Objective: The aim of the study was to observe and compare the effects of cryopreservation and thawing meth- ods on rat ovarian tissues. Methods: Twenty 5-6 weeks old SPF-SD female rats were randomly divided into two ...Objective: The aim of the study was to observe and compare the effects of cryopreservation and thawing meth- ods on rat ovarian tissues. Methods: Twenty 5-6 weeks old SPF-SD female rats were randomly divided into two groups, with ten rats in each group. Freshly isolated ovaries saved as a control (group 1: fresh ovaries) in formalin-fixed or vitrified immediately after dissection (group 2: vitrified ovaries). Ovaries in vitrified group were processed into thin slices then cryo- preserved, stored in liquid nitrogen for 21 days, rapidly thawed and grossly examined. All of the collected ovaries underwent hematoxylin and eosin-stained paraffin serial sections and observed the microscopic evaluation in vitrified ovaries. Results: Grossly the vitrified ovaries turned pale color and the size was same as before freeze. The vitrified ovarian tissue had normal anatomical structures of cortex and medulla under the microscope and had no difference with the fresh control ovarian tis- sue. The number and distribution of the follicles were similar with the fresh ovarian tissue, but had smaller size and the gap between oocyte and the surrounding granulosa cells was increased. Few ooctyes were in irregular appearance however the morphology of follicular cells did not give a different appearance as compared to the fresh control ovarian tissue. Conclusion: Cryopreservation of ovarian tissues by vitrification method has some detrimental effect on the morphology of follicles but does not induce negative impact on the number, density and survival of the primordial ovarian follicles. However the whole follicle anatomical structures also had no significant changes.展开更多
Objective: The aim of our study was to measure and compare the serum hormone level of transplant group with blank control and castrated control groups after heterotopic autotransplantation of cryopreserved-thawed ovar...Objective: The aim of our study was to measure and compare the serum hormone level of transplant group with blank control and castrated control groups after heterotopic autotransplantation of cryopreserved-thawed ovarian tissues into back muscles. Methods: A total of 40 SPF-SD female rats(5–6 week-old) were randomly divided into three groups: blank control group(group A), castration control group(group B) and transplant group(group C). Ovaries were removed by surgical procedure, then after cryopreservation and thawing procedures the ovarian tissues were implanted into the back muscles of mice in group C. After 4 weeks of ovarian tissues transplantation, all rats blood sampling were measured for E2, LH and FSH hormone levels by ELISA. Results: E2 level was significantly higher in group C and group A than group B [(38.98 ± 5.66) pg/mL,(8.14 ± 3.24) pg/mL; P < 0.05) and [(36.30 ± 6.90) pg/mL,(8.14 ± 3.24) pg/mL; P < 0.05)]. However, E2 level in group C and group A had no significant difference. FSH level in group B, group A and group C was(18.87 ± 2.54) nmol/L,(7.77 ± 0.87) nmol/L and(9.39 ± 2.12) nmol/L respectively. FSH level increased significantly in group B compared with group A, and the difference had statistical significance(P < 0.05). FSH level was slightly increased in group C compared with group A, and the difference was not statistically significant(P > 0.05), but compared with group B, FSH level was significantly reduced and being statistically significant(P < 0.05). Conclusion: Autotransplantation of cryopreserved-thawed ovarian tissue into back muscles can sustain follicular development and re-establish endogenous hormone production by restoring the factors such as angiogenesis and innervations at the graft site.展开更多
