13 research outputs found

    Dietary effects of cottonseed and vitamin E on greenhouse gas emissions from cattle feces analyzed in biodigesters

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    O objetivo deste trabalho foi avaliar o efeito de dietas com caroço de algodão e vitamina E nas emissões de gases de efeito estufa (GEE) de fezes de bovinos analisadas em biodigestores. As fezes dos animais foram avaliadas em 18 digestores anaeróbios de laboratório, por meio dos seguintes tratamentos: controle, fezes de vacas alimentadas com dieta controle; CS, fezes de vacas alimentadas com caroço de algodão; e CSVitE, fezes de vacas alimentadas com caroço de algodão e vitamina E. As produções de biogás e GEE foram medidas, e não foram observadas diferenças para as emissões de biogás, metano (CH4) e óxido nitroso (N2O). A partição da energia bruta do processo de digestão anaeróbica foi semelhante entre os tratamentos. Em média, 28% da energia bruta alimentada foi liberada como CH4, 47% na digestão e 25% como outros gases e calor. A adição de caroços de algodão na dieta de bovinos aumenta a concentração de CH4 e reduz a de CO2, mas não afeta a produção total de CH4, CO2 e N2O nos biodigestores. A inclusão de vitamina E na dieta não tem efeito na produção de GEE nos biodigestores.The objective of this work was to evaluate the effect of diets with cottonseed and vitamin E on the greenhouse gas (GHG) emissions of cattle feces analyzed in biodigesters. Animal feces were evaluated in 18 laboratory-scale anaerobic digesters through the following treatments: control, feces from cows fed with a control diet; CS, feces from cows fed with a cottonseed diet; and CSVitE, feces from cows fed with a cottonseed and vitamin E diet. The production of biogas and GHG was measured, and no differences were observed for the emissions of biogas, methane (CH4), and nitrous oxide (N2O). The partition of the gross energy of the anaerobic digestion process was similar among treatments. On average, 28% of gross energy fed was released as CH4, 47% during digestion, and 25% as other gases and heat. The addition of cottonseeds to the cattle diet increases the concentration of CH4 and reduces that of CO2, but it does not affect the total production of CH4, CO2, and N2O in the biodigesters. The inclusion of vitamin E in the diet has no effect on GHG production in the biodigesters

    Emissão de gases de efeito estufa (GEE) pela pecuária: situação e perspectivas para criação de um mercado de crédito de carbono

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    O mercado mundial de carbono desenvolveu-se rapidamente com\ud a ratificação do Protocolo de Quioto, sendo considerado como base para a\ud implantação deste mercado. O objetivo do presente estudo foi analisar a\ud situação e perspectivas da emissão de gases de efeito estufa (GEE) pela\ud pecuária, e a possível criação de um mercado de crédito de carbono. Pelos\ud dados levantados, pode-se afirmar que o Brasil tem um grande potencial para\ud a criação deste mercado, por possuir o maior rebanho bovino comercial do\ud mundo e importante participação na emissão de GEE, principalmente o metano.\ud Com um horizonte de crescimento para o setor, a melhora da eficiência técnica\ud e a utilização dos preceitos de sustentabilidade são fatores importantes para\ud quem almeja um desenvolvimento sólido e sustentável.Os organizadores autorizam a reprodução total ou parcial deste trabalho, para qualquer meio convencional ou eletrônico, para fins de estudo e pesquisa, desde que citada a fonte

