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

La présente étude visait à déterminer les effets de l'âge de sevrage sur les performances de croissance, le développement du rumen et le microbiote chez les chevreaux alpins. Soixante-douze chevreaux ont été assignés au hasard par paires mâle et femelle à l'un des trois traitements. 1) sevrage précoce (EW), à l'âge de 6 semaines, 2) sevrage moyen (MW), à l'âge de 8 semaines (MW) et 3) sevrage tardif (LW), à 10 semaines d’âge (LW). Le lait de remplacement a été proposé ad libitum jusqu'à la semaine de sevrage, où le lait a été réduit de 12.5 % par jour pendant sept jours. Deux semaines après la naissance, du concentré, du foin et de l'eau ont été offerts à volonté jusqu'à l'âge de 12 semaines où les chevreaux ont été abattus. La consommation a été enregistrée quotidiennement et le poids corporel (PC) a été enregistré chaque semaine. Pour évaluer le développement du rumen, des échantillons de sang ont été prélevés pendant tout l'essai et analysés pour le β-hydroxybutyrate sanguin (BHB) et les acides gras non estérifiés (NEFA). Les mesures ruminales ont été prises à la semaine 12 avec les trente-six mâles seulement. Le contenu du rumen a été obtenu pour l'analyse de la composition bactérienne en utilisant la région V4 du gène de l'ARNr 16S et la qPCR a été utilisée pour quantifier les bactéries, les protozoaires et les champignons. Les papilles du rumen ont été analysées dans 4 régions du rumen : atrium du rumen (RA), sac ventral (VS), Cul-de-sac caudo-dorsal (DS) et Cul-de-sac caudo-ventral (VBS). Les chevreaux sevrés à 10 semaines, contre 8 semaines et 6 semaines avaient un gain quotidien moyen plus élevé pour la semaine post-sevrage (0.35 vs. 0.24 vs. 0.24 kg/j) et étaient plus lourds à la 12e semaine (27.44 vs. 25.45 vs. 24.07 kg, P< .05). Chez les animaux LW, le PC n'a pas été affecté pendant la période post-sevrage; probablement en raison d'un apport élevé en énergie métabolisable (EM) causé par la stratégie de sevrage contrairement à ce que nous observons chez les animaux EW et MW. Les taux sanguins de BHB ont augmenté au moment du sevrage pour tous les traitements, mais étaient plus élevés chez les chevreaux EW par rapport aux chevreaux MW (+ 21 %) et LW (+ 41 %) (P< .05). Les taux sanguins de NEFA chez les chevreaux EW ont augmenté au sevrage et étaient plus élevés que MW (+ 40 %) et LW (+ 101 %) chez les chevreaux (P< .05), suggérant une mobilisation plus prononcée du tissu adipeux chez les chevreaux sevrés précocement au sevrage. La population de microbiote en post-sevrage a montré que si le sevrage était retardé, l'abondance des bactéries totales semblait augmenter (P< .05) par rapport aux animaux EW, tandis que les protozoaires et les champignons diminuaient pour les LW. L'âge du sevrage semble induire des modifications du microbiote ruminal au cours du post-sevrage. Les mesures d'absorptiométrie à rayons X à double énergie (DEXA) des carcasses ont été utilisées comme indication de la récupération de la composition des graisses après le sevrage. Le tissu adipeux (%) dans LW était (9.4%) et EW était (7.2%) plus élevé par rapport à MW (P< .05). La surface totale de la papille était plus grande chez les chevreaux LW, par rapport aux chevreaux MW (+ 49 %) et EW (+ 22 %) (P< .05). Globalement, le sevrage des chevreaux à l'âge de 10 semaines a limité l'impact négatif d'un sevrage précoce sur la croissance et le développement du rumen de la race alpine. / The present study aimed to determine the effects of weaning age on growth performance, rumen development and microbiota composition in Alpine goat kids. Seventy-two kids were randomly assigned in pairs male and female to one of three treatments. 