Objective: To investigate the factors that might influence the success of an embryo freezing and thawing program. Method: The relationship between the pregnancy rate in 73 cycles of embryo freezing and thawing progr...Objective: To investigate the factors that might influence the success of an embryo freezing and thawing program. Method: The relationship between the pregnancy rate in 73 cycles of embryo freezing and thawing program and the following factors was analyzed: maternal age, E 2 level at the time of HCG trigger, embryo storage time, number of thawed embryos transferred, presence of sponsoring embryos and intact embryos. And the survival rate of thawed embryos with different morphology, cell stage and storage time was evaluated. Result: Transfer with three or more than three thawed embryos resulted in pregnancy rates of 38.5% and 35.7%, respectively, compared with 5.3% for transfer of fewer than three embryos. The presence of sponsoring embryos and intact embryos significantly increases pregnancy rate in embryo freezing and thawing program. No other factor examined had any effect on pregnancy outcome. The survival rate of good morphology embryos was higher than poor ones, but was not influenced by cell stage and storage time. Conclusion: Embryo morphology before freezing, number of thawed embryos transferred and the presence of intact embryos are important to the outcome of embryo freezing and thawing program.展开更多
The aim of the present study was to evaluate effect of 3 levels of butylated hydroxytoluene （BHT） supplemented in extender for fighting bull semen. Ejaculates were obtained from 10 healthy fighting bulls by artifici...The aim of the present study was to evaluate effect of 3 levels of butylated hydroxytoluene （BHT） supplemented in extender for fighting bull semen. Ejaculates were obtained from 10 healthy fighting bulls by artificial vagina. Semen with motility of more than 70% was included in the experiment. Tris-fructose-glycerol-egg yolk extender containing 0, 1, 3 and 7 mM BHT was used to dilute semen to final concentration of 200 × 10^6 cells/ml. Diluted semen was frozen in 0.25 mL straws and frozen semen was thawed in a 37 ℃ water bath for 15 sec before being evaluated at 0 and 1 hour. Sperm motility at 0 and 1 hour after thawing did not differ among groups tested （ranged 47.86-53.57 and 21.79-24.29 respectively）. At 0 hour after thawing, percentage of live sperm of 3 mM BHT （37.39% ± 2.91%） was lower than those of 0 and 1 mM BHT （43.71%± 2.76% and 41.46% ±2.59% respectively, P 〈 0.05） but not different from 7 mM BHT （40.89%± 2.50%）. The effect was more significant at 1 hour after thawing and 3 mM BHT （32.07% ± 2.45%） suppressed live cells more than other concentrations （ranged 35.07%-37.46%, P 〈 0.05）. At 0 hour after thawing, percentage of membrane integrity （hypo-osmotic swelling test） was not affected by BHT concentration （ranged 23.89%-28.54%）. However, at 1 hour after thawing, percentage of membrane integrity of 3 mM BHT （19.75% ± 1.41%） was lower that of 7 mM BHT （23.29% ± 1.88%, P 〈 0.05） but not different from those of 0 and 1 mM BHT （20.32% ±1.81% and 22.07% ± 2.27% respectively）. No significant effect was found on percentage of abnormal sperm. There was no effect of BHT supplementation in extender on most of the Hamilton-thorn motility analyser parameters. It may be concluded that 3 mM BHT can be harmful to fighting bull spermatozoa and lower concentrmion can be used without detrimental effect. Further study may be needed to verify use of BHT in lower range of concentrations.展开更多
Objective： The aim of our study was to observe the survival and morphological changes of thawed ovarian tis- sues after heterotopic transplantation. Methods： Twenty SPF-SD female rats （5-6 weeks old） were equally ...Objective： The aim of our study was to observe the survival and morphological changes of thawed ovarian tis- sues after heterotopic transplantation. Methods： Twenty SPF-SD female rats （5-6 weeks old） were equally randomized into the control group and experimental group. In control group, the freshly isolated ovaries were fixed in formalin. In experimental group, the freshly isolated ovaries were vitrified immediately and cut into thin slices. After stored in liquid nitrogen for 21 days, the tissues of experimental group were rapidly thawed and transplanted into back muscles of rats for 2 or 4 weeks, respectively. After that, all rats in experimental group were sacrificed and the ovarian tissues were collected and fixed in 4% formaldehyde solution. Then the ovarian tissues were stained with