Methane is a potent greenhouse gas with a global warming potential 21 times that of carbon dioxide. Globally, ruminants are the main anthropogenic contributors to methane release to the atmosphere. Methane is produced in the gastrointestinal tract of ruminants, mostly within the rumen by methanogenic archaea. However, methane production represents a loss of 2 to 12% of dietary gross energy for the animal, which could otherwise be available for growth or milk production. Therefore, mitigation of methane production by ruminants could produce both economic and environmental benefits, with more sustainable and energy efficient livestock, and offering a promising way of slowing global warming. Despite extensive research undertaken to find ways of reducing methane emissions from ruminants, progress has been relatively limited. Furthermore, there is still a lack of studies linking rumen microbiology and ruminant nutrition and production. The central purpose of this research was to investigate feed additives to reduce methane emissions and to understand associated changes that occur in the rumen microbiota. For the first experiment (Chapter 2), biochar was evaluated as an antimethanogenic compound for beef cattle. The in vitro gas production technique was used to study the effects of biochar on rumen fermentation and methane production. Overall, methane production was reduced by 5% by the addition of biochar compounds (10 g/kg of substrate). The observed reduction in methane produced was not associated with a change in volatile fatty acid profile suggesting biochar primarily inhibited fermentation. Ammonia concentration was significantly reduced with biochar inclusion. Because different biochars had different effects on methane production, further investigation of relationships between the physicochemical properties of biochars and antimethanogenic effects are necessary. However, due to the small reduction in methane production recorded, research with biochar was discontinued. Encapsulated nitrate was then explored as an antimethanogenic additive and as an alternative non-protein nitrogen source to urea (Chapter 3). The effect of using encapsulated nitrate as a replacement for urea or dietary protein, plus the addition of inorganic sulphur, on enteric methane emissions, nutrient digestibility, nitrogen utilization and microbial protein synthesis from crossbred beef steers were studied. In addition, nitrate toxicity and eating behaviour were investigated. The inclusion of encapsulated nitrate reduced methane production compared to urea and a true protein source, with no adverse effects on rumen fermentation or nitrogen metabolism and no effects with the inclusion of elemental sulphur. The level of addition of encapsulated nitrate (14.3 g nitrate /kg DM) and the time of adaptation chosen for this study (14 days) were adequate to avoid nitrate toxicity. Finally, the effects of adding nitrate inclusion to different basal diets on rumen microbial populations and relationships of these populations with methane production were investigated (Chapter 4). The V4 hypervariable regions of the bacterial and archaea 16S rRNA genes were amplified and sequenced. Effects on microbial population induced by nitrate were dependant on the basal diet but nitrate altered specific archaeal and bacterial OTUs consistently between studies. A direct and strong correlation between some archaea taxonomic groups and OTUs with methane production was observed.

A preocupação com a segurança alimentar e o aquecimento global pressiona a cadeia de produção de ruminantes a buscar alternativas para incremento na produtividade e redução nas emissões dos gases de efeito estufa, uma vez que essa atividade é responsável por grande parte das emissões de metano (CH4) entérico. O desenvolvimento de produtos enzimáticos para uso na nutrição de ruminantes torna-se de grande valia para contribuir com a demanda alimentar e reduzir os impactos ambientais. O objetivo do presente estudo foi avaliar o uso de um extrato de enzimas fibrolíticas (EEF), composto por endoglucanases, exoglucanases e xilanases, produzido pelo fungo Trichoderma reesei utilizando como substrato resíduos da indústria canavieira, sobre a digestibilidade, fermentação ruminal e desempenho de ovinos em crescimento, conduzidos através de dois experimentos. No experimento 1, foi avaliado a adição de doses crescentes (0, 10, 100, 1000 e 10000 µL g-1 de substrato) do EEF sobre a produção total de gases e CH4, degradabilidade e fermentação ruminal de três forrageiras tropicais (Cynodon spp., Panicum maximum e Cenchrus ciliares L.) em um ensaio in vitro, com o objetivo de verificar a possibilidade de uso do EEF na dieta de ruminantes sem prejudicar os parâmetros fermentativos. Verificou-se que os resultados foram dependentes da dose e do substrato utilizados. Contudo, foi verificado aumento na produção total de gases e CH4 e na degradabilidade de todas as forrageiras utilizadas com a dose mais alta do EEF. Conclui-se que o EEF pode ser utilizado na dieta de ruminantes como alternativa para incremento na degradabilidade de forrageiras tropicais. O experimento 2 foi dividido em duas fases conduzidas com o objetivo de avaliar o efeito do EEF sobre a digestibilidade aparente dos nutrientes, fermentação ruminal, desempenho animal e produção de CH4 entérico de 19 ovinos em crescimento divididos em dois