Chapter 4.8.

Collection and processing of
in vitro fertilised bovine embryos/
in vitro maturing oocytes

Article 4.8.1.

1. The team should be supervised by a team veterinarian.

2. The team veterinarian is responsible for all team operations which include hygienic collection of ovaries and oocytes and all other procedures involved in the production of embryos intended for international movement.

3. The team veterinarian should be specifically approved for this purpose by an official Veterinarian.

4. Team personnel should be adequately trained in the techniques and principles of disease control. High standards of hygiene should be practised to preclude the introduction of infection.

5. The production team must have adequate facilities and equipment for:

   1. collecting oocytes;

   2. processing of oocytes and embryos;

   3. storing embryos.

6. The collection team must keep a record of its activities, which must be maintained for inspection by the approving authority for a period of at least 2 years after the embryos have been exported.

7. The production team should be subjected to regular inspection by an Official Veterinarian to ensure compliance with sanitary collection and processing of oocytes and production and storage of embryos.

8. The production team must not operate in an infected zone for foot and mouth disease and rinderpest.

Article 4.8.2.

1. The laboratory should be under the direct supervision of the team veterinarian and regularly inspected by an Official Veterinarian.

2. While embryos for export are being produced prior to their storage in ampules, vials or straws, no oocyte/embryo of a lesser health status should be recovered or processed in the laboratory.

3. The laboratory should be protected against rodents and insects.

4. The processing laboratory should be constructed with materials which permit its effective cleansing and disinfection. This should be done following each occasion on which embryos are processed.

5. The laboratory must not be situated in an infected zone for foot and mouth disease or rinderpest.

Article 4.8.3.

Individual collection usually involves the aspiration of oocytes from the ovaries of live animals on the farm where the donor animal resides or at the laboratory. Occasionally oocytes may also be recovered from individual live donors by aspiration from surgically excised ovaries. When oocytes are recovered from individual live animals, the procedures for these donors should follow the recommendations set out in Article 4.7.4.

Cleaning and sterilisation of equipment is especially important and must be carried out between each donor in accordance with the requirements of the Procedures Manual of the International Embryo Transfer Society (IETS)².

Batch collection usually involves the removal of ovaries from slaughtered animals at an abattoir but may alternatively involve the surgical removal of ovaries from live donors; these ovaries are then transported to the laboratory where oocytes are removed by aspiration. Batch collection involving abattoir derived ovaries has the disadvantage that it is usually impractical to relate ovaries which are transported to the laboratory to the donors which were slaughtered at the abattoir. Nevertheless, it is critical to ensure that only healthy tissues are obtained and that they are removed from the donors in a hygienic manner.

Additionally:

1. The Veterinary Authority should have knowledge of, and authority over, the herd(s) of origin of the donor animals.

2. The donor females should not originate from an infected zone for foot and mouth disease or rinderpest and the removal of any tissue should not take place in an infected zone for foot and mouth disease or rinderpest.

3. The abattoir should be officially approved and under the supervision of a veterinarian whose responsibility it is to ensure that ante-mortem and post-mortem inspections of potential donor animals are carried out and to certify them to be free of signs of contagious diseases of concern transmissible to cattle.

4. The donor females should not have been designated for compulsory slaughter for a notifiable disease and other animals of a lesser health status must not be slaughtered at the same time as donors from which ovaries and other tissues will be removed.

5. Records of the identities and origins of all donors must be kept.

6. Batches of ovaries should not be transported to the processing laboratory before confirmation has been obtained that ante and post-mortem inspection of donors has been satisfactorily completed.

7. Equipment for removal and transport of ovaries and other tissues should be cleaned and sterilised before use and exclusively used for these purposes.

Article 4.8.4.

Tests may be carried out on the following materials to confirm the absence of pathogenic organisms which are of concern to the importing country:

1. non-viable oocytes/embryos: all non-viable oocytes/embryos at any stage of the production line from batches intended for export should be pooled for testing;

2. in vitro maturation medium prior to mixing the oocytes with semen for the fertilisation process;

3. embryo culture medium taken immediately prior to embryo storage.

These samples should be stored at 4°C and tested within 24 hours. If this is not possible, then the samples should be stored frozen at -70°C or lower.

In the case of oocyte recovery from individual animals or batch collection from live donors, monitoring of clinical health status and post collection testing of donors for diseases of concern may be considered.

Additionally:

1. Semen used to fertilise oocytes in vitro should meet the health requirements and standards set out in Chapter  4.5.

   When the donor of the semen used to fertilise the oocytes is no longer living, and when the health status of the semen donor concerning a particular infectious disease or diseases of concern was not known at the time of semen collection, additional tests on the spare IVF embryos may be required to verify that these infectious diseases were not transmitted. An alternative may be to subject an aliquot of semen from the same collection date to testing.

2. Any biological product of animal origin, including co-culture cells and media constituents, used in oocyte recovery, maturation, fertilisation, culture, washing and storage should be free of living pathogenic micro-organisms. Media should be sterilised by approved methods according to the IETS Manual² and handled in such a manner as to ensure that sterility is maintained. Antibiotics should be added to all fluids and media as recommended in the IETS Manual².

3. All equipment used to recover, handle, culture, wash, freeze and store oocytes/embryos should be cleaned and sterilised prior to use as recommended in the IETS Manual².

Article 4.8.5.

1. After the culture period is finished but prior to freezing, storage and transport, the embryos should be subjected to washing and other treatments similar to those specified for in vivo derived embryos in accordance with the IETS Manual².

2. Only embryos from the same donor, in the case of individual animal recovery, or from the same batch collection, should be washed together.

3. The zona pellucida of each embryo must be examined over its entire surface area at not less than 50X magnification and certified to be intact.

4. The IVF embryos should be stored in sealed sterile ampules, vials or straws and then frozen in fresh liquid nitrogen or other cryoprotectant in cleaned and sterilised containers under strict hygienic conditions at a storage place, approved by the Veterinary Authority of the exporting country, where no risk of contamination of the embryos can occur.

5. Only embryos from the same individual donor or batch collection should be stored together in the same ampule, vial or straw.

6. Ampules, vials or straws must be sealed at the time of freezing and should be labelled according to the IETS Manual².

7. Liquid nitrogen containers should be sealed prior to shipment from the exporting country.

8. Embryos must not be exported until the appropriate veterinary certification documents are completed.

Article 4.8.6.

When micromanipulation of the embryos is to be carried out, this should be done after completion of the treatments described in Article 4.8.5. and conducted in accordance with Chapter 4.7.

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1. Where transportation of in vitro maturing (IVM) oocytes is intended, the conditions outlined in this Chapter are also applicable.

2. Manual of the International Embryo Transfer Society (1998).

   An up-to-date list of relevant scientific publications is available on the IETS Website Homepage at http://www.iets.uiue.edu where the link entitled 'Embryo-Pathogen Research and Reference Lists' may be visited.