| Ovarian cortical cryopreservation,which is an important way to preserve livestock germplasm resources,but at present,ovarian cortical cryopreservation technology of swine is still in the research stage,and there are mainly problems such as low survival rate of ovarian cortical tissue follicles and easy apoptosis after cryothawing.Current approaches to solving these problems mainly focus on the development of cryocarriers and cryoprotective agents(CPAs).In this study,porcine ovarian cortex was used as the test object,and an effective porcine ovarian cortical tissue freezing technology was established.In order to obtain the best method of preserving the primordial follicles of the ovarian cortex,this test vitrified the porcine ovarian cortical tissue(10 mm×10 mm×1 mm),and the suitable carrier and CPAs for porcine ovarian cortical tissue freezing were screened by counting the normal proportion of follicle structure,the survival rate of oocytes and the Apoptosis conditions of TUNEL experiment after thawing.At the same time,the effect of antioxidant isoglycyrrhizene on the cryoppreservation of porcine ovarian cortical tissue was studied.The main findings are as follows:1)Using cryopreservation tubes as the freezing carrier,0.1 M sucrose(15%ethylene glycol(Ethylene Glycol,EG)+ 15% dimethyl sulfoxide(Dimethyl Sulfoxide,DMSO)+ 0.1 M sucrose),0.5 M sucrose(15% EG + 15% DMSO + 0.5 M sucrose),0.1 M trehalose(15% EG + 15% DMSO + 0.1 M trehalose)and 0.5 M trehalose(15%EG + 15% DMSO + 0.5 M trehalose)were the CPAs of experimental groups.There were also negative controls(fresh group)and active controls(unprotected group).The results showed that the normal structure of follicles in ovarian cortex tissue in fresh group,unprotected group,0.1 M sucrose group,0.5 M sucrose group,0.1 M trehalose group and 0.5 M trehalose group were 68.61 ± 6.27%,14.24 ± 1.58%,21.77 ± 2.63%,31.65 ± 2.84%,24.83 ± 3.86%,respectively.The preservation effect of the 0.1 M trehalose group was significantly higher than that in the unprotected group and the 0.1M sucrose group(P<0.05),indicating that the 0.1 M trehalose group had the best preservation effect.2)0.1 M trehalose(15% EG + 15% DMSO + 0.1 M trehalose)was used as CPAs,and cryopreservation tube group,acupuncture needle group and thin metal group were designed with cryopreservation tubes,acupuncture needles and thin metal strips as freezing carriers.The results showed that the normal structure of follicles of ovarian cortical tissue in the cryopreservation tube group,acupuncture needle group and thin metal strip group were 33.53 ± 1.88%,18.16 ± 2.90%,20.27 ± 4.38%,respectively,and there was no significant difference between the thin metal strip group and the acupuncture needle group(P>0.05),but the preservation effect of the cryopreservation tube group was significantly higher than that of the thin metal strip group and the acupuncture needle group(P<0.05),indicating that the preservation effect of cryopreservation tubes as the freezing carrier was the best.3)With cryopreservation tubes as cryopreservation carriers,Different concentrations of isoglycyrrhizene(0 μM,10 μM,100 μM,1 m M)were added to 0.1M trehalose(15% EG + 15% DMSO + 0.1 M trehalose)as CPAs.Negative controls(fresh group)and positive controls(unprotected group)were also designed.The results showed that the proportions of normal structure of follicles in ovarian cortical tissues in the fresh group,unprotected group,0 μM,10 μM,100 μM and 1 m M isoglycyrrhizin group were 69.12 ± 1.73%,14.46 ± 2.20%,28.76 ± 4.63%,32.75 ±1.55%,46.12 ± 5.02%,25.65 ± 4.38%,respectively.The preservation effect of the100 μM isoglycyrrhizin group was significantly higher than that in the 0 μM,10 μM and 1 m M isoglycyrrhizin groups(P<0.05),indicating that the addition of 100 μM isoglycyrrhizin helped to improve the cryopreservation effect of porcine ovarian cortex tissue.4)The overall ROS level of ovarian tissue in the 0 μM and 100 μM ISL groups,the negative control(fresh group)and the positive control(unprotected group)were then detected by ROS experiment.The fluorescence intensity of ROS(5.59 ± 0.50)in the 100 μM ISL group was significantly lower than that in the 0 μM ISL group(3.83 ±0.15)(P<0.05),and there was no significant difference between it and the fresh group(P>0.05),indicating that the addition of 100 μM ISL could effectively inhibit the overall ROS level of ovarian tissue.5)The apoptosis of ovarian stromal cells in the 0 μM and 100 μM isoglycyrrhizin group,negative control(fresh group)and positive control(unprotected group)was detected by TUNEL experiment.It was found that the fluorescence intensity of TUNEL(1.60 ± 0.17)in the 100 μM isoglycyrrhizin group was significantly lower than that in the 0 μM isoglycyrrhizin group(2.45 ± 0.02)(P<0.05),and there was no significant difference from the fresh group(P>0.05),indicating that the addition of 100 μM isoglycyrrhizene to CPAs could effectively inhibit cell apoptosis in cortical regions6)The ability of follicles to continue development in thawed ovarian tissue was determined by in vitro culture experiments of ovarian cortical tissue.The ratios of primordial,primary,secondary,and luminal follicles in thawed ovarian cortical tissue were 72.33 ± 3.59%,24.82 ± 2.80%,2.00 ± 0.59%,and 0.85 ± 0.36%,respectively.The ratios of primordial follicles,primary follicles,secondary follicles and luminal follicles in thawed ovarian cortical tissue were 11.38 ± 1.84%,9.19 ± 2.89%,75.17 ±5.47% and 4.26 ± 0.82%,respectively.Suggesting that follicles in the thawed ovarian cortical tissue remain viable and able to continue development.In summary,when the porcine ovarian cortical tissue was vitrified,cryopreservation tubes were used as freezing carriers,and 15% EG + 15% DMSO +0.1 M trehalose + 100 μM isoglycyrrhizin was used as the CPAs,and the thawed follicles on the ovarian cortex still have developmental potential. |
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