TPC NEWS Vol.13,
No.2, Winter, 1994 (Whole Number 23)
(
English Summary)
Page-3
Message from the chief director of the Corporation for Production and
Research of Laboratory Primates (CPLP)
Dr. Matsuura, chief director of the Corporation for Production and
Research of Laboratory Primates (CPLP), is
describing the present state and
future role of the CPLP, reviewing
the performance of the CPLP.
Page-4
Project of a research resource bank for the primate starts
The project for the research and development of techniques for germ cells
of the primate has started. The
following research items are going to be investigated:
1)
Superovulation, 2) ovum collection, 3) in vitro culture of oocyte for maturation
(IVM), 4) cryopreservation of inmature
eggs, 5) sperm collection, 6) sperm pre-incubation, 7) artificial insemination,
8) cryopreservation of spermatozoa, 10) in
vitro fertilization (IVF), 11) micro injection of spermatozoa, 12) in vitro
culture of embryo development, 13) cryopreservation of embryos, 14) embryo
transfer, 15) establishment of transgenic
monkeys, 16) in vitro culture of male
germ cells, 17) establishment of ES cells and EG cells, 18) bacteriological
monitoring, and 19) establishment
of SPF primates.
Page-5
Breeding of squirrel monkeys (Saimiri
sciureus) under indoor-caged conditions
from 1981 to 1994
F. Cho, M. Hamano & H. Ohto
The squirrel monkey (Saimiri
sciureus) is one of the small-sized non-human
primates living in the central and south America.
The monkey has long been used for various fields of
research. We can find
literature reporting that breeding colonies of
squirrel monkeys were already established in the 1940s.
The monkey began to be used for cardiovascular and metabolic studies in
the 1960s. Nowadays,
supported by extended fields of use, increase in number of
users and highly advanced research precision, supply of the squirrel
monkey of good quality is strongly required.
Supply of laboratory animals which satisfy biomedical use can be realized
by the implementation of artificial breeding/rearing, and it makes contribution
to conservation of wild animals.
For the purpose of reproduction
under indoor-caged conditions, Tsukuba Primate Center for Medical Science (TPC)
imported squirrel monkeys from the U.S.A in February 1980, April 1981, and
November 1982. These animals had been imported
from
their countries of origin to the States, and there they were being
kept temporarily.
On completion of quarantine, males and females were 9 and 49 in number,
respectively, but among which some animals were immature as a breeder.
After being quarantined, the animals were individually observed their
health conditions for about three months, and then they were housed in groups
consisting of one male and one to six females in an animal room of
14-hour light cycle, temperature 25 + 2o
C, and relative humidity 50 +10%.
They were given fruit (apples, oranges and bananas) and boiled quail eggs
in the morning, monkey pellets (AS, Oriental Yeast Co., Ltd.) in the afternoon.
A cage for individual housing measured
W: 43 cm x D: 60 cm x H: 60 cm. A
polyvinyl chloride pipe (25 mm in diameter) was built across the cage as
a perch. Two individual cages
were piled up so as to make a unit cage (W: 43 cm x D: 60 cm x H: 120 cm) for
group housing. Side walls of unit
cages were connected to form the
cage for group housing. The cages
being used this time measured W: 86 cm x D: 60 cm x H: 120 cm,
W: 129 cm x D:60 cm x H: 120
cm and W: 172 cm x D: 60 cm x H: 120 cm, accordingly.
Cages were hooked on the walls of the animal room.
Feces/urine and leftovers were allowed to drop down from the cage to the
floor which was slanted so as to wash them away easily.
Results of breeding of the squirrel monkeys for 14 years from the 1981
breeding season to the 1994 breeding season are reported below.
Table 1 shows pregnancies and outcomes.
The number of pregnancies was 185 for wild-born females.
The outcomes of the pregnancies were 36 (19.5%) abortions/stillbirths and
149 (80.5%) normal births. Pregnancies
of laboratory-bred females numbered in 106 for ten years from 1985 to 994, and
the outcomes of the pregnancies were 17 (16.0%) abortions/stillbirths and
89(84.0%) normal births. Fig. 1
shows yearly outcomes of pregnancies (Nos. of abortions, stillbirths and normal
births ). The numbers of
pregnancies of the wild-born females were rather constant between 23 and 30 from
1984 to 1988, whereas that of the laboratory-bred females increased steadily
between 1985 and 1994.
