C S I R O P U B L I S H I N G

Australian
Journal of
Agricultural
Research
Volume 50, 1999
© CSIRO Australia 1999

A journal for the publication of original contributions

towards the understanding of an agricultural system

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 Aust. J. Agric. Res., 1999, 50, 889–908

Amino acid availability in poultry—in vitro and in vivo measurements

V. RavindranA and Wayne L. Bryden

Department of Animal Science, The University of Sydney, Camden, NSW, 2570, Australia.
A
Corresponding author; present address: Monogastric Research Centre, Institute of Food, Nutrition and Human
Health, Massey University, Private Bag 11222, Palmerston North, New Zealand.
Email: V.Ravindran@massey.ac.nz

Abstract. Methodology to evaluate the protein quality or amino acid availability in feed ingredients for poultry
using in vitro (enzymic, chemical, or microbiological assays), indirect in vivo (plasma amino acid assays), or direct
in vivo (growth or digestibility assays) measurements has been reviewed. The specific applications and limitations
of these methods are examined. In vitro assays are useful in providing information on heat damage in selected
protein sources under defined conditions, and on relative ranking of different samples, but they cannot form the
basis of practical feed formulations. While growth assays remain the only direct means of confirming nutritional
relevance of values obtained by other procedures, in vivo digestibility assays appear to be most useful, at present,
to estimate amino acid availability. Amino acid digestibility assays in poultry should be based on the analysis of
digesta from the terminal ileum rather than excreta, because of the variable and modifying effects of hindgut
microflora. Techniques used to estimate endogenous amino acid losses in poultry are discussed. The needs for cor-
rection of endogenous losses in amino acid digestibility calculations and the relative merits of apparent and true
digestible amino acid systems are still being debated. It is, however, clear that both digestible amino systems are
superior to the total amino acid system currently employed to formulate practical diets. Digestible amino acid values
are likely to form the basis of poultry feed formulations in the future. In particular, there is an urgent need for more
precise information on the variation in digestible amino acid contents of locally grown ingredients and on the factors
causing this variation (e.g. variety, location, season, agronomic practices, processing, etc.).

Additional keywords: amino acids, digestibility, endogenous protein losses, homoarginine.

Introduction tion of amino acids used for protein synthesis (Friedman

The bioavailability of nutrients is an important factor in
the efficient utilisation of food for the growth, maintenance,
and production of domestic animals. Ideally, nutrient
requirements should be expressed in terms of bioavailable
units, but bioavailability is difficult to measure. A nutrient is
bioavailable if it can be used for normal metabolic functions.
The proteins as a nutritional entity are sharply distinguished
from the carbohydrates and fats. For the latter a quantitative
assessment of digestibility is sufficient, because they will
serve chiefly as energy sources. For proteinaceous con-
stituents, however, their qualitative composition is also of
great importance. The basic function of dietary protein is to
supply adequate amounts of required amino acids. Thus, the
quality of a feed protein depends not only on its nitrogen
content, but also on its constituent amino acids, their
digestibility, physiological utilisation of specific amino acids
after digestion, and minimal obligatory rates of oxidation.
The utilisation of amino acids is determined by the propor-
1996). Therefore the bioavailability of a protein-bound
amino acid can be defined as an amino acid being in a form
appropriate for digestion, absorption, and utilisation
(Batterham 1992a). The term ‘digestibility’, often used syn-
onymously with ‘availability’, refers only to the process of
digestion and absorption and should not be confused with
availability.
The concept of essentiality of amino acids underlies all
protein quality evaluation measurements. There are 22 amino
acids in body proteins, and all are physiologically essential.
Of these amino acids, however, 10 cannot be synthesised by
poultry and must be supplied in the diet in accordance with
established requirements for maintenance, growth, and pro-
duction. The 10 essential amino acids are lysine, methionine,
tryptophan, threonine, arginine, isoleucine, leucine, histi-
dine, phenylalanine, and valine. In addition, glycine is con-
sidered to be essential for the modern broiler chicken, since
the rate of synthesis of this amino acid is not adequate to

