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Nutritional value of different dietary proteins to juvenile lobsters, Homarus americanus
Aquaculture, 22 (1981) 343-351 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
343
NUTRITIONAL VALUE OF DIFFERENT DIETARY PROTEINS TO JUVENILE LOBSTERS, HOMARUS AMERICANUS
ANDREW D. BOGHEN and JOHN D. CASTELL* Department
of Biology, Uniuersit8 de Moncton, Moncton,
N.B. ElA 3E9 (Canada)
*Department of Fisheries and Oceans Fisheries and Marine Service, P.O. Box 550, Halifax, N.S. B3J 2S7 (Canada) (Accepted
19 May 1980)
ABSTRACT Boghen, A.D. and Castell, J.D., 1981. Nutritional value of different dietary proteins to ,juvenile lobsters, Homarus americanus. Aquaculture, 22: 343-351. A comparative study of the nutritional value of four proteins (casein, egg, feather and shrimp) was conducted using test diets for juvenile lobsters. Growth and survival patterns were similar for each of two groups of lobsters held at 15” C and 20” C. In general, the shrimp protein diet produced significantly better lobster growth and survival. While the feather based diet also resulted in relatively good survival, it caused the poorest growth. Amino acid analysis revealed that the shrimp protein contained higher levels of histidine than did the egg or feather proteins and higher concentrations of methionine than did the feather protein. The superiority of the shrimp-based diet, especially in relation to the casein diet, may be a result of factors other than the amino acid composition of the protein.
INTRODUCTION
There are two general approaches to developing nutritionally adequate diets for feeding any cultured animal. One can either employ empirically formulated diets based on the application of crude raw materials, or conduct carefully controlled nutrition research using purified or semipurified test diets. Attempts to develop culture diets for lobsters, Homarus americanus (Conklin et al., 1975,1977; Gallagher et al., 1976) and for other crustaceans (Myers and Zein-Eldin, 1972) have largely involved the first approach. The second approach, although requiring much more research time,, has proven to be of greater value for culture of the domesticated animals and more recently in the field of crustacean culture. Information, for example, on the lipid levels and types required by juvenile and adult lobsters is now available (Caste11and Covey, 1976). Caste11and Budson (1974) have shown that adult lobsters displayed a high 0044-8486/81/0000-0000/$02.50
0 1981 Elsevier Scientific Publishing Company
344
protein requirement (40-60% of the total diet) when casein was used as the only dietary protein. Similar high protein requirements were suggested for certain species of prawn (Deshimaru and Kuroki, 1974,1975; Colvin, 1976). Moreover, the optimal protein level (54% casein and 6% egg protein) reported for the prawn Penaeus japonicus (Deshimaru and Kuroki, 1974) was almost the same as that suggested for lobster. While casein is widely used as a protein source in test diets in animal nutrition studies, some supplementation with limiting essential amino acids is often necessary (Halver, 1957; Frost, 1959). More recently casein has proven to be limiting in essential amino acids for lobsters (Mason and Castell, 1980). In an attempt to find a protein that more closely satisfies all of the essential amino acid requirements of juvenile lobsters, we assessed the effects of several semi-purified test proteins on juvenile lobsters. MATERIALS
AND METHODS
Lobster larvae were hatched from two egg-bearing females and raised to 4th and 5th stage juveniles at 20°C using the method previously described by Caste11(1977). Juveniles were randomly distributed among four diet groups in each of two identical closed rearing systems and acclimated to 15” C and 20°C respectively. Water quality was closely monitored and maintained stable for both rearing units (Boghen and Castell, 1979). Concurrent studies were conducted on members of the two families, each family originating from one of the two females mentioned above. Diet groups were made up of 19-20 juveniles from family 1 and 9-12 lobsters from family 2. The latter were introduced into the culture basins.a week later and were, on the average, slightly smaller i.e. mostly fourth stage. Diets were prepared and contained all the ingredients previously described by Caste11and Budson (1974). Only casein was replaced by other proteins in diets 2-4 (Table I). Each of the test diets containing casein, egg, feather or shrimp were fed to juvenile lobsters ad libitum daily at 9:00 and 16:30 for lo-12 weeks. Uneaten food was removed prior to feeding time. Every 2 weeks, the lobsters were dried lightly on filter paper and weighed in plastic weighing boats on a Mettler balance. The percentages of moisture and ash content of the dietary proteins were derived by drying samples for 24 h at 104°C to constant weight and subsequently by determining the weight of the residue remaining after burning at 500°C respectively (Table I). The semi-purified proteins and the gelatin fraction of the diets (see Table I) and the bodies of juvenile lobsters were hydrolyzed by the method of Matsubara and Sasaki (1969). Separation and isolation of amino acid (Table IV) hydrolysates was achieved by a modification of the method of Moore and Stein (1963). A one column (0.15 X 30 cm) analyser packed with
345 TABLE I Summary of diet formulation and percent ash, moisture and purity of proteins used in this study Dieta
Proteinb
% Ash
1 2 3 4 -
Casein Whole egg Feather Shrimp Gelatin
0.99 2.55 2.28 17.27 2.3
+ + f + c
0.6 0.3 0.1 0.1 0.1
% Moisture
% Protein purity
9.88 7.39 8.45 6.96 10.25
91.5 93.2 71.9 43.1 86.5
f 0.1 f 0.3 + 0.2 f. 0.1 ?: 0.3
aProteins included in diet at 50% of dry weight. Other ingredients used as reported by Caste11 and Budson (1974): Corn starch 5%, Minerals 3% (Bernhart-Tormarelli salt mix), Cellulose l&8%, Gelatin lo%, Vitamins 2%, Alpha tocopherol 0.2%, Cholesterol l%, Cod liver oil 9%, Choline chloride 1%. bAll dietary ingredients obtained from ICN, Nutritional Biochemicals Corp., Cleveland, OH except feather protein (Can. Col. Mar. Ltd., Winnipeg, Man,) and shrimp protein prepared from Penaeus aztecus (Hercules Inc., Wilmington, DE).
