Effect Of Freeze Drying On The Structure And DNA Of Human Spermatozoa

Objectiveâ‘ To explore the effect of freeze drying on the structure and DNA of human spermatozoa.â‘¡To explore the protective function of the trehalose and egg yolk for the structure and membrane of sperm in freeze drying.Method Forty eight qualified semen samples were obtained from healthy volunteers.Among them,40 samples were divided randomly into 4 groups. Three semen sample groups were added with freeze drying protective agents—ETBS, ETBS+trehalose,or ETBS+trehalose+egg yolk respectively,and then conserved in a refrigerator at 4℃for 3 weeks.Another group of semen sample was used as a fresh sperm control.According to the hypoosmotic swelling test and observation by microscopy,the protective functions of these protective agents to the structure and membrane of the sperm were compared. In situ nick-end labeling(TUNEL) test and comet assay were used to detect the sperm DNA damage.Eight samples were detected by TUNEL and comet assay after treated by H₂O₂ and acted as positive control.Resultâ‘ In the hypoosmotic swelling test,the percentage of the hypoosmotic swelling sperm in ETBS group,ETBS+trehalose group,and ETBS+trehalose +egg group were 4%,12%,36%,96%respectivelyâ‘¡Under the electron microscopy,the sperm morphology was best in ETBS+ trehalose+egg group,worse in ETBS+trehalose group,and worst in the ETBS group.â‘¢The percentage of TUNEL-positive spermatozoa in the ETBS group,ETBS +trehalose group,ETBS+trehalose+egg yolk group and the fresh control group were(6.39±1.46)%,(5.75±1.29)%,(5.20±1.38)%,and(4.94±1.86)%respectively;while these detected by Comet assay were(6.48±1.58)%,(5.83±1.48)%,(5.28±1.42)%,and(5.12±1.65)%respectively. No significant difference was found in the percentage of spermatozoa with damaged DNA between fresh and freeze dried spermatozoa(P＞0.05).Conclusionâ‘ Trehalose added into ETBS can reduce the damage to membrane and the tail of sperm in freeze drying,and if trehalose combined with egg yolk can further reduce the damage to the sperm morphology.â‘¡Freeze drying using ETBS,ETBS+trehalose,or ETBS+trehalose+egg yolk as protective agents have no damage to the DNA of human spermatozoa.â‘¢The Freeze drying protective agents modified by trehalose could improve the protection to sperm,and the media supplemented with trehalose and egg yolk was the best.