Abstract

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.

Key words:
antioxidant enzymes; erythrocyte; reactive oxygen species (ROS); lipoperoxidation; Nelore

Resumo

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.

Palavras chaves:
enzimas antioxidantes; eritrócito; espécies reativas de oxigênio (ERO); lipoperoxidação; Nelore

Introduction

Brazilian livestock has been developing a lot in recent years, which resulted in a contribution of 491.2 billion reais to the economy, with an expressive valuation of 24.56% of the Brazilian GDP in 2020(¹1 CEPEA 2020 - CEPAE/USP. Centro de Estudos Avançados em Economia Aplicada. Desenvolvido pela Universidade de São Paulo, 2020 [Internet]. Disponível em: https://cepea.esalq.usp.br/br/releases/cepea-retrospectivas-de-2020.aspx Acesso em: 09 out 2021.
https://cepea.esalq.usp.br/br/releases/c... ). One of the competitive advantages of the Brazilian meat production chain is the extensive system, but this system of raising cattle on pasture can lead to some inefficiencies in production(²2 Seixas JN, Pinto CA, Rodrigues A, Tokarnia CH, França TN, Graça F.S, d´Avila MS, Peixoto PV. Comparative study between Brachiaria spp. and Pithomyces chartarum poisoning in cattle. Rev. Bras. Med. Vet. 2016; 38(Supl.2):1-10. https://rbmv.org/BJVM/article/view/192
https://rbmv.org/BJVM/article/view/192... ). To minimize these losses, the incorporation of technologies related to animal nutrition, such as the addition of some microminerals and vitamins to the diet(³3 Sordillo LM. Nutritional strategies to optimize dairy cattle immunity. J. Dairy Sci. 2016; 99(6):4967-4982. https://doi.org/10.3168/jds.2015-10354
https://doi.org/10.3168/jds.2015-10354... ), in order to improve the antioxidant and immune response of animals and improve the efficiency of the production chain, focusing on sustainability(⁴4 Brasil. Ministério da Agricultura, Pecuária e Abastecimento. Projeções do Agronegócio: Brasil 2012/2013 a 2022/2023. Assessoria de Gestão Estratégica, 4ª ed., Brasília, 2013. 96 p.,⁵5 Soares CO, Rosinha GMS. Segurança alimentar, sustentabilldade e produção de proteína de origem animal. In. Vilela, E. F.; Callegaro, G. M.; Fernandes, G. W. Coord. Biomas e agricultura: oportunidades e desafios. Rio de Janeiro: Vertente edições, 2019. p.149-162.).

The species of the Brachiaria genus are important forages considered as the salvation of national livestock⁴4 Brasil. Ministério da Agricultura, Pecuária e Abastecimento. Projeções do Agronegócio: Brasil 2012/2013 a 2022/2023. Assessoria de Gestão Estratégica, 4ª ed., Brasília, 2013. 96 p.. However, some species such as B. brizantha, B. humidicola, and, especially, B. decumbens have been described as causing hepatogenous photosensitization in ruminants(⁶6 Clayton MJ, Davis TZ, Knoppel EL, Stegelmeier BL. Hepatotoxic Plants that Poison Livestock. Vet. Clin. North Am. Food Anim. Pract. 2020; 36(3):715-723. https://doi.org/10.1016/j.cvfa.2020.08.003
https://doi.org/10.1016/j.cvfa.2020.08.0... ). The main cause of hepatogenous photosensitization in cattle is the ingestion of the toxin sporidesmin, present in the spores of the fungus Pithomyces chartarum(⁷7 Motta AC, Riet-Correa Rivero G, Schild AL, Riet-Correa F, Méndez MC, Ferreira JL. Fotossensibilização hepatógena em bovinos no sul do Rio Grande do Sul. Ciênc. Rural. 2000; 30(1):143-149. https://doi.org/10.1590/S0103-84782000000100023
https://doi.org/10.1590/S0103-8478200000... ,⁸8 Macedo MF, Bezerra MB, Soto Blanco B. Fotossensibilização em animais de produção na região semi-árida do Rio Grande do Norte. Arq. Inst. Biol. 2006; 73(2):251-254. https://www.researchgate.net/publication/242143971
https://www.researchgate.net/publication... ).

