Aquaculture Nutrition 2007 13; 17–34
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A feed is only as good as its ingredients – a review of
ingredient evaluation strategies for aquaculture feeds
B.D. GLENCROSS1, M. BOOTH2 & G.L. ALLAN2
1
Department of Fisheries – Western Australia, Research Division, North Beach, WA, Australia; 2 New South Wales Department
of Primary Industries, Port Stephens Fisheries Centre, Nelson Bay, NSW, Australia
Abstract
The evaluation of feed ingredients is crucial to nutritional
research and feed development for aquaculture species. In
evaluating ingredients for use in aquaculture feeds, there are
several important knowledge components that should be
understood to enable the judicious use of a particular
ingredient in feed formulation. This includes information on
(1) ingredient digestibilities, (2) ingredient palatability and (3)
nutrient utilization and interference.
Diet design, feeding strategy, faecal collection method and
method of calculation all have important implications on the
determination of the digestible value of nutrients from any
ingredient. There are several ways in which palatability of
ingredients can be assessed, usually based on variable inclu-
sion levels of the ingredient in question in a reference diet and
feeding of those diets under an apparent satietal or self-
regulating feeding regimes. However, the design of the diets,
the parameters of assessment and the feeding regime can all
be subject to variation depending on subtleties of the
experimental design. Clearly, issues relating to feed intake are
the key performance criteria in palatability assessments, and
it is important that such experiments maintain sufficient
stringency to allow some self-discrimination of the test feeds
by the fish. The ability of fish to use nutrients from the test
ingredient, or defining factors that interfere with that process,
is perhaps the most complex and variable part of the ingre-
dient evaluation process. It is crucial to discriminate effects
on feed intake from effects on utilization of nutrients from
ingredients (for growth and other metabolic processes). To
allow an increased focus on nutrient utilization by the ani-
mals, there are several experimental strategies that can be
adopted, which are based on variations in diet design and
feeding regime used. Other issues such as ingredient func-
tionality, influence on immune status and effects on
organoleptic qualities are also important consideration in
determining the value of ingredients in aquaculture feed
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 2007 Blackwell Publishing Ltd
formulations. A key aspect to note is the need to design all
experiments with sufficient experimental capacity to detect
significant effects.
KEY WORDS: nutrition, fishmeal replacement, methodology
Received 31 August 2005, accepted 26 June 2006
Correspondence: Dr Brett Glencross, PO Box 20, North Beach, WA 6920,
Australia. E-mail: bglencros@fish.wa.gov.au
Introduction
Fish diets of the future will include a wider range of alterative
ingredients to fishmeal than is currently the case. Many of
these ingredients are more complex than fishmeal and require
thorough evaluation in order to determine their nutritional
value and appropriate use levels in prospective diets. In the
evaluation of specific ingredients, ideally, the science of
nutrition should endeavour to gain knowledge on the nutri-
tional implications of use of those ingredients, and once this
knowledge is gained then to seek apply it to commercial feed
formulation. While it could be stated that there is no single
strategy by which ingredient evaluation should be under-
taken, there are clearly pitfalls and problems to be wary of in
undertaking the evaluation process. This nutritional evalua-
tion process has several key facets that need to be undertaken
to provide a clear indication of the potential that any
ingredient may have for use in an aquaculture feed.
The fishmeal and oil problem and ingredient risk
reduction
Fishmeal has traditionally been considered an important
protein source for use in aquaculture diets for both carni-
vorous and omnivorous species, and many aquaculture for-
mulations still have fishmeal included at levels in excess of
50%. However, being too reliant on any one ingredient
17
18 B. D. Glencross et al.
presents considerable risk associated with supply, price and
quality fluctuations. As a strategy to reduce risk, the identi-
fication, development and use of alternatives to fishmeal and
oil in aquaculture diets remains a high priority. As a result of
the volumes of fishmeal and oil used in aquaculture, espe-
cially for carnivorous species, aquaculture of these species is
still perceived as a net fish consumer rather than producer,
and this practice has raised concerns about the long-term
sustainability of these industries (Naylor et al. 2000).
Substantial effort has been expended over the past decades
in evaluating a wide range of potential alternatives to fish-
meal and fish oils for use in aquaculture diets. Those ingre-
dients can generally be classified into those being derived
from either plant origin or terrestrial animal origin.
Plant derived resources include soybean meals, protein
concentrates and oils (Kaushik et al. 1995; Refstie et al. 1998,
1999), canola meals, protein concentrates and oils (Higgs
et al. 1982; Mwachireya et al. 1999; Burel et al. 2000; Forster
et al. 2000; Glencross et al. 2003b, 2004a,b) and lupin meals
and protein concentrates (Burel et al. 1998; Booth et al. 2001;
Farhangi & Carter 2001; Glencross et al. 2003a, 2004c).
Key potential terrestrial animal ingredients have included
resources such as rendered meat meals (Bureau et al. 1999,
2000; Stone et al. 2000; Sugiura et al. 2000; Williams et al.
2003a,b), blood meals (Allan et al. 1999a,b; Bureau et al.
1999) and poultry meals (Bureau et al. 1999; Nengas
et al. 1999).
Key evaluation components
We consider that there are several key components in
ingredient assessment including ingredient characterization,
ingredient digestibility, palatability, nutrient utilization and
functionality.
1. Ingredient characterization is the first part of any evalua-
tion process. Chemical composition, variability in com-
position, source and species of origin are all important
factors that need to be documented so as to allow any
meaningful assessment and reporting of that assessment.
2. Ingredient digestibility is the measurement of the propor-
tion of energy and nutrients, which an animal can obtain
from a particular ingredient through its digestive and
absorptive processes. Several methods have been used to
determine diet and ingredient digestibilities in aquaculture
species. This review will examine these methods and
explore some of their key strengths and weaknesses.
3. Determination of ingredient palatability is the second key
component of knowledge required about an ingredient
before it can be successfully used. Palatability being
defined as the combination of both attractiveness and
ingestion of a diet and therefore of most relevance to feed
development. This is important because, irrespective of
how digestible and available the nutrients and energy from
an ingredient might be, if the ingredient reduces feed intake
then it will have limited value. A range of methods have
been used to explore palatability and feed intake issues
in aquaculture feeds and a summary of some of these
methods used in this aspect of ingredient evaluation is
presented in this review.
4. The determination of nutrient utilization or interference
with nutrient utilization because of incorporation of any
one ingredient is perhaps the most complex step in the
ingredient evaluation process. This complexity is largely
related to the wide variety of factors that may impact on
nutrient or energy utilization. The methods employed to
examine issues relating to nutrient utilization are also
diverse and this review highlights some of the problems
that occur with their use.
5. Ingredient functionality is another crucial aspect of ingre-
dient evaluation. Irrespective of the compositional or
nutritional attributes of an ingredient, if it cannot be
functionally introduced into a feed in a manner that allows
its processing in a suitable manner then it is of diminished
value as a feed ingredient. Alternatively, some ingredients
may add additional value to a diet, based on some func-
tionality features that they contribute to a formulation.
This is particularly the case with modern extruded feeds.
Characterization and preparation of
ingredients
Detailed compositional information on test samples of all
ingredients to be evaluated is critical. High level of variability
between common ingredients is well recognized and this
variability can affect the nutritional value of the ingredient
and determination of the best strategies to determine the
nutritional value of the ingredient (Jiang 2001). As variability
can exist within ingredient samples, it is also important that
samples are adequately mixed to ensure that what is ulti-
mately evaluated is representative. The preparation of diets
and ingredients is also important, as is their long-term stor-
age if they are to provide useful information over extended
periods of time.
Characterizing ingredients
A key reason for comprehensively characterizing ingredients
is so others can use the findings from the study. Simple
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 Feed ingredient evaluation strategies for aquaculture feeds 19

identification factors such as the species of origin and whe-
ther a genotype or cultivar classification exists (if relevant)
should be detailed. Differences between production site,
season or year can also be important. For example, within
the lupin species of Lupinus angustifolius, there are more than
a dozen different commercial cultivars being grown and there
is substantial variation in key nutrient parameters such as
protein, amino acid and energy content among those culti-
vars (Table 1). The implications of such variability have been
noted. For example, Glencross et al. (2003c) found that there
was substantial protein and energy digestibility variation
among cultivars.
The source of the ingredient may also be important. For
example, significant problems relating to antinutritional
factors (ANF) in canola and rapeseed meals were identified
from Canadian-produced meal in studies by Higgs et al.
(1982, 1983) and Mwachireya et al. (1999) and in European-
produced meals by Burel et al. (2001). However, Glencross
et al. (2004a,b) found no significant problems attributable to
ANF in Australian-produced rapeseed meals. Identification
of the origins of the ingredient being studied at least to
country should be considered as a minimum. This is especi-
ally important for plant ingredients, as it is well known that
soil type and climate can affect the nutritional composition of
many grains.
The nature of processing of the ingredient sample prior to
addition to the experimental diets also has important impli-
cations. Booth et al. (2001) noted clear differences in the
chemical composition and also the nutritional value of a
range of grain legume meals produced from either whole-seed
or seed kernels. Similarly, clear differences in rapeseed meal
produced through different oil extraction methods have been
noted on not only the composition of the meal, but also on
the nutritional value of the respective meals (Glencross et al.
2004a,b). Accordingly, some characterization of the pro-
cessing methods/technologies used to produce the ingredient
from its raw or natural state would be useful. It is well known
that protein damage sustained during ingredient or diet
processing will affect the value of an ingredient (Peres et al.
2003). For example, heat damage to canola meals affects
their usefulness when fed to fish (Glencross et al. 2004a), and
it is well known that nutritional loss of some amino acids
occurs through such heat damage and the influence of
protein, carbohydrates and moisture on Malliard reactions
(Oste 1984; Anderson et al. 1993). There are potential in vitro
assays that can be employed to characterize such
protein damage (e.g. reactive lysine assay) (Rutherfurd et al.
1997).
In addition to the clear identification of the ingredient of
concern, its origins and processing, a detailed analysis of
the compositional characteristics should be provided. Ideally,
this analysis should be as comprehensive as possible, but key
variables such as crude protein (nitrogen · 6.25), total lipids,
ash, moisture and gross energy should be considered man-
datory for all test ingredients and preferably all ingredients
used in any experimental diets. Crude fibre is one traditional
element of proximate analysis, which is losing favour to more
useful analyses such as acid-detergent fibre and neutral-
detergent fibre, which relate more closely to levels of cellu-
lose, hemicellulose and lignins (Petterson et al. 1999). A more
comprehensive guide to key compositional parameters to be
considered is provided in Table 2. In the absence of clear
information on the ingredient species, cultivar, origin and
processing level, the importance of the compositional

Table 1 Composition variability within the kernel meals of some narrow-leaf lupin, Lupinus angustifolius, cultivars

Cultivar type Gungurru Tanjil Kalya Merrit Warrah Myallie

Dry matter 873 907 899 903 911 912

Crude protein (N · 6.25) 397 402 429 459 423 469
Phosphorus 4 5 4 4 6 5
Ash 33 33 31 31 33 37
Crude fat 59 58 55 66 56 57
Energy (MJ kg)1 DM) 20.2 20.6 20.7 21.1 20.0 20.4
Arginine 44 47 49 39 45 47
Histidine 12 10 12 10 11 10
Isoleucine 16 17 18 14 17 16
Leucine 28 30 30 23 32 29
Lysine 18 16 20 16 14 12
Methionine 3 2 3 2 5 2
Phenylalanine 16 17 17 14 18 16
Threonine 14 17 15 12 17 16
Valine 16 17 17 14 17 16

All values are g kg)1 DM unless otherwise stated. Data from Glencross (unpublished).

