Effect of N-Methylacetamide Concentration and Thawing Rate on Chicken Sperm Quality after Cryopreservation

SIMPLE SUMMARY: The semen cryopreservation technology is still the only efficient method for the ex situ conservation of genetic diversity in birds. This study investigates the effect of different concentrations (6% and 9%) of the cryoprotectant N-Methylacetamide and of different thawing temperatures (at 5 °C for 100 s; 38 °C for 30 s) on chicken semen quality after cryopreservation. The cryoprotectant concentration significantly affected sperm membrane integrity, total and progressive motility after cryopreservation and this effect was dependent by the thawing temperature. The treatment that provided the best cryoprotective action and decreased the cellular cryodamage was the concomitant use of 6% N-Methylacetamide and thawing at 5 °C for 100 s. These results can contribute to improve the efficacy of the current chicken semen cryopreservation technology. ABSTRACT: In seeking alternative cryoprotectants to glycerol for a reference chicken semen freezing procedure, the aim of the present study was to compare the effect of two concentrations of N-Methylacetamide (MA) and two thawing rates on the quality of frozen-thawed semen. Semen samples were diluted in Lake pre-freezing extender, including 0.1 M trehalose in presence of 6% or 9% MA, loaded into straws, frozen in nitrogen vapors, and stored in liquid nitrogen. The following thawing treatments were used: 5 °C for 100 s and 38 °C for 30 s. Sperm quality (cell membrane integrity, motility and kinetic parameters) was assessed before and after cryopreservation. The decrease of MA concentration from 9 to 6% improved sperm quality after freezing/thawing and this effect was dependent on thawing temperature. Decreasing the MA concentration from 9 to 6% improved the proportion of undamaged membrane, motile, and progressive motile sperm recovered after thawing at 5 °C for 100 s; in contrast, no effect of the MA concentration was observed thawing at 38 °C for 30 s. Therefore, the treatment with 6% MA and thawing at 5 °C for 100 s has given the best cryoprotective action. These results contribute to improve the efficacy of the current chicken semen cryopreservation procedures.