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Aus dem Institut für Tierzucht und Tierhaltung
der Agrar- und Ernährungswissenschaftlichen Fakultät
der Christian-Albrechts-Universität zu Kiel
___________________________________________________________________________
RAPESEED PROTEIN PRODUCTS AS FISH MEAL
REPLACEMENT IN FISH NUTRITION
Dissertation
zur Erlangung des Doktorgrades
der Agrar- und Ernährungswissenschaftlichen Fakultät
der Christian-Albrechts-Universität zu Kiel
vorgelegt von
Master of Science
HANNO SLAWSKI
aus Neustadt in Holstein
Kiel, 2011
___________________________________________________________________________
Dekanin: Prof. Dr. K. Schwarz
Erster Berichterstatter: Prof. Dr. C. Schulz
Zweiter Berichterstatter: Prof. Dr. A. Susenbeth
Tag der mündlichen Prüfung: 14.07.2011
___________________________________________________________________________
Die Dissertation wurde mit dankenswerter finanzieller Unterstützung aus dem Europäischen Fischereifond und
dem Zukunftsprogramm Fischerei des Landes Schleswig-Holsteins angefertigt
II
Gedruckt mit Genehmigung der Agrar- und Ernährungswissenschaftlichen Fakultät der
Christian-Albrechts-Universität zu Kiel
III
Table of Contents
General Introduction ................................................................................................................... 1 Chapter 1: Replacement of fish meal with rapeseed protein concentrate in diets fed to common carp (Cyprinus carpio L.) ............................................................................................ 4 Chapter 2: Replacement of fish meal with rapeseed protein concentrate in diets fed to wels catfish (Silurus glanis L.) ......................................................................................................... 16 Chapter 3: Austausch von Fischmehl durch Rapsproteinkonzentrat in Futtermitteln für Steinbutt (Psetta maxima L.) .................................................................................................... 34 Chapter 4: Total fish meal replacement with rapeseed protein concentrate in diets fed to rainbow trout (Oncorhynchus mykiss W.) ................................................................................ 47 Chapter 5: Replacement of fish meal with albumin and globulin rapeseed protein fractions in diets fed to rainbow trout (Oncorhynchus mykiss W.) ............................................................. 69 Chapter 6: Total fish meal replacement with canola protein isolate in diets fed to rainbow trout (Oncorhynchus mykiss W.) .............................................................................................. 85 General Discussion ................................................................................................................. 102 General Summary ................................................................................................................... 111 Zusammenfassung .................................................................................................................. 114 Danksagung ............................................................................................................................ 117 Lebenslauf .............................................................................................................................. 118
IV
List of Tables
Table 1.1 Proximate composition and amino acid profiles of fish meal and
rapeseed protein concentrate and concentration of antinutritional factors detected in RPC
9
Table 1.2 Formulation, amino acid profiles and proximate composition of experimental diets for common carp
10
Table 1.3 Growth response, feed efficiencies and survival of carp fed experimental diets
11
Table 1.4 Proximate whole body composition of carp fed the experimental diets
11
Table 2.1 Nutrient composition and essential amino acid profiles of fish meal and rapeseed protein concentrate and concentration of antinutritional factors detected in RPC
20
Table 2.2 Formulation, essential amino acids composition and proximate composition of experimental diets
21
Table 2.3 Growth response, feed intake, feed efficiencies, condition factor and survival of wels catfish fed experimental diets
23
Table 2.4 Proximate whole body composition of wels catfish fed the experimental diets
24
Table 2.5 Blood haematocrit content and blood serum values of wels catfish fed experimental diets
25
Table 3.1 Nährstoff- und Aminosäurenzusammensetzung von Fischmehl und Rapsproteinkonzentrat
37
Table 3.2 Formulierung der Versuchsfuttermittel
38
Table 3.3 Nährstoff- und Aminosäurenzusammensetzung der Versuchsfuttermittel
39
Table 3.4 Ganzkörperzusammensetzung der Steinbutt nach der Fütterungsperiode
41
Table 3.5 Wachstumsparameter und Futterverwertung der Steinbutt nach dem Fütterungsversuch
41
Table 4.1 Proximate and amino acid composition of fish meal and rapeseed protein concentrate and concentration of antinutritional factors determined
51
V
Table 4.2 Formulation of experimental diets
52
Table 4.3 Proximate and amino acid composition of experimental diets
53
Table 4.4 Growth response, feed efficiencies and survival of rainbow trout fed experimental diets
56
Table 4.5 Proximate whole body composition of rainbow trout fed experimental diets
56
Table 4.6 Blood parameters of trout fed experimental diets
57
Table 5.1 Nutrient composition and amino acid profiles of fish meal, albumin concentrate and globulin concentrate
68
Table 5.2 Formulation and nutrient composition and amino acid profiles of experimental diets used in the digestibility trial
69
Table 5.3 Formulation, proximate nutrient composition and amino acid composition of experimental diets for rainbow trout
72
Table 5.4 Apparent digestibility coefficients
73
Table 5.5 Growth performance, feed intake and feed efficiencies of rainbow trout fed experimental diets
74
Table 5.6 Proximate whole body composition of rainbow trout fed experimental diets
75
Table 6.1 Nutrient composition and essential amino acid profiles of fish meal and canola protein isolate
83
Table 6.2 Formulation, nutrient composition and essential amino acid profiles of experimental diets used in the digestibility trial
84
Table 6.3 Formulation, proximate composition and essential amino acid profiles of experimental diets
86
Table 6.4 Apparent digestibility coefficients
88
Table 6.5 Growth response, feed intake and feed efficiencies of rainbow trout fed experimental diets
89
Table 6.6 Proximate whole body composition of rainbow trout fed experimental diets
89
1
General Introduction
In 2009 aquaculture production hit a landmark: half of all fish and shellfish destined for
human consumption were cultured, and production of farmed fish eclipsed that of wild caught
fish. But, the increased aquaculture production also accounted for 68 % of the worldwide fish
meal consumption (Naylor et al. 2009). However, fish meal, the most important source of
protein in fish feeds, is a limited resource with an annual production volume between 5 to 6.5
Mio t (FAO 2004). Tremendous price increases for fish meal together with environmental
concerns therefore force the aquaculture sector to find alternative protein sources to be
included in fish feeds. Presently, most relevant alternatives are protein concentrates derived
from vegetables. Among them, soybean protein concentrates have become a commonly
accepted fish feed ingredient and fish meal alternative (Gatlin et al. 2007). While soybean
ranks as number one oilseed worldwide (222.2 Mio t/a), protein products derived from
rapeseed, which ranks as number three oilseed worldwide (61.6 Mio t/a) (FAO 2010), are less
commonly used as fish feed ingredients. However, simple oilcakes or rapeseed meals with
increased protein content produced from oilcakes that were de-oiled with organic solvents
have been widely tested as protein sources in feeding trials with several fish species.
Experiments with rainbow trout (Burel et al. 2000a,c; Shafaeipour et al. 2008), Nile tilapia
(Davies et al. 1990), common carp (Dabrowski and Kozlowska 1981) and turbot (Burel et al.
2000a,b) have shown, that the nutritional quality of simple rapeseed products is below that of
fish meal although they contained a well balanced amino acid profile. Particularly
antinutritional factors (ANF) determine the quality of rapeseed products for fish nutrition. The
most prominent ANF in rapeseed products are glucosinolates, phytic acid, phenolic
constituents and indigestible carbohydrates (Francis et al. 2001). By several processing
techniques the level of antinutrients in rapeseed products can be reduced and their value for
fish nutrition can be improved. Dehulling of seeds and utilisation of high temperatures and
organic solvents during oil extraction as well as sieving of meal decrease content of
glucosinolates, phytate, fibre, cellulose, hemicellulose, sinapin and tannins (Fenwick et al.
1986; Anderson-Haferman et al. 1993). Protein extraction from meals by methanol-ammonia-
treatment or ethanol-treatment will increase protein level and effectively remove
glucosinolates, phenolic compounds, soluble sugars, such as sucrose, and some
oligosaccharides (Naczk and Shahidi 1990; Chabanon et al. 2007). In different countries,
rapeseed protein products of high quality were produced for application in animal nutrition.
2
However, these products were made for test purposes in small volumes until their potential as
protein source in animal nutrition is clarified. Besides nutritive quality, their costs of
production must decrease to make rapeseed protein products available at a competitive price
compared to other protein sources, especially fish meal.
In the present study, different protein protein products derived from rapeseed (including
canola) were tested as fish meal replacement in diets for several fish species. A high quality
rapeseed protein concentrate (RPC) with a protein content of 71 % was evaluated as fish meal
replacement in diets for common carp (chapter 1), wels catfish (chapter 2), turbot (chapter 3)
and rainbow trout (chapter 4). Based on the results presented in chapter 4, in chapter 5 the
potential of two rapeseed protein concentrates partitioned in albumin and globulin fractions as
fish meal alternatives was evaluated in a digestibility study and a consecutive growth trial
with rainbow trout. Compared to the RPC, the fractionized protein concentrates were
produced under lower cost and time effort. In chapter 6 a canola protein isolate with a crude
protein content of 81 % was evaluated as fish meal alternative in diets for rainbow trout. The
nutritional quality of the raw material was determined in a digestibility experiment followed
by a growth trial.
References
Anderson-Hafermann, J.C., Zhang, Y., Parsons, C.M., 1993. Effects of processing on the
nutritional quality of canola meal. Poultry Science 72, 326-333.
Burel, C., Boujard, T., Tulli, F., Kaushik, S.J., 2000a. Digestibility of extruded peas, extruded
lupin, and rapeseed meal in rainbow trout (Oncorhynchus mykiss) and turbot (Psetta
maxima). Aquaculture 188, 285–298.
Burel, C., Boujard, T., Kaushik, S.J., Boeuf, G., van der Geyten, S., Mol, K.A., Kühn, E.R.,
Quinsac, A., Krouti, M., Ribaillier, D., 2000b. Potential of plant-protein sources as fish
meal substitutes in diets for turbot (Psetta maxima): growth, nutrient utilisation and
thyroid status. Aquaculture 188, 363-382.
Burel, C., Boujard, T., Escaffre, A.M., Kaushik, S.J., Boeuf, G., Mol, K., Van Der Geyten, S.,
Kühn, E.R., 2000c. Dietary low glucosinolate rapeseed meal affects thyroid status and
nutrient utilization in rainbow trout (Oncorhynchus mykiss). Brit. J. Nutr. 83, 653–664.
Chabanon, G., Chevalot, I., Framboisier, X., Chenu, S., Marc, I., 2007. Hydrolysis of
rapeseed protein isolates: Kinetics, characterization and functional properties of
hydrolysates. Process Biochem. 42, 1419–1428.
3
Dabrowski, K., Kozlowska, H., 1981. Rapeseed meal in the diet of common carp reared in
heated waters. I. Growth of fish and utilization of the diet. In: K. Tiews (ed.).
Aquaculture in Heated Effluents and Recirculation Systems. Heenemann, Hamburg, pp.
263-274.
Davies, S.J., McConnel, S., Bateson, R.I., 1990. Potential of rapeseed meal as an alternative
protein source in complete diets for tilapia (Oreochromis mossambicus Peters).
Aquaculture 87, 145-154.
Food and Agriculture Organization (FAO), United Nations (2004). FAO Fisheries
Department, Fishery Information, Data and Statistics Unit. Fishstat Plus: Universal
software for Fishery Statistical Time series, version 2.30 (www.fao.org).
FAO (2010): http://faostat.fao.org/site/567/default.aspx#ancor
Fenwick, G.R., Spinks, E.A., Wilkinson, A.P., Henry, R.K., Legoy, M.A., 1986. Effect of
processing on the antinutrient content of rapeseed. J. Sci. Food Agr. 37, 735-741.
Francis, G., Makkar, H.P.S., Becker, K., 2001. Antinutritional factors present in plant-derived
alternate fish feed ingredients and their effects in fish. Aquaculture 199, 197–227.
Gatlin III, D.M., Barrows, F.T., Brown, P., Dabrowski, K., Gaylord, T.G., Hardy, R.W.,
Herman, E., Hu, G., Krogdahl, Å., Nelson, R., Overturf, K., Rust, M., Sealey, W.,
Skonberg, D., Souza, E.J., Stone, D., Wilson, R., Wurtele, E., 2007. Expanding the
utilization of sustainable plant products in aquafeeds: a review. Aquaculture Research
38, 551-579.
Naczk, M., Shahidi, F., 1990. Carbohydrates of canola and rapeseed. In: F. Shahidi (ed.).
Canola, Rapeseed: Production, Chemistry, Nutrition & Processing Technology. Van
Nostrand Reinhold, New York, pp211-220.
Naylor, R.L., Hardy, R.W., Bureau, D.P., Chiu, A., Elliott, M., Farrell, A.P., Forster, I., Gatlin
III., D.M., Goldburg, R.J., Hua, K., Nichols, P.D., 2009. Feeding aquaculture in an era
of finite resources. PNAS 106, 15103-15110.
Shafaeipour, A., Yavari, V., Falahatkar, B., Maremmazi, J.G.H., Gorjipour, E., 2008. Effects
of canola meal on physiological and biochemical parameters in rainbow trout
(Oncorhynchus mykiss). Aquaculture Nutrition 14, 110–119.
4
Chapter 1: Replacement of fish meal with rapeseed protein concentrate in
diets fed to common carp (Cyprinus carpio L.)
H. Slawski1,2, H. Adem3, R.-P. Tressel3, K. Wysujack4, U. Koops4 and C. Schulz1,2
1Gesellschaft für Marine Aquakultur mbH, Hafentörn 3, D-25761 Büsum
2Institute of Animal Breeding and Husbandry, Christian-Albrechts-Universität zu Kiel,
D-24098 Kiel
3Pilot Pflanzenöltechnologie Magdeburg e.V., Berliner Chaussee 66, D-39114 Magdeburg
4Johann Heinrich von Thünen-Institut, Federal Research Institute of Rural Areas, Forestry and
Fisheries; Institute of Fisheries Ecology, Wulfsdorfer Weg 204, D-22926 Ahrensburg
Published in: The Israeli Journal of Aquaculture (2011) 63, 605-611.
5
Abstract
The potential of rapeseed protein concentrate (RPC) as fish meal alternative in diets for
common carp (initial average weight 26.7 ± 0.8 g) was evaluated. Triplicate groups of fish
were fed isonitrogenous (40.4 ± 0.2 % crude protein) and isocaloric (21.4 ± 0.1 kJ g-1)
experimental diets with 0% (R0), 33% (R33), 66% (R66) or 100 % (R100) of fish meal
replaced with rapeseed protein concentrate. At the end of the 56 days feeding period, growth
parameters and feed efficiencies were not significantly different between fish fed on diet R0
and R33. Diets R66 and R100 led to reduced diet intake and feed efficiencies resulting in
lower growth performances. It appears that diet taste and amino acid profiles were negatively
affected by high inclusion levels of rapeseed protein concentrate resulting in reduced diet
acceptance and protein value. It is concluded, that the used rapeseed protein concentrate can
effectively replace 33 % of fish meal in diets for carp without using palatability enhancers or
amino acid supplements.
6
Introduction
Wide availability, high protein content and a desirable amino acid profile have caused an
interest in rapeseed products as fish meal alternative in fish feeds. Rapeseed and canola
products have been tested as protein sources in diets for several fish species, including
rainbow trout (Thiessen et al. 2004), Coho salmon (Higgs et al. 1979), Chinook salmon
(Satoh et al. 1998), tilapia (Yigit and Olmez 2009), channel catfish (Lim et al. 1998), silver
perch (Booth and Allan 2003), carp (Dabrowski and Kozlowska 1981), red sea bream
(Glencross et al. 2004), and turbot (Burel et al. 2000ac). It was found, that the nutritional
quality of rapeseed products largely depends on their levels of antinutritional factors.
Prominent antinutritional factors in rapeseed are glucosinolates, phytic acid, phenolic
constituents (e.g. tannins), and indigestible carbohydrates (Francis et al. 2001). Several
processing techniques have been adapted to reduce the level of antinutrients in rapeseed in
order to improve its value for fish nutrition. Dehulling of seeds and utilisation of high
temperatures and organic solvents (hexane) during oil extraction as well as sieving of meal
decrease content of glucosinolates, phytate, fibre, cellulose, hemicellulose, sinapin and
tannins (Anderson-Haferman et al. 1993; Mawson et al. 1993, 1994ab, 1995; Leming et al.
2004) and increase protein level in meals (Mwachireya et al. 1999). In addition, protein
extraction from meals by methanol-ammonia-treatment or ethanol-treatment will further
increase protein level and effectively remove glucosinolates, phenolic compounds, soluble
sugars, such as sucrose, and some oligosaccharides (Naczk and Shahidi 1990; McCurdy and
March 1992; Chabanon et al. 2007) but will also increase levels of non-digestible fibre
(Mwachireya et al. 1999). In the present study liquid water extractions combined with
ultrafiltration were used to further increase the protein concentration of the final product and
at the same time deposit non-digestible fibres. The resulting rapeseed protein concentrate
(RPC) contained 71 % crude protein. Momentarily, rapeseed and canola protein products of
similar quality are being produced in different countries for application in animal nutrition.
However, these products are produced for test purpose until their potential as protein source in
animal nutrition is clarified. Besides nutritive quality, their costs of production will have to
become low enough to make rapeseed and canola protein concentrates available at a
competitive price compared to other protein sources, e.g. fish meal. As basic trial in a series
of consecutive feeding trials in order to optimize the produced RPC for application in fish
feeds, the product was tested as fish meal replacement in pelleted diets, using juvenile
7
common carp as model species. By this, we intended to determine the fundamental native
limitations of our RPC as fish feed ingredient.
Materials and methods
Diet preparation and experimental procedures
Four experimental diets were formulated to replace fish meal with rapeseed protein
concentrate (RPC) at 0, 33, 66, or 100 % (designated as R0, R33, R66 and R100,
respectively). Vitamins and minerals were added to diets to meet the dietary requirements of
carp (NRC, 1993). Diets were manufactured to give pellets 4 mm in diameter (L 14-175,
AMANDUS KAHL, Reinbek, Germany). The diets were formulated to be isonitrogenous
(40.4 ± 0.2 % crude protein) and isocaloric (21.4 ± 0.1 kJ g−1). Since this is the first trial in a
series of consecutive feeding trials investigating our RPC as fish meal replacement, we
intended to highlight direct effects on feed quality resulting from dietary RPC incorporation.
Therefore diets were formulated without palatability enhancers or crystalline amino acids.
Diet formulations, nutritional compositions and amino acid profiles are given in Table 1.1.
Solvent extracted RPC was obtained from PPM, Magdeburg, Germany. For oil extraction,
rapeseed was cold pressed and residual oil removed by a hexane treatment. Glucosinolates
were extracted with an ethanol solution. Liquid water extraction as well as dia- and
ultrafiltration of proteins followed by spray drying provided a protein concentrate with 71 %
crude protein content (Table 2.1).
The growth trial was conducted at the Johann Heinrich von Thünen Institute of Fisheries
Ecology, Ahrensburg, Germany. In the growth trial, common carp (Cyprinus carpio L.) was
used as model fish. In its juvenile stage, common carp has a high dietary protein requirement
(Fine et al. 1996) making this relatively modest fish an ideal model species for fish meal
replacement studies. Juvenile common carp that had been hatched in the institute were used.
One week before the experiment started 12 fish were stocked in each of twelve experimental
tanks (70 L; bottom surface 480 cm2), being part of a freshwater recirculation system. Tanks
were provided with water at 1 L min-1 (temperature: 23.8 ± 0.7 °C; O2: 6.5 ± 0.7 mg L-1; pH:
7.0 ± 0.7; NH4+: <0.1 mg L-1; NO2
-: <0.2 mg L-1). Photoperiod was in accordance to natural
rhythmic from February to April at our latitude (53° 41' 0" N). For a one week adaptation
period fish were fed the control diet (Table 1.1) in 4 daily meals until apparent satiation. After
the adaptation period, fish were fasted for one day and initial average weight was determined
(26.7 ± 0.8 g). Triplicate groups of 10 fish were fed the experimental diets in four daily meals
8
(at 8.00 a.m., 11.00 a.m., 2.00 p.m., 5.00 p.m.) to apparent satiation for 56 days. At the
beginning and at end of the experiment, 2 fish per tank were removed and analyzed for
proximate body composition.
Chemical analysis and laboratory procedures
Experimental diets and homogenized fish bodies were analysed for dry matter (DM) (105°C,
until constant weight), crude ash (550°C, 2 hours), crude fat (Soxtec HT6, Tecator, Höganäs,
Sweden) and crude protein content (N x 6.25; Kjeltec Auto System, Tecator, Höganäs,
Sweden). Raw material and dietary amino acid concentrations were analysed as described by
Tzovenis et al. (2009).
Calculations and statistical analysis
Weight gain (WG): (final weight – initial weight) / initial weight × 100; Specific growth rate
(SGR) (% per day): (ln final body weight – ln initial body weight) × 100 / days fed; Feed
conversion ratio (FCR): g dry feed intake / g wet body weight gain; Protein efficiency ratio
(PER): g wet body weight gain / g protein intake; Gross energy intake (GEI): gross energy
content in diet × g dry feed intake; Survival (%): initial fish count – dead fish count × 100 /
initial fish count.
All diets were assigned by a completely randomized design. The data were checked for
normal distribution using Kolmogoroff Smirnov Test and eventually subjected to
transformation. Data were analyzed by one-way analysis of variance (ANOVA) with “R“
statistical software. When differences among groups were found, the differences in means
were made with Tukey’s Honestly Significant Difference test. Statistical significance was
determined by setting the aggregate type I error at 5 % (P<0.05) for each set of comparisons.
Data are presented as means±SD.
9
Table 1.1: Formulation (g kg-1), amino acid profiles (% of dietary protein) and proximate composition (% of dry
matter) of experimental diets for common carp
Ingredients R0 R33 R66 R100
Herring meala 240 160 80 0
Soyprotein concentrateb 160 160 160 160
Rapeseed protein concentratec 0 75 155 235
Wheat glutend 150 150 150 150
Sunflower oil 53 57 58 60
Rapeseed oil 27 28 29 30
Dextrose 150 150 148 145
Maize starch 200 200 200 200
Vit/MinMixe 20 20 20 20
Amino acids
Aspartic acid 7.40 7.28 7.33 7.29
Threonine 3.38 3.37 3.56 3.68
Serine 4.43 4.29 4.67 4.76
Glutamic acid 21.45 22.01 22.92 23.80
Prolin 7.40 7.34 7.57 7.76
Glycin 5.57 5.12 4.72 4.39
Alanine 4.59 4.31 4.14 3.95
Cystine 1.35 1.51 1.71 1.92
Valine 4.38 4.53 4.38 4.52
Methionine 1.84 1.75 1.69 1.63
Isoleucine 3.89 4.02 3.85 4.02
Leucine 6.94 7.07 7.25 7.52
Phenylalanine 4.51 4.56 4.67 4.87
Histidine 2.27 2.37 2.48 2.63
Lysine 4.67 4.34 3.80 3.24
Arginine 5.38 5.53 5.75 6.00
Proximate composition
Dry matter (%) 91.7 92.4 93.4 94.0
Crude protein 40.4 40.1 40.6 40.5
Crude fat 11.0 11.1 10.4 9.4
Ash 7.6 6.3 5.3 4.1
NfEf 41.0 42.5 43.7 46.0
Gross energyg (kJ g-1) 21.2 21.5 21.5 21.5 aVFC GmbH, Cuxhaven, Germany; bIMCOSOY 60 Piglet, IMCOPA, Araucaria, Brasil; cPPM, Magdeburg, Germany; dEuroduna-Technologies GmbH, Barmstedt, Germany; eAA-Mix 517158 & 508240, Vitfoss, Gråsten, Denmark; fNitrogen
free extract = 100 – (%crude protein + %crude fat + %ash + %fibre); gCalculated by: crude protein = 23.9 kJ g-1; crude fat =
39.8 kJ g-1; NfE, fibre: 17.6 kJ g-1
10
Table 1.2: Proximate composition (% dry matter) and amino acid profiles (% of dietary protein) of fish meal
(FM) and rapeseed protein concentrate (RPC) and concentration of antinutritional factors detected in RPC
FM RPC
Dry matter (%) 91.6 94.2
Crude protein 69.0 71.0
Crude fat 7.0 2.2
Ash 20.7 9.2
Crude fibre 0.5 4.8
NfEa 2.8 12.8
Gross energyb (kJ g-1) 19.9 25.2
Amino acids
Aspartic acid 8.29 7.60
Threonine 3.90 4.44
Tryptophane 0.84 1.42
Serine 4.06 4.40
Glutamic acid 12.47 17.80
Prolin 4.69 5.89
Glycine 8.13 5.29
Alanine 6.41 4.70
Cystine 0.80 2.18
Valine 4.45 5.43
Methionine 2.37 2.03
Isoleucine 3.63 4.29
Leucine 6.46 7.81
Tyrosine 2.62 3.28
Phenylalanine 3.52 4.28
Histidine 2.00 2.99
Lysine 6.55 5.70
Arginine 5.84 7.49
Glucosinolates (µmol g-1) 0.2
Phytic acid (mg kg-1) < 500
Tannins (g 100g-1) < 0.005 aNitrogen free extract = 100 – (%crude protein + %crude fat + %ash + %fibre) bCalculated by: crude protein = 23.9 kJ g-1; crude fat = 39.8 kJ g-1; NfE, fibre: 17.6 kJ g-1
11
Results
Final weight, weight gain and standard growth rate were not significantly different between
fish fed on the control diet and diet R33. In addition, feed and gross energy intake as well as
feed conversion and protein efficiency ratio were not significantly different between control
diet and diet R33 fed fish. On the contrary, fish fed on diets R66 and R100 showed
significantly reduced growth performance values, lower feed intake and decreased feed
efficiencies (Table 1.3). No significant differences in whole body composition were detected
between fish fed control diet and fish receiving test diets (Table 1.4).
