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| Ecology and Reproduction
of Rare Wild Animal Species |
| Studies
on the regulation of reproductive functions in Hokkaido Sika deer |
| Motoki SASAKI |
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The reproductive functions are maintained by the regulatory factors
such as hormone and growth factor and the complicated regulatory mechanism.
As results of investigations of several domestic, experimental and wild
animals, it has clarified that the expression and localization of regulatory
factors and the regulatory mechanism were different among species or
during developmental stage. Thus, the comprehensive studies on reproductive
functions are necessary to understand the reproductive strategy in some
animal species. The fully understanding the characteristic of normal
reproductive functions must be useful as standard data against the occurrence
of an environmental pollution, because the reproductive functions are
easy to be influenced by changes of environment.
In Japan, Sika deer (Cervus nippon) which is large seasonally breeding
mammal inhabits broad area from Hokkaido until Kerama Islands, Okinawa.
The Hokkaido Sika deer (C. nippon yesoensis), one of sub-species in
Sika deer is living only in Hokkaido. At present, any problems about
other environmental pollutions are not brought in Hokkaido except lead
poisoning (plumbism) against Raptores by lead ammunition. For the assessment
of environmental pollution, it would be necessary to collect basic data
on Hokkaido Sika deer, typical large mammal in Japan. The present studies
are carried out for two purposes, as follows.
- Determination of the regulatory mechanism in testis
The expression and localization of the steroidogenic enzymes and growth
factors and the receptors of sex steroid hormones, gonadotropin and
growth factors in the testis of Hokkaido Sika deer are immnohistochemically
and molecularbiologically examined with seasonal changes and during
developmental stage.
- Determination of the morphological and physiological characteristics
in pituitary gland
The mobility of LH, FSH, TSH, ACTH, PRL, GH and Leptin producing cells
in the adenohypophysis of Hokkaido Sika deer is immnohistochemically
and molecularbiologically examined with seasonal changes.
In these studies of Hokkaido Sika deer, the regulatory mechanism of
pituitary-gonadal (testis) axis and paracrine and/or autocrine in the
testis would be defined.
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| Promote
Breeding Programs in Zoological and Endangered Birds |
| Yoh-Ichi MIYAKE |
It has been known that many avian species
cannot be sexually identified by their external appearances. For this
reason, it is important to establish a method to identify their genetic
sexes to promote the captive breeding program for wild bird species, especially
endangered birds, and birds bred in the zoo. Recently, Cpolymerase chain
reaction (PCR) has been available as a sex identification tool in the
bird. By this technique,213 birds were sexually identified. In conclusion,
this PCR protocol is useful in sex identification because various parts
of body tissue could be utilized as DNA source, and because this protocol
is applicable to a variety of avian species. This PCR technique has a
potential to promote the breeding programs in zoological birds bred in
the zoo and wild birds.
But it is necessary to promote another new technique because of difficulty
in PCR technique. And it is expected to promote the breeding programs
by using artificial insemination. LAMP (Loop-Mediated Isothermal Amplification)
method is a new technique to amplify the genes. This experiment will focus
to establish the optimum PCR conditions and primer set for sex identification
of avian species and to investigate the its usefulness of sex identification.
Also, it is investigated about optimum time of insemination, collection
of semen and method of artificial insemination.
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| Study
on Preservation of Wild Animals in the Tohoku Area of Japan Location and
Method of Survey |
| Kosuke OKADA |
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Through the Japanese serow project of Iwate University, seven heads
of Japanese serow have been confirmed to be living in Takizawa Forest,
Iwate University's research forest. A one-year old male was captured
and blood, fecal, lesion , and ectoparasite samples were taken and the
serow was tagged with a telemetry transmitter and GPS device. This project
has furthered comprehensive research which combines wildlife medicine
(including blood type, DNA individual identification, parasites, viruses,
and medical examinations) and ecology (inhabitable numbers, food property,
territory and behavior), and conservation plan. Eventually, we plan
to capture the entire population of Japanese serow in the research forest.
Then, wildlife medicine and ecology research will be performed on these
samples from Takizawa Forest. In this project, all individual Japanese
serow that have died within Morioka City are kept at Iwate University.
Blood and DNA samples were taken from these Japanese serow to determine
the sex and to uniquely identify each individual. In addition to identifying
the dead samples, we identified each individual serow in Takizawa Forest.
