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Purdue University, Department of Forestry and Natural Resources, West Lafayette, IN 47907-1159
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • yellow perch • methionine • sulfur amino acid requirement • cyst(e)ine
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Total sulfur amino acid
(TSAA)3
requirements of several species of fish have been reported. Requirement estimates range from 1.9 g of TSAA/100 g of dietary protein for
rainbow trout to 4.0 g of TSAA/100 g dietary protein for gilthead
sea bream (Sparus aurata) (NRC 1993
).
Cyst(e)ine can spare the dietary methionine requirement of fishes. Most
of those values are in the range of 40–60% (Griffin et al. 1994a
, Harding et al. 1977
, Moon and Gatlin 1991
). However, TSAA requirements of fish have been
estimated using diets that contained graded concentrations of
L-methionine and a constant amount of cyst(e)ine, usually
in relatively high ratios of methionine/cyst(e)ine. Thus, it is
difficult to state that any of the sulfur amino acid requirement
estimates for fish are actually requirements for total sulfur amino
acids and not simply methionine. Further, there are no estimates of the
methionine requirement of yellow perch (Perca flavescens)
and only one other estimate for a coolwater species of fish
(Griffin et al. 1994a
).
Brown et al. (1996)
reported that yellow perch fed a purified diet grew
as rapidly as those fed commercial trout feeds. Results of that study
indicated that a purified diet containing relatively high levels of
crystalline L-amino acids was appropriate for further
nutritional research with yellow perch. The basal diet was subsequently
used to determine the dietary choline (Twibell and Brown, in press),
lysine (unpublished data from our laboratory) and arginine requirements
of yellow perch (Twibell and Brown 1997
). The basal diet
used in those experiments provided ~33 g of crude protein/100 g diet
(23 g of crude protein supplied by crystalline L-amino
acids and 10 g of crude protein supplied by casein and gelatin)
and 12 g of lipid/100 g diet. The utility of this diet in
quantifying dietary essential amino acid requirements of fish had been
demonstrated previously in studies with hybrid striped bass in our
laboratory (Brown et al. 1993
, Griffin et al. 1992
, 1994a
, 1994b
).
The objectives of these studies were to determine the dietary methionine requirement, the cyst(e)ine replacement (CR) value and the TSAA requirement of yellow perch using diets that provided the maximum proportion of cyst(e)ine.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The basal diet used in each experiment was formulated to provide
33.6 g of crude protein/100 g diet (Table 1
). Casein and gelatin served as intact protein sources and provided a
total of 10.1 g of crude protein/100 g diet, and 0.28 g of
TSAA/100 g diet [0.25 g of methionine/100 g diet and 0.03 g of
cyst(e)ine/100 g diet] based on NRC (1993)
values. A methionine- and
cyst(e)ine-free L-amino acid mixture (Table 2
) supplied 22.0 g of crude protein/100 g diet. The
L-glutamic acid was used to maintain isonitrogenous diets
and was incorporated at varying concentrations as additions of sulfur
amino acids were added to experimental diets. The L-amino
acid mixture was formulated so the basal diet contained 1.41 g of
arginine/100 g diet (Twibell and Brown 1997
) and
1.81 g of lysine/100 g diet (unpublished data from our
laboratory), thus meeting the dietary requirement of yellow perch for
these amino acids. The remaining essential amino acid concentrations
met or exceeded the highest known requirements for fish (NRC 1993
). The basal diet contained 12 g of lipid (menhaden
oil)/100 g diet and 20 g of carbohydrate (dextrin)/100 g diet.
Vitamins and minerals were added to the diets as nutritionally complete
premixes (Griffin et al. 1992
) at levels that met or
exceeded the highest reported requirements for fish. Dietary choline
concentration was maintained at 3730 mg/kg diet as choline chloride.
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).
