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No description available.

Yes / Buruli ulcer (BU) is an insidious neglected tropical disease. Cases are reported around the world but the rural regions of West and Central Africa are most affected. How BU is transmitted and spreads has remained a mystery, even though the causative agent, Mycobacterium ulcerans, has been known for more than 70 years. Here, using the tools of population genomics, we reconstruct the evolutionaryhistoryofM. ulceransbycomparing165isolatesspanning48yearsandrepresenting11endemiccountriesacrossAfrica. The genetic diversity of African M. ulcerans was found to be restricted due to the bacterium’s slow substitution rate coupled with its relatively recent origin. We identified two specific M. ulcerans lineages within the African continent, and inferred that M. ulcerans lineage Mu_A1 existed in Africa for several hundreds of years, unlike lineage Mu_A2, which was introduced much more recently, approximately during the 19th century. Additionally, we observed that specific M. ulcerans epidemic Mu_A1 clones were introduced during the same time period in the three hydrological basins that were well covered in our panel. The estimated time span of the introduction events coincides with the Neo-imperialism period, during which time the European colonial powers divided the African continent among themselves. Using this temporal association, and in the absence of a known BU reservoir or—vector on the continent, we postulate that the so-called "Scramble for Africa" played a significant role in the spread of the disease across the continent. / K.V. was supported by a PhD-grant of the Flemish Interuniversity Council—University Development Cooperation (Belgium). B.d.J. and C.M. were supported by the European Research Council-INTERRUPTB starting grant (no. 311725). T.P.S. was supported by a fellowship from the National Health and Medical Research Council of Australia (1105525). Funding for this work was provided by the Department of Economy, Science and Innovation of the Flemish Government, the Stop Buruli Consortium supported by the UBS Optimus Foundation, and the Fund for Scientific Research Flanders (Belgium) (FWO grant no. G.0321.07N). The computational resources used in this work were provided by the HPC core facility CalcUA and VSC (Flemish Supercomputer Center), funded by the University of Antwerp, the Hercules Foundation and the Flemish Government—department EWI. Aspects of the research in Cameroon and Benin were funded by the Raoul Follereau Fondation France.

South Africa produces an average of 154 million broilers (Gallus gallus domesticus) annually, arising to an estimated 886 million kg of broiler litter. The largest population of broilers are reared in the North West province. Various applications for this largely underexploited resource have been published, including forming part of ruminant diets and direct land application. This however has several disadvantages, as it could lead to eutrophication of fresh water sources and faecal contamination of produce. Windrow composting of broiler litter has previously been studied, and found to deliver a stabilized product free of pathogenic and phytotoxic effects, therefore making it an excellent soil conditioner. This study aimed to characterize the microbial community present during the windrow composting of broiler litter. Four different formulations of substrate were tested; these being broiler litter (Windrow 1), Windrow 1 with previously composted material (Windrow 2), Windrow 2 amended with woodchips (Windrow 3) and Windrow 3 with an additional 12.5% (w/w) zeolite (Windrow 4). Broiler litter used in this experiment had a C:N ration of 10.3:1, whilst the blue gum woodchips added as an amendment had a C:N ratio of 172:1. Windrow and environmental temperatures were monitored on a regular basis. Windrow 1 largely mimicked environmental temperature, and could not sustain a true thermophilic phase during the experimental period. Windrow 2 did achieve a short lived thermophilic phase during the first few days of the composting process, however could not sustain its temperature over the whole period. In contrast Windrows 3 and 4 sustained temperature above 40°C for the largest part of the experimental period, regardless of environmental temperature. No significant difference (p < 0.05) could be observed between average moisture levels in the 4 windrows. Internal moisture profiles were however found to differ significantly, especially on the surface of the windrows. Moisture was also lost faster in Windrows 1 and 2 compared to Windrows 3 and 4. Chemical analysis showed differences between the four windrows constructed. A higher amount of nitrogen was lost in Windrows 1 and 2, mostly due to a sub-optimal initial C:N ratio in these windrows. Windrow 2 contained the highest values for plant nutrients P, Mg, Ca, Mn and Cu. Microbial population dynamics were observed using PCR-DGGE of samples collected throughout the composting of various