Aquaculture Nutrition 2001 7; 237^245

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Apparent digestibility comparison in rainbow trout

(Oncorhynchus mykiss) assessed using three methods
of faeces collection and three digestibility markers
G.W. VANDENBERG & J. DE LA NOUÈE
Groupe de recherche en recyclage biologique et aquiculture, DeÂpartement des sciences animales, Universite Laval, Ste-Foy, QueÂbec
G1K-7P4, Canada

Introduction
Abstract
Information related to speci®c nutrient availability is
Experiments were undertaken to investigate the apparent
required to carry out nutrient requirement research, evaluate
digestibility coecient (ADC) of a range of macro- and
feed ingredients as potential candidates for dietary inclusion,
micronutrients in rainbow trout using three external digest-
develop least-cost feed formulations and to minimize envi-
ibility markers (chromic oxide, acid insoluble ash and
ronmental impact of animal production. The need for
titanium dioxide, alone or in combination), and three
reliable tools to study ingredient utilization has lead to the
methods of faeces collection (modi®ed Guelph system, St-Pee
development of a number of methods to understand the
system and abdominal massage; hereafter referred to as
degree by which nutrients are absorbed, including those to
`column', 'collect' and `strip', respectively). For each method
measure apparent nutrient digestibility.
of faeces collection, three replicate tanks of ®sh were
The utilization of internal indigestible components or
acclimated for 5 days to a practical diet containing the
addition of external indigestible markers eliminates the
external marker followed by 5 days of faeces collection. Both
need for quantitative faeces collection but requires that a
marker type and collection method signi®cantly in¯uenced
representative faeces sample be collected (Maynard &
ADC estimate in a similar manner for dry matter, protein,
Loosli 1969). A number of external markers have been
nitrogen-free extract, ash and energy. The ADC was consis-
suggested for nutritional studies, with chromic oxide being
tently highest for the column and lowest for the strip
the most widely used in work with aquatic animals.
methods of collection, and titanium dioxide gave higher
However, a variety of alternative markers have been
ADCs vs. the other markers. Lipid ADC did not di€er
evaluated as a result of concerns related to the validity of
between the column and collect methods, but collection of
chromic oxide resulting from analytic variability and poor
faeces by the strip method gave lower ADCs, irrespective of
recovery (Riche et al. 1995), di€erential marker intestinal
marker type. In general, nutrient ADC was higher with the
transit (Leavitt 1985), and digestive and metabolic e€ects
combined markers than when single markers were employed.
(Shiau & Chen 1993). Alternative markers have included
The in¯uence of marker type and collection method was less
sources of acid insoluble ash, such as acid-washed sand
clear for the individual minerals studied.
2 (Tacon & Rodrigues 1984), Celiteä (Atkinson et al. 1984;
3 Bureau et al. 1999; Morales et al. 1999) and Sipernat
KEY WORDS: digestibility, ®sh faeces, markers, nutrient
3 (Degussa AG, Frankfurt, Germany) (Rodehutscord et al.
absorption
1996), polyethylene (Tacon & Rodrigues 1984), oxides of
yttrium (Refstie et al. 1997; Storebakken et al. 1998;
1 Received 19 February 2001, accepted 17 April 2001
Sugiura et al. 1998a, b), Yb (Refstie et al. 1998), La
Correspondence: Joel de la NouÈe, DeÂpartement des sciences animales,
(Hillestad et al. 1999), Ti (Lied et al. 1982; Weatherup &
Pavillon Paul Comtois, Universite Laval, Ste Foy, QC G1K 7P4 Canada.
E-mail: joel.de-la-Noue@san.ulaval.ca McCracken 1998) as well as barium oxide (Riche et al.
1995), 5-a cholestane (Sigurgisladottir et al. 1990) and the
microtracer Fe-Ni (Kabir et al. 1998). In addition, several
studies have simultaneously mixed external markers within

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Ó 2001 Blackwell Science Ltd 237

