UNIVERSIDADE ESTADUAL PAULISTA – UNESP CÂMPUS DE JABOTICABAL IDENTIFICATION AND CONTROL EFFECTS OF MYCOTOXINS IN NELLORE BULLS FINISHED IN FEEDLOT M.Sc. Letícia Custódio Animal Scientist 2018 UNIVERSIDADE ESTADUAL PAULISTA – UNESP CÂMPUS DE JABOTICABAL IDENTIFICATION AND CONTROL EFFECTS OF MYCOTOXINS IN NELLORE BULLS FINISHED IN FEEDLOT M.Sc. Letícia Custódio Advisor: Prof. Ph.D Gustavo Rezende Siqueira Co-Advisor: Ph.D Laura Franco Prados Thesis presented to the Faculdade de Ciências Agrárias e Veterinárias – Unesp, Campus of Jaboticabal in partial fulfillment of requirements for the degree of doctor in Animal Science. 2018 C987i Custódio, Letícia Identification and control effects of mycotoxins in nellore bulls finished in feedlot / Letícia Custódio. -- Jaboticabal, 2018 79 p. Tese (doutorado) - Universidade Estadual Paulista (Unesp), Faculdade de Ciências Agrárias e Veterinárias, Jaboticabal Orientador: Gustavo Rezende Siqueira Coorientadora: Laura Franco Prados 1. Adsorbent. 2. Carcass gain. 3. Meat quality. 4. Performance. 5. Survey. I. Título. Sistema de geração automática de fichas catalográficas da Unesp. Biblioteca da Faculdade de Ciências Agrárias e Veterinárias, Jaboticabal. Dados fornecidos pelo autor(a). Essa ficha não pode ser modificada. DADOS CURRICULARES DA AUTORA Letícia Custódio, nascida em 23 de março de 1988, em Barretos, São Paulo. Filha de Jaime José Custódio e Sueli Aparecida Ferreira Custódio. Graduada em Zootecnia pelo Centro Universitário da Fundação Educacional de Barretos, em Barretos - SP (2006-2010) sob a orientação da Professora Doutora Marcella de Toledo Piza Roth. Desenvolveu estágio extracurricular durante a graduação na Agência Paulista de Tecnologia dos Agronegócios – APTA, em Colina – SP (2007- 2010), sob a orientação do professor Gustavo Rezende Siqueira. Em 2010 desenvolveu o estágio curricular na University of Florida em Ona – FL, EUA, sob a orientação do professor João Vendramini. Mestre em Ciência Animal e Pastagens pela Escola Superior de Agricultura Luiz de Queiroz da Universidade de São Paulo, em Piracicaba - SP (2011-2013) na área de Conservação de forragens e bovinos leiteiros, sob a orientação do Professor Doutor Luiz Gustavo Nussio. Em fevereiro de 2015 ingressou no Curso de Doutorado em Zootecnia na Faculdade de Ciências Agrárias e Veterinárias da Universidade Estadual Paulista, em Jaboticabal – SP, sob a orientação do Professor Doutor Gustavo Rezende Siqueira. ―O destino não é uma questão de sorte, é uma questão de escolha. Não é algo para se esperar, é algo para se conquistar‖. William Jennings Bryan A Deus, à Nossa senhora Aparecida e à minha família, Dedico!!! AGRADECIMENTOS À Deus e a Nossa Senhora Aparecida, por acompanhar meus caminhos e me permitir ter saúde e alegria para enfrentar os obstáculos que surgem no dia a dia com leveza e sensatez. À minha família que sempre está por trás de tudo, todos os momentos, todas as decisões, sempre me apoiando da melhor forma possível. Mamãe, Papai, Lá, Thá, Vovó eu não tenho palavras pra agradecer cada conselho, cada ―independente da sua decisão estamos aqui para te dar o apoio que precisar‖, vocês são perfeitos. Aos professores Gustavo Rezende Siqueira e Flávio Dutra de Resende, que além de serem ótimos profissionais, são pessoas maravilhosas e fizeram que esses anos de doutorado se tornassem a melhor etapa da minha vida acadêmica. À Universidade Estadual Paulista por me proporcionar uma ótima condição para o desenvolvimento do doutorado. À fazenda (Agência Paulista de Tecnologia dos Agronegócios) que me proporcionou toda e estrutura e funcionários necessários para o desenvolvimento do projeto de pesquisa. À Laura Franco Prados, que foi uma professora incrível, me ajudou imensamente com o trabalho e o mais importante de tudo, sempre com muito carinho e paciência. Ao Matheus, por todo amor, carinho, paciência, apoio e compreensão. Você faz com que tudo se torne mais alegre. À Danúbia que me ajudou muito durante o experimento de campo e com todo o resto, minha companheira de todas as horas. Às meninas mais lindas desse mundo, Cleisy, Aline, Verônica, Ivanna, Val, Naiara, Paloma, Bia e Cíntia que estão sempre por perto, sempre cuidando, ajudando. Eu sei que posso contar com vocês, pra sempre! Aos meninos, Renan, Felipe, Alexandre e Flávio, que me ajudaram muito nessa etapa, tudo era mais fácil, porque sabia que podia sempre contar com vocês. Aos estagiários da APTA, Jaque, Rebeca, Duda, Willian e Hugo, por toda a ajuda de sempre! A todos os funcionários da APTA pela ajuda na realização do trabalho, em especial ao Toizinho, Lori, Seu João, Seu Alcino, Toga, Chico, Luizão, Nandi, Deley, Rogério, Sueli, Flora e Neia. À Regina, sempre aconselhando sobre análises laboratoriais. Ao Professor Eduardo da ESALQ, que teve papel fundamental para que o trabalho de campo fosse realizado. À Professora Elizabeth Santin e ao Adrien da Universidade Federal do Paraná que ajudaram com análises essenciais para o trabalho. O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Código de Financiamento 001. Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) pela bolsa concedida por um período do Doutorado. À outorgante, processo 2015/21416-6, Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) e a CAPES, pela bolsa concedida. À Alltech pelo apoio financeiro e pelo imenso apoio intelectual. i SUMMARY CHAPTER 1 – GENERAL CONSIDERATIONS ........................................................ 1 INTRODUCTION.......................................................................................... 1 REVIEW OF LITERATURE .......................................................................... 3 LITERATURE CITED ................................................................................. 11 CHAPTER 2: SURVEY OF MYCOTOXIN CONTAMINATION OF DIETS FOR BEEF CATTLE FINISHED IN FEEDLOT ........................................................................... 22 ABSTRACT ................................................................................................ 23 INTRODUCTION........................................................................................ 23 RESULTS .................................................................................................. 24 DISCUSSION ............................................................................................. 32 CONCLUSION ........................................................................................... 35 MATERIAL AND METHODS ...................................................................... 35 REFERENCES .......................................................................................... 36 CHAPTER 3: MYCOTOXIN CONTAMINATED DIETS AND MYCOSORB A+ AFFECT NELLORE CATTLE PERFORMANCE FINISHED IN FEEDLOT .............. 38 ABSTRACT ................................................................................................ 39 INTRODUCTION........................................................................................ 40 MATERIAL AND METHODS ...................................................................... 41 RESULTS .................................................................................................. 47 DISCUSSION ............................................................................................. 49 CONCLUSION ........................................................................................... 53 LITERATURE CITED ................................................................................. 54 CHAPTER 4: DO MYCOTOXIN CONTAMINATED DIETS AND MYCOSORB A+ AFFECT MEAT QUALITY OF FINISHED NELLORE CATTLE IN FEEDLOT? ....... 68 ABSTRACT ................................................................................................ 69 INTRODUCTION........................................................................................ 69 MATERIAL AND METHODS ...................................................................... 70 RESULTS .................................................................................................. 72 DISCUSSION ............................................................................................. 72 CONCLUSION ........................................................................................... 73 REFERENCES .......................................................................................... 