treatments. Various commercial DNA extraction kits where tested in a previous study for their ability to remove PCR inhibitory substances, such as humic acids. The Machery-Nagel Soil DNA isolation kit was used as it gave amplifiable DNA from all samples. Samples were amplified using a nested PCR approach primer sets 27f-1492r \ 341f(GC)-907r and EF3-EF4 \ EF4(GC)-fung5 (where “GC” indicates a GC-rich clamp) for prokaryotic and eukaryotic species respectively. The PCR products were analyzed by agarose gel electrophoresis, and equal amounts of product were subjected to denaturing gradient gel electrophoresis (DGGE). Bands obtained from these polyacrylamide gels where then re-amplified using the same secondary primer sets (without the GC-clamp), and sequenced. A total of 454 prokaryotic bands in 55 distinct rf-positions were observed. Seven distinct rf-positions were observed in eukaryotic DGGE profiles. Prokaryotic profiles were aligned and the microbial diversity was analyzed by means of Ward’s clustering algorithm and the dice coefficient of similarity, as well as Simpson’s reciprocal, Shannon-Weaver and Species richness indices. Canonical correspondence analysis (CCA) was also performed on both the banding patterns as well as the bands present, together with the physico-chemical results obtained. Several bands were successfully identified as being influenced by physico-chemical parameters. Temperature, C:N ratio, ash, and moisture showed a correlation on CCA bi-plots. Sixteen bands were sequence identified. These sequences were compared to two different databases. The 16S rRNA database for Bacteria and Archaea gave identities to genus level, however maximum identity scores were low. Of the 16 sequences, 12 sequences were identified as uncultured bacteria when compared to the nucleotide collection database. In comparing the sequences with sequences collected in the nucleotide collection database, 12 were either first described in composts and soils, or animal manures. Results indicated mostly members of the genus Bacillus and Paenibacillus. The addition of a carbon source greatly affected the microbial metabolism, resulting in a thermophilic phase being achieved in amended windrows. As no thermophilic phase was observed in windrows that were not amended with woodchips, it could be concluded that the use of a carbon source is irremissible when composting broiler litter. A zeolite amendment is also strongly advised, as this further increased temperatures within the windrow. / Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013.

South Africa produces an average of 154 million broilers (Gallus gallus domesticus) annually, arising to an estimated 886 million kg of broiler litter. The largest population of broilers are reared in the North West province. Various applications for this largely underexploited resource have been published, including forming part of ruminant diets and direct land application. This however has several disadvantages, as it could lead to eutrophication of fresh water sources and faecal contamination of produce. Windrow composting of broiler litter has previously been studied, and found to deliver a stabilized product free of pathogenic and phytotoxic effects, therefore making it an excellent soil conditioner. This study aimed to characterize the microbial community present during the windrow composting of broiler litter. Four different formulations of substrate were tested; these being broiler litter (Windrow 1), Windrow 1 with previously composted material (Windrow 2), Windrow 2 amended with woodchips (Windrow 3) and Windrow 3 with an additional 12.5% (w/w) zeolite (Windrow 4). Broiler litter used in this experiment had a C:N ration of 10.3:1, whilst the blue gum woodchips added as an amendment had a C:N ratio of 172:1. Windrow and environmental temperatures were monitored on a regular basis. Windrow 1 largely mimicked environmental temperature, and could not sustain a true thermophilic phase during the experimental period. Windrow 2 did achieve a short lived thermophilic phase during the first few days of the composting process, however could not sustain its temperature over the whole period. In contrast Windrows 3 and 4 sustained temperature above 40°C for the largest part of the experimental period, regardless of environmental temperature. No significant difference (p < 0.05) could be observed between average moisture levels in the 4 windrows. Internal moisture profiles were however found to differ significantly, especially on the surface of the windrows. Moisture was also lost faster in Windrows 1 and 2 compared to Windrows 3 and 4. Chemical analysis showed differences between the four windrows constructed. A higher amount of nitrogen was lost in Windrows 1 and 2, mostly due to a sub-optimal initial C:N ratio in these windrows. Windrow 2 contained the highest values for plant nutrients P, Mg, Ca, Mn and Cu. Microbial population dynamics were observed using PCR-DGGE of samples collected throughout the composting of various treatments. Various