Questions which we sought to answer were: Which individuals were depositing
their feces in which part of the forest? What is the relation between
an individual's territory and the area where they deposit their feces,
shown through telemetry? Does their utilization of the area where they
deposit their feces change according to season? We hope to solve these
questions and would like to develop new techniques of serow protection
management.
In 2003 and 2004, we performed a study of serum protein polymorphism
(albumin and transferrin) and sex distinction method by DNA. However,
there is still improvement needed in the sex distinction method. Therefore,
in 2005 and 2006, we will extract DNA from the feces of Japanese serow
kept at Iwate University. In addition to performing examinations to
more accurately distinguish sex, we will use DNA fingerprinting or microsatellites
as a method of individual identification of Japanese serow. Additionally,
we will capture Japanese serow in Takizawa Forest and extract DNA from
blood and fecal samples, and identify each individual by DNA polymorphism.
We will combine data about where the serow deposit their feces with
GPS data. We will then identify individual serow by collecting their
feces. We will then examine the relationship between territory and the
area where the serow deposit feces using collected feces and data about
already-identified individuals.
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| The
fundamental and applied behavioral studies on the dispersion and metapopulational
structure in wild animals |
| Yoshiaki OBARA |
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It is widely known that ecology and behavioral biology of wild animals,
including migration, dispersal, social structure and reproductive behavior,
are necessitated for the conservation of wild animals. Evolutionary
biology on the new trait evolution and its fixation in the population
are also needed for the investigation of some aspects of conservation.
Present study is, thus, designed from these biological viewpoint to
investigate how wild animal conservation should be achieved.
For the animals with larger scale of migration, we investigate male
and female migration between rookeries in Steller's sea lion by analyzing
mitochondrial and nuclear DNA polymorphism. Same approach and additional
MHC analysis are applied for sea otter with sorter scale of migration,
which has experienced bottleneck by excessive hunting and therefore
is supposed to be genetically endangered.
Terrestrial animals are contrastively susceptible to geographical barriers
and consequently have shorter range of dispersion. We apply above described
approach to a filed mouse, Apodemus speciosus, as a representative of
these animals. This species is ideal to evaluate the possible influence
of human activities on the migration and dispersion, because it has
a broad habitat including woodland and urbanized area.
By the same method, we investigate also invertebrates that are different
from mammals in some life historical aspects. Cabbage butterfly, Pieris
rapae crucivora, represents these animals, and we investigate their
distributional pattern and migration. It could provide knowledge of
global range of dispersion, since it inhabits over the Eurasian continent.
They are also expected to provide the knowledge of evolution and dispersion
of the female wings with ultra violet color (Fig.1), which is established
to play an essential role in mate recognition by the conspecific males
(Fig.2). Different from cabbage butterfly, Japanese small carpenter
ant, Camponotus yamaokai, is polygynous and reproduce colonies by budding,
so their dispersal ability is extremely restricted. We analyze genetic
differentiation and dispersal pattern of this species, compared with
those of closely related monogynous ant, C. nawai.
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| Development
of the monitoring method of hormones in eyes to evaluate reproductive
status in wild animals. |
| Kazuyoshi TAYA |
Monitoring humoral biological information, such as hormones
in circulation, is quite valuable to understand reproductive status of
wild animals together with behavioral observation. To obtain blood samples
from wild animals, stressful restraint and anesthesia is necessary. To
avoid stress for animals, measuring hormones in feces to estimate circulating
hormones is available, though this has problems due to food change and
bacterial metabolism in feces. Although information of wild animals is
limited, some carcasses, obtained by hunting, population control, accident
and diseases, available to get valuable information about reproductive
status of wild animals by anatomical, histological or pathological method.
To assess reproductive status from carcass, eyeball may be possible candidate
as a capsule containing humoral biological information without bacterial
contamination.
Purpose of this research is establishing a method to obtain hormonal reproductive
information by evaluating hormonal levels in fluid of eyeball of wild
animals. Firstly, we will investigate correlations of hormonal levels
in eyeballs and peripheral circulation using experimental animals. Secondly,
we will evaluate samples obtained from wild animals. In the first part
of the research, we will use hamsters and quails as animal models to study
correlation of hormonal levels between in eyeballs and in peripheral circulation.
