590 μm, Copisces, Toledo, Brazil) and soy protein concentrate as the main source of 107
protein, without the inclusion of linseed protein concentrate (0 g/kg LPC). In the other 108
treatments the fish meal protein was replaced by levels of 100, 200, 300 or 400 g / kg LPC. The 109
other ingredients (maize starch, soybean oil, soy protein concentrate, vitamin and mineral 110
mixture) were kept at very similar levels in the experimental diets. To prepare the rations, the 111
dry ingredients were mixed manually until complete homogenization, with concomitant 112
addition of oil and water. The diets were pelleted, dried in an oven with air circulation at 55 °C 113
for 24 hours, crushed, placed in plastic bags and stored in a freezer (-18 °C) until the beginning 114
of fish feeding. 115
116
2.4 Fish farming and feeding trial 117
The biological assay was carried out at the Fish Farming Laboratory, Department of 118
Animal Science, UFSM, after approval by the Animal Experimentation Ethics Committee of 119
said university, under protocol N. 8015120816. 120
A water recirculation system with water reservoir (500L), waste separation tank and 121
biological water filtration system was used. The silver catfish (initial weight of 6.13 ± 0.97 g) 122
were randomly distributed in 20 tanks (70L) with individual inlet and outlet of water, with a 123
density of 25 fish per tank (four tanks per treatment). During the experimental period, water 124
quality parameters were monitored and maintained as follows: temperature: 25.6±1.49° C; 125
dissolved oxygen: 7.10±0.70 mg/L; pH: 7.5±0.24 units; total ammonia: 0.30±0.11 mg/L; nitrite: 126
0.2±0.17 mg/L; alkalinity: 33.6±13.60 mg/L of CaCO3; and hardness: 34.40±18.45 mg/L of 127
CaCO3. The biological assay lasted 60 days and during this time the fish were fed three times 128
daily until apparent satiety. Daily siphoning of the experimental units was performed for the 129
removal of residual debris. 130
131
2.5 Data collection and assessed variables 132
At the beginning and at the end of the experimental period (60 days of treatment), 133
biometric analyzes of the animals were performed, fasted for 24 hours and anesthetized with 134
benzocaine (100 mg/L), to estimate the final weight (FW) (g); survival (%) = [(total number of 135
fish harvested/total number of fish stocked) x 100] and the hepatosomatic index (HSI) (%): 136
[(liver weight / whole fish weight) x 100]. 137
Body composition was determined in samples of eight fish per treatment which were 138
slaughtered (10%, ≥250 mg/L) of benzocaine (AVMA, 2013) and ground. For the crude protein, 139
the micro-Kjeldahl (N. 960.52) method was used with factor N x 6.25 (AOAC, 1995) and for 140
body fat, the Bligh and Dyer (1959) method was used. The nutrients retention was calculated 141
according to the following equations: 142
- Body protein deposition: BPD (g) = (FW x (% final body crude protein/100)) – (IW x (% 143
initial body crude protein/100)); 144
- Body fat deposition: BFD (g) = (FW x (% final body fat/100)) – (IW x (% initial body 145
fat/100)); 146
- Protein retention coefficient: (PRC) (%) = 100 x ((FW x final body crude protein) – (IW x 147
initial body crude protein)) / feed consumed x crude protein diet (%). Where FW, final weight; 148
IW, initial weight. 149
150
2.6 Plasma biochemistry and hepatic metabolism assay 151
At the end of the experimental period, after a 24-hour fast, twelve fish per treatment 152
were randomly captured. The blood was rapidly collected from the caudal vein using 153
heparinized syringes and the fish were slaughtered by immersion in water with high dose of 154
benzocaine (10%, ≥250 mg/L) (AVMA, 2013) and were eviscerated to remove the liver. The 155
livers after weighing were immediately frozen (-20 °C) for analysis of biochemical parameters. 156
Plasma aliquots were separated after centrifugation (10 min at 1200xg) of the blood at room 157
temperature for further determination of plasma metabolic parameters. 158
For hepatic glycogen analysis (Park and Johnson, 1949), 50 mg of tissue were weighed 159
and 1 and 3 mL of KOH and ethanol were added for hydrolysis and glycogen precipitation. For 160
