Made available in DSpace on 2015-03-26T13:55:30Z (GMT). No. of bitstreams: 1 texto completo.pdf: 283092 bytes, checksum: 26a551be6ca2a30c3d887ce7b402e49f (MD5) Previous issue date: 2007-03-14 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The aim of this work was to evaluate the effects of supplementation with nitrogenous compounds on intake, digestibility, nitrogenous compounds balance, efficiency of microbial synthesis, and neutral detergent fiber (NDF) rumen dynamics in cattle fed low-quality forage. Five crossbred heifers, with average live weight of 191 kg and fitted with rumen canullae, were used. The animals were fed ad libitum with signal grass (Brachiaria decumbens Stapf.) hay, which had crude protein (CP) content of 4.86% of dry matter (DM). The five treatments were defined according to the level of CP in the diet (0, 2, 4, 6, and 8 percentile points above the CP level of the roughage). The supplement was a mixture of urea, ammonium sulfate, and albumin (4.5:0.5:1.0, respectively). The experiment was carried out according to a 5 x 5 Latin square design, with five experimental periods. The average CP levels in the diets were: 5.19, 7.11, 8.60, 11.67, and 13.02% in DM basis. The intakes of DM, organic matter (OM), NDF, and NDF corrected for ash and nitrogenous compounds (NDFom(n)) showed a quadratic profile according to CP levels in the diet (P<0.10) with maximum responses on 10.24%, 10.27%, 10.12%, and 10.05% of CP, respectively. The digestibility coefficients of DM and NDFom(n) were not affected by CP levels (P>0.10). The average concentration of rumen ammonia nitrogen (RAN) was linearly increased with CP levels (P<0.10). The RAN estimate associated with the maximum DM intake was 9.64 mg/dL. The microbial nitrogen flow in small intestine was linearly increased with the CP level in the diet (P<0.10). The efficiency of microbial synthesis was not affected by supplementation (P>0.10). The potentially degradable NDF was linearly increased (P<0.10) according to CP levels in diet until 6.97% CP. From this point, there was stabilization of estimates (47.87% of NDF). The degradation rate of potentially degradable NDF has been increased linearly with CP levels in diet (P<0.10). The rumen flow of fibrous particles (L) had a linear response-plateau according to CP levels in the diet (P<0.10). The plateau (maximum) for the estimates has begun on 7.24% of CP. The mean retention time in rumen-reticulum and the rumen fill effect of undegradable NDF was affect by CP levels similarly to (L) (P<0.10), with plateau (minimum) beginning on 6.90 and 6.97% of CP, respectively. / Objetivou-se avaliar os efeitos da suplementação com compostos nitrogenados sobre o consumo, a digestibilidade, o balanço dos compostos nitrogenados, a eficiência de síntese de proteína microbiana e sobre a dinâmica ruminal da fibra em detergente neutro (FDN) em bovinos alimentados com forragem de baixa qualidade. Foram utilizadas cinco novilhas mestiças Holandês x Zebu, com peso vivo médio inicial de 191 kg, fistuladas no rúmen. A alimentação volumosa basal dos animais foi constituída por feno de capim-braquiária (Brachiaria decumbens Stapf.) com 4,86% de proteína bruta (PB) com base na matéria seca (MS), fornecido ad libitum. Os cinco tratamentos avaliados foram definidos de acordo com o nível de suplementação protéica (0, 2, 4, 6 e 8 pontos percentuais acima do nível de PB da forragem). Como fonte de compostos nitrogenados empregou-se mistura de uréia, sulfato de amônia e albumina, nas proporções de 4,5:0,5:1,0, respectivamente. O experimento foi constituído de 5 períodos experimentais, segundo delineamento em quadrado latino 5 x 5. Os níveis médios de PB nas dietas foram de 5,19, 7,11, 8,60, 11,67 e 13,02%, com base na MS. Verificou-se efeito quadrático (P<0,10) dos níveis de PB na dieta sobre os consumos de MS, matéria orgânica (MO), FDN e FDN corrigida para cinzas e proteína (FDNcp) (kg/dia), com respostas máximas sobre os níveis de 10,24%, 10,27%, 10,12% e 10,05% de PB, respectivamente. Os coeficientes de digestibilidade da MS e da FDNcp não foram afetados pelos níveis de PB na dieta (P>0,10). A concentração média diária de nitrogênio amoniacal ruminal apresentou comportamento linear crescente (P<0,10) em função dos níveis de PB na dieta, com valor estimado de 9,64 mg/dL equivalente ao máximo consumo de MS. O fluxo de compostos nitrogenados microbianos no intestino delgado relacionou-se linear e positivamente (P<0,10) com os dos níveis de PB da dieta. Observou-se falta de relação (P>0,10) entre os níveis de PB e a eficiência de síntese de proteína microbiana. Verificou-se elevação linear (P<0,10) da fração potencialmente degradável da FDN até o nível de 6,97% de PB, com platô em 47,87% da FDN. A taxa de degradação da fração potencialmente degradável da FDN elevou-se linearmente (P<0,10) com os níveis de PB da dieta. O fluxo ruminal de partículas fibrosas (L) apresentou relação linear response-plateau (P<0,10), com estabelecimento do platô (máximo) em 7,24% de PB. O tempo médio de retenção no rúmen-retículo e a repleção ruminal da fração indegradável da FDN apresentaram comportamento semelhante a L (P<0,10), com ponto crítico para o início do platô (mínimo) em 6,90 e 6,97% de PB, respectivamente.

