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a USDA, ARS, LPSI, Germplasm and Gamete Physiology Laboratory, Beltsville, Maryland 20705
b Patuxent Wildlife Research Center, Laurel, Maryland 20708
c Conservation & Research Center, National Zoological Park, Smithsonian Institution, Front Royal, Virginia 22630
d Center for Studies on Iberian Raptors, C.E.R.I, Toledo, Spain
Potential factors influencing spermatozoa survival to cryopreservation and thawing were analyzed across a range of the following avian species: domestic chicken (Gallus domesticus), domestic turkey (Meleagris gallopavo), golden eagle (Aquila chrysaetos), Bonelli's eagle (Hieraaetus fasciatus), imperial eagle (Aquila adalberti), and peregrine falcon (Falco peregrinus). Studies focused on spermatozoa tolerance to the following: 1) osmotic stress, 2) different extracellular concentrations of the cryoprotectant dimethylacetamide (DMA), 3) equilibration times of 1 versus 4 h, 4) equilibration temperature of 4 versus 21°C, and 5) rapid versus slow cooling before cryopreservation and standard thawing. Sperm viability was assessed with the live/dead stain (SYBR-14/propidium iodine). Sperm viability at osmolalities 
800 mOsm was higher (P < 0.05) in raptor than poultry semen. Return to isotonicity after exposure to hypertonicity (3000 mOsm) decreased (P < 0.05) number of viable spermatozoa in chicken, turkey, and golden and Bonelli's eagle spermatozoa but not in imperial eagle or peregrine falcon spermatozoa. Differences were found in spermatozoa resistance to hypotonic conditions, with eagle species demonstrating the most tolerance. Semen, equilibrated for 1 h (4°C) in diluent containing DMA (2.06 M), experienced decreased (P < 0.05) spermatozoa survival in all species, except the golden eagle and peregrine falcon. Number of surviving spermatozoa diminished progressively with increasing DMA concentrations in all species. Increased equilibration temperature (from 4 to 21°C) markedly reduced (P < 0.05) spermatozoa survival in all species except the Bonelli's eagle and turkey. Rapid cooling was detrimental (P < 0.05) to spermatozoa from all species except the imperial eagle and the chicken. These results demonstrate that avian spermatozoa differ remarkably in response to osmotic changes, DMA concentrations, equilibration time, temperature, and survival after fast or slow freezing. These differences emphasize the need for species-specific studies in the development and enhancement of assisted breeding for poultry and endangered species.
1 This study was supported by the USDA Foreign Agricultural Services Project SP 33, the Patuxent Wildlife Research Center, USGS, the Junta de Comunidades de Castilla-La Mancha, and the Women's Committee of the Smithsonian Institution/British Airways Partnership.
2 Correspondence: Ann Donoghue, PPPSR, ARS, USDA, O-304 Poultry Science Center, University of Arkansas, Fayetteville, AR 72701.
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