The main toxic effect of sporidesmin is due to the occurrence of lipid peroxidation and protein carboxylation present in the cells, resulting from the excessive amount of free radicals(⁹9 Vivanco RHC, Menge FGW, Barriga PAC. Variations of the erythrocyte osmotic fragility in cattle grazing on pastures with low selenium content with or without supplement with selenium. Rev. Cient. 2006; 16(3):227-231. https://www.researchgate.net/publication/286828714
https://www.researchgate.net/publication... ). However, some researchers have attributed the cause of intoxication to the lithogenic steroid saponins present in Brachiaria sp.(¹⁰10 Cruz C, Driemeier D, Pires VS, Schenkel EP. Experimentally induced cholangiopathy by dosing sheep with fractionated extracts from Brachiaria decumbens. J. Vet. Diagn. Invest. 2001; 13(2):170-172. http://doi.org/10.1177/104063870101300215
http://doi.org/10.1177/10406387010130021... ,¹¹11 Brum KB, Haraguchi M, Garutti MB, Nóbrega FN, Rosa B, Fioravanti MCS. Steroidal saponin concentrations in Brachiaria decumbens and B. brizantha at diferent develomental stages. Ciênc. Rural. 2009; 39(1):279-281. https://doi.org/10.1590/S0103-84782008005000034
https://doi.org/10.1590/S0103-8478200800... ). These saponins, when metabolized in the animal organism, form insoluble salts that are deposited as crystals in the biliary system(¹²12 Miles CO, Munday SC, Holland PT, Smith BL, Embling PP, Wilkins AL. Identification of a sapogenin glucoronide in the bile of sheep affected by Panicum dichotomiflorum toxicosis. N Z Vet J. 1991; 39(4):150-152. http://doi.org/10.1080/00480169.1991.35684
http://doi.org/10.1080/00480169.1991.356... ). These crystals cause inflammation and obstruction of the biliary system, causing necrosis of periportal hepatocytes and resulting in jaundice, photosensitization, and hepatitis(¹³13 Santos JCA, Riet-Correa F, Simiões SV, Barros CSL. Patogênese, sinais clínicos e patologia das doenças causadas por plantas hepatotóxicas em ruminantes e equinos no Brasil. Pesqui. Vet. Bras. 2008; 28(1):1-14. https://doi.org/10.1590/S0100-736X2008000100001
https://doi.org/10.1590/S0100-736X200800... ).

Oxidative stress can be defined as an imbalance between oxidants and antioxidant in favor of oxidants(¹⁴14 Sies. H. 2019. Oxidative Stress: Eustress and Distress in Redox Homeostasis. In: Fink G. (Ed.) Stress: Physiology, Biochemistry, and Pathology, Handbook of Stress Series, London: Academic Press, Vol. 3, Chapter 13, p.153-163. https://doi.org/10.1016/B978-0-12-813146-6.00013-8
https://doi.org/10.1016/B978-0-12-813146... ), causing changes in redox balance and control or molecular damage, with consequent functional alteration and impairment of vital functions in several organs and tissues(¹⁵15 Droge W. Free radicals in the physiological control of cell function. Physiol. Rev. 2002; 82(1):47-95. https://doi.org/10.1152/physrev.00018.2001
https://doi.org/10.1152/physrev.00018.20... ). From a functional point of view, oxidative damage promotes alterations in fluidity, permeability, and metabolic function, which results in an increase in the fragility of the erythrocyte membrane(⁹9 Vivanco RHC, Menge FGW, Barriga PAC. Variations of the erythrocyte osmotic fragility in cattle grazing on pastures with low selenium content with or without supplement with selenium. Rev. Cient. 2006; 16(3):227-231. https://www.researchgate.net/publication/286828714
https://www.researchgate.net/publication... ). Among mammals, bovines have a particularity, in which they have a lower susceptibility to the action of free radicals(¹⁶16 Brezezinska-Slebodziska E. Species differences in the susceptibility of erythrocytes exposed to free radicals in vitro. Vet. Res. Comm. 2003; 27(3):211-217. https://doi.org/10.1023/a:1023344607691
https://doi.org/10.1023/a:1023344607691... ), due to the composition and organization of the erythrocyte membrane that contains low amounts of phosphatidylcholine(¹⁷17 Oliveira S, Saldanha C. An overview about erythrocyte membrane. Clin. Hemorheol. Microcirc. 2010; 44(1):63-74. https://doi.org/10.3233/CH-2010-1253
https://doi.org/10.3233/CH-2010-1253... ,¹⁸18 Pandey KB, Rizvi SI. Biomarkers of oxidative stress in red blood cells. Biomed. Pap. Med. Fac. Univ. Palacky Olomouc Czech Repub. 2011; 155(2):131-136. https://doi.org/10.5507/bp.2011.027
https://doi.org/10.5507/bp.2011.027... ), a highly peroxidable phospholipid(¹⁹19 Florin-Christensen J, Suarez CE, Florin-Christensen M, Wainszelbaum M, Brown WC, Mcelwain TF, Palmer GH. A unique phospholipid organization in bovine erythrocyte membranes. Proc. Natl. Acad. Sci. USA. 2001; 98(14):7736-7741. https://doi.org/10.1073/pnas.131580998
https://doi.org/10.1073/pnas.131580998... ).