commercial DNA extraction kits where tested in a previous study for their ability to remove PCR inhibitory substances, such as humic acids. The Machery-Nagel Soil DNA isolation kit was used as it gave amplifiable DNA from all samples. Samples were amplified using a nested PCR approach primer sets 27f-1492r \ 341f(GC)-907r and EF3-EF4 \ EF4(GC)-fung5 (where “GC” indicates a GC-rich clamp) for prokaryotic and eukaryotic species respectively. The PCR products were analyzed by agarose gel electrophoresis, and equal amounts of product were subjected to denaturing gradient gel electrophoresis (DGGE). Bands obtained from these polyacrylamide gels where then re-amplified using the same secondary primer sets (without the GC-clamp), and sequenced. A total of 454 prokaryotic bands in 55 distinct rf-positions were observed. Seven distinct rf-positions were observed in eukaryotic DGGE profiles. Prokaryotic profiles were aligned and the microbial diversity was analyzed by means of Ward’s clustering algorithm and the dice coefficient of similarity, as well as Simpson’s reciprocal, Shannon-Weaver and Species richness indices. Canonical correspondence analysis (CCA) was also performed on both the banding patterns as well as the bands present, together with the physico-chemical results obtained. Several bands were successfully identified as being influenced by physico-chemical parameters. Temperature, C:N ratio, ash, and moisture showed a correlation on CCA bi-plots. Sixteen bands were sequence identified. These sequences were compared to two different databases. The 16S rRNA database for Bacteria and Archaea gave identities to genus level, however maximum identity scores were low. Of the 16 sequences, 12 sequences were identified as uncultured bacteria when compared to the nucleotide collection database. In comparing the sequences with sequences collected in the nucleotide collection database, 12 were either first described in composts and soils, or animal manures. Results indicated mostly members of the genus Bacillus and Paenibacillus. The addition of a carbon source greatly affected the microbial metabolism, resulting in a thermophilic phase being achieved in amended windrows. As no thermophilic phase was observed in windrows that were not amended with woodchips, it could be concluded that the use of a carbon source is irremissible when composting broiler litter. A zeolite amendment is also strongly advised, as this further increased temperatures within the windrow. / Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013.

Thesis (M.A.)--University of South Florida, 2006. / Title from PDF of title page. Document formatted into pages; contains 86 pages. Includes bibliographical references.

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.

The fitness function developed in this thesis directly links the physicochemical properties of an enzyme to evolutionary fates in a quantitative and predictive manner through a comparative study of empirical and simulated data. The success or failure of organisms during evolution is dictated by changes in molecular structure that give rise to changes in fitness revealed by evolutionary dynamics within a population. While the conceptual link between genotype, phenotype and fitness is clear, the ability to relate these in a quantitative manner remains difficult. I show here that predicting success during adaptation can depend critically upon enzyme kinetic and folding models. We used a 'weak link' method to favor mutations to an essential, but maladapted adenylate kinase gene within a microbial population that resulted in the identification of five mutants that arose nearly simultaneously and competed for success. The unique catalytic role of adenylate kinase in vivo is to maintain adenylate homeostasis by catalyzing the reaction: ATP + AMP [imaginary] ADP. The stabilizing substitutions retained this essential function and were shown to be necessary for viability at higher temperatures. Physicochemical characterization of these mutants demonstrated that, although steady-state enzyme activity is important, success within the population is critically dependent on resistance to denaturation and aggregation thus emphasizing the importance of proper folding in adaptation. In vitro activity is a product of critical catalytic and folding pathways, and hence is a valuable proxy for fitness. A fitness function relating in vitro measurements of enzyme activity and reversible and irreversible unfolding to growth rate must impose an activity threshold above which there is no added fitness benefit in order to reproduce in vivo evolutionary fates in an in silico population. The fitness function thereby links organismal adaptation to the properties of a single gene. Understanding the physical basis for adaptation of an organism is the first step in the development of approaches that can accurately model, and someday predict, the manner in which organisms would respond to new antibiotics and improve upon the current clinical regimens.

