Of chromic oxide aredue mainly to incomplete mixing of the digesta and marker in the

gastro intestinal tract. Study of ileal digestibility in the chicken also seemed to show that
improper mixing had occurred with the digesta of some birds (taylor,1998).furthermore,
chromic oxide probably passes out of the stomach of a pig at a faster rate than the
particulate fraction of the digesta (Asche et al, 1989). Chromic oxide is a very fine
powder compared with the diet. To ensure that the marker is of similar particle size and
density to the diet, the use of chromium in a form of chromic bread has been advised (
taylor, 1988; Achinewu and Hewitt, 1979).

As an alternative to chromium oxide, chromium mordanted to cell walls has been

used widely as a marker for measurement of digesta flow (uden,colluci and van
soest,1980;pond,deswysen,schelling and ellis,1981;van soest, uden and wrick,
1983;ehle,jeraci,Robertson and van soest,1984; pond,pond, ellis and matis, 1986;asche
et al.,1989). Uden et al.(1980) stated that chromium mordant fulfilled most of the
criteria necessary for a particulate marker as it formed sufficiently strong complexes
with plant cell walls to withstand the digestive process.this would be advantageous
because it would provide the bonding to ensure that the chromium remained
mordanted to the digesta residues during the transit through the gastrointestinal tract,
as has been observed in cattle (uden et al.,1980) and pigs (pond et al.,1986). This
condition is also needed for a good marker in digestibility studies. (Asche et al., 1989)
used chromium-mordanted cellulose (solka floc) as a marker for both digesta flow and
nitrogen digestibilities studies in pigs . any discrepancy caused by the possibility of a
faster movement of the marker than the chime in the stomatch can be avoided by using
this type of marker.

2.7. procedures for the determination of Ileal true digestibility of protein and amino
acids in rats many studies have been performed using rats as an experimental animal
model to study aspects of animal nutrition. In the study of ileal digestibility
of dietary protein in the pig, the use of rats as an animal model is favourable because of
the ease of collecting ileal contents after slaughter (moughan, smith and
kies,1987).although the 1989 joint FAO-WHO Expert consultation on protein quality
evaluation acknowledged the usefulness of ileal digestibility values and noted that there
is a similarity between humans and rats in the digestion of foods (FAO/WHO,1990),
there has not been a great deal of study of ileal digestibility using rats as an
experimental animal for humans.

The study of ileal digestibility does not overcome the problem of measuring the
correction factor necessary for true digestibility determinations. All of the problems
mentioned under endogenous or metabolic secretions attend this procedure, plus
others such as the physical difficulties of obtaining samples and the consequent
likelihood of sample contamination during extraction of the ileal contents. Therefore it
seems that a combination of ileal sampling and varying the input of nitrogen (regression
procedure) would provide a method combining the advantages of current techniques.
This would allow determination of true digestibility, avoiding measurement of
endogenous amino acid losses. Although this method has been performed with the
chicken (beilorai et al.,1985b; taylor, 1988), it has not been used for assessment of
human foods by rats.

The basic relationship which demonstrates the derivation of true digestibility

from nitrogen absorbed and nitrogen intake is as follows: true ileal digestibility of
protein or amino acid (Dt) can be defined as

( )
Dt =

Where: Ni = Nitrogen or amino acid intake

Nd = Nitrogen or amino acid output (in the digesta)

Ne = Endogenous losses of nitrogen or amino acid

Therefore: = +
if nitrogen or amino acid absorbed is considered as (Na) :

=
Therefore :

= +

Or

If it is assumed that Dt and Ne are constants, the equation is a linear relationship and
can be quantified by regression procedures. The slope of the regression indicates the
value of true digestibility while endogenous losses of nitrogen or an amino acid is the
intercept of the regression. This method needs some knowledge of the intake range in
wich the response is linear. Combination of the simple linear regression equation above
with absorption using a marker for ileal contents enable the measurement of ileal true
digestibility of protein or amino acid, without the need for estimating of endogenous
losses. This method will be described further with refrence to the methods used in
study.

