Erythrogram and oxidative stress in confined cattle fed with Brachiaria sp hay and supplemented with antioxidants / Eritrograma e estresse oxidativo em bovinos confinados alimentados com feno de Brachiaria sp. e suplementados com antioxidantes

Brachiaria sp contains sporidesmin that can be oxidized by lipoperoxidation and cause oxidative stress. In the present study we evaluated the effects of different antioxidants on lipoperoxidation of erythrocytes from Nelore cattle fed with Brachiaria sp hay. The experimental design was entirely randomized, in which 40 whole male cattle were divided into five treatments (G1: control - no supplementation; G2: selenium and vitamin E supplementation; G3: zinc supplementation; G4: selenium supplementation and G5: vitamin E supplementation) and allocated in feedlot pens for 105 days. The samples heparinized and withethylenediaminetetraacetic acid (EDTA) were obtained every 28 days for hematological and oxidative stress evaluation (0, 28 56, 84 and 105 days). In the erythrogram total erythrocyte count, hemoglobin, and hematocrit (Ht) were measured. For the evaluation of oxidative stress, in order to analyze the characteristics of the erythrocyte membrane, the thiobarbituric acid reactive substances (TBARS), total glutathione (GSH-T), glutathione peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD) were determined. The results showed that regardless of the treatment there was no oxidative stress during the experimental confinement period and that the joint association of selenium and vitamin E in the bovine diet provided a lower incidence of deleterious alterations on erythrocytes.

As Brachiaria sp contêm esporidesminas que podem ser oxidadas por lipoperoxidação e ocasionar estresse oxidativo. No presente estudo foram avaliados os efeitos de diferentes antioxidantes na lipoperoxidação dos eritrócitos de bovinos da raça Nelore, alimentados com feno de Brachiaria sp. O delineamento experimental foi inteiramente casualizado, em que 40 bovinos machos, inteiros, foram divididos, em cinco tratamentos (G1: controle - sem suplementação; G2: suplementação de selênio e vitamina E; G3: suplementação de zinco; G4: suplementação de selênio e G5: suplementação de vitamina E) e alocados em baias de confinamento, por 105 dias. As amostras de plasma heparinizado ou com ácido etilenodiamino tetra-acético (EDTA) foram obtidas a cada 28 dias para avaliação hematológica e de estresse oxidativo (0, 28 56, 84 e 105 dias). No eritrograma foi mensurado a contagem total de eritrócitos, a hemoglobina e o volume globular (VG). Para a avaliação do estresse oxidativo, com o objetivo de analisar as características da membrana do eritrócito foram determinadas as substâncias reativas ao ácido tiobarbitúrico (TBARS), glutationa total (GSH-T), glutationa peroxidase (GSH-Px), catalase (CAT) e superóxido dismutase (SOD). Os resultados demonstraram que independente do tratamento não houve estresse oxidativo durante o período do confinamento experimental e que a associação conjunta de selênio e vitamina E na dieta dos bovinos proporcionaram menor incidência de alterações deletérias sobre os eritrócitos.

Efeitos da suplementação com anetol durante a MIV e CIV na maturação oocitária e na produção e qualidade de embriões bovinos

