ABSTRACT
La criopreservación espermática induce daño por estrés oxidativo en las células, lo que conlleva a un deterioro de la calidad del semen descongelado. Los espermatozoides pueden ser protegidos de este daño, por la adición de antioxidantes al medio de congelación. El objetivo de este estudio fue determinar el efecto de la adición de extracto de hojas de arándano (EHA) al medio de congelación, sobre la calidad de espermatozoides de canino criopreservados. Espermatozoides desprovistos del plasma seminal fueron congelados con diferentes concentraciones de EHA (0 %, control; 1 %, EHA1; 2 %, EHA2; 4 %, EHA4 y 6 %, EHA6) adicionadas al medio de congelación. Post descongelación se evalúo la motilidad progresiva; la viabilidad e integridad de la membrana plasmática (SYBR-14/PI) e integridad de la membrana acrosomal (FITC-PNA/PI) por citometría de flujo. La motilidad progresiva fue similar al control con las concentraciones de EHA1y EHA4 (P >0,05), mientras que con las concentraciones de EHA2 y EHA6 se observó una disminución significativa de este parámetro comparado con el control (P <0,01 y P <0,001 respectivamente). La adición de EHA1, EHA2 y EHA4 al medio de congelación no presentó diferencias significativas respecto al control sobre la viabilidad e integridad de la membrana plasmática (P >0,05); por el contrario, con la adición de EHA6 se observaron valores significativamente menores (P <0,001). Los valores de integridad de la membrana acrosomal, con las diferentes concentraciones de EHA no presentaron diferencias significativas respecto al control. En conclusión, los resultados obtenidos en este estudio revelaron que las concentraciones de EHA utilizadas no fueron eficaces en mejorar la calidad del semen canino descongelado.
During cryopreservation, oxidative stress damage leads to a deterioration of the quality of thawed semen, which could be reduced by the addition of antioxidants to freezing extender. This study was designed to determine the effect of the addition of blueberry leaf extract (EHA) to freezing extender, on quality of cryopreserved canine sperms. Sperm devoid from seminal plasma were frozen with different concentrations of EHA (0 %, control; 1 %, EHA1; 2 %, EHA2; 4 %, EHA4 y 6 %, EHA6) added to freezing extender. Post-thawing progressive motility was evaluated; the viability and plasma membrane integrity (SYBR-14/PI) and acrosomal membrane integrity (FITC-PNA/PI) were assessed by flow cytometry. Progressive motility was similar to the control with concentrations of EHA1 and EHA4 (P >0.05); at concentrations of EHA2 and EHA6 a significant decrease of this parameter compared to control (P <0.01and P <0.001, respectively) was observed. The addition of EHA1, EHA2 and EHA4 to the freezing extender showed no significant differences with respect to the control on viability and plasma membrane integrity (P >0.05); however with the addition of EHA6 values significantly lower (P <0.001) were exhibited. The concentrations of EHA used showed no significant differences with respect to the control on acrosome membrane integrity. In conclusion, the results of this study revealed that none of the concentrations of EHA used were effective in improving canine thawed semen quality.
Subject(s)
Animals , Male , Dogs , Cryopreservation/methods , Cryoprotective Agents/chemistry , Plant Extracts/chemistry , Spermatozoa , Antioxidants/chemistryABSTRACT
Lymphocyte subset analysis is widely used in clinical laboratories, and more than two levels of daily QC materials are required for reliable results. Commercially available, expensive QC materials have short shelf lives and may not be suitable in resource-poor settings. We compared different methods for preparing homemade QC material, including fixation with 1%, 2%, or 4% paraformaldehyde (PFA); freezing with 10% dimethylsulfoxide (DMSO), 0.1% bovine serum albumin-phosphate buffered saline, or after ethanolic dehydration; and using cryopreservation temperatures of -20℃, -80℃, or -196℃. We found an optimal experimental condition, which is 'fixation with 4% PFA, freezing with 10% DMSO, and storage at 80℃'. To evaluate long-term stability of QC materials prepared in this optimal condition, two levels of QC materials (QM1 and QM2) were thawed after 30, 33, 35, 37, 60, 62, 64, and 67 days of cryopreservation. Lymphocyte subset was analyzed with BD Multitest IMK kit (BD Biosciences, USA). QM1 and QM2 were stable after 1-2 months of cryopreservation (CV <3% for CD3, CD4, and CD8 and 5-7% for CD16/56 and CD19). We propose this method as an alternative cost-effective protocol for preparing homemade internal QC materials for lymphocyte subset analysis in resource-poor settings.
