- Email: [email protected]
Effect of diet composition and protein level on growth, body composition, haematological characteristics and cost of production of rainbow trout (Salmo gairdneri)
Aquaculture, 58 (1986) 75-85
Elsevier Science ~blishers
75
B.V., Amsterdam - Printed in The Netherl~~
Effect of Diet Composition and Protein Level on Growth, Body Composition, Haematological Characteristics and Cost of Production of Rainbow Trout (Salmo gairdneri) MARIA N. ALEXIS, VASSILIKI THEOCHAR12 and ELLI PAPAPA~SK~VAPAPO~O~LOU’ ‘National Centre for Marine Research, GR-166 04 Elliniko (Greece) %NationalHatchery ojLouros, Hani Terouou, Zoannina (Greece)
(Accepted 2 June 1986)
ABSTRACT Alexis, M.N., Theochari, V. and Papaparaskeva-Papoutsoglou, E., 1986. Effect of diet composition and protein level on growth, body composition, haematological characteristics and cost of production of rainbow trout ( SaLmo gairdneri) . Aquac~t~re, 58: 75-85. Rainbow trout of approximately 1 g initial average weight were fed eight experimental diets and a commercial diet for 196 days. Four different combinations of by-products were used as the main ingredient in formulating four pairs of diets differing in their protein content ( 40 and 49% ) . The response of the fish towards the protein increase in the diet was not uniform, a better performance being observed for two of the mixtures used at the higher protein content and a worse performance for the other two. The growth of the fish was negatively correlated with the carob seed germ meal content of the diet. The differences in the protein content of the fish fed the experimental diets were small while the lipid content was more variable. A large proportion of the body lipids was contributed by the visceral lipids. Haematological characteristics of all of the fish tested were within the normal values reported in the literature.
INTRODUCTION
Protein is considered to be the most expensive component of fish diets. For this reason the study of the protein requirements of rainbow trout has been extensive and levels of 3550% in the diet have been found to produce maximum growth, depending on diet composition, level of feeding, and the age and physiological condition of the fish (Lee and Putnam, 1973; Satia, 1974; Cho et al., 1976; Zeitoun et al., 1976; Watanabe et al.; 1979, Cowey and Sargent, 1979; Steffens, 1981) _ Under specified conditions, the growth maxima are usually identified within a range of dietary protein concentrations that depends on the
~44-8486/86/$03.50
0 1986 Elsevier Science Publishers B.V.
76
method of calculation used and the experimental error. There is generally an improvement in growth and food conversion from the low to the high protein levels of this range (Zeitoun et al., 1976). The choice of dietary protein to be used in practical rations is, therefore, an economic decision, which depends on the cost of the protein source as well as on the expected returns from fish growth and value. The particular characteristics of the protein sources used in fish diets are another factor that has to be considered when selecting an economic protein level. The response of the fish to an increase in the protein content of the diet could vary depending on whether more digestible protein is provided or whether the levels of antigrowth compounds increase unfavourably for the growth of the fish, especially in mixtures containing plant by-products. Eight diets containing different combinations of animal and plant by-products at two protein levels (40 and 49%) were tested in the present experiment on rainbow trout having an initial average weight of 1 g. A commercial feed was also used for comparison. Fish performance, body composition and haematological characteristics are presented and discussed. MATERIALS
AND METHODS
Eighteen groups of 250 rainbow trout fry having an average weight of 1 g/fish were used for the experiment. They were maintained in 2 x 0.45 x 0.40-m fibreglass tanks, with a water flow of 0.5 l/s, up to a weight of about 10 g/fish and were then transferred to concrete raceways (with dimensions of 6 x 1 x 1.2 m and a water flow of 1.5 l/s). The water was 11-13”C, with a pH of 7.4-7.8, conductivity of 235-265 ,uMhos/cm and hardness of 220-260 ppm CaCO,. Four protein-rich mixtures were prepared (Table 1) and used for formulating eight experimental diets (Table 2). Two diets were formulated from each mixture used, one with a low (40%) and one with a high (49%) protein level. The four diets containing 40% protein (1,2,3 and 4) were similar to some of the diets (3, 5,6 and 7 respectively) tested in a previous experiment (Alexis et al., 1985) with larger fish ( 20 g initial weight). All of these diets contained carob seed germ meal (CSGM) in various proportions. On the basis of the previous experiment, diet 4 was expected to result in the best growth rate, diet 3 in the lowest growth rate and diets 1 and 2 to give intermediate growth rates. The present arrangement, therefore, allowed the comparison of diets of different nutritional characteristics at two protein levels, as well as the extension of the results obtained in the first experiment to fish of a smaller size (1 g initial weight). The proximate composition of the by-products used was as previously presented (Alexis et al., 1985). The carob seed germ meal used in this experiment was, however, slightly different, containing a lower level of protein (38% ) . Each diet was assigned to duplicate tanks of fish and two commercial diets (diets gAand gB in Table 2) were used for comparison. Diet QAwas fed to fish
TABLE 1
