Terrestrial Animal Health Code
Terrestrial Animal Health Code |
Collection and processing of laboratory rodent and rabbit oocytes or embryos
Microbial status of laboratory animal colonies
Colonies of the various species and genotypes of laboratory animals are usually kept within specialised premises and their microbial status depends largely on the system whereby the colony was formed and is maintained. In this chapter the microbial status of colonies is considered to be of three main types: ‘defined’, ‘conventional’ and ‘undefined’. Colonies of defined status are those where, at least initially, the animals are totally free of pathogenic and non-pathogenic agents (i.e. gnotobiotic), although sometimes a cocktail of known, non-pathogenic agents has been given subsequently. In either case defined colonies are kept in highly controlled environments in barrier maintained rooms, with strict protocols in place to exclude all potential sources of unwanted microbiological contamination. Colonies of conventional status are those where the animals are kept in closed colonies but where known (‘specific’) pathogenic agents as well as non-pathogenic agents may exist. While management protocols for conventional colonies may be less rigid than those for defined colonies, they are designed to control potential sources of microbial contamination. Simple aseptic precautions (e.g. the autoclaving of food and bedding) are taken to ensure that the animals do not become infected with any unwanted microflora. Finally, laboratory animals may be kept in microbiologically undefined colonies which are unrestricted and may include free ranging animals. Details of these different types of colony can be found in the FELASA Report1..
The health status of defined and conventional colonies should be confirmed at least quarterly by bacteriological, virological, parasitological, serological and other tests on pre-designated sentinel animals or other representative members of the colony. Older breeding males which have sired multiple litters are often selected for this purpose.
The purpose of official sanitary control of laboratory rodent and rabbit embryos intended for movement internationally is to ensure that specific pathogenic agents, which could be associated with such embryos, are controlled and transmission of infection to recipient animals, progeny and colonies, is avoided. Requirements for the management of donors and processing of embryos vary depending on the microbial status of the colony, i.e. whether it is defined (including gnotobiotic), conventional, or undefined.
Conditions applicable to the embryo collection team
The embryo collection team is a group of competent technicians including at least one experienced professional to perform the collection, processing and storage of oocytes or embryos.
The following conditions should apply:
The team should be supervised by a team professional.
The team professional is responsible for all team operations which include verification of colony and donor health status, sanitary handling and surgery of donors, disinfection and hygienic procedures. The team professional should be responsible to the institute veterinarian.
The institute veterinarian should be certified or accredited in laboratory animal care and should be specifically approved for the purpose of embryo collection for export. It is the responsibility of the institute veterinarian to ensure that required health profiling procedures appropriate for the colony status are implemented. He or she is responsible for certifying that the embryo handling procedures and laboratory facilities conform to the requirements laid down in this chapter.
Team personnel should be adequately trained in the techniques and principles of disease control and in the use of aseptic techniques in embryo handling. The zoonotic potential of specific pathogenic agents affecting the various laboratory animal species should be identified and understood so as to avoid contamination of colonies via human vectors, and vice versa.
High standards of hygiene should be practiced to preclude the introduction of infection to the donor animals, colonies, facilities, and equipment. Restrictions should be established to prevent free access of personnel into the embryo collection and handling facilities especially after such personnel have been exposed to other animal facilities.
The team should have adequate facilities and equipment for:
collecting embryos;
processing and treatment of embryos at a permanent or mobile laboratory;
storing embryos.
It is the responsibility of the institute veterinarian to ensure that complete animal and embryo records, including records of collection, processing and storage of embryos are maintained. Record sheets of the type shown in the IETS Manual2. for livestock species should be used where applicable, and data such as genotypic identification of the donors, embryo quality grading, morphological stage and should be given. The embryo collection team should keep a record of its activities which should be maintained for inspection by the Veterinary Authority for at least two years after the embryos have been exported.
The embryo collection team, if involved in the export of embryos, should be approved by the Competent Authority and be subject to regular inspection by an Official Veterinarian to ensure compliance with procedures for the sanitary collection, processing and storage of embryos.
Conditions applicable to the processing laboratory
A processing laboratory used by the embryo collection team is a facility in which embryos are recovered from donors (or from their excised reproductive tracts), and from the collection media. Here also the embryos are examined and subjected to any required treatments such as washing, cryopreservation for storage and quarantine pending results of any diagnostic procedures. The processing laboratory may be part of a specifically designed collection and processing unit, or a suitably adapted part of an existing building. It may be on the premises where the donor animals are kept but in this case should be physically separated from animals.
