Aquaculture, 13 (1978) 266-272

265
o Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
DI GESTI BI LI TY DETERMI NATI ON I N FI SH USI NG CHROMI C OXI DE
MARKI NG AND ANALYSI S OF CONTENTS FROM DI FFERENT
SEGMENTS OF THE GASTROI NTESTI NAL TRACT
ERLAND AUSTRENG
Department of Poultry and Fur Animal Science, Agricultural University of Norway,
1432 AS-NLH, AS (Norway)
(Received 14 October 1977)
ABSTRACT
Austreng, E., 1978. Digestibility determination in fiih using chromic oxide marking and
analysis of contents from different segments of the gastrointestinal tract. Aquaculture,
13: 26.5-272.
Methods of feed digestibility determination in fish were reviewed. The indicator
method, using chromic oxide, was judged most suitable, but the best method of sampling
faeces was uncertain. Rainbow trout (Salmo gairdneri, Richardson) were fed an experi-
mental diet containing 1% chromic oxide. Faeces were collected by two stripping
methods, and gut contents were obtained by dissection from five zones of the alimentary
canal. Analyses of feed, faeces, and gut contents were made, and digestibility of protein,
fat, carbohydrate, ash and gross energy calculated. I t was concluded that absorption oc-
curs in all parts of the alimentary tract, and that it is important to limit the stripping of
faeces for analysis in digestibility studies to the hindmost part of rectum, i.e. from the
ventral fin to anus.
I NTRODUCTI ON AND LI TERATURE REVI EW
In feed evaluation it is desirable to have a quick and simple method for
determination of digestibility, and for domestic animals digestion trials are
carried out routinely. For fish the aquatic environment makes measurement
of feed intake and faeces output very difficult, and relatively few estimates
of digestibility by fish have been attempted.
Tunison et al. (1942) adapted a direct method for determination of
digestibility using brook trout (SaZueZims fontinalis, Mitchell). They col-
lected faeces by filtering the water and also sampled the water itself for ana-
lysis. The method required much analysis and calculation and was not very
accurate. In addition, errorscould have arisen because of contamination of
faeces by N-compounds excreted in urine or across the gills.
Phillips et al. (1948) force-fed trout with gelatin capsules containing
known amounts of a test diet. After various intervals fish were killed and the
contents of their digestive tracts analysed. The usefulness of this method is
266
limited by the fact that it is neceassary to kill the fish, and by the difficulty
of estimating how far digestion has proceeded in an individual before it is
killed. Also, calculation of results requires a knowledge of the amounts of
feed eaten and faeces produced, which is difficult to obtain in fish studies.
A method for measuring digestibility using a metabolism chamber (Post et
al., 1965) was described by Smith (1971). Using this chamber collection of
gill excretions and urine as well as faeces is possible. However, the test fish
suffer much stress and restriction of swimming activity, and force-feeding
is necessary. Analyses and calculations are also laborious.
Recently, most digestion experiments with fish have used the indicator
method. Hirao et al. (1960) and Yamada et al. (1962) used diets labelled
with 32P in the form of water-insoluble ammonium phosphomolybdate for
determination of digestibility of commercial feeds for rainbow trout.
However, use of isotopes as markers has not been usual. Ever since Edin
(1918) proposed the use of chromic oxide ( Cr203) most indicator trials in
domestic animals have been carried out with this indicator. Nose (1960,
1961) and Inaba et al. (1962) used chromic oxide to investigate protein
digestibility in fish. The latter authors found that estimates of digestibility in
rainbow trout obtained from analyses of faeces recovered after being voided
into pond water were consistently about 10% units higher than those from
analysis of faeces stripped manually from the fish, indicating that some N-
compounds are lost in the water. Later Pierre (undated) found a difference of
about 5% units between the two methods of faeces collection. Inaba et al.
(1963) and Kitamikado et al. (1964a, b) used the same indicator technique to
investigate the digestibility of protein and starch and the effect of starch and
oil on protein digestibility. Similar investigations were carried out by Singh and
Nose (1967) and Nose (1967), who sampled faeces of rainbow trout by
applying slight pressure with the fingers between the ventral fin and anus.
Several other authors have also used chromic oxide in digestion studies with
fish (Nehring, 1963; Cho et al., 1974,1976; Lall and Bishop, 1976).