Table 1.3: Growth response, feed efficiencies and survival of carp fed experimental diets
Ingredients R0 R33 R66 R100
Initial weight (g) 26.4 ± 0.6 26.7 ± 0.6 26.5 ± 0.8 27.2 ± 1.3
Final weight (g) 73.5a ± 4.1 71.2a ± 4.3 60.6b ± 3.2 49.7c ± 2.3
Weight gain (%) 178.4a ± 20.3 167.0a ± 17.3 128.7b ± 18.3 83.3c ± 16.3
SGR (% day-1) 1.83a ± 0.13 1.75a ± 0.12 1.47b ± 0.15 1.08c ± 0.16
DM Feed intake 51.5a ± 4.11 49.43a ± 3.82 42.20b ± 2.47 34.80c ± 3.80
FCR 1.09a ± 0.04 1.11a ± 0.02 1.24b ± 0.07 1.56c ± 0.14
PER 36.96a ± 1.23 36.09a ± 0.69 32.72b ± 1.95 26.18c ± 2.36
GEI (kJ) 109.2a ± 10.7 106.3ab ± 10.1 90.7b ± 6.5 74.8bc ± 10.0
Survival (%) 96.7 ± 5.8 96.7 ± 5.8 93.3 ± 5.8 96.7 ± 5.8
Means in the same row with different superscript letters are significantly different determined by Tukey's test (P<0.05)
Table 1.4: Proximate whole body composition of carp fed the experimental diets
Ingredients R0 R33 R66 R100
Moisture (%) 76.3 ± 0.3 75.9 ± 0.3 75.5 ± 0.4 75.3 ± 0.4
Crude protein (% w.w.a) 16.7 ± 0.3 16.9 ± 0.8 17.2 ± 0.5 17.3 ± 0.3
Crude fat (% w.w.) 4.4 ± 0.4 4.3 ± 0.5 4.4 ± 0.3 4.2 ± 0.3
Ash (% w.w.) 2.1 ± 0.2 2.3 ± 0.1 2.4 ± 0.1 2.5 ± 0.1
Initial body composition: moisture 75.7 %, crude protein 12.6 %, crude fat 2.6 %, ash 1.3 % aw.w.: wet weight
12
Discussion
Rapeseed protein concentrate has been found to be a viable alternative to fish meal in fish
feeds. Different replacement levels of fish meal have been achieved by inclusion of rapeseed
and canola protein concentrate in feeding trials with several fish species (Dabrowski and
Kozlowska 1981; Lim et al. 1998; Burel et al. 2000; Booth and Allan 2003; Glencross et al.
2004; Thiessen et al. 2004; Yigit and Olmez 2009). It was found, that antinutritional factors
present in rapeseed and canola particularly determine its value for fish nutrition. It was
therefore recommended to greatly reduce antinutritional factors in rapeseed protein
concentrates in order to achieve higher fish meal replacement levels in fish diets. In the
present study a rapeseed protein concentrate (RPC) with 71 % crude protein content and
particularly low levels of glucosinolates, phytic acid and tannins (Table 1.2) was tested as fish
meal alternative in diets for common carp. The RPC successfully replaced 33 % of fish meal
protein from a control diet without retarding fish growth performance, feed intake or feed
efficiencies. At 66 % and 100 % fish meal replacement with RPC, however, fish growth
performance, feed intake and feed efficiency decreased compared to the control group. This is
in contrast to findings by Dabrowski and Kozlowska (1981) who successfully replaced 100 %
of fish meal protein from diets for common carp with rapeseed meal protein without reducing
fish weight gain or standard growth rate. We believe that a 66 and 100 % fish meal
replacement with our RPC negatively affected diet palatability and protein quality and
therefore limited fish growth performance.
We observed, that increasing dietary levels of RPC as fish meal replacement diminished diet
acceptance by common carp. This is documented in lower feed intake in fish fed diet R66 and
R100 compared to diet R0 and R33 (Table 1.3). It is known that the bitter taste exuded by
glucosinolates and their breakdown products present in rapeseed and canola meals can
potentially retard diet acceptance by fish. This was found in rainbow trout and turbot at
dietary glucosinolate levels of 7.3 µmol g-1 or 18.7 µmol g-1, respectively (Burel et al.
2000b,c). Because the RPC used in our study contained about 0.2 µmol g-1 (Table 1.2) the
highest dietary glucosinolate concentration was therefore 0.05 µmol g-1 in R100.
Nevertheless, we noticed a mustard smell in diets R66 and R100 resulting from high RPC
inclusion. This smell clearly influenced diet acceptance by carp as RPC was the dominant
protein source in diets R66 and R100. In Dabrowski and Kozlowska (1981) diet acceptance
by carp was probably equalized by the usage of several protein sources (blood meal, yeast,
soyabean meal, barley meal and rapeseed meal) giving a more versatile diet taste.
13
Beside palatability problems, high dietary inclusion of RPC led to insufficient dietary amino
acid concentrations for common carp. Table 1.1 shows the dietary amino acid profiles. Diets
high in RPC are low in lysine, because of low fish meal inclusion and no inclusion of other
protein sources of animal origin. As reported by Ogino (1980) the lysine requirement of
common carp is 5.7 % of dietary protein. But, although diets R0 and R33 contained 4.67 %
and 4.34 % lysine respectively, good growth results obtained with these diets counteract the
observation by Ogino (1980). However, the fast drop in growth rates in fish fed on diet R66
and R100 confirms an inappropriate quality of their dietary protein.
In conclusion, 33 % of fish meal can be replaced by rapeseed protein concentrate from diets
for common carp. Consecutive feeding trials will clarify if higher fish meal replacement with
our RPC can be achieved by using palatability enhancers and amino acid supplements.
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16
Chapter 2: Replacement of fish meal with rapeseed protein concentrate in
diets fed to wels catfish (Silurus glanis L.)
H. Slawski1,2, H. Adem3, R.-P. Tressel3, K. Wysujack4, U. Koops4,
S. Wuertz1 and C. Schulz1,2
1Gesellschaft für Marine Aquakultur mbH, Hafentörn 3, D-25761 Büsum
2Institute of Animal Breeding and Husbandry, Christian-Albrechts-Universität zu Kiel,
D-24098 Kiel
3Pilot Pflanzenöltechnologie Magdeburg e.V., Berliner Chaussee 66, D-39114 Magdeburg
4Johann Heinrich von Thünen-Institut, Federal Research Institute of Rural Areas, Forestry and
Fisheries; Institute of Fisheries Ecology, Wulfsdorfer Weg 204, D-22926 Ahrensburg
Published in: Aquaculture Nutrition (2011) DOI: 10.1111/j.1365-2095.2011.00857.x
17
Abstract
The potential of rapeseed protein concentrate as fish meal alternative in diets for wels catfish
(initial average weight 86.5 ± 1.9 g) was evaluated. Sixteen fish were stocked into each of
twelve experimental tanks being part of a freshwater recirculation system. Fish were
organized in triplicate groups and received isonitrogenous (603 ± 3 g CP kg-1) and isocaloric
(23.0 ± 0.3 kJ g-1) experimental diets with 0, 25, 50, and 75 % of fish meal replaced with
rapeseed protein concentrate (710 g CP kg-1). At the end of the 63 days feeding period, weight
gain, standard growth rate, feed intake, feed conversion ratio and protein efficiency showed
no significant difference between control group and fish fed on diets with 25 % reduced fish
meal content by inclusion of rapeseed protein concentrate. Higher dietary fish meal
replacement negatively affected diet quality and palatability resulting in reduced feed intake,
feed efficiencies and fish performance. However, blood serum values of triglycerides, glucose
and protein were not significantly different between treatment groups, still indicating a
favourable nutrient supply from all experimental diets.
18
Introduction
In 2009, worldwide production of rapeseed (including canola) was 61.6 Mio t. Thus,
rapeseed, commonly produced in temperate regions, ranked as number three oilseed
worldwide, only surpassed by soybean (222.2 Mio t) and cotton seed (64.0 Mio t) (FAO
2010). For soybean, a crop mainly cultivated in warm regions, efforts and research have been
undertaken to make it a commonly accepted fish feed ingredient and fish meal alternative
(Gatlin et al. 2007). The usage of rapeseed products as fish feed ingredients, however, is
limited. Either simple oilcakes or rapeseed meals with increased protein content produced
from oilcakes that were de-oiled with organic solvents have been tested as protein sources in
feeding trials with several fish species, among them Oncorhynchus mykiss (Burel et al.
2000ac, 2001; Thiessen et al. 2003, 2004; Shafaeipour et al. 2008), Oreochromis
mossambicus (Davies et al. 1990), Ictalurus punctatus (Webster et al. 1997), Cyprinus carpio
(Dabrowski and Kozlowska 1981), Pagrus auratus (Glencross et al. 2004) and Psetta maxima
(Burel et al. 2000ab). In general, experimental results showed that the nutritional quality of
simple rapeseed products is below that of fish meal although they contained a well balanced
amino acid profile. Particularly antinutritional factors (ANF) determine the quality of
rapeseed products for fish nutrition. Prominent ANF in rapeseed products are glucosinolates,
phytic acid, phenolic constituents (e.g. tannins) and indigestible carbohydrates (Mawson et al.
1995; Francis et al. 2001). Several processing techniques can be adapted to reduce the level of
antinutrients in rapeseed products and improve their value for fish nutrition. Dehulling of
seeds and utilisation of high temperatures and organic solvents (hexane) during oil extraction
as well as sieving of meal decrease content of glucosinolates, phytate, fibre, cellulose,
hemicellulose, sinapin and tannins (Fenwick et al. 1986; Anderson-Haferman et al. 1993;
Tripathi et al. 2000) and increase protein level in meals (Mwachireya et al. 1999). Protein
extraction from meals by methanol-ammonia-treatment or ethanol-treatment will increase
protein level and effectively remove glucosinolates, phenolic compounds, soluble sugars, such
as sucrose, and some oligosaccharides (e.g. raffinose and stachyose) (Naczk and Shahidi
1990; Chabanon et al. 2007). Last but not least water treatment appears to be a cost effective
method for removing glucosinolates from rapeseed meals (Tyagi 2002). Sporadically,
rapeseed protein products of high quality are being produced in different countries for
application in animal nutrition. However, these products are produced for test purposes in
small volumes until their potential as protein source in animal nutrition is clarified. Besides
nutritive quality, their costs of production will have to become low enough to make rapeseed
19
protein products available at a competitive price compared to other protein sources, especially
fish meal. In the present study, a high quality rapeseed protein concentrate containing 710 g
CP kg-1 was tested as fish meal replacement in fish diets. Diets were fed to wels catfish
(Silurus glanis L.), a carnivorous species that is believed to have potential for indoor
recirculation farming in Europe as a high value product for local markets (Mazurkiewicz et al.
2008). Fish performance and blood serum parameters were investigated to evaluate rapeseed
protein concentrate as fish meal alternative in diets for wels catfish.
Materials and methods
Diet preparation and experimental procedures
Four experimental diets were formulated in which fish meal was replaced with rapeseed
protein concentrate (RPC) at 0, 25, 50, and 75 % level (designated as R0, R25, R50, or R75,
respectively). Solvent extracted RPC was obtained from the PPM e.V., Magdeburg, Germany.
For the production of RPC a batch of rapeseed (variety Lorenz, Norddeutsche Pflanzenzucht,
Germany) was conditioned in a vacuum dryer for 15 minutes at 60-70 °C to inactivate the
enzyme myrosinase. Then rapeseed was cold pressed. To remove residual oil from the oilcake
(129 g kg-1 oil, 313 g kg-1 protein) it was crushed into 1-5 mm particle size followed by a
hexane treatment. The treatment lasted for two hours and the incubation temperature was 60
°C. Hexane treated rapeseed meal extract was desolventised under pressure to remove hexane
(< 300 ppm), then rapeseed meal extract was further crushed to a particle size of 0.2 to 0.1
mm. A four step treatment using a 75 % ethanol solution (35 min at 60 °C) aimed to remove
glucosinolates from rapeseed meal extract. This resulted in a residual oil content of the nearly
glucosinolate free rapeseed meal extract of 11 g kg-1 and a protein content of 398 g kg-1. In the
following, protein was gained through liquid water extraction (rapeseed meal extract 1:15
water). For this, the suspension was heated to 40-45 °C followed by two hours of constant
agitation. Afterwards the suspension was decanted. Following decantation the solvent was
collected and residue material was secondly extracted (residue 1:10 water, 5 % NaCl) at 40-45
°C and one hour contact time under constant agitation. Following extraction the suspension
was decanted. Solvent was collected and residue prepared for a third extraction. Then solvents
of extraction 1, 2 and 3 were collected to remove low-molecular compounds and to
concentrate dissolved proteins by dia- and ultrafiltration. During filtration conductivity was
checked. Protein washing ended, when conductivity was 5-6 mS cm-1, corresponding to a
protein content of 600 g kg-1. The gained material was spray dried at 70-80 °C, which led to a
20
rapeseed protein concentrate with 710 g kg-1 protein content (Table 2.1). Vitamins and
minerals were added to diets to meet the dietary requirements of freshwater fish (NRC 1993).
The diets were formulated to be isonitrogenous (603 ± 3 g CP kg-1) and isocaloric (23.0 ± 0.3
MJ kg-1). Essential amino acid concentrations did not differ considerably between
experimental diets. The diets were manufactured to give pellets 4 mm in diameter (L 14-175,
AMANDUS KAHL, Reinbek, Germany). Diet formulations, proximate compositions and
amino acid profiles are given in Table 2.2.
Table 2.1: Nutrient composition (g kg-1 dry matter) and essential amino acid profiles (g kg-1 protein) of fish meal
(FM) and rapeseed protein concentrate (RPC) and concentration of antinutritional factors detected in RPC
FM RPC
Dry matter 916 942
Crude protein 690 710
Crude fat 70 22
Ash 207 92
Phosphorus 29 21
Crude fibre 5 48
NfEa 28 128
Gross energyb (kJ g-1) 19.9 25.2
Essential amino acids
Arginine 58.4 74.9
Histidine 20.0 29.9
Isoleucine 36.3 42.9
Leucine 64.6 78.1
Lysine 65.5 57.0
Methionine 23.7 20.3
Phenylalanine 35.2 42.8
Threonine 39.0 44.4
Tryptophane 8.4 14.2
Valine 44.5 54.3
Glucosinolates (µmol g-1) 0.2
Phytic acid (g kg-1) < 0.5
Tannins (g 100g-1) < 0.005 aNitrogen free extract = 100 – (%crude protein + %crude fat + %ash + %fibre). bCalculated by: crude protein = 23.9 MJ kg-1; crude fat = 39.8 MJ kg-1; NfE, fibre: 17.6 MJ kg-1.
21
Table 2.2: Formulation (g kg-1), essential amino acids composition (g kg-1 crude protein) and proximate
composition (g kg-1 dry matter) of experimental diets
Ingredients R0 R25 R50 R75
Herring meala 500 375 250 125
Rapeseed protein concentrate 0 122 243 362
Soyprotein concentrateb 110 110 110 110
Blood mealc 105 105 105 105
Wheat glutend 65 65 65 65
Fish oila 120 126 132 138
Dextrosec 80 77 75 75
Vit/MinMixe 20 20 20 20
Essential amino acids
Arginine 52.9 55.6 58.3 59.1
Histidine 30.9 33.0 33.9 34.6
Isoleucine 30.2 31.7 33.2 32.1
Leucine 75.9 79.1 81.3 81.7
Lysine 59.3 59.2 57.7 54.7
Methionine + Cysteine 27.5 28.9 30.4 31.1
Phenylalanine 42.9 44.9 46.4 47.1
Threonine 34.1 35.6 36.6 37.1
Valine 49.9 52.0 53.5 52.6
Proximate composition
Dry matter (g kg-1) 922 931 939 944
Crude protein 607 603 600 600
Crude fat 158 165 158 148
Ash 131 114 99 83
Phosphorus 14.8 14.1 13.0 12.7
NfE + fibref 104 118 143 169
calculated Glucosinolates
(µmol g-1)
0 0.02 0.05 0.07
Gross energyg (MJ kg-1) 22.6 23.1 23.1 23.2 aVFC GmbH, Cuxhaven, Germany; bIMCOSOY 60 Piglet, IMCOPA, Araucaria, Brasil.; cEuroduna-Technologies GmbH,
Barmstedt, Germany; dCargill Deutschland GmbH, Krefeld, Germany; eAA-Mix 517158 & 508240, Vitfoss, Gråsten,
Denmark; fNitrogen free extract = 100 – (%crude protein + %crude fat + %ash).; gCalculated by: crude protein = 23.9 MJ kg-
1; crude fat = 39.8 MJ kg-1; NfE, fibre: 17.6 MJ kg-1.
Tryptophane was not analyzed.
22
The growth trial was conducted at the Johann Heinrich von Thünen Institute of Fisheries
Ecology, Ahrensburg, Germany. Juvenile wels catfish (Silurus glanis L.) were obtained from
the Ahrenhorster Edelfisch GmbH & CO KG (Ahrenhorst, Germany). Two weeks before the
experiment started 17 fish were stocked in each of nine experimental tanks (96 L; bottom
surface 480 cm2), being part of a recirculation system. Tanks were provided with freshwater
at 2 L min-1 (temperature: 26.9 ± 0.7 °C; O2: 6.8 ± 0.5 mg L-1; pH: 7.3 ± 0.5; NH4+: <0.6 mg
L-1; NO2-: <0.2 mg L-1). Photoperiod was in accordance to natural rhythmic. In respect of the
fishes’ light sensitivity, tanks were half covered with translucent plastic lids. For a two week
adaptation period fish were fed the control diet in four daily meals until apparent satiation.
After the adaptation period, initial average fish weight was determined (86.5 ± 1.9 g). For an
experimental period of 63 days, triplicate groups of fish were fed the experimental diets in
four daily meals (8:00 and 11:00 a.m., 2:00 and 5:00 p.m.) until apparent satiation. At the
beginning and at end of the experiment, two fish per tank were removed and stored at -23 ºC
for the determination of initial and final body composition.
Sampling
At the end of the feeding period, blood samples from the caudal vein and artery of eight fish
per experimental treatment were taken with a heparinized syringe (1 ml). Blood haematocrit
percentage was determined after centrifugation (3500 rpm, 6 min) of glass tubes filled with
fresh blood in a haematocrit centrifuge (Haematokrit 210, Andreas Hettich GmbH & Co.KG,
Tuttlingen, Germany). Remaining fresh blood was filled in Eppendorf tubes and centrifuged
(4000 rpm, 5 min). Supernatant blood plasma was separated into Eppendorf tubes and stored
at -84 °C in a freezer.
Chemical analysis and laboratory procedures
Diets and homogenised fish bodies were analysed in duplicate for proximate composition.
Dry matter was calculated from weight loss after drying in an oven at 105 °C until constant
weight. Fat content was determined after HCl hydrolysis (Soxtec HT6, Tecator, Höganäs,
Sweden) and total nitrogen content by the Kjeldahl technique (protein = N × 6.25; Kjeltec
Auto System, Tecator, Höganäs, Sweden). Ash content was calculated from weight loss after
incineration of samples in a muffle furnace for 2 hours at 550 °C. Raw material and dietary
amino acid concentrations were analysed as described by Tzovenis et al. (2009). Blood
plasma concentrations of triglycerides, glucose and protein were determined using a
23
microplate reader (Infinite 200®, TECAN Group Ltd., Männedorf, CH) and commercial kits
(Triglycerides GPO and Glucose GOD-PAP, Greiner Diagnostic GmbH, Bahlingen,
Germany; Roti®-Quant, CARL ROTH GmbH + Co.KG, Karlsruhe, Germany).
Calculations and statistical analysis
Fish performance was determined, using the following formulae:
Specific growth rate (SGR, % day-1) = (ln final body weight – ln initial body weight) × 100 /
days fed
Feed intake as % body weight day-1 = the mean feed consumption per fish per day as a
percentage of the daily fish body weight for the experimental period. The daily fish body
weight was calculated using daily SGR values equal to the final SGR of each tank.
Feed conversion ratio (FCR) = g dry feed intake / g wet body weight gain
Protein efficiency ratio (PER) = g wet body weight gain / g protein intake
Condition factor (CF) = g body weight / cm total length3 × 100
Survival (%) = (initial fish count - dead fish count) / initial fish count × 100
All diets were assigned by a completely randomized design. Biological and analytical data
were checked for normal distribution using the Kolmogoroff Smirnov Test and eventually
subjected to transformation. Data were subjected to regression and one-way analysis of
variance (ANOVA) using SPSS 17.0 for Windows. When differences among groups were
identified, multiple comparisons among means were made using Tukey’s HSD test. Statistical
significance was determined by setting the aggregate type I error at 5% (P<0.05) for each set
of comparisons.
Results
No significant differences in growth performance parameters and feed efficiencies were
detected between control diet and R25 diet fed fish. Compared to the control group, fish
growth performance, voluntary feed intake and feed efficiencies declined at fish meal
replacement levels above 25 %. Feed intake as % body weight was not affected up to 50 %
fish meal replacement level (Table 2.3). While fish growth performance, voluntary feed intake
and feed efficiencies significantly correlated with the dietary inclusion level of RPC (Table
2.3) no correlation was found between feed intake as % body weight and dietary inclusion of
RPC. No significant differences in whole body composition were detected between fish fed
24
on the control diet and fish receiving RPC diets (Table 2.4). Significant correlations were
found between dietary RPC and phosphorus level and whole-body moisture (R2=0.50 and
0.48, P<0.05), fat (R2=0.54 and 0.53, P<0.01) and ash (R2=0.56 and 0.58, P<0.01) content.
Table 2.3: Growth response, feed intake, feed efficiencies, condition factor and survival of wels catfish fed
experimental diets
R0 R25 R50 R75 *R2 P
Initial weight (g) 87.8 ± 2.7 85.5 ± 1.2 86.1 ± 1.5 86.7 ± 2.0
Final weight (g) 279.8a ± 9.1 264.0a ± 7.7 218.4b ± 15.7 159.4c ± 4.4 0.92 <0.01
SGR (%) 1.84a ± 0.10 1.79a ± 0.06 1.48b ± 0.10 0.97c ± 0.04 0.86 <0.01
Feed intake (g DM) 102.9a ± 3.8 97.1a ± 3.2 85.7b ± 5.3 60.9c ± 3.1 0.88 <0.01
Feed intake as %
body weight day-1
0.96a ± 0.04 1.02a ± 0.03 0.97a ± 0.02 0.81b ± 0.01 0.36 ns
FCR 0.53a ± 0.03 0.56a ± 0.02 0.68b ± 0.04 0.86c ± 0.04 0.84 <0.01
PER 1.13a ± 0.06 1.11a ± 0.02 0.92b ± 0.05 0.72c ± 0.02 0.88 <0.01
GEI (MJ) 2.33a ± 0.09 2.24a ± 0.07 1.98b ± 0.12 1.41c ± 0.07 0.85 <0.01
CF 0.57 ± 0.04 0.62 ± 0.04 0.61 ± 0.05 0.61 ± 0.04 0.08 ns *R2: parameter values are regressed to the dietary level of rapeseed protein concentrate.
Values are given as mean ± standard deviation. Values in the same row with common superscript letters are not significantly
different (P<0.05).
Table 2.4: Proximate whole body composition (g kg-1 wet weight) of wels catfish fed the experimental diets
Parameter R0 R25 R50 R75
Moisture 740 ± 12 746 ± 10 755 ± 10 763 ± 11
Crude protein 153 ± 6 152 ± 7 150 ± 8 144 ± 5
Crude fat 85 ± 5 82 ± 7 76 ± 6 72 ± 6
Ash 24 ± 2 23 ± 3 21 ± 1 20 ± 2
Initial body composition: moisture 823 g kg-1, crude protein 131 g kg-1, crude fat 28 g kg-1, ash 18 g kg-1.
25
Haematocrit values as well as blood serum values determined showed no significant
difference between treatment groups and were not correlated to the dietary inclusion level of
RPC (Table 2.5).
