Células fetais bovinas de cultivo primário submetidas a diferentes pressões negativas antes do congelamento em palhetas / Fetal bovine primary culture cells submitted to different negative pressures before straw freezing

O congelamento de células é uma importante ferramenta na preservação de espécies ameaçadas de extinção. Células fetais de cultivo primário obtidas de um bovino clone foram submetidas à pressão negativa (PN) de 200, 500 ou 800 mbar, imediatamente (PN0h) ou três horas antes (PN3h) do congelamento em palhetas finas, com 10% de DMSO como crioprotetor. Células frescas e congeladas sem submissão à PN foram utilizadas como controles. Avaliou-se a viabilidade pós-descongelamento, a curva de proliferação celular, assim como o tempo de duplicação da população (PDT) celular, a cada 24 horas, durante oito dias. Os dados obtidos foram submetidos ao teste de Tukey ou Qui quadrado (P≤0,05). A sobrevivência média dos grupos controle (89,8%) e PN500 0h (88,1%) foi superior aos outros grupos; o tempo de PDT foi semelhante nos grupos fresco (27,5 ± 0,35 h), controle congelado (30,1 ± 2,3 h) e PN500 0h (32,4 ± 1,6 h). O menor tempo foi observado no grupo PN800 0h (21,9 h). O congelamento de células fetais bovinas de cultivo primário, realizado em palhetas de 0,25 mL, com 10% de DMSO, possibilita elevadas taxas de sobrevivência após o descongelamento. A PN modifica a curva de crescimento de células criopreservadas, sendo que as intensidades de 200 ou 500 mbar, aplicadas imediatamente antes do congelamento das células, possibilitam curvas de proliferação semelhantes às obtidas com células frescas.

Cell cryopreservation is an important tool in the preservation of endangered species. Fetal bovine primary culture-obtained cells were subjected to negative pressure (NP) 200, 500 or 800 mbar, just before (NP0h) or 3 hours before (NP3h) freezing into fine (0.25 mL) straws, using 10% DMSO as cryoprotectant. Fresh and frozen fibroblasts non submitted to NP were used as controls. We evaluated cell viability after freezing, cell proliferation curve, and population doubling time (PDT) every 24 hours during 8 days. Data were submitted to Tukey or Chi square test (P≤ 0.05). The average survival of control group (89.8%) and NP500-0h (88.1%) was higher than other groups; the time of PDT was similar in the fresh (27.5 ± 0.35 h), frozen control (30.1 ± 2.3 h) and NP500-0h groups (32.4 ± 1.6 h). The lowest time was observed in the NP800-0h (21.9 h) group. Freezing of bovine fibroblasts into 0.25 mL straws with 10% DMSO enables high survival rates after thawing. The NP modifies the growth curve of cryopreserved cells and the intensities of 200 or 500 mbar, applied just before freezing the cells, allow proliferation curves similar to those obtained with fresh cells.

Células fetais bovinas de cultivo primário submetidas a diferentes pressões negativas antes do congelamento em palhetas / Fetal bovine primary culture cells submitted to different negative pressures before straw freezing

O congelamento de células é uma importante ferramenta na preservação de espécies ameaçadas de extinção. Células fetais de cultivo primário obtidas de um bovino clone foram submetidas à pressão negativa (PN) de 200, 500 ou 800 mbar, imediatamente (PN0h) ou três horas antes (PN3h) do congelamento em palhetas finas, com 10% de DMSO como crioprotetor. Células frescas e congeladas sem submissão à PN foram utilizadas como controles. Avaliou-se a viabilidade pós-descongelamento, a curva de proliferação celular, assim como o tempo de duplicação da população (PDT) celular, a cada 24 horas, durante oito dias. Os dados obtidos foram submetidos ao teste de Tukey ou Qui quadrado (P≤0,05). A sobrevivência média dos grupos controle (89,8%) e PN500 0h (88,1%) foi superior aos outros grupos; o tempo de PDT foi semelhante nos grupos fresco (27,5 ± 0,35 h), controle congelado (30,1 ± 2,3 h) e PN500 0h (32,4 ± 1,6 h). O menor tempo foi observado no grupo PN800 0h (21,9 h). O congelamento de células fetais bovinas de cultivo primário, realizado em palhetas de 0,25 mL, com 10% de DMSO, possibilita elevadas taxas de sobrevivência após o descongelamento. A PN modifica a curva de crescimento de células criopreservadas, sendo que as intensidades de 200 ou 500 mbar, aplicadas imediatamente antes do congelamento das células, possibilitam curvas de proliferação semelhantes às obtidas com células frescas.(AU)