1) early weaning, at 6 wk of age (EW), 2) medium weaning, at 8 wk of age (MW) and 3) late weaning, at 10 wk of age (LW). Milk replacer (MR) was offered ad libitum until the step-down wk, when milk was reduced by 12.5% per day for seven days. Two wk after birth, starter ration, hay, and water were offered ad libitum until 12 wk of age where kids were slaughtered. Feed intake was recorded daily, and body weight (BW) was recorded weekly. To evaluate rumen development, blood samples were taken during the whole trial and analyzed for blood β-hydroxybutyrate (BHB) and non-esterified fatty acids (NEFA). Ruminal measurements such as papillae, VFAs and microbiota were taken at wk 12 with only thirty-six males. Rumen contents were obtained for bacteria composition analysis using the V4 region of the 16S rRNA gene and qPCR was used to quantify bacteria, protozoa and fungi. Rumen papillae were obtained for histological analysis in 4 rumen areas: rumen atrium (RA), ventral sac (VS), caudodorsal blind sac (DS), and caudoventral blind sac (VBS). Kids weaned at 10 wk, compared with 8 wk and 6wk had higher average daily gain for the wk postweaning (0.35 vs. 0.24 vs. 0.24 kg / d) and were heavier at wk 12 (27.44 vs. 25.45 vs. 24.07 kg, P< .05). In LW animals, BW was not affected during post weaning period; possibly due to high metabolizable energy (ME) intake caused by the weaning strategy contrary to what was observed in EW and MW animals. Blood levels of BHB increased at weaning time for all treatments but were higher in EW compared to MW (+21%) and LW (+41%) kids (P< .05). Blood levels of NEFAs in EW kids spiked at weaning and were higher than MW (+ 40%) and LW (+101%) in kids (P< .05), suggesting a more pronounced adipose tissue mobilization in early weaned kids at weaning. Microbiota population in postweaning showed that while weaning was delayed, abundance of total bacteria seemed to increase (P< .05) compared with EW animals, whereas protozoa and fungi decreased for LW. Weaning age influences the ruminal microbiota during postweaning. Dual-energy X-ray absorptiometry (DEXA) measurements of carcasses were used as an indication of fat composition recovery in post-weaning. Fat tissue (%) in LW was (9.4%), and EW was (7.2%) higher compared to MW (P< .05). The total papilla surface area was greater in LW, compared to MW (+49%) and EW (+22%) kids (P< .05). Overall weaning kids at 10 wk of age limited the negative impact of earlier weaning on growth and rumen development in the Alpine breed.

Call number: LD2668 .T4 1984 S876 / Master of Science

No description available.

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).

Call number: LD2668 .T4 1986 T39 / Master of Science / Animal Sciences and Industry

Objetivou-se avaliar o uso de nitrato de cálcio (NC) na alimentação de ruminantes com vistas à mitigação da emissão de metano. Foram utilizadas quatro fêmeas de cada subespécie, Bos taurus taurus (Holandês) e vBos taurus indicus (Nelore), com peso médio inicial de 909,0 kg &#177; 72,0 e 387,0 kg &#177; 25,5, respectivamente, alimentadas com diferentes níveis de nitrato de cálcio na dieta (0%, 1%, 2% e 3%), sendo utilizado delineamento quadrado latino 4X4 replicado. O experimento teve duração de 4 períodos de 28 dias cada. Foram avaliados, a ingestão, excreção e digestibilidade de nutrientes, por meio de marcador externo dióxido de titânio, o comportamento ingestivo, com monitoramento visual por 24 horas, parâmetros sanguíneos, dinâmica ruminal, com esvaziamento total do rumen, contagem de protozoários, fermentação ruminal, pela técnica ex-situ, parâmetros urinários e balanço de nitrogênio, com coleta manual de urina tipo spot, e a biodigestão dos dejetos, por biodigestores do tipo batelada. Os taurinos apresentaram valores superiores para CMS, ingestão e digestibilidade de nutrientes e excreção de MS, PB, FDN, FDA, N, MO e EB, e inferiores para CMS em relação ao PV e PM. Os zebuínos apresentaram maiores taxas de ingestão, ruminação e mastigação de MS e FDN em min/kg, tendo valores inferiores para as mesmas taxas em kg/min. A genética e a dose de nitrato influenciaram as variáveis séricas, porém sem valores fora da normalidade. Quanto à dinâmica ruminal, os taurinos apresentaram valores absolutos superiores, e valores em relação ao PV e PM inferiores. Os zebuínos apresentaram maiores pH médio e mínimo e menos tempo de pH abaixo de 6,2, e maior contagem de protozoários. As concentrações de N-NH3, volume e produção de CH4 foram maiores para os zebuínos. Os taurinos apresentaram valores mais elevados para N microbiano (g/dia) e balanço de N