© CSIRO 1999 10.1071/AR98174 0004-9409/99/050889

890
support maximum growth. Tyrosine and cystine are consid-
ered as semi-essential since they can be formed in the tissues
from phenylalanine and methionine, respectively. In practi-
cal diets, methionine (or methionine + cystine) is the first and
lysine the second amino acid limiting growth and production
for poultry.
Amino acid analysis
Amino acid analysis is routinely carried out using ion-
exchange chromatography after acid hydrolysis with
6 N hydrochloric acid for 24 h. The sensitivity, resolution,
versatility, and reliability of the measurement of amino acids
in proteins have increased due to significant advances in
chromatography during the past 30 years. Several in-depth
reviews on the methods of, and developments in, amino acid
analysis are available (Blackburn 1978; Rattenbury 1981;
Williams 1982, 1994; Sibbald 1987; Eggum and Sorensen
1989). However, the analysis of amino acids in a protein
remains difficult and complicated. Unlike many analytical
procedures for single components, amino acid analysis
requires the determination of 18 compounds with a wide
variety of chemical characteristics. The diversity of these
compounds and varying sensitivities to chemical conditions
demand that at least 3 separate and specific hydrolyses and
chromatographic analyses be conducted: a method for tryp-
tophan, one for sulfur-containing amino acids, and a standard
method based on acid hydrolysis for the reminder. Because
tryptophan is destroyed by acid hydrolysis, it has to be anal-
ysed separately using alkaline hydrolysis procedures which
render tryptophan stable. Similarly, methionine and cystine
are partially degraded during acid hydrolysis. Methionine
plus cysteine is usually determined after oxidation with per-
formic acid. Performic acid oxidation of cysteine to cysteic
acid and methionine to methionine sulfone yields acid-stable
products that can be separated chromatographically and mea-
sured accurately.
Amino acid analysis involves 4 essential steps: hydroly-
sis, separation, identification, and quantification. As shown
in a review by Ambler (1981), all 4 steps can introduce error
and variability in the results. However, hydrolysis conditions
are probably the major source of variation in amino acid
determination (AFRC 1987).
In vitro methods
The measurement of bioavailable amino acids using in
vitro techniques has been attractive to many workers because
such assays are relatively simple, rapid, and reproducible and
avoid the use of animals. However, data obtained from in
vitro assays have not found acceptance as a basis for practi-
cal feed formulations. Some assays appear to be satisfactory
for selected feedstuffs under defined conditions. In vitro
assays may provide useful information on heat damage to
proteins, and on relative ranking of different protein sources
V. Ravindran and Wayne L. Bryden
or samples of a given protein source. Detailed accounts of
in vitro assay procedures may be found in the reviews of
Carpenter (1973), Sibbald (1987), Whitacre and Tanner
(1989), and Fuller (1991).
Chemical assays
Chemical methods of accessing the availability of amino
acids have been almost invariably associated with lysine
(Low 1990) for two reasons. Firstly, lysine is the second lim-
iting amino acid in cereals and most practical diets for
poultry; secondly, its e-amino group is highly sensitive to
maillard reaction during heat treatment. The 1-fluoro-2,4-
dinitrobenzene (FDNB) procedure, proposed by Carpenter
(1960), is widely used to measure available lysine. The
method is based on the premise that only the lysine
molecules with free e-amino groups are nutritionally avail-
able to the animal (Finot and Hurrell 1985). The method
involves reaction between the free e-amino group of lysine in
the test protein and FDNB to form e-dinitrophenyl-lysine (e-
DNPL), which is released after subsequent hydrolysis and
measured colorimetrically. For feedstuffs high in carbohy-
drates and low in protein, e-DPNL is unstable during acid
hydrolysis and a modification (Roach et al. 1967) has been
developed. In the modified procedure, e-DPNL is not mea-
sured but is calculated by the difference between the total
lysine content and lysine regenerated after acid hydrolysis of
the dinitrophenylated protein (inaccessible, bound lysine).
The FDNB method has been reported to estimate efficiently
available lysine in heat-damaged proteins, especially from
animal products (Waibel et al. 1977; Rayner and Fox 1978).
The lysine availability data generated by this method were
found to be in good agreement with growth assays in chicks
(Major and Batterham 1981; Nordheim and Coon 1984).
Alternative methods for chemically measuring the reac-
tive lysine have been outlined by Carpenter (1973) and Finot
and Hurrell (1985). These include the use of 2,4,6-trini-
trobenzenesulfonic acid (TNBS, Kakade and Liener 1969),
O-methylisourea (OMIU) or guanidination reaction (Mauron
and Bujard 1964), furosine (Finot et al. 1977), dye-binding
with sulfonated dyes such as acid orange 12 (Hurrell and
Carpenter 1974), and total lysine determination after reduc-
tion with borohydrite (NaBH4) treatment (Finot et al. 1977).
In a comparison of these methods of measuring reactive
lysine in heat-damaged proteins, Hurrell and Carpenter
(1974) found that FNDB and OMIU procedures were the
most suitable methods for a range of possible heat damage.
Other methods, although useful for detecting particular types
of heat damage, were of less general application. However,
as noted by Sibbald (1987), the conclusion of research
studies with chemical assays to measure chemically reactive
lysine has been somewhat inconsistent.
No chemical methods are available for the determination
of the availability of other individual amino acids, with the
Amino acid availability in poultry
exception of methionine. The chemical determination of
available methionine is based on the assumption that methio-
nine present in proteins as the sulfoxide form is biologically
unavailable (Smith 1972), although this premise has been
questioned (Mackenzie 1985). Several methods to distin-
guish between methionine and its sulfoxide in proteins have
been developed (Ellinger and Duncan 1976; Bos et al. 1983).
However, the values for chemically available methionine
generated from these assays are yet to be correlated with in
vivo results.
A chemical assay that measures protein solubility in 0.2%
KOH is widely used by the feed industry in North America
as an indicator of overprocessing of oilseed meals. The assay
is based on the principle that, depending on the degree of
heating, the e-amino groups of lysine react with other moi-
eties such as reducing sugars to form enzyme-resistant inter-
and intra-molecular linkages (Mauron 1981), thereby reduc-
ing the solubility and digestibility of protein (Parsons 1996).
Protein solubility in KOH has been shown to be a sensitive
index of overprocessing of soybean meal (Araba and Dale
1990; Parsons et al. 1991), canola meal (Anderson-
Haferman et al. 1993), and sunflower meal (Zhang and
Parsons 1994). Results indicate that protein solubility values
<70% for soyabean meal, 65% for sunflower meal, and 50%
for canola meal (in 0.5% KOH) are suggestive of overpro-
cessing and reduced protein quality. Many of the adverse
effects of overprocessing were associated with decreased in
vivo lysine digestibility (Parsons et al. 1992). This procedure
has attracted considerable interest because the solubility
values are significantly correlated with weight gain and feed
efficiency in chickens (Araba and Dale 1990; Parsons et al.
1991, 1992; Whittle and Araba 1992).
Enzymic assays
There is considerable appeal in evaluating proteins by
means of in vitro enzymic assays, either with a single enzyme
(usually pepsin) or a mixture of gastric, pancreatic, and
intestinal enzymes; this mixture can be relatively pure or it
may be digesta from the small intestine (Low 1990). The
assays involve the incubation of feedstuffs with proteolytic
enzymes under controlled conditions, and undigested mate-
rial is separated from released amino acids and peptides by
techniques such as precipitation, dialysis, and gel or mem-
brane filtration. The end products of enzymic digestion of
protein may include free amino acids, peptides of various
lengths, and undigested protein. The amino acid concentra-
tion of both dialysate and filtrate can be measured and
digestibility calculated. Several critical reviews on in vitro
protein digestibility methods based on enzymic digestion
have been published (Hsu et al. 1977; Assoumani and
Nguyen 1991; Boisen and Eggum 1991; Swaisgood and
Catignani 1991).
Digestibility of nitrogen by a pepsin hydrolysis method
has been used for evaluation of animal protein feedstuffs,
891
which are subject to heat damage during heat processing, for
many years. The pepsin assay is widely accepted by the feed
industry because it is relatively simple, inexpensive, and
rapid, and many samples can be compared at the same time.
The concentration of the enzyme appears to be a critical
factor in the use of this assay. Parsons (1997) reported that a
0.002% pepsin assay is superior to the 0.2% pepsin assay
recommended by AOAC (1984) in predicting in vivo protein
and amino acid quality of meat meals and feather meals. It
was suggested that the 0.2% pepsin concentration is exces-
sive to assess differences in quality among ingredient
samples. Johnston and Coon (1979) similarly noted that
assays using 0.2% concentration failed to discriminate
between good and heat-damaged fish meal, whereas those
using 0.02 and 0.002% clearly showed quality differences.
However, application of this assay, with a single enzyme, to
mixed diets could give misleading results because some pro-
teins are more susceptible to attack by one particular enzyme
(Sibbald 1987).
Compared with pepsin assay, assays that use $2 enzymes
with different peptide specificities resulted in greater diges-
tion of proteins (Akeson and Stahmann 1964; Saunders and
Kohler 1972). Among the in vitro multi-enzymic assays, the
pH change method of Hsu et al. (1977) has proved most
promising for the evaluation of heat damage in proteins and
is widely used by food and feed industries. This method
involves the incubation of test ingredients in an aqueous sus-
pension with a combination of trypsin, chymotrypsin, and
intestinal peptidase. As ionisable carboxyl groups of the pep-
tides are released by proteolysis, the pH usually decreases
rapidly during the first few minutes of the incubation and
then gradually stabilises by about 10 min. The decline in pH
after 10 min of incubation is a good indicator of protein
quality, and has been shown to be correlated to in vivo protein
digestibility in rats (Hsu et al. 1977) and in vivo lysine
digestibility in adult cockerels (Parsons 1988, 1991a). The
method, however, has been criticised for its sensitivity to the
buffering capacity of the protein suspensions, phenolic acids,
and other ionisable compounds (Hahn et al. 1982). To over-
come the buffering capacity problems, a modification known
as the pH-stat method was proposed by Pedersen and Eggum
(1983). In this method, pH is kept constant by titration with
0.1 M sodium hydroxide. The amount of alkali added is a
direct measure of the extent of proteolysis.
The major criticism of enzymic assays involves the tech-
nicality of simulating the complex mechanisms for protein
digestion in the laboratory to that actually happening within
the digestive tract of animals. In general, most in vitro diges-
tion methods suffer from various degrees of uncertainty
(Swaisgood and Catignani 1991). Estimates of amino acid
availability by these assays will be underestimated if the
hydrolysis is incomplete. The usefulness of enzymatic assays,
however, lies in the fact that they do allow meaningful dis-
crimination between feedstuffs of varying digestibilities.
892
Microbiological assays
The proteolytic activity of certain microorganisms offers
a further possibility for the estimation of protein quality and
amino acid availability. Before the advent of ion exchange
chromatography, microbiological assays were routinely used
to determine the total amino acid concentrations in proteins.
Subsequently such assays, all based on microbial growth as
the response criterion, have been used to assess the avail-
ability of proteins and they have been a satisfactory means of
quality control, ranking materials of similar composition
(Low 1990).
Microbiological assays employing Streptococcus fae-
calis, Streptococcus durens, and Lactobacillus arabinosus
(Ford 1964; Dvorak 1968), Streptococcus zymogenes (Miller
et al. 1965), and Tetrahymena pyriformis (Stott and Smith
1966) amongst others have been suggested. The relationship
between microbiological and chick assays for available
amino acids varied with the nature of the test material but
was generally good (Carpenter and Woodham 1974; Boyne
et al. 1975). As noted by Low (1990), the availability of
amino acids or short peptides to bacteria is not necessarily
the same as it is to poultry and this is a major weakness of
these assays.
Near infrared reflectance (NIR) spectroscopy
An alternative method using NIR spectroscopy has the
potential to be developed into a reliable method for predict-
ing in vivo amino acid digestibility (Harrison et al. 1991; van
Leeuwen et al. 1991; Jackson et al. 1996; van Kempen and
Jackson 1996). This method is clearly much more rapid than
in vitro methods, but is dependent on calibration of the equip-
ment with a relatively large number of samples with known
in vivo values which is the major constraint to further devel-
opment. In addition, calibrated data sets need to be validated
by statistical comparison with independent data sets.
Immunological assays
There is an increasing interest in the use of enzyme-linked
immunosorbent assay (ELISA) technology as an analytical
tool in the human food industry (Skerritt and Hill 1991). As
yet, it has not been applied to animal nutrition. It should be
possible to apply such technology to the in vitro assessment
of protein digestibility. For example, ELISAs could be devel-
oped for the determination of the indigestible peptide com-
ponents of dietary proteins (J. H. Smith, pers. comm.).
Indirect in vivo methods
Plasma amino acid assays
The principle of this method is that blood serves as a
carrier of products from digestion and absorption of dietary
proteins (free amino acids and peptides) and transports them
to the target tissues for further anabolic processes. Therefore,
V. Ravindran and Wayne L. Bryden
the level of free amino acids in blood can reflect the avail-
ability in the dietary protein. Whether this method is appro-
priate depends on the relationship between the free amino
