Effects of venoms on neutrophil respiratory burst: a major inflammatory function

Neutrophils play a pivotal role in innate immunity and in the inflammatory response. Neutrophils are very motile cells that are rapidly recruited to the inflammatory site as the body first line of defense. Their bactericidal activity is due to the release into the phagocytic vacuole, called phagosome, of several toxic molecules directed against microbes. Neutrophil stimulation induces release of this arsenal into the phagosome and induces the assembly at the membrane of subunits of the NAPDH oxidase, the enzyme responsible for the production of superoxide anion that gives rise to other reactive oxygen species (ROS), a process called respiratory burst. Altogether, they are responsible for the bactericidal activity of the neutrophils. Excessive activation of neutrophils can lead to extensive release of these toxic agents, inducing tissue injury and the inflammatory reaction. Envenomation, caused by different animal species (bees, wasps, scorpions, snakes etc.), is well known to induce a local and acute inflammatory reaction, characterized by recruitment and activation of leukocytes and the release of several inflammatory mediators, including prostaglandins and cytokines. Venoms contain several molecules such as enzymes (phospholipase A2, L-amino acid oxidase and proteases, among others) and peptides (disintegrins, mastoporan, parabutoporin etc.). These molecules are able to stimulate or inhibit ROS production by neutrophils. The present review article gives a general overview of the main neutrophil functions focusing on ROS production and summarizes how venoms and venom molecules can affect this function.(AU)

Effects of venoms on neutrophil respiratory burst: a major inflammatory function

Neutrophils play a pivotal role in innate immunity and in the inflammatory response. Neutrophils are very motile cells that are rapidly recruited to the inflammatory site as the body first line of defense. Their bactericidal activity is due to the release into the phagocytic vacuole, called phagosome, of several toxic molecules directed against microbes. Neutrophil stimulation induces release of this arsenal into the phagosome and induces the assembly at the membrane of subunits of the NAPDH oxidase, the enzyme responsible for the production of superoxide anion that gives rise to other reactive oxygen species (ROS), a process called respiratory burst. Altogether, they are responsible for the bactericidal activity of the neutrophils. Excessive activation of neutrophils can lead to extensive release of these toxic agents, inducing tissue injury and the inflammatory reaction. Envenomation, caused by different animal species (bees, wasps, scorpions, snakes etc.), is well known to induce a local and acute inflammatory reaction, characterized by recruitment and activation of leukocytes and the release of several inflammatory mediators, including prostaglandins and cytokines. Venoms contain several molecules such as enzymes (phospholipase A2, L-amino acid oxidase and proteases, among others) and peptides (disintegrins, mastoporan, parabutoporin etc.). These molecules are able to stimulate or inhibit ROS production by neutrophils. The present review article gives a general overview of the main neutrophil functions focusing on ROS production and summarizes how venoms and venom molecules can affect this function.(AU)

Antioxidant and antifungal activities of Camellia sinensis (L.) Kuntze leaves obtained by different forms of production

Abstract The antioxidant and anticandidal activities of leaves obtained from Camellia sinensis by non-fermentation (green and white teas), semi-fermentation (red tea) and fermentation method (black tea) were investigated. It was evaluated the total phenolic content by Folin-Ciocalteau assay; antioxidant capacities were evaluated in vitro using DPPH and ABTS radicals, hypochlorous acid and superoxide anion scavenger assays, induced hemolysis, lipid peroxidation by conjugated diene formation and myeloperoxidase activity. Anticandidal activity was performed on three strains of Candida spp. The results showed that non-fermented teas have a higher concentration of phenolic compounds, and then presented the best inhibitory activity of AAPH-induced hemolysis, the best inhibition of conjugated diene formation and more pronounced antioxidant activity in all tests. The highest anticandidal activity was obtained from fermented tea, followed by non-fermented tea. These results indicate that the antioxidant activity demonstrated has no direct relation with the anticandidal activity.

Resumo A atividade antioxidante e antifúngica das folhas obtidas da Camellia sinensis pelos métodos de não-fermentação (chás verde e branco), semi-fermentação (chá vermelho) e fermentação (chá preto) foram investigadas. Foi avaliado o conteúdo total de compostos fenólicos pelo método de Folin-Ciocalteau; a capacidade antioxidante foi avaliada in vitro usando os radicais artificiais DPPH e ABTS, o ácido hipocloroso, ensaios do ânion superóxido, hemólise induzida, peroxidação lipídica por formação de dienos conjugados e atividade da Mieloperoxidase. A atividade antifúngica foi obtida sobre três cepas de Candida spp. Os resultados obtidos mostram que os chás não fermentados apresentam a maior concentração de compostos fenólicos e também, apresentam a melhor atividade inibitória, sobre hemólise induzida por APPH, sobre a formação de dienos conjugados e a mais pronunciada atividade antioxidante sobre todos os testes. A maior atividade antifúngica foi obtida pelo chá fermentado, seguido pelo semi-fermentado e não-fermentados. Os resultados obtidos demonstram que a atividade antioxidante observada não apresenta relação com a atividade antifúngica.

Antioxidant and antifungal activities of Camellia sinensis (L. ) Kuntze leaves obtained by different forms of production / Atividade antioxidante e antifúngica das folhas de Camellia sinensis (L. ) Kuntze, obtidas por diferentes formas de produção

Abstract The antioxidant and anticandidal activities of leaves obtained from Camellia sinensis by non-fermentation (green and white teas), semi-fermentation (red tea) and fermentation method (black tea) were investigated. It was evaluated the total phenolic content by Folin-Ciocalteau assay; antioxidant capacities were evaluated in vitro using DPPH and ABTS radicals, hypochlorous acid and superoxide anion scavenger assays, induced hemolysis, lipid peroxidation by conjugated diene formation and myeloperoxidase activity. Anticandidal activity was performed on three strains of Candida spp. The results showed that non-fermented teas have a higher concentration of phenolic compounds, and then presented the best inhibitory activity of AAPH-induced hemolysis, the best inhibition of conjugated diene formation and more pronounced antioxidant activity in all tests. The highest anticandidal activity was obtained from fermented tea, followed by non-fermented tea. These results indicate that the antioxidant activity demonstrated has no direct relation with the anticandidal activity.(AU)