Le microbiote du rumen bovin est un écosystème très diversifié et efficace pour la dégradation de substrats complexes, notamment issus de la biomasse végétale. Composé majoritairement de microorganismes non cultivés, il constitue un réservoir très riche de nouvelles enzymes d’intérêt potentiel pour les biotechnologies industrielles, en particulier les bioraffineries et la bioremédiation. Dans le cadre de cette thèse, nous avons mis en œuvre une approche de criblage fonctionnel du métagenome ruminal pour accélérer la découverte d’enzymes de dégradation des lignocelluloses, mais aussi de divers polluants synthétiques. En particulier, de nouvelles estérases capables de dégrader un insecticide de la famille des carbamates, le fenobucarb, ainsi qu’un polyuréthane commercial, l’Impranil DLN, ont pu être identifiées. De plus, le développement d’une nouvelle stratégie de criblage d’oxydoréductases nous a permis d’isoler trois enzymes bactériennes originales, très polyspécifiques, ne requérant ni cuivre ni manganèse pour dégrader différents substrats polycycliques tels que des polluants majeurs de l’industrie textile, mais aussi des dérivés de lignine. Enfin, le criblage de deux banques issues d’enrichissements in vivo et in vitro du microbiome du rumen sur paille de blé a permis d’isoler des cocktails d’enzymes lignocellulolytiques au profil fonctionnel et d’origine taxonomique différents, constitués de glycoside-hydrolases, estérases et oxydoréductases. Quinze nouveaux modules CAZy, correspondant à des familles enzymatiques jamais caractérisées, ont été identifiés. L’ensemble de ces résultats met en lumière l’immense potentiel d’innovation biotechnologique contenu dans les écosystèmes microbiens, en particulier dans le microbiote du rumen bovin / Bovine rumen microbiota is a highly diverse and efficient ecosystem for the degradation of complex substrates, especially those issued from plant biomass. Predominantly composed of uncultivated microorganisms, it constitutes a rich reservoir of new enzymes of potential interest for industrial biotechnologies, especially biorefineries and bioremediation. As part of this thesis, we used the functional screening of the ruminal metagenome to increase the discovery of enzymes able to degrade lignocelluloses, as well as different synthetic pollutants. In particular, new esterases able to degrade a carbamate insecticide, fenoucarb, and a commercial polyurethane, Impranil DLN, have been identified. Moreover, the development of a new screening strategy for oxidoreductases allowed the isolation of three original bacterial enzymes that are very polyspecific, and do not need copper nor manganese to degrade different polycyclic substrates, like major pollutants of the textile industry, as well as lignin derivatives. Finally, the screening of two libraries from in vivo and in vitro enrichments of the ruminal microbiome on wheat straw allowed the isolation of lignocellulolytic enzymatic cocktails, with different functional profiles and taxonomical origins, comprising glycoside-hydrolases, esterases and oxidoreductases. Fifteen novel CAZy modules, related to enzymatic families never characterized, were identified. All these results highlight the vast potential of microbial ecosystems, in particular the bovine rumen microbiota, for biotechnological innovation

Orientador: Telma Teresinha Berchielli / Abstract: The use of rumen undegradable protein (RUP) sources in high-energy diets may be an alternative to increase the nitrogen use efficiency (NUE) in ruminants. In this study we had two main objectives: 1) to understand how the protein content of the diet and the rumen degradable protein (RDP) intake can affect urea recycling and its utilization. 2) how the protein content of the diet and the amino acid profile of the metabolizable protein (MP) can affect the efficiency of the use of amino acids (AA) in feedlot Nellore cattle receiving high-energy density diets. Thus, we evaluated the effects of different sources and protein levels in the diet of feedlot Nellore cattle receiving high-concentrate diets. We had two major hypotheses: 1) reducing dietary N associated with RUP sources can increase NUE by reducing ruminal NH3 concentration, urea production in the liver and urinary N excretion, while maintaining N available for microbial protein (MICP) synthesis through urea N recycling. 2) differences in AA use efficiency exists and the gross AA use efficiency is affected by dietary factors such as N sources and levels. These hypotheses were tested using six Nellore steers, cannulated in the rumen, duodenum and ileum with initial body weight (BW) of 354 ± 11.8 kg and 18 months of age. The animals were randomly assigned to receive each diet once over the 6 periods in a 6 × 6 Latin square design. Experimental diets consisted of 80% concentrate and 20% roughage (DM basis), where fresh chopp... (Complete abstract click electronic access below) / Resumo: O uso de fontes de proteína não degradável no rúmen (PNDR) em dietas de alta energia pode ser uma alternativa para aumentar a eficiência do uso de nitrogênio (ENU) em ruminantes. Neste estudo, tivemos dois objetivos principais: 1) compreender como o teor de proteína da dieta e a ingestão de proteína degradável no rúmen (PDR) podem afetar a reciclagem de uréia e sua utilização. 