acids and amino acid absorption. In this method, constant
plasma free amino acid concentrations during starvation are
used as a reference and compared with concentrations of
post-prandial limiting amino acids (Ng and Pascaud 1990).
Hill and Olsen (1963) used this method in chickens, employ-
ing a protein-free diet as the reference standard that,
however, tended to reduce plasma free amino acids below the
fasting levels (Bergen and Purser 1968).
Although plasma amino acid assays have been considered
by nutritionists as a quick, convenient, and simple method to
determine amino acid availability, the technique suffers from
several limitations. Free amino acid levels in the blood
depend not only on the ingested protein but also on the nutri-
tional status of the animal. In a positive nitrogen balance, free
amino acids are removed from the circulation for protein
synthesis. On the other hand, when catabolism is high,
excess amino acids will be released into the plasma.
Moreover, free amino acid levels in the plasma are influ-
enced by a multitude of factors including circadian rhythm,
age, species, physiological status, meal size, site and time of
blood sampling, frequency of feeding, the supply of energy-
yielding nutrients during starvation, amino acid imbalances,
and ambient temperature (Sibbald 1987; Low 1990). It is
therefore difficult to interpret the observed changes in
plasma amino acids as a quantitative measure of amino acid
availability (Papadopoulos 1985). Because of these limita-
tions, it is unlikely that this assay will ever be widely
accepted. However, the levels of plasma free amino acids
may be used to identify the limiting amino acid in poultry
diets and to demonstrate the point at which the requirement
is met (Hill et al. 1961; Fernandez-Figares et al. 1997). The
most limiting amino acids remain at a low concentration in
the plasma irrespective of the severity of the deficiency and
do not increase until the dietary concentration exceeds that
required for maximum growth. In growing chickens fed on
diets containing graded levels of an essential amino acid, low
concentrations of plasma free amino acids were observed
until the actual dietary requirement is reached beyond which
point the plasma free concentrations increase rapidly
(Zimmerman and Scott 1965).
Direct in vivo methods
The ‘bioavailable’ form of amino acids may be defined as
amino acids which can be released by digestion, absorbed
and utilised by animals. The two methods used for the direct
estimation of available amino acids are growth assays and
digestibility assays. The growth assays are attractive because
the response criteria are similar to those used under practical
conditions. Digestibility assays are applied with the assump-
tion that the difference between input and output is a valid
Amino acid availability in poultry
indicator of bioavailability. Availability and digestibility are
not necessarily identical, since the latter takes no account of
the response of the animal to the absorbed amino acid(s).
Growth assays
Growth bioassays have been developed to determine how
much of the dietary amino acid is digested, absorbed, and
incorporated into body proteins or metabolites for other body
uses. Growth bioassays are the only direct means of evaluat-
ing nutritional values obtained by other procedures
(Carpenter 1973). The principle is to measure the ability of
protein to replace a specific amino acid in supporting growth,
which is a function of protein accretion. The growth assays
for an available amino acid involve the addition of graded
levels of the amino acid to a basal diet deficient in the amino
acid under study, in order to establish a relationship between
the growth response and the level of the amino acid in the
diet (Carpenter et al. 1972). This ability is normally com-
pared with the response obtained with a crystalline amino
acid which is assumed to be fully available. The response cri-
teria may not be limited to growth rate as other biological
responses such as feed efficiency, amino acid intake, or
carcass gain have also been used to evaluate the availability
(Batterham et al. 1990). Critical reviews on various aspects
of the methodology of growth assays have been published
(Carpenter 1973; Sibbald 1987; Parsons 1991b; Batterham
1992a; Lewis and Bayley 1995).
Chick growth assays have been used to determine
bioavailability of lysine (Major and Batterham 1981;
Nordheim and Coon 1984; Parsons 1986; Parsons et al.
1997), digestible lysine (Fernandez and Parsons 1996),
methionine (Carpenter et al. 1972), digestible valine
(Fernandez and Parsons 1996), and tryptophan (Harwood
and Shrimpton 1969) in various protein concentrates. The
use of growth assays to determine bioavailable amino acids
is attractive because growth assays measure a response
(growth) that encompasses all components that can affect
bioavailability (digestion, absorption, and utilisation). On the
other hand, growth assays have several serious limitations.
Apart from being expensive and time-consuming, they can
measure the availability of only one amino acid at a time and
the large inherent variation among animals often makes it
difficult to achieve a satisfactory level of precision. The
design and conduct of a growth assay can have a large effect
on the result obtained (Lewis and Bayley 1995). Growth
assays involve several assumptions, the most important of
which is that the growth response to supplementation of the
test ingredient is due only to the deficient amino acid.
However, it is now known that when the amino acid intake
approaches and exceeds the requirement, the growth
response curve is usually curvilinear and this requires appro-
priate mathematical analysis (Sibbald 1987). It is also known
that growth is influenced by a number of nutritional factors
893
(other than available amino acids) including feed intake,
dietary protein level, and interactions among amino acids.
This raises questions as to whether growth is the appropriate
response criteria in availability determinations.
Using practical feed ingredients, it is often impossible to
formulate rations that are deficient enough in specific amino
acids. Imbalance in amino acids may also occur (Whitacre
and Tannner 1989). The addition of extra amino acids
designed to stimulate the test protein has been shown to
depress the growth of chicks, presumably due to dietary
imbalance, resulting in artificially high amino acid availabil-
ity values (Smith and Scott 1965). Most importantly growth
assays fail to meet the criteria for an ideal technique, namely
that it should be simple, rapid, reproducible, and accurate.
Digestibility assays
The assay of digestibility has become the most favoured
technique for measuring availability, largely because the
values apply directly to the animal and all amino acids can be
measured in one assay. Digestibility assays may be divided
into excreta and ileal digestibilities. Excreta digestibility
involves the collection of excreta from intact, caecectomised
or colostomised birds, depending on the purpose of each
technique. Ileal digestibility can be subdivided into 2 tech-
niques, depending on whether the digesta are collected from
the ileum of the animal after death or from a cannula inserted
at the terminal ileum.
Excreta digestibility
In an attempt to measure the proportion of amino acids in
the ingested protein that had disappeared from the gastroin-
testinal tract, Kuiken and Lyman (1948) were the first to
develop a faecal analysis method. This assay is based on the
premise that availability can be determined by measuring
digestibility which is obtained by subtracting the ‘unab-
sorbed’ amino acids in the faeces from those ingested. The
method involves the total collection of excreta for an appro-
priate time after feeding or the use of markers such as
chromic oxide (Keith and Bell 1988), acid insoluble ash
(McCarthy et al. 1974), ytterbium nitrate (Leibholz 1992), or
ferric oxide (Bragg et al. 1969).
Excreta collection may not be accurate for poultry since
there is contamination of faeces by urine, feathers, scales,
and foreign materials (Ivy et al. 1968). This assay thus does
not measure ‘digestibility’ as classically defined, but rather
the ‘metabolisability’, since faeces and urine are excreted
together in birds. Methods have been developed for separat-
ing faeces and urine in birds prior to excretion using such
techniques as colostomy (Imbayashi et al. 1955) and exteri-
orisation of the ureters (Dixon and Wilkinson 1957).
However, the urinary contribution of amino acids to excreta
is generally overlooked, the rationale being that, as the con-
centrations of amino acids in urine is exceedingly small, it
894
has negligible effect on digestibility estimates (Terpstra
1978). Some support for this argument comes from the find-
ings of Yamazaki (1983) that there were no differences
between the true digestibility values measured with
colostomised hens and intact adult cockerels. However, it has
been suggested that in the case of overheated or heat-
damaged proteins, amino acids may be voided in the urine as
metabolites (McNab 1995; Fernandez and Parsons 1996),
resulting in misleading estimates.
Most published values currently available on digestible
amino acids for poultry are based on excreta analysis because
of its simplicity and the assay can be carried out on a large
number of birds without sacrificing the birds or surgical
modification (McNab 1994). In particular, research in the
area of amino acid digestibility has increased greatly since
the development of a rapid assay (Likuski and Dorrell 1978;
Sibbald 1979), which has proved popular in North America
and Europe. The assay, commonly known as precision-fed
cockerel assay, involves fasting adult cockerels for 24–48 h,
placing a known quantity (usually 30–50 g) of test ingredient
directly into the crop, and subsequent quantitative collection
of excreta (faeces and urine) for 48 h. Endogenous amino
acid losses are estimated from excretion of fasted birds or of
birds fed protein-free diets.
Excreta-based digestibility measurements are criticised
because of the possible effects of microorganisms in the
hindgut on dietary protein utilisation and their contribution
of microbial proteins to amino acid excretion in the faeces
(Bryden et al. 1990). Although the presence of microflora in
the caeca and large intestine of poultry is established, the
actual impact of these microflora on protein nutrition is not
clearly understood.
Most microorganisms in the avian gut are concentrated in
the caeca. Avian caeca are a pair of intestinal sacs located at
the boundary between the ileum and colon. The caeca excrete
sticky and chocolate-coloured digesta (‘caecal faeces’)
several times a day. Since this part of the intestine has an
anaerobic, liquid milieu with a pH range 6.5–7.5 and an
intermittent evacuation of contents every 6–8 h (Jane-
Williams and Fuller 1974), the conditions favour the prolif-
eration of bacteria. The major microbial activity in the
chicken’s caeca is fermentative (McNab 1973) with Gram-
positive anaerobic cocci as the predominant type. A large
population of uric acid degrading bacteria is also found. The
caeca contributes to the reabsorption of water and utilisation
of non-protein nitrogen from the retro-peristalsis of urine
(Mead 1989). McBee (1977) reported that no cellulose or
xylan fermentation occurred in avian caecum. In terms of
amino acid utilisation, it is proposed that caecal microbes
metabolise large amounts of undigested dietary amino acids
(Parsons 1986; Mead 1989). Studies based on a limited range
of feedstuffs show that intestinal microflora have little influ-
ence on excreta amino acid digestibility (Sibbald 1979;
V. Ravindran and Wayne L. Bryden
Parsons et al. 1981). However, using in vitro studies, Moreto
and Planas (1989) showed that the avian caeca has the ability
to absorb valine, phenylalanine, and leucine, although its
contribution to overall nutrient absorption is probably negli-
gible.
Activity of caecal microorganisms may modify the amino
acid composition of the excreta and this can influence the
digestibility values calculated based on excreta analysis.
Several studies have demonstrated the possible role of caeca
in digestibility estimations. Nitsan and Alumot (1963) have
provided evidence for proteolytic activity by caecal
microflora. This was later supported by the studies of Isshiki
et al. (1974), who showed that caecal contents can hydrolyse
protein. Payne et al. (1971) observed lower apparent
digestibilities of several amino acids in fish meal when cae-
cectomised chicks were used. The differences were substan-
tial for all amino acids and were most striking for threonine.
Nesheim and Carpenter (1967) found that heat-damaged fish
fillet had reduced nitrogen digestibility and that the reduction
was marked in caecectomised birds compared to the intact
group. This indicated that the contribution of microflora may
become significant when evaluating denatured proteins.
Green and Kiener (1989) found no differences in the true
digestibility of amino acids in soyabean meal and sunflower
meal but a significant reduction in caecectomised chickens
fed on meat and bone meal. Similar results have been
reported by Parsons (1986). Caecectomy had no influence on
the amino acid digestibility for cereal grains, but there were
slight differences in oilseed meals (Green et al. 1987) and
significant differences for some animal protein supplements
(Green and Kiener 1989). Salter and Coates (1971) also
demonstrated the influence of microbial changes on the
chemical form of nitrogen in the lower gut. Microbial alter-
ation of amino acids may influence the microbial protein
content in excreta and this protein may contribute up to 25%
of the total excreta protein (Parsons et al. 1982). For these
reasons, the use of caecectomised birds is now a well-
accepted procedure for studying amino acid digestibility
using excreta analysis (Johns et al. 1986a; Green et al. 1987;
Johns 1990; Parsons 1991b) so as to overcome the influence
of the caecal microflora.
Ileal digestibility
As discussed above, a major concern with faecal
digestibility assays is the effect of the intestinal microflora on
faecal amino acid output. Payne et al. (1968) were the first to
suggest that analysis of ileal contents rather than excreta
might be a reliable method for assessing protein and amino
acid digestibility in poultry. Few studies have been conducted
to compare the ileal digesta v. excreta analysis for determina-
tion of amino acid digestibility in chickens. In an earlier study
at Camden, Wallis and Balnave (1984a) found that the amino
acid digestibility in soybean meal was underestimated by
Amino acid availability in poultry 895

