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Optimum dietary protein level and protein to energy ratio for growth of grouper (Epinephelus malabaricus)
Aquaculture Aquaculture 145 ( 1996) 259-266
Optimum dietary protein level and protein to energy ratio for growth of grouper ( E’inephelus malabaricud Shi-Yen Shiau *, Ching-Wan Lan Depwnnent
ofMarine Food Science, National Taiwan Ocean Universi~, Keelung, Taiwan Accepted 29 March 1996
Abstract A series of two experiments was conducted to study the optimum dietary protein level and protein to energy ratio of juvenile groupers. In Experiment 1, eight isoenergetic fish meal based semi-purified diets ranging from 0 to 56% protein in 8% increments were fed for 8 weeks to triplicate groups of grouper (initial weight: 9.22 f 0.11 g per fish). Weight gain of fish was proportional to the protein content of the diet up to an incorporation rate of 48%. Feed efficiency (FE) increased with increasing dietary protein level, and the diet with 48% protein yielded the lowest FE. Fish fed the 56% protein diet had the highest body protein and lipid contents, and the lowest body moisture content. ‘Ihe dietary protein level that yielded maximum growth was 50.2% based on a broken-line model estimation of weight gain. In Experiment 2, two dietary protein levels (50% and 44%) were used along with four energy levels at each protein level (305, 340, 375 and 410 kcal per 100 g diet). The experimental diets were fed for 8 weeks to triplicate groups of grouper (initial weight: 10.70 f 0.14 g per fish). Weight gain of fish was not significantly (P > 0.05) different at 50% dietary protein regardless of energy level. However, the 44% protein diet at energy levels of 340 and 375 kcal per 100 g improved weight gain of fish. Fish fed the 44% protein with 340 or 410 kcal per 100 g diet had higher body lipid content. These data suggest that when the energy level of the diet is maintained at 340-375 kcal per 100 g, the dietary protein level for juvenile grouper can be lowered from 50% to 44%. Keywords:
l
Epinephelus
spp.; Feeding and nutrition-fish;
Protein; Protein energy ratio; Grouper
Corresponding author.
CW48486/%/$15.00
Copyright 0 1996 Elsevier Science B.V. All rights reserved.
PII SOO44-8486(96)01324-S
S.-Y. Shim, C.-W. Lnn/Aquuculrure
145 (1996) 259-266
1. Introduction Groupers are a good candidate for intensive aquaculture because of their desirable taste, hardiness in a crowded environment and rapid growth. They are valued as one of the highest quality seafoods in many parts of the world (Chen and Tsai, 1994). In Taiwan, the importance of grouper culture has become more important because of the shortage of freshwater resources. Little is known about their nutritional requirements, especially the species Epinephelus malabaricars, which is one of the most important cultured groupers in Asia. Protein is the most expensive component in fish feed. Thus, knowledge of the protein requirement is important for the formulation of well-balanced and low cost feeds. A study using casein as the protein source has indicated an optimal protein level for E. malabarcius (Chen and Tsai, 1994). Currently, raw trash fish supplemented with compounded feed formulated mainly for eel is the main food source for E. malabaricus in Taiwan’s commercial production. The optimum dietary protein level of E. malabaricus fed a fish meal based diet is not known. Studies have shown that providing adequate energy from dietary lipid can minimize the use of protein as an energy source (Reinitz et al., 1978; Shiau and Huang, 1990; Takeuchi et al., 1992). However, excess energy may produce fatty fish, reduce feed consumption (reducing total protein intake) and reduce the utilization of other nutrients (Page and Andrews, 1973; Prather and Lovell, 1973; Takeda et al., 1975; Shiau and Huang, 1990). Therefore, it is important to obtain the proper protein to energy ratio in a diet for the most economical production of grouper. The objectives of this study were two-fold. Firstly, to assess the optimum dietary protein level of juvenile grouper fed fish meal based diets and, secondly, to determine the optimun protein to energy ratio when dietary protein level is optimum and suboptimum.
2. Materials and methods 2.1. Diet preparation Formulations and proximate analysis (AOAC, 1984) of the diets fed in Experiment 1 and Experiment 2 are given in Tables 1 and 2, respectively. White fish meal (blue whiting, imported from Norway), cod liver oil and corn oil, and corn starch were used as dietary protein, lipid and carbohydrate sources, respectively. In Experiment 1, the diets were formulated to give 8% increments of protein from 0 to 56%, for a total of eight diets in all. Diets were made isocaloric by adjusting the corn starch and cellulose content. In Experiment 2, two dietary protein levels, 50% and 44%, were used. For each protein level, diets were formulated with four energy values of 305, 340, 375 and 410 kcal per 100 g diet for a total of eight experimental diets. The values of used for calculating energy levels were 4.5 kcal g- ’ for protein, 3.49 kcal g- ’ for carbohydrate and 8.51 kcal gg ’ for lipid (Shiau and Huang, 1989). The diets were prepared and handled as previously described (Shiau et al., 1987).
S.-Y. Shiau. C.-W. Lan/Aquuculrure Table 1 Formulation
of proximate
composition
of experimental
261
145 (1996) 259-266
diets (% dry wt.) in Experiment
1
Diet code:
1
2
3
4
5
6
7
8
Ingredient Fish meal ’ Cod liver oil Corn oil Corn starch Vitamin mix b Mineral mix b Carboxymethylcellulose Cellulose Calculated protein level Gross energy (kcal per 100 g) ’
0 7 3 78.8 2.7 0.3 3 5.2 0 360
11.7 6.3 3 68.4 2.7 0.3 3 4.6 8 360
23.4 5.5 3 58.1 2.7 0.3 3 4.0 16 360
35.1 4.8 3 47.8 2.7 0.3 3 3.3 24 360
46.8 4.0 3 37.5 2.7 0.3 3 2.7 32 360
58.5 3.3 3 27.2 2.7 0.3 3 2.0 40 360
70.2 2.5 3 16.9 2.7 0.3 3 1.4 48 360
81.9 1.8 3 6.5 2.7 0.3 3 0.8 56 360
Proximate composition Crude protein Ether extract Ash Crude fiber
(% dry wt.) 0.72 4.65 1.33 5.30
9.45 5.07 3.89 5.55
17.49 6.46 5.81 4.88
25.77 6.72 7.40 4.61
33.25 6.62 8.95 3.30
41.26 7.11 10.91 2.53
a Norsemink, Norwegian Herring Oil and Meat Industries, Bergen, Norway. b According to Teng et al. (1978). 3-49 kcal g- ‘; lipid, 8.51 kcal g- ’ as reported ’ Protein, 4.5 kcal g- ‘; carbohydrate, Huang, 1989).