The chromic oxide indicator technique has a number of advantages over
other methods of digestibility estimation for fish, i.e. quantitative feeding
and collection of faeces are inessential, experimental fish do not have to be
killed to obtain results, and they can live undisturbed in normal culture con-
ditions until the end of the experiment. In addition, a large number of fish
can be included in the experiment, which will eliminate a possible effect of
differences between individuals and easily procure enough faeces for the
analyses. Manual stripping is the most convenient and accurate way of col-
lecting faeces.
The present experiment was designed to find out (a) how far forward in
the rectum and intestine of rainbow trout faeces can be taken without
including material from which absorption is not completed, and which
would therefore bias digestibility estimated, and (b) whether digestibility
estimates obtained from stripped faeces were biased by contamination with
urine.
267
MATERIALS AND METHODS
The experiment was carried out in February 1977 at the Fish Breeding
Experimental Station, Sunndals$ra, Norway.
Thirty-eight rainbow trout with individual weights of between 500 and
900 g were used. They were kept in a 2 m2 fibreglass tank supplied with
fresh water at about 1lC. The fish received dry, pelleted food in excess
from an electrically-driven automatic feeder.
Composition and results of chemical analysis of the experimental diet are
shown in Tables I and I I . 1.00% chromic oxide (Cr,03) was included as an
inert digestion indicator.
TABLE I
Composition of the experimental diet
Ingredients
g/kg
Capelin meal
Torula yeast
Soya-bean meal
Wheat meal
Capelin oil
Vitamin mixture II*
Chalk meal
Cr,O,
salt
Micromineral*
Methionine
350.0
100.0
100.0
313.5
100.0
10.0
10.0
10.0
5.0
0.5
1.0
*Austreng (1976).
TABLE II
Chemical analyses of the experimental diet
Chemical component
Dry matter (96) 91.4
Crude protein (%) 37.0
Crude fat (HCl-sther extract) (%) 15.3
Crude fibre ( W) 1.3
Nitrogen-free extracts (%) 30.9
Ash (%) 6.9
Cr,O, (4%) 0.99
Gross energy (kcal/kg) 4851
The results of Windell and Norris (1969) and Pierre (undated) indicate
that at temperatures higher than El-10C feeding with the experimental diet
for 48 hours before sampling faeces from rainbow trout is sufficient. How-
ever, to be certain that the indicator-loaded feed was present throughout
268
the alimentary canal the fish were allowed to feed for a week before any
samples were taken.
Faeces were stripped from fish in two ways (Fig. la). I n method I fish
were stroked firmly down the belly, using much the same technique as for
stripping eggs from ripe fish, from half way between the pectoral and
ventral fins to the anus. I n method I I the fish were pressed only from the
Fig. 1. (a) Areas of belly of rainbow trout pressed to expel faeces. (b) Diagram of the
ventral side of a rainbow trout dissected to show the alimentary canal. Segments of the
canal from which gut contents were taken are shown.
ventral fins to the anus. About half the fish were treated in each way, and all
fish anaesthetized with chlorobutanol before handling.
After faeces-stripping the fish were returned to their tank and allowed to
feed on the experimental diet for three more days. All fish were then killed.
Each fish was cut open along the belly, and the alimentary canal was dis-
sected out. The gastrointestinal tube was cut into five parts. These are shown
in Fig. lb as I I I : the stomach, I V: the anterior half of the small intestine in-
cluding caeca, V: the posterior half of the small intestine, VI : the anterior
half of the rectum, and VI I : the posterior half of the rectum. The gut con-
tents were squeezed out from each of these segments with the back of a
knife. Samples from each alimentary canal segment from all fish were
pooled, weighed wet, then dried for 24 hours at 60PC before analysis. Faeces
samples were similarly treated. The pooled samples, and samples of the ex-
perimental diet were subjected to the following analysis.
(1) Dry matter was measured by drying for 20 hours at 105C.
(2) Total nitrogen was measured using the micro-Kjeldahl method, and
269
protein content calculated as N X 6.25. I n four samples also water soluble
nitrogen was determined as described by AOAC (1975).
(3) Fat was determined by ether extraction after HCl-hydrolysis. Prin-
cipally, the method was as described by Stoldt (1952) for feedstuffs and
Hartfiel(l960) for faeces.
(4) Ash content was determined using an oven at 600C for 15 hours.
