Sections of 5 μm were cut on a microtome and the tissue
slides were stained with Haematoxylin and Eosin staining
in an automated histological stainer. The histopathological
assessments were graded as no observable organ damage
(−), mild organ damage (1+), moderate organ damage
(2+) and severe organ damage (3+), where damage is a deviation
from normal morphology.
The serum of blood samples from two chickens of each
replicate was analysed for free T3 and T4 concentration by
Chemiluminescence Enzyme Immunoassay (CLIA) (Kricka
et al., 1987).
In the T3 CLIA, as explained by Rongen et al. (1994), a certain
amount of anti-T3 antibody was coated on micro titer
wells. A measured amount of chicken serum and a constant
amount of T3 conjugated with horseradish peroxidase were
added to the micro titer wells. During incubation, T3 in the
samples and conjugated T3 competed for the limited binding
sites on the anti-T3 antibody of the wells. After 60 min of incubation
at room temperature, the wells were washed 5
times by wash solution to remove unbound T3 conjugate. A
solution of chemiluminescent substrate was then added and
relative light units in a Luminometer were read. The intensity
of the emitting light was proportional to the amount of enzyme
present and was inversely related to the amount of
unlabelled T3 in the sample. By reference to a series of T3
standards assayed in the same way, the concentration of T3
in the unknown sample was quantified (Rongen et al.,
1994). The precise method for T3 was repeated for T4.
2.6. Statistical evaluation
The GLM procedure of the SAS (Statistical Analyses
System®) software system was used in the statistical analysis.
The significance of differences between treatments and exposure
periods was established by means of the Fisher's test at
Pb0.05.