238 G.W. Vandenberg & J. de la NoÏe

the same test diet (Sugiura et al. 1998; Hillestad et al. 1999)
Diets and faeces collection
either to verify apparent digestibility coecient (ADC)
values using two markers, or for particular applications A practical diet (Table 1) was formulated to meet the
(e.g. the use of 5-a cholestane for lipid digestibility nutrient requirements (NRC 1993) and three external mark-
calculation). ers were incorporated, alone or in combination, at the levels
An additional source of variation between studies is related indicated in Table 1. Micro-ingredients were ®rst mixed and
to the method of faeces collection. To date, three techniques then slowly added to the macro-ingredients to ensure a
(with slight variations) have been adopted, including: homogenous mixture. The mash was steam conditioned and
(1) collection of un-defecated intestinal contents using either pelleted using a laboratory mill equipped with a 3.5 mm die.
abdominal pressure, anal suction or dissection (Nose 1967; The resulting pellets were dried in a forced air oven at room
Austreng 1978; Windell et al. 1978), (2) collection of decan- temperature for 24 h, sieved and stored in airtight containers
ted faeces within the tank (Smith & Lovell 1971; Windell at 5 °C until required. The proximate composition of the diet
et al. 1978), from the e‚uent water via ®ltration (Ogino et al. is given in Table 2.
1973) or decantation column (Cho & Slinger 1979), and Tanks were divided into two groups of three tanks with
(3) continuous e‚uent ®ltration/faeces removal (Choubert e‚uent water being directed to a series of automatic faeces
et al. 1979, 1982). While several studies have attempted to collection devices (collect method) (Choubert et al. 1982), or
directly compare the various methods of faeces collection
(Spyridakis et al. 1989; Hajen et al. 1993; Weatherup &
McCracken 1998), many of these studies have focused on Table 1 Experimental diet formulation (as-is basis)
only a narrow range of possible nutrients. Ingredient1 Inclusion (gákg diet)1)
The objective of the current study was to compare three
Fish meal 325.0
external indigestible markers (alone and in combination) Wheat middlings 150.0
and three methods of faeces collection on the ADC of Soyabean meal 130.0
a range of dietary components, including macro- and Corn gluten meal 100.0
Whey 125.0
microminerals.
Blood meal 40.0
Fish oil 80.0
Carboxymethyl cellulose 20.0
Materials and methods Vitamin premix2 5.0
Mineral premix3 5.0
Fish and experimental conditions Chromic oxide4 5.0
Sipernat 505 10.0
Rainbow trout (n ˆ 150; initial mass 42.3 ‹ 1.4 g) from a Titanium dioxide4 5.0

certi®ed disease-free hatchery (Pisciculture des Alleghanys, 1

St. Philemon, Quebec, Canada) were randomly distributed
among 6±60 L cylindroconic tanks and fed a commercial diet
(Martin Feed Mills, Elmira, Ontario, Canada) for 7 days
prior to the start of the experiment. Each tank was supplied
with water from a recirculation system receiving approxi-
mately 25% daily make-up water. Suspended solids were
removed using a sand ®lter and e‚uent ammonia removed
using a trickling bio®lter; ammonia and nitrite concentrations
were monitored twice weekly to assess bio®lter performance.
Water temperature and dissolved oxygen were continuously
monitored by a computer and automatically maintained at
15.0 ‹ 0.5 °C and 10.3 ‹ 0.5 mg L±1, respectively. Water
pH was measured daily and maintained by the addition of
calcium carbonate to the sand ®lter and sodium carbonate to
the e‚uent water. The ®sh were held in accordance with the
guidelines of the Canadian Council of Animal Care and the
Comite de Protection des Animaux, Universite Laval.
Major feed ingredients obtained from Martin Feed Mills, Elmira, Ontario,
Canada.
2
Based on Ontario Ministry of Natural Resources formulation #VIT-9608.
Provides per kg diet: vitamin A (as acetate): 3750 IU; vitamin D3 (as
cholecalciferol): 3000 IU; vitamin E (as dL-a-tocopheryl-acetate): 75 mg;
vitamin K (as menadione Na-bisulphate): 1.5 mg; vitamin B12
(as cyanocobalamin): 0.03 mg; ascorbic acid (as ascorbyl
polyphosphate): 75 mg; d-biotin: 0.21 mg; choline (as chloride):
1500 mg; folic acid: 1.5 mg; niacin (nicotinic acid): 15 mg; pantothenic
acid: 30 mg; pyridoxine: 7.5 mg; ribo£avin: 9 mg; thiamin: 1.5 mg.
Provided by BASF Corp., Georgetown, Ontario, Canada.
3
Based on Ontario Ministry of Natural Resources formulation #MIN-
9504. Provides per kg diet: sodium chloride (39%, 61% Cl): 3075 mg;
ferrous sulphate (FeSO4á7H2O, 20% Fe): 65 mg; copper sulphate
(CuSO4á5H2O, 25% Cu): 30 mg; manganese sulphate (MnSO4, 36% Mn):
90 mg; potassium iodide (24% K, 76% I): 10 mg; zinc sulphate
(ZnSO4á7H2O, 40% Zn): 150 mg. Provided by BASF Corp., Georgetown,
Ontario, Canada.
4
Sigma-Aldrich, Mississauga, Ontario, Canada; a-cellulose added as ¢ller
for diets employing single markers.
5
Sipernat 50: source of acid insoluble ash comprised of 98.50% SiO2 with
an average particle size of 50 lm from Degussa-HÏls Canada Inc.,
Brampton, Ontario, Canada.

