. Webster, 1997 . Several nutritional studies have been conducted with striped bass and its
hybrids, particularly focusing on practical diet evaluations, nutrient requirements and nutrient availability. To our knowledge, there is only one published study on feeding
Ž .
Ž .
stimulants FS for striped bass Hughes, 1997 . The identification of FS is valuable both from a practical and a scientific standpoint. Previous research has demonstrated that
FS supplementation can enhance the acceptance of artificial feeds by larvae and fry ŽMeteiller et al., 1983; Person-Le Ruyet et al., 1983; Hughes, 1991; Kamstra and
. Ž
Heinsbroek, 1991 , increase the consumption of poorly palatable feeds Takeda and .
Ž . Ž
Takii, 1992 , increase feed consumption thus leading to higher growth rates Heinsbroek .
and Krueger, 1992 , and may be included in lures for commercial and recreational Ž
. fishing Jones, 1992 . Using FS to increase the palatability of formulated feeds for fish
can therefore result in an increase in total feed intake leading to a reduction of overall feeding time and subsequently feed wastage.
Compounds that have been associated with gustation in carnivorous fish are amino acids, nucleotides, quaternary ammonium compounds, organic acids and other low
Ž .
molecular weight components of animal tissues Carr et al., 1996 . Mixtures of FS have Ž
been reported to be more effective than individual compounds Takeda et al., 1984; Carr .
and Derby, 1986 . In addition, natural prey extracts, as well as their synthetic counter- parts, are very effective FS for a variety of fish species and have been used as references
in studies where the goal was to identify the simplest mixture or individual compounds Ž
that would yield a maximum feeding response Carr, 1976; Mackie et al., 1980; Ikeda et .
al., 1988; Fukuda et al., 1989 . Of great importance when screening for FS is the sensitivity and duration of the bioassays, especially when a large number of compounds
are to be tested. Although it is difficult to separate responses due to olfaction from those due to gustation in fish, it is believed that the final step in the feeding behavior, that of
Ž ingestion, is a response to the palatability of the food item and is gustatory based Marui
. and Caprio, 1992; Hara, 1994 . The purpose of this study was to identify the amino acid
Ž component responsible for the feeding stimulatory activity of a menhaden BreÕoortia
. tyrannus homogenate prepared in our laboratory; to evaluate the use of an agar-gel
carrier as a rapid and sensitive assay; to screen a number of compounds for FS activity; and to develop a simple yet effective FS mixture for striped bass.
2. Materials and methods
2.1. EnÕironmental All fish were obtained from the University of Maryland’s Crane Aquaculture facility
Ž .
Baltimore, MD and were acclimated to our environment for 30 days before the onset of the experiments. 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 Ž
activated carbon filter to remove chlorine. An electrolyte solution of CaCl :NaCl 9r1,
2
. wrw was added continuously to increase the electrolyte concentration and maintain a
hardness of approximately 170 mgrl and a Ca
2q
concentration of approximately 40 mgrl. Supplemental aeration was provided via airstone diffusers to reach a minimum
dissolved oxygen concentration of 7.0 mgrl. The temperature was maintained at 20–228C and the levels of hardness, pH, ammonia, chlorine, temperature and dissolved
oxygen were measured weekly. Water conditions were closely maintained at levels
Ž .
recommended for 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. Experiment series A Ž
. Striped bass, Morone saxatilis initial mean weight: 21 g were stocked in twelve 200
Ž .
l round fiberglass tanks, with an average density of 17 fishrtank 1.79 grl . Four Ž
. experiments A1, A2, A3 and A4 were conducted to evaluate the 20 common amino
Ž .
acids AA for their efficacy as feeding stimulants for striped bass, using an aqueous Ž
. extract of a menhaden homogenate MHI as a reference. A nutritionally complete basal
Ž .
diet was formulated based on plant feedstuffs basal I, Table 1 . In experiment A4, the Ž
. basal diet was modified basal II, Table 1 , mainly by the incorporation of a higher level
of casein, in order to adjust for the increased acceptance of basal I over time. Both diets Ž
were formulated to meet all known nutrient requirements for Morone species Gatlin, .
