| Testicular tissue cryopreservation is a developing modern reproductive technology, and it provides a new method to preserve animal germplasm resources. Trehalose is a kind of nonreducing disaccharide, which has the properties of anti-freezing, anti-hypertonic and low toxicity. Meanwhile, it has been widely used in the cryopreservation of tissues and cells.However, the research about the effects of trehalose on the quality of frozen-thawed dairy goat testicular tissue is scanty. To investigate the optimum permeating cryoprotectant for dairy goat testicular tissue and the effects of trehalose on the quality of frozen-thawed dairy goat testicular tissue, the testis of Guanzhong dairy goat was collected and cut into pieces, then cryopreserved in the cryomedia containing different concentrations of dimethyl sulfoxide(DMSO), glycerol and propanedio respectively. The total cell viability, interstitial cell viability and tubular epithelial cell viability of fresh and frozen-thawed dairy goat testicular tissue were evaluated respectively. The permeating cryoprotectant and the optimal concentration of the permeating cryoprotectant which were suitable for dairy goat testicular tissue were determined in this step. Then, different concentrations of trehalose were added in the selected cryomedia. The total cell viability, interstitial cell viability and tubular epithelial cell viability of fresh and frozen-thawed dairy goat testicular tissue were evaluated respectively. Frozen-thawed dairy goat testicular tissue was cultured and the tissue culture media was collected to evaluate the testosterone content. Paraffin-embedded tissue slices of the fresh and frozen-thawed dairy goat testicular tissue were made to evaluate the morphological structure. The cell apoptosis of the fresh and frozen-thawed dairy goat testicular tissue was detected by TUNEL. On the other hand, T-AOC activity, SOD activity,CAT activity, GSH content and MDA content of fresh and frozen-thawed dairy goat testicular tissue were also evaluated. Meanwhile, real-time PCR was performed to evaluate the m RNA expression level of Bax, Bcl-2, HSP70-2, Boule and CREM respectively. The main results of this research were as following:1. After freezing and thawing, the total cell viability, interstitial cell viability and tubular epithelial cell viability of dairy goat testicular tissue was decreased significantly(P < 0.05).The total cell viability, interstitial cell viability and tubular epithelial cell viability of the dairy goat testicular tissue cryopreserved in the cryomedia containing DMSO was significantly higher than those of cryopresered in the cryomedia containing glycerol and propeandio respectively(P < 0.05). The total cell viability, interstitial cell viability and tubular epithelial cell viability of the dairy goat testicular tissue cryopreserved in the cryomedia containing propanedio was significantly higher than those of cryopresered in the cryomedia containing glycerol(P < 0.05). The total cell viability, interstitial cell viability and tubular epithelial cell viability of the dairy goat testicular tissue cryopreserved in the cryomedia containing 10%DMSO was the highest in all treatments, they were 66.41%, 66.76% and 65.87% respectively.2. The total cell viability, interstitial cell viability and tubular epithelial cell viability of the dairy goat testicular tissue cryopreserved in the cryomedia containing trehalose was significantly higher than those of the control group(P < 0.05). The total cell viability,interstitial cell viability and tubular epithelial cell viability of the dairy goat testicular tissue cryopreserved in the cryomedia containing 15% trehalose was the highest in all treatments(P< 0.05). On the other hand, the trehalose in the cryomedia had protective effects on the capacity of frozen-thawed dairy goat testicular tissue testosterone secretion. Meanwhile, the testosterone content of the group added 15% trehalose was higher than that of other groups(P< 0.05). Meanwhile, compared with the fresh group, the process of freezing and thawing had bad effects on the morphological structure of dairy goat testicular tissue. The morphological structure of the frozen-thawed dairy goat testicular tissue was improved by adding trehalose in the cryomedia, and the group added 15% trehalose showed positive effects on the morphological structure. On the other hand, the process of freezing and thawing lead cell apoptosis in dairy goat testicular tissue(P < 0.05). The cryomedia containing trehalose could decrease the cell apoptosis of frozen-thawed dairy goat testicular tissue significantly(P <0.05). Meanwhile, the group added 15% trehalose showed the lowest apoptosis cell number in all treatments, and showed no difference compared with the group added 20% trehalose(P >0.05).3. Compared with the fresh dairy goat testicular tissue, the T-AOC activity, SOD activity,CAT activity and GSH content of the frozen-thawed dairy goat testicular tissue was decreased significantly(P < 0.05), the MDA content was increased significantly(P < 0.05). After freezing and thawing, the T-AOC activity, SOD activity, CAT activity and GSH content of the groups added trehalose were higher than those of the control group significantly(P < 0.05),MDA content was lower than those of the control group significantly(P < 0.05). Meanwhile,the group added 15% trehalose showed the highest T-AOC activity, SOD activity, CAT activity and GSH content and the lowest MDA content in all treatments.4. Compared with the fresh dairy goat testicular tissue, the Bax expression level of the frozen-thawed groups was increased significantly(P < 0.05), the expression level of Bcl-2,HSP70-2, Boule and CREM of frozen-thawed groups was decreased significantly(P < 0.05).After freezing and thawing, the Bax expression level of the groups added trehalose was lower than that of control group(P < 0.05), the expression level of Bcl-2, HSP70-2, Boule and CREM expression level of the groups added trehalose was higher than those of control group(P < 0.05). Meanwhile, the group added 15% trehalose showed the lowest Bax expression level in all treatments(P < 0.05) and the Bcl-2, HSP70-2, Boule and CREM expression level of the group added 15% trehalose was the highest in all treatments respectively. |
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