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Identification of feeding stimulants for striped bass,
Morone saxatilis
Elias Papatryphon, Joseph H. Soares Jr.
)Graduate Program in Marine – Estuarine and EnÕironmental Sciences and Department of Animal and AÕian
Sciences, UniÕersity of Maryland, College Park, MD, 20742-2311, USA
Received 9 August 1999; received in revised form 28 October 1999; accepted 29 October 1999
Abstract
A series of experiments were conducted to screen amino acids and other compounds, presented
Ž . Ž
individually or in mixtures, as feeding stimulants FS for striped bass. Two bioassays experiment
.
series A and B were conducted. The FS carrier in series A was a nutritionally complete plant feedstuff-based diet whereas in series B, the basal was an agar-gel matrix. The second bioassay proved to be the more sensitive testing and delivery system. Striped bass had a maximum response to the neutral amino acids, and particularly toL-alanine andL-serine. Inosine-5X-monophosphate
and betaine were the only non-amino acid compounds that elicited a significant positive response. All combinations tested ofL-alanine, L-serine, inosine-5X-monophosphate and betaine stimulated
feed intake for striped bass, the maximum response being attained when all four compounds were included together in the feed. Additive andror synergistic effects between the compounds may have accounted for the enhanced response of the mixtures. q2000 Elsevier Science B.V. All
rights reserved.
Keywords: Feeding; Stimulants; Striped bass; Palatability
1. Introduction
Striped bass is an important food fish in the US and is currently being cultured
Ž
commercially for human consumption along with hybrid striped bass Harrell and
)Corresponding author. Tel.:q1-301-405-5785; fax:q1-301-314-9059.
Ž .
E-mail address: [email protected] J.H. Soares Jr. .
0044-8486r00r$ - see front matterq2000 Elsevier Science B.V. All rights reserved. Ž .
.
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
ŽBaltimore, 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
.
hardness of approximately 170 mgrl and a Ca2qconcentration 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
ŽAla , valine Val , methionine Met , isoleucine Ile , leucine Leu , tyrosine Tyr ,. Ž . Ž . Ž . Ž . Ž .
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
ŽTable 1 : all 20 AA 20AA , basic AA BAA: Lys, His, Arg , acidic AA AAA: Glu,. Ž . Ž . Ž
. Ž . Ž .
Asp , aromatic AA ARAA: Tyr, Trp, Phe , sulphur AA SAA: Met, Cys , amide
Ž . Ž .
Table 1
Percent composition of basala,bdiets fed to striped bass in Experiment series A
Ingredients Basal
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.
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
Ž500,000 Urg , 2750; cholecalciferol, 5 ugrkg; cerelose as a filler..
Ž . Ž
non-essential neutral AA NENAA: Ala, Ser, Pro, Gly and neutral AA NAA:
.
ENAAqNENAA . 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 .
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 RFIs 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 .
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 .
ŽTau , United States Biochemical, Cleveland, OH and menhaden fish solubles at 2%. Ž . Ždonated 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
Ž40–428C , 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-.
3. Results
3.1. Experiment series A
Table 3 summarizes the effects of the various amino acid based test solutions on the
Ž .
FI of striped bass. Significant P-0.05 increases in feed intake over the basal were
Ž .
achieved with the MHI and NAA treatments RFIs161.4 and 174.5%, respectively . When the NAA group was further subdivided to the NENAA and the ENAA groups,
Ž . Ž
both yielded a significant P-0.05 increase in feed intake RFIs128.3 and 124.3%,
. Ž .
respectively over the basal, equal to that of the NAA group 121.5% . Significant
ŽP-0.05 decreases in feed intake over the basal were observed with the SAA and.
Ž .
AAA treatments RFIs71.5 and 64.8%, respectively .
3.2. Experiment series B
Ž .
In experiment B1 Table 4 , significant increases in feed intake over the agar-gel
Ž . Ž .
basal were observed in fish fed 0.5% FM P-0.03 , AMAA P-0.03 , AAA
ŽP-0.01 , ENAA. ŽP-0.05 , fish solubles. ŽP-0.0003 , NAA. ŽP-0.0001 , NE-.
Ž . Ž .