Table 2.5: Blood haematocrit content and blood serum values of wels catfish fed experimental diets
Parameter R0 R25 R50 R75
Haematocrit (Proportion of 1) 0.23 ± 0.04 0.24 ± 0.04 0.25 ± 0.02 0.24 ± 0.02
Triglycerides (mM L-1) 4.72 ± 2.21 6.55 ± 4.89 7.20 ± 4.52 8.47 ± 1.46
Glucose (mM L-1) 6.54 ± 1.33 7.10 ± 1.27 6.66 ± 1.11 6.43 ± 1.27
Protein (g L-1) 36.0 ± 2.8 35.5 ± 2.1 36.1 ± 2.8 37.1 ± 1.9
Discussion
While usability and limitations of simple rapeseed products as fish feed ingredients have been
widely investigated (Dabrowski and Kozlowska 1981; Davies et al. 1990; Webster et al. 1997;
Burel et al. 2000a,b,c, 2001; Thiessen et al. 2003, 2004; Glencross et al. 2004; Shafaeipour et
al. 2008), lack of information exists about the benefits of high quality products originating
from rapeseed oilcakes with protein contents comparable to or above that of fish meal. Higgs
et al. (1982) successfully replaced 25 % of dietary protein from a fish meal control diet for
juvenile Oncorhynchus tshawytscha with rapeseed protein concentrate (613 g CP kg-1)
without reducing growth rate and food (protein) utilization. In the study, however, higher fish
meal replacement levels with rapeseed protein concentrate were not evaluated.
The results of our study demonstrate that 25 % of dietary fish meal can be replaced with RPC
in diets fed to wels catfish without negative effects on feed efficiencies and fish growth.
When 50 % of dietary fish meal was replaced with RPC the feed intake as % of fish body
weight was not significantly different from the control group but feed efficiencies and fish
growth were reduced. At 75 % fish meal replacement level fish showed reduced diet
acceptance and reluctant feed intake as a result of unfavourable diet taste. It appears therefore,
that the level of blood meal incorporated into diets as feed attractant did not effectively
counteract the negative effects on diet taste resulting from rapeseed protein concentrate. It is
known that the bitter taste exuded by glucosinolate metabolites, such as isothiocyanates and
26
vinyloxazolidinethiones, present in rapeseed meals can potentially retard diet acceptance by
fish. This was found in O. mykiss and P. maxima at dietary glucosinolate levels of 7.3 µmol g-
1 or 18.7 µmol g-1, respectively (Burel et al. 2000bc). Because the RPC used in our study
contained 0.2 µmol glucosinolates g-1 (Table 2.1) the highest calculated dietary glucosinolate
concentration was 0.07 µmol g-1 in diet R75 (Table 2.2). This value is far below the level
when glucosinolates become detrimental on food intake of O. mykiss and P. maxima (Burel et
al. 2000bc) but to our observation the typical mustard smell of glucosinolates was still
noticeable in diets R50 and R75. It seems, therefore, that wels catfish is more sensitive
towards a bitter diet taste than other carnivorous fish species. This goes together with the
fish’s excellent developed olfactory organ (Jakubowski and Kunysz 1979). Reduced feed
intake in fish fed on diets R50 and R75 resulted in lower growth rates and reduced feed
conversion compared to the control group (Table 2.3). For prospective feeding trials with
rapeseed protein products in wels catfish it appears recommendable to use other feed
attractants than blood meal. Fish protein hydrolysate, squid hydrolysate, stick water or krill
meal at dietary levels from 30 to 50 g kg-1 have shown to be effective feed attractants and
sources of amino acids and minerals when diets low in fish meal were fed to carnivorous fish
(Espe et al. 2006, 2007; Torstensen et al. 2008; Kousoulaki et al. 2009). Since fish behaved
calm in all treatment groups increased energy expenditure due to feed searching activity in
high RPC groups did not deplete feed conversion. Thus, lower feed efficiency might be a
result of reduced diet digestibility due to RPC inclusion.
In the present study we did not determine the digestibility of nutrients and minerals from RPC
in wels catfish. We discovered that faeces collection from wels catfish in order to determine
nutrient and mineral digestibility appears hardly possible. On the one hand, faeces of wels
catfish are slimy and rapidly dilute in water. This precludes faeces collection with an
automatic collector. On the other hand, faeces stripping, even when fish are anaesthetized,
will stress the sensitive fish. As a result, wels catfish will stop feed intake for days. Killing
fish, as a last alternative to gain faeces, requires a high number of individuals to collect
enough faeces for laboratory analysis. In the present study, fish count was not sufficient to
gain required amounts of faeces for laboratory analysis. Therefore, assumptions regarding
nutrient and mineral digestibility of RPC in wels catfish are based on studies conducted with
rapeseed protein products in other fish species. Mwachireya et al. (1999) stated that fibre
levels, either alone or together with phytate, can have greatest adverse effects on the
digestibility of canola protein products for O. mykiss. The authors reported that among
27
different canola products tested only canola protein isolate (908 g CP kg-1) met nutrient
digestibility coefficients corresponding to fish meal. In our study, the applied processing
techniques to produce RPC from rapeseed oilcake led to relatively low levels of phytic acid in
the final RPC (0.5 g kg-1). Accordingly, calculated dietary phytic acid concentrations
originating from RPC were 0.1 and 0.2 g kg-1 in diets R50 and R75, respectively. In fish
nutrition studies, phytic acid concentrations that negatively influence mineral and nutrient
availability are commonly higher. Spinelli et al. (1983) observed decreased growth rates in
rainbow trout fed a diet containing 5 g kg-1 synthetic phytic acid. Synthetic phytic acid at
concentrations of 5 and 10 g kg-1 feed resulted in lower growth performance in common carp
(Hossain and Jauncey 1993). Due to insignificant phytic acid concentrations in diets R50 and
R75, we assume, that diet digestibility was mainly reduced by fibre and other complex
carbohydrates. However, lack of information exists about the influence of complex
carbohydrates on nutrient digestibility in wels catfish. But it is known from other carnivorous
fish that complex carbohydrates can greatly reduce mineral and nutrient availability from
aquafeeds, thereby reducing feed efficiencies as observed in Salmo salar and P. maxima
(Storebakken et al. 1998; Burel et al. 2000). The RPC used in our study contained 48 g kg-1
fibre and calculated NfE was 128 g kg-1. This resulted in a dietary fibre + NfE content of 143
or 169 g kg-1 compared to 102 g kg-1 in the control group. We therefore assume, that diets
R50 and R75 were less digestible than the control diet for wels catfish, due to increased
dietary fibre and NfE contents originating from RPC. In contrast, Hayen et al. (1993) found
that rapeseed protein concentrate (600 g CP kg-1) was as digestible as LT herring/capelin meal
when fed to O. tshawytscha. According to our findings, it seems that wels catfish is highly
sensitive towards dietary fibre and NfE.
The amino acid requirement of wels catfish, to our knowledge, is not known. We therefore
assume that it is comparable to other carnivorous fish such as rainbow trout. Accordingly,
experimental diets were formulated to contain amino acid concentrations above established
requirement levels (NRC 1993). However, due to antinutritional factors present in RPC,
digestibility of amino acids could have been negatively affected as it is known from other
protein sources of vegetable origin (Francis et al. 2001). In particular lower dietary levels of
lysine in diets R50 and R75 compared to the control diet together with reduced lysine
digestibility might have negatively influenced feed efficiencies and fish growth in the present
study.
28
Fish body composition was not significantly different between treatments (Table 2.4).
Regression analysis, however, revealed a correlation between the dietary level of RPC and/or
phosphorus and the moisture, fat and ash content in fish bodies. Sinking ash levels in fish
body indicate that the levels of available phosphorus in diets were not sufficient to meet the
dietary requirement of wels catfish. It is known that whole-body ash can be reduced when
carnivorous fish are fed a diet deficient in available phosphorus (Skonberg et al. 1997; Shao et
al. 2008). Although dietary levels of phosphorus were 12.7 to 14.8 g kg-1 and therefore above
established requirement levels for many fish species (NRC 1993) it seems possible that
phosphorus availability from RPC was lower than from fish meal. Antinutritional factors such
as phytic acid, fibre and other complex carbohydrates present in RPC are known to influence
phosphorus availability in fish (Francis et al. 2001). However, as shown above, phytic acid
concentrations in diets R50 and R75 were insignificant. We assume therefore, that phosphorus
availability was mainly reduced by fibre and other complex carbohydrates. The often
increased whole-body lipid content with high dietary levels of vegetable proteins that has
been reported in several fish species (Adelizi et al. 1998; Kaushik et al. 2004) was not
observed here. Instead, the whole-body lipid level tendentially decreased with increasing
dietary level of vegetable protein as reported by Espe et al. (2006) when feeding Atlantic
salmon a diet devoid of fish meal. According to Espe et al. (2006), it seems possible therefore,
that the substitution of fish meal with plant proteins may not give the same results in different
fish species.
In prospective feeding trials with wels catfish and rapeseed protein concentrate it appears
advisory to supplement diets with a phosphorus source such as dicalcium phosphate in order
to overcome problems regarding phosphorus availability. This has been shown to positively
affect dietary phosphorus supply, feed efficiencies and fish growth when diets rich in plant
based proteins are applied (Lee et al. 2010).
In the present study, blood serum parameters were not significantly different between
treatment groups and no correlation with dietary RPC levels was found (Table 2.5). As
suggested by Caruso and Schlumberger (2002) blood serum parameters can be used to
estimate the health status of fish. Our results indicate that fish did not suffer from malnutrition
and that dietary nutrient supply was sufficient to support growth and maintain average body
development in all feeding groups. However, found individual blood values of wels are highly
variable as attested by a high standard deviation. This was also found by Caruso and
Schlumberger (2002) who established a baseline blood haematocrit value of 0.25 ± 0.01 for
29
wels catfish. The fish (individual weight 55 to 250 g) were reared in a closed warm water
system (26-27°C) and were fed at 1 % of their biomass per day. The haematocrit baseline
value corresponds to the value determined in our study (Table 2.5). Detected blood serum
values of triglycerides, glucose and protein, however, differ from values cited by Jirásek et al.
(1998). They monitored blood serum values of one-year old wels catfish (individual weight
752 and 1288 g) held in heated effluent water of a power station. Fish were fed on different
pelleted diets that contained 400 to 490 g CP kg-1 and up to 220 g CF kg-1. Average blood
serum values determined were total lipids 9.95 mM L-1, glucose 49.39 mM L-1 and total
protein 29.89 g L-1. We assume that differences between values published by Jirásek et al.
(1998) and in the present study reflect the different compositions of used diets and possible
differences in starvation time before sampling. It is known that blood serum values generally
represent the nutrient composition of a diet (Jirásek et al. 1998) and that starvation time
before sampling can have a significant influence on plasma glucose, triglycerides and protein
concentrations (Shi et al. 2010).
In conclusion, wels catfish accept diets formulated to contain 122 g kg-1 of the used RPC as
fish meal replacement. At higher dietary RPC inclusion diet taste became undesirable for wels
catfish, thereby reducing feed intake and fish growth. Antinutritional factors present in RPC
might also have reduced dietary phosphorus and amino acid availability with negative effects
on feed efficiencies and fish growth. To overcome difficulties with diet taste and nutrient
availability we suggest the use of ANF free rapeseed protein isolates in prospective feeding
trials.
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34
Chapter 3: Austausch von Fischmehl durch Rapsproteinkonzentrat in
Futtermitteln für Steinbutt (Psetta maxima L.)
H. Slawski1,2, H. Adem3, R.-P. Tressel3, K. Wysujack4,
Y. Kotzamanis5 und C. Schulz1,2
1Gesellschaft für Marine Aquakultur mbH, Hafentörn 3, D-25761 Büsum
2Institut für Tierzucht und Tierhaltung, Christian-Albrechts-Universität zu Kiel, D-24098 Kiel
3Pilot Pflanzenöltechnologie Magdeburg e.V., Berliner Chaussee 66, D-39114 Magdeburg
4Johann Heinrich von Thünen-Institut, Federal Research Institute of Rural Areas, Forestry and
Fisheries; Institute of Fisheries Ecology, Wulfsdorfer Weg 204, D-22926 Ahrensburg
5Hellenic Centre for Marine Research, Institute of Aquaculture, Ag.Kosmas, Helleniko,
Athens, Hellas, Greece
Published in: Züchtungskunde (2011) 83, (6), 451-460.
35
Zusammenfassung
Rapsproteinkonzentrat wurde als Alternative zu Fischmehl in Futtermitteln für juvenile
Steinbutt getestet. Triplikate Fischgruppen erhielten isonitrogene (58,1 ± 0,9 % RP)
Futtermischungen mit gleichem Gehalt an Bruttoenergie (21,5 ± 0,3 MJ kg−1), in denen 0, 33
und 66 % (bezeichnet als R0, R33 oder R66) Fischmehl durch Rapsproteinkonzentrat ersetzt
wurde. Am Ende des Fütterungszeitraumes (84 Tage) zeigten Fische, welche die Mischung
R0 oder R33 erhalten hatten, signifikant bessere Zuwachsraten und Futteraufnahmen als
Fische, die Mischung R66 erhielten. Verringerte Futteraufnahme und folglich schlechteres
Fischwachstum in der R66 Gruppe beruhen vermutlich auf geschmacklichen
Beeinträchtigungen der Futtermischung hervorgerufen durch Glucosinolate im
Rapsproteinkonzentrat. Für die Kontrollfuttermischung wurde gegenüber den -mischungen
R33 und R66 eine signifikant bessere Futterverwertung festgestellt. Dies geht vermutlich auf
geringere Protein-, Aminosäuren- und Phosphorverfügbarkeit in den Mischungen R33 und
R66 zurück.
36
Einleitung
Der stagnierenden Produktionsmenge an Fischmehl, der wichtigsten Proteinquelle in
Fischfuttermitteln, steht eine steigende Nachfrage des Aquakultursektors nach diesem
Rohstoff gegenüber. Um der dadurch eintretenden Verteuerung des Fischmehls und folglich
der Fischfuttermittel entgegenzuwirken, benötigt die Industrie für die Herstellung von
Futtermitteln hochwertige Fischmehlalternativen. Hierbei sind pflanzliche Proteinquellen von
höchster Relevanz (Gatlin et al. 2007). Mit einer weltweiten Produktionsmenge von 61,6 Mio
t in 2009 ist Raps (Brassica napus L., B. campestris L.) eine der bedeutendsten Ölsaaten
(FAO 2010). Entsprechend ist der bei der Rapsölgewinnung entstehende Rapsölkuchen ein
weit verfügbares Nebenprodukt. Dieser wurde sowohl in unbehandelter Form als auch nach
Bearbeitung mit organischen Lösungsmitteln in Futtermitteln für verschiedene Fischarten
getestet, darunter Regenbogenforelle (Burel et al. 2000ab; Thiessen et al. 2004; Shafaeipour et
al. 2008), Tilapia (Davies et al. 1990), Karpfen (Dabrowski und Kozlowska 1981) und
Steinbutt (Burel et al. 2000ac). Es zeigte sich, dass insbesondere antinutritive Faktoren (ANF)
wie Glucosinolate, Phytinsäure, und unverdauliche Kohlenhydrate die Eignung einfacher
Rapsprodukte für die Fischernährung beeinträchtigen (Francis et al. 2001). Der Gehalt an
ANF in einfachen Rapsprodukten kann allerdings durch verschiedene Bearbeitungsverfahren
verbessert werden. Hierzu zählen eine Wärmebehandlung der Saat, das Schälen der Saat, die
Nutzung organischer Lösungsmittel (z.B. Hexan) nach dem Ölpressen,
Dekantierungsverfahren sowie Ultrafiltration in Wasser gelöster Proteinbestandteile (Fenwick
et al. 1986; Anderson-Hafermann et al. 1993; Mawson et al. 1995; Mwachireya et al. 1999;
Tyagi 2002). In der vorliegenden Untersuchung wurde ein eigens angefertigtes
Rapsproteinkonzentrat mit einem Proteingehalt von 71 % als Fischmehlersatz in Futtermitteln
für Steinbutt (Psetta maxima L.) getestet. Anhand eines Fütterungsversuches sollte untersucht
werden, ob das hochaufgereinigte Rapsproteinkonzentrat einen höheren Fischmehlaustausch
im Futtermittel als bisher untersuchte, relativ einfach verarbeitete Rapsprodukte ermöglicht.
Material und Methoden
Herstellung der Futtermischungen
Es wurden drei Versuchsfuttermischungen formuliert. In diesen wurde Fischmehl zu 0, 33
und 66 % (bezeichnet als R0, R33 oder R66) durch Rapsproteinkonzentrat (RPK)
ausgetauscht. Das RPK wurde im PPM e.V., Deutschland, hergestellt (Slawski et al. im
Druck). Die Nährstoffzusammensetzung des RPK ist in Tab. 3.1 aufgeführt. Basierend auf
37
den Aminosäurengehalten der verwendeten Rohstoffe wurden die Mischungen so konzipiert,
dass der Aminosäurenbedarf mariner Fische gedeckt war (NRC 1993). Vitamine und
Mineralstoffe wurden den Mischungen dem Bedarf mariner Fische entsprechend beigefügt
(NRC 1993). Die drei Futtermischungen waren isonitrogen (58,1 ± 0,9 % RP) und wiesen
gleiche Gehalte an Bruttoenergie auf (21,5 ± 0,3 MJ kg−1). Die Mischungen wurden zu Pellets
mit 4 mm Durchmesser gepresst (L 14-175, AMANDUS KAHL, Reinbek, D).
Futtermittelformulierung und -zusammensetzung sind in Tab. 3.2 und 3.3 dargestellt.
Tabelle 3.1: Nährstoff- und Aminosäurenzusammensetzung von Fischmehl und Rapsproteinkonzentrat
Fischmehl Rapsproteinkonzentrat
Rohwasser
(% der lufttrockenen Substanz)
8,4 5,6
Nährstoffzusammensetzung (% der Trockensubstanz)
Rohprotein 69,0 71,2
Rohfett 7,0 0,6
Rohasche 20,7 16,1
Phosphor 2,9 0,9
Rohfaser 0,5 0,5
NfEa 2,9 11,6
Bruttoenergieb (MJ kg-1) 19,9 19,4
Aminosäurenzusammensetzung (% der Trockensubstanz)
Arginin 4,03 4,83
Histidin 1,38 2,75
Isoleucin 2,50 2,70
Leucin 4,45 5,39
Lysin 4,52 5,60
Methionin + Cystein 2,19 4,64
Phenylalanin 2,43 2,56
Threonin 2,69 2,97
Valin 3,07 3,67
Antinutritive Faktoren
Glucosinolate (µmol g-1) 1,32
Phytinsäure (g 100g-1) 1,77 aN-freie Extraktstoffe = 100 – (%Rohprotein + %Rohfett + %Asche + %Rohfaser) bBerechnung: Rohprotein = 23,9 MJ kg-1; Rohfett = 39,8 MJ kg-1; Rohfaser, NfE: 17,6 MJ kg-1
38
Aufbau des Fütterungsversuchs
Der Fütterungsversuch wurde am Institut für Fischereiökologie (Außenstelle Ahrensburg) des
Johann Heinrich von Thünen Institutes, Bundesinstitut für Ländliche Räume, Wald und
Fischerei, durchgeführt. Juvenile Steinbutt aus der Ejsing Seafarm (Vinderup, DK) wurden
verwendet. Jeweils 14 Fische wurden in eines der neun Versuchsbecken (96 L; Grundfläche
4800 cm2) gesetzt. Angeschlossen an eine Salzwasserkreislaufanlage wurden die Becken mit
2 L min-1 künstlichem Meerwasser versorgt (Temperatur: 17,3 ± 1,1 °C; O2: 7,8 ± 0,2 mg L-1;
Salinität: 25,8 ± 0,5 ‰; pH-Wert: >7,8; NH4+: <0,07 mg L-1; NO2
-: <0,13 mg L-1). Die
Photoperiode entsprach der für den Standort (53° 41' 0" N) natürlichen Rhythmik von Juli bis
Oktober. Für eine zweiwöchige Anpassungsphase erhielten die Fische die Mischung R0 in
einer täglichen Fütterung bis zur scheinbaren Sättigung. Nach der Anpassungsphase wurden
die Fische für zwei Tage genüchtert und ihr Anfangsgewicht wurde bestimmt (73,5 ± 0,7 g).
Für einen Zeitraum von 84 Tagen wurden dann in drei Wiederholungen je Behandlung
Gruppen der Fische mit den Versuchsfuttermischungen einmal täglich (8:30 Uhr) bis zur
scheinbaren Sättigung gefüttert. Zu Beginn und am Ende des Versuches wurden zwei Fische
pro Becken für die Bestimmung der Ganzkörperzusammensetzung entnommen.
Tabelle 3.2: Formulierung der Versuchsfuttermittel (g kg-1)
Futterkomponenten R0 R33 R66
Heringsmehl 450 300 150
Rapsproteinkonzentrat 0 145 295
Sojaproteinkonzentrat 100 100 100
Garnelenmehl 85 85 85
Blutmehl 95 95 95
Kartoffelstärke 90 90 90
Dextrose 75 70 60
Fischöl 55 65 75
Vitamin/Mineralmixa 20 20 20
Weizengluten 30 30 30 aAA-Mix 507101, Vitfoss, Gråsten, Dänemark
39
Tabelle 3.3: Nährstoff- und Aminosäurenzusammensetzung der Versuchsfuttermittel
Inhaltsstoffe R0 R33 R66
Nährstoffzusammensetzung (% der Trockensubstanz)
Rohprotein 57,5 57,7 59,1
Rohfett 10,8 11,1 10,8
Rohasche 13,6 12,3 10,8
Phosphor 1,7 1,4 1,1
Rohfaser 0,8 0,7 0,5
NfEa 17,4 18,2 18,7
GEb (MJ kg-1) 21,2 21,6 21,8
Aminosäurenzusammensetzung (g 100g Rohprotein-1)
Arginin 5,97 6,13 5,91
Histidin 2,47 3,15 3,16
Isoleucin 3,15 3,23 3,07
Leucin 7,94 8,25 7,93
Lysin 5,81 6,21 5,99
Methionin + Cystein 2,39 2,66 2,84
Phenylalanin 4,37 4,49 4,23
Threonin 3,75 3,85 3,66
Valin 5,19 5,34 5,05
Antinutritive Faktorenc
Glucosinolate (µmol g-1) - 0,20 0,40
Phytinsäure (g 100g-1) - 0,25 0,52 aN-freie Extraktstoffe = 100 – (%Rohprotein + %Rohfett + %Asche + %Rohfaser) bBerechnung: Rohprotein = 23,9 MJ kg-1; Rohfett = 39,8 MJ kg-1; Rohfaser, NfE: 17,6 MJ kg-1
ckalkuliert anhand der Konzentrationen in den Rohstoffen (Tabelle 1)
Durchführung der Nährstoffanalysen
Futtermittel und homogenisierte Fischkörper wurden in Duplikaten auf ihren Nährstoffgehalt
untersucht. Der Feuchtegehalt wurde über Trocknung der Materialien in einem Ofen bei 105
°C bis zu einem konstanten Gewicht ermittelt. Der Aschegehalt wurde mittels
Gewichtsverlust nach zweistündiger Veraschung im Muffelofen bei 550 °C bestimmt. Der
Fettgehalt wurde über Etherextraktion (Soxtec HT6, Tecator, Höganäs, S) und der
Gesamtstickstoffgehalt mit dem Kjeldahl-Verfahren (Rohprotein = N × 6,25; Kjeltec Auto
System, Tecator, Höganäs, S) erfasst. Die Aminosäurenzusammensetzung wurde nach dem
bei Tzovenis et al. (2009) geschilderten Verfahren ermittelt. Dabei wurde der
40
Tryptophangehalt nicht bestimmt. Der Glucosinolatgehalt im Rapsproteinkonzentrat wurde
anhand der Methodik in der EC-Gazette (1864/90 Nr. L 170/28, 3.7.90) bestimmt. Der Gehalt
an Phytinsäure wurde anhand des Verfahrens von Harland und Oberleas (1986) ermittelt.
Berechnungen und statistische Verfahren
Fischwachstum, Futterverwertung und physiologische Parameter wurden anhand folgender
Formeln bestimmt:
Spezifische Wachstumsrate (SWR, % Tag-1) = (ln Endgewicht – ln Anfangsgewicht) × 100 /
Fütterungstage
Futterquotient (FQ) = g Trockenfutteraufnahme / g Gewichtszunahme
Proteinwirkungsverhältnis (PER) = g Gewichtszunahme / g Proteinaufnahme
Fultonscher Konditionsfaktor (K) = g Körpergewicht / cm Gesamtlänge3 × 100
Überlebensrate (%) = (Anzahl Fische Versuchsbeginn – Anzahl Totfische) / Anzahl Fische
Versuchsbeginn × 100
Erhobene Daten wurden mithilfe der Statistiksoftware SPSS 17.0 ausgewertet. Die Daten
wurden mittels Kolmogoroff Smirnov Test auf Normalverteilung untersucht und falls nötig
transformiert. Nach der Varianzanalyse der Mittelwerte in einer ANOVA wurden signifikant
unterschiedliche Mittelwerte mithilfe des Tukey Tests identifiziert (P<0,05).