    Tannins as feed additive to mitigate methane emissions in ruminants

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    Embora algumas estratégias alimentares tenham sido propostas para diminuir a emissão de metano (CH4) provenientes de ruminantes, poucas têm mostrado diminuição persistente, principalmente in vivo. O objetivo do presente estudo foi avaliar o uso de diferentes níveis de taninos como aditivo alimentar para ruminantes como mitigador das emissões de CH4 ruminal e dos dejetos. Utilizou-se o delineamento experimental quadrado latino 4x4 duplicado (4 períodos de 28 dias cada), em arranjo fatorial 2x4, sendo testados dois grupos distintos de bovinos (taurinos ou zebuínos) e quatro níveis de inclusão de taninos (extrato comercial de Acacia mearnsii, contendo 82,3% de taninos totais - equivalente em ácido tânico e 32,3% de taninos condensados - equivalente em leucocianidina) na dieta (0; 0,5; 1,0 e 1,5% da MS da dieta). Para avaliar a produção de CH4 dos dejetos utilizaram-se biodigestores anaeróbios experimentais do tipo batelada, em delineamento inteiramente casualizado. Os níveis utilizados do aditivo promoveram mudanças no comportamento de ruminação dos animais e apesar de causarem redução linear da digestibilidade dos nutrientes, mantiveram o equilíbrio entre a síntese e consumo de N-NH3 ruminal, reduziram linearmente a síntese de proteína microbiana e a produção de CH4 ruminal sem, contudo, reduzir a eficiência microbiana e a produção de ácidos graxos de cadeia curta (AGCC), demonstrando que estes taninos interferem diretamente e indiretamente sobre as archeas metanogênicas. Através do modelo Broken-line encontrou-se o limiar de 0,72% de taninos na dieta como ponto de inflexão para iniciar a diminuição da produção de CH4 ruminal. O processo de biodigestão dos dejetos não foi modificado pelo uso dos taninos, no entanto, as velocidades das produções de CH4 e CO2 foram influenciadas pela composição dos dejetos dos diferentes grupos de bovinos avaliados, sendo mais acentuada para os taurinos, podendo ser um fator positivo para redução do tempo de biodigestão. A utilização de extrato de Acacia mearnsii até níveis de 1,5% da MS da dieta demonstrou ser uma opção segura como aditivo alimentar para bovinos de grupos distintos, com potencial para mitigação de CH4 ruminal sem afetar a eficiência de conversão nos processos de tratamento de resíduos, com potencial para minimização dos volumes de dejetos.Although some dietary strategies have been proposed to reduce the emission of methane (CH4) from ruminants, few have shown persistent decline, mainly in vivo. The objective of the present study was to evaluate the use of different levels of tannins as feed additive for ruminants as a mitigator of ruminal CH4 and waste emissions. A double 4x4 Latin square experimental design (4 periods of 28 days each) was used in a 2x4 factorial arrangement, with two different groups of cattle (taurine or zebu) and four levels of tannins inclusion (commercial extract of Acacia mearnsii, containing 82.3% of total tannins - tannic acid equivalent, 32.3% of condensed tannins - leucocianidine equivalent) in the diet (0, 0.5, 1.0 and 1.5% of dietary DM). To evaluate the CH4 production of the wastes, anaerobic experimental batch biodigesters were used in a completely randomized design. The used additive levels promoted changes in rumination behavior of the animals and, although they caused linear reduction of nutrient digestibility, they maintained the balance between the synthesis and consumption of ruminal NH3-N, linearly reduced protein microbial synthesis and the ruminal CH4 production without, however, reducing the microbial efficiency and the short chain fatty acids production (SCFA), demonstrating that these tannins interfered directly and indirectly on the methanogenic archeas. Through the Broken-line model, the threshold of 0.72% of tannins in the diet was found as inflection point to initiate a decrease in ruminal CH4 production. The biodigestion process of the waste was not modified by the tannins use, however, the velocities of CH4 and CO2 production were influenced by the composition of the different bovines group evaluated, being more accentuated for the taurine, which can be a positive factor to reduce biodigestion time. The use of Acacia mearnsii extract up to 1.5% dietary DM levels proved to be a safe option as a feed additive for cattle of different groups, with potential for rumen CH4 mitigation without affecting conversion efficiency in waste treatment processes, with potential to minimize waste volumes