Composition and proximate analysis of the protein-rich mixtures used for formulating the diets Mixture A Composition ( % ) Herring meal Pouftry by-products CSGM” CGM” Methionine Lysine Proximate analysis ( % f Protein Lipid Ash Moisture NFE’ Fibre Cost ( dr.,&g )
B
c
D
29.8 39.5 29.8 0.9
13.8 36.7 48.5 -
-
-
50.3 47.4 -
0.7 0.3
0.9 1.4
47.7 18.0 30.6 0.8 2.9
57.9 10.8
52.7 9.5 9.2 7.3 19.1 2.2 33.6
53.5 10.0 7.8 7.9 18.5 2.4 33.5
60.5 11.7 5.5 8.2 12.6 1.6 45.8
10.3 7.4 12.0 1.6 40.9
“Carob seed germ meal. Vorn gluten meal. ‘Nitrogen free extract. up to an average weight of 10 g/fish and 9n after that weight, according to the producer’s specifications. The fish were fed by hand, at a daily rate of about 3% of their body weight, three times a day, seven days a week. A sample of 50 fish from each tank was weighed every fortnight. Fish were starved for 24 h before each weighing. The whole population of each tank was weighed at the end of the experiment, which lasted from 25 May to 5 December 1984. A sample of 140 fish of the initial population, as well as a sample of 10 fish from each tank removed at the end of the experiment, was used for proximate analysis. The fish removed at the end of the experiment were also used for the determination of the hepatosomatic index and haematological characte~stics. The methods used for the proximate analyses and the determination of the haematologicai characteristics were as previously described (Alexis et al., 1985). Comparison of the averages was carried out using the Student’s t-test. The results are reported at the 5% probability level. RESULTS
Diets 4 and 8, containing the same protein mixture ( D ) , resulted in the best growth rates produced by the experimental diets (Fig. 1 and Table 3) +Similar
78 TABLE 2 Formulations, proximate analysis, energy content and cost of the experimental (l-8) and commercial (9* and 9,) diets and % CSGM in each experimental diet Diet 1
2
Formulationh ( %f
lwixtun? A’ MixweB’ Mixture c” Mixture D’ Wheat middlings Bone meal Linseed oil Proximate analysis Protein Lipid Ash Moisture NFE Fibre Gross energy f k&/kg diet)” Cost (dr./kg) CSGM (%)
62.4 -
69.2
3
4
-
-
5 82.7
7
91.7
69.4 -
30.7 0.3 5.7
22.4 1.0 6.4
22.3 1.7 5.8
61.3 28.5 3.8 5.5
40.5 13.7 8.2 8.3 24.6 3.7
39.7
14.0 8.5 7.7 25.9 3.6
40.3 13.7 8.2 8.9 25.4 3.6
41.1 13.9 8.5 8.4 23.8 3.6
4598 50.1 20
4623 47.7 35
4606 46.9 34
4614 53.1 12
-
6
8
-
-
-
-
90.4
-
9,”
5.2
2.2 1.9 4.7
80.0 10.7 4.3 4.1
14.0 9.2 7.6 16.6 2.4
48.7 14.0 8.7 7.0 18.7 2.2
48.6 13.8 9.2 1.4 17.9 2.3
49.9 14.0 9.3 7.8 16.1 2.3
51.3 5.4 14.3 10.0 15.6 2.9
46.8 7.2 12.6 10.0 19.8 3.6
4806 33.3 25
4839 49.4 45
4787 48.8 44
4799 56.5 15
4048 70.0
4136 65.0
-
-
Qh*
2.2
11.8 4.6
t %) 49.6
“Feed gAwas used up to a weight of about 10 g/fish and 9s for the rest of the rearing period. “Each diet also contained 0.6% choline chloride (50% I, 0.08% vitamin premix and 0.2% mineral premix. The composition of the premixes used was given in a previous paper (Alexis et al., 1985). “The composition of the mixture is given in Table 1. “Based on 5.65 kcal/g protein. 9.45 kcal/g fat and 4.1 kcal/g carbohydrate.
growth rates were also exhibited by the groups fed diet 5 and the commercial feed. Diets 6 and 7 resulted in worse growth rates than their respective lower protein counterparts (diets 2 and 3). However, the differences in growth between diets 2 and 6 were not si~i~~ant. The general performance of the fish is presented in Table 3. The values for feed conversion closely followed the growth data with diets 4,5,8 and 9 showing the best feed utilization. The highest mortality rate was exhibited by fish fed diet 7, followed by fish fed diets 8,6 and 5, the rest of the groups showing a similar low mortality rate. Some of the fish in both groups fed diet 1 appeared to suffer from lens cataracts after 160 days of rearing. Protein retention ( % ) was generally lower for the high protein diets: increasing dietary protein levels generally results in decreased protein retention in fish ( Steffens, 1981). Energy retention decreased significantly with the higher protein level for fish showing a lower growth rate at the higher protein level. All of the parameters related to fish performance were correlated with the CSGM content of the diet (Table 4). A negative correlation was apparent for all of these with the exceptions of feed conversion and visceral weight where a positive correlation was found. The proximate analysis of the whole fish carcasses is also given in Table 3. The protein content of the fish correlated positively with the fish weight
79
a
I
L
)
L
I,,.,,
1
too
50
150
,,.‘I‘
200
Days Fig. 1. Average body weight throughout the experiment (diet 1,O; diet 2,O; diet 3, A; diet 4, V; diet5,@;diet6,@diet?,A;diet&V;diet9,+).
( r= 0.685, P= 0.05) and negatively with the CSGM content of the diet (Table 4). No correlation between the body lipid content and the two previous parameters could be estabhshed. The body lipid and moisture contents had an inverse relationship ( r = - 0.915, P=O.Ol ) . The ash content did not change significantly among the groups with the exception of the fish fed the commercial diet, which had a significantly higher ash content. The lean body and visceral compositions of the rainbow trout are given in Table 5. The visceral composition indicated a slightly lower level of protein but a considerably higher (about 4 times more with the experimental diets)
1.3 53.Qb 1.2 1.63& 8.8’ 24.9” 25.9t” 13.8” 76.4 16.3ab 10.Sd 2.3” 70.3&b
16.0” 11.0” 2 4”b 70kk
3
1.2 60.8b 0.4 1.56b” 9.7b 25.9” 27.2cd 14.1” 74.4
2
17.2” 9.7b 2.4* 70.7”
1.3 71.1” 1.6 1.41* 12.3”d 29.7d 29.3d 10.2b 74.9
4
‘Values on the same line with the same superscript are not significantly different.
