be 47 or 22 g digestible proteinrMJ digestible energy. q 2000 Elsevier Science B.V. All rights reserved.
Keywords: American eel; Anguilla rostrata; Protein requirement; Elver; Optimum growth; Digestibility
1. Introduction
American eel culture is a relatively new industry in Atlantic Canada. Due to environmental constraints, particularly low winter water temperature in the Atlantic
provinces, a recirculation system must be used to rear wild elvers to market size. Feeds used for this system should be water-stable and highly digestible to minimize nitrogen,
phosphorous and solid output to the aquatic environment. Unfortunately, information on the nutrient requirements of American eel is limited and there are no published data on
nutrient bioavailability from locally available feed ingredients.
Dietary protein supply is one of the major factors that influence the productivity of fish and the production of nitrogenous waste material that is excreted in water. Like
other fish species, the reported protein requirement of Japanese eel and European eel is Ž
. relatively high compared to terrestrial animals
National Research Council, 1993 . Ž
Although the protein requirement of Japanese eel has been investigated Arai et al., .
1971, 1972; Nose and Arai, 1973 and is 44.5 of the diet, reported values for European Ž
eel range from 30 to 48 Spannhof and Kuhne, 1977; Degani et al., 1984, 1985; Arai .
et al., 1986 . Dietary requirement for protein is, in fact, a requirement for the essential amino acids contained in the dietary protein. The quantitative amino acid requirement
Ž .
values for Japanese eel Nose, 1979 are commonly used for feed formulation of both American eel and European eel.
Dietary energy concentration has a profound effect on how well protein is utilized. Ž
. Studies have indicated that American eels Otwell and Rickards, 1981 , Japanese eels
Ž .
Ž .
Takeuchi et al., 1980 and European eels Dave et al., 1974, 1976 efficiently utilize fat as an energy source.
The present study was designed to determine the quantitative dietary crude protein requirement for optimum growth of juvenile American eel using a practical fish-meal-
based diet and to measure the digestibility of nutrients from the experimental diets. This information is necessary for cost-effective feed formulations, to optimize growth and
protein retention and reduce the soluble and solid load of nitrogenous compounds in the water used for eel culture systems, particularly in recirculation systems.
2. Materials and methods
2.1. Rearing systems, diets and experimental design Ž
. American eels
Anguilla rostrata obtained from Springhill Fish Farms, Springhill, Nova Scotia were randomly distributed into 15, 40-l cylindrical fibreglass tanks. Elvers
were acclimated for a 10-day period and during that time fed a commercial pelleted diet
twice daily. The experiment was conducted according to a randomized complete block design and the tanks were arranged in three blocks of five tanks. Each of the five
experimental diets were fed to three tanks, each containing 45 fish and individual diets were represented in each block of tanks. The initial biomass density in each tank was
approximately 18 kgrm
3
. Water was supplied to each tank at a flow rate of 1 lrmin, constantly aerated and maintained thermostatically at 22 0.2
8C. Photoperiod was controlled automatically on a 15 h light:9 h dark schedule. During the 84-day experi-
mental period, fish were fed at a rate judged to be near satiety twice daily during the week and once daily on weekends. Daily feed intake was recorded by weighing feed
containers at the beginning of each day and ensuring that all feed offered was consumed. All mortalities were collected, weighed and recorded on a daily basis. When necessary,
these weights were used to adjust tank biomass for calculation of feed conversion ratio Ž
. FCR .
Ž .
Five isoenergetic 19 MJ DErkg experimental diets were formulated to supply crude Ž
. protein levels of 35, 39, 43, 47 and 51 on an as fed basis Table 1 . Digestible protein
Ž .
Ž .
Ž DP and digestible energy DE values of feed ingredients for salmonids National
. Research Council, 1993 were used for feed formulation. Dry feed ingredients were
finely ground, mixed with micronutrient and lipid supplements, steam-pelleted in a Ž
. laboratory pellet mill California Pellet Mill, Crawfordsville, IN and stored in air-tight
containers until used. Fish from each tank were individually weighed on days 0, 28, 56 and 84 and the
average weight was determined. Growth was estimated from average weight in each of Ž
. the three tanks receiving each dietary treatment. Specific growth rate
SGR was
Ž .
calculated using the equation of Ricker 1979 . FCRs were calculated from weight of Ž
. Ž
. feed consumed grams of apparent DM feed intake divided by wet weight gain grams .
After the termination of the growth experiment, 10 fish from each tank were killed with Ž
. an overdose of MS222 tricaine methane sulfonate after 48 h food deprivation and
immediately frozen at y60 8C until analyzed for body composition.
2.2. Nutrient digestibility After the growth trial, remaining fish were fed the experimental diets supplemented
Ž .
Ž .
with an inert marker, chromium III oxide Cr O , 5 grkg for an additional 4 weeks
2 3
Ž .
Austreng, 1978 . It was obvious from our preliminary work designed to evaluate faecal collection
methods that it is very difficult to separate faeces from water. In addition, escapement of eels from the tanks was a major problem. Other common methods using collection of
rectal contents by manually stripping, anal suction, metabolic chambers or dissecting the fish could not be applied to eels. Moreover, forced evacuation of the rectum results in
the addition of enzymes, bodily fluids and intestinal epithelial cells to the rectal contents
Ž .
resulting in under-estimated digestibility coefficients Cho et al., 1982 . Ž
. Fish were housed in 10 glass aquariums 60 l for digestibility measurements. After
the last feeding of the day, the tanks were completely cleaned of any feed and faeces Ž
. that had accumulated on the bottom. Each morning 17 hours after the previous feeding ,
the faeces were siphoned into a container, centrifuged, drained of water and frozen at
Table 1 Composition of experimental diets used to determine optimum dietary protein requirement of juvenile
Ž .
