Effects Of Different Cryoprotectants On Calves Testicular Tissue Cryopreservation

A new approach was applied for the preservation of the male reproductive cells, theprotection of rare and endangered species and the recovery of adolescent male fertility inhuman assisted reproductive technology by testicular tissue cryopreservation. Testicular tissuecryopreservation could avoid low the cell viability resulted from enzymolysis and cells anoxiaaggravation. Compared with common cell cryopreservation, testicular tissue cryopreservationwas difficult because of complex structure and function.The aim of this research was to explore the effects of different cryoprotectants on calvestesticular tissue cryopreservation and select suitable cryoprotectants for testicular tissuecryopreservation. Dimethyl sulfoxide, glycerol, propylene glycol, sucrose, grape seedprocyanidin were chose as cryoprotectants to cryostorage8months calf testicular tissue. NO,NOS, SOD, MDA, testosterone level and cell viability of calf testicular tissue were measured.Key factors of testicular tissue cryopreservation were revealed, the aim was to establish a lowdamage system and provide reliable basis for further study of calf testicular tissuecryopreservation. The results of the study were as follows:1. The NO content was0.45±0.02μmol/mg，the NOS activity was0.86±0.05U/mg, theSOD activity was8.85±0.3U/mg, the MDA content was1.95±0.1nmol/mg, testosteronelevels was10.37±1.2nmol/l in fresh calf testicular tissue.2. After frozen-thawed of testicular tissue by10%DMSO, the NO content, the NOSactivity and the MDA content was0.52±0.01μmol/mg,1.03±0.03U/mg and2.56±0.6nmol/mg, respectively. The NO content, the NOS activity and the MDA content weresignificantly lower than that of other DMSO concentration groups (P<0.05). The SOD activity,testosterone levels and cell viability was8.34±0.9U/mg,8.85±0.7nmol/L and77.00±0.01%,respectively. The SOD activity and cell viability were significantly higher than that of otherDMSO concentration groups (P<0.05). DMSO could supply good effects for testicular cellsand testicular tissue structure and function, suitable for use in calf testicular tissuecryopreservation.3. After frozen-thawed of testicular tissue by1mg/ml GSP, the NO content, the NOSactivity and the MDA content was0.49±0.01μmol/mg,0.90±0.08U/mg,2.22±0.4nmol/mg,respectively. The NO content, the NOS activity and the MDA content were significantlylower than that of other GSP concentration groups (P<0.05). The SOD activity was 8.77±0.9U/mg, significantly higher than that of other GSP concentration groups (P<0.05).GSP could reduce the NO content and the NOS activity significantly in testicular tissue,protect antioxidant capacity of testicular tissue preferably, and reduce oxygen free radicalattacks on tissue.4. After frozen-thawed of testicular tissue by10%PG, the NO content, the NOS activityand the MDA content was0.66±0.01μmol/mg,1.08±0.06U/mg and2.39±0.3nmol/mg,respectively. The NO content, the NOS activity and the MDA content were significantlylower than that of other PG concentration groups (P<0.05). The SOD activity and cellviability was8.22±0.6U/mg and62.00±0.04%, the SOD activity and cell viability weresignificantly higher than that of other PG concentration groups (P<0.05).