Due to the oxygen transport carried out by hemoglobin, erythrocytes are constantly exposed to reactive oxygen species (ROS)(¹⁹19 Florin-Christensen J, Suarez CE, Florin-Christensen M, Wainszelbaum M, Brown WC, Mcelwain TF, Palmer GH. A unique phospholipid organization in bovine erythrocyte membranes. Proc. Natl. Acad. Sci. USA. 2001; 98(14):7736-7741. https://doi.org/10.1073/pnas.131580998
https://doi.org/10.1073/pnas.131580998... ). The erythrocyte presents in its membrane many sulfhydryl groups, which, when oxidized, results in denaturation of membrane proteins. In this process, intracellular damage can occur, with oxidation of hemoglobin to methemoglobin, which precipitates and forms Heinz bodies(¹⁸18 Pandey KB, Rizvi SI. Biomarkers of oxidative stress in red blood cells. Biomed. Pap. Med. Fac. Univ. Palacky Olomouc Czech Repub. 2011; 155(2):131-136. https://doi.org/10.5507/bp.2011.027
https://doi.org/10.5507/bp.2011.027... ,²⁰20 Caldin M, Carli E, Furlanello T, Solano-Gallego L, Tasca S, Patron C, Lubas G. A retrospective study of 60 cases of eccentrocytosis in the dog. Vet. Clin. Pathol. 2005; 34(3):224-231. https://doi.org/10.1111/j.1939-165x.2005.tb00045.x
https://doi.org/10.1111/j.1939-165x.2005... ,²¹21 Tang X, Xia Z, Yu J. An experimental study of hemolysis induced by onion (Allium cepa) poisoning in dogs. J. Vet. Pharmacol. Ther. 2008; 31(2):143-149. https://doi.org/10.1111/j.1365-2885.2007.00930.x
https://doi.org/10.1111/j.1365-2885.2007... ).

To prevent the damage caused by peroxidation, cells have an antioxidant defense system consisting of the enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px)(²²22 Barbosa KBF, Costa NMB, Alfenas RCG, Paula SO, Minim VPR, Bressan J. Oxidative stress: concept, implications and modulating factors. Rev. Nutr. 2010; 23(4):629-643, https://doi.org/10.1590/S1415-52732010000400013
https://doi.org/10.1590/S1415-5273201000... ). The non-enzymatic antioxidant agents consist of ascorbic acid, vitamin E, glutathione reductase (GSH-Rd), reduced glutathione (GSH), carotenes, uric acid, transition metal-chelating proteins, among others(²³23 Cimen MY. Free radical metabolism in human erythrocytes. Clin. Chim. Acta. 2008; 390(1-2):1-11. https://doi.org/10.1016/j.cca.2007.12.025
https://doi.org/10.1016/j.cca.2007.12.02... ).

One of the methods for evaluating lipid peroxidation in the biomembranes of erythrocytes is the measurement of thiobarbituric acid reactive substances (TBARS) that quantifies the content of malondialdehyde (MDA) generated in lipid peroxidation of polyunsaturated fatty acids(²⁴24 Todovora I, Simeonova G, Kyuchukova D, Dinev D, Gadjeva V. Reference values of oxidative stress parameters (MDA, SOD, CAT) in dogs and cats. Comp. Clin. Pathol. 2005; 13(4):190-194. https://doi.org/10.1007/s00580-005-0547-5
https://doi.org/10.1007/s00580-005-0547-... ,²⁵25 Machado LP, Watanabe MJ, Kohayagawa A, Saito ME, Da Silveira VF, Yonezawa LA. Malondialdeído eritrocitário como índice de estresse oxidativo em equinos da raça Árabe. Rev. Bras. Hematol. Hemoter. 2007; 29:237. https://doi.org/10.1590/S0103-84782010005000094
https://doi.org/10.1590/S0103-8478201000... ,²⁶26 Ciampi F, Sordillo LM, Gandy JC, Caroprese M, Sevi A, Albenzio M, Santillo A. Evaluation of natural plant extracts as antioxidants in a bovine in vitro model of oxidative stress. J. Dairy Sci. 2020; 103(10):8938-8947. https://doi.org/10.3168/jds.2020-18182
https://doi.org/10.3168/jds.2020-18182... ).

The use of antioxidants added to the feed offered to animals has been an efficient option for improving the production system. Its use has provided the animals with improvements in health, reproduction, and production (feed conversion and weight gain)(²⁷27 Spears JW, Weiss WP. Role of antioxidants and trace elements in health and immunity of transition dairy cows, Vet J. 2008; 176(1):70-76. https://doi.org/10.1016/j.tvjl.2007.12.015
https://doi.org/10.1016/j.tvjl.2007.12.0... ,²⁸28 Mattioli GA, Rosa DE, Turic E, Picco SJ, Raggio SJ, Minervino AHH, Fazzio LE. Effects of Parenteral Supplementation with Minerals and Vitamins on Oxidative Stress and Humoral Immune Response of Weaning Calves. Animals (Basel). 2020; 10(8):1298. https://doi.org/10.3390/ani10081298
https://doi.org/10.3390/ani10081298... ).

