Dissertationen zum Thema „Rumen Microbiology“

The rumen microbial populations involved in the degradation of barley straw and clover/ryegrass forage during incubation in sacco were studied by the analysis of microbial phospholipids. The results suggested that the adherent populations differed from those in the liquid phase of the rumen contents, and that the microorganisms attached to barley straw differed from those attched to clover/ryegrass. In addition, the population adherent to barley straw appeared to change during the degradative process. The latter point was supported by observations using the electron microscope. When barley straw was incubated in vitro with Ruminococcus flavefaciens prior to incubation in the rumen, phospholipid analysis suggested that R.flavefaciens persisted during 72 h incubation in the rumen, although as a declining component of the mixed population. Ruminococcus flavefaciens was possibly displaced or other 'free' sites of attachment were occupied by different species. The in vitro incubation of Ruminococcus flavefaciens and Fibrobacter succinogenes on clover/ryegrass and barley straw showed that the presence of F.succinogenes reduced the population size of R.flavefaciens and the degradability of clover/ryegrass suggesting a competitive or antagonist interaction betweeen these species. The analysis of phospholipid marker components and viable counts showed that R.flavefaciens rapidly outgrew F.succinogenes. Ruminococcus flavefaciens and Fibrobacter succinogenes differed in the quantity and nature of the soluble plant components that accumulated in the culture liquids. After training Ruminococcus flavefaciens strain 17 to grow on different forages, adaptation through enhanced substrate degradation was detected when cultures were grown repeatedly on ryegrass. Significant increases in specific xylanase and beta-xylosidase activities were detected. It is concluded that the increase in dry matter solubilization and enzyme activities during prolonged subculture on ryegrass probably resulted from forward mutations.

O objetivo deste trabalho foi avaliar o efeito de um preparado de anticorpos policlonais (PAP) contra bactérias ruminais específicas, Streptococcus bovis e Fusobacterium necrophorum, em parâmetros ruminais da fermentação, em vacas canuladas, adaptadas ou não a uma dieta de alta proporção de carboidratos prontamente fermentescíveis, após indução à acidose. O delineamento experimental utilizado foi o quadrado latino 3X3 replicado em arranjo fatorial de tratamentos 3X2, sendo 2 aditivos alimentares (PAP na apresentação em pó - PAPP e PAP na apresentação líquida - PAPL) mais um grupo controle (CON) e dois manejos de adaptação à dieta, resultando em seis tratamentos. O primeiro quadrado latino foi submetido a um protocolo de adaptação à dieta do tipo gradual ou step-up: dos dias D0 a D4 os animais receberam 100% de forragem; do D5 ao D9, 30% de concentrados e do D10 ao D14, 60% de concentrados. O segundo quadrado latino recebeu 100% de forragem do D0 ao D14 (sem adaptação). Nos D15 e D16, todos os animais receberam dieta com 80% de concentrados. Para as análises foram coletadas amostras de líquido ruminal a cada 3 horas a partir da 0h antes da alimentação até as 36h (D15 e D16) durante o desafio com uma dieta de 80% de concentrados. Os dados foram analisados pelo procedimento Mixed do SAS com nível de significância de 0,05. Foi observada interação entre tempo e adaptação (P<0,05) para pH ruminal com diferença entre método de adaptação nas 0, 3, 6, 9, 12 e 36 horas pós alimentação, quando o grupo não adaptado teve valores maiores que o grupo adaptado, sendo que na hora 24 ocorreu o contrário. Para a concentração de ácidos graxos de cadeia curta (AGCC), nas horas 0, 3, 6, 9 e 36 pós alimentação o grupo adaptado obteve maiores valores comparado ao grupo não adaptado. Para proporção molar de acetato, a 0 hora o grupo sem adaptação obteve valores maiores comparado ao grupo adaptado. Já nas horas 24, 27 e 30 o grupo com adaptação que obteve maiores valores. Para a proporção molar de propionato o grupo sem adaptação teve valores mais altos em comparação ao outro grupo das 3 às 36 horas pós alimentação. Quanto à proporção acetato:propionato (Ac:Pr) às 6, 12, 24, 27, 30 e 36 horas pós alimentação, o grupo de animais adaptados teve valores mais altos que o grupo não adaptado. Na proporção molar de butirato, o grupo de animais adaptados obteve maiores valores nas horas 0, 3, 6, 9, 12, 33 e 36. Para os valores de nitrogênio amoniacal (N-NH3), às 6 horas pós alimentação, o grupo não adaptado obteve maiores valores que o grupo adaptado (26,1 vs. 19,3, respectivamente). Nas horas 9, 30, 33 e 36 ocorreu o contrário. Observou-se também interação entre tempo e aditivo (P=0,0430) para a proporção molar de butirato. Porém, quando a análise foi realizada por tempo, nenhum efeito foi observado. Para os valores relativos de protozoários mensurados (Dasytricha, Isotricha, Epidinium, Diplodinium e Entodinium) apenas o Entodinium apresentou efeito de adaptação (P<0,0236) tendo sua proporção maior no grupo adaptado. Os valores de haptoglobina também não foram influenciados nem por aditivo nem por adaptação. O preparado de anticorpos policlonais não foi tão eficaz quanto a adaptação gradual à dieta de alto concentrado para controlar alterações dos parâmetros ruminais.
The objective of this trial was to evaluate the effects of polyclonal antibodies preparation (PAP) against specific rumen bacteria Streptococcus bovis and Fusobacterium necrophorum on rumen fermentation parameters in ruminally cannulated cows adapted or not to highly fermentable carbohydrates diets (HFC) after an acidosis challenge. The experimental design was two 3X3 Latin squares in a factorial arrangement of treatments 3X2 regarding two feed additives (PAP in powder presentation - PAPP and PAP in liquid presentation - PAPL) plus control group (CON) and two managements of diets adaptation, resulting in six treatments. The first Latin square had a step-up diet adaptation: from D0 to D4 100% forage; D5 to D9 30% of concentrates and D10 to D14 60% of concentrates. The second Latin square received 100% forage from D0 to D14. On D15 and D16, all animals received a diet with 80% of concentrates. For analysis, rumen fluid was sampled at 0 and every 3 h posfeeding totaling 36 h (D15 and D16) of challenge with a diet with 80% of concentrates. Data were analyzed by MIXED procedure with a significance level of 0.05. An interaction between time and adaptation (P<0,05) was observed for ruminal pH. At 0, 3, 6, 9, 12 and 36 h postfeeding, the non-adapted group had higher values compared to the adapted group and at 24 h postfeeding, the inverse was observed. For total short-chain fatty acids concentration, at 0, 3, 6, 9 and 36 h postfeeding, the adapted group had higher values compared to non-adapted group. For molar proportion of acetate at 0h postfeeding, the non-adapted group had higher values than the adapted group, and at 24, 27 and 30h, the adapted group had greater values than the non-adapted group. For molar proportion of propionate the non-adapted group had greater values compared to the adapted group from 3 to 36h postfeeding. For acetate:propionate (Ac:Pr) ratio at 6, 12, 24, 27, 30 and 36 h postfeeding, the adapted group had greater values compared to the nonadapted group. For butyrate molar proportion at 0, 3, 6, 9, 12, 33 and 36h postfeeding the adapted group had greater values than the non-adapted group. For ammonia nitrogen (NH3- N) concentration at 6h, the non-adapted group had greater values than the adapted group (26.1 vs. 19.3, respectively), however at 9, 30, 33 and 36h postfeeding, the adapted group had higher values compared to the non-adapted group. It was also observed an interaction between time and additive (P=0.0430) for butyrate molar proportion, but when the analysis was performed by time no effect was observed. For the relative values of protozoa measured (Dasytricha, Isotricha, Epidinium, Diplodinium and Entodinium) only Entodinium presented adaptation effect (P<0.0236) with a higher proportion in the adapted group. Haptoglobin values was also not influenced (P>0.05) by additive or adaptation effect. Polyclonal antibodies preparation was not as effective as the gradual adaptation to the diet high concentrate to control changes of ruminal parameters.

Bibliography: leaves 122-138. Investigates the use of microbial inoculants to prevent the onset of acidosis in acutely grain fed animals; and, the most effective combination of virginiamycin and lactic acid utilising bacteria (selenomonas ruminantium subsp. lactilytica and Megasphaera elsdenii) in controlling lactic acid accumulations in vitro.

Videorecording has title: Effect of antibodies on the motility of rumen ciliates. Bibliography: leaves 197-259. Consists of a review of rumen ciliates, their implications in ruminant nutrition and a description of the research methods, the results and the conclusions drawn with regard to the prospects of establishing an immunological basis for the manipulation of rumen ciliates.

Bibliography: leaves 139-167 The aim of this project was to identify an enzyme responsible for the metabolism of oxalate which would be suitable for degrading oxalate in the rumen, and clone and characterise that gene.

Rumen microbiota enable dairy cattle to breakdown fiber into useable energy for milk production. Rumen bacteria, protozoa, and fungi ferment feedstuff into volatile fatty acids (VFA), the main energy source, while methanogens utilize fermentation by-products to produce methane. Milk fat contains several bioactive rumen-derived fatty acids (FA), including odd-chain FA (OCFA) and branched-chain FA (BCFA), important for maintenance of human health. The overarching dissertation goal was to determine which factors affect rumen methanogen and protozoal community structures and their metabolism products, while defining relationships between rumen microbiota and animal performance. Results presented contribute to the goals of providing new knowledge to dairy farmers, maintaining ruminant health, and enhancing bioactive FA in milk. The first objective was to use next-generation sequencing techniques to determine if lactation stage and dairy breed affect rumen methanogen and protozoal community structures and protozoa cell FA compositions in Jersey, Holstein, and Holstein-Jersey crossbred cows at 3, 93, 183, and 273 days in milk (DIM). A core methanogen community persisted by lactation stage and breed. At 3 DIM, methanogen 16S rRNA gene sequences formed distinct clusters apart from 93, 183, and 273 DIM, reflective of the dietary transition period post-partum. The starch-utilizing protozoal genus Entodinium, was more abundant in Holsteins than in Jerseys and Holstein-Jersey crossbred cows and positively correlated with milk yield. Jerseys had greater iso-BCFA contents in protozoa and milk and protozoa of the genus Metadinium. The second objective was to determine if supplementation of mixed cool-season grasses with annual forages (AF) alters the forage, microbial, and milk FA contents during typical periods of decreased pasture growth in Northeastern US. In short-term grazing (21d) of AF, ruminal VFA and major rumen-derived FA were not altered in bacterial and protozoal cells, suggesting little alteration of biohydrogenation and maintenance of ruminant health. In spring, milk contents of iso-15:0 and 17:0 per serving of whole milk were greater in control (CON)-fed cows, while contents of 12:0 and 14:0 per serving were greater in AF-fed cows. Contents of de novo FA and OCFA per serving of whole milk were greater in summer AF-fed cows than CON-fed cows, while total contents and BCFA did not differ, suggesting post-ruminal FA modifications in adipose tissue and the mammary gland. The third objective was to characterize and relate the rumen microbiota from CON- and AF-fed cows to animal performance. Rumen protozoal taxa were not altered, while less abundant bacterial taxa (< 5%) were different in both periods. The protozoal genus Diplodinium was positively correlated with feed efficiency and milk fat yield. In spring, AF-fed cows had greater abundances of the methanogen species Methanobrevibacter millerae, whereas CON-fed cows had greater abundances of the methanogen species Methanobrevibacter ruminantium, potentially as a result of differences in substrate availability. In conclusion, the work presented identifies several factors that influence rumen microbiota, rumen microbial FA, and milk FA, while providing new information to dairy farmers, researchers, and consumers.