NAA P-0.0001 and 20AA P-0.0001 . Fish fed the 20AA supplemented diet had a significantly higher intake when compared to all treatments except to the NENAA treatment. In experiment B2, from the neutral amino acid groups, only the NENAA
Ž .
group elicited a significant positive response P-0.003 , although the full complement
Table 3
Ž . Ž .a
Average feed intake FI in % body weightrday and relative feed intake RFI of striped bass fed either the
Ž . Ž .
basal or the test diets in series A experiments A1, A2, A3 and A4 . Means ns3 sharing the same letter are
Ž .
not significantly different by LSD comparison P-0.05 b
Diet Experiment A1 Experiment A2 Experiment A3 Experiment A4
Ž . Ž . Ž . Ž .
FI RFI % FI RFI % FI RFI % FI RFI %
Basal 1.53a 100.0 2.27b 100.0 2.70b 100.0 2.47a 100.0
MHI 2.47b 161.4 – – – – – –
Pooled SEM 0.21 0.17 0.23 0.17
a
Ž .
RFIstest diet FIrbasal FI=100; no statistical analyses conducted. b
Table 4
Ž . Ž . Ž .a
Feed intake FI , probability of being significantly different from the basal P and relative feed intake RFI
Ž . Ž .
of striped bass fed either the basal or the test diets experiment B1 . Geometric means ns4; mean"SE
Ž .
sharing the same letter are not significantly different by LSD comparison P-0.05
b Ž . Ž .
Diet FI % body weightrday P Relative intake %
Basal 0.25–0.40a – 100.0
FM 0.5% 0.49–0.79 b 0.0275 196.9
AMAA 0.50–0.80b 0.0265 196.9
Fish solubles 2% 0.82–1.86cde 0.0003 371.9
NAA 1.01–1.63de 0.0001 403.1
NENAA 1.50–2.42ef 0.0001 596.9
20AA 2.02–3.27f 0.0001 803.1
a Ž .
RFIsmean test diet FIrmean basal FI=100; no statistical analyses conducted. b
Average feed intake FI , probability of being significantly different from the basal P and relative feed intake
ŽRFI.a of striped bass fed either the basal or the test diets experiment B2 . Geometric meansŽ . Žns7;
. Ž .
mean"SE sharing the same letter are not significantly different by LSD comparison P-0.05
b Ž . Ž .
Diet FI % body weightrday P Relative intake %
Basal 1.16–1.86bcd – 100.0
RFIsmean test diet FIrmean basal FI=100; no statistical analyses conducted. b
Abbreviations: see text. c
Table 6
Ž . Ž .
Average feed intake FI , probability of being significantly different from the basal P and relative feed intake
ŽRFI.aof striped bass fed either the basal or the test diets experiment B3 . Means ns6 sharing the sameŽ . Ž .
Ž .
letter are not significantly different by LSD comparison P-0.05
b Ž . Ž .
Diet FI % body weightrday P Relative intake %
Basal 2.77a – 100.00
RFIsmean test diet FIrmean basal FI=100; no statistical analyses conducted. b
Abbreviations: see text. c
Omega Protein, Reedville, VA.
Ž .
of the NAA also increased FI marginally P-0.06, Table 5 . When the NENAA group was further subdivided to its constituents, significant increases in FI over the basal were
Ž . Ž .
observed in fish fed Ala P-0.003 and Ser P-0.02 . In addition, only IMP
ŽP-0.006 and Bet P. Ž -0.05 significantly increased the FI of bass from the individ-.
Ž . Ž .
ual compounds tested Table 5 . Fish fed FM 15% had the highest feed intake, being
Ž .
significantly higher P-0.05 than all other treatments except from fish fed the
Ž . Ž .
complete set of the amino acids 20AA . Results from experiment B3 Table 6 indicate that all treatments were significantly different from the basal. Fish fed a mixture of Ala,
Ž .
Ser, IMP and Bet or FM 15% performed better than fish fed 20AA, NENAA or a
Ž . Ž .
mixture of Ala and Ser P-0.05 . In experiment B4 Table 7 , the highest feed consumption was obtained with bass fed the Ala-Ser-IMP-Bet mixture. Omission of
Ž .
either IMP or Bet resulted in non-significant decreases in feed intake P)0.05 , while
Ž .
omission of both Ala and Ser resulted in a significant decrease in feed intake P-0.05 .
Table 7
Ž . Ž .
Average feed intake FI , probability of being significantly different from the basal P and relative feed intake
Ž .a Ž . Ž .