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20 B. D. Glencross et al.

Table 2 Typical composition of commonly used aquaculture feed ingredients (values are g kg)1 DM unless otherwise detailed)
1 2 1
Nutrient Fishmeal Yellow lupin NL lupin LPC Expeller canola meal SE canola meal SE soybean meal SPC

Dry matter content (g kg ) 917

)1
903 885 942 898 962 909 939
Crude protein 770 547 415 690 381 431 518 590
Total lipids 68 87 53 93 136 22 47 54
Ash 142 44 33 31 66 86 69 79
Phosphorus 22 6 4 5 24 23 8 9
Gross energy (MJ kg)1 DM) 21.3 20.9 20.4 22.2 23.1 19.6 19.6 20.3
Arginine 43 61 47 78 39 32 42 45
Histidine 25 15 10 15 28 26 14 15
Isoleucine 28 20 15 27 3 3 23 26
Leucine 55 45 29 51 28 25 44 48
Lysine 46 23 14 25 46 41 28 28
Methionine 21 4 3 5 37 30 9 9
Phenylalanine 29 21 16 28 29 27 27 30
Threonine 32 20 16 23 18 16 24 25
Valine 34 19 14 23 66 78 24 27

NL lupin, narrow-leaf lupin Lupinus angustifolius (mixed cultivars) kernel meal; LPC, Lupinus angustifolius (mixed cultivars) protein concentrate; SE,
solvent-extracted. SPC, soybean protein concentrate; HP300, Hamlet-protein, Horsens, Denmark; EHC, enzymatically hydrolyzed casein. Data from
Glencross (unpublished).
1
Chilean anchovetta meal. 2 Lupinus luteus (cv. Wodjil) kernel meal.

analysis increases. As many modern aquaculture feeds are
now being formulated on a digestible amino acid basis, the
importance of ingredient amino acid composition is import-
ant (Sorenson et al. 2002).
Details of ingredient composition should include moisture
content but other variables should be expressed on a g kg)1
dry matter basis, to help standardize the ingredient infor-
mation. This is because most feeds are prepared with the
addition of water to ingredients followed by a drying process,
which dehydrates them to a relatively uniform dry matter
content. Therefore, it is more practical to provide a stan-
dardized assessment of composition, such as that on a dry
matter basis. The moisture content of the original raw
material is important when considering ingredient storage
and in the feed manufacturing process, but as pointed out
will not necessarily have an important bearing on the final
moisture content of the feed.
The methods used for composition analysis should be
consistent with those recommended by the Association of
Official Analytical Chemists (Association of Official Analy-
tical Chemists (AOAC) 1993). Specific recommendations
pertaining to the evaluation of grain products were made by
Petterson et al. (1999) who identified several opportunities
for improvement or modifications to methods that made
them more suitable for grain products.
The presence and concentration of ANF or bioactive
compounds such as protease inhibitors, saponins, glucosi-
nolates and alkaloids have important implications for
potential ingredient use and as such form an important part
of the characterization of the ingredient. This too must be
recorded, as this information can influence the strategy used
to evaluate the potential for use of an ingredient, or even
whether it is worth pursuing at all. For a comprehensive
review on ANF, refer Francis et al. (2001). Availability and
price are key determinants of the potential for an ingredient
to be used. When characterizing ingredient availability, vol-
ume, time of availability (e.g. is it available every day or week
or is it only available during one season each year), source
(e.g. is it available everywhere or only in a particular city or
region) and accessibility (i.e. supply chain mechanism) are all
important. Ingredient pricing is obviously critical, and it is
important to explain what drives price change. Weather
(especially droughts) will affect supply and price of grains
and other agricultural products, as will the changing patterns
in ingredient use for other purposes.
Ingredient preparation prior to evaluation
To ensure that any assessment of feed ingredients is under-
taken on representative samples, it is important that due
consideration is given to the physical preparation of all the
feed ingredients with respect to particle size. Fine grinding
(200–300 lm) is important to ensure homogeneity in the
finished diet. This extends beyond the test ingredients to all
those used in any experimental diets. Particle size has been
implicated as an important factor in affecting the ingredient
evaluation process (Kaushik 2001; Nir & Ptichi 2001). A
recommendation of 250 lm for maximum particle size was
made by the National Research Council (NRC 1993).
Application of this recommendation has not been widely

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 Feed ingredient evaluation strategies for aquaculture feeds
adopted with a maximum particle size of 600–800 lm being
more typical (Burel et al. 2000; Glencross & Hawkins 2004).
Most studies provide little indication of particle size for any
of the ingredients used.
The sample size required will vary according to many
factors and every effort should be made to ensure homo-
geneity of the batch used, irrespective of any samples taken
for analysis (Jiang 2001). Principally, a large enough sample
or a number of replicate samples should be taken to account
for prospective variation within a batch of an ingredient. The
specific sample size required, however, will vary according
to many factors (Jiang 2001). Notably, any intrasample
variability can be largely minimized by thorough mixing of
the ingredient sample prior to allocation to any diet and
should be considered routine practice in experimental diet
preparation.
Evaluation of ingredient digestibility
Modern aquaculture diets are routinely formulated based on
the digestible nutrient and energy criteria (Cho & Kaushik
1990). Measuring digestible energy and digestibility of
ingredients and diets simply means measuring that amount of
the energy or nutrient that is not excreted in faeces. Energy or
nutrients in faeces are clearly unavailable for maintenance or
growth and represent one of the major ÔlossesÕ from intake to
tissue growth.
In assessing diet digestibilities, the two key methodological
approaches are the direct and indirect assessment methods
(Maynard & Loosli 1969). In the direct assessment method, a
complete account of both feed inputs and faecal outputs is
required. The digestible value of the feeds is then determined
on a mass-balance basis. Unfortunately, this method is
fraught with problems, largely because of the difficulty and
errors involved with collection of accurate data on feed
intake and faecal production. Indirect assessment is the
alternative. Here, a representative sample of both the feed
and the faeces is required and an indigestible marker is added
to the diet. The ratio of the marker in the feed and faeces
determines dry matter digestibility and is used to calculate
digestibility of energy and other nutrients. Indirect assess-
ment gives Ôapparent digestibilityÕ.
Feed issues in ingredient digestibility assessment
Ideally, assessments of the digestibility of ingredients would
be made by feeding single ingredients. However, this is rarely
possible, because aquatic animals will refuse to eat many
diets comprising a single ingredient. Instead, an ingredient
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21
substitution approach is usually used, where test diets com-
prise the test ingredient plus a reference diet component.
The reference diets used for digestibility studies with most
aquaculture species have usually been simple, practical diets.
Typically, these diets have been based on fishmeal as a key
protein and energy source, although other ingredients have
also been routinely used, such as soybean meals and wheat
flours (Cho & Slinger 1979; Gomes et al. 1995; Glencross &
Hawkins 2004). While reference diets are usually formulated
to meet requirements for energy and other nutrients, it is
acknowledged that diet composition can influence ingredient
digestibility (Lupatsch et al. 1997). To address this issue,
some attempts at formulating test diets to equivalent protein
levels through the complementary substitution of an addi-
tional ingredient have been made (Glencross et al. 2003c).
Essentially, there are two methods of ingredient inclusion
for specific ingredient digestibility assessment. These are
usually referred to as the diet replacement method (DRM) or
the ingredient replacement method (IRM) (Aksnes et al.
1996). With the DRM method, a test ingredient is added to
replace a portion of the reference diet to create a test diet.
The digestibility values for both the reference and test diets
are then determined and, based on proportionality factors,
the digestibility of the ingredient, or any of its nutrients can
be calculated. It is important to note that, with this method,
the portion of the reference diet within any test diet must be
fully representative of the complete reference diet. For
example, all ingredients, including additives and the marker,
must be included in the portion of the reference diet used and
not added to the test diet at equivalent levels as those in the
reference diet.
The IRM also uses a reference diet, but differs in that the
reference diet usually has a single, well-defined reference
ingredient at a fixed, moderately high inclusion level (Aksnes
et al. 1996). This single ingredient is then replaced with the
test ingredients to create the test diets. The assessment of the
digestibility of any ingredient is then based on the relative
diet digestibility with regard to the reference ingredient. With
this method, the basis of the digestible value of the test
ingredient is largely dependent on the choice of the reference
ingredient and its assigned or measured digestibility values
(Morales et al. 1994). Furthermore, with the choice of a
reference ingredient as one of the test ingredients in the DRM
method, it effectively becomes possible to capture the
strengths of both methods (Glencross & Hawkins 2004;
Glencross et al. 2004d).
The amount of the test ingredient that is included into a
test diet also has important implications for the rigor of the
digestibility assessment made (Smith & Tabrett 2004).
22 B. D. Glencross et al.
Typically, it is more reliable where there is a larger con-
tribution to the diet made by the test ingredient. It is useful to
evaluate ingredients at levels more typical to those used in
practical diets and a 20–40% inclusion level on an as-received
basis is common (Gomes et al. 1995; Allan et al. 1999a).
There are also potential benefits from examining the inclu-
sion of a particular ingredient at more than one inclusion
level, as it allows the examination of potential interactive
effects of ingredients within a feed formulation (Allan et al.
1999a,b). This is especially important for carbohydrate
sources where effects of inclusive content on digestibility tend
to be greater than for protein on lipid sources.
A wide variety of marker types have been used in aqua-
culture nutrition digestibility studies. While chromic oxide
(Cr2O3) is perhaps the most commonly used marker, rare
earth metal oxides such as ytterbium oxide, yttrium oxide and
other rare earth metal oxides are gaining favour (Austreng
1978; Ringo 1995; Austreng et al. 2000). For studies focusing
on lipid utilization, hydrocarbon markers such as cholestane
have proved useful (Carter et al. 2003). While endogenous
markers such as acid-insoluble ash and crude fibre have been
used, they are somewhat less reliable and more prone to
producing data with larger variance (Morales et al. 1999).
Collecting faeces for digestibility assessment
The method of ÔfaecalÕ collection used in aquaculture nutri-
tion research has been well debated. Essentially, there are
three methods adopted by most researchers; dissection,
stripping and collection of voided faeces. Where digesta (not
called faeces until actually excreted) are collected by dissec-
tion or stripping, there is the potential to underestimate
digestibility because of incomplete digestion and potential
contamination of digesta with endogenous material. In con-
trast, when faeces are collected from the water column or
following settlement, there is the potential to overestimate
digestibility because of leaching losses of organic matter.
Early studies by Austreng (1978) examined the changes
in diet digestibility when assessments were made from
digesta collected from different parts along the gastro-
intestinal tract (GIT) of rainbow trout using a dissection
approach. In that study, substantial increases in digestibility
were noted throughout the GIT except between the prox-
imal and distal intestine. Austreng (1978) argued that this
was supportive of the use of faecal stripping techniques,
where gentle abdominal pressure is applied to the abdomen
of the fish, approximately over the distal intestine, to expel
its faecal contents. It should be noted, however, that it is
not always possible to collect faeces from all species of fish,
especially juveniles, using stripping and it is impossible for
crustaceans.
Cho & Slinger (1979) collected rainbow trout faeces after
defaecation using a steep-sided conical tank with the faeces
collecting in a small settling chamber. Allan et al. (1999a)
adopted a similar approach to collect faeces from silver
perch, Bidyanus bidyanus, but used steeply sloped cylindro-
conical tanks with a terminal collection chamber. Choubert
et al. (1982) developed a modification to the settlement
approach of Cho & Slinger (1979), which involved the settled
faeces being removed from the water onto a moving screen
before being deposited onto a collection tray.
A study by Vandenberg & de la Noue (2001) compared the
influences of three faecal collection methods (Austreng 1978;
Cho & Slinger 1979; Choubert et al. 1982) of the digestibility of
a practical diet containing a range of protein sources (e.g.
fishmeal, soybean meal, whey, blood meal) when fed to rain-
bow trout. The findings of that study suggested that there was
essentially no difference in diet digestibility assessments
between the methods of Cho & Slinger (1979) and Choubert
et al. (1982), but that both of these settlement methods resulted
in significantly different (higher) diet digestibilities than those
determined from the faecal stripping collection method.
Glencross et al. (2005) compared the digestibilities of a
series of ingredients when faeces were collected using either
settlement (Cho & Slinger 1979) or stripping techniques
(Austreng 1978) (Table 3). Significant differences were
observed between the two faecal collection methods as to
their effects on ingredient digestibility. Notably, the effect
was more pronounced on ingredients high in indigestible
carbohydrates. Faecal stripping provides a more conservative
estimate of both diet and ingredient digestibilities than that
provided using settlement techniques.
The duration of the faecal collection period is usually
largely dependent on obtaining sufficient sample to be able to
undertake the required chemical analyses although in gen-
eral, a longer period of collection e.g. >5 days will minimize
variability in faeces composition and improve reliability of
results; although arguably this can also be resolved using a
larger number of fish from which to collect faecal samples.
Reduction in variance has the potential to substantially
improve the experimental power and capacity to detect sig-
nificant effects (Searcy-Bernal 1995).
Experiment management issues in ingredient
digestibility assessment
As with any experimental strategy, management of the
operational conditions is critical. The experimental condi-
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 Feed ingredient evaluation strategies for aquaculture feeds 23

