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Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan 202, Republic of China
2To whom correspondence and reprint request should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • choline • fish • tilapia
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Several of the quantitative
vitamin requirements of tilapia were determined over the past several
years, including niacin (Shiau and Suen 1992
), vitamin D
(Shiau and Hwang 1993
), vitamin B-12 (Shiau and Lung 1993
), vitamin C (Shiau and Hsu 1995
and
1999
), vitamin B-6 (Shiau and Hsieh 1997
)
and biotin (Shiau and Chin 1999
).
Choline, a vitamin-like nutrient, is an important component of the
phospholipid lecithin and certain other complex lipids. It serves as a
source of labile methyl groups for the synthesis of various methylated
metabolites and as a precursor of acetylcholine. Most animals can
synthesize choline if adequate methyl donors such as methionine are
present in the diet. However, the studies with fish identified choline
as essential for maximal weight gain (Craig and Gatlin 1996
, Griffin et al. 1994
, Hung 1989
). Thus, choline is an essential nutrient for fish.
The quantitative requirement of choline has been studied in only a few
species of fish. For example, 1,000, 400, 1700–3200, 500, 588 and
714–813 mg/kg were reported to be the amount of choline required in
the diet for lake trout (Ketola 1976
), channel catfish
(Wilson and Poe 1988
), white sturgeon (Hung 1989
), striped bass (Griffin et al. 1994
), red
drum (Craig and Gatlin 1996
) and rainbow trout
(Rumsey 1991
), respectively. Information on the choline
requirement of tilapia is lacking. Physiological age is an important
factor of the dietary choline requirement for the teleostean species,
such as birds and mammals (Woodward 1994
). The purpose
of the present study was to estimate the dietary choline requirement of
juvenile tilapia, Oreochromis niloticus x O.
aureus, using growth indices supported by body choline
concentration and liver lipid concentration.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental diet formulation is given in Table 1
. The formulation is similar to that of Shiau and Chin (1999)
, which was
shown to be adequate for tilapia. Vitamin-free casein (Sigma
Chemical Co., St. Louis, MO) was used as the protein source. The
choline concentration (Venugopal 1985
) of casein, fish
oil and corn oil were 370, 43 and 36 mg/kg, respectively. The vitamin
mixture was similar to that used by Shiau and Hsieh (1997)
, except that
it did not contain choline. Choline chloride (Sigma) was added to the
test diets at the expense of cellulose to provide concentrations of 0,
100, 200, 400, 600, 800, 1,000 and 2,000 mg/kg diet. Diets were
analyzed for choline (Venugopal 1985
), and supplemental
levels were confirmed by analysis. The diets were prepared by
thoroughly mixing the dry ingredients with oil and then adding cold
water until a stiff dough resulted. This was then passed through a
mincer with die, and the resulting strings were dried using an
electrical fan at 28°C. After drying, the diets were broken up and
sieved into pellets and stored at -20°C.
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Male hybrid tilapia, O. niloticus x O. aureus, were supplied from the Far East Hatchery (Cha-Yi, Taiwan). Upon arrival, they were acclimated to laboratory conditions for 4 wk in a plastic tank [74 cm (w) x 95 cm (l) x 45 cm (h)] and fed a commercial diet (Hung Kuo Industrial, Taipei, Taiwan). The choline concentration of the commercial diet was measured as 1,092 mg/kg. At the beginning of the experiment, 15 fish (mean weight, 0.62 ± 0.01 g) were stocked in each aquarium (30.5 x 61.0 x 55.5 cm3). There was a total of eight treatments. Each experimental diet was fed to fish in three aquariums. The fish were chosen for the experiment and the diets were assigned to groups of fish randomly. Each aquarium was part of a closed-recirculated system with a common water reservoir maintained at 26 ± 1°C. The water was circulated at 2 L/min through two separate biofilters to remove impurities and reduce ammonia concentrations.
The fish were fed 5 g/(100 g body wt · d). This amount was close to the maximal daily rations consumed by the tilapia during the acclimation period. The daily ration was subdivided into two equal feedings and fed at 0900 and 1700 h. Fish were weighed once every 2 wk and the daily ration adjusted accordingly. A photoperiod of 12 h light, 12 h dark (0800–2000 h) was used. The fish were fed the test diets for an 8-wk period.
At the end of the feeding trial, the fish were weighed. Weight gain (as
measured by the percentage of body weight gain) and feed efficiency
(FE)3
were calculated as described previously (Chou and Shiau 1999
). After the final weighing, four fish were
randomly removed from each aquarium, blood samples were collected from
the caudal vein and pooled for blood triglyceride, cholesterol and
phospholipid concentration estimation (Carson and Goldfard 1979
). Liver samples were removed and pooled for total lipid
determination (Folch et al. 1957
). Four other fish were
then taken randomly from each aquarium and pooled for body choline
determination (Venugopal 1985
).
Statistical analysis.
Each experimental diet was fed to three groups of fish according to a
completely randomized design. Results were analyzed by one-way
analysis of variance (ANOVA). When the ANOVA identified differences
among groups, multiple comparisons among means were made with Duncan’s
new multiple range test. Statistical significance was determined by
setting the aggregate type I error at 5% (P < 0.05) for each set of comparisons. Dietary choline requirements for
juvenile tilapia were estimated by the broken-line regression
method (Robbins 1986
) and the polynomial regression
method (Zeitoun et al. 1976
).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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200 mg
choline/kg, followed by fish fed the diet with 100 mg of choline/kg and
they were all significantly higher than fish fed the control diet.
Blood triglyceride, cholesterol and phospholipid concentrations were
higher in fish fed diets supplemented with choline than in fish fed the
control diet (data not shown).