Resumo A atividade antioxidante e antifúngica das folhas obtidas da Camellia sinensis pelos métodos de não-fermentação (chás verde e branco), semi-fermentação (chá vermelho) e fermentação (chá preto) foram investigadas. Foi avaliado o conteúdo total de compostos fenólicos pelo método de Folin-Ciocalteau; a capacidade antioxidante foi avaliada in vitro usando os radicais artificiais DPPH e ABTS, o ácido hipocloroso, ensaios do ânion superóxido, hemólise induzida, peroxidação lipídica por formação de dienos conjugados e atividade da Mieloperoxidase. A atividade antifúngica foi obtida sobre três cepas de Candida spp. Os resultados obtidos mostram que os chás não fermentados apresentam a maior concentração de compostos fenólicos e também, apresentam a melhor atividade inibitória, sobre hemólise induzida por APPH, sobre a formação de dienos conjugados e a mais pronunciada atividade antioxidante sobre todos os testes. A maior atividade antifúngica foi obtida pelo chá fermentado, seguido pelo semi-fermentado e não-fermentados. Os resultados obtidos demonstram que a atividade antioxidante observada não apresenta relação com a atividade antifúngica.(AU)

A toxina urêmica ácido guanidinicoacético inibe o metabolismo oxidativo dos neutrófilos de cães / Uremic toxin guanidine acetic acid inhibits the oxidative metabolism of neutrophils in dogs

Among the uremic toxins proven to affect the neutrophil function in humans with chronic kidney disease(CKD), guanidine compounds stand out. To achieve a clearer understanding of the mechanisms thataffect the immunity of uremic patients, the hypothesis that guanidine acetic acid (GAA) contributesto the inhibition of oxidative metabolism and an increase in neutrophil apoptosis in healthy dogswas investigated in vitro. To this end, neutrophils isolated from ten healthy dogs were incubated inpure RPMI 1640 (control) and enriched with 5 mg/L of GAA. Capillary flow cytometry was usedto quantify superoxide production in neutrophils with the probe (hydroethidine), in the presenceand absence of phorbol-12-myristate-13-acetate (PMA), in order to assess oxidative metabolism.Apoptotic indices were quantified using the Annexin V-PE system, with and without the inductiveeffect of camptothecin. Neutrophils isolated and incubated in a GAA-enriched medium producedsmaller amounts of superoxide (p 0.001) when activated with PMA, however, this inhibition ofoxidative metabolism occurred without significantly altering their viability or rate of apoptosis. Thus, the results show guanidine compounds contribute to immunosuppression in dogs with CKD.

Dentre as toxinas urêmicas que comprovadamente afetam a função neutrofílica na doença renalcrônica (DRC) em humanos, destacam-se os compostos guanidínicos. A fim de melhor entenderos mecanismos que afetam a imunidade de pacientes urêmicos, no presente estudo foi investigadain vitro a hipótese de que o composto guanidínico ácido guanidinicoacético (AGA) contribui parainibição do metabolismo oxidativo, aumentando a apoptose dos neutrófilos de cães saudáveis. Paratal, neutrófilos isolados de dez cães saudáveis foram incubados em meio de cultura RPMI 1640puro (controle) e enriquecido com 5 mg/L de AGA. Utilizando-se citometria de fluxo capilar paraa avaliação do metabolismo oxidativo, quantificou-se a produção de superóxido dos neutrófilosempregando-se a sonda hidroetidina, com e sem a presença do estímulo com acetato miristato deforbol (PMA). O índice apoptótico foi quantificado utilizando-se o sistema Anexina V-PE, com e semo efeito indutor da camptotecina. Os neutrófilos isolados e incubados em meio enriquecido com AGA,quando ativados com PMA, produziram uma menor quantidade de superóxido (p 0,001), porém talinibição do metabolismo oxidativo ocorreu sem alterar significativamente a viabilidade e a taxa deapoptose. Assim, os resultados evidenciam que os compostos guanidínicos podem contribuir paraimunossupressão de cães com DRC.

A toxina urêmica ácido guanidinicoacético inibe o metabolismo oxidativo dos neutrófilos de cães / Uremic toxin guanidine acetic acid inhibits the oxidative metabolism of neutrophils in dogs

Among the uremic toxins proven to affect the neutrophil function in humans with chronic kidney disease(CKD), guanidine compounds stand out. To achieve a clearer understanding of the mechanisms thataffect the immunity of uremic patients, the hypothesis that guanidine acetic acid (GAA) contributesto the inhibition of oxidative metabolism and an increase in neutrophil apoptosis in healthy dogswas investigated in vitro. To this end, neutrophils isolated from ten healthy dogs were incubated inpure RPMI 1640 (control) and enriched with 5 mg/L of GAA. Capillary flow cytometry was usedto quantify superoxide production in neutrophils with the probe (hydroethidine), in the presenceand absence of phorbol-12-myristate-13-acetate (PMA), in order to assess oxidative metabolism.Apoptotic indices were quantified using the Annexin V-PE system, with and without the inductiveeffect of camptothecin. Neutrophils isolated and incubated in a GAA-enriched medium producedsmaller amounts of superoxide (p 0.001) when activated with PMA, however, this inhibition ofoxidative metabolism occurred without significantly altering their viability or rate of apoptosis. Thus, the results show guanidine compounds contribute to immunosuppression in dogs with CKD.(AU)

Dentre as toxinas urêmicas que comprovadamente afetam a função neutrofílica na doença renalcrônica (DRC) em humanos, destacam-se os compostos guanidínicos. A fim de melhor entenderos mecanismos que afetam a imunidade de pacientes urêmicos, no presente estudo foi investigadain vitro a hipótese de que o composto guanidínico ácido guanidinicoacético (AGA) contribui parainibição do metabolismo oxidativo, aumentando a apoptose dos neutrófilos de cães saudáveis. Paratal, neutrófilos isolados de dez cães saudáveis foram incubados em meio de cultura RPMI 1640puro (controle) e enriquecido com 5 mg/L de AGA. Utilizando-se citometria de fluxo capilar paraa avaliação do metabolismo oxidativo, quantificou-se a produção de superóxido dos neutrófilosempregando-se a sonda hidroetidina, com e sem a presença do estímulo com acetato miristato deforbol (PMA). O índice apoptótico foi quantificado utilizando-se o sistema Anexina V-PE, com e semo efeito indutor da camptotecina. Os neutrófilos isolados e incubados em meio enriquecido com AGA,quando ativados com PMA, produziram uma menor quantidade de superóxido (p 0,001), porém talinibição do metabolismo oxidativo ocorreu sem alterar significativamente a viabilidade e a taxa deapoptose. Assim, os resultados evidenciam que os compostos guanidínicos podem contribuir paraimunossupressão de cães com DRC.(AU)