Table 2 Formulation
and proximate
Diet code: Protein (%)/lipid
composition
(%):
Ingredient Fish meal a Cod liver oil Corn oil Corn starch Vitamin mix b Mineral mix b Carboxymethylcellulose Cellulose Gross energy (kcal per 100 g) Proximate composition Crude protein Ether extract Ash Crude fiber Nitrogen-free extract Calculated energy Protein/energy ratio (mg kcal- ’ ) Lb As for Table 1.
of experimental
diets (% dry wt.) in Experiment
50.82 7.87 12.62 1.91
57.21 8.88 14.57 1.95
for tilapia (Shiau and
2
1 50/S
2 44/8
3 50/12
4 44/12
5 50/16
6 44/16
7 50/20
8 44/20
75.1 0.7 2 3.4 2.7 0.3 2 13.8 305
66.1 1.3 2 11.1 2.7 0.3 2 14.5 305
75.1 4.7 2 3.7 2.7 0.3 2 9.5 340
66.1 5.3 2 11.4 2.7 0.3 2 10.2 340
75.1 8.7 2 4.0 2.7 0.3 2 5.2 375
66.1 9.3 2 11.7 2.7 0.3 2 5.9 375
75.1 12.7 2 4.2 2.7 0.3 2 1.0 410
66.1 13.3 2 12.0 2.7 0.3 2 1.6 410
49.07 7.37 14.46 14.17 5.69 303.4 161.7
44.62 6.76 13.35 14.93 11.63 298.9 149.3
49.49 13.24 15.46 10.06 3.33 347 142.6
45.06 -12.88 13.76 10.64 9.03 343.9 131.0
50.97 14.37 15.64 5.% 3.97 365.5 139.5
45.29 14.01 14.61 5.13 12.09 365.2 124.0
51.06 17.69 16.59 1.86 4.04 394.4 129.5
46.66 17.05 15.36 1.56 10.71 392.4 118.9
262
2.2. Experimental
S.-Y. Shim, C.-W. Lan/Aquaculture
14.5 (1996) 259-266
procedures
Wild E. malabaricus juveniles obtained from a local fish fry dealer were used in the study. Upon arrival, they were acclimated to laboratory conditions for 3 weeks in a plastic tank and fed a commercial diet (Lucky Star; Hunh Kuo Industrial Co., Taipei, Taiwan). The proximate composition (%I of the commercial diet was as follows: moisture, 6.0; crude protein, 53.9; lipid, 4.9; ash, 10.8. At the beginning of the experiment, eight fish (mean weight: 9.2 k 0.1 g per fish in Experiment 1 and 10.7 + 0.1 g per fish in Experiment 2) were stocked in each experimental aquarium (30.5 cm wide X 61.0 cm long X 55.5 cm high). There was a total of eight treatments in both Experiments 1 and 2. Each experimental diet was fed to fish in three aquaria. Each aquarium was part of a closed recirculating system with a common reservoir of water at 29-32%0 salinity maintained at 28 + 1°C. The water was circulated at 2 1 min- ’ through two separate biofilters to remove impurities and reduce ammonia concentrations. The fish were fed 3% of body weight per day. This amount was close to the maximum daily ration for grouper according to the feed consumption during the adjustment period of the study. The daily ration was subdivided into two equal feedings and fed at 09:OO h and 17:00 h. Fish were weighed every 2 weeks and the daily ration adjusted accordingly. The diurnal cycle was 12-h light/l2-h dark. The fish were fed the test diets for as g-week period in both experiments. Growth (as measured by the percentage of body weight gain) and FE ratio were calculated as described previously (Shiau et al., 1990). At the end of the experimental period, all fish were taken for analysis of body composition (AOAC, 1984). 2.3. Statistical analysis Data were analyzed by one-way analysis of variance @AS/PC program) in both experiments. Multiple comparisons among means were made with the Duncan’s new multiple range test. The optimum dietary protein level in Experiment 1 was determined by a broken-line model (Robbins, 1986) using growth results.
Table 3 Effect of dietary protein levels on weight gain, feed efficiency containing graded levels of protein for 8 weeks Protein level (%o) 0
8 16 24 32 40 48 56
(FE) and survival of juvenile grouper fed diets
Final body weight (g)
Weight gain (g)
FE (%I
Survival rate (W)
6.33 f 0.35 6.60* 0.20 16.68 f 0.78 24.84f 1.81 32.16 &-1.75 44.91 * 1.39 61.49f3.07 57.86 + 2.22
-32.4k1.9 -29.0f3.0 48.2*0.2 b 172.1+20.1’ 247.5 f 14.0 408.2* 10.2 559.0+ 27.8 526.3 f 35.0
_
45.8 41.6 58.3 75.0 75.0 66.6 70.8 75.0
Values represent meanf SEM (n = 3). Means significantly different(P < 0.05).
within
a a
d ’ f f
the same
_ 35.7f7.1 a 64.1 f2.3 b 76.9 f 10.2 b 105.2+4.3 bc 126.6 f 2.7 ’ 123.4k7.0’ column
with different
superscripts
are
S.-Y. Shim, C.-W. Lan/Aqunculture Table 4 Body composition levels of protein
145 (1996) 259-266
263
(%) of juvenile grouper before and after 8 weeks of feeding the test diets containing
Protein level (%I
Moisture
Protein
Lipid
Initial fish 0 8 16 24
74.5 82.5 78.5 74.0 72.2
g f d b
17.2 f 0.2 13.2kO.7 a 14.4+0.1 b 17.3f0.7 c 16.4+0.2’
32 40 48 56
75.OkO.2 72.6 f 0.4 73.0 f 0.5 70.5 f 0.9
’ bc c a
15.7+0.7 18.0 + 0.3 16.6+cO.5 19.250.5
2.2kO.I 0.6 + 0.2 2.7+0.1 4.6+0.1 5.5 *Il.1 4.9f0.1 4.8fO.l 5.5fO.l 6.6kO.l
f f + f f
0.2 0.3 0.4 0.3 0.3
Values represent mean f SEM (R = 3). Means significantly different (P < 0.05).
