Orientador: Prof. Dr.Ivanor Nunes do Prado

Data da Defesa: 22/02/2021


INTRODUCTION: Raising efficiency in beef cattle is one of the main objectives of recent studies on the nutrition of farm animals handling rumen fermentation; for this, the use of additives such as monensin and virginiaminicin in food is frequent. Many works have already proven their efficiency, however modern society, concerned with their well-being and quality of life, presses for the use of natural additives having as option mineral micros being components of enzymes, vitamins, gene expression, stability of membranes between others.
OBJECTIVES: The objective of the present work was to evaluate the synergism of monensin + virginiamycin and monensin in combination with micro minerals + yeasts by assessing blood biochemistry and oxidative stress of liver, meat and blood plasma.
MATERIALS AND METHODS: 36 cattle (European vs. Nellore) were used, with an average age of 24 ± 3.2 months and an initial average live weight of 385.5 ± 3.84 kg. They were distributed in a completely randomized design, with four diets and nine repetitions per diet. The basal diet consisted of 850 g / kg DM of concentrate and 150 g / kg DM of corn silage, provided ad libitum for 84 days. The four diets were: CONT - basal diet; MONE - basal diet and inclusion of 30 mg / kg of dry matter of monensin; MO + VI - basal diet and inclusion of 30 mg / kg of monensin + 30 mg / kg of dry virginiamycin; MO + AD - basal diet and inclusion of 30 mg / kg of monensin + 3.0 g / kg of dry matter from micro minerals and Saccharomyces cerevisiae (Advantage ™ Confinement). Blood samples were collected at the beginning (day 0) and at the end of the experiment (day 84) in 10 ml vacutainner® tubes, centrifuged at 1,000 x g for 10 minutes and were immediately transported to the laboratory for analysis. The animals were slaughtered in a commercial refrigerator respecting the rules of humane slaughter. Soon after slaughter, the liver samples were collected, encapsulated in ice, stored in liquid nitrogen and transported to the biochemical analysis laboratory of UEM. The liver was homogenized in a van Potter homogenizer with 10 volumes of dry ice 0.1 M potassium phosphatase buffer (pH 7.4) and the aliquots were separated for use with the total homogenous. The rest of the homogeneous was centrifuged at 11,000 g for 15 min and the supernatant was separated as a soluble fraction of the homogeneous. 24 hours after slaughter, meat samples were taken for analysis. They were performed on the Longissumus lumborum muscle between the 12th and 13th ribs. After collection, the samples were placed on dry ice and transported to the Meat Quality laboratory at the State University of Maringá and frozen at -20 ºC until the analyzes were performed. The values of the components of the blood count (erythrocytes, hemoglobin, hematocrit, MCV and MCHC) and the leukogram (leukocytes, myelocytes, metamielocytes, neutrophils, segmented, lymphocytes, monocytes, eosinophils and basophils) were determined according to the methodologies described by Jain & Jain. Urea was determined by ultraviolet photometry using two-point kinetics (fixed time), creatinine was evaluated according to the Jaffe technique and the total protein was measured by the biuret method. The plasma iron reduction capacity (FRAP) was measured by spectrophotometry (595 nm) using tripyridyltriazine (TPTZ) and ferric chloride (FeCl3). Aspartate aminotransferase (AST) and alanine amino transferase (ALT) activities were measured in plasma to assess liver damage using commercial kits (Gold Analisa®). Plasma thiol content was measured by spectrophotometry (412 nm) using DTNB (5,5'-dithiobis 2-nitrobenzoic acid). The thiol content was calculated using the molar extinction coefficient (ε) of 1.36 × 104 · M-1 cm-1. The carbonylated protein groups were measured by spectrophotometry using 2,4-dinitrophenylhydrazine. The levels of carbonylated protein groups were calculated using the molar extinction coefficient (ε) of 2.20 × 104 M-1 cm-1. For the preparation of the homogenate (liver and beef), the portion of tissue clamped by freezing was homogenized in a Van Potter- Elvehjem homogenizer, with 10 volumes of cold 0.1 M potassium phosphate buffer (pH 7.4) and a aliquot was separated for use as total homogenate. The remaining homogenate was centrifuged at 11,000 g for 15 min and the supernatant was separated as a soluble fraction from the homogenate. Carbonylated proteins were measured spectrophotometrically in the liver and beef homogenate supernatant with 2,4-dinitrophenyl hydrazine as described above for plasma. The lipoperoxide content was measured using the TBARS test (substances reactive to thiobarbituric acid). TBARS levels were calculated from the standard curve prepared with 1,1 ', 3,3'-tetraethoxypropane. The reduced content (GSH) was measured spectrofluorimetrically (excitation at 350 nm and emission at 420 nm) using the o-phthalaldehyde (OPT) assay. The activities of catalase and superoxide dismutase (SOD) were analyzed by spectrophotometry in the liver homogenate supernatant. Catalase activity was estimated at 240 nm using H2O2 as a substrate. SOD activity was estimated according to the pyrogallol auto-oxidation method.
RESULTS AND DISCUSSION: The results of the CBC, total proteins, urea and blood creatinine on day 0 were within normal standards for animals that were grazing. The analysis of the erythrogram (day 84) showed a difference (P <0.05) in blood hemoglobin and hematocrit levels, with the MO + AD diet having the lowest results. The number of platelets decreased when the combination of monensin and virginiamycin was included in the diet. For the analysis of the leukogram, there was an effect (P <0.05) in the percentage of segmented, in which the MONE diet presented the highest value for this variable. Total protein, urea and creatinine were similar (P> 0.05) between diets. The concentration of urea and creatinine were lower (P <0.05) when the number of feedlots increased, being directly related to the diets and can be explained by the fact that the animals were previously being recreated in pasture systems in Brachiaria, at the end of winter, presenting low levels of crude protein and reduced digestibility. The inclusion of monensin, or the combination of monensin with virginiamycin or with micro minerals + yeasts did not change (P> 0.05) the values of FRAP and Tiois group in the animals' blood plasma. On the other hand, the levels of carbonylated protein groups were approximately 30% (P <0.05) lower in the blood plasma of animals fed with the addition of additives compared to animals in the control group. Furthermore, the levels of carbonylated proteins were similar in the blood plasma of cattle fed the diets MONE, MO + VI and MO + AD, respectively. These results show that the inclusion of ionophores, antibiotics, micro minerals and yeasts reduces oxidative damage in animals fed with additives. The inclusion of monensin alone or in combination with virginiamycin or with micro minerals + yeasts did not alter the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the animals' blood plasma. The levels of TBARS and carbonylated proteins in the liver were not altered (P> 0.05) with the inclusion of additives to the bovine diets, as well as the antioxidant markers GSH, SOD and CAT did not change. In meat, TBARS levels remained unchanged when monensin, virginiamycin and micro minerals + yeasts were included in the diet (P> 0.05). The test shows that the inclusion of additives reduced (P <0.05) oxidative damage and decarbonylation of muscle protein. Cattle fed diets supplemented with monensin + virginiamycin or monensin associated with micro minerals + yeasts increased the muscle concentration of GSH compared to the CONT and MONE diet (P <0.05). GSH plays an important role in the defense of cells against oxidative stress and, when present in low concentration, there is a greater risk of oxidative imbalance.
CONCLUSION: The inclusion of monensin sodium combined with virginiamycin and the micro minerals associated with yeasts (Advantage ™ Confinement) showed similar results and did not show oxidative damage in plasma, liver and beef. In addition, no treatment has been associated with liver disorders.
KEY WORDS: Natural additives; Antioxidants; Protein carbonylation; Glutathione peroxidase;

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