EFEITO DE DIFERENTES DILUENTES NA QUALIDADE E FERTILIDADE DO SÊMEN REFRIGERADO DE JUMENTO PÊGA (Equus asinus)

Frente aos resultados menos expressivos no uso de sêmen refrigerado e o interesse no comércio de muares, o aprimoramento das biotecnologias é um passo importante para a espécie asinina. Dentre os principais fatores relacionados com o sucesso no uso do sêmen refrigerado destaca-se a presença do plasma seminal e a composição do diluente utilizado para garantir a viabilidade espermática durante o processo de fertilização. Os objetivos do presente estudo visam avaliar o efeito de três diluentes sobre a qualidade do sêmen asinino, com ou sem plasma seminal, bem como os efeitos da fertilidade in vivo. Foram utilizados 2 ejaculados de 6 jumentos da raça Pêga, entre 4 e 12 anos. Cada ejaculado foi diluído em meios comerciais a base de leite desnatado (BotuSêmen Special®), caseína (BotuSêmen Gold®) e gema de ovo (BotuCrio®) e as amostras processadas na presença e remoção de plasma seminal, conforme os 6 tratamentos: leite, caseína e gema de ovo com plasma na concentração de 50 milhões de espermatozoides/mL; leite, caseína e gema de ovo centrifugado e ressuspendido na concentração de 100 milhões de espermatozoides/mL. As amostras foram avaliadas a fresco (T0), 24 (T24) e 48 (T48) horas pós-refrigeração. Os parâmetros avaliados foram: cinética espermática pelo método computadorizado CASA e desestabilização da membrana, produção de espécies reativas ao oxigênio e atividade mitocondrial por citometria de fluxo. Para o teste de fertilidade foram utilizados 6 ciclos de 15 éguas em sistema crossover e um jumento Pêga com fertilidade comprovada. O diluente à base de leite apresentou resultados inferiores para motilidade total (MT), progressiva (MP) e rápidos (RAP) sem plasma seminal em todos os momentos avaliados e maior produção de ânion superóxido em T48, na presença do plasma seminal (P<0,05). Já o meio à base de caseína sem plasma seminal, apresentou resultados superiores para todos os parâmetros avaliados em todos os momentos (P<0.05), com exceção dos valores de desestabilização de membrana em T24 que não apresentou diferença significativa. Os resultados com meio à base de gema de ovo não sofreram efeito do plasma seminal e também mostraram-se superiores, com exceção de MP e RAP em T48 (P<0,05). A caseína e a gema de ovo apresentaram elevada taxa de fertilidade tanto com (73% vs 93%) quanto sem plasma seminal (93% vs 73%), respectivamente. Já o leite desnatado apresentou valores similares sem plasma seminal (60%), porém diferiu estatisticamente na presença de plasma (20%). Conclui-se que apesar de demonstrar resultados satisfatórios na refrigeração do sêmen equino, o leite desnatado apresenta pior interação com o sêmen asinino refrigerado. Já a gema de ovo e a caseína, demonstraram ser as melhores alternativas para a refrigeração de sêmen asinino por até 48 horas.

In view of the less expressive results using cooled semen and the interest in breeding mules, the improvement of biotechnologies is an important step for the donkey species. Among the main factors related to the successful use of cooled semen, the presence of seminal plasma and the composition of the extender used to guarantee sperm viability during the fertilization process stands out. The aims of this study were to evaluate the effect of three extenders on the quality of donkey semen, with or without seminal plasma, as well as the effects of in vivo fertility. Two ejaculates from 6 donkeys of the Pêga breed (4 -12y), were used. Each ejaculate was extended in commercial extender based on skimmed milk (BotuSemen Special®), casein (BotuSemen Gold®) and egg yolk (BotuCrio®) and the samples processed in the presence and removal of seminal plasma, according to the 6 treatments: milk , casein and egg yolk with plasma at a final concentration of 50 million sperm per mL; milk, casein and egg yolk centrifuged and resuspended at a final concentration of 100 million sperm per mL. The samples were evaluated fresh (T0), 24 (T24) and 48 (T48) hours after refrigeration. The parameters evaluated were: sperm kinetics by the CASA and membrane destabilization, reactive oxygen species production and mitochondrial activity by flow cytometry. For fertility test, 6 cycles of 15 mares were used in a crossover system and semen from a Pêga donkey with proven fertility. Milk-based extender had lower results for total (TM), progressive (PM) and rapid (RAP) motility without seminal plasma at all evaluated times and higher production of superoxide anion in T48, in the presence of seminal plasma (P < 0.05). Casein-based extender without seminal plasma had superior results for all parameters evaluated at all times (P <0.05), except for the values of membrane destabilization at T24, with no statistical difference. Results of egg yolk-based extender were not affected by seminal plasma and were also superior, with the exception of PM and RAP at T48 (P<0.05). Casein and egg yolk had a high fertility rate with (73% vs 93%) and without seminal plasma (93% vs 73%), respectively. On the other hand, skimmed milk had similar values without seminal plasma (60%), but it differed statistically in the presence of plasma (20%). In conclusion, despite satisfactory results for equine cooled semen, skimmed milk extender had a worse interaction with donkey cooled semen. Egg yolk and casein proved to be the best alternatives for cooling donkey semen for up to 48 hours.

