Aim: This study was undertaken to compare the reduction in color of two dyes methylene blue (MBRT) and resazurindye (RRT) with other tests of bull semen quality and to examine their relationship to fertility. Methods:...Aim: This study was undertaken to compare the reduction in color of two dyes methylene blue (MBRT) and resazurindye (RRT) with other tests of bull semen quality and to examine their relationship to fertility. Methods: One hundredsixty-four ejaculates from 59 bulls were examined, processed, and used for 30 016 inseminations. Results: Bulls usedin artificial insemination have been selected for high semen quality and fertility, and semen from these bulls averaged80.6 % unstained sperm, only 11% had abnormalities, and fertility ranged from 64 %to 76 %. The MBRT and RRTwere run with standardized sperm numbers to prevent sperm concentration from influencing the dye reduction time. Ashort RRT was correlated with higher fertility ( r = -0.26) but MBRT was not correlated ( r = -0.05, P >0.0,5).The correlations were low, but axe typical and reflect the statistical effect of the large binomial variance associated withpregnancy or nonpregnancy being coded as 1 or O. Conclusion: The fact that the RRT was significantly correlated withfertility when sperm numbers were standardized, and published reports that resazurin is useful for monitoring other semencharacteristics, indicate that this dye may provide valuable information not given by other simple laboratory tests. (AsianJ Androl 1999 Sep; 1: 109 - 114 )展开更多
Aim： To assess that metabolic status of spermatozoa could provide a useful tool for evaluation of semen quality. Methods： The accuracy of the spectrophotometric application of the resazurin reduction assay was asses...Aim： To assess that metabolic status of spermatozoa could provide a useful tool for evaluation of semen quality. Methods： The accuracy of the spectrophotometric application of the resazurin reduction assay was assessed using receiver operating characteristic （ROC） analysis. Results： Areas under ROC curves （AUC） for motile sperm concentration and sperm index （SI） （sperm concentration multiplied by the square root of percentage sperm motility multiplied by the percentage normal sperm morphology） were 0.922. The best discrimination between poor and good semen samples according to the SI was achieved at a cut-off point of A610 = 0.209, where high sensitivity （94.1%） and specificity （91.7%） were calculated. The assay was less accurate when motile sperm concentration was used as the criterion value, yielding sensitivity of 88.2% and specificity of 87.5%, respectively. Likelihood ratios （LR） indicate that absorbances lower than 0.209 were at least 11.3 times as likely to be found in good semen samples than those in poor according to the SI, whereas in the case of motile sperm concentration, the LR was calculated to be 7.06. Conclusion： These results show that the resazurin reduction assay combined with spectrophotometry is an accurate method of assessing the quality of boar semen.展开更多
Development of viable cell estimation method without sacrificing proliferation and functions of cells cultured on regenerative biomaterials is essential for regenerative engineering.Cytotoxicity and depletion of resaz...Development of viable cell estimation method without sacrificing proliferation and functions of cells cultured on regenerative biomaterials is essential for regenerative engineering.Cytotoxicity and depletion of resazurin are critical but often overlooked limitations that hindered applications of resazurin in viable cell estimation.The present work found that cytotoxicity and depletion of resazurin depended on cell concentration,resazurin concentration and resazurin incubation time.A simple strategy which only allowed cells to incubate with resazurin during each measurement was developed to eliminate negative effects of resazurin.This strategy was verified by monitoring proliferation of MC3T3-E1 preosteoblasts on poly(D,L-lactic acid)scaffold during a continuous 3D culture process for up to 21 days,comparing the accuracy with MTT assay which is a destructive assay with high sensitivity and accuracy and commonly used in regenerative engineering and comparing viability,proliferation and differentiation functions of MC3T3-E1,which were treated with/without this strategy for nondestructive evaluation.This method showed comparable linearity of standard curve and characteristics of growth curve to MTT assay.No major negative effects of this method on MC3T3-E1 viability and functions were found.Our work highlighted the importance of the concentration and incubation time of resazurin in designing application-specific nondestructive viability assay and would be helpful in improving the implanted medical devices as well as in regenerative engineering.展开更多
