Species Variation in Osmotic, Cryoprotectant, and Cooling Rate Tolerance in Poultry, Eagle, and Peregrine Falcon Spermatozoa1

Abstract

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 degrees 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 degrees C) in diluent containing DMA (> or =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 degrees 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.

Kinetic Vitrification of Spermatozoa of Vertebrates: What Can We Learn from Nature?

“…For the all 3 raptor species, gyrfalcon, the golden and the imperial eagles, slow freezing (using the slow freezing protocol in [Blanco et al, 2000]) worked very poorly in our hands despite the fact that artificial insemination with fresh sperm is a routine and successful procedure in that Center. And finally, kinetic vitrification using the Isachenko's "droplet" method failed completely.…”

Flow cytometry for the assessment of animal sperm integrity and functionality: state of the art

“…Propidium iodide is normally used as the viability probe of choice in FC as this supravital stain rapidly penetrates non-viable spermatozoa when their plasma membrane is disrupted. 30 This FC procedures (available as a live/dead kit from Invitrogen (Carlsbad, CA, USA)) have been successfully applied for many species, as human, 31 bovine, 32 porcine, 13,31,32 ovine, 31 Lagomorpha, 31 murine, 31,33 avian, 34,35 honey bees 36,37 and fish. 38 Furthermore, it is able to simultaneously evaluate sperm cell viability together with some other attributes, e.g., in combination with fluorescently labeled plant lectins for simultaneous assessment of plasma membrane and acrosome integrity.…”

Specific features of in vivo and in vitro sperm storage in birds

“…Indeed, avian sperm share properties that are common to sperm of most animal species with internal fertilisation (Massip et al, 2004). Their low cytoplasmic content and relatively large surface of membranes exposed to adverse micro-environmental conditions (Lake, 1984;Etches, 1996) explain why the overall biophysical characteristics of membranes, including resistance to osmotic shock and membrane fluidity, appear critical for the success of cryopreservation (Blanco et al, 2000;Blesbois et al, 2005, 2006a andb). As a consequence, membrane damage induced by cryopreservation also results in impaired motility and decreased concentrations of various metabolic factors, including ATP concentration (Long, 2006).…”