Subject(s)
Cryopreservation , Cryoprotective Agents/chemistry , Flow Cytometry/standards , Lymphocyte Subsets/cytology , Quality Control , Reagent Kits, Diagnostic , Time FactorsABSTRACT
Con la finalidad de determinar la mejor solución de vitrificación (SV), usando Etilenglicol (EG), Glicerol (G) y Sucrosa (SUC), se evaluó la apariencia morfológica post vitrificación de embriones murinos (Mus musculus). Ocho mezclas diferentes de crioprotectores fueron evaluadas, con las siguientes concentraciones: SV1: 0% de crioprotectores; SV2: 50% EG; SV3: 50% G; SV4: 50% SUC; SV5: 25% EG + 25% G; SV6: 50% EG + 0,3M SUC; SV7: 50% G + 0.3M SUC y SV8: 25% EG + 25% G + 0,3M SUC. El mayor número de embriones (94,7%) con apariencia morfológica normal, fueron los equilibrados con SV5. No hubo diferencia significativa entre la SV8 (88,9%) y la SV5. Mientras que los embriones criopreservados con las soluciones restantes, presentaron viabilidad morfológica más baja (p<0,05). Estos resultados sugieren que la SV5 provee mejor tolerancia al proceso de vitrificación, observándose en los embriones la más alta viabilidad y la más baja frecuencia de anormalidades morfológicas. Estos hallazgos contribuyen de manera importante, en la selección de los crioprotectores para la vitrificación.
In order to determine the best vitrification solution (VS), using ethylene glycol (EG), glycerol (G) and sucrose (SUC), the post vitrification morphology in murine embryos (Mus musculus) was evaluated. Eight different mixtures of these chemicals, with the following concentrations: VS1: 0%; VS2: 50% EG; VS3: 50% G; VS4: 50% SUC; VS5: 25% EG + 25% G; VS6: 50% EG + 0.3M SUC; VS7: 50% G + 0.3M SUC and SV8: 25% EG + 25% G + 0.3M SUC, were used. VS5 was the solution with the highest percentage (94,7%) of embryos with normal morphology. There were no significant differences between the VS8 (88.9%) and VS5. However, embryos cryopreserved with the other solutions had a lower morphological viability (p<0.05). These results suggest that VS5 provides embryos with better tolerance to the vitrification process, because a higher viability and lower frequency of morphological abnormalities were present. These findings provide details of great importance to the selection of cryoprotectants for vitrification.
Subject(s)
Animals , Male , Female , Mice , Rodentia/growth & development , Cryoprotective Agents/chemistry , Embryonic Structures/abnormalities , Embryonic Structures/metabolism , Public Health , Muridae/classificationABSTRACT
BACKGROUND: The red blood cell (RBC) deformability test is a useful method for measuring the ability of RBCs to adapt their shape to the flow conditions. Using this test, several investigators have shown the relationship between RBC deformability and numerous clinical conditions. For the quality control (QC) of RBC deformability test, we evaluated whether frozen-thawed-deglycerolized RBCs can be used as QC materials. METHODS: Packed RBCs were frozen with 40% (wt/vol) glycerol and stored at -80degrees C for 3 months. For 10 different frozen RBC panels, RBCs were thawed, deglycerolized and stored at 4degrees C for 4 weeks. Using microfluidic ektacytometer, we measured RBC deformability of the thawed RBCs. The stability of thawed RBCs was tested once a day for 28 days of storage time and was analyzed by simple regression analysis. The precision of the test using thawed RBCs was analyzed for 7 days of storage time by calculation of CV values of intra-assay (10 measurements/assay) and between-day measurements. RESULTS: Frozen-thawed-deglycerolized RBCs were stable for 1 week. Within-run and between-day precisions of the RBC deformability test during 7 days of storage of thawed RBCs were 1.4-2.9%, and 1.9-2.8%, respectively. CONCLUSIONS: Frozen-thawed-deglycerolized RBCs used in RBC deformability test showed satisfactory within-run and between-run precisions and stability for one week after thawing, and may be used as QC materials for this test.