To analyze correlations in hormonal levels, progesterone, testosterone
and estradiol-17β -will be measured in eyeballs and sera at various
reproductive status of both sexes. To evaluate temporal correlation of
hormonal change, we will check the effect of removal gonads and replacement
of hormones, also. In the second part of the research, we will evaluate
reproductive hormones and assess correlation in eyeballs and sera in wild
animals, such as shika deer and great cormorants obtained to control population.
We will analyze relationship between reproductive status and hormonal
levels in eyeballs and sera. Cut off values of hormonal levels in eyeballs
could be calculated to estimate reproductive status of wild animals. As
a goal of our research, we would like to develop a method to understand
reproductive status of wild animals by assessing hormones in eyeballs. |
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| Studies
on the reproductive characteristics of marine mammals |
| Kazuyoshi TAYA |
The protection of the endangered marine mammals and the
improvement their reproduction are one of the most important international
problem. However, information of reproductive physiology of marine mammals
is limited. We previously monitored changes in urinary progesterone and
estradiol-17β of a captive female dugong, Serena, has been
kept in captivity at Toba aquarium since 1987. Results obtained are as
followed. Levels of urinary progesterone and estradiol-17β
were measured twice a week in a female dugong, Dugong dugon, in captivity
for two years from Aprol 1996 to April 1998. The dugong showed 14 ovarian
cycles during the period of study. Concentrations of progesterone ranged
from 0.01ng/mg creatinine (Cr) to 1.94ng/mg Cr and the length of estrous
cycle was 53.6 ± 8.6 (mean ± SEM) days based on intervals of urinary
progesterone peak-to-peak measurements. Concentrations of urinary estradiol-17β
ranged from 0.9pg/mgCr to 23.7pg/mgCr, and tended to peak just prior to
elevations of progesterone during the first year of study. This is the
first report demonstrates that the ovulatory cycle of the dugong is about
50 days. The present findings suggest that measurement of urinary progesterone
is a useful method to detect ovarian cycle of the dugong in captivity.
In the present study, we monitor steroid hormones in blood, urine or feces
of endangered marine mammals, such as killer whales, bottlenose dolphins,
dugongs, manatees and Baikal seals to clarify their reproductive physiology.
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| Studies
on reproductive characteristics in elephants. |
| Kazuyoshi Taya |
Not so many cases were reported for breeding of elephants
at zoos and safari parks not only in Japan but also in the world, because
many facilities have only females. The major reason why only the female
elephants are kept at zoos is so called Musth, which is the aggressive
period, typical for mature males lasting from several weeks to months
per year. This is very dangerous for keepers. Therefore artificial insemination
would be suitable for elephant breeding, however success rate is still
low due to less information available for reproductive physiology in male
and female elephants. We have successfully evaluated reproductive hormones
during the estrous cycle in female elephants in collaboration with zoos
in Japan. Especially we have published the data of estradiol-17β
in peripheral blood during estrous cycle in the female African and Asian
elephant for the first time in the world. In the first step of COE project,
we have set up assay methods for measuring inhibin in elephants. During
the estrous cycle in African and Asian female elephants, circulating inhibin
increased in the luteal phase as compared with the follicular phase, suggesting
that corpus luteum is a source of circulating inhibin. In a male African
elephant, circulating inhibin increased paralleling with testosterone
during the period of musth. These results suggest that inhibin is a marker
of ovarian and testicular function as well as steroid hormones in elephants.
The goal of our research is development of artificial breeding techniques
by exploring the characteristics of reproduction in elephants, and successful
breeding of such an internationally endangered animal in Japan. In order
to do so, we further investigate the function of the hypothalamus-pituitary-gonadal
axis of elephants. |
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| Studies
on the reproductive characteristics of primates |
| Kazuyoshi TAYA |
| The primate listed as endangered species is increasing and
the international project of artificial breeding is required to keep population
of these species. Although some mechanisms of reproductive physiology
in primates are similar to human, data concerning with reproductive characteristics
of each primate are limited. In the first step of COE research, mechanisms
responsible for reproductive physiology of primates were investigated
using limited samples of Japanese monkeys and chimpanzees. These results
clearly demonstrated that a critical difference among Japanese monkeys,
chimpanzees and human was observed in reproductive endocrinology during
pregnancy, though that of menstrual cycle was quite similar in three species.
