A Method for Cryopreserving Semen from Yakido Roosters Using N-Methylacetamide as a Cryoprotective Agent

A method of freezing semen using N-methylacetamide (MA) as a cryoprotective agent was applied to Yakido, a chicken breed designated as a “Natural Monument” under the “Law for the Protection of Cultural Property” (Law No. 214, May 30, 1950) in Japan. Semen was collected from Yakido roosters, and pooled semen was diluted 1: 1 using semen diluent containing no cryoprotective agent. After equilibration at 5°C for 30min, diluted semen was further diluted 1: 1 using semen diluent containing MA at a final concentration of 9%. Diluted semen was packaged into 0.5-mL plastic straws and frozen by placing the straws in liquid nitrogen vapor followed by plunging into liquid nitrogen at -196°C. After storage in liquid nitrogen for about one month, frozen semen was thawed in ice water. Intravaginal artificial insemination (AI) was performed immediately after thawing semen without removing the cryoprotective agent. In Fertility Trial 1, AI was performed once a week for four consecutive weeks. Weekly fertility rates using frozen semen were 83.8±3.8%, 77.1±8.8%, 88.6±5.1%, and 77.3±5.7% for weeks 1, 2, 3, and 4, respectively. Hatchability was over 90% throughout the experimental period. In Fertility Trial 2, the duration of fertility was examined after intravaginal insemination for 2 consecutive days. Daily fertility rates using frozen-thawed semen were 90.0%, 90.0%, 91.7%, 88.9%, 100.0%, 100.0%, 100.0%, 55.6%, 60.0%, 25.0%, 30.0%, 30.0%, and 9.1% for days 1 through 13 after AI, respectively. Overall hatchability was 89.5%. These results indicated that freezing chicken spermatozoa using MA as a cryoprotective agent is a practical method of preserving chicken semen collected from genetically valuable chicken breeds.