2. Materials and methods
2.1. EnÕironmental Municipal water was supplied to each tank via a flow through system, where
incoming water was filtered through an in-line particulate filter and an activated carbon Ž
. filter to remove chlorine. An electrolyte solution of CaCl :NaCl 9r1, wrw was added
2
continuously to increase the electrolyte concentration and maintain a hardness of approximately 170 mgrl and a Ca
2q
concentration of approximately 40 mgrl. Supple- mental aeration was provided via airstone diffusers to reach a dissolved oxygen
concentration of at least 7.0 mgrl dissolved oxygen. The temperature was maintained at 22–24
8C. Hardness, pH, ammonia, chlorine, temperature and dissolved oxygen were measured weekly. Water conditions were closely maintained at levels recommended for
Ž .
the striped bass Nicholson et al., 1990 . The photoperiod was set at 12-h light:12-h dark. Tanks were cleaned of solid debris twice a week. Animal handling and rearing
conditions followed an approved protocol by the University of Maryland Animal Care and Use Committee.
2.2. Diets Three dietary treatments were fed to 10 tanks of striped bass for a period of 6 weeks
Ž .
in a completely randomized design. The basal diet Table 1 , which was fed to four replicate tanks of fish, was nutritionally complete and was based on plant feedstuffs. The
two test diets were fed to three replicate tanks each and were identical to the basal diet Ž
. Ž
. Ž
with the exception that 2 2 FS and 4 4 FS of the plant feedstuffs isolated .
soy protein, soybean meal, corn gluten meal and wheat middlings were replaced by an Ž
.
X
isocaloric and isonitrogenous by calculation mixture of
L
-alanine,
L
-serine, inosine-5 - Ž
. monophosphate and betaine Table 1 . The diets were formulated to meet all known
Ž .
nutrient requirements for Morone species Gatlin, 1997; Small and Soares, 1998 . The Ž
. nutrient requirements established for chinook salmon NRC, 1993 were used where
information on striped bass was lacking. The dietary ingredients were mixed and cold Ž
. pelleted in a laboratory pellet mill CPM, San Francisco, CA . The four chemicals were
Ž added to the diets at equimolar concentrations to reach 2 and 4 of the diet 0.03 and
. 0.06 M, respectively . Just prior to pelleting, the chemicals were dissolved in distilled
Ž .
water 20 by weight of the diet and mixed into the diet. Following pelleting, the diets were dried overnight in a forced-draft oven at 60
8C. 2.3. Experimental animals
Ž .
Striped bass Morone saxatilis were obtained from the Blackwater Fisheries Re-
Ž .
search and Development Center Holt, FL and were acclimated in our tanks for a period Ž
. of 30 days. Striped bass initial mean weight: 11.8 g were stocked in ten 400 l tanks at
Ž .
a density of 38 fishrtank biomass: 1.12 grl . Fish were fed twice daily to satiation Ž
. 0900 and 1700 h
and feed intake was recorded. The fish were weighed at the
Table 1 Ž
. Ž
. Ingredient composition , as fed and calculated protein and digestible energy levels dry matter of the
Ž .
Ž .
basal, 2 feeding stimulant mix 2 FS , and 4 feeding stimulant mix 4 FS supplemented diets fed to striped bass for 6 weeks
Ingredients Basal
2 SM 4 SM
Wheat middlings 21.00
20.50 20.00
Soybean meal 20.70
20.20 19.70
Corn gluten meal 20.25
19.75 19.25
Ž .
Soybean protein concentrate 70 6.00
5.50 5.00
Feather meal 10.00
10.00 10.00
Blood flour 5.00
5.00 5.00
a
Fish oil 11.00
11.00 11.00
Lignin sulfonate 1.00
1.00 1.00
b
Mineral mix 1.20
1.20 1.20
c
Vitamin mix 1.00
1.00 1.00
Calcium phosphate 1.50
1.50 1.50
Ž .
Choline chloride 77 0.25
0.25 0.25
Lysine HCl 0.60
0.60 0.60
DL Methionine 0.50
0.50 0.50
d,e
Stimulant mix –
2.00 4.00
Ž . Protein
48.13 48.82
49.52
f
Ž .
Digestible energy kcalrkg 3827
3829 3830
a
Ž .
Omega Protein Reedville, VA .
b
Ž .
Mineral mix contains mgrkg of diet : KCl, 5200; NaCl, 3600; MgSO , 1640; FeC H O , 92; MnSO ,
4 6
5 7
4
80; ZnCO , 100; CuSO , 3.2; KI, 0.4; Na SeO , 0.328.
3 4
2 3
c
Ž .
Vitamin mix contains mgrkg of diet : ascorbic acid, 400.0; inositol, 440.0; Santoquin, 140.0; niacin, 170.0; alpha-tocopheryl acetate, 500.0; phylloquinone, 15.0; riboflavin, 23.0; thiamin, 14.0; pantothenic acid,
Ž .
56.0; pyridoxine, 14.0; folic acid, 6.0; biotin, 5.0; cyanocobalamin 3000 ugrg , 19.5 ugrkg; retinol ester Ž
. 500,000 Urg , 2750; cholecalciferol, 5 ugrkg; cerelose as a filler.
d
Ž .
X
Stimulant mix in 2 FS contains grkg of diet :
L
-alanine, 2.71;
L
-serine, 3.20; inosine-5 -monophos- phate, 10.53; betaine, 3.56.
e
Ž .
X
Stimulant mix in 4 FS contains grkg of diet :
L
-alanine, 5.42;
L
-serine, 6.40; inosine-5 -monophos- phate, 21.06; betaine, 7.12.
f
Ž .
Calculated from published values NRC, 1993 .
beginning of the study and every 2 weeks thereafter. Feed intake, growth and feed Ž
. conversion ratios FCR were calculated throughout the course of the study.
2.4. Experimental design and statistical analyses A completely randomized design was used. Data were analyzed by one-way ANOVA
Ž .
using SAS software SAS Institute, 1992 . The data were examined for meeting the Ž
. Ž .
assumptions of the analysis of variance ANOVA Sokal and Rohlf, 1987 . For the feed
intake and weight gain data, a repeated measures ANOVA was performed and LSD pairwise contrasts were used to compare the means at each period. Pairwise contrasts
Ž .
LSD were also used for the FCR data. Results were considered statistically significant at the 5 level.
3. Results
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