Durrum DC4A cation exchange resin was used. Protein purity data for the test proteins was provided by the various suppliers. Statistical analysis of the weight and growth data was conducted using analysis of variance with Duncan’s test for significant differences at the 5% level (Steel and Torrie, 1960). Survival differences between the lobsters on the shrimp diet and those on the other diets were determined by using the Chi-square test for significant differences at the 1% level. RESULTS
The apparent superiority of shrimp protein is supported by the weight gain and survival data (Table II and Fig. 1). For family 1, weight gain ranged from 42.3 mg for lobsters on the feather protein diet and maintained at 15°C to 204.6 mg for lobsters receiving shrimp protein and maintained at 20°C. Analysis of variance with significant differences at the 5% level (PG 0.05) indicated that shrimp resulted in a significantly higher weight gain than did casein-fed lobsters maintained at 15” C and lobsters fed the feather protein and maintained at 15°C and 20°C (Table II). Despite the fact that there was no significant difference in mean weight gain between lobsters fed on shrimp and those receiving whole egg (15” C and 20” C) or casein (20” C), the significantly lower survival rates (P < 0.01) of animals in the latter diet groups (Fig. la and lc) attest to their inferiority. Similar results are observed for lobsters belonging to family 2 (Table II). Mean weight gain ranged from 12.5 mg for lobsters fed feather protein (15°C) to 125.8 mg for lobsters receiving shrimp and maintained at 20°C. Irrespective of temperature, feather protein always produced significantly
346 TABLE
II
Weight gain data of lobsters formulated diets containing Diet No.
belonging to families 1 and 2 maintained at 15’C and 20°C and receiving either semi-purifed casein, whole egg, feather or shrimp Protein
Protein
Temperature (“C)
Avg. initial weight (mg) Family 1
Mean weighta gain (mg) Family 1
Avg. initial weight (mg) Family 2
Mean weighta gain (mg) Family 2
Casein
15 20
50.8 * 9.2 46.4 f 5.3
108.0 164.3
f 43.3BC ?: 34.SAB
34.0 29.1
i: 16.0 f 10.0
-
Whole egg
15 20
46.6 49.6
132.0 128.0
+ 51.5ABC f 17.0ABC
31.0 29.9
+ 6.0 ?: 5.0
Feather
15 20
48.4 + 8.1 44.3 f 7.0
Shrimp
15 20
44.3 47.1
aDifferent superscripts as determined through interpretation.
f 9.0 f 10.0
42.3 * 25.4D 58.4 i 29.7CD
* 4.4 f 7.2
184.8 204.6
f 25.2BC
72.0 * ll.BABCD 12.5 f 6.tiD 42.3 f 29.0DC
28.0 f 5.2 $32.9 f 13.1
+ 41.6A f 10.IA
66.5
34.2 f 7.3 30.3 * 5.8
107.0 125.8
* 44.1AB + 43.SA
indicate significant differences in mean weight gain (P Q 0.05) between an analysis of variance. A dash indicates too few samples for statistical
diet groups
,s.
25
0
a 2
4
6
8
10
12
6 Weeks
egg--= 2
4
6
8
10
12
Weeks
Weeks
4
0
8
10
12 Weeks
Fig. 1. Percent survival of lobsters in families 1 and 2 maintained at 15°C (Fig. la and lb respectively) and 20” C (Fig. lc and Id respectively) and receiving semi-purified casein, egg, feather or shrimp diets. Arrows indkate significant differences (P < 0.01) in survival between lobsters fed either casein or whole egg and those receiving shrimp protein.
