Submitted to: Cryobiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/11/2007
Publication Date: 4/1/2008
Citation: Blanco, J.M., Long, J.A., Gee, G., Donoghue, A.M., Wildt, D.E. 2008. Osmotic tolerance of avian spermatozoa: influence of time, temperature, cryoprotectant and membrane ion pump function on sperm viability. Cryobiology. 56:8-14.
Interpretive Summary: In an effort to understand species-specific tolerance to cryopreservation, a comparative approach was used to evaluate novel cryopreservation protocols. Specifically, the benefits of incorporating osmoprotectants and supplemental ATP were investigated. The results were interesting in that both turkey and crane sperm responded differently to the various treatments. For example, turkey sperm motility was greatly improved when both sucrose and trehalose were included in the cryodiluent; whereas crane sperm motility was only improved by the addition of ATP. Also interesting was that treatments which improved sperm motility did not always improve sperm viability post-thaw. These data support the idea that cryopreservation protocols should be tailored for each species and present possible avenues for improving turkey and crane sperm cryopreservation protocols.
Technical Abstract: A comparative approach was used to evaluate the viability of avian spermatozoa frozen in a dimethylacetamide (DMA) cryodiluent supplemented with osmoprotectants or ATP. Turkey and crane semen was cryopreserved with a final concentration of 6, 10, 18, 24 or 26% of the permeating cryoprotectant DMA either alone or in combination with ATP (1.5, 3 or 6%) or one of the following osmoprotectants: 1) sucrose (turkey, 8%; crane, 5%); 2) 5% sucrose and 5% trehalose; or 3) betaine hydrochloride (1.25, 2.5 or 5%). After thawing, sperm viability was assessed using the eosin-nigrosin stain and sperm motility was determined using the hanging drop technique. For semen frozen only with DMA, post-thaw sperm motility was highest (p<0.05) for 6% and 10%, regardless of species. Turkey sperm frozen with the combination of sucrose and trehalose had higher (p<0.05) post-thaw motility for all DMA treatments compared to DMA alone; however, for all DMA treatments, only the osmoprotectant betaine (1.25%) improved turkey sperm viability post-thaw compared to DMA alone or other treatments (p<0.05). Conversely, the post-thaw motility of crane sperm was only improved with the combination of 24% DMA and 1.5% ATP; in the presence of osmoprotectants, crane sperm motility decreased as the osmoprotectant concentration increased. The lower concentration of ATP also improved crane sperm viability post-thaw, especially for DMA concentrations 18% or greater. The combination of sucrose and trehalose improved crane sperm viability only with 6% and 10% DMA. These data further confirm species-specific differences in sperm survival after cryopreservation and suggest potential strategies to improve cryopreservation protocols.