Performance factors’ Initial weight of fish (g) 1.1 Weight gain (g ) 52.3& Mortality ( % ) 1.4 Feed conversion 1.5Qb: Protein retained (g) 6.5”b Protein retained ( % ) 25.3” Energy retained ( % ) 27.1kd Visceral weight (% body weight) 14.8f Cost of fish produced (dr./kg ) 79.7 Proximate analysis ( % whole body composition ) ’ Crude protein 16.3”b Crude fat 11.0” Ash 2 4”b Moisture 70:4*b”
1
Diet
Performance and whole body composition of rainbow trout fed experimental (l-8)
TABLE 3
1.0 53.3b 6.0 1.65” 8.9b 20.8b 24.6b 15.6K 81.5 16.4b 10.4cd 2.3” 70.6bc
16.2”b 9.8b 2.4”b 71.Sd
6
1.2 68.9” 4.4 1.36” 11.1” 24.0’ 28.4cd 13.od 72.5
5
16.1ab 10.2’ 2.3” 71.5d
1.4 45.7” 28.0 1.82d 7.4” 18.3* 21.9” 16.3h 88.8
7
and commercial (9) diets for 196 days
17.2” 10.4”d 2.3” 70.2”
1.4 75.4” 14.8 1.35” 13.od 25.5” 30.4d 11.3” 76.3
8
18.3d 5.9” 2.6b 73.3”
1.4 72.7” 0.8 1.45”b 13.4d 26.5” 26.8t” 8.6” 95.8
9
81 TABLE 4 Linear correlations between %CSGM in the diet and various experimental significant correlations are not included) Parameter
r
P
Weight gain Feed conversion Protein retained (g) Protein retained ( % ) Energy retained ( %) Visceral weight (% body weight) Crude protein (whole body) Hepatosomatic index Haematocrit Haemoglobin
-0.791 +0.743 - 0.847
0.05 0.05
-0.710 -0.682 $0.916 -0.640 f0.736 -0.824 -0.609
0.05 0.05 0.01
parameters
(non-
0.01
0.01 0.05 0.01 0.01
level of lipids compared with the lean body composition of the fish. The crude protein and fat levels of the viscera were about the same for all of the groups of fish (with the exception of the commercial feed). The contribution of the visceral lipids to the total body lipid content depended mainly on the % visceral weight attained by each group of fish (Table 3 1. As mentioned above, the visceral weight correlated positively with the CSGM content of the diet (Table 4). The hepatosomatic indices and haematological characteristics of the rainbow trout are given in Table 6. The hepatosomatic index correlated positively, TABLE 5 Lean body and visceral composition of rainbow trout fed experimental (l-8) diets for 196 days
and commercial (9)
Diet 1
2
Lean body composition ( % ) Crude protein 16.9” 16.6” Crude-fat 7.2” 7.6f Ash 2.5ab 2.4” Moisture 73.6d ‘?3.2& Visceral composition ( % ) Crude protein 12.gb 12.1b 31.3’ Crude fat 33.5e’ Ash l.lb 2.3’ Moisture 51.9& 53.8’
3
4
5
6
7
8
9
16.7” 6.gcd 2.5ab 73.&d
17&b 7.0de 2.4” 73.0”b
16.8” 6.6b 2.5”b 74.4”
17.4b 7.Od” 2.5sb 73.4Cd
16.7” 6.1b” 2.5”b 74.2”
17.4b 7.6f 2.3” 72.7
18.7’ 5.2” 2.7b 73.6d
13.9’ 33.4” 1.6b 51.1ab
13.8 33.7f 2.Sd 49.9”
14.0” 31.5cd 1.8s 52.7”
11.6” 28.8b 1.3a 56.2’
13.0s 28.4b 1.2” 57.5f
15.ld 32.5d” l.Sb 50.7”b
15.P 13.8” 1.7b 69.4g
‘Values on the same line with the same superscript are not significantly different.
82 TABLE 6 Hepatosomatic index and haemstological characteristics of rainbow trout fed experimental (I-8) and commercial (9) diets for 196 days’ Diet
Hepatosomatic index’
Haematocrit
Haemoglabin (g/loo ml)
Cholesterol (mg/lOO ml)
Lipids (mg/lOO ml)
GlUClXE (mg/lOO ml)
Protein (g/l00 ml)
1 2 3 4 5 6 7 8 9
0.97’” 1.12’ 0.92* 0.82” 0.85”” 1.05d’ 1.11* 0.84”” 0.9o”h
37.4” 36.4”” 37.5” 40.7 40.5” 35.5”” 34.7” 36.5”” 44.5”
7.6’” 6.7” 7.F 8.Zrd 8.7*’ 6.4” 6.3” 7.0”” 9.4”
404.9 324.Skd 292.9b 245.3” 348.!F’ 343.2’” 350.6’d 318.7hc 361.Sde
1868 1643” 1592”h 1396” 1750” 1646” 1693h 1561nb 1680”
142.4b’ 136.gb’ 104.8” 125.1”” 137.8” 137.tib” 154.5’ 142.4be 144.Oh
4.09d@ 3.81”M 3.63-b 3.50 3.7Th 3.86b”’ 4.06+ 3.99Cd’ 4.32’
‘Values in the same column with the same superscript are not significantly different. “Liver weight as a 7%of the body weight.
and the haematocrit and haemoglobin negatively, with the CSGM content of the diet (Table 4). No correlation with the CSGM content of the diet or the protein level could be established for the other blood components. Certain consistent changes in the values of some of the blood parameters appeared to exist between the respective pairs of diets based on the same protein mixture. Thus, the haemoglobin values decreased, and the plasma cholesterol, glucose and protein values increased, when the protein content of the diet increased from diets 2 to 6,3 to 7 and 4 to 8, while an opposite trend was observed between diets 1 and 5. The differences observed in these parameters between diets 2 and 6 were not significant. The lipid content of the plasma did not change significantly between each pair of diets. The values of all of the blood parameters of the fish fed the commercial feed were among the highest observed. All of the haematological characteristics were within the range of the normal values reported in the literature (McCarthy et al., 1973,1975; Hille, 1982). DISCUSSION
The protein concentrations used in the present study cover the range of optimal values for rainbow trout growth reported by Zeitoun (1976) for fish of similar size. The change in the growth characteristics between the groups of fish fed the respective pairs of diets was not, however, uniform indicating a remarkable contribution of the protein sources used to the results obtained. The growth of the fish was positively affected by an increase in the protein content from diets 1 to 5 and diets 4 to 8. Diet 4 was found to give the best results in terms of growth rate, feed conversion, % protein retention and flesh quality when fed to fish having an initial average weight of 20 g (Alexis et al., 1985). Similar results were also obtained in the present study with fish having an initial average weight of 1 g. The slight improvement in growth observed at the highest protein level was accompanied by an increased use of the dietary
83
protein for body fat accumulation since the % protein retention decreased and the lipid content of the fish increased. The differences in growth observed between the groups fed diets 1 and 5 were of higher magnitude, indicating a different response of the fish to this pair of diets. The different response observed could be the result of different digestibilities of the mixtures used. If the digestible protein content of diets 1 and 5 was lower than 40 and 50% of the diet respectively, then the changes in growth would include a range outside the protein optima, where growth responses are more sensitive to protein increases in the diet (Zeitoun et al,, 19761, The difference in growth between diets I and 5 might, however, have been amplified by the factor causing the lens cataracts in the low protein group. The reduced growth of the fish fed diets 6 and 7, compared with those fed diets 2 and 3, despite their higher protein content, appeared to be mainly the result of the increased CSGM content in these diets since fish growth rates and CSGM content of the diets correlated negatively. Condensed tannins are the main antigrowth compounds reported for carobs (Nachtomi and Alumot, 1963). In vitro studies with carob tannins indicated that these are non-competitive inhibitors of many digestive enzymes (Tamir and Alumot, 1969). CSGM has been found to contain about 0.4% tannins (Drouliscos and Malfaki, 1980)) an amount which is much lower than that contained in whole carobs, The susceptibility of rainbow trout digestive enzymes to inhibition by tannins has not so far been studied. Studies on trypsin inhibitors, however, have shown that trout enzymes are much more susceptible to inhibition than those of many other animals tested (Krogdahl and Holm, 1983). The same could also occur with inhibition by tannins. Therefore, increasing the level of CSGM in diets 6 and 7 might have reduced considerably the active enzyme levels in the rainbow trout digestive system, resulting in an overall poorer utilization of the feed. The negative effect of the CSGM content of the diet on fish growth was apparent when its level exceeded 25% of the diet, while levels lower than this did not appear to reduce markedly the performance of the fish. Fish fed diets with a lower nutritional value appear to have a higher visceral. weight and, therefore, less useful flesh, as indicated by the positive correlation found between the CSGM content of the diet and visceral weight. The contribution of the final fish weight to the differences in the visceral weights observed is expected to be small since a change from about 16 to 13% of the total fish weight has been found in relation to fish size during the first 14 months of rainbow trout life (Denton and Yousef, 1976). Higher visceral weights did not appear to result from increased lipid accumulation since no correlation between these two parameters could be found. Visceral weight has been found to correlate positively with the lipid content of the diet f Lee and Putnam, 1973 1.