Distribution of the births by month is shown in Fig. 2.
Normal births of the wild-born females most often occurred in May,
thereafter the frequency kept on decreasing until October.
The laboratory-bred females had normal births from April to September,
the most in June. Table 2 shows the
length from arrival of the wild-born females to their parturition, frequency of
their parturition (parity) and interbirth intervals. The earliest birth occurred 11 months after arrival.
The length from arrival to
the first birth averaged 30 months for 32 females.
One female delivered nine times. Interbirth
intervals were calculated excluding abortions.
Copulation was often found immediately after abortion; two wild-born
females conceived twice a year.
Both females had normal births by the
Nursing conditions are shown in the far right columns of Table 1.
Normal nursing behavior was found in 145 (97.3%) of the 149 wild-born
mothers. Wild-born females who rejected their newborn babies numbered in four;
their newborn babies died during artificial nursing.
On the other hand, nursing behavior was found in 29 (32.6%) of the 89
laboratory-bred mothers, who had normal births, and the remaining 60 (67.4%)
mothers rejected nursing. Newborn
babies rejected by their mothers were reared artificially;
but those rejected in 1994
were not subjected to artificial nursing.
Table 4 shows the numbers of the
cases of artificial nursing, causes
thereof and
outcomes
therefrom.
Among those numbers, the following cases were included: a baby whose
mother died immediately after parturition, a baby with thoracic subsidence,
therefore, being separated from its mother.
Two other newborns, whose mothers were too exhausted by hard labor to
nurse them, underwent artificial nursing , however, the mothers recovered four
days later and began to nurse their babies normally. Cases of nursing rejection by the laboratory-bred females
whose offspring are F2 and/or F3 numbered 52 in total between 1986 and 1993.
Twenty-nine (55.8%) of the 52 rejected newborn babies were reared
artificially and weaned. Some of the rejected F2 newborns were low in body weight, and
one of them was physiologically
handicapped.
Summing up the above results of breeding of the squirrel monkeys in TPC,
we can conclude as follows: Parturition of the wild-born females was found
constantly between 1984 and 1988 as shown in Fig. 1. If mating had not been stopped intentionally, we could have
known until what age the squirrel monkeys can continue breeding, and how many
times they can give birth in their
life.
Next, we cannot help but recognize that there are many problems with
nursing conditions of the laboratory-bred females in comparison with those of
the wild-born females. Reviewing
individually the results of nursing
conditions of 38 laboratory-bred females in total, neither successful nor
unsuccessful nursing is biased toward certain individuals.
The key to the success in self-sustaining
reproduction of squirrel monkeys must be to eliminate
nursing rejection right at birth, and it can be achieved by adequate management of
individual females during the final period of pregnancy.
Another helpful measure is provide
fully equipped environments for parturition, in which, for example, body
temperature dropping of newborns whose ability in maintaining their body
temperature is extremely low, can
be prevented.
( English translation by F. Cho)
Page-8
Laboratory Tests: Detection of B
virus antibody by enzyme-linked immunosorbent assay (EIA)
Toyoko Narita
We developed an enzyme-linked immunosorbent assay (EIA) for the detection
of antibodies to B virus in monkey sera by using inactivated purified B virus
antigens licensed by Microbiological Associate Inc., U.S.A.
We adopted indirect method, in which purified antigens were diluted 3.000
times and fixed to a plate. HRP-labeled anti-macaque IgG antibody diluted 10.000 times was used as
the secondary antibody(Fig .1) . Test
sera were diluted 100 times. The
sera, which showed more than 0.3 OD difference in
antibody levels between B
virus antigen and normal cell antigen, were considered to be positive (Fig. 2).
Other results obtained are shown in
Figs. 3 - 6.
We could detect by this EIA
method some positive animals among those which we had considered to be negative.
This method was more sensitive to detect the antibodies to B virus than
the compliment fixation test (CF) or EIA method using HSV-1 as antigen, which we
used to do.
Page-11
P3-Laboratory
Masao Takasaka
At the TPC, we have built a facility for Biosafety Level 3
experiment (P3- Laboratory). The
Lab, which mainly consisted of two animal rooms and two laboratories for
cultural experiment, was remodeled from a corner of our quarantine building.