protein analysis, tissues were heated to 100 °C with KOH and centrifuged at 1000xg for 10 min. 161
The supernatant was used to estimate the total protein level by the method described by 162
Bradford (1976). To measure hepatic amino acids and transaminases, 1 ml of 20 mM phosphate 163
buffer, pH 7.5 were added to samples and the homogenate was centrifuged at 1000xg for 10 164
min. Amino acid determination was performed in neutral supernatant according to Spies (1957). 165
Part of these aliquots were diluted (two and ten times, respectively) and used to measure alanine 166
aminotransferase (ALA) (EC 2.6.1.2) and alkaline phosphatase (ALP) (EC 3.1.1.1). The 167
enzymes were determined according to the protocols described in the reagents produced by 168
Labtest Diagnóstica S.A. (Lagoa Santa, Minas Gerais, Brazil). 169
For the analyzes of total protein, albumin, free amino acids and glucose plasma 170
metabolites, Labtest Diagnóstica S.A. (Lagoa Santa, Minas Gerais, Brazil) reagents were used. 171
2.7 Histological evaluation 172
The intestine (10 cm initial) of four fish/treatment were collected (after slaughter) and 173
prepared for optical microscopy. The histological samples were fixed in 10% formalin, 174
preserved in 70% ethanol and submitted to histological routine. The material was dehydrated 175
in ethanol (70% -99% alcohol) and incorporated into the glycol methacrylate resin (Technovit 176
7100). From this material, 2μm slits were obtained per rotary microtome (LEICA RM2245), 177
until subsequent staining with hematoxylin-eosin. For morphological examination, the slides 178
were observed and documented by optical microscopy (ZEISS PrimoStar with AxioCam 179
ERc5s) and analyzed using ZEN LITE (Carl Zeiss) software. Goblet cells were counted in 180
500μm villi. The slides were carefully examined for the presence of histopathological changes. 181
182
2.8 Statistical analysis 183
Data were submitted to normality test of the residues (Shapiro-Wilk) and analysis of 184
regression until third order. In the absence of adjustment of the data to the regressions, the 185
results were compared at the probability level of 5%, using the Tukey test. Differences were 186
considered statistically significant when P<0.05. Values are expressed as mean ± standard error 187 of the average. 188 189 3. Results 190 191
3.1 Protein concentrate obtaining and in vitro protein digestibility 192
Protein concentration by isoelectric pH provided a product (LPC) with 532.4 g/kg of 193
crude protein. Even so, the protein level was lower (P <0.05) than that determined for fish meal 194
(596.5 g/kg) used as standard in experimental diets (Figure 1). On the other hand, LPC showed 195
higher (P <0.05) in vitro protein digestibility (889.8 g/kg) when compared to fish meal (824.5 196
g/kg) (Figure 1). 197
198
3.2 Growth, protein utilization and survival rate 199
There was no difference (P> 0.05) in final weight and survival of fish fed with increasing 200
levels of LPC in replacement of fish meal protein in diets (Table 2). The LPC protein included 201
in the experimental diets did not promote differences (P> 0.05) in protein and fat deposition 202
and fish protein retention coefficient (Table 2). 203
204
3.3 Plasma biochemistry assay 205
Increasing levels of LPC in substitution of fish meal protein in diets did not cause 206
significant changes in total protein, albumin and plasma glucose concentrations of silver catfish 207
(P > 0.05) (Table 3). The concentration of free amino acids was higher in the plasma of the fish 208
fed with higher levels of LPC (300 and 400 g/kg) (P < 0.05), in substitution of fish meal protein. 209
210
3.4 Liver biochemistry assay 211
The inclusion of LPC protein did not cause significant differences (P> 0.05) in the 212
hepatic concentration of free amino acids, glycogen, alanine aminotransferase (ALT) and 213
phosphatase alkaline (ALF) (Table 4). Hepatic protein concentration was influenced (P <0.05) 214
by the substitution of fish meal protein for 300 and 400 g / kg LPC differing from the 0% LPC 215
treatment, which presented lower protein in the liver. The hepatic ammonia concentration was 216
higher (P <0.05) in fish fed with the 0 g/kg LPC in diet, without significantly different from 217