Transplante ovariano: Destaques na reprodução de primatas não humanos / Ovarian transplantation: highlight to non-human primates reproduction

Background: Ovarian transplantation in nonhuman primates (NHP) has been used as a strategy for the development of experimental models for biomedical research in the reproductive area. The prospects for application of this technique range from the restoration of female fertility to the conservation of endangered wild animals. However, studies with NHP were performed mostly focusing on the ovarian transplantation of cryopreserved tissue, in order to simulate the reality of human species, as well as aiming to obtain experimental models suitable for comparative studies. On the other hand, ovarian transplantation could be applied also in NHP species preservation.Review: According to the last census (2012-2014) of the International Union for Conservation of Nature (IUCN), currently, more than half of the 633 types of primates known around the world are in danger of disappearing forever due to the continued destruction of their natural habitat by human activities. Thus, there is an interest to expose the possible methods of ovarian preservation followed by transplantation that can be employed to promote the conservation of endangered NHP. Despite the positive results obtained with avascular autograft of fresh ovarian tissue in NHP, it is important to bring in mind the significant loss of follicles as a result of this procedure due to ischemia and reperfusion during the first days after grafting. This phenomenon leads to the production of reactive oxygen species (ROS) such as hydrogen peroxide, superoxide anion and hydroxyl radical, which are responsible by lipid peroxidation, cell membrane damage and subsequent follicular atresia. An alternative to counteract this oxidative stress consists in the application/administration of different sources of antioxidants previously or during grafting, in the grafted tissue or in the animal receiving the transplant. The most used compounds with a claimed radical scavenger activity are catalase, trolox and some vitamins.[...](AU)

Transplante ovariano: Destaques na reprodução de primatas não humanos / Ovarian transplantation: highlight to non-human primates reproduction

Background: Ovarian transplantation in nonhuman primates (NHP) has been used as a strategy for the development of experimental models for biomedical research in the reproductive area. The prospects for application of this technique range from the restoration of female fertility to the conservation of endangered wild animals. However, studies with NHP were performed mostly focusing on the ovarian transplantation of cryopreserved tissue, in order to simulate the reality of human species, as well as aiming to obtain experimental models suitable for comparative studies. On the other hand, ovarian transplantation could be applied also in NHP species preservation.Review: According to the last census (2012-2014) of the International Union for Conservation of Nature (IUCN), currently, more than half of the 633 types of primates known around the world are in danger of disappearing forever due to the continued destruction of their natural habitat by human activities. Thus, there is an interest to expose the possible methods of ovarian preservation followed by transplantation that can be employed to promote the conservation of endangered NHP. Despite the positive results obtained with avascular autograft of fresh ovarian tissue in NHP, it is important to bring in mind the significant loss of follicles as a result of this procedure due to ischemia and reperfusion during the first days after grafting. This phenomenon leads to the production of reactive oxygen species (ROS) such as hydrogen peroxide, superoxide anion and hydroxyl radical, which are responsible by lipid peroxidation, cell membrane damage and subsequent follicular atresia. An alternative to counteract this oxidative stress consists in the application/administration of different sources of antioxidants previously or during grafting, in the grafted tissue or in the animal receiving the transplant. The most used compounds with a claimed radical scavenger activity are catalase, trolox and some vitamins.[...]

Effects of Lactobacillus Probiotic, P22 Bacteriophage and Salmonella Typhimurium on the Heterophilic Burst Activity of Broiler Chickens

Due to the constant evolution of industrial poultry production and the global emergence of bacterial resistance to antibiotics there has been an increasing interest in alternatives for the treatment of poultry salmonellosis, such as phage therapy and probiotics. The present study evaluated the effects of the oral administration of the bacteriophage P22 and of a probiotic, consisting of four Lactobacillus species, on the level of circulating heterophils containing a superoxide anion of one-day-old broilers challenged with Salmonella Typhimurium for seven days. It was concluded that the treatment with a probiotic with lactobacilli of broilers experimentally infected with Salmonella spp eliminates this pathogen by increasing the circulating levels of reactive heterophils. When chicks are treated with a probiotic and a bacteriophage, the agent is eliminated with no changes in circulating reactive heterophil counts. It is also concluded that the heterophils of day-old chicks are not capable of producing superoxide anion. However, this capacity is detected after 48 h of life, indicating that heterophils mature as birds age.

Effects of Lactobacillus Probiotic, P22 Bacteriophage and Salmonella Typhimurium on the Heterophilic Burst Activity of Broiler Chickens

Due to the constant evolution of industrial poultry production and the global emergence of bacterial resistance to antibiotics there has been an increasing interest in alternatives for the treatment of poultry salmonellosis, such as phage therapy and probiotics. The present study evaluated the effects of the oral administration of the bacteriophage P22 and of a probiotic, consisting of four Lactobacillus species, on the level of circulating heterophils containing a superoxide anion of one-day-old broilers challenged with Salmonella Typhimurium for seven days. It was concluded that the treatment with a probiotic with lactobacilli of broilers experimentally infected with Salmonella spp eliminates this pathogen by increasing the circulating levels of reactive heterophils. When chicks are treated with a probiotic and a bacteriophage, the agent is eliminated with no changes in circulating reactive heterophil counts. It is also concluded that the heterophils of day-old chicks are not capable of producing superoxide anion. However, this capacity is detected after 48 h of life, indicating that heterophils mature as birds age.

Effect of serum from dogs with renal failure on apoptosis and oxidative metabolism of polymorphonuclear / Efeito do soro de cães portadores de insuficiência renal sobre a apoptose e o metabolismo oxidativo dos polimorfonucleares

In recent years, uremic toxins have been widely investigated as an immunosuppressive factor for nephropathic patients. This study aimed to test the hypothesis that, as it was similarly observed in humans, the rate of apoptosis and superoxide production in polymorphonuclear leukocytes are changed in dogs treated with uremic serum. The superoxide production of polymorphonuclear leukocytes and apoptotic index of 10 healthy dogs incubated with autologous and homologous serum from healthy and uremic dogs was compared. Thus, there was an effect of partial inhibition of oxidative metabolism in uremia without correlation with the acceleration of polymorphonuclear leukocytes apoptosis in dogs.

Nos últimos anos, as toxinas urêmicas têm sido amplamente investigadas como elemento imunossupressor em pacientes nefropatas. Este trabalho objetivou testar a hipótese de que, à semelhança do que ocorre em humanos, a taxa de apoptose e a produção de superóxido em leucócitos polimorfonucleares de cães tratados com soro urêmico se alteram. Foi comparada a produção de superóxido e o índice apoptótico de leucócitos polimorfonucleares de 10 cães sadios incubados com o soro autólogo e homólogo de indivíduos sadios e urêmicos. Com isso, verificou-se efeito parcial de inibição do metabolismo oxidativo na uremia sem correlação com a aceleração da apoptose dos leucócitos polimorfonucleares de cães.