grupos experimentais, controle (CTL) e enzima (ENZ), recebendo dieta composta de 70% de feno de Tifton-85 e 30% de mistura concentrada. O EEF foi aplicado diretamente no feno 24 horas antes do fornecimento da refeição. Na Fase I, avaliou-se o efeito de doses crescentes (0, 20, 40 e 80 µL) do EEF sobre a hidrólise da fibra e posteriormente a dose com melhor resposta foi utilizada nos estudos de digestibilidade aparente dos nutrientes e fermentação ruminal. A dose de 40 µL foi selecionada para ser utilizada no ensaio in vivo, pois apresentou melhores respostas sobre a redução nas frações fibrosas. Os animais suplementados com o EEF apresentaram maior digestibilidade da fibra em detergente ácido e maior produção de valerato. Na Fase II, foi avaliado o efeito do EEF sobre o desempenho, saúde e produção de CH4 entérico de ovinos em crescimento. Os procedimentos utilizados foram os mesmos descritos na Fase I. A suplementação do EEF proporcionou maior percentual médio de ganho de peso e menor produção de CH4 entérico por kg de matéria seca ingerida. As análises sanguíneas demonstraram que o EEF não prejudicou a saúde dos animais. Conclui-se que o EEF pode ser utilizado na dieta animal sem trazer riscos à saúde, além de ser uma estratégia para aumento na produtividade animal e contribuir com a mitigação de CH4 entérico de ovinos em crescimento / Concern about food security and global warming is pressing the ruminant production chain to seek alternatives to increase productivity and reduce greenhouse gas emissions, as this activity accounts for most of the enteric methane (CH4) emissions. The development of enzyme products for use in ruminant nutrition is of great value in order to contribute to food demand and reduce environmental impacts. The aim of the present study was to evaluate the use of a fibrolytic enzyme extract (FEE), produced by the fungus Trichoderma reesei using as substrate residues from the sugar cane industry, on the digestibility, ruminal fermentation and performance of growing sheep, conducted through two experiments. In the experiment 1, the addition of increasing doses (0, 10, 100, 1000 and 10000 ?L g-1) of the FEE on the total gas and CH4 production, degradability and ruminal fermentation of three tropical forages (Cynodon spp., Panicum maximum and Cenchrus ciliares L.) in an in vitro assay, with the objective of verifying the possibility of use of the FEE in the diet of ruminants without harming the fermentative parameters and, consequently, animal health. The results were found to be dose and substrate dependent. However, there was an increase in total gas and CH4 production and in the degradability of all forages used with the highest dose of FEE. It is concluded that the EPS can be used in the diet of ruminants as an alternative to increase the degradability of tropical forages. The experiment 2 was divided in two phases, with the objective of evaluating the effect of the FEE on the apparent digestibility of nutrients, ruminal fermentation, animal performance and enteric CH4 production of 19 growing sheep divided in two experimental groups, control (CTL) and enzyme (ENZ), receiving a diet composed of 70% of Tifton-85 hay and 30% of concentrated mixture. The FEE was applied directly to the hay 24 hours before the meal. In Phase I, the effect of increasing doses (0, 20, 40 and 80 ?L) of the FEE on fiber hydrolysis was evaluated and the dose with the best response was then used in the studies of apparent digestibility of nutrients and ruminal fermentation. The 40 ?L dose was selected to be used in the in vivo assay because it presented better responses on the reduction in fibrous fractions. The animals supplemented with FEE showed higher acid digestible fiber digestibility and higher valerate production. The other parameters were not influenced by the addition of FEE. In Phase II, the effect of FEE was evaluated on the performance, health and production of Enteric CH4 of growing sheep. The procedures used were the same as described in Phase I. The supplementation of the FEE provided a higher percentage of weight gain and lower enteric CH4 production per kg of ingested dry matter. Blood tests showed that FEE did not affect animal health. It is concluded that FEE can be used in the animal diet without presenting health risks, besides being a strategy to increase animal productivity and contribute to the mitigation of enteric CH4 of growing sheep

A preocupação com a segurança alimentar e o aquecimento global pressiona a cadeia de produção de ruminantes a buscar alternativas para incremento na produtividade e redução nas emissões dos gases de efeito estufa, uma vez que essa atividade é responsável por grande parte das emissões de metano (CH4) entérico. O desenvolvimento de produtos enzimáticos para uso na nutrição de ruminantes torna-se de grande valia para contribuir com a demanda alimentar e reduzir os impactos ambientais. O objetivo do presente estudo foi avaliar o uso de um extrato de enzimas fibrolíticas (EEF), composto por endoglucanases, exoglucanases e xilanases, produzido pelo fungo Trichoderma reesei utilizando como substrato resíduos da indústria canavieira, sobre a digestibilidade, fermentação ruminal e desempenho de ovinos em crescimento, conduzidos através de dois experimentos. No experimento 1, foi avaliado a adição de doses crescentes (0, 10, 100, 1000 e 10000 µL g-1 de