2) como o teor de proteína da dieta e o perfil de aminoácidos da proteína metabolizável (MP) podem afetar a eficiência do uso de aminoácidos (AA) em bovinos Nelore em confinamento recebendo dietas de alta densidade energética. Assim, avaliamos os efeitos de diferentes fontes e níveis proteína na dieta de bovinos Nelore confinados que recebendo dietas de alto concentrado. Tivemos duas hipóteses principais: 1) reduzir o teor de N na dieta associado às fontes de PNDR pode aumentar a ENU por uma redução na concentração de NH3 ruminal, produção de uréia no fígado e excreção urinária de N, enquanto mantém o N disponível para a síntese de proteína microbiana (MICP) através da reciclagem de ureia. 2) existem diferenças na eficiência do uso de AA e a eficiência bruta de uso de AA é afetada por fatores dietéticos, como fontes e níveis de N. Essas hipóteses foram testadas utilizando seis novilhos Nelore, canulados no rúmen, duodeno e íleo com peso corporal inicial (PC) de 354 ± 11,8 kg e 18 meses de idade. Os animais foram distribuídos aleatoriamente para receber cada dieta por uma vez ao longo dos 6 períodos, em ... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23934号 / 農博第2483号 / 新制||農||1089(附属図書館) / 学位論文||R4||N5369(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 廣岡 博之, 教授 松井 徹, 教授 吉田 天士 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

[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.

La biohydrogénation (BH) ruminale des acides gras polyinsaturés (AGPI) est à l'origine de la production d'intermédiaires trans retrouvés dans les productions de ruminants (essentiellement le lait). Il existe deux voies de BH produisant des acides gras (AG) trans qui auraient des propriétés différentes : les isomères t11 auraient des effets bénéfiques pour la santé des consommateurs et les isomères t10 seraient responsables d'une forte diminution du taux butyreux du lait, représentant une contrainte majeure pour les éleveurs. Dans des conditions physiologiques normales, la voie t11 est fortement majoritaire, par contre avec des rations à base d'ensilage de maïs, riches en concentrés et surtout si elles comprennent des suppléments lipidiques riches en AGPI, une déviation de la voie t11 à la voie t10 peut se produire avec une augmentation significative des isomères t10 au détriment des isomères t11. L'objectif de cette thèse est d'expliquer les modalités de cette déviation, afin de mieux la maîtriser en élevage. Nos travaux permettent de conclure que les facteurs alimentaires de maîtrise de la déviation de la voie t11 vers la voie t10, sont la teneur en amidon rapidement fermentescible et la teneur en c9,c12-C18:2. Lorsque la quantité de c9,c12-C18:2 présente dans le rumen est faible, même avec une ration riche en amidon et un pH bas dans le rumen, la déviation n'a pas lieu, la voie t11 suffisant à assurer l'hydrogénation des AGPI puisque dans ces conditions, la ∆9 isomérisation est elle aussi peu efficace à pH bas. En revanche, lorsqu'en plus de l'amidon, du c9,c12-C18:2 est ajouté dans la ration, la voie t11 devient insuffisante et c'est la voie t10 qui prend le relais. Le pyroséquençage 454 couplé à une régression multiple SPLS nous ont permis d'établir des corrélations entre les taxons identifiés et la proportion d'AG (t10 ou t11) dans le rumen. Il s'avère que les genres bactériens corrélés fortement et positivement aux AG t10 sont plus ou moins impliqués dans le métabolisme ruminal du lactate ainsi qu'au faible pH ruminal. Cependant, l'identification des taxons les plus corrélés aux AG t11 était moins précise, elle s'arrête à l'ordre des Clostridiales. Enfin, dans des conditions de déviation de la voie t11 à la voie t10, l'addition de vitamine E dans la ration des vaches n'a pas permis de restaurer un ratio déjà élevé. Ces résultats ont abouti à une meilleure compréhension de cette déviation et orientent vers une meilleure maîtrise en élevage. / Rumen biohydrogenation (BH) of polyunsaturated fatty acids (PUFA) is responsible of the production of trans intermediates found in ruminant products (mainly milk). There are two BH pathways leading to trans fatty acids (FA) with different biological properties: t11 isomers have beneficial effects for consumer's health and t10 isomers result in low milk fat content, representing a major constraint for farmers. In most conditions, t11 FA are the major trans FA, but in some conditions, especially with diets based on corn silage and including lipid supplements rich in PUFA, a shift from t11 to t10 pathway can occur with a significant increase of t10 isomers at the expense t11 isomers. The objective of this work was to explain modalities of this shift to better control it in animal production. Our results demonstrated that dietary factors responsible of the shift from t11 to t10 pathway are starch rapidly fermentable and c9, c12-C18:2 contents. When the amount of c9, c12-C18:2 present in the rumen is low, even though the diet is rich in starch and the pH is low in the rumen, the shift does not occur, t11 pathway being sufficient to ensure the hydrogenation of PUFA since in these conditions, the 9 isomerization is also poorly effective at low pH. However, when c9, c12-C18:2 is supplemented to the diet in addition to starch, t11 pathway becomes insufficient for FA BH, and t10 pathway becomes dominant. 