A produção de metano entérico está entre as principais fontes de emissão de gases de efeito estufa dentre as atividades agropecuárias, além de gerar perda energética ao animal de até 12% da energia bruta consumida. Assim, o objetivo deste trabalho foi avaliar o uso de nitrato de cálcio encapsulado na alimentação de ruminantes como estratégia nutricional a mitigação de metano entérico. O experimento consistiu de duas fases. Fase I: Foram testadas dietas suplementadas com produto comercial de nitrato de cálcio encapsulado utilizando a técnica semiautomática de produção de gases in vitro. Meio grama de substrato com 50 mL de meio de incubação e 25 mL de inóculo ruminal foram incubados em frascos de vidro (160 mL) à 39 ºC por 24 horas para determinação da melhor dieta a ser testada in vivo. O primeiro ensaio testou a associação entre a monensina (dietas com e sem adição de monensina) e doses de nitrato encapsulado (0; 1,5 e 3% da matéria seca (MS)) para mitigação de metano in vitro. Não foi observada interação entre monensina e nitrato para as variáveis testadas. O segundo ensaio in vitro testou a interação do tipo de dieta com duas relações concentrado:volumoso, 20:80 e 80:20, e a inclusão de doses de nitrato encapsulado (0; 1,5; 3 e 4,5% MS). Embora não foi observado efeito associativo entre dieta e nitrato para redução de metano, foi observada mudança nos produtos da fermentação ruminal, com redução de propionato, em decorrência da concorrência de nitrato e propianogênicas por hidrogênio mais escasso em dietas com menor fermentação. Fase II: Conforme os resultados obtidos na Fase I, na segunda fase foi avaliado o efeito associativo da relação de concentrado:volumoso da dieta e a dose de nitrato sobre a emissão de metano, constituintes ruminais e toxicidade do nitrato in vivo. Utilizou-se seis borregos canulados no rúmen, distribuídos em delineamento experimental quadrado latino 6 x 6, em fatorial 2 x 3. Os fatores foram tipo de dieta (relação concentrado:volumoso 20:80 e 80:20) e inclusão de doses de nitrato encapsulado na dieta (0; 1,5 e 3% MS) em substituição gradual ao farelo de soja, totalizando seis tratamentos. Os teores de substituição do farelo de soja pelo nitrato foram em equivalente proteico de maneira a deixar as dietas isonitrogenadas. Os animais foram adaptados gradualmente a oferta de nitrato dietético para evitar problemas com toxidez. A análise de toxicidade foi avaliada pela taxa de metahemoglobina no sangue dos ovinos 3 horas após a alimentação. Nitrato reduziu a produção de metano em ambas as dietas. Os níveis de metahemoglobina no sangue dos animais não foram alterados pela adição de nitrato. Foi observado efeito associativo entre o tipo de dieta e nitrato para os produtos da fermentação ruminal, como acetato, que aumentou linearmente nas dietas com 80% de concentrado quando nitrato foi adicionado. Concluí-se que nitrato, utilizado de forma segura, é uma promissora estratégia para redução de metano entérico independentemente do tipo de dieta com que está sendo suplementado / Production of enteric methane is among the leading sources of greenhouse gas emissions from agricultural activities and generate energy loss to the animal up to 12% of gross energy consumption. The objective of this study was to evaluate the use of calcium nitrate encapsulated in ruminant feed as a nutritional strategy for mitigation of enteric methane. The experiment consisted of two phases. Phase I: tested diets were supplemented with encapsulated calcium nitrate using a semi-automatic in vitro gas production technique Half gram of substrate with 50 mL of incubation medium, and 25 mL of rumen fluid were incubated in glass bottles (160 ml) at 39 °C for 24 hours to determine the best diets to be tested in vivo. The first trial tested the association between