    Tannins as feed additive to mitigate methane emissions in ruminants

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    Embora algumas estratégias alimentares tenham sido propostas para diminuir a emissão de metano (CH4) provenientes de ruminantes, poucas têm mostrado diminuição persistente, principalmente in vivo. O objetivo do presente estudo foi avaliar o uso de diferentes níveis de taninos como aditivo alimentar para ruminantes como mitigador das emissões de CH4 ruminal e dos dejetos. Utilizou-se o delineamento experimental quadrado latino 4x4 duplicado (4 períodos de 28 dias cada), em arranjo fatorial 2x4, sendo testados dois grupos distintos de bovinos (taurinos ou zebuínos) e quatro níveis de inclusão de taninos (extrato comercial de Acacia mearnsii, contendo 82,3% de taninos totais - equivalente em ácido tânico e 32,3% de taninos condensados - equivalente em leucocianidina) na dieta (0; 0,5; 1,0 e 1,5% da MS da dieta). Para avaliar a produção de CH4 dos dejetos utilizaram-se biodigestores anaeróbios experimentais do tipo batelada, em delineamento inteiramente casualizado. Os níveis utilizados do aditivo promoveram mudanças no comportamento de ruminação dos animais e apesar de causarem redução linear da digestibilidade dos nutrientes, mantiveram o equilíbrio entre a síntese e consumo de N-NH3 ruminal, reduziram linearmente a síntese de proteína microbiana e a produção de CH4 ruminal sem, contudo, reduzir a eficiência microbiana e a produção de ácidos graxos de cadeia curta (AGCC), demonstrando que estes taninos interferem diretamente e indiretamente sobre as archeas metanogênicas. Através do modelo Broken-line encontrou-se o limiar de 0,72% de taninos na dieta como ponto de inflexão para iniciar a diminuição da produção de CH4 ruminal. O processo de biodigestão dos dejetos não foi modificado pelo uso dos taninos, no entanto, as velocidades das produções de CH4 e CO2 foram influenciadas pela composição dos dejetos dos diferentes grupos de bovinos avaliados, sendo mais acentuada para os taurinos, podendo ser um fator positivo para redução do tempo de biodigestão. A utilização de extrato de Acacia mearnsii até níveis de 1,5% da MS da dieta demonstrou ser uma opção segura como aditivo alimentar para bovinos de grupos distintos, com potencial para mitigação de CH4 ruminal sem afetar a eficiência de conversão nos processos de tratamento de resíduos, com potencial para minimização dos volumes de dejetos.Although some dietary strategies have been proposed to reduce the emission of methane (CH4) from ruminants, few have shown persistent decline, mainly in vivo. The objective of the present study was to evaluate the use of different levels of tannins as feed additive for ruminants as a mitigator of ruminal CH4 and waste emissions. A double 4x4 Latin square experimental design (4 periods of 28 days each) was used in a 2x4 factorial arrangement, with two different groups of cattle (taurine or zebu) and four levels of tannins inclusion (commercial extract of Acacia mearnsii, containing 82.3% of total tannins - tannic acid equivalent, 32.3% of condensed tannins - leucocianidine equivalent) in the diet (0, 0.5, 1.0 and 1.5% of dietary DM). To evaluate the CH4 production of the wastes, anaerobic experimental batch biodigesters were used in a completely randomized design. The used additive levels promoted changes in rumination behavior of the animals and, although they caused linear reduction of nutrient digestibility, they maintained the balance between the synthesis and consumption of ruminal NH3-N, linearly reduced protein microbial synthesis and the ruminal CH4 production without, however, reducing the microbial efficiency and the short chain fatty acids production (SCFA), demonstrating that these tannins interfered directly and indirectly on the methanogenic archeas. Through the Broken-line model, the threshold of 0.72% of tannins in the diet was found as inflection point to initiate a decrease in ruminal CH4 production. The biodigestion process of the waste was not modified by the tannins use, however, the velocities of CH4 and CO2 production were influenced by the composition of the different bovines group evaluated, being more accentuated for the taurine, which can be a positive factor to reduce biodigestion time. The use of Acacia mearnsii extract up to 1.5% dietary DM levels proved to be a safe option as a feed additive for cattle of different groups, with potential for rumen CH4 mitigation without affecting conversion efficiency in waste treatment processes, with potential to minimize waste volumes

    Effect of feed additives on ruminal methane production using the technique of ex situ ruminal fermentation (micro-rumen), total tract apparent digestibility of nutrients and excretion in cattle