Table 3 Ingredient digestibility when compared using rainbow trout, but different collection methods (data derived from Glencross et al. 2004e)

Fish meal Narrow-leaf lupin LPC LPI Soybean meal SPC EHC Pooled SEM

Stripping
a
Organic matter 93.1a a
44.6c a
70.7b a
87.6a a
61.0b a
67.2b a
89.1a 2.95
a
Phosphorus 35.1de a
346.0a a
138.5bc a
120.9cd a
27.7e a
76.3d a
92.3cd 14.15
a
Energy 99.0a a
53.1c a
84.2ab a
91.3ab a
72.1bc a
87.3ab a
91.5ab 2.94
a
Nitrogen/protein 87.5b a
85.3b a
98.4a a
95.1ab a
92.1b a
97.9a a
92.2b 1.21
Settlement
a
Organic matter 94.5ab b
64.8c a
76.7c a
94.8a b
77.3c b
82.0c a
98.5a 1.62
a
Phosphorus 36.2d b
272.2a b
87.2c b
71.7c a
56.7cd a
58.9cd a
85.4c 10.73
a
Energy 96.4ab b
70.5e a
86.6cd a
93.8ab a
83.3d a
85.6d a
98.8a 1.27
a
Nitrogen/protein 89.3d b
97.2c a
101.0b a
98.6bc a
99.0bc b
106.9a a
96.0c 0.67

Different presuperscripts within columns indicate significant differences between mean values of collection method, but within nutrients
and ingredients (P < 0.05). Different postsuperscripts within rows indicate significant differences between mean values of ingredients, but within
collection method and nutrients (P < 0.05).

tions used can affect digestibility and need to be managed
accordingly. Key considerations include environmental con-
ditions, fish size and feeding ration structure.
Environmental conditions, notably water temperature,
seem to have minor effects on digestibility; Windell et al.
(1978) noted little influence of water temperature (7, 11 and
15 C) on dry matter, protein, lipid, carbohydrate or energy
digestibility of a diet fed to rainbow trout of three size classes
(19, 207 and 585 g), except for the lowest temperature and
the smallest fish. Conversely, substantial differences were
noted in the digestibility of starch of varying levels of gela-
tinization between rainbow trout (Oncorhynchys mykiss) held
at either 8 C or 18 C (Kaushik 2001) and Kim et al. (1998)
reported temperature effects on lipid and energy digestibility
for common carp (Cyprinus carpio) at 18 and 25 C. Ideally,
conducting experiments in thermal regimes that are optimal
for each species is the most desired regime, as it will maximize
feed intake and also faecal/digesta sample collection.
Feed allocation or ration size has been shown to influence
digestibility assessment, but only at the highest feeding rates
(Windell et al. 1978). In that study, rainbow trout were fed
varying rations of 0.4%, 0.8% and 1.6% of live weight per
day, with fish fed the highest feed ration, yielding signifi-
cantly lower digestibility values for dry matter, carbohydrate
and energy, but not for protein or lipid. It could be argued
that restricted pair-feeding is the most suitable approach for
standardizing digestibility experiments because of this, but
practicality and the need for application of the data to
commercial conditions mean that feeding to apparent satiety
is a more useful strategy.
Substantial variability has been noted in the digestibili-
ties of diets during the period when fish are first fed a new
diet (Wybourne & Carter 1999). Because of this, a period
of acclimation to diets prior to collection has been sug-
gested and adopted by most researchers (Allan et al.
1999a; Burel et al. 2000). The length of this period varies
among researchers, although the data from Wybourne &
Carter (1999) suggest that, as there was sufficient reduction
in variability of digestibility assessments of most diets after
4 days, that collection should be commenced from day 5.
For an additional degree of conservatism, most researchers
use a minimum of 7 days acclimation (Allan et al. 1999b;
Sorenson et al. 2002).
Calculating diet and ingredient digestibilities
The calculation of diet and subsequently ingredient digesti-
bilities are largely derived from methods developed from ter-
restrial animal nutrition science (Maynard & Loosli 1969). The
use of digestibility assessments for aquaculture was first
adapted by Cho & Slinger (1979). These researchers developed
much of the original methodology for faecal collection based
on the ÔGuelph-styleÕ faecal settlement tank system and
adopted the calculation methods being used by the terrestrial
nutrition sectors. The calculation allows the determination of
the apparent digestibility, based on the ratio of marker in the
diet and faeces. The apparent digestibility coefficient (ADCdiet)
of each specific nutritional variable is based on Equation 1
 
Markerdiet  Nutrientfaeces
ADCdiet ¼ 1  ð1Þ
Markerfaeces  Nutrientdiet
In this equation, the terms Markerdiet and Markerfaeces rep-
resent the marker content of the diet and faeces, respectively,
and Nutrientdiet and Nutrientfaeces represent the nutritional
parameter of concern (e.g. protein or energy) in the diet and
faeces, respectively. With this formula, values would typically
range from 0 to 1. To achieve a percent apparent digestibility,
the equation should be multiplied by 100. To calculate the
digestibility of the test ingredient, Cho & Slinger (1979) used

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24 B. D. Glencross et al.
½ADNtest  ðADNbasal  70%Þ
Nutr:ADingredient ¼ ð2Þ
ð30%Þ
In Equation 2, the Nutr.ADingredient is the digestibility of a
given nutrient (or energy) from the test ingredient included in
the test diet at 30%. ADNtest is the apparent digestibility of
the nutrient of interest in the test diet. ADNbasal is the
apparent digestibility of the same nutrient from the basal
diet, which makes up 70% of the test diet. A progression of
this equation was reported by Sugiura et al. (1998) who used
Nutr.ADingredient
½ADtest  Nutrtest  ðADbasal  Nutrbasal  70%Þ ð3Þ
¼ The first key assumption is that digestible coefficients are
ð30%  NutrIngredient Þ
In Equation 3, the Nutr.ADingredient is the digestibility of a
given nutrient from the test ingredient included in the test
diet at 30%. ADtest is the apparent digestibility of the test
diet. ADbasal is the apparent digestibility of the basal diet,
which makes up 70% of the test diet. Nutringredient, Nutrtest
and Nutrbasal are the levels of the nutrient of interest in the
ingredient, test diet and basal diet, respectively (Sugiura et al.
1998). A similar equation (Equation 4), also based on the
nutrient contribution of the test ingredient to the digestibility
assessment, was proposed by Forster (1999).
Nutr:ADingredient
 
ð70%  Nutrbasal þ Nutringredient  30%Þ  ADtest  ð70%  Nutrbasal  ADbasal Þ
¼
Nutringredient  30%
In Equation 4, Nutr.ADingredient is the digestibility of a given
nutrient from the test ingredient included in the test diet at
30%. ADtest is the apparent digestibility of the test diet.
ADbasal is the apparent digestibility of the basal diet, which
makes up 70% of the test diet. Nutringredient, Nutrtest and
Nutrbasal are the levels of the nutrient of interest in the
ingredient, test diet and basal diet, respectively (Forster
1999). Essentially, this equation is the same as that of Sugiura
et al. (1998). More recently, prudent amendments have been
suggested by Bureau (2006) to ensure that any differences in
dry matter content of the reference diets and test ingredient
are accounted for.
Of the last three equations, the latter two are the more
appropriate ones for determining ingredient digestibilities,
because they account for relative contribution from test
ingredient and reference diet to energy or nutrient digesti-
bility being estimated. For example, if an ingredient with
90% protein is added to a reference diet at a ratio of
30% : 70% and the reference diet contains 50% protein, then
the added test ingredient actually contributes 270 g kg)1 of
protein to the total protein of the diet (>40% of total dietary
protein) and the reference diet contributes 350 g kg)1 of
protein to the test diet. This is substantially more than
the 30% indicated by the direct proportion of ingredient
substituted.
Assumptions inherent in use of digestibility data
There are several key assumptions in the digestibility
assessment process.
additive, which is, if you sum the proportional digestibility
values for each ingredient in a diet, then it will equal the
measured digestibility of the diet. This assumes that there are
no interactions among ingredients that differentially affect
digestibility, and that changing the inclusion content of a
particular ingredient does not change its digestibility either.
Neither of these assumptions holds true all the time. Several
studies using a range of practical ingredients have been used
to validate the assumption of additivity [Cho et al. (1982)
with rainbow trout, Watanabe et al. (1996) with carp, rain-
bow trout, tilapia and ayu, Wilson & Poe (1985) with channel
ð4Þ
catfish and Allan et al. (1999a,b) with silver perch]. However,
it has been shown conclusively that digestibility of some
ingredients, particularly ingredients dominated with carbo-
hydrate sources, varies considerably with inclusive content
(Stone et al. 2003). It is recommended, therefore, that
digestibility of carbohydrate sources is assured at several
inclusion levels to improve the robustness of the assessment
of digestibility of such ingredients.
A second key assumption is that the marker used to cal-
culate apparent digestibility is inert, that is it passes through
the digestible tract without influencing digestion at approxi-
mately the same rate as digesta. Chromic oxide, perhaps the
most commonly used marker, has been claimed to affect
carbohydrate digestibility (Shiau & Liang 1995) but effects
are relatively minor and probably do not reduce the value of
digestibility results calculated with that marker (Ng & Wilson
1997) and did not affect carbohydrate digestibility with gilt-
head sea bream (Fernandez et al. 1999).
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 Feed ingredient evaluation strategies for aquaculture feeds 25