74 ii iii IDENTIFICATION AND CONTROL EFFECTS OF MYCOTOXINS IN NELLORE BULLS FINISHED IN FEEDLOT ABSTRACT: The study was divided into two phases. The first phase was designed to identify which mycotoxin was present in Brazillian feedlot diets, and the second phase was designed to evaluate the performance of Nellore bulls finished in feedlot fed mycotoxins, and the effects of mycotoxin adsorbent (ADS). Thus, the objective of the first phase was to identify which mycotoxins were present in ingredients used in diets fed to feedlot cattle and its concentrations. The survey covered 30 Brazillian feedlots located in the 5 largest beef-producing states. Samples of total mixed ration (TMR) and ingredients were collected on-site and sent to the 37+® Analytical Services Laboratory (KY, USA) for analysis of mycotoxins. The quantification of 38 different mycotoxins was performed using ultra-performance liquid chromatography coupled to tandem mass spectrometry. The mycotoxin concentrations were further interpreted according to known species- specific sensitivities and normalized according to the principles of toxic equivalent factors, determining the Risk Equivalent Quantities (REQ) expressed in µg/kg of aflatoxin B1-equivalent. Descriptive statistics were obtained using the UNIVARIATE procedure of SAS and multivariate statistics were obtained using STATISTICA. The toxins identified in TMR were: fumonisins, trichothecenes A, trichothecenes B, fusaric acid, aflatoxins and ergot (means of 2,330, 104.3, 79.5, 105, 10.5, and 5.5 µg/kg, respectively). Fumonisins were the primary mycotoxins found and at highest concentrations in TMR samples. Peanut meal was the most contaminated ingredient. The objective of the second phase was to evaluate the effect of mycotoxins and ADS on performance and meat quality of Nellore cattle finished in feedlot. One-hundred 24-mo-old Nellore bulls (430 ± 13 kg of body weight (BW)) were used in a randomized block design with a 2 × 2 factorial arrangement of treatments. The factors consisted of two diets (Factor 1) with natural contamination (NC) or exogenous contamination (EC) and presence (10g/d/animal; ADS) or absence of ADS (Factor 2). The NC and EC diets had, respectively, the following contaminations: aflatoxin 0 and 10 µg/kg, fumonisin 5114 and 5754 µg/kg, trichothecenes A 0 and 22.1 µg/kg, trichotecenes B 0 and 42.1 µg/kg and fusaric acid 42.9 and 42.9 µg/kg. At the beginning of the experiment, all iv animals were weighed, and 4 were randomly selected to be slaughtered to evaluate initial carcass weight. After 97 days of experiment, all animals were weighed and slaughtered. Steaks from Longissumus thoracics harvested between 11th to 13th ribs, in which three steaks were randomly assigned to aging times of 7, 14 and 28 days at 4°C. The meat quality was analyzed. There was no interaction among factors for DMI (P = 0.92), however there was a tendency for EC- diets decrease DMI by 650 g/d (P = 0.09). The ADG was greater for NC- when compared to EC- fed cattle (P = 0.04) and there was a tendency for interaction among factors (P = 0.08) being 1.77, 1.65, 1.51 and 1.63 kg for NC-, NC+ADS, EC- and EC+ADS, respectively. There was a tendency for interaction among factors for carcass gain (P = 0.08). Daily carcass gain was 1.20, 1.14, 1.05 and 1.12 kg/d, respectively, for cattle receiving NC-, NC+ADS, EC- and EC+ADS. Then, the NC had greater carcass gain compared to EC- and the addition of ADS recovered part of the gain when used in EC diets. The chemical composition, color, cooking loss and shear force of meat were not affected (P ≥ 0.38) by the factors. In conclusion, mycotoxin affects the performance of beef cattle, and the ADS may alleviate part of this damage when animals were fed diets containing higher contamination. However, these factors did not negatively affect meat quality. Key words: adsorbent, carcass gain, meat quality, performance, survey v CARACTERIZAÇÃO E CONTROLE DE MICOTOXINAS NA ALIMENTAÇÃO DE BOVINOS CONFINADOS RESUMO: O experimento foi dividido em duas fases. A primeira fase foi desenvolvida para identificar quais micotoxinas estavam presentes nas dietas de confinamentos brasileiros e a segunda fase foi para verificar o desempenho de bovinos confinados alimentados com micotoxinas e efeito do Mycosorb A+ ADS. Assim, o objetivo da primeira fase foi identificar quais micotoxinas e em quais concentrações estavam presentes em ingredientes utilizados em dietas de bovinos de cortes confinados. A pesquisa abrangeu 30 confinamentos brasileiros localizados em 5 diferentes estados. Amostras de ração total (RT) e ingredientes foram coletadas no local e enviadas para o 37+® Analytical Services Laboratory (KY, USA) para análise de micotoxinas. A quantificação de 38 micotoxinas diferentes foi realizada utilizando cromatografia líquida de ultra-desempenho acoplada a espectrometria de massa. As concentrações de micotoxinas foram interpretadas de acordo com sensibilidades específicas de espécies conhecidas e normalizadas de acordo com os princípios de fatores equivalentes tóxicos, determinando o Risk Equivalency Quantities (REQ) expressas em µg/kg equivalente de aflatoxina B1. Estatísticas descritivas foram obtidas utilizando o procedimento UNIVARIATE do SAS e estatísticas multivariadas foram obtidas utilizando o STATISTICA. As toxinas identificadas nas RT foram: fumonisinas, tricotecenos A, tricotecenos B, ácido fusárico, aflatoxinas e ergot (média de 2330; 104,3; 79,5; 105; 10,5 e 5,5 µg/kg). As fumonisinas foram as micotoxinas mais encontradas e em maiores concentrações nas amostras de RT. O amendoim foi o ingrediente mais contaminado. O objetivo da segunda fase foi avaliar o efeito de micotoxinas e ADS sobre o desempenho e a qualidade da carne de bovinos confinados. Foram utilizados 100 bovinos (430 ± 13 kg de peso corporal (PC) e 24 meses). O delineamento foi em blocos casualizados, em esquema fatorial 2 × 2 de tratamentos. Os tratamentos consistiram de dois fatores: Fator 1: RT com contaminação natural (CN) ou contaminação exógena (CE) e Fator 2 presença (10g/d/animal; ADS) ou ausência do ADS. As dietas CN e CE apresentaram, respectivamente, as seguintes contaminações: aflatoxina 0 e 10 µg/kg, fumonisina 5114 e 5754 µg/kg, tricotecenos A 0 e 22,1 µg/kg, tricotecenos B 0 vi e 42,1 µg/kg e ácido fusárico 42,9 e 42,9 µg/kg. No início do experimento, todos os animais foram pesados e 4 animais selecionados aleatoriamente foram abatidos para avaliar o peso inicial da carcaça. Após 97 dias de experimento, todos os animais foram pesados e abatidos. Bifes do Longissumus thoracics foram retirados entre a 11ª e a 13ª costelas, sendo três bifes aleatoriamente designados para tempos de maturação de 7, 14 e 28 dias a 4°C. A qualidade da carne foi analisada. Não houve interação entre os fatores para o consumo de matéria seca (CMS); (P = 0,92), porém houve tendência de redução para dietas CE- em 650 g/dia (P = 0,09). O ganho médio diário (GMD) foi maior para CN- em relação ao CE- (P = 0,04) e houve tendência de interação entre fatores (P=0,08) sendo 1,77, 1,65, 1,51 e 1,63kg para CN-, CN+ADS, CE-, CE+ADS, respectivamente. Os animais da CN- apresentaram maior PC final (596 kg) do que CE- (582 kg, P = 0,04). Houve tendência de interação entre os fatores para ganho de carcaça (P = 0,08). O ganho médio diário de carcaça foi de 1,20, 1,14, 1,05 e 1,12 kg, respectivamente, para CN-, CN+ADS, CE-, CE+ADS. Assim, o CN- apresentou maior ganho de carcaça em relação ao CE- e, além disso, o ADS recuperou parte do ganho quando usado em dietas CE. A composição química, cor, perda por cocção e maciez da carne não foram afetadas (P ≥ 0,38) pelos fatores. Em conclusão, a micotoxina afeta o desempenho de bovinos de corte, e o ADS pode recuperar parte desse dano quando os animais consomem dietas com uma contaminação mais alta. Porém, esses fatores não afetam a qualidade da carne. Palavras-chave: adsorvente, desempenho, ganho de carcaça, levantamento de dados qualidade de carne 1 CHAPTER 1 - GENERAL CONSIDERATIONS 1. INTRODUCTION Health issues related to the production system intensification may frequently occur, and these problems can cause disorders to the animals and impact the immune system, performance and consequently increase production costs. Some feedstuff contaminants can cause serious damage to animals, such as mycotoxins (Friend et al., 1992; Merril et al., 1996; Blank et al., 2003; Iqbal et al., 2013). Fungi and mycotoxins can occur in feedstuffs used in beef cattle, such as grains, silages, hay and by-products (Mallmann et al., 2009) and, this issue becomes important regarding feedlot diets. If losses related to mycotoxins in animal feeds were scaled, the range of economic losses would be huge in Brazil (Trail et al.,1995; Jobim et al., 2001). Mycotoxins are substances naturally produced by fungi and usually a form of microorganisms defense (Jouany, 2001). Most natural feedstufs are susceptible to contamination. The fungi growth is typically stimulated by environmental factors, as high temperature and humidity, both pre and post-harvest (Binder et al., 2007). However, toxin production is dependent on factors such as microbial competition, nutrient availability and substrate structure, water activity, pH, temperature, relative humidity, presence of bugs, and application of fungicides and pesticides (Hameed et al., 