RFI of striped bass fed either the basal or the test diets experiment B4 . Means ns10 sharing the same
Ž .
letter are not significantly different by LSD comparison P-0.05
b Ž . Ž .
Diet FI % body weightrday P Relative intake %
Basal 0.90a – 100.0
RFIsmean test diet FIrmean basal FI=100; no statistical analyses conducted. b
4. Discussion
Results from both bioassays indicate that the neutral amino acids stimulate the feeding behavior of striped bass. As indicated by the first bioassay, all groups containing neutral amino acids were as effective in stimulating feeding as the MHI prepared in our laboratory. Neutral amino acids have been identified as FS in a number of marine
Ž
teleosts Mackie and Mitchell, 1982; Mackie et al., 1980; Takeda et al., 1984; Takaoka
.
et al., 1990 . In addition, results from the second bioassay suggest that the effect of the neutral amino acids was due primarily to the non-essential group, namely Ala, Ser, Gly and Pro. Gly, Ala and Pro have all been implicated as major FS in a number of fish
Ž .
species Carr et al., 1996 . However, of the four amino acids mentioned above, only Ala and Ser resulted in a significant response when tested individually with striped bass.
Ž .
In another study conducted with striped bass Hughes, 1997 , Ala, Arg, Glu and Gly were tested for their effect on feed intake. From these compounds only Glu and Gly enhanced feed consumption when incorporated into a fish meal-based diet. In our experiments, Glu was fed in combination with Asp, forming the acidic amino acid group. In the first bioassay, the acidic amino acids resulted in a significant decrease in feed intake compared to the basal diet, whereas in the second bioassay they elicited a significant positive response over the agar-gel basal. Nevertheless, the response to the neutral amino acids was significantly higher than the response to the acidic amino acids in all our experiments, and therefore they were regarded as having a higher potential as practical FS.
It is important to emphasize that the response to chemical stimulation may be modified, to some extent, by the use of different carriers. A more complex carrier, such as a complete diet of variable composition, may mask the effects of certain compounds and enhance the effects of others. Such an effect would account, perhaps, for differences in the magnitude of the response to the 20AA, AAA, SAA, AMAA, BAA and ARAA between the two bioassays in our study and between different studies conducted by different researchers. The differences in intake between the basal and the test diets
Žrelative feed intake were more pronounced in the second bioassay when compared to.
the first, indicating that the complete diet was more palatable than the agar-gel. The basal diet in the first bioassay has inherent baseline palatability provided by its
Ž .
constituents including amino acids . The above information would suggest that the agar-gel might indeed act as a ‘‘neutral’’ carrier, as demonstrated by minimal
consump-Ž .
tion when presented unsupplemented this study; Adams and Johnsen, 1986 . It is also of interest to note that supplementation with the AAA increased feed intake in
Ž .
from the agar-gel bioassay, in complete diets before making recommendations for practical applications.
The responses of striped bass to individual compounds indicate that Ala is the most effective FS, followed by IMP, Ser and Bet. Each of the above mentioned compounds
Ž
have been reported as FS or attractants in a number of species Jones, 1992; Takeda and
.
Takii, 1992; Carr et al., 1996 . However, striped bass mostly resembles the puffer Fugu
Ž
pardalis, which is stimulated by Ala, Ser and Bet but also Gly and Pro Hidaka et al.,
.
1978; Ohsugi et al., 1978 , and the marbled rockfish Sebastiscus marmoratus, which is
Ž .
stimulated by Ala, Ser, IMP but also Met, Pro, and inosine Takaoka et al., 1990 . It is noteworthy that Ser has been reported as a feeding stimulant or attractant in all species that are also stimulated by Ala. This might be due to the similarity in the structures of the two compounds, differing only in a hydroxyl side group.
The adenosine containing nucleotides had a neutral to negative activity for striped bass. The negative activity seems to increase with increasing phosphate side groups, with the worst effect being observed with ATP. Similar results were obtained in a study
Ž .
with jack mackerel Trachurus japonica Ikeda et al., 1988 , where AMP had no FS activity whereas ADP and ATP acted as deterrents. Likewise, in a study with eel
ŽTakeda et al., 1984 , AMP was reported as a feeding deterrent..
Citric acid also reduced feed intake of striped bass in our study, although not significantly. By contrast, citric acid was the most potent FS in the herbivorous tilapia T.