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 Comparing digestibility markers and faeces collection methods 239

Table 2 Proximate analysis of the experimental diet (100% dry Feed and faeces were analysed for dry matter (vacuum oven
matter basis) at 65 °C for 18 h) and ash (incineration at 550 °C for 18 h)
Component1 g kg)1 using standard methods (AOAC 1990) and crude protein
(% N ´ 6.25) quanti®ed using the semiautomatic Kjedahl
Dry matter 954
Crude protein 433 4 method (Foss Electric, Hillerùd, Denmark; AOAC method
Lipid 134 7.B01±7.B04). Lipid was analysed via ethyl ether extraction
Ash 105 (Soxtec System HT12, Foss Tecator AB; Hoganas, Sweden)
Nitrogen-free extract2 328
Gross energy (MJ kg ^1)3 197 following acid hydrolysis (Lynch et al. 1963), and energy
Phosphorus 13 content was determined using an adiabatic bomb calorimeter
Calcium 14 (Parr Instrument Co., Moline, IL, USA). Nitrogen-free
Magnesium (´10^1) 20
Manganese (´10^3) 36
extract (NFE) was estimated by di€erence. Phosphorus was
Copper (´10^3) 20 determined by the vannadate/molybdate method of analysis
Iron (´10^2) 22 (Varley 1966) using a Technicon AutoAnalyzerÒ apparatus
Zinc (´10^2) 10
(Technicon Corporation, TarryTown, NY, USA) following
1
Analysed according to AOAC methods. ash digestion in 10% nitric acid. Other minerals (Ca, Mg,
2
Nitrogen-free extract obtained by di¡erence.
3
Mn, Cu, Zn, Fe) were analysed using atomic absorption
Gross energy obtained by adiabatic bomb calorimetry.
spectrophotometry (AOAC 1990), following ash digestion in
decantation columns (column method) (Cho & Slinger 1979). 2 N hydrochloric acid. Chromic oxide was measured by
The decantation columns were assembled in a manner such titration (Christian & Coup 1954) and titanium quanti®ed
that e‚uent water from separate tanks was redirected away spectrophotometrically according to Short et al. (1996) with
from the automatic faeces collector mechanism, and into a 6¢¢ the exception that ashed samples were boiled in 7.4 M H2SO4
diameter settling column with a height of 18¢¢ and having an for 3 h rather than 1 h as suggested in the original article.
angled bottom to facilitate faeces decantation. Tests were The alternation of the original protocol improved the
carried out to include all combinations of marker type recovery of the titanium and reduced the variability between
[chromic oxide (Cr2O3), acid insoluble ash (AIA) and titanium samples. Acid insoluble ash was measured according to
dioxide (Ti) alone and three markers combined] and collection Atkinson et al. (1984) with particular care taken to thor-
method (collect, column and strip). Each treatment combina- oughly rinse the ®ltrate with boiling, de-mineralized water.
tion was randomly assigned to three tanks and was fed for a The ADC was calculated as described by Cho & Slinger
5-day acclimation period, which was followed by a 5-day faeces (1979).
collection period. Diets were fed to apparent satiation once
daily at 8:00 hours; the tanks were ¯ushed and the e‚uent
Statistical analyses
pipes cleaned at 16:00 hours to remove any uneaten feed.
Faeces from the collect and column treatments were collected Data are presented as means of duplicate analyses from three
until just prior to the morning feeding. Faeces collected in the replicate tanks. Apparent digestibility values were veri®ed for
decantation columns were further centrifuged (5000 ´ g) for normality and subjected to analysis of variance using marker
15 min and the supernatant discarded. The strip treatment was type, collection method, tank replicate and their interactions
performed twice daily, 60 min following the morning feeding in the statistical model as appropriate. Means were compared
and again at 16:00 hours. Fish were anaesthetized in using the Duncan's multiple range post hoc test and di€er-
100 p.p.m. MS222 (tricane methanesulphonate; Argent Labor- ences were considered signi®cant at P < 0.05.
atories, Vancouver, British Columbia, Canada) prior to
stripping, and intestinal contents gently expressed by massa-
Results and discussion
ging the ventral surface three times from the anal ®ns toward
the anus. For all treatment groups, daily faeces collections The in¯uence of faeces collection method, marker type and
were pooled and stored at )80 °C until further processing. marker inclusion method (i.e. the use individual vs. combined
external markers) followed similar trends for dry matter,
protein, lipid, NFE, ash and energy (Tables 3 and 4). In all
Sample analysis
cases, the stripping method consistently gave the lowest and
Frozen faeces samples were freeze-dried, ground using a the column method the highest ADCs, with the collect
1 mm screen, and stored at ±30 °C until required for analysis. method being intermediate, irrespective of maker type and