Table 1. Differences between excreta and ileal amino acid digestibilities (%) in selected feedstuffs for broilers
Difference = excreta digestibility – ileal digestibility (data from Hew et al. 1996 and Ravindran et al. 1996a)

Amino acid Maize Wheat Sorghum Soybean meal Cottonseed meal Meat meal Fish meal Feather meal

Aspartic acid –0.7 –18.3* –3.9 +3.7* +0.8 +20.5* +8.9* +36.8*
Threonine +6.7 –19.8** –2.9 +2.4* +1.3 +11.8* +7.3** +24.5*
Serine +4.8 –11.3* +0.1 +4.2* +1.5 +13.5* +9.1** +21.4*
Glutamic acid –2.0* –5.6** –4.0 +0.1 –2.2* +7.1† +0.2 +18.2†
Alanine –5.0** –25.2* –8.1† –9.4* –8.3* +6.3† –3.9 +7.6†
Valine –5.8** –14.6* –7.1 –5.8* –3.1† +5.3† –2.9 +14.0†
Methionine +1.4 –7.8* –1.0 –1.4 –6.3* +5.8† –0.2 +12.0†
Isoleucine –8.2* –13.5† –7.8† –4.9** –3.4** +5.6† –4.1** +11.1†
Leucine –1.3 –10.9† –3.0 –1.1 –3.2* +5.5† +0.8 +14.6*
Tyrosine +1.1 –4.5** –4.1 –1.3 –1.1 +1.7 +1.8 +12.8†
Phenylalanine –0.5† –9.7† –3.6 +1.7 –1.9 –3.6† +1.0 +8.1
Histidine — –17.5† –12.0* — –0.8 –2.8 +4.5† +12.0†
Lysine +1.1† –17.5* –9.8† –0.6 –6.4** +14.0* +1.1 +19.2*
Arginine –0.6 –7.6* –4.2 +1.6 –0.5 +5.6† +2.1** +15.8*
Mean –1.1 –13.1* –3.3 –1.0 –2.4† +6.9 +1.1 +16.5*

†
P < 0.10; * P < 0.05; ** P < 0.01.

