The dilution rate of 1:4 was found to be more effective than 1:3, probably as the consequence of the difference in
sperm concentration (number of sperm cells per mL of sem- inal plasma and extender). Hence, in a previous study [29] in which we assessed the post-thaw quality of turkey semen cryopreserved by the pellet method, the prefreezing sperm concentration was noted to affect the cryosurvival of the sperm cells. This could be attributed to a different amount of DMA per sperm cell or to the lower content of seminal plasma in the total volume of sperm sample. Seminal plasma seems to negatively affect turkey spermatozoa during both liquid storage [30,31] and cryopreservation. In effect, im- proved fertility rates have been obtained by removing the seminal plasma from turkey spermatozoa before freezing, as reviewed by Blesbois [3]. In our study, a DMA concentration of 8% worked better than one of 6% as a cryoprotectant during freezing. It is well known that when cells are frozen at high cooling rates, for example by direct plunging into liquid nitrogen (from 2,000 to 30,000 °C/min), a higher concentra- tion of permeable cryoprotectant in the medium is needed to increase osmolarity. This osmolarity helps to dehydrate the cells thus avoiding damage due to ice crystal formation dur- ing cryostorage, minimizing the cryodamage observed after thawing [32]. Although turkey spermatozoa are highly sen- sitive to osmotic shock [10], 8% DMA in the medium seems better at preserving the sperm cells than a lower concentra- tion of this cryoprotectant.
Another step that emerged here as critical in the cryopreservation process was the thawing temperature. Hence, a higher warming temperature (75 °C) over a shorter period (10 –12 s) was better than slightly longer exposure to a lower temperature (60 °C for 18–20 s). Studies on human sperm have shown that very rapid cooling and warming rates improve the cryosurvival of spermatozoa [33].
The proportion of viable spermatozoa recovered af- ter freezing/thawing using the best procedure was 42%, sperm mobility was 60% and sperm membrane func- tional integrity was 40%.