A third assumption is that digestibility coefficients will fall
between 0% and 100%. Sometimes this is not the case.
Possible causes are analytical errors for markers or nutrients,
poor mixing of the marker in the diet or Ônon-representativeÕ
samples of diet or faeces or, as mentioned earlier, interaction
between ingredients. Even when all possible sources of error
are checked and minimized, digestibility coefficients above
100 or below 0 do occur (Table 4). We recommend that these
values be reported but rounded to 0 or 100 when used to
formulate diets on digestible energy or nutrient basis.
A fourth assumption is that faeces collected will be Ôrep-
resentativeÕ of those excreted over time. Allan et al. (1999a,b)
showed that digestibility coefficients varied when taken from
faeces collected at different periods after the last meal. To
obtain a representative sample, faeces must either be collec-
ted over a sufficiently long period after feeding ceases. For
stripping (or dissection) faeces (digested) need to either be
obtained from different animals at different periods after
feeding ceases or animals should be fed continuously and
digesta sampled at a single time.
Use of a standard reference ingredient in each digestibility
experiment provides a useful way to standardize and assist in
the assessment of the temporal and intralaboratory variance.
Ingredients such as vitamin-free casein, enzymatically
hydrolysed casein and wheat gluten have been used for this
purpose (Morales et al. 1994; Glencross & Hawkins 2004;
Glencross et al. 2005) to help standardize digestibility
assessments over time.
Ingredient palatability assessment
Formally, palatability is defined as acceptable to the taste or
sufficiently agreeable in flavour to be eaten. While it may be
difficult to ascertain whether or not a fish ÔlikesÕ some flavour
or not, it is certainly possible to determine differences in the
amounts of feed eaten. It is this context that we refer to
palatability of a feed and by inference an ingredient. This
is important because, irrespective of how digestible the
nutrients and energy from a particular ingredient are, if the
ingredient reduces feed intake it is of limited use in a feed
formulation.
While there may be strategies to avert or resolve palata-
bility issues of feed ingredients using ingredient processing
or feeding stimulants, clearly it is the best if these can be
avoided. For research to be clearly categorized as nutritional
research, it primarily has to be based on the ingestion of
nutrients by an organism; therefore one of the key assessment
criteria of good nutritional research should be some
demonstration of the level of food intake by the target
organism. Such an assessment then allows some manner of
measurement of a response relative to that feed intake.
However, assessing palatability, particularly for aquatic
animals, is not necessarily straightforward. This topic is a
large issue in its own right and therefore will only be sum-
marily examined here. For further details, examination of the
reviews and texts by Jobling et al. (1995, 2001) and Ruoho-
nen et al. (2001) are suggested.
For an animal to demonstrate a feed intake response, it
must be given the opportunity to refuse feed, therefore
feeding beyond apparent satiety is an imperative. Regardless
of whether an ingredient affects attractiveness in palatability,
its effect on feed intake must be assured independently of
effects on utilization of energy and nutrients. Feed preference
studies are one way of assessing effects on intake. The use of
self-feeding through computer-managed feedback response
mechanisms is another option that has been frequently used
to allow discrimination of feeds by fish and certainly assists
in removing human error from the feed intake assessment
process (Juell 1991; Boujard & Le Gouvello 1997; Burel et al.
1997).

Table 4 Typical representation of ingredient digestibilities and digestible nutrient levels. Note that those digestibility values of over 100% have
been rationalized to absolute (100%) digestibility for calculation of digestible nutrient levels in each ingredient. (Data derived from Glencross
et al. 2004d)

Nutrient Fishmeal Narrow-leaf lupin APC API Soybean meal SPC EHC

Digestibility
Organic matter 93.1 44.6 70.7 87.6 61.0 67.2 89.1
Phosphorus 35.1 346.0 138.5 120.9 27.7 76.3 92.3
Energy 99.0 53.1 84.2 91.3 72.1 87.3 91.5
Nitrogen/Protein 87.5 85.3 98.4 95.1 92.1 97.9 92.2
Digestible nutrient
Organic matter 799 431 685 850 568 619 828
Phosphorus 8 4 5 5 2 7 8
Energy 21.1 10.8 18.7 20.6 14.1 17.7 19.4
Protein 673 354 679 770 477 578 774

APC, lupin protein concentrate; API, lupin protein isolate; SPC, soybean protein concentrate; EHC, enzymatically hydrolysed casein.

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26 B. D. Glencross et al.
One of the key aspects to examine is variability in feed
intake over time. Adaptation to diets has been noted with
some species (Wybourne & Carter 1999). To enable such
examination in variability, an assessment of daily feed intake
of individual replicates is advantageous.
Ingredient inclusion trials are probably the simplest way to
examine effects on feed intake. An ingredient can be included
in a reference diet to create a test diet and then the reference
and test diets are fed to apparent satiety to replicate groups
of fish for each diet. Significant differences in feed intake
between the reference and test diets reflect the apparent
palatability because of the test ingredient. The issue of how
much ingredient to include in the test diets is somewhat
subjective. Ideally, a range of test ingredient inclusion levels
that cover what would be the practical inclusion levels should
be used, as this also allows examination of critical palata-
bility levels or break points (Shearer 2000). Unfortunately,
one of the often-identified limitations with such experimental
designs is both the sensitivity and capacity for the experi-
ments to actually detect significant differences. This high-
lights the need for stringent experimental design, sufficient
experimental power and the appropriate use of control
treatments (Ruohonen et al. 2001).
The additional use of positive control treatments, such as
those with palatability inhibitors like sulfamerazine sodium
(Boujard & Le Gouvello 1997), provides an added degree of
confidence to experiments examining intake effects. Notably,
many experiments designed to examine serial inclusion effects
of a particular ingredient end up with no significant effects.
While this can be used to argue that the ingredient is palat-
able to the test animal up to the inclusion level used, it is
often difficult to determine the degree of confidence in such
results without positive controls designed to demonstrate a
specific effect, such as a decrease in feed palatability.
Nutrient utilization value or interference
Once the variables of digestibility and palatability of an
ingredient have been defined, the remaining key issue to
resolve is based on the capacity of the animal to utilize the
digested nutrients for growth. There can be many aspects to
this issue depending on the nature of the ingredient. For
example, problems associated with metabolic modifiers, such
as glucosinolates, are one such issue that will often not be
identified through digestibility or palatability studies per se
and are more suited to a study that defines the specific nature
of the problem through controlling for digestibility- and
palatability-related variability. In addition, subtle effects of
differences in things such as amino acid composition and
availability are often lost in the ÔnoiseÕ of experimental vari-
ance, if appropriate controls are not employed.
Measuring growth
The initial weight and size variability of the animals used in
the study has an important bearing on the capacity of a study
to determine significant effects. Studies should be conducted
on animals of an appropriate size for the intended applica-
tion technology. For instance, there is limited value in con-
ducting alternative ingredient utilization evaluation on larval
or juvenile fish with the intention of the data for grow-out
production implications (Windell et al. 1978).
With most nutrient utilization studies, the response vari-
able is growth. Accordingly, this is often simply defined as
the difference between initial and final live weights. More
specifically, this should be defined as live-weight gain. As
such the live-weight gain of the animals from each replicate
should be determined, using the same equipment as that used
to determine their initial weights, and averaged with data
from other replicates to form a treatment final weight
assessment. Live-weight gain is also often reported as per-
centage gain, which is usually expressed as a percentage
of the final weight divided by the initial weight. For
such measures as this, it is imperative that replicate specific
initial weights are used in any statistical analysis such as a
covariance.
Technically, for a measure to be a rate it has to be time
specific. The three most routinely used growth rate assess-
ments are daily gain (DG), daily growth coefficient (DGC)
and specific growth rate (SGR). DG is perhaps the simplest
of the three rates and is merely the live-weight gain over time
and is often given in units of g day)1. It is also perhaps the
most practical of all growth rate assessments in that it pro-
vides information in a more tangible assessment. DGC in
contrast is calculated based on a percentage of the one-third
root transformation of the final (Wf) and initial (Wi) live
weights over time (t) (Equation 5) (Kaushik 1998):
1=3 1=3
DGC ¼ ½ðWf  Wi Þ=t  100 ð5Þ
The SGR is calculated based on the percentage of the natural
logarithm transformation of the final (Wf) and initial (Wi)
live weights over time (t) (Equation 6) (Kaushik 1998):
SGR ¼ ½ðln Wf  ln Wi Þ=t  100 ð6Þ
A further growth rate parameter gaining use is the thermal
growth coefficient (TGC), which is derived from the DGC,
but the time component is expanded to be considered on a
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temperature basis. In this regard, the time component of the
TGC is multiplied by the average temperature (C) over the
period of the study (t) (Cho & Bureau 1998) (Equation 7):
1=3 1=3
TGC ¼ ½ðWf  Wi Þ=ðt  CÞ  100
The point of using a growth rate descriptor is essentially to
try and standardize the assessment and allow for some
comparability of performance across experiments. To
achieve this, such a rate should ideally be independent of size.
Kaushik (1998) reviewed the applicability of both DGC and
SGR for growth assessment of a variety of non-salmonid
species and noted that SGR did not provide a very good
transformation of growth rates when compared with that
achieved using DGC. It was noted that an inverse logarith-
mic relationship was apparent with the SGR transformation,
but that DGC provided a more uniform rate across the entire
fish live-weight range studied. In this regard, if such a growth
rate descriptor is required, then DGC is perhaps more
appropriate than SGR, but if the initial weights of the ani-
mals are provided, then gain per day is perhaps just as, if not
more practical.
Survival
Stock losses sustained during an experiment are usually
presented as a percentage survival. This is determined based
on the percentage basis of the number of individuals survi-
ving at the end of a study relative to the number included in
the study at the beginning. Unless the percentage is divided
by the time of the experiment, survival should not be
reported as a rate.
Feed intake and conversion efficiency
For an assessment to be made on the nutrient utilization of a
diet, and by reference an ingredient, there is a clear need to
measure feed intake. Feed intake by fish is often reported as
both an amount (g fish)1) and rate (g fish)1 day)1). How-
ever, accurate assessment of feed intake by fish is one of the
more difficult aspects of aquaculture nutrition research to
achieve.
The efficiency of food use by fish is usually reported as
either feed conversion efficiency (FCE: Equation 8) or feed
conversion ratio (FCR: Equation 9). These assessments are
usually made on a dry weight of food and live weight of fish
basis. Because these variables rely on both live-weight gain
and feed intake assessment, they assume the errors of both
assessments.
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Feed ingredient evaluation strategies for aquaculture feeds 27
FCE ¼ ðweightgainÞ=ðfeedconsumedÞ ð8Þ
FCR ¼ ðfeedconsumedÞ=ðweightgainÞ ð9Þ
ð7Þ
Nutrient retention
The efficiency by which nutrients and energy are retained
from feeds provides a useful assessment of the efficiency of
nutrient utilization from diets (Cho & Kaushik 1990; Booth
& Allan 2003; Glencross et al. 2004b). To determine this,
assessment of the nutrient and energy composition of both
the feed and the fish is required on as fed and live-weight
basis, respectively. However, such data can be strongly
influenced by animal size, with smaller animals typically
being far more efficient at retaining both nutrients and energy
than larger fish of the same species (Lupatsch et al. 2002).
Apparent biological value (ABV) is another parameter
often quoted and is largely a derivation of nutrient and
energy retention values (Morales et al. 1994). Typically, ABV
is the retention percentage but on a digestible nutrient basis.
In this regard, it provides some assessment of the proportion
of the nutrients or energy absorbed from the diet that is
actually used for somatic growth. Clearly, for this parameter
to be estimated, an assessment of the diet nutrient and energy
digestibilities is required.
Additional response variables
The range of response variables that can be examined is quite
substantial and increasing. Clearly, there are some variables
that are critical for an objective assessment of the perform-
ance of fish fed specific diets. However, there are additional
variables that can provide useful additional information and
are worth considering in the appropriate circumstances,
depending on the specific objectives of the study.
Biochemical alternatives, such as changes in blood glucose
levels or thyroid hormone levels (triiodothyronine and thy-
roxine), or enzyme activities have also been used to provide
an indication of disturbance by ANF to the metabolic
function and nutrient utilization by fish (Burel et al. 2001).
Biochemical factors such as homeostasis hormones (e.g.
thyroxine and triiodothyronine) have the potential to be
sensitive indicators of disruption to metabolic function and
nutrient utilization by fish. Other hormone assays used more
recently include insulin-like growth factor 1 (Dyer et al.
2004). More recently, the examination of protein expression
using two-dimensional electrophoresis has allowed the
assessment of the influences of specific diet types on hepatic
28 B. D. Glencross et al.

