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Management of the breeding and rearing facility for microbiologically and genetically regulated cynomolgus macaques. Establishment of breeding technology for efficient breeding and research of assisted reproductive technology. Research of animal models for human diseases.
Establishment of breeding technology for efficient breeding and research of assisted reproductive technology (ART)
Improving the breeding efficiency is crucial for the colony maintenance and the scale of research use. Reproductive physiologic research has been continuously executed from the time this primate research center was established to the present, and a vast amount of basic data has been accumulated to date. The extent of this data is reflected, for example, in the selection of suitable monkeys for a breeding colony, selection of breeding methods, monitoring of embryo growth, and prevention of accidents such as a stillborn. Acquired physiologic research data can reveal indicators that can be used to decide the direction of future breeding. Moreover, applying artificial reproductive technology not only improves breeding efficiency, but also enables preservation and supply of monkeys as a research resource. The basis of development biotechnology is to create fertilized eggs via in vitro fertilization and micro-insemination, for example, and to obtain offspring via embryo transfer. Research into frozen preservation technology of sperm and eggs is accomplished concurrently with research into development biotechnology. Current hot research topics are regenerative medicine, development of disease models, and evaluation of medicines. Other current topics include cloned monkeys and gene-manipulated monkeys.

Breeding and Rearing
Mating
During ovulation, a female is paired with a male for several days. Females with irregular menstrual cycles cohabit with a male for two days or longer. In all pairing, only one female cohabits with a single male, thus ensuring no ambiguity in the parents of the offspring. The photograph shows a cynomolgus monkey couple.

Pregnancy diagnosis at early stage
Two methods have been established to determine pregnancy in monkeys. The first is ultrasonography, which can be used to determine pregnancy five weeks after mating. The second is measurement ofβ-CG concentration discharged from the placenta, and is used to determine pregnancy three weeks after mating. Measurement of the E2 concentration can be used to accurately predict the day of ovulation and to clearly determine the stage of the embryo during pregnancy. The photograph shows pregnancy diagnosis by ultrasonography and shows an embryo on day 36 of pregnancy.


Suckling and Rearing
When unexpected accidents occur at birth or when a mother rejects her newborn, artificial nursing is executed. The increase in weight of newborn raised under artificial nursing is similar to those under nursing by the mother. Artificial nursing is conducted in a clean environment, and the maintenance management is done as SPF monkey. All offspring are weaned about six months after birth. Multiple immature monkeys of similar weight have been bred in a cage. The left photograph shows a mother and her offspring, the center photograph shows a monkey under artificial nursing, and the right shows two immature monkeys.

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Development of ART

ART in monkeys is needed to maintain a colony that is genetically and microbiologically controlled. ART is also used in the preservation, supply, and improvement of monkeys used as research resources. In cynomolgus monkeys, however, ovulation typically produces only a single oocyte at a time, thus limiting in vitro research. Administration of hormones enables collection of multiple oocytes at a time, and thus enables embryology such as in vitro fertilization and microinsemination. Offspring can be obtained by transferring the embryos produced by such technique into foster mothers. ART also includes the cryopreservation of embryos and gametes and in vitro maturation culture of immature gametes. Cryopreservation not only enables sequentially embryological research, become also provides “backup” monkeys when serious infection symptoms are present in a colony. In addition, the technique that can promote maturation of immature gametes from deceased monkeys is crucial for obtaining new individuals with the same genetic lineage. Furthermore, research is now directed towards production of gene-manipulated monkeys and cloned monkeys. Because cloned monkeys derived from somatic cells have the same genome between individuals, the use of such monkeys is expected in a wide range of research. Because monkeys are also the same primates as humans, preclinical research into regenerative medicine using ES cells from monkeys is critical to the advancement of medical science. Establishing and developing ART in monkeys are vital for accurately determining the effect and safety of medicines in humans.


1) Collection of mature oocytes from ovaries 2) Intracytoplasmic sperm injection (ICSI)
Mature oocytes with follicular fluid can be recovered from ovaries by inserting a needle into grown follicles. Administration of hormones enables collection of multiple mature oocytes. ICSI is a technique in which spermatozoon is injected into the cytoplasm of mature oocytes by using a glass pipette (0.7-0.8 mm in inner diameter). This method is commonly used to artificially produce fertilized eggs.
3) Colony of ES (Embryonic Stem) cells 4) Blastocyst stage embryo from ICSI
An ES cell colony is a cell line established from inner cell mass cells of a blastocyst. This cell line maintains a normal karyotype for the long-term culture and can differentiate into all types of cells. The blastocyst stage embryo is a preimplantation stage embryo developed via in vitro culture after ICSI. This embryo consists of “inner cell mass cells” and trophoectoderm cells. The dense part of an inner cell is called the inner cell mass and is generated to the individual.
5) Offspring derived from in vitro fertilization
Offspring can be born as result of in vitro fertilization and embryo transfer. The preimplantation stage embryo is transferred to the oviduct of a foster mother. For confirmation of successful implantation, pregnancy of the foster mother is monitored and finally the offspring is delivered by Caesarean section.

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Reproductive endocrinology

Comparison of physiological ages between humans and cynomolgus monkeys
The age of Japanese females versus that of female cynomolgus monkeys are shown here. Also shown are the ages at which several physiological indexes occur. The origin of the coordinate axes is the birthday, and is connected to the age at menopause by a dotted line. Most of the data is below the dotted line, indicating the relatively rapid growth rate of female cynomolgus monkeys compared with that of female humans.


Distribution of ovulation days
The days of ovulation in young female monkeys and older female monkeys were shown here. Ovulation occurs after serum concentration of estradiol-17b (E2) secreted from the follicle decreases suddenly. The average ovulation day for the old group (12.8 days after onset of menstruation) was similar to that for the young group (13.3 days). The old group, however, showed wider variation in the day of ovulation.

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