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240 G.W. Vandenberg & J. de la NoÏe

Table 3 Apparent digestibility of dry matter (DM), protein (Prot) and lipid using di€erent external markers added alone or in combination and
3 methods of faeces collection

Single Combined

Method Cr2O3 AIA Ti Cr2O3 AIA Ti

DM
Collect 68.3 þ 0.2a m x 68.5 þ 0.3a m x 71.8 þ 0.4a n x 74.2 þ 0.2a m y 70.1 þ 0.4a n x 78.1 þ 0.4a o y
Column 75.5 þ 0.4b m x 73.8 þ 0.6b n x 78.3 þ 0.3b o x 78.7 þ 0.4b m y 74.3 þ 0.4b n x 83.0 þ 0.2b o y
Strip 48.0 þ 0.8c m x 58.1 þ 1.0c n x 64.4 þ 0.5c o x 61.9 þ 0.5c m y 58.5 þ 0.8c n x 69.7 þ 0.5b o y
Prot
Collect 87.4 þ 0.4a m x 88.2 þ 0.1a m x 89.7 þ 0.2a n x 89.9 þ 0.1a m y 88.3 þ 0.1a n x 91.4 þ 0.1a o y
Column 91.9 þ 0.3b m x 90.9 þ 0.4b m x 91.9 þ 0.5b m x 92.1 þ 0.3b m x 90.5 þ 0.3b n x 93.7 þ 0.1b o y
Strip 80.0 þ 0.3c m x 83.1 þ 0.40c n x 85.7 þ 0.1c o x 84.1 þ 0.3c m y 82.7 þ 0.5c n x 87.4 þ 0.3b o y
Lipid
Collect 84.3 þ 0.8a m x 85.1 þ 0.3a m x 86.9 þ 0.3a n x 90.3 þ 0.4a m y 88.8 þ 0.5a m y 91.8 þ 0.3a m y
Column 81.7 þ 0.6b m x 84.3 þ 0.4a n x 86.8 þ 0.5a o x 88.8 þ 0.5a m y 86.6 þ 0.5b n y 91.1 þ 0.2a o y
Strip 75.0 þ 0.8c m x 75.4 þ 0.9b m x 81.8 þ 0.6b n x 84.8 þ 0.7 b m y 83.5 þ 0.6c m y 87.9 þ 0.7b n y
abc
Within marker type, di¡erent superscripts are signi¢cantly di¡erent (P < 0.05).
mno
Wthin collection method, di¡erent superscripts are signi¢cantly di¡erent (P < 0.05).
xy
Di¡erent di¡erent superscripts indicate signi¢cant di¡erences between single and combined markers.
Mean þ standard error, n = 3.

Table 4 Apparent digestibility of nitrogen free extract (NFE), ash and energy (En) using di€erent external markers added alone or in
combination and three methods of faeces collection