2013; Anfossi et al., 2016). However, the occurrence of fungal growth does not indicate the presence of mycotoxins (Cheeke and Shull, 1985). Late harvest may aggravate mycotoxin production in the crop, because the longer grain stays in the field, the more susceptible to stress factors it becomes (Duncan et al., 1994). In addition, no-proper storage of grains or forages may also allow fungi growth and mycotoxins (Motta et al., 2015). According to Santos and Fink-Gremmels (2014), the mycotoxin problem may be related to preharvest infestation of cereals and grains by toxinogenic Fusarium species, as well as post- harvest contamination of stored/ensiled materials by Penicillium (P. roqueforti and P. carnosum a.o.) and Aspergillus species. Thus, contamination can be avoided through good management practices, but it is difficult to ensure that all material coming from the field is contamination free. 2 The effects generated by mycotoxins on animals depend on the amount, time of exposure and synergistic action (Smith and Korosteleva, 2010). These effects may be reproductive, immunological and performance disorders (Mallmann et al., 2009). Most of mycotoxin studies evaluated its effects on monogastric animals, since these animals are more susceptible to the toxic effects of mycotoxins compared to ruminant (Di´az-Llano and Smith, 2014; Kong et al., 2016). In ruminants, the harmful effects of mycotoxin may be less aggressive, because ruminal microorganisms can inactivate some of these compounds (Upadhaya et al.,2010). However, it is not all mycotoxins that are inactivated in the rumen and, in addition, they may affect ruminal microorganisms due to their antibiotic effect (Fink-Gremmels, 2008) and others can be transformed in products more dangerous than the mycotoxin. According to Marczuk et al. (2014), some mycotoxins have antibacterial properties, they modify the ruminal microflora and minimize detoxicating effects of ruminal digesta. A serious consequence of the mycotoxins may be related to animal products, such as meat, milk and eggs, which may contain residues of these compounds, and harm human health (Bruerton, 2001). Thus, it is important to study the hygienic and sanitary quality of the feedstuffs used in cattle diets and how these impact health, performance and quality of the final product. First of all, to avoid the contamination of feedstuffs used in animal diets, it is necessary to identify which is the most frequently contaminated ingredient and which mycotoxins are often found in these materials. After these identifications, it becomes possible to seek strategies to minimize the harmful effects to animals. As mentioned above, the best way to avoid contamination would be through proper management of the crop, forages or by-products used to feed animals. However, since this is not always possible, there are other strategies that can be adopted when feed is already contamination, such as the use of adsorbents in the diets. The use of adsorbents in feeds is an strategy that may reduce the absorption of mycotoxins by the animals, since these compounds contain substances that complex with toxins, preventing them to be absorbed in the gastrointestinal tract, as they form an adsorbent-mycotoxin complex and they are eliminated in feces (Yiannikouris and Jouany, 2002). 3 We hypothesized that mycotoxins could decrease performance of Nellore bulls finished in feedlot, but the use of yeast cell wall adsorbent can attenuate this damage. In addition, mycotoxins may negatively impact the meat quality of Nellore cattle. Thus, the objective of this study was to identify contaminated ingredients, measuring which type of mycotoxins, as well as the level of contamination of diets used in feedlots in Brazil. Further, the effects of these mycotoxins and the use of adsorbent (Mycosorb A+) in performance and meat quality of Nellore cattle finished in feedlot were evaluated as well. 2. LITERATURE REVIEW Main mycotoxins and their effects on animals Aflatoxins Aflatoxin is the most aggressive mycotoxin for animal health and is produced by fungi Aspergillus flavus, Aspergillus parasiticus, and Aspergillus nomius (Battacone et al., 2012). These fungi invade plant tissue, in particular when damaged and it is produced in warm climates (Cotty and Jaime-Garcia, 2007). The contamination can occur before or after harvest mainly on starch cereal, cottonseeds, and peanuts crops (Richard, 2007). These toxins can be identified as aflatoxin B1, B2, G1 and G2 and mainly affect the liver, forming abscesses, which are represented by primary biochemical lesions (Foster et al., 1983). The aflatoxin B1 (AFB1) is the most important toxin in this group, acting as mutagen and carcinogen (McLean and Dutton, 1995). These are associated with the worldwide incidence of liver cancer, which is one of the most lethal cancers (Liu et al., 2012). Aflatoxins are not altered by rumen microorganisms, but, when it occurs, only 10% of the ingested aflatoxin can be transformed in aflatoxicol and this substance maintains the same toxic power as the original molecule (Upadhaya et al., 2010). Thus, the rumen does not provide protection against aflatoxins. Studies have indicated that values between 0.3% and 6.2% of AFB1 in animal feedstuffs are metabolized, biotransformed, and secreted in milk in the form of aflatoxin M1 (AFM1) (Creepy, 2002; Unusan, 2006; Iqbal et al., 2013; Duarte et al., 2013). The AFM1 may be considered more dangerous than AFB1, because AFB1 is 4 dependent a metabolic activation to result in its carcinogenicity acute (Neal et al., 1998). However, the AFM1 has toxic effects without metabolic activation (Caloni et al., 2006). Furthermore, this toxin is included in the category of carcinogenic agents to humans (Iarc, 2002). In ruminants, chronic exposure to aflatoxins reduces appetite, and leads to poor feeding efficiency and low milk production (Rossi et al., 2009; Whitlow, 2010). Furthermore, death due to aflatoxicosis in calves has been reported (Lynch, 1972). Ochratoxin A (OTA A) Ochratoxin A is produced by Aspergillus and Penicillium fungi (Halasz et al., 2009). Aspergillus species predominate in warm and temperate regions while Penicillium is frequently found in colder areas (Futagami et al., 2011). This toxin seriously affects monogastric animals, because it can cause inhibition of protein synthesis, lipid peroxidation, DNA changes, respiratory chain inhibition, cellular apoptosis, and inhibition of enzymes involved in kidney and liver metabolism (Kruger, 2006). However, in ruminant animals, due mainly to the activity of protozoa (Mobashar et al., 2012), the OTA A is almost all degraded by ruminal microorganisms in a less toxic compound: ochratoxin α (Battacone et al.,2010). However, the detoxification capacity of the rumen can be exceeded in situations of high contamination (Ribelin et al., 1978). These authors indicated that the lethal single oral dose of OTA A in cattle is probably higher than 13 mg/kg of body weight (BW). In addition, when animals are receiving ingredients that are able to maintain low rumen pH, which is common in feedlot diets, the inactivation capacity of the microorganisms on OTA A may be reduced, which facilitates the direct uptake of toxin from the rumen into the blood (Marquardt and Frohlich, 1992). Blank et al. (2003) observed increased of ochratoxin α in serum of sheep when they fed increased doses of OTA (0, 9.5, 19.0 and 28.5 μg OTA/kg of BW). Besides this, small amounts of these toxins were detected in plasma, suggesting OTA A could bypass the rumen (Denli and Perez, 2010). Höhler et al. (1999) observed that when sheep were fed 14 mg of OTA/kg of diet, animals reduced dry matter intake (DMI) compared to sheep fed no contaminated diet. In addition, the 5 authors reported residues of OTA in milk, which suggests a ruminal escape of this toxin. Fumonisins Fumonisins are mainly produced by Fusarium verticillioides and Fusarium proliferatum fungi (Tiemann and Danicke, 2007). These fungi are important cereal pathogens, mainly corn, at various stages of development, including post-harvest periods, when the grains are stored (Diaz and Boermans, 1994). Fumonisins are diesters of tricarballylic acid and polyhydric alcohols which are very similar to sphingosine structure (Norred, et al., 1992). The variations of the hydroxyl group of the fumonisin molecules determine their different types, which can be B1, B2, and B3 (Visconti et al., 1995). Sphingolipids are strongly influenced by fumonisins, since this mycotoxin may block their synthesis, due to the inhibition of ceramide synthetase and prolonged inhibition that can promote cell death induced by free sphingoid bases (Riley et al., 1998). These toxins affect more monogastric animals. The symptoms in horses are necrotic brain lesions (Kellerman et al., 1990), in swine and chickens occurs cellular