A produção in vitro de embriões (PIV) vem apresentando um crescimento significativo, representando 73,7% dos embriões produzidos mundialmente em 2019. Apesar de sua viabilidade comercial, a PIV necessita de aprimoramento para expandir ainda mais seu potencial. Desta forma, o aumento nas taxas de produção e qualidade embrionária proporcionaria ganhos substanciais na pecuária. Objetivou-se avaliar o efeito do anetol na MIV, nas doses de 300 e 3000µg/mL (Experimento 1) e avaliar o efeito do anetol na MIV e/ou CIV, na dose de 300µg/mL, na produção e qualidade de embriões bovinos (Experimento 2). Foram coletados ovários provenientes de frigoríficos utilizados nos dois experimentos. Folículos de 2 a 8mm foram puncionados e submetidos à maturação com meio maturação in vitro (MIV) suplementado com SFB 10%. No experimento 1, foram divididos em 3 grupos experimentais, sendo o Grupo Controle composto por (meio base); Grupo M300 (meio base suplementado com 300 µg/ml de anetol) e Grupo M3000 (3000 µg/ml). Após a MIV, a maturação nuclear foi avaliada por meio da técnica de análise da configuração da cromatina para obtenção dos resultados de metáfase II. No experimento 2, a MIV foi realizada com meio base (Grupo Controle) ou suplementado com (300 µg/ml de anetol). Posteriormente, os oócitos foram submetidos à fecundação in vitro (FIV) e cultivo in vitro (CIV), sendo divididos em 04 grupos: Grupo Controle M0-C0 maturados e cultivados em meio sem adição de anetol, Grupo M0-C300 maturados sem anetol e cultivados em meio com 300 µg/ml, Grupo M300-C0 µg/ml maturados com 300 µg/ml de anetol e cultivados sem anetol e o Grupo M300-C300 com meio contendo 300 µg/ml de anetol na maturação e no cultivo. No 3º dia do cultivo (D3), após a FIV foi avaliado a clivagem, no 7º dia após a FIV (D7), foi avaliado a produção de blastocistos e no 8º dia (D8) foi avaliado e classificado os blastocistos por meio de analise morfológica em estereomicroscópico. Como análise estatística foi utilizado a análise de variância, com on way ANOVA, utilizando o software GraphPad Prism versão 6.0 (GraphPad Software Inc, La Jolla, CA, USA), e testes de Tukey (dados paramétricos). Observou-se que 300 µg/mL de anetol no meio MIV não promoveu diferença nas taxas de MII quando comparado ao grupo controle. Entretanto, a adição de 3000 µg/mL de anetol reduziu significativamente a porcentagem de CCOs que chegaram à metáfase II em relação aos demais grupos (P0,05). Em relação ao índice de clivagem, observamos que a adição de 300 µg/ml de anetol durante a MIV e a CIV aumentou a porcentagem de embriões clivados quando comparado aos demais grupos experimentais (P 0,05). Entretanto, nenhuma diferença foi encontrada entre os grupos quanto a produção embrionária, avaliada pela taxa de formação de blastocistos no D8 (P>0,05), nem sobre a qualidade embrionária, obtida por meio da contagem do número total de células embrionárias (P>0,05). Apesar das evidencias dos efeitos antioxidantes do anetol, encontradas na literatura, neste estudo o teste de TBARS realizado no meio de produção de embriões não apresentou significância estatística. Os genes SOD 1, CAT e GST relacionados a resistência ao estresse oxidativo, apresentaram aumento em sua abundancia nas células da granulosa quando foi utilizado o anetol. Alem disso, os genes COX2 e HAS também se apresentaram elevados, ressaltando o efeito benéfico do anetol sobre o metabolismo das CCs. Os embriões produzidos com ócitos maturados em presença de anetol também apresentaram elevação de transcritos do gene GPX1 que está envolvido na metabolização do peróxido de hidrogenio. A elevação da abundancia destes transcritos demonstra a contribuição do anetol para a neutralização do efeito das EROS. Por outro lado, apesar do alto número de embriões produzido no grupo maturado e cultivado com anetol, os genes IFNT2 e HASPA1A se encontravam diminuídos. Conclui se que a adição de 300 µg/mL de anetol durante a MIV e a CIV melhora a clivagem embrionária sem comprometer o desenvolvimento posterior do blastocisto.