Ergebnisse
Wachstumsleistung, Futterverwertung und Körperzusammensetzung
Während Wachstumsleitungen und Futteraufnahme in den Gruppen R0 und R33 keine
signifikanten Unterschiede zeigten, waren Wachstum und Futteraufnahme in der Gruppe R66
signifikant schlechter. Die Futterverwertung war in den Gruppen R33 und R66 signifikant
schlechter als bei der Kontrollfuttermischung. Allerdings war die Verwertung der Mischungen
R33 und R66 einheitlich. Der Konditionsfaktor war signifikant geringer in Gruppe R66
gegenüber den Gruppen R0 und R33. Die Überlebensrate zeigte genauso wie die
Körperzusammensetzung der Tiere keine gruppenübergreifenden signifikanten Unterschiede
(Tab. 3.4 und 3.5).
41
Tabelle 3.4: Ganzkörperzusammensetzung der Steinbutt nach der Fütterungsperiode
R0 R33 R66
Rohwasser 79,1 ± 1,2 78,4 ± 1,5 79,5 ± 3,0
Rohprotein 14,5 ± 0,8 15,0 ± 0,9 13,9 ± 1,3
Rohfett 2,6 ± 0,2 2,8 ± 0,5 3,4 ± 0,8
Rohasche 4,1 ± 0,9 3,9 ± 0,5 3,7 ± 0,7
Ganzkörperzusammensetzung zu Versuchsbeginn: Wasser 76,8 %, Rohprotein 15,%, Rohfett 3,6%, Rohasche 3,5%
Tabelle 3.5: Wachstumsparameter und Futterverwertung der Steinbutt nach dem Fütterungsversuch
R0 R33 R66
Anfangsgewicht (g/Fisch) 73,1 ± 1,0 74,1 ± 0,4 73,3 ± 0,4
Endgewicht (g/Fisch) 147,5a ± 10,3 145,0a ± 7,5 122,2b ± 4,5
SWR (%/Tag) 0,83a ± 0,07 0,80a ± 0,07 0,61b ± 0,05
Futteraufnahme (g/Fisch) 73,9a ± 8,2 82,6a ± 10,8 58,2b ± 1,1
FQ (g/g) 1,00a ± 0,06 1,16b ± 0,03 1,20b ± 0,09
PER 1,75a ± 0,11 1,49b ± 0,04 1,42b ± 0,11
K 1,80a ± 0,16 1,76a ± 0,12 1,56b ± 0,13
Überlebensrate (%) 94,4 ± 9,6 100 ± 0,0 100 ± 0,0
Werte in denselben Zeilen mit unterschiedlichen Indices sind signifikant verschieden (Tukey Test; P<0,05)
Diskussion
Während die Einsetzbarkeit einfacher Rapsprodukte als Inhaltsstoff in Fischfuttermitteln
hinlänglich untersucht wurde (Dabrowski und Kozlowska 1981; Davies et al. 1990; Burel et
al. 2000a,b,c; Thiessen et al. 2004; Shafaeipour et al. 2008), ist über die Eignung qualitativ
hochwertiger Rapsprodukte, die mit Fischmehl vergleichbare Proteingehalte besitzen, wenig
bekannt. Higgs et al. (1982) ersetzten erfolgreich 25 % des Futtermittelproteins aus einer
Fischmehlkontrollmischung für juvenile Königslachse durch Rapsproteinkonzentrat (61,3 %
RP) ohne die Wachstumsraten und Proteinverwertung der Fische gegenüber einer
Kontrollgruppe zu beeinträchtigen. Die Ergebnisse der vorliegenden Untersuchung zeigen,
dass 33 % des Fischmehls im Futtermittel für Steinbutt durch RPK ersetzt werden kann, ohne
die Futteraufnahme oder das Fischwachstum zu verringern. Bei einem 66 %igen
42
Fischmehlaustausch durch RPK zeigten die Fische geringere Futterakzeptanz und reduzierte
Futteraufnahme. Dies geht vermutlich auf geschmackliche Beeinträchtigung des Futters durch
RPK zurück. Es scheint, dass das in den Mischungen als Geschmacksverbesserer eingesetzte
Blutmehl und Garnelenmehl nicht ausreichte, um durch RPK auftretende negative Effekte auf
den Futtermittelgeschmack auszugleichen. Der bittere Geschmack von
Glucosinolatmetaboliten wie Isothiocyanaten und Vinyloxazolidinethionen, die in
Rapsmehlen enthalten sind, kann die Futtermittelakzeptanz von Fischen verschlechtern. Dies
wurde für Regenbogenforelle und Steinbutt bei Futterglucosinolatgehalten von 7,3 bzw. 18,7
µmol g-1 festgestellt (Burel et al. 2000bc). Das RPK in unserer Studie enthielt 1,3 µmol
Glucosinolate g-1 (Tab. 3.1). Entsprechend betrug der höchste Futterglucosinolatgehalt 0,4
µmol g-1 in Mischung R66 (Tab. 3.3). Dieser Wert unterschreitet die laut Burel et al.
(2000b,c) problematische Futterglucosinolatmenge für Regenbogenforelle und Steinbutt.
Allerdings ging trotz der niedrigen Glucosinolatkonzentrationen in den Mischungen von
Futtermittel R66 ein für Glucosinolate typisch senfiger Geruch aus. Für weitere
Fütterungsversuche mit RPK erscheint es sinnvoll, Futtermischungen mit zusätzlichen
Geschmacksverbesserern anzureichern. Beispielsweise wurden Fischproteinhydrolysat,
Tintenfischhydrolysat oder Krillmehl als effektive Geschmacksverbesserer und Quellen von
Aminosäuren und Mineralien identifiziert, wenn Futtermittel mit niedrigem Fischmehlanteil
an carnivoren Fischarten verfüttert wurden (Espe et al. 2006; Torstensen et al. 2008).
Während die verringerte Akzeptanz gegenüber der Mischung R66 vermutlich auf
geschmackliche Beeinträchtigungen zurückging, dürfte für die schlechtere Verwertung der
Mischungen R33 und R66 eine Beeinträchtigung der Nährstoff- und
Mineralstoffverfügbarkeit ursächlich sein.
Rohfaser und komplexe Kohlenhydrate können die Verdaulichkeit und Energieverfügbarkeit
von Futtermitteln für Steinbutt verringern (Burel et al. 2000a,c). Der Anteil an Rohfaser +
NfE des in der vorliegenden Untersuchung verwendeten RPK war mit 12,1 % deutlich höher
als bei dem verwendeten Fischmehl (3,4 %). Es erscheint daher möglich, dass mit steigendem
RPK-Anteil in den Futtermischungen R33 und R66 der Gehalt an verdaulicher Energie
zurückging, was zu schlechterer Futterverwertung gegenüber der Kontrollmischung führte.
Neben dem Gehalt an verdaulicher Energie könnte auch das geringere Phosphorangebot durch
den Austausch von Fischmehl mit RPK ursächlich für verringerte Futterverwertung sein.
Obgleich der Futtermittel-Phosphorbedarf des Steinbutts unserem Wissen nach bisher nicht
ermittelt wurde, lagen die Phosphorgehalte in den Futtermitteln mit über einem Prozent
43
oberhalb des bekannten Bedarfswertes für die meisten Fische (NRC 1993). Es ist aber
bekannt, dass erhöhte Phosphorgehalte im Futtermittel die Futterverwertung bei Lachs,
Dorsch und Wolfsbarsch verbessern können (Vielma und Lall 1998; Roy und Lall 2003;
Oliva-Teles und Pimentel-Rodrigues 2004). Dagegen können antinutritive Faktoren wie
Phytinsäure, Rohfaser und andere komplexe Kohlenhydrate in Rapsproteinprodukten die P-
Verfügbarkeit in Fischfuttermitteln verringern (Mwachireya et al. 1999; Francis et al. 2001).
Das in der vorliegenden Studie getestete RPK enthielt 1,77 g Phytinsäure 100g-1, was 0,25
und 0,52 g Phytinsäure 100g-1 in den Mischungen R33 bzw. R66 entspricht (Tab. 3.3).
Spinelli et al. (1983) beobachteten verringertes Wachstum bei Regenbogenforellen, die
Futtermittel mit 0,5 g Phytinsäure 100g-1 erhielten. In Futtermitteln für Karpfen führte
synthetische Phytinsäure bei Konzentrationen von 0,5 bis 1,0 g 100g-1 zu geringeren
Wachstumsleistungen (Hossain und Jauncey 1993).
Es scheint daher möglich, dass die Phytinsäurekonzentrationen in den Mischungen R33 und
R66 negativen Einfluss auf die P-Verfügbarkeit hatten und somit die Futterverwertung und
das Wachstum verschlechterten. Der signifikant geringere Konditionsfaktor in Gruppe R66
veranschaulicht zusätzlich die schlechtere körperliche Entwicklung der Steinbutt. Tendenziell
variierende Asche- und Fettgehalte in der Körperzusammensetzung bekräftigen die
Vermutung einer geringen P-Verfügbarkeit bei hohem Fischmehlaustausch (Tab. 3.4).
Erhalten carnivore Fische ein Futtermittel, das reich an pflanzlichen Proteinen ist und relativ
geringe Mengen an verfügbarem Phosphor enthält, können sinkende Körperaschegehalte und
steigende –fettgehalte die Folge sein (Skonberg et al. 1997; Adelizi et al. 1998; Kaushik et al.
2004). Der für diesen Effekt verantwortliche Mechanismus ist unklar. Es wird angenommen,
dass eine Akkumulation von Fettsäuren durch eine gestörte β-Oxidation (Takeuchi und
Nakazoe 1981) oder oxidative Phosphorylierung durch P-Mangel bewirkt wird. Dies könne zu
einer Beeinträchtigung des Citratzyklus und einer Anreicherung von Acetyl-CoA und
schliesslich zu einer gesteigerten Fettsäuresynthese führen (Skonberg et al. 1997). Um die
getroffenen Annahmen zum Einfluss der Phosphorverfügbarkeit auf die Futterverwertung
beim Steinbutt zu bekräftigen, wären Informationen zum tatsächlichen Phosphorbedarf dieser
Fischart vonnöten.
Zusammenfassend ist festzustellen, dass Steinbutt bei Aufnahme eines Futtermittels, bei dem
33 % des Fischmehls durch Rapsproteinkonzentrat ersetzt wurden, keine Verschlechterung
der Wachstumsleistungen zeigten. Bei höherem Einsatz von RPK als Fischmehlersatz wurde
die Futtermittelakzeptanz für Steinbutt stark beeinträchtigt und folglich das Wachstum
44
negativ beeinflusst. Zudem dürften Phytinsäuregehalte im RPK zu einer Beeinträchtigung der
P-Verfügbarkeit geführt haben, weshalb zusätzlich Futterverwertung und Fischwachstum
verringert wurden. In folgenden Fütterungsversuchen mit RPK sollten daher zusätzliche
Geschmacksverbesserer eingesetzt werden, um eine bessere Futteraufnahme zu erzielen.
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47
Chapter 4: Total fish meal replacement with rapeseed protein concentrate
in diets fed to rainbow trout (Oncorhynchus mykiss W.)
H. Slawski1, 2, H. Adem3, R.-P. Tressel3, K. Wysujack4, U. Koops4, Y. Kotzamanis5,
S. Wuertz1 and C. Schulz1, 2
1Gesellschaft für Marine Aquakultur mbH, Hafentörn 3, D-25761 Büsum
2Institute of Animal Breeding and Husbandry, Christian-Albrechts-Universität zu Kiel,
D-24098 Kiel
3Pilot Pflanzenöltechnologie Magdeburg e.V., Berliner Chaussee 66, D-39114 Magdeburg
4Johann Heinrich von Thünen-Institut, Federal Research Institute of Rural Areas, Forestry and
Fisheries; Institute of Fisheries Ecology, Wulfsdorfer Weg 204, D-22926 Ahrensburg
5Hellenic Centre for Marine Research, Institute of Aquaculture, Ag.Kosmas, Helleniko,
Athens, Hellas, Greece
Published in: Aquaculture International (2011), http://www.doi 10.1007/s10499-011-9476-2
48
Abstract
The potential of rapeseed protein concentrate as fish meal alternative in diets for rainbow
trout (initial average weight 37.8 ± 1.4 g) was evaluated. Nine experimental tanks of a
freshwater flow through system were stocked with 12 fish each. Triplicate groups of fish
received isonitrogenous (47.9 ± 0.5 % CP) and isoenergetic (22.4 ± 0.2 kJ g-1) experimental
diets with 0, 66 and 100 % of fish meal substituted with rapeseed protein concentrate (71.2 %
CP). As the amino acid profile of rapeseed protein concentrate was comparable to fish meal
there was no need to supplement experimental diets with synthetic amino acids. At the end of
the 84 days feeding period, fish growth performance, feed intake and feed efficiencies were
not compromised when 100 % of fish meal in the control diet was replaced with rapeseed
protein concentrate, revealing a SGR of 1.19 or 1.10, a FCR of 1.09 or 1.18 and a feed intake
of 78.5 g or 74.7 g in fish fed on the control diet or fed the diet devoid of fish meal,
respectively. Intestinal morphology did not reveal any histological abnormalities in all dietary
groups. Blood parameters including haematocrit, haemoglobin as well as glucose,
triglycerides and total protein in the plasma were not different between treatment groups.
Thus, the rapeseed protein concentrate tested here has great potential as an alternative to fish
meal in rainbow trout diets.
49
Introduction
In search of fish meal alternatives in aqua feeds, rapeseed (including canola) products have
been widely tested as protein sources in diets for several fish species, among them rainbow
trout (Stickney et al. 1996; Burel et al. 2001; Thiessen et al. 2003; Shafaeipour et al. 2008),
tilapia (Davies et al. 1990), wels catfish (Slawski et al. in press), gilthead sea bream (Kissel et
al. 2000) and turbot (Burel et al. 2000ac). In general, experimental results demonstrated that
the nutritional quality of simple rapeseed products such as oilcakes or rapeseed meals is
below that of fish meal despite a well balanced amino acid profile. It was reported, that the
nutritional quality of these simple rapeseed products largely depends on the level of
antinutritional factors. Glucosinolates, phytic acid, phenolic constituents and indigestible
carbohydrates have been recognized as predominant antinutritional factors in rapeseed
products (Francis et al. 2001). The contents of these antinutritional factors in rapeseed
products can be reduced by several processing techniques thereby improving its value for fish
nutrition. Dehulling of seeds and application of high temperatures and organic solvents (e.g.
hexane) during oil extraction as well as sieving of meal decrease content of glucosinolates,
phytate, fibre, cellulose, hemicellulose, sinapin and tannins (Fenwick et al. 1986; Anderson-
Haferman et al. 1993; Mawson et al. 1995; Tripathi and Agrawal 2000). Extraction processes
from meals with methanol-ammonia or ethanol will increase protein level and effectively
remove glucosinolates, phenolic compounds, soluble sugars, such as sucrose and some
oligosaccharides (Naczk and Shahidi 1990; Chabanon et al. 2007).
Sporadically, rapeseed protein products of high quality are being produced in different
countries for use in animal nutrition. However, these products are only produced for test
purposes in small volumes until their potential as alternative protein source is documented.
Besides nutritive quality, costs of production need to be to sufficiently reduced to turn
rapeseed protein products an economically feasible alternative to other protein sources, in
particular fish meal. In the present study, a high quality rapeseed protein concentrate (71 %
CP) gained after hexane and ethanol extraction as well as ultrafiltration was tested as fish
meal replacement in diets of rainbow trout, thereby investigating fish growth performance,
feed efficiencies, and the potential impacts on blood parameters and histological intestinal
morphology.
50
Materials and methods
Preparation of experimental diets
Three experimental diets were formulated to replace fish meal with rapeseed protein
concentrate (RPC) at 0, 66, or 100 % (designated as R0, R66, or R100, respectively). Solvent
extracted RPC was obtained from PPM, Magdeburg, Germany. For the production of RPC a
batch of rapeseed (variety Lorenz, Norddeutsche Pflanzenzucht, Hohenlieth, Germany) was
conditioned in a vacuum dryer for 15 minutes at 60-70 °C to inactivate the enzyme
myrosinase. Then rapeseed was cold pressed. To remove residual oil from the oilcake (12.9 %
crude lipid, 31.3 % crude protein) it was crushed into 1-5 mm particle size followed by a
hexane treatment. The treatment lasted for two hours and the incubation temperature was 60
°C. Hexane treated rapeseed meal extract was desolventised under pressure to remove hexane
(< 300 ppm), then rapeseed meal extract was further crushed to a particle size of 0.2 to 0.1
mm. A four step treatment using a 75 % ethanol solution (35 min at 60 °C) aimed to remove
glucosinolates from rapeseed meal extract. This resulted in a residual oil content of the nearly
glucosinolate free rapeseed meal extract of 1.1 % and a protein content of 39.8 %. In the
following, protein was gained through liquid water extraction (rapeseed meal extract 1:15
water). For this, the suspension was heated to 40-45 °C followed by two hours of constant
agitation. Afterwards the suspension was decanted. Following decantation the solvent was
collected and residue material was secondly extracted (residue 1:10 water, 5 % NaCl) at 40-45
°C and one hour contact time under constant agitation. Following extraction the suspension
was decanted. Solvent was collected and residue prepared for a third extraction. Then solvents
of extraction 1, 2 and 3 were collected to remove low-molecular compounds and to
concentrate dissolved proteins by dia- and ultrafiltration. During filtration conductivity was
checked. Protein washing ended, when conductivity was 5-6 mS cm-1, corresponding to a
protein content of 60 %. The gained material was spray dried at 70-80 °C, which led to a
rapeseed protein concentrate with 71 % crude protein content (Tab. 4.1).
51
Table 4.1: Proximate and amino acid composition of fish meal and rapeseed protein concentrate and
concentration of antinutritional factors determined
Fish meal Rapeseed protein concentrate
Proximate composition (% of dry weight)
Crude protein 69.0 71.2
Crude fat 7.0 0.6
Ash 20.7 16.1
Phosphorus 2.89 0.94
Crude fibre 0.5 0.5
NfEa 2.8 11.6
Gross energyb (MJ kg-1) 19.9 19.4
Essential amino acids (g 100g-1 crude protein)
Arginine 5.84 6.78
Histidine 2.00 3.86
Isoleucine 3.62 3.79
Leucine 6.45 7.57
Lysine 6.55 7.87
Methionine
(+ Cysteine)
2.36
3.17
2.36
6.52
Phenylalanine 3.52 3.60
Threonine 3.90 4.17
Valine 4.45 5.15
Antinutritional factors
Glucosinolates (µmol g-1) - 1.32
Phytic acid (g 100g-1) - 1.77 aNitrogen free extract = 100 – (%crude protein + %crude fat + %ash + %fibre) bCalculated by: crude protein = 23.9 MJ kg-1; crude fat = 39.8 MJ kg-1; NfE, fibre: 17.6 MJ kg-1
Tryptophane was not analyzed
52
Since the available amount of RPC was limited due to high cost of production, not more than
two test diets containing RPC were applied in the feeding trial. Diets were formulated to be
isonitrogenous (47.9 ± 0.5 % CP) and isocaloric (22.4 ± 0.2 MJ g-1). Vitamins and minerals
were added to diet mixes to meet the dietary requirements of rainbow trout (NRC 1993).
Because the amino acid profile of RPC was similar to fish meal, the concentrations of
essential amino acids did not differ considerably between experimental diets. Therefore,
supplementation of synthetic amino acids in experimental diets was not required. The diets
were manufactured to give pellets 4 mm in diameter (L 14-175, AMANDUS KAHL,
Reinbek, Germany).
Based on raw material analysis, dietary concentrations of the prominent antinutritional factors
were calculated. Accordingly, concentrations of glucosinolates were 0.26 or 0.39 µmol g-1 and
concentrations of phytic acid were 0.35 or 0.52 g 100g-1 in diets R66 or R100, respectively.
Dietary formulations, proximate and amino acid compositions are given in Tab. 4.2 and 4.3.
Table 4.2: Formulation of experimental diets (%)
Ingredients R0 R66 R100
Herring meal 30 10 0
Rapeseed protein concentrate 0 20 29.5
Soyprotein concentrate 10 10 10
Blood meal 10 10 10
Crustacean meal 9.5 9 9
Potato starch 12 12 12
Dextrose 12.5 11.5 11.5
Fish oil 11 12.5 13
Vit/MinMixa 2 2 2
Wheat gluten 3 3 3 aAA-Mix 517158 & 508240, Vitfoss, Gråsten, Denmark
53
Table 4.3: Proximate and amino acid composition of experimental diets (% of dry weight)
R0 R66 R100
Proximate composition (% of dry weight)
Crude protein 48.1 48.2 47.3
Crude fat 15.8 16.0 15.7
Ash 11.2 9.7 9.3
Phosphorus 1.27 0.97 0.82
Crude fibre 0.9 0.7 0.7
NfEa 24.1 25.4 27.1
Gross energyb (MJ kg-1) 22.2 22.5 22.4
Essential amino acids
Requirementc
Arginine 2.61 2.73 2.62 1.15
Histidine 1.31 1.51 1.42 0.58
Isoleucine 1.38 1.39 1.38 1.37
Leucine 3.72 3.77 3.80 1.36
Lysine 2.78 2.80 2.84 2.77
Methionine
(+ Cysteine)
0.86
1.05
0.78
1.15
0.78
1.13
0.80
Phenylalanine 2.06 2.03 2.08 1.20
Threonine 1.65 1.66 1.70 1.03
Valine 2.41 2.42 2.41 1.57
200 295
Antinutritional factorsd
Glucosinolates (µmol g-1) 0.26 0.39
Phytic acid (g 100g-1) 0.35 0.52 aNitrogen free extract = 100 – (%crude protein + %crude fat + %ash + %fibre) bCalculated by: crude protein = 23.9 MJ kg-1; crude fat = 39.8 MJ kg-1; NfE, fibre: 17.6 MJ kg-1 cRodehutscord et al. (1997) dcalculations based on ANF levels in raw materials (Tab. 1)
Design of the feeding trial
The feeding trial was conducted at the Institute of Fisheries Ecology of the Johann Heinrich
von Thünen Institute, Federal Research Institute of Rural Areas, Forestry and Fisheries,
Germany, using rainbow trout (Oncorhynchus mykiss W.) that had been hatched in the
institute. Fish were randomly distributed into nine 40 L tanks arranged as flow through system
(water exchange 0.5 % min-1; 10.9 ± 0.5 °C), providing a triplicate (n=12 fish) per diet. For a
54
two week acclimatisation period fish were fed the control diet until apparent satiation once a
day. After the adaptation period, fish were fasted for two days and initial individual weight
was determined (37.8 ± 1.4 g). At the beginning and at the end of the experiment, 2 fish per
tank were bulk sampled for the determination of the initial and final body composition and
stored at -23 ºC. During the feeding trial, fish were fed to apparent satiation (at 8.00 a.m.) for
84 days. Uneaten feed was siphoned from the bottom of the tank, separated from faeces,
weighed back and frozen. Based on the dry matter quantification of waste feed the daily feed
intake was calculated. All diets were assigned by a completely randomized design.
Photoperiod was in accordance to the local natural photoperiod from July to October (53° 41'
0" N). Oxygen concentration (>8 mg L-1) and pH (6.5-7) in the effluent water of the flow
through system were measured daily, ammonia (<0.1 ppm), nitrite (<0.2 ppm) and nitrate
(<50 ppm) were determined photometrically twice a week.
Sampling
At the end of the feeding trial, 8 fish per treatment were randomly sampled: Blood was taken
from the caudal vein with a heparinized syringe (1 ml). The haematocrit was determined in
heparinised micro capillaries upon centrifugation (10000 g, 6 min) in a Haematokrit 210
centrifuge (Hettich, Tuttlingen, Germany). Blood haemoglobin content was determined
photometrically (540 nm) within 48 h from whole blood stored 4°C as haemoglobincyanide
with the haemoglobin FS kit (DiaSys, Germany). Plasma was separated, (5000 g, 5 min)
shock frozen and stored at -80 °C until analysed.
Seven fish from each treatment were killed by intersection of the spinal cord. After careful
dissection, 0.5 cm pieces of intestinal tissue were taken from each of four locations: (1)
stomach; (2) central part of the pyloric caecae; (3) mid-intestine; and (4) distal-intestine.
Histological samples were fixed in Histofix®, dehydrated and embedded in paraffin. Sections
of approximately 5 µm were cut and stained with haematoxylin and eosin (H&E) using
standard histological procedures (Sheehan and Hrapchak 1980). Sections of gastrointestinal
tract were evaluated qualitatively by light microscopy by comparing with experimental
controls for the presence of inflammation and alterations in the architecture of the mucosa,
eosinophilic granular layer and goblet cell count and size (Adelizi et al. 1998).
