Vol.18, No.
7 July 1996 V
Continuing Education Article
FOCAL POINT
★ As more species are
characterized according to
analysis of excretory steroids, the
normal reproductive patterns of
exotic and zoo animals can be
identified and applied toward
artificial breeding techniques.
KEY FACTS
■ Many of the techniques for
monitoring estrous cycles,
reproductive readiness, and
reproductive disorders in
domesticated animals are
not readily transferable to
nondomesticated animals.
■ The practical advantage of
monitoring urine or feces is that
the samples can be collected
even under the most adverse
conditions.
■ Because hormone levels are
measured on a concentration
basis and urine production is
not constant, total hormone
production can only be assessed
by total urine collection.
■ Numerous studies illustrate the
differences in the excretory
hormone profiles between taxa,
sometimes even between
related species.
Analysis of Sex
Steroid Metabolites
Excreted in the
Feces and Urine of
Nondomesticated
Animals
Purdue University Cryobiology Research Institute
A. T. Peter, BVSc, MVSc, Methodist Hospital of Indiana
Indianapolis, Indiana
MSc, PhD
J. K. Critser, PhD
Veterinary Hospital
Indianapolis Zoo
Indianapolis, Indiana
N. Kapustin, DVM
A rtificial breeding of nondomesticated animals has great potential and
offers the best hope of maintaining viable populations. To achieve such
a goal, the normal reproductive patterns of nondomesticated animals
need to be characterized. Many of the techniques available for monitoring es-
trous cycles, reproductive readiness, and reproductive disorders in domesticated
animals are not readily transferable to nondomesticated animals. The large
number and intractability of nondomesticated animals preclude researchers
from obtaining repeated blood samples to monitor reproductive cycles, as is
done with domesticated animals. Human handling of many nondomesticated
animals causes stress, which may alter production of the hormones being mon-
itored.1,2 Furthermore, extrapolation of veterinary knowledge of reproductive
physiology from domesticated animals to related or unrelated nondomesticated
species without good monitoring techniques is unreliable and may be mis-
leading. Collection of urine and feces can usually be accomplished, however,
without restraining or capturing the animal; and analysis of urinary or fecal
metabolites of sex steroids has proven to be useful for monitoring reproductive
status in many species.3,4
Small Animal
MEASURING SEX STEROIDS IN
PERIPHERAL BLOOD AND THEIR
METABOLITES IN FECES AND URINE
The principal advantage of measuring sex steroids in
peripheral blood is that the rapid secretory dynamics of
the hormones can be studied. The major disadvantage
is the requirement for blood collection, which is not al-
ways possible with nondomesticated animals. For ex-
ample, the amount of blood available for collection is
limited in small, fragile nondomesticated species.
In contrast, the primary benefit of measuring sex
steroid metabolites in feces and urine is that the collec-
tion protocol can be imposed for prolonged periods
without manipulating or stressing (noninvasive) the an-
imals, which is essential when events to be studied are
infrequent and take place over long periods.4 A sec-
ondary advantage is that metabolite concentrations
frequently are two to four times higher than the con-
centration of the parent steroid in blood. For example,
measurement of urinary conjugates of estrogen can pro-
vide equal or superior information compared with the
more conventional evaluation of serum or plasma estra-
diol.5 A practical advantage of monitoring urine or fe-
ces is that the samples can be collected even under the
most adverse conditions and from the most pugnacious
of subjects without risk to either the subject or the in-
vestigator. In addition, a close relationship exists be-
tween changes in plasma estradiol and urinary estrone
conjugates.6 Finally, the assays currently used to mea-
sure sex steroid metabolites are relatively simple, effi-
cient, and easily adapted from one species to another.4
The disadvantages are as follows:
■ If the animal excretes estrogen in feces rather than in
urine,7 collection of urine is not beneficial. Thus,
the excretory route of the steroid metabolite should
be known.
■ Collecting feces from the ground is easier than col-
lecting urine from nondomesticated animals, as
urine collection in some nondomesticated animals
requires restraint in a metabolic cage, surgical inter-
ference, or rigorous surveillance to collect midflow
samples during urination.
■ The entire amount of the steroid or its metabolites
might not be excreted in feces. In this regard, a re-
cent study has shown that only 32% of progesterone
and 11% of estradiol were excreted in feces.8 Using
high-performance liquid chromatography and im-
munoreactivity, the same researchers have demon-
strated that baboons excrete estradiol in feces as
estradiol (36%), estrone (44%), and a conjugated
metabolite that co-eluted with estrone sulfate
(20%). Progesterone is excreted as eight different
FRAGILE NONDOMESTICATED SPECIES ■ RADIOIMMUNOASSAY
The Compendium July 1996
free forms. These researchers have suggested that
currently available immunoassays can adequately
characterize fecal estrogen profiles while caution
should be exercised in selecting a progesterone anti-
serum for the assays, as there is high variability in
cross-reactivities of various progesterone antisera to
progesterone metabolites.9
METHODOLOGIES
Numerous procedures for collecting and analyzing
excreted steroids and gonadotropins have been de-
veloped, although increasing evidence indicates that
species differences necessitate customized approaches.1
Some commonality does, however, exist among the
procedures; the following discussion is an introduction
to some of the general principles.
Most studies to date have dealt with analysis of sex
steroid metabolites in urine. Only a few studies have
characterized the concentrations of sex steroid metabo-
lites in feces (Table I). Except for the initial sample col-
lection and processing, much of the methodology for
these two approaches is the same. Fecal samples are col-
lected fresh from animals housed separately overnight
or from special cages where individual animals are con-
fined for urination and defecation. Samples are frozen
(–20°C) with no preservatives and stored until ana-
lyzed. In cases in which urine might have contaminated
the fecal sample, addition of ethanol to the sample is
recommended.10 Ethanol also prevents storage-depen-
dent changes to steroid content in the feces. Thawed
samples are suspended and centrifuged, and the super-
natant is used in further analyses. Urinary samples are
collected by midstream catch, aspiration from cage
floors, or use of special cages. As with fecal samples,
urinary samples also are frozen without preservatives
and stored until analyzed.
Quantitative analyses vary with the metabolites of in-
terest. Many studies have used a nonspecific radio-
immunoassay (RIA) for total excreted estrogens. Other
researchers use more specific antisera to identify estrone
or one of its major conjugates: estrone sulfate or es-
trone glucuronide. Some of the more recent analysts
used hydrolyzing procedures to separate these conju-
gates before assay. Pregnanediol-3-glucuronide (PDG)
is commonly assayed as an indicator of progesterone
levels. A few researchers have implemented enzyme-
linked immunosorbent assay (ELISA) techniques as an
alternative to radioimmunoassay in an attempt to avoid
the special requirements involved in the use of radio-
isotopes.11,12 In some primate species, commercially
available human pregnancy test kits for chorionic go-
nadotropin (CG) can be used.13,14 Overall, analysis and
sample handling are less laborious and demanding than
The Compendium July 1996 Small Animal