2.8 biological availability of amino acid

2.8.1 Background

The terms digestibility and biological availability (or bioavailability or availability)

are frequently used synonymously when referring to amino acids (sarwar, 1987).
However some recent studies have shown that these two terms are not always
equivalent because some absorbed amio acids may not be utilized by the living
organism. In such cases digestibility would over estimate the value of availability. By
definition, every amino acid wich is potentially utilizable form and wich is absorbed by
the gut is available.

A wide variety methods have been used to measure the availability of amino
acids in protein. These include various chemical, microbial and biological methods.
While the first two methods are still being developed, the
biological assay is needed to asses the validity of the chemical and microbial methods.

2.8.2. Growth assay for the determination of amino acid availability

Most investigators would agree that some form of growth assay works best for
estimating the availability of nutriens in food ingredients (baker, 1978). However,
inconsistent results are often associated with growth assay and this may be attributed
to a variety causes. Protein level, amino acid balance, delayed realese of amino acids
from intact protein as opposed to the readily absorbed crystalline amino acids, and
energy density of the diet are all known to affect growth (baker, 1978). Undoubtedly
these confounding and other factors have led to many inconsistencies reported in the
literature. Soares and kifer (1971) contended that growth assay overestimate laysin
availability. Robel and frobish (1977) suggested than short-term bio assays may
underestimate bioavailability of amino acids, including lysine in soyabean meal.
Carpenter (1973) however concluded that growth assay, though expensive, slow and
somewhat imprecise, remained the only direct means of determining the nutritional
relevance of values obtained by other procedures.

2.8.2.1. procedures of the growth assay

There are two major procedures used in the growth assay for the determination
of amino acid availability, namely standard curve and slope-ratio analysis. In both
procedures, diets are prepared to contain all required nutrients except the amino acid
being evaluated.

In the standard curve procedure, a basal deficient diet and range of standard
diets containing increasing amount of the amino acid being evaluated (standard amino
acid) are used to develop a standard curve. The amino acid standard is assumed to be
fully available for utilization. At levels of amino acid below the requirement a linear plot
of weight gain against amino acid consumed is then derived. Once the standard curve is
constructed, each observation using a test protein substituted into the diet in place of
the amino acid standard becomes an estimate of availability. This is accomplished by
substituting an observed weight gain datum from animals receiving the test protein into
the equation describing the standard curve and solving for the amount of effective or
available amino acid consumed by the animals. The availability estimate is then
calculated as the portion of the total amino acid consumed that was found to be
effective.

The standard curve procedure used by some investigators is often critised. Sasse
and Baker (1973) stated that the slope-ratio technique gives a more statistically valid
estimate of availability than the standard method because the latter assumes there is no
experimental error in determining the standard curve when, in fact, there usually is.
Bjarnason and carpenter (1969) also criticized the standard curve procedure because no
valid statistical test could be made of the precision of the amino acid availability
estimated by feedingone experimental diet containing one level of amino acid being
evaluated. However , the standard curve method has the advantages of simplicity and
straight-forwardness.

Slope-ratio analysis relates the slope of the regression equation describing the
test response with that describing the standard amino acid response. In this assay, diets
are formulated to contain graded levels of standard (free) and test amino acid being
examined. Simple linear regression coefficients of response to increasing level or intake
of amino acid in test protein and standard amino acid are calculated. Thus when the
slop of the test response is divided by the slop of the standard amino acid response, an
estimate of availability of the amino acid in the test protein is obtained. Carpenter
(1973) suggested that with slope-ratio assays at least two levels of the test protein and
two for the standard amino acid response, together with basal diet are required,
provided that these lie within the linear range.

The regression analysis used in the traditional slope-ratio analysis (finney, 1964)
relates the response to the increasing dose level of substance (s) to be assessed. In this
method, three criteria are used to establish