The visceral weight of the fish fed the commercial diet containing a low level of lipid was lower than that of all of the other experimental groups. The whole body composition of fish fed the commercial diet was also significantly differ-
ent from that of all of the other experimental groups (Table 3 ) . The principal difference in the total body lipids appeared to result from the much smaller contribution of the visceral lipids of the fish fed the commercial feed to the total body composition, while differences in the lipid content of the lean body were of a smaller magnitude (Table 5). A considerable increase in the visceral lipids, and a smaller increase in the muscle lipids, with increasing lipid content of the diet was also found by Watanabe et al. (1979) for rainbow trout of the same initial average weight. The negative correlation found between the CSGM content of the diet and the haematocrit and haemoglobin levels indicates that the haema~logi~al characteristics are affected by diet quality. Increased haematoerit values with diets prompting faster growth have also been reported in the literature (Barnhart, 1969). The positive correlation observed between the CSGM content of the diet and the hepatosomatic index cannot be explained in terms of lower available protein with increasing CSGM content of the diets since the hepatosomatic index and the protein content of the diet were found to correlate positively (Lee and Putnam, 1973). The correlation observed might result either from glycogen accumulation created by a high digestibility of CSGM carbohydrates or from some other factor contained in CSGM that contributes to an enlargement of the liver. The final cost of the fish produced is determined by both the feed cost and the feed conversion. The marginal differences in feed conversion between diets 4 and 8 resulted in a slightly higher cost/kg fish produced (Table 3) with the high protein diet since the protein source used (D) was one of the most expensive (Table 1). To the contrary, the much improved feed conversion of diet 5 makes it preferable to its lower protein counterpart. The adverse effects of increased CSGM inclusion in the diet on fish growth resulted in a higher production cost of fish fed diets 6 and 7 containing a high protein content, compared with their respective lower protein diets, since both feed price and feed conversion increased. The nutritional characteristics of the by-products used as protein sources for form~ating practical diets should, therefore, be carefully determined and taken into account when selecting an economic protein level, so that increased levels of antigrowth factors and adverse effects on growth and final cost of production are avoided. Information regarding the nutritional potential of many plant by-products of high protein content is, however, limited, a factor which restricts their use as ingredients in fish diets and, therefore, their maximum exploitation as cheap protein sources. ACKNOWLEDGEMENTS
We wish to thank Mr M. Filioglou and Miss M. Galani for their valuable assistance with the samplings and Mr G. Nanos for the care and feeding of the
85
fish. Thanks are also due to Miss E. Valla and Mrs E. Papoutsi for their careful laboratory assistance.
REFERENCES Alexis, M.N., Papaparaskeva-Papoutsoglou, E. and Theochari, V., 1985. Formulation of practical diets for rainbow trout (Salmo guirdneri) made by partial or complete substitution of fish meal by poultry by-products and certain plant by-products. Aquaculture, 50: 61-73. Barnhart, R.A., 1969. Effects of certain variables on hema~lo~c~ characteristics of rainbow trout. Trans. Am. Fish. Sot., 98: 411-418. Cho, C.Y., Slinger, S.J. and Bayley, H.S., 1976. Influence of level and type of dietary protein, and of level of feeding on feed utilization by rainbow trout. J. Nutr., 106: 1547-1556. Cowey, C.B. and Sargent, J.R., 1979. Nutrition, In: W.S. Hoar, D.J. Randall and J.R. Brett (Editors), Fish Physiology. Vol. 8: Bioenergetics and Growth. Academic Press, New York, pp. 58-69. Denton, J.E. and Yousef, M.K., 1976. Body composition and organ weights of rainbow trout, Salmo gairdneri. J. Fish Biol., 8: 489-499. Drouliscos, N.J. and Malefaki, V., 1980. Nutritionaf evaluation of the germ meal and its protein isolate obtained from the carob seed (Ceratonia &qua) in the rat. Br. J. Nutr., 43: 115-123. Hille, S., 1982. A literature review of the blood chemistry of rainbow trout, Salmogatrdneri Rich. J. Fish Biol., 20: 535-569. Krogdahl, A. and Holm, H., 1983. Pancreatic proteinases from man, trout, rat, pig, cow, chicken, mink and fox. Enzyme activities and inhibition by soybean and lima bean proteinase inhibitors. Comp. Biochem. Physiol., 74B: 403-409. Lee, D.J. and Putnam, G.B., 1973. The response of rainbow trout to varyingprotein/energy ratios in a test diet. J. Nutr., 103: 916-922. McCarthy, D.H., Stevenson, J.P. and Roberts, MS., 1973. Some blood parameters of the rainbow trout (Salmo gairdneri Richardson), I. The Kamloops variety. J. Fish Biol.,.5: l-8. McCarthy, D.H., Stevenson, J.P. and Roberts, MS., 1975. Some blood parameters of the rainbow trout (Salmo gu~rd~ri Richardson). II. The Shasta variety. J. Fish Biol., 7: 215219. Nachtomi, E. and Alumot, E., 1963. Tannins and polyphenols in carob pods (Ceratonia siliqua) . J. Sci. Food Agric., 14: 464-468. Satia, B.P., 1974. Quantitative protein requirementsof rainbow trout. Prog. Fish. Cult., 36: 80-85. Steffens, W., 1981. Protein utilization by rainbow trout (Salmo gairdneri) and carp (Cyprinus carpio) : A brief review. Aquaculture, 23: 337-345. Tamir, M. and Alumot, E., 1969. Inhibition of digestive enzymes by condensed tannins from green and ripe carobs. J. Sci. Food Agric., 20: 199-202. Watanabe, I‘., Takeuchi, T. and Ogino, C.H., 1979. Studies on the sparing effect of lipids on dietary protein in rainbow trout (Salmo gairdneri) . In: J.E. Halver and K. Tiews (Editors), Finfish Nutrition and Fishfeed Technology. Vol. I. Heeneman, Berlin, pp. 113-125. Zeitoun, I.H., Ullrey, D.E., Magee, W.T., Gill, J.L. and Bergen, W.G., 1976. Quantifying nutrient requirements of fish. J. Fish. Res. Board Can., 33: 167-172.