American eel as fed basis Ž .
Dietary crude protein 35
39 43
47 51
Ingredients Herring meal
37.1 42.2
47.4 52.5
57.7 Whey powder
7.0 7.0
7.0 7.0
7.0 Blood meal
5.0 5.0
5.0 5.0
5.0 Ž
. Corn starch pre-gel.
31.6 28.2
24.8 21.4
18.9
1
Vitamin premix 1.0
1.0 1.0
1.0 1.0
L
-methionine 0.6
0.6 0.6
0.6 0.6
L
-isoleucine 0.5
0.4 0.2
0.0 0.0
Glutamic acid 0.0
0.1 0.3
0.5 0.5
2
Mineral premix 1.0
1.0 1.0
1.0 1.0
Choline chloride 0.2
0.2 0.2
0.2 0.2
Herring oil 3.0
3.0 3.0
3.0 3.0
Corn oil 13.0
11.3 9.5
7.8 5.1
Analysis Ž .
Moisture 8.0
7.5 7.3
6.7 7.3
Ž . Crude protein
34.6 39.1
43.1 47.5
50.6 Ž .
Lipid 19.5
17.6 16.3
15.6 13.5
Ž . Ash
4.8 5.3
5.8 6.5
6.9 Ž .
Carbohydrate 33.1
30.5 27.5
23.7 21.7
Ž .
Gross energy MJrkg 21.3
21.3 21.3
21.3 20.9
Ž . Digestible protein
29.4 35.0
39.1 43.0
46.6 Ž
. Digestible energy MJrkg
18.2 18.9
19.2 19.3
19.2 Ž
. PrE ratio g DPrMJ DE
16.2 18.5
20.4 22.3
24.3
1
Ž .
Vitamin premix mgrkg or IU : vitamin A, 6000 IU; vitamin D, 4000 IU; vitamin E, 250 IU; vitamin K Ž
. menadione sodium bisulphite , 30 mgrkg; thiamin, 40 mgrkg; riboflavin, 50 mgrkg;
D
-calcium pantothen- Ž
. Ž
. ate, 150 mgrkg; biotin 1 , 0.8 mgrkg; folic acid, 15 mgrkg; vitamin B
0.1 , 0.05 mgrkg; niacin, 200
12
Ž .
mgrkg; pyridoxine, 30 mgrkg; ascorbic acid phosphate, 15 , 200 mgrkg; inositol, 400 mgrkg; ethoxyquin, 125 mgrkg.
2
Ž .
Ž .
Ž .
Mineral premix mgrkg of diet : MnSO PH O 32.5 Mn , 40.0; CuSO P5H O 25.4 Cu , 10.0;
4 2
4 2
Ž .
Ž .
Ž .
Ž .
ZnSO P7H O 22.7 Zn , 50.0; MgSO P7H O 9.95 Mg , 0.04; KI 76.4 I , 5.0; Na SeO 45.6 Se ,
4 2
4 2
2 3
Ž .
Ž .
1.0; CoCl P6H O 24.8 Co , 10.0; NaF 45.2 F , 4.5.
2 2
Ž y60
8C. For individual tanks, faeces were pooled, lyophilized Edwards High Vacuum .
Freeze-Dryer, Manor Royal, Crawley, West Sussex, England and finely ground.
Ž .
Apparent digestibility coefficients ADC of organic matter, protein and energy were Ž
. calculated by the formula of Cho et al. 1974 .
2.3. Chemical analysis In preparation for chemical analysis, fish carcasses were thawed, coarsely ground,
re-frozen, lyophilized and coarsely ground again. Lyophilized fish carcasses, diets and faecal samples were analyzed by similar procedures. Ash was determined by ignition at
Ž .
Ž .
550 8C AOAC, 1984 , organic matter calculated as 100y percentage ash , lipid by
Ž .
Ž ether extraction Tecator Soxtec System HT2 1045 Extraction Unit following acid 4N
. Ž
. Ž
HCl hydrolysis 1047 Hydrolyzing Unit , total nitrogen by the Dumas method Ebling, .
Ž .
1968 using a Leco nitrogen determinator model FP-228, Leco, St. Joseph, MI with protein calculated as N
6.25, moisture by weight loss after drying for 24 h at 105 8C
Ž .
Ž AOAC, 1984 and gross energy by an adiabatic bomb calorimeter Parr Instrument,
. Moline, IL . Chromium content of diets and faeces was measured spectrophotometri-
Ž .
cally using a micro-method outlined by Suzuki and Early 1991 . 2.4. Statistical procedures
All statistical analysis on growth data were performed according to Steel and Torrie Ž
. 1960 . A 5 level of probability was chosen in advance to sufficiently demonstrate a
statistically significant difference. Statistical analysis was performed using Statistical Ž
. Analysis System 1992 . Treatment means were differentiated using least square means
SEM if ANOVA showed significant differences. Mortality data were compared by Ž
. G-test based on the intrinsic null hypothesis a y 2 degrees of freedom that treatment
Ž .
had no significant effect on mortality Sokal and Rohlf, 1969 . Where the null
hypothesis was rejected, pair-wise comparisons were made by calculating the 95 Ž
. confidence interval by use of the Bonferroni Z statistic Neu et al., 1974 . Within the
Ž .
51 protein treatment, one of the tanks had significantly P - 0.05 higher mortality than the other two replications, therefore, data from this tank were removed from the
analysis of the growth data.
3. Results and discussion