metabolism (Vilhelmsson et al. 2004). Studies such as these
are significantly advancing the understanding of specific
ingredient effects on functional metabolism.
Effects of dietary treatments on whole somatic or organ-
specific composition are another means of assessment of
potential ingredient effects (Ruyter et al. 2000; Booth &
Allan 2003). Notably, whole somatic composition analysis is
required for the examination of nutrient/energy utilization
efficiency and/or ABV assessments.
Organoleptic properties, while not a common assessment,
have been used to evaluate the potential impact of novel
ingredients on product quality aspects. Such assessments
have been more prevalent in studies on fish oil replacement
than for fishmeal replacement, but some reports on influences
of products such as rendered meat meals do exist
(Thomassen & Rosjo 1989; Williams et al. 2003a,b).
An evaluation of immune responses and parameters
associated with an immune challenge have also been effect-
ively used in recent times (Hardy 1999; Krogdhal et al. 2000;
Montero et al. 2003).
Strategies for evaluating the inclusion of ingredients
Limited-inclusion trials are perhaps the most typical ingre-
dient evaluation trials used (Hughes 1988; Refstie et al. 1998;
Carter & Hauler 2000; Table 5). In these studies, the test
ingredient is substituted at either one or two levels only,
usually for fishmeal, on a basis to maintain equivalent pro-
tein and energy levels. Advancements in this research strategy
involve the substitutions being made on an equivalent
digestible level. Such trials can be useful where experimental
treatment options are limited, and the test ingredient com-
prises a substantial amount of the diet (e.g. 30%). While such
studies provide a limited assessment of the utilization of the
ingredient, the use of only a single inclusion level provides a
relatively risky assessment with little capacity for extrapola-
tion of effects.
An advancement on the limited-inclusion trial is the serial-
inclusion trial, where multiple (three or more) levels of the
ingredient are evaluated (Burel et al. 1998; Farhangi &
Carter 2001; Glencross et al. 2004c; Table 5). Typically, such
trials have used a series of inclusion levels such as 0%, 10%,
20%, 30%, 40% and 50% inclusion of the test ingredient
into a reference diet (0%). In these studies, two key diet
mixes, usually the reference (0%) and the highest inclusion
level are made in sufficiently large quantities to allow the
creation of the other treatment diets by blending at the
appropriate ratios. This strategy reduces diet preparation
risk. This style of utilization trial provides substantially more
information than the single-inclusion level trials. However,
the practicality of the upper level of test ingredient inclusion
has varied substantially with some attempts made at single
ingredient inclusion levels as high as 70% of the diet. How-
ever, inclusion levels of new ingredients in commercial diets

Table 5 Experimental trial types used to study nutrient utilization and ingredient use limitations

Trial type Diet design specifics Ingredient inclusion Feeding strategy Examples

Limited inclusion All diets formulated to same One or two levels of Apparent satiety or Hughes (1988),
relevant practical protein and inclusion fixed ration Refstie et al. (1998),
energy levels (usually on a Carter & Hauler (2000)
digestible basis) for use with
respective aquaculture species
Serial inclusion All diets formulated to relevant Included in a series Apparent satiety or Robaina et al.
practical protein and energy levels of test diets at fixed ration (1995), Burel et al.
(usually on a digestible basis) for varying inclusion (1998), Farhangi &
use with respective aquaculture levels Carter (2001)
species
Summit dilution Reference diet formulated to Included in a series Apparent satiety or Williams et al.
relevant practical protein and of test diets at fixed ration (2003a,b), Booth &
energy levels for use with varying inclusion Allan (2003)
respective aquaculture species, levels
but protein and energy
specifications not maintained
with progressive inclusion of test
ingredients
Limiting constraint All diets formulated to equivalent Included in a series Restrictively pair-fed Glencross et al.
digestible protein and energy of test diets at (2003d, 2004b)
levels, but with protein provided varying inclusion
at a reduced proportion of levels
estimated requirements