TABLE I
Fecal and Urinary Excretory Steroids and Gonadotropin by Species

Hormone Species

Urinary pregnanediol-3-glucuronide Addax,24 bison,15,25 black mangabey,14 cynomolgus

monkey,26 Eld’s deer,27 giant panda,28 giraffe,2 gorilla,1,13,29
Indian rhinoceros,30 killer whale,16,31 okapi,21 ungulates,1
white-faced saki,32 zebra12

20α-hydroxyprogesterone Killer whale16

Urinary estrogens Asian elephant,17,33 black rhinoceros,34 black and white

rhinoceros,35 brown hyena,36 gorilla,1,29,37,38 Indian
rhinoceros,39 lion-tailed macaque,40 orangutan,37 ruffled
lemur,41 saddle-backed tamarins,42 ungulates1

Urinary estrone sulfate Black rhinoceros,34 gorilla,1 Harmann’s mountain ze-

bra,43 Indian rhinoceros,30 Prezewalski’s horse,43 tapir,19
ungulates1

Urinary estrone conjugates Black mangabey,14 cotton-top tamarins,44,45 cynomolgus

monkey,26,46–48 Eld’s deer,27 Goeldi’s monkey,49,50 goril-
la,13 Indian rhinoceros,30 killer whale,16,31 long-tailed
macaque,40,51 macaque,11 rhesus macaque,22 white-faced
saki32

Urinary luteinizing hormone Addax,24 African elephant,52 cotton-top tamarins,44,45

gorilla,29 killer whale31

Urinary follicle-stimulating hormone Killer whale16,31

Urinary progesterone Black and white rhinoceros35

Urinary androgens African elephant,52,54 free-ranging zebra53

Fecal estrogens Baboons,10 bald eagle,55 black-footed ferret,56 macaca,58

muskoxen,59 tigers,60 lions,60 snow leopards,60 caracals,60
white-crowned sparrow,61 white-faced saki32