retido (g/kg N ingerido), e menores valores para balanço de N nas fezes (g/kgN ingerido) e N microbiano (mg/kgPM.d-1). O aumento no nível de NC na dieta aumentou a digestibilidade de EE e ENN, tempo médio ruminando e taxa de mastigação de MS e FDN (min/kg), e diminuiu o CMS em relação ao PV e PM e excreção de MS, EE, ENN, MO e EB. A taxa de ingestão de MS e FDN (kg/min), a concentração de creatinina na urina (mg/kgPV), o tempo total comendo e balanço de nitrogênio amoniacal o rúmen sofreram efeito quadrático com a adição de NC na dieta. A porcentagem de FDN, PB e EE nos afluentes dos bidigestores sofreram influência da dose de nitrato na alimentação das vacas. Os biodigestores contendo os dejetos de taurinos tiveram os afluentes com maiores valores de FDN e FDA e menor teor de sólidos totais (ST), com menor volume de biogás e CH4 e menor relação de CH4/quantidade de fezes. Os taurinos apresentaram melhor aproveitamento do alimento, produzindo menos CH4, com menor rendimento verdadeiro de produção de metano nos biodigestores. A adição de NC afetou o consumo, digestibilidade e utilização do nitrogênio porém, sem afetar a produção de CH4 nem a eficiencia dos biodigestores. / The aim of the present study was evaluate the use of calcium nitrate in the feeding of ruminants to mitigate of methane. Four females cattle from each subspecies of cattle, Bos taurus taurus (Holstein) and Bos taurus indicus (Nellore), with initial average weight of 909,0 kg &#177; 72,0 e 387,0 kg &#177; 25,5, respectively, were used, and they were fed with distinct levels of calcium nitrate in the diet (0%, 1%, 2% e 3%) at four periods of 28 days each. Experimental design was composed by a replicated Latin square 4X4. Were avaluated: ingestion, excretion and digestibility of nutrients, by means of external marker titanium dioxide; the observation of behavior parameters during 24 hours; serum analysis; rumen content collection, aiming assessment of fermentation; protozoa count; ruminal fermentation by the ex-situ technique; manual urine collection for measurement of urinary compounds and nitrogen balance; and the anaerobic digestion of feces using batch digesters. Holsteins presented higher values for the DMI, ingestion and digestibility of the nutrients and excretion of DM, CP, NDF, ADF, N, OM e GE, and lower values for DMI at body weight (BW) and metabolic weight (MW) basis. Nellore cattle showed higher rates of ingestion, rumination and chewing of DM e NDF min/kg, having lower values for the same rates in kg/min. Serum variables were influenced by genetics and nitrate levels, but without values beyond normality. As rumen dynamics, Holsteins presented superior absolute values and inferior values when these parameters were calculated in BW and MW basis. The zebu cattle presented medium and minimum pH larger and less time pH below 6.2, having larger count for protozoa count. Nellore had higher concentrations for NH3, CH4 and production of CH4. Holstein presented higher values for microbial N (g/day) and N retained balance (g/kg N ingested) and lower values for N balance in feces (g/kgN ingested) and microbial N (mg/kgMW.d-1). The increase in the level of CN in the diet increased linearly the digestibility of EE and NNE, average time ruminating, chewing rate for DM and NDF (min/kg), and decreased the DMI in BW and MW basis and the excretion of DM, EE, NNE, OM and GE. The intake rate of DM and NDF kg/min, creatinine in urine (mg/kgBW), the total time eating and NH3 balance showed quadratic effect by increasing the addition of CN in the diet. The levels of nitrate on animal feed influenced the percentage of NDF, CP and EE in the batch digesters afluents. The biodigestors containing the taurine feces presented the highest values of NDF/ ADF affluent and lowest values for total solids (TS), lower volume of biogas and CH4 and lower ratio of CH4/amount of feces. Taurines presented better use of the feed, producing lesser CH4, and lower ultimate methane yield at batch digesters. The CN levels affected the intake, digestibility and nitrogen metabolism, however without affecting the production of CH4 or the batch digester efficiency.