INTRODUCTION: In vitro embryo production (IVP) has shown significant rise, reaching 68.7% of the embryos produced worldwide in 2018. Despite its commercial viability, IVP needs improvement to further expand its potential. Thus, livestock would benefit with substantial gains from increased production rates and embryo quality. OBJECTIVE: The objective was to evaluate the effect of anethole on in vitro maturation (IVM) at doses of 300 µg/mL and 3,000 µg/mL (Experiment 1) and to evaluate the effect of anethole on IVM and/or in vitro culture (IVC) at a dose of 300 µg/mL on the production and quality of bovine embryos (Experiment 2). MATERIAL AND METHODS: A total of 700 oocytes from ovaries from slaughterhouses were collected to be used in the two experiments. The 2 to 8 mm follicles were punctured and submitted to maturation with IVM medium supplemented with 10 % fetal bovine serum (FBS). In Experiment 1, a total of 300 oocytes were used and divided into 3 experimental groups: the Control Group, composed of basal medium; Group M300 (basal medium supplemented with 300 µg/ml anethole); and Group M3000 (basal medium supplemented with 3,000 µg/ml). After IVM, nuclear maturation was evaluated using chromatin configuration analysis technique to obtain the results of metaphase II (MII) with the best concentration, which was used in Experiment 2. In Experiment 2, IVM was performed either with basal medium only (Control Group) or with supplementation (Group M300 300 µg/ml anethole). Subsequently, 400 oocytes were subjected to in vitro fertilization (IVF) and IVC, and then divided into four groups: Control Group M0-C0: matured and cultivated in medium without addition of anethole; Group M0-C300: matured without anethole and cultivated in medium with 300 µg/ml anethole; Group M300-C0: matured with 300 µg/ml anethole and cultivated without anethole; and Group M300-C300: matured and cultivated in medium containing 300 µg/ml anethole. On the 3rd day of culture (D3), cleavage was evaluated after IVF; on the 7th day after IVF (D7), blastocyst production was evaluated; and on the 8th day (D8), blastocysts were evaluated and classified through morphological analysis in a stereomicroscope. Statistical analysis was carried out by using the one-way analysis of variance (one-way ANOVA), as well as Tukey tests for parametric data. RESULTS: When 300 µg/mL anethole were added to the MIV medium, there was no difference in MII rates in comparison to the control group. However, the addition of 3,000 µg/mL anethole significantly reduced the percentage of cumulus-oocyte complex (COC) that reached MII in relation to the other groups (P 0.05). Regarding the cleavage index, the addition of 300 µg/ml anethole during IVM and IVC was observed to increase the percentage of cleaved embryos when compared to the other experimental groups (P 0.05). Nevertheless, no difference was found between the groups regarding embryo production, which was assessed by blastocyst formation rate on D8 (P > 0.05), nor related to embryo quality, which was obtained by counting the total number of embryonic cells (P > 0.05). In spite of the evidence of antioxidant effects of anethole pointed out in the literature, the TBARS test performed in this study in the embryo production medium did not show any statistical significance. The SOD1, CAT, and GST genes related to resistance to oxidative stress presented increased abundance in granulosa cells when anethole was used. In addition, the COX2 and HAS genes were also elevated, highlighting the beneficial effect of anethole on the metabolism of COCs. Similar to in embryos produced with cells matured in the presence of anethole, a rise was detected in transcripts of the GPX1 gene, which is involved in the metabolization of hydrogen peroxide. The increased abundance of these transcripts demonstrates the role of anethole in neutralizing the effect of ROS. On the other hand, regardless of the large number of embryos produced in the group matured and cultivated with anethole, the IFNT2 and HASPA1A genes were decreased. CONCLUSION: The addition of 300 µg/mL anethole during IVM and IVC improves embryonic cleavage without compromising blastocyst development.

Development of innate immune response in healthy Holstein calves from birth until weaning.

The general aim of this research was to evaluate the hematological and innate immune profile in Holstein calves naturally exposed to microorganisms during pre-weaning phase. It was performed a transversal study including 175 dairy calves distributed in three age groups: Group 1 - 1 to 7 days of life (n=65), Group 2 - 30 to 40 days of life (n=52) and Group 3 - 60 to 89 days of life (n=58) and also distributed in 2 subgroups, healthy and unhealthy, according to clinical examination findings. Blood samples were harvested in tubes with and without EDTA anticoagulant and heparin for Red Blood Cells (RBC) and White Blood Cell (WBC) count determination by using ADVIA 2120 system, biochemical markers and for immune response assays. The flow cytometry technique was used to assess polymorphonuclear (PMN) and mononuclear (MN) cells phagocytosis of pre labeled Escherichia coli, Staphylococcus aureus and Mannheimia haemolytica. Production of Reactive Oxygen Species (ROS) was assessed in leucocytes by use of the fluorescent dye dihydrorhodamine 123 and then the response ratio (RR) was calculated to compare data between the groups/subgroups. The predominant disease detected in unhealthy neonate calves (G1) was diarrhea (46%), while Bovine Respiratory Disease (BRD) was the most important illness in G2 (63%) and G3 (59%). Regard to age physiological response, the most differences were observed contrasting G1 with G2 and G3 observing in neonates a decrease trend of RBC and serum iron also a pro inflammatory profile under natural challenges. Most of phagocytosis parameters (MN and PMN) were independent of age or healthy groups, although the amount of ROS (AFU) decreased through advance of age. G1 unhealthy subgroup presented low concentration of total solids and total protein associated with low number of RBC and leukocytosis due to neutrophilia and lymphopenia compared with healthy subgroup. Production of ROS (AFU) for M. haemolytica was higher in unhealthy than healthy neonates. G2 unhealthy subgroup presented subtle changes in hematological and innate immune response, however the concentration of total solids, protein and ROS production and RR for all antigens were lower in the unhealthy group. G3 unhealthy animals presented increase of MN cells in WBC count, and both MN and PMN cells were more phagocytic responsive for all antigens, especially M. haemolytica, also in ROS assay, compared with healthy calves. This research has evidences that neonate had a high innate immune response intensity against natural exposition to pathogens, while the intermediate heifers (G2) presented immunologically less responsive to antigens associated with the high rate of BRD. G3 group seen to be more prepared to face the challenges presented during pre-weaning phase as the immune response was more effective against the antigens. The assumption is that the differences reported are associated with the concentration of colostrum maternal factors that act enhancing the innate immune response of neonate calves, with a gradual decrease of immune response in animals with 30 to 40 days of life (G2) and subsequently development of the active adaptative response in G3

