Annex

NOTE: Veterinary certificates are negotiated directly between relevant government authorities. In cases where a veterinary certificate has not been negotiated, this annex can be used as the basis for developing a suitable document.

1. Embryo production and collection team(s) and team veterinarian(s)

Note: The embryo production team is a group of competent veterinarians and technicians and includes the team veterinarian, to perform the collection and processing of ovaries/oocytes and the production and storage of in vitro produced embryos. The embryo collection team is a group of competent veterinarians and technicians and includes the team veterinarian, to perform final processing of fresh embryos created by the embryo production team, and storage of frozen in vitro produced embryos.

1.1. The embryo production team and the embryo collection team was approved by USDA–APHIS for export of bovine in vitro produced embryos.

1.2. The team veterinarian is:

• certified by the American Embryo Transfer Association (AETA) as a competent embryo transfer practitioner, and
• deemed to be competent in the production and final processing of in vitro produced embryos.

1.3. The embryo production team was supervised by the team veterinarian. The team veterinarian was responsible for all team operations which include the hygienic collection of oocytes and all other procedures involved in the production of embryos intended for international movement.

The embryo production team personnel were adequately trained in the techniques and principles of disease control. High standards of hygiene were practiced to preclude the introduction of infection.

1.4. The embryo production team had adequate facilities and equipment for

• collecting ovaries and/or oocytes;
• processing of oocytes and production of embryos at a permanent or mobile laboratory; and
• storing oocytes and/or embryos.

These facilities need not necessarily be at the same location.

1.5. The embryo production team keeps records of its activities, which are maintained for inspection by USDA–APHIS for a period of at least two years after the embryos have been exported.

2. Oocyte collection facility

Note: The oocyte collection facility is the premises consisting of an oocyte recovery area and a permanent or mobile laboratory for the processing of oocytes and in vitro maturation before transporting to the in vitro produced embryo processing laboratory. The premises may also include the in vitro produced embryo processing laboratory.

The oocyte recovery area is the area dedicated to the ultrasonographically guided aspiration of oocytes and includes facilities for the safe handling of donor cows.

2.1. The oocyte collection facility:

• was on a property not subject to any restriction or quarantine measure with respect to contagious and infectious animal diseases;
• was under the supervision of the team veterinarian;
• was built and maintained in accordance with the recommendations in the current IETS Manual to permit the sanitary collection, handling and processing of the oocytes for maturing;
• was subjected to, and passed, inspection at least once a year by the team veterinarian; and
• was subjected to review by USDA–APHIS confirming approval at least once a year.

2.2. Only animals associated with oocyte collection and meeting health requirements as specified in this certificate were permitted to enter the oocyte recovery area during collection of oocytes for processing of in vitro produced embryos for export to Australia.

3. Oocyte donors

3.1. Only live animals permanently identified according to an identification system recognised by USDA–APHIS were used for oocyte collection.

3.2. To the knowledge of the team veterinarian, donors showed no clinical signs of contagious and infectious diseases for 30 days prior to, at the time of, and for 30 days after, each collection.

3.3. The team veterinarian or another veterinarian who is a member of the embryo production team inspected each female donor on each day that the oocytes were collected for this consignment and found the donor to be free of clinical signs from contagious and infectious diseases.

3.4. Donors resided in the United States for at least 90 days prior to oocytes collection for this consignment.

4. Oocyte collection, processing and in vitro maturation

Note: An oocyte collection is defined as oocytes collected during a single ovum pickup from a live donor.

4.1. Only oocytes from the same female donor were washed and processed together.

4.2. All equipment/materials were disposed of and replaced with new items, or sterilized or disinfected in accordance with the current IETS Manual, before use and between different donors.

4.3. No oocytes of a lesser health status were processed within the laboratory at the same time as the germplasm for this consignment.

4.4. Any biological product of animal origin, including media constituents, used in oocyte recovery, maturation, washing and storage presented no animal disease risk. Media were sterilized prior to use by approved methods in accordance with the current IETS Manual and handled in such a manner as to ensure that sterility is maintained. Antibiotics were added to all fluids and media as recommended in the current IETS Manual.

5. Transport of oocytes from oocyte collection facility

5.1. The oocytes were processed, stored and transported to the in vitro produced embryo processing laboratory in a hygienic manner in accordance with recommendations of the current IETS Manual.

5.2. Only oocytes from the same individual donor were stored together in the same ampoule, vial or straw.

5.3. Ampoules, vials or straws were capped or sealed before transport.

5.4. Where a third party was used for transport, the storage container was sealed at the oocyte collection facility by the team veterinarian or another veterinarian who is a member of the embryo production team and the seal was not broken until receipt by the team veterinarian or a member of the embryo production team at the in vitro produced embryo processing laboratory.

6. Semen donors

6.1. The semen donor must be resident in the United States for 90 days prior to the collection of semen used to fertilise the oocytes in this consignment, or

6.2. If the semen was collected in another country, the semen importer provided a copy of certification from the country of origin to USDA–APHIS as evidence that the semen met Australian import requirements

7. Semen

Only semen certifiable for export to Australia was used to fertilise the oocytes. Evidence was provided by the team veterinarian to USDA–APHIS for review at the time of endorsement of the health certificate.