Foram desenvolvidos cinco experimentos com o objetivo de avaliar o efeito do fornecimento de óleos essenciais sobre a fermentação ruminal e a digestibilidade dos nutrientes em borregos. No experimento I, avaliou-se um produto comercial tendo como princípio ativo óleo essencial de pimenta (Activo&reg;), os tratamentos experimentais foram: controle negativo ração base (30% de volumoso e 70% de concentrado, % da MS); controle positivo - ração base adicionada com 25 mg de monensina sódica por kg de matéria natural e ração base adicionada com 100, 200 ou 300 ppm de Activo&reg; (%MS). Nos quatro experimentos restantes, além dos tratamentos controle negativo e controle positivo descritos para o primeiro experimento, avaliou-se também a inclusão na dieta de 1,09; 2,18 ou 3,27 mL/kg de matéria seca (MS) de óleo essencial extraído do capim cidreira, Cymbopogon citratus (Experimento II); Óleo essencial extraído do fruto da aroeira-vermelha, Schinus terebinthifolius (Experimento III); Óleo essencial extraído do capim limão, Cymbopogon flexuosus (Experimento IV) ou óleo essencial extraído das folhas da aroeira vermelha; Schinus terebinthifolius (Experimento V). No experimento I, não houve efeito dos tratamentos sobre a concentração ruminal de ácidos graxos de cadeia curta (AGCC). No entanto, quando comparados ao controle positivo, o fornecimento de Activo&reg; (%MS) aumentou o consumo de MS e reduziu a digestibilidade dos nutrientes. No experimento II, não houve efeito dos tratamentos sobre as características de fermentação ruminal, contudo, a digestibilidade da PB foi maior nas dietas contendo monensina sódica ou óleo essencial de capim cidreira em comparação ao tratamento controle. Nos experimentos III, IV e V, não observou-se efeito dos tratamentos sobre nenhuma das variáveis estudadas. Desta forma, os resultados mais promissores foram observados para o experimento com capim cidreira, devido ao benéfico observado sobre a digestibilidade da proteína bruta. / Five experiments were developed in order to evaluate the effect of providing essential oils on ruminal fermentation and nutrient digestibility in lambs. In the first experiment (Experiment I) it was evaluated a commercial product having as active principle pepper essential oil (Activo&reg;), the experimental treatments were: negative control - basal diet (30% roughage and 70% concentrate,% DM); positive control - basal diet plus 25 mg monensin per kg of natural matter (NM) and basal diet plus 100, 200 or 300 ppm Activo&reg; (% DM). The remaining four experiments, besides of the treatments negative and positive control described in the first experiment, were also evaluated the inclusion in the diet 1.09; 2.18 or 3.27 mL/kg dry matter of essential oil extracted from West Indian lemongrass, Cymbopogon citratus (Experiment II); Essential oil extracted from the fruit of the Brazilian peppertree; Schinus terebinthifolius (Experiment III); essential oil extracted from Lemon grass; Cymbopogon flexuosus (Experiment IV) or essential oil extracted from the leaves of the Brazilian peppertree; Schinus terebinthifolius (Experiment V). In the first experiment, there was no effect of treatments on ruminal concentration of short-chain fatty acids (SCFA). However, when compared to the positive control, the supply of Activo&reg; increased dry matter intake (DMI) and reduced nutrient digestibility. In the second experiment, there was no effect of treatments on ruminal fermentation characteristics, however, the digestibility of CP was higher in diets containing monensin or essential oil of lemon grass compared to the control treatment. In the experiments III, IV and V, there was no effect of treatments on any of the variables studied. Thereby, the most promising results were observed for the experiment with essential oil of lemon grass, because of the beneficial on the digestibility of crude protein.