HE and observed under the light confocal microscope. Re- suits： With the naked eyes, there was no specific alteration except the size reduction with color changing. Under microscopy, we found normal cortex and medulla in the ovary, and the primordial follicles and follicles in various stages were observed in the cortex. The normal oocytes in ovarian tissues of experimental group were significant decreased than in the control group. Conclusion： The ovarian tissues survive well in experimental group and there is no significant difference in the proportion of follicles between different times （2 and 4 weeks） after grafting. Our results suggest that thawed ovarian tissues could survive after heterotopic transplantation into back muscles of rat models and maintain their morphology and function.展开更多
文摘Objective: The aim of the study was to observe and compare the effects of cryopreservation and thawing meth- ods on rat ovarian tissues. Methods: Twenty 5-6 weeks old SPF-SD female rats were randomly divided into two groups, with ten rats in each group. Freshly isolated ovaries saved as a control (group 1: fresh ovaries) in formalin-fixed or vitrified immediately after dissection (group 2: vitrified ovaries). Ovaries in vitrified group were processed into thin slices then cryo- preserved, stored in liquid nitrogen for 21 days, rapidly thawed and grossly examined. All of the collected ovaries underwent hematoxylin and eosin-stained paraffin serial sections and observed the microscopic evaluation in vitrified ovaries. Results: Grossly the vitrified ovaries turned pale color and the size was same as before freeze. The vitrified ovarian tissue had normal anatomical structures of cortex and medulla under the microscope and had no difference with the fresh control ovarian tis- sue. The number and distribution of the follicles were similar with the fresh ovarian tissue, but had smaller size and the gap between oocyte and the surrounding granulosa cells was increased. Few ooctyes were in irregular appearance however the morphology of follicular cells did not give a different appearance as compared to the fresh control ovarian tissue. Conclusion: Cryopreservation of ovarian tissues by vitrification method has some detrimental effect on the morphology of follicles but does not induce negative impact on the number, density and survival of the primordial ovarian follicles. However the whole follicle anatomical structures also had no significant changes.
文摘Objective: The aim of our study was to measure and compare the serum hormone level of transplant group with blank control and castrated control groups after heterotopic autotransplantation of cryopreserved-thawed ovarian tissues into back muscles. Methods: A total of 40 SPF-SD female rats(5–6 week-old) were randomly divided into three groups: blank control group(group A), castration control group(group B) and transplant group(group C). Ovaries were removed by surgical procedure, then after cryopreservation and thawing procedures the ovarian tissues were implanted into the back muscles of mice in group C. After 4 weeks of ovarian tissues transplantation, all rats blood sampling were measured for E2, LH and FSH hormone levels by ELISA. Results: E2 level was significantly higher in group C and group A than group B [(38.98 ± 5.66) pg/mL,(8.14 ± 3.24) pg/mL; P < 0.05) and [(36.30 ± 6.90) pg/mL,(8.14 ± 3.24) pg/mL; P < 0.05)]. However, E2 level in group C and group A had no significant difference. FSH level in group B, group A and group C was(18.87 ± 2.54) nmol/L,(7.77 ± 0.87) nmol/L and(9.39 ± 2.12) nmol/L respectively. FSH level increased significantly in group B compared with group A, and the difference had statistical significance(P < 0.05). FSH level was slightly increased in group C compared with group A, and the difference was not statistically significant(P > 0.05), but compared with group B, FSH level was significantly reduced and being statistically significant(P < 0.05). Conclusion: Autotransplantation of cryopreserved-thawed ovarian tissue into back muscles can sustain follicular development and re-establish endogenous hormone production by restoring the factors such as angiogenesis and innervations at the graft site.