Fecal progesterone Baboons,10 black-footed ferret,56 macaque,58 muskoxen,59

tigers,60 lions,60 snow leopards,60 caracals,60 white-faced
saki32

Fecal androgens Bald eagle,55 cranes,57 white-crowned sparrow61

procedures used for serum hormones.3 The assays can
be performed as rapidly as radioimmunoassays. Some
workers have used high-performance liquid chromatog-
raphy to confirm the secretory pattern of steroids in
urine,5 a procedure that may not be practical.
Because hormone levels are measured on a concentra-
tion basis and urine production is not constant, total
hormone production can only be assessed by total urine
collection. A practical alternative is to index urinary
hormone concentrations by urinary creatinine concen-
trations in small samples.16 Furthermore, the cyclic
profiles also improved when daily estrone and estradiol
values were indexed by creatinine concentrations in
Asian elephants.17 Because the total daily production
and excretion of creatinine is relatively constant, the
ratio of hormone to creatinine in urine should remain

HORMONE LEVELS ■ CYCLIC PROFILES ■ CREATININE PRODUCTION

Small Animal The Compendium July 1996

constant unless hormone prediction of estrus because

production changes. In Applications of Excretory Steroid Analysis of the relatively short follic-
some cases, the need for in- ular phase; optimum mating
dexing has been lessened by ■ Pregnancy diagnosis 10,19,37,40,41,43,62–64 times are better predicted on
strict adherence to a sched- the basis of declining preg-
ule of urine collections in ■ Determination of maternal and fetal status47 nanediol-3-glucuronide lev-
the early morning, when ■ Determination of intrauterine fetal death62 els, which signal luteolysis
urinary hormone levels are ■ Determination of ovulation6,15,29,40,42,46,48,51,65,66 and the start of the next fol-
highest; but indexing re- ■ Seasonal hormone profiling (birds) 55 licular phase.1
mains important for assur- Care must always be tak-
ance of standardization. 1 ■ Timing of artificial insemination14,24 en to ensure that urinary
The lack of an indexing ■ Determination of implantation6,46 levels of hormonal metabo-
compound in feces is one ■ Characterization of normal ovarian cycles11,30,36,67–69 lite reflect circulating levels
of the drawbacks to this ■ Characterization of normal menstrual cycle 10 of biologically active com-
approach; however, at least pounds. This concern has
in the case of fecal proges- ■ Timing of parturition2,11 been satisfied in domesticat-
terone, indexing can be ■ Determination and tentative diagnosis of ed cows by comparing hor-
expressed in terms of cho- reproductive cycle abnormalities11 monal levels in plasma with
lestanone concentrations. ■ Correlation of hormone levels and social those in urine1; however, be-
Such indexing helps to over- cause of differences in the
come the effects of pro- behaviors38,54 metabolism of other species,
found variations in dietary ■ Characterization of male reproductive cycles52 researchers should not as-
fiber.18 This type of research ■ Sex determination (birds)57,70–72 sume that the levels in urine
has been conducted for ba- and in blood are uniform.
boons. Further study is need- Inactivation of sex steroid
ed for other species. hormones in the liver and
kidneys before excretion and bacterial action in the gut
REPRODUCTIVE APPLICATIONS before elimination of feces may make such an assump-
AND POTENTIAL PROBLEMS WITH tion problematic. Furthermore, hormones may be ex-
EXCRETORY STEROID ANALYSIS creted in high concentrations in feces and low levels in
Reproductive applications involving the analysis of urine,20,21 and the hormone may be missed if only one
urinary and fecal levels of hormones have been used in type of assay is used. The patterns of excretion also
numerous situations, as evidenced by the boxed listing seem to vary between species. Of interest is the 24-hour
of applications. Some potential pitfalls in the whole- delay in the excretion of hormonal metabolites in urine
sale application of excretory steroid analysis must be of the rhesus macaque.22 Linear correlation between
noted. urinary estrone conjugate and serum estradiol during
Numerous studies illustrate the differences in the ex- nonconceptive cycles aligned by the preovulatory es-
cretory hormone profiles between taxa, sometimes even trone conjugate improved when daily hormone values
between related species,1,19 thereby emphasizing the were realigned to account for the delay. 22 In both
danger in extrapolating between species and the neces- conceptive and nonconceptive cycles, urinary estrone
sity of establishing profiles for each species studied. conjugate paralleled serum estradiol. Researchers also
Furthermore, the selection of hormonal metabolites to believe that dietary fiber can influence the levels of sex
be assayed and the quantitative method to be used steroid hormone in feces; however, if adjustment is
must be chosen carefully for each species or group of made for the water content of feces, this effect can be
related species. While some related species (e.g., giraffe kept minimal.10 Other points to be considered with re-
and okapi) show similar patterns of hormonal physiolo- gard to free-ranging nonhuman primates are proper
gy and react similarly to assay methods,2 other groups identification of animal source and collection of urine
of species (e.g., primates) do not.14 In some species samples before they soak into dirt or ground vegeta-
(e.g., primates, Indian rhinoceros), detection of the LH tion.
surge or rising preovulatory estrogens allows prediction The number and timing of samples must be carefully
of fertile periods and scheduling of timed matings or considered. Many species require daily sampling to de-
inseminations. In numerous ungulate species, such termine the reproductive stage accurately. Other species
monitoring of preovulatory estrogen does not allow may require only a few samples to assess fertility; but