Subject(s)
Humans , Blood Preservation , Cryopreservation , Cryoprotective Agents/chemistry , Erythrocyte Deformability , Erythrocytes/immunology , Glycerol/chemistry , Hematologic Tests/standards , Quality ControlABSTRACT
OBJECTIVE: To compare the bone graft cryopreservation method (at -80°C) with a preservation method using a 98 percent glycerol solution at room temperature (10°C-35°C), by testing the antibacterial and fungal effects of 98 percent glycerol and comparatively analyzing the observed histological changes resulting from the use of both methods. METHOD: This study was of 30 samples of trabecular bone tissue from 10 patients undergoing total hip arthroplasty. Each femoral head provided 3 samples that were randomized into 3 groups, namely, the control group, the cryopreserved group, and the group preserved in a 98 percent glycerol at room temperature for 1 year. The samples were submitted to histomorphologic, cell feasibility, and microbiologic analyses. The results were statistically analyzed using the McNemar test, with a statistical significance index of 0.05. RESULTS: Values obtained using the McNemar test to compare probability distributions of histomorphologic variables (mature or lamellar bone, immature bone, and necrosis) and cell feasibility (osteoblasts and osteoclasts) indicated that there is no difference between the distributions of variables under the 3 experimental conditions. Microbiological analysis of the 98 percent glycerol solution and bone fragments from samples stored for 1 year at room temperature did not show bacterial or fungal growth. The histological and microbiological investigation were performed at 2 different time points: immediately after the sample processing and after 1 year. CONCLUSION: The method used to preserve bone grafts kept in 98 percent glycerol at room temperature (10°C-35°C) was similar to cryopreservation in terms of bone matrix preservation; no bacteria or fungi were found in the samples.
OBJETIVO: Comparar o método da criopreservação de enxertos ósseos (- 80° C) com o da conservação em glicerol a 98 por cento em temperatura ambiente (10° C a 35° C), testando os efeitos antibacterianos e antifúngicos do glicerol a 98 por cento e analisando comparativamente as alterações histológicas verificadas e decorrentes do emprego dos dois métodos. MÉTODO: Este estudo foi constituído de 30 amostras de tecido ósseo trabecular provenientes de 10 pacientes, submetidos a Artroplastia Total do Quadril. Cada cabeça femoral forneceu 3 amostras e estas foram divididas aleatoriamente em 3 grupos, a saber: controle, criopreservado e conservado em glicerol a 98 por cento à temperatura ambiente durante um ano. As amostras foram encaminhadas à Anatomia Patológica para estudo histomorfologico, de viabilidade celular, e microbiológico. Os resultados foram analisados estatisticamente pelo método de McNemar, com índice de significância de 0,05. RESULTADOS: A análise dos valores obtidos no teste de McNemar na comparação das distribuições de probabilidades das variáveis da histomorfologia (osso maduro ou lamelar, osso imaturo e necrose) e da viabilidade celular (osteoblastos e osteoclastos) indica não haver diferença entre as distribuições das variáveis nas três condições experimentais. A análise microbiológica da solução de glicerol a 98 por cento e dos fragmentos ósseos das amostras armazenadas durante um ano em temperatura ambiente não apresentou crescimento bacteriano ou de fungos. As espécimens do grupo controle foram analisadas histológica e microbiologicamente logo após a coleta das mesmas. CONCLUSÃO: O método de conservação de enxertos ósseos mantidos no glicerol a 98 por cento em temperatura ambiente (10°C a 35°C) foi similar ao da criopreservação quanto à preservação da matriz óssea e à ausência de crescimento de bactérias ou fungos.