Ação dos crioprotetores glicose, trealose e quitosana na manutenção da viabilidade de células de Escherichia coli, Saccharomyces cerevisiae após liofilização / Action of cryoprotectors glucose, trealose and quitosan in maintaining viability of Escherichia coli, Saccharomyces cerevisiae cells after lyophilization

A liofilização é considerada uma das técnicas mais seguras para obtenção de estabilidade de cepas de cultura. Para este processo, podem ser utilizados crioprotetores, que são substâncias que protegem as estruturas celulares durante o período de congelamento, descongelamento e desidratação. O objetivo do presente trabalho foi avaliar o desempenho da glicose, trealose e quitosana como crioprotetores para manutenção de cepas de Escherichia coli e Saccharomyces cerevisiae. Para tratamento estatístico, utilizou-se ANOVA e teste de Tukey com o software Bioestat, versão 5.3. Para Escherichia coli, a Trealose apresentou melhores resultados após a liofilização, porém nenhum dos tratamentos mostrou-se eficaz em prolongar a viabilidade até os 60 dias de armazenamento. Para Saccharomyces cerevisiae, todos os tratamentos apresentaram-se satisfatórios ao longo dos 60 dias avaliados.

Lyophilization is considered one of the safest techniques for acquiring stability of culture strains. For this process, cryoprotectants, which are substances that protect cell structures during the period of freezing, thawing and dehydration, may be used. The objective of the present work was to evaluate the performance of glucose, trehalose, and chitosan as cryoprotectants for Escherichia coli e Saccharomyces cerevisiae. For statistical treatment, ANOVA and Tukey’s test were used with the software Bioestat, version 5.3. For Escherichia coli Trealose presented better performance after lyophilization, but none of the treatments proved to be effective in prolonging viability up to 60 days of storage. For Saccharomyces cerevisiae all treatments were satisfactory throughout the 60 days evaluated.

Ação dos crioprotetores glicose, trealose e quitosana na manutenção da viabilidade de células de Escherichia coli, Saccharomyces cerevisiae após liofilização / Action of cryoprotectors glucose, trealose and quitosan in maintaining viability of Escherichia coli, Saccharomyces cerevisiae cells after lyophilization

A liofilização é considerada uma das técnicas mais seguras para obtenção de estabilidade de cepas de cultura. Para este processo, podem ser utilizados crioprotetores, que são substâncias que protegem as estruturas celulares durante o período de congelamento, descongelamento e desidratação. O objetivo do presente trabalho foi avaliar o desempenho da glicose, trealose e quitosana como crioprotetores para manutenção de cepas de Escherichia coli e Saccharomyces cerevisiae. Para tratamento estatístico, utilizou-se ANOVA e teste de Tukey com o software Bioestat, versão 5.3. Para Escherichia coli, a Trealose apresentou melhores resultados após a liofilização, porém nenhum dos tratamentos mostrou-se eficaz em prolongar a viabilidade até os 60 dias de armazenamento. Para Saccharomyces cerevisiae, todos os tratamentos apresentaram-se satisfatórios ao longo dos 60 dias avaliados.(AU)

Lyophilization is considered one of the safest techniques for acquiring stability of culture strains. For this process, cryoprotectants, which are substances that protect cell structures during the period of freezing, thawing and dehydration, may be used. The objective of the present work was to evaluate the performance of glucose, trehalose, and chitosan as cryoprotectants for Escherichia coli e Saccharomyces cerevisiae. For statistical treatment, ANOVA and Tukeys test were used with the software Bioestat, version 5.3. For Escherichia coli Trealose presented better performance after lyophilization, but none of the treatments proved to be effective in prolonging viability up to 60 days of storage. For Saccharomyces cerevisiae all treatments were satisfactory throughout the 60 days evaluated.(AU)

Trehalose addition to a Tris-fructose egg yolk extender on quality of ram sperm preserved at 0 °C