347
(P < 0.05) lower mean weight gain that did shrimp protein (Table II). No significant difference in mean weight gain could be shown between shrimp-fed lobsters and those animals receiving egg or casein at similar temperatures. A significant difference in survival (P G O.Ol), however, was noted at 1.5”C between shrimp and egg and at 20” C between shrimp and casein (Fig. lb and Id). In all instances, if survival and weight gain were incorporated into a common mathematical expression (Normalized Biomass Increase, mg/day, Conklin et al., 1975) the shrimp protein would prove to be superior. The amino acid composition has been tabulated for each of the proteins and all of our diets (Tables III and IV). A brief comparison of the amino acid content of the shrimp and that of the other proteins is considered in the following section.
TABLE III Amino acid composition (g/100 g sample)a Amino acid Ala Arg* ASP GYS GIY Glu His* 1so* Leu* Lys* Met* Phe* Pro Ser Tau Thr* TrP* Tyr VaI* NH, c
Juvenile lobsters 6.0 6.8 10.9 0.9 7.6 20.6 2.1 3.8 6.4 4.0 2.8 3.9 5.3 3.0 1.2 3.8 2.3 3.4 4.6 0.6 100
of casein, egg, feather, shrimp, gelatin and juvenile lobsters
Vit. free casein 2.4 3.0 6.7 0.4 1.5 23.1 2.0 4.2 7.7 6.0 2.7 378 7.2 5.0 3.6 1.4 4.7 4.8 90.2
Feather
-
3.0 4.8 4.7 5.0 4.6 7.5 0.1 4.0 6.7 1.1 0.6 3.2 6.7 7.1 3.3 0.9 2.2 4.0
69.5
Shrimp
Egg 5.8 5.8 7.0 /2.7 14.3 0.2 4.4 7.9 7.3 1.0 4.8 4.9 4.5 3.8 ’ 2.6 3.2 5.4 85.6
’
-
2.4 2.9 3.5 0.4 1.7 7.5 0.5 2.2 4.4 3.8 1.2 2.2 2.2 1.9 2.0 0.5 1.7 1.5
44.5
Gelatin
8.7 3.2 3.4 0.2 25.8 5.8 1.0 -
-
’ -
7.1 5.9 1.4 9.5 0.4
14.1 86.5
%Smail differences in total amino acid composition as presented above and overall purity of the test proteins (Table I) may be a result of a partial loss of certain bound proteins during processing.
348 TABLE IV Amino acid composition
of casein, egg, feather and shrimp protein diets?
Amino acid
Diet 1 Casein
Diet 2 Egg
Diet 3 Feather
Diet 4 Shrimp
Ala Arg ASP CYS GIY Glu His Len LYS Met Phe Pro Ser Tau
2.0 1.8 3.7 0.2 3.4 12.1 1.1 2.1 4.6 3.6 1.4 2.0 4.6 2.5 -
3.8 3.2 3.8 4.0 7.8 0.2” 2.2 4.7 4.3 0.5 2.5 3.9 2.3 -
2.4 2.1 2.7 2.5 4.9 4.4 0.2” 2.0 4.0 1.2 0.3* 1.7 4.4 3.6 -
2.1 1.8 2.1 0.2 3.5 4.4 0.4 1.1 2.9 2.5 0.6 1.2 2.1 1.0 -
Thr Trp Tyr Val
1.8 0.7 2.4 3.8
1.9 1.3 1.6 4.1
1.7 0.4 1.1 3.4
1.0 0.2 0.9 2.2
IS0
-
aThe data reflects the adjusted amino acid values from Table III for each diet based on protein purity as presented in Table I and the combination of gelatin (10%) and the test protein (50%) (g/lOOg dry feed). DISCUSSION
Due to the variation in protein purity (Table I), it cannot be stated with certainty that the observed differences in growth and survival for lobster in the four diet groups resulted from the differing protein compositions. If, however, we assume that the shrimp protein is “balanced” in essential amino acids, then the optimal protein concentration of an effective artificial diet may be considerably less than that which was previously believed necessary (Caste11and Budson, 1974; Deshimaru and Kuroki, 1974). A number of other feeding studies have been conducted in which crustaceans represented a major dietary component. Colvin (1976) showed that prawn meal was superior to fish meal in the diet of the prawn Penaeus in&us. Sick et al. (1972) fed several rations of fish meal and shrimp meal to Penaeid shrimp Penaeus setiferus and Penaeus aztecus. The formulated feed with the highest levels of shrimp meal resulted in the best growth and survival. Forster and Beard (1973) also found that shrimp meal was the best of several protein-rich food meals tested in feeds for Palaemon serratus. Thus it appears that crustaceans provide the best protein combinations for feedin,g to other carnivorous crustaceans and in this respect our findings are consistent with those of other workers. The above conclusion must however be qualified by indicating that “shrimp meals” consist of more than just one
349