monensin (diets with and without monensin) and encapsulated nitrate levels (0, 1.5 and 3% of dry matter (DM)) for in vitro methane mitigation. There was no interaction between monensin and nitrate for the tested variables. The second in vitro assay tested the interaction of diet type with two concentrate:forage ratios, 20:80 and 80:20, and the inclusion of encapsulated nitrate levels (0, 1.5, 3 and 4.5% DM). Although it was not observed associative effect between diet and nitrate for reduction of methane, it was observed change in the ruminal fermentation products. Phase II: According to the results obtained in Phase I, in the second phase we evaluated the associative effect of concentrate: forage ratio of diet and the dose of nitrate on the methane emission, ruminal constituents and nitrate toxicity in vivo. We used six lambs cannulated in the rumen, distributed in Latin square design 6 x 6 in factorial 2 x 3. The factors were type of diet (concentrate:forage ratios 20:80 and 80:20) and inclusion of encapsulated nitrate doses in the diet (0, 1.5 and 3% DM) in the gradual replacement of soybean meal, a total of six treatments. The replacement levels of soybean meal by nitrate were in protein equivalent so as to leave the diets isonitrogenous. The animals were gradually adapted to dietary nitrate supply to avoid problems with toxicity. The analysis of toxicity were evaluated by the rate of methemoglobin in the blood of sheep 3 hours after feeding. Methemoglobin levels in blood of animals were not changed by the addition of nitrate. Associative effect was observed between the type of diet and nitrate for ruminal fermentation products such as acetate, which decreased linearly in the diets with 80% forage when nitrate was added. It can be concluded that nitrate used securely is a promising strategy for reducing enteric methane independently of the type of diet being supplemented

A produção de metano entérico está entre as principais fontes de emissão de gases de efeito estufa dentre as atividades agropecuárias, além de gerar perda energética ao animal de até 12% da energia bruta consumida. Assim, o objetivo deste trabalho foi avaliar o uso de nitrato de cálcio encapsulado na alimentação de ruminantes como estratégia nutricional a mitigação de metano entérico. O experimento consistiu de duas fases. Fase I: Foram testadas dietas suplementadas com produto comercial de nitrato de cálcio encapsulado utilizando a técnica semiautomática de produção de gases in vitro. Meio grama de substrato com 50 mL de meio de incubação e 25 mL de inóculo ruminal foram incubados em frascos de vidro (160 mL) à 39 ºC por 24 horas para determinação da melhor dieta a ser testada in vivo. O primeiro ensaio testou a associação entre a monensina (dietas com e sem adição de monensina) e doses de nitrato encapsulado (0; 1,5 e 3% da matéria seca (MS)) para mitigação de metano in vitro. Não foi observada interação entre monensina e nitrato para as variáveis testadas. O segundo ensaio in vitro testou a interação do tipo de dieta com duas relações concentrado:volumoso, 20:80 e 80:20, e a inclusão de doses de nitrato encapsulado (0; 1,5; 3 e 4,5% MS). Embora não foi observado efeito associativo entre dieta e nitrato para redução de metano, foi observada mudança nos produtos da fermentação ruminal, com redução de propionato, em decorrência da concorrência de nitrato e propianogênicas por hidrogênio mais escasso em dietas com menor fermentação. Fase II: Conforme os resultados obtidos na Fase I, na segunda fase foi avaliado o efeito associativo da relação de concentrado:volumoso da dieta e a dose de nitrato sobre a emissão de metano, constituintes ruminais e toxicidade do nitrato in vivo. Utilizou-se seis borregos canulados no rúmen, distribuídos em delineamento experimental quadrado latino 