55
Analysis
Feeds and homogenized fish body samples were analysed for dry matter (105°C, until
constant weight), crude lipid (Soxtec HT6, Tecator, Höganäs, Sweden), crude protein
(N×6.25; Kjeltec Auto System, Tecator, Höganäs, Sweden) and ash (550°C, 2h). Amino acids
were determined after acid hydrolysis (6 N, 110°C, 24 h) and derivatisation by AccQ-Tag
according to the amino acid analysis application solution (Waters, Eschborn, Germany). DL-
Norvaline (Sigma) 2.5 mM was used as standard. UPLC was performed on an Acquity system
(Waters, Eschborn, Germany) equipped with PDA detector set at 260 nm. Column used was
BEH C18 column (100 mm × 2.1 mm i.d., 1.7 µm) from Waters. Flow rate was 0.7 mL min−1
and column temperature was kept at 55°C. Peak identification and integration was performed
with software Empower 2 (Waters, Eschborn, Germany) using an Amino Acid Standard H
(Pierce, USA). Tryptophan was not quantified due to its susceptibility to acid hydrolysis.
Plasma glucose and triglycerides were determined by the enzymatic colorimetric GPO-PAP
method with commercial kits (Greiner, Bahlingen, Germany). Plasma protein was quantified
according to Bradford with the Roti-Quant kit and a BSA standard dilution (CARL ROTH,
Karlsruhe, Germany) series. All colorimetric assays were measured with a microplate reader
Tecan® Infinite 200 (Crailsheim, Germany) and calculated from a standard dilution series.
Calculations and statistical analysis
Fish performance was determined using the following formulae:
Specific growth rate (SGR, % day-1) = (ln final body weight – ln initial bw) × 100 / days fed
Feed conversion ratio (FCR) = g dry feed intake / g wet body weight gain
Protein efficiency ratio (PER) = g wet body weight gain / g protein intake
Survival (%) = (initial fish count - dead fish count) / initial fish count × 100
All diets were assigned by a completely randomized design. Data are presented as mean ±
standard deviation (SD) of n samples. Biological and analytical data were checked for normal
distribution using the Kolmogoroff Smirnov Test and eventually subjected to transformation.
Data were subjected to one-way analysis of variance (ANOVA) using SPSS 17.0 for
Windows (SPSS Inc., Chicago, US). When differences among groups were identified,
multiple comparisons among means were made using Tukey’s HSD test. Statistical
significance was determined by setting the aggregate type I error at 5% (P<0.05) for each set
of comparisons.
56
Results
Growth performance, feed efficiencies and body composition
Final growth, SGR, feed intake and survival of fish were not compromised when RPC
replaced up to 100 % of dietary fish meal in the control diet (Tab. 4.4). The FCR and PER
were significantly lower in fish fed on diet R66 (1.22±0.03 and 1.70±0.04) compared to the
control group (1.09±0.02 and 1.90±0.04) but similar to fish fed diet R100 (1.18±0.06 and
1.80±0.08).
Table 4.4: Growth response, feed efficiencies and survival of rainbow trout fed experimental diets
R0 R66 R100
Initial weight 37.6 ± 1.9 37.4 ± 1.6 38.5 ± 1.0
Final weight 102.8 ± 11.5 94.0 ± 2.4 97.0 ± 6.6
SGR 1.19 ± 0.08 1.10 ± 0.06 1.10 ± 0.08
Feed intake (DM) 78.5 ± 10.98 75.8 ± 3.39 74.7 ± 5.12
FCR 1.09a ± 0.02 1.22b ± 0.03 1.18ab ± 0.06
PER 1.90a ± 0.04 1.70b ± 0.04 1.80ab ± 0.08
Survival (%) 100 ± 0.0 97.2 ± 4.8 91.7 ± 8.3
Means in the same row with different superscript letters are significantly different (Tukey's test, P<0.05)
Dietary treatment did not influence whole body moisture or protein content. Although not
significantly different from the control group, whole body fat content revealed a slight
increase with increasing dietary RPC inclusion. Percentages of body ash were significantly
lower in trout feeding diets R66 and R100 compared to those fed the control diet (Tab. 4.5).
Table 4.5: Proximate whole body composition (% of original substance) of rainbow trout fed experimental diets
R0 R66 R100
Moisture 68.8 ± 1.1 69.5 ± 0.4 67.9 ± 0.2
Crude protein 15.7 ± 0.4 16.0 ± 0.2 16.1 ± 0.1
Crude fat 13.2 ± 1.3 13.6 ± 0.4 14.9 ± 0.5
Ash 2.0a ± 0.1 1.7b ± 0.2 1.4b ± 0.2
Initial body composition: moisture 72.3%, crude protein 16.1%, crude fat 9.5%, ash 2.6%
Means in the same row with different superscript letters are significantly different (Tukey's test, P<0.05)
57
Blood features
Neither blood haematocrit values nor haemoglobin concentrations displayed significant
differences between the treatment groups. Additionally, investigated blood serum values were
not significantly different between treatment groups (Tab. 4.6).
Table 4.6: Blood parameters of trout fed experimental diets
R0 R66 R100
Haemoglobin (mM L-1) 3.78 ± 0.49 3.22 ± 0.68 3.72 ± 0.62
Haematocrit (Proportion of 1) 0.38 ± 0.06 0.34 ± 0.04 0.36 ± 0.04
Triglycerides (mM L-1) 7.32 ± 2.70 5.74 ± 1.68 5.44 ± 2.13
Glucose (mM L-1) 10.98 ± 3.82 9.82 ± 2.55 9.82 ± 2.94
Protein (g L-1) 33.6 ± 1.9 34.1 ± 1.4 33.7 ± 1.7
Histology features
No changes in histological morphology were observed between dietary treatments for any of
the intestinal regions examined and no pathological reactions were identified.
Discussion
In several fish feeding trials rapeseed products have been found to be viable alternatives to
fish meal (Davies et al. 1990; Burel et al. 2000, 2001; Kissel et al. 2000; Thiessen et al. 2003;
Shafaeipour et al. 2008). However, when replacing relatively high percentages of dietary fish
meal, it was found, that antinutritional factors present in rapeseed products such as
glucosinolates, phytic acid or polysaccharides can negatively affect feed taste, feed intake and
nutrient absorption and consequently deplete fish growth (Francis et al. 2001). Despite a
favourable amino acid profile, the nutritional quality of rapeseed products tested so far was
below that of fish meal. The rapeseed protein concentrate tested in the present study was
produced to contain a protein level comparable to fish meal. Processing techniques applied
(Slawski et al. in press) led to a RPC with relatively low levels of glucosinolates (1.32 µmol
g-1), phytic acid (1.77 g 100g-1), polysaccharides and other antinutritional factors. In
comparison, rapeseed meals tested by Burel et al. (2000a,b,c; 2001) in fish meal replacement
studies with rainbow were either pressure cooked or directly oil extracted. These meals
contained 26 µmol g-1 or 40 µmol g-1 glucosinolates and 4.43 or 4.15 g 100g-1 phytic acid,
58
respectively, and led to reduced growth performance when replacing 33 % of dietary fish
meal. With our rapeseed protein product, it was possible to replace 100 % of fish meal from a
rainbow trout diet without negative effects on fish growth or feed efficiencies.
It has been reported (Burel et al. 2000; Slawski et al. in press) that fish meal replacement with
rapeseed products reduces feed intake even at low dietary concentrations, most probably due
to the bitter taste of glucosinolates. In the present study, feed intake did not vary significantly
between treatment groups thereby indicating the elimination of bitter flavour as well as a
suitable diet taste at high dietary RPC inclusion. Blood meal and particularly crustacean meal
incorporated into the diets potentially contributed to maintaining appetence and diet taste as
well as nutritional quality as reported in studies on alternative plant protein sources fed to
carnivorous fish (Espe et al. 2006, 2007; Torstensen et al. 2008).
While diet taste and feed intake were not compromised by RPC inclusion, slightly negative
tendencies in fish growth and feed efficiencies in treatment groups R66 and R100 still
indicate some unfavourable effects, though not significant here. Although nutrient
digestibility was not determined in the current study, it has been reported that even products at
low replacement, high protein content and subsequent low levels of antinutrients have adverse
effects on nutrient digestibility. Mwachireya et al. (1999) found that fibre levels - either alone
or together with phytates - negatively affects digestibility of canola protein products in
rainbow trout. The authors reported that among different canola products tested only a protein
isolate (90.8 % CP) met nutrient digestibility coefficients corresponding to fish meal.
Therefore, it appears possible, that remaining levels of antinutritional factors present in the
RPC might have a slightly negative impact on nutrient digestibility, thereby depressing feed
efficiencies in treatment groups R66 and R100 compared to the control group.
Besides reduced diet digestibility due to antinutritional factors, experimental result suggested,
that dietary phosphorus content and phosphorus availability were reduced in treatment groups
R66 and R100. This is indicated by decreasing body ash levels in respective groups as it is
known that body ash level can be reduced when fish are fed a diet deficient in available
phosphorus (Skonberg et al. 1997; Shao et al. 2008). As shown in Tab. 4.3, dietary
phosphorus levels decreased from 1.27 to 0.82 % with increasing dietary level of RPC.
Although these values are above established requirement levels for rainbow trout (Ogino and
Takeda 1978) it seems possible that better phosphorus availability in diet R0 positively
affected feed efficiencies. Antinutritional factors such as phytic acid, fibre and other complex
carbohydrates present in RPC might have furthermore reduced phosphorus availability in fish
59
(Francis et al. 2001). The assumption, that phosphorus availability was lower in diets R66 and
R100 is supported by slight differences in fish whole-body lipid content (Tab. 4.5). Although
not significantly different between treatment groups a tendency for increased body lipid
content with higher dietary RPC content can be detected. Increased whole-body lipid content
with high dietary levels of vegetable proteins has been reported in several fish species
(Adelizi et al. 1998; Kaushik et al. 2004). It is believed that this is caused by the accumulation
of fatty acids due to impaired β-oxidation (Takeuchi and Nakazoe 1981) or oxidative
phosphorylation due to phosphorus deficiency, thereby inhibiting the TCA cycle and leading
to an accumulation of acetyl-CoA and an increased fatty acid synthesis (Skonberg et al.
1997). For prospective feeding trials dietary phosphorus supplementation might support
phosphorus availability as shown by Lee et al. (2010).
With regard to the plasma parameters, no significant differences between the control group
and fish fed diets containing RPC were found. Consistent blood haemoglobin, haematocrit
and serum values therefore indicate an equal nutritional status among feeding groups
(Congleton and Wagner 2006).
In this study, no histopathological abnormalities compared to the control were observed. The
use of RPC did not cause any detectable histological alterations as found in Atlantic salmon
when receiving diets that contained certain amounts of solvent extracted soybean meals,
which have been identified to potentially induce enteritis (van den Ingh et al. 1991;
Baeverfjord and Krogdahl 1996).
In conclusion, the rapeseed protein concentrate processed and tested here showed great
potential as an alternative to fish meal in rainbow trout diets. With fish meal prices on the rise
it remains a matter of time until high quality rapeseed protein products can be produced in
large quantities at a competitive price and become a common fish feed ingredient.
References
Adelizi, P.D., Rosati, R.R., Warner, K., WuÆ� &vR� � �V çF—F–
W �2 � B� � � �6ö× WF—F—fR� � &– �6R � æB� &V6ö � �ÖR � �6öÖÖöâ f—
� �6‚ fVVB –æw&VF– �VçBàÐÕ&VfW&Væ6W0Ô FVÆ—
� �¦’ äBâÂ� � � � �&÷6 F’ "å"â v &æ � �W" ²â wR � ’ �åbâ ×VVæ6‚Â� Bå"â
� v†—FR � � � � Òå"â '&÷vâ ä"âÂ� � ““ � �‚â Wf ÇV� F– � �öâ öb f—
6‚ÖÖV� Â� g&VR� F– �WG2 f÷"
60
arge quantities at a competitive price and become a common fish feed ingredient.
References
Adelizi, P.D., Rosati, R.R., Warner, K., Wu, Y.V., Muench, T.R., White, M.R., Brown,
P.B., 1998. Evaluation of fish-meal f �ree diets for Æ &vR� � �V çF—F–
� � � � � �W2 B 6ö× WF—F—fR� � &– � � � � � �6R æB &V6öÖR 6öÖÖ �öâ f—
6‚� fV �VB –æw&VF–VçBàÐÕ&VfW&Væ6W0Ô� FVÆ—
¦’Â� � �äBâ &÷6� F’Â� �"å"â v� &æ � �W" ²â wR � ’åbâ � ×VVæ6 �‚ Bå"â
� v†— � � � � � � � � �FR Òå"â '&÷vâ ä"â ““‚â Wf ÇV F– � �öâ öb f—
� � �6‚ÖÖV Â g&VR F– �WG2 f÷"
61
arge quantities at a competitive price and become a common fish feed ingredient.
RefepC., Boujard, T., Escaffre, A.M., Kaushik, S.J., Boeuf, G., Mol, K., Van Der
Geyten, S., Kühn, E.R., 2000b. Dietary low glucosinolate rapeseed meal affects thyroid
status and nutrient utilization in rainbow trout (Oncorhynchus mykiss). British Journal of
Nutrition 83, 653–664.
� � � �R Òå"â '&÷vâ ä"âÂ� � “ � �“‚â Wf Ç �V F– � �öâ öb f—6‚ÖÖV� Â� g&VR� F–
�WG2 f÷"
62
arge quantities at a competitive price and become a common fish feed ingredient.
References
Adelizi, P.D., Rosati, R.R., Warner, K., Wu, Y.V., Muench, T.R., White, M.R., Brown,
P.B., 1998. Evaluation of fish-meal f �ree diets for Æ &vR� � �V çF—F–
� � � � � �W2 B 6ö× WF—F—fR� � &– � � � � � �6R æB &V6öÖR 6öÖÖ �öâ f—
6‚� fV �VB –æw&VF–VçBàÐÕ&VfW&Væ6W0Ô� FVÆ—
¦’Â� � �äBâ &÷6� F’Â� �"å"â v� &æ � �W" ²â wR � ’åbâ � ×VVæ6 �‚ Bå"â
� v†— � � � � � � � � �FR Òå"â '&÷vâ ä"â ““‚â Wf ÇV F– � �öâ öb f—
� � �6‚ÖÖV Â g&VR F– �WG2 f÷"
63
arge quantities at a competitive price and become a common fish feed ingredient.
RefepC., Boujard, T., Escaffre, A.M., Kaushik, S.J., Boeuf, G., Mol, K., Van Der
Geyten, S., Kühn, E.R., 2000b. Dietary low glucosinolate rapeseed meal affects thyroid
status and nutrient utilization in rainbow trout (Oncorhynchus mykiss). British Journal of
Nutrition 83, 653–664.
erences
Adelizi, P.D., Rosati, R.R., Warner, K., Wu, Y.V., Muench, T.R., White, M.R., Brown,
P.B., 1998. Evaluation of fish-meal f �ree diets for Æ &vR� � �V çF—F–
� � � � � �W2 B 6ö× WF—F—fR� � &– � � � � � �6R æB &V6öÖR 6öÖÖ �öâ f—
6‚� fV �VB –æw&VF–VçBàÐÕ&VfW&Væ6W0Ô� FVÆ—
¦’Â� � �äBâ &÷6� F’Â� �"å"â v� &æ � �W" ²â wR � ’åbâ � ×VVæ6 �‚ Bå"â
� v†— � � � � � � � � �FR Òå"â '&÷vâ ä"â ““‚â Wf ÇV F– � �öâ öb f—
� � �6‚ÖÖV Â g&VR F– �WG2 f÷"
64
arge quantities at a competitive price and become a common fish feed ingredient.
RefepC., Boujard, T., Escaffre, A.M., Kaushik, S.J., Boeuf, G., Mol, K., Van Der
Geyten, S., Kühn, E.R., 2000b. Dietary low glucosinolate rapeseed meal affects thyroid
status and nutrient utilization in rainbow trout (Oncorhynchus mykiss). British Journal of
Nutrition 83, 653–664.
� FVÆ—
¦’Â� � �äBâ &÷6� F’Â� �"å"â v� &æ � �W" ²â wR � ’åbâ � ×VVæ6 �‚ Bå"â
� v†— � � � � � � � � �FR Òå"â '&÷vâ ä"â ““‚â Wf ÇV F– � �öâ öb f—
� � �6‚ÖÖV Â g&VR F– �WG2 f÷"
65
arge quantities at a competitive price and become a common fish feed ingredient.
RefepC., Boujard, T., Escaffre, A.M., Kaushik, S.J., Boeuf, G., Mol, K., Van Der
Geyten, S., Kühn, E.R., 2000b. Dietary low glucosinolate rapeseed meal affects thyroid
status and nutrient utilization in rainbow trout (Oncorhynchus mykiss). British Journal of
Nutrition 83, 653–664.
RefepC., Boujard, T., Escaffre, A.M., Kaushik, S.J., Boeuf, G., Mol, K., Van Der
Geyten, S., Kühn, E.R., 2000b. Dietary low glucosinolate rapeseed meal affects thyroid
status and nutrient utilization in rainbow trout (Oncorhynchus mykiss). British Journal of
Nutrition 83, 653–664.
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meal substitutes in diets for turbot (Psetta maxima): growth, nutrient utilisation and
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67
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69
Chapter 5: Replacement of fish meal with albumin and globulin rapeseed
protein fractions in diets fed to rainbow trout (Oncorhynchus mykiss W.)
H. Slawski1, 2, H. Adem3, R.-P. Tressel3, K. Wysujack4,
F. Nagel1 and C. Schulz1, 2
1Gesellschaft für Marine Aquakultur mbH, Hafentörn 3, D-25761 Büsum
2Institute of Animal Breeding and Husbandry, Christian-Albrechts-Universität zu Kiel,
D-24098 Kiel
3Pilot Pflanzenöltechnologie Magdeburg e.V., Berliner Chaussee 66, D-39114 Magdeburg
4Johann Heinrich von Thünen-Institut, Federal Research Institute of Rural Areas, Forestry and
Fisheries; Institute of Fisheries Ecology, Wulfsdorfer Weg 204, D-22926 Ahrensburg
…submitted to!
70
Abstract
The potential of two rapeseed protein concentrates partitioned in albumin and globulin
fractions as fish meal alternative was evaluated. In a digestibility experiment with juvenile
rainbow trout apparent digestibility coefficients were determined by indirect marker method.
ADCs of protein from fish meal (89.2±1.1 %) and globulin concentrate (88.8±0.6 %) were
significantly higher than from albumin concentrate (77.7±1.4 %). ADCs of dietary dry matter
were similar between the control diet (62.5±4.7 %) and the globulin concentrate diet
(62.3±0.5 %), but significantly lower in the albumin concentrate diet (56.2±1.5 %). In a
consecutive growth trial, ten rainbow trout (initial average weight 31.5±0.5 g) were stocked
into each of 21 experimental tanks of a freshwater flow-through system. Fish were organized
in triplicate groups and received experimental diets with 0, 50, 75, or 100 % of fish meal
replaced with albumin or globulin concentrate on the basis of digestible protein. At the end of
a 70 day feeding period it was found that only in treatment group A50, fish growth
performance and feed intake were not negatively affected by dietary treatment. However, feed
efficienies were not significantly different compared to the control group at 100 % or 75 %
fish meal replacement level with albumin or globulin concentrate, respectively. Significant
lower fish survival rates were observed when fish received diets A75, A100, G50, G75, or
G100 compared to the control diet or diet A50. For the whole body composition, the crude
protein content was significantly lower in fish fed diet G75 or G100 compared to the control
diet, while fish fed on diet A50, A75, or A100 were lower in body fat content than fish fed on
the control diet. It is concluded, that the used albumin concentrate can effectively replace 50
% of dietary fish meal in rainbow trout diets. The used globulin concentrate negatively
influenced diet palatability, thereby reducing diet intake and subsequently fish growth.
71
Introduction
Fish meal, the most important source of animal protein for fish diets, is a limited resource.
Due to increasing prices for fish meal together with environmental concerns the aquaculture
sector is forced to find alternative protein sources to be included in fish feeds. Wide
availability, relatively high protein contents and a desirable amino acid profile have caused an
interest in rapeseed products as ingredients for fish feed production. But, the nutritional
quality of rapeseed products largely depends on their levels of antinutritional factors,
particularly glucosinolates, phytic acid, phenolic constituents and indigestible carbohydrates,
as it was found in feeding trials with several fish species (Webster et al. 1997; Burel et al.
2000a,b,c, 2001; Thiessen et al. 2003, 2004; Glencross et al. 2004; Shafaeipour et al. 2008). It
was generally observed, that the nutritional quality of simple rapeseed products was below
that of fish meal, mainly due to antinutritional factors present in rapeseed products (Mawson
et al. 1995; Francis et al. 2001). By several processing techniques the level of antinutrients in
rapeseed products can be decreased and their value for fish nutrition improved (Fenwick et al.
1986; Anderson-Haferman et al. 1993; Tripathi et al. 2000). Protein extraction from meals by
ethanol-treatment will increase protein level and effectively remove glucosinolates, phenolic
compounds, soluble sugars and some oligosaccharides (Naczk and Shahidi 1990; Chabanon et
al. 2007). However, particularly ethanol-treatments are costly and time consuming (Slawski et
al. in press a,b).
In the present study, two rapeseed protein products either consisting mainly of albumin or
globulin fractions were derived from rapeseed after a hexane treatment and ultrafiltration. The
aim of the study was to clarify, if fractionized protein concentrates produced with less effort
than high quality protein concentrates or isolates are valuable fish meal alternatives in the
nutrition of rainbow trout. For this, nutrients digestibility was determined by indirect marker
method and compared to fish meal and the concentrates were evaluated as fish meal
replacement in rainbow trout diets.
Materials and methods
Digestibility trial
Three diets were produced for the digestibility trial. A quantity of 10g kg-1 of titanium oxide
was added to a control diet mixture as inert marker for determination of apparent digestibility
coefficients. For the determination of apparent digestibility coefficients of rapeseed albumin
and rapeseed globulin concentrate diets were formulated that consisted of 700 g kg-1 of the
72
control diet and of 300 g kg-1 rapeseed albumin or globulin concentrate, respectively (on as is
basis). The rapeseed albumin and globuline concentrates were produced by the PPM,
Magdeburg, Germany. For the production of the concentrates a batch of rapeseed (variety
Lorenz, Norddeutsche Pflanzenzucht, Hohenlieth, Germany) was conditioned in a vacuum
dryer for 15 minutes at 70-80 °C to inactivate the enzyme myrosinase. Then rapeseed was
cold pressed. To remove residual oil from the oilcake (12.9 % crude lipid, 31.3 % crude
protein) it was crushed into 1-5 mm particle size followed by a hexane treatment. The
treatment lasted for two hours and the incubation temperature was 60 °C. Hexane was
removed from rapeseed meal extract by ventilation until the material contained not more than
1.5 % hexane. Then rapeseed meal extract was further crushed to a particle size of 0.2 to 0.1
mm. In the following, protein was gained through liquid water extraction (rapeseed meal
extract 1:10 water). For this, the suspension was heated to 40-45 °C followed by one hour of
constant agitation. Afterwards the suspension was decanted. Following decantation the
solvent was collected and residue material was secondly extracted (residue 1:10 water, 5 %
NaCl) at 40-45 °C and one hour contact time under constant agitation. Following extraction
the suspension was decanted. Then solvents of extraction 1 and 2 were separately treated in
order to receive mainly rapeseed globulin or albumin molecules after dia- and ultrafiltration
(membrane size: 10 kDa). During filtration conductivity was checked. Protein washing ended,
when conductivity was 5-6 mS cm-1. The gained materials were spray dried at 70-80 °C,
which led to a globulin or albumin concentrate with a crude protein content of 56.3 or 70.1 %,
respectively (Table 5.1). Diet mixes were manufactured to give pellets 4 mm in diameter (L
14-175, AMANDUS KAHL, Reinbek, Germany). Diet formulations, nutritional compositions
and amino acid profiles are presented in Table 5.2.
73
Table 5.1: Nutrient composition (% dry matter) and amino acid profiles (g 100g-1 CP) of fish
meal, albumin concentrate and globulin concentrate
Fish meal Albumin Globulin
Dry matter (%) 91.6 94.6 94.8
Crude protein 69.0 70.1 56.3
Crude fat 7.0 0.38 0.37
Ash 20.7 20.8 8.6
NfEa 3.3 8.7 29.5
Gross energyb (MJ kg-1) 19.9 18.4 18.8
Amino acids
Arginine 5.84 6.50 6.24
Histidine 2.00 3.28 2.62
Isoleucine 3.62 4.00 3.87
Leucine 6.45 7.55 7.04
Lysine 6.55 6.95 5.07
Methionine
(+ Cysteine)
2.37
3.17
1.86
5.22
2.02
4.18
Phenylalanine 3.52 3.89 4.16
Threonine 3.90 4.24 4.20
Valine 4.45 5.31 5.00
Phytic acid (g 100g-1) 2.04 1.53
Glucobrassicanapin 0.29
Glucobrassicin
Gluconapin 0.86
Gluconapoleiferin
Progoitrin 1.16
4-Hydroxyglucobrassicin
∑ Glucosinolates (µmol g-1) <0.10 2.31 aNitrogen free extract = 100 – (%crude protein + %crude fat + %ash). bCalculated by: crude protein = 23.9 MJ kg-1; crude fat = 39.8 MJ kg-1; NfE, fibre: 17.6 MJ kg-1.