apoptosis, affecting part of normal organ development and tissue maintenance (Merril et al., 1996). Although this mycotoxin is less toxic to ruminants, it normally passes by the rumen, and if it is ingested in high amounts, can affect different organs, but liver and kidneys are the most affected. According to Fink-Gremmels (2008), one of the signs of intoxication in cattle are: high enzymatic activity of serum hepatic enzymes (aspartate aminotransferase and gammaglutamyl transferase). Furthermore, the toxicity of fumonisin B1 may induce the initiation of carcinogenic tumors in the liver (Gelderblom et al., 2001). Zearalenone (ZEA) Zearalenone is a lactone produced by Fusarium graminearum, Fusarium culmorum, Fusarium equiseti and Fusarium crookwellense fungi (Kummar et al., 2008). The ZEA contamination has been reported in cereal grains, mainly in temperate climates (Hagler et al., 2001). Typically, this toxin is found at low 6 concentrations in contaminated grains in the field but, it increases under high moisture storage conditions (30 to 40%); (Gupta, 2007). This toxin is frequently detected in grains with another mycotoxin (deoxynivalenol), and despite it is heat stable, it may be partially destroyed during extrusion of cereals (Castells et al., 2005). The zearalenone can cause reproductive and estrogenic problems in animals (Minervini and Dell’aquila, 2008) In monogastric, it is rapidly absorbed and distributed to the ovary, uterus, adipose tissue and interstitial tissue (Kuipergoodman et al., 1987). Kurtz and Mirocha (1978) observed that dietary concentrations of ZEA as low as 1.0 mg/kg may lead to hyperestrogenic syndromes in swines, and higher concentrations can lead to disrupted conception, abortion, and other reproductive problems. In ruminants, about 90% of ingested ZEA is converted into α-zearalenol and β- zearalenol by the ruminal microorganisms (Cruz, 2012). The α-zearalenol has higher estrogenic potency than ZEA because it can be converted to zeranol, which acts as a growth promoter, but its toxic effect causes less damage because it is less absorbed, and when absorbed it is converted to β-zearalenol in the liver. The β-zearalenol has toxic activity on endometrial cells, but its affinity for estrogen receptors is smaller (Bottalico et al., 1985). In this way, ruminants are less sensitive to ZEA exposure than nonruminant animals, because of the metabolization of this toxin in the rumen (Seeling et al., 2005; Fink-Gremmels and Malekinejad, 2007). However, if rumen acidosis occurs in cattle, it is expected that the microflora will fail to eliminate ZEA (Takagi et al., 2011). This fact occurs because one of the consequencies of rumen acidosis are destruction of a large percentage of the normal rumen microflora, so less chance to toxins metabotization. Trichothecenes Trichothecenes are produced by Fusarium sporotrichioides, Fusarium graminearum, Fusarium poae, and Fusarium culmorum (Upadhaya et al., 2010) and they can also be produced by Trichothecium (Jones and Lowe, 1960). This toxin can be classified in type A, which are: T-2, HT-2, neosolaniol, 15-monoacetoxiscirpenol (15-MAS) and diacetoxiscirpenol (DAS); and in type B, which are: desoxinilvalenol 7 (DON or vomitoxin), 15-acetildesoxinivalenol (15aDON), fusarenona X (FX) and the nivalenol (NIV) (Santin et al., 2001). These toxins may be present in most of the cereals during harvest and storage (Yiannikouris and Jouany, 2002). However, DON is considered the most usual mycotoxin in silages and other forages (Storm et al., 2008) Swines and chickens have been shown to be very sensitive to T-2 toxin and DON (Friend et al., 1992). The toxic effects may reach nervous, immune and digestive systems (Lazzari, 1997). According to Upadhaya et al. (2010), ruminants are less susceptible to these mycotoxins. However, Swanson et al. (1987) demonstrated that when DAS, DON, and T-2 toxin were incubated with rumen fluid, all three were rapidly metabolized by the microflora producing monoacetoxyscirpenol (MAS) that is also a toxic compound. The effect of these mycotoxins is to inhibit the initiation, elongation and termination of protein synthesis in microrganisms cells, making them more potent than other mycotoxins (Ehrliche and Daigle, 1987). Therefore, they cause mucosal lesions, weight loss, interference in motor coordination and cutaneous ulcers (Cavan et al., 1988). Others mycotoxins Toxins from Penicillium are produced by Penicillium roqueforti and Penicillium paneum (Storm et al., 2008). These metabolites can cause immunosuppressive and antibacterial effects in animals (Fink-Gremmel et al., 2008). Moreover, animals consuming forages contaminated by Penicillium strains may present loss of appetite, influence on nutrient efficiency, ketosis, abomasal ulcer, gastroenteritis, laminitis, paralysis and abortion (Nout et al., 1993; Nielsen et al., 2006; Fink-Gremmel et al., 2008; Pereyra et al., 2008). These effects are probably due to the production of their toxic metabolites. For ruminants, however, no adverse effects on blood parameters were detected when sheep were fed 300 mg/kg/day of mycotoxins from Penicillium (Mohret al., 2007). µg/kg Fusaric acid is a toxin produced by Fusarium species (Bacon et al., 1995). This toxin is often present in cereals (Yiannikouris and Jouany, 2002) and cause loss of appetite, lethargy, and loss of muscle coordination in swine (Smith and McDonald, 8 1991). Fusaric acid is highly recognized for its synergistic power with other mycotoxins. This toxin increases the toxicity of trichothecenes (Smith et al., 1997), fumonisin (D’Mello and McDonald, 1996) and ZEA (Porter et al., 1996). Tapia et al. (2005) observed that patulin, produced by Penicillium paneum, interfered on rumen activity. Sabater-Vilar et al. (2004) reported severe cases of neurotoxicosis, comprising tremors, ataxia, paresis, recumbence and death concomitantly occurred in beef cattle because of this mycotoxin. The citrinin are produced by species of the genus Aspergillus and Penicillium and often occur concomitantly with OTA in feedstuffs (Bouslimi et al., 2008). Stec et al. (2008), evaluating this mycotoxin in vitro studies, reported immunotoxic effects of citrinin only at very high doses. Furthermore, Griffiths and Done (1991) conducting in vivo studies, observed that dairy cows fed citrinin and OTA contaminated diets presented signs of pruritus, pyrexia, hemorrhagic syndrome, fever, diarrhea and uremia. Alternaria derived toxins are alternariol and alternariol monomethyl ether. They are toxic for bacteria and mammalian cells in vitro, whereas altertoxins are mutagenic for bacteria and induce cell transformation (Wang et al., 1996; Ostry, 2008). Studies about these mycotoxins are limited. Tall fescue straw is a source of forage widely used in USA for ruminants (Hovermale and Craig, 2001). However, the use is limited because of ergot alkaloids contaminations (Morgan-Jones and Gams, 1982). Cattle consuming high-ergot alkaloid in tall fescue straw have presented lower feed intake, excitability, increased rectal temperature and respiration rate, decreased reproductive efficiency and lighter body weight (Mizinga et al., 1992; Aldrich et al., 1993; Zain, 2011). Mycotoxins Synergism Synergistic effect may occur among different mycotoxins. Feeds are frequently contaminated simultaneously by several fungi that can produce several toxins and there may be synergetic effects (Yiannikouris and Jouany, 2002). Mycotoxins severely affect health and performance of animals as discussed above; however, there are few studies that cover the effects of more than one mycotoxins and the 9 complementary effect of one over the other (D’Mello and McDonald, 1996; Porter et al., 1996; Smith et al., 1997; Bouslimi et al., 2008). Fusaric acid is highly recognized for its synergistic power with other mycotoxins. This toxin increases the toxicity of trichothecenes (Smith et al., 1997), fumonisin (D’Mello and McDonald, 1996) and ZEA (Porter et al., 1996). In turkeys, a combination of fumonisin B1 and DAS reduced 46% of body weight and a combination of fumonsin B1 and OTA reduced 37% (Kubena et al., 1997). Other combinations, involving DON and DAS, DAS and aflatoxins and aflatoxins and fumonisin B1 may also have synergetic effects (Harvey et al., 1995a; Harvey et al., 1995b). There are no studies in the literature reporting the combined and synergistic effect of mycotoxins for beef cattle. Mycotoxin Adsorbents Prevention through preharvest and harvest management is the best method for controlling mycotoxin contamination. There are some strategies that can be applied to minimize the contamination of mycotoxins in feedstuffs used in animal diets, such as: crop rotation, time of irrigation, planting and harvesting, plant breeding for resistance to toxigenic fungi, genetically modified crops resistant to insect penetration, and competitive exclusion by using of non-toxigenic strains in the field (Duncan et al., 1994). When the material are stocked, there are some strategies as additives composed by either organic acids or bacteria that produced organic acids, to prevent fungi growth and in this way, mycotoxin production can be avoid. However, when feedstuffs are already contaminated, it is possible to add adsorbents in diets prior to feeding animals avoiding the mycotoxin absorption. The adsorbents can be divided into two main groups: inorganics and organics. The inorganic adsorbents may be carbon, zeolites, bentonites, clays, calcium hydrated sodium aluminosilicates and diatomaceous soil (Wyatt, 1991; Piva et al., 1995). Organic adsorbents may be oat bark, wheat bran, alfalfa fiber, yeast cell wall, cellulose, hemicellulose and pectin (Dilkin and Mallmann, 2004). A good adsorbent may have a broad spectrum of adsorption acting on a large amount of mycotoxins (Mallmann et al., 2006) and it can not react with other substances. 