within
c cd c d the same
column
graded
Ash
a b ’ e d d c f
5.1 kO.2 5.9kO.l 6.0+0.1 4.6 f 0.3 5.2+0.4 4.1 kO.3 4.6kO.l 4.3 f 0.4 4.5 + 0.4
with different
c ’ a b a a a =
superscripts
arc
3. Results The growth characteristics of fish fed the different dietary protein levels in Experiment 1 are presented in Table 3. Weight gain was highest for fish fed diets containing > 48% protein. The differences among the groups were significant (P C 0.05). The FE data followed the same general pattern as weight gain. Broken-line analysis of the weight gain data detected a break point in the regression lines (dietary protein level versus weight gain) when the dietary protein was 50.2%, suggesting that this level of protein was optimum for the growth of juvenile groupers. After the 8 weeks of Experiment 1, the lipid composition of fish showed much change from the initial composition (Table 4). The lipid content of the fish generally Table 5 Weight gain, feed efficiency (FE) and survival of juvenile grouper containing two protein levels with various energy contents
after 8 weeks of feeding with the test diets
Energy (kcal per 100 g diet) 305
340
375
410
46.21 f 2.66 42.13f2.16
48.89 f 3.67 46.72k3.17
44.39 f 4.20 43.35 f 3.61
47.7 * 3.9 40.6f 3.9
340.3*31.1 x 299.1 f 17.1 y
35 1.9 f 37.8 330.9 f 33.7
334.2rt31.3 303.6 f 29.1
350.9 + 34.3 x 286.0 f 27.7 y
FE (%I 50% protein 44% protein
lOl.O&S.l 94.3k5.2
99.2 f 3.3 98.Ok7.0
98.0 * 7.2 92.6 f 10.3
lOO.lk6.5 ’ 86.2 + 7.3 y
Survival (o/o) 50% protein 44% protein
87.5 66.7
87.5 75.0
87.5 83.3
75.0 79.2
Final body weight (gl 50% protein 44% protein Weight gain (o/o) 50% protein 44% protein
Values represent mean + SEM ( n = 3). xy Significant (P < 0.05) difference between protein levels within energy level; no significant observed between energy levels within protein level.
differences
were
264
S.-Y.
Shiau,
C.-W.
Lan/Aquacubure
145 (1996)
259-266
Table 6 Body composition (%) of juvenile grouper after 8 weeks of feeding the test diets containing with various energy contents
two protein levels
Energy (kcal per 100 g diet)
Moisture 50% protein 44% protein Protein 50% protein 44% protein Lipid 50% protein 44% protein Ash 50% protein 44% protein
305
340
375
410
68.2 f 0.3 67.8 + 0.6
66.4 + 0.3 66.6 rt 0.4
67.9 f 0.3 66.2 f 0.4
68.3 f 0.2 66.5 k 0.5
18.4kO.l 19.6 + 0.5
19.5 * 0.3 19.0 &-0.4
18.9f0.4 19.8 + 0.6
17.8 + 0.6 1s.5+0.4
7.9kO.l x 6.5 f 0.3 ‘Y
8.1 +0.4 8.5 f 0.3 ’
8.4kO.l x 7.7 f 0.2 by
8.8 f 0.5 8.9 f 0.2 ’
5.0+0.3 6.1 *0.5
5.9kO.6 b 6.2 * 0.2
4.6 + 0.6 a’ 6.0 k 0.6
4.6kO.l 5.7kO.4
ab
a’ y
Values represent mean f SEM (n = 3). xy Significant (P < 0.05) difference between protein levels within energy level. ab Significant (P < 0.05) difference between energy levels within protein level.
increased with increasing dietary protein level. Fish fed the lower protein diets had a higher moisture content than those fed higher protein diets. The fish fed different test diets had different protein and ash contents. The effect of different protein to energy ratios on weight gain and FE are presented in Table 5. The weight gain of fish fed diets containing 44% protein with 305 or 410 kcal per 100 g was significantly lower (P < 0.05) than that of fish fed diets containing 50% protein regardless of energy level. FE followed the same pattern as weight gain. The body composition of fish fed diets with various protein to energy ratios is shown in Table 6. The lipid content of fish fed different diets fell into three clusters: highest for fish fed 50% protein with 375 or 410 kcal per 100 g, and 44% protein with 340 or 410 kcal per 100 g; intermediate for those fed 50% protein with 305 or 340 kcal per 100 g and 44% protein with 375 kcal per 100 g; and lowest for those fed 44% protein with 305 kcal per 100 g. The fish fed the 50% protein with 375 or 410 kcal per 100 g showed lower ash content. Protein and moisture contents were not affected by the different dietary protein to energy ratios.