Additionally:
The processing laboratory should be under the supervision of the institute veterinarian and be inspected by an Official Veterinarian.
While embryos for export are being handled prior to their storage in ampoules, vials or straws, no embryos of lesser health status should be processed.
The processing laboratory should be constructed with materials which permit its effective cleansing and disinfection. This should be done frequently, and always before and after each occasion on which embryos for export are processed.
Risk management
With regard to disease transmission, transfer of in vivo derived embryos is a very low risk method for moving the genetic material of laboratory animals. Irrespective of animal species, there are three phases in the embryo transfer process that determine the final level of risk:
The first phase comprises the risk potential for embryo contamination and depends on:
the disease situation in the exporting country or zone;
the microbial status of the colony (i.e. defined, conventional or undefined) and the donors from which the embryos are collected;
the characteristics of the specified pathogenic agents that are of concern to the Veterinary Authority of the importing country.
The second phase covers risk mitigation by use of internationally accepted procedures for processing of embryos which are set out in the IETS Manual2.. These include the following:
Depending on microbial status of the colony, the embryos should be washed up to ten times with at least 100-fold dilutions between each wash, with a fresh pipette being used for transferring the embryos through each wash.
Only embryos from the same donor should be washed together, and no more than ten embryos should be washed at any one time.
Sometimes, for example when removal of certain viruses (e.g. herpesviruses) is required, the standard washing procedure should be modified to include additional washes with the enzyme trypsin, as described in the IETS Manual2..
The zona pellucida of each embryo, after washing, should be examined over its entire surface area at not less than 50X magnification to ensure that it is intact and (apart from the mucin layer in the case of rabbit embryos) free of adherent material.
The third phase, which is applicable to diseases of concern to the Veterinary Authority of the importing country, encompasses risk mitigation resulting from:
post-collection surveillance of the microbial status of the donor colony based on the recognised incubation periods of the diseases of concern to determine retrospectively the health status of the colony whilst the embryos are stored (in species where effective storage by cryopreservation is possible) in the exporting country;
post-mortem testing of the donors or other samples such as blood, embryo-collection (flushing) fluids and non-viable embryos, in a laboratory for presence of specified disease agents.
Conditions applicable to the embryo team or institute veterinarian
It is the responsibility of the institute veterinarian to ensure that required health testing procedures are implemented to demonstrate microbial status of the colony (i.e. defined, conventional or undefined). Colony microbial status should be reviewed by the institute veterinarian before shipment of the embryos.
The veterinarian is responsible for certifying that the embryo handling procedures and laboratory conditions were maintained in accordance with Articles 4.10.2. and 4.10.3.
The veterinarian is responsible for the risk management procedures outlined in Article 4.10.4.
The veterinarian should authorise all embryo shipments, ensuring that the correct embryo collection records and veterinary certification documents have been completed and are included in the shipments.
Conditions applicable to donors from animal colonies of different microbial status
It should be noted that the conditions applicable
to donor animals vary in accordance with the microbial status of
the colony from which they originate, i.e. defined, conventional
or undefined.
Sentinel animals in each donor colony of defined and conventional status should be subjected to routine microbial screening, preferably monthly, but at least quarterly. Testing for specific pathogenic agents depends on the animal species and may be influenced by geographical location. Recommendations regarding specific pathogenic agents to be tested for in different laboratory animal species have been published elsewhere1..
Defined microbial status
Microbiologically defined colonies (Article 4.10.1.) represent the cleanest sources of gametes, and the embryos recovered from these animals can be regarded as pathogen free.
Since the male and female donors are pathogen free, dissection of the female reproductive tract and embryo collection procedures should be performed under aseptic laboratory conditions, using a biological safety cabinet if appropriate.
Embryo washed as described in point 2) of Article 4.10.4. is not necessary but it is recommended that embryos are washed two or three times. In each wash, embryos should be gently agitated in the medium.
The embryos should be recorded as coming from a germfree or microbiologically defined, barrier maintained colony, thus indicating that special risk management procedures (Article 4.10.4.) for the removal of pathogenic agents are not necessary. The need to quarantine the embryo recipients is a matter for the importing institute.
Conventional conditions
Colonies of conventional microbial status are usually closed and their health status is routinely monitored (Article 4.10.1.). The animals may have been exposed to various pathogenic agents, resulting in infection, with positive antibody titres or even active clinical disease, but the pathogenic agents of concern in each individual colony should be well known.
