028 Successful cryopreservation of avian germplasm: Why a multifaceted approach is required

The value of the ability to cryopreserve and store germplasm has long been recognized for indefinite preservation of genetic material, especially for “at-risk” populations. For domestic livestock species, options for preserving genetic material in frozen form range from sperm to oocytes to embryos. More than sixty years ago, the discovery of glycerol’s cryoprotective properties for rooster sperm pioneered the success of modern cryobiology and led to the development of semen cryopreservation for a wide range of species. Despite decades of research, the overall fertility rates of frozen/thawed poultry sperm are highly variable and not reliable enough for preservation of genetic stocks. The relatively low fertilizing ability most likely results from physiological sensitivity to the cryogenic process coupled with the requirement for prolonged sperm functionality in the hen reproductive tract. We have conducted systematic experiments to understand how and why poultry sperm lose functional competence after cryopreservation, and have shown that the glycocalyx, the carbohydrate-rich zone on the sperm surface, is altered by the cryogenic cycle. The amount of sialic acid, a terminal sugar critical for sperm transport in the female reproductive tract, was reduced after cryopreservation; however, we have shown that sperm cells can incorporate exogenous sialic acid, suggesting a strategy for sperm freezing protocols. Second, sperm lipids such as phosphotidylcholine also are altered during cold storage. The ratio of lipids in the sperm membrane determine the overall fluidity of the membrane and impact the ability of sperm to remain viable during the wide temperature fluctuations that are part of the cryogenic cycle. We have shown that supplemental lipids can circumvent the loss of phospholipids during cryopreservation. Finally, we have conducted the first proteomic analysis of poultry sperm and have identified three proteins not previously found in sperm from any species to date (dihydropyrimidinase, mitofilin and mitochondrial tri-functional protein). We currently are evaluating the proteomic profile of frozen/thawed sperm to determine the impact of cryopreservation on sperm proteins. Taken together, these data suggests innovative strategies for developing reliable poultry semen cryopreservation methods. For most species, sperm cryopreservation effectively captures the entire genome; however, in birds, the female is the heterogametic sex. Cryopreservation of avian oocytes and embryos is problematic because of the macrolecithal characteristics of the egg, where the surface-to-volume ratio poses a significant barrier to the movement of solutes/cryoprotectants and impedes effective protection of the cells from freeze/thaw damage. Recent advances for manipulation of poultry primordial germ cells and culture of embryonic stem cells suggests that cryopreservation of these cell types, followed by re-implantation in recipient embryos, could provide a mechanism to preserve the entire genome; albeit the efficiency rates of reconstitution associated with the current technologies are rather low. A novel innovation of cryopreservation and transplantation of ovarian tissue offers an alternative for whole genome preservation in birds (Song and Silversides, 2008); although chemical treatment to reduce the growth of host germ cells does not completely prevent the production of host-derived offspring. Reconstitution of poultry lines will most likely rely on more than one type of cryopreserved material. Source of funding: USDA ARS Project Number: 1245-31000-105-00. Conflict of interest: None declared. Julie.Long@ars.usda.gov