Bibliography: leaves 150-183.
As lignocellulose represents an abundant renewable resource, research is in progress to obtain a better understanding of the natural mechanisms whereby this resource is utilised. Of particular interest is the degradation of forage in the rumen and one research goal is to ultimately increase animal productivity through an improvement in lignocellulose utilisation. However, although the mechanisms behind lignocellulose utilisation are reasonably well understood, relatively little is known about the mechanisms which occur in the rumen. Thus, the aim of this thesis was to gain more insight into the mechanisms of lignocellulose utilisation which occur in the rumen. Initially this research was focused on the poorly characterised exo-acting cellulases from rumen bacteria. Preliminary enzymology studies on one cellulase from Ruminococcus flavefaciens FD-1, previously isolated in this laboratory, indicated that an exo-acting cellodextrinase, CelA, had been isolated. In this report, the enzyme was purified and biochemically characterised and was shown to be an exo-acting cellodextrinase.

Thesis (Ph. D.)--Virginia Polytechnic Institute and State University, 1990.
Vita. Abstract. No film copy made for this title. Includes bibliographical references (leaves 149-165). Also available via the Internet.

The yeast expression system Pichia pastoris was investigated as an encapsulation technology capable of serving as a rumen escape vehicle. Cellularly encapsulated protein is protected from the ruminal environment so long as the cell membrane, which surrounds and isolates the intracellular protein is physically intact. Intracellular expression of Green Fluorescent Protein (GFP) allows for the monitoring of cellular integrity as necessary for the protection of encapsulated protein from ruminal proteases. Upon cellular lysis GFP is exposed to extracellular proteases which result in both the proteolytic degradation of the protein-based GFP chromophore and its associated fluorescence. Visualization of rumen fluid under epifluorescent microscopy revealed a high level of background autofluorescence owing to the fluorescent plant particles, microbes, and fluorescent compounds therein. Visualization of GFP in rumen fluid can be optimized through GFP variant selection, filter set design, and light source selection based on bulb emission spectra. Incubation of intracellular GFP expressing P. pastoris in batch culture ruminal in vitro simulations demonstrated that 93%, 97%, and 25% of the P. pastoris inoculum maintained cellular integrity in clarified rumen fluid, bacterial fraction of rumen fluid, and whole rumen fluid, respectively, when incubated over 36 to 48 h. Continuous fermentation in vitro rumen simulations (Rusitec) demonstrated a P. pastoris escape rate of 19% when added daily to fully adapted Rusitec vessels having a dilution rate of 0.75d-1. Abomasal in vitro simulations demonstrated that 84% of the P. pastoris inoculum was lysed within 12 h, as necessary for the release of encapsulated protein. P.pastoris may be an effective post-fuminal delivery vehicle, provided that similar results are obtained in vivo.
xiv, 120 leaves : ill. ; 28 cm.

The goal of the work presented herein was to further our understanding of the rumen microbiota and microbiome of wild moose, and to use that understanding to improve other processes. The moose has adapted to eating a diet of woody browse, which is very high in fiber, but low in digestibility due to the complexity of the plant polysaccharides, and the presence of tannins, lignin, and other plant-secondary compounds. Therefore, it was hypothesized that the moose would host novel microorganisms that would be capable of a wide variety of enzymatic functions, such as improved fiber breakdown, metabolism of digestibility-reducing or toxic plant compounds, or production of functional metabolites, such as volatile fatty acids, biogenic amines, etc. The first aim, naturally, was to identify the microorganisms present in the rumen of moose, in this case, the bacteria, archaea, and protozoa. This was done using a variety of high-throughput techniques focusing on the SSU rRNA gene (see CHAPTERS 2-5). The second aim was to culture bacteria from the rumen of the moose in order to study their biochemical capabilities (see CHAPTERS 6-7). The final aim was to apply those cultured bacterial isolates to improve other systems. Specifically, bacteria from the rumen of the moose was introduced to young lambs in order to colonize the digestive tract, speed the pace of rumen development, and improve dietary efficiency (see CHAPTER 8).

CAPES
O objetivo do presente trabalho foi identificar e quantificar os protozoários ciliados e bactérias do rúmen de bovinos cruzados (Europeu x Nelore), com média de 2,5 anos, alimentados em pastagens de inverno (Azevém e Aveia + Azevém) e verão (Brachiaria spp. e Brachiaria spp. + Esrela Africana). Amostras de conteúdo ruminal foram obtidas do centro da massa ruminal, após o abate dos animais. A contagem de bactérias totais (CBT), para as frações sólida e para líquida, foi realizada em placas com auxílio de contador de colônias manual. Para a análise morfológica empregou-se a técnica de coloração de Gram e a leitura das lâminas foi feita em microscópio óptico com objetiva de 100x, sendo as bactérias classificadas em cocos e bacilos, gram positivos e gram negativos. A avaliação da atividade fermentativa foi evidenciada pelo crescimento de colônias com coloração rósea atravéz de uma fonte de carboidrato. A quantificação e identificação dos gêneros de ciliados foram realizadas em câmara Sedgewick-Rafter em microscopia ótica. Foi determinado o teor de Matéria Seca, Proteína Bruta, Matéria Mineral, Fibra em Detergente Neutro e Fibra em Detergente Ácido, Digestibilidade In Vitro na Matéria Seca, Lignina, Celulose, Hemicelulose. O delineamento experimental foi inteiramente casualizado, com quatro tratamentos e 10 repetições para cada tratamento. Os dados foram submetidos à análise por meio da metodologia de Modelos Lineares, generalizados, com distribuição Poisson (1%) e foi utilizado o procedimento GENMOD. Foram observadas bactérias Gram positivas e negativas, nas formas cocos e bacilos e 11 gêneros de protozoários ciliados, sendo Diplodinium o gênero predominante. A maior concentração de bactérias foi encontrada nas forrageiras de inverno (494,8 x1010 mL-1), com maior intensidade na pastagem de Azevém. Nos protozoários ciliados, maior concentração foi observada no verão (200,70 x104 mL-1), principalmente na pastagem de Brachiaria spp.
The objective of the present work was to identify and quantify the ciliate protozoa and crossbovine rumen bacteria (European x Nellore), with an average of 2.5 years, fed on winter pastures (Azevém and Aveia + Azevém) and summer (Brachiaria spp And Brachiaria spp. + Esrala Africana). Samples of ruminal contents were obtained from the center of the ruminal mass, after the slaughter of the animals. The total bacterial count (CBT), for solid and liquid fractions, was performed in plates with the aid of a manual colony counter. For the morphological analysis the Gram staining technique was used and the slides were read under an optical microscope with objective of 100x, being the bacteria classified in cocci and bacilli, gram positive and gram negative. The evaluation of the fermentative activity was evidenced by the growth of colonies with pink coloration through a carbohydrate source. The quantification and identification of ciliate genera were performed in Sedgewick-Rafter chamber under optical microscopy. The content of Dry Matter, Crude Protein, Mineral Matter, Neutral Detergent Fiber and Acid Detergent Fiber, In Vitro Digestibility in Dry Matter, Lignin, Cellulose and Hemicellulose were determined. The experimental design was completely randomized, with four treatments and 10 replicates for each treatment. The data were submitted to analysis using the methodology of Linear Models, generalized with Poisson distribution (1%) and the GENMOD procedure was used. Gram positive and negative bacteria were observed, in the forms cocci and bacilli and 11 genera of ciliate protozoa, being Diplodinium the predominant genus. The highest concentration of bacteria was found in winter forages (494.8 x1010 mL-1), with higher intensity in the Azevém pasture. In the ciliate protozoa, higher concentration was observed in the summer (200.70 x 10 4 mL-1), mainly in Brachiaria spp.

The rumen microbiome constitutes a unique genetic resource of plant fiber degrading microbial enzymes that could be used for agricultural and industrial purposes. Anaeromyces mucronatus is a poorly characterized anaerobic lignocellulolytic fungus in the rumen. This thesis aimed at better understanding A. mucronatus YE505 and the particle associated rumen microbiota based on transcriptomic and metatranscriptomic approaches. High quality RNA was isolated from the fiber-associated rumen sample based on an improved RNA extraction method. A transcriptomic study was performed to investigate the expression of the fiber degrading system of A. mucronatus YE505, and the functional diversity of the fiber-associated eukaryotes from the rumen of muskoxen (Ovibos moschatus) was explored by a metatranscriptomic study. Much carbohydrate degradation related protein modules were detected. This study established effective approaches to characterizing the functional contents of rumen eukaryotic microbiome as well as rumen fungi, and identified several candidate genes that merit further investigation.
xiv leaves : ill. (some col.) ; 29 cm

[Truncated abstract] In Australia, there is pressure to reduce the amount of methane produced by ruminant livestock because they are the single largest source of methane emitted from anthropogenic sources, accounting for 70.7% of agricultural methane emissions. In addition, methane production represents a loss of gross energy intake to the animal. The organisms that are responsible for methane production in the animal gut are a distinct group of Archaea called methanogens. Methanogens occupy three different niches within the rumen. Some live freely in the rumen digesta (planktonic), others are attached to the outer surface of the rumen ciliates (ectosymbiotic), and some reside within the ciliates (endosymbiotic). The types and number of methanogens, as well as rumen ciliates and their symbiotic interactions, influence the amount of methane produced from the rumen. These factors in turn are affected by many factors, including diet and ruminal retention time. In this thesis, I tested the general hypothesis that increasing the amount of grain in the diet and reducing the retention time would affect the abundance and diversity of methanogens in their different niches, including their association with ruminal ciliates. Twenty-four fistulated sheep were used in a complete factorial design with the sheep randomly divided into four groups. ... The change in DGGE banding patterns and Shannon indices when sheep were fed grain indicated that the types of methanogens changed when sheep were fed low and high grain diets, but their diversity did not. In contrast, the diversity of rumen ciliates decreased when sheep were fed a high grain diet. A total of 18 bands from the DGGE analysis of the ciliates were sequenced. All except one, which was 98% similar to Cycloposthium sp. not found previously in the rumen, matched the sequences for previously identified rumen ciliates. Some of the rumen ciliates identified were not present in sheep fed the high grain diet. On a high grain diet, methanogens associate endosymbiotically with rumen ciliates to get better access to hydrogen. It appears that the association between methanogens and rumen ciliates is dictated by the availability of hydrogen in the rumen and not the generic composition of the ciliate population. Furthermore, endosymbiotic methanogens appear to produce less methane than methanogens in other niches. The pot scrubbers did not change ruminal retention time but they did reduce the acetate/propionate measurements observed in sheep on the high grain treatment. The reason why pot scrubbers had this effect remains unknown, but it is interesting to consider that some physical interaction has occurred between the pot scrubbers, the grain and the sheep that has improved the fermentation parameters in sheep fed a high grain diet. The results from this study have advanced our understanding of the interaction between methanogens and ruminal ciliates, and methanogenesis in the rumen in response to dietary changes and mechanical challenges. Extending this work to look more specifically at the species of methanogens that are most closely linked to high methane production and how they interact with the ruminal ciliates will be critical for manipulating enteric greenhouse gas emissions.