When the polynomial (second-order) regression model (Zeitoun 1976
) was employed to express the relationship between weight
gain, body choline concentration and dietary choline concentration, a
growth peak and maximal body choline concentration were reached when
dietary choline concentrations were 880 and 920 mg/kg diet,
respectively (Fig. 1
). The relationship between liver lipid concentration and dietary
choline concentration is best expressed by a broken-line analysis
(Robbins et al. 1986
). Because the breakpoint at 923 mg
choline/kg diet gave the least mean square error (Fig. 2
), this value is estimated to be the requirement. Collectively, these
data suggest that the dietary choline requirement for tilapia is about
900 mg/kg diet. Since the casein, fish oil and corn oil contained 370,
43 and 36 mg choline/kg, respectively, they thus, contributed 140 (370
x 0.38), 2 (43 x 0.05) and 2.5 (36 x 0.07) mg
choline/kg diet, respectively. Therefore, the estimated dietary choline
requirement for rapid growing tilapia is about 1,000 mg/kg diet.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Shiau and Liang 1995
, Shiau and Shy 1998
). The estimated choline
requirement from the present study is different from those reported for
channel catfish (Wilson and Poe 1988
), hybrid striped
bass (Griffin et al. 1994
) and red drum (Craig and Gatlin 1996
) in which the sulfur amino acid levels in their
diets were adjusted to the minimal level. Whereas, in our study the
dietary sulfur amino acid was provided from vitamin-free casein
which was used as the protein source providing 36.67% crude protein in
the basal diet (Table 1)
. The sulfur amino acid concentrations from
casein were confirmed by analysis. The methionine and cystine
concentrations calculated by the analysis were 0.46 g/100 g protein
(methionine concentration in casein: 1.26 g/100 g protein) and 0.07
g/100 g protein (cystine concentration in casein: 0.19% g/100 g
protein) of the diet, respectively, providing total of 0.53 g/100 g
protein of sulfur amino acids which almost meets the total sulfur amino
acid (methionine + cystine) requirement of tilapia (0.54 g/100 g of the
protein; Santiago and Lovell 1988). Thus, the choline requirement for
tilapia obtained in the present study may not represent a minimal
requirement. It may still represent the maxmal dietary choline
requirement, i.e., the requirement under conditions which minimize de
novo synthesis of choline. This information may be broadly applicable
to the needs of practical, least-cost feed formulation than is an
estimate of minimal requirement.
A choline deficiency does not cause liver lipid accumulation in tilapia
as reported for other fish (Griffin et al. 1994
,
Ketola 1976
, Wilson and Poe 1988
).
Indeed, tilapia fed choline-free diet had the lowest liver lipid
concentration (Fig. 2)
. Similar observation was also reported for red
drum (Craig and Gatlin 1996
). Unlike red drum however,
the liver lipid concentration in tilapia is associated with dietary
choline supplementation level, with reduced value for fish fed the
basal diet, intermediate values for fish fed diets supplemented with
100–600 mg choline/kg, and highest values for fish fed diets with
>800 mg choline/kg diet (Fig. 2)
. The altered liver lipid
concentrations observed in the present study suggest that this variable
may permit an evaluation of choline status of the fish. Zeitoun et al. (1976)
suggested the use of polynomial regression analysis as a means
of approximating the relationship of weight gain and essential nutrient
intake. As indicated by Zeitoun, the value corresponding to maximal
gain estimated by quadratic regression is defined as the optimal
concentration of dietary nutrient that produces optimal growth, and
beyond which growth is depressed. In the present study, weight gain and
choline concentrations of fish reached a maximum at 800 mg of choline
supplementation/kg diet and decreased thereafter. Thus, the requirement
was estimated by a second-order regression analysis (Zeitoun et al. 1976
). It should be noted, however, that the liver lipid
concentration did not decline when the supplemental choline levels were
higher than 800 mg/kg diet. No statistical significance among 800,
1,000 and 2,000 mg/kg groups (Fig. 2)
indicates a plateau was reached
when dietary choline supplementation level >800 mg/kg. Thus, the
broken-line model (Robbins 1986
) appears to better
fit the data (Fig. 2)
. It also suggests that liver lipid concentration
can be used to estimate the dietary choline requirements of tilapia.
In rats fed choline-deficient diets, plasma total lipid was low,
probably due to the alteration in production of choline-containing
phospholipids (Lombardi 1971
). In the present study,
tilapia exhibited similar trends; dietary choline significantly
affected triglycerides, cholesterol and phospholipid in plasma. As
dietary choline increased above the basal level, the concentrations of
these lipid classes also increased. The lower concentrations of
phospholipid in plasma of tilapia fed choline-deficient diet are
not surprising due to the substantial contribution of choline to the
synthesis of phospholipid (Lombardi 1971
). Due to the
important role of phospholipid in membrane formation as well as
lipoprotein synthesis in the liver, the reduced concentrations of
phospholipid in plasma of fish fed choline-deficient diets would
likely have contributed to their reduced weight gain. It is noted,
however, that the plasma lipid indices assessed in this investigation
did not exhibit sensitivity to dietary choline level (above the
supplemented level of 100 mg/kg), suggesting that the requirement for
these indices is below the choline concentration of the diet containing
the supplement of 100 mg choline/kg diet.
In summary, weight gain of tilapia was consistently improved as the level of choline increased in the diet, until reaching a peak after the requirement was met. A similar trend was observed in total body choline concentrations. Liver lipid concentration of tilapia was also responsive to dietary choline level as a plateau was reached when the dietary choline requirement was met. The study provided an estimation of the dietary choline requirement of rapidly growing juvenile tilapia fed casein-based diet.
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FOOTNOTES |
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3 Abbreviations used: FE, feed efficiency.
Manuscript received June 30, 1999. Revision accepted August 31, 1999.
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REFERENCES |
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