excreta analysis. These two methods of amino acid digestibil-
ity determination have been recently compared in our labora-
tory (Hew et al. 1996: Ravindran et al. 1996a, 1996b) using
3 cereals (maize, sorghum, and wheat), 4 plant protein
sources (soybean meal, canola meal, cottonseed meal, and
sunflower meal), and 5 animal protein sources (meat meal,
meat and bone meal, feather meal, blood meal, and fish meal).
Selected data from these studies are presented in Table 1. The
influence of site of measurement, in terms of magnitude and
direction, was inconsistent, varying among feed ingredients,
among samples within an ingredient, and among different
amino acids within an ingredient. Ileal amino acid digestibil-
ity values in some feed ingredients were similar to the corre-
sponding excreta values, but significantly lower or higher in
some others. Average ileal and excreta amino acid digestibil-
ities in sorghum and maize were similar, although significant
differences were noted for some individual amino acids. In
contrast, the ileal amino acid digestibility values were 10–25
percentage units higher than the corresponding excreta amino
acid digestibility values in wheat. The average ileal and
excreta digestibilities of amino acids in the 3 soyabean meal
samples were similar, although small, but significant, differ-
ences were noted for individual amino acids. Site of measure-
ment had no effect on the digestibility of amino acids in
canola meal. In the case of sunflower meal and cottonseed
meal, excreta digestibilities of some individual amino acids
were underestimated by excreta analysis. Amino acid
digestibilities in animal protein meals, except for highly
digestible blood meal and fish meal, were consistently over-
estimated by excreta analysis. Ileal-excreta differences in
individual amino acids digestibilities were more evident in
feather meal, meat meal, and meat and bone meal. Among the
indispensable amino acids, threonine and valine were more
frequently modified by microbial fermentation in the hindgut.
Of the dispensable amino acids, aspartic acid, serine, glu-
tamic acid, and alanine were the most affected. Differences
observed between ileal and excreta digestibilities in these
studies clearly demonstrated that amino acid metabolism by
hindgut microflora in chickens may be substantial and that
digestibilities determined at the terminal ileum are more accu-
rate measures of amino acid availability than those deter-
mined at the excreta level.
The addition of an indigestible marker to the diet and its
measurement in both the diet and ileal digesta are required to
relate the amino acid contents in the ileum to that in the diet.
A number of substances have been used as indigestible
markers. The most commonly used are chromic oxide and
acid insoluble ash. The use of reference markers in nutri-
tional studies has been reviewed by Kotb and Luckey (1972).
Ileal digestibility can be determined in two ways, depend-
ing on the technique of digesta collection. The simplest
method for collection of ileal digesta is to kill the bird and the
alternative is to use an cannula inserted into the distal ileum
(Thomas and Crissey 1983; Raharjo and Farrell 1984). In
early studies, chickens were killed by cervical dislocation;
but this method of killing has been criticised because it
increases losses of endogenous protein due to increased
shedding of mucosal cells into the gut lumen at the time of
slaughter. Euthanasia of birds, using substances such as pen-
tobarbitone sodium, is currently preferred since this min-
imises both peristalsis, which could result in movement of
digesta, and mucosal shedding, which occur with conven-
tional slaughter techniques (Badaway 1964). Summer and
Robblee (1985) showed that the ileal apparent amino acid
digestibility was underestimated in birds killed by cervical
dislocation as compared to euthanased birds.
To overcome the disadvantages associated with the killing
of birds, ileal cannulation has been used by some researchers
896
(Raharjo and Farrell 1984; Gurnsey and James 1985). A
comparison between slaughter (anaesthetised) and cannula-
tion techniques by Johns et al. (1986b) showed that apparent
ileal amino acid digestibility values determined with cannu-
lated cockerels were significantly lower for all amino acids
when compared with those determined with anaesthetised
growing chickens, with the exception of arginine and glu-
tamic acid. The usefulness of ileal cannulation is limited by
problems arising from the rejection of the cannula and varia-
tions in the flow of digesta through the cannula and the need
to use appropriate markers. It has also been suggested that
the physical alteration to the intestine from ileal cannulation
may interfere with normal physiological processes of the
animal (Tanksley et al. 1981).
Apparent v. true amino acid digestibility
A question often posed by commercial nutritionists is
which digestible amino acid system is most appropriate for
use in the formulation of poultry diets: apparent or true
digestibility values (Low 1990; Batterham 1992b; Johnson
1992). A brief comparison of these 2 systems is given below.
Apparent digestibility
Apparent digestibility measures the digestibility of amino
acids of both dietary and endogenous origins. The major crit-
icism in the use of apparent digestibility values is that they
are influenced by the level of feed intake and dietary protein
(or amino acid) concentrations (Bielorai and Iosif 1987; Li et
al. 1993; Fan et al. 1994; Angkanaporn et al. 1997a). At low
dietary protein (or amino acid) intakes, greater proportions of
amino acids from endogenous sources will be present in the
digesta, relative to protein of dietary origin. Thus in assay
diets containing low protein (or amino acid) levels, the cal-
culated apparent amino acid digestibility will underestimate
actual digestibility. As the dietary protein intake increases,
the proportion of endogenous sources decreases, and the
apparent digestibility of amino acids increases and
approaches that of true digestibility (McNab 1989).
True digestibility
True digestibility on the other hand, includes a correction
for endogenous amino acid secretions, and is considered to
be a fundamental characteristic of the feedstuff that is rela-
tively constant across varying protein intakes (McNab 1989).
Therefore, using true digestibility data permits feed ingredi-
ents to be compared even if they are assayed under varying
dietary conditions. Uncertainty exists, however, regarding
the reliability of traditional methods for determining endoge-
nous amino acid losses under a given set of dietary circum-
stances and this is a major concern limiting the usefulness of
true digestibility estimates.
A brief mention must also be made of the term ‘real
digestibility’, which includes the correction for the actual
endogenous losses which are related to the feeding of a spe-
V. Ravindran and Wayne L. Bryden
cific ingredient (Boisen and Moughan 1996; Gabert et al.
1997). The separation of endogenous amino acids from undi-
gested dietary amino acids at the terminal ileum, after
feeding a specific protein, has become possible in recent
years with developments in isotope-dilution (Schulze 1994)
and the homoarginine (Bryden et al. 1996) techniques.
Endogenous losses of amino acids determined with these
techniques are much higher than those estimated by tradi-
tional methods (Roos et al. 1994; Siriwan et al. 1994). Real
digestibility may be preferable to, and more reliable than,
true digestibility (Batterham 1992a), but at this stage insuffi-
cient data are available to use this system. In addition, the
merit of real v. true or apparent digestibilities in practical
nutrition remains to be elucidated.
Endogenous amino acid secretions
Apart from dietary sources, proteins are also supplied to
the gut in the form of endogenous secretions. It has been
reported that up to 25% of the daily protein synthesis may be
secreted into the gut (Simon et al. 1983). Endogenous amino
acid secretions must be quantified to correct apparent amino
acid digestibility to obtain true digestibility values.
Endogenous proteins or amino acids predominantly orig-
inate from various digestive secretions (saliva, bile, pancre-
atic secretions, gastric secretions, and intestinal secretions),
mucoproteins and desquamated intestinal epithelial cells
(Scott et al. 1982), and reflux of metabolites released by
protein catabolism (in forms of peptides or free amino acids)
from the lower gut into the small intestine (Simon et al.
1986). Exocrine pancreatic secretions are the principal
sources of non-dietary nitrogen in the gut (Greene et al.
1963). Endogenous proteins in the intestine represent the
algebraic sum of both secretion and digestion (Boorman
1976). The source of endogenous proteins seems to be criti-
cal in determining this balance. When pancreatic secretions
predominate, the proteins must pass through the jejunum and
ileum where digestion and absorption may occur. In contrast,
if desquamation is significant, the opportunity for digestion
is lower and some endogenous amino acid loss seems
inevitable (Nasset 1972).
Buraczewska (1979) indicated that of the nitrogenous
compounds secreted in the small intestine of pigs, 86–90%
are soluble nitrogen compounds consisting of a-amino nitro-
gen (free amino acids and peptides) and the remaining nitro-
gen are represented by amides, aminosugars, and urea.
Moughan and Schuttert (1991) used an ultrafiltration proce-
dure to differentiate the composition of ileal digesta in pigs
fed protein-free diets. They found that the major fraction
(69%) of nitrogen in the ultrafiltrate was peptide nitrogen
whereas free amino acids plus small peptides constituted a
small fraction.
The amino acid composition of endogenous proteins has
been studied by several researchers. Taverner et al. (1981)
reported that the 4 most abundant amino acids in the ileal
Amino acid availability in poultry
digesta of pigs were proline, glycine, glutamic acid, and
aspartic acid. Since these amino acids are found in large
amounts in mucin protein, their results indicated that mucins
were probably the major source of endogenous protein. In
chickens, high concentrations of aspartic acid, serine, and
glutamic acid were determined in ileal endogenous secre-
tions (Bielorai and Iosif 1987; Siriwan et al. 1994;
Angkanaporn et al. 1997b). Salter and Fulford (1974) found
that threonine and serine, major amino acids found in diges-
tive enzymes and mucoprotein, were considerably high in the
endogenous secretions of germ-free chicks. Studies in pigs
have shown that the amino acid composition of ileal digesta
varied from that of dietary origin, but compared closely with
that of endogenous protein while the amino acid composition
of faeces was similar to that of bacterial proteins (Darcy-
Vrillon et al. 1991).
Procedures for determining endogenous amino acids
Several extensive reviews of the procedures available to
measure endogenous losses of amino acids have been pub-
lished (Sibbald 1987; Nyachoti et al. 1997; Moughan et al.
1998). The general principles of these procedures are exam-
ined below.
Classical methods
Various techniques have been evaluated to determine the
output of endogenous amino acids. The classical approaches
use a nitrogen-free diet, regression analysis, or fasted
animals. The first method involves the measurement of
amino acids in the excreta of animals fed a diet free of nitro-
gen. The regression method is performed by determining
amino acids in the excreta or digesta of animals fed graded
levels of dietary protein and extrapolating to calculate the
endogenous output at zero intake. These techniques have
been employed in chickens by several workers (Okumura
et al. 1981; Bielorai and Iosif 1987). The third method
involves fasting of cockerels for 24–48 h and determining the
amino acid content in excreta (Likuski and Dorrel 1978;
Sibbald 1979).
Yamazaki (1983) used fasted cockerels and a nitrogen-
free diet to determine endogenous amino acid secretions and
found the values to be similar. In contrast, the findings of
Muztar and Slinger (1980) indicate that endogenous amino
acid determinations should be derived from birds fed a nitro-
gen-free diet rather than from unfed birds. It has been sug-
gested that a meal devoid of protein provides adequate
stimulus for the gastrointestinal tract to secrete endogenous
proteins (Nasset 1965). However, these techniques have
been criticised because during starvation or the absence of
dietary protein, the body will be in negative nitrogen balance
and the rate of whole-body protein synthesis will fall. This,
in turn, may influence the entry of protein into the gut. Low
(1990) was of the view that the use of nitrogen-free diets is
897
inappropriate because the absence of dietary protein will pro-
foundly alter metabolism and the animal can no longer be
regarded as physiologically normal. Due to these reasons,
corrections based on traditional methods underestimate true
digestibility estimates (Batterham 1994). Several recent
studies have shown that endogenous amino acid losses vary
depending on the protein source (Siriwan et al. 1991), dietary
protein level (Siriwan et al. 1989; Brannon 1990), dietary
fibre concentrations (Siriwan et al. 1989; Schulze 1994), and
the presence of anti-nutritional factors (Angkanaporn et al.
1994). These findings suggest that the standard practice of
using a single output value generated by conventional
methods for endogenous correction across different feed-
stuffs lacks reliability.
The use of regression analysis where graded amounts of
protein are given to animals has also been criticised. In this
method, increasing levels of protein are fed and amino acid
excretion is determined. The increased excretion of amino
acids, which may be from undigested feed and/or endoge-
nous proteins, is assumed to be directly proportional to the
increased intake. A regression equation is then used to calcu-
late the amino acid excretion at zero protein intake and this is
considered to be an estimate of endogenous losses. This
methodology, however, assumes that there are no changes in
the amount of endogenous amino acid secretions and that the
increase of ileal amino acid flow is attributed entirely to
increases in undigested food proteins. Although the method
overcomes the constraint of physiological abnormality, it
incorrectly assumes that the flow of endogenous amino acid
secretions does not vary with the amount of protein given. It
has been shown that part of the increased ileal amino acid
flow results from an increase in unabsorbed endogenous
amino acids (Fuller 1988).
Peptide alimentation ultrafiltration technique
A method for estimating ileal endogenous amino acid
flow that involves feeding the animal peptides (from enzymi-
cally hydrolysed casein), followed by ultrafiltration of the
ileal digesta, has been proposed (Moughan et al. 1990) and
evaluated with rats (Butts et al. 1991) and pigs (Butts et al.
1993). The peptide alimentation ultrafiltration technique is
based on the differences in physical properties of nitrogenous
fractions in the digesta, and the dietary free amino acids and
small peptides are separated from the large undigested
endogenous proteins in ileal digesta based on molecular
weights. In this method, a semi-synthetic diet containing
enzymically hydrolysed casein as the sole source of protein
is fed to the animal. Ileal digesta is collected and the nitroge-
nous fractions are separated using centrifugation and ultrafil-
tration. Two major fractions are separated: a high molecular
weight (MW >10 000 Da) fraction that provides a measure of
endogenous amino acid flow and a low molecular weight
fraction which contains unabsorbed dietary amino acids and
898 V. Ravindran and Wayne L. Bryden