Three duodenally cannulated lactating Holstein cows fed cotton-seed meal (CSM), corn gluten meal (CGM) or blood meal (BM) as protein supplement were used in a 3 x 3 Latin Square experiment to determine microbial crude protein (MCP) in duodenal digesta. Diets, formulated to contain 15% crude protein (CP) on a dry matter basis, consited of 60% concentrate, 31% corn silage and 9% alfalfa hay. Chromium oxide was employed as flow marker. Microbial protein fraction of digesta CP (MCP/DCP) was estimated by three microbial markers: ¹⁵N, diaminopimelic acid (DAP) and ribonucleic acid (RNA). The isotopic method gave the most reliable results. Variability was higher with DAP and RNA. Results from RNA were lower (P < .01) and unreasonable. Based on ¹⁵N, MCP/DCP differed among treatments (P < .10) with means of 61.5, 59.4, and 50.0% for CSM, CGM, and BM, respectively, but differences were not significant for absolute amounts of total CP and MCP in duodenal digesta.

CAPES
O objetivo do presente trabalho foi identificar e quantificar os protozoários ciliados do rúmen de dois grupos genéticos de bovinos de corte (Nelore e Cruzados Nelore x Europeu), sob três sistemas de alimentação (confinado, à pasto e à pasto com suplemento). Amostras de conteúdo ruminal foram obtidas do centro da massa ruminal, após o abate dos animais. A quantificação e identificação dos gêneros de ciliados foram realizadas em câmara de contagem Sedgewick-Rafter em microscopia ótica. Foi determinado o conteúdo de matéria seca (MS), proteína bruta (PB), extrato etéreo (EE), matéria mineral (MM), fibra em detergente neutro (FDN) e fibra em detergente ácido (FDA) dos alimentos analisados. Os dados foram submetidos a analise por meio da metodologia de Modelos Lineares Generalizados, com distribuição Poisson (1%). Adicionalmente os dados foram submetidos a analise dos Componentes Principais. Houve efeito da interação (P<0,001) para as dietas e as raças analisadas. Verificou-se a ocorrência de 14 gêneros, sendo o gênero Entodinium o predominante em todos os animais analisados. Os ciliados pertencentes à ordem Entodiniomorphida, Eodinium, Epidinium, Eremoplastron, Eudiplodinium, Metadinium e Ostracodinium apresentaram maior prevalência nos animais da raça Nelore, quando comparados com o grupo racial cruzado Nelore x Europeu. Protozoários da família Isotrichidae (Dasytricha e Isotricha) foram observados em maior quantidade nos animais à pasto e a pasto recebendo suplemento. Em relação ao tipo de alimentação, animais alimentados exclusivamente a pasto apresentaram maior densidade de ciliados em relação aos animais confinados e/ou a pasto recebendo suplemento. Registrou-se a ocorrência do gênero Buestchlia, em um animal (prevalência de 1,66%) sendo este um dos poucos registros deste gênero em ruminantes.
The aim of this study was to identify and quantify the rumen ciliated protozoa of two genetic groups cattle (Nellore and crossbred Nellore x European) under three feeding systems (confined, pasture and pasture with supplement). The rumen fluid samples were obtained from the center of the rumen mass, after … The quantification was done in Sedgewick-Rafter counting chambre in light microscopy. It was determined the content of dry matter (DM), mineral matter (MM), ether extract (EE), crude protein (CP), neutral detergent fiber (NDF) and acid detergent fiber (ADF) to determine the chemical quality of the feed supplied to the animals. The data were submitted to analysis by Generalized Linear Models methodology, assuming Poisson distribution with logarithmic link function, with significance level of 1%. In addition, data were submitted to principal component analysis. The first two principal components were analyzed by least squares. There was a significant interaction (P<0.001) for diets and analyzed races. Occurrence of 14 ciliate genera was observed, being Entodinium the predominant in all animals analyzed. The ciliates belonging to order Entodiniomorphida, Eodinium, Epidinium, Eremoplastron, Eudiplodinium, Metadinium e Ostracodinium had a higher prevalence in Nellore cattle, compared with the racial group crossbred Nellore x European. Protozoa of the family Isotrichidae (Dasytricha e Isotricha) were observed in greater quantities in animals to pasture and pasture with supplement. Regarding the type of food, animals on pasture had higher ciliates density compared to confined animals and pasture with supplement. Occurrence of gender Buestchlia only one animal (prevalence 1,66%), which is one of the few records of this kind in ruminants.

Bibliography: leaves 226-261. Investigates nitrogen utilization in some species of rumen bacteria with the object of understanding the role of ammonia versus exogenous amino acids in relation to microbial growth.

[Truncated abstract] Antimicrobial growth promoters are added to feed to prevent lactic acidosis in ruminant animals by selectively inhibiting rumen bacteria that produce lactic acid. However, recently imposed or impending bans on the use of antimicrobial growth promoters in animal production have lead to a critical need to find practical alternatives that are safe for the animal and consumer and that obtain similar production benefits. I investigated bioactive plants of Australia for their potential to prevent lactic acidosis in ruminants. The unifying hypothesis tested was that plants would be identified that selectively inhibit lactic acid-producing bacteria and consequently protect against lactic acidosis. This hypothesis was tested in a three phase process: phase 1, plant selection and collection; phase 2, a three stage protocol for screening plants and essential oils; phase 3, in vivo experiments and chemical fractionation of the most promising plant. I developed an in vitro bioassay that simulated acidosis by adding glucose to rumen fluid in Bellco tubes and incubating for 5 h (Chapter 4). The pH and gas production were used as indicators of acidosis and fermentation activity. I used this bioassay to screen ninety-five plants (dried and ground material from 79 species) and ten essential oils and included a negative control (oaten chaff) and a positive control (virginiamycin). One plant, Eremophila glabra, produced a similar pH (5.63) to the positive control (5.43) although it inhibited gas production to a moderate extent (P < 0.05). ... Seven serrulatane diterpenes were identified to be the major secondary metabolites in E. glabra. The metabolites were screened using a broth dilution and microtitre spectrophotometry method and were selective against S. bovis at between 320 and 1077 [mu]g/ mL. The serrulatanes from E. glabra were probably responsible for the activity against acidosis that I observed in vitro, because they selectively inhibited lactateproducing bacteria. It is also possible that a synergy between serrulatanes and possibly other metabolites are responsible for the activity observed in vitro. The results from my experiments support the role that bioactive plants may have to replace the antibiotics that are added to livestock feed. Australian plants were identified containing compounds that were active against the bacterial processes responsible for ruminant acidosis. To my knowledge this is the first work undertaken to identify bioactive plants of Australia for their potential to prevent acidosis. I developed in vitro screening bioassays that targeted key indicators of acidosis. These bioassays enabled me to identify 5 plants from the 104 screened that could potentially control acidosis. One of these plants in particular, E. glabra, showed a level of activity in vitro that was comparable to antibiotic protection against acidosis. The exciting in vitro results were not demonstrated in vivo but only one dose level of E. glabra was used, which was based on the in vitro work. In contrast to the in vitro system the rumen is a continuous flow system with greater complexity and it is possible that the concentration of E. glabra that I used in vivo was not optimum. This places importance on future dose response experiments to confirm the efficacy of E. glabra in vivo.