74
Table 5.2: Formulation and nutrient composition (g kg-1) and amino acid profiles (g kg crude protein-1) of
experimental diets used in the digestibility trial
Control Albumin Globulin
Herring meal 685.6 480 480
Albumin 300
Globulin 300
Maizestarch 144.9 101 101
Fishoil 99.5 70 70
Vit/MinMixa 20.0 14 14
Wheat Gluten 40.0 28 28
Titaniumdioxide 10.0 7 7
Nutrient composition
Moisture (% wet weight) 66.1 69.1 84.8
Crude protein 525.1 559.1 542.5
Crude fat 162.7 135.2 127.8
Ash 139.7 185.8 129.8
NfEb 172.5 119.9 199.9
Gross energyc (MJ kg-1) 22.1 20.8 21.6
Amino acids
Arginine 57.2 59.8 58.2
Histidine 20.7 25.3 22.6
Isoleucine 37.2 37.9 37.0
Leucine 66.0 68.5 66.4
Lysine 62.0 64.4 58.0
Methionine
(+ Cysteine)
24.4
32.7
21.3
39.0
22.4
35.2
Phenylalanine 36.4 36.6 37.4
Threonine 37.4 39.0 37.8
Valine 45.3 47.6 46.2 aAA-Mix 507101, Vitfoss, Gråsten, Denmark. bNitrogen free extract = 100 – (%crude protein + %crude fat + %ash).
cCalculated by: crude protein = 23.9 MJ kg-1; crude fat = 39.8 MJ kg-1; NfE, fibre: 17.6 MJ kg-1.
75
Digestibility trial
The digestibility trial was conducted in the experimental facilities of the Gesellschaft für
Marine Aquakultur (GMA) in Büsum, Germany. Fifty rainbow trout (average weight
41.9±3.3 g), obtained from Fischzucht Reese (Sarlhusen, Germany) were stocked into each of
9 cylindrical tanks (350 L) of a freshwater recirculation system. Photoperiod was artificially
controlled (6.00 a.m. to 6.00 p.m.). Tanks were provided with filtered water at 9 L min-1
(temperature: 15.5±0.7 °C; O2: 9.2±0.5 mg L-1; pH: 7.2±0.5; NH4+: <1.0 mg L-1; NO2
-: <0.5
mg L-1). Fish were fed at 2 % of their body weight per day in three portions to assure effective
nutrient utilization. Beside an overflow, tanks had a funnel shaped bottom were faeces
accumulated. The funnel was connected to a pipe outlet. Faeces were obtained by continuous
sieving of pipe outlet water. After a one week adaptive period to the experimental diets
readily excreted faeces were collected for 7 days. Faecal samples were freeze dried before
analysis. Following dietary and faeces nutrient and marker analysis apparent digestibility
coefficients (ADCs) of dry matter and protein of the reference diet were calculated according
to Maynard & Loosly (1969):
ADC of dry matter of diet (%) = 100 × [1 – (dietary TiO2/faecal TiO2)]
ADC of protein of diet (%) = 100 × [1 – (dietary TiO2/ faecal TiO2) × (faecal protein
concentration / dietary protein concentration)]
ADCs of dry matter and protein in albumin or globulin concentrate were calculated as follows
(Sugiura et al. 1998):
ADC of dry matter of CPI (%) = (ADC of the test diet – 0.7 × ADC of the reference diet) / 0.3
ADC of protein of albumin or globulin concentrate (%) = [(protein concentration in test diet ×
protein ADC of the test diet) – (0.7 × protein concentration in reference diet × protein ADC of
the reference diet)] / (0.3 × protein concentration in ingredient)
Data are presented as means with standard deviation (Table 5.4). Means were compared with
Student’s t-test (P<0.05).
76
Growth trial
Based on the results from the digestibility trial five experimental diets were formulated in
which 0, 50, 75 or 100 % of digestible fish meal protein was replaced with digestible protein
from albumin or globulin concentrate (designated as Control, A50, A75, A100, G50, G75,
G100, respectively). Diets were supplemented with vitamins and minerals to meet the dietary
requirements of freshwater fish (NRC 1993). Since essential amino acid concentrations did
not differ considerably between experimental diets supplementation of synthetic amino acids
appeared unnecessary. The diet mixtures were manufactured to give pellets 4 mm in diameter
(L 14-175, AMANDUS KAHL, Reinbek, Germany). Diet formulations, proximate
compositions and amino acid profiles are presented in Table 5.3.
The growth trial was conducted in the experimental facilities of Johann Heinrich von Thünen-
Institut, Institute of Fisheries Ecology (Ahrensburg, Germany). Juvenile rainbow trout that
had been hatched in the institute were used. One week before the experiment started ten fish
were stocked in each of fifteen experimental tanks (40 L), being part of a flow-through
system. Photoperiod was in accordance to natural rhythmic from August to October at our
latitude (53° 41' 0" N). Tanks were provided with well freshwater at 1 L min-1 (temperature:
12.1±0.5 °C; O2: 7.8±0.2 mg L-1; pH: 7.2±0.5; NH4+: <0.1 mg L-1; NO2
-: <0.2 mg L-1). For a
one week adaptation period fish were fed the control diet in two daily meals until apparent
satiation. After the adaptation period, fish were fasted for two days and initial average weight
was determined (31.5 ± 0.5 g). Triplicate groups of fish were fed the experimental diets twice
daily (at 8.30 a.m. and 16.00 p.m.) to apparent satiation for 70 days. At the beginning and at
end of the experiment, samples of the initial and final fish population (21 x 2 fish) were taken
and stored at -23 ºC to determine initial and final body composition.
Chemical analysis and laboratory procedures
Diets and homogenised fish bodies were analysed in duplicate for proximate composition.
Dry matter was calculated from weight loss after drying in an oven at 105 °C until constant
weight. Fat content was determined after HCl hydrolysis (Soxtec HT6, Tecator, Höganäs,
Sweden) and total nitrogen content by the Kjeldahl technique (protein = N × 6.25; Kjeltec
Auto System, Tecator, Höganäs, Sweden). Ash content was calculated from weight loss after
incineration of samples in a muffle furnace for 2 hours at 550 °C. Raw material and dietary
amino acid concentrations were analysed as described by Tzovenis et al. (2009). Tryptophane
was not analyzed.
77
Table 5.3: Formulation, proximate nutrient composition (g kg-1) and amino acid composition
(g 100g-1 dietary protein) of experimental diets for rainbow trout
Control A50 A75 A100 G50 G75 G100
Herring meal 325 162.5 81.3 0 162.5 81.3 0
Albumin concentrate 0 183.6 275.4 367.2
Globulin concentrate 0 200 300.2 400.2
Blood meal 150 150 150 150 150 150 150
Soyprotein concentrate 135 135 135 135 135 135 135
Potato starch 232 199.9 183.3 167.8 183.5 158.5 134.8
Fish oil 118 129 135 140 129 135 140
Vit/MinMixa 20 20 20 20 20 20 20
Wheat gluten 20 20 20 20 20 20 20
Nutrient composition
Moisture (% wet weight) 71 69 68 71 75 70 79
Crude protein 489 526 545 551 507 518 536
Crude fat 159 165 162 167 157 156 154
Ash 80 84 82 69 65 54 47
NfEb 272 225 212 213 271 272 263
Gross energyc (MJ kg-1) 22.8 23.1 23.2 23.6 23.1 23.4 23.6
Phytic acid (g kg-1) 3.74 5.62 7.50 3.06 4.59 6.12
Glucobrassicanapin
Glucobrassicin
Gluconapin 0.17 0.21
Gluconapoleiferin
Progoitrin 0.23 0.30 0.37
4-Hydroxyglucobrassicin
∑ Glucosinolates (µmol g-1) 0.23 0.47 0.58
Amino acids
Arginine 5.59 5.74 5.73 5.87 5.59 5.70 5.68
Histidine 3.98 4.27 4.26 4.30 4.04 4.01 4.13
Isoleucine 2.88 2.95 2.97 3.03 2.89 2.95 2.89
Leucine 8.89 9.09 8.96 9.08 8.92 8.78 8.99
Lysine 7.00 7.12 7.03 6.98 6.54 6.30 6.21
Methionine
(+ Cysteine)
1.72
2.56
1.59
3.14
1.52
3.33
1.51
3.49
1.56
2.73
1.54
2.87
1.47
2.94
Phenylalanine 4.91 4.92 4.79 4.99 4.97 4.97 5.08
Threonine 3.50 3.69 3.64 3.77 3.54 3.56 3.57
Valine 6.16 6.28 6.15 6.22 6.18 6.16 6.21 aAA-Mix 507101, Vitfoss, Gråsten, Denmark bNitrogen free extract = 100 – (%crude protein + %crude fat + %ash) cCalculated by: crude protein = 23.9 MJ kg-1; crude fat = 39.8 MJ kg-1; NfE, fibre: 17.6 MJ kg-1
78
Calculations and statistical analysis
Fish performance was determined, using the following formulae:
Specific growth rate (SGR, % day-1) = (ln final body weight – ln initial bw) × 100 / days fed
Feed conversion ratio (FCR) = g dry feed intake / g wet body weight gain
Protein efficiency ratio (PER) = g wet body weight gain / g protein intake
Survival (%) = (initial fish count - dead fish count) / initial fish count × 100
All diets were assigned by a completely randomized design. Biological and analytical data
were checked for normal distribution using the Kolmogoroff Smirnov Test and eventually
subjected to transformation. Data were subjected to a one-way analysis of variance (ANOVA)
using SPSS 17.0 for Windows (SPSS Inc., Chicago, US). When differences among groups
were identified, multiple comparisons among means were made using Tukey’s HSD test.
Statistical significance was determined by setting the aggregate type I error at 5% (P<0.05)
for each set of comparisons.
Results
Digestibility coefficients
As shown in Table 5.4, the ADC of dry matter in the control diet (62.5±4.7 %) was not
significantly different towards the globulin concentrate diet (62.3±0.5 %), but significantly
higher compared to the albumin concentrate diet (56.2±1.5 %). The ADC of protein from fish
meal (89.2±1.1 %) – the predominant protein source in the control diet - was similar to the
ADC of protein in the globulin concentrate diet (88.8±0.6 %) but significantly lower in the
albumin concentrate diet (77.7±1.4 %) (P<0.05).
Table 5.4: Apparent digestibility coefficients
Fish meal diet Albumin
concentrate diet
Globulin
concentrate diet
ADC of dry matter 62.5a ± 4.7 56.2b ± 1.5 62.3a ± 0.5
ADC of test ingredient 41.6b ± 4.9 62.0a ± 1.6
Crude protein 89.2a ± 1.1 77.7b ± 1.4 88.8a ± 0.6
Values in the same row with different superscript letters are significantly different (P<0.05).
79
Growth performance, feed efficiencies and body composition
Fish growth response, feed intake and feed efficiencies were not negatively affected when 50
% of digestible fish meal protein was replaced with protein from albumin concentrate (Table
5.5). At higher fish meal replacement with albumin concentrate, final weight, specific growth
rate and feed intake were significantly lower compared to the control group and fish receiving
diet A50. Feed conversion ratio and protein efficiency ratio, however, were not significantly
different towards the control group at 100 % fish meal replacement level with albumin
concentrate. When fish meal was replaced with globulin concentrate, growth performance and
feed intake were significantly reduced at 50 % fish meal replacement level (Table 5.5). Feed
efficiencies were similar to the control group up to 75 % fish meal replacement with globulin
concentrate (Table 5.5). Significant lower fish survival rates were observed when fish
received diets A75, A100, G50, G75, or G100 compared to the control diet or diet A50 (Table
5.5). For the whole body composition, significant higher body moisture contents were
determined for fish fed diet A75 or A100 compared to the control diet. The whole body crude
protein content was significantly lower in fish fed diet G75 or G100 compared to the control
diet, while fish fed on diet A50, A75, or A100 were lower in whole body fat content than fish
fed on the control diet (Table 5.6). Body ash content was not significantly different among
dietary treatments.
Table 5.5: Growth performance, feed intake and feed efficiencies of rainbow trout fed
experimental diets
Control A50 A75 A100 G50 G75 G100
Initial weight 31.4±0.3 31.3±0.9 31.0±0.6 31.9±0.2 31.8±0.1 31.4±0.2 31.4±0.2
Final weight 91.3a±7.6 90.6a±2.8 79.7b±5.7 79.9b±4.6 77.7b±5.4 66.3c±3.9 60.8c±4.0
SGR 1.57a±0.12 1.57a±0.02 1.39b±0.08 1.35b±0.07 1.31b±0.11 1.09c±0.10 0.97c±0.11
Feed intake (DM) 67.6a±3.9 60.3ab±3.7 52.7bc±3.9 54.3bc±8.0 57.1bc±3.7 49.5c±6.9 44.0cd±2.1
FCR 1.14ab±0.08 1.02a±0.04 1.09a±0.04 1.04a±0.08 1.26b±0.07 1.41c±0.05 1.39c±0.02
PER 1.80ab±0.13 1.92a±0.07 1.72b±0.07 1.65b±0.11 1.61b±0.09 1.38c±0.04 1.44c±0.03
Survival (%) 96.6a±5.7 96.6a±5.7 76.7b±11.5 50.0b±26.4 46.6b±31.1 50.0b±36.0 46.6b±28.9
Values are given as mean ± standard deviation. Values in the same row with different superscript letters are
significantly different (P<0.05). SGR, Specific growth rate; FCR, Feed conversion ratio; PER, Protein efficiency ratio.
80
Table 5.6: Proximate whole body composition (g kg-1 wet weight) of rainbow trout fed
experimental diets
Control A50 A75 A100 G50 G75 G100
Moisture 696a ± 5 708ab ± 8 711b ± 6 717b ± 6 702a ± 1 707ab ± 9 708ab ± 7
Crude protein 164a ± 2 164a ± 4 165a ± 4 163ab ± 5 158ab ± 6 155b ± 4 155b ± 5
Crude fat 117a ± 7 96b ± 9 95b ± 8 93b ± 8 109ab ± 8 110ab ± 9 110ab ± 9
Ash 33 ± 3 32 ± 4 31 ± 2 29 ± 3 29 ± 3 27 ± 3 28 ± 2
Initial body composition: moisture 782 g kg-1, crude protein 141 g kg-1, crude fat 50 g kg-1, ash 28 g kg-1.
Means in the same row with different superscript letters are significantly different (Tukey's test, P<0.05)
Discussion
In the conducted feeding trial 50 % of dietary fish meal was successfully replaced with
albumin concentrate. Higher fish meal replacement levels with albumin concentrate or fish
meal replacement with globulin concentrate failed and resulted in reduced feed intake,
decreased growth performance and high mortalities.
The digestibilty of protein from albumin and globulin concentrate was determined and
compared to fish meal protein. While protein from globulin concentrate (88.8±0.6 %) was as
efficiently digested as fish meal protein (89.2±1.1 %), the digestibility of protein from
albumin concentrate was lower (77.7±1.4 %). The ADC of protein from albumin concentrate
is comparable to that of canola meal protein (74.0 %) determined in Atlantic salmon
(Anderson et al. 1992). Furthermore, Mwachireya et al. (1999) observed ADCs of protein
between 77.4 to 88.1 % from differently processed canola meals. Different ADCs for protein
can result from the raw material NfE content, which can negatively influence protein
digestibility in carnivorous fish (Storebakken et al. 1998; Burel et al. 2000a; Francis et al.
2001). Mwachireya et al. (1999) stated that fibre levels, either alone or together with phytate,
can have greatest adverse effects on the digestibility of canola protein products for rainbow
trout. The albumin concentrate (CP: 70.1 %) used in the present study contained 20.8 % crude
ash, 8.7 % NfE and 2.04 g 100g-1 phytic acid while the globulin concentrate (CP: 56.3 %)
contained 8.6 % crude ash, 29.5 % NfE and 1.53 g 100g-1 phytic acid. It appears possible
therefore, that the negative effect on protein digestion from the combination of NfE and
phytic acid in albumin concentrate was more severe than in globulin concentrate. Simple
81
rapeseed products have been widely investigated as feed ingredients in fish growth studies
(Webster et al. 1997; Burel et al. 2000a,b,c, 2001; Thiessen et al. 2003, 2004; Glencross et al.
2004; Shafaeipour et al. 2008). However, the usability of high quality rapeseed protein
products originating from rapeseed oilcakes with protein contents comparable to or above that
of fish meal was seldomly evaluated. Higgs et al. (1982) successfully replaced 25 % of dietary
protein from a fish meal control diet for juvenile Chinook salmon with rapeseed protein
concentrate (CP: 61.3 %) without reducing growth rate and protein utilization. In the present
study, feed intake and fish growth were similar between fish fed on the control diet and fish
receiving diet A50. In all other treatments, feed intake and growth performance were
significantly reduced compared to fish fed on the control diet (Table 5.6). Results indicate
therefore, that diet palatability was negatively influenced at high fish meal replacement with
albumin (diets A75 and A100) or globulin concentrate (diets G50, G75 and G100). Reduced
diet palatability and decreased feed intake when using rapeseed protein concentrate at high
inclusion levels in diets for common carp or wels catfish has also been reported by Slawski et
al. (in press, a,b). This was referred to glucosinolates present in rapeseed, which are known to
negatively influence diet taste (Burel et al. 2000a,b,c). In the present experiment, low diet
intake in respective treatment groups also led to aggressive fish behaviour, which resulted in
further reduction of feed intake, consequently low growth performance and high mortalities.
Although applied processing techniques led to levels of glucosinolates in albumin and
globulin concentrate which were until now assumed to be to low to have detrimental effects
on food intake in rainbow trout (Burel et al. 2000c), it appears that neither albumin nor
globulin concentrate can effectively replace fish meal. The poor fish development in treatment
groups with low feed intake is further indicated in different whole body composition. As
presented in Table 5.6, in respective treatment groups fish bodies were relatively low in
protein (G75 and G100) or body fat (A50, A75 and A100) compared to normal developed fish
from the control group, pointing out to nutritional imbalances (Jobling 1994).
In conclusion, albumin concentrate appeared to be more suitable as fish meal replacement in
rainbow trout diets. However, feed intake of diets containing globulin concentrate or high
levels of albumin concentrate was significantly reduced possibly by reduced diet palatability.
This resulted in aggressive fish behaviour and consequently poor growth and high mortalities.
82
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and mineral availabilities in various feed ingredients for salmonid feeds. Aquaculture
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211–216.
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meal. Aquaculture 150, 103-112.
85
Chapter 6: Total fish meal replacement with canola protein isolate in diets
fed to rainbow trout (Oncorhynchus mykiss W.)
H. Slawski1, 2, F. Nagel1, K. Wysujack3, D. T. Balke4, P. Franz5 and C. Schulz1, 2
1Gesellschaft für Marine Aquakultur mbH, Hafentörn 3, D-25761 Büsum
2Institute of Animal Breeding and Husbandry, Christian-Albrechts-Universität zu Kiel,
D-24098 Kiel
3Johann Heinrich von Thünen-Institut, Federal Research Institute of Rural Areas, Forestry and
Fisheries; Institute of Fisheries Ecology, Wulfsdorfer Weg 204, D-22926 Ahrensburg
4BioExx Speciality Proteins Ltd., 219 (North) Dufferin Street, Toronto, Ontario, Canada
5HELM AG, Business Unit Animal Nutrition, Nordkanalstrasse 28, D-20097 Hamburg
…submitted!
86
Abstract
The potential of canola protein isolate as fish meal alternative in diets for rainbow trout was
evaluated. Apparent digestibility coefficients for protein from fish meal (89.2±1.1 %) and
canola protein isolate (84.6±1.8 %) were determined by indirect marker method in a
digestibility experiment with juvenile rainbow trout. ADC of dietary dry matter was slightly
lower for the control diet (62.5±4.7 %), but not significantly different to the test diet
(65.9±3.1 %). In a consecutive growth trial, twenty fish (initial average weight 31.5±0.5 g)
were stocked into each of fifteen experimental tanks of a freshwater flow-through system.
Fish were organized in triplicate groups and received experimental diets with 0, 25, 50, 75
and 100 % of fish meal replaced with canola protein isolate on the basis of digestible protein.
At the end of a 70 days feeding period it was found that growth performance (individual final
weight: 91.3±7.6 g to 107.9±5.8 g), feed intake (65.4±4.4 g to 69.8±2.5 g) and feed
efficiencies (FCR: 0.92±0.04 to 1.14±0.08) in treatment groups receiving diets devoid of fish
meal were not negatively affected compared to the control group. The tested canola protein
isolate was therefore identified to be a highly valuable fish meal alternative, not affecting diet
taste, feed intake and feed efficiencies.
87
Introduction
Worldwide, 61.6 Mt of rapeseed/canola (Brassica napus L., B. campestris L.) were farmed as
sources of vegetable oil (FAO 2010). Thus, following oil extraction, enormous amounts of
oilcake become available. Canola Meal is widely used in livestock feed systems and Canola
concentrates have been developed also for use in feed system. Recently Canola isolates have
been developed for the food industry with the first producer nearing commercial release.
Although the amino acid profile of canola is suitable for fish nutrition (Higgs et al. 1996), the
oilcake or processed products that were de-oiled with organic solvents retain a variety of
antinutritional factors namely glucosinolates, phytic acid, phenolic constituents and
indigestible carbohydrates (Mawson et al. 1995; Francis et al. 2001). These antinutritional
factors potentially limit the suitability of simple canola products as a protein source and fish
meal alternative in finfish diets at relatively high inclusion levels as shown in experiments
with Oncorhynchus mykiss (Burel et al. 2000a,c 2001; Thiessen et al. 2003, 2004; Shafaeipour
et al. 2008), Oreochromis mossambicus (Davies et al. 1990), Ictalurus punctatus (Webster et
al. 1997), Cyprinus carpio (Dabrowski and Kozlowska 1981), Pagrus auratus (Glencross et
al. 2004) and Psetta maxima (Burel et al. 2000a,b). While several processing techniques such
as dehulling of seeds, heat and water treatments, utilisation of organic solvents and
ultrafiltration will increase protein levels and reduce levels of antinutrients in canola products
(Fenwick et al. 1986; Anderson-Hafermann et al. 1993; Tripathi et al. 2000; Tyagi 2002;
Chabanon et al. 2007) the benefits for fish nutrition are variable.
In previous work, a rapeseed protein concentrate with a crude protein content of 710 g kg-1
and extremely low levels of glucosinolates was tested as fish meal replacement in diets for
Cyprinus carpio and Silurus glanis (Slawski et al. in press, a,b). Replacement of fish meal
with rapeseed protein concenrate at levels above 33 % in carp and 25 % in wels catfish with
rapeseed protein concentrate negatively affected diet taste and feed intake leading to reduced
feed efficiencies.
In the present study, a canola protein isolate with a crude protein content of 812 g kg-1 was
tested as fish meal replacement in the nutrition of rainbow trout. Consecutive to a digestibility
trial the isolate was evaluated as fish meal replacement on the basis of digestible protein in a
growth trial. By this, we aimed to demonstrate the limitless potential of canola protein isolate
as protein source in fish diets.
88
Materials and methods
Digestibility trial
A quantity of 10 g kg-1 of titanium oxide was added to the control diet mixture as inert marker
for estimation of apparent digestibility coefficients. To estimate apparent digestibility
coefficients of canola protein isolate (CPI) a second diet was formulated. This consisted of
700 g kg-1 of the control diet and 300 g kg-1 of CPI. The isolate was produced by BioExx
Specialty Proteins Ltd. (Saskatoon, Canada) (Tab. 6.1) using a novel cold processing
sequence. It consisted of low temperature conditioning, cold oil pressing, low temperature
solvent extraction and desolventization followed by aqueous processing for isolation of the
soluble proteins. The resulting purified protein solution was spray dried to limit thermal
damage. Diet mixes were manufactured to give pellets 4 mm in diameter (L 14-175,
AMANDUS KAHL, Reinbek, Germany). Diet formulations, nutritional compositions and
amino acid profiles are given in Table 6.2.
