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*
Department of Zoology, University of Bergen, N-5007 Bergen, Norway and
CCMAR, Universidade do Algarve, Campus de Gambelas, 8000-810 Faro, Portugal
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: • Solea senegalensis • fish larvae • absorption • protein • free amino acids.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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examined the
developmental nutrient assimilation in six fish species by using a
method of controlled tube feeding of radiolabeled nutrients. His
results indicated that larvae that develop gastric digestion during
metamorphosis (most marine fish of commercial interest belong in this
group) initially assimilate simple forms of amino acids
(AA)3
more efficiently than more complex forms [assimilation order: free AA
(FAA) > peptides > protein]. His results (Rust 1995
) also indicated that the differences in assimilation
decreased as the larvae approached metamorphosis.
Due to their fast growth rate, fish larvae have a high requirement for
AA, the building blocks for the protein synthesis. In addition, in
marine fish larvae, including Senegal sole (Parra et al. 1999
), AA are an important energy source
(Conceição et al. 1998
, Finn 1994
, Finn et al. 1996
, Fyhn 1989
, Rønnestad and Fyhn 1993
,
Rønnestad and Naas 1993
, Rønnestad et al. 1999
, Sivaloganathan et al. 1998
). Therefore,
there is an exceptional demand for AA by fish larvae. The need implies
that the delivery of AA from intestinal protein digestion in marine
fish larvae may be insufficient to cover the demand.
It has been proposed that freely dissolved AA may prevent nutritional
deficiency during the developmental window from first feeding until the
intestine is fully differentiated and able to digest ingested proteins
(Fyhn 1989
, Rønnestad et al. 1999
,
Walford et al. 1991
).
To specifically test the assimilation rates of FAA and protein in the early stages of marine fish, postlarval Senegal sole (Solea senegalensis) were tube fed controlled amounts of diets containing [35S] and [14C]AA. To our knowledge, this is the first report on the kinetics of absorption of AA during the early life stages of marine fish.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The larvae originated from a group of naturally spawned eggs collected
from a tank of wild-caught Senegal sole (S.
senegalensis) maintained at the University of Algarve. The
larvae were reared according to the protocol described by Dinis et al. (1999),
in which the larvae were kept in tanks (100 L)
at the University of Algarve in a recirculating system (temperature
21°C, salinity 34 g/L). Larvae opened their mouths 2 d after
hatching (DAH) and started to feed on rotifers (Brachionus
plicatilis, small strain) enriched with
Tetraselmis sp. and Isochrysis sp.
Artemia nauplii (Bass Entrée; INVE,
Dendermonde, Belgium) were introduced at 3 DAH and became the only prey
at 6 DAH. From d 10 on, the larvae were fed Artemia
metanauplii (RH; INVE) enriched for 24 h with
Tetraselmis sp. and Isochrysis sp.
On 20 DAH, larvae were transferred to flat-bottomed tanks at the
University of Algarve. On the day of experimentation (23 DAH), groups
of larvae (2.45 ± 0.87 mg dry weight, means ± SD, n = 12; weight determined from
larvae from the main group) were transferred under temperature control
(21°C) to the radioisotope laboratory, where radioactive diets were
tube fed. Only larvae with an empty gut at the time of injection were
used. All animal procedures and handling were in compliance with the
Guide for the Care and Use of Laboratory Animals (NRC 1996
).
Experimental set-up.
To specifically test the functionality of the digestive system, we used
an in vivo method to control tube feeding of fish larvae that was
described by Rust et al. (1993)
and modified by
Rønnestad et al. (2000)
with additional modifications
as described here.
The in vivo set-up consisted of a stereo dissecting microscope with
a micromanipulator. A nanoliter injector (World Precision Instruments,
Sarasota, FL) was fastened to the micromanipulator. A plastic capillary
(0.19-mm diameter; Sigma Chemical Co., St. Louis, MO) was fastened to
the nanoliter injector. Before the injection, the larvae were
tranquilized (33 µg/L MS-222; Sigma Chemical Co.) and thereafter
gently placed in a droplet of clean seawater. With the fish in
position, the capillary was gently passed through the mouth and the
esophagus into the stomach. This operation was controlled visually,
because all of the larvae used in the experiments had transparent and
semitransparent gut/stomach areas. A single injection of the test diet
was then deposited into the stomach lumen with the nanoliter injector.
After capillary withdrawal, the larvae were gently rinsed for any
spillage through three successive transfers to wells (20 mL) that
contain clean seawater. The larvae were transferred between the wells
with a pipette with as little water as possible and then transferred to
single-larva incubation wells with 2 mL of clean seawater. Visual
observations of a diet that contains food coloring in a pilot study
showed that the ventilation movements of the opercula effectively
removed any contamination in the mouth during the first steps of the
rinsing series. The total transfer time was 
75 s.
Assimilation study.