Elsevier Science ~blishers
75
B.V., Amsterdam - Printed in The Netherl~~
Effect of Diet Composition and Protein Level on Growth, Body Composition, Haematological Characteristics and Cost of Production of Rainbow Trout (Salmo gairdneri) MARIA N. ALEXIS, VASSILIKI THEOCHAR12 and ELLI PAPAPA~SK~VAPAPO~O~LOU’ ‘National Centre for Marine Research, GR-166 04 Elliniko (Greece) %NationalHatchery ojLouros, Hani Terouou, Zoannina (Greece)
(Accepted 2 June 1986)
ABSTRACT Alexis, M.N., Theochari, V. and Papaparaskeva-Papoutsoglou, E., 1986. Effect of diet composition and protein level on growth, body composition, haematological characteristics and cost of production of rainbow trout ( SaLmo gairdneri) . Aquac~t~re, 58: 75-85. Rainbow trout of approximately 1 g initial average weight were fed eight experimental diets and a commercial diet for 196 days. Four different combinations of by-products were used as the main ingredient in formulating four pairs of diets differing in their protein content ( 40 and 49% ) . The response of the fish towards the protein increase in the diet was not uniform, a better performance being observed for two of the mixtures used at the higher protein content and a worse performance for the other two. The growth of the fish was negatively correlated with the carob seed germ meal content of the diet. The differences in the protein content of the fish fed the experimental diets were small while the lipid content was more variable. A large proportion of the body lipids was contributed by the visceral lipids. Haematological characteristics of all of the fish tested were within the normal values reported in the literature.
INTRODUCTION
Protein is considered to be the most expensive component of fish diets. For this reason the study of the protein requirements of rainbow trout has been extensive and levels of 3550% in the diet have been found to produce maximum growth, depending on diet composition, level of feeding, and the age and physiological condition of the fish (Lee and Putnam, 1973; Satia, 1974; Cho et al., 1976; Zeitoun et al., 1976; Watanabe et al.; 1979, Cowey and Sargent, 1979; Steffens, 1981) _ Under specified conditions, the growth maxima are usually identified within a range of dietary protein concentrations that depends on the
~44-8486/86/$03.50
0 1986 Elsevier Science Publishers B.V.
76
method of calculation used and the experimental error. There is generally an improvement in growth and food conversion from the low to the high protein levels of this range (Zeitoun et al., 1976). The choice of dietary protein to be used in practical rations is, therefore, an economic decision, which depends on the cost of the protein source as well as on the expected returns from fish growth and value. The particular characteristics of the protein sources used in fish diets are another factor that has to be considered when selecting an economic protein level. The response of the fish to an increase in the protein content of the diet could vary depending on whether more digestible protein is provided or whether the levels of antigrowth compounds increase unfavourably for the growth of the fish, especially in mixtures containing plant by-products. Eight diets containing different combinations of animal and plant by-products at two protein levels (40 and 49%) were tested in the present experiment on rainbow trout having an initial average weight of 1 g. A commercial feed was also used for comparison. Fish performance, body composition and haematological characteristics are presented and discussed. MATERIALS
AND METHODS
Eighteen groups of 250 rainbow trout fry having an average weight of 1 g/fish were used for the experiment. They were maintained in 2 x 0.45 x 0.40-m fibreglass tanks, with a water flow of 0.5 l/s, up to a weight of about 10 g/fish and were then transferred to concrete raceways (with dimensions of 6 x 1 x 1.2 m and a water flow of 1.5 l/s). The water was 11-13”C, with a pH of 7.4-7.8, conductivity of 235-265 ,uMhos/cm and hardness of 220-260 ppm CaCO,. Four protein-rich mixtures were prepared (Table 1) and used for formulating eight experimental diets (Table 2). Two diets were formulated from each mixture used, one with a low (40%) and one with a high (49%) protein level. The four diets containing 40% protein (1,2,3 and 4) were similar to some of the diets (3, 5,6 and 7 respectively) tested in a previous experiment (Alexis et al., 1985) with larger fish ( 20 g initial weight). All of these diets contained carob seed germ meal (CSGM) in various proportions. On the basis of the previous experiment, diet 4 was expected to result in the best growth rate, diet 3 in the lowest growth rate and diets 1 and 2 to give intermediate growth rates. The present arrangement, therefore, allowed the comparison of diets of different nutritional characteristics at two protein levels, as well as the extension of the results obtained in the first experiment to fish of a smaller size (1 g initial weight). The proximate composition of the by-products used was as previously presented (Alexis et al., 1985). The carob seed germ meal used in this experiment was, however, slightly different, containing a lower level of protein (38% ) . Each diet was assigned to duplicate tanks of fish and two commercial diets (diets gAand gB in Table 2) were used for comparison. Diet QAwas fed to fish
TABLE 1
Composition and proximate analysis of the protein-rich mixtures used for formulating the diets Mixture A Composition ( % ) Herring meal Pouftry by-products CSGM” CGM” Methionine Lysine Proximate analysis ( % f Protein Lipid Ash Moisture NFE’ Fibre Cost ( dr.,&g )
B
c
D
29.8 39.5 29.8 0.9
13.8 36.7 48.5 -
-
-
50.3 47.4 -
0.7 0.3
0.9 1.4
47.7 18.0 30.6 0.8 2.9
57.9 10.8
52.7 9.5 9.2 7.3 19.1 2.2 33.6
53.5 10.0 7.8 7.9 18.5 2.4 33.5
60.5 11.7 5.5 8.2 12.6 1.6 45.8
10.3 7.4 12.0 1.6 40.9
“Carob seed germ meal. Vorn gluten meal. ‘Nitrogen free extract. up to an average weight of 10 g/fish and 9n after that weight, according to the producer’s specifications. The fish were fed by hand, at a daily rate of about 3% of their body weight, three times a day, seven days a week. A sample of 50 fish from each tank was weighed every fortnight. Fish were starved for 24 h before each weighing. The whole population of each tank was weighed at the end of the experiment, which lasted from 25 May to 5 December 1984. A sample of 140 fish of the initial population, as well as a sample of 10 fish from each tank removed at the end of the experiment, was used for proximate analysis. The fish removed at the end of the experiment were also used for the determination of the hepatosomatic index and haematological characte~stics. The methods used for the proximate analyses and the determination of the haematologicai characteristics were as previously described (Alexis et al., 1985). Comparison of the averages was carried out using the Student’s t-test. The results are reported at the 5% probability level. RESULTS