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). Total sulfur amino acid concentrations were ~90% of the
requirement determined in the first experiment. As the cys/met were
calculated on an equal sulfur basis, the TSAA levels varied from
0.86 g to 0.96 g/100 g diet (2.6 g to 2.9 g/100 g dietary protein,
respectively).
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), which was determined to be the maximum cyst(e)ine replacement value
for methionine in the CR experiment. The diets contained TSAA
concentrations ranging from 0.5 to 1.2 g/100 g diet in gradations of
0.1 g/100 g diet.
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). The mixed ingredients were then made into 3.1-mm
diameter strands using the pelleting attachment of the Hobart mixer.
The diets were air-dried for 72 h and stored under
air-tight conditions at -20°C until needed. The diets were
ground and sifted into the appropriate size for feeding each day. Fish and experimental design.
Juvenile, all-female yellow perch used in the methionine requirement and CR experiments were obtained from a commercial producer (Coolwater Farms, Cambridge, WI) and transported to the Purdue University Aquaculture Research Facility. All fish were acclimated to laboratory conditions for 6 wk prior to initiation of the methionine requirement experiment. The fish not used in the methionine requirement experiment were maintained at 20°C for an additional 6 mo until needed for the CR experiment. All-female perch used in the TSAA experiment were obtained from the same source the following year and were acclimated to laboratory conditions for 6 mo prior to the experiment. Procedures used during transport, quarantine and experimental period were approved by the Purdue Animal Care and Use Committee (PACUC No. 89–060-98, "Nutritional Studies with Aquatic Animals," Principal Investigator Qualification No. BRO-249).
Two similar experimental systems were used during the studies. The closed recirculating systems contained either 24 or 28 individual 38-L aquaria. Each system was equipped with two submerged filtration tanks for solid material removal and denitrification of the water. Water was pumped through a sand filter to each aquarium at a rate of ~1 L/min. Water temperature was 23 ± 2°C throughout each experiment. The diurnal light/dark cycle remained at 16 h light/8 h dark throughout the study.
In each experiment, groups of 20 randomly chosen fish were stocked into each aquarium. Following an acclimation period, the number of fish in each tank was reduced to 15 so that fish of a similar weight remained in each tank. Mean weight per fish ranged from 4.4 to 4.9 g in the methionine requirement experiment, 19.1 to 19.4 g in the CR experiment and 7.9 to 8.2 g in the TSAA experiment. Fish were acclimated to the experimental systems for a minimum of 2 wk prior to each experiment and were fed a commercial trout diet during the first week of the acclimation period. Following the first week of the acclimation period, fish were fed their respective experimental diets for the remainder of each study. Dietary treatments were randomly assigned to triplicate aquaria in all studies. In the methionine requirement and CR experiments, fish were weighed every 14 d to adjust food allotment which was 3 g/(100 g body weight·d) offered in two equal meals. Food allotment was reduced to 2 g/(100 g body weight·d) after 4 wk in the CR experiment to more closely approximate satiation of the larger fish used in that study. The methionine requirement and CR experiments were conducted for 12 wk. Fish were fed to apparent satiation in the TSAA experiment, which was conducted for 10 wk.
Water-quality conditions were monitored daily and were similar to those
reported in previous research with yellow perch (Twibell and Brown 1997
). Dissolved oxygen was monitored with a YSI Model 55
oxygen meter (YSI, Yellow Springs, OH). Ammonia-nitrogen and
nitrite-nitrogen concentrations were measured with a HACH DREL/1C
portable colorimeter (HACH, Loveland, CO) using methods provided by the
manufacturer. Dissolved oxygen concentrations were not below 7.2 mg/L,
ammonia-nitrogen concentrations were not >0.4 mg/L and
nitrite-nitrogen did not exceed 0.2 mg/L at any point during the
study.
Serum and liver collection.