10 Inorganic additives as zeolites, bentonites and montmorillonites adsorb more aflatoxin, which has a strong positive charge (Buragas, 2005). Adsorbents based on sodium and calcium aluminosilicate are derived from zeolites, so they are effective in reducing the effects of aflatoxin. Phillips et al. (1988) observed an increase in the weight gain of broilers fed aflatoxin-contaminated feed containing sodium and calcium aluminosilicate. Inorganic materials were used for reducing the toxic effect of aflatoxins (Galvano et al., 2001; Huwig et al., 2001; Lemke et al., 2001). However, it has limited efficacy when there are other mycotoxins in the diet (Jouany et al., 2005). The use of yeast cell wall as organic adsorbents has the advantages of surface area for adsorption of a large number of mycotoxins (Yiannikouris et al., 2004; Shetty and Jespersen, 2006). The addition of yeast cell wall as adsorbent in contaminated diets has been used as dietary approach to reduce effects of mycotoxins (Raymond et al., 2003; Diaz et al., 2004; Diaz-Llano and Smith, 2006; Firmin et al., 2011; Marson, 2014). Raymond et al. (2003) observed an increase in intake and reduction of the gammaglutamyl transferase activity in horses fed grain contaminated with DON, 15aDON, fusaric acid and ZEA containing yeast cell wall as organic adsorbent. Diaz- Llano and Smith (2006) reported reduction of stillborn piglets when gilts were fed yeast cell wall in diets contaminated with Fusarium mycotoxins. In studies with ruminants Diaz et al. (2004) observed a decrease of 58.5% of aflatoxin M1 in milk with the use of yeast cell wall. Firmin et al. 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Journal of Saudi Chemical Society 15:129–144. 22 CHAPTER 2 The following article is in accordance with Toxins Journal's publication guidelines. 23 Article Survey of mycotoxin contamination of diets for beef cattle finishing in feedlot Abstract: The objective of this survey was to identify which mycotoxins were present in ingredients used in diets fed to beef cattle in feedlots and their concentrations. The survey covered 30 Brazilian feedlots located into the 5 largest beef-producing states. Samples of total mixed ration (TMR) and ingredients were collected on-site and sent to the 37+® Analytical Services Laboratory (KY, USA) for analysis of mycotoxins. The quantification of 38 different mycotoxins was performed using ultra- performance liquid chromatography coupled to tandem mass spectrometry. The mycotoxin concentrations were further interpreted according to known species - specific sensitivities and normalized according to the principles of toxic equivalent factors, determining the Risk Equivalent Quantities (REQ) expressed in µg/kg of aflatoxin B1 (AFB1)-equivalent. Descriptive statistics were obtained using the UNIVARIATE procedure of SAS and multivariate statistics were obtained using STATISTICA. The toxins identified in TMR were: fumonisins, trichothecenes A, trichothecenes B, fusaric acid, aflatoxins and ergot (mean values: 2,330; 104.3; 79.5; 105; 10.5; 5.5 µg/kg, respectively). All samples presented at least one mycotoxin contamination, and 65.5% of the samples were classified as low contamination, 27.6% medium contamination and 6.90% high contamination. In conclusion, fumonisins were the mycotoxin most frequently found and at highest concentrations in TMR samples, and peanut meal was the most contaminated ingredient. Keywords: aflatoxin; beef cattle; feedlots; fumonisin; ingredients; REQ Key Contribution: The study evaluated the occurrence of mycotoxins in diets of beef cattle. These findings are of importance in the monitoring and management of mycotoxins in beef cattle systems since mycotoxins can limit the optimal performance of animals. 1. Introduction Mycotoxin contamination occurs in many materials, including animal feed, animal products and soil. As these toxins affect animal production and health, they can cause substantial economic losses. Factors that can affect the mycotoxins production by fungi include abiotic factors, such as temperature and humidity, and biotic factors, such as fungal load at the time of transportation and storage. Besides that, there are two types of fungi, those that acts before harvest, commonly called field fungi, and those that occur only after harvest, called storage fungi [1]. The most important genus of mycotoxigenic fungi are Aspergillus, Alternaria, Claviceps, Fusarium, Penicillium and Stachybotrys [2]; while the most common mycotoxins investigated and found in ingredients of animal diets worldwide are: aflatoxin, fumonisin, zearalenone, ochratoxin and trichothecenes [3]. However, as the presence of these mycotoxins relates to specific environmental conditions and type of material, the characteristics of contamination could vary regionally. Nevertheless, the trading of ingredients among regions, countries and continents could also play a role in the contamination dynamic and change mycotoxins distribution patterns. Thus, the objectives of this survey were to identify the mycotoxins and their concentrations present in ingredients used in typical total mixed rations (TMR) fed at Brazilian feedlots, and to relate these results to characteristics of the feedlot. 24 2. Results 2.1. Information about the visited feedlots Eighteen feedlots (60%) had less than 5,000 animals, whereas 7 (23%) had from 5,000 to 10,000 animals and 5 feedlots (17%) had more than 10,000 animals on feeding. The observed average daily gain (ADG) ranged from 0.70 kg to 1.85 kg in these feedlots, however most of feedlots (63%) had animals with ADG of about 1.53 kg; (Table 1). Table 1. General characteristics of the feedlots surveyed. Item Mean Min1 Max1 Number of animals 7,085 324 50,000 ADG, kg 1.53 0.70 1.85 Days on feed 107 85 155 Feeding frequency (times/day) 5 3 8 1Min = Minimum; Max = maximum. Most of the feedlots (83%) stored the ingredients in a common barn, and 57% of the feedlots cleaned up the storage barns (Table 2). Forty percent of the visited feedlots had apparent fungi in TMR, however only one feedlot (3%) used a mycotoxin adsorbent (Table 2). Besides that, from the twelve diets with apparent fungi, six showed moderate mycotoxin contamination, while other six had low contamination. On the other hand, diets with high contamination (6.9%) presented no apparent fungi. Table 2. Information about barns, apparent fungi in TMR and the use of mycotoxin adsorbents of feedlots. Item N of responses % of responses Type of barn for feed storage Common 25 83.3 Double side 3 10.0 Silo 1 3.33 No barn 1 3.33 Barn cleaning Yes 17 56.7 No 13 43.3 Apparent fungi in TMR Yes 12 40.0 No 18 60.0 Use mycotoxin adsorbent Yes 1 3.33 No 29 96.7 25 2.2. Characterization of TMR samples of visited feedlots Almost all TMR samples (93.3%) presented fumonisin (B1+B2), 80% had fusaric acid and 66.7% had trichothecenes A (T-2, H-T2, diacetoxiscirpenol, and neosolaniol); whereas aflatoxin (B1+B2+G1+G2), trichothecenes B (DON, 15-acetyl DON, 3-acetyl DON, fusarenol X, nivalenol, and DON 3-glicoside), ergot and others mycotoxins produced by Penicillium (patulin, penicillic acid, roquefortine C, mycophenolic acid, and wortmannin) were present in fewer samples (6.67, 20.0, 33.3, and 6.67, respectively). Ochratoxin A, zearalenone and other mycotoxins produced by Aspergillus (Gliotoxin, Sterigmatocystin, Verruculogen) were not found (Figure 1). The levels of samples contamination are presented in the Figure 2. Figure 1. Mycotoxin occurrence (%) in total mixed ration (TMR) samples of 30 feedlots collected in five Brazilian states (Aflatoxins = Aflatoxin B1+B2+G1+G2; Ochratoxin = OTA; Trichothecenes B = DON; Trichothecenes A = Toxin T-2 + HT-2; Fumonisins = Fumonisins B1+B2; Others Penicillium = Patulin, Penicillic Acid, Roquefortine C, Mycophenolic Acid, Wortmannin; Others Aspergillus = Gliotoxin, Sterigmatocystin, Verruculogen.). 