454 pyrosequencing coupled to a multiple sPLS regression allowed us to establish correlations between some identified taxa and FA proportions (t10 or t11) in the rumen. It appears that bacterial genera that are strongly and positively correlated with t10 FA are more or less involved in the metabolism of ruminal lactate and also positively correlated with a low ruminal pH. However, identification of taxa correlated with t11 FA was less accurate, stopping at Clostridiales order. Finally, once the shift occurred, the subsequent addition of vitamin E was not able to counteract this process. These results lead to a better understanding of this shift to better control it in animal livestock.

Le veau nouveau-né possède un rumen peu développé et non fonctionnel. Au cours des premiers mois, les fonctions digestives s’établissent, avec l’implantation du microbiote composé majoritairement de bactéries, archées et protozoaires. Les objectifs de ce travail étaient doubles : i) caractériser et comprendre la séquence d’implantation taxonomique des microorganismes du rumen chez le veau par des techniques de biologie moléculaire et de dénombrement, ainsi que la séquence de mise en place des paramètres fermentaires (AGV et ammoniac) et des activités principales enzymatiques chez le veau en périodes pré- et post-sevrage, ii) étudier l’effet de l’addition de levures vivantes sur la mise en place de cet écosystème ruminal en périodes pré- et post-sevrage. D’une part, nos travaux ont permis de confirmer qu’à la naissance, le rumen chez le veau, est dépourvu de micro-organismes, d’AGV, d’activité xylanasique et amylasique, avec un pH proche de la neutralité et un Eh fortement positif. La colonisation du rumen se fait dès la naissance, pendant les 15 premiers jours de la vie de l’animal par un microbiote complexe prédominé par les bactéries (phyla Proteobacteria et Bacteroidetes) et comprenant aussi des archées (majoritairement Methanobrevibacter). En même temps, le Eh devient fortement négatif. Ces communautés entraînent la production de produits fermentaires grâce à leurs activités enzymatiques. Entre 15 jours et le sevrage, avec l’ingestion d’aliments solides, la composition du microbiote du rumen évolue pour se rapprocher de celle de ruminants adultes, sans atteindre pour autant la maturité en termes de densités et abondances relatives. A cette période, le phylum Bacteroidetes est majoritaire, avec le genre Prevotella. Après le sevrage de légers changements apparaissent sur certains paramètres fermentaires comme les AGV sans doute en raison d’une évolution du microbiote qui devient moins diversifié et plus adapté à la dégradation d’aliments solides. L’apparition, à partir de 90 jours, des protozoaires ciliés dans le rumen semble conditionnée par la présence d’animaux adultes à proximité. A 4 mois d’âge, l’écosystème ruminal tend à devenir proche de celui observé chez les animaux adultes en matière de paramètres fermentaires, activités enzymatiques et composition taxonomique du microbiote. D’autre part, nos travaux ont permis de conclure que, avant sevrage, une supplémentation en levures vivantes (Saccharomyces cerevisiae) diminue l’ingestion de concentrés, conduit à une apparition plus précoce de la communauté des protozoaires et une plus grande densité d’archées, mais a peu d'effets sur la densité et la diversité de la communauté bactérienne, à l'exception de variations d’abondances de quelques taxa mineurs. L’apport de levures entraîne une diminution de la protéolyse, une augmentation de la proportion d'acétate ruminal et une diminution de la proportion de propionate. Au cours de la période post-sevrage, les veaux supplémentés en levures consomment plus de foin et la densité en archées est plus importante alors qu’une réduction de la diversité et de la densité de la communauté bactérienne est observée, mais accompagnée d’une augmentation de l’abondance relative des bactéries dégradant l’amidon, les pectines, les protéines et majoritairement les parois cellulaires en fonction des substrats présents dans le rumen. Ces changements sont probablement à relier aux augmentations de l'activité xylanasique et de la proportion d'acétate. L’ensemble des résultats acquis dans ce travail de thèse a permis d’apporter un certain nombre de connaissances et