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 Comprehensive
science: Non-invasive monitoring of hormones
Monitoring of hormones means the measurement of hormones, which
regulate homeostasis and reproduction in humans or animals over a longer
period of time. All hormones are synthesised from specific organs, e.g.
the gonads, the adrenal or pituitary gland. They are secreted into the
blood plasma and distributed by the circulatory blood stream. When they
reach their target organs they induce their biological effects. Reproduction
is unalterable dependent on regulatory hormones. The measurement of hormone
concentrations in blood plasma over a prolonged time period (covering
days and weeks) allows conclusions about the reproductive activity in
male or female individuals. Characteristic hormones for female animals
are the gonadotropins: follicle stimulating hormone (FSH) and luteinising
hormone (LH), and the steroids estradiol (estrogens) and progesterone
(gestagens). Other hormones, however, can be essential for the maintenance
of cyclicity and pregnancy (like relaxin, prolactin). In males, the most
important hormones are the gonadotrophins LH and FSH and the steroid testosterone
. Measurements of these hormones were performed by radio- or enzyme-immunoassays
(RIA, ELISA). It is necessary to mention that in between different species
steroids are mostly identical, whereas proteohormones (FSH, LH, prolactin,
relaxin) are species specific. This fact complicates the monitoring and
comparison of different species. Therefore, in zoo and wildlife animals
monitoring of cycle and pregnancy in females and reproductive season in
males concentrates on the determination of steroid hormones .
In females, the corpus luteum (C.l.) of the ovary secretes the
hormone progesterone, which is responsible for maintaining pregnancy and
also reflects cyclic ovarian activity. The periodic growth and regression
of the C.l. (luteolysis) is accompanied by increasing and decreasing progesterone
concentrations, respectively.
In case of a successful (fertile) mating the C.l. persists and high amounts
of progesterone are secreted to maintain pregnancy. Without fertilisation,
the C.l. regresses. This so called follicular phase of the female reproductive
cycle is characterised by low progesterone concentrations. At the end
of the follicular phase ovulation occurs within one or two days. After
ovulation a new C.l. grows (luteal phase of the reproductive cycle) and
without mating another cycle starts. The time period in between ovulations
is defined as cycle length. It can easily be calculated from the progesterone
profile (see figure below).
Analytical methods to characterise stress are of increasing importance
for zoo and wildlife scientist . Animals exposed to stress secrete the
glucocorticoid cortisol which is produced in the adrenal glands. Therefore,
cortisol measurements can be used to demonstrate stress, e.g., induced
due to transportation of an animal from one zoo to another. However, one
must keep in mind that high cortisol levels cannot solely be attributed
to stress. Cortisol has also metabolic functions and increases during
energy deficiency and starvation to mobilise energy. Furthermore, blood
sampling from animals induces stress due to catching, fixation, and anaesthesia;
consequently, it is nearly impossible to obtain blood samples from unstressed
individuals (this is also true for humans).
Non-invasive methods for
measuring hormones are the best "way out" in this respect. For
non-invasive monitoring degradation of hormone products (metabolites)
are determined in substrates, which the animal gives away voluntarily
without stress. Such substrates are urine, saliva, sweat or faeces. Collecting
faeces, for instance, can be easily performed in captivity or in the field
without disturbing the animals. In the laboratory, the specific metabolites
can be extracted by using chemical methods. The non-invasive determination
of hormone metabolites are necessary for different scientific projects:
- Investigations on seasonal reproduction (limited to a period of several
months).
Comparison of different animal species revealed specific seasonal reproductive
strategies (see figure below).
Continuous reproductive activity throughout the whole year (non-seasonal,
polyestrous), is shown e.g. in elephants and human beings. Some species,
like the Przewalski's horse, are seasonal breeders with an extended
period of cyclic ovarian activity of approximately 9 months followed
by a short non-cycling period during the winter months (seasonal, polyestrous).
The European wild boar revealed reproductive activity during autumn
and early winter and a second facultative period in spring. The boar
can also be regarded as seasonal polyestrous breeder. In contrast to
the polyestrous strategies, the roe deer and the giant panda are characterised
as seasonal monoestrous breeders. There is only a single chance (one
estrous / one ovulation) for successful reproduction in late summer
or spring, respectively. The overall aim of all these different strategies
is to time parturition into a season with proper environmental conditions
(temperature, nutrition) to guarantee an optimal survival rate of the
young.
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See also research projects on non-invasive monitoring of hormones
in roe deer, elephants , and
and giant panda)
- Determination of cycle length in various mammalian species
The estrous cycle varies in length and may cover a period of a few days
in small rodents (rat, mouse) up to 16 weeks in elephants
- Investigations on male reproduction
Reproductive activities of males are usually linked to female reproduction
and - if seasonal - gonadal activity of males coincides with those of
the females. To asses male gonadal activity testosterone, synthesised
in the testis, and its urinary or faecal metabolites are monitored.
The correlation of male reproductive hormone secretion with season,
dominance status, and conditions in captivity (nutrition, domestication)
is one of the main aims of our studies.
- Diagnosis of reproductive failure
There are many reasons for the failure of cyclic ovarian activity in
females. While immature animals have no cyclic ovarian activity, in
adult females reproductive disorders may occur. The diagnosis of reproductive
disorders can be performed by hormonal analyses in blood, or better
by non-invasive monitoring. A persistent low progesterone metabolite
level in an adult female may be attributed to follicular cysts. Follicular
cysts are defined as cystic structures derived from non-ovulated follicles
They prevent the generation of a new C.l. and thus suppress cyclic ovarian
activity. Cystic females can recover to normal conditions, e.g. after
the administration of gonadotropins. The effect of such treatments can
be judged with non-invasive methods.
- Failure of male reproductive behaviour
Many reports demonstrate that male zoo animals do not show any sexual
interest in estrous females or natural mating. In these cases artificial
insemination carried out by zoo veterinarians is an alternative to fertilise
the female. Based on the short fertile life span of sperms (several
hours) and the life span of oocytes (2-3 days) knowledge of the females
reproductive status to predict the period of ovulation is of great importance.
An expensive procedure like artificial insemination must be performed
just before or directly after ovulation (after the LH peak and during
estrogen increase).
- Contraception in zoo animals
Some zoo animals, e.g. the brown bear reproduce so well in captivity
that due to restricted place reproduction must be controlled to avoid
a "surplus" of animals. The best contraception methods are
the application of an "anti-baby-pill" (hormonal contraception:
interaction with the hormonal system to prevent activity in the ovary)
or the immunisation with zona pellucida proteins (immunocontraception:
avoids fertilisation of the oocyte), rather than irreversible castration
or separation of the genders. The effect of contraceptive treatments
can be monitored by non-invasive methods.
- Stress monitoring in wild and zoo animals
There is an increasing demand for methods to monitor stress in zoo and
wild animals. Different laboratories are very busy developing analytical
methods to measure faecal and urinary stress hormone metabolites (glucocorticoids
secreted by the adrenal gland). The aim is to objectivate stress as
a consequence of anthropogenic disturbances in habitats, to evaluate
stress due to translocation of animals (often part of international
breeding programs), to monitor social stress dependent on ranking and
territorial defence, and to measure glucocorticoids as metabolic hormones
for seasonal studies. This information cannot be obtained from a single
blood sample and frequent blood sampling counteracts with the welfare
of the animal (which is not a voluntary donor). The only alternative
is to measure faecal and urinary metabolites of stress hormones using
non-invasive methods.
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