Thesis (MScAgric (Animal Sciences))--University of Stellenbosch, 2009.
ENGLISH ABSTRACT: Ruminants have a compound stomach system that enables them to utilize forages more efficiently than monogastric animals. However, forages alone do not contain sufficient nutrients to meet the requirements of high producing dairy cows. Forages are high in fibre and their nutrient availability depends on the degree of cell wall degradability. Improvements in forage fermentation would increase energy intake and subsequently milk production and performance by dairy cows. It is therefore important to find ways to improve forage degradation and utilization in the rumen. The use of different non-fibre carbohydrate (NFC) sources has different effects on animal performance. Supplementing forage based diets with energy sources containing sugar, starch or pectin results in variation in performance measurements such as milk yield, milk composition and dry matter intake (DMI). This thesis reports on two studies in which the effect of energy supplementation on forage fermentation and digestion parameters was investigated. In the first study an in vitro gas production protocol was used to determine the effect of sugar (molasses), starch (maize meal) and pectin (citrus pulp) on total gas production and rate of gas production of different forages. The forage substrates included wheat straw (WS), oat hay, (OH) lucerne hay (LUC), ryegrass (RYE) and kikuyu grass (KIK). The three energy sources, as well as a control (no energy source) were incubated in vitro with each of the above mentioned forages. Rumen fluid was collected from two lactating Holstein cows receiving a diet consisting of oat hay, lucerne, wheat straw and a concentrate mix. Forages alone (0.25 g DM) and/or together (0.125 g DM) with either molasses (0.1412 g DM), citrus pulp (0.1425 g DM) or maize meal (0.125 g DM) were weighed into glass vials and incubated for 72 hours. The weights of the energy sources were calculated on an energy equivalent basis. Blank vials, that contained no substrates, were included to correct for gas production from rumen fluid alone. The substrates were incubated in 40 ml buffered medium, 2 ml of reducing solution and 10 ml rumen fluid. Gas pressure was recorded automatically every five minutes using a pressure transducer system and the method based on the Reading Pressure Technique (Mauricio et al., 1999). Gas pressure was converted to gas volume using a predetermined regression equation. In the first gas production trial, the gas production included gas produced by the energy sources, while in the second gas production trial, the energy source gas production was deducted from the total gas production to determine the effect of energy source on gas production of respective forage substrates per se. Data were fitted to two non-linear models adapted from Ørskov and McDonald (1979). Significant forage x energy interactions were observed for the non-linear parameter gas production (b) in Model 1 and for b and lag phase (L) in Model 2 in both trials. In the first gas production trial, the higher fermentability of the energy sources supplemented to forage substrates, increased b (Model 1 & 2) of the LUC and WS. The gas production rate was affected in different ways for different forages, with the most noticeable effect on WS when it was supplemented with energy sources. All the energy sources increased c of WS irrespective of the model used. Energy sources had no effect on the L of LUC, OH or RYE, but decreased the L of WS and KIK. In the second trial, maize meal had no effect on b for any of the forages (Model 1 & 2), while molasses (Model 1 & 2) decreased b for all forage substrates, and citrus pulp (Model 1 & 2) decreased b of OH and RYE, to lower values than those of the control treatments. Gas production rate was not affected by molasses for any of the forage substrates, while citrus pulp (Model 1 & 2) increased c of OH and maize meal increased c of OH and KIK. Lag phase was only affected by energy sources in WS and KIK, where all the energy sources had lower L values than the control treatment. It was concluded that forage fermentability is affected differently by different energy sources. These observations may have important implications, in practice, on rumen health and milk production, and the data obtained can potentially be used as guidelines in feed formulations. In the second study, in vitro digestibility trials were undertaken to determine the effect of sugar (molasses and sucrose), starch (maize meal and maize starch) and pectin (citrus pulp and citrus pectin) on neutral detergent fibre (NDF) and dry matter (DM) degradability of forages. Forage substrates used included wheat straw, oat hay, lucerne hay, ryegrass and kikuyu grass. Rumen fluid was collected from two lactating Holstein cows receiving a diet consisting of oat hay, wheat straw and a concentrate mix. In vitro degradability was done with an ANKOM Daisy II incubator and forage substrates were incubated with or without the respective energy sources for 24, 48 and 72 hours. The substrates were incubated in 1076 ml buffered medium, 54 ml of reducing solution and 270 ml rumen fluid. The residues were washed, dried and analyzed for NDF. In the study with the applied energy sources (molasses, maize meal and citrus pulp) there were a forage x energy source interactions. Supplementation with the applied energy sources all improved dry matter degradability (DMD) of forages (24 and 72 hours), when compared to the control treatment, except for RYE supplemented with maize meal and citrus pulp at 24 hours. Molasses seemed to have had the biggest effect on DMD in all forage substrates. Supplementation with maize meal had no effect on neutral detergent fibre degradability (NDFD) of any forage substrate, except for an improvement in NDFD of LUC at 72 hours. Molasses improved NDFD of LUC at 24h, but had no effect on the other forage substrates. Citrus pulp improved NDFD of OH (72 hours), as well as LUC and WS (24 and 72 hours). It is postulated that the NDF of the energy sources was more digestible than that of the respective forages, and that the improved NDFD values could be ascribed to the contribution of the energy source NDFD. Overall, pasture grasses had a higher NDFD than the hays and straw, and appear to be more readily fermentable by rumen microbes than the low quality hays and straw explaining the higher NDFD. In the study involving the purified energy sources (sucrose, maize starch and citrus pectin), forage x energy source interactions were observed. In general, supplementation with these energy sources improved DMD at 24 and 72 hours except for RYE and KIK (72 hours). Pasture grasses (RYE and KIK) had a higher NDFD than LUC, OH and WS. At 72 hours, NDFD was 37.1% for LUC, 42.5% for OH and 40.3% for WS, compared to 70.5% for KIK and 64.9% for RYE. A possible explanation is that KIK and RYE samples came from freshly cut material, harvested after a 28d re-growth period. In general, sucrose (24 and 72 hours) and citrus pectin (72 hours) had no effect on NDFD of forage substrates. However, supplementing oat hay (24 hours) with starch and citrus pectin, and wheat straw (24 and 72 hours) with starch lowered NDFD, when compared to the control treatment. It is hypothesized that microbes fermented the easily fermentable energy sources first, before attacking forage NDF. The study suggested that forage NDFD values are not fixed, and may be altered by type of energy supplementation.
AFRIKAANSE OPSOMMING: Die meervoudige maagsisteem van herkouers stel hulle in staat om ruvoer meer effektief te benut as enkelmaagdiere. Ruvoere alleen bevat egter nie genoeg voedingstowwe om die behoeftes van hoogproduserende melkbeeste te bevredig nie. Ruvoere is ryk aan vesel en hul voedingstofbeskikbaarheid word bepaal deur die graad van selwand degradeerbaarheid. ‘n Verhoging in ruvoerfermentasie sal energieinname verhoog en gevolglik ook melkproduksie en prestasie. Dit is dus belangrik om maniere te vind om ruvoerdegradeerbaarheid en -verbruik in die rumen te verbeter. Die gebruik van verskillende nie-vesel koolhidraat (NFC) bronne het verskillende uitwerkings op die prestasie van diere. Energie-aanvullings soos suiker, stysel en pektien tot ruvoer-gebasseerde diëte, beïnvloed prestasiemaatstawwe soos melkproduksie, melksamestelling en droëmateriaalinname (DMI) op verskillende maniere. Hierdie tesis lewer verslag oor twee studies waar die invloed van energie-aanvullings op ruvoerfermentasie en verteringsmaatstawwe ondersoek is. In die eerste studie is ‘n in vitro gasproduksieprotokol gebruik om die invloed van suiker (melasse), stysel (mieliemeel) en pektien (sitruspulp) op totale gasproduksie (b) en tempo van gasproduksie (c) van verskillende ruvoersubstrate te bepaal. Ruvoersubstrate wat gebruik is, was koringstrooi (WS), hawerhooi (OH), lusernhooi (LUC), raaigras (RYE) en kikuyugras (KIK). Die drie energiebronne, sowel as ‘n kontrole (geen energiebron), is in vitro geïnkubeer saam met elk van die genoemde ruvoere. Rumenvloeistof is verkry van twee lakterende Holsteinkoeie, wat ‘n dieet ontvang het bestaande uit hawerhooi, koringstrooi en ‘n kragvoermengsel. Ruvoere is alleen en/of in kombinasie met melasse (0.1412 g DM), sitruspulp (0.1425 g DM) of mieliemeel (0.125 g DM) in glasbottels afgeweeg en vir 72 uur geïnkubeer. Die massas van die energiebronne is op ‘n energie-ekwivalente basis bereken. Leë bottels wat geen substraat bevat het nie, is ingesluit om te korrigeer vir gasproduksie afkomstig vanaf rumenvloeistof alleen. Substrate is in 40 ml van ‘n buffermedium, 2 ml reduserende oplossing en 10ml rumenvloeistof geïnkubeer. Gasdruk is elke vyf minute outomaties aangeteken deur gebruik te maak van ‘n drukmetersisteem en die metode is gebasseer op die Reading gasdruktegniek. Gasdruk is omgeskakel na gasvolume deur gebruik te maak van ‘n voorafbepaalde regressievergelyking. In die eerste proef het totale gasproduksie die gas wat deur die onderskeie energiebronne geproduseer is, ingesluit. In die tweede proef is gasproduksie afkomstig van die energiebronne afgetrek van totale gasproduksie, om sodoende die invloed van die energiebronne per se op die gasproduksie van die onderskeie ruvoersubstrate, te bepaal. Data is met behulp van twee nie-liniëre modelle gepas. Betekenisvolle ruvoer x energie-interaksies is in albei proewe waargeneem vir die nie-liniëre parameter b (gasproduksie) in Model 1, en vir b en L (sloerfase) in Model 2. In die eerste proef het die energiebronne se hoë fermentasie gelei to ‘n verhoging in b (Model 1 & 2) van LUC en WS. Energie-aanvullings het die c-waarde van die onderskeie ruvoere verskillend beïnvloed, met WS wat die mees opvallende effek gehad het. Al die energiebronne het die c-waarde van WS verhoog, ongeag watter model gebruik is. Energiebronne het geen invloed op die L-waarde van LUC, OH of RYE gehad nie, maar het wel die L-waarde van WS en KIK verlaag. In die tweede proef het mieliemeel geen invloed op die b-waarde van enige van die ruvoere gehad nie (Model 1 & 2), terwyl melasse (Model 1 & 2) die b-waarde van alle ruvoere verlaag het, en sitruspulp (Model 1 & 2) OH en RYE se b waardes verlaag het tot laer as die kontroles. Melasse het geen invloed op die c-waarde van die onderskeie ruvoersubstrate gehad nie, terwyl sitruspulp (Model 1 & 2) die c-waarde van OH, en mieliemeel die c-waarde van OH en KIK, verhoog het. Energiebronne het slegs ‘n invloed op die sloerfase in WS en KIK gehad, waar dit L verlaag het tot laer waardes as dié van die kontroles. Daar is gevind dat ruvoer-fermenteerbaarheid verskillend beïnvloed word deur verskillende energiebronne. Bogenoemde resultate kan in die praktyk betekenisvolle invloede hê op rumengesondheid en melkproduksie en die data wat verkry is, kan potensieël gebruik word as riglyne in voerformulerings. In die tweede studie is in vitro verteerbaarheidsproewe gedoen om die effek van suiker (molasse en sukrose), stysel (mieliemeel en mieliestysel) en pektien (sitruspulp en sitrus-pektien) op neutraalonoplosbare vesel (NDF) en droë materiaal (DM) degradeerbaarheid van ruvoere, te bepaal. Ruvoersubstrate wat gebruik is, was WS, OH, LUC, RYE en KIK. Rumen vloeistof is verkry van twee lakterende Holstein koeie, wat ‘n dieet ontvang het bestaande uit hawerhooi, koringstrooi en ‘n konsentraat mengsel. Die in vitro degradeerbaarheidsproef is gedoen met ‘n ANKOM Daisy II inkubator. Ruvoersubstrate is geïnkubeer met of sonder die onderskeie energiebronne vir 24, 48 en 72 uur. Die substrate is geïnkubeer in 1076 ml buffer medium, 54 ml reduserende oplossing en 270 ml rumen vloeistof. Residue is gewas, gedroog en geanaliseer vir NDF. In die proef met toegepaste energiebronne (molasse, mieliemeel en sitruspulp), was daar ruvoer x energiebron interaksies. Toegepaste energiebron aanvullings het almal DMD van ruvoersubstrate (24 en 72 uur) verbeter, uitsluitend vir RYE wat aangevul is met mieliemeel (24 uur) en sitruspulp (24 uur). Van al die ruvoersubstrate het molasse die grootste effek gehad op DMD. Mieliemeel aanvullings het geen effek gehad op neutraal-onoplosbare vesel degradeerbaarheid (NDFD) van ruvoersubstrate nie, behalwe vir ‘n verbetering in NDFD van LUC by 72 uur. Molasse het NDFD van lucern by 24 uur verbeter, maar geen effek gehad op ander ruvoersubstrate nie. Sitruspulp het NDFD van OH (72 uur), asook LUC en WS (24 & 72 uur) verbeter. Daar word beweer dat die NDF van energiebronne meer verteerbaar is as die van ruvoersubstrate, en dat die verbetering in NDFD waardes toegeskryf kan word aan die bydraes van energiebronne se NDFD. Weidingsgrasse (RYE & KIK) het oor die algemeen ‘n hoër NDFD as hooie en strooi gehad. Rumen mikrobes blyk ook om dié grasse vinniger te verteer as lae kwaliteit hooie en strooi, wat gevolglik die hoër NDFD verduidelik. In die proef met suiwer energiebronne (sukrose, mieliestysel en sitrus-pektien) is ruvoer x energiebron interaksies waargeneem. Energiebronaanvullings het DMD by 24 en 72 uur verbeter, buiten vir RYE en KIK (72 uur). Weidingsgrasse het hoër NDFD as LUC, OH en WS. By 72 uur was die NDFD van LUC 37.1%, OH 42.5%, WS 40.3%, in vergelyking met 70.5% vir KIK en 64.9% vir RYE. ‘n Moontlike verklaring vir die hoër NDFD van KIK en RYE, is omdat dit vars gesnyde material is, geoes na slegs 28 dae hergroei. Oor die algemeen het sukrose (24 & 72 uur) en sitrus-pektien (72 uur) geen effek gehad op NDFD van ruvoersubstrate nie, terwyl stysel en pektien aanvullings tot OH (24 uur), en stysel aanvullings tot WS (24 & 72 uur) NDFD verlaag het. Daar word hipotetieseer dat mikrobes eers die vinnig fermenteerbare energiebronne fermenteer, voordat hulle ruvoer NDF aanval. Hierdie studie beweer dat ruvoer NDFD waardes nie vas is nie, en dat dié waardes beïnvloed mag word deur energiebron aanvullings.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico
This work aimed to evaluate the effects of ruminal and/or abomasal protein supplementation in cattle fed tropical forages on nutritional variables, degradation of insoluble fiber, activity of fibrolytic enzymes and rumen bacterial communities profile. Two experiments were carried out sequentially, and in the same way, differing only on the forage quality that was defined based on crude protein content: medium quality (784 g of crude protein/ kg of dry matter) and high quality (986 g of crude protein/ kg of dry matter). Four Nellore young bulls, averaging 227±11 kg body weight and fitted with ruminal and abomasal cannullas, were used. The following treatments were evaluated: control (only forage), ruminal nitrogenous compounds supplementation (daily supply of 230 g of crude protein in the rumen), abomasal nitrogenous compounds supplementation (daily supply of 230 g of crude protein in the abomasum), and ruminal and abomasal nitrogenous compounds supplementation (daily supply of 230 g of protein, being 115 g in the rumen and 115 g in the abomasum). Casein was used as protein source for supplementation and basal diet consisted of Tifton 85 (Cynodon spp) hay. Two 4 x 4 Latin squares experimental trials, balanced for residual effects, with four treatments, four animals and four experimental periods lasting 29 days each, were implemented sequentially, one for each forage as stated above. There were no effects (P>0.05) of supplementation scheme or forage quality on organic matter (OM) intake. On the other hand, neutral detergent fiber (NDF) intake was higher (P<0.05) when animals were fed high quality forage. The total digestibility of OM was increased (P<0.05) by supplementation and by increasing forage quality. The digestibility coefficient of NDF was not affected by supplementation scheme (P>0.05), but was increased by the enhancing in forage quality. The apparent ruminal nitrogen balance (RNB) was increased (P<0.05) when animals were supplemented in rumen or in both rumen and abomasum cannula. It is worth highlighting that no supplementation or exclusively abomasal supplementation led to negative values of RNB. The apparent nitrogen balance (NB) was increased (P>0.05) when high quality forage was fed to the animals and by supplementation, no matter if the supplement was infused into rumen, abomasum or both. Ruminal supplementation enhanced (P<0.05) the concentration of rumen ammonia nitrogen (RAN). Although, animals supplemented in rumen and abomasum or exclusively in abomasum did not exhibit increase in RAN concentrations (P>0.05) when compared to no supplemented animals. The bacterial richness and diversity (Shannon-Wiener index) associated with the solid phase of rumen digesta were not affected (P>0.05). However, richness and diversity associated with the liquid phase were increased (P<0.05) by the improvement in forage quality. NDF degradation rate (kp) was enhanced (P<0.05) by ruminal supplementation. Carboximetilcellulase activity was decreased (P<0.05) by the abomasal and ruminal/abomasal supplementation. Xylanase activity was decreased (P<0.05) by both post-ruminal supplementation schemes, when high quality forage was fed to the animals. It was not detected correlations among variables associated with bacterial diversity and activity and variables associated with nutritional performance, excepted for the positive correlation between kp and RNB Clustering analyses based on the Unweighted Pair Group Method (UPGMA) evidenced that distinct bacterial communities are associated with liquid and solid fraction, independently of the forage quality. Clustering analyses also showed that forage quality affects the diversity of liquid phase associated bacteria, even though, effects on solid phase was not clear. None of the evaluations related to the microbial community were able to detect clear effects of supplementation on the microbial community diversity. Improving forage quality enhances fiber digestibility, nitrogen balance and modifies the bacterial community associated with liquid phase. On the other hand, protein supplementation enhances the nitrogen retention in animal body.
Objetivou-se avaliar os efeitos da suplementação proteica ruminal e/ou abomasal em bovinos consumindo forragens tropicais sobre as características nutricionais, a atividade enzimática fibrolítica, a degradação in vitro da fibra insolúvel e o perfil da comunidade bacteriana ruminal. Dois experimentos foram realizados sequencialmente diferindo apenas na qualidade do feno ofertado, sendo de média qualidade (78,4 g de proteína bruta/kg de matéria seca) e alta qualidade (98,6 g de proteína bruta/kg de matéria seca), respectivamente. Os procedimentos experimentais foram os mesmos para ambas as forragens. Foram utilizados quatro novilhos Nelore, não castrados, com peso corporal médio de 227±11 kg, fistulados no rúmen e no abomaso. Foram avaliados os seguintes esquemas de suplementação: controle (somente forragem); suplementação nitrogenada ruminal (fornecimento diário de 230 g de proteína suplementar no rúmen); suplementação nitrogenada abomasal (fornecimento diário de 230 g de proteína suplementar no abomaso) e suplementação nitrogenada ruminal e abomasal (fornecimento diário de 230 g de proteína suplementar, sendo 115 g no rúmen e 115 g no abomaso). A alimentação volumosa basal foi constituída por feno de tifton 85 (Cynodon spp) e como fonte de compostos nitrogenados suplementares foi utilizada a caseína (caseína pura, Labsynth). O experimento foi conduzido segundo delineamento em quadrado latino 4 x 4 balanceado para efeitos residuais, sendo dois quadrados sequenciais, um para cada forragem, com quatro esquemas de suplementação, quatro animais e quatro períodos experimentais com 29 dias em cada quadrado. Não foram observados efeitos (P>0,05) da qualidade da foragem ou do esquema de suplementação sobre o consumo de matéria orgânica (MO). Por outro lado, o consumo de fibra em detergente neutro (FDN) foi superior (P<0,05) quando forragem de alta qualidade foi ofertada aos animais. O coeficiente de digestibilidade total da MO foi incrementado (P>0,05) pela suplementação e pela melhoria na qualidade da forragem. O coeficiente de digestibilidade da FDN não foi afetado pelo esquema de suplementação (P>0,05), mas foi superior quando os animais receberam forragem de alta qualidade (P<0,05). O balanço aparente de compostos nitrogenados no rúmen (BNR) foi ampliado (P<0,05) pela suplementação no rúmen e no rúmen-abomaso. Ressalta-se que a ausência de suplementação ou a suplementação exclusiva no abomaso proporcionam valores negativos de BNR. O balaço aparente de compostos nitrogenados no organismo animal foi ampliado (P<0,05) pelo fornecimento de suplemento, independentemente do local de suplementação, e pelo fornecimento de forragem de melhor qualidade A suplementação no rúmen incrementou (P<0,05) a concentração de nitrogênio amoniacal ruminal (NAR). Por outro lado, animais recebendo suplementação no rúmen e abomaso ou exclusivamente no abomaso apresentaram concentrações de NAR similares (P>0,05) a animais não suplementados. A riqueza e a diversidade microbiana da fase sólida da digesta ruminal não foram afetadas (P>0,05). Entretanto, a riqueza e a diversidade bacteriana associada à fase líquida da digesta ruminal foram incrementadas (P<0,05) pela melhoria na qualidade da forragem. A taxa de degradação da FDN foi ampliada (P<0,05) pela suplementação no rúmen. A atividade da carboximetilcelulase foi reduzida (P<0,05) pela suplementação no abomaso e rúmen-abomaso. A atividade da xilanase foi deprimida (P<0,05) pela suplementação pós-ruminal somente quando forragem de alta qualidade foi fornecida aos animais. Não foi observada correlação (P>0,05) entre as variáveis associadas à atividade enzimática e diversidade microbiana e as características ligadas ao desempenho nutricional, exceção feita à correlação positiva (P<0,05) entre a taxa de degradação da FDN e o BNR. A avaliação multivariada da diversidade permitiu evidenciar que comunidades bacterianas distintas estão associadas às frações líquida e sólida, independente da qualidade da forragem ofertada. Percebeu- se que a variação na qualidade da forragem influencia a diversidade microbiana associada à fração líquida do fluido ruminal, embora não tenha exercido tal efeito sobre a fração sólida. Em nenhuma avaliação percebeu-se efeito claro do esquema de suplementação sobre o perfil da comunidade microbiana ruminal. A melhoria da qualidade da forragem amplia a digestibilidade da fibrae a retenção de nitrogênio e é capaz de modificar a comunidade bacteriana associada à fração liquida do conteúdo ruminal. A suplementação proteica, por sua vez, independentemente do local de suplementação, amplia a retenção de nitrogênio no organismo animal.