small peptides, non-protein nitrogen, and low concentrations Table 2. Endogenous amino acid losses (g/kg dry matter intake)
of endogenous free amino acids. in ileal digesta of broilers determined using a nitrogen-free diet
(NFD), regression analysis (REG), and guanidinated casein (GC)
Although not subject to the criticisms of the traditional From Siriwan et al. (1994)
methods, this approach generates estimates applicable only
to correction of ileal flows for protein sources, such as Amino acid NFD REG GC s.e.m.
animal protein meals, which do not contain fibre and/or anti-
nutritional factors (Donkoh et al. 1995). This technique may Aspartic acid 0.61 0.81 1.69 0.08
Threonine 0.54 0.61 1.48 0.10
also underestimate endogenous flow because some endoge- Serine 0.51 0.97 2.11 0.15
nous free amino acids and endogenous small peptides may be Glycine 0.74 1.78 3.08 0.24
discarded in the low molecular weight fraction (Butts et al. Glutamic acid 0.29 0.31 0.72 0.05
1993; Leterme et al. 1996a). Alanine 0.27 0.27 0.69 0.04
Valine 0.42 0.50 1.02 0.07
Techniques involving isotope markers Methionine 0.07 0.08 0.11 0.01
Isoleucine 0.20 0.52 0.91 0.06
During the past decade, numerous studies have been Leucine 0.47 0.48 1.04 0.08
undertaken to measure endogenous amino acids using either Tyrosine 0.32 0.15 0.49 0.04
stable (15N) or radioactive isotopes (14C, 35S, 75Se). The 15N Phenylalanine 0.29 0.21 0.51 0.04
isotope dilution technique of Souffrant et al. (1982) has been Histidine 0.14 0.13 0.31 0.03
Lysine 0.24 0.19 0.56 0.05
employed by several researchers (de Lange et al. 1990; Roos Arginine 0.25 0.18 0.54 0.05
et al. 1994; Schulze 1994) to differentiate endogenous pro-
teins from non-digested dietary proteins in the ileal digesta of Total 5.36 7.19 15.26 0.55
pigs. Their work showed that the recovery of endogenous
proteins in the ileal digesta was higher than those determined
by feeding a protein-free diet. The method involves the per- ditions such as feeding frequency, diet type, infusion proto-
fusion of 15N-labelled leucine into the blood of pigs consum- col, rate of tracer infusion, sampling procedures, sample
ing the test protein. The labelled amino acid is incorporated preparation, and choice of precursor pool(s) is necessary if
in the endogenous secretions, and the dilution of 15N mea- reliable comparisons of data are to be made (Gabert et al.
sured in the digesta gives an estimation of the amount of 1997).
endogenous nitrogen.
Homoarginine technique
Intravenous infusion of 14C-lysine or 14C-phenylalanine
has also been used in pigs (Simon et al. 1983, 1986; Another interesting approach, using homoarginine as a
Zebrowska et al. 1986). The amounts of radioactive labelled marker, to determine endogenous amino acid secretions was
amino acids are detected in the digesta and the flux rate of proposed by Hagemeister and Erbersdobler (1985). Lysine
amino acids entering the intestine is quantified. Simon et al. residues in dietary proteins are transformed into homoargi-
(1983) suggested that the dilution of in-flowing labelled nine by guanidination which involves treatment with O-
amino acids may change by mixing with unlabelled amino methylisourea under alkaline conditions (Mauron and Bujard
acids derived from intracellular protein degradation, thus 1964). After the labelled protein is fed, endogenous losses of
affecting the specific radioactivity of amino acids. amino acids are determined by comparing amino
Other isotopes used for measuring endogenous amino acid : homoarginine ratios in the diet and ileal digesta.
acid secretions are 75Se and 35S. Ochoa-Solano and Gitler Homoarginine is not found in normal feedstuffs (Rao et al.
(1968a, 1968b) simultaneously fed trace amounts of 35S 1963). However, homoarginine is digested and absorbed in a
methionine and 75Se selenomethionine labelled ovalbumin to manner similar to other amino acids (Siriwan et al. 1994), but
rats and differentiated exogenous and endogenous proteins in does not reappear in endogenous secretions into the gut. This
the gastrointestinal tract. There was evidence for appreciable characteristic of homoarginine is an important advantage
quantities of endogenous proteins and the amounts substan- compared to the isotope labelling discussed previously. This
tially exceeded those of exogenous origin in the intestine. approach has been employed to measure endogenous amino
Although attractive, this technique suffers from several acid losses and to estimate true ileal amino acid digestibility
constraints, because the 15N enrichment of the endogenous in poultry (Angkanaporn et al. 1994; Siriwan et al. 1994;
secretions is not easy to determine. The inability to measure Angkanaporn 1995; Angkanaporn et al. 1996a). Siriwan
the recovery of all individual amino acids in ileal digesta (de et al. (1994) reported that values for endogenous amino acid
Lange et al. 1990; Lien et al. 1994) and the rapid precursor losses obtained by the use of guanidinated casein were about
pool recycling (Souffrant et al. 1982; Leterme et al. 1996b) 2-fold greater than those estimated either by feeding a nitro-
are other drawbacks. These limitations have been elegantly gen-free diet or by extrapolation to zero nitrogen intake
discussed by Leterme et al. (1996b). Standardisation of con- (Table 2). Interestingly, the values obtained by homoarginine
technique have been reported to be of similar magnitude to
Amino acid availability in poultry
those measured using the 15N-dilution technique (Roos et al.
1994).
The homoarginine method is based on a number premises
(Bryden et al. 1996) which are outlined below:
1. Homoarginine must not be recycled into the gut. An
assumption implicit in the use of this technique is that the
absorbed homoarginine is not incorporated into tissue
proteins and therefore is not re-cycled into the small intes-
tine. This has been confirmed in studies with broiler
chickens, where homoarginine was not detected in intesti-
nal contents after the intravenous infusion of 18 mM
homoarginine for 3 h (Angkanaporn et al. 1997c).
2. The dietary protein must be homogenously labelled with
homoarginine. An essential criterion for the application of
homoarginine technique is that the transformation of
lysine to homoarginine in guanidinated proteins occurs
randomly. This assumption has been validated by sequen-
tial proteolysis in vitro of guanidinated materials where
the ratios of homoarginine to other amino acids were
found to remain unchanged for casein and soybean
protein, though somewhat aberrant values were noted for
some amino acids in cereals and other protein supple-
ments (Siriwan et al. 1994).
3. Homoarginine must behave within the digestive tract like
other amino acids. Homoarginine incorporated into
dietary proteins is released during digestion and then
absorbed at rates similar to those of other amino acids.
Studies have also shown that guanidination has only
minor effects on the structure of protein and, in particular,
on its susceptibility to proteolysis (Schmitz et al. 1991; de
Vrese et al. 1994; Siriwan et al. 1994). Research from this
laboratory (Ravindran et al. 1998a) and elsewhere
(Moughan and Rutherfurd 1996) has shown that guanidi-
nation does not cause significant modification in the con-
centrations of any of the acid-stable amino acids, except
lysine, when O-methylisourea is used as the guanidinating
agent. In most feed proteins, the recoveries of amino acids
were close to 100%. Recent observations in our labora-
tory indicating that the guanidination process has no influ-
ence on the in vivo digestibility of amino acids (except
lysine) for broiler chickens (Ravindran et al. 1998a) also
suggest that protein racemisation is not a practical
problem.
4. Homoarginine per se must not influence endogenous
amino acid losses. While direct evidence of absence of
homoarginine influence on endogenous protein losses has
not been obtained, the fact that homoarginine behaves in
the small intestine like a typical amino acid suggests that
homoarginine per se has no effect on endogenous protein
output.
5. Homoarginine must not be preferentially metabolised by
gut microflora. The conversion of homoarginine to lysine
and urea within the digestive tract, catalysed by microbial
899
arginase, must be negligible. As shown by Schuttert et al.
(1991) and Siriwan et al. (1994), homoarginine is no more
susceptible to microbial action than other amino acid.
6. Homoarginine must be easily and accurately determined.
Homoarginine may be determined by ion-exchange chro-
matography as a typical amino acid during routine amino
acid analysis without specific hydrolysis or separation
procedures.
Since the homoarginine method enables the separation of
endogenous and exogenous components of amino acid flow
at the terminal ileum, after the feeding of specific ingredients,
it can be used to determine endogenous amino acid secretions
in animals given protein sources which contain fibre and/or
anti-nutritional factors. Endogenous protein output is variable
and may be influenced by several factors including dry matter
and protein intake, protein quality, and dietary fibre content.
The homoarginine technique presents a unique opportunity to
investigate the various factors influencing endogenous amino
acid secretions and a series of experiments has been con-
ducted in our laboratory with poultry (Siriwan et al. 1989;
Siriwan 1990). Dietary levels of protein were found to have a
marked effect on endogenous amino acid losses. Endogenous
amino acids increased significantly when dietary protein
levels were increased from 0 to 250 g/kg. As expected, dietary
fibre (rice hulls) levels also had a significant effect on endoge-
nous amino acid secretions. The output of endogenous amino
acids increased as fibre levels in the diet increased from 0 to
120 g/kg. In a separate study where different types of guanid-
inated protein sources were investigated, it was found that the
extent of endogenous protein secretion was inversely related
to the quality of protein. Endogenous losses were higher when
low quality proteins (e.g. meat meal, cottonseed meal) were
fed and lower when medium (e.g. soybean meal, sunflower
meal) and high (casein, isolated soyabean) quality proteins
were fed. Angkanaporn et al. (1994) investigated the effect of
wheat non-starch polysaccharides (arabinoxylans) on
endogenous amino acid secretions using the homoarginine
technique. Semi-purified diets containing guanidinated
casein plus 0, 15, and 35 g/kg of arabinoxylans were preci-
sion-fed to 5-week-old broilers. Addition of arabinoxylans
was found to markedly increase endogenous losses of amino
acid.
As noted earlier, faecal amino acid digestibility has been
used by many workers because of its simplicity. Thus the
application of homoarginine technique to measure endoge-
nous amino acid losses in the excreta of caecectomised birds
rather than at the ileal level would have obvious advantages.
This possibility was investigated by Angkanaporn et al.
(1996b) using guanidinated casein. They observed a 20-fold
increase in the level of homoarginine in the excreta
(38 ± 2.9 g/kg DM) compared with that in the ileal digesta
(2.1 ± 1.2 g/kg DM) resulting in negative endogenous amino
acid values. The source of homoarginine in excreta is more
 900 V. Ravindran and Wayne L. Bryden