文摘Objective: To investigate the factors that might influence the success of an embryo freezing and thawing program. Method: The relationship between the pregnancy rate in 73 cycles of embryo freezing and thawing program and the following factors was analyzed: maternal age, E 2 level at the time of HCG trigger, embryo storage time, number of thawed embryos transferred, presence of sponsoring embryos and intact embryos. And the survival rate of thawed embryos with different morphology, cell stage and storage time was evaluated. Result: Transfer with three or more than three thawed embryos resulted in pregnancy rates of 38.5% and 35.7%, respectively, compared with 5.3% for transfer of fewer than three embryos. The presence of sponsoring embryos and intact embryos significantly increases pregnancy rate in embryo freezing and thawing program. No other factor examined had any effect on pregnancy outcome. The survival rate of good morphology embryos was higher than poor ones, but was not influenced by cell stage and storage time. Conclusion: Embryo morphology before freezing, number of thawed embryos transferred and the presence of intact embryos are important to the outcome of embryo freezing and thawing program.
文摘The aim of the present study was to evaluate effect of 3 levels of butylated hydroxytoluene （BHT） supplemented in extender for fighting bull semen. Ejaculates were obtained from 10 healthy fighting bulls by artificial vagina. Semen with motility of more than 70% was included in the experiment. Tris-fructose-glycerol-egg yolk extender containing 0, 1, 3 and 7 mM BHT was used to dilute semen to final concentration of 200 × 10^6 cells/ml. Diluted semen was frozen in 0.25 mL straws and frozen semen was thawed in a 37 ℃ water bath for 15 sec before being evaluated at 0 and 1 hour. Sperm motility at 0 and 1 hour after thawing did not differ among groups tested （ranged 47.86-53.57 and 21.79-24.29 respectively）. At 0 hour after thawing, percentage of live sperm of 3 mM BHT （37.39% ± 2.91%） was lower than those of 0 and 1 mM BHT （43.71%± 2.76% and 41.46% ±2.59% respectively, P 〈 0.05） but not different from 7 mM BHT （40.89%± 2.50%）. The effect was more significant at 1 hour after thawing and 3 mM BHT （32.07% ± 2.45%） suppressed live cells more than other concentrations （ranged 35.07%-37.46%, P 〈 0.05）. At 0 hour after thawing, percentage of membrane integrity （hypo-osmotic swelling test） was not affected by BHT concentration （ranged 23.89%-28.54%）. However, at 1 hour after thawing, percentage of membrane integrity of 3 mM BHT （19.75% ± 1.41%） was lower that of 7 mM BHT （23.29% ± 1.88%, P 〈 0.05） but not different from those of 0 and 1 mM BHT （20.32% ±1.81% and 22.07% ± 2.27% respectively）. No significant effect was found on percentage of abnormal sperm. There was no effect of BHT supplementation in extender on most of the Hamilton-thorn motility analyser parameters. It may be concluded that 3 mM BHT can be harmful to fighting bull spermatozoa and lower concentrmion can be used without detrimental effect. Further study may be needed to verify use of BHT in lower range of concentrations.
文摘Objective： The aim of our study was to observe the survival and morphological changes of thawed ovarian tis- sues after heterotopic transplantation. Methods： Twenty SPF-SD female rats （5-6 weeks old） were equally randomized into the control group and experimental group. In control group, the freshly isolated ovaries were fixed in formalin. In experimental group, the freshly isolated ovaries were vitrified immediately and cut into thin slices. After stored in liquid nitrogen for 21 days, the tissues of experimental group were rapidly thawed and transplanted into back muscles of rats for 2 or 4 weeks, respectively. After that, all rats in experimental group were sacrificed and the ovarian tissues were collected and fixed in 4% formaldehyde solution. Then the ovarian tissues were stained with HE and observed under the light confocal microscope. Re- suits： With the naked eyes, there was no specific alteration except the size reduction with color changing. Under microscopy, we found normal cortex and medulla in the ovary, and the primordial follicles and follicles in various stages were observed in the cortex. The normal oocytes in ovarian tissues of experimental group were significant decreased than in the control group. Conclusion： The ovarian tissues survive well in experimental group and there is no significant difference in the proportion of follicles between different times （2 and 4 weeks） after grafting. Our results suggest that thawed ovarian tissues could survive after heterotopic transplantation into back muscles of rat models and maintain their morphology and function.