Léquilibre de l'écosystème ruminal est sous la dépendance des interactions microbiennes. Parmi les bactéries du rumen, nous avons choisi d'étudier celles responsables de la dégradation des matières azotées. Nos expérimentations ont abouti à la sélection de bactéries protéolytiques : bactéroides ruminicola, anaérobie strict; streptococcus bovis, anaérobie facultatif; bacillus sp2, aérobie. L'activité protéasique de ces microorganismes se manifeste d'abord sur les unités de haut poids moléculaire des protéines testées (caséine, blé, maïs et soja). Toutefois, l'intensité de la protéolyse est directement liée à la nature même des protéines soumises aux potentialités propres à chaque bactérie. Un modèle est proposé en coculture. Il souligne la complémentarité des métabolismes respiratoires et l'interdépendance trophique de streptococcus bovis et de bacteroides ruminicola. L'ensemble de ces données s'applique aux bactéries citées. Elles peuvent aussi s'étendre aux deux groupes bactériens auxquels elles pourraient appartenir: les microorganismes à caractère hydrolytique et fermentaire. Par cette extrapolation nous pouvons proposer une hypothèse quant à la place de la protéolyse dans l'ensemble des mécanismes fermentaires comme l'amylomyce et la cellulolyse. Ainsi, la mise en évidence des interactions microbiennes responsables de la dégradation des matières azotées permet de valoriser au mieux la ration des ruminants en optimisant le fonctionnement des processus lytiques rencontres dans l'écosystème étudié