The floor plan of the Lab is shown in Fig.1.
The Lab has one access door and only the staff registered can open it
with a magnetic card. Air
pressure is graded to create directional airflow: pressure deference between the
outside and the corridor, and the access rooms, and
P3 laboratory rooms are -2, -4 and -6 mm H2O, respectively. On
emergency, a safety system for the
negative pressure works.
The exhaust air from the laboratory rooms is
discharged to the outside through a
high efficiency particulate air (HEPA) filter, and the air from the
negative-pressure isolator for monkeys and from the Class III
3 safety cabinets is filtered twice by
HEPA filters. Fresh
air is supplied into the Lab through a HEPA filter, too. The operation of these system is watched closely
from the control center.
Fifty-three negative-pressure isolators for monkeys are provided in the
Lab. Animal room waste and
carcasses are sterilized in a double-doored autoclave and then incinerated.
Laboratory clothing and experimental instruments used in the rooms are
sterilized by a small-sized autoclave before removing from the facility.
Waste water used for hand
washing is disinfected with chemicals in the liquid waste decontamination tank
in each access room before being released to the TPC's sewage treatment
facility.
Page-12
Clinography: Hypertension
Fumiko Ono
For humans, analyzing the mechanism of hypertension is one of the most
important tasks to prevent senile diseases.
Hypertension may be is induced by multiple risk factors such as
gene and environment. Spontaneous hypertension rats (SHR) and various
kinds of spontaneous rats are used as the animal model for the study of
hypertension. Transgenic mice with elevated blood pressure by introduction of
rat renin and angiotenshinogen genes are tested as the animal model.
However, development of nonhuman primate hypertension model is more
needed for gene analysis and understanding of the pathogenesis of human
hypertension.
Now, we showed the preliminary data of blood pressure of cynomolgus
monkeys. At TPC, we are taking care of the cynomolgus monkeys over 20 years old
as the aged monkeys. Sixty eight
monkeys, including 34 aged monkeys, were measured their blood pressure.
The blood pressure was measured by oscillometric method at the right
brachium of the animals under ketamin hydrochloride anesthesia. It is well
established that the ketamin hydrochloride has little influence for the blood
pressure, since ketamin hydrochloride anesthesia does not change the resistance
of peripheral vessel .
In this study, there was no
significant change of the blood pressure between the awake and anesthetized
condition. Figure showed the blood pressure of the monkeys with different ages.
The average of systolic blood pressure was 97.6(+12.8) mmHg and
diastolic blood pressure was 50.1 (+7.4) mmHg before 10 years old (n=12),
106.2(+11.6) mmHg and 58.4 (+7.0) mmHg from 10 to 19 years old
(n=34), 103.9 (+15.4) mmHg and 59.1 (+10.0) mmHg over 20 years old
(n=34). There was no significant
difference in the blood pressure with age. Only one monkey, that is the wild
female monkey about 29 years old, was hypertension complicated with obesity(B.W.
7kg), hyperglycemia (294 mg/dl) and hypercholesterolemia (214 mg/dl).
In man, these hyperlipemia and diabetes are considered as risk factors
for hypertension or arteriosclerosis. However,
in our colony, few lesion of blood vessel was observed with the monkeys died of
diabetes or
hyperlipemia .
Extensive study, loading other risk factors such as hypersodium, will be
required to establish the
hypertension animal model of the
primate.
Page-13
Comment on my research: AIDS virus and macrophage
Kazuyasu
Mori
An experimental inoculation of molecularly cloned SIVmac239 caused AIDS
in rhesus monkeys. By using this animal model for AIDS we have been studying the
importance of cells of monocyte/macrophage lineage in AIDS pathogenesis.
SIVmac239 replicates very poorly in alveolar macrophage culture.
Forty percent of animals died of SIVmac239 infection showed lesions
associated with tissue macrophage infection such as SIV-related encephalitis and
granulomatous interstitial pneumonia. Macrophage-tropic variant viruses were
isolated from these animals just before they died. However, the rest of 60%
animals died of SIVmac239 infection had neither obvious SIV infected tissue
macrophage nor emergence of macrophage-tropic variant virus.