Ž
zillii, which was found to respond primarily to acidic compounds Johnsen and Adams,
.
1986; Adams et al., 1988 .
The agar-gel supplemented with 0.5% FM was not effective enough to serve as a positive control, although it elicited significantly higher feed intake when compared to the basal. However, the 15% FM diet was very palatable, rendering it an adequate positive control. The palatability of the 15% FM supplemented diet could also be attributed to its texture, which was clearly different from the other gels. Supplementation with 2% fish solubles resulted in a significant increase in feed consumption over the basal, approaching in magnitude the effect of the NENAA, but not that of the complete 20AA mix. Fish solubles have been used in the past in diet formulations for carnivorous fish as flavor enhancers with varying success. A 2% fish solubles supplementation in a fish meal based diet for the red drum did not have any beneficial effect on performance
ŽDavis et al., 1995 . On the other hand, 2% or 4% supplementation with fish solubles. Ž
improved feed intake of red drum fed a soybean meal based diet McGoogan and Gatlin,
.
1997 . Fish solubles might be a potential candidate as a flavor enhancer in the absence of more detailed information, but it was not as effective as other stimulants for striped bass. Furthermore, there are no data on its effectiveness when incorporated in a complete diet.
It is demonstrated here that the effects of Ala and Ser can simulate the effects of the NENAA and 20AA. Combining Ala and Ser did not seem to result in any further increase in feed intake than when either substance was fed individually at the levels tested. Combinations of Ala, Ser, IMP and Bet, however, yielded the highest feed intake,
Ž .
comparable to the intake of fish fed the positive control diet 15% FM . Omitting either
Ž .
Ž . Ž .
fraction Ala and Ser or the non-amino acid fraction IMP and Bet reduced the feed
Ž .
consumption rates significantly by 26.3 and 42.3%, respectively . It appears that combining Ala, Ser, IMP and Bet resulted in sufficient increases in feed intake that can be attributed to additive effects or synergistic interactions between some or all of the compounds. Further work is currently being conducted in our laboratory to determine the optimal combination and possible interactions of these compounds. However, our data appear to be consistent with the suggestion that mixtures of compounds rather than
Ž
single substances are more effective in stimulating feeding behavior in fish Carr and
.
Derby, 1986 .
Ž . Ž .
We suggest that two simple, neutral amino acids Ala and Ser , a nucleotide IMP
Ž .
and a quaternary ammonium base Bet produce maximum feeding stimulation in striped bass at the concentrations tested. It is of interest that Ala and Bet are among the most prominent organic solutes in marine mollusks and crustaceans, while Ala and IMP are
Ž .
very prominent in fish Carr et al., 1996 . The above information might indicate that these substances serve as gustatory cues to the carnivorous striped bass, which naturally
Ž .
feeds on a fishrmolluskrcrustacean-based diet Hartman and Brandt, 1995 . It is noteworthy, that there are a certain number of compounds that stimulate the feeding behavior of most species; but specificity and sensitivity varies for each individual species, possibly due to differences in their evolutionary and ecological backgrounds. As a result, it is important that studies on each species are conducted to identify the most potent FS, the concentrations at which they are most effective, as well as any multi-factorial effects or interactions that might exist. Nevertheless, it should be noted that some of the experimental differences might be a result of using different carriers rather than actual biological responses.
Although a substantial number of compounds were screened in these assays, it should be noted that our intent was not to identify every possible compound that stimulates the feeding behavior of striped bass, but rather to identify FS from a number of compounds commonly found in fish and marine invertebrates and attempt to develop the most effective stimulant mixture of these substances. It is of significance that similar results were obtained in the studies described herein with fish of different age and size and with the use of different carriers. In addition, Ala, Ser, IMP and Bet were repeatedly effective in eliciting enhanced feed intake, validating their positive effects. We believe that the stimulants identified herein may be of importance in enhancing the feed intake of striped bass, especially when fed diets of low palatability.
Acknowledgements
Special thanks to Rachel Howell, Dan Brougher and Brian Small for their help during the conduct of these experiments. Appreciation is extended to Larry Douglass for his valuable comments on statistical analysis, to Curry Woods for providing the fish and to Tim Shellem for performing the amino acid analyses. Thanks also goes to Omega Protein for providing us with fish meal, fish solubles and fish oil and Hilton Davis for donating the dye. This research was supported by a grant from the Maryland Agriculture
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