substrato) do EEF sobre a produção total de gases e CH4, degradabilidade e fermentação ruminal de três forrageiras tropicais (Cynodon spp., Panicum maximum e Cenchrus ciliares L.) em um ensaio in vitro, com o objetivo de verificar a possibilidade de uso do EEF na dieta de ruminantes sem prejudicar os parâmetros fermentativos. Verificou-se que os resultados foram dependentes da dose e do substrato utilizados. Contudo, foi verificado aumento na produção total de gases e CH4 e na degradabilidade de todas as forrageiras utilizadas com a dose mais alta do EEF. Conclui-se que o EEF pode ser utilizado na dieta de ruminantes como alternativa para incremento na degradabilidade de forrageiras tropicais. O experimento 2 foi dividido em duas fases conduzidas com o objetivo de avaliar o efeito do EEF sobre a digestibilidade aparente dos nutrientes, fermentação ruminal, desempenho animal e produção de CH4 entérico de 19 ovinos em crescimento divididos em dois grupos experimentais, controle (CTL) e enzima (ENZ), recebendo dieta composta de 70% de feno de Tifton-85 e 30% de mistura concentrada. O EEF foi aplicado diretamente no feno 24 horas antes do fornecimento da refeição. Na Fase I, avaliou-se o efeito de doses crescentes (0, 20, 40 e 80 µL) do EEF sobre a hidrólise da fibra e posteriormente a dose com melhor resposta foi utilizada nos estudos de digestibilidade aparente dos nutrientes e fermentação ruminal. A dose de 40 µL foi selecionada para ser utilizada no ensaio in vivo, pois apresentou melhores respostas sobre a redução nas frações fibrosas. Os animais suplementados com o EEF apresentaram maior digestibilidade da fibra em detergente ácido e maior produção de valerato. Na Fase II, foi avaliado o efeito do EEF sobre o desempenho, saúde e produção de CH4 entérico de ovinos em crescimento. Os procedimentos utilizados foram os mesmos descritos na Fase I. A suplementação do EEF proporcionou maior percentual médio de ganho de peso e menor produção de CH4 entérico por kg de matéria seca ingerida. As análises sanguíneas demonstraram que o EEF não prejudicou a saúde dos animais. Conclui-se que o EEF pode ser utilizado na dieta animal sem trazer riscos à saúde, além de ser uma estratégia para aumento na produtividade animal e contribuir com a mitigação de CH4 entérico de ovinos em crescimento / Concern about food security and global warming is pressing the ruminant production chain to seek alternatives to increase productivity and reduce greenhouse gas emissions, as this activity accounts for most of the enteric methane (CH4) emissions. The development of enzyme products for use in ruminant nutrition is of great value in order to contribute to food demand and reduce environmental impacts. The aim of the present study was to evaluate the use of a fibrolytic enzyme extract (FEE), produced by the fungus Trichoderma reesei using as substrate residues from the sugar cane industry, on the digestibility, ruminal fermentation and performance of growing sheep, conducted through two experiments. In the experiment 1, the addition of increasing doses (0, 10, 100, 1000 and 10000 ?L g-1) of the FEE on the total gas and CH4 production, degradability and ruminal fermentation of three tropical forages (Cynodon spp., Panicum maximum and Cenchrus ciliares L.) in an in vitro assay, with the objective of verifying the possibility of use of the FEE in the diet of ruminants without harming the fermentative parameters and, consequently, animal health. The results were found to be dose and substrate dependent. However, there was an increase in total gas and CH4 production and in the degradability of all forages used with the highest dose of FEE. It is concluded that the EPS can be used in the diet of ruminants as an alternative to increase the degradability of tropical forages. The experiment 2 was divided in two phases, with the objective of evaluating the effect of the FEE on the apparent digestibility of nutrients, ruminal fermentation, animal performance and enteric CH4 production of 19 growing sheep divided in two experimental groups, control (CTL) and enzyme (ENZ), receiving a diet composed of 70% of Tifton-85 hay and 30% of concentrated mixture. The FEE was applied directly to the hay 24 hours before the meal. In Phase I, the effect of increasing doses (0, 20, 40 and 80 ?L) of the FEE on fiber hydrolysis was evaluated and the dose with the best response was then used in the studies of apparent digestibility of nutrients and ruminal fermentation. The 40 ?L dose was selected to be used in the in vivo assay because it presented better responses on the reduction in fibrous fractions. The animals supplemented with FEE showed higher acid digestible fiber digestibility and higher valerate production. The other parameters were not influenced by the addition of FEE. In Phase II, the effect of FEE was evaluated on the performance, health and production of Enteric CH4 of growing sheep. The procedures used were the same as described in Phase I. The supplementation of the FEE provided a higher percentage of weight gain and lower enteric CH4 production per kg of ingested dry matter. Blood tests showed that FEE did not affect animal health. It is concluded that FEE can be used in the animal diet without presenting health risks, besides being a strategy to increase animal productivity and contribute to the mitigation of enteric CH4 of growing sheep