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    Problemática mundial levantada nas últimas duas décadas, a geração de gases de efeito estufa (GEE) tem parte devida à emissão de metano por ruminantes. O metano, um potente GEE, é produto final do processo fermentativo de bovinos e, por constituir perda no potencial produtivo destes, tem sido objeto de estudo por nutricionistas do mundo todo. Na busca por estratégias para diminuírem essas perdas, diferentes dietas, aditivos e manejos nutricionais têm sido empregados. Portanto, o objetivo deste trabalho foi avaliar o efeito dos aditivos alimentares, monensina ou tanino, sobre a produção de metano ruminal em bovinos, utilizando-se a técnica de fermentação ruminal ex situ (micro-rúmen), e sobre os parâmetros da fermentação ruminal, a digestibilidade aparente total e a excreção de nutrientes da dieta. Seis vacas (873 ± 81 kg) canuladas no rúmen foram utilizadas e distribuídas a três dietas, que diferiram quanto ao aditivo utilizado, seguindo-se delineamento experimental em quadrado latino 3x3 replicado (n= 18 unidades experimentais): Controle (CON): sem aditivo; Monensina (MON): adição de 300 mg de monensina sódica por animal por dia; Tanino (TAN): adição de 100 g de extrato concentrado de tanino condensado obtido da Acácia-negra (Acacia mearnsii) por animal por dia. Cada período experimental foi constituído de 21 dias, sendo que, entre o dia 5 e o dia 15, 2 g do marcador óxido crômico por kg de MS de alimento consumido foi administrado via cânula ruminal, para determinação da digestibilidade aparente total da MS e suas frações, bem como da excreção dos nutrientes da dieta. O ensaio de digestibilidade foi constituído por duas fases, sendo os cinco primeiros dias para adaptação ao marcador e os cinco últimos para coleta de fezes. A excreção da MS e dos nutrientes, bem como a excreção de nitrogênio, foi calculada a partir dos dados de coeficiente de digestibilidade da MS e suas frações. Para cada período experimental, os últimos 6 dias foram destinados para coleta de dados do consumo de matéria seca (CMS). No dia 21 coletou-se líquido ruminal, antes, 3, 6, 9 e 12 h após a alimentação matinal, para determinação da concentração de ácidos graxos de cadeia curta (AGCC) e metano (CH4). As concentrações de CH4 e AGCC foram determinadas por cromatografia gasosa. O pH ruminal foi mensurado por um dispositivo contínuo de mensuração, durante 24 horas no 21º dia de cada período experimental. A técnica de fermentação ex situ consiste em incubar frascos tipo penicilina com conteúdo ruminal sólido e líquido, em banho termostático por 30 minutos, com posterior mensuração da produção de metano por cromatografia gasosa, sendo estimada a perda de energia relativa (PER). A PER avalia a eficiência da fermentação dos alimentos, ou seja, verifica a perda de metano quando comparada aos outros produtos da fermentação, tais como, acído acético, propiônico e butírico. Os dados foram analisados pelo programa SAS (Versão 9.2, 2010) através do procedimento MIXED. No modelo, o efeito de tratamento foi considerado fixo e os efeitos de período, quadrado e animal dentro de quadrado considerados aleatórios. Não houve diferenças significativas (P0,05) entre os tratamentos para o consumo, digestibilidade aparente total e excreção da MS, PB, EB, FDN, FDA, EE, ENN, MO ou P, nem na digestibilidade do NDT e na excreção de N. Não houve efeito signifivativo dos aditivos (P>0,05) sobre a concentração de N-NH3, pH ruminal, para os ácidos acético, propiônico e butírico, nem para o AGCC total. Para a variável metano houve diferença significativa (P0.05) of additives on rumen pH, concentration of total SCFA, acetic, propionic and butyric acids, as well as, NH3- N. Monensin reduced (P<0.05) methane production by 10.7%, whereas tannin reduced by 8.0%, when compared to control treatment. Relative energy loss was significantly decreased by 20.3% and 23.8% (P=0.0387) with administration of monensin and tannin when compared to control. Therefore, the use of monensin or tannin in diets with forage to concentrate ratio of 50%, shows to be an interesting option in catlle diets aiming to improve energy efficiency in animals, not interfering on intake, digestibility and nutrient excretion with consequent reduction in methane emissions

    Nutrient digestibility and changes in feeding behavior of cattle fed cottonseed and vitamin E