Effect of serum from dogs with renal failure on apoptosis and oxidative metabolism of polymorphonuclear / Efeito do soro de cães portadores de insuficiência renal sobre a apoptose e o metabolismo oxidativo dos polimorfonucleares

In recent years, uremic toxins have been widely investigated as an immunosuppressive factor for nephropathic patients. This study aimed to test the hypothesis that, as it was similarly observed in humans, the rate of apoptosis and superoxide production in polymorphonuclear leukocytes are changed in dogs treated with uremic serum. The superoxide production of polymorphonuclear leukocytes and apoptotic index of 10 healthy dogs incubated with autologous and homologous serum from healthy and uremic dogs was compared. Thus, there was an effect of partial inhibition of oxidative metabolism in uremia without correlation with the acceleration of polymorphonuclear leukocytes apoptosis in dogs.(AU)

Nos últimos anos, as toxinas urêmicas têm sido amplamente investigadas como elemento imunossupressor em pacientes nefropatas. Este trabalho objetivou testar a hipótese de que, à semelhança do que ocorre em humanos, a taxa de apoptose e a produção de superóxido em leucócitos polimorfonucleares de cães tratados com soro urêmico se alteram. Foi comparada a produção de superóxido e o índice apoptótico de leucócitos polimorfonucleares de 10 cães sadios incubados com o soro autólogo e homólogo de indivíduos sadios e urêmicos. Com isso, verificou-se efeito parcial de inibição do metabolismo oxidativo na uremia sem correlação com a aceleração da apoptose dos leucócitos polimorfonucleares de cães.(AU)

Effects of Lactobacillus Probiotic, P22 Bacteriophage and Salmonella Typhimurium on the Heterophilic Burst Activity of Broiler Chickens

Due to the constant evolution of industrial poultry production and the global emergence of bacterial resistance to antibiotics there has been an increasing interest in alternatives for the treatment of poultry salmonellosis, such as phage therapy and probiotics. The present study evaluated the effects of the oral administration of the bacteriophage P22 and of a probiotic, consisting of four Lactobacillus species, on the level of circulating heterophils containing a superoxide anion of one-day-old broilers challenged with Salmonella Typhimurium for seven days. It was concluded that the treatment with a probiotic with lactobacilli of broilers experimentally infected with Salmonella spp eliminates this pathogen by increasing the circulating levels of reactive heterophils. When chicks are treated with a probiotic and a bacteriophage, the agent is eliminated with no changes in circulating reactive heterophil counts. It is also concluded that the heterophils of day-old chicks are not capable of producing superoxide anion. However, this capacity is detected after 48 h of life, indicating that heterophils mature as birds age.(AU)

Extraction and in vitro antioxidant activity of exopolysaccharide by Pleurotus eryngii SI-02

The extraction parameters for Pleurotus eryngii SI-02 exopolysaccharide (EPS) produced during submerged culture were optimized using response surface methodology (RSM). The optimum conditions for EPS extraction were predicted to be, precipitation time 20.24 h, ethanol concentration 89.62% and pH 8.17, and EPS production was estimated at 7.27 g/L. The actual yield of EPS under these conditions was 7.21 g/L. The in vitro antioxidant results of the EPS showed that the inhibition effects of EPS at a dosage of 400 mg/L on hydroxyl, superoxide anion and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals were 59.63 ± 3.72%, 38.69 ± 2.59%, and 66.36 ± 4.42%, respectively, which were 12.74 ± 1.03%, 8.01 ± 0.56%, and 12.19 ± 1.05% higher than that of butylated hydroxytoluene (BHT), respectively. The reducing power of EPS of P. eryngii SI-02 was 0.98 ± 0.05, 60.66 ± 5.14% higher than that of BHT. The results provide a reference for large-scale production of EPS by P. eryngii SI-02 in industrial fermentation and the EPS can be used as a potential antioxidant which enhances adaptive immune responses.

INFLUÊNCIA DA CATALASE NO MEIO DE CONGELAÇÃO NAS CARACTERÍSTICAS DO SÊMEN CRIOPRESERVADO DE TOUROS DA RAÇA NELORE SUPLEMENTADOS OU NÃO COM GORDURA PROTEGIDA

Os ácidos graxos poliinsaturados (AGPs) são integrantes da membrana celular espermática, e embora favoreçam sua fluidez, são extremamente susceptíveis ao estresse oxidativo. Nesse contexto, o presente estudo objetivou avaliar a influência da catalase no meio de congelação para criopreservação do sêmen de machos da raça Nelore suplementados com gordura protegida, a fim de evitar a produção das espécies reativas de oxigênio (EROs). Foram utilizados 12 machos da raça Nelore mantidos em pastagem de capim-marandu em lotação contínua e taxa de lotação fixa. Os animais foram divididos em dois grupos: suplementados dos 14 aos 24 meses de idade (S+), ou não suplementados (S-). A colheita de sêmen foi realizada aos 24 meses, e as amostras de sêmen a fresco foram submetidas a analise computadorizada de cinética espermática (CASA). Posteriormente, as amostras foram divididas em duas frações e foram criopreservadas após a adição de meio de congelação, de igual composição, contendo catalase (C+) ou não (C-). Após o descongelamento do sêmen foram realizadas: avaliações de CASA; teste de termorresistência rápido (TTR); integridade das membranas plasmática e acrossomal; potencial mitocondrial, desestabilização da membrana e produção da ERO ânion superóxido (O2 -); produção da EROs; produção in vitro de embriões (PIVE) e quantificação intracitoplasmática de lipídio dos embriões. O delineamento experimental utilizado foi um esquema fatorial 2X2 (suplementação x catalase), totalizando 4 tratamentos (S+, S-, C+ e C-). A análise dos dados foi realizada utilizando o procedimento PROC MIXED do SAS. Os parâmetros de cinética espermática do sêmen a fresco foram maiores para o grupo S- (motilidade total e porcentagem de células rápidas) quando comparados com o grupo S+ (MT: 91,33 x 85,83%; RAP: 89,00 x 84,33% respectivamente) (P<0,05). Houve interação entre SUP*CAT para a frequência de batimento de cabeça (BCF) (P<0,05). O grupo S+, apresentou valores maiores, quando comparado com o grupo S- para: membrana plasmática e acrossomal lesadas (MPAL) (11,29 X 2,89% respectivamente); mediana da população total de células totais (5,66 x 4,41) (P<0,05); células lesadas (6,54 X 5,39 respectivamente) (P<0,05) e produção de O2- depois do TTR; (6,47 X 5,36 respectivamente) (P<0,05); quantificação lipídica intracitoplasmática, quando avaliado média/área de blastocistos (0,323 X 0,300 respectivamente) (P<0,05).O grupo S+ com catalase apresentou menores valores de MPAL após o TTR (10,11 X 17,69) (P<0,05). Não foram observadas diferenças entre os grupos para os valores de TTR na cinética espermática; produção de EROs e no desenvolvimento embrionário na PIVE. Os resultados do presente estudo sugerem que a dieta influenciou negativamente na qualidade seminal e possivelmente contribuiu para a peroxidação lipídica causada pelo O2-. Além disso, a dieta influenciou fortemente na quantificação lipídica intracitoplasmática. Por outro lado, a catalase no meio de congelação foi benéfica evidenciando que a suplementação com gordura protegida pode ser vantajosa se utilizada em associação com antioxidantes.