The present study investigated the effects of various concentrations of trehalose in Tris-fructose egg yolk diluent on ram semen preservation at 0 ℃. Semen was collected by artificial vagina ejaculation from six rams of proven fertility. High-quality ejaculates were diluted with 0 (control), 5, 10, 15, and 20 mM trehalose of Tris-fructose egg yolk extender and control (tris-fructose egg yolk extender without trehalose), respectively. Then, the ejaculates were diluted to a concentration of 5×108 sperm/mL, cooled to 0 ℃ for 90 min, and maintained at that temperature for twelve days. The diluted semen samples were examined, and their sperm progressive motility, membrane functionality, and acrosome integrity recorded at 0, 24, 72, 144, 216, and 288 h. Two hundred ninety-six ewes were transcervically inseminated with the 216-h control (without trehalose) or the optimal trehalose concentration group semen, and the pregnancy and lambing rates were measured. No significant differences were established in the sperm progressive motility and membrane functionality among the control and 5, 10, 15, and 20 mM groups. The sperm samples of trehalose addition groups had no significant difference in the acrosome integrity of sperm, but they were, nonetheless, significantly higher than those in the control. No significant difference was detected in the lambing and pregnancy rates between the 5 mM and control groups. These results suggest that ram sperm is capable of fertilization after cooling and preservation at 0 ℃ by the use of 5 mM trehalose for Tris-fructose egg yolk diluent. Under these conditions, ram sperm can be more effectively preserved than under other four concentrations of diluents.(AU)

Effects of trehalose and catalase on the viability and kinetic parameters of cryopreserved ram sperm

Background: Most part of ram spermatozoa membrane has unsaturated fatty acids (phospholipids). Membrane structure of cells is composed of double ordered phospholipid layers adorned with mosaic-like protein, glycoprotein and glycolipids. Sperm freezing protocols could be negatively affected on ram sperm motility, viability and acrosome integrity during cryopreservation. For these reasons, researchers were designed their topics has led to the search for effective antioxidant systems against peroxidative damage and spermatozoon dysfunction. There are three protective enzymatic systems against reactive oxygen species (ROS) damage in sperm. These include superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase / reductase cycles. Catalase is a hemo-protein in the enzyme tetramer structure. The aim of this study was to investigate the effects of trehalose, catalase and their combinations on ram sperm parameters after the cryopreservation/ thawing process.Materials, Methods & Results: At the out of breeding season (March-May) seven rams (1-3 years of age) were used in this study. Ejaculates were collected by electro-ejaculator twice a week. Pooled ejaculates were kept at 37°C, divided into six aliquots, diluted with the Tris based extender containing Trehalose 25 mM (Group-1), Trehalose 50 mM (Group-2), Catalase 200 µg (Group-3), Catalase 400 µg (Group-4), Trehalose 50 mM + Catalase 400 µg (Group-5) and no anti-oxidant (control), respectively, were cooled to 5°C than frozen in 0.25 mL French straws on the nitrogen vapour and stored in liquid nitrogen. The extender supplemented with Group 1 (54.1 ± 1.53; 73.1 ± 4.37), Group 2 (58.3 ± 4.01; 63.1 ± 0.30) and Group 5 (56.6 ± 1.05; 58.3 ± 0.55) resulted in higher subjective motility in comparison to the control (40.0 ± 3.87; 40.5 ± 0.22) group respectively (P < 0.05).[...]

Effects of trehalose and catalase on the viability and kinetic parameters of cryopreserved ram sperm