To specifically study the assimilation of FAA and proteins, solutions were injected according to the described system for in vivo tube feeding. The series consisted of six groups of six larvae that were incubated for 15 min, 30 min, 1 h, 2 h, 4 h, 8 h and 24 h (FAA only) after being fed a single injection (36 nL; 4.08 g/L) of an FAA-containing or a protein-containing diet (see later). The injected volume was based on a pilot study in which we gently injected increasing volumes while we observed the distention of the stomach wall. The volume (not nutrient concentration) is equivalent to three Artemia nauplii.
FAA assimilation rates were determined by using an aqueous diet that consists of FAA (FAA diet: 32.35 mmol/L) that contain L-[35S]methionine (Amersham Pharmacia Biotech AB, Uppsala, Sweden) and the following L-AA (analytical grade, Sigma Chemical Co.): 4.21 mmol/L alanine, 1.74 mmol/L arginine, 2.24 mmol/L aspartic acid, 3.14 mmol/L glutamic acid, 2.61 mmol/L glycine, 0.51 mmol/L histidine, 1.52 mmol/L isoleucine, 2.70 mmol/L leucine, 2.35 mmol/L lysine, 1.31 mmol/L methionine, 1.14 mmol/L phenylalanine, 1.53 mmol/L proline, 2.22 mmol/L serine, 1.57 mmol/L threonine, 0.37 mmol/L tryptophan, 0.69 mmol/L tyrosine and 1.96 mmol/L valine.
For studies on protein assimilation rates, an aqueous diet that contains bovine serum albumin (BSA; Sigma Chemical Co.) added L-[methylated-14C]BSA (DuPont New England Nuclear, Boston, MA) was injected (Prot-diet). Both diets were prepared using seawater diluted 1:3 in distilled water. The final activity was 43.7 and 0.87 Bq/nL for the FAA diet and the Prot-diet, respectively.
The larvae were tube fed and incubated individually as described earlier. After the set time, the larvae were gently lifted by the tail with a forceps and rapidly transferred to a slide on ice for dissection under the microscope. The larvae were killed by spinal cord lesioning, and the digestive tract and the liver were dissected. The operation lasted 4–5 min. Any liquid remnant on the slide was included with the gut compartment. Once the gut and the liver was dissected, each compartment, including the carcass/body, was transferred to individual 6-mL scintillation vials, and 1.0 mL tissue solubilizer (35% H2O2) was added. When the solution was clear (>24 h at 60°C, including two shakings), 4.0 mL scintillation cocktail (Optiphase HiSafe2; Wallac, Milton Keynes, U.K.) was added. The well incubation water (2.0 mL) was transferred to a 20-mL scintillation vial, and 8 mL scintillation cocktail was added. Samples were counted with a Beckman LS 6000IC liquid scintillation counter (Fullerton, CA).
The data were converted from counts/min to Bq after the subtraction of
blanks and correction for counting efficiency (93%).
Retention in the larval carcass/body, liver and gut compartment, as
well as the water, were calculated with the formula
 |
where Rx is the retention of compartment X, B is Bq of the larval carcass/body, L is Bq of the liver, G is Bq of the gut and W is Bq of the incubation water.
Statistical analysis.
Results are given as mean ± SD (n = 6). Mean values were compared with a t test after a check for equality of variances through F-test (Microsoft Excel 97; Microsoft Corporation, Redmond, WA). Differences were considered statistically significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The FAA diet deposited into the digestive tract was absorbed with a
3.5-times-higher transfer rate (P < 0.001) from the
gut lumen into the larval body compartment compared with the
Prot-diet (Fig. 1
). This is based on data from 15 min after tube feeding, when only 20.6
± 4.7% of the injected 35S-labeled FAA
diet remained in the gut compartment (Fig. 1C
). Of the 80%
that was absorbed, 20% was found in the liver, and the remaining 60%
were distributed among the remaining body tissues. In comparison, for
protein-fed larvae at the same time (i.e., 15 min after tube
feeding), 72.0 ± 6.1% of the protein remained in the gut
compartment (Fig. 1D
).
|
). For
protein, the intestinal processing took much longer; the data indicate
that it may take as long as 8 h to complete absorption (Fig. 1D
).
Assimilation efficiency assessed as the sum of liver and body
compartments at 8 h after tube feeding was calculated for the two
diets. These calculations showed that FAA were absorbed with higher
efficiency than AA from the Prot-diet (80% versus 58%,
P = 0.001). If we assume that the label present in the
gut at this time represents AA incorporated into the intestinal tissue
and this compartment is included in the calculations, the values are 89
and 64%, respectively.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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indicated a higher rate of assimilation for methionine than for a
mixture of AA in striped bass (Morone saxatilis).
Rønnestad et al. (2000)
studied the assimilation
efficiency for a mixture of 20
L-[14C]AA in Japanese flounder
larvae and found comparable values (79.5 ± 7.1%) to those for
the assimilation of
L-[35S]methionine in the present
study (80 or 89%, depending on assumptions for calculation). Further
studies are necessary to determine the absorption rates of individual
FAA.