une meilleure compréhension de la mise en place de l’écosystème ruminal chez le veau de race laitière en périodes pré- et post-sevrage, ainsi que quelques pistes pour orienter ou améliorer cette implantation pour une meilleure maîtrise de l’élevage des veaux d’élevage. / The newborn calf has a little and non-fonctional rumen. During the first months of life, digestive functions establish, in relationship with the colonization by a microbiota mainly composed of bacteria, archaea and protozoa. This study had two objectves: i) characterize and understand the sequence of establishment of ruminal microbiota in calves by molecular biology and counting techniques abd describe the appearance of fermentation parameters (VFA and ammonia) and enzyme activities during the pre- and post-weaning periods, ii) define the effect of yeast supplementation on the establishment of the ruminal ecosystem in pre-and post-weaning periods. On the one hand, our work confirmed that at birth, calf rumen is devoid of micro-organisms, AGV, xylanase and amylase activities, with a pH close to neutrality and a strongly positive Eh. From 2 to 15 days of age, the rumen is colonized by a complex microbiota dominated by bacteria (Proteobacteria and Bacteroidetes phyla) and also containing archaea (with mainly the Methanobrevibacter genus), and Eh becomes strongly negative. These communities result in the production of fermentation products due to their enzymatic activities. Between 15 days and weaning, with the ingestion of solid food, the composition of rumen microbiota changes to become closer to that of adult ruminants without reaching maturity in term of densities and relative abundances. At this time, the phylum Bacteroidetes is predominant with the Prevotella genus. After weaning, slight differences occurs on some fermentative parameters such as VFA, probably related to a change in the microbiota that becomes less diversified and more adapted to the degradation of solid food. From 90 days, the establishment of ciliated protozoa in the calf rumen seems conditioned by the proximity with adult animals. From 4 months of age, considering fermentation, enzymatic and taxonomic composition of the microbiota, the ruminal ecosystem tends to be similar to that observed in adult animals. On the other hand, our work showed that during the pre-weaning period, the supplementation with live yeast (Saccharomyces cerevisiae) results in a lower concentrate intake, an earlier establishment of ciliated protozoa and a higher archaeal community density, but poorly affects the density and diversity of the bacterial community, with the exception of changes in abundances of some minors taxa. Yeast supplementation reduces proteolysis, increases the proportion of acetate and decreases that of propionate. During the post-weaning period, yeast supplemented calves consumed more hay, had a higher archaeal density, but a lower diversity and density of the bacterial community with an increased relative abundance of fiber, protein, starch, pectine degrading bacteria compared to control according to the substrates present in the rumen. These changes are probably related to increases of the xylanolytic activity and the proportion of acetate. Taken together, results obtained in his thesis have improved knowledge and understanding of the establishment of the ruminal ecosystem in the dairy calf in pre- and post-weaning periods, and carried some possibilities to orientate or improve the ruminal establishment for better control of calves rearing.

Made available in DSpace on 2015-03-26T13:55:23Z (GMT). No. of bitstreams: 1 texto completo.pdf: 443287 bytes, checksum: 08ce9d8927b6aa909671e81fc2aba183 (MD5) Previous issue date: 2009-05-08 / The present study presents as speculation the possibility of existence of interactions between both chemical and physical characteristics of forage plants acting on performance of small ruminants. The literature presents evidences of individual actions of those factors, but there is a lack on informations in respect to interactive effects. The objective of our work was to evaluate possible variations on feed intake, on rumen fiber mass of goats in different moments of the day according to alteration on source of forage offered as well as the particle size of that feed. We analyzed the combinations obtained among three levels of quality, named as high, medium and low based on age of the plant which was considered as 35, 50 and 60 days for haymaking of coast-cross (Cynodon dactylon), chopped to obtain three particle size (small, medium and long) by using an experimental randomized design in a 3 x 3 factorial arrangement with 6 repeats. Animals were non-castrated male Saanen and Alpine kids averaging 191 ± 4 days of age and body mass of 24.68 ± 3.17 kg. Granulometric profile of feed, refusals and ruminal content was evaluated by use of sieve openings of 4.76 mm, 2.38 mm, 1.19 mm, 0.70 mm and 0.297 mm. Mass and composition of fiber in the rumen