26 Figure 2. Mycotoxin concentration (µg/kg) in total mixed ration (TMR) samples of 30 feedlots collected in five Brazilian states (Aflatoxins = Aflatoxin B1+B2+G1+G2; Ochratoxin = OTA; Trichothecenes B = DON; Trichothecenes A = Toxin T-2 + HT-2; Fumonisins = Fumonisins B1+B2; Others Penicillium = Patulin, Penicillic Acid, Roquefortine C, Mycophenolic Acid, Wortmannin; Others Aspergillus = Gliotoxin, Sterigmatocystin, Verruculogen.). In this study, 65.5% of TMR samples were classified as having low Risk Equivalent Quantities (REQ), 27.6% were classified as intermediate REQ and 6.9% were classified as high REQ (Figure 3). The maximum REQ found in TMR used in the feedlots evaluated in this survey was 230 µg/kg AFB1- equivalent and the minimum REQ was 1 µg/kg AFB1-equivalent. Figure 3. Percent of total mixed ration (TMR) samples of 30 feedlots in five Brazilian states with Risk Equivalent Quantities (REQ) at Low (0 – 50 µg/kg), Medium (51 -100 µg/kg) and High (>101 µg/kg) risk to beef cattle. Low 65.5% Medium 27.6% High 6.9% 27 The analysis of principal components allowed the construction of the two-dimensional biplot formed by the first two major components (Factor 1 and Factor 2); (Figure 4). In the Figure 4, the samples contaminated with aflatoxins were separated from the other samples and the REQ. As the REQ computes a multi-contamination complex situation pertaining to feedstuffs into one single value [4]. It is possible to observe that the combination of other mycotoxins can be more dangerous than aflatoxin by itself. 28 Figure 4. Principal components of Risk Equivalent Quantity (REQ) of total mixed ration (TMR) samples of 30 feedlots collected in five Brazilian states. 29 0 50 100 150 200 250 1 -G O 2 -G O 3 -G O 4 -G O 5 -G O 6 -G O 7 -G O 1 -M G 2 -M G 3 -M G 4 -M G 1 -M S 2 -M S 3 -M S 4 -M S 5 -M S 6 -M S 1 -M T 2 -M T 3 -M T 4 -M T 5 -M T 6 -M T 7 -M T 8 -M T 1 -S P 2 -S P 3 -S P 4 -S P 5 -S P R E Q , p p b It is possible to observe, according to the contamination of the TMR samples per region, that Mato Grosso do Sul state presented the highest contaminations in some of the TMR (above 200 µg/kg; Figure 5). Figure 5. Contamination per state of total mixed ration (TMR) samples of 30 feedlots in five Brazilian states (GO = Goiás; MG = Minas Gerais; MS = Mato Grosso do Sul; MT = Mato Grosso; SP = São Paulo). 2.3. Characterization of ingredients of ten most contaminated TMR samples of visited feedlots The ingredients from the 10 most contaminated diets were analyzed to determine which ingredients were responsible for the high contamination and which mycotoxins were associated with each ingredient. It was possible to observe that the source of roughage most often used in these feedlots was corn silage and the most common sources of concentrates were corn and cottonseed (Table 3). The Table 4 shows the mean levels of contamination of ingredients from the 10 most contaminated diets, indicating which ingredients were most responsible for TMR contamination and which mycotoxin stood out in each source. 30 Table 3. Roughage and concentrate sources used in diets of visited feedlots. Item N of responses % of responses Roughage source Corn silage 13 43.3 Corn residue 2 6.67 Grass silage 2 6.67 Sugarcane straw + Tifton hay 2 6.67 Others1 11 36.7 Concentrate source Corn grain 23 76.7 Whole Cottonseed 16 53.3 Cottonseed cake 11 36.7 Citrus pulp 7 23.3 Soybean meal 7 23.3 Soybean hulls 6 20.0 Corn germ 5 16.7 High moisture corn 5 16.7 Peanut meal 4 13.3 Sorghum grain 4 13.3 Corn gluten feed 3 10.0 Soybean residue 3 10.0 Others2 12 40.0 1Other roughages source: 3,33% each: Brachiaria hay, Corn silage + Ear corn silage, Corn silage + Grass silage, Corn silage + Sugarcane bagasse, Cotton residue, Ear corn silage + Sugarcane bagasse, Cotton residue, Ear corn silage + Sugarcane bagasse, Grass, millet and sorghum silage, Sorghum silage, Sugarcane bagasse, Sugarcane bagasse + Brachiaria hay, Sugarcane silage. 2Others concentrate sources: 3,33% each: Dehydrated cottonseed, Crambe meal, Corn DDGS, Micro algae, Orange Bagasse, Peanut starch, Rice meal, Rice residue, Rehydrated sorghum, Sunflower cake, Sunflower meal, Tomato + corn residue. 31 Table 4. Mean mycotoxin contamination (µg/kg) of ingredients of ten most contaminated total mixed ration (TMR). Item1 n REQ AFB1 Other aflatoxins Tricho B Tricho A Fumo ZEA F. Acid Penic Asper Ergot Roughages Corn silage 1 50.0 - - - - 7,116 - 619 - - 22.0 Sugarcane bagasse 2 6.00 - - 25.0 - 1,774 - 13.0 - - - Sugarcane straw 1 134 - - 2,276 21.0 - - 87.0 - - - Tifton hay 1 12.5 - - - - 270 - 25.5 13.0 1.50 0.50 Concentrates and by-products Citrus pulp 4 14.0 - - - - - - 88.3 - - 125 Corn 9 58.4 - - 12.1 2,536 18,402 5.60 104 - - - Corn germ 1 103 - - 613 - 25,801 - 152 - - - Cottonseed cake 2 21.0 - - - - - - 409 - 0.50 - Cottonseed 3 4.67 - - - - 1,042 - 39.7 - - - Crambe meal 1 6.00 - - - - 1,192 - 54.0 - - 2.00 DDGS by corn 1 118 6.00 6.00 150 - 12,184 206 693 11.0 - 3.00 Peanut meal 2 1,018 471 578 - - - - 483 - 555 - Peanut starch 1 58.0 - - - - 3,362 - 676 - 21.0 - Corn gluten feed 1 277 - - - - 48,828 - 2,347 30.0 - 201 Soybean hulls 2 8.50 - - - 8.50 217 - 15.0 - 1.00 - Soybean meal 3 63.7 - - - 84.3 28.3 - 13.0 - - 4.70 Soybean residue 1 239 - - - 306 243 - - - - 3.00 1DDGS by corn: Dry destilled grain plus solubles; REQ = Risk equivalent quantities; AFB1 = Aflatoxin B1; Other Aflatoxins = B1+B2+G1+G2; Tricho B = DON; Tricho A = Toxin T-2 + HT-2; Fumonisins = Fumonisins B1+B2; ZEA = Zearalenone; F. Acid = Fusaric Acid; Penic = Patulin, Penicillic Acid, Roquefortine C, Mycophenolic Acid, Wortmannin; Asper = Gliotoxin, Sterigmatocystin, Verruculogen. 32 The cluster analysis of all contaminated ingredients allowed the construction of a dendrogram resulting from hierarchical grouping analysis (a) and non-hierarchical method (b) (Figure 6). In both Figures “a” and “b”, three different groups could be characterized. In Figure 6(a), groups were characterized by contamination levels while in Figure 6(b), groups were clustered according to the contamination levels and to the mycotoxin type. Euclidian distance Euclidian distance (a) (b) Figure 6. Dendrogram of the cluster analysis by the hierarchical method and non-hierarchical method of groups profile 1, 2 and 3 constructed by the K-means algorithm of the ingredients from the 10 most contaminated diets. (a): Group 1: Most contaminated ingredients, Group 2 Intermediate contaminated ingredients and Group 3: Less contaminated ingredients. (b): Aflas: Aflatoxins B1+B2+G1+G2, Tricho B = DON; Tricho A = Toxin T-2 + HT-2; Fumo = Fumonisins B1+B2; ZEA = Zearalenone; Fus Acid = Fusaric Acid; Penic = Patulin, Penicillic Acid, Roquefortine C, Mycophenolic Acid, Wortmannin; Asper = Gliotoxin, Sterigmatocystin, Verruculogen. 3. Discussion Mycotoxins are an important issue, because they can affect production cost and could also impact health of animals and humans [5]. This study assessed what types and amount of mycotoxins are present in the diets of beef cattle at Brazilian feedlots. The diets of visited feedlots showed different levels of contamination, but all TMR samples had some sort of contamination. The type of mycotoxins varied largely by the type of diet component, but the level of contamination varied within ingredient. One example is a sample of peanut meal and one of corn that were extremely contaminated, whereas other samples of the same materials collected from other feedlots had low contamination, indicating the dependency of more than one factor. The high prevalence of fumonisin, trichothecene A and fusaric acid in the samples can be attributed to the type of feedstuffs used in the diets. Fumonisin is produced by species of Fusarium and these toxins occur very often in corn [6]. In this survey 76.7% of the feedlots used corn, 43.4% used corn silage, 16.7% used corn germ, 10% used refinazil and 3.33% used corn dry destilled grain plus solubles (DDGS), which is consistent with the high frequency of fumonisin in TMR samples of this survey. More broadly, corn is the primary source of grain used in feedlot diets [7,8], so this toxin can be very important for cattle in feedlots, since the ingestion of highly contaminated diets can cause lower intake, the variable that most influences performance, [9] besides, affect metabolic organs, such as liver and kidneys [10]. Trichothecenes A and B are also produced by species of Fusarium and they are present in corn and forages [11] widely used in TMR samples analyzed in this survey. These toxins are not very toxic for ruminants [12], however, [13] reported that ruminants ingesting feed contaminated by DON Group 3 Group 2 Group 1 33 presented mycotoxin biotransformation and excretion in fluids, such as blood and milk, which classifies this mycotoxin as a risk to human health. Fusaric