In this context, this present study aimed to evaluate whether the supplementation of different sources of antioxidants in the diet of confined cattle fed Brachiaria sp. hay could reduce the occurrence of oxidative stress on erythrocytes.

Material and methods

The project was approved by the Ethics Committee on Use of Animals (CEUA) of the Federal University of Goiás (UFG) and was registered under number 360/2010. The study was developed at Fazenda Tomé Pinto, owned by the School of Veterinary and Animal Science at UFG, located in the municipality of São Francisco de Goiás, State of Goiás, 110 km from the capital.

Forty uncastrated male cattle were used, with an average initial weight of 360 kg and age between 24 and 36 months, raised on Brachiaria sp. pastures. The animals were divided into five groups, with eight animals each, which received the following treatments: Group 1 (G1 = CG) - control (no supplementation); Group 2 (G2 = Se + Vit. E) - supplementation with 2g α-tocopherol acetate (1000 UI Vitamin E/animal/day) and 10g methionine selenium (10 mg selenium/animal/day); Group 3 (G3 = Zn) - supplementation with 6g in the form of zinc methionine (600mg zinc/animal/day); Group 4 (G4 = Se) - supplementation with 10g methionine selenium (10mg selenium/animal/day) and Group 5 (G5 = Vit. E) - supplementation with 2g α-tocopherol acetate (1000 UI Vitamin E/animal/day).

After the groups were formed, the animals were taken to their respective enclosures, with an area of 15m²/animal, with shaded area, troughs and drinking fountains, and diet twice a day, at 8:00am and 3:00 pm. Feed (Brachiaria sp. hay) and water were supplied ad libitum. The concentrate (feed) was balanced according to the bromotological analysis of the hay offered (Chart 1), based on an estimated daily gain of 0.880 kg/day and to meet the nutritional requirements of the animals(²⁹29 NRC - NATIONAL RESEARCH COUNCIL. Nutrient requirements of beef cattle. 7.ed. Washington: National Academy Press, 1996. 242p.). The formulated concentrate (Integral Nutrição Animal, Goiânia, GO, Brazil) had a concentration of 34% protein and 71% of total digestible nutrients (NDT), being the main constituent’s soybean meal, corn meal, and a mineral mixture (Chart 2). The cattle remained confined for 119 days, with 14 days of adaptation and 105 days of experiment.

During the experimental period, antioxidants supplementation was added to the diet for a period of 105 days. The antioxidants were weighed daily on precision scale (Shimadzu^® AY 220), in disposable cups, so that each animal received the daily amount of antioxidant, according to its treatment in the trough. To improve consumption of the amount supplied, a small amount of feed was placed on the antioxidant, with individual supply per animal. The animals were monitored until the antioxidant and concentrate were fully ingested, preventing any other bovine from approaching the trough. The food was provided as a total diet, and the leftover food was weighed and discarded daily before the morning feeding, to determine food consumption by the group and to avoid that the leftovers did not exceed more than 10% of the total provided.

For sample collection, the cattle were kept at station and restrained in the stall. Blood samples were obtained every 28 days (0, 28, 56, 84, and 105 days) by jugular puncture in tubes containing 10% ethylenediaminetetraacetic acid, disodium salt (EDTA) and tubes with heparin.

After collection, the tubes were stored in thermal boxes at 10°C and sent to the laboratory for a maximum period of two hours to perform the erythrogram and obtain the hemolysate. After obtaining aliquots to measure TBARS and antioxidants: SOD, CAT, thiol groups and glutathione), the samples were stored in the freezer at -80°C.

The samples destined for the erythrogram were processed as they arrived at the laboratory, not exceeding six hours from the time of collection. The erythrocyte count and hemoglobin were evaluated in a semiautomatic equipment (BC 2800 VET, Mindray, Shenzhen). The hematocrit was obtained by microcentrifugation using the microhematocrit technique(³⁰30 Jain NC. Essentials of veterinary hematology. 4. ed. Philadelphia: Lea & Febiger, 1993. 407p.).

TBARS analysis in the samples was determined according to the methodology proposed by Esterbauer and Cheeseman(³¹31 Esterbauer H, Cheeseman KH. Determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hydroxynonenal. Methods Enzymol. 1990; 186:407-21. https://doi.org/10.1016/0076-6879(90)86134-h
https://doi.org/10.1016/0076-6879(90)861... ). GST and GSH-Px were determined by the techniques described by Tietze(³²32 Tietze F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal. Biochem. 1969; 27(3):502-22. https://doi.org/10.1016/0003-2697(69)90064-5
https://doi.org/10.1016/0003-2697(69)900... ) and Bayoumi and Rosalk(³³33 Bayoumi RA, Rosalki SB. Evaluation of methods of coenzyme activation of erythrocyte enzymes for detection of deficiency of vitamins B1, B2, and B6. Clin. Chem. 1976; 22(3):327-335.), respectively. SOD activity was performed according to the procedures proposed by Beutler(³⁴34 Beutler E. Superoxide dismutase. In: Beutler E (Editor), Red Cell Metabolism. A Manual of Biochemical Methods. Philadelphia: Grune & Stratton, 1984. p.83-85.). CAT was established by the method proposed by Aebi(³⁵35 Aebi H. Catalase in vitro. Methods Enzymol. 1984; 105:121-126. https://doi.org/10.1016/s0076-6879(84)05016-3
https://doi.org/10.1016/s0076-6879(84)05... ).