Les champignons anaerobies du rumen produisent et secretent un systeme d'enzymes hydrolytiques capables de degrader les parois cellulaires vegetales de la nourriture des ruminants. Notre travail a porte sur le genre le plus etudie jusqu'a present: neocallimastix frontalis. Ce champignon produit toute une panoplie d'enzymes lytiques. Parmi ces enzymes, 7 glycosidases et 3 polysaccharidases ont ete etudiees. Ces enzymes lytiques se caracterisent par leur grande efficacite dans l'hydrolyse de substrats cellulosiques ou hemicellulosiques, liberant des sucres simples directement assimilables. D'autre part, elles sont produites sous la forme de multiples formes moleculaires, qui presentent pour certaines d'entre elles, une aspecificite de substrat. Toutes ces enzymes presentent un faible niveau de synthese, dit constitutif, lors de cultures effectuees en presence d'un sucre simple (glucose ou xylose). Mais toutes ces activites augmentent considerablement dans les cultures effectuees sur des inducteurs complexes (polysaccharides) ou simples (methyl--glucose). Cette induction est donc aspecifique, cependant une activite enzymatique peut presenter des niveaux d'induction variables en fonction de l'inducteur. Enfin, la production de ces enzymes est soumise a une repression catabolique par le glucose. Trois enzymes ont ete purifiees par des techniques chromatographiques: les endo--1,4-xylanases i et ii et une -glucosidase minoritaire. Elles ont ete caracterisees par leurs proprietes physico-chimiques, l'influence de certains effecteurs et leurs specificites vis-a-vis de differents substrats. Nous avons determine la sequence n-terminale de la xylanase i et produit des anticorps polyclonaux. Les anticorps polyclonaux ont permis d'etudier la production et la secretion de ces enzymes au cours de cinetiques effectuees en conditions de non-induction, d'induction et de repression de la synthese des enzymes. Les effets d'un antibiotique ionophore (monensine) et d'un antibiotique bloquant les n-glycosylations (tunicamycine) ont ete analyses au cours de la production de ces enzymes. Enfin, la localisation subcellulaire des xylanases i et ii a ete etudiee au cours de leur maturation et de leur secretion. Le clonage du gene correspondant a la xylanase i a ete aborde par la technique de pcr, a partir d'une banque d'adnc construite dans le vecteur zap ii

Etude de la protéosynthèse dans un rusitec (qui est un rumen artificiel en écoulement semi-continu) en comparant le taux d'azote total de bactéries isolées à partir d'effluents journaliers avec le taux d'azote des aliments. La protéosynthèse microbienne augmente de façon linéaire avec le temps dans le rusitec

La nutrition et la physiologie des ruminants sont étroitement liées au résultat des interactions entre les bactéries du rumen. En effet, la composition des régimes alimentaires affecte directement la dégradation des protéines contenues dans la ration, conséquemment à une modification de la flore dominante du contenu ruminal. La fourniture de 2 rations à base de pois a 4 brebis a montré une augmentation de 30% de l'activité protéolytique totale dans le rumen avec le régime conduisant a la plus faible teneur an ammoniac. Cet accroissement est du a une augmentation des activités exopeptidasiques libérées dans la phase liquide ainsi qu'à une diversification de la composition en endopeptidases dans la phase particulaire. In vitro, des doses croissantes de métabolites azotes issus du catabolisme des protéines (ammoniac et acides amines AA) ont montré des effets distincts sur l'activité protéolytique de 3 souches de bactéries du rumen cultivées avec de la caséine : S. Bovis JB1, P. Albensis et B. Fibrisolvens 3701. L’activité protéolytique totale de S. Bovis et de P. Albensis est inhibée tandis qu'elle est stimulée chez B. Fibrisolvens. La distribution et la composition en endopeptidases de l'activité protéolytique totale entre les fractions fixée aux cellules libérée dans le milieu sont également affectées, de manière distincte selon la forme azotée exogène et l'espèce considérée. Dans une étude in vitro des interactions entre les 3 souches cultivées deux par deux sur caséine, les effets sur l'activité protéolytique par rapport aux monocultures ont pu être mis en relation essentiellement avec la proportion des petits peptides résiduels ainsi qu'avec la concentration en AA libres, dont l'accumulation dans le milieu découle d'une activation des exopeptidases. Enfin, le remplacement de la caséine par des protéines de pois a montré que l'utilisation de cette source protéique, moins dégradable, favorise les coopérations bactériennes quelle que soit la coculture considérée.

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O rúmen é um ecossistema aberto, no qual o alimento consumido pelos ruminantes é fermentado a ácidos graxos de cadeia curta e proteína microbiana, os quais servem respectivamente como fonte de energia e de proteína para o animal. Espécies de micro-organismos têm desenvolvido no rúmen uma série de interações complexas, o qual é um dos melhores exemplos de simbiose entre micro-organismos na natureza. Os métodos convencionais para a taxonomia baseada em técnicas de cultivo vêm sendo substituídas por técnicas moleculares, que são rápidas e mais precisas. O fundamento das técnicas moleculares está na sequência do 16S rRNA que fornece a classificação filogenética usadas na identificação e quantificação da comunidade bacteriana. Para avaliar a diversidade bacteriana ruminal neste estudo, foram utilizados 3 bovinos da raça Nelore, canulados no rúmen. As frações líquida e sólida do conteúdo ruminal foram processadas para extração de DNA metagenômico, após a extração foi verificado a quantidade e integridade das amostras. Em seguida, foi feita a PCR baseada nas regiões hipervariáveis V1 e V2 do 16S rRNA, e em sequência procedeu-se a construção da biblioteca e sequenciamento utilizando a plataforma Illumina. Os dados foram analisados pelos softwares MG-RAST e MOTHUR para filiações bacterianas. Aproximadamente 11.407.000 reads foram geradas com qualidade e 812 e 752 UTOs foram encontrados no nível de espécie e gênero respectivamente. Foram identificados 27 filos no conteúdo ruminal de bovinos Nelore através do sequenciamento do gene 16S rRNA pela plataforma Illumina. Os conhecimentos gerados a partir do presente estudo são informações primárias e primordiais para o entendimento da composição bacteriana ruminal. Assim, nos proporciona vislumbrar futuro promissor no desenvolvimento de novos métodos e tecnologias aplicáveis na nutrição animal
The rumen is an open ecosystem in which the food consumed by ruminant is fermented to yield short-chain fatty acids and microbial protein, that in turn serve as a source of energy and protein for the animal, respectively. Species of microorganisms into rumen have developed a series of complex interactions that become one of the best examples of symbiosis between microorganisms in nature. Conventional methods for microbial taxonomy based on culture techniques have been replaced by molecular techniques which are quickly and more accurate. The majority of phylogenetic molecular tools for bacteria classification are based on ribosomal 16S rRNA gene that provides resources for identification and also quantification of bacterial communities. In order to evaluate the ruminal bacterial diversity present in three Nelore cannulated cattle, the rumen content was fractionated in liquid and solid samples after its collect. Both parts were processed for metagenomic DNA extraction which in turn was evaluated according quantity and integrity parameters. Next stage consisted in PCR technique based on V1 and V2 hypervariable 16S rRNA regions to generate amplicons used for DNA sequencing performed at Illumina platform. Data were processed by MG-RAST and MOTHUR softwares to deduce bacterial affiliations. Approximately 11,407,000 reads were generated showing quality for indication of 812 and 752 OTUs at the level of species and genus, respectively. Twenty-seven phyla were successfully identified in Nellore ruminal contents by 16S rRNA sequencing in the powerful Illumina platform. The knowledge generated from this study are primary and essential for the wide understanding of rumen bacterial composition information. Thus, it provides us resources to be explored in a promising future focusing the development of new methods and technologies applied to animal nutrition

Etude des hydrolases de trois especes de phycomycetes anaerobies du rumen de mouton, neocallimastix frontalis, sphaeromonas communis et piromonas communis: caracterisation physicochimique, regulation de la synthese et de la secretion. Purification de la beta -glucosidase, de la beta -fucosidase et de la beta -xylosidase de n. Frontalis

Orientador: Telma Teresinha Berchielli
Coorientador: Giovani Fiorentini
Banca: Saulo da Luz e Silva
Banca: Otavio Rodrigues Machado Neto
Banca: Paulo Henrique Moura Dian
Banca: Juliana Duarte Messana
Resumo: O glicerol é um substrato utilizado por bactérias que metabolizam o lactato ruminal e a virginiamicina é um antibiótico não ionóforo que inibe o crescimento de bactérias gram-positivas produtoras de lactato. Foram realizados dois experimentos para avaliar os efeitos da glicerina bruta (GB) e da virginiamicina (VM) na ingestão, digestibilidade, fermentação ruminal, população microbiana, desempenho, características de carcaça e perfil de ácidos graxos da carne de bovinos Nelore. Os tratamentos experimentais foram organizados em um arranjo fatorial 2 × 2: dietas sem virginiamicina (VM-) ou 25 mg de virginiamicina/kg de matéria seca (VM+) combinadas com dietas sem glicerina bruta (GB-) ou 100 g de glicerina bruta/kg de matéria seca (GB+). O bagaço de cana-de-açúcar foi usado como forragem na proporção de 20% na matéria seca (MS) da dieta e a GB substituiu o milho na formulação da dieta. No primeiro experimento, foram utilizados oito bovinos Nelore fistulados no rúmen (Peso corporal = 600 ± 34 kg, 26 ± 3 meses) em um quadrado latino 4×4 replicado (período= 21 dias) para se avaliar a digestibilidade dos nutrientes, fermentação ruminal e população microbiana. A ingestão de MS teve uma tendência a aumentar em dietas com GB (P = 0,07). As digestibilidades aparentes totais dos nutrientes foram semelhantes entre as dietas (P ≥ 0,10). As dietas com GB ou VM apresentaram valores de pH similares (média = 6,15; P ≥ 0,10). A proporção de propionato aumentou 27,5% nas dietas com GB+, independ... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Glycerol is a substrate used for bacteria that metabolize ruminal lactate and virginiamicyn is a non-ionophore antibiotic that inhibits the growth of gram-positive lactate-producing bacteria. Two experiments were conducted to evaluate the effects of crude glycerin (CG) combined with virginiamycin (VM) on intake, digestibility, ruminal fermentation, microbial population, performance, carcass traits and fatty acid profile of meat from feedlot Nellore cattle. Treatments were arranged in 2 × 2 factorial design: diets without virginiamycin (VM-) or virginiamycin at 25 mg/kg DM (VM+) combined with diets without crude glycerin (CG-) or CG (80% glycerol) at 100 g/kg DM (CG+). The sugar cane bagasse was used as the exclusive roughage in the proportion of 20% in the dry matter (DM) of diet and crude glycerin replaced corn in the diet formulation. In the first experiment, eight rumen fistulated bulls (BW= 600 ± 34 kg; 26 ± 3 months) were used in a replicated 4 × 4 Latin square (21-d periods) to evaluate the digestibility, ruminal fermentation and microbial population. The intake of DM had a tendency to be greater in CG+ than CG- diets (P = 0.07). Apparent total tract digestibilities of nutrients were similar among diets (P ≥ 0.10). Diets with CG or VM had similar values of pH (mean=6.15; P ≥ 0.10). The proportion of propionate increased 27.5% in CG+ diets, regardless of VM inclusion (P = 0.01). In the second experiment, forty-eight bulls with initial BW (408.4 ± 22.2 kg; 21 ± 2 months) ... (Complete abstract click electronic access below)
Doutor