4. Discussion The dietary protein level that resulted in maximum weight gain of juvenile grouper determined in this study is very similar to the value of 47.8 % reported by Chen and Tsai (1994). The slight difference between the two studies may be due to different protein sources used (white fish meal versus casein) and/or different fish sizes (9.22 g vs. 3.79 g>. Mortalities in both experiments were reasonable and were not associated with a
S.-Y. Shim,
C.-W. Lan/Aqumulrure
145 (1996) 259-266
26.5
disease. Grouper is a rather sensitive species to work with. They get frightened easily. Normally a few fish died after weighing. The body lipid content generally increased as the dietary protein level increased ( y = 2.0667 + 0.083x, r = 0.75; Table 4). This is in contrast to the results obtained by Chen and Tsai (1994). In their study with juvenile grouper, increased dietary protein resulted in a decrease in body lipid concentration. While carbohydrate was used in both studies to replace protein to achieve protein gradation, the source of carbohydrate used was different. Corn starch was used in the present study, whereas dextrin was used by Chen and Tsai (1994). It has been reported for several fish species that different types of carbohydrates may not be utilized equally. For example, Furuichi and Yone (1982) fed common carp and red sea bream diets containing dextrin or starch and found that growth and FE of both species were highest when they were fed starch, followed by dextrin. Tilapia has been reported to utilize starch better than glucose (Anderson et al., 1984; Shiau and Chen, 1993. Shiau and Lin, 1993). In our previous study, higher body lipid was observed in tilapia fed starch diets than fish fed glucose diets (Shiau and Lin, 1993). This may provide an explanation for the differences in body lipid content of groupers in the two studies. However, carbohydrate nutrition of grouper has not yet been studied. The other possible explanation is that the lipid content of the test diets was different at the beginning of the experiment. There was a difference of as much as 4% in the lipid content between the highest and lowest value (Table 1). The gross energy content was not analyzed in the present study. The difference in lipid content may well be the reason for the lipid variation in the fish. In the first experiment, about 50% protein was found to produce maximum growth of grouper. The energy value of the diet was kept at 360 kcal per 100 g. It has been reported that the protein-sparing action of dietary carbohydrate is less marked than that of dietary lipid for certain species (Adron et al., 1976). In Experiment 2, lipid was used to adjust the dietary energy level. In this experiment, as well as the optimum protein level which was obtained from Experiment 1 (50%), a suboptimum dietary protein level of 44% was also included. In both 50% and 44% protein diets, 8, 12, 16 and 20% lipid was included to form diets containing 305, 340, 375 and 410 kcal energy per 100 g, respectively. Similar to previous results from studies of this type (Winfree and Stickney, 1981; Machiels and Henken, 1985; Shiau and Huang, 19901, the high dietary non-protein energy content restricted feed consumption which affected growth performance (Page and Andrews, 1973; Prather and Lovell, 1973; Takeda et al., 1975). In the present study, this phenomenon was observed in fish fed the suboptimum protein diets (Table 51. The non-significant change in growth of juvenile grouper, despite a reduction in dietary protein content from 50% to 44% while maintaining an energy level of 340 kcal per 100 g, suggests that protein may be spared by lipid as long as the caloric requirements are met, thus permitting more efficient utilization of protein. Acknowledgements This work was supported by the Council of Agriculture, nos. 83 ASP-2.15~FID-07 and 84 ASP-2.15-FID-0.6(051).
Republic
of China, grant
266
S.-Y. Shiau, C.-W. L.an/Aquaculture
I45 (1996) 259-266
References Adr0n;J.W.. Blair, A., Cowey, C.B. and Shanks, A.M., 1976. Effects of dietary energy level and dietary energy source on growth, feed conversion and body composition of turbot (Scophthtdmus marimus L.). Aquaculture, 7: 125 132. Anderson, J., Jackson, A.J., Matty, A.J. and Copper, B.S., 1984. Effects of dietary carbohydrate and fiber on the tilapia (Oreochromis niloticus). Aquaculture, 37: 303-3 14. AOAC, 1984. Official Methods of Analysis, 14th edn. Association of Official Analytical Chemists, Washington, DC, 1018 pp. Chen, H.Y. and Tsai., J.C., 1994. Optimal dietary protein level for the growth of juvenile grouper, Epinephefus malaharicus, fed semipurified diets. Aquaculture, I 19: 265-271. Furuichi, M. and Yone, Y., 1982. Availability of carbohydrate in nutrition of carp and red sea bream. Bull. Jpn. Sot. Sci. Fish., 48: 945-948. Machiels, M.A.M. and Henken, A.M., 1985. Growth rate, feed utilization and energy metabolism of the African catfish, Clarias guriepinus (Burchell, 1822). as affected by dietary protein and energy content. Aquaculture, 44: 271-284. Page, J.W. and Andrews, J.W., 1973. Interactions of dietary levels of protein and energy on channel catfish Icralurus punctatuu. J. Nutr., 103: 1339-1346. Prather, E.E. and Lovell, R.T., 1973. Response of intensively fed channel catfish to diets containing various protein-energy ratios. Proc. Annu. Conf. S. E. Assoc. Game Fish Comm., 27: 455-459. Reinitz, G.L., Orme, L.E., Lemm, C.A. and Hitzel, F.N., 1978. Influence of varying lipid concentration with two protein concentrations in diets for rainbow trout (S&no gairdneri). Trans. Am. Fish. Sot., 107: 75 l-754. Robbins, K.R., 1986. A method, SAS program, and example for fitting the broken-line to growth data. University of Tennessee Agricultural Experimental Station Research Report 86-09, pp. l-8. Shiau, S.Y. and Chen, M.J., 1993, Carbohydrate utilization by tilapia (Oreochromis nibticus X 0. aureus) as influenced by different chromium sources. J. Nutr., 123: 1747-1753. Shiau, S.Y. and Huang. S.L., 1989. Optimal dietary, protein level for hybrid tilapia (Oreochromis niloricus X 0. aureus) reared in seawater. Aquaculture, 81: 119- 127. Shiau. S.Y. and Huang, S.L., 1990. Influence of varying energy levels with two protein concentrations in diets for hybrid tiliapia (Oreochromis niloticus X 0. aureus) reared in seawater. Aquaculture, 91: 143- 152. Shiau, S.Y. and Lin, S.F., 1993. Effect of supplemental dietary chromium and vanadium on the utilization of different carbohydrates in tilapia, Oreochromis niloticus X 0. crureus. Aquaculture, 110: 321-330. Shiau, S.Y., Chuang, J.L. and Sun, C.L., 1987. Inclusion of soybean meal in tilapia (Oreochromis niloticus X 0. aurrus) diets at two protein levels. Aquaculture, 65: 25 l-261. Shiau, S.Y., Lin, S.F., Yu, S.L., Lin, A.L. and Kwok, C.C., 1990. Defatted and full-fat soybean meal as partial replacements for tishmeal in tilapia (Oreochromis niloticus X 0. oureus) diets at low protein level. Aquaculture, 86: 40 I-407. Takeda, M., Shimeno, S., Hosokawa, H., Hedetoshi, K. and Kaisyo, T., 1975. The effect of dietary calorie-to-protein ratio on the growth, feed conversion and body composition of young yellowtail. Bull. Jpn. Sot. Sci. Fish., 41: 443-447. Takeuchi, T., Shiina, Y., Watanabe, T., Sekiya, S. and Imaizumi, K., 1992. Suitable protein and lipid levels in diet for fingerlings of yellowtail. Nippon Suisan Gakkaishi, 58: 1333-1339. Teng, S.K., Chua, T.E. and Lim, P.E., 1978. Preliminary observation on the dietary protein requirement of estuary grouper, Epinephelus salmonides Maxwell, cultured in floating netcages. Aquaculture, 15: 257-271. Winfree, R.A. and Stickney, R.R., 1981. Effects of dietary protein and energy on growth, feed conversion efficiency and body composition of Tilapia aurecr. J. Nutr., 1 I I: 1001-1012.