8. In vitro produced embryo processing laboratory

Note: The in vitro produced embryo processing laboratory is the facility/ies at which the in vitro produced embryos were processed through, at minimum, in vitro fertilization, in vitro culture, embryo washing, freezing and dispatch. Final processing of fresh embryos may also be done in a USDA–APHIS approved mobile laboratory.

8.1. Each permanent facility:

• was on a property not subject to any restriction or quarantine measure with respect to contagious or infectious animal disease;
• was under the supervision of a team veterinarian;
• is a permanent structure that was built and maintained in accordance with the recommendations of the current IETS Manual; and
• was subjected to inspection at least once a year by USDA–APHIS.

Date of last inspection……………………………………………………..

8.2. Each mobile laboratory processing fresh in vitro produced embryos:

• is under the supervision of a team veterinarian;
• is maintained in accordance with the recommendations of the current IETS Manual;
• is associated with a permanent facility to sterilize equipment, maintain records, and for storage of embryos;
• was subjected to inspection at least once a year by USDA–APHIS; and
• has not been on any property subject to restrictions or quarantine measures with respect to contagious or infectious animal disease since the last USDA–APHIS inspection.

Date of last inspection…………………………………………………………

Note: If the mobile laboratory processing fresh in vitro produced embryos has been on any property subject to restrictions or quarantine measures with respect to contagious or infectious animal disease since the last USDA–APHIS inspection, it must be reinspected and approved by USDA–APHIS to be eligible to process embryos for export to Australia.

8.3. Where more than one facility was involved in the processing, evaluation, storage and dispatch of embryos for export, all transfers between facilities were hygienically transported under seal, with the seal/s applied and removed by a member of the relevant embryo production/collection team.

9. Production and storage of embryos

9.1. During the production of embryos for export to Australia and prior to their storage, no oocytes or embryos of a lesser health status were processed at the same time using the same equipment and materials.

9.2. All equipment/materials were disposed of and replaced with new items, or disinfected in accordance with the recommendations of the current IETS Manual between different donors.

9.3. Any biological product of animal origin, including co-culture cells and media constituents, used in fertilisation, culture, washing and storage presented no animal disease risk. Media were sterilised prior to use by approved methods in accordance with the current IETS Manual and handled in such a manner as to ensure that sterility is maintained. Antibiotics were added to all fluids and media as recommended in the current IETS Manual.

9.4. Cleaning, and sterilisation or disinfection of, equipment were carried out in accordance with the recommendations of the current IETS Manual.

10. Embryos

The embryos were handled in accordance with the current IETS Manual:

• All embryos were identified and can be traced to the male and female donors.
• Only embryos from the same female donor were washed together, and no more than ten embryos were washed at any one time.
• The zona pellucida of each embryo, before washing, was examined over its entire surface area at not less than 50X magnification to ensure that it is intact and free of adherent material.
• The embryos were washed at least ten times with at least 100–fold dilutions between each wash, and a new sterile micropipette was used for transferring the embryos through each wash.
• The standard washing procedure includes additional washes with the enzyme trypsin.
• Eligible embryos include those subject to micromanipulation only for the purpose of collecting biopsy samples for genetic analysis. No other micromanipulations such as splitting, transgene injection, intracytoplasmic sperm injection, nuclear transfer or other interventions that breach the integrity of the zona pellucida of embryos are permitted. If performed, micromanipulation was carried out only on embryos with intact zona pellucida after the standard washing procedure, and in suitable laminar-flow facilities which were properly cleaned and disinfected between batches.
  

11. Diagnostic testing

11.1. The diagnostic samples were collected by a USDA–APHIS accredited veterinarian.

11.2. Tests for disease were carried out at a laboratory approved by the competent authority to perform the required test.

11.3. The tests were conducted in accordance with the current OIE Manual.

11.4. The test reports provided to USDA to support certification must display the dates of sampling for the tests required, the type of test used and the test results. This information must be contained in a table against donor information, annexed to the health certificate, and verified and certified correct by the USDA–APHIS certifying officer.

12. Disease freedom

At the time of, and for 30 days after, each oocyte collection for this consignment, the United States was officially recognised by the OIE as a:

• foot and mouth disease free country where vaccination is not practised
• contagious bovine pleuropneumonia free country

AND meets the current OIE Code Article definitions for country freedom from:

• lumpy skin disease
• Rift valley fever
• Brucellosis due to B. melitensis

AND was recognised by the Australian Government as a country free from foot and mouth disease where vaccination is not practised.

13. Vesicular stomatitis

During the 30 days prior to, and at the time of, each collection of oocytes, there was no clinical signs or reports of vesicular stomatitis at the premises where donor cows were kept and at the oocyte collection facility.

14. Bovine brucellosis

14.1. During the 30 days prior to, and at the time of, each collection of oocytes, the donor cows were from a herd that was located in a country or zone free from infection with bovine brucellosis (B. abortus) in accordance with the current OIE Code.