Les bactéries protéolytiques sont essentiellement du genre bactéroïdes rumincola, streptococcus bovis butyrivibrio fibrisolvens et clostridium bifermentans. Les enzymes protéolytiques synthétisées par clostridium bifermentans s 17/2 sont essentiellement des cystéines protéases. Une part de l'activité est due à des métallo et serine proteases. L'activite enzymatique est maximale entre ph 7 et 8,5. Elle se maintient à un niveau élevé (sup. 79%) sur le plage de ph assez large (ph 5 a 9). L'activité protéolytique est activée par le glucose et réprimée par les sources azotées (acides amines, peptides et ammoniac). Cette souche a une énergie de maintenance égale à 0,304 g de glucose/g de bactéries/h. Les taux de dégradation in sacco de l'arabinose et du galactose sont fortement corrélés à la disparition de l'azote pariétal. La disparition au préalable d'une partie de ces deux oses semble nécessaire à la dégradation de l'azote pariétal. Les cocultures in vitro des bactéries protéolytiques et cellulolytiques améliorent la dégradation de l'azote pariétal qui est maximale lors de la fermentation de la luzerne par ruminococcus albus associé à bactéroïdes ruminicola. Le galactose et l'arabinose provoquent une diminution de l'activité protéolytique de bactéroïdes ruminicola. L'activité protéolytique de butyrivibrio fibrisolvens est réduite en présence de concentrations croissantes en glucose, cellobiose, xylose, galactose et arabinose. Au contraire des concentrations croissantes en glucose et en cellobiose améliorent l'activité protéolytique de s. Bovis. Les acides phénoliques ont un effet inhibiteur

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Objetivou-se com este estudo caracterizar as alterações na microbiota ruminal, parâmetros ruminais, consumo, digestibilidade das dietas e a eficiência de síntese microbiana, em novilhos Nelore confinados alimentados com diferentes relações volumoso:concentrado, utilizando como fontes de volumoso o feno de Tifton 85 ou cana-de-açúcar. Foram realizados dois experimentos, no experimento 1 utilizou a cana-de-açúcar como fonte de volumoso e no experimento 2 utilizou-se o feno de tifton 85 como volumoso. Em ambos os experimentos foram testadas diferentes relações V:C (70:30; 60:40; 40:60 e 20:80). Em cada experimento, utilizaram-se oito novilhos Nelore (331±8 kg PV), cânulados no rumen, distribuídos em duplo quadrado latino 4x4 balanceados para o controle do efeito residual. No experimento 1 (cana-de-açúcar), o aumento da proporção de concentrado na dieta reduziu a população de Fibrobacter succinogenes e Ruminococus flavefaciens, e aumentou a população de Selenomonas ruminantium e Megasphaera elsdenii, porém o CDFDN não foi alterado. O aumento da participação de carboidratos não estruturais na dieta favoreceu a síntese de proteína microbiana e reduziu a população de bactérias fibrolíticas. A cana-de-açúcar como fonte de volumoso em dieta com proporções crescente de concentrado pode otimizar a síntese de proteína microbiana sem alterar digestibilidade da fibra. No experimento 2 (feno de Tifton 85), o aumento da proporção de concentrado na dieta reduziu a população de Fibrobacter succinogenes, Ruminococus flavefaciens e Ruminococcus albus e aumentou a população de Selenomonas ruminantium e Megasphaera elsdenii e Streptococcus bovis e o CDFDN diminuiu com o aumento da proporção de concentrado na dieta. Feno de Tifton 85 em dietas com altas proporções de concentrado pode minimizar o risco de distúrbios ruminais em novilhos confinados
This trial aimed to characterize the changes in ruminal microbiota, ruminal fermentation, intake, diet digestibility and microbial efficiency in Nellore steers fed with different forage:concentrate proportions, using as sources of forage Tifton 85 hay or sugar cane. Two experiments were conducted: in experiment 1 the sugar cane was used as forage source and in Experiment 2 the Tifton 85 hay was used as forage source. In both experiments were tested different F:C proportions (70:30, 60:40, 40:60 and 20:80). On each experiment were usedeight Nellore steers ( 331 ± 8 kg BW) cannulated in the rumen, in a double latin square 4x4 balanced to control the residual effect. In experiment 1 (sugar cane), increasing the proportion of concentrate in the diet reduced the population of Fibrobacter succinogenes and Ruminococus flavefaciens, and increased the population of Selenomonas ruminantium and Megasphaera elsdenii, but the digestibility of NDF has not changed. The increased participation of non-structural carbohydrates in the diet favored microbial protein synthesis and reduced the population of fibrolytic bacteria. The use of sugar cane as forage source associated with the increases of concentrate proportions in the diet can optimize microbial protein synthesis without change the fiber digestibility. In experiment 2 (Tifton 85 hay), increasing the proportion of concentrate in the diet reduced the population of Fibrobacter succinogenes, Ruminococcus albus and Ruminococus flavefaciens and increased the population of Selenomonas ruminantium, Megasphaera elsdenii and Streptococcus bovis and the digestibility of NDF decreased when increases the proportion of concentrate in the diet. Tifton 85 hay in diets with high proportions of concentrate can minimize the risk of ruminal disorders in feedlot steers

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Os microrganismos ruminais têm um papel central na nutrição de ruminantes. Eles têm a capacidade de fermentar componentes do alimento para produzir ácidos graxos voláteis (AGV’s) e crescer (sintetizar proteína microbiana), os quais fornecem a maior parte da energia e aminoácidos exigidos pelos animais. No entanto, quando são fornecidos carboidratos em excesso, a eficiência de crescimento dos microrganismos torna-se baixa porque estes direcionam a energia para outras funções, ao invés de a utilizarem para o crescimento. Diferentes microrganismos respondem a esse excesso de maneiras diferentes. Certas espécies respondem armazenando energia (sintetizando carboidratos de reserva), mas outras espécies respondem dissipando a energia na forma de calor. Para determinar a importância relativa dessas respostas na comunidade microbiana do rúmen, este estudo foi cinduzido com o objetivo de quantificar como os protozoários ciliados e as bactérias responderam à glicose. Teve-se como hipótese que os protozoários ciliados direcionariam mais glicose para a síntese de carboidratos de reserva e desperdiçariam menos energia na forma de calor, em relação as bactérias. Ciliados e bactérias foram isolados do líquido ruminal por filtração e centrifugação, respectivamente. Posteriormente, os ciliados e as bactérias foram suspensos em tampão isento de nitrogênio para limitar o crescimento e dosados com 5 mM de glicose. As amostras foram coletadas ao longo do tempo e, posteriormente, divididas por centrifugação em pellets (células) e sobrenadante. Amostras de pellets foram analisadas quanto à reserva de carboidratos e proteínas, enquanto amostras de sobrenadante foram analisadas para glicose livre, ácido D-L lático, ácido acético, propionato e butirato. Adicionalmente, foi analisado a produção de calor e gases de fermentação (H 2 , CH 4 e CO 2 ). O metabolismo endógeno, a síntese de carboidratos de reserva e o desperdício na forma de calor foram calculados a partir dos dados das análises. A maior parte dos dados foi analisada usando o PROC GLIMMIX do SAS. Teste t de Student foi usado para separar as médias ou determinar se as médias diferiam de 100%. Regressão local (pacote LOCFIT de R; Loader, 1999) foi usada para ajustar os dados das séries no tempo. Em comparação com as bactérias, os ciliados consumiram três vezes mais glicose e sintetizaram carboidratos de reserva quatro vezes mais rápido. Eles incorporaram 53% da glicose em carboidratos de reserva, quase o dobro do valor (27%) obtido para as bactérias. Desperdício de energia na forma de calor não foi detectado para os ciliados, uma vez que toda a produção de calor foi contabilizada pela síntese de reserva de carboidratos e pelo metabolismo endógeno. Em bactérias, a síntese de carboidratos de reserva e o metabolismo endógeno representaram apenas 68% da produção total de calor, assim, elas desperdiçaram grande quantidade de energia por meio da produção de calor (32% da produção total de calor). Esses resultados sugerem que os protozoários ciliados ruminais alteram o curso do metabolismo de carboidratos no rúmen, consumindo glicose mais rapidamente, limitando o uso do excesso de carboidratos pelas bactérias. Essa ação dos ciliados no rúmen provavelmente maximiza a síntese carboidratos de reserva, enquanto minimiza a ocorrência de desperdício de energia na forma de calor.
Rumen microbes hold a central role in ruminant nutrition. They ferment feed components to produce volatile fatty acids (VFA) and grow (synthesize microbial protein), which supplies the greater part of energy and amino acids required by the animals. However, when given excess carbohydrate, microbes growth efficiency becomes low because microbes direct energy to non-growth functions, instead of using it for growth. Different microorganisms respond to this excess in different ways. Certain species respond by storing energy (synthesizing reserve carbohydrate), but other species respond by dissipating the energy as heat (spilling energy). To determine the relative importance of these responses in the microbial community of the rumen, this study aims to quantify how mixed ciliate protozoa and bacteria respond to glucose. It was hypothesized that ciliate protozoa would direct more glucose to synthesis of reserve carbohydrate and less to energy spilling than would bacteria. Ciliates and bacteria were isolated from rumen fluid using filtration and centrifugation, respectively. Posteriorly, ciliates and bacteria were resuspended in nitrogen-free buffer to limit growth and dosed with 5 mM glucose. Samples were collected over time and were subsequently divided in pellet (cells) and supernatant by centrifugation. Pellet samples were analyzed for reserve carbohydrate and protein, while supernatant sample were analyzed for free glucose, D- /L-lactic acid, acetic acid, propionate and butyrate. Additionally, were analyzed heat production and fermentation gases (H 2 , CH 4 and CO 2 ). Endogenous metabolism, reserve carbohydrate synthesis and energy spilling were calculated from the data obtained from the analysis data. Most data were analyzed using PROC GLIMMIX of SAS. Student’s t- test was used to separate means or determine if means differed from 100%. Local regression (LOCFIT package of R; Loader, 1999) was used to fit time-series data to smooth curves. Compared to bacteria, ciliates consumed glucose more than 3-fold faster and synthesized reserve carbohydrate 4-fold faster. They incorporated 53% of glucose carbon into reserve carbohydrate, nearly double the value (27%) for bacteria. Energy spilling was not detected for ciliates, as all heat production was accounted by synthesis of reserve carbohydrate and endogenous metabolism. For bacteria, reserve carbohydrate and endogenous metabolism accounted for only 68% of heat production, thus they spilled large amounts of energy (32% of total heat production). These results suggest that rumen ciliates protozoa alter the course of carbohydrate metabolism in the rumen by consuming glucose more rapidly and outcompeting bacteria for excess carbohydrate. This action of the ciliates in the rumen likely maximizes reserve carbohydrate synthesis while minimizing spilling.