Optimum dietary protein level and protein to energy ratio for growth of grouper ( E’inephelus malabaricud Shi-Yen Shiau *, Ching-Wan Lan Depwnnent
ofMarine Food Science, National Taiwan Ocean Universi~, Keelung, Taiwan Accepted 29 March 1996
Abstract A series of two experiments was conducted to study the optimum dietary protein level and protein to energy ratio of juvenile groupers. In Experiment 1, eight isoenergetic fish meal based semi-purified diets ranging from 0 to 56% protein in 8% increments were fed for 8 weeks to triplicate groups of grouper (initial weight: 9.22 f 0.11 g per fish). Weight gain of fish was proportional to the protein content of the diet up to an incorporation rate of 48%. Feed efficiency (FE) increased with increasing dietary protein level, and the diet with 48% protein yielded the lowest FE. Fish fed the 56% protein diet had the highest body protein and lipid contents, and the lowest body moisture content. ‘Ihe dietary protein level that yielded maximum growth was 50.2% based on a broken-line model estimation of weight gain. In Experiment 2, two dietary protein levels (50% and 44%) were used along with four energy levels at each protein level (305, 340, 375 and 410 kcal per 100 g diet). The experimental diets were fed for 8 weeks to triplicate groups of grouper (initial weight: 10.70 f 0.14 g per fish). Weight gain of fish was not significantly (P > 0.05) different at 50% dietary protein regardless of energy level. However, the 44% protein diet at energy levels of 340 and 375 kcal per 100 g improved weight gain of fish. Fish fed the 44% protein with 340 or 410 kcal per 100 g diet had higher body lipid content. These data suggest that when the energy level of the diet is maintained at 340-375 kcal per 100 g, the dietary protein level for juvenile grouper can be lowered from 50% to 44%. Keywords:
l
Epinephelus
spp.; Feeding and nutrition-fish;
Protein; Protein energy ratio; Grouper
Corresponding author.
CW48486/%/$15.00
Copyright 0 1996 Elsevier Science B.V. All rights reserved.
PII SOO44-8486(96)01324-S
S.-Y. Shim, C.-W. Lnn/Aquuculrure
145 (1996) 259-266
1. Introduction Groupers are a good candidate for intensive aquaculture because of their desirable taste, hardiness in a crowded environment and rapid growth. They are valued as one of the highest quality seafoods in many parts of the world (Chen and Tsai, 1994). In Taiwan, the importance of grouper culture has become more important because of the shortage of freshwater resources. Little is known about their nutritional requirements, especially the species Epinephelus malabaricars, which is one of the most important cultured groupers in Asia. Protein is the most expensive component in fish feed. Thus, knowledge of the protein requirement is important for the formulation of well-balanced and low cost feeds. A study using casein as the protein source has indicated an optimal protein level for E. malabarcius (Chen and Tsai, 1994). Currently, raw trash fish supplemented with compounded feed formulated mainly for eel is the main food source for E. malabaricus in Taiwan’s commercial production. The optimum dietary protein level of E. malabaricus fed a fish meal based diet is not known. Studies have shown that providing adequate energy from dietary lipid can minimize the use of protein as an energy source (Reinitz et al., 1978; Shiau and Huang, 1990; Takeuchi et al., 1992). However, excess energy may produce fatty fish, reduce feed consumption (reducing total protein intake) and reduce the utilization of other nutrients (Page and Andrews, 1973; Prather and Lovell, 1973; Takeda et al., 1975; Shiau and Huang, 1990). Therefore, it is important to obtain the proper protein to energy ratio in a diet for the most economical production of grouper. The objectives of this study were two-fold. Firstly, to assess the optimum dietary protein level of juvenile grouper fed fish meal based diets and, secondly, to determine the optimun protein to energy ratio when dietary protein level is optimum and suboptimum.
2. Materials and methods 2.1. Diet preparation Formulations and proximate analysis (AOAC, 1984) of the diets fed in Experiment 1 and Experiment 2 are given in Tables 1 and 2, respectively. White fish meal (blue whiting, imported from Norway), cod liver oil and corn oil, and corn starch were used as dietary protein, lipid and carbohydrate sources, respectively. In Experiment 1, the diets were formulated to give 8% increments of protein from 0 to 56%, for a total of eight diets in all. Diets were made isocaloric by adjusting the corn starch and cellulose content. In Experiment 2, two dietary protein levels, 50% and 44%, were used. For each protein level, diets were formulated with four energy values of 305, 340, 375 and 410 kcal per 100 g diet for a total of eight experimental diets. The values of used for calculating energy levels were 4.5 kcal g- ’ for protein, 3.49 kcal g- ’ for carbohydrate and 8.51 kcal gg ’ for lipid (Shiau and Huang, 1989). The diets were prepared and handled as previously described (Shiau et al., 1987).
S.-Y. Shiau. C.-W. Lan/Aquuculrure Table 1 Formulation
of proximate
composition
of experimental
261
145 (1996) 259-266
diets (% dry wt.) in Experiment
1
Diet code:
1
2
3
4
5
6
7
8
Ingredient Fish meal ’ Cod liver oil Corn oil Corn starch Vitamin mix b Mineral mix b Carboxymethylcellulose Cellulose Calculated protein level Gross energy (kcal per 100 g) ’
0 7 3 78.8 2.7 0.3 3 5.2 0 360
11.7 6.3 3 68.4 2.7 0.3 3 4.6 8 360
23.4 5.5 3 58.1 2.7 0.3 3 4.0 16 360
35.1 4.8 3 47.8 2.7 0.3 3 3.3 24 360
46.8 4.0 3 37.5 2.7 0.3 3 2.7 32 360
58.5 3.3 3 27.2 2.7 0.3 3 2.0 40 360
70.2 2.5 3 16.9 2.7 0.3 3 1.4 48 360
81.9 1.8 3 6.5 2.7 0.3 3 0.8 56 360
Proximate composition Crude protein Ether extract Ash Crude fiber
(% dry wt.) 0.72 4.65 1.33 5.30
9.45 5.07 3.89 5.55
17.49 6.46 5.81 4.88
25.77 6.72 7.40 4.61
33.25 6.62 8.95 3.30
41.26 7.11 10.91 2.53
a Norsemink, Norwegian Herring Oil and Meat Industries, Bergen, Norway. b According to Teng et al. (1978). 3-49 kcal g- ‘; lipid, 8.51 kcal g- ’ as reported ’ Protein, 4.5 kcal g- ‘; carbohydrate, Huang, 1989).