At the end of each study, all fish were anesthetized (tricaine
methanesulfonate; Argent Chemical, Redmond, WA) and weighed 24 h
after the final feeding. At the conclusion of the CR experiment, livers
from three randomly chosen fish were frozen at -20°C for subsequent
determination of lipid concentration. Lipid concentration of the livers
was determined by chloroform/methanol extraction (Folch et al. 1957
). In each experiment, blood was collected from a minimum
of three fish in each aquarium and pooled by replicate for
determination of serum methionine concentration. Serum was obtained
from pooled blood samples by centrifuging at 3000 x g for 20 min and deproteinized with HPLC-grade
acetonitrile. Serum amino acids were separated and quantified using a
Waters PicoTag system (Waters Chromatography Division, Millipore Corp.,
Milford, MA) following derivatization with phenylisothiocyanate
(Sarwar and Botting 1990
).
Statistical analyses.
Data were analyzed as a completely randomized design using the
Statistical Analysis System (1990)
and each aquarium as the
experimental unit. Analyses were conducted with dietary treatment as
the independent variable; accepted level of significance was 0.05.
Dietary methionine and TSAA requirements were estimated using
broken-line regression analyses of weight gain, feed efficiency
(FE) and feed consumption data (Robbins et al. 1979
). In
the CR experiment, broken-line analysis of feed consumption failed
to provide a realistic value and, while a reasonable value was
generated by broken-line analysis of weight gain, convergence
criteria were not met. Thus, Student-Newman-Keuls test separated mean
values in the CR experiment when significant differences were detected
by ANOVA.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. However, analysis of covariance indicated there was no significant
effect of initial fish weight on weight gain (P > 0.20) or FE (P > 0.27). Initial weights of fish were
not significantly different in the other two studies.
Graded additions of dietary L-methionine to the basal diet
resulted in significantly increased weight gain and FE of juvenile
yellow perch (Table 3)
. Broken-line analyses of weight gain and FE
data indicated the dietary requirement to be 1.0 and 1.1 g of
methionine/100 g diet (3.1 and 3.4 g of TSAA/100 g dietary
protein), respectively. Survival was lower in fish fed 0.37 g of
methionine/100 g diet compared to fish fed higher concentrations. Serum
methionine levels were not significantly affected by dietary methionine
concentration.
In the second experiment, varying dietary cys/met significantly
affected weight gain, FE, survival and liver lipid concentration (Table 4)
. Weight gain and FE of fish fed a dietary cys/met of 51:49 were
significantly higher than in fish fed cys/met of 70:30 and 64:36.
Survival increased as the cyst(e)ine concentration decreased to a
cys/met of 58:42. Liver lipid concentration of fish fed the highest
level of cyst(e)ine (cys/met 70:30) was lower than in fish fed any
other ratio. Serum methionine concentrations were not significantly
affected by dietary cys/met.
In the final experiment, graded additions of dietary TSAA resulted in
significant increase in weight gain, FE, feed intake and survival
(Table 5)
. Broken-line analysis of weight gain, FE and feed intake
data indicated that the dietary TSAA requirement of yellow perch was
0.85 g/100 g diet (2.5 g of TSAA/100 g dietary protein), 1.0 g/100 g
diet (3.0 g of TSAA/100 g of dietary protein) and 0.87 g/100 g diet
(2.6 g TSAA/100 g of dietary protein), respectively. Survival was
reduced in fish fed 0.5 and 0.6 g of TSAA/100 g diet. Serum
methionine concentration was not significantly affected by dietary
TSAA.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Jackson and Capper 1982
, Keembiyehetty and Gatlin 1993
, Moon and Gatlin 1991
, Nose 1979
, Rodehutscord et al. 1995
, Santiago and Lovell 1988
). However,
several reported methionine requirements for fish are in the range of
1.9–2.3 g/100 g of protein (Cowey et al. 1992
,
Griffin et al. 1994a
, Harding et al. 1977
). Given these lower values and the variety of species
represented, there are no clear predictors of methionine needs in fish
as a function of growth rates, optimal environmental conditions, food
habits or other variables. Thus, it appears that methionine needs of
each new aquaculture candidate must be quantified independently.