Diets 4 and 8, containing the same protein mixture ( D ) , resulted in the best growth rates produced by the experimental diets (Fig. 1 and Table 3) +Similar
78 TABLE 2 Formulations, proximate analysis, energy content and cost of the experimental (l-8) and commercial (9* and 9,) diets and % CSGM in each experimental diet Diet 1
2
Formulationh ( %f
lwixtun? A’ MixweB’ Mixture c” Mixture D’ Wheat middlings Bone meal Linseed oil Proximate analysis Protein Lipid Ash Moisture NFE Fibre Gross energy f k&/kg diet)” Cost (dr./kg) CSGM (%)
62.4 -
69.2
3
4
-
-
5 82.7
7
91.7
69.4 -
30.7 0.3 5.7
22.4 1.0 6.4
22.3 1.7 5.8
61.3 28.5 3.8 5.5
40.5 13.7 8.2 8.3 24.6 3.7
39.7
14.0 8.5 7.7 25.9 3.6
40.3 13.7 8.2 8.9 25.4 3.6
41.1 13.9 8.5 8.4 23.8 3.6
4598 50.1 20
4623 47.7 35
4606 46.9 34
4614 53.1 12
-
6
8
-
-
-
-
90.4
-
9,”
5.2
2.2 1.9 4.7
80.0 10.7 4.3 4.1
14.0 9.2 7.6 16.6 2.4
48.7 14.0 8.7 7.0 18.7 2.2
48.6 13.8 9.2 1.4 17.9 2.3
49.9 14.0 9.3 7.8 16.1 2.3
51.3 5.4 14.3 10.0 15.6 2.9
46.8 7.2 12.6 10.0 19.8 3.6
4806 33.3 25
4839 49.4 45
4787 48.8 44
4799 56.5 15
4048 70.0
4136 65.0
-
-
Qh*
2.2
11.8 4.6
t %) 49.6
“Feed gAwas used up to a weight of about 10 g/fish and 9s for the rest of the rearing period. “Each diet also contained 0.6% choline chloride (50% I, 0.08% vitamin premix and 0.2% mineral premix. The composition of the premixes used was given in a previous paper (Alexis et al., 1985). “The composition of the mixture is given in Table 1. “Based on 5.65 kcal/g protein. 9.45 kcal/g fat and 4.1 kcal/g carbohydrate.
growth rates were also exhibited by the groups fed diet 5 and the commercial feed. Diets 6 and 7 resulted in worse growth rates than their respective lower protein counterparts (diets 2 and 3). However, the differences in growth between diets 2 and 6 were not si~i~~ant. The general performance of the fish is presented in Table 3. The values for feed conversion closely followed the growth data with diets 4,5,8 and 9 showing the best feed utilization. The highest mortality rate was exhibited by fish fed diet 7, followed by fish fed diets 8,6 and 5, the rest of the groups showing a similar low mortality rate. Some of the fish in both groups fed diet 1 appeared to suffer from lens cataracts after 160 days of rearing. Protein retention ( % ) was generally lower for the high protein diets: increasing dietary protein levels generally results in decreased protein retention in fish ( Steffens, 1981). Energy retention decreased significantly with the higher protein level for fish showing a lower growth rate at the higher protein level. All of the parameters related to fish performance were correlated with the CSGM content of the diet (Table 4). A negative correlation was apparent for all of these with the exceptions of feed conversion and visceral weight where a positive correlation was found. The proximate analysis of the whole fish carcasses is also given in Table 3. The protein content of the fish correlated positively with the fish weight
79
a
I
L
)
L
I,,.,,
1
too
50
150
,,.‘I‘
200
Days Fig. 1. Average body weight throughout the experiment (diet 1,O; diet 2,O; diet 3, A; diet 4, V; diet5,@;diet6,@diet?,A;diet&V;diet9,+).
( r= 0.685, P= 0.05) and negatively with the CSGM content of the diet (Table 4). No correlation between the body lipid content and the two previous parameters could be estabhshed. The body lipid and moisture contents had an inverse relationship ( r = - 0.915, P=O.Ol ) . The ash content did not change significantly among the groups with the exception of the fish fed the commercial diet, which had a significantly higher ash content. The lean body and visceral compositions of the rainbow trout are given in Table 5. The visceral composition indicated a slightly lower level of protein but a considerably higher (about 4 times more with the experimental diets)
1.3 53.Qb 1.2 1.63& 8.8’ 24.9” 25.9t” 13.8” 76.4 16.3ab 10.Sd 2.3” 70.3&b
16.0” 11.0” 2 4”b 70kk
3
1.2 60.8b 0.4 1.56b” 9.7b 25.9” 27.2cd 14.1” 74.4
2
17.2” 9.7b 2.4* 70.7”
1.3 71.1” 1.6 1.41* 12.3”d 29.7d 29.3d 10.2b 74.9
4
‘Values on the same line with the same superscript are not significantly different.
Performance factors’ Initial weight of fish (g) 1.1 Weight gain (g ) 52.3& Mortality ( % ) 1.4 Feed conversion 1.5Qb: Protein retained (g) 6.5”b Protein retained ( % ) 25.3” Energy retained ( % ) 27.1kd Visceral weight (% body weight) 14.8f Cost of fish produced (dr./kg ) 79.7 Proximate analysis ( % whole body composition ) ’ Crude protein 16.3”b Crude fat 11.0” Ash 2 4”b Moisture 70:4*b”
1
Diet
Performance and whole body composition of rainbow trout fed experimental (l-8)
TABLE 3
1.0 53.3b 6.0 1.65” 8.9b 20.8b 24.6b 15.6K 81.5 16.4b 10.4cd 2.3” 70.6bc
16.2”b 9.8b 2.4”b 71.Sd
6
1.2 68.9” 4.4 1.36” 11.1” 24.0’ 28.4cd 13.od 72.5
5
16.1ab 10.2’ 2.3” 71.5d
1.4 45.7” 28.0 1.82d 7.4” 18.3* 21.9” 16.3h 88.8
7
and commercial (9) diets for 196 days
17.2” 10.4”d 2.3” 70.2”
1.4 75.4” 14.8 1.35” 13.od 25.5” 30.4d 11.3” 76.3
8
18.3d 5.9” 2.6b 73.3”
1.4 72.7” 0.8 1.45”b 13.4d 26.5” 26.8t” 8.6” 95.8
9
81 TABLE 4 Linear correlations between %CSGM in the diet and various experimental significant correlations are not included) Parameter
r
P
Weight gain Feed conversion Protein retained (g) Protein retained ( % ) Energy retained ( %) Visceral weight (% body weight) Crude protein (whole body) Hepatosomatic index Haematocrit Haemoglobin
-0.791 +0.743 - 0.847
0.05 0.05
-0.710 -0.682 $0.916 -0.640 f0.736 -0.824 -0.609
0.05 0.05 0.01
parameters
(non-
0.01
0.01 0.05 0.01 0.01
level of lipids compared with the lean body composition of the fish. The crude protein and fat levels of the viscera were about the same for all of the groups of fish (with the exception of the commercial feed). The contribution of the visceral lipids to the total body lipid content depended mainly on the % visceral weight attained by each group of fish (Table 3 1. As mentioned above, the visceral weight correlated positively with the CSGM content of the diet (Table 4). The hepatosomatic indices and haematological characteristics of the rainbow trout are given in Table 6. The hepatosomatic index correlated positively, TABLE 5 Lean body and visceral composition of rainbow trout fed experimental (l-8) diets for 196 days
and commercial (9)
Diet 1
2
Lean body composition ( % ) Crude protein 16.9” 16.6” Crude-fat 7.2” 7.6f Ash 2.5ab 2.4” Moisture 73.6d ‘?3.2& Visceral composition ( % ) Crude protein 12.gb 12.1b 31.3’ Crude fat 33.5e’ Ash l.lb 2.3’ Moisture 51.9& 53.8’
3
4
5
6
7
8
9
16.7” 6.gcd 2.5ab 73.&d
17&b 7.0de 2.4” 73.0”b
16.8” 6.6b 2.5”b 74.4”
17.4b 7.Od” 2.5sb 73.4Cd
16.7” 6.1b” 2.5”b 74.2”
17.4b 7.6f 2.3” 72.7
18.7’ 5.2” 2.7b 73.6d
13.9’ 33.4” 1.6b 51.1ab
13.8 33.7f 2.Sd 49.9”
14.0” 31.5cd 1.8s 52.7”
11.6” 28.8b 1.3a 56.2’
13.0s 28.4b 1.2” 57.5f
15.ld 32.5d” l.Sb 50.7”b
15.P 13.8” 1.7b 69.4g
‘Values on the same line with the same superscript are not significantly different.