Table 2 Formulation
and proximate
Diet code: Protein (%)/lipid
composition
(%):
Ingredient Fish meal a Cod liver oil Corn oil Corn starch Vitamin mix b Mineral mix b Carboxymethylcellulose Cellulose Gross energy (kcal per 100 g) Proximate composition Crude protein Ether extract Ash Crude fiber Nitrogen-free extract Calculated energy Protein/energy ratio (mg kcal- ’ ) Lb As for Table 1.
of experimental
diets (% dry wt.) in Experiment
50.82 7.87 12.62 1.91
57.21 8.88 14.57 1.95
for tilapia (Shiau and
2
1 50/S
2 44/8
3 50/12
4 44/12
5 50/16
6 44/16
7 50/20
8 44/20
75.1 0.7 2 3.4 2.7 0.3 2 13.8 305
66.1 1.3 2 11.1 2.7 0.3 2 14.5 305
75.1 4.7 2 3.7 2.7 0.3 2 9.5 340
66.1 5.3 2 11.4 2.7 0.3 2 10.2 340
75.1 8.7 2 4.0 2.7 0.3 2 5.2 375
66.1 9.3 2 11.7 2.7 0.3 2 5.9 375
75.1 12.7 2 4.2 2.7 0.3 2 1.0 410
66.1 13.3 2 12.0 2.7 0.3 2 1.6 410
49.07 7.37 14.46 14.17 5.69 303.4 161.7
44.62 6.76 13.35 14.93 11.63 298.9 149.3
49.49 13.24 15.46 10.06 3.33 347 142.6
45.06 -12.88 13.76 10.64 9.03 343.9 131.0
50.97 14.37 15.64 5.% 3.97 365.5 139.5
45.29 14.01 14.61 5.13 12.09 365.2 124.0
51.06 17.69 16.59 1.86 4.04 394.4 129.5
46.66 17.05 15.36 1.56 10.71 392.4 118.9
262
2.2. Experimental
S.-Y. Shim, C.-W. Lan/Aquaculture
14.5 (1996) 259-266
procedures
Wild E. malabaricus juveniles obtained from a local fish fry dealer were used in the study. Upon arrival, they were acclimated to laboratory conditions for 3 weeks in a plastic tank and fed a commercial diet (Lucky Star; Hunh Kuo Industrial Co., Taipei, Taiwan). The proximate composition (%I of the commercial diet was as follows: moisture, 6.0; crude protein, 53.9; lipid, 4.9; ash, 10.8. At the beginning of the experiment, eight fish (mean weight: 9.2 k 0.1 g per fish in Experiment 1 and 10.7 + 0.1 g per fish in Experiment 2) were stocked in each experimental aquarium (30.5 cm wide X 61.0 cm long X 55.5 cm high). There was a total of eight treatments in both Experiments 1 and 2. Each experimental diet was fed to fish in three aquaria. Each aquarium was part of a closed recirculating system with a common reservoir of water at 29-32%0 salinity maintained at 28 + 1°C. The water was circulated at 2 1 min- ’ through two separate biofilters to remove impurities and reduce ammonia concentrations. The fish were fed 3% of body weight per day. This amount was close to the maximum daily ration for grouper according to the feed consumption during the adjustment period of the study. The daily ration was subdivided into two equal feedings and fed at 09:OO h and 17:00 h. Fish were weighed every 2 weeks and the daily ration adjusted accordingly. The diurnal cycle was 12-h light/l2-h dark. The fish were fed the test diets for as g-week period in both experiments. Growth (as measured by the percentage of body weight gain) and FE ratio were calculated as described previously (Shiau et al., 1990). At the end of the experimental period, all fish were taken for analysis of body composition (AOAC, 1984). 2.3. Statistical analysis Data were analyzed by one-way analysis of variance @AS/PC program) in both experiments. Multiple comparisons among means were made with the Duncan’s new multiple range test. The optimum dietary protein level in Experiment 1 was determined by a broken-line model (Robbins, 1986) using growth results.
Table 3 Effect of dietary protein levels on weight gain, feed efficiency containing graded levels of protein for 8 weeks Protein level (%o) 0
8 16 24 32 40 48 56
(FE) and survival of juvenile grouper fed diets
Final body weight (g)
Weight gain (g)
FE (%I
Survival rate (W)
6.33 f 0.35 6.60* 0.20 16.68 f 0.78 24.84f 1.81 32.16 &-1.75 44.91 * 1.39 61.49f3.07 57.86 + 2.22
-32.4k1.9 -29.0f3.0 48.2*0.2 b 172.1+20.1’ 247.5 f 14.0 408.2* 10.2 559.0+ 27.8 526.3 f 35.0
_
45.8 41.6 58.3 75.0 75.0 66.6 70.8 75.0
Values represent meanf SEM (n = 3). Means significantly different(P < 0.05).
within
a a
d ’ f f
the same
_ 35.7f7.1 a 64.1 f2.3 b 76.9 f 10.2 b 105.2+4.3 bc 126.6 f 2.7 ’ 123.4k7.0’ column
with different
superscripts
are
S.-Y. Shim, C.-W. Lan/Aqunculture Table 4 Body composition levels of protein
145 (1996) 259-266
263
(%) of juvenile grouper before and after 8 weeks of feeding the test diets containing
Protein level (%I
Moisture
Protein
Lipid
Initial fish 0 8 16 24
74.5 82.5 78.5 74.0 72.2
g f d b
17.2 f 0.2 13.2kO.7 a 14.4+0.1 b 17.3f0.7 c 16.4+0.2’
32 40 48 56
75.OkO.2 72.6 f 0.4 73.0 f 0.5 70.5 f 0.9
’ bc c a
15.7+0.7 18.0 + 0.3 16.6+cO.5 19.250.5
2.2kO.I 0.6 + 0.2 2.7+0.1 4.6+0.1 5.5 *Il.1 4.9f0.1 4.8fO.l 5.5fO.l 6.6kO.l
f f + f f
0.2 0.3 0.4 0.3 0.3
Values represent mean f SEM (R = 3). Means significantly different (P < 0.05).