AIM: To investigate collagen patches seeded with mesenchymal stem cells(MSCs) and/or tenocytes(TCs) with regards to their suitability for anterior cruciate ligament(ACL) repair. METHODS: Dynamic intraligamentary stabi...AIM: To investigate collagen patches seeded with mesenchymal stem cells(MSCs) and/or tenocytes(TCs) with regards to their suitability for anterior cruciate ligament(ACL) repair. METHODS: Dynamic intraligamentary stabilization utilizes a dynamic screw system to keep ACL remnants in place and promote biological healing, supplemented by collagen patches. How these scaffolds interact with cells and what type of benefit they provide has not yet been investigated in detail. Primary ACL-derived TCs and human bone marrow derived MSCs were seeded onto two different types of 3D collagen scaffolds, Chondro-Gide?(CG) and Novocart?(NC). Cells were seeded onto the scaffolds and cultured for 7 d either as a pure populations or as "premix" containing a 1:1 ratio of TCs to MSCs. Additionally, as controls, cells were seeded in monolayers and in co-cultures on both sides of porous high-density membrane inserts(0.4 μm). We analyzed the patches by real time polymerase chain reaction, glycosaminoglycan(GAG), DNA and hydroxyproline(HYP) content. To determine cell spreading and adherence in the scaffolds microscopic imaging techniques, i.e., confocal laser scanning microscopy(c LSM) and scanning electron microscopy(SEM), were applied.RESULTS: CLSM and SEM imaging analysis confirmed cell adherence onto scaffolds. The metabolic cell activity revealed that patches promote adherence and proliferation of cells. The most dramatic increase in absolute metabolic cell activity was measured for CG samples seeded with tenocytes or a 1:1 cell premix. Analysis of DNA content and c LSM imaging also indicated MSCs were not proliferating as nicely as tenocytes on CG. The HYP to GAG ratio significantly changed for the premix group, resulting from a slightly lower GAG content, demonstrating that the cells are modifying the underlying matrix. Real-time quantitativepolymerase chain reaction data indicated that MSCs showed a trend of differentiation towards a more tenogenic-like phenotype after 7 d.CONCLUSION: CG and NC are both cyto-compatible with primary MSCs and TCs; TCs seemed to perform better on these collagen patches than MSCs.展开更多
文摘Aim: This study was undertaken to compare the reduction in color of two dyes methylene blue (MBRT) and resazurindye (RRT) with other tests of bull semen quality and to examine their relationship to fertility. Methods: One hundredsixty-four ejaculates from 59 bulls were examined, processed, and used for 30 016 inseminations. Results: Bulls usedin artificial insemination have been selected for high semen quality and fertility, and semen from these bulls averaged80.6 % unstained sperm, only 11% had abnormalities, and fertility ranged from 64 %to 76 %. The MBRT and RRTwere run with standardized sperm numbers to prevent sperm concentration from influencing the dye reduction time. Ashort RRT was correlated with higher fertility ( r = -0.26) but MBRT was not correlated ( r = -0.05, P >0.0,5).The correlations were low, but axe typical and reflect the statistical effect of the large binomial variance associated withpregnancy or nonpregnancy being coded as 1 or O. Conclusion: The fact that the RRT was significantly correlated withfertility when sperm numbers were standardized, and published reports that resazurin is useful for monitoring other semencharacteristics, indicate that this dye may provide valuable information not given by other simple laboratory tests. (AsianJ Androl 1999 Sep; 1: 109 - 114 )
文摘Aim： To assess that metabolic status of spermatozoa could provide a useful tool for evaluation of semen quality. Methods： The accuracy of the spectrophotometric application of the resazurin reduction assay was assessed using receiver operating characteristic （ROC） analysis. Results： Areas under ROC curves （AUC） for motile sperm concentration and sperm index （SI） （sperm concentration multiplied by the square root of percentage sperm motility multiplied by the percentage normal sperm morphology） were 0.922. The best discrimination between poor and good semen samples according to the SI was achieved at a cut-off point of A610 = 0.209, where high sensitivity （94.1%） and specificity （91.7%） were calculated. The assay was less accurate when motile sperm concentration was used as the criterion value, yielding sensitivity of 88.2% and specificity of 87.5%, respectively. Likelihood ratios （LR） indicate that absorbances lower than 0.209 were at least 11.3 times as likely to be found in good semen samples than those in poor according to the SI, whereas in the case of motile sperm concentration, the LR was calculated to be 7.06. Conclusion： These results show that the resazurin reduction assay combined with spectrophotometry is an accurate method of assessing the quality of boar semen.