Made available in DSpace on 2016-08-10T10:44:16Z (GMT). No. of bitstreams: 1 PATRICIA RABELO DE FREITAS.pdf: 2747031 bytes, checksum: 9bc40ace4655abf6757b02c63ae2a6e0 (MD5) Previous issue date: 2012-05-15 / Avaliou-se o efeito de uma solução de amilase produzida por Aspergillus awamori sobre a digestibilidade in vitro da matéria seca (DIVMS) de milho. Foram realizados dois experimentos, onde o primeiro a solução de enzima amilase foi aplicada por pulverização em 24g de milho moído (2 mm) e o segundo a solução de enzima amilase foi aplicado no fluido ruminal. Os tratamentos foram: controle (0 enzima), T1 (5Ml de enzima) e T2 (10Ml de enzima) para cada experimento. O ensaio da DIVMS foi obtido usando a técnica de rúmens artificiais adapatada durante os períodos de 15 ; 1,30 , 3, 6, 12 e 24 horas. Para a coleta de líquido ruminal foi utilizado um bovino de peso aproximado de 380 kg. O animal foi mantido em baia e adaptado a dieta durante um período de 10 dias antes do recolhimento do líquido ruminal com acesso livre à água e sal mineral. Para os dois experimentos foi adotado o delineamento inteiramente casualizado, em esquema de parcelas subdivididas 3 x 6, com quatro repetições (jarros). As parcelas foram constituídas por milho tratado com três diferentes níveis de enzima e as subparcelas por seis momentos de digestão. Para enzima amilase aplicada no líquido ruminal o resultado de DIVMS para os três tratamentos nos períodos de 3, 6 e 12 horas não diferiram estatisticamente entre si. Entre o tratamento controle e T1 houve diferença significativa nos tempos 15 e 1,30 horas. Foi observado maior DIVMS para o tratamento controle, em relação ao T1, com valores de 54,54% e 49,05 , e não houve diferença nos tempos 3, 6, 12 e 24 horas. Entre o tratamento controle e T2 não houve diferença no tempo 15 e 24 horas. O controle foi superior a T2 28,74% e 10,53%, respectivamente. A DIVMS foi superior para o tratamento controle, indicando que os níveis de 5 e 10 ml de enzimas injectados no fluido ruminal não aumentaram a DIVMS. Para amilase aplicada por pulverização em 24g de milho moído, no tempo de 15 , observou-se que o tratamento controle e T1 não diferiram. No entanto, o T2 melhorou a DIVMS em 55,54%, comparado ao grupo controle. O tratamento T1 aumentou a DIVMS apenas em tempos de 3 e 24 horas de incubação, em relação ao controle. Com a aplicação de 10 ml de enzima, a DIVMS aumentou em todos os tempos de incubação, em comparação com o controle.