Table 6.1: Nutrient composition (g kg-1 dry matter) and essential amino acid profiles (g kg-1 protein) of fish meal
and canola protein isolate
Fish meal Canola protein isolate Dry matter (g kg-1) 916 946 Crude protein 690 812 Crude fat 70 11 Crude ash 207 27
Phosphorus 24 6 NfEa 34 150 Gross energyb (MJ kg-1) 19.9 22.5 Essential amino acids Arginine 58.4 70.9 Histidine 20.0 27.6 Isoleucine 36.2 41.8 Leucine 64.5 75.4 Lysine 65.5 51.0 Methionine (+ Cysteine)
23.7 31.7
19.4 40.5
Phenylalanine 35.2 41.9 Threonine 39.0 41.8 Valine 44.5 51.7 Glucosinolates (µmol g-1) 0.13 Phytate (g kg-1) 6.90 aNitrogen free extract = 100 – (%crude protein + %crude fat + %ash + %fibre). bCalculated by: crude protein = 23.9 MJ kg-1; crude fat = 39.8 MJ kg-1; NfE, fibre: 17.6 MJ kg-1
89
Table 6.2: Formulation, nutrient composition (g kg-1) and essential amino acid profiles (g kg-1 crude protein) of
experimental diets used in the digestibility trial
Ingredients Fish meal control
diet
Canola protein
isolate test diet
Herring meala 685.6 480
Canola protein isolate 300
Maizestarchb 144.9 101
Fish oila 99.5 70
Vit/MinMixc 20.0 14
Wheat glutend 40.0 28
Titaniumdioxidee 10.0 7
Proximate composition
Dry matter (g kg-1) 934 933
Crude protein 525 594
Crude fat 163 114
Crude ash 140 128
NfEf 172 164
Gross energyg (MJ kg-1) 22.0 21.6
Essential amino acids
Arginine 57.2 62.3
Histidine 20.7 23.6
Isoleucine 37.2 38.4
Leucine 66.0 68.9
Lysine 62.0 56.9
Methionine
(+ Cysteine)
24.4
32.7
22.0
35.4
Phenylalanine 36.4 38.2
Threonine 37.4 38.6
Valine 45.3 47.1 aVFC GmbH, Cuxhaven, Germany; bEuroduna-Technologies GmbH, Barmstedt, Germany; cAA-Mix 517158 & 508240,
Vitfoss, Gråsten, Denmark; dCargill Deutschland GmbH, Krefeld, Germany; enaturhaus, Neckarwestheim, Germany; fNitrogen free extract = 100 – (%crude protein + %crude fat + %ash).; gCalculated by: crude protein = 23.9 MJ kg-1; crude fat
= 39.8 MJ kg-1; NfE, fibre: 17.6 MJ kg-1. Tryptophane was not analyzed.
The digestibility trial was conducted in the experimental facilities of the Gesellschaft für
Marine Aquakultur (Büsum, Germany). Fifty rainbow trout (average weight 41.9±3.3 g),
obtained from Fischzucht Reese (Sarlhusen, Germany) were stocked into each of 6 cylindrical
90
tanks (350 L) of a freshwater recirculation system. Photoperiod was artificially controlled
(6.00 a.m. to 6.00 p.m.). Tanks were provided with filtered water at 9 L min-1 (15.5±0.7 °C;
O2: 9.2±0.5 mg L-1; pH: 7.2±0.5; NH4+: <1.0 mg L-1; NO2
-: <0.5 mg L-1). Fish were fed at 2 %
of their body weight per day in three portions to assure effective nutrient utilization. Beside an
overflow, tanks had a funnel shaped bottom where faeces accumulated. The funnel was
connected to a pipe outlet. Faeces were obtained by continuous sieving of pipe outlet water.
After a one week adaptive period to the experimental diets readily excreted faeces were
collected for 7 days. Faecal samples were freeze dried before analysis. Following dietary and
faeces nutrient and marker analysis apparent digestibility coefficients (ADCs) of dry matter
and protein of the reference diet were calculated according to Maynard and Loosly (1969):
ADC of dry matter of diet (%) = 100 × [1 – (dietary TiO2/faecal TiO2)]
ADC of protein of diet (%) = 100 × [1 – (dietary TiO2/ faecal TiO2) × (faecal protein
concentration / dietary protein concentration)]
ADCs of dry matter and protein in CPI were calculated as follows (Sugiura et al. 1998):
ADC of dry matter of CPI (%) = (ADC of the test diet – 0.7 × ADC of the reference diet) / 0.3
ADC of protein of CPI (%) = [(protein concentration in test diet × protein ADC of the test
diet) – (0.7 × protein concentration in reference diet × protein ADC of the reference diet)] /
(0.3 × protein concentration in ingredient)
Data are presented as means with standard deviation (Tab. 6.4). Means were compared with
Student’s t-test (P<0.05).
Growth trial
Based on the results from the digestibility trial five experimental diets were formulated in
which 0, 25, 50, 75 or 100 % of digestible fish meal protein was replaced with digestible
protein from canola protein isolate (designated as I0, I25, I50, I75, or I100, respectively).
Diets were supplemented with vitamins and minerals to meet the dietary requirements of
freshwater fish (NRC 1993). Since essential amino acid concentrations did not differ
considerably between experimental diets supplementation of synthetic amino acids appeared
unnecessary. The diet mixtures were manufactured to give pellets 4 mm in diameter (L 14-
175, AMANDUS KAHL, Reinbek, Germany). Diet formulations, proximate compositions
and amino acid profiles are presented in Table 6.3.
91
Table 6.3: Formulation, proximate composition (g kg-1) and essential amino acid profiles (g kg-1 crude protein)
of experimental diets
Ingredients Control I25 I50 I75 I100
Herring meala 325 243.8 162.5 81.3 0
Canola protein isolate 0 72.8 145.5 218.3 291.1
Blood mealb 150 150 150 150 150
Soyprotein concentratec 135 135 135 135 135
Potato starchb 232 235.5 239 242.4 245.9
Fish oila 118 123 128 133 138
Vit/MinMixd 20 20 20 20 20
Wheat glutene 20 20 20 20 20
Proximate composition
Dry matter (g kg-1) 929 935 933 934 938
Crude protein 489 488 481 512 506
Crude fat 159 157 156 157 159
Crude ash 80 72 47 57 37
Phosphorus 10.3 8.0 5.0 6.0 4.0
NfEf 272 283 316 274 298
Glucosinolatesg
(µmol g-1)
0.01 0.02 0.03 0.04
Phytateg (g kg-1) 0.50 1.00 1.50 2.00
Gross energyh (MJ kg-1) 22.8 22.9 23.3 23.3 23.7
Essential amino acids
Arginine 55.9 58.0 57.5 60.4 60.4
Histidine 39.8 40.8 43.0 40.0 42.0
Isoleucine 28.8 29.8 27.6 30.8 30.4
Leucine 88.9 90.9 92.9 90.1 91.3
Lysine 70.0 68.4 65.1 65.0 62.1
Methionine
(+ Cysteine)
17.2
25.6
17.0
27.1
14.3
26.6
16.3
29.1
14.5
29.2
Phenylalanine 49.1 50.5 50.8 49.7 50.1
Threonine 35.0 36.0 35.2 37.1 36.3
Valine 61.6 62.9 63.1 60.8 62.5 aVFC GmbH, Cuxhaven, Germany; bEuroduna-Technologies GmbH, Barmstedt, Germany; cIMCOSOY 60 Piglet, IMCOPA,
Araucaria, Brasil.; dAA-Mix 517158 & 508240, Vitfoss, Gråsten, Denmark; eCargill Deutschland GmbH, Krefeld, Germany; fNitrogen free extract = 100 – (%crude protein + %crude fat + %ash).; gCalculated according to concentration in raw
material: 0.13 µmol g-1; hCalculated by: crude protein = 23.9 MJ kg-1; crude fat = 39.8 MJ kg-1; NfE, fibre: 17.6 MJ kg-1.
Tryptophane was not analyzed.
92
The growth trial was conducted in the experimental facilities of Johann Heinrich von Thünen-
Institut, Federal Research Institute of Rural Areas, Forestry and Fisheries; Institute of
Fisheries Ecology (Ahrensburg, Germany). Juvenile rainbow trout that had been hatched in
the institute were used. One week before the experiment started twenty fish were stocked in
each of fifteen experimental tanks (40 L), being part of a flow-through system. Photoperiod
was in accordance to natural rhythmic from August to October at our latitude (53° 41' 0" N).
Tanks were provided with well freshwater at 1 L min-1 (temperature: 12.1±0.5 °C; O2: 7.8±0.2
mg L-1; pH: 7.2±0.5; NH4+: <0.1 mg L-1; NO2
-: <0.2 mg L-1). For a one week adaptation
period fish were fed the control diet (Table 6.4) in two daily meals until apparent satiation.
After the adaptation period, fish were fasted for two days and initial average weight was
determined (31.5 ± 0.5 g). Triplicate groups of fish were fed the experimental diets twice
daily (at 8.30 a.m. and 16.00 p.m.) to apparent satiation for 70 days. At the beginning and at
end of the experiment, a sample of 15 x 2 fish of the initial fish population was taken and
stored at -23 ºC to determine initial and final body composition.
Chemical analysis and laboratory procedures
Diets and homogenised fish bodies from each tank were analysed in duplicate for proximate
composition. Dry matter was calculated from weight loss after drying in an oven at 105 °C
until constant weight. Fat content was determined after HCl hydrolysis (Soxtec HT6, Tecator,
Höganäs, Sweden) and total nitrogen content by the Kjeldahl technique (protein = N × 6.25;
Kjeltec Auto System, Tecator, Höganäs, Sweden). Ash content was calculated from weight
loss after incineration of samples in a muffle furnace for 2 hours at 550 °C. Raw material and
dietary amino acid concentrations were analysed as described by Tzovenis et al. (2009).
Calculations and statistical analysis
Fish performance was determined, using the following formulae:
Specific growth rate (SGR, % day-1) = (ln final body weight – ln initial body weight) × 100 /
days fed
Feed conversion ratio (FCR) = g dry feed intake / g wet body weight gain
Protein efficiency ratio (PER) = g wet body weight gain / g protein intake
Survival (%) = (initial fish count - dead fish count) / initial fish count × 100
93
All diets were assigned by a completely randomized design. Biological and analytical data
were checked for normal distribution using the Kolmogoroff Smirnov Test and eventually
subjected to transformation. Data were subjected to linear regression analysis in order to
detect correlations between diet formulation and fish performance and/or fish body
composition. Data were also subjected to a one-way analysis of variance (ANOVA) using
SPSS 17.0 for Windows (SPSS Inc., Chicago, US). When differences among groups were
identified, multiple comparisons among means were made using Tukey’s HSD test. Statistical
significance was determined by setting the aggregate type I error at 5% (P<0.05) for each set
of comparisons.
Results
Digestibility coefficients
As shown in Table 6.4, ADC of dry matter in the control diet (62.5±4.7 %) was slightly lower
than in the test diet (65.9±3.1 %). The ADC of protein from fish meal (89.2±1.1 %) – the
single protein source in the control diet - was significantly higher than from canola protein
isolate (84.6±1.8 %) (P<0.05). Accordingly, amino acid digestibility followed this trend.
Table 6.4: Apparent digestibility coefficients
Fish meal control diet Canola protein isolate test diet
ADC of dry matter 62.5 ± 4.7 65.9 ± 3.1
ADC of test ingredient 73.9 ± 10.3
Crude protein 89.2a ± 1.1 84.6b ± 1.8
Amino acids
Arginine 94.4 88.6
Histidine 91.8 89.9
Isoleucine 92.1a 81.8b
Leucine 93.2a 83.6b
Lysine 94.4a 83.0b
Methionine
(+ Cysteine)
92.3a
80.7a
79.0b
89.2b
Phenylalanine 89.2a 79.3b
Threonine 90.8a 80.4b
Valine 92.2a 83.1b
Values in the same row with different superscript letters are significantly different (P<0.05).
94
Growth performance, feed efficiencies and body composition
Fish growth response, feed intake and feed efficiencies were not negatively affected when 100
% of digestible fish meal protein was replaced with protein from CPI (Table 6.5). In contrast,
with a final weight of 107.9±5.8 g and a SGR of 1.80±0.10, fish receiving diet I75 grew
significantly better than fish fed the control diet (final weight: 91.3±7.6 g; SGR: 1.57±0.12).
In addition, diet I75 gave a better feed conversion ratio (0.92±0.04) and protein efficiency
ratio (2.13±0.09) than the control diet (FCR: 1.14±0.08; PER: 1.80±0.13). Regression
analysis revealed significant positive correlations between dietary level of CPI and FCR
(R2=0.53, P<0.05) as well as PER (R2=0.37, P<0.05) indicating a relation between dietary
level of CPI and feed efficiencies. Survival of fish was not negatively affected by any
treatment. No significant differences in whole body composition were detected between fish
fed control diet and fish receiving CPI diets (Table 6.6). Furthermore, no correlations between
dietary level of CPI/ash/phosphorus and fish body parameters were identified.
Table 6.5: Growth response, feed intake and feed efficiencies of rainbow trout fed experimental diets
Control I25 I50 I75 I100 *R2 P
Initial weight (g) 31.4 ± 0.3 31.6 ± 0.2 31.3 ± 0.4 31.7 ± 0.4 31.7 ± 0.1
Final weight (g) 91.3a ± 7.6 101.2ab ± 6.2 100.9ab ± 3.3 107.9b ± 5.8 99.5ab ± 3.5 0.27 ns
SGR (%) 1.57a ± 0.12 1.71ab ± 0.10 1.72ab ± 0.03 1.80b ± 0.10 1.68ab ± 0.04 0.27 ns
Feed intake (g DM) 67.6 ± 3.9 69.4 ± 5.7 68.4 ± 3.5 69.8 ± 2.5 65.4 ± 4.4 0.02 ns
FCR 1.14a ± 0.08 1.00ab ± 0.09 0.98ab ± 0.03 0.92b ± 0.04 0.97ab ± 0.02 0.53 <0.05
PER 1.80a ± 0.13 2.06ab ± 0.17 2.11ab ± 0.07 2.13b ± 0.09 2.05ab ± 0.04 0.37 <0.05
R2: parameter values are regressed to the dietary level of canola protein isolate.
Values are given as mean ± standard deviation. Values in the same row with different superscript
letters are significantly different (P<0.05).
SGR, Specific growth rate; FCR, Feed conversion ratio; PER, Protein efficiency ratio.
Table 6.6: Proximate whole body composition (g kg-1 wet weight) of rainbow trout fed experimental diets
Control I25 I50 I75 I100
Moisture 696 ± 5 700 ± 5 694 ± 11 704 ± 1 707 ± 6
Crude protein 164 ± 2 162 ± 9 162 ± 7 161 ± 7 156 ± 3
Crude fat 117 ± 7 117 ± 6 119 ± 8 109 ± 4 111 ± 5
Crude ash 33 ± 3 32 ± 2 30 ± 3 32 ± 2 28 ± 2
Initial body composition: moisture 782 g kg-1, crude protein 141 g kg-1, crude fat 50 g kg-1, ash 28 g kg-1.
95
Discussion
Results show that canola protein isolate can be used as a valuable source of protein in diets for
rainbow trout. In the present feeding trial it was possible to replace all fish meal without
negative effects on feed intake, feed efficiencies and fish growth. In general, specific growth
rates ranged from 1.57 to 1.80 and feed conversion ratios varied between 0.92 and 1.14. This
is in line with results from other experiments investigating fish meal alternatives in rainbow
trout diets (Adelizi et al. 1998; Drew et al. 2007).
In several studies, the ADCs of canola protein products in fish diets have been determined. In
experiments with Atlantic salmon an ADC of protein from canola meal of 74.0 % has been
found (Anderson et al. 1992). However, the canola meal tested had a protein content of 390 g
kg-1 and therefore contained significant amounts of NfE. These are known to negatively
influence protein digestibility in carnivorous fish (Storebakken et al. 1998; Burel et al. 2000a;
Francis et al. 2001). Mwachireya et al. (1999) evaluated the protein digestibility of a canola
protein isolate in rainbow trout diets. The digestibility trial was conducted as described by
Hayen et al. (1993), using a settling column for the collection of fish faeces. For the canola
protein isolate tested Mwachireya et al. (1999) determined an ADC of protein of 97.6 %. This
value was regarded as one of the highest ever reported in fish nutrition studies. The authors
ascribed this high protein digestibility to the high level of protein (908 g kg-1) and low levels
of all antinutritional factors and indigestible carbohydrates present in the canola protein
isolate compared to canola concentrates. Mwachireya et al. (1999) concluded that fibre levels,
either alone or together with phytate, can have greatest adverse effects on the digestibility of
canola protein products for rainbow trout. The CPI used in our study contained 812 g kg-1 of
crude protein and 150 g kg-1 of NfE. It appears possible, that NfE negatively affected protein
digestibility of our CPI. Accordingly, the amino acid digestibility was also lower in CPI than
in the fish meal control diet. It has to be noted, however, that the ADC of protein (84.6±1.8
%) was in a range with other rapeseed/canola protein products tested in rainbow trout. In
example, ADCs of protein of 90.9±2.3 % from solvent extracted and 88.5±1.5 % of protein
from heat treated rapeseed meal were reported by Burel et al. (2000a) using an automatic
faecal collector as described by Choubert et al. (1982) being similar to the system applied in
the present study. Furthermore, Mwachireya et al. (1999) observed ADCs for protein between
77.4 to 88.1 % for differently processed canola meals.
The limitations of simple rapeseed products as feed ingredients in fish growth studies have
been widely investigated (Dabrowski and Kozlowska 1981; Davies et al. 1990; Webster et al.
96
1997; Burel et al. 2000a,b,c, 2001; Thiessen et al. 2003, 2004; Glencross et al. 2004;
Shafaeipour et al. 2008). However, lack of information exists about the benefits of high
quality products originating from rapeseed/canola oilcakes with protein contents comparable
to or above that of fish meal. Higgs et al. (1982) successfully replaced 25 % of dietary protein
from a fish meal control diet for juvenile Chinook salmon with rapeseed protein concentrate
(613 g CP kg-1) without reducing growth rate and food (protein) utilization. In that study,
however, higher fish meal replacement levels with rapeseed protein concentrate were not
evaluated. Rapeseed protein concentrate with a protein content of 710 g kg-1 was evaluated as
fish meal replacement in diets for wels catfish and common carp (Slawski et al. in press, a,b).
It was found, that diet taste and feed intake were negatively influenced by high dietary
inclusion of rapeseed protein concentrate probably due to glucosinolates present in rapeseed.
In addition, feed efficiencies and consequently fish growth decreased when wels catfish or
carp received diets with more than 25 % or 33 % of fish meal replaced with rapeseed protein
concentrate. This was referred to dietary levels of NfE and insufficient phosphorus
availability (Slawski et al. in press, a,b).
In the present study, feed intake was similar in all feeding groups. This indicates that the taste
of canola protein isolate was well accepted by rainbow trout. In addition, we found no
negative influence from dietary inclusion of CPI on fish growth performance and feed
efficiencies. Interestingly, fish receiving diet I75 grew significantly better than fish receiving
the control diet. A reason for this might be an increased dry matter digestibility with
increasing dietary incorporation of CPI for fish meal. As shown in Table 6.4, dietary
incorporation of CPI led to slightly higher dry matter digestibility (65.9±3.1 %) than in a fish
meal control diet (62.5±4.7). The slightly higher but not significantly different growth
performance and feed efficiencies of fish in treatment group I75 compared to other groups
could be attributed to varying dietary levels of phosphorus and NfE. Diet I75 (6.0 g P kg-1)
contained more phosphorus than diet I50 (5.0 g P kg-1) or I100 (4.0 g P kg-1). Dietary
phosphorus requirements ranging from 5.0 to 8.0 g kg-1 have been reported for rainbow trout
(Ogino and Takeda 1978). Antinutritional factors such as phytic acid, fibre and other complex
carbohydrates present in CPI may have contributed to reduced phosphorus availability in fish
(Francis et al. 2001). Severe phosphorus deficiencies from any dietary treatment, however,
appear unlikely. It is known that whole-body ash is reduced when carnivorous fish are fed a
diet deficient in available phosphorus (Skonberg et al. 1997; Shao et al. 2008) and that whole-
body lipid content can be increased due to high dietary levels of vegetable protein (Adelizi et
97
al. 1998; Kaushik et al. 2004). Latter is believed to be caused by the accumulation of fatty
acids due to impaired β-oxidation (Takeuchi and Nakazoe 1981) or oxidative phosphorylation
due to phosphorus deficiency, thereby inhibiting the TCA cycle and leading to an
accumulation of acetyl-CoA and an increased fatty acid synthesis (Skonberg et al. 1997).
In the present study, neither differences in whole body composition nor correlations between
dietary phosphorus content and ash levels in fish body indicating insufficient dietary
phosphorus supply were detected. It has been reported, however, that increased dietary
phosphorus levels can improve feed efficiencies and consequently growth in Atlantic salmon,
cod and sea bass (Vielma and Lall 1998; Roy and Lall 2003; Oliva-Teles and Pimentel-
Rodrigues 2004). Accordingly, a slightly higher phosphorus level in diet I75 compared to diet
I100 may have resulted in tendentially better feed efficiencies and fish growth.
Besides different dietary phosphorus supply, varying dietary levels of NfE might also have
contributed to slight differences in growth performances and feed efficiencies among
treatment groups. Since dietary NfE can potentially reduce nutrient and mineral digestibility
in fish (Storebakken et al. 1998; Burel et al. 2000a; Mwachireya et al. 1999; Francis et al.
2001) lower dietary levels of NfE in diet I75 (274 g kg-1) compared to diet I50 (316 g kg-1) or
diet I100 (298 g kg-1) might have resulted in slightly better growth performance and feed
efficiencies.
In conclusion, the canola protein isolate tested has shown great potential as fish meal
replacement in diets for rainbow trout. High dry matter and protein digestibility together with
unaffected palatability make canola protein isolate a promising candidate as protein source in
fish diets.
Acknowledgements
We gratefully acknowledge the financial assistance provided by HELM AG and BioExx for
this project.
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General Discussion
Simple rapeseed products have been widely tested as protein source in feeds for several fish
species (Dabrowski and Kozlowska 1981; Davies et al. 1990; Burel et al. 2000; Shafaeipour
et al. 2008). In general it was found that the nutritional quality of simple rapeseed products is
below that of fish meal. In particular, antinutritional factors (ANF) determine the quality of
rapeseed products for fish nutrition, among them glucosinolates, phytic acid, phenolic
constituents and indigestible carbohydrates (Francis et al. 2001). Different processing
techniques have been identified to reduce the level of antinutrients in rapeseed products
(Fenwick et al. 1986; Naczk and Shahidi 1990; Anderson-Haferman et al. 1993; Chabanon et
al. 2007) and potentially increase their value for fish nutrition. But, lack of information exists
about the benefits of high quality rapeseed products with protein contents comparable to or
above that of fish meal. However, results obtained by Higgs et al. (1982) indicate the
applicability of rapeseed protein concentrate (RPC) in fish nutrition. The authors successfully
replaced 25 % of dietary protein from a fish meal control diet for juvenile Oncorhynchus
tshawytscha with RPC (CP: 61 %) without reducing growth rate and food (protein) utilization.
In the present study, different protein products derived from rapeseed (including canola) were
tested as fish meal replacement in fish diets. A high quality rapeseed protein concentrate
(RPC) with a protein content of 71 % was evaluated as fish meal replacement in diets for
common carp (chapter 1), wels catfish (chapter 2), turbot (chapter 3) and rainbow trout
(chapter 4). Advanced processing techniques applied led to a RPC with relatively low levels
of glucosinolates (1.32 µmol g-1), phytic acid (1.77 g 100g-1), polysaccharides and other
antinutritional factors. In comparison, rapeseed meals tested by Burel et al. (2000a,b,c; 2001)
in fish meal replacement studies with rainbow trout were either pressure cooked or directly oil
extracted. These meals contained 26 or 40 µmol g-1 glucosinolates and 4.43 or 4.15 g 100g-1
phytic acid, respectively, and led to reduced growth performance when replacing 33 % of
dietary fish meal.
In common carp, the RPC successfully replaced 33 % of fish meal protein from a control diet
without retarding fish growth performance, feed intake or feed efficiencies. At 66 % and 100
% fish meal replacement with RPC, however, fish growth performance, feed intake and feed
efficiencies decreased compared to the control group. In wels catfish, 25 % of dietary fish
meal was successfully replaced with RPC without negative effects on feed efficiencies and
fish growth. When 50 % of dietary fish meal was replaced with RPC the feed intake as % of
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fish body weight was not significantly different from the control group but feed efficiencies
and fish growth were reduced. In turbot, 33 % of dietary fish meal was replaced with RPC. At
66 % fish meal replacement level, feed intake and fish growth were significantly reduced. In
rainbow trout, feed intake and fish growth were not compromised when RPC replaced up to
100 % of dietary fish meal in the control diet.
Based on the results presented in chapter 4, in chapter 5 the potential of two rapeseed protein
concentrates partitioned in albumin and globulin fractions as fish meal alternatives was
evaluated in a digestibility study and a consecutive growth trial with rainbow trout. Compared
to the RPC, production of the fractionized protein concentrates was simplified. At 75 % fish
meal replacement with albumin concentrate, feed intake and fish growth were significantly
lower compared to the control group and fish receiving diet A50. Feed conversion ratio and
protein efficiency ratio, however, were not significantly different towards the control group at
100 % fish meal replacement level with albumin concentrate. When fish meal was replaced
with globulin concentrate, growth performance and feed intake were significantly reduced at
50 % fish meal replacement level. Feed efficiencies were similar to the control group up to 75
% fish meal replacement with globulin concentrate.
In chapter 6 a canola protein isolate (CPI) with a crude protein content of 81 % was evaluated
as fish meal alternative in diets for rainbow trout. The CPI was produced using a novel cold
processing sequence. It consisted of low temperature conditioning, cold oil pressing, low
temperature solvent extraction and desolventization followed by aqueous processing for
isolation of the soluble proteins. The resulting purified protein solution was spray dried to
limit thermal damage. Fish growth response, feed intake and feed efficiencies were not
negatively affected when 100 % of digestible fish meal protein was replaced with protein
from CPI.