likely to be of urinary origin rather than of dietary origin in
view of the low concentrations of homoarginine in the ileal
digesta. This was confirmed in a subsequent study where
homoarginine was found to be excreted via urine
(Angkanaporn et al. 1997b). These results demonstrated that
homoarginine is not a suitable marker to estimate endoge-
nous amino acid secretions in poultry excreta but is applica-
ble to studies where measurements are made in the small
intestine. The high urinary excretion of homoarginine was
unexpected since previous reports with rats suggest that a
significant proportion of homoarginine is rapidly converted
to lysine after absorption (Ryan et al. 1968). Recent studies,
however, have demonstrated that homoarginine has no lysine
bioactivity in chickens (Aoyagi and Baker 1994;
Angkanaporn et al. 1997d).
The usual procedure for measuring endogenous amino
acid output using homoarginine labelling involves fasting the
animals overnight, followed by a single meal of diets con-
taining guanidinated protein as the only protein source. This
procedure, however, does not represent normal feeding con-
ditions and may influence endogenous protein losses. It is
known that endogenous secretions are higher when digesta
flows continuously along the digestive tract. A series of
studies has been conducted to apply the homoarginine tech-
nique under continuous feeding conditions. As the first step,
a simple procedure for large-scale preparation of guanidi-
nated proteins (5–10 kg batches) has been developed
(Imbeah et al. 1996). The conversion rate of lysine to
homoarginine in casein during large-scale preparations was
similar (93–98%) to those obtained during laboratory scale
preparations, indicating that large-scale guanidination of
proteins is feasible and efficient. Two major problems were
noted when continuous feeding of guanidinated proteins was
attempted. Firstly, feeding diets containing guanidinated
casein resulted in marked depressions in feed intake of
chicks. Subsequent studies found that the reduced feed intake
may reflect a direct effect of lysine deficiency and/or HA on
feed intake regulation (Angkanaporn et al. 1997d). Secondly,
preliminary observations indicated that low dietary elec-
trolyte balance (DEB; Na + K–Cl) is a problem in diets con-
taining guanidinated proteins due to a chloride overload and
that the diets need to be balanced to contain at least
250 meq DEB/kg to prevent the occurrence of watery
excreta. The model developed from these findings is cur-
rently used in the determination of endogenous amino acid
output using the homoarginine technique under continuous
feeding regimes. Diets based on guanidinated proteins are
now balanced with electrolytes and lysine, and such diets
have been found to maintain satisfactory feed intake levels of
guanidinated proteins in amino acid digestibility assays
(Bryden et al. 1996). Alternatively, diets containing
50 : 50 mixture of normal and guanidinated forms of protein
(Schmitz et al. 1991) may be used to overcome partly the
feed intake problems associated with the continuous feeding
of guanidinated proteins.
In our laboratory, the homoarginine technique has been
used to measure the true digestibilities of a range of proteins,
including casein, gelatin, soybean meal, cottonseed meal,
canola meal, sunflower meal, and meat and bone meal.
Based on the assumptions of this procedure, the digestibility
of homoarginine is considered representative of the true
digestibility values of all amino acids within the ingredient.
An interesting approach extending the use of homoargi-
nine-labelling to estimate the digestible reactive lysine
content in heat-damaged foods has been recently reported
(Moughan and Rutherfurd 1996). The method combines the
guanidination reaction with conventional true ileal amino
acid digestibility assays, wherein homoarginine labelling is
used to determine the reactive lysine contents of the diet and
the ileal digesta from digestibility assays. Further research is
required to evaluate and validate the assay.
Digestible amino acid systems
A large volume of published values, including several
compilations (Sibbald 1986; Green 1987; Parsons 1991b;
Rhone-Poulenc 1993, 1995; NRC 1994; Heartland Lysine

Table 3. Terminology used to describe amino acid digestibility in feed ingredients for poultry — selected examples

Reference Terminology Type of bird Method of feeding Site of sampling Endogenous output
measurement