Considerando a dieta como um fator modulador do microbioma ruminal, neste trabalho objetivou-se investigar o impacto do bagaço da cana-de-açúcar sobre a composição e funcionalidade das espécies microbianas residentes no rúmen de carneiros (Ovis aries). Foram utilizados seis animais machos fistulados de O. aries, dos quais três foram alimentados com uma dieta composta por 70% de volumoso e 30% de concentrado (tratamento controle) e outros três animais alimentados com uma dieta similar a anterior, mas com 14% do volumoso substituído por bagaço de cana-de-açúcar (tratamento bagaço). O conteúdo ruminal (líquido e fibra) foram amostrados quinzenalmente durante 60 dias. A partir dessas amostras foram acessadas a estrutura e a composição da comunidade microbiana pela extração de DNA total e amplificação das regiões V3 e V6-V7 do gene 16S rRNA bacteriano e a região intergênica fúngica (ITS2). Além disso, foram feitas análises metagenômicas e metatranscriptômicas de comunidade microbianas enriquecidas em fibra ruminal para identificar enzimas lignocelulolíticas expressas. As frações líquida e fibrosa do conteúdo ruminal de O. aries revelaram uma comunidade bacteriana dominada principalmente por Bacteroidetes e Firmicutes ao longo de todo período experimental. Dois gêneros, Prevotella e Ruminococcus representaram 20% e 4% da comunidade bacteriana ruminal, respectivamente. Para a comunidade fúngica o filo Neocallimastigomycota representou 91% das sequências e os principais gêneros deste filo foram Piromyces, Neocallimastix, Orpinomyces, Anaeromyces, Caecomyces e Cyllamyces aderidos a fibra ruminal. O gênero Caecomyces, foi significativamente mais abundante na fibra ruminal de animais que se alimentaram de bagaço de cana-de açúcar. Além disso, foi observado um aumento significativo na frequência de enzimas como, por exemplo, 1,4-&alpha;-glucano, &alpha;-galactosidase, endo 1,4-&beta;-xilanase, &beta;- xilosidase, xilose isomerase, celobiose fosforilase e &alpha;-N-arabinofuranosidase no tratamento com bagaço de cana-de-açúcar. Considerando que a recuperação de enzimas a partir de comunidades microbianas naturalmente selecionadas para a degradação de biomassa é uma estratégia promissora para superar a atual ineficiência da ação enzimática na produção industrial de biocombustíveis, os resultados deste trabalho representam a possibilidade de aumentar a capacidade de recuperação ou descoberta de enzimas a partir de ruminantes, ou ainda, a possibilidade de manipular a estrutura do microbioma do rúmen para usá-lo como fonte de inóculo enriquecido em processos industriais de degradação de biomassa. / Considering the diet as a modulator of ruminal microbiome, this work aimed to investigate the impact of sugarcane bagasse on the composition and function of microbial species residents in the sheep (Ovis aries) rumen. Six cannulated male animals were used in the experiment, where three individuals were fed on a diet consisting of 70% forage and 30% concentrate (control treatment), and three were fed on a similar diet, but with sugarcane bagasse replacing 14% of the forage portion (bagasse treatment). The ruminal content (i.e., liquid and fiber) were sampled every two weeks during 60 days. From these samples, the structure and composition of the microbial community were assessed by total DNA extraction and amplification of V3 and V6-V7 regions of 16S rRNA gene from bacteria and the fungal intergenic region (ITS2). Furthermore, metagenomics and metatranscriptomics approaches were used to evaluate the enrichment of specific members of the microbial community in the ruminal fiber and genes related to lignocellulolytic enzymes. The liquid and fiber fractions of the O. aries rumen revealed a microbial community dominated mainly by Firmicutes and Bacteroidetes throughout the experimental period. The genera Prevotella and Ruminococcus accounted for 20% and 4% of the bacterial community of rumen, respectively. In the fungal community, the phylum Neocallimastigomycota accounted for 91% of sequences and its main genera adhered on the ruminal fiber were Piromyces, Neocallimastix, Orpinomyces, Anaeromyces, Caecomyces and Cyllamyces. The genus Caecomyces was significantly more abundant in the ruminal fiber in animals fed on sugarcane bagasse. Furthermore, there was a significant increase in the frequency of enzymes, such as &alpha;-1,4-glucan, &alpha;-galactosidase, endo- 1,4-&beta;-xylanase, &beta;-xylosidase, xylose isomerase, cellobiose phosphorylase and &alpha;- Narabinofuranosidase in the bagasse treatment. Considering that the recovery of enzymes from ecosystems naturally evolved for degradation of biomass is a promising strategy to overcome the current inefficient enzymatic action in industrial production of biofuels, the results of this study bring great possibilities to increase the discovery and or recovery of enzymes from ruminants, as well as the possibility of the ruminal microbiome structure manipulation to be used as source of an enriched inoculum for biomass degradation in industrial processes.