1997; Small and Soares, 1998 . The nutrient requirements established for chinook Ž
. salmon NRC, 1993 were used where information on striped bass was lacking. The
Ž dietary ingredients were mixed and extruded using a 1-mm die benchtop extruder,
. model 544rPE, C.W. Brabender Instruments, South Hackensack, NJ . Prior to extru-
Ž .
sion, distilled water was added at 15 of the diet w:w . The pellets were cut to similar Ž
. sizes approximately 1 cm in length before the application of the test solutions.
An aqueous menhaden homogenate was prepared to serve as the reference and provided the ratios of the individual amino acid concentrations used in the test solutions.
Ž .
Menhaden fish were ground in water Osterizer; 1:1, w:v , until a homogeneous,
liquified mixture was reached. The homogenate was centrifuged at 3000 = g for 10 min at 48C to remove the solids. The supernatant was then stored at y208C until analyzed.
The sample was hydrolyzed in duplicate in 6 M HCl at 1108C followed by chromato- Ž
. graphic separation using a Dionex D-600 amino acid analyzer Dionex, Smyrna, GA .
Ž .
Ž .
Ž .
The homogenate was analyzed Table 1
for cysteine Cys , aspartic acid
Asp , Ž
. Ž
. Ž
. Ž
. Ž
. threonine Thr , serine Ser , glutamic acid Glu , proline Pro , glycine Gly , alanine
Ž .
Ž .
Ž .
Ž .
Ž .
Ž .
Ala , valine Val , methionine Met , isoleucine Ile , leucine Leu , tyrosine Tyr , Ž
. Ž
. Ž
. Ž
. Ž
. phenylalanine Phe , lysine Lys , histidine His , arginine Arg and ammonia NH .
3
Ž .
Ž .
Ž .
Values for asparagine Asn , glutamine Gln and tryptophan Trp were estimated from Ž
. published literature values Carr et al., 1996 . To facilitate the screening process, amino
acids were categorized in nine groups according to their molecular structure and pH Ž
. Ž
. Ž
. Ž
Table 1 : all 20 AA 20AA , basic AA BAA: Lys, His, Arg , acidic AA AAA: Glu, .
Ž .
Ž .
Asp , aromatic AA ARAA: Tyr, Trp, Phe , sulphur AA SAA: Met, Cys , amide Ž
. Ž
. containing AA AMAA: Gln, Asn , essential neutral AA ENAA: Leu, Ile, Val, Thr ,
Table 1 Percent composition of basal
a,b
diets fed to striped bass in Experiment series A Ingredients
Basal I
II Soybean meal
13.25 –
Corn gluten meal 10.00
25.00 Wheat middlings
23.00 15.00
Wheat flour 10.00
15.75 Isolated soy protein
19.00 –
Casein 10.00
30.00
c
Fish oil 8.00
8.00 Lignin sulfonate
2.00 2.00
d
Mineral mix 2.00
2.00
e
Vitamin mix 1.00
1.00 Calcium phosphate, dibasic
1.50 1.00
Choline chloride, 77 0.25
0.25
a
For the preparation of the experimental diets the amino acids were added at final concentrations based on Ž
. MHI as diet : Ala, 0.18; Ser, 0.10; Pro, 0.11; Gly, 0.14; Leu, 0.20; Ile, 0.13; Val, 0.17; Thr, 0.11; Glu,
0.25; Asp, 0.23; Lys, 0.20; His, 0.10; Arg, 0.09; Tyr, 0.07; Trp, 0.03; Phe, 0.12; Met, 0.07; Cys, 0.10; Gln, 0.05; Asn, 0.02.
b
The amino acids were grouped in nine categories for preparing the respective test solutions according to molecular structure and pH: AAA, BAA, ARAA, SAA, AMAA, ENAA, NENAA, NAA, 20AA. For the
abbreviations see text.
c
Omega, Reedville, VA, USA.
d
Ž .
Mineral mix contains as mgrkg of diet : KCl, 5200; NaCl, 3600; MgSO , 1640; FeC H O , 92;
4 6
5 7
MnSO , 80; ZnCO , 100; CuSO , 3.2; KI, 0.4; Na SeO , 0.328.
4 3
4 2
3 e
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 3,000 ugrg , 19.5 ugrkg; retinol ester Ž
. 500,000 Urg , 2750; cholecalciferol, 5 ugrkg; cerelose as a filler.