L'objectif de ce travail est de contribuer a l'etude de l'etiologie de la necrose du cortex (ncc) en determinant les effets de l'acidose ruminale et d'une quantite elevee de sulfate sur le metabolisme de la thiamine dans le rumen. La premiere partie du travail experimental a ete consacree a l'adaptation d'une technique de dosage hplc pour determiner les concentrations de thiamine et de ses esters phosphates dans le milieu ruminal. Dans une seconde partie, l'effet de l'acidose chronique et d'une dose elevee de soufre (sous forme de sulfate), en presence ou en l'absence de thiamine, a ete etudie in vitro a l'aide d'un fermenteur semi-continu de type rusitec et d'un aliment synthetique. Pour cela, la mise au point d'un modele d'acidose chronique en rusitec a ete prealablement realisee en diminuant la quantite des sels tampons apportes au fermenteur et en augmentant la proportion d'amidon dans l'aliment. La supplementation en thiamine n'a pas modifie l'activite fermentaire des micro-organismes du rumen. Elle a provoque une diminution de la synthese nette mais une augmentation de la production totale de thiamine. L'acidose chronique n'a pas diminue la production ruminale de thiamine ni augmente l'activite thiaminasique. Une dose de 0,5% de soufre dans l'aliment n'a pas modifie le metabolisme energetique et azote des micro-organismes du rumen mais a diminue la production de thiamine de 20%, sans entrainer d'augmentation de l'activite thiaminasique. Enfin, dans une derniere partie, nous avons montre, chez le mouton nourri avec un aliment semi-synthetique totalement depourvu de thiamine, qu'une dose de soufre de 0,6% ne modifie pas la concentration en thiamine et l'activite des thiaminases dans le rumen, l'activite de la transcetolase erythrocytaire et l'effet tpp. En revanche, cette dose peut provoquer des signes cliniques et des lesions de ncc qui sont, en fait, dus a l'effet toxique direct des sulfures provenant de la reduction des sulfates du rumen.

L'objectif de ce travail est de caractériser l'effet des levures vivantes Sc47 sur le métabolisme ruminal et l'utilisation digestive de la ration en lien avec le statut oxydo-réducteur du rumen chez des vaches laitières recevant des rations qui diffèrent dans leur composition. En effet, le milieu ruminal est très anaérobie et réducteur : les micro-organismes qui s'y développent sont à l'origine de ces conditions physico-chimiques particulières caractérisées par des valeurs de potentiel rédox (Eh) ou indice de Clark (rH) basses : respectivement dans cette étude entre –213 et –147 mV et entre 6.04 et 7.48 unités rH. L'impact de trois constituants du régime des vaches laitières d'une part sur le statut réducteur du milieu ruminal et d'autre part sur son métabolisme et/ou l'utilisation digestive de la ration a été testé en interaction avec l'addition de levures vivantes : un fourrage sec chez la vache tarie, deux concentrés azotés différant par le niveau de solubilité ruminal des protéines qu'il contient et enfin la nature de l'amidon, rapidement ou lentement dégradable dans le rumen chez la vache en lactation. Il apparaît nettement que le régime alimentaire ainsi que le niveau d'ingestion des animaux influencent directement le statut réducteur du rumen : un niveau réducteur bas étant favorable à l'activité d'une flore cellulolytique alors que des niveaux plus élevés s'avèrent être un indicateur de l'apparition de troubles métaboliques du rumen. En effet, le pouvoir réducteur ruminal est corrélé avec l'activité fermentaire qui s'y développe ainsi qu'à la structure des communautés bactériennes en présence. D'après nos résultats, l'effet de la levure vivante sur les conditions réductrices du rumen semble fortement conditionné par le niveau du statut réducteur induit par le régime alimentaire : les conditions réductrices ruminales peuvent être renforcées dans la mesure où le Eh initial se trouve, dans notre étude, entre –174 et –152 mV. Ce renforcement serait favorable à l'activité de la flore fibrolytique si celle-ci n'est pas dominante, ce groupe fonctionnel participant alors également au maintien des conditons réductrices. En outre, la levure vivante aurait un impact direct sur les bactéries protéolytiques favorisant la quantité de protéines "by pass" si bien que la valeur azotée de la ration peut être améliorée en particulier en termes de PDIA
The objective of this work is to characterize the effect of live yeast Sc47 on ruminal metabolism and ration digestibility in relation to ruminal redox status of dairy cows fed diets that differed in their composition. Indeed, the rumen environment is very anaerobic and very reducing : the inhabiting micro-organisms are the main sources of particular physic-chemical conditions characterized by low values of redox potential (Eh) or Clark’s exponent (rH): in this study, values were between –213 and –147 mV and between 6.04 and 7.48 units for Eh and rH, respectively. The impact of three constituting ingredients of the diet of dairy cows was investigated. On the one hand, the reducing status of rumen was observed and on the other hand, the metabolism and/or digestibility of the diet with or without addition of live yeast was explored. The tested constituents were: hay for dry dairy cows, two concentrates differing in nitrogen levels of ruminal solubility, and two energetic concentrates differing in rate of ruminal degradation of starch – quickly or slowly degradable in the rumen of lactating dairy cows. It clearly appeared that the diet and the level of dry matter intakes of animals directly influenced the reducing ruminal status: low levels being favorable to the activity of cellulolytic microflora whereas higher levels appeared to be indicators of metabolic disorders occuring in the rumen. Indeed, the reducing power was correlated to rumen fermentative activity and the structure of bacterial communities involved. According to our results, the effect of live yeast on rumen reducing conditions appeared strongly influenced by the level of the intrinsic reducing status induced by diet: reducing conditions in the rumen could be strengthened to the extent that the original values ranged between –174 and –152 mV. This reinforced ruminal conditions would uphold the activity of fibrolytic microflora if it is not dominant, this functional group then participating to the maintenance of reducing conditions. In addition, live yeast has a direct impact on proteolytic bacteria promoting by-pass proteins so that the protein value of the ration may be improved especially in terms of PDIA

L'acidose ruminale est l'une des préoccupations majeures des exploitations laitières actuelles. Les levures vivantes (LV) ont été largement étudiées et utilisées chez les vaches laitières pour stabiliser la fermentation ruminale. Récemment, la mesure du potentiel redox ruminal (Eh, en mV) a été considérée comme un outil intéressant pour indiquer le trouble de la fermentation ruminale. L'effet positif de LV sur Eh ruminal a été rapporté, mais il reste variable selon les conditions expérimentales. Les objectifs de ce travail étaient de fournir une meilleure compréhension du mode d'action de LV et de définir la condition optimale de l'utilisation de LV chez les vaches laitières. La première partie de ce travail a consisté en une analyse quantitative des résultats de 22 expériences avec des vaches laitières canulées. La deuxième partie de ce travail a consisté à vérifier certains des résultats de l'analyse quantitative par une expérience chez des vaches en lactation. En utilisant l'analyse quantitative de données existantes provenant d'expériences antérieures, nous avons clarifié la relation entre le Eh ruminal et d'autres paramètres ruminaux principaux tels que le pH et le profil VFA, et suggéré que les variations de Eh pourraient être liées au transfert d'électrons dans les réactions dans le rumen. En outre, la réponse du Eh après la supplémentation en LV était également liée à celle du profil AGV ruminal, suggérant que l'effet de LV sur le profil VFA était atteint par l'augmentation du pouvoir réducteur, reflétant un meilleur transfert d'électrons dans le rumen. L'analyse a en outre démontré que la régulation du Eh ruminal par LV serait particulièrement efficace lorsque le risque de troubles digestifs est élevé. Puisque l'influence des caractéristiques de la ration sur le Eh ruminal a été quantifiée, l'effet de LV dans un régime donné pourrait être estimé indirectement. En outre, l'analyse quantitative a également révélé que la réponse de Eh suite à la supplémentation en LV était associée à la quantité de sucres solubles ingérée. L'expérience in vivo chez des vaches en début de lactation a confirmé un effet plus important de LV sur Eh ruminal avec une ration riche en sucres solubles, et a démontré que la supplémentation en LV avait un impact sur la richesse des bactéries, et que les métabolites ont également été influencés par la supplémentation en LV, probablement associée à la diminution du Eh ruminal
Ruminal acidosis is one of the major concerns of current dairy farms. Live yeasts (LY) have been extensively studied and used in dairy cows for stabilization of rumen fermentation. Recently, measurement of ruminal redox potential (Eh, in mV) has been considered as an interesting tool to indicate ruminal fermentation disorder. The positive effect of LY on ruminal Eh has been reported, but it remains variable according to the experimental conditions. The aims of this work was to provide better understanding of mode of actions of LY, and to define the optimal condition of LY utilization in dairy cows. The first part of this work consisted to quantitative analysis of existing results from 22 experiments with cannulated dairy cattle. The second part of this work consisted to verify some of the results from quantitative analysis by an in vivo experiment in lactating cows. By using quantitative analysis of existing data from previously conducted experiments, we clarified the relationship between ruminal redox and other main ruminal parameters such as pH and VFA profile, and suggested that Eh variations might be related to the transfer of electrons in the reactions producing VFAs in the rumen. Moreover, response of ruminal Eh following live yeast supplementation was also related to that of ruminal VFA profile, which suggested that the effect of LY on VFA profile was achieved via the increase of reducing power, possibly reflected improved electron transfer and use in the rumen. The analysis further demonstrated that the regulation of ruminal Eh by LY would be particularly effective when risk of digestive disorder is high. Since the influence of dietary characteristics on ruminal Eh was quantified, the effect of LY in a given diet could be indirectly estimated. In addition, quantitative analysis also associated the response of ruminal Eh following LY supplementation to the intake of soluble sugars. The in vivo experiment in early-lactating cows confirmed greater effect of LY on ruminal Eh in diet rich in soluble sugars, and further demonstrated that i) LY supplementation tended to impact the richness of ruminal bacteria, and ii) some unidentified metabolites were also influenced by LY supplementation, probably associated to the decrease of ruminal Eh