100 and 400 g/kg LPC treatments in substitution of fish meal protein. The hepatosomatic index 218
(HSI) of the fish was not influenced by the diets tested (Table 4). 219
3.5 Count of goblet cells 221
The intestinal goblet cell count was positively influenced (P <0.05) by the inclusion of 222
300 g/kg LPC in the diet in substitution of fish meal protein when compared to the treatment 223
without addition of the LPC ingredient (Figure 2). 224
225
4. Discussion 226
The LPC was presented as an ingredient with adequate protein level (532.4 g/kg) for 227
use in diets of aquatic organisms, comparable to the usual protein sources such as soybean meal 228
(450 g/kg) and flours of fish waste (600 g/kg), more easily found in the Brazilian market. In 229
addition, the digestibility of LPC (889.8 g/kg) emphasizes the protein quality of the source 230
obtained. In a work developed by Oliveira Filho and Fracalossi (2006), the apparent 231
digestibility of fish meal (waste) for silver catfish was 777 g/kg, lower than in vitro digestibility 232
(824.5 g/kg) found in this study. The lower digestibility of fish meal in relation to LPC can be 233
explained by the high content of mineral material (250 g/kg) present in this ingredient (Oliveira 234
Filho and Fracalossi, 2006). 235
Even the methionine content being elevated with the protein concentration of 5.4 g/kg 236
(linseed meal) for 9.9 g/kg in the LPC (data not shown), it was necessary to supplement this 237
amino acid to meet the requirement of the species (Montes-Girao and Fracalossi, 2006). Still 238
with the inclusion of 408.8 g/kg of fish meal in the 0 g/kg diet, we did not obtain adequate levels 239
of methionine, and supplementation was performed at similar levels in all diets (Table 1). 240
According to Ndou et al. (2018), methionine is one of the least abundant amino acids in linseed 241
meal. 242
The highest in vitro protein digestibility of LPC and the zootechnical indexes (FW and 243
survival) statistically equal in all tested treatments indicate that LPC exhibits nutritional 244
characteristics equivalent to fish meal and can be used as a parcial substitute for this protein 245
source. The total fiber content of the LPC (291 g/kg) (data not shown) is 21% lower than that 246
found in linseed meal in natura (370.6 g/kg), studied by Goulart et al. (2015). The concentration 247
of fiber present in linseed meal and its water retention capacity are considered as responsible 248
for the depletion of growth and reduction of food intake (Ndou et al., 2017). According to 249
Eastwood et al. (2009), this is due to the viscosity of the mucilage and the cell wall 250
polysaccharides present in linseed meal. These components exhibit limited degradability, high 251
capacity to absorb water and increase volume by restricting intestinal capacity and digestion of 252
nutrients (Eastwood et al., 2009). It is possible to infer that the lower amount of LPC fibers 253
reduced the effects of the antinutritional factors bound to the fibrous fraction, regarding the 254
nutrient utilization and fish growth. 255
The absorption of nutrients depends on the rate at which they can be absorbed from the 256
intestinal lumen and the time they remain in contact with the absorptive epithelium (Shiau et 257
al., 1988). Thus, the ultimate response to dietary use is reflected in the body's deposition of 258
nutrients. We did not observe differences in the protein and fat body deposition of the LPC-fed 259
fish, which is a positive result for the use of the ingredient in the nutrition of aquatic animals. 260
According to Pratoomyot et al. (2010), there may be an increase in body fat that is usually 261
associated with the inefficient use of protein sources and amino acids in the diet. As observed 262
by Gaber (2006), where the inclusion of plant sources reduced the energy available for protein 263
synthesis, resulting in lower growth and nutrient utilization. The increase in body fat deposition 264
of fish with the use of vegetable proteins in the diet was explained by imbalances in amino acid 265
concentrations (Kaushik et al., 2004), which interfere with energy metabolism (Hansen et al., 266