The data were submitted to descriptive statistics and were later analyzed by analysis of variance (ANOVA), using Tukey’s test, by means of the Statistical Analysis System software (SAS v.9.3, Cary, North Carolina), with a 5% significance level.

Results

The mean values of erythrocytes, hemoglobin, and hematocrit are described in Table 1. There was no difference in the supplementation between the experimental groups (p<0.05). The results from the fourth collection were discarded because the equipment was not properly calibrated on the day the laboratory was performed.

In the hematological evaluation (erythrocytes, hemoglobin and hematocrit) numerical alterations were found in the parameters measured, but these alterations did not produce differences between the different treatments. The differences were observed only within each group, between the experimental times. At the 2nd harvest, the erythrocyte count in G4 showed lower results than G1 and G3 (p<0.05). In the determination of hemoglobin, in this same harvest, there were significant variations (p<0.05) in which the highest value was found in G3 and the lowest in G4. The hematocrit did not show significant variations (p>0.05) in the experimental period.

In the TBARS determination (Table 2) when analyzing the harvests performed, it was verified that there was an interaction groups/time (p<0.05), in which G2 presented lower results in relation to the other groups 28, 56 and 105 days. Differences were identified (p<0.05) between the means of the evaluated, so that G3 showed the highest mean TBARS concentration among the treatments and G2 the lowest. and G2 the lowest value. The marginal means between collections were similar (p>0.05).

In the enzymatic activity of GSH-T (Table 3), no interaction was observed in relation group/time (p>0.05). There was a significant difference between group means of the groups (p<0.05), in G5 had the lowest mean value and G1 had the highest mean value. However, between harvest there was no significant difference (p>0.05).

In the determination of the enzymatic activity of GSH-Px there was an interaction regarding group/time (p<0.05), with a significant difference at all times of harvest. There was a significant difference (p<0.05) between the experimental groups, with G3 showing the lowest mean value and G2 the highest (Table 4). Considering the times of harvest, the highest means were found on day 56 and the lowest on days 28 and 84 (p<0.05), however, without significant difference (p>0.05) between the beginning and the end of the study.

In the measurement of SOD interaction was observed in relation group/time (p<0.05) in the mean values, there was significant difference between the experimental groups 56, 84 and 105 days (Table 5). Differences were identified (p<0.05) among the groups evaluated, in which G3 presented the mean value lower than the other treatments, and G1 and G4 presented the highest values. The marginal means between harvest were similar (p>0.05).

The determination of CAT showed interaction in relation group/time (p<0.05) in the experimental groups only at 84 and 105 days (Table 6). It was found that in G3, the mean value was higher (p<0.05) than the other treatments, while G2 presented the lowest values (p<0.05). The marginal means between harvest were similar (p>0.05).

Discussion

The erythrocyte profile of the animals during the whole experimental remained within the reference limits(³⁶36 Fagliari JJ, Santana AE, Lucas FA, Campos Filho E, Curi PR. Constituintes sanguíneos de bovinos lactantes, desmamados e adultos das raças Nelore (Bos indicus) e Holandesa (Bos taurus), e de bubalinos (Bubalus bubalis) da raça Murrah. Arq. Bras. Med. Vet. Zootec. 1998; 50(3):263-271. https://repositorio.unesp.br/handle/11449/38249
https://repositorio.unesp.br/handle/1144... ). In previous studies with cattle fed Brachiaria sp. grass, similar results to the hematological profile for erythrocyte count(³⁷37 Machado LP, Kohayagawa A, Saito ME, Da Silveira VF, Yonezawa LA. Lesão oxidativa eritrocitária e mecanismos antioxidantes de interesse na Medicina Veterinária. Rev. Ciênc. Agrovet. 2009; 8(1):84-94. https://www.revistas.udesc.br/index.php/agroveterinaria/article/view/5317/3523
https://www.revistas.udesc.br/index.php/... ) and hemoglobin concentration were observed for animals receiving selenium(⁹9 Vivanco RHC, Menge FGW, Barriga PAC. Variations of the erythrocyte osmotic fragility in cattle grazing on pastures with low selenium content with or without supplement with selenium. Rev. Cient. 2006; 16(3):227-231. https://www.researchgate.net/publication/286828714
https://www.researchgate.net/publication... ) and vitamin E supplementation in the feed(³⁸38 Sharma N, Singh NK, Singh OP, Pandey V, Verma PK. Oxidative stress and antioxidant status during transition period in dairy cows. Asian-Aust. J. Anim. Sci. 2011; 24(4):479-484. https://doi.org/10.5713/ajas.2011.10220
https://doi.org/10.5713/ajas.2011.10220... ). However, divergent results were observed in the study(³⁹39 Calamari L, Petrera F, Abeni F, Bertin G. Metabolic and hematological profiles in heat stressed lactating dairy cowsfed diets supplemented with different selenium sources and doses. Livest. Sci. 2011; 142(1-3):128-137. https://doi.org/10.1016/j.livsci.2011.07.005
https://doi.org/10.1016/j.livsci.2011.07... ) using supplementation of different selenium sources (organic and inorganic) in dairy cow diets, where mean erythrocyte, hemoglobin and hematocrit values were higher than those obtained in the control group. Assays evaluating erythrocyte oxidative damage(³⁷37 Machado LP, Kohayagawa A, Saito ME, Da Silveira VF, Yonezawa LA. Lesão oxidativa eritrocitária e mecanismos antioxidantes de interesse na Medicina Veterinária. Rev. Ciênc. Agrovet. 2009; 8(1):84-94. https://www.revistas.udesc.br/index.php/agroveterinaria/article/view/5317/3523
https://www.revistas.udesc.br/index.php/... ) concluded that, antioxidant association promotes less erythrocytes alterations with potential benefits of treatment by antioxidants supplementation on erythrocytes.