Reproductive tracts (uteri, oviducts or ovaries) should be removed at a separate site and then taken into the embryo processing laboratory. These procedures should be performed by different technicians or, at the minimum, their protective clothing should be changed between locations. If animals should be handled in the laboratory, the tracts should be dissected out within a biological safety cabinet. This will help protect against the possible shedding of pathogenic agents into the laboratory itself.
Once the reproductive tracts have been removed, embryo recovery should be performed under aseptic conditions. Depending on which, if any, pathogenic agents are known to occur in the colony, embryos should be processed in accordance with the risk management procedures, including washing, as described in Article 4.10.4., and in the IETS Manual2..
Embryos derived from animals that have positive antibody titres or other evidence of specific pathogenic agents should only be transferred into a new colony via a quarantine system, using microbiologically defined recipient females. Quarantine may also be appropriate if there is any uncertainty about the microbial status of the donor colony or the donors. In situations where the embryos could have been exposed to bacterial infection, they should be cultured in a medium containing appropriate antibiotic for 24 h before cryopreservation, or in the interval between thawing and transfer into recipients.
If the recipient institution does decide to quarantine the recipient dam and offspring until their health status is confirmed, the recipients should be tested post-weaning for pathogenic agents of concern, and introduction of offspring into the colony should only take place if the test results are satisfactory.
Undefined microbial conditions
Embryos from free ranging animals or from colonies of unknown health status require the full range of risk management procedures that are described in Article 4.10.4. and in the IETS Manual2.. The procedures resemble those used for embryos of livestock as recommended in Chapters 4.7. and 4.8. of this Terrestrial Code. Ideally, the breeder males and donor females should be separated from other animals and tested 15 days before and on the day of breeding (for males) or at embryo collection (for females). Alternatively, the animals could be incorporated into a conventional colony, where, over time, a health history can be documented to reduce the strict monitoring and embryo handling requirements.
Biological safety cabinet should be used for handling donors and reproductive tissues, and for processing embryos.
Post-mortem testing of the donor females for diseases or pathogenic agents of concern to the importing country may be appropriate after the oocytes or embryos have been collected. Alternatively if embryos are collected surgically an aliquot of flush fluid from each donor, or a pooled sample, should be tested for the presence of specific pathogenic agents of concern.
Embryos should be washed at least ten times in accordance with the protocols in the IETS Manual2. and trypsin treatment should be used if presence of certain pathogenic herpesviruses is of concern.
Cryopreserved embryos should be stored in the exporting laboratory until such time as the necessary disease screening of colonies, tissues or fluids is completed and the supporting documents for certification completed and signed by the institute veterinarian.
On arrival in the importing country the embryos should be transferred into recipients in a quarantine system. Recipients should be tested at intervals appropriate to recognised incubation periods of the diseases of concern. In addition to testing recipients after transfer, the offspring should be tested at 12 weeks of age and before their introduction into breeding colonies outside the quarantine facility
Conditions applicable to the storage and transport of embryos
Embryos for export should
be frozen in fresh liquid nitrogen and then stored in fresh liquid
nitrogen in cleaned and disinfected tanks or containers.
The embryos should be stored in sealed sterile ampoules, vials or straws under strict hygienic conditions at a storage place approved by the Veterinary Authority of the exporting country. Only embryos from the same donor should be stored together in the same ampoule, vial or straw.
Ampoules, vials or straws should be sealed at the time of freezing (or prior to export where cryopreservation is not possible) and they should be clearly identified in accordance with or similar to the system recommended in the IETS Manual2.. Identification should include details of the species or genotype of the donors, microbial status (e.g. defined, conventional or undefined), collection or cryopreservation date, number and developmental stage of the embryos, container number and details of any specialised procedure such as in vitro fertilisation, micromanipulation.
Liquid nitrogen storage containers should be sealed under the supervision of the Official Veterinarian prior to shipment from the exporting country.
Embryos should not be exported until the appropriate veterinary certificates are completed.
Procedures for invitro fertilisation and micromanipulation
If embryos are to be produced by invitro fertilisation of oocytes, it is advised that the washed sperm should be used so as to minimise the risk of possible exposure to pathogenic agents. If embryos are to undergo micromanipulation procedures that involve penetration of the zona pellucida, any required risk management steps (including washing) should be carried out first, as described in Chapter 4.9.
nb: first adopted in 1998; most recent update adopted in 2010.
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