within
c cd c d the same
column
graded
Ash
a b ’ e d d c f
5.1 kO.2 5.9kO.l 6.0+0.1 4.6 f 0.3 5.2+0.4 4.1 kO.3 4.6kO.l 4.3 f 0.4 4.5 + 0.4
with different
c ’ a b a a a =
superscripts
arc
3. Results The growth characteristics of fish fed the different dietary protein levels in Experiment 1 are presented in Table 3. Weight gain was highest for fish fed diets containing > 48% protein. The differences among the groups were significant (P C 0.05). The FE data followed the same general pattern as weight gain. Broken-line analysis of the weight gain data detected a break point in the regression lines (dietary protein level versus weight gain) when the dietary protein was 50.2%, suggesting that this level of protein was optimum for the growth of juvenile groupers. After the 8 weeks of Experiment 1, the lipid composition of fish showed much change from the initial composition (Table 4). The lipid content of the fish generally Table 5 Weight gain, feed efficiency (FE) and survival of juvenile grouper containing two protein levels with various energy contents
after 8 weeks of feeding with the test diets
Energy (kcal per 100 g diet) 305
340
375
410
46.21 f 2.66 42.13f2.16
48.89 f 3.67 46.72k3.17
44.39 f 4.20 43.35 f 3.61
47.7 * 3.9 40.6f 3.9
340.3*31.1 x 299.1 f 17.1 y
35 1.9 f 37.8 330.9 f 33.7
334.2rt31.3 303.6 f 29.1
350.9 + 34.3 x 286.0 f 27.7 y
FE (%I 50% protein 44% protein
lOl.O&S.l 94.3k5.2
99.2 f 3.3 98.Ok7.0
98.0 * 7.2 92.6 f 10.3
lOO.lk6.5 ’ 86.2 + 7.3 y
Survival (o/o) 50% protein 44% protein
87.5 66.7
87.5 75.0
87.5 83.3
75.0 79.2
Final body weight (gl 50% protein 44% protein Weight gain (o/o) 50% protein 44% protein
Values represent mean + SEM ( n = 3). xy Significant (P < 0.05) difference between protein levels within energy level; no significant observed between energy levels within protein level.
differences
were
264
S.-Y.
Shiau,
C.-W.
Lan/Aquacubure
145 (1996)
259-266
Table 6 Body composition (%) of juvenile grouper after 8 weeks of feeding the test diets containing with various energy contents
two protein levels
Energy (kcal per 100 g diet)
Moisture 50% protein 44% protein Protein 50% protein 44% protein Lipid 50% protein 44% protein Ash 50% protein 44% protein
305
340
375
410
68.2 f 0.3 67.8 + 0.6
66.4 + 0.3 66.6 rt 0.4
67.9 f 0.3 66.2 f 0.4
68.3 f 0.2 66.5 k 0.5
18.4kO.l 19.6 + 0.5
19.5 * 0.3 19.0 &-0.4
18.9f0.4 19.8 + 0.6
17.8 + 0.6 1s.5+0.4
7.9kO.l x 6.5 f 0.3 ‘Y
8.1 +0.4 8.5 f 0.3 ’
8.4kO.l x 7.7 f 0.2 by
8.8 f 0.5 8.9 f 0.2 ’
5.0+0.3 6.1 *0.5
5.9kO.6 b 6.2 * 0.2
4.6 + 0.6 a’ 6.0 k 0.6
4.6kO.l 5.7kO.4
ab
a’ y
Values represent mean f SEM (n = 3). xy Significant (P < 0.05) difference between protein levels within energy level. ab Significant (P < 0.05) difference between energy levels within protein level.
increased with increasing dietary protein level. Fish fed the lower protein diets had a higher moisture content than those fed higher protein diets. The fish fed different test diets had different protein and ash contents. The effect of different protein to energy ratios on weight gain and FE are presented in Table 5. The weight gain of fish fed diets containing 44% protein with 305 or 410 kcal per 100 g was significantly lower (P < 0.05) than that of fish fed diets containing 50% protein regardless of energy level. FE followed the same pattern as weight gain. The body composition of fish fed diets with various protein to energy ratios is shown in Table 6. The lipid content of fish fed different diets fell into three clusters: highest for fish fed 50% protein with 375 or 410 kcal per 100 g, and 44% protein with 340 or 410 kcal per 100 g; intermediate for those fed 50% protein with 305 or 340 kcal per 100 g and 44% protein with 375 kcal per 100 g; and lowest for those fed 44% protein with 305 kcal per 100 g. The fish fed the 50% protein with 375 or 410 kcal per 100 g showed lower ash content. Protein and moisture contents were not affected by the different dietary protein to energy ratios.