(5) Chromic oxide ( CraOJ ) was measured photometrically after a method
described by Petry and Rapp (1971).
(6) Energy content was determined by bomb calorimetry.
The calculation of the digestibility was made as follows (Maynard and
Loosli, 1969)
Digestion coefficient =
100 -- (100 x
indicator in feed (%) X nutrient in faeces (%)
indicator in faeces (%) nutrient in feed (Y&o)
)
RESULTS AND DISCUSSION
Table I I I shows the calculated digestibility for crude protein, crude fat,
crude carbohydrate, ash and gross energy.
TABLE III
Digestion coefficients calculated for different parts of the intestinal tube in rainbow trout
Sampling method Protein Fat Carbohydrate Ash (X.&O,) Gross energy
(see Fig. 1)
I 86.5 90.9 25.8 39.0 72.2
II 87.2 91.4 28.2 39.9 73.4
III 16.5 8.5 0.4 18.7 12.7
IV 47.8 47.9 2.6 16.6 34.4
V 72.2 75.6 14.4 23.1 57.8
VI 83.5 85.9 20.9 33.3 67.3
VII 84.5 89.9 25.9 37.4 71.3
Comparison between estimates from faeces obtained by the two stripping
methods (Fig. la), and between samples obtained by dissection from the
anterior and posterior parts of the rectum (sections VI and VI I in Fig. lb)
shows that higher digestion coefficients were obtained in samples from the
hindmost part of the rectum for all nutrients. This indicates that, in digesti-
bility studies of this type, it is advisable to take faeces samples from as close
to the anus as possible.
Samples obtained by stripping faeces and by dissection from the rectum
were also analysed for water soluble nitrogen, and digestibility was re-cal-
culated discounting this N-fraction. This resulted in digestion coefficients
about 9% units greater and, along with the results of I naba et al. (1963) and
270
Pierre (undated), indicates that calculations based on analysis of faeces col-
lected from water will overestimate protein digestibility.
I f the technique of faeces stripping resulted in significant contamination
of samples with urine the estimates of protein digestibility from samples ob-
tained by dissection (from section VI I , Fig. lb) would be expected to be
higher than from samples obtained by stripping (method I I , Fig. la). I n fact
the reverse of this was found (Table I I I ). This must be due to experimental
error, and it is possible that samples squeezed out of the intestinal segments
were contaminated with digestive juices and small particles of intestinal epi-
thelium. However, Forster and Goldstein (1969) established that only a
small fraction of the total nitrogen excreted by fish appears in the urine. I n
addition Smith (1971) found that about 80% of non-faecal waste nitrogen
was exceted as ammonia through the gills of fish. Therefore, in experiments
on digestibility designed to help in practical evaluation of feed ingredients,
the theoretical source of error of contamination of faeces samples with urine
can probably be ignored.
For protein and fat the digestibility estimates from faeces samples
stripped by method I I (Table I I I ) are in good agreement with values
generally accepted in calculating metabolizable energy (Phillips and Brock-
way, 1959). However, in the present study carbohydrate was less well
digested than expected.
The digestibility coefficients obtained from different segments of the ali-
mentary canal (Table I I I ) indicate the sites of absorption. Though the results
may be biased by the presence of digestive juices, it seems that absorption
occurs in the stomach, while protein is absorbed mainly from the small intes-
tine. A little fat is absorbed from the stomach, but most from the intestine.
Carbohydrate absorption was low in the stomach and anterior part of the
small intestine, but increased thereafter.
I n this experiment the digestibility was determined in two zones of the
rectum. The differences in digestibility between these zones did not indicate
where the absorption ceases. Therefore, a supplementary experiment was
carried out with the aim of studying this question. Gut contents were in the
latter experiment sampled from four parts of the rectum. -The samples were
analysed for chromic oxide and protein. The calculated protein digestibility
increased backwards and indicated that absorption of protein occurs far
backwards in the rectum. This supports the conclusion that it is advisable to
take faeces samples from as close to the anus as possible. However, in ex-
periments designed to provide practical evaluation of feedstuffs, the error of
extending faecal collection forward to the ventral fins may be negligible.
The indicator method using chromic oxide will in future be used to esti-
mate digestibility of feedstuffs in our nutrition studies on salmonoid fish. I n
view of the above results, the procedure for collecting faeces by stripping
from the ventral fins to the anus will be used.
271
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