Collection and processing of micromanipulated oocytes or embryos from livestock and horses

Article 4.9.1.

Introduction

Neither Chapter 4.7. which recommends official sanitary control measures for the international movement of in vivo derived embryos nor Chapter 4.8. which recommends measures for in vitro produced embryos or in vitro maturing oocytes covers embryos which have been subjected to biopsy, splitting, transgene injection, intracytoplasmic sperm injection (ICSI), nuclear transfer or other interventions which breach the integrity of the zona pellucida. Such oocytes or embryos are those referred to here as having been ‘micromanipulated’.

It should be noted that complete removal of granulosa cells or other adherent material from the outer surface of the zona pellucida of oocytes, zygotes and embryos is necessary prior to micromanipulation to avoid lowering their health status.

Removal of such material from the zona pellucida of immature oocytes can be difficult. However, to bring micromanipulated oocytes or embryos within the scope of the above mentioned chapters, the following conditions should apply.

Article 4.9.2.

1. Prior to any micromanipulation which involves breaching the zona pellucida, all oocytes or embryos should be collected and processed in accordance with the sanitary conditions laid down in Chapter 4.7. (in vivo derived embryos), or produced in accordance with the sanitary conditions laid down in Chapter 4.8. (in vitro produced oocytes or embryos).
   

2. Responsibility for the oocytes or embryos remains with the embryo collection team (in vivo derived embryos) or with the embryo production team (in vitro produced embryos), and all processing involving micromanipulation should be carried out in an approved processing laboratory under supervision of an approved team veterinarian (see Articles 4.7.2. and 4.7.3., and Articles 4.8.2. and 4.8.3., as appropriate).
   

3. Donor animals should comply with the conditions laid down in Article 4.7.4. (in vivo derived embryos) or Article 4.8.4. (in vitro produced embryos), whichever is appropriate. Risk management and criteria for testing samples to ensure that embryos are free from pathogenic agents are laid down in Articles 4.7.5. and Article 4.7.7. and in Articles 4.8.5. and 4.8.6. respectively, and these should be followed.

4. All embryos to be micromanipulated should be washed in accordance with the protocols laid down in the IETS Manual^1. and they should be observed to have an intact zona pellucida before and after washing. Only embryos from the same donor, or, in the case of some in vitro produced embryos, embryos originating from the same batch of ovaries from a slaughterhouse/abattoir (see Chapter 4.8.), should be washed together at the same time. After washing, but before micromanipulation, the zona pellucida of each embryo should be examined over its entire surface area at not less than 50X magnification and certified to be intact and free of adherent material.

5. If surrogate zonae are used, they should be from the same species and the oocytes or embryos from which they are obtained should be treated in the same manner as if they were in vivo derived or in vitro produced embryos intended for international movement.

Article 4.9.3.

Procedures for micromanipulation

The term ‘micromanipulation’ covers several different procedures and a variety of specialised microsurgical instruments and other equipment may be used. However, from the standpoint of animal health, any cutting, penetrating or breaching of the integrity of the zona pellucida is an action that can alter the health status of an embryo. To maintain health status during and after micromanipulation, the following conditions should apply:

1. Media

   Any product of animal origin, including co-culture cells and media constituents, used in the collection or production of oocytes, embryos or other cells, and in their micromanipulation, culture, washing and storage should be free from pathogenic agents (including transmissible spongiform encephalopathy agents, sometimes called prions). All media and solutions should be sterilised by approved methods in accordance with the IETS Manual^1. 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 Manual^1..

2. Equipment

   Equipment (e.g. microsurgical instruments which have direct contact with embryos) should either be of the single-use type (disposed of after each oocytes or embryos batch) or should be effectively sterilised between oocytes or embryos batch in accordance with recommendations in the IETS Manual^1..

3. Nuclei for transplantation (‘nuclear transfer’)

   1. Where it is intended to transplant nuclei derived from pre-hatching stage (i.e. zona pellucida intact) embryos, the parent embryos from which those nuclei are derived should fulfil the conditions of this chapter. Where nuclei derived from other types of donor cell (e.g. post-hatching stage embryos, embryonic, foetal and adult cells, including spermatozoa or spermatids for ICSI) are to be transplanted, the parent embryo, foetus or animal from which those donor cells originate, and the methods whereby they are derived, including cell culture, should comply with the relevant animal health standards recommended elsewhere in this Terrestrial Code and in the Terrestrial Manual.

   2. Where it is intended to transplant a nucleus into an intact oocyte (e.g. for ICSI), or into an enucleated oocyte (for nuclear transfer), those oocytes should be collected, cultured and manipulated in accordance with the recommendations in this chapter.

Article 4.9.4.

Optional tests and treatments

The importing country may request that tests be carried out on certain samples or that embryos be treated to ensure that specified pathogenic agents are absent.

1. Samples

   Samples to be tested may include those referred to in Article 4.7.7. and in Article 4.8.5. Where cells other that from zona pellucida-intact embryos (e.g. somatic or sperm cells) are used as donors of nuclei for transplantation, then samples or cultures of those donor cells may also be tested.

2. Treatments

   Treatments of embryos with the enzyme trypsin or other substances proven to inactivate or remove pathogenic agents may be requested when pathogenic agents that are not removed by washing may be present. If used, such treatments should also be applied prior to any micromanipulation, and in accordance with the IETS Manual^1..

Article 4.9.5.

Conditions applicable to storage, quarantine and transport

Micromanipulated embryos should be stored, quarantined and transported in accordance with the conditions laid down in Article 4.7.8. or in Article 4.8.7. as appropriate. Veterinary certification documents should identify all micromanipulations, where and when they were carried out.

nb: first adopted in 1992; most recent update adopted in 2009.

1. Manual of the International Embryo Transfer Society.