The increase in the mean values erythrocytes in G3 could be explained by the function that zinc plays in maintaining the integrity of erythrocyte cell membranes(⁴⁰40 Kouryi JC, Donangelo CM. Zinco, estresse oxidativo e atividade física. Rev. Nutr. 2003; 16(4):433-441. https://doi.org/10.1590/S1415-52732003000400007
https://doi.org/10.1590/S1415-5273200300... ). The action of this microelement favors the association of proteins present in the membrane with other components of the cell cytoskeleton(⁴¹41 Bian X, Teng T, Zhao H, Qin J, Qiao Z, Sun Y, Liun Z, Xu Z. Zinc prevents mitochondrial superoxide generation by inducing mitophagy in the setting of hypoxia/reoxygenation in cardiac cells. Free Radic. Res. 2018; 52(1):80-91. https://doi.org/10.1080/10715762.2017.1414949
https://doi.org/10.1080/10715762.2017.14... ), which in turn provides antioxidants protection to the membranes against lipid and protein oxidation(⁴²42 Kloubert V, Rink L. Zinc as a micronutrient and its preventive role of oxidative damage in cells. Food Funct. 2015;6(10):3195-3204. https://doi.org/10.1039/c5fo00630a
https://doi.org/10.1039/c5fo00630a... ) and antagonizes possible deleterious effects by ionic elements(⁴³43 Oteiza PI. Zinc and the modulation of redox homeosta- sis. Free Radic. Biol. Med. 2012; 53(9):1748-1759. https://doi.org/10.1016/j.freeradbiomed.2012.08.568
https://doi.org/10.1016/j.freeradbiomed.... ).

The measurement of TBARS in the erythrocytes throughout the experiment showed that during the harvest the means of the animals did not present significant difference. However, there was a significant difference between the proposed treatments (p<0.05). Malondialdehyde (MDA) is one of the main markers of lipid peroxidation and it is used to measured TBARS. The increased blood concentration of this marker is related to an increase lipid peroxidation and oxidative damage(⁴⁴44 Draper HH, Hadley M. Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol. 1990; 186:421-431. https://doi.org/10.1016/0076-6879(90)86135-i
https://doi.org/10.1016/0076-6879(90)861... ). Animals that received selenium and vitamin E supplementation showed higher lipid stability. The animals that received selenium and vitamin E showed higher lipid stability. In an experiment with isolated and associated supplementation of selenium and vitamin E in cattle(⁴⁵45 Liu ZL, Yang P, Chen P, Dong WX, Wang DM. Supplementation with selenium an vitamin E improves milk fat depression an fatty acid composition in dairy cows fed fat diet. Asian-Aust. J. Anim. Sci. 2008; 21(6): 38-844. https://doi.org/10.5713/ajas.2008.70618
https://doi.org/10.5713/ajas.2008.70618... ), an improvement in the redox state of the blood was observed when association of antioxidants occurred.

