Genetic preservation through cryopreservation

Dr. Carl Lessard 1,2, Erl Svendsen 1, Crissandra Auckland 1, Pamela Hind 1 - 1 Agriculture and Agri‐Food Canada, Saskatoon, SK - 2 University of Saskatchewan, Saskatoon, SK Canada

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With agriculture intensification, genetic diversity of livestock and poultry breeds has significantly declined over the last few decades. To fight against its erosion of animal genetic resources, Agriculture and AgriFood Canada launched the Canadian Animal Genetic Resources (CAGR). The mission of CAGR is to preserve the genetic diversity of livestock and poultry breeds by preserving germplasm and gonadal tissue.
For poultry breeds, CAGR is developing a method to preserve gonadal tissues (by vitrification) from 1‐day old donor chicks and then restore them by transferring the gonads into a recipient of same age but with different genetic background.

Fertilized eggs from the targeted breed to be preserved were incubated in our facility at Saskatoon. Testes or ovary were harvested from newly hatched chicks and submitted to a standard vitrification procedure (15 min in VS1 (7.5% Ethylene glycol and DMSO); 3 min in VS2 (15% Ethylene glycol and DMSO; plunged in liquid nitrogen).
On the day of the transplant (restoration) surgery, vitrified gonads were warmed in a series of solutions of decreasing sucrose concentrations (from 100% to 0%). Recipients (different genetic background from donor breed) were orally administrated with MetaCam to minimize pain after surgery. They were anesthetized with isoflurane gas and the incision area was shaved and cleaned. A small incision (around 2 cm) into the left abdomen was made.

The yolk sac was carefully removed to provide space to reach the recipient gonad. Fine forceps of 45° angle tips were used to carefully remove the recipient gonad and to introduce the vitrified‐warmed gonad. An orthotopic transfer (put at the same location) was performed for the graft and it was not attached into the recipient. After closing the incision, saline and antibiotic were administrated to the recipients before removing the isoflurane gas. On average, it took 5 minutes for the recipient to wake up after surgery. Post‐surgery and until reproductive maturity, recipient birds initially received an immunosuppressant (mycophenolate mofetil, 100 mg/Kg) administrated daily for the first three weeks and then subsequently twice a week. Female recipients laying eggs were inseminated with semen from a rooster with a genetic background identical to the graft and male recipients were sacrificed to recover sperm cells from the graft.

In 2015, 22 recipients received grafts (15 ovary and 7 testes); only one male successfully grew its graft to maturity. Matured sperm cells were recovered from this graft. For these experiments, CAGR has used three different recipient lines to test their ability to support the growth of grafts. Only one of them demonstrated ovarian growth; however, folliculogenesis was not observed on the recovered graft. Growing testis graft does not show the same difficulties as ovarian grafts; 50% of male recipients will support the growth of testis grafts. Moving forward, other recipient lines are currently being tested to find the best recipient to support ovarian growth.

From the Genetic Preservation Summit, Alberta, Canada