In general, results obtained in the course of the present study demonstrate the high potential of
rapeseed protein products as fish meal alternative in fish nutrition. In the following, results of
the different trials are compared and possible limitations when using rapeseed protein
products are discussed.
104
Diet palatability
We observed a diminished diet acceptance at 66 % (and 100 %) fish meal replacement level
in carp and turbot and at 75 % in wels catfish, which is documented in lower feed intake in
respective fish groups. In trout, diet palatability appeared to be negatively affected at 75 %
(and 100 %) fish meal replacement level with albumin and at 50 % (as well as 75 and 100 %)
with globulin concentrate, also resulting in reduced feed intake. It is known that the bitter
taste exuded by glucosinolate metabolites, such as isothiocyanates and
vinyloxazolidinethiones, present in rapeseed meals can potentially retard diet acceptance by
fish. This was found in rainbow trout and turbot at dietary glucosinolate levels of 7.3 µmol g-
1 or 18.7 µmol g-1, respectively (Burel et al. 2000bc). Because the rapeseed protein products
used in our study contained 0.2-2.3 µmol glucosinolates g-1, dietary glucosinolate
concentrations were 0.07-0.6 µmol g-1. These values are far below the level when
glucosinolates were assumed to become detrimental on food intake of rainbow trout and
turbot (Burel et al. 2000bc). However, to our observation the typical mustard smell of
glucosinolates was still noticeable in respective experimental diets with high inclusion of
rapeseed protein products. When feeding rainbow trout with RPC and CPI however, feed
intake did not vary significantly between treatment groups thereby indicating the elimination
of bitter flavour as well as a suitable diet taste at high dietary RPC inclusion. It can only be
speculated, that carp, turbot and wels catfish are more sensitive towards a bitter diet taste than
rainbow trout. For prospective feeding trials with carp, turbot and wels catfish it appears
recommendable to use strong feed attractants, such as fish protein hydrolysate, squid
hydrolysate, stick water or krill meal to maintain high feed intake (Espe et al. 2006, 2007;
Torstensen et al. 2008; Kousoulaki et al. 2009).
Nutrient availability
Lower feed efficiencies observed at high RPC inclusion levels particularly in wels catfish,
turbot and trout might be a result of reduced dietary nutrient availability. Because of
insignificant phytic acid concentrations in respective diets, it is assumed that nutrient
availability was mainly reduced by fibre and other complex carbohydrates. It is known from
carnivorous fish that complex carbohydrates can greatly reduce mineral and nutrient
availability from aquafeeds, thereby reducing feed efficiencies as observed in Atlantic salmon
and turbot (Storebakken et al. 1998; Burel et al. 2000). The RPC used in our study contained
4.8 % fibre and calculated NfE was 12.8 %. In comparison, the NfE content of the used fish
105
meal was 3.4 %. High dietary inclusion of RPC therefore lead to relatively higher dietary fibre
+ NfE contents than in respective experimental control feeds. It can be assumed, that nutrient
availability from diets with high content of RPC was reduced.
Protein digestibility
Protein digestibility of rapeseed products has been determined in several studies. In Atlantic
salmon, the ADC of canola meal protein was 74.0 % (Anderson et al. 1992). ADCs of protein
of 90.9±2.3 % from solvent extracted and 88.5±1.5 % of protein from heat treated rapeseed
meal were reported by Burel et al. (2000a) in studies with rainbow trout. In addition,
Mwachireya et al. (1999) observed ADCs of protein between 77.4 to 97.6 % for differently
processed canola products. Digestibility experiments undertaken in the present study revealed
that protein from globulin concentrate (88.8±0.6 %) was as efficiently digested as fish meal
protein (89.2±1.1 %), while the ADCs of protein from albumin concentrate (77.7±1.4 %) and
canola protein isolate (84.6±1.8 %) were significantly lower. Different ADCs for protein can
result from the raw material NfE content, which can negatively influence protein digestibility
in carnivorous fish (Storebakken et al. 1998; Burel et al. 2000a; Francis et al. 2001). The
albumin concentrate contained less NfE (8.7 %) than the globulin concentrate (NfE: 29.5 %)
and the CPI (15.0 %). Accordingly, Mwachireya et al. (1999) stated, that high protein
digestibility demands lowest levels of all antinutritional factors and indigestible carbohydrates
present in canola products.
Body composition
Tendentially sinking body ash levels suggest reduced phosphorus availability from diets high
in RPC when fed to wels catfish, turbot and rainbow trout. It is known that body ash levels
can be reduced when fish are fed a diet deficient in available phosphorus and rich in vegetable
protein (Skonberg et al. 1997; Adelizi et al. 1998; Kaushik et al. 2004; Shao et al. 2008). In
example, the phosphorus levels of diets for rainbow trout decreased from 1.27 to 0.82 % with
increasing dietary level of RPC. Although these values are above established requirement
levels for rainbow trout and other fish species (Ogino and Takeda 1978; NRC 1993) it can not
be excluded that better phosphorus availability in diets devoid of RPC positively affected feed
efficiencies. It is known that excessive dietary phosphorus content can improve the feed
efficiency of diets for Atlantic salmon, cod and European sea bass (Vielma und Lall 1998;
Roy und Lall 2003; Oliva-Teles und Pimentel-Rodrigues 2004). Antinutritional factors such
106
as phytic acid, fibre and other complex carbohydrates present in RPC can potentially reduce
the phosphorus availability in fish (Mwachireya et al. 1999; Francis et al. 2001). In the
present studies, dietary phytic acid concentrations originating from RPC were up to 0.2 g kg-1
in diets for wels catfish and up to 5.2 g 100g-1 phytic acid in diets for turbot. In comparison,
Spinelli et al. (1983) observed decreased growth rates in rainbow trout fed a diet containing 5
g kg-1 synthetic phytic acid. Synthetic phytic acid at concentrations of 5 and 10 g kg-1 feed
resulted in lower growth performance in common carp (Hossain and Jauncey 1993). While
negative effects resulting from phytic acid concentrations in diets for wels catfish appear
marginal, for turbot, however, dietary phytic acid concentrations have probably reduced
phosphorus availability and finally reduced feed efficiencies and growth. In prospective
feeding trials with rapeseed protein products it appears advisory to supplement diets with a
phosphorus source such as dicalcium phosphate in order to overcome problems regarding
phosphorus availability (Lee et al. 2010). In comparison, no significant differences in whole
body composition were detected between carp or rainbow trout fed control diet and fish
receiving RPC or CPI diets, respectively. Furthermore, no correlations between dietary level
of CPI/ash/phosphorus and fish body parameters were identified.
Amino acid content
In the experiments with carp and wels catfish, high dietary inclusion of RPC may have led to
insufficient dietary supply of lysine. This might have negatively influenced feed efficiencies
and fish growth. Diets high in RPC were low in lysine, because of low fish meal inclusion and
no inclusion of other protein sources of animal origin. Additionally, ANF in RPC could have
negatively affected amino acid digestibility as it is known from other protein sources of
vegetable origin (Francis et al. 2001).
Blood features and histopathology
Certain blood values were determined in wels catfish and in rainbow trout fed the RPC.
Investigated blood values were not significantly different between treatment groups.
Consistent blood haemoglobin, haematocrit and serum values therefore indicate an equal
nutritional status among feeding groups (Congleton and Wagner 2006). In addition, a
histological investigation of the digestive tract of rainbow trout fed the RPC did not cause any
detectable histological alterations as found in Atlantic salmon when receiving diets that
107
contain high amounts of solvent extracted soybean meal (van den Ingh et al. 1991;
Baeverfjord and Krogdahl 1996).
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juvenile black seabream, Sparus macrocephalus. Aquaculture 277, 92–100.
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phosphorus intake in rainbow trout (Oncorhynchus mykiss). Aquaculture 157, 11–24.
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other elements in fishmeal, soy protein concentrate and phytase-treated soy protein-
concentrate-based diets to Atlantic salmon, Salmo salar. Aquaculture 161, 365–379.
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Torstensen, B.E., Espe, M., Sanden, M., Stubhaug, I., Waagbø, R., Hemre, G.-I., Fontanillas,
R., Nordgarden, U., Hevrøy, E.M., Olsvik, P., Berntssen, M.H.G., 2008. Novel
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Effects of soybean-containing diets on the proximal and distal intestine in Atlantic
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111
General Summary
Fish meal, the most important source of marine protein for fish diets, is a limited resource.
Increasing prices for fish meal together with environmental concerns force the aquaculture
sector to find alternative protein sources to be included in fish feeds. Wide availability,
relatively high protein contents and a desirable amino acid profile have caused an interest in
rapeseed (and canola) products as ingredients for fish feed production. However, the
nutritional quality of rapeseed products largely depends on their levels of antinutritional
factors, particularly glucosinolates, phytic acid, phenolic constituents and indigestible
carbohydrates. Several processing techniques can be adapted to reduce the level of
antinutrients in rapeseed in order to improve its value for fish nutrition. In the present study a
high quality rapeseed protein concentrate (RPC) with a protein content of 71 % was evaluated
as fish meal replacement in diets of different fish species.
In chapter 1 the RPC was tested as fish meal replacement in diets for juvenile common carp
(Cyprinus carpio L.). Triplicate groups of fish were fed isonitrogenous (40.4 ± 0.2 % CP) and
isocaloric (21.4 ± 0.1 kJ g-1) experimental diets with 0 %, 33 %, 66 % or 100 % of fish meal
replaced with RPC. Results from a 56 day feeding trial showed, that growth parameters and
feed efficiencies were not significantly different between fish fed on the control diet and the
diet with 33 % of fish meal replaced with RPC. At higher RPC inclusion levels, diet intake
and feed efficiencies were reduced resulting in lower growth performances. It appeared that
diet taste and amino acid profiles were negatively affected by high dietary inclusion levels of
RPC.
In chapter 2 the potential of RPC as fish meal alternative in diets for juvenile wels catfish
(Silurus glanis L.) was evaluated. Fish were organized in triplicate groups and received
isonitrogenous (60.3 ± 0.3 % CP) and isocaloric (23.0 ± 0.3 kJ g-1) experimental diets with 0
%, 25 %, 50 % and 75 % of fish meal replaced with RPC. At the end of the 63 day feeding
period, growth performance, feed intake and feed efficiencies were not significantly different
between the control group and fish fed on diets with 25 % of fish meal replaced with RPC.
Higher dietary RPC inclusion negatively affected diet quality and palatability resulting in
reduced feed intake, feed efficiencies and fish performance.
In chapter 3 RPC was tested as fish meal alternative in diets for juvenile turbot (Psetta
maxima L.). Triplicate groups of fish were fed isonitrogenous (58.1 ± 0.9 % CP) experimental
diets with equal gross energy content (21.5 ± 0.3 MJ kg−1) where 0 %, 33 % and 66 % of fish
112
meal were replaced with RPC. At the end of the feeding period (84 days), fish fed the control
diet or the diet with 33 % of fish meal replaced with RPC showed significantly higher weight
gain and feed intake than fish fed diet with the highest RPC inclusion. This was mostly
attributed to negative effects on diet taste resulting from the glucosinolate content of RPC.
Furthermore, the control diet gave significantly better feed efficiencies than diets containing
RPC, probably due to lower protein, amino acid and phosphorus availability in these diets.
In chapter 4 RPC was tested as fish meal alternative in diets for juvenile rainbow trout.
Triplicate groups of fish received isonitrogenous (47.9 ± 0.5 % CP) and isoenergetic (22.4 ±
0.2 kJ g-1) experimental diets with 0, 66 and 100 % of fish meal substituted with RPC. At the
end of the 84 day feeding period, fish growth performance, feed intake and feed efficiencies
were not compromised when 100 % of fish meal in the control diet was replaced with RPC. In
addition, intestinal morphology did not reveal any histological abnormalities in all dietary
groups. Blood parameters including haematocrit, haemoglobin as well as glucose,
triglycerides and total protein in the plasma were not different between treatment groups.
In chapter 5 the potential of two rapeseed protein concentrates partitioned in albumin and
globulin fractions as fish meal alternatives was evaluated. These fractionized protein
concentrates were produced under lower cost and time effort compared to the rapeseed protein
concentrate in the experiments presented above. In a digestibility experiment with juvenile
rainbow trout apparent digestibility coefficients were determined by indirect marker method.
ADCs of protein from fish meal (89.2±1.1 %) and globulin concentrate (88.8±0.6 %) were
significantly higher than from albumin concentrate (77.7±1.4 %). In a consecutive growth
trial, juvenile rainbow trout were organized in triplicate groups and received experimental
diets with 0, 50, 75 or 100 % of fish meal replaced with albumin or globulin concentrate on
the basis of digestible protein. It was found that only in treatment group A50, fish growth
performance and feed intake were not negatively affected by dietary treatment. However, feed
efficienies were not significantly different compared to the control group at 100 % or 75 %
fish meal replacement level with albumin or globulin concentrate, respectively. Significant
lower fish survival rates were observed when fish received diets A75, A100, G50, G75, or
G100 compared to the control diet or diet A50. The experiment showed that the quality of the
fractionized protein concentrates was below that of the rapeseed protein concentrate used in
chapter 4.
In chapter 6 the potential of a canola protein isolate (CP: 81 %) as fish meal alternative in
diets for juvenile rainbow trout was evaluated. Apparent digestibility coefficients for protein
113
from fish meal (89.2±1.1 %) and canola protein isolate (84.6±1.8 %) were determined by
indirect marker method in a digestibility experiment with juvenile rainbow trout. In a
consecutive growth trial, fish organized in triplicate groups received experimental diets with 0
%, 25 %, 50 %, 75 % and 100 % of fish meal replaced with canola protein isolate on the basis
of digestible protein. At the end of a 70 day feeding period it was found that growth
performance, feed intake and feed efficiencies in treatment groups receiving diets devoid of
fish meal were not negatively affected compared to the control group.
Experimental results from all feeding trials conducted demonstrate an enormous potential of
high quality rapeseed protein products as protein source in fish feeds. Particularly for the
nutrition of rainbow trout, rapeseed protein concentrate and canola protein isolate appear to be
highly valuable fish meal alternatives.
114
Zusammenfassung
Fischmehl ist die wichtigste marine Proteinquelle für Fischfuttermittel und zugleich ein
limitierter Rohstoff. Steigende Fischmehlpreise sowie ökologische Bedenken bei dessen
Nutzung veranlassen die Aquakulturindustrie nach alternativen Proteinquellen für die
Nutzung in Fischfuttermitteln zu suchen. Weite Verfügbarkeit, ein relativ hoher Proteingehalt
sowie ein wünschenswertes Aminosäurenprofil führen zu wachsendem Interesse an
Rapsprodukten (einschliesslich Canola) als Rohstoff für die Fischfutterproduktion. Allerdings
hängt die nutritive Qualität von Rapsprodukten für die Fischernährung stark von deren Gehalt
an antinutritiven Faktoren ab, insbesondere Glucosinolaten, Phytinsäure, phenolischen
Verbindungen und unverdauliche Kohlenhydraten sind hierfür von Bedeutung. Verschiedene
Verarbeitungstechniken erlauben eine Senkung des Gehaltes der Antinutritiva in
Rapsprodukten und erhöhen deren Wert für die Fischernährung.
In der vorliegenden Studie wurde ein qualitativ hochwertiges Rapsproteinkonzentrat (RPK)
mit einem Rohproteingehalt von 71 % als Fischmehlersatz in Futtermitteln für Karpfen
(Cyprinus carpio L.), Wels (Silurus glanis L.), Steinbutt (Psetta maxima L.) und
Regenbogenforelle (Oncorhynchus mykiss W.) getestet.
In Kapitel 1 wurde das RPK als Fischmehlersatz in Futtermitteln für juvenile Karpfen
eingesetzt. Triplikate Fischgruppen erhielten isonitrogene (RP: 40.4 ± 0.2 %) Futtermittel, in
denen 0 %, 33 %, 66 % oder 100 % des Fischmehls durch RPK ausgetauscht worden war.
Nach einer 56-tägigen Fütterungsperiode waren Gewichtszunahme und Futterverwertung bis
zu einem Fischmehlaustausch von 33 % durch RPK nicht signifikant verschieden gegenüber
der Kontrollgruppe. Bei höherem Einsatz von RPK waren Futteraufnahme und
Futterverwertung signifikant verringert, was zu geringeren Gewichtszunahmen in den
betreffenden Gruppen führte. Dies geht vermutlich auf eine negative Beeinflussung des
Futtermittelgeschmacks sowie verschlechterter Aminosäuremuster in den Futtermitteln mit
sehr hohem RPK-Anteil zurück.
In Kapitel 2 wurde die Eignung von RPK als Fischmehlalternative in Futtermitteln für
juvenile Welse untersucht. Die Fische waren in triplikate Versuchsgruppen eingeteilt und
erhielten isonitrogene (RP: 60.3 ± 0.3 %) Futtermittel, in denen 0 %, 25 %, 50 % oder 75 %
des Fischmehls durch RPK ausgetauscht worden war. Am Ende der 63-tägigen
Fütterungsperiode waren Wachstumsleistungen, Futteraufnahme und Futterverwertung bis zu
einem Fischmehlaustausch von 25 % nicht signifikant verschieden zur Kontrollgruppe.
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Fischmehlaustausch mit RPK von 50 % und 75 % hatte offensichtlich negative Effekte auf
Futtermittelqualität und –geschmack, wodurch es zu geringerer Futteraufnahme, verringerter
Futterverwertung und abnehmenden Wachstumsleistungen kam.
In Kapitel 3 wurde RPK als Fischmehlersatz in Futtermitteln für juvenile Steinbutt getestet.
Triplikate Fischgruppen wurden mit isonitrogenen (RP: 58.1 ± 0.9 %) Futtermitteln gefüttert,
bei denen 0 %, 33 % oder 66 % des Fischmehls durch Rapsproteinkonzentrat ausgetauscht
worden war. Am Ende des 84-tägigen Fütterungsszeitraums, zeigten Fische, die das
Kontrollfutter oder das Futter mit 33 %igem Fischmehlaustausch erhalten hatten, signifikant
höhere Gewichtszunahmen und Futteraufnahme als Fische, die das Futter mit 66 %igem
Fischmehlaustausch gefressen hatten. Die Unterschiede in der Futteraufnahme gehen
vermutlich auf negative Effekte auf den Futtermittelgeschmack durch Glucosinolate im RPK
zurück. Desweiteren erzielte das Kontrollfuttermittel bessere Futterverwertung gegenüber den
Futtermitteln, die RPK enthielten, was mit geringerer Protein-, Aminosäuren- und
Phosphorverfügbarkeit zusammenhängen dürfte.
In Kapitel 4 wurde RPK als Fischmelalternative in Futtermitteln für juvenile
Regenbogenforellen eingesetzt. Triplikate Fischgruppen erhielten isonitrogene (RP: 47.9 ±
0.5 %) Futtermittel, in denen 0 %, 66 % oder 100 % des Fischmehls durch RPK ersetzt
worden war. Am Ende des 84-tägigen Fütterungszeitraums zeigten die Wachstumsleistungen,
die Futteraufnahme und die Futterverwertung zwischen den Versuchsgruppen keine
signifikanten Unterschiede. Auch der lichtmikroskopisch untersuchte Verdauungstrakt zeigte
zwischen den Gruppen keine Unterschiede. Desweiteren waren Blutparameter wie Hämatokrit
und Hämoglobin sowie Glucose, Triglyceride und Gesamtprotein gruppenübergreifend
einheitlich.
In Kapitel 5 kamen zwei Rapsproteinkonzentrate zur Anwendung. Die in eine Albumin- und
eine Globulinfraktion aufgeteilten Proteinkonzentrate wurden unter geringerem Kosten- und
Zeitaufwand als das in vorangegangenen Experimenten verwendete RPK hergestellt. In einer
Verdaulichkeitsuntersuchung an juvenilen Regenbogenforellen wurde die
Nährstoffverdaulichkeit der Proteinkonzentrate mittels der indirekten Markermethode erfasst.
Die Verdaulichkeit des Proteins aus Fischmehl (89.2±1.1 %) und Globulinkonzentrat
(88.8±0.6 %) war signifikant höher als bei dem Albuminkonzentrat (77.7±1.4 %). In einem
folgenden Wachstumsversuch erhielten in triplikate Gruppen eingeteilte Forellen Futtermittel,
bei denen 0 %, 50 %, 75 % oder 100 % des Fischmehls auf der Basis verdaulichen Proteins
durch Albumin- oder Globulinkonzentrat ausgetauscht worden war. Lediglich in der
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Versuchsgruppe mit 50 % Fischmehlaustausch durch Albuminkonzentrat waren die
Wachstumsleistungen, die Futteraufnahme und die Überlebensrate einheitlich mit den
Ergebnissen aus der Kontrollgruppe. Allerdings war die Futterverwertung nicht signifikant
schlechter gegenüber der Kontrollgruppe bis zu 100 % bzw. 75 % Fischmehlaustausch durch
Albumin- oder Globulinkonzentrat. Die Untersuchung zeigte, dass die nutritive Qualität der
kostengünstiger hergestellten Proteinkonzentrate unter der des RPK aus Kapitel 4 liegt.
In Kapitel 6 wurde Canolaproteinisolat (RP: 81 %) als Fischmehlalternative in Futtermitteln
für juvenile Regenbogenforellen eingesetzt. Die Verdaulichkeit von Protein aus Fischmehl
(89.2±1.1 %) und Canolaproteinisolat (84.6±1.8 %) wurde über die indirekte Markermethode
in einem Verdaulichkeitsexperiment erfasst. In einem darauf aufbauenden Wachstumsversuch
wurden triplikate Fischgruppen mit Futtermitteln gefüttert, bei denen 0 %, 25 %, 50 %, 75 %
oder 100 % des Fischmehls auf der Basis verdaulichen Proteins durch Canolaproteinisolat
ersetzt worden war. Am Ende der 70-tägigen Fütterungsperiode zeigten die
Wachstumsleistungen, Futteraufnahme und Futterverwertung keine negative Beeinträchtigung
trotz 100 %igem Fischmehlaustauschs.
Die Ergebnisse aus allen Fütterungsversuchen demonstrieren das große Potenzial von
qualitativ hochwertigen Rapsproteinprodukten für die Fischernährung. Besonders für die
Ernährung von Regenbogenforellen wurden RPK und Canolaproteinisolat als hervorragende
Fischmehlalternativen identifiziert.
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Danksagung
An dieser Stelle möchte ich den Menschen Dank entgegenbringen, die zum Gelingen der
vorliegenden Arbeit beigetragen haben.
Ich bedanke mich bei meinem Betreuer Herrn Prof. Dr. Carsten Schulz für die Überlassung
des interessanten Themas und das mir geschenkte Vertrauen bei der Projektbearbeitung. Die
gute fachliche Betreuung und die ermöglichte Teilnahme an nationalen und internationalen
Konferenzen förderten ein positives Arbeitsklima.
Herrn Simon Kreft und Frau Nina Bajdura danke ich für die verwaltungstechnische
Bearbeitung des Projektes.
Dank gilt meinen lieben Kolleginnen und Kollegen vom vTI für die Unterstützung bei der
Versuchsdurchführung und die überaus schöne Zeit in Ahrensburg. Besonders bei Herrn Prof.
Dr. Hilge bedanke ich mich, dass ich als Gast in Ahrensburg arbeiten durfte.
Den Kollegen vom PPM danke ich für die gute Zusammenarbeit.
Ich bedanke mich bei Herrn Dr. Florian Nagel für die Hilfsbereitschaft bei der
Versuchsdurchführung und die angenehme Büronachbarschaft.
Meinen Eltern danke ich für die wie immer selbstlose Unterstützung jeglicher Art.
Und ganz besonders danke ich Dir, Sophie, weil Du mir allzeit Kraft gibst.
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Lebenslauf
Name Hanno Slawski
Geburtstag 28.09.1981
Geburtsort Neustadt in Holstein, Schleswig-Holstein
Staatsangehörigkeit Deutsch
Familienstand ledig
Schulbildung
1988 – 1992
1992 - 2001
Hochtorgrundschule Neustadt in Holstein
Kreisgymnasium Neustadt in Holstein
Zivildienst
09/2001 – 07/2002
Karl-Schütze-Heim für Menschen mit geistiger Behinderung,
Merkendorf
Studium
10/2002 – 08/2004
03/2005 – 08/2006
09/2006 – 10/2008
Bachelorstudiengang Agrarwissenschaften an der
Christian-Albrechts-Universität zu Kiel
Bachelorstudiengang Agrarwissenschaften an der
Humboldt-Universität zu Berlin
Masterstudiengang Fishery Science and Aquaculture an der
Humboldt-Universität zu Berlin
Berufliche Tätigkeiten
11/2008 – 03/2011
01/2011 – 03/2011
seit 04/2011
Wissenschaftlicher Mitarbeiter bei der Gesellschaft für Marine
Aquakultur mbH, Büsum, bei Herrn Prof. Dr. C. Schulz
Wissenschaftlicher Mitarbeiter am Institut für Tierzucht und
Tierhaltung der Christian-Albrechts-Universität zu Kiel bei
Herrn Prof. Dr. C. Schulz
R&D Manager bei Aller Aqua A/S
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