2007). 267
Plasma evaluation of the animals revealed similarity in metabolite content (total protein, 268
albumin and glucose) indicating that nutrients were metabolized without impairing hepatic 269
synthesis, as reported in other studies (Lovatto et al., 2016). The replacement of the fish meal 270
protein by 300 or 400 g / kg LPC in the diet promoted an increase in the concentration of free 271
amino acids in the plasma, a result that is related to the faster digestion of proteins of vegetal 272
origin in relation to animal protein. Larsen et al. (2012) reported that the time at which amino 273
acids reached the bloodstream was impaired in diets with the inclusion of plant sources in 274
relation to the use of fish meal. This imbalance in the plasma amino acid profile favors 275
deamination and reduces protein synthesis, impairing the growth of the animals (Pretto, 2013). 276
Our results suggest that there was a synchronism in the availability of nutrients from the diet 277
that culminated in adequate utilization of these for nutrient deposition and growth. Probably the 278
LPC levels used were not high enough to impair the productive performance of the fish. 279
The composition of diets not only affects the growth and quantity of internal nutrients 280
in the body, but also interferes with the enzymatic activity of the liver (Lovatto et al., 2016). In 281
this study, no differences were found in hepatic metabolites evaluated (amino acids, glycogen, 282
ALT and ALF). Currently, researches in the area of fish nutrition are seeking to relate variables 283
of hepatic metabolism with the performance of animals submitted to different diets. These 284
results may indicate the best use of nutrients, since the liver acts to receive and distribute 285
nutrients from the digestive process (Pretto, 2013). In some studies, for example, ALT activity 286
may suggest increased protein catabolism and gluconeogenic activity (Metón et al., 1999; 287
Lovatto et al., 2016), followed by changes in the concentration of free amino acids and other 288
hepatic and plasmatic metabolites. Obviously, these changes should be analyzed with caution, 289
relating them to performance responses, since animals submitted to nutritional deficiencies 290
usually present metabolic alterations that lead to unsatisfactory growth (Pianesso et al., 2015). 291
The alterations in protein and ammonia in this study do not allow us to infer decisively about a 292
metabolic modification of the animals with the levels of LPC included in the diets. Mainly 293
when we observed that they presented satisfactory growth without alterations in the corporal 294
composition. It should be noted that, even with the inclusion of LPC, the diets tested were 295
formulated according to the requirement of amino acids for the species (Montes-Girao and 296
Fracalossi, 2006), enhancing the protein quality of the tested ingredient. We observed in diet 297
without protein substitution of fish meal by LPC, that the of fish showed lower and higher 298
concentration of protein and ammonia hepatic, respectively. This result may indicate inefficient 299
use of the dietary protein, however, the growth results were similar among all the treatments. 300
Another result that underscores our discussion of the positive effects of LPC on fish 301
nutrition is the lack of changes in HSI. According to Cheng and Hardy (2004), Pacific white 302
prawns (Litopenaeus vannamei) fed with fish meal in the diet had lower HSI than those fed 303
with plant proteins. Similarly, the results of Mundheim et al. (2004) demonstrated that high 304
levels of plant protein sources in the salmon diet (Salmo salar L.) resulted in elevated liver size. 305
The authors note that this increase is usually due to energy storage by fat deposition. Our 306
findings do not follow this trend, as we did not observe differences in HSI, neither increase in 307
liver glycogen content nor greater deposition of body fat in fish fed with LPC levels. The 308
balance between protein and energy, food energy density and the quality of the sources used in 309
the experimental diets favored the use of nutrients and the similarity observed in the animal