acid also can be present in cereals and forages, and it was present in almost all TMR samples. Moreover, this toxin increases the toxicity of trichothecenes through a synergistic mechanism [14]. The synergism is common because mycotoxins are seldom found in isolation, and when multiple mycotoxins are consumed, they may have strong interactions that increase the risk to animal performance and health. As a result, the animal may have a stronger response than what would be expected if it was consumed only a single mycotoxin. Other mycotoxins, including aflatoxins, ergot and mycotoxins produced by Penicillium, were found. Aflatoxins are produced by Aspergillus flavus and Aspergillus parasiticus [2]. Peanut is one substrate that is most commonly contaminated with aflatoxin, because it is one of the most susceptible foods to contamination by fungi that produce this toxin [15]. And although aflatoxin was present in only 6.7% of the TMR samples, this mycotoxin is very important for animal health [16], because it is hepatotoxic, carcinogenic, and immunosuppressive [17], besides it is not metabolized in the rumen, which negatively affects the ruminant. Ergot and mycotoxins produced by Penicillium can also be dangerous for ruminants. According to [18], ergot alkaloids are produced by a group of fungi of the genus Claviceps and frequently found in cereal grains. When ingested by ruminants this toxin can decrease feed intake, elevate body temperature, lead to excessive salivation, increase respiration rate and decrease peripheral circulation [19]. Mycotoxins produced by Penicillium are mostly found in materials stored in bad conditions, such as silages and hays, and can reduce appetite, impact nutrient efficiency, and increase abomasal ulcers, laminitis, gastroenteritis and paralysis [20]. These toxins are not always present in materials with obvious fungal growth, because the microorganisms may produce mycotoxins only if they suffer some type of stress [21]. This occurred in this study, since diets with high contamination of mycotoxin did not presented apparent fungi and diets with apparent fungi, had low or moderate contamination. When the general information about the feedlots and the contamination levels of the samples were contrasted, it was observed that the contamination had no relationship with the performance of the animals. The mean of ADG of the animals in the feedlots with high or moderate risk diets was 1.56 kg, whereas animals in feedlots with low risk diets were 1.52 kg. The animals in feedlots with the highest ADG (1.85 kg) consumed moderate risk diet, whereas the animals in feedlots with the lowest ADG (0.70 kg), consumed low risk diet. This difference can be explained, probably because the variability of the genetic potential, management of the animals and the different diets. Studies to confirm the impact of mycotoxins on beef cattle performance need to be conducted. An important issue in this study, which is related to what was stated above, is the real risk of each mycotoxin in the feed. The concentration of each mycotoxins does not always demonstrate the real risk of the sample, since some mycotoxins are present in small concentrations, but may present high risks for ruminants and the reverse, or the combination of different mycotoxins could be more dangerous than one single mycotoxin [14]. In this way, besides identifying which mycotoxins were present in TMR and which levels, it is also important to estimate the risks associated with the presence of different types and concentration of mycotoxins. It was possible to estimate the equivalent risk of the feed through the REQ (Risk Equivalent Quantity), created by [4] based on the concepts of chemistry, which generates a real risk of the mycotoxin. The risk assessment calculates a risk equivalency quantity (REQ) expressed in µg/kg of AFB1-equivalent, which computes a multi-contamination complex situation pertaining to feedstuffs into one single value [4]. According to [4], the REQ, expressed as µg/kg of AFB1- equivalent can be classified and the range between 0 to 50 µg/kg is considered low, 51 to 100 µg/kg is considered intermediate and above 100 µg/kg is considered high for beef cattle. In this study, the maximum REQ 34 found in TMR used in the feedlots evaluated was 230 µg/kg and the minimum REQ was 1 µg/kg AFB1-equivalent. Besides that, through the detection of multiple mycotoxins, it is possible to estimate the risk of all mycotoxins together [14]. The principal components showed part of this combination (Figure 4), since while aflatoxin is the most aggressive single mycotoxin, REQ is along with the other mycotoxins, probably because the combination of mycotoxins and their high concentrations could be more dangerous than aflatoxin by itself. So, these data demonstrated that it is important to consider the combined occurrence of different types of mycotoxins in ingredients and TMR samples. However, this is often neglected in other analytical approaches. Co-occurrences are important since mycotoxins could have an additive effect, potentially further increasing their negative impact in animal. Other important issue to observe about mycotoxins contamination is that contamination by fungi is environmentally dependent, as these microorganisms overcome in more humid and warmer environments. Furthermore, the production of mycotoxins depends on environmental factors that are able to cause fungi stress, allowing them to produce the toxins. In this sense, aflatoxin contamination occurs most often in the Southern United States [3]. The Mato Grosso do Sul (MS) state in Brazil is characterized by a tropical climate, high temperature, rain in summer and dry and cold winter. High temperatures and humid it may provide condition for development of fungi, and dry and cold winter can cause some stress, which stimulates mycotoxin production. Because of these factors, the MS state presented the greatest contaminations in TMR samples. After all analyzes of TMR samples, it was possible to further investigate into diets through the analyses performed in the ingredients used in the TMR composition. Through this, we observed that from all ingredients, the most used source of roughage was corn silage and the most used sources of concentrate was corn grain and cottonseed. These ingredients are very common in Brazilian feedlots. In a survey conducted by [7], about management practices and nutritional recommendations used by feedlot nutritionists in Brazil, they observed that corn silage was used by 28.5% of the respondents as roughage source. They also observed that corn was the primary source of grain, whereas cottonseed was the primary by-product used in feedlot diets. Through the results of ingredient contamination from the 10 most contaminated diets, it was possible to observe more specifically which ingredient was responsible for TMR contaminations, and which mycotoxin stood out in each source. As aflatoxin is a dangerous toxin for beef cattle, the ingredient that showed high concentrations of this mycotoxin, was peanut meal, which presented the highest REQ as well. Aflatoxin is commonly found in peanut samples. In a study conducted by [15], aflatoxin was found in 40.4% of peanuts analyzed. The occurrence of aflatoxins is higher in peanuts because it is the preferred product for the fungi that produces this toxin, and there are also delays and rains in the post-harvest drying period. Another form of contamination is when the peanut is stored at high humidity [22]. In addition to peanut, aflatoxin can be found in many other products widely used in Brazil, such as corn and oilseeds [13]. These ingredients presented considerable contamination with high REQ, like corn and its by-products (refinazil, DDGS), as well as soybean and soybean residues. However, only peanut and DDGS by corn presented aflatoxin contamination. Sugarcane straw also presented high REQ values. This contamination was due trichothecenes B, trichothecenes A and fusaric acid. These contaminations occurred because this material is the residue of the sugarcane that stays in the field after the harvest and is more subject to contamination. The cluster analysis of all contaminated ingredients allowed the construction of a dendrogram resulting from hierarchical grouping analysis. In the dendrogram it is possible to observe the 35 formation of three groups: group 1 was characterized by the most contaminated ingredients, group 2 by medium contaminated ingredients and group 3 the less contaminated ingredients. Through the results of the cluster analysis by non-hierarchical method it was possible to observe the formation of three different groups. Group 1 was that had high contamination of aflatoxin and other mycotoxins produced by Aspergillus, group 2 was the group that has high contamination of fumonisin, fusaric acid, mycotoxins produced by Penicillium and ergot while group 3 were the samples that had low contamination for all mycotoxins. The clustering factors found in these analyses were the level of ingredients contamination. However, it was interesting that most of mycotoxins of the same group are produced by the same fungal genus, as in group 1, where the mycotoxins were produced by fungi of Aspergillus genus. In group 2, we observed fumonisin and fusaric acid, which are produced by Fusarium genus, although there is also the presence of ergot mycotoxins produced by Penicillium. In the group 3, we verified represents low contaminated samples for all mycotoxins. In this way, we can define through these analyses is that the groups characterize the different ingredients, since each ingredient has a higher concentration of each mycotoxin. As example, corn and its by-products present concentrations of fumonisin and fusaric acid, whereas feeds, such as peanuts, have high concentrations of aflatoxin and mycotoxins by Aspergillus. 