Effects Of Added BSA To DMSO And EG On Activity Of Cryopreserved Lamb Testis Tissue

The cryopreservation of testicular tissue not only become a part of reproductive performance preservation in wild animals and fine livestock, but also benefit to prepubertal boys who have to undergo chemotherapy or radiotherapy. Cryoprotectant plays an important role in protecting testicular tissue from cryodamage during cryopreservation, however, the research about cryoprotective agents was focused on permeable cryoprotectants previously. Permeating cryoprotectant could reduce ice crystals formation by penetrate into cells, however, permeating cryoprotectant have cell toxicity because it could destroy intracellular signaling pathways, denaturation of enzymes and dissolving DNA struture. In order to test the effect of bovine serum albumin(BSA) as an addition in permeating cryoprotectants(dimethyl sulfoxide and ethylene glycol) on the activity of cryopreserved lamb testicular tissue, this experiment researched the effect of different cryoprotective agents(10% DMSO, 10% EG, 10% DMSO combine with 5 g/L BSA and 10% EG combine with 5 g/L BSA) on morphology, level of hormone production, activity of marker enzymes, antioxidant ability and key genes relative expression in cryopreserved lamb testicular tissue. The main results as follows:1. The cell viability, rate of seminiferous tubule integrity, level of inhibin and testosterone production in the group of 10% DMSO + 5 g/L BSA were 79.97%, 88.08%, 137.73 nmol/L, 1507.92 ng/L, 198.63 U/gprot and 33.04 U/gprot respectively, which all were significantly higher than other groups(P < 0.05), but less than fresh testes significantly(P < 0.05). The cell viability, rate of seminiferous tubule integrity, level of inhibin and testosterone production of cryopreserved lamb testicular tissue in groups of 10% DMSO and 10% EG + 5 g/L BSA were lower than the group of 10% DMSO + 5 g/L BSA(P < 0.05), and there were no significant difference between these two groups(P > 0.05).2. The activity of alkaline phosphatase(AKP), acid phosphatase(ACP), γ-glutamyl transpeptidase(γ-GT) and total antioxidant capacity(T-AOC) in the group of 10% DMSO + 5 g/L BSA were 198.63 U/gprot, 33.04 U/gprot, 137.23 U/gprot and 1.62 U/mgprot respectively, which all were higher than other groups significantly(P < 0.05), but less than fresh testes significantly(P < 0.05). The activity of lactic dehydrogenase(LDH) in the group of 10% DMSO + 5 g/L BSA and 10% EG + 5 g/L BSA were 1444.50 U/gprot and 1324.20 U/gprot respectively, which were higher than other groups significantly(P < 0.05), and there was no significant difference between these two groups(P > 0.05). The activity of malondialdehyde(MDA) in the group of 10% DMSO + 5 g/L BSA and 10% EG + 5 g/L BSA were 1.63 U/mgprot and 1.80 U/mgprot respectively, which were lower than other groups significantly(P < 0.05) and there was no significant difference between these two groups(P > 0.05).3. The relative expression of Caspase-3 in the group of 10% DMSO + 5 g/L BSA was lower than other groups but higher than fresh testis significantly(P < 0.05). The relative expression of CD90 in fresh group was higher than other group significantly(P < 0.05), and there was no significant difference among four freezing groups(P > 0.05). The relative expression of GDNF in the group of 10% DMSO + 5 g/L BSA were higher than the groups of 10% DMSO and 10% EG significantly(P < 0.05), but there was no significant difference with the group of 10% EG + 5 g/L BSA(P > 0.05). The relative expression of Oct-4 in the group of 10% DMSO + 5 g/L BSA were higher than the group of 10% EG significantly(P < 0.05), but there was no significant difference with 10% EG + 5 g/L BSA group and 10% DMSO group(P > 0.05).In conclusion, the morphology, level of inhibin and testosterone production, activity of marker enzymes, antioxidant ability and key genes relative expression were enhanced by add 5 g/L BSA into 10% DMSO in cryopreserved lamb testicular tissue. These results indicated that add BSA into DMSO is feasible for testicular tissue cryopreservation. This research offers a new direction for choosing cryoprotectants, and provides theoretical references for cryopreservation of testis tissue.