4. Discussion The dietary protein level that resulted in maximum weight gain of juvenile grouper determined in this study is very similar to the value of 47.8 % reported by Chen and Tsai (1994). The slight difference between the two studies may be due to different protein sources used (white fish meal versus casein) and/or different fish sizes (9.22 g vs. 3.79 g>. Mortalities in both experiments were reasonable and were not associated with a
S.-Y. Shim,
C.-W. Lan/Aqumulrure
145 (1996) 259-266
26.5
disease. Grouper is a rather sensitive species to work with. They get frightened easily. Normally a few fish died after weighing. The body lipid content generally increased as the dietary protein level increased ( y = 2.0667 + 0.083x, r = 0.75; Table 4). This is in contrast to the results obtained by Chen and Tsai (1994). In their study with juvenile grouper, increased dietary protein resulted in a decrease in body lipid concentration. While carbohydrate was used in both studies to replace protein to achieve protein gradation, the source of carbohydrate used was different. Corn starch was used in the present study, whereas dextrin was used by Chen and Tsai (1994). It has been reported for several fish species that different types of carbohydrates may not be utilized equally. For example, Furuichi and Yone (1982) fed common carp and red sea bream diets containing dextrin or starch and found that growth and FE of both species were highest when they were fed starch, followed by dextrin. Tilapia has been reported to utilize starch better than glucose (Anderson et al., 1984; Shiau and Chen, 1993. Shiau and Lin, 1993). In our previous study, higher body lipid was observed in tilapia fed starch diets than fish fed glucose diets (Shiau and Lin, 1993). This may provide an explanation for the differences in body lipid content of groupers in the two studies. However, carbohydrate nutrition of grouper has not yet been studied. The other possible explanation is that the lipid content of the test diets was different at the beginning of the experiment. There was a difference of as much as 4% in the lipid content between the highest and lowest value (Table 1). The gross energy content was not analyzed in the present study. The difference in lipid content may well be the reason for the lipid variation in the fish. In the first experiment, about 50% protein was found to produce maximum growth of grouper. The energy value of the diet was kept at 360 kcal per 100 g. It has been reported that the protein-sparing action of dietary carbohydrate is less marked than that of dietary lipid for certain species (Adron et al., 1976). In Experiment 2, lipid was used to adjust the dietary energy level. In this experiment, as well as the optimum protein level which was obtained from Experiment 1 (50%), a suboptimum dietary protein level of 44% was also included. In both 50% and 44% protein diets, 8, 12, 16 and 20% lipid was included to form diets containing 305, 340, 375 and 410 kcal energy per 100 g, respectively. Similar to previous results from studies of this type (Winfree and Stickney, 1981; Machiels and Henken, 1985; Shiau and Huang, 19901, the high dietary non-protein energy content restricted feed consumption which affected growth performance (Page and Andrews, 1973; Prather and Lovell, 1973; Takeda et al., 1975). In the present study, this phenomenon was observed in fish fed the suboptimum protein diets (Table 51. The non-significant change in growth of juvenile grouper, despite a reduction in dietary protein content from 50% to 44% while maintaining an energy level of 340 kcal per 100 g, suggests that protein may be spared by lipid as long as the caloric requirements are met, thus permitting more efficient utilization of protein. Acknowledgements This work was supported by the Council of Agriculture, nos. 83 ASP-2.15~FID-07 and 84 ASP-2.15-FID-0.6(051).
Republic
of China, grant
266
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I45 (1996) 259-266
References Adr0n;J.W.. Blair, A., Cowey, C.B. and Shanks, A.M., 1976. Effects of dietary energy level and dietary energy source on growth, feed conversion and body composition of turbot (Scophthtdmus marimus L.). Aquaculture, 7: 125 132. Anderson, J., Jackson, A.J., Matty, A.J. and Copper, B.S., 1984. Effects of dietary carbohydrate and fiber on the tilapia (Oreochromis niloticus). Aquaculture, 37: 303-3 14. AOAC, 1984. Official Methods of Analysis, 14th edn. Association of Official Analytical Chemists, Washington, DC, 1018 pp. Chen, H.Y. and Tsai., J.C., 1994. Optimal dietary protein level for the growth of juvenile grouper, Epinephefus malaharicus, fed semipurified diets. Aquaculture, I 19: 265-271. Furuichi, M. and Yone, Y., 1982. Availability of carbohydrate in nutrition of carp and red sea bream. Bull. Jpn. Sot. Sci. Fish., 48: 945-948. Machiels, M.A.M. and Henken, A.M., 1985. Growth rate, feed utilization and energy metabolism of the African catfish, Clarias guriepinus (Burchell, 1822). as affected by dietary protein and energy content. Aquaculture, 44: 271-284. Page, J.W. and Andrews, J.W., 1973. Interactions of dietary levels of protein and energy on channel catfish Icralurus punctatuu. J. Nutr., 103: 1339-1346. Prather, E.E. and Lovell, R.T., 1973. Response of intensively fed channel catfish to diets containing various protein-energy ratios. Proc. Annu. Conf. S. E. Assoc. Game Fish Comm., 27: 455-459. Reinitz, G.L., Orme, L.E., Lemm, C.A. and Hitzel, F.N., 1978. Influence of varying lipid concentration with two protein concentrations in diets for rainbow trout (S&no gairdneri). Trans. Am. Fish. Sot., 107: 75 l-754. Robbins, K.R., 1986. A method, SAS program, and example for fitting the broken-line to growth data. University of Tennessee Agricultural Experimental Station Research Report 86-09, pp. l-8. Shiau, S.Y. and Chen, M.J., 1993, Carbohydrate utilization by tilapia (Oreochromis nibticus X 0. aureus) as influenced by different chromium sources. J. Nutr., 123: 1747-1753. Shiau, S.Y. and Huang. S.L., 1989. Optimal dietary, protein level for hybrid tilapia (Oreochromis niloricus X 0. aureus) reared in seawater. Aquaculture, 81: 119- 127. Shiau. S.Y. and Huang, S.L., 1990. Influence of varying energy levels with two protein concentrations in diets for hybrid tiliapia (Oreochromis niloticus X 0. aureus) reared in seawater. Aquaculture, 91: 143- 152. Shiau, S.Y. and Lin, S.F., 1993. Effect of supplemental dietary chromium and vanadium on the utilization of different carbohydrates in tilapia, Oreochromis niloticus X 0. crureus. Aquaculture, 110: 321-330. Shiau, S.Y., Chuang, J.L. and Sun, C.L., 1987. Inclusion of soybean meal in tilapia (Oreochromis niloticus X 0. aurrus) diets at two protein levels. Aquaculture, 65: 25 l-261. Shiau, S.Y., Lin, S.F., Yu, S.L., Lin, A.L. and Kwok, C.C., 1990. Defatted and full-fat soybean meal as partial replacements for tishmeal in tilapia (Oreochromis niloticus X 0. oureus) diets at low protein level. Aquaculture, 86: 40 I-407. Takeda, M., Shimeno, S., Hosokawa, H., Hedetoshi, K. and Kaisyo, T., 1975. The effect of dietary calorie-to-protein ratio on the growth, feed conversion and body composition of young yellowtail. Bull. Jpn. Sot. Sci. Fish., 41: 443-447. Takeuchi, T., Shiina, Y., Watanabe, T., Sekiya, S. and Imaizumi, K., 1992. Suitable protein and lipid levels in diet for fingerlings of yellowtail. Nippon Suisan Gakkaishi, 58: 1333-1339. Teng, S.K., Chua, T.E. and Lim, P.E., 1978. Preliminary observation on the dietary protein requirement of estuary grouper, Epinephelus salmonides Maxwell, cultured in floating netcages. Aquaculture, 15: 257-271. Winfree, R.A. and Stickney, R.R., 1981. Effects of dietary protein and energy on growth, feed conversion efficiency and body composition of Tilapia aurecr. J. Nutr., 1 I I: 1001-1012.