Ž .
Ž non-essential neutral AA
NENAA: Ala, Ser, Pro, Gly and neutral AA
NAA: .
ENAA q NENAA . The test solutions were prepared by mixing the corresponding Ž
. concentrations for each amino acid purchased from Sigma, St. Louis, MO in deionized
water and subsequently adjusting the pH to 6.8–7.0 with 0.1 N NaOH or 4 M acetic acid. The mixtures were then stored at y208C until 24 h before being used in an
experiment, when they were thawed and sprayed onto the diets. For each test diet, 100 g
Ž .
of the basal diet were sprayed with 10 ml of the test solution or deionized water basal Ž
. to reach final concentrations as found in MHI Table 1 . The diet was then allowed to
dry overnight at room temperature. The MHI was also added at 10 of the diet, and as a result the levels of the amino acids in the MHI supplemented diet were ten times lower
than the diets supplemented with the synthetic mixtures of FS. The MHI was, however, very effective as a FS for striped bass at this level.
Ž .
In all experiments, four treatments three replicates per treatment were fed twice daily to striped bass for a five day period. Feed was administered via a spatula to
minimize contamination, one pellet at a time. It was assumed that the fish had become Ž .
satiated when three consecutive pellets were either rejected or avoided. Feed intake FI Ž
. was recorded at each feeding g fedrtank . Fish were weighed at the end of each
Ž .
experiment in order to express FI as a percent of body weight per day BWrday to Ž
Ž account for differences in feeding based on weight. Relative feed intake RFI s test diet
. .
FIrbasal diet FI = 100 was also calculated as a means of standardizing FI across the Ž
. different assays. The specific treatments at each experiment were: A1 basal, 20AA,
Ž .
Ž .
NAA and MHI; A2 basal, BAA, AAA and ARAA; A3 basal, 20AA, SAA and Ž
. AMAA; A4 basal, NAA, ENAA and NENAA. In experiment A4 the NAA group was
further broken down to ENAA and NENAA, because the NAA was found to be the only group which elicited a positive feeding response.
2.3. Experiment series B Ž
. A series of four experiments B1, B2, B3 and B4 were conducted utilizing an agar
Ž .
Ž .
gel matrix as a carrier Table 2 . Striped bass mean weight, 56.7 g were stocked in Ž
. 200-l tanks at a density of 11 fish per tank for experiment B1 biomass: 3.12 grl .
Ž .
Experiments B2, B3 and B4 were conducted with striped bass mean weight, 2.7 g Ž
. stocked in 10-l tanks at a density of eight fish per tank biomass: 2.16 grl .
In experiment B1, the amino acid groups mentioned above were screened to evaluate Ž
. the use of the agar-gel carrier Noble, Difco Lab., Detroit, MI which had previously
Ž .
been used for Tilapia zillii Adams and Johnsen, 1986 , as a rapid and sensitive assay for striped bass. The purpose of experiment B2 was to isolate the ‘‘active’’ compounds
Ž .
from the most potent amino acid mixture NENAA
and to screen 10 additional
X
Ž .
X
compounds as FS for striped bass: adenine-5 -monophospate AMP , adenine-5 -diphos- Ž
.
X
Ž .
Ž .
Ž .
phate ADP , adenine-5 -triphosphate ATP , citric acid Cit , lactic acid Lac and Ž
. Ž .
X
Ž trimethylamine N-oxide TMAO , Sigma ; inosine-5 -monophosphate IMP; disodium
. Ž .
Ž .
salt, hydrated , Fisher Scientific, Pittsburgh, PA ; betaine Bet; anhydrous and taurine
Table 2 Ž
. Design of experiments for series B experiments B1, B2, B3 and B4 : diets fed, number of tanks used, density
Ž . of fish, duration of each trial and number of replicates n
a,b
Ž .
Ž .
Experiment Diets
Tanks Density grl
Duration days n
Ž .
B1 basal, FM 0.5 , 20AA, AAA, BAA,
12 3.1
4 4
AMAA, ARAA, SAA, NAA, ENAA, Ž
. NENAA, FS 2
Ž .
