Clinical Biochemistry of Domestic Animals (Sixth Edition) - UMK ...

644
Chapter | 21 Clinical Reproductive Endocrinology
steroids in the sample, are given equal opportunities to interact
with the antibody used in the assay. The synthetic steroid
danazol (17 α ,2,4-pregnandien-20-yno(2,3-d)isoxazol-17ol)
can be used to displace progesterone from the binding
proteins ( Carrière and Lee, 1994 ).
Certain immunoassay systems may require purification
of the plasma extract to achieve an acceptable specificity.
The main problem in the immunoassay of primary
prostaglandins is that they can continue to be formed in
large amounts by platelets after the blood sample has been
obtained ( Samuelsson et al. , 1975 ), thus the concentrations
of PGF 2 α reported in blood serum or plasma can appear
much higher than the actual values. The primary prostaglandins
have a very short half-life in the circulation ( Hamberg
and Samuelsson, 1971 ) and are rapidly converted to their
corresponding 15-keto-13,14-dihydro derivatives. The latter
have considerably longer half-lives and occur in higher
concentrations than the parent compounds ( Beguin et al. ,
1972 ). Analysis of metabolites of prostaglandin F 2 α avoids
the problem of the overestimation observed for the parent
compound in that the metabolites are formed only within the
body and values thus remain stable once a blood sample has
been obtained. Radioimmunoassay systems utilizing antibodies
to 9 α ,11α -dihydroxy-15-ketoprost-5-enoic acid and
5 α ,7α -dihydroxy-11-ketotetranorprosta-1,16-dioic acid have
been developed ( Granström and Samuelsson, 1972 ). Most
problems involved in the determination of the primary prostaglandins
are avoided if their main metabolites, the 15-keto-13,
14-dihydro compounds, are measured.
b . Sensitivity
The sensitivity of immunoassays is defined as the smallest
quantity of hormone that the assay can reliably distinguish
from a zero amount of the hormone. Usually two kinds of
sensitivity are evaluated. The sensitivity of the standard
curve is defined as the smallest amount of hormone that
is significantly different from zero at the 95% confidence
limit. However, the most meaningful sensitivity to establish
is the smallest amount of hormone that can be measured per
unit of biological fluid (e.g., per milliliter of blood plasma).
c . Accuracy
The accuracy of an assay is defined as the extent to which
the measurement of a hormone agrees with the exact
amount of the hormone. Accuracy is often determined by
comparing immunoassay data with values determined by
other procedures such as gravimetry, gas liquid chromatography,
and mass spectrometry. For steroid hormones and
prostaglandins, accuracy is also often determined by recovery
experiments in which different amounts of hormones
are added to a biological fluid (e.g., blood plasma), which
contains low concentrations of the hormone; the amount of
hormone measured in the assay is then compared with the
amount of hormone added.
d . Precision
Two types of precision are usually evaluated. The withinassay
precision is determined from duplicate measurements
of the same sample within the same assay. The betweenassay
precision is determined from replicate analyses of
the same sample in different assays. Usually the betweenassay
variance is greater than the within-assay variance.
Assay variance should be checked continuously with each
assay of a certain hormone by use of plasma pools containing
set amounts of the hormone. Usually three different
plasma sets containing low, medium, and high hormone
concentrations are used. Within- and between-assay variations
in immunoassay procedures are usually greater than
for ordinary routine procedures used in clinical chemistry.
III . PHYSIOLOGY OF REPRODUCTIVE
HORMONES IN THE FEMALE
This presentation is relatively brief as to its coverage of
endocrinological events during the reproductive cycle.
Readers interested in a more complete presentation of hormonal
events involved in reproduction are referred to texts
dealing specifically with the subject. Also, some hormones
with some influence on reproductive processes, such as
those of the thyroid, adrenal, and pineal glands, are not
covered in this presentation.
A . Estrus Cycle
The major endocrine events that precede ovulation have been
well documented in the cow ( Chenault et al. , 1975 ), ewe
(Nett et al. , 1974 ), sow (Shearer et al. , 1972 ), mare (Evans
and Irvine, 1975 ; Palmer and Jousset, 1975 ; Stabenfeldt et al. ,
1975 ), dog ( Concannon et al. , 1975 ), and cat ( Shille et al. ,
1979b ). In large domestic animals (cattle, horse, pig, sheep,
and goat), follicle growth occurs during the luteal phase in
spite of the inhibitory nature of progesterone, the main secretory
product of the CL. Although follicles are usually not
ovulated during the luteal phase in most species, the mare
occasionally ovulates during the luteal phase.
With regression of the CL, follicles grow rapidly before
ovulation because of gonadotropin stimulation. The follicles
secrete increasing amounts of estrogen during
development, which is important for the onset of sexual
receptivity as well as for the initiation of the surge release
of gonadotropins that is essential for the ovulatory process
( Fig. 21-4 ). Estrogens initiate the surge of LH and
FSH through the release of GnRH ( Moenter et al. , 1990 ).
In most species, the preovulatory surge of gonadotropins
begins approximately 24 h before ovulation and is usually
of short duration, (e.g., 8 to 10 h in the cow). The mare
is an exception in that large amounts of LH are released
during an 8- to 9-day period with ovulation occurring on
the third day ( Geschwind et al. , 1975 ). Another important