基金supported by the National Natural Science Foundation of China(31771019,11172031,11827803,and 31470901)The National Key Research and Development Plan(2016YFC1101101)International Joint Research Center of Aerospace Biotechnology and 344 Medical Engineering from Ministry of Science and Technology of China,111 Project 345(B13003).
文摘Development of viable cell estimation method without sacrificing proliferation and functions of cells cultured on regenerative biomaterials is essential for regenerative engineering.Cytotoxicity and depletion of resazurin are critical but often overlooked limitations that hindered applications of resazurin in viable cell estimation.The present work found that cytotoxicity and depletion of resazurin depended on cell concentration,resazurin concentration and resazurin incubation time.A simple strategy which only allowed cells to incubate with resazurin during each measurement was developed to eliminate negative effects of resazurin.This strategy was verified by monitoring proliferation of MC3T3-E1 preosteoblasts on poly(D,L-lactic acid)scaffold during a continuous 3D culture process for up to 21 days,comparing the accuracy with MTT assay which is a destructive assay with high sensitivity and accuracy and commonly used in regenerative engineering and comparing viability,proliferation and differentiation functions of MC3T3-E1,which were treated with/without this strategy for nondestructive evaluation.This method showed comparable linearity of standard curve and characteristics of growth curve to MTT assay.No major negative effects of this method on MC3T3-E1 viability and functions were found.Our work highlighted the importance of the concentration and incubation time of resazurin in designing application-specific nondestructive viability assay and would be helpful in improving the implanted medical devices as well as in regenerative engineering.
基金Supported by A grant of the Swiss Orthopaedics Society(SGOT)to Ahmad SS,No.S99083814080618560
文摘AIM: To investigate collagen patches seeded with mesenchymal stem cells(MSCs) and/or tenocytes(TCs) with regards to their suitability for anterior cruciate ligament(ACL) repair. METHODS: Dynamic intraligamentary stabilization utilizes a dynamic screw system to keep ACL remnants in place and promote biological healing, supplemented by collagen patches. How these scaffolds interact with cells and what type of benefit they provide has not yet been investigated in detail. Primary ACL-derived TCs and human bone marrow derived MSCs were seeded onto two different types of 3D collagen scaffolds, Chondro-Gide?(CG) and Novocart?(NC). Cells were seeded onto the scaffolds and cultured for 7 d either as a pure populations or as "premix" containing a 1:1 ratio of TCs to MSCs. Additionally, as controls, cells were seeded in monolayers and in co-cultures on both sides of porous high-density membrane inserts(0.4 μm). We analyzed the patches by real time polymerase chain reaction, glycosaminoglycan(GAG), DNA and hydroxyproline(HYP) content. To determine cell spreading and adherence in the scaffolds microscopic imaging techniques, i.e., confocal laser scanning microscopy(c LSM) and scanning electron microscopy(SEM), were applied.RESULTS: CLSM and SEM imaging analysis confirmed cell adherence onto scaffolds. The metabolic cell activity revealed that patches promote adherence and proliferation of cells. The most dramatic increase in absolute metabolic cell activity was measured for CG samples seeded with tenocytes or a 1:1 cell premix. Analysis of DNA content and c LSM imaging also indicated MSCs were not proliferating as nicely as tenocytes on CG. The HYP to GAG ratio significantly changed for the premix group, resulting from a slightly lower GAG content, demonstrating that the cells are modifying the underlying matrix. Real-time quantitativepolymerase chain reaction data indicated that MSCs showed a trend of differentiation towards a more tenogenic-like phenotype after 7 d.CONCLUSION: CG and NC are both cyto-compatible with primary MSCs and TCs; TCs seemed to perform better on these collagen patches than MSCs.