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 Feed ingredient evaluation strategies for aquaculture feeds
are unlikely to ever be greater than 25% of the diet, therefore
increasing the number of inclusion levels over a practical
range, rather than going to extreme inclusion levels adds
greater value to a study. Therefore, we propose upper
inclusion levels closer to 40–50% of the diet, will better focus
on ingredient inclusion levels more likely to be used com-
mercially.
A similar strategy was used by Higgs et al. (1983) to
evaluate the nutritional value of different types of canola
meals. However, they also adopted the use of a series of
isoenergetic diets of various protein levels, with one diet
specifically designed to be protein limited as a means of
exacerbating nutrient utilization effects of fish fed the diets.
In this study, the diets were fed to apparent satiety, and some
of the capacity of the study to detect limitations to nutrient
utilization was lost through variability in feed intake in the
protein-limited diets, although significant effects of canola
meal inclusion were noted in the higher protein diets, but not
the protein-limited diets.
To avert the potentially confounding issue of unregulated
feed intake of nutrient-limited diets, the use of limiting-
constraint trials was advocated by Glencross et al. (2003d);
Table 5) as a means of distinguishing the difference in
nutritional value between two closely related varieties of
lupin kernel meal. In this study, earlier experiments using
commercially analogous diets and apparent satiety feeding
strategies demonstrated no significant difference between the
two lupin varieties, but subsequent re-evaluation based on
diets formulated to equivalent digestible energy levels and
reduced protein levels, with a pair-feeding regime used to
minimize dietary intake differences, allowed clear significant
differentiation in nutritional value between the two lupin
varieties.
Summit-dilution trials (SDT) are another ingredient
nutrient utilization assessment trial, which use a serial dilu-
tion principle with increasing amounts of a reference diet
being replaced by a test ingredient (Booth & Allan 2003;
Table 5). In these trials, the ingredient is substituted for
whole diet, similar in principle to the DRM used for digest-
ibility diets, without balancing of nutrients or energy. An
important additional component of the SDT is the use of a
series of controls, where the ingredient inclusion levels are
matched with treatments where a similar amount of non-
nutritional value filler (e.g. cellulose or diatomaceous earth)
is incorporated into diets. These controls provide a reference
to each respective test ingredient treatment by demonstrating
the relative nutrient utilization achieved from that incor-
poration of the ingredient. Feeding of fish in such an
experiment is performed on a restricted basis, so animals
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29
cannot simply increase intake to account for reductions in
energy or nutrients (Booth & Allan 2003). The limitation
with the SDT is that without balancing the diets to an
equivalent digestible nutrient and energy levels, interpreta-
tion of the response effects exhibited may be confounded by
nutrient and energy intake limitations.
Factors likely to affect nutrient and energy
utilization of ingredients
Antinutritional factors have the capacity to have significant
deleterious effects on nutrient utilization by fish. Notably,
substantial effects of a range of ANF have already been
noted on fish using a wide range of experimental techniques
(Krogdhal et al. 1994; Bureau et al. 1998; Refstie et al. 1998;
Burel et al. 2001; Francis et al. 2001; Glencross et al. 2003a,
2006). In defining the effects of ANF on fish, a variety of
experimental strategies have been adopted.
Limiting-constraint trials are another way of defining the
potential impacts of ANF on nutrient utilization by fish. By
limiting the capacity of the fish to self-regulate protein and
energy intake, any influences of the ANF on nutrient util-
ization are manifested as growth effect differences.
Assessment of tissue histology is one additional parameter
that has been useful in examining some of the more long-
term and chronic effects of ingredient and ANF inclusion in
fish diets (Robaina et al. 1995; Krogdhal et al. 2000; Store-
bakken et al. 2000). Some specific parameters such as ente-
ritic problems of the distal intestine, associated with use of
soybean meals are a particular case in example (Krogdhal
et al. 2000, 2003).
Ingredient functionality
Ingredient functionality is another facet of ingredient eva-
luation that is increasing in importance. Functionality of feed
ingredients relates to their effects on the physical properties
of the processed feed (Thomas & van der Poel 2001). For
example, during extrusion processing, starch increases the
porosity of the pellet through expansion of the starch struc-
ture during the starch gelatinization and expansion processes.
However, there are numerous other physical attributes of
aquaculture feeds, which can be influenced by ingredient
choice. Similarly, there are also several methods that can be
used to assess these attributes.
Clearly, the attributes sought are those where pellets
produced from the formulation result in a product with
properties that provide advantages for feeding aquatic spe-
cies. These properties include aspects such as sink rates, pellet
30 B. D. Glencross et al.
durability, degree of starch gelatinization and oil absorption
capacity.
Experimental extrusion processing is clearly one of the
better ways to evaluate ingredient functionality, as the results
will have direct implications for a final product. In these
studies, a hypothetical formulation including a test ingredient
is run through an extruder and the properties of the pellets
produced are compared with either a reference formulation
or a series of target specifications.
Analysis of ingredient or formulation viscosity is a rela-
tively new technique that has been used as a basis for
examining the potential functionality of ingredients and their
possible attributes under feed extrusion conditions (Interna-
tional Association for Cereal Science and Technology (ICC).
1995). Assessment of this has largely made use of rapid
viscosity analysis (RVATM, Newport Scientific, Sydney,
Australia) equipment, and is based on an examination of the
gelling/pasting characteristics of an ingredient/formulation at
varying temperatures, the peak viscosity and relative deteri-
oration of that viscosity with time and temperature. From an
ingredient evaluation perspective, the technique lends itself to
a relatively rapid throughput, provided some relation is made
between the RVA observations and those achieved under
extrusion conditions.
Emerging technologies and issues for
ingredient evaluation
Improving our basis for the assessment of ingredient nutrient/
energy digestibility and availability is perhaps one of the key
issues in maximizing opportunities for optimal use of feed
ingredients for the aquaculture feed sector. While there have
been substantial advances in this area in recent years, there is
a clear need to improve our understanding of the processes of
interactions among ingredients, nutrients and processing and
how these influence digestibility. As the adoption of alterna-
tives to fishmeal increases, there will probably be increasingly
complex interactions among feed ingredients. The nature of
such ingredient interactions may also have important impli-
cations for the study of ingredient functionality.
While there have been several in vitro assays developed for
ingredient digestibility evaluation, there is yet to be a suc-
cessful assay validated against in vivo digestibilities, which
has gained widespread acceptance (Carter et al. 1999). Fur-
ther progress in this area could provide a much-valued test
for the assessment of ingredient quality.
The use of rapid analysis techniques for ingredient com-
position, such as near infrared spectroscopy (NIRS), has
considerable potential to improve the basis for diet formu-
lation from variable batches of raw ingredients. Although
NIRS has gained almost routine use in many feed companies
for the evaluation of crude protein, moisture and fat com-
position of ingredients and products the technology is now
also beginning to be used for the assessment of digestible
nutrients and energy from ingredients (Aufrere et al. 1996).
Presently, the widespread adoption of the use of NIRS for
such digestible nutrient and energy assessments is limited by
the availability of robust digestibility data, on enough sam-
ples of specific ingredients, to develop competent calibration
curves.
The use of exogenous enzymes has been partly explored in
aquaculture nutrition for sometime, but has been largely
limited to the evaluation of phytase enzyme preparations
applied to some plant protein meals to improve phosphorus
utilization and other minor effects (Mwachireya et al. 1999;
Forster et al. 2000). However, substantial scope exists for the
use of other enzymes, many of which are being used by the
terrestrial animal feed sectors (Cowan et al. 1996; Castañón
et al. 1997). The potential of some of these enzymes, such as
amylase, a-galactosidase, b-glucanase and xylanase, has been
explored (Glencross et al. 2003a; Stone et al. 2003). While
these enzymes certainly improve nutrient (mostly carbohy-
drate) absorption across the gut, the effective utilization of
these absorbed nutrients is yet to be fully studied. Other
enzymes such as pectinases, hemicellulases, cellulases and
their potential mode of application are yet to be examined.
To realize this scope, significantly different strategies to those
used with terrestrial animal feeds will be required to allow
application of the enzymes with extrusion-produced fish
feeds. Opportunities such as thermostable enzyme prepara-
tions and preprocessing of ingredients have been recognized
(Bedford 2000).
There is some information on the mode of action of bio-
logically active compounds on fish (beneficial and negative).
Some of the more notable biologically active compounds, or
ANF, such as glucosinolates, oligosaccharides, saponins,
gossypol and trypsin-inhibitors have been studied to an
extent (reviewed in Francis et al. 2001). However, most
studies examined a specific ANF and focus on whether they
exert an effect on the animal, but not the specific mode of
action by which the compound affects the animal.
There is also limited information on the efficiencies of
amino acid and energy utilization and how these differ
between ingredients. While the determination of the digest-
ible value of ingredients is the first basis for improving our
understanding of the nutritional utilization of feeds and
ingredients, this could be further improved by refining the
understanding to a level specific to nutrient and energy
..............................................................................................
 2007 Blackwell Publishing Ltd Aquaculture Nutrition 13; 17–34
 Feed ingredient evaluation strategies for aquaculture feeds
utilization from feeds and ingredients. This would not only
serve as a basis for improving our understanding of the
nutritive values, but also for estimating an economic value on
specific ingredients for inclusion in aquaculture feeds.
Conclusions
As aquaculture continues to develop, there will be an
increasing need to use alternative raw materials in aqua-
culture diets. This fact alone places great importance on the
need for careful and constructive experiment design in eva-
luating these raw materials. However, there is a clear and
present need to ensure that ingredient evaluation experiments
are designed to answer specific questions. By formulating
succinct strategies based on the key aspects of ingredient use
and choice, digestibility, palatability, utilization and func-
tionality then the development of sustainable alternatives will
be ensured.
Acknowledgements
We acknowledge the editorial support of Greg Maguire in