The [14C]BSA used in the present study was
produced through methylation with the use of formaldehyde and sodium
cyanohydroboride. This radiolabeling procedure will produce a BSA that
has chemical properties similar to unlabeled BSA. The labeling modifies
primary amines (mainly lysine) in the protein and has the advantage
that the charge properties of the modified residues are preserved
(Jentoft and Dearborn 1979
). It is therefore reasonable
to assume that the results obtained for the Prot-diet are
representative of both the digestion and absorption of dietary BSA.
The tested FAA diet showed that FAA seems to be an amino acid source
with 40% better total assimilation efficiency than the tested protein
diet (BSA) for Senegal sole postlarvae. This is in line with the
results from Rust (1995),
who found better assimilation
efficiencies for FAA than for Escherichia coli protein for
striped bass, especially in the initial larval stages. The present
results are intriguing because the Senegal sole stage that was tested
had a well-defined stomach that contained the first gastric glands
(Ribeiro et al. 1999a
) and a mature intestinal enzyme
profile (Ribeiro et al. 1999b
). Therefore, even though
Senegal sole early postlarvae have an apparently well-developed
digestive system, have completed metamorphosis, and have acquired a
benthic behavior, their protein digestive capacity seems to still be
somewhat limited. This may explain at least in part the historic
difficulties in the adaptation of young sole to artificial diets
(Dinis et al. 1999
, Howell 1997
). This
limited protein digestive capacity supports the findings that sole
postlarvae benefit from having at least part of their dietary AA in a
simpler molecular form (Day et al. 1997
). This is also
supported by the fact that in nature, Solea postlarvae, as
older stages, feed on polychaete worms and other invertebrates
(Dinis et al. 1999
), which, like all marine
invertebrates, have a high content of FAA (Fyhn 1989
).
In addition to the overall effect on AA assimilation efficiency, this
study also shows that there are large differences in AA absorption
rates between FAA and protein in Senegal sole postlarvae. Based on the
data at 15 min after tube feeding, the FAA was absorbed into the larval
tissues/body carcass 3.5 times faster. The data for assimilation
efficiency show the differences between diets at the time when diet
processing is complete, whereas the absorption rate calculated here
describes the flow rate of nutrients from the gut into body tissues.
The data for absorption rates are more relevant for continuously
feeding fish larvae. Their high potential growth rates (30–100%/d;
Kamler 1992
), together with their dominant use of AA as
a fuel (Rønnestad and Naas 1993
), imply a large demand
for precursor AA. The larval growth rate may therefore depend on the
flow of a balanced AA solution from the intestine and into the tissue
cells. In the present study, the injected AA constituted only 0.01%
of the protein in the Senegal sole larva and 0.40% of the FAA pool.
Therefore, the composition of the AA mixture that was tube fed to the
fish is not likely to significantly influence the composition of the
fish FAA pool. However, when fish are fed a larger meal (the real
situation), a faster absorption of FAA may lead to transient AA
imbalances and consequently to decreased protein utilization if
crystalline AA are used to supplement dietary protein. Such effects
have been shown in pigs and other animals (Batterham 1974
, Rérat 1993
and 1995
); faster
absorption has been documented when FAA are the source of AA rather
than protein. Poor utilization of dietary FAA has also been observed in
white sturgeon (Ng et al. 1996
). The observations from
the present study may explain 1) the poor results verified
when crystalline AA are used in microdiets for larval fish with
inclusion levels above 10% (López-Alvarado and Kanazawa 1995
), 2) the limitations in the use of high levels
of protein hydrolysates in diets for larval and juvenile fish
(Cahu et al. 1999
, Carvalho et al. 1997
,
Espe et al. 1999
) and 3) the poor results
when alternative protein sources are supplemented with crystalline AA
in juvenile and adult fish (Davies and Morris 1997
,
Murai et al. 1986
).
The liver serves an important function in screening and processing the
nutrients absorbed from the gut. The high initial retention of labeled
methionine in the liver in comparison with the low liver retention of
labeled AA from protein (Fig. 1D
) suggests that the liver
has some buffering capacity for high AA absorption rates. However, the
liver retention of the absorbed FAA reported here is only about half of
the peak value of 42% reported in pigs (Rérat 1993
). The high rates of whole body protein synthesis that seem
to characterize fish larvae and postlarvae
(Conceição et al. 1997a
and 1997b
,
Houlihan et al. 1993
) compared with older fish probably
also contribute to an high overall retention efficiency of dietary FAA.
Postlarval sole had a higher retention efficiency and absorption rate
for FAA than for intact protein, which supports the important role of
AA sources that are simpler than protein, such as FAA
(López-Alvarado and Kanazawa 1995
,
Rønnestad et al. 1999
) or protein hydrolysates
(Cahu et al. 1999
, Carvalho et al. 1997
,
Day et al. 1997
) in the nutrition of fish larvae and
postlarvae. The challenge is to find proper feed production
technologies and the appropriate inclusion levels so the buffering
capacity of the larvae for rapidly absorbed AA can be used to the
maximum extent without reducing protein utilization, growth or survival
rates.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: AA, amino acids; BSA, bovine
serum albumin; DAH, days after hatch; FAA, free amino acids.
Manuscript received May 16, 2000. Revision accepted July 25, 2000.
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