Three common
amino acid changes in SU (surface protein in env)
were found in the three cases that we
examined and they were found to be primarily responsible for the high
replication capability of variant viruses.
While it has been demonstrated that genetic determinants of
macrophage-tropism in HIV-1 were mapped in V3 sequence, no mutation was found in
the corresponding region of SIV. The
ensuing study on the restricted replication of SIVmac239 in macrophages
demonstrated that SIVmac239 enters into macrophage efficiently and replicates
normally. However, spreading
infections to neighboring cells seems to be blocked.
Further investigation on the noble mechanism of the replication of
SIVmac239 in macrophages might elucidate the relationship between its strong
pathogenic potential in animals and the cell-tropism of SIV for AIDS.
Page-17
Announcement: Seminar '95
by the CPLP
The first seminar by the
Corporation for Production and Research of Laboratory Primates (CPLP) was held
on 23 and 24 February, 1995, in Tsukuba.
The theme was " the present state and understanding on the
laboratory primate." This note
announces the Seminar.
Page-17
Introduction of New Technology: Instrument for the embryo technology
Takahiro Ono
We are trying
to establish the embryo technology such as artificial insemination, embryo
transplantation and collecting method of vaginal mucus, by using cynomolgus and
African green monkeys. The
instruments, therefore, specially designed for the monkeys are indispensable.
We have developed two kinds of vaginal speculums, cylinder type of
plastic and beak-shaped of stainless, and stainless
vaginal dilators with five different diameters from 3 to 12 mm and round apexes.
These instruments are used as follows:
The vaginal dilator is inserted into the vagina to loosen (Fig. 1).
Next, the vaginal speculum is inserted into the vagina (Fig. 2, 3).
When the ostium uteri is observed, the ostium uteri dilator is inserted
into the uterus so that a probe for sperm infusion or curettage can be
introduced in the uterus(Fig. 4). However,
in practice, it is difficult to insert the probe, because the structure of the
canal of cervix of monkeys is very complicated (Fig. 5).
The technical renovation and the development of more suitable instruments
will be required.
Page-18
From the Front of Animal Feeding: The
cynomolgus monkeys with
growth disorder
Koji Hanari
We here report on the cynomolgus monkeys with growth disorder which we
have hardly ever seen in natural environment.
Fig.1 shows a nine-year-old
female cynomolgus monkey with growth disorder (Left)
and
a normally grown animal of the same age.
The spine of some disordered animals abnormally curved, resulting in
hunchback. The females have no menstruation even after they became
three and a half years old, the age of sexual maturation of the normally
growns. The volume of the
testis of the disordered (10 cm in length and 15 cm in width) was
one eighth of the normals'
(20 cm x 30 cm) .
Fig. 2 shows the body weight change.
There were no deference in body weight change between the animals with
growth disorder and the normally grown until
several months after weaning.
But they did not show any
growth after they became one and half years old.
At TPC, we have had about
3.200 offsprings including still birth, of which nine males and eleven females
were growth disordered. Some
of the disordered animals were found successively in the descendants of some
males. Heredity
may be taking part of the onset of this disorder.
But, these animals can not produce next generation.
We have about 200 to 300
newborns every year, of which about
one percent animals suffers from growth disorder.
It is possible that we constantly maintain 10 to 20 growth disordered animals.
Page-19
Report on a Congress: Report
on the 15th Congress of the International
Primatological
Society (IPS) in Bali
Dr. Koji Fujimoto went to Bali, Indonesia to attend the 15th Congress of
the International Primatological Society
(IPS). He describes his impression
on the Congress , listing up some sessions he was interested in, and summed up
that the IPS Congress which covers various themes of
wide range is very informative for him, a researcher of the laboratory
primates.
Page-20
Report on a Conference: Report
on the Tenth International Conference
on AIDS
Dr.
Akari attended at "The Tenth International Conference on AIDS", held
in Yokohama, Japan,
from 7 to 12 in August, 1994. In
this report, he reviews the presentation, and discusses about the progress of
the clinical and basic research on AIDS.
Page-23
A Comment from a Trainee:
A trainee from a company studied how to detect viruses in the tissue
culture at the TPC. He said
in this note that his experience was very fruitful for him,
expressing his thanks to the TPC's people.