were kept unaltered throughout the day. Values of fresh ruminal content, of DM, of fiber, of physically effective fiber, and of indigestible material did not differ among the different combinations between quality and particle size studied, nor to the average turnover time of dry matter and of fiber in the rumen. Average time of retention for lignin varied according to the quality of forage offered, with lower values for the hay of low quality, which implies in higher rate of ruminal passage of lignin to compensate the intake of lignified material. The higher intake of dry matter and NDF with the use of hay of low quality occurred as particles of higher size were offered allowing for selection of feed. Conversely, higher intake of NDF and lignin were observed as kids were offered high and medium quality hay chopped to present particles of small size. The higher intake of physically effective fiber was associated to hay with large particle size independent of the quality of the forage. We concluded that composition and fiber mass on rumen content of goats is not altered throughout of day and that the particle size associated with the higher intake varies with the quality of fiber which in turn influence the average turnover time of lignin in the rumen. / O presente estudo apresenta como especulação a possibilidade da existência de interações entre as características químicas e físicas de plantas forrageiras atuando sobre o desempenho de pequenos ruminantes. A literatura apresenta evidências das ações individuais destes fatores, mas existe uma lacuna nas informações quando se busca seus efeitos interativos. Para implantar esta linha de pesquisa objetivou-se avaliar possíveis variações no consumo de alimento bem como variações da massa fibrosa no rúmen de caprinos, em diferentes horários, quando da alteração na qualidade da fonte forrageira fornecida, bem como do tamanho da partícula deste alimento. Analisamos as combinações obtidas entre três níveis de qualidade (alta, média e baixa, em função da idade, respectivamente de 35, 50 e 65 dias) de feno de coast-cross (Cynodon dactylon), desintegrado para obter três tamanhos de partículas (pequeno, médio e grande) em delineamento experimental inteiramente casualizado, em arranjo fatorial 3x3, com 6 repetições . Os animais utilizados eram cabritos machos, não-castrados, das raças Saanen e Alpina, com idade média de 191 ± 4 dias, e massa corporal de 24,68 ± 3,17 kg, . O perfil granulométrico dos alimentos, sobras e conteúdo ruminal foi avaliado com uso de peneiras com abertura de 4,76 mm; 2,38 mm; 1,19 mm; 0,70 mm e 0,297 mm. A massa e a composição de fibra no rúmen se mantiveram inalteradas ao longo do dia. Os valores de conteúdo ruminal fresco, de MS, de fibra, de fibra fisicamente efetiva e de material indigestível não diferiram entre as diferentes combinações entre qualidade e tamanho de partículas estudadas, tampouco para o tempo médio de renovação da matéria seca e da fibra no rúmen. O tempo médio de retenção da lignina variou com a qualidade da forragm oferecida, sendo menor para o feno de baixa qualidade, o que implica em maior taxa de passagem ruminal da lignina para compensar o consumo de material lignificado. O maior consumo de MS e FDN com o feno de baixa qualidade ocorreu quando da oferta de partículas de maior tamanho permitindo a seleção do alimento. Contrariamente, maiores consumos de FDN e lignina foram observados quando os cabritos recebiam o feno de alta e média qualidade em partículas de tamanho pequeno. O consumo maior de fibra fisicamente efetiva, para feno dos três níveis de qualidade esteve associado aos maiores tamanhos de partícula. Concluímos que a composição e a massa de fibra no conteúdo ruminal de caprinos não é alterada ao longo do dia; o tamanho de partícula associado ao maior consumo varia com a qualidade da forragem, e que por sua vez influencia o tempo médio de renovação da lignina no rúmen.

La biohydrogénation (BH) ruminale des acides gras polyinsaturés (AGPI) est à l’origine de la production d’AG trans pouvant se retrouver dans les productions de ruminants, dont le lait. Parmi ceux-ci, les isomères t11 auraient des effets bénéfiques pour la santé des consommateurs alors que les isomères t10 sont potentiellement défavorables. En élevage, l’apport de graines oléagineuses dans la ration des vaches permet d’augmenter la teneur du lait en ces acides gras. Or ces graines sont souvent distribuées chauffées pour améliorer leur valeur nutritionnelle. Expérimentalement, les effets des graines chauffées sur les teneurs en isomères t11 dans le rumen ou le lait sont variables, mais généralement elles permettent une augmentation des isomères t11 et une protection des AGPI. Au contraire, des huiles très oxydées par chauffage diminuent fortement les isomères t11 et augmentent parfois les isomères t10. Les produits d’oxydation des lipides générés pendant le chauffage des graines ou des huiles pourraient être incriminés. Les objectifs de