The general aim of this research was to evaluate the hematological and innate immune profile in Holstein calves naturally exposed to microorganisms during pre-weaning phase. It was performed a transversal study including 175 dairy calves distributed in three age groups: Group 1 - 1 to 7 days of life (n=65), Group 2 - 30 to 40 days of life (n=52) and Group 3 - 60 to 89 days of life (n=58) and also distributed in 2 subgroups, healthy and unhealthy, according to clinical examination findings. Blood samples were harvested in tubes with and without EDTA anticoagulant and heparin for Red Blood Cells (RBC) and White Blood Cell (WBC) count determination by using ADVIA 2120 system, biochemical markers and for immune response assays. The flow cytometry technique was used to assess polymorphonuclear (PMN) and mononuclear (MN) cells phagocytosis of pre labeled Escherichia coli, Staphylococcus aureus and Mannheimia haemolytica. Production of Reactive Oxygen Species (ROS) was assessed in leucocytes by use of the fluorescent dye dihydrorhodamine 123 and then the response ratio (RR) was calculated to compare data between the groups/subgroups. The predominant disease detected in unhealthy neonate calves (G1) was diarrhea (46%), while Bovine Respiratory Disease (BRD) was the most important illness in G2 (63%) and G3 (59%). Regard to age physiological response, the most differences were observed contrasting G1 with G2 and G3 observing in neonates a decrease trend of RBC and serum iron also a pro inflammatory profile under natural challenges. Most of phagocytosis parameters (MN and PMN) were independent of age or healthy groups, although the amount of ROS (AFU) decreased through advance of age. G1 unhealthy subgroup presented low concentration of total solids and total protein associated with low number of RBC and leukocytosis due to neutrophilia and lymphopenia compared with healthy subgroup. Production of ROS (AFU) for M. haemolytica was higher in unhealthy than healthy neonates. G2 unhealthy subgroup presented subtle changes in hematological and innate immune response, however the concentration of total solids, protein and ROS production and RR for all antigens were lower in the unhealthy group. G3 unhealthy animals presented increase of MN cells in WBC count, and both MN and PMN cells were more phagocytic responsive for all antigens, especially M. haemolytica, also in ROS assay, compared with healthy calves. This research has evidences that neonate had a high innate immune response intensity against natural exposition to pathogens, while the intermediate heifers (G2) presented immunologically less responsive to antigens associated with the high rate of BRD. G3 group seen to be more prepared to face the challenges presented during pre-weaning phase as the immune response was more effective against the antigens. The assumption is that the differences reported are associated with the concentration of colostrum maternal factors that act enhancing the innate immune response of neonate calves, with a gradual decrease of immune response in animals with 30 to 40 days of life (G2) and subsequently development of the active adaptative response in G3

Espécies reativas do oxigênio e as doenças respiratórias em grandes animais

The reactive oxygen species (ROS) are unstable and highly reactive molecules capable of transforming other molecules with which they collide. The ROS are generated in large numbers during the oxidative stress, a condition in which molecules are affected such as proteins, carbohydrates, lipids and nucleic acid. This research discussed the main concepts of the free radicals and ROS: main types, their training and how they act on the cellular structures causing significant tissue injury. The main antioxidant defense systems and the influence of the increase in the production of ROS in the respiratory tract of large animals are also discussed, with emphasis on the involvement of ROS in diseases such as pneumonia in ruminants and recurrent obstruction of the airways and pulmonary hemorrhage induced by exercise in horses.