Background: Most part of ram spermatozoa membrane has unsaturated fatty acids (phospholipids). Membrane structure of cells is composed of double ordered phospholipid layers adorned with mosaic-like protein, glycoprotein and glycolipids. Sperm freezing protocols could be negatively affected on ram sperm motility, viability and acrosome integrity during cryopreservation. For these reasons, researchers were designed their topics has led to the search for effective antioxidant systems against peroxidative damage and spermatozoon dysfunction. There are three protective enzymatic systems against reactive oxygen species (ROS) damage in sperm. These include superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase / reductase cycles. Catalase is a hemo-protein in the enzyme tetramer structure. The aim of this study was to investigate the effects of trehalose, catalase and their combinations on ram sperm parameters after the cryopreservation/ thawing process.Materials, Methods & Results: At the out of breeding season (March-May) seven rams (1-3 years of age) were used in this study. Ejaculates were collected by electro-ejaculator twice a week. Pooled ejaculates were kept at 37°C, divided into six aliquots, diluted with the Tris based extender containing Trehalose 25 mM (Group-1), Trehalose 50 mM (Group-2), Catalase 200 µg (Group-3), Catalase 400 µg (Group-4), Trehalose 50 mM + Catalase 400 µg (Group-5) and no anti-oxidant (control), respectively, were cooled to 5°C than frozen in 0.25 mL French straws on the nitrogen vapour and stored in liquid nitrogen. The extender supplemented with Group 1 (54.1 ± 1.53; 73.1 ± 4.37), Group 2 (58.3 ± 4.01; 63.1 ± 0.30) and Group 5 (56.6 ± 1.05; 58.3 ± 0.55) resulted in higher subjective motility in comparison to the control (40.0 ± 3.87; 40.5 ± 0.22) group respectively (P < 0.05).[...](AU)

Avaliação da eficácia de crioprotetores permeantes e não permeantes no descongelamento rápido e lento do sêmen canino / Evaluating the effectiveness of permeating and non-permeating cryoprotectants in fast and slow defrosting of canine semen

The aim of this study was to evaluate the association between cryoprotectants little studied in Brazil such as dimethylformamide and trehalose amid thinner, using protocols of fast and slow defrosting. Three adult Labrador Retrievier male, healthy dogs, weekly submitted to one semen collection during five-weeks period, were used. The base diluent medium used in this study was tris-citrate added with 3% of dimethylformamide + 3% glycerol (D1), 3%  dimethylformamide and trehalose (D2) and 4% glycerol (D3). At defrosting, half of the semen samples from each diluent medium was defrosted by rapid method in water-bath at 75 C for seven minutes, followed by a new immersion at 37 C for 1 minute. The other half of the samples was defrosted by slow method, in water-bath at 37 C for 1 minute. The semen was evaluated for sperm progressive motility and vigor, besides membrane integrity. For this, the semen samples were submitted to either hyposmotic and membrane integrity tests of the plasmatic membrane and acrosome  (fluorescence). The results indicated that the use of glycerol as cryoprotector in TRIS diluter provides greater efficacy in cryopreserving spermatozoa of the canine species, when compared to dimethylformamide associated with trehalose or glycerol.

O objetivo do trabalho foi avaliar o efeito da associação entre crioprotetores ainda pouco estudados no Brasil como a dimetilformamida e a trealose em meio diluidor, por meio de protocolos de descongelamento rápido e lento. Foram utilizados três cães machos, adultos, sadios da raça Labrador do Retrievier, que foram submetidos a uma coleta de sêmen semanal durante o período de cinco semanas. Os três meios diluentes utilizados neste estudo foram: (D1)-tris-citrato, acrescido de 3% de dimetilformamida + 3% de glicerol, (D2)-3% de dimetilformamida e de trealose e (D3)-4% de glicerol. No descongelamento, metade das amostras de cada meio diluente foi descongelada pelo método rápido, em banho-maria a 75 ºC por sete segundos, seguido de nova imersão a 37 ºC por 1 minuto. A outra metade foi descongelada pelo método lento, em banho-maria à 37 ºC por 1 minuto. O sêmen foi avaliado quanto à motilidade progressiva, vigor espermáticos e integridade de membrana. Pra isso, as amostras foram submetidas aos testes hipo-osmótico e de integridade da membrana plasmática e do acrossoma (fluorescência). Os resultados indicam que o uso do glicerol como crioprotetor em diluidor TRIS proporciona maior eficácia na criopreservação dos espermatozoides da espécie canina, quando comparados com a dimetilformamida associada ao glicerol ou trealose.