preparation of this manuscript.
References
Aksnes, A., Hjertnes, T. & Opstvedt, J. (1996) Comparison of two
assay methods for determination of nutrient and energy digest-
ibility in fish. Aquaculture, 140, 343–359.
Allan, G.L., Rowland, S.J., Parkinson, S., Stone, D.A.J. &
Jantrarotai, W. (1999a) Nutrient digestibility for silver perch
Bidyanus bidyanus: development of methods. Aquaculture, 170,
131–145.
Allan, G.L., Parkinson, S., Booth, M.A., Stone, D.A.J., Rowland,
S.J., Frances, J. & Warner-Smith, R. (1999b) Replacement of fish
meal in diets for Australian silver perch Bidyanus bidyanus: I.
Digestibility of alternative ingredients. Aquaculture, 186, 293–310.
Anderson, J.S.L., Anderson, D.M. & McNiven, M.A. (1993) Eva-
luation of protein quality in fish meals by chemical and biological
assays. Aquaculture, 115, 305–323.
Association of Official Analytical Chemists (AOAC). (1993) Official
Methods of Analysis of the Association of Official Analytical
Chemists, 16th edn. Association of Official Analytical Chemists,
Washington, DC, USA.
Aufrere, J., Graviou, D., Demarquilly, C., Perewz, J.M. & Andrieu,
J. (1996) Near infrared reflectance spectroscopy to predict energy
value of compound feeds for swine and ruminants. Anim. Feed Sci.
Technol., 62, 77–90.
Austreng, E. (1978) Digestibility determination in fish using chromic
oxide marking and analysis of different segments of the gastro-
intestinal tract. Aquaculture, 13, 265–272.
Austreng, E., Storebakken, T., Thomassen, M.S., Refstie, S. &
Thomassen, Y. (2000) Evaluation of selected trivalent metal oxides
as inert markers used to estimate apparent digestibility in salmo-
nids. Aquaculture, 188, 65–78.
..............................................................................................
 2007 Blackwell Publishing Ltd Aquaculture Nutrition 13; 17–34
31
Bedford, M.R. (2000) Exogenous enzymes in monogastric nutrition –
their current value and future benefits. Anim. Feed Sci. Technol.,
86, 1–13.
Booth, M.A. & Allan, G.L. (2003) Utilization of digestible nitrogen
and energy from four agricultural ingredients by juvenile silver
perch Bidyanus bidyanus. Aquac. Nutr., 9, 317–326.
Booth, M.A., Allan, G.L., Frances, J. & Parkinson, S. (2001)
Replacement of fishmeal in diets of silver perch: VI. Effects of
dehulling and protein concentration on the digestibility of four
Australian grain legumes in diets for silver perch (Bidyanus
bidyanus). Aquaculture, 196, 67–85.
Boujard, T. & Le Gouvello, R. (1997) Voluntary feed intake and
discrimination of diets containing a novel fluoroquinone in self-fed
rainbow trout. Aquat. Living Resour., 10, 343–350.
Bureau, D.P. (2006) Letter to the Editor of Aquaculture. Aqua-
culture, 252, 103–105.
Bureau, D.P., Harris, A.M. & Cho, C.Y. (1998) The effects of
purified alcohol extracts from soy products on the feed intake
and growth of chinhook salmon (Oncorhynchus tshawytscha)
and rainbow trout (Oncorhynchus mykiss). Aquaculture, 161, 27–
43.
Bureau, D.P., Harris, A.M. & Cho, C.Y. (1999) Apparent digest-
ibility of rendered animal protein ingredients for rainbow trout
(Oncorhynchus mykiss). Aquaculture, 180, 345–358.
Bureau, D.P., Harris, A.M. & Cho, C.Y. (2000) Feather meals and
bone meals from different origins as protein sources in rainbow
trout (Oncorhynchus mykiss). Aquaculture, 181, 281–291.
Burel, C., Robin, J. & Boujard, T. (1997) Can turbot, Psetta maxima,
be fed with self-feeders? Aquat. Living Resour., 10, 381–384.
Burel, C., Boujard, T., Corraze, G., Kaushik, S.J., Boeuf, G.,
Mol, K.A., Van der Geyten, S. & Kuhn, E.R. (1998) Incorporation
of high levels of extruded lupin in diets for rainbow trout
(Oncorhynchus mykiss): nutritional value and effect on thyroid
status. Aquaculture, 163, 325–345.
Burel, C., Boujard, T., Tulli, F. & Kaushik, S. (2000) Digestibility of
extruded peas, extruded lupin, and rapeseed meal in rainbow trout
(Oncorhynchus mykiss) and turbot (Psetta maxima). Aquaculture,
188, 285–298.
Burel, C., Boujard, T., Kaushik, S.J. et al. (2001) Effects of rapeseed
meal glucosinolates on thyroid metabolism and feed utilisation in
rainbow trout. Gen. Comp. Endocrinol., 124, 343–358.
Carter, C.G. & Hauler, R.C. (2000) Fish meal replacement by plant
meals in extruded feeds for Atlantic salmon, Salmo salar L.
Aquaculture, 185, 299–311.
Carter, C.G., Bransden, M.B., van Barneveld, R.J. & Clarke, S.M.
(1999) Alternative methods for nutrition research on the southern
bluefin tuna, Thunnus maccoyii: in vitro digestibility. Aquaculture,
179, 57–70.
Carter, C.G., Lewis, T.E. & Nichols, P.D. (2003) Comparison of
cholestane and yttrium oxide as digestibility markers for lipid
components in Atlantic salmon (Salmo salar L.) diets. Aquaculture,
225, 341–351.
Castañón, J.I.R., Flores, M.P. & Petterson, D. (1997) Mode of
degradation of non-starch polysaccharides by feed enzyme pre-
parations. Anim. Feed Sci. Technol., 68, 163–167.
Cho, C.Y. & Bureau, D.P. (1998) Development of bioenergetic
models and the Fish-PrFEQ software to estimate production,
feeding ration and waste output in aquaculture. Aquat. Living
Resour., 11, 199–210.
Cho, C.Y. & Kaushik, S.J. (1990) Nutritional energetics in fish:
energy and protein utilisation in rainbow trout (Salmo gairdnerii).
World Rev. Nutr. Diet., 61, 132–172.
32 B. D. Glencross et al.
Cho, C.Y. & Slinger, S.J. (1979) Apparent digestibility measurement
in feedstuff for rainbow trout. In: Finfish Nutrition and Fishfood
Technology. (Halver, J.E. & Tiews, K. eds), Vol. 2, pp. 239–247.
Heenemann GmbH, Berlin.
Cho, C.Y., Slinger, S.J. & Bayley, H.S. (1982) Bioenergetics of
Salmonid fishes: Energy intake, expenditure and productivity.
Comp. Biochem. Physiol, 73B, 25–41.
Choubert, G., De la Noue, J. & Luquet, P. (1982) Digestibility in
fish: improved device for the automatic collection of feces. Aqua-
culture, 29, 185–189.
Cowan, W.D., Korsbak, A., Harstrup, T. & Rasmussen, P.B. (1996)
Influence of added microbial enzymes on energy and protein
availability of selected feed ingredients. Anim. Feed Sci. Technol.,
60, 311–319.
Dyer, A.R, Barlow, C.G., Bransden, M.P., Carter, C.G., Glencross,
B.D., Richardson, N., Thomas, P.M., Williams, K.C. & Carra-
gher, J.F. (2004) Correlation of plasma IGF-I concentrations and
growth rate in aquacultured finfish: a tool for assessing the
potential of new diets. Aquaculture, 236, 583–592.
Farhangi, M. & Carter, C.G. (2001) Growth, physiological and
immunological responses of rainbow trout (Oncorhynchus mykiss)
to different dietary inclusion levels of dehulled lupin (Lupinus
angustifolius). Aquac. Res., 32, 329–340.
Fernandez, F., Miquel, A.G., Martinez, R., Serra, E., Guinea, J.,
Narbaiza, F.J., Caseras, A. & Baanante, I.V. (1999) Dietary
chromic oxide does not affect the utilization of organic compounds
but can alter the utilization of mineral salts in gilthead sea bream
Sparus aurata. J. Nutr., 129, 1053–1059.
Ford, J.E., Shorrock, C. (1971) Metabolism of heat-damaged protein
in the rat. Influence of heat-damage on the excretion of amino
acids and peptides in the urine. British Journal of Nutrition, 26,
311–322.
Forster, I. (1999) A note on the method of calculating digestibility
coefficients of nutrients provided by single ingredients to feeds of
aquatic animals. Aquac. Nutr., 5, 143–145.
Forster, I., Higgs, D.A., Donsanjh, B.S., Rowshandeli, M. & Parr, J.
(2000) Potential for dietary phytase to improve the nutritive value
of canola protein concentrate and decrease phosphorus output in
rainbow trout (Oncorhynchus mykiss) held in 11C fresh water.
Aquaculture, 179, 109–125.
Francis, G., Makkar, H.P.S. & Becker, K. (2001) Anti-nutritional
factors present in plant-derived alternate fish feed ingredients and
their effect in fish. Aquaculture, 199, 197–227.
Glencross, B.D. & Hawkins, W.E. (2004) A comparison of the
digestibility of several lupin (Lupinus spp.) kernel meal varieties
when fed to either rainbow trout (Oncorhynchus mykiss) or red
seabream (Pagrus auratus). Aquac. Nutr., 10, 65–73.
Glencross, B.D., Boujard, T.B. & Kaushik, S.J. (2003a) Evaluation
of the influence of oligosaccharides on the nutritional value of
lupin meals when fed to rainbow trout, Oncorhynchus mykiss.
Aquaculture, 219, 703–713.
Glencross, B.D., Hawkins, W.E. & Curnow, J.G. (2003b) Eva-
luation of canola oils as alternative lipid resources in diets for
juvenile red seabream, Pagrus auratus. Aquac. Nutr., 9, 305–
315.
Glencross, B.D., Curnow, J.G. & Hawkins, W.E. (2003c) Evaluation
of the variability in chemical composition and digestibility of dif-
ferent lupin (Lupinus angustifolius) kernel meals when fed to
rainbow trout (Oncorhynchus mykiss). Anim. Feed Sci. Technol.,
107, 117–128.
Glencross, B.D., Curnow, J.G., Hawkins, W.E., Kissil, G.W. &
Petterson, D.S. (2003d) Evaluation of the feed value of a
transgenic strain of the narrow-leaf lupin (Lupinus angustifolius)
in the diet of the marine fish Pagrus auratus. Aquac. Nutr., 9,
197–206.
Glencross, B.D., Hawkins, W.E. & Curnow, J.G. (2004a) Nutritional
assessment of Australian canola meals. I. Evaluation of canola oil
extraction method, enzyme supplementation and meal processing
on the digestible value of canola meals fed to the red seabream
(Pagrus auratus, Paulin). Aquac. Res., 35, 15–24.
Glencross, B.D., Hawkins, W.E. & Curnow, J.G. (2004b) Nutritional
assessment of Australian canola meals. II. Evaluation of the
influence of canola oil extraction method on the protein value of
canola meals fed to the red seabream (Pagrus auratus, Paulin).
Aquac. Res., 35, 25–34.
Glencross, B.D., Evans, D., Jones, J.B. & Hawkins, W.E. (2004c)
Evaluation of the dietary inclusion of yellow lupin (Lupinus luteus)
kernel meal on the growth, feed utilisation and tissue histology of
rainbow trout (Oncorhynchus mykiss). Aquaculture, 235, 411–422.
Glencross, B.D., Carter, C.G., Duijster, N., Evans, D.E., Dods, K.,
McCafferty, P., Hawkins, W.E., Maas, R. & Sipsas, S. (2004d) A
comparison of the digestive capacity of Atlantic salmon (Salmo
salar) and rainbow trout (Oncorhynchus mykiss) when fed a range
of plant protein products. Aquaculture, 237, 333–346.
Glencross, B.D., Hawkins, W.E., Evans, D., McCafferty, P.,
Dods, K., Maas, R. & Sipsas, S. (2005) Evaluation of the
digestible value of lupin and soybean protein concentrates and
isolates when fed to rainbow trout, Oncorhynchus mykiss, using
either stripping or settlement faecal collection methods. Aqua-
culture, 245, 211–220.
Glencross, B.D., Hawkins, W.E., Evans, D., McCafferty, P., Dods,
K., Jones, J.B., Sweetingham, M., Morton, L., Harris, D. &
Sipsas, S. (2006) Evaluation of the influence of the lupin alkaloid,
gramine when fed to rainbow trout (Oncorhynchus mykiss).
Aquaculture, 253, 512–522.
Gomes, E.F., Rema, P. & Kaushik, S.J. (1995) Replacement of fish
meal by plant proteins in the diet of rainbow trout (Oncorhynchus
mykiss): digestibility and growth performance. Aquaculture, 130,
177–186.
Hardy, R.W. (1999) Collaborative opportunities between fish nutri-
tion and other disciplines in aquaculture: an overview. Aqua-
culture, 177, 217–230.
Higgs, D.A., McBride, J.R., Markert, J.R., Dosanjh, B.S., Plotnik-
off, M.D. & Clarke, W.C. (1982) Evaluation of tower and candle
rapeseed protein concentrate as protein supplements in practical
dry diets for juvenile chinook salmon (Oncorhynchus tshawytscha).
Aquaculture, 29, 1–31.
Higgs, D.A., Fagerlund, U.H.M., McBride, J.R., Plotnikoff, M.D.,
Dosanjh, B.S., Markert, J.R. & Davidson, J. (1983) Protein quality
of Altex canola meal for juvenile chinook salmon (Oncorhynchus
tshawytscha) considering dietary protein and 3,5,3¢-triiodo-L-
thyronine content. Aquaculture, 34, 213–238.
Hughes, S.G. (1988) Assessment of lupin flour as a diet ingredient for
rainbow trout (Salmo gairdneri). Aquaculture, 71, 379–385.
International Association for Cereal Science and Technology (ICC).
(1995) Rapid Pasting Method using the Newport Rapid Visco
Analyser. ICC Standard No. 162. International Association of
Cereal Science and Technology, Vienna, Austria.
Jiang, Z. (2001). Ingredient variation: its impact and management.