4. Conclusion In conclusion, current data obtained in this study evidence that 100% of TMR are contaminated and some strategies need to be implement to minimize the risk for beef cattle. In addition, fumonisins were the mycotoxins found most frequently and at highest concentrations in TMR fed at Brazilian feedlots. Peanut meal was the most contaminated ingredient and more aggressive for beef cattle. Moreover, the greatest risk of contamination is in the combination of different mycotoxins instead of an isolated one. 5. Materials and methods The survey was applied in the years 2015/2016. Thirty Brazilian feedlots located in five different Brazillian states: eight in Mato Grosso (MT), six in Mato Grosso do Sul (MS), seven in Goiás (GO), four in Minas Gerais (MG) and five in São Paulo (SP) (Table 5) were surveyed. These states are the 5 largest beef-producing states, responsible for 81.3% of all animals finished in feedlot in Brazil [23], and therefore were they chosen for the evaluation. Each feedlot was visited by the authors, where samples of ingredients and TMR were collected and a questionnaire about concerning aspects of the feedlot and its management was completed. Table 5. Visited Brazilian states and their number of animals at feedlot. Brazilian State Number of Animals1 % Total Mato Grosso 977,131 24.4 Goiás 817,442 20.4 Mato Grosso do Sul 636,395 15.9 São Paulo 628,940 15.7 Minas Gerais 197,906 4.90 Total 4,008,764 100 1 [23] 36 The questionnaire contained 61 questions that were categorized into the following topics: general information about the facilities (12 questions), general cattle management (21 questions), diets (18 questions), concentrates and co-products used (4 questions), and roughage used (6 questions). Samples of total mixed ration (TMR) (n=30) and ingredients were sent to the research facility (Colina, SP, BRA). These samples were lyophilized and ground in the laboratory. Subsequently, TMR samples were vacuum packed and sent to the 37+® Analytical Services Laboratory (Lexington, KY, USA) for mycotoxin analysis. The evaluation of mycotoxins comprised two distinct steps: in a first step, the absolute quantification of 38 different mycotoxins was performed using a validated and ISO/IEC 17025:2005 accredited method by means of ultra-performance liquid chromatography (UPLC) electrospray ionization tandem mass spectrometry (ESI-MSMS) involving an isotopic dilution step and data normalization process. In a second step, the mycotoxin concentrations were further interpreted according to known species-specific sensitivities and normalized according to the principles of toxic equivalent factors, determining the risk equivalent quantities (REQ) expressed in µg/kg of AFB1- equivalent [4]. After the evaluation of TMR samples, the ingredients of the 10 most contaminated TMR (n=41) were sent to the 37+® Analytical Services Laboratory (Lexington, KY, USA) for analysis of mycotoxins. This procedure was done to evaluate which ingredients were most responsible for TMR contamination. The responses generated from the questionnaire and the analytical data were submitted to descriptive analysis using the UNIVARIATE procedure of SAS (SAS Institute, Inc., Cary, NC, USA) and multivariate statistics using STATISTICA (STATSOFT, Inc, Tulsa, OK, USA). The multivariate analyses were performed using cluster analysis, which allows grouping of the variables using the Ward method and considers the Euclidean distance for group establishment. Starting of the number of groups adopted in the cluster analysis by hierarchical method, the analysis of grouping has developed using a non-hierarchical method using the k-means algorithm, which complements the results of the previous analysis. Finally, through the analysis of principal components it was possible to evaluate the importance of each component and the discriminatory power of each variable. Acknowledgements: The authors acknowledge the support of FAPESP for the scholarship of the first author (2015/21416-6). 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Nutrients, 2017, 9, 1287. doi:10.3390/nu9116287. 23. Anualpec. Anuário da pecuária brasileira. São Paulo, SP, Brazil: Informa Economics FNP, 2015. 38 CHAPTER 3 The following article is in accordance with Journal of Animal Science publication guidelines. 39 Running head: Mycotoxins and adsorbent on finishing cattle Mycotoxin contaminated diets and adsorbent affect performance of Nellore bulls finished in feedlot 1 1 This study was made possible by grants from: #2015/21416-6, São Paulo Research Foundation (FAPESP). ABSTRACT: Mycotoxins are present in almost all feedstuffs used in animal nutrition, but are often ignored in beef cattle systems, although they can affect animal performance. The objective of this study was to evaluate the effects of mycotoxins and mycotoxin adsorbent on performance of Nellore cattle finished in feedlot. One hundred Nellore cattle (430 ± 13 kg and 24 months) were used in a randomized block design with a 2 × 2 factorial arrangement of treatments. The factors consisted of two diets (Factor 1) with either natural contamination (NC) or exogenous contamination (EC) and presence (10 g/animal/d; ADS) or absence of mycotoxin adsorbent (Factor 2). The NC and EC diets had, respectively, the following contaminations: aflatoxins 0.00 and 10.0 µg/kg, fumonisins 5,114 and 5,754 µg/kg, trichothecenes B 0.00 and 42.1 µg/kg, trichothecenes A 0.00 and 22.1 µg/kg, and fusaric acid 42.9 µg/kg for both diets. At the beginning of the experiment, all animals were weighed, and 4 randomly selected animals were slaughtered to evaluate initial carcass weight. After 97 days of experiment, all animals were weighed and slaughtered. There was no interaction among factors for dry matter intake (DMI; P = 0.92). However there was a tendency for exougenous contamination diets (EC) to decrease DMI by 650 g/d (P = 0.09). There was a trend for interaction among factors (P = 0.08) for ADG, where the highest ADG was observed for natural contamination diets without adsorbent (NC-) (1.77 kg) and the lowest was observed 40 for EC without adsorbent (1.51 kg). The NC+ADS and EC+ADS treatments presented intermediate values (1.65 and 1.63 kg, respectively) and they did not differ significantly from NC- and EC- treatments. The animals fed NC diet had greater final BW (596 kg) than those at the EC diet (582 kg; P = 0.04). There was a tendency for interaction among factors for carcass gain (P = 0.08). Similar to ADG, the highest carcass gain was observed for NC without adsorbent (1.20 kg/d) and the lowest was observed for EC without adsorbent (1.05 kg/d). The NC+ADS and EC+ADS treatments presented intermediate values (1.14 and 1.12 kg/d, respectively) but they did not differ significantly from NC- and EC- treatments. So, the NC- had greater carcass gain compared to EC- and the use of ADS recovered part of the gain when used in EC diets. In conclusion, mycotoxin affects the performance of beef cattle and adsorbent may mitigate its impact. Key words: carcass gain, dressing, fungi, mycotoxin production INTRODUCTION The effects of mycotoxins in animals depend of the amount consumed, exposure time, and interaction among different toxins (Upadhaya et al., 2010). These effects may impact reproduction, immune system and performance (Zain, 2011). Since 1968, it is known that mycotoxins affect beef cattle (Garrett et al., 1968), in addition, a recent study of Custodio et al. (2017) reported that at least one type of mycotoxin is presented in the samples of feedlot diets collected in a survey conducted in Brazil. However, studies evaluating the effects of mycotoxin on ruminant were developed mainly with dairy cattle (Fink-Gremmels, 2008; Chaiyotwittayakun, 2010; Santos and Fink-Gremmels, 2014). The main effects involved reduced dry matter intake and consequently reduced performa