type of protein while in the current study the P. aztecus protein is an alkali derivative of the chitin/chitosan process and as a result constitutes the membrane protein from the shrimp shell and exoskeleton only. Several radio-tracer studies with crustaceans have suggested that arginine, methionine, valine, threonine, isoleucine, leucine, lysine histidine, phenylalanine and tryptophan are amino acids which are not synthesized de novo and can thus be assumed to be essential (Cowey and Forster, 1971; Shewbart et al., 1972; Gallagher and Brown, 1975; Miyajima et al., 1976; Lasser and Allen, 1976). Despite its relatively low level of purity (43.1%) the shrimp protein contained higher levels of certain essential amino acids than did the feather or egg proteins. For example, shrimp contained five times and twoand-a-half times the amounts of histidine found in feather and egg respectively (Table IV). Even after adjusting the figures to take into consideration the relative amino acid contributions provided by the gelatin in each diet (Table IV), the histidine content in the shrimp protein was two times greater than in egg and feather. In a similar manner, the methionine concentration in the shrimp diet was twice that of the feather diet. In a feeding study with lobsters, Mason and Caste11(1980) were able to demonstrate a growth promoting effect in supplementing casein with several essential amino acids including glycine and threonine. In our study the total glycine concentration of the shrimp protein diet exceeded that of the casein diet by only 3% and threonine was more abundant in the casein than in the shrimp diet (Table IV). It is therefore quite likely that factors other than just the amino acid composition (e.g. mineral content, digestibility, etc.) play a significant role in the superiority of the shrimp protein diet as compared to the casein, egg and feather meal protein diets. This likelihood is furthermore enhanced by the relatively low purity level and high ash content of the shrimp protein. Preliminary studies in our laboratory on the importance of mineral content for work in lobster nutrition, lend credence to this idea. ACKNOWLEDGEMENTS
We appreciate the assistance provided by Mr. D. Trider in the preparation of diets for this study. This work is, in part, a result of research financed by the National Research Council of Canada (A9366) and the Research Council, Universite de Moncton (CRU79) through grants awarded to Dr. A. Boghen.
REFERENCES Boghen, A.’ and Castell, J., 1979. A recirculating system for smallscale experimental work on juvenile lobsters Homarus americanus. Aquaculture, 18: 383-387. Castell, J.D., 1977. Productjon of juvenile lobsters Homarus americanus for nutrition research. Actes Colloq. CNEXO, 4: 277-281.
Castell, J.D. and Budson, S.D., 1974. Lobster nutrition: The effects on Homarus americanus of dietary protein levels. J. Fish. Res. Board Can., 32: 1363-1370. C&tell, J.D. and Covey, J.F., 1976. Dietary lipid requirements of adult lobsters Homarus americanus M.E.). J. Nutr., 106: 1159-1165. .Castell, J.D., Mauviot, J.C. and Covey, J.F., 1976. The use of eyestalk ablation in nutrition studies with American lobsters (Homarus americanus). Proc. World Maricult. Sot. 7th Annu. Workshop, pp. 431-443. Colvin, P.M., 1976. Nutritional studies on penaeid prawns: Protein requirements in compounded diets for juvenile Penaeus indicus (Mime Edwards). Aquaculture, 7: 315-326. Conklin, D.E., Devers, K. and Shleser, R.A., 1975. Initial development of artificial diets for the lobster, Homarus americanus. Proc. World Maricult. Sot. 6th Annu. Workshop, pp. 237-248. Conklin, D.E., Devers, K. and Bordner, C., 1977. Development of artificial diets for the lobster Homarus americanus. Proc, World Maricult. Sot. 8th Annu. Workshop, pp. 841-852. Cowey, C.B. and Forster, J.R.M., 1971. The essential amino acid‘requirements of the prawn (Palaemon serratus). The growth of prawns on diets containing proteins of different amino acid compositions. Mar. Biol., 10: 77-81. Deshimaru, 0. and Kuroki, K., 1974, Studies on a purified diet for prawn: I. Basal composition of