In the present study, reproductive physiology of endangered primates such
as orangutan, bonobos and chimpanzees, will be investigated using limited
samples. In addition, mechanisms responsible for seasonal breeding of
Japanese monkey will be investigated. |
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| Development
of the novel noninvasive contraceptive method in transfusion animals |
| Kazuyoshi TAYA |
In recent years, the damage to crops due to the increase
in the number of wild animals such as monkeys, deer and wild boar and
transfusion animals such as goats has become a major social problem in
Japan. Deforestation has progressed and the coexistence of human society
and wild animals is becoming ever more complicated. Conversely, there
is yet no scientific means to regulate their numbers and parties concerned
are strongly requesting the introduction of new contraceptive methods.
In Thailand, the number of inhabitant monkeys has shown a sharp increase
over the last 10 years and the country is striving to devise countermeasures,
being a Buddhist country, clearance of such monkeys through slaughter
is not an option and the parties involved are demanding alternative measures
of our co-researcher; Assistant Professor Malaivijitnond. Furthermore,
the many street-dogs inhabiting the urban areas are also a matter of social
concern, one which is also crying out for a solution. This project aims
to develop a completely novel contraceptive method through missile-therapy
and to put this to use in controlling the excessive increase of wild animals
in Japan and Thailand, and consequently contribute to the conservation
of nature and its apposite coexistence with human society.
In this project, a plant derived cell-destructing substance (saporin)
will be bonded with gonadotropin releasing hormone (GnRH) or Inhibin to
create 2 novel contraceptives (GnRH-T and Inhibin-T). Both GnRH-T and
Inhibin-T when administered will bond to receptors unique to gonadotrophs
in the pituitary, killing the cell and thus interrupting the secretion
of LH and FSH. This in turn will cause atrophy of the ovaries in females,
thus terminating ovulation. In males, atrophy of the testes occurs, resulting
in suspended sperm production. Both GnRH-T and Inhibin-T can be classified
and missile-therapy in that they will only bond to gonadotrophs in possession
of their corresponding receptors, and therefore will exert no influence
on other cells,. Furthermore, a lasting effect can be expected from a
single shot. |
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| A
new method to increase the population of endangered wild animals |
| Kazuyoshi TAYA |
| Recently, number of endangered wild animals
is increasing in the world. To prevent extinction the artificial breeding
is now applying for increasing their population around the world. In the
artificial breeding, the most important factor is to get many oocyte from
females and sperm from males. In the first step of the COE project, we
developed a new method for induction of superovulation in female mammals
"Inhibin Vaccine Method", by immunoneutralization of endogenous
inhibin. We also demonstrated that this new method is effective in most
of mammalians. In the present study, further experiments will be done
for application of this method in male animals for increasing number of
sperm. In addition, it will be applied in avian species. |
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| Analysis
of the central nervous system (CNS) changes and endocrine kinetics of
hibernators and the establishment of assessment biomarkers |
| Tadashi TAKEWAKI |
It is well known that global warming and exposure to endocrine
disrupting chemicals influence reproduction and hibernation of wildlife.
For example, raccoons become inactive and hibernate during winter season
in Hokkaido are but this is not the case in Honsyu area. However, factors
responsible for such behavioral differences are not well recognized. Therefore,
our target is to reveal the changes in the CNS and the endocrine kinetics
of hibernators and to establish the bio-markers that are essential for
objective environmental assessment. The following experimental plan is
proposed:
In the first experiment, hibernation will be induced artificially in hamsters.
To clarify changes in the CNS that are related to hibernation, the amounts
of neurotransmitters and biologically active substances in the brain will
be measured using the microdialysis method. Additionally the functional
changes in the heart, which is influenced by the CNS, will be analyzed.
To clarify the changes in the endocrine kinetics, the amounts of the hormones
such as inhibin in the blood and tissues will be measured.
In the second experiment, hamsters will be divided into two main groups:
a low temperature group (suitable environment for hibernation) and a room
temperature group. The influence of such variance in environmental temperatures
on behavior as well as on changes in the CNS and endocrine kinetics will
be assessed. Additionally, the effects of endocrine disrupting chemicals,
such as bisphenol A (which will be delivered to the animals through drinking
water), on hibernation will be tested.
Based on these two experiments it will be decided whether environmental
alterations and changes in neurotransmitters and hormones could be bio-markers
for hibernation. |