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    High lipid concentration in ruminant diets often harms nutrient digestibility and feed intake; thus, a protected lipid and antioxidant source can be considered as an alternative for improving diet energy without putting animal production at a disadvantage. The aim of this study was to evaluate the dry matter intake (DMI), nutrient digestibility and feeding behavior of cattle fed cottonseed and vitamin E. Six cannulated cows, non-pregnant, non-lactating were distributed in a replicated 3 × 3 Latin Square design. Feed was offered ad libitum twice daily. Treatments were: 1) Control, 2) CS: 30 % cottonseed included; and 3) CSVitE: 30 % cottonseed plus 500 IU VitE included. Data were analyzed by SAS (Statistical Analysis System, v.9.3) and the significance was declared at p &lt; 0.05. Diets with cottonseed had 22 % greater digestibility of ether extract and 9 % lower digestibility of non-fiber-carbohydrates compared to the control. Treatments with cottonseed had 13 % higher time eating, 48 % more ruminating, 34 % more chewing and 17 % lower time idling compared to the control. Molar proportion of propionate was 36 % higher and the butyrate and acetate:propionate ratio were 27 % and 30 % lower, respectively, for the cottonseed treatments compared to the control. Including cottonseed up to 30 % can be used to increase diet energy density leading to improvements in feeding behavior and ruminal parameters. The inclusion of Vitamin E did not result in benefits to cattle when it was combined with cottonseed. Further studies should be undertaken to evaluate vitamin E levels in association with different amounts and lipid sources

    Nutrient digestibility and changes in feeding behavior of cattle fed cottonseed and vitamin E

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    ABSTRACT: High lipid concentration in ruminant diets often harms nutrient digestibility and feed intake; thus, a protected lipid and antioxidant source can be considered as an alternative for improving diet energy without putting animal production at a disadvantage. The aim of this study was to evaluate the dry matter intake (DMI), nutrient digestibility and feeding behavior of cattle fed cottonseed and vitamin E. Six cannulated cows, non-pregnant, non-lactating were distributed in a replicated 3 × 3 Latin Square design. Feed was offered ad libitum twice daily. Treatments were: 1) Control, 2) CS: 30 % cottonseed included; and 3) CSVitE: 30 % cottonseed plus 500 IU VitE included. Data were analyzed by SAS (Statistical Analysis System, v.9.3) and the significance was declared at p < 0.05. Diets with cottonseed had 22 % greater digestibility of ether extract and 9 % lower digestibility of non-fiber-carbohydrates compared to the control. Treatments with cottonseed had 13 % higher time eating, 48 % more ruminating, 34 % more chewing and 17 % lower time idling compared to the control. Molar proportion of propionate was 36 % higher and the butyrate and acetate:propionate ratio were 27 % and 30 % lower, respectively, for the cottonseed treatments compared to the control. Including cottonseed up to 30 % can be used to increase diet energy density leading to improvements in feeding behavior and ruminal parameters. The inclusion of Vitamin E did not result in benefits to cattle when it was combined with cottonseed. Further studies should be undertaken to evaluate vitamin E levels in association with different amounts and lipid sources

    Effect of tannins and monensin on feeding behaviour, feed intake, digestive parameters and microbial efficiency of nellore cows

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    This study aimed to evaluate the associative effect of monensin and tannins on intake, feeding behaviour, digestibility, rumen kinetics, microbial protein synthesis and nitrogen balance. In a 2 × 4 factorial arrangement, 8 rumen cannulated Nellore cows were distributed in 2 contemporary 4 × 4 Latin squares and received 8 diets that differed in the level of tannins (0.00, 0.75, 1.50 and 2.25% DM) and presence of monensin. Monensin was daily administered to each cow in one square (about 32 mg/kg DM). No interaction between monensin and tannins was observed (p > .05). Tannins linearly reduced feed intake, but linearly increased daily eating time (p < .05), although these did not alter the number of meals. Monensin increased CP digestibility by 6% (p = .0387) while tannins linearly reduced digestibility of DM, CP, OM and TDN, whereas the reduction was quadratic for ADF and NDF. Tannins linearly reduced the rumen disappearance rate by linearly reducing both passage and digestion rates. Tannins also linearly reduced urinary urea, though neither additive affected microbial protein synthesis. Monensin reduced the proportion of N excreted in faeces, whereas tannins linearly increased faecal N and linearly reduced both urinary and retained N. Monensin and tannins have shown independent effects on feeding behaviour, feed intake, digestive parameters, microbial protein synthesis and N balance, but they did not improve nutrient usage, although monensin alone has shown to have potential to promote N utilisation. Tannins may play an important role in reducing the excretion of N in urine.Highlights Tannins reduce the efficiency of nutrient usage in cattle. Tannins change the pathway of the excretion of the feeding nitrogen. The emission of N2O from the urine may be reduced by the use of tannins in cattle feeding