la présente étude étaient d’explorer les éventuels effets des produits d’oxydation du c9,c12-C18:2 sur sa BH ruminale. In vitro, la protection des AGPI dans des graines de soja chauffées a été liée aux aldéhydes et en particulier à l’hexanal. L’augmentation des isomères t11 a été observée avec un aldéhyde long et insaturé, le t2,t4-décadiénal, suite à une inhibition de la dernière étape de la BH. Cet effet était concomitant à une modification marquée de la communauté bactérienne du rumen induite par cet aldéhyde. Les hydroperoxydes issus du c9,c12-C18:2 sont le 13HPOD et le 9HPOD. L’augmentation des isomères t10 a systématiquement été reliée au 13HPOD dans nos différentes expérimentations. Aucun mécanisme d’action n’a pu être formellement démontré mais l’effet du 13HPOD s’exercerait plutôt sur le microbiote, car il ne module pas l’activité de la Δ9-isomérase. Quant à la diminution des isomères t11 observée avec les huiles chauffées, elle n’a pas pu être expliquée. Elle pourrait également être liée, au moins partiellement, aux 13HPOD et 9HPOD, capables d’inhiber la Δ12-isomérase. Des modifications des fermentations ruminales, sans altération mesurable de l’abondance, de la diversité ou de la structure de la communauté bactérienne, suggèrent aussi un effet des AGPI chauffés sur l’activité des bactéries. / Ruminal biohydrogenation (BH) of polyunsaturated fatty acids (PUFA) produces some trans FA which can be found in ruminant products. Among them, t11 isomers would be beneficial for human health while t10 isomers are potentially deleterious. In farms, addition of oilseeds to the diet of dairy cows increases these fatty acids in milk. Oilseeds are often heated before incorporation to cow’s diets, to enhance their nutritional value. Experiments investigating effects on t11 isomers content in rumen and milk of cows receiving heated oilseeds provided variable results, but they usually increased t11 isomers and protected PUFA from BH. On the contrary, highly oxidized oil decrease t11 isomers and sometimes increase t10 isomers. Lipid oxidation products generated during heating of oilseeds and oils could be incriminated. The objectives of the present study were to investigate the effects of c9,c12-C18:2 oxidation products on its BH. Protection of PUFA in heated soybeans was linked to aldehydes, mainly hexanal. An increase of t11 isomers was observed with a long and unsaturated aldehyde, the t2,t4-decadienal, due to an inhibition of the last BH step. This effect was concomitant with a modification of bacterial community by t2,t4-decadienal. Hydroperoxides formed during c9,c12-C18:2 heating are 13HPOD and 9HPOD. The increase of t10 isomers in all of our experimentations was systematically linked with 13HPOD. No definitive explanation about the mechanism of action could be proposed, but 13HPOD would most probably act on microbiote since it had no effect on Δ9-isomerase. Our experiments did not explain the decrease of t11 isomers observed with heated oils, which could, at least in part, be due to 13HPOD and 9HPOD, which were able to inhibit Δ12-isomerase Some modifications of ruminal fermentation without measurable alteration of bacterial community abundance, diversity or structure also suggest an action of heated PUFA on bacterial activity.

Introduction: Microbial fermentation of carbohydrates in forestomach of ruminants produces large amounts of short-chain fatty acids (SCFA, mainly acetic acid, propionic acid, and n-butyric acid). The majority of these substrates is taken up directly across the ruminal wall. After luminal uptake into the epithelial cells, SCFA mainly occur in the dissociated form due to the intracellular pH of ~7.4. Moreover, a big portion of SCFA is metabolised within the cytosol. Main end products of epithelial SCFA metabolism are ketone bodies (D-3-hydroxybutyric acid and acetoacetic acid) and lactic acid. Both intact SCFA and ketone bodies and lactate need to be efficiently extruded from the ruminal epithelial cells to prevent a lethal drop of intracellular pH and counteract osmotic load of the cytosol. All these substances are less lipophilic in comparison to the undissociated form of SCFA. Thus, dissociated SCFA (SCFA-) and their metabolites need Protein mediated mechanisms for the extrusion across the basolateral side of ruminal epithelium. One mechanism suggested to be involved in the extrusion of SCFA- across basolateral membrane of the ruminal epithelium is the monocarboxylate transporter 1 (MCT1). Functionally, MCT1 was first assumed to operate as proton-coupled transporter for monocarboxylates including SCFA. Nonetheless, a recent study found a bicarbonate dependent anion exchange mechanism which turned out to be sensitive to MCT1 Inhibitors at the basolateral side of the ruminal epithelium pointing to the ability of MCT1 to act as an anion exchanger. However, in these experiments the inhibition of MCT1 abolished bicarbonate dependent transport only by half. This suggests the