Polyunsaturated fatty acids (PUFA) comprise an important component of a sperm cell membrane. Although improving the membrane fluidity, they are extremely susceptible to oxidative stress. In this context, this study aimed to evaluate the catalase influence on extender for semens cryopreservation of Nellore Bulls supplemented with sources of PUFA in order to avoid reactive species of oxygen (ROS) production. Therefore, we have included twelve Nellore bulls kept in Marandu grass pasture in continuous stocking and fixed stocking rate. Bulls were divided in two groups: supplemented at 14 to 24 months old (S+), or non-supplemented (S-). The sperm collections were performed at 24 months old and fresh semen samples were analyzed by computer-assisted sperm analysis (CASA). Subsequently, we have divided these samples into two equal portions, which were both cryopreserved after same composition extender medium addition, containing catalase (C+) or not (C-). After thawing semen, computer-assisted sperm analysis (CASA); rapid test of thermo resistance (TTR); plasma membrane integrity, acrosomal membrane integrity; mitochondrial membrane potential, membrane destabilization; production of ROS superoxide anion (O2 -); ROS ; in vitro embryo production (IVEP) and intracellular lipid content of embryos were performed. The experimental design used was a 2X2 factorial (supplementation x catalase), with 4 treatments (SUPL, CONTR, C+ e C-). Data analysis was performed using SAS PROC MIXED procedure. Sperm kinetic parameters of fresh semen were higher for S- (total motility and percentage of rapid sperm) than S+ (MT: 91.33 x 85.83%; RAP: 89.00 x 84.33% respectively) (P<0.05). We have also observed an interaction between SUP*CAT for beat cross frequency (BCF) (P<0.05). The S+ group has presented higher values compared to S- for damage percentage of plasma and acrosomal membranes (DPAM) (11.29 X 2.89%, respectively); median for total population of total cells (5.66 x 4.41, respectively) (P<0.05); damaged cells (6.54 x 5.39, respectively) (P<0.05) and O2- production after TTR 96.47 x 5.36, respectively) (P<0.05); intracellular lipid content, when evaluated the mean/area of blastocysts (0.323 X 0.300 respectively) (P<0.05). The S+ group with catalase presented lower DPAM values after TTR (10.11 X 17.69 respectively) (P <0.05). No differences were observed between the groups for TTR values on spermatic kinetics, ROS production and on embryonic development in IVEP. Our results suggest that diet has negatively influenced seminal quality and possibly contributed to lipid peroxidation caused by O2-. In addition, diet strongly influenced intracytoplasmic lipid quantification. On the other hand, catalase in the extent was beneficial in showing that supplementation with protected fat may be advantageous if used in combination with antioxidants.

Estresse oxidativo seminal em cães: Estudo da susceptibilidade dos espermatozoides e possíveis terapias durante a criopreservação

Resumo: O desequilíbrio entre a capacidade antioxidante e a produção de espécies reativas de oxigênio (EROs), também conhecido como estresse oxidativo (EO), pode ser extremamente prejudicial aos espermatozoides. Essa célula é altamente susceptível ao EO devido ao seu citoplasma reduzido e consequente limitação na reserva antioxidante citoplasmática. Além disso, os espermatozoides exibem em sua membrana uma grande quantidade de ácidos graxos polinsaturados (PUFAs), facilmente oxidadas e vulneráveis a peroxidação lipídica. Por outro lado, esses ácidos graxos, conferem fluidez a membrana plasmática, desempenhando um importante papel nos processos de fertilização além de proteger os espermatozoides durante a criopreservação. Com base nessas afirmações, o tratamento com ácidos graxos polinsaturados durante a criopreservação parece ser uma terapia interessante. No entanto, parece plausível associar essa terapia com um tratamento antioxidante para prevenir a exacerbada peroxidação lipídica. Portanto, o objetivo deste estudo foi associar o tratamento antioxidante com suplementação de PUFA, o ácido docosaexaenoico (DHA), ao diluidor para a criopreservação de sêmen canino. Para isso, foram desenvolvidos dois experimentos visando a melhora da qualidade do sêmen criopreservado, utilizando-se em ambos, o sêmen de oito cães saudáveis e sexualmente maduros e avaliações das amostras para motilidade, membrana plasmática e integridade acrossomica, integridade do DNA, atividade e função mitocondrial e susceptibilidade da peroxidação lipídica. No primeiro estudo avaliou-se a susceptibilidade espermática a diferentes desafios de indução oxidativa (ânion superóxido [O2-], peróxido de hidrogênio [H2O2], radical hidroxil [OH-]e malondialdeído [MDA]) na presença ou ausência de plasma seminal (PS). Sêmen com PS teve função mitocondrial preservada contra EROs. No entanto, na ausência de PS, H2O2 reduziu o potencial da membrana mitocondrial. Além do mais, independentemente do PS, H2O2 foi deletério para a cinética espermática e para as membranas plasmática/acrossomal, e OH- reduziu a atividade mitocondrial e aumentou a fragmentação do DNA. No entanto, amostras com PS foram mais resistentes para a peroxidação lipídica. O segundo estudo foi feito testando o DHA em três concentrações diferentes, para isso foi dividido em quatro grupos: controle, 1M, 5M e 10M DHA. Foram adicionados ao diluidor de sêmen, congelados, descongelados e analisados. Foram constatadas diferenças significativas entre o grupo controle e 1M, com este último apresentando menores valores para motilidade progressiva e espermatozoides rápidos; o grupo 5M obteve maiores valores de espermatozoides lentos e menores para espermatozoides estáticos e; o grupo 10M maiores valores para espermatozoides estáticos. Desta forma, selecionamos a concentração de 5M DHA para associar com os antioxidantes catalase e vitamina E no diluidor (que combatem o radical OH- e o H2O2, respectivamente), baseando-se no primeiro estudo. Foram adicionados ao diluidor de sêmen e divididos em quatro grupos (todos contendo 5m DHA): controle, vitamina E, catalase e vitamina E mais catalase. As amostras foram congelados, descongelados e avaliadas. Os resultados mostraram que 5M DHA mais vitamina E teve efeitos benéficos nas características cinéticas espermáticas, como velocidade média de percurso (VAP), velocidade linear (VSL), motilidade, motilidade progressiva e espermatozoides rápidos; enquanto a suplementação de 5M DHA mais catalase (300U/mL) não mostrou o efeito benéfico potencializado esperado, indicando que esse antioxidante parece ter toxicidade para o espermatozoide na concentração utilizada em nosso experimento. Finalmente, 5M DHA associado a vitamina E (0,6mM) mais catalase (300U/mL), não aumentou a qualidade da cinética e estrutura espermáticas, mas melhorou a resistência ao estresse oxidativo.