Single Combined

Method Cr2O3 AIA Ti Cr2O3 AIA Ti

NFE
Collect 44.6 þ 0.5a m x 43.9 þ 0.4a m x 50.2 þ 0.9a n x 57.0 þ 0.4a m y 50.3 þ 0.6a n y 63.4 þ 0.7a o y
Column 56.3 þ 0.2b m x 53.3 þ 0.8b n x 63.2 þ 0.6b m x 66.0 þ 0.6b m y 59.1 þ 0.7b n y 72.9 þ 0.4b o y
Strip 4.9 þ 1.8c m x 24.0 þ 1.9c n x 36.7 þ 0.8c o x 34.7 þ 1.1c m y 28.9 þ 1.6c n y 48.2 þ 0.8c o y
Ash
Collect 50.7 þ 0.4a m x 48.9 þ 0.7a m x 55.6 þ 0.5a n x 52.4 þ 0.6a m x 45.0 þ 0.6a n y 59.6 þ 0.7a o y
Column 63.7 þ 0.3b m x 57.0 þ 0.6b n x 65.2 þ 0.9b m x 57.5 þ 0.6b m y 48.9 þ 0.3b n y 66.1 þ 0.5b o x
Strip 33.5 þ 0.8c m x 44.0 þ 1.4c n x 52.9 þ 1.0c o x 34.9 þ 0.6cm x 29.1 þ 1.2c n y 48.2 þ 1.0c o y
En
Collect 74.8 þ 0.2a m x 75.7 þ 0.2a m x 78.0 þ 0.3a n x 81.4 þ 0.1a m y 78.5 þ 0.2a n y 84.2 þ 0.2a o y
Column 79.5 þ 0.4b m x 78.9 þ 0.4b m x 82.1 þ 0.3b n x 83.9 þ 0.4b m y 80.5 þ 0.4b n x 87.2 þ 0.2b o y
Strip 58.1 þ 0.9c m x 65.9 þ 0.8c n x 71.5 þ 0.5c o x 72.1 þ 0.4c m y 69.7 þ 0.6c n y 77.8 þ 0.4c o y
abc
Within marker type, di¡erent superscripts are signi¢cantly di¡erent (P < 0.05).
mnc
Within collection method, di¡erent superscripts are signi¢cantly di¡erent (P < 0.05).
xy
Di¡erent di¡erent superscripts indicate signi¢cant di¡erences between single and combined markers.
Mean þ standard error. n = 3.

inclusion method. The exception was lipid ADC, where little
di€erence was observed between the collect and the column
methods (Table 3). Regarding di€erent external markers,
Cr2O3 and AIA provided the most similar ADCs, whereas Ti
gave signi®cantly higher values, irrespective of collection and
inclusion method. Combining the three markers resulted in
higher ADCs than when equivalent single markers were used.
The current study rearms the signi®cant in¯uence that
faeces collection method has on macronutrient ADC. A
number of studies have previously shown that the collection
of undefecated faeces via stripping (Kabir et al. 1998;
Storebakken et al. 1998), anal suction (Spyridakis et al.
1989) or dissection (Hajen et al. 1993; Storebakken et al.
1998) results in reduced ADC values vs. the collection of
faeces using either decantation columns of mechanical faeces
removal; a number of factors have been suggested as playing
a role in this observation. This includes the possibility of
endogenous products (blood, urine, semen) contaminating
the samples. Care was taken to eliminate contamination of
this kind, although, a number of ®sh were observed as