In: Advances in Nutritional Technology 2001 (van der Poel, A.F.B.,
Vahl, J.L. & Kwakkel, R.P. eds), pp. 47–56. Proceedings of the 1st
World Feed Conference, Utrecht, Netherlands, 7–8 November.
Wageningen Pers, Wageningen.
Jobling, M., Arnesen, A.M., Baardvik, B.M., Christiansen, J.S. &
Jorgensen, E.H. (1995) Monitoring feeding behaviour and food
intake: methods and applications. Aquac. Nutr., 1, 131–143.
..............................................................................................
 2007 Blackwell Publishing Ltd Aquaculture Nutrition 13; 17–34
 Feed ingredient evaluation strategies for aquaculture feeds
Jobling, M., Coves, D., Damsgard, B., Kristiansen, H.R., Koskela,
J., Petursdottir, T.E., Kadri, S. & Gudmundsson, O. (2001)
Techniques for measuring feed intake. In: Food Intake in Fish
(Houlihan, D. Boujard, T. & Jobling, M. eds), pp. 49–87. Black-
well Science, Oxford.
Juell, J.E. (1991) Hydroacoustic detection of food waste – A method
to estimate maximum food intake of fish populations in sea cages.
Aquacult. Eng., 10, 207–217.
Kaushik, S.J. (1998) Nutritional bioenergetics and estimation of
waste production in non-salmonids. Aquat. Living Resour., 11,
311–318.
Kaushik, S.J. (2001) Feed technologies and nutrient availability in
aquatic feeds. In: Advances in Nutritional Technology 2001 (van der
Poel, A.F.B., Vahl, J.L. & Kwakkel, R.P. eds), pp. 187–196.
Proceedings of the 1st World Feed Conference, Utrecht, Nether-
lands, 7–8 November, Wageningen Pers.
Kaushik, S.J., Cravedi, J.P., Lalles, J.P., Sumpter, J., Fauconneau,
B. & Laroche, M. (1995) Partial or total replacement of fish meal
by soybean protein on growth, protein utilization, potential
estrogenic or antigenic effects, cholesterolemia and flesh quality in
rainbow trout, Oncorhynchus mykiss. Aquaculture, 133, 257–274.
Kim, J.D., Breque, J. & Kaushik, S.J. (1998) Apparent digestibilities
of feed components from fish meal or plant protein based diets in
common carp as affected by water temperature. Aquat. Living
Resour., 11, 269–272.
Krogdhal, A., Lea, T.B. & Olli, J.J. (1994) Soybean proteinase
inhibitors affect intestinal trypsin activities and amino acid
digestibilities in rainbow trout (Oncorhynchus mykiss). Comp.
Biochem. Physiol., 107A, 215–219.
Krogdhal, A., Bakke-McKellep, A.M., Roed, K.H. & Baeverfjord,
G. (2000) Feeding Atlantic salmon Salmo salar L. soybean pro-
ducts: effects on disease resistance (furunculosis), and lysozyme
and IgM levels in intestinal mucosa. Aquac. Nutr., 6, 77–84.
Krogdhal, A., Bakke-McKellep, A.M. & Baeverfjord, G. (2003)
Effect of graded levels of standard soybean meal on intestinal
structure, mucosal enzyme activities, and pancreatic response in
Atlantic salmon (Salmo salar). Aquac. Nutr., 9, 361–371.
Lupatsch, I., Kissil, G.Wm., Sklan, D. & Pfeffer, E. (1997) Apparent
digestibility coefficients of feed ingredients and their predictability
in compound diets for gilthead seabream (Sparus aurata). Aquac.
Nutr., 3, 81–90.
Lupatsch, I., Kissil, G.Wm. & Sklan, D. (2002) Comparison of
energy and protein efficiency among three fish species Sparus
aurata, Dicentrarchus labrax and Epinephelus aeneus: energy
expenditure for protein and lipid deposition. Aquaculture, 225,
175–189.
Maynard, L.A. & Loosli, J.K. (1969) Animal Nutrition, 6th edn.
McGraw-Hill Book Co, New York, NY.
Montero, D., Kalinowski, T., Obach, A., Robaina, L., Tort, L.,
Caballero, M.J., & Izquierdo, M.S. (2003) Vegetable lipid sources
for gilthead seabream (Sparus aurata): effect on fish health.
Aquaculture, 225, 353–370.
Morales, A.E., Cardenete, G., De la Higuera, M. & Sanz, A. (1994)
Effects of dietary protein source on growth, feed conversion and
energy utilisation in rainbow trout, Oncorhynchus mykiss. Aqua-
culture, 124, 117–126.
Morales, A.E., Cardenete, G., Sanz, A. & De la Higuera, M. (1999)
Re-evaluation of crude fibre and acid-insoluble-ash as inert mar-
kers, alternative to chromic oxide, in digestibility studies with
rainbow trout, Oncorhynchus mykiss. Aquaculture, 179, 71–79.
Mwachireya, S.A., Beames, R.M., Higgs, D.A. & Dosanjh, B.S.
(1999) Digestibility of canola protein products derived from the
physical, enzymatic and chemical processing of commercial canola
..............................................................................................
 2007 Blackwell Publishing Ltd Aquaculture Nutrition 13; 17–34
33
meal in rainbow trout, Oncorhynchus mykiss (Walbaum) held in
freshwater. Aquac. Nutr., 5, 73–82.
National Research Council (1993) Nutrient requirement of fish.
National Academy Press, Washington, DC, USA. pp. 114.
Naylor, R.L., Goldburg, R.J., Primavera, J.H., Kautsky, N.,
Beveridge, M.C.M., Clay, J., Folke, C., Lubchenco, J., Mooney,
H. & Troell, M. (2000) Effect of aquaculture on world fish
supplies. Nature, 405, 1017–1024.
Nengas, I., Alexis, M.N. & Davies, S.J. (1999) High inclusion levels
of poultry meals and related byproducts in diets for gilthead
seabream Sparus aurata L. Aquaculture, 179, 13–23.
Ng, W.K. & Wilson, R.P. (1997) Chromic oxide inclusion in the diet
does not affect glucose utilization or chromium retention by
channel catfish, Ictalurus punctatus. J. Nutr., 127, 2357–2362.
Nir, I. & Ptichi, I. (2001) Feed particle size and hardness: influence
on performance, nutritional, behavioural and metabolic aspects.
In: Advances in Nutritional Technology 2001 (van der Poel, A.F.B.,
Vahl, J.L. & Kwakkel, R.P. eds), pp. 157–186. Proceedings of the
1st World Feed Conference, Utrecht, Netherlands, 7–8 Novem-
ber., Wageningen Pers, Wageningen.
Oste, R.E. (1984) Effect of Maillard reaction products on protein
digestion. In vivo studies in rats. J. Nutr., 114, 2228–2234.
Peres, H., Lim, C. & Klesius, P.H. (2003) Nutritional value of heat-
treated soybean meal for channel catfish (Ictalurus punctatus).
Aquaculture, 225, 67–82.
Petterson, D.S., Harris, D.J., Rayner, C.J., Blakeney, A.B. & Choct,
M. (1999) Methods for the analysis of premium livestock grains.
Aust. J. Agric. Res., 50, 775–787.
Refstie, S., Storebakken, T. & Roem, A.J. (1998) Feed consumption
and conversion in Atlantic salmon (Salmo salar) fed diets with fish
meal, extracted soybean meal or soybean meal with reduced con-
tent of oligosaccharides, trypsin inhibitors, lectins and soya anti-
gens. Aquaculture, 162, 301–312.
Refstie, S., Svihus, B., Shearer, K.D. & Storebakken, T. (1999)
Nutrient digestibility in Atlantic salmon and broiler chickens
related to viscosity and non-starch polysaccharide content in
different soyabean products. Anim. Feed Sci. Technol., 79, 331–
345.
Ringo, E. (1995) Does chromic oxide (Cr2O3) affect faecal lipid and
intestinal bacterial flora in Artic charr, Salvelinus alpinus (L.)?
Aquac. Fish. Manag., 24, 767–776.
Robaina, L., Izquierdo, M.S., Moyano, F.J., Socorro, J., Vergara,
J.M., Montero, D. & Fernandez-Palacios, H. (1995) Soybean and
lupin seed meals as protein sources in diets for gilthead seabream
(Sparus aurata): nutritional and histological implications. Aqua-
culture, 130, 219–233.
Ruohonen, K., Kettunen, J. & King, J. (2001) Experimental design in
feeding experiments. In: Food Intake in Fish (Houlihan, D.,
Boujard, T. & Jobling, M. eds), pp. 88–107. Blackwell Science,
Oxford.
Rutherfurd, S.M., Moughan, P.J. & van Osch, L. (1997) Digestible
reactive lysine in processed feedstuffs: application of a new
bioassay. J. Agric. Food Chem., 45, 1189–1194.
Ruyter, B., Rosjo, C., Einen, O. & Thomassen, M.S. (2000) Essential
fatty acids in Atlantic salmon: effects of increasing dietary doses of
n-6 and n-3 fatty acids on growth, survival and fatty acid
composition of liver, blood and carcass. Aquaculture Nutrition, 6,
119–128.
Searcy-Bernal, R. (1995) Statistical power and aquacultural research.
Aquaculture, 127, 371–388.
Shearer, K.D. (2000) Experimental design, statistical analysis and
modelling of dietary nutrient requirement studies for fish: a critical
review. Aquac. Nutr., 6, 91–102.
34 B. D. Glencross et al.
Shiau, S.Y. & Liang, H.S. (1995) Carbohydrate utilization and
digestibility by tilapia Oreochromis niloticus · O. aureus, are affec-
ted by chromic oxide inclusion in the diet. J. Nutr., 125, 976–982.
Smith, D.M. & Tabrett, S.J. (2004) Accurate measurement of in vivo
digestibility of shrimp feeds. Aquaculture, 232, 563–580.
Sorenson, M., Ljkjel, K., Storebakken, T., Shearer, K.D. & Skrede,
A. (2002) Apparent digestibility of protein, amino acids and energy
in rainbow trout (Oncorhynchus mykiss) fed a fishmeal based diet
extruded at different temperatures. Aquaculture, 211, 215–225.
Stone, D.A.J., Allan, G.L., Parkinson, S. & Rowland, S.J. (2000)
Replacement of fish meal in diets for Australian silver perch,
Bidyanus bidyanus. III. Digestibility and growth using meat meal
products. Aquaculture, 186, 311–326.
Stone, D.A.J., Allan, G.L. & Anderson, A.J. (2003) Carbohydrate
utilisation by juvenile silver perch, Bidyanus bidyanus. IV. Can
dietary enzymes increase digestible energy from wheat starch,
wheat and dehulled lupin? Aquac. Res., 34, 135–147.
Storebakken, T., Shearer, K.D., Baeverfjord, G., Nielsen, B.G.,
Asgard, T., Scott, T.M. & De Laporte, A. (2000) Digestibility of
macronutrients, energy and amino acids, absorption of elements
and absence of intestinal enteritis in Atlantic salmon, Salmo salar,
fed diets with wheat gluten. Aquaculture, 184, 115–132.
Sugiura, S.H., Dong, F.M., Rathbone, C.K. & Hardy, R.W. (1998)
Apparent protein digestibility and mineral availabilities in various
feed ingredients for salmonid feeds. Aquaculture, 159, 177–202.
Sugiura, S.H., Babbit, J.K., Dong, F.M. & Hardy, R.W. (2000)
Utilization of fish and animal by-product meals in low-pollution
feeds for rainbow trout Oncorhynchus mykiss (Walbaum). Aquac.
Res., 31, 585–593.
Thomas, M. & van der Poel, A.F.B. (2001) Functional properties of
diet ingredients: manufacturing and nutritional implications. In:
Advances in Nutritional Technology 2001 (van der Poel, A.F.B.,
Vahl, J.L. & Kwakkel, R.P. eds), pp. 109–122. Proceedings of the
1st World Feed Conference, Utrecht, Netherlands, 7–8 November,
Wageningen Pers, Wageningen.
Thomassen M.S. & Rosjo, C. (1989) Different fats in feed for sal-
mon: influence on sensory parameters, growth rate and fatty acids
in muscle and heart. Aquaculture, 79, 129–135.
Vandenberg, G.W. & de la Noue, J. (2001) Apparent digestibility
comparison in rainbow trout (Oncorhynchus mykiss) assessed using
three methods of faeces collection and three digestibility markers.
Aquac. Nutr., 7, 237–245.
Vilhelmsson, O.T., Martin, S.A.M., Medale, F., Kaushik, S.J. &
Houlihan, D.F. (2004) Dietary plant-protein substitution affects
hepatic metabolism in rainbow trout (Oncorhynchus mykiss). Br. J.
Nutr., 92, 71–80.
Watanabe, T., Takeuchi, T., Satoh, S. & Kiron, V. (1996) Digestible
energy: methodological influences and mode of calculation. Fish.
Sci., 62, 288–292.
Williams, K.C., Barlow, C.G., Rodgers, L.J. & Ruscoe, I. (2003a)
Potential of meat meal to replace fish meal in extruded dry diets
for barramundi, Lates calcarifer (Bloch). I. Growth performance.
Aquac. Res., 34, 23–32.
Williams, K.C., Patterson, B.D., Barlow, C.G., Ford, A. &
Roberts, R. (2003b) Potential of meat meal to replace fish meal
in extruded dry diets for barramundi, Lates calcarifer (Bloch).
II. Organoleptic characteristics and fatty acid composition.
Aquac. Res., 34, 33–42.
Wilson, R.P. & Poe, W.E. (1985) Apparent digestible protein and
energy coefficients of common feed ingredients for channel catfish.
Prog. Fish. Cult., 47, 154.
Windell, J.T., Foltz, J.W. & Sarokan, J.A. (1978) Effect of body size,
temperature and ration size on the digestibility of a dry pelleted
diet by rainbow trout. Trans. Amer. Fish. Soc., 107, 613–616.
Wybourne, B.A. & Carter, C.G. (1999) The effect of plant meal
inclusion on feed intake and nutritional adaptation by Atlantic
salmon, Salmo salar L. In: Fishmeal Replacement in Aquaculture
Feeds for Atlantic Salmon. pp. 100–126. Project 93/120.
Fisheries Research and Development Corporation, Canberra,
Australia.
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