involvement of further anion exchanger(s) in the transport of SCFA across the basolateral membrane of ruminal epithelium. Promising candidates to underlie this exchange are the putative Anion exchanger 1 (PAT1) and a transport protein designated „down-regulated in adenoma“ (DRA). Materials and Methods: Sheep rumen epithelium was mounted in Ussing Chambers under short-circuit conditions. Radioactively labelled acetate (ac) was added to the serosal side. Serosal to mucosal flux of ac (Jsm ac) was measured with or without anion Exchange inhibitors (50 mM NO3- or 1 mM DIDS) or the MCT1 inhibitor p-hydroxy mercuribenzoic acid (pHMB; 1.5 mM) in the serosal buffer solution. The inhibitors were added alone or in combination with each other. Furthermore, mucosal to serosal flux of radioactivelly labelled ac or butyrate (bu) (Jms ac, bu) was measured in the presence or absence of SO42-, Cl- or NO3- (50 mM respectively) as exchange substrate in the serosal buffer solution. Immunohistochemical staining was conducted to locate PAT1 and DRA by use of commercially available antibodies. Results: NO3- and pHMB significantly reduced Jsm ac by 57 % and 51 %, respectively. When pHMB was applied after pre-incubation with NO3- an additional inhibition of Jsm ac was observed. Vice versa, NO3- further inhibited Jsm ac when epithelia were pre-incubated with pHMB before. DIDS had no inhibitory effect on SCFA flux. Serosal presence of SO42- or Cl- enhanced Jms ac significantly. Regarding bu, Cl- or SO4 2- also enhanced Jms bu significantly. The different anions available in the serosal buffer solution numerically enhanced Jms in the order of SO4 2- > Cl- for both ac and bu, which corresponds to the known affinity sequence of PAT1 and DRA. Immunohistochemistry revealed localization of PAT 1 in the stratum basale, whereas DRA was not detectable using this method. Conclusions: Basically, this study supports the suggestion that MCT1 works as an Anion exchanger in ruminal epithelium. In addition, it clearly shows that there is at least one further anion exchanger involved in the basolateral extrusion of SCFA and their metabolites. The functional and immunohistochemical findings suggest that PAT1 holds a significant role in this respect.:1 Introduction 1 2 Literature Review 3 2.1 Importance of short-chain fatty acid production of ruminants 3 2.2 Apical uptake of short-chain fatty acids from the rumen 5 2.2.1 Apical uptake of undissociated SCFA from the rumen 6 2.2.2 Apical uptake of dissociated fatty acids from the rumen 8 2.3 Intraepithelial metabolism of short-chain fatty acids 9 2.4 Mechanisms for the basolateral discharge of the short-chain fatty acids 11 2.4.1 Basolateral extrusion of short-chain fatty acids in other gastrointestinal tract epithelia 12 2.4.2 Basolateral extrusion of short-chain fatty acids in ruminal epithelium 14 2.4.3 Further candidate proteins for extrusion of SCFA- in exchange for HCO3 - 19 2.4.3.1 Putative Anion transporter 1 (PAT1 = SLC26A6) 19 2.4.3.2 Down-regulated in adenoma (DRA = SLC26A3) 21 2.4.3.3 Anion exchanger 2 (AE2 = SLC4A2) 22 2.5 Literature implications for this study 23 3 Materials and Methods 24 3.1 Animals 24 3.2 Ussing chamber studies 24 3.2.1 Buffer solutions 24 3.2.2 Preparation of ruminal epithelium 25 3.2.3 Incubation 25 3.2.4 Electrophysiological parameters 26 3.3 Experimental procedure 27 3.3.1 Determination of the unidirectional SCFA flux rate 29 3.4 Experimental Setups 30 3.4.1 Sensitivity of Jsm ac to inhibitors 30 3.4.1.1 Effect of nitrate and pHMB on Jsm ac 30 3.4.1.2 Effect of DIDS, NO3 - and pHMB on Jsm ac 31 3.4.2 Effect of the basolateral replacement of the anions on the extrusion of SCFA 32 3.4.2.1 Effect of Cl- and NO3 - on Jms of acetate and butyrate 32 3.4.2.2 Effect of SO4 2- on Jms of acetate and butyrate 32 3.4.3 Effect of different anions available in the serosal solution on Jms of acetate and butyrate 33 3.5 Immunohistochemistry 34 3.5.1 Preparation of the samples. 34 3.5.2 Fixation and staining of the samples. 34 3.5.3 Evaluation 35 3.6 Statistical analysis 36 4 Results 37 4.1 Inhibitors sensitivity 37 4.1.1 Effect of nitrate and pHMB on Jsm ac 37 4.1.2 Effect of DIDS, pHMB and NO3 - on Jsm ac 41 4.2 Effect of Cl- and NO3 - on Jms of acetate and butyrate 43 4.2.1 Effect of SO4 2- on Jms of acetate and butyrate 44 4.3 Effect of Cl-, NO3 - or SO4 2- when present in the serosal solution for 150 min 49 4.4 Immunohistochemistry 52 5 Discussion 54 5.1Ussing chamber experiments 56 5.1.1 Effect of Cl- and NO3 - on Jms of acetate 56 5.1.2 Effect of nitrate and pHMB on Jsm of acetate 57 5.1.3 Effect of DIDS, pHMB or NO3 - on Jsm of acetat 58 5.1.4 Effect of SO4 2- on Jms of acetate 59 5.1.5 Comparison between different anions as exchange substrate for the basolateral extrusion of acetate 60 5.2 Immunohistochemistry 62 5.3 Comparison between basolateral extrusion of butyrate and acetate 62 5.4 Conclusions 64 6 Summary 66 7 Zusammenfassung 68 8 References 70 Ac Aknowledgements