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 Comparing digestibility markers and faeces collection methods
showing secondary signs of precocious sexual development,
and produced semen upon the application of abdominal
pressure. Any obvious contaminated samples were discarded,
however, the contribution of endogenous contamination to
reduced ADC values cannot be discounted. As well, the
expression of un-defecated faeces provides a sample for
which all of the absorption processes may not have been
complete. For example, studies have reported the uptake of
intact proteins by the posterior intestine in a number of ®sh
species including salmonids (Sire & Vernier 1992; McLean
et al. 1999), thus the reduced protein ADC observed in the
strip group (Table 3) may be because of incomplete protein
absorption.
A number of additional criticisms have been directed at
the stripping method of faeces collection. Inherent in the
stripping method is the repeated handling of ®sh, which
imparts an increased level of stress, and may a€ect ADCs
(Hajen et al. 1993); the use of anaesthetics to minimize stress
has been reported to induce spontaneous defecation (Spy-
ridakis et al. 1989), thus accelerating intestinal transit,
perhaps further altering nutrient absorption dynamics.
Finally, repeated stripping of ®sh may induce injury to the
intestinal mucosal membrane and a€ect ADC values over
time. These factors combined with the observation that
nitrogen-free extract ADC values vary considerably with
time (de la NouÈe et al. 1980) along with the possibility of an
e€ect of previous diet history, may explain the extremely low
ADC value for this dietary component when the strip
method was used with Cr2O3 as the single external marker
(Table 4), given that this was the initial treatment combina-
tion employed in the study.
For all macronutrients with the exception of lipids, ADC
values for the column method of faeces collection were
signi®cantly higher vs. the collect method. The increased
nutrient ADCs with the column method is probably because
of nutrient leaching between the time that the faeces is
expelled and sample collection. Although Cho et al. (1985)
claimed that nutrient leaching is minimal when the faeces
remain undisturbed and unbroken faecal pellets are collected,
a number of reports have demonstrated increased ADCs of
nutrients when this system of faeces collection is employed
(Spyridakis et al. 1989; Hajen et al. 1993; Kabir et al. 1998).
Windell et al. (1978) reported that the ADC of dry matter,
crude protein and lipid increased with time up to 4 h when
faeces were stored in water, with the largest changes taking
place in the ®rst hour. Negligible changes in ADC values
were observed between 4 and 16 h of incubation. Similarly,
protein and dry matter ADC values were unchanged in faeces
samples that were subjected either to 3 and 15-h immersion in
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Ó 2001 Blackwell Science Ltd Aquaculture Nutrition 7; 237^245
241
water (Satoh et al. 1992). However Hajen et al. (1993)
reported that the majority of nutrient losses in faeces from
chinook salmon occurred between 6 and 18 h exposure to sea
water. Working in fresh water, similar results were obtained
for protein and dry matter from rainbow trout faeces
between 6 and 24-h exposure to fresh water (Kabir et al.
1998).
In the current study, there was no di€erence in lipid
digestibility between the collect and column methods of
faeces collection (Table 3). Previous work has demonstrated
that lipid ADC values are altered to a lesser degree
when di€erent faeces collection methods are employed
(Spyridakis et al. 1989; Storebakken et al. 1998; Weatherup
& McCracken 1998). This may be the result of the hydro-
phobic nature and reduced solubility of dietary lipids relative
to other nutrients resulting in lower nutrient leaching.
In the present study, for diets containing single external
markers, the ADCs for macronutrients, dry matter and
energy were similar between AIA and Cr2O3 markers
(Tables 3 and 4) with the exception of nitrogen-free extract.
Although sources of AIA are widely used for nutritional
studies of aquatic animals, some reports have questioned its
use. For example, Tacon & Rodrigues (1984) found that AIA
signi®cantly underestimated ADC whereas Morales et al.
(1999) and De Silva & Perera (1983) found this marker
signi®cantly overestimated ADC vs. Cr2O3; the di€erent
sources of AIA between the studies may have contributed to
the observed inconsistencies. Bowen (1981) indicated that a
signi®cant percentage of the AIA is absorbed, thus negating
its utility as an external digestibility marker. However, the
source of AIA in the present study comprised over 98% SiO2,
which is considered insoluble.
A number of external markers have been reported as
having di€erential rates passage vs. the feed (De Silva &
Perera 1983). The degree to which digestibility markers and
feed move through the digestive tract can be veri®ed by
comparing the ratio of two markers in the feed and faeces
(Owens & Hanson 1992); in a steady-state situation, the ratio
of the two markers in the feed and faeces should be similar.
In diets containing single or combined markers, those
incorporating Ti in the calculation had larger di€erences in
this regard (Table 5), suggesting that Ti may di€erentially
pass through the digestive system.
For a number of macronutrients, diets employing AIA,
and to a lesser degree Cr2O3, as the marker provided the
most repeatable results when ADC values from single and
combination marker diets are compared. The observed
di€erences between Ti and the other two markers may be
because of the fact that Ti is not a free-¯owing powder and
242 G.W. Vandenberg & J. de la NoÏe

tends to aggregate if care is not taken during the mixing
process. Thus it is possible that there was a lack of uniform
dispersion of Ti in the feed, leading to erroneous results. For
diets containing single external markers, the ADC values for
macronutrients, dry matter and energy were consistently
higher when Ti is employed as the marker vs. Cr2O3 and
AIA. This is in contrast to Weatherup & McCracken (1998)
who reported that ADC values were signi®cantly lower vs.
Cr2O3. The reasons for this discrepancy is unknown, how-
ever, it may be related to di€erences in marker inclusion, as
the aforementioned study included Ti at a level of 1 g kg±1
whereas the current study incorporated it at 5 g kg±1.
In general, the e€ect of combining the three markers into a
single diet resulted in consistently elevated ADCs for
macronutrients. The reason for this is unknown, however,
it suggests the possibility of marker interaction, although this
was not investigated directly. Further work is warranted to
determined possible analytic errors caused by interactions
between external markers.
The trends observed for macronutrient ADCs resulting
from changing collection method and marker type were not
as consistent when mineral ADCs were compared (Tables 6
and 7). The exception is observed in the case of Cu and Fe
when single markers were mixed individually (Table 7).
Digestibility values for Fe were greatly a€ected by collection
method, with negative digestibility values noted when the
strip method was employed, which is probably the result of
trace amounts of blood contaminating the faeces. The
in¯uence of collection method and marker type on mineral
digestibility has not been widely reported, however, some
work has provided similar results as the current study.
Brown (1993) reported large di€erences in phosphorus
ADCs from rainbow trout fed soybean-based diets when
faeces were collected using a decantation column and by

Table 5 Mean ratio of external digestibility markers in the feed and faeces for diets with single or combined markers