The values obtained in the measurement of TBARS in the supplemented groups showed results similar to those observed in a study with Simmental cattle(⁴⁶46 Križanović D, Sušić V, Božić P, Štoković I, Ekert-Kabalin A. Changes of bovine blood lipid peroxides and some antioxidants in the course of growth. Veterinarski Arhiv. 2008; 78 (4):269-278. https://hrcak.srce.hr/26534
https://hrcak.srce.hr/26534... ) aged around 20 months fed with feed and roughage. Tests on the influence of some antioxidants (zinc and vitamin E, isolated and associated) on lipid peroxidation in the blood of cattle(⁴⁷47 Chandra G, Aggarwal A, Singh K, Kumar M, Upadhyay RC. Effect of vitamin E and zinc supplementation on energy metabolites, lipid peroxidation, and milk production in peripartum sahiwal cows. Asian-Aust. J. Anim. Sci. 2013; 26(11):1569-1576. https://doi.org/10.5713/ajas.2012.12682
https://doi.org/10.5713/ajas.2012.12682... ), it was observed that the association of antioxidants resulted in lower lipid peroxidation indices. However, the results obtained in the present study were higher than those found in the literature in animals supplemented with antioxidants when evaluating lipid peroxidation in bovines(³⁸38 Sharma N, Singh NK, Singh OP, Pandey V, Verma PK. Oxidative stress and antioxidant status during transition period in dairy cows. Asian-Aust. J. Anim. Sci. 2011; 24(4):479-484. https://doi.org/10.5713/ajas.2011.10220
https://doi.org/10.5713/ajas.2011.10220... ,⁴⁸48 Castillo C, Hernandez J, Bravo A, Lopez-Alonso M, Pereira V, Benedito JL. Oxidative status during late pregnancy and early lactation in dairy cows. Vet J. 2005; 169(2):286-292. https://doi.org/10.1016/j.tvjl.2004.02.001
https://doi.org/10.1016/j.tvjl.2004.02.0...

49 Castillo C, Hernandez J, Valverde I, Pereira V. Plasma malonaldehyde (MDA) and total antioxidant status (TAS) during lactation in dairy cows. Res. Vet. Sci. 2006; 80(2):133-139. https://doi.org/10.1016/j.rvsc.2005.06.003
https://doi.org/10.1016/j.rvsc.2005.06.0... -⁵⁰50 Kumar A, Gyanendra Singh BV, Meur SK. Modulation of antioxidant status and lipid peroxidation in erythrocyte by dietary supplementation during heat stress in buffaloes. Livest. Sci. 2011; 138(1-3):299-303. https://doi.org/10.1016/j.livsci.2010.12.021
https://doi.org/10.1016/j.livsci.2010.12... ). This fact probably occurred because different methodologies and concentration units to measure MDA as an indicator of oxidative stress.

It is also worth mentioning that the maintenance of GSH-t levels was due to the fact that the glutathione in higher concentration in most cells, including mammalian erythrocytes, is in the reduced form (GSH). The depletion of GSH levels can occur directly, by conjugation with free radicals, and indirectly by inhibitors of its synthesis and regeneration(⁵¹51 Halliwell B, Gutteridge JMC. Free radicals in biology and medicine. Oxford: Clarendon Press, 2007, 543p.,⁵²52 Ma X, Deng D, Chen W. Inhibitors and Activators of SOD, GSH-Px, and CAT. In: Murat Senturk. Enzyme inhibitors and activators. 1nd ed. Croatia: IntechOpen; 2017. Chapter 9. https://doi.org/10.5772/65936
https://doi.org/10.5772/65936... ).

As the consumption of GSH probably occurred only because of its antioxidant property, the permanence of high GSH concentrations in erythrocytes contributed to the maintenance of GSH-T levels. Allied to this, the maintenance of GSH-T values may have also occurred due to the “compensatory increase” in GSH production by other organs, in an attempt to help the body, combat the increase in ROS production(⁵³53 Machefer G, Groussard C, Ranou-Bekono F, Zouhal H, Faure H, Vicent S, Cillard J, Gratas-Delamarche A. Extreme running competition decrease blood antioxidant defense capacity. J. Am. Coll. Nutr. 2004; 23(4):358-364. https://doi.org/10.1080/07315724.2004.10719379
https://doi.org/10.1080/07315724.2004.10... ). This excessive generation of ROS may increase the degree of lipid peroxidation and cell damage. Similar behavior to what occurred in this study was reported by other authors(⁴⁵45 Liu ZL, Yang P, Chen P, Dong WX, Wang DM. Supplementation with selenium an vitamin E improves milk fat depression an fatty acid composition in dairy cows fed fat diet. Asian-Aust. J. Anim. Sci. 2008; 21(6): 38-844. https://doi.org/10.5713/ajas.2008.70618
https://doi.org/10.5713/ajas.2008.70618... ), when they described that the associations of selenium and vitamin E provided to the analyzed bovines a synergism between the effects of antioxidants administered resulting in less deleterious effects on erythrocyte cells.