(Note: this includes donor cows from a herd that was located in a State or area officially free from infection with bovine brucellosis (B. abortus) and the herd was recognized as officially free of brucellosis by USDA–APHIS in accordance with the USDA Brucellosis Eradication Program.)

14.2. Oocytes were not collected within six months of vaccination of donors against brucellosis.

15. Bovine tuberculosis

During the 30 days prior to, and at the time of, each collection of oocytes, the donor cows were from a herd that was located in a country or zone free from bovine tuberculosis in accordance with the current OIE Code.

(Note: this includes donor cows from a herd that was located in a State or zone officially free from bovine tuberculosis and the herd was recognized as officially free of tuberculosis by USDA–APHIS in accordance with the USDA Tuberculosis Eradication Program.)

16. Bluetongue

Blood samples drawn from each donor:

• were subjected to a cELISA test to detect antibodies to the BTV group between 28 and 60 days after each collection of oocytes with negative results
  

  or

• were subjected to an agent identification test on a blood sample taken on the day of collection with negative results.
  

17. Epizootic haemorrhagic disease (EHD)

Blood samples drawn from each donor:

• were subjected to the cELISA test to detect antibodies to the EHDV group between 28 and 60 days after each collection of the oocytes for this consignment with negative results
  

  or

• were subjected to an agent identification test on a blood sample taken on the day of collection with negative result.
  

18. Infectious bovine rhinotracheitis (IBR) and infectious pustular vulvovaginitis (IPV)

For all oocytes collected from the donors:

• The donor was kept in a herd where all eligible animals including the donor were vaccinated against IBR/IPV with a vaccine approved by the USDA at least 30 days prior to collection of oocytes. The vaccine was administered as per manufacturer’s instructions for vaccination and revaccination; AND
• A vaginal mucus sample# was collected from the donor immediately prior to washing/disinfecting the vagina in preparation for oocyte collection and tested for bovine herpesvirus-1 by the qRT-PCR for bovine herpesvirus-1 with negative results.

#Double guarded culture swabs, appropriately prepared for testing samples, were used to hygienically collect samples of vaginal mucus from the donors.

19. Bovine viral diarrhoea virus (BVDV)

19.1. At the time of each collection of oocytes, each female donor gave a negative result to one of the following tests for BVDV:

• an antigen-capture ELISA on peripheral blood leucocytes
  

  or

• a monoclonal immunoperoxidase or other virus isolation test on blood or serum.

  AND

  19.2. If vaccinated, the donors were kept in a herd where all eligible animals including the donors were vaccinated against both BVDV-1 and BVDV-2 with a vaccine approved by the USDA at least 30 days prior to collection of oocytes. The vaccine was administered as per manufacturer’s instructions for vaccination and revaccination.
  

20. Schmallenberg viruses

No cases of disease caused by Schmallenberg virus has been detected or reported in the United States.

21. Storage and transport

From the time of embryo freezing until export, the in vitro produced embryos in this consignment were stored for at least 30 days:

• in sealed sterile containers (e.g. straws, ampoules or vials) and identified in a legible and non-erasable manner as specified in the current IETS Manual. Goblets and canes were also identified as specified in the current IETS Manual;
• only with other bovine germplasm collected for export to Australia, or of equivalent health status;
• in storage or shipping containers containing only new, unused liquid nitrogen;
• in a secure place within an approved centre or laboratory; and
• under the supervision of the team veterinarian.
  

22. Shipping containers (liquid nitrogen shippers/tanks)

EITHER

The shipping container was new.

OR

Immediately prior to loading, the shipping container was emptied and inspected and any loose straws removed. The shipping container, including all surfaces in contact with the straws, ampoules or vials was then disinfected with one of the following disinfectants: 2% available chlorine (e.g. chlorine bleach), 2% Virkon, 2.4% Prevail or irradiated at 50 kGy.

Date of disinfection/ irradiation……………………………………………………..

Disinfectant used/ active ingredient……………………………………………………..

[The veterinary certificate must indicate the option that applies. For used shipping containers, the date of disinfection, the disinfectant used and its active chemical must be recorded on the health certificate.]

23. Further processing

23.1. For transport of embryos for further processing (e.g., DNA testing) at another facility, the in vitro produced embryos were:

• stored in sealed sterile containers (e.g. incubators, straws, ampoules or vials) and identified in a legible and non-erasable manner as specified in the current IETS Manual, and
• processed hygienically under veterinary supervision.

(Delete clause if embryos not transported for further processing)

24. Official government seals

24.1. Under the supervision of a USDA–APHIS Veterinarian prior to export to Australia:

• the containers (e.g. straws, ampoules or vials) for reproductive material in this consignment were checked as being sealed; and
• the identity of the reproductive material was checked prior to being placed into new, unused liquid nitrogen in a shipping container for export that was new or disinfected as specified in this veterinary certificate.

24.2. Only bovine reproductive material (that is, semen and/or in vivo derived embryos and/or in vitro produced embryos) that met Australian import conditions can be transported in the same shipping container.

24.3. An official government seal was applied by a USDA–APHIS Veterinarian to the shipping container and the number or mark on the seal recorded on the certificate.

Shipping container official government seal number…………………………………………..