82 TABLE 6 Hepatosomatic index and haemstological characteristics of rainbow trout fed experimental (I-8) and commercial (9) diets for 196 days’ Diet
Hepatosomatic index’
Haematocrit
Haemoglabin (g/loo ml)
Cholesterol (mg/lOO ml)
Lipids (mg/lOO ml)
GlUClXE (mg/lOO ml)
Protein (g/l00 ml)
1 2 3 4 5 6 7 8 9
0.97’” 1.12’ 0.92* 0.82” 0.85”” 1.05d’ 1.11* 0.84”” 0.9o”h
37.4” 36.4”” 37.5” 40.7 40.5” 35.5”” 34.7” 36.5”” 44.5”
7.6’” 6.7” 7.F 8.Zrd 8.7*’ 6.4” 6.3” 7.0”” 9.4”
404.9 324.Skd 292.9b 245.3” 348.!F’ 343.2’” 350.6’d 318.7hc 361.Sde
1868 1643” 1592”h 1396” 1750” 1646” 1693h 1561nb 1680”
142.4b’ 136.gb’ 104.8” 125.1”” 137.8” 137.tib” 154.5’ 142.4be 144.Oh
4.09d@ 3.81”M 3.63-b 3.50 3.7Th 3.86b”’ 4.06+ 3.99Cd’ 4.32’
‘Values in the same column with the same superscript are not significantly different. “Liver weight as a 7%of the body weight.
and the haematocrit and haemoglobin negatively, with the CSGM content of the diet (Table 4). No correlation with the CSGM content of the diet or the protein level could be established for the other blood components. Certain consistent changes in the values of some of the blood parameters appeared to exist between the respective pairs of diets based on the same protein mixture. Thus, the haemoglobin values decreased, and the plasma cholesterol, glucose and protein values increased, when the protein content of the diet increased from diets 2 to 6,3 to 7 and 4 to 8, while an opposite trend was observed between diets 1 and 5. The differences observed in these parameters between diets 2 and 6 were not significant. The lipid content of the plasma did not change significantly between each pair of diets. The values of all of the blood parameters of the fish fed the commercial feed were among the highest observed. All of the haematological characteristics were within the range of the normal values reported in the literature (McCarthy et al., 1973,1975; Hille, 1982). DISCUSSION
The protein concentrations used in the present study cover the range of optimal values for rainbow trout growth reported by Zeitoun (1976) for fish of similar size. The change in the growth characteristics between the groups of fish fed the respective pairs of diets was not, however, uniform indicating a remarkable contribution of the protein sources used to the results obtained. The growth of the fish was positively affected by an increase in the protein content from diets 1 to 5 and diets 4 to 8. Diet 4 was found to give the best results in terms of growth rate, feed conversion, % protein retention and flesh quality when fed to fish having an initial average weight of 20 g (Alexis et al., 1985). Similar results were also obtained in the present study with fish having an initial average weight of 1 g. The slight improvement in growth observed at the highest protein level was accompanied by an increased use of the dietary
83
protein for body fat accumulation since the % protein retention decreased and the lipid content of the fish increased. The differences in growth observed between the groups fed diets 1 and 5 were of higher magnitude, indicating a different response of the fish to this pair of diets. The different response observed could be the result of different digestibilities of the mixtures used. If the digestible protein content of diets 1 and 5 was lower than 40 and 50% of the diet respectively, then the changes in growth would include a range outside the protein optima, where growth responses are more sensitive to protein increases in the diet (Zeitoun et al,, 19761, The difference in growth between diets I and 5 might, however, have been amplified by the factor causing the lens cataracts in the low protein group. The reduced growth of the fish fed diets 6 and 7, compared with those fed diets 2 and 3, despite their higher protein content, appeared to be mainly the result of the increased CSGM content in these diets since fish growth rates and CSGM content of the diets correlated negatively. Condensed tannins are the main antigrowth compounds reported for carobs (Nachtomi and Alumot, 1963). In vitro studies with carob tannins indicated that these are non-competitive inhibitors of many digestive enzymes (Tamir and Alumot, 1969). CSGM has been found to contain about 0.4% tannins (Drouliscos and Malfaki, 1980)) an amount which is much lower than that contained in whole carobs, The susceptibility of rainbow trout digestive enzymes to inhibition by tannins has not so far been studied. Studies on trypsin inhibitors, however, have shown that trout enzymes are much more susceptible to inhibition than those of many other animals tested (Krogdahl and Holm, 1983). The same could also occur with inhibition by tannins. Therefore, increasing the level of CSGM in diets 6 and 7 might have reduced considerably the active enzyme levels in the rainbow trout digestive system, resulting in an overall poorer utilization of the feed. The negative effect of the CSGM content of the diet on fish growth was apparent when its level exceeded 25% of the diet, while levels lower than this did not appear to reduce markedly the performance of the fish. Fish fed diets with a lower nutritional value appear to have a higher visceral. weight and, therefore, less useful flesh, as indicated by the positive correlation found between the CSGM content of the diet and visceral weight. The contribution of the final fish weight to the differences in the visceral weights observed is expected to be small since a change from about 16 to 13% of the total fish weight has been found in relation to fish size during the first 14 months of rainbow trout life (Denton and Yousef, 1976). Higher visceral weights did not appear to result from increased lipid accumulation since no correlation between these two parameters could be found. Visceral weight has been found to correlate positively with the lipid content of the diet f Lee and Putnam, 1973 1.
