A solution is therefore needed, preferably one that allows for the preservation of as much avian genetic diversity as possible. This will allow for genes from heritage breeds to be fully examined and characterized – genes which may hold great future promise in commercial breeding in terms of important traits like resistance to disease. American geneticist Dr. Janet Fulton has already demonstrated that there are some genes present in heritage poultry breeds that are not present in commercial breeds, and some of this heritage DNA (very much at risk of being lost at this point in time) may become crucial in future commercial poultry breeding enhancements.
But how is a central, efficient and secure way to preserve poultry genes to be developed? Cryopreservation (slow freezing) was tried because it works for mammalian sperm, eggs, embryos and more. But it turned out that cryopreservation of avian sperm significantly lowers its ability to fertilize eggs, and avian sperm doesn’t contain the entire bird genome anyway. While avian embryonic cells do, cryopreservation doesn’t work with them either.
Finding a reliable way to preserve poultry genetics is also challenging because of the trickiness involved with manipulating bird eggs and sperm, explains Dr. Carl Lessard, curator of the Canadian Animal Genetic Resources program (CAGR) at the University of Saskatoon in Saskatchewan. “What’s required is to open a small spot in an egg shell and deposit desired embryonic cells into the host embryo without killing it,” he notes. “That’s very difficult. So, while freezing embryonic blastodermal cells is a good way to preserve the entire genome of a species, it just doesn’t allow for easy usability of that genome in poultry.”
In 2006, Dr. Fred Silversides (now retired from Agriculture and Agri-Food Canada) tried some new thinking. What about preserving the gonadal tissue (testicular and ovarian tissue) where sperm and eggs are created and stored? Might it be possible to develop a relatively efficient way to remove gonads, chill and store them, and then thaw and transfer them, resulting in the hatching of a chick with the desired genetics and not any from the surrogate mother hen?
Instead of the slow freezing involved with cryopreservation, Silversides tried vitrification, where a gonad is removed from a day-old chick, treated with lots of cryoprotectant and chilled rapidly through a plunge in liquid nitrogen. The gonad is never technically frozen (there’s no ice crystal formation) but maintained in a glass-like (vitreous) state at a very low temperature. Once thawed, the gonad is surgically transferred to a day-old chick recipient that has had its gonad totally or partially removed.
At the same time, Silversides and his team developed ways to preserve the viability of the tissues during and after thawing and transplantation, such as treating the recipient chick with immunosuppressants to avoid rejection of the graft.
Success was achieved! Over time, the work of Silversides and his colleagues at AFFC was transferred to CAGR, where Lessard became curator in 2014. Since that point, Lessard and his team have been working hard to move all aspects of poultry genetics preservation forward.
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What’s happening now
The technique for chicken testicular tissue is now well-established, and Lessard and colleagues are currently optimizing Silversides’ technique for ovarian tissue. “The ovarian grafts are not growing the way we need them to, so we are now trying to find a new chicken line recipient,” Lessard explains. “The bird line we were using likely has an immune response that’s too high. We didn’t see this with the testicular tissue grafts in that line.”
With turkeys, Lessard has established a reliable protocol for freezing gonads from newly-hatched chicks, with the next step to optimize the surgical procedures and immunosuppressive treatment to obtain successful growth of the grafts. In terms of the team’s preliminary genetic analysis, they’ve found turkey breeds have a lot of genome ‘admixture’ (many shared genes alleles between breeds), but more samples are needed to confirm this finding. Shared alleles, says Lessard, make it harder to characterize the entire genetic diversity of turkeys and establish what is, and what is not, pure turkey genetics.
Once vitrification of male and female gonadal tissue for chicken and turkeys is complete, the team will launch a national call in 2017 to request genetic samples of fertilized eggs from commercial and heritage breeds. They will also move on to other poultry breeds such as ducks.
Lessard and his colleagues are also creating a germplasm repository (sperm, eggs, gonads, embryos) for other types of livestock from all across Canada. “We are looking for donations from purebred animals in all areas of the country,” he says, “including bison, cattle, sheep, goat, horse, pig, deer, elk and more. It’s going well, and we’re getting more and more participation from livestock associations and individual producers. Right now (in September and October 2016), we are in Ontario and Quebec gathering samples from sheep, goat and beef cattle.” A website letting the public know what has been contributed is being developed and Lessard is looking for more Canadian and international graduate students to tackle all the work.
“We need many samples for poultry and everything else produced in Canada,” he explains. “Genetic characterization of commercial and heritage poultry breeds is extremely important and we need to establish the true diversity of the different poultry breeds produced here. The number of heritage breed birds is shrinking every year, and it’s very important to capture genetics as soon as possible.”
Silversides’ vitrification preservation technique has so far been adopted by the United States Department of Agriculture ‘Agricultural Research Service’ Germplasm Resources Information Network (GRIN). Lessard says individuals at that organization have already used the technique to preserve the genetics of several U.S. commercial and heritage breeds. In terms of other groups beyond CAGR working on gonadal transfer, a team in Hungary is currently working to master it.
To make is easier for them and other researchers around the world learn how to successfully complete surgical transfer of vitrified gonads, Lessard has been working on a free tutorial e-book featuring detailed video and audio descriptions of each step. “This strategy (vitrification and gonadal transfer technique) has great potential to preserve the entire genome of a poultry breed and also use that genome fairly easily,” he explains. “We want it to be available to everyone.”