diet. Bull. Jpn. Sot. Sci. Fish., 40: 413-419. Deshimaru, 0. and Kuroki, K., 1975. Studies on a purified diet for prawn -IV. Evaluation of protein, free amino acids and their mixture as nitrogen source. Bull. Jpn. Sot. Sci. Fish., 41: 101-103. Forster, J.R.M. and Beard, T.W., 1973. Growth experiments with prawn Palaemon serratus fed with fresh and compounded foods. Fish. Invest. Minist. Agric. Fish. Food G.B. Ser. II Salmon Freshwater Fish., 27 (7), 16 pp. Frost, D.V., 1959. Measuring the nutritional value of proteins. In: A.A. Albanese (Editor) Protein and Amino Acid Nutrition. Academic Press, New York and London, pp. 225-279. Gallagher, M.L. and Brown, W.D., 1975. Amino acid requirements of the lobster, (Homarus americanus). Fed. Proc. Fed. Am. Sot. Exp. Biol., 34: 880 (abstract). Gallagher, M.L., Conklin, D.E. and Brown, W.D., 1976. The effects of pelletized protein diets on growth, molting and survival of juvenile lobsters. Proc. World Maricult. Sot. 7th Annu. Workshop, pp. 363-369. Halver, J.E., 1957. Nutrition of salmonid fishes. III. Water-soluble vitamin requirements of chinook salmon. J. Nutr., 62: 225-243. Lasser, G.W. and Allen, W.A., 1976. The essential amino acid requirements of the Dungeness crab, Cancer magister. Aquaculture, 7: 235-244. Mason, E.G. and Castell, J.D., 1980. Essential amino acid studies with juvenile lobsters, Homarus americanus, using purified amino acid test diets. Proc. World Maricult. Sot. 11th Annu. Workshop. Matsubara, H. and Sasaki, R.M., 1969. High recovery of tryptophan from acid hydrolysis of proteins. Biochem. Biophys. Res. Commun., 35(2): 175-181. Miyajima, L.S., Broderick, G.A. and Reimer, R.D., 1976. Identification of the essential amino acids of the freshwater shrimp, Macrobrachium ohione. Proc. World Maricult. Sot. 7th Annu. Workshop, pp. 699-704. Moore, S. and Stein, W.H., 1963. Chromatographic determination of amino acids by the use of automatic recording equipment. In: S.P. Colowick and H.O. Kaplin (Editors), Methods in Enzymology. Vol. VI. Academic Press, New York, pp. 819-831. Myers, S.P. and Zein-Eldin, Z.P., 1972. Binders and pellet stability in development of crustacean diets. Proc. World Maricult. Sot. 3rd Annu. Workshop, pp. 351-364. Shewbart, K.L., Mies, W.L. and Ludwig, P.D., 1972. Identification and quantitative analysis of the amino acids present in protein of the brown shrimp Penaeus aztecus. Mar. Biol., 16: 64-67.
351 Sick, L.V., Andrews, J.W. and White, D.B., 1972. Preliminary studies of selected environmental and nutritional requirements for the culture of penaeid shrimp. Fish. Bull., 70(l): 101-109. Steel, R.G.D. and Torrie, J.H., 1960. Principles and Procedures of Statistics. McGraw-Hill, New York, p. 481.
343
NUTRITIONAL VALUE OF DIFFERENT DIETARY PROTEINS TO JUVENILE LOBSTERS, HOMARUS AMERICANUS
ANDREW D. BOGHEN and JOHN D. CASTELL* Department
of Biology, Uniuersit8 de Moncton, Moncton,
N.B. ElA 3E9 (Canada)
*Department of Fisheries and Oceans Fisheries and Marine Service, P.O. Box 550, Halifax, N.S. B3J 2S7 (Canada) (Accepted
19 May 1980)
ABSTRACT Boghen, A.D. and Castell, J.D., 1981. Nutritional value of different dietary proteins to ,juvenile lobsters, Homarus americanus. Aquaculture, 22: 343-351. A comparative study of the nutritional value of four proteins (casein, egg, feather and shrimp) was conducted using test diets for juvenile lobsters. Growth and survival patterns were similar for each of two groups of lobsters held at 15” C and 20” C. In general, the shrimp protein diet produced significantly better lobster growth and survival. While the feather based diet also resulted in relatively good survival, it caused the poorest growth. Amino acid analysis revealed that the shrimp protein contained higher levels of histidine than did the egg or feather proteins and higher concentrations of methionine than did the feather protein. The superiority of the shrimp-based diet, especially in relation to the casein diet, may be a result of factors other than the amino acid composition of the protein.