As espécies reativas do oxigênio (ERO) são moléculas instáveis e extremamente reativas capazes de transformar outras moléculas com as quais colidem. As ERO são geradas em grande quantidade durante o estresse oxidativo, condição em que são afetadas moléculas como proteínas, carboidratos, lipídeos e ácido nucleicos. Neste trabalho, são discutidos os principais conceitos sobre os radicais livres e as ERO: principais tipos, sua formação e a forma como atuam sobre as estruturas celulares, provocando lesão tecidual significativa. Os principais sistemas de defesa antioxidantes e a influência do aumento na produção dessas ERO no trato respiratório de grandes animais também são discutidos, dando ênfase ao envolvimento das ERO em doenças como a pneumonia em ruminantes e na obstrução recorrente das vias aéreas e a hemorragia pulmonar induzida por exercício em equinos.

Espécies reativas do oxigênio e as doenças respiratórias em grandes animais

The reactive oxygen species (ROS) are unstable and highly reactive molecules capable of transforming other molecules with which they collide. The ROS are generated in large numbers during the oxidative stress, a condition in which molecules are affected such as proteins, carbohydrates, lipids and nucleic acid. This research discussed the main concepts of the free radicals and ROS: main types, their training and how they act on the cellular structures causing significant tissue injury. The main antioxidant defense systems and the influence of the increase in the production of ROS in the respiratory tract of large animals are also discussed, with emphasis on the involvement of ROS in diseases such as pneumonia in ruminants and recurrent obstruction of the airways and pulmonary hemorrhage induced by exercise in horses.

As espécies reativas do oxigênio (ERO) são moléculas instáveis e extremamente reativas capazes de transformar outras moléculas com as quais colidem. As ERO são geradas em grande quantidade durante o estresse oxidativo, condição em que são afetadas moléculas como proteínas, carboidratos, lipídeos e ácido nucleicos. Neste trabalho, são discutidos os principais conceitos sobre os radicais livres e as ERO: principais tipos, sua formação e a forma como atuam sobre as estruturas celulares, provocando lesão tecidual significativa. Os principais sistemas de defesa antioxidantes e a influência do aumento na produção dessas ERO no trato respiratório de grandes animais também são discutidos, dando ênfase ao envolvimento das ERO em doenças como a pneumonia em ruminantes e na obstrução recorrente das vias aéreas e a hemorragia pulmonar induzida por exercício em equinos.

Espécies reativas do oxigênio e as doenças respiratórias em grandes animais

The reactive oxygen species (ROS) are unstable and highly reactive molecules capable of transforming other molecules with which they collide. The ROS are generated in large numbers during the oxidative stress, a condition in which molecules are affected such as proteins, carbohydrates, lipids and nucleic acid. This research discussed the main concepts of the free radicals and ROS: main types, their training and how they act on the cellular structures causing significant tissue injury. The main antioxidant defense systems and the influence of the increase in the production of ROS in the respiratory tract of large animals are also discussed, with emphasis on the involvement of ROS in diseases such as pneumonia in ruminants and recurrent obstruction of the airways and pulmonary hemorrhage induced by exercise in horses.

As espécies reativas do oxigênio (ERO) são moléculas instáveis e extremamente reativas capazes de transformar outras moléculas com as quais colidem. As ERO são geradas em grande quantidade durante o estresse oxidativo, condição em que são afetadas moléculas como proteínas, carboidratos, lipídeos e ácido nucleicos. Neste trabalho, são discutidos os principais conceitos sobre os radicais livres e as ERO: principais tipos, sua formação e a forma como atuam sobre as estruturas celulares, provocando lesão tecidual significativa. Os principais sistemas de defesa antioxidantes e a influência do aumento na produção dessas ERO no trato respiratório de grandes animais também são discutidos, dando ênfase ao envolvimento das ERO em doenças como a pneumonia em ruminantes e na obstrução recorrente das vias aéreas e a hemorragia pulmonar induzida por exercício em equinos.