The visceral weight of the fish fed the commercial diet containing a low level of lipid was lower than that of all of the other experimental groups. The whole body composition of fish fed the commercial diet was also significantly differ-
ent from that of all of the other experimental groups (Table 3 ) . The principal difference in the total body lipids appeared to result from the much smaller contribution of the visceral lipids of the fish fed the commercial feed to the total body composition, while differences in the lipid content of the lean body were of a smaller magnitude (Table 5). A considerable increase in the visceral lipids, and a smaller increase in the muscle lipids, with increasing lipid content of the diet was also found by Watanabe et al. (1979) for rainbow trout of the same initial average weight. The negative correlation found between the CSGM content of the diet and the haematocrit and haemoglobin levels indicates that the haema~logi~al characteristics are affected by diet quality. Increased haematoerit values with diets prompting faster growth have also been reported in the literature (Barnhart, 1969). The positive correlation observed between the CSGM content of the diet and the hepatosomatic index cannot be explained in terms of lower available protein with increasing CSGM content of the diets since the hepatosomatic index and the protein content of the diet were found to correlate positively (Lee and Putnam, 1973). The correlation observed might result either from glycogen accumulation created by a high digestibility of CSGM carbohydrates or from some other factor contained in CSGM that contributes to an enlargement of the liver. The final cost of the fish produced is determined by both the feed cost and the feed conversion. The marginal differences in feed conversion between diets 4 and 8 resulted in a slightly higher cost/kg fish produced (Table 3) with the high protein diet since the protein source used (D) was one of the most expensive (Table 1). To the contrary, the much improved feed conversion of diet 5 makes it preferable to its lower protein counterpart. The adverse effects of increased CSGM inclusion in the diet on fish growth resulted in a higher production cost of fish fed diets 6 and 7 containing a high protein content, compared with their respective lower protein diets, since both feed price and feed conversion increased. The nutritional characteristics of the by-products used as protein sources for form~ating practical diets should, therefore, be carefully determined and taken into account when selecting an economic protein level, so that increased levels of antigrowth factors and adverse effects on growth and final cost of production are avoided. Information regarding the nutritional potential of many plant by-products of high protein content is, however, limited, a factor which restricts their use as ingredients in fish diets and, therefore, their maximum exploitation as cheap protein sources. ACKNOWLEDGEMENTS
We wish to thank Mr M. Filioglou and Miss M. Galani for their valuable assistance with the samplings and Mr G. Nanos for the care and feeding of the
85
fish. Thanks are also due to Miss E. Valla and Mrs E. Papoutsi for their careful laboratory assistance.
REFERENCES Alexis, M.N., Papaparaskeva-Papoutsoglou, E. and Theochari, V., 1985. Formulation of practical diets for rainbow trout (Salmo guirdneri) made by partial or complete substitution of fish meal by poultry by-products and certain plant by-products. Aquaculture, 50: 61-73. Barnhart, R.A., 1969. Effects of certain variables on hema~lo~c~ characteristics of rainbow trout. Trans. Am. Fish. Sot., 98: 411-418. Cho, C.Y., Slinger, S.J. and Bayley, H.S., 1976. Influence of level and type of dietary protein, and of level of feeding on feed utilization by rainbow trout. J. Nutr., 106: 1547-1556. Cowey, C.B. and Sargent, J.R., 1979. Nutrition, In: W.S. Hoar, D.J. Randall and J.R. Brett (Editors), Fish Physiology. Vol. 8: Bioenergetics and Growth. Academic Press, New York, pp. 58-69. Denton, J.E. and Yousef, M.K., 1976. Body composition and organ weights of rainbow trout, Salmo gairdneri. J. Fish Biol., 8: 489-499. Drouliscos, N.J. and Malefaki, V., 1980. Nutritionaf evaluation of the germ meal and its protein isolate obtained from the carob seed (Ceratonia &qua) in the rat. Br. J. Nutr., 43: 115-123. Hille, S., 1982. A literature review of the blood chemistry of rainbow trout, Salmogatrdneri Rich. J. Fish Biol., 20: 535-569. Krogdahl, A. and Holm, H., 1983. Pancreatic proteinases from man, trout, rat, pig, cow, chicken, mink and fox. Enzyme activities and inhibition by soybean and lima bean proteinase inhibitors. Comp. Biochem. Physiol., 74B: 403-409. Lee, D.J. and Putnam, G.B., 1973. The response of rainbow trout to varyingprotein/energy ratios in a test diet. J. Nutr., 103: 916-922. McCarthy, D.H., Stevenson, J.P. and Roberts, MS., 1973. Some blood parameters of the rainbow trout (Salmo gairdneri Richardson), I. The Kamloops variety. J. Fish Biol.,.5: l-8. McCarthy, D.H., Stevenson, J.P. and Roberts, MS., 1975. Some blood parameters of the rainbow trout (Salmo gu~rd~ri Richardson). II. The Shasta variety. J. Fish Biol., 7: 215219. Nachtomi, E. and Alumot, E., 1963. Tannins and polyphenols in carob pods (Ceratonia siliqua) . J. Sci. Food Agric., 14: 464-468. Satia, B.P., 1974. Quantitative protein requirementsof rainbow trout. Prog. Fish. Cult., 36: 80-85. Steffens, W., 1981. Protein utilization by rainbow trout (Salmo gairdneri) and carp (Cyprinus carpio) : A brief review. Aquaculture, 23: 337-345. Tamir, M. and Alumot, E., 1969. Inhibition of digestive enzymes by condensed tannins from green and ripe carobs. J. Sci. Food Agric., 20: 199-202. Watanabe, I‘., Takeuchi, T. and Ogino, C.H., 1979. Studies on the sparing effect of lipids on dietary protein in rainbow trout (Salmo gairdneri) . In: J.E. Halver and K. Tiews (Editors), Finfish Nutrition and Fishfeed Technology. Vol. I. Heeneman, Berlin, pp. 113-125. Zeitoun, I.H., Ullrey, D.E., Magee, W.T., Gill, J.L. and Bergen, W.G., 1976. Quantifying nutrient requirements of fish. J. Fish. Res. Board Can., 33: 167-172.