Abstract: The imbalance between the antioxidant capacity and the production of reactive oxygen species (ROS), also known as oxidative stress (EO), can be extremely harmful to mammalian sperm. This cell is highly susceptible to EO due to its reduced cytoplasm and consequent limitation on the enzymatic antioxidant reserve. In addition, sperm exhibit a large amount of polyunsaturated fatty acids (PUFAs), which confer fluidity to the plasm membrane, playing an important role in the fertilization processes. Also, PUFAs are known to protect the sperm during the cryopreservation process. However, PUFAs are easily oxidized and vulnerable to lipid peroxidation. Based on these statements, treatment with polyunsaturated fatty acids during cryopreservation seems to be an interesting therapy. However, it seems plausible to associate this therapy with an antioxidant treatment to prevent exacerbated lipid peroxidation. Therefore, the objective of this study was to associate the antioxidant treatment with docosaexaenoic acid (DHA) supplementation to the cryopreservation extender of canine semen. Towards this aim, two experiments were developed aiming to improve the quality of cryopreserved semen. For both experiments, semen samples of eight healthy and sexually mature dogs were collected. Post-thaw semen evaluations consisted of motility, plasma and acrosome membrane integrity, DNA status, mitochondrial activity and function, and susceptibility of lipid peroxidation. In the first study, we aimed to assess which ROS is the most deleterious for canine semen in the absence of seminal plasma (condition required for the cryopreservation). This was performed in order to define the ideal antioxidant for semen cryopreservation. Therefore, semen samples were incubated with different oxidative induction challenges (superoxide anion [O2-], hydrogen peroxide [H2O2], hydroxyl radical [OH-] and malondialdehyde [MDA]) in the presence or absence of seminal plasma (PS). Semen with PS had a mitochondrial function preserved against ROS. However, in the absence of PS, H2O2 reduced the potential of the mitochondrial membrane. Moreover, regardless of the PS, H2O2 was deleterious for the sperm kinetics and for the plasmatic/acrossomal membranes, and OH- reduced mitochondrial activity and increased DNA fragmentation. However, PS samples were more resistant to lipid peroxidation. The second study was performed by testing DHA in three different concentrations. Ejaculates were divided into four groups: control, 1M, 5M and 10M DHA added to the semen extender. Samples were cryopreserved, thawed and analyzed. Significant differences were found between the control group and 1M, with the latter presenting lower values for progressive motility and rapid spermatozoa; the 5M group showed higher values of slow and lower percentage of static sperm; the group 10M presented higher values for static sperm. Therefore, we selected the concentration of 5M DHA to associate with the antioxidants catalase and vitamin E in the extender (specific for OH- and the H2O2, respectively), based on the first study. Antioxidants were added to the semen extender and divided into four groups (all containing 5M DHA): control, vitamin E, catalase and vitamin E plus catalase. Samples were cryopreserved, thawed and evaluated. The results showed that 5M of DHA plus vitamin E (0,6mM) had beneficial effects on the sperm kinetic characteristics, such as mean route velocity (VAP), linear velocity (VSL), motility, progressive motility and percentage of rapid sperm. On the other hand, 5M of DHA associated with vitamin E (0,6mM) plus catalase (300u/ML), did not increase the quality of the kinetics and spermatic structure, but improved the resistance to oxidative stress.

SUPLEMENTAÇÃO DIETÉTICA DE GLUTAMINA NO DESEMPENHO PRODUTIVO, MORFOLOGIA INTESTINAL, RESPOSTAS HEMÁTICAS, ENZIMÁTICAS E IMUNOLÓGICAS DA TILAPIA-DO-NILO SUBMETIDA A DESAFIO BACTERIANO