Avaliação da eficácia de crioprotetores permeantes e não permeantes no descongelamento rápido e lento do sêmen canino / Evaluating the effectiveness of permeating and non-permeating cryoprotectants in fast and slow defrosting of canine semen

The aim of this study was to evaluate the association between cryoprotectants little studied in Brazil such as dimethylformamide and trehalose amid thinner, using protocols of fast and slow defrosting. Three adult Labrador Retrievier male, healthy dogs, weekly submitted to one semen collection during five-weeks period, were used. The base diluent medium used in this study was tris-citrate added with 3% of dimethylformamide + 3% glycerol (D1), 3%  dimethylformamide and trehalose (D2) and 4% glycerol (D3). At defrosting, half of the semen samples from each diluent medium was defrosted by rapid method in water-bath at 75 C for seven minutes, followed by a new immersion at 37 C for 1 minute. The other half of the samples was defrosted by slow method, in water-bath at 37 C for 1 minute. The semen was evaluated for sperm progressive motility and vigor, besides membrane integrity. For this, the semen samples were submitted to either hyposmotic and membrane integrity tests of the plasmatic membrane and acrosome  (fluorescence). The results indicated that the use of glycerol as cryoprotector in TRIS diluter provides greater efficacy in cryopreserving spermatozoa of the canine species, when compared to dimethylformamide associated with trehalose or glycerol.(AU)

O objetivo do trabalho foi avaliar o efeito da associação entre crioprotetores ainda pouco estudados no Brasil como a dimetilformamida e a trealose em meio diluidor, por meio de protocolos de descongelamento rápido e lento. Foram utilizados três cães machos, adultos, sadios da raça Labrador do Retrievier, que foram submetidos a uma coleta de sêmen semanal durante o período de cinco semanas. Os três meios diluentes utilizados neste estudo foram: (D1)-tris-citrato, acrescido de 3% de dimetilformamida + 3% de glicerol, (D2)-3% de dimetilformamida e de trealose e (D3)-4% de glicerol. No descongelamento, metade das amostras de cada meio diluente foi descongelada pelo método rápido, em banho-maria a 75 ºC por sete segundos, seguido de nova imersão a 37 ºC por 1 minuto. A outra metade foi descongelada pelo método lento, em banho-maria à 37 ºC por 1 minuto. O sêmen foi avaliado quanto à motilidade progressiva, vigor espermáticos e integridade de membrana. Pra isso, as amostras foram submetidas aos testes hipo-osmótico e de integridade da membrana plasmática e do acrossoma (fluorescência). Os resultados indicam que o uso do glicerol como crioprotetor em diluidor TRIS proporciona maior eficácia na criopreservação dos espermatozoides da espécie canina, quando comparados com a dimetilformamida associada ao glicerol ou trealose.(AU)

Evaluation of stress tolerance and fermentative behavior of indigenous Saccharomyces cerevisiae

Sixty six indigenous Saccharomyces cerevisiae strains were evaluated in stressful conditions (temperature, osmolarity, sulphite and ethanol tolerance) and also ability to flocculate. Eighteen strains showed tolerant characteristics to these stressful conditions, growing at 42 ºC, in 0.04% sulphite, 1 mol L-1 NaCl and 12% ethanol. No flocculent characteristics were observed. These strains were evaluated according to their fermentative performance in sugar cane juice. The conversion factors of substrates into ethanol (Yp/s), glycerol (Yg/s) and acetic acid (Yac/s), were calculated. The highest values of Yp/s in sugar cane juice fermentation were obtained by four strains, one isolated from fruit (0.46) and the others from sugar cane (0.45, 0.44 and 0.43). These values were higher than the value obtained using traditional yeast (0.38) currently employed in the Brazilian bioethanol industry. The parameters Yg/s and Yac/s were low for all strains. The UFLA FW221 presented the higher values for parameter related to bioethanol production. Thus, it was tested in co-culture with Lactobacillus fermentum. Besides this, a 20-L vessel for five consecutive batches of fermentation was performed. This strain was genetically stable and remained viable during all batches, producing high amounts of ethanol. The UFLA FW221 isolated from fruit was suitable to produce bioethanol in sugar cane juice. Therefore, the study of the biodiversity of yeasts from different environmental can reveal strains with desired characteristics to industrial applications.(AU)