INTRODUCTION
There are two general approaches to developing nutritionally adequate diets for feeding any cultured animal. One can either employ empirically formulated diets based on the application of crude raw materials, or conduct carefully controlled nutrition research using purified or semipurified test diets. Attempts to develop culture diets for lobsters, Homarus americanus (Conklin et al., 1975,1977; Gallagher et al., 1976) and for other crustaceans (Myers and Zein-Eldin, 1972) have largely involved the first approach. The second approach, although requiring much more research time,, has proven to be of greater value for culture of the domesticated animals and more recently in the field of crustacean culture. Information, for example, on the lipid levels and types required by juvenile and adult lobsters is now available (Caste11and Covey, 1976). Caste11and Budson (1974) have shown that adult lobsters displayed a high 0044-8486/81/0000-0000/$02.50
0 1981 Elsevier Scientific Publishing Company
344
protein requirement (40-60% of the total diet) when casein was used as the only dietary protein. Similar high protein requirements were suggested for certain species of prawn (Deshimaru and Kuroki, 1974,1975; Colvin, 1976). Moreover, the optimal protein level (54% casein and 6% egg protein) reported for the prawn Penaeus japonicus (Deshimaru and Kuroki, 1974) was almost the same as that suggested for lobster. While casein is widely used as a protein source in test diets in animal nutrition studies, some supplementation with limiting essential amino acids is often necessary (Halver, 1957; Frost, 1959). More recently casein has proven to be limiting in essential amino acids for lobsters (Mason and Castell, 1980). In an attempt to find a protein that more closely satisfies all of the essential amino acid requirements of juvenile lobsters, we assessed the effects of several semi-purified test proteins on juvenile lobsters. MATERIALS
AND METHODS
Lobster larvae were hatched from two egg-bearing females and raised to 4th and 5th stage juveniles at 20°C using the method previously described by Caste11(1977). Juveniles were randomly distributed among four diet groups in each of two identical closed rearing systems and acclimated to 15” C and 20°C respectively. Water quality was closely monitored and maintained stable for both rearing units (Boghen and Castell, 1979). Concurrent studies were conducted on members of the two families, each family originating from one of the two females mentioned above. Diet groups were made up of 19-20 juveniles from family 1 and 9-12 lobsters from family 2. The latter were introduced into the culture basins.a week later and were, on the average, slightly smaller i.e. mostly fourth stage. Diets were prepared and contained all the ingredients previously described by Caste11and Budson (1974). Only casein was replaced by other proteins in diets 2-4 (Table I). Each of the test diets containing casein, egg, feather or shrimp were fed to juvenile lobsters ad libitum daily at 9:00 and 16:30 for lo-12 weeks. Uneaten food was removed prior to feeding time. Every 2 weeks, the lobsters were dried lightly on filter paper and weighed in plastic weighing boats on a Mettler balance. The percentages of moisture and ash content of the dietary proteins were derived by drying samples for 24 h at 104°C to constant weight and subsequently by determining the weight of the residue remaining after burning at 500°C respectively (Table I). The semi-purified proteins and the gelatin fraction of the diets (see Table I) and the bodies of juvenile lobsters were hydrolyzed by the method of Matsubara and Sasaki (1969). Separation and isolation of amino acid (Table IV) hydrolysates was achieved by a modification of the method of Moore and Stein (1963). A one column (0.15 X 30 cm) analyser packed with
345 TABLE I Summary of diet formulation and percent ash, moisture and purity of proteins used in this study Dieta
Proteinb
% Ash
1 2 3 4 -
Casein Whole egg Feather Shrimp Gelatin
0.99 2.55 2.28 17.27 2.3
+ + f + c
0.6 0.3 0.1 0.1 0.1
% Moisture
% Protein purity
9.88 7.39 8.45 6.96 10.25
91.5 93.2 71.9 43.1 86.5
f 0.1 f 0.3 + 0.2 f. 0.1 ?: 0.3
aProteins included in diet at 50% of dry weight. Other ingredients used as reported by Caste11 and Budson (1974): Corn starch 5%, Minerals 3% (Bernhart-Tormarelli salt mix), Cellulose l&8%, Gelatin lo%, Vitamins 2%, Alpha tocopherol 0.2%, Cholesterol l%, Cod liver oil 9%, Choline chloride 1%. bAll dietary ingredients obtained from ICN, Nutritional Biochemicals Corp., Cleveland, OH except feather protein (Can. Col. Mar. Ltd., Winnipeg, Man,) and shrimp protein prepared from Penaeus aztecus (Hercules Inc., Wilmington, DE).
Durrum DC4A cation exchange resin was used. Protein purity data for the test proteins was provided by the various suppliers. Statistical analysis of the weight and growth data was conducted using analysis of variance with Duncan’s test for significant differences at the 5% level (Steel and Torrie, 1960). Survival differences between the lobsters on the shrimp diet and those on the other diets were determined by using the Chi-square test for significant differences at the 1% level. RESULTS
The apparent superiority of shrimp protein is supported by the weight gain and survival data (Table II and Fig. 1). For family 1, weight gain ranged from 42.3 mg for lobsters on the feather protein diet and maintained at 15°C to 204.6 mg for lobsters receiving shrimp protein and maintained at 20°C. Analysis of variance with significant differences at the 5% level (PG 0.05) indicated that shrimp resulted in a significantly higher weight gain than did casein-fed lobsters maintained at 15” C and lobsters fed the feather protein and maintained at 15°C and 20°C (Table II). Despite the fact that there was no significant difference in mean weight gain between lobsters fed on shrimp and those receiving whole egg (15” C and 20” C) or casein (20” C), the significantly lower survival rates (P < 0.01) of animals in the latter diet groups (Fig. la and lc) attest to their inferiority. Similar results are observed for lobsters belonging to family 2 (Table II). Mean weight gain ranged from 12.5 mg for lobsters fed feather protein (15°C) to 125.8 mg for lobsters receiving shrimp and maintained at 20°C. Irrespective of temperature, feather protein always produced significantly
346 TABLE
II
Weight gain data of lobsters formulated diets containing Diet No.