6 x 6, em fatorial 2 x 3. Os fatores foram tipo de dieta (relação concentrado:volumoso 20:80 e 80:20) e inclusão de doses de nitrato encapsulado na dieta (0; 1,5 e 3% MS) em substituição gradual ao farelo de soja, totalizando seis tratamentos. Os teores de substituição do farelo de soja pelo nitrato foram em equivalente proteico de maneira a deixar as dietas isonitrogenadas. Os animais foram adaptados gradualmente a oferta de nitrato dietético para evitar problemas com toxidez. A análise de toxicidade foi avaliada pela taxa de metahemoglobina no sangue dos ovinos 3 horas após a alimentação. Nitrato reduziu a produção de metano em ambas as dietas. Os níveis de metahemoglobina no sangue dos animais não foram alterados pela adição de nitrato. Foi observado efeito associativo entre o tipo de dieta e nitrato para os produtos da fermentação ruminal, como acetato, que aumentou linearmente nas dietas com 80% de concentrado quando nitrato foi adicionado. Concluí-se que nitrato, utilizado de forma segura, é uma promissora estratégia para redução de metano entérico independentemente do tipo de dieta com que está sendo suplementado / Production of enteric methane is among the leading sources of greenhouse gas emissions from agricultural activities and generate energy loss to the animal up to 12% of gross energy consumption. The objective of this study was to evaluate the use of calcium nitrate encapsulated in ruminant feed as a nutritional strategy for mitigation of enteric methane. The experiment consisted of two phases. Phase I: tested diets were supplemented with encapsulated calcium nitrate using a semi-automatic in vitro gas production technique Half gram of substrate with 50 mL of incubation medium, and 25 mL of rumen fluid were incubated in glass bottles (160 ml) at 39 °C for 24 hours to determine the best diets to be tested in vivo. The first trial tested the association between monensin (diets with and without monensin) and encapsulated nitrate levels (0, 1.5 and 3% of dry matter (DM)) for in vitro methane mitigation. There was no interaction between monensin and nitrate for the tested variables. The second in vitro assay tested the interaction of diet type with two concentrate:forage ratios, 20:80 and 80:20, and the inclusion of encapsulated nitrate levels (0, 1.5, 3 and 4.5% DM). Although it was not observed associative effect between diet and nitrate for reduction of methane, it was observed change in the ruminal fermentation products. Phase II: According to the results obtained in Phase I, in the second phase we evaluated the associative effect of concentrate: forage ratio of diet and the dose of nitrate on the methane emission, ruminal constituents and nitrate toxicity in vivo. We used six lambs cannulated in the rumen, distributed in Latin square design 6 x 6 in factorial 2 x 3. The factors were type of diet (concentrate:forage ratios 20:80 and 80:20) and inclusion of encapsulated nitrate doses in the diet (0, 1.5 and 3% DM) in the gradual replacement of soybean meal, a total of six treatments. The replacement levels of soybean meal by nitrate were in protein equivalent so as to leave the diets isonitrogenous. The animals were gradually adapted to dietary nitrate supply to avoid problems with toxicity. The analysis of toxicity were evaluated by the rate of methemoglobin in the blood of sheep 3 hours after feeding. Methemoglobin levels in blood of animals were not changed by the addition of nitrate. Associative effect was observed between the type of diet and nitrate for ruminal fermentation products such as acetate, which decreased linearly in the diets with 80% forage when nitrate was added. It can be concluded that nitrate used securely is a promising strategy for reducing enteric methane independently of the type of diet being supplemented