Also regarding GSH, the results obtained in this assay for the isolated supplementation of selenium and vitamin E were lower than those found in an evaluation of the benefits of selenium supplementation in cattle(⁹9 Vivanco RHC, Menge FGW, Barriga PAC. Variations of the erythrocyte osmotic fragility in cattle grazing on pastures with low selenium content with or without supplement with selenium. Rev. Cient. 2006; 16(3):227-231. https://www.researchgate.net/publication/286828714
https://www.researchgate.net/publication... ) on the erythrocyte membrane stability. In an experiment to analyze the effects of vitamin E supplementation in cows in transition(³⁸38 Sharma N, Singh NK, Singh OP, Pandey V, Verma PK. Oxidative stress and antioxidant status during transition period in dairy cows. Asian-Aust. J. Anim. Sci. 2011; 24(4):479-484. https://doi.org/10.5713/ajas.2011.10220
https://doi.org/10.5713/ajas.2011.10220... ), they found higher results and a negative correlation between enzyme activity and lipid peroxidation marker. It is believed that this divergence occurred due to the gender, the not concentration of antioxidant supplied and the physiological state of the animals, besides the methodology employed, which the use of commercial reagents different from those employed in this study.

The means of SOD between the harvest showed significant difference (p<0.05) for the analyzed groups due to the variation in the concentration of H₂O₂ caused by the action of SOD, indicating that there was activation of the glutathione-reducing cycle, in which the groups supplemented with selenium and vitamin E (G2), selenium (G4), and vitamin E (G5) showed increasing values throughout the harvest. Researchers working with measurements of oxidative stress in cattle(⁵⁴54 Abd Ellah MR, Okada K, Goryo M, Oishi A, Yasuda J. Superoxide dismutase activity as a measure of hepatic oxidative stress in cattle following ethionine administration. Vet. J. 2009; 182(2):336-341. https://doi.org/10.1016/j.tvjl.2008.05.013
https://doi.org/10.1016/j.tvjl.2008.05.0... ) on hay-based diet, verified through activities of SOD and CAT similar results to this study, which demonstrates that high concentrations of H₂O₂ do not produce an imbalance with the antioxidants. In an evaluation of the influence of metabolic disorders on oxidative stress(⁵⁵55 Dobbelaar P, Bouwstra RJ, Goselink RMA, Jorritsma R, Van Den Borne JJGC, Jansen EHJM. Effects of vitamin e supplementation on and the association of body condition score with changes in peroxidative biomarkers and antioxidants around calving in dairy heifers. J. Dairy Sci. 2010; 93(7):3103-3113. https://doi.org/10.3168/jds.2009-2677
https://doi.org/10.3168/jds.2009-2677... ), they verified that the inclusion of vitamin E did not provide alterations in SOD and GSH-Px.

The evaluation of the enzymatic activity of CAT promoted an increase in the means of the group that received zinc supplementation (G3) at 84 a 105 days. This enzyme plays an important role in the evaluation of oxidative stress, as it acts to neutralize H₂O₂ at high concentrations, formed by the dismutation of the O₂^- radical promoted by SOD. However, in low concentrations of this free radical, GSH acts in the neutralization of H₂O₂(⁵¹51 Halliwell B, Gutteridge JMC. Free radicals in biology and medicine. Oxford: Clarendon Press, 2007, 543p.). Some authors(⁵⁶56 Felton GW, Summers CB. Antioxidant systems in insects. Arch. Insect Biochem. Physiol. 1995; 29(2):187-197. https://doi.org/10.1002/arch.940290208
https://doi.org/10.1002/arch.940290208... ) have reported the importance of determining the interrelationship between the activities of these enzymes to evaluate the defense mechanisms of antioxidants present in the body. The measurement of the activity of the enzymes SOD, GSH-Px, and CAT is commonly used to assess the defense capacity of the organism against the action of ROS(⁵⁷57 Linke A, Adams V, Schulze PC, Erbs S, Gielen S, Fiehn E. Antioxidative effects of exercise training patients with chronic heart failure increase in radical scavenger enzyme activity in skeletal muscle. Circulation. 2005; 111(14):1763-1770. https://doi.org/10.1161/01.CIR.0000165503.08661.E5
https://doi.org/10.1161/01.CIR.000016550... ). The observations of variations between harvest for CAT were analogous to the study for ruminants(⁵⁰50 Kumar A, Gyanendra Singh BV, Meur SK. Modulation of antioxidant status and lipid peroxidation in erythrocyte by dietary supplementation during heat stress in buffaloes. Livest. Sci. 2011; 138(1-3):299-303. https://doi.org/10.1016/j.livsci.2010.12.021
https://doi.org/10.1016/j.livsci.2010.12... ), indicating that this variation is probably attributed to the stress condition.

The joint evaluation of the oxidative stress biomarkers evaluated in this study reveals that erythrocytes did not undergo lipid peroxidation, which was mainly confirmed by the no change in TBARS values in erythrocyte cells.

Conclusion

The supplementation of confined Nelore cattle fed with Brachiaria sp. hay with, containing antioxidants, in form isolated (zinc, selenium, and vitamin E) or associated (selenium and vitamin E), does not alter the oxidative stress in erythrocytes.

Acknowledgments

To the National Council of Science and Technology (CNPq) for granting funding to the research project and doctoral scholarship.

References

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