Cell cryopreservation is an important tool in the preservation of endangered species. Fetal bovine primary culture-obtained cells were subjected to negative pressure (NP) 200, 500 or 800 mbar, just before (NP0h) or 3 hours before (NP3h) freezing into fine (0.25 mL) straws, using 10% DMSO as cryoprotectant. Fresh and frozen fibroblasts non submitted to NP were used as controls. We evaluated cell viability after freezing, cell proliferation curve, and population doubling time (PDT) every 24 hours during 8 days. Data were submitted to Tukey or Chi square test (P≤ 0.05). The average survival of control group (89.8%) and NP500-0h (88.1%) was higher than other groups; the time of PDT was similar in the fresh (27.5 ± 0.35 h), frozen control (30.1 ± 2.3 h) and NP500-0h groups (32.4 ± 1.6 h). The lowest time was observed in the NP800-0h (21.9 h) group. Freezing of bovine fibroblasts into 0.25 mL straws with 10% DMSO enables high survival rates after thawing. The NP modifies the growth curve of cryopreserved cells and the intensities of 200 or 500 mbar, applied just before freezing the cells, allow proliferation curves similar to those obtained with fresh cells.(AU)

Negative pressure in the pre-freezing of ram semen

Background: The effect of controlled stress on cells has been widely studied and there is strong evidence that positivepressure improves the survival of cryopreserved gametes and embryos. Positive pressure resulted in higher motility offrozen boar semen, and increased bull semen cryotolerance. Looking for similar effects, we developed a piece of equipment (Nitrocooler) to apply negative pressure. The “Nitrocooler” increased the cryotolerance in bovine IVP blastocysts.Nevertheless, until now, negative pressure has not been tested in sperm cells. This study aimed to determine the effectsand the best negative pressure intensity to apply in pre-freezing ram semen.Material, Methods & Results: The ram semen obtained with artificial vagina was immediately diluted (1 + 1.5) in Trisegg yolk, and fractioned into 4 experimental groups, Control: Without treatment; P200: Submission to 200 mBar negativepressure; P500: Submission 500 mBar negative pressure; P800: Submission to 800 mBar negative pressure. Then, Tris-yolkglycerol was added to all groups to obtain a dilution of 1 + 3, and a sperm concentration of 180 million per straw. Thecooling and freezing procedure was identical for all groups. After thaw, progressive motility (PM) was assessed at zero,1, 2 and 3 h of TRT and after Percoll selection (PMPP). Acrosome integrity (ACI) was assessed by FITC PNA staining;plasmatic membrane integrity (PMI) by hypo-osmotic test, acrosome integrity post Percoll (ACIPP), membrane integritypost Percoll (PMIPP), and cleavage rate after heterologous IVF with bovine oocytes. Data were analyzed by t test (P <0.05). Just after thawing, control group showed higher PM (49%) than P200 (40.9%), P500 (38.9%) and P800 (38.9%),without differences among them. There were no differences between PM in the Control, P200, P500 and P800 groupsat 1 h, 2 h and 3 h of TRT, and in ACI...(AU)

Negative pressure in the pre-freezing of ram semen

Background: The effect of controlled stress on cells has been widely studied and there is strong evidence that positivepressure improves the survival of cryopreserved gametes and embryos. Positive pressure resulted in higher motility offrozen boar semen, and increased bull semen cryotolerance. Looking for similar effects, we developed a piece of equipment (Nitrocooler) to apply negative pressure. The “Nitrocooler” increased the cryotolerance in bovine IVP blastocysts.Nevertheless, until now, negative pressure has not been tested in sperm cells. This study aimed to determine the effectsand the best negative pressure intensity to apply in pre-freezing ram semen.Material, Methods & Results: The ram semen obtained with artificial vagina was immediately diluted (1 + 1.5) in Trisegg yolk, and fractioned into 4 experimental groups, Control: Without treatment; P200: Submission to 200 mBar negativepressure; P500: Submission 500 mBar negative pressure; P800: Submission to 800 mBar negative pressure. Then, Tris-yolkglycerol was added to all groups to obtain a dilution of 1 + 3, and a sperm concentration of 180 million per straw. Thecooling and freezing procedure was identical for all groups. After thaw, progressive motility (PM) was assessed at zero,1, 2 and 3 h of TRT and after Percoll selection (PMPP). Acrosome integrity (ACI) was assessed by FITC PNA staining;plasmatic membrane integrity (PMI) by hypo-osmotic test, acrosome integrity post Percoll (ACIPP), membrane integritypost Percoll (PMIPP), and cleavage rate after heterologous IVF with bovine oocytes. Data were analyzed by t test (P <0.05). Just after thawing, control group showed higher PM (49%) than P200 (40.9%), P500 (38.9%) and P800 (38.9%),without differences among them. There were no differences between PM in the Control, P200, P500 and P800 groupsat 1 h, 2 h and 3 h of TRT, and in ACI...