Abstract: 【Objective】 This study was aimed to explored the influence of different glycerol removal methods on the artificial insemination efficacy of thawed chicken semen,aiming to simplify the glycerol removal procedure for chicken semen. 【Method】 Semen was collected from Wenchang roosters using the abdominal-dorsal combined massage method,frozen,thawed,and then diluted with extender at ratios of 1∶2,1∶4,1∶6,and 1∶8 (V/V) to remove glycerol.The optimal dilution ratio was determined by assessing sperm motility after deglycerolization.The optimal proportions were used for one-step,two-step,and six-step dilution methods for deglycerolization.The effectiveness of each method was evaluated by assessing sperm motility,sperm recovery rate,and glycerol content in both seminal plasma and sperm.Subsequently,the deglycerolized semen from the three methods was used for artificial insemination trials to measure fertilization and hatching rates. 【Result】 Among various dilution ratios,the 1∶4 six-step dilution method for deglycerolization demonstrated superior sperm motility both post-deglycerolization and at 30 minutes post-deglycerolization compared with 1∶2,1∶6,and 1∶8 six-step dilution methods,although the differences were not statistically significant (P＞0.05).Among different deglycerolization methods with a total dilution ratio of 1∶4,the six-step dilution method achieved the highest post-deglycerolization sperm motility at 68.34%,but the differences among the three methods were not significant (P＞0.05).The glycerol removal rates of both seminal plasma and sperm in the two-step dilution method were extremely significantly higher than those of the other two methods (P＜0.01).The sperm recovery rate was higher with the six-step dilution method,but no significant differences were found among the three methods(P＞0.05).In terms of artificial insemination outcomes,the fertilization rate,live embryo rate,and hatching rate of hatching eggs in non-glycerol-removed group were all 2.25%,which were extremely significantly lower than those of the fresh semen group and the three glycerol-removed method groups (P＜0.01).Among the three glycerol removal methods,the six-step dilution method showed relatively higher fertilization rate,live embryo rate,and hatching rate of hatching eggs,but no significant differences were observed compared with the two-step dilution method (P＞0.05).The hatching rate of fertilized eggs in the two-step dilution method was similar to that of fresh semen,and the live embryo emergence rate was the highest,reaching 95.71%,but the overall differences were not significant (P＞0.05). 【Conclusion】 The 1∶4 two-step dilution method efficiently removed glycerol from frozen chicken semen and achieved favorable sperm recovery rate,sperm motility,and artificial insemination outcomes.Meanwhile,compared with the traditional six-step dilution method,the 1∶4 two-step dilution method had a simpler operational process,making it more suitable for application in chicken breeding production.

Key words: chicken frozen semen; deglycerin removal; sperm vitality; sperm recovery rate; fertilization rate; hatching rate