Faeces

Format Marker ratio Feed Collect Column Strip

a a a
Single Cr2O3:AIA 0.37 þ 0.01 0.37 þ 0.01 0.39 þ 0.01 0.34 þ 0.02a
AIA:Ti 3.14 þ 0.12a 2.84 þ 0.08b 2.61 þ 0.06c 2.69 þ 0.03c
Ti:Cr2O3 0.86 þ 0.08a 0.96 þ 0.02a 0.97 þ 0.02a 1.22 þ 0.05b
Combined Cr2O3:AIA 0.21 þ 0.01a 0.23 þ 0.02a 0.24 þ 0.02a 0.22 þ 0.01a
AIA:Ti 5.66 þ 0.21a 4.12 þ 0.02b 3.72 þ 0.02c 4.08 þ 0.01b
Ti:Cr2O3 0.89 þ 0.12a 1.06 þ 0.01a 1.13 þ 0.03b 1.14 þ 0.01b
abc
Within marker ratio, di¡erent superscripts are signi¢cantly di¡erent (P < 0.05).
Mean þ standard error, n = 3.

Table 6 Apparent digestibility of phosphorus (P), calcium (Ca) and magnesium (Mg) using di€erent external markers added alone or in
combination and three methods of faeces collection

Single Combined

Method Cr2O3 AIA Ti Cr2O3 AIA Ti

P
Collect 44.3 þ 0.6a m x 43.2 þ 1.6a m x 49.3 þ 0.6a n x 51.0 þ 0.9a m y 43.3 þ 0.9a n x 58.4 þ 0.7a o y
Column 55.6 þ 0.6b m x 50.6 þ 1.0b n x 55.1 þ 1.5b m x 52.7 þ 0.6a m x 43.1 þ 0.6a n y 62.2 þ 0.9b o y
Strip 45.2 þ 1.4a m x 58.6 þ 1.2c n x 62.6 þ 1.1c o x 44.4 þ 1.7b m x 39.4 þ 1.9b n y 55.8 þ 1.8a o y
Ca
Collect 15.0 þ 1.1a m x 14.1 þ 1.4a m x 20.3 þ 0.6a n x 28.8 þ 1.7a m y 17.7 þ 1.9a n x 39.5 þ 1.6a o y
Column 32.7 þ 0.8b m x 23.7 þ 1.3b n x 32.6 þ 1.7b m x 34.0 þ 1.8a m x 20.7 þ 2.0a n x 47.4 þ 0.8b o y
Strip 17.9 þ 2.2a m x 32.5 þ 2.0c n x 38.5 þ 2.5c o x 12.4 þ 1.6b m y 4.5 þ 2.5b n y 30.3 þ 2.0c o y
Mg
Collect 55.3 þ 1.0a m x 49.9 þ 1.0a n x 43.4 þ 0.7a o x 65.1 þ 0.4a m y 59.6 þ 0.5a n y 70.3 þ 0.6a o y
Column 77.2 þ 0.5b m x 72.0 þ 0.5b n x 70.1 þ 0.8b n x 75.3 þ 0.4b m x 70.3 þ 0.4b n x 80.3 þ 0.2b o y
Strip 26.6 þ 2.3c m x 56.6 þ 1.7c n x 54.4 þ 1.8c n x 41.7 þ 0.9c m y 36.4 þ 1.7c n y 53.6 þ 0.9c o x
abc
Within marker type, di¡erent superscripts are signi¢cantly di¡erent (P < 0.05).
mno
Within collection method, di¡erent superscripts are signi¢cantly di¡erent (P < 0.05).
xy
Di¡erent di¡erent superscripts indicate signi¢cant di¡erences between single and combined markers.
Means þ standard error, n = 3.

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 Comparing digestibility markers and faeces collection methods 243

Table 7 Apparent digestibility of manganese (Mn), copper (Cu), iron (Fe) and zinc (Zn) using di€erent external markers added alone or in
combination and three methods of faeces collection