However, the maximum level of cyst(e)ine for sparing the dietary
methionine requirement has been within a relatively narrow range of
40–60% (Griffin et al. 1994a
, Harding et al. 1977
, Moon and Gatlin 1991
). When formulating
diets for new culture species, assuming a maximum contribution of
cyst(e)ine to the TSAA requirement of 30–40% appears a relatively
safe starting point.
Some variation among dietary methionine requirements is not surprising
considering the different feeding habits of fish studied thus far and
different dietary formulations used. All of the TSAA requirements for
fish were determined with diets that contained relatively little or no
cyst(e)ine. When methionine and cyst(e)ine are supplied in the diet at
ratios that maximize sparing of methionine, the requirement for TSAA
can be lower than the methionine requirement (Chung and Baker 1992
). When perch were fed graded levels of methionine in a
diet that contained 0.03 g of cyst(e)ine/100 g diet, the dietary
TSAA requirements were 1.0 and 1.1 g/100 g diet (3.1 and 3.4 g of
TSAA/100 g dietary protein). In the final experiment, dietary TSAA
requirements were 0.85 to 1.0 g/100 g diet (2.5 and 3.0 g/100 g dietary
protein) when fish were fed graded levels of TSAA in a cys/met of
51:49. Although the TSAA requirement determined in the final experiment
was lower than that observed in the methionine requirement experiment,
these studies were not designed for direct comparison of results.
Furthermore, other factors could have contributed to the different
estimates, including initial fish size and method of feeding (fixed
rate vs. satiation feeding). Methionine is often limiting in dietary
formulations for fish, and any factor that might spare part of the
requirement would be beneficial in formulating diets. Further studies
on factors other than cyst(e)ine would be beneficial.
Dietary TSAA requirements of fish encompass the entire range of values
reported for terrestrial vertebrates. Dietary TSAA requirements range
from 2.8 to 3.9 g/100 g of dietary protein for avians (NRC 1994
) and from 2.6 to 2.9 g of TSAA/100 g of dietary
protein for swine (NRC 1988
). However, estimates for
rodents are higher, ranging from 3.6 to 5.0 g of TSAA/100 g of
dietary protein (NRC 1978
). Thus, the range of TSAA
requirements for birds is on the higher end of the range established
for fishes, whereas the values for swine are on the lower end; rodents
appear to have higher dietary needs for these amino acids.
Plasma and serum methionine levels in fish have been used to confirm
dietary TSAA requirements derived from weight gain and FE data in some
species of fish (Griffin et al. 1994a
, Harding et al. 1977
), but not in others (Cowey et al. 1992
,
Keembiyehetty and Gatlin 1992
, Walton et al. 1986
). Serum amino acid concentrations in fish have not
indicated higher dietary requirements than those derived from weight
gain and FE, only confirmation of the requirement. Thus, measuring
those values at a single postprandial sampling time, as has been the
general approach, is not the best method for understanding amino acid
intake, absorption and requirements in fishes. Rates of absorption or
catabolism, as measured by excretion of carbon dioxide originating from
the amino acid (Walton et al. 1986
), might be better
indicators of nutritional status. However, to date, those data have
only confirmed the requirement based on weight gain and FE.
Juvenile yellow perch have a clear requirement for dietary methionine, and cyst(e)ine can spare ~50% of that requirement in diets containing ~3.3 g/kg of choline. Supplying other nutrients in this catabolic pathway as dietary supplements may spare some portion of the requirement. Further, a reduction in the dietary choline level might result in an increased dietary methionine requirement.
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FOOTNOTES |
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3 Abbreviations used: CR, cyst(e)ine replacement; cys/met, cyst(e)ine/methionine; FE, feed efficiency; TSAA, total sulfur amino acid.
Manuscript received July 12, 1999. Initial review completed August 17, 1999. Revision accepted November 5, 1999.
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