belonging to families 1 and 2 maintained at 15’C and 20°C and receiving either semi-purifed casein, whole egg, feather or shrimp Protein
Protein
Temperature (“C)
Avg. initial weight (mg) Family 1
Mean weighta gain (mg) Family 1
Avg. initial weight (mg) Family 2
Mean weighta gain (mg) Family 2
Casein
15 20
50.8 * 9.2 46.4 f 5.3
108.0 164.3
f 43.3BC ?: 34.SAB
34.0 29.1
i: 16.0 f 10.0
-
Whole egg
15 20
46.6 49.6
132.0 128.0
+ 51.5ABC f 17.0ABC
31.0 29.9
+ 6.0 ?: 5.0
Feather
15 20
48.4 + 8.1 44.3 f 7.0
Shrimp
15 20
44.3 47.1
aDifferent superscripts as determined through interpretation.
f 9.0 f 10.0
42.3 * 25.4D 58.4 i 29.7CD
* 4.4 f 7.2
184.8 204.6
f 25.2BC
72.0 * ll.BABCD 12.5 f 6.tiD 42.3 f 29.0DC
28.0 f 5.2 $32.9 f 13.1
+ 41.6A f 10.IA
66.5
34.2 f 7.3 30.3 * 5.8
107.0 125.8
* 44.1AB + 43.SA
indicate significant differences in mean weight gain (P Q 0.05) between an analysis of variance. A dash indicates too few samples for statistical
diet groups
,s.
25
0
a 2
4
6
8
10
12
6 Weeks
egg--= 2
4
6
8
10
12
Weeks
Weeks
4
0
8
10
12 Weeks
Fig. 1. Percent survival of lobsters in families 1 and 2 maintained at 15°C (Fig. la and lb respectively) and 20” C (Fig. lc and Id respectively) and receiving semi-purified casein, egg, feather or shrimp diets. Arrows indkate significant differences (P < 0.01) in survival between lobsters fed either casein or whole egg and those receiving shrimp protein.
347
(P < 0.05) lower mean weight gain that did shrimp protein (Table II). No significant difference in mean weight gain could be shown between shrimp-fed lobsters and those animals receiving egg or casein at similar temperatures. A significant difference in survival (P G O.Ol), however, was noted at 1.5”C between shrimp and egg and at 20” C between shrimp and casein (Fig. lb and Id). In all instances, if survival and weight gain were incorporated into a common mathematical expression (Normalized Biomass Increase, mg/day, Conklin et al., 1975) the shrimp protein would prove to be superior. The amino acid composition has been tabulated for each of the proteins and all of our diets (Tables III and IV). A brief comparison of the amino acid content of the shrimp and that of the other proteins is considered in the following section.
TABLE III Amino acid composition (g/100 g sample)a Amino acid Ala Arg* ASP GYS GIY Glu His* 1so* Leu* Lys* Met* Phe* Pro Ser Tau Thr* TrP* Tyr VaI* NH, c
Juvenile lobsters 6.0 6.8 10.9 0.9 7.6 20.6 2.1 3.8 6.4 4.0 2.8 3.9 5.3 3.0 1.2 3.8 2.3 3.4 4.6 0.6 100
of casein, egg, feather, shrimp, gelatin and juvenile lobsters
Vit. free casein 2.4 3.0 6.7 0.4 1.5 23.1 2.0 4.2 7.7 6.0 2.7 378 7.2 5.0 3.6 1.4 4.7 4.8 90.2
Feather
-
3.0 4.8 4.7 5.0 4.6 7.5 0.1 4.0 6.7 1.1 0.6 3.2 6.7 7.1 3.3 0.9 2.2 4.0
69.5
Shrimp
Egg 5.8 5.8 7.0 /2.7 14.3 0.2 4.4 7.9 7.3 1.0 4.8 4.9 4.5 3.8 ’ 2.6 3.2 5.4 85.6
’
-
2.4 2.9 3.5 0.4 1.7 7.5 0.5 2.2 4.4 3.8 1.2 2.2 2.2 1.9 2.0 0.5 1.7 1.5
44.5
Gelatin
8.7 3.2 3.4 0.2 25.8 5.8 1.0 -
-
’ -
7.1 5.9 1.4 9.5 0.4
14.1 86.5
%Smail differences in total amino acid composition as presented above and overall purity of the test proteins (Table I) may be a result of a partial loss of certain bound proteins during processing.