Dois experimentos foram realizados para avaliar a suplementação de glutamina (Gln) em dietas para tilápia-do-nilo. Estudo I objetivou determinar o nível ótimo de suplementação dietética de Gln por meio das respostas de desempenho produtivo, composição corporal, morfometria intestinal e capacidade antioxidante. Foram distribuídos 440 juvenis de tilápia-donilo (4,4 ± 0,1g) em 40 aquários de 250 L, em sistema de recirculação. Dietas práticas foram formuladas contendo níveis de suplementação de Gln de 0; 0,5; 1,0; 1,5 e 2,0 % em delineamento inteiramente casualizado com oito repetições. Os peixes foram arraçoados quatro vezes ao dia, por 90 dias. A suplementação desse aminoácido em concentrações entre 1 e 2% influenciou positivamente o peso e comprimento relativos do intestino, altura de vilosidades, relação vilo:cripta e parâmetros de desempenho produtivo. Houve aumento na concentração de glutationa reduzida (GSH) no intestino de animais que receberam a suplementação de Gln. O Estudo II avaliou a contribuição da suplementação de Gln nas respostas hematológicas, imunológicas e bioquímicas, antes e após o desafio bacteriano por Aeromonas hydrophila. Após o ensaio de desempenho, 60 peixes (244,5 ± 10,2 g) foram inoculados intraperitonealmente com agente patógeno e transferidos para a sala experimental de desafio. Ao final de 15 dias foram analisados os parâmetros hematológicos, imunológicos e bioquímicos. Os parâmetros hematológicos e índices hematimétricos não foram afetados pelos tratamentos. A suplementação dietética de 1,5% de Gln resultou em aumento significativo na concentração sérica de lisozima. Os demais parâmetros imunológicos não foram afetados pelos tratamentos, somente pelo desafio bacteriano comparando-se os períodos. De forma geral, a suplementação dietética de Gln promoveu o desenvolvimento e funcionamento intestinal, além de apresentar efeitos positivos na atividade de lisozima sérica, sistema antioxidante e de desempenho produtivo. O nível estimado de suplementação de Gln em dietas para juvenis de tilápia-do-nilo foi de 1,40%.

- Glutamine supplementation of culture media for Nile tilapia immune cells ABSTRACT: Under appropriate conditions, glutamine (Gln) is essential for cell proliferation, acting as a respiratory fuel for enterocytes and lymphocytes, with positive effects on the function of stimulated immune cells. Thus, specific components of both innate and adaptive immune systems of Nile tilapia were evaluated after supplementation of culture media with glutamine. Primary cell cultures of head-kidney macrophages were used for respiratory burst and phagocytic activity assessment. The ability of macrophages to kill Streptococcus iniae was also evaluated. Additionally, proliferation assays were conducted with peripheral blood lymphocytes (PBL) and head-kidney leukocytes exposed to non-specific mitogens. Macrophage phagocytosis, anion superoxide production and bactericidal capacity were significantly (P < 0.05) enhanced by Gln supplementation to culture media. Proliferation of naïve T- and B lymphocytes upon mitogenic exposure was also significantly (P < 0.05) enhanced by supplementing Gln to the media. These results suggest that in vitro, Gln is essential as substrate and immunomodulator of both innate and adaptive responses in Nile tilapia leukocytes, highlighting its potential application as an immunonutrient. Dietary glutamine supplementation on growth performance, intestinal morphology, hematic, enzymatic and immunological responses of Nile tilapia juveniles subjected to bacterial challenge Abstract: Two experiments were conducted to evaluate the effect of glutamine (Gln) supplementation in Nile tilapia diets. Study I: aimed to determine the optimal level of Gln supplementation through productive performance responses, body composition, intestinal morphology and antioxidant capacity. Four hundred Nile tilapia juveniles (4.4 ± 0.1 g) were distributed in 40 aquaria (250 L each), in a recirculating water system. The experimental design utilized was completely randomized with five treatments (0, 0.5, 1.0, 1.5 and 2.0 % Gln) and eight replicates. The fish were hand fed four times a day for 90 days. Glutamine supplementation improved relative weight and length of intestine, as well as villus length and villus:crypt ratio, which could be related to the lower feed conversion ratio found on fish fed Gln-supplemented diets. Final body weight, weight gain and specific growth rate were also positively affected by Gln supplementation. Fish fed Gln diets showed increased gluthatione (GSH) concentrations in the intestine. Study II: the contribution of Gln supplementation in hematological, immunological and biochemical responses was assessed under bacterial challenge by Aeromonas hydrophila. After the feeding trial, 60 fish (244.5 ± 10.2 g) were inoculated intraperitoneally with the pathogen and transferred to the experimental challenge room. After 15 days, hematological, immunological and biochemical parameters were analyzed. The bacterial challenge affected the hematological and immunological parameters. However, Gln supplementation presented limited effect on these parameters, except for lysozyme concentration, which significantly increased on fish fed 1.5% Gln diets. In sum, dietary Gln supplementation promoted intestinal development and function, improved immune response and antioxidant system, as well as the growth performance. The dietary Gln supplementation level was estimated to be 1.40%.

Composto homeopático reduz a liberação de ânion superóxido pelas células mononucleares de ema (Rhea americana) / Homeopathic compound reduces the release of superoxide anion by mononuclear cells of rhea (Rhea americana)

The action of the "Stress Factor Ostrich (Arenales - Fauna and Flora)" was tested in the release of superoxide anion by cells in the peripheral blood of rhea (Rhea americana). Sixteen samples of 0.5mL of venous blood were collected through the jugular vein in the morning and placed in heparinized tubes. The leukocytes were separated into polymorphonuclear (PMN) and mononuclear (MN). The production of superoxide anion by phagocytes of peripheral blood was determined using the chromogen ferricytochrome C. There was a reduction of superoxide by MN cells in the presence of "Stress Factor Ostrich" indicating a positive influence of product against oxidative stress. Furthermore, future researches, such as the evaluation of other reactive oxygen intermediates and antioxidant enzymes, researches.

Composto homeopático reduz a liberação de ânion superóxido pelas células mononucleares de ema (Rhea americana) / Homeopathic compound reduces the release of superoxide anion by mononuclear cells of rhea (Rhea americana)

The action of the "Stress Factor Ostrich (Arenales - Fauna and Flora)" was tested in the release of superoxide anion by cells in the peripheral blood of rhea (Rhea americana). Sixteen samples of 0.5mL of venous blood were collected through the jugular vein in the morning and placed in heparinized tubes. The leukocytes were separated into polymorphonuclear (PMN) and mononuclear (MN). The production of superoxide anion by phagocytes of peripheral blood was determined using the chromogen ferricytochrome C. There was a reduction of superoxide by MN cells in the presence of "Stress Factor Ostrich" indicating a positive influence of product against oxidative stress. Furthermore, future researches, such as the evaluation of other reactive oxygen intermediates and antioxidant enzymes, researches.(AU)