B2 basal, FM 15 , 20AA, NAA, ENAA,
19 2.2
7 7
NENAA, Ala, Ser, Pro, Gly, Bet, IMP, AMP, ADP, ATP, Cit, Lac, Tau, TMAO
Ž .
B3 basal, FM 15 , 20AA, NENAA,
6 2.2
6 6
Ala-Ser-IMP-Bet, Ala-Ser B4
basal, Ala-Ser-IMP-Bet, Ala-Ser-IMP, 5
2.2 10
10 Ala-Ser-Bet, IMP-Bet
a
Abbreviations: see text.
b
Ž .
The amino acids in mixtures were added at final concentrations based on MHII as diet : Ala, 0.15; Ser, 0.10; Pro, 0.10; Gly, 0.13; Leu, 0.19; Ile, 0.11; Val, 0.12; Thr, 0.11; Glu, 0.34; Asp, 0.23; Lys, 0.19; His,
0.06; Arg, 0.11; Tyr, 0.08; Trp, 0.03; Phe, 0.10; Met, 0.07; Cys, 0.02; Gln, 0.05; Asn, 0.02. All compounds added individually where included at a final concentration of 0.1 M each. In experiments B3 and B4 Ala, Ser,
IMP and Bet were included at 0.1 M each.
Ž . Ž
. Tau , United States Biochemical, Cleveland, OH and menhaden fish solubles at 2
Ž .
donated by Omega Protein, Reedville, VA . Finally, in experiments B3 and B4 an attempt was made to develop the most effective FS mixture based on results from
Ž .
previous experiments. Menhaden fish meal FM, Omega Protein at 0.5 was used as a positive control in experiment B1, but because of its low stimulant activity it was
Ž .
replaced by 15 FM in subsequent experiments B2, B3 and B4 . Ž
. A second menhaden homogenate MHII was prepared and analyzed as previously
described. The ratios from the two homogenates were similar for most amino acids and the new amino acid ratios were taken into consideration when formulating the test
Ž .
solutions for the amino acid groups Table 2 . The test gels were prepared by adding 5 ml from the prepared stock solutions to 45 ml of 2 agar before reaching gelatinization
Ž .
40–428C , to yield a 10-fold dilution of the test solutions. Single compounds were added at a final concentration of 0.1 M in experiment B2, B3 and B4 in order to
compare them on the same basis and to approximate the concentrations of the more complex mixtures.
Ž .
A brown dye Brown Shade 216, Hilton Davis, Cincinnati, OH was added at 8 mgrml in order to make the pellets visible to the fish and the person feeding them. In
contrast to experiment series A, the pH of the solutions was not neutralized in order to examine the effects of the compounds at their respective pH. The gel was poured into
petri dishes and allowed to completely gelatinize. The disk was subsequently cut to similar sized pieces, 2–3 mm for small fish and 5–7 mm for larger fish. The gels were
freshly prepared before each experiment and during the experiments when more was needed. All gels were stored at 48C prior to feeding. Fish were weighed at the end of
each experiment. Fish were fed twice daily, the gel test diets in the morning and a generic fish meal based diet in the afternoon. Feed intake was recorded in the morning
and expressed as BWrday and RFI, whereas fish were fed to satiation in the afternoon to ensure high consumption compliance.
2.4. Experimental design and statistical analyses The experiments in series A were one-way completely randomized designs. The
experiments in series B were designed as Latin rectangles, the test diets being rotated among the tanks every day. Data from all experiments were analyzed using analysis of
Ž . Ž
. variance ANOVA
SAS Institute, 1992 . Day and tank were included as random effects in the model for the experiments in series B in order to exclude the variance of
the responses due to random effects. The data were examined for meeting the assump- Ž
. tions of the ANOVA Sokal and Rohlf, 1987 . The FI data of experiments B1 and B2
were not normally distributed so they were log transformed and comparisons were made Ž
. on the transformed data. The geometric means inverse log are thus presented for the
transformed data. For the proper presentation of the standard errors they were first added in the logarithmic scale and then the inverse log was calculated. When the ANOVA
Ž yielded a significant effect of the dependent variable, pairwise contrasts LSD compari-
. son were used to identify significant differences at the 5 level for all data. In addition,
for series B, the level of significance is presented for the pairwise comparisons of all treatments to the basal.
3. Results