348 TABLE IV Amino acid composition
of casein, egg, feather and shrimp protein diets?
Amino acid
Diet 1 Casein
Diet 2 Egg
Diet 3 Feather
Diet 4 Shrimp
Ala Arg ASP CYS GIY Glu His Len LYS Met Phe Pro Ser Tau
2.0 1.8 3.7 0.2 3.4 12.1 1.1 2.1 4.6 3.6 1.4 2.0 4.6 2.5 -
3.8 3.2 3.8 4.0 7.8 0.2” 2.2 4.7 4.3 0.5 2.5 3.9 2.3 -
2.4 2.1 2.7 2.5 4.9 4.4 0.2” 2.0 4.0 1.2 0.3* 1.7 4.4 3.6 -
2.1 1.8 2.1 0.2 3.5 4.4 0.4 1.1 2.9 2.5 0.6 1.2 2.1 1.0 -
Thr Trp Tyr Val
1.8 0.7 2.4 3.8
1.9 1.3 1.6 4.1
1.7 0.4 1.1 3.4
1.0 0.2 0.9 2.2
IS0
-
aThe data reflects the adjusted amino acid values from Table III for each diet based on protein purity as presented in Table I and the combination of gelatin (10%) and the test protein (50%) (g/lOOg dry feed). DISCUSSION
Due to the variation in protein purity (Table I), it cannot be stated with certainty that the observed differences in growth and survival for lobster in the four diet groups resulted from the differing protein compositions. If, however, we assume that the shrimp protein is “balanced” in essential amino acids, then the optimal protein concentration of an effective artificial diet may be considerably less than that which was previously believed necessary (Caste11and Budson, 1974; Deshimaru and Kuroki, 1974). A number of other feeding studies have been conducted in which crustaceans represented a major dietary component. Colvin (1976) showed that prawn meal was superior to fish meal in the diet of the prawn Penaeus in&us. Sick et al. (1972) fed several rations of fish meal and shrimp meal to Penaeid shrimp Penaeus setiferus and Penaeus aztecus. The formulated feed with the highest levels of shrimp meal resulted in the best growth and survival. Forster and Beard (1973) also found that shrimp meal was the best of several protein-rich food meals tested in feeds for Palaemon serratus. Thus it appears that crustaceans provide the best protein combinations for feedin,g to other carnivorous crustaceans and in this respect our findings are consistent with those of other workers. The above conclusion must however be qualified by indicating that “shrimp meals” consist of more than just one
349
type of protein while in the current study the P. aztecus protein is an alkali derivative of the chitin/chitosan process and as a result constitutes the membrane protein from the shrimp shell and exoskeleton only. Several radio-tracer studies with crustaceans have suggested that arginine, methionine, valine, threonine, isoleucine, leucine, lysine histidine, phenylalanine and tryptophan are amino acids which are not synthesized de novo and can thus be assumed to be essential (Cowey and Forster, 1971; Shewbart et al., 1972; Gallagher and Brown, 1975; Miyajima et al., 1976; Lasser and Allen, 1976). Despite its relatively low level of purity (43.1%) the shrimp protein contained higher levels of certain essential amino acids than did the feather or egg proteins. For example, shrimp contained five times and twoand-a-half times the amounts of histidine found in feather and egg respectively (Table IV). Even after adjusting the figures to take into consideration the relative amino acid contributions provided by the gelatin in each diet (Table IV), the histidine content in the shrimp protein was two times greater than in egg and feather. In a similar manner, the methionine concentration in the shrimp diet was twice that of the feather diet. In a feeding study with lobsters, Mason and Caste11(1980) were able to demonstrate a growth promoting effect in supplementing casein with several essential amino acids including glycine and threonine. In our study the total glycine concentration of the shrimp protein diet exceeded that of the casein diet by only 3% and threonine was more abundant in the casein than in the shrimp diet (Table IV). It is therefore quite likely that factors other than just the amino acid composition (e.g. mineral content, digestibility, etc.) play a significant role in the superiority of the shrimp protein diet as compared to the casein, egg and feather meal protein diets. This likelihood is furthermore enhanced by the relatively low purity level and high ash content of the shrimp protein. Preliminary studies in our laboratory on the importance of mineral content for work in lobster nutrition, lend credence to this idea. ACKNOWLEDGEMENTS
We appreciate the assistance provided by Mr. D. Trider in the preparation of diets for this study. This work is, in part, a result of research financed by the National Research Council of Canada (A9366) and the Research Council, Universite de Moncton (CRU79) through grants awarded to Dr. A. Boghen.
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