STANDARDIZATION ■ EXCRETORY HORMONE PROFILES

 Small Animal The Compendium July 1996

Your Animal even in these species, continuous monitoring is desir-

Health Collection able because the stress imposed by human handling at
artificial insemination may alter a predicted cycle.1 For
pregnancy diagnosis and sex determination, one sample
Isn’t Complete may be adequate. It is important to ascertain from the
laboratory the duration of the assay procedure and the

Without laboratory schedules (e.g., once a week) to run the as-

CE CREDIT FROM MICHIGAN STATE UNIVERSITY
says.
Finally, most studies to date have necessarily involved
Volume 21 Number 9 September 2000
very few individuals. In many cases, the number of ani-
mals available is extremely limited. Great care must be

Veterinary Breathing Easy

(page 509)
taken in interpreting results based on a few samples,
and application of new knowledge and techniques must
Technician
The Complete Journal for the Veterinary Hospital Staff
®
proceed with caution.

504 CONCLUSION
HOTLINE HEROES
at the NAPCC
New
The use of excretory steroid analysis in exotic and
506
TOXICOLOGY BRIEF:
Column!
zoo animal reproduction is a rapidly expanding field.
Permethrin in Cats
As more species are accurately characterized and assay
508
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12 issues only $38 systems are refined, the use of such techniques should
Join VETTEAM

518
significantly expand species propagation by establish-
Get a Handle
on HIRING
ing normal reproductive patterns. Extrapolating in-
526 formation from one species to other closely related
The Golden Years—
A Roundtable on species may be possible.
SENIOR CARE
Check out TechMart.
See page 541.
Studies involving exotic species in the past few
Dana Farbman, CVT
National Animal Poison
Control Center
decades have allowed researchers to characterize the
reproductive cycles in some of the species. Most of the
processes adopted to characterize the hormones and
their metabolites in urine and feces are acceptable.
The Most Widely Read Some of the procedures involved may entail more
time. Those procedures, however, are widely accepted;
Journal in Its Field in general, the results are informative.23 Finally, simi-
lar research has been conducted in the areas of human
■ The technician’s right hand—the source clinical medicine and epidemiology; there is great po-
they reach for first tential for application of those procedures in animal
and human reproduction.
■ Articles of interest to animal health
professionals of all kinds: breeders, ACKNOWLEDGMENT
caretakers, trainers… The authors thank Dr. B. L. Lasley of the Institute of
Toxicology and Environmental Health, University of
■ Medical and management articles of California, for his comments on this article.
interest to the dedicated pet owner

About the Authors

SUBSCRIBE TODAY! Drs. Peter, Critser, and Kapustin are affiliated with
CALL 800-426-9119 the Department of Veterinary Clinical Sciences, School
of Veterinary Medicine, Purdue University, West
Veterinary Technician is published by Lafayette, Indiana. Dr. Critser also is president and sci-
Veterinary Learning Systems entific director of the Biodiversity Research Institute at
275 Phillips Blvd. Indianapolis, Indiana. Dr. Peter is a Diplomate of the
Trenton, NJ 08618-1496 American College of Theriogenologists.
Price is in US dollars and is subject to change.
The Compendium July 1996
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The Compendium July 1996
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