Raharjo and Farrell (1984) Apparent ileal digestibility 10-week-old cockerels Allowance feeding 100 g Ileal digesta —
Raharjo and Farrell (1984) Apparent ileal digestibility Adult cockerels ileal cannulated Allowance feeding 100 g Ileal digesta —
Sibbald (1986) True digestibility Adult cockerels intact Precision feeding 25–40 g Excreta Fasting
Green (1987) True digestibility Adult cockerels caecectomised Precision feeding 50 g Excreta Protein–free diet
Rhone–Poulenc (1993) True digestibility Adult cockerels caecetomised Precision feeding 50 g Excreta Protein–free diet
Rhone Poulenc (1995) True ileal digestibility Adult cockerels caecetomised Precision feeding 50 g Excreta Protein–free diet
Parsons (1991b) True digestibility Adult cockerels Precision feeding 25–50 g Excreta Fasting/protein–free
caecectomised/intact diet
Angkanaporn et al. (1996a) True ileal digestibility Adult cockerels caecetomised Precision feeding 30 g Ileal digesta Homoarginine
labelling
Ravindran et al. (1998b) Apparent ileal digestibility 5-week-old broilers Ad libitum feeding Ileal digesta —
Amino acid availability in poultry
1996; Ravindran et al. 1998) on amino acid digestibility
coefficients for poultry are now available. However, confu-
sion about the terminology used to describe the amino acid
digestibility estimates will soon become clear to anyone
perusing the available digestibility data (see Table 3). For
each amino acid in a feedstuff, there are at least 5 possible
values to describe the digestibility for poultry: apparent or
true for excreta (from intact or caecectomised) or ileal
digestibility.
For reasons discussed earlier, analysis of digesta from the
terminal ileum or analyses of excreta from caecectomised
birds are the superior procedures to describe the amino acid
digestibility in feed ingredients for poultry. However, debate
will continue regarding the need for correction of endoge-
nous amino acid losses in amino acid digestibility calcula-
tions (Sibbald 1987; Low 1990; Batterham 1994; McNab
1995; Boisen and Moughan 1996). One school of thought is
that, as there is no reliable method for measuring endogenous
secretions under a given dietary situation, a system based on
apparent digestibility is a better practical basis for diet for-
mulations. In addition, as noted by Blair (1996), endogenous
correction and the use of true digestibility values appear to
overestimate the digestibilities of several amino acids in
animal by-products such as meat meal, poultry by-product
meal, and feather meal. The need for endogenous output cor-
rection is also dictated by the magnitude of dietary protein
(or amino acid) intake during the assay relative to the amino
acid requirements of the bird. Clearly apparent digestibility
values are invalid if generated using methods that involve
low levels of feed intake (such as the precision-feeding tech-
nique) since this will not support a positive protein balance.
Therefore, to obtain meaningful results, apparent digestibil-
ity values must be determined with birds which have free
access to assay diets.
Correcting apparent digestibility for endogenous losses
can introduce artifacts and mask important differences
between feed ingredients. Although digestibility is often con-
sidered to be a characteristic of a diet or feed ingredient, it is,
in reality, the property of the ingredient in relation to the
animal to which the diet is given (McNab 1989). If a feed
ingredient increases endogenous amino acid flow out of the
small intestine, that represents a loss to the animal and must
be realistically ‘charged’ against the feed ingredient as
lowered amino acid digestibility (Fuller 1988). This would
be a valid statement if apparent digestibility values of indi-
vidual ingredients are additive when combined in diet for-
mulations. The additivity of apparent digestibility values has
been demonstrated by several researchers. Imbeah et al.
(1988) showed that apparent ileal digestibility coefficients
are additive for barley mixed with soybean meal or rapeseed
meal. In studies with pigs and adult cockerels, Fuller et al.
(1994) found that apparent digestibility of amino acids deter-
mined for a mixture of barley and skimmed milk powder was
901
essentially equal to those predicted from the values deter-
mined for the individual ingredients. Similarly, Angkanaporn
et al. (1996b) found that apparent digestibility values are
additive and that the digestible amino acid supply in a com-
plete diet can be predicted, with reasonable accuracy, based
on apparent amino acid digestibilities determined for indi-
vidual feed ingredients in cockerels. In all these studies, the
predicted values were similar or slightly lower than those
observed and this will ensure that the digestible amino acid
supply in mixed diets is not overestimated. However, further
research on this topic is warranted, since some studies have
suggested that there may be associative effects when some
ingredients are combined (Kadim and Moughan 1997).
Two problems that may be encountered when diets are
formulated using apparent digestibility data need mention at
this point. Firstly, for feedstuffs with low protein content
(e.g. cereals, grain legumes), the apparent digestibility values
are underestimated relative to feedstuffs with high protein
content because of the relatively greater proportion of
endogenous amino acids in the digesta or excreta. Secondly,
the applicability of ideal protein concept (Baker 1994) in for-
mulation based on apparent digestibility estimates may be
questioned. Because of the way in which ideal protein ratios
are determined, the patterns reflect true ileal digestibility
rather than apparent ileal digestibility (Baker 1996).
However, using apparent ileal digestible amino acids and
ideal protein ratios of Baker (1994) in formulations, several
recent studies (Ravindran et al. 1998c; Ravindran and
Bryden 1999a, 1999b) have demonstrated the beneficial
effects of formulating broiler diets when poorly digestible
ingredients are incorporated. Ideal protein concept therefore
appears valid for the apparent digestibility values as well, but
further studies are needed to confirm this.
According to Batterham (1994), choice of the appropriate
system of digestible amino acids depends on the method of
formulating diets. If diets are being formulated to least-cost
using linear programming, then apparent ileal digestibility
values are the most appropriate as they take into account of
the endogenous cost of digestion. On the other hand, if diets
are being formulated in computer simulation models, then
true digestibility values will be relevant as the model should
correct for the endogenous cost of digestion.
Most amino acid digestibility values reported for feed-
stuffs in the literature have been determined with adult cock-
erels using the rapid assay procedure of Sibbald (1979) or
modifications thereof. These values are generally applied to
all classes of poultry, irrespective of age difference. Although
it is known that age-effects may exist in terms of efficiency
of digestion, absorption, and endogenous secretions, few
studies have been done to determine the amino acid
digestibility for poultry of different ages (Wallis and Balnave
1984b). To our knowledge, no studies have examined
whether the widely accepted application of a single value
902
(generated using adult cockerels) to laying hens and broilers
is valid. Amino acid digestibility is not the same for all feed
ingredients, with some ingredients have lower digestibility
than others. It may be hypothesised that for good quality
proteins such as soybean meal and fish meal, age-effects
will be minimal and that for less digestible proteins (such as
canola meal, cottonseed meal, lupins, rice pollards) the
digestibility values will be influenced by the poultry model
employed.
Conclusions
Methodology to evaluate the protein quality or amino acid
availability using in vitro (enzymic, chemical or microbio-
logical assays), indirect in vivo (plasma amino acid assays),
or direct in vivo (growth or digestibility assays) measure-
ments have been reviewed. While growth assays remain the
only direct means to confirm the validity of claims for the
nutritional relevance of values obtained by other procedures
(Carpenter 1973), digestibility assays appear to be most
useful to determine amino acid availability because values
apply directly to the animal and all amino acids can be mea-
sured in one assay. While it is possible to visualise circum-
stances where an amino acid could be digested but not
available for use by the host animal, it is also obvious that
undigested amino acids (those appearing in the terminal
ileum or excreta) make no contribution to the requirements
of the animal. Therefore, describing the proteins in feed
ingredients in terms of their digestible amino acids, although
perhaps not ideal, is clearly closer than total amino acids in
reflecting the amount that actually becomes available for
maintenance and production purposes (McNab 1989).
While it is generally accepted that analysis of ileal con-
tents rather than excreta might be a reliable method for
assessing amino acid digestibility in poultry, debate will
obviously continue among nutritionists about the relative
merits of apparent and true digestible amino acid systems. It
should be appreciated, however, that both digestible amino
systems are superior to the total amino acid system currently
employed in practical feed formulations. All present methods
of amino acid evaluation have specific applications and
shortcomings, and ‘the perfect technique for measuring the
bioavailabilities of amino acids in feedstuffs is yet to be
developed’. As stated by Low (1990), ‘feed evaluation in its
practical sense is and always will be a compromise between
the need for simplicity, speed and low cost on the one hand,
and the great complexity of feedstuffs and of the living
organisms on the other’.
Digestible amino acid values are likely to form the basis of
poultry feed formulations in the future. Despite this, currently
few commercial nutritionists formulate diets based solely on
digestible amino acids. The major reasons contributing to this
limited acceptance have been discussed by Johnson and
Richards (1994): (1) wide variations in published digestible
V. Ravindran and Wayne L. Bryden
amino acid values from different sources, arising from differ-
ences in sample variation, type of birds, assay diets, and assay
methodology; (2) insufficient knowledge of the batch-to-
batch variation of amino acid digestibility values; and (3)
limited published information on broiler responses to diets
formulated on the basis of digestible amino acids. Thus there
is an urgent need for more precise information on the varia-
tion in digestible amino acid contents of locally grown ingre-
dients and on the factors causing this variation (e.g. variety,
location, season, agronomic practices, processing, etc.). The
majority of the amino acid digestibility data currently used by
the Australian feed industry originate from North America
and Europe. While such information could serve as a guide,
experience shows that it may be inadequate for accurate feed
formulations. Interactions of cultivars, soil, climate, and agro-
nomic factors could cause appreciable differences in nutrient
profiles and digestibilities between locally grown ingredients
and those available elsewhere.
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