    Dry Matter Intake of Nellore Steers in Different Pasture Production Systems Compared to Cajanus Cajan Legume-Grass Mixed Pasture

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    Brazil has one of the largest pasture areas for livestock on the planet, and estimates of dry matter intake (DMI) are necessary for adequate nutritional plans to improve animal performance. This study evaluated the dry matter intake (DMI) of Nellore cattle in different production systems, including a Cajanus cajan legume-grass mixed pasture. The experiment was carried out at the experimental farm of Embrapa Southeast Livestock, São Carlos, SP, Brazil, in two seasons of 2021: rainy (January) and dry (July). Eighteen Nellore steers (approximately 445 ± 44 kg and 15 ± 16 months old) were randomly distributed in three treatments with three replications (1.5 ha each): 1) degraded pasture of Urochloa decumbens cv. Basilisk (DEG); 2) mixture of grasses and legumes, U. decumbens Stapf cv. Basilisk, U. brizantha Stapf cv. Marandu and Cajanus cajan cv. BRS Mandarin (MIX); and 3) pasture with a mixture of U. decumbens cv. Basilisk and U. brizantha cv. Marandu with liming, P, K, S, micronutrient fertilization of 200 kg N-urea ha-1 year-1; and three applications of 66.7 kg N ha-1 every 60 days in the rainy season (REC). All pastures were grazed by Nellore cattle and submitted to stocking rate adjustments using the “put and take†technique. The animals received 15 g of the titanium dioxide (TiO2) orally for nine consecutive days. Feces were collected at 6:00 a.m. by spontaneous defecation during four days after the beginning of TiO2 administration until the tenth day. Daily the samples were frozen, and at the end of the experimental period were dried in a forced-air oven (65 ºC - 72 h), ground to 1 mm in a Wiley mill and analyzed by ICP-OES (Thermo iCAP 6000 series - Dual View Thermo Fisher Scientific, Waltham, Massachusetts, USA). The statistical model considered treatment, season and treatment x season interaction as fixed effects. Data were submitted to analysis of variance (PROC MIXED) and means were compared by the Fisher test at 5%. Significance (P = 0.0168) was observed for treatment x season interaction. The DMI (kg of DM day-1) in the rainy season presented values of 11.8, 10.0 and 13.8, and in the dry season 4.9, 7.8 and 4.7 for DEG, MIX and REC, respectively. In the dry season, the MIX treatment differed significantly from the others.  In the rainy season, the MIX was different from the REC, while the DEG treatment showed no difference in relation to REC and MIX. In the dry season, Cajanus cajan consumption was higher than the other treatments. This behavior can be explained by the greater availability of forage mass and the inferior quality of the grass and by the grazing behavior of the animals, which consumed more legume as the dry season extended

    Methane mitigation and ruminal fermentation changes in cows fed cottonseed and vitamin E

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    Inherently, ruminant production of methane (CH4), a greenhouse gas (GHG), causes animal energy losses. Cottonseed is a lipid source and is used sometimes to enhance energy density in cattle diets. It also can mitigate enteric CH4. Lipids release peroxides in the rumen, and antioxidants have the ability to neutralize them. Thus, a lipid and antioxidant source can benefit rumen fermentation. The aim of this study was to evaluate rumen fermentation parameters from cows fed cottonseed and vitamin E. Six cannulated cows were arranged in a replicate 3 × 3 latin square. Treatments were: 1) Control, 2) CS (30 % corn replaced by cottonseed) and 3) CSVitE (30 % corn replaced by cottonseed, plus 500 IU VitE). Results were compared by orthogonal contrast. When compared to the control diet, cottonseed inclusion reduced enteric CH4 emissions by 42 %. Production of acetate, butyrate and the acetate to propionate ratio were respectively 34 %, 47 % and 36 % lower with the cottonseed treatments. Energy lost in the rumen as CH4 and energy release as butyrate were reduced by 26 % and 32 % respectively. Propionate and intestinal energy release were, respectively, 43 % and 35 % higher with cottonseed treatments. Furthermore, as a nutritional strategy to mitigate enteric CH4, cottonseed has positive effects on short chain fatty acid (SCFA) production and gastrointestinal energy release. Vitamin E did not result in improvements in ruminal fermentation. Further studies evaluating levels of vitamin E in association with different amounts and sources of lipids are required
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