Single Combined

Method Cr2O3 AIA Ti Cr2O3 AIA Ti

Mn
Collect 41.4 þ 2.4a m x 46.3 þ 0.9a n x 43.7 þ 0.5a n x 64.9 þ 0.6a m y 59.6 þ 0.5a n y 70.2 þ 0.7a o y
Column 47.4 þ 1.1b m x 41.9 þ 1.0b n x 51.7 þ 1.0b o x 47.2 þ 0.8b m x 36.5 þ 0.6b n y 57.9 þ 0.7b o y
Strip 43.3 þ 1.7a b m x 60.4 þ 2.2c n x 61.6 þ 1.2c n x 45.1 þ 1.7b m x 40.2 þ 1.9b n y 56.3 þ 1.7b o y
Cu
Collect 69.2 þ 1.2a m x 73.1 þ 0.4a n x 74.7 þ 0.5a n x 89.7 þ 0.7a m y 88.0 þ 0.9a m y 91.2 þ 0.6a m y
Column 81.0 þ 0.8b m x 79.1 þ 1.1b n x 86.4 þ 0.2b o x 83.8 þ 0.9b m x 80.6 þ 1.0b m x 87.1 þ 0.5b n x
Strip 35.4 þ 2.9c m x 53.3 þ 2.4c n x 60.3 þ 2.4c o x 66.0 þ 0.9c m y 62.9 þ 1.0c m y 72.9 þ 0.8c n y
Fe
Collect 12.9 þ 6.2a m x 9.5 þ 3.6a m x 9.9 þ 2.6a m x 39.7 þ 1.6a m y 30.1 þ 2.4a n y 48.7 þ 1.4a o y
Column 16.4 þ 1.1a m x 5.9 þ 2.6a m x 27.0 þ 1.4b n x 25.8 þ 1.8b m y 10.8 þ 2.3b n x 40.9 þ 0.8a o y
Strip )29.1 þ 3.5b m x )16.1 þ 5.7b n x )2.2 þ 1.4c o x )9.9 þ 4.3c m y )19.7 þ 4.8c n x 12.7 þ 3.6b o y
Zn
Collect 66.8 þ 0.8a m x 67.5 þ 1.4a m x 60.4 þ 0.4a n x 92.9 þ 0.3a m y 91.7 þ 0.4a m y 93.9 þ 0.3a m y
Column 58.8 þ 0.5b m x 49.4 þ 1.0b n x 59.4 þ 1.6a m x 76.5 þ 0.7b m y 71.7 þ 1.2b n y 81.2 þ 0.8b o y
trip 47.9 þ 1.6c m x 59.6 þ 2.1c n x 57.8 þ 1.7a n x 74.3 þ 2.7b m y 72.0 þ 3.0b m y 79.6 þ 1.9b n y
abc
Within marker type, di¡erent superscripts are signi¢cantly di¡erent (P < 0.05).
mno
Within collection method, di¡erent superscripts are signi¢cantly di¡erent (P < 0.05).
xy
Di¡erent di¡erent superscripts indicate signi¢cant di¡erences between single and combined markers.
Mean þ standard error, n = 3.

dissection. Di€erences in phosphorus ADCs were not as
marked when ®shmeal diets were fed. Using ®shmeal-based
diets and comparing faeces stripping and decantation,
Sugiura et al. (1998) observed di€erences in availability of
Fe, Cu, Mn, Sr, whereas those for Ca, P, Mg and Zn were
una€ected by faeces collection method. This may be
partially explained by the fact that certain mineral fractions
may be sparingly soluble in water, and thus not susceptible
to leaching-related alteration of ADC values. For example,
soluble forms of dietary P are absorbed in the small
intestine, with the quantity above that required by the ®sh
being excreted in the urine (Sugiura 1998). Thus the
remaining P fraction in the faeces is likely relatively
insoluble in water (complexes of P, hydroxyappatite, bound
phytate-P, etc.), and would be largely una€ected by the
e€ects of leaching.
The observed inconsistencies in mineral ADCs may be
related to a number of factors. Diets employed in the present
study were supplemented with minerals to NRC recommen-
dations. Insucient reduction of mineral particle size and/or
inadequate mixing results in an inhomogeneous diet. As well,
given that minerals comprise a small percentage of the overall
ration, they are more susceptible to errors vs. macro-
ingredients. Interaction between minerals may also promote
analytical errors, and di€erential solubilities of alternate
mineral forms may make certain fractions more sensitive to
leaching under certain faeces collection regimes.
The results of this study underline to need for well-de®ned
methods that allow objective, repeatable experiments, thus
permitting the accurate interpretation of digestibility data
between di€erent studies.
Acknowledgements
This work was supported by the Natural Sciences and
Engineering Research Council (NSERC Strategic Projects
Program) and BASF Canada Inc (JlN). GW Vandenberg is
the recipient of an NSERC Postgraduate Scholarship. The
authors are grateful to Martin Feed Mills who generously
supplied the dietary ingredients, Richard Prince and Pierre
Castonguay for their help with the feed formulation, Andre
Roy, Jean Bricault and Francine GigueÁre for their skilled
analytical assistance, and the sta€ of the Laboratoire Regional
des Sciences Aquatiques for their assistance with the feeding
experiments. The authors appreciate the helpful comments of
Drs D. Bureau and F. Moyano related to the manuscript.
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