Ruminant livestock maintain an important role in meeting the nutrient requirements of the global population through their unique ability to convert plant fiber into human-edible meat and milk products. Volatile fatty acids (VFA) produced by rumen microbial fermentation of feed substrates represent around 70% of the ruminant animal's metabolic energy supply. Rumen fermentation profiles may directly impact productivity because the types of VFA produced are utilized at differing efficiencies by the animal. Improving our understanding of factors that control these fermentative outcomes would therefore aid in optimizing the productive efficiency of ruminant livestock. Improvements in animal efficiency are now more important than ever as the livestock industry must adapt to continue meeting the nutritional needs of a growing global population in the context of increased resource restrictions and requirements to lower the environmental impact of production. The relationship between diet and VFA ultimately supplied to the animal is complex and poorly understood due to the influence of numerous nutritional, biochemical, and microbial variables. The central aim of this body of work was therefore to explore and characterize how fermentation dynamics, rumen environmental characteristics, and the rumen microbiome behave in response to variations in the supply of fermentative substrate. The objective of our first experiment was to describe a novel in vitro laboratory technique to rank livestock feeds based on their starch degradability. This experiment also compared the starch degradation rates estimated by the in vitro method to the rates estimated by a traditional in situ method using sheep. A relationship between the degradation rates determined by these two procedures was observed, but only when feed nutrient content was accounted for. While this in vitro approach may not be able to reflect actual ruminal starch degradation rates, it holds potential as a useful laboratory technique for assessing relative differences in starch degradability between various feeds. Our second experiment aimed to measure changes in VFA dynamics, rumen environmental characteristics, and rumen epithelial gene expression levels in response to dietary sources of fiber and protein designed to differ in their rumen availabilities. Conducted in sheep, this study utilized beet pulp and timothy hay as the more and less available fiber source treatments, respectively, and soybean meal and heat-treated soybean meal as the more and less available protein source treatments, respectively. Results indicated that rumen environmental parameters and epithelial gene expression levels were not significantly altered by treatment. However, numerous shifts in response to both protein and fiber treatments were observed in fermentation dynamics, especially in interconversions of VFA. The objective of the third investigation was to assess whether the rumen microbiome can serve as an accurate predictor of beef and dairy cattle performance measurements and compare its predictive ability to that of diet explanatory variables. The available literature was assembled into a meta-analysis and models predicting dry matter intake, feed efficiency, average daily gain, and milk yield were derived using microbial and diet explanatory variables. Comparison of model quality revealed that the microbiome-based predictions may have comparable accuracy to diet-based predictions and that microbial variables may be used in combination with diet to improve predictions. In our fourth experiment, the objective was to investigate rumen microbial responses to the fiber and protein diet treatments detailed in Experiment 2. Responses of interest included relative abundances of bacterial populations at three taxonomic levels (phylum, family, and genus) in addition to estimations of community richness and diversity. Numerous population shifts were observed in response to fiber treatment. Prominent fibrolytic population abundances as well as richness and diversity estimations were found to be greater with timothy hay treatment and lower with beet pulp whereas pectin degraders increased in abundance on beet pulp. Microbial responses associated with protein treatment were not as numerous but appeared to reflect taxa with roles in protein metabolism. These four investigations revealed that significant changes can occur in VFA fermentation and rumen microbial populations when sources of nutrient substrates provided in a ruminant animal's diet are altered and that a new approach may be useful in investigating degradation of another important substrate for fermentation (starch) in a laboratory setting. Our findings also determined that animal performance can be predicted to a certain extent by rumen microbial characteristics. Collectively, these investigations offer an improved understanding of factors that influence the process of converting feed to energy sources in the ruminant animal. / Doctor of Philosophy / Ruminant animals, such as beef cattle, dairy cattle, and sheep, play a major role in delivering essential nutrients to the human population through their provision of meat and dairy products. The current growth projections of the global population, in addition to increased concerns surrounding greenhouse gas emissions and restrictions on resources such as land and water make it important for us to consider ways of optimizing the productivity of these animals. A unique feature of ruminants is their ability to conduct microbial fermentation of large amounts of plant matter in their rumens to produce energetically valuable compounds called volatile fatty acids (VFA), which are the primary source of energy that the animals use for growth, reproduction, and milk production. One promising way of improving animal productivity is to increase the amount of energy from the diet that becomes available to fuel the animal's body processes; however, the process of converting feed to VFA is complicated and currently not well understood. The overall aim of this body of work was therefore to explore various nutritional, ruminal, and microbial factors that are known to impact fermentation in order to 1) increase our understanding of how these factors interconnect and 2) put us in a better position to manipulate these factors for optimal animal performance. The goal of our first experiment was to devise and use a novel laboratory technique to rank livestock feeds based on the degradability of their starch content, which is an important substrate for VFA fermentation. Our observations indicate that this technique may be a useful tool to help us determine relative differences between feeds based on their starch degradabilities in a laboratory setting. Our second experiment investigated the effects of feeding varying sources of fiber (beet pulp and timothy hay) and protein (heat-treated and untreated soybean meals) to sheep in terms of their VFA fermentation, rumen conditions, and the expression of certain key genes in the epithelial tissue of the rumen wall. While rumen environmental characteristics and epithelial gene expression remained largely unchanged, numerous key aspects of VFA fermentation, predominantly carbon exchanges between different VFA, were altered in response to nutrient source. The third investigation described in this work examined the ability of the microbial populations responsible for rumen fermentation to explain variation in beef and dairy cow productivity compared with the ability of diet characteristics to explain this variation. Using statistical methods to analyze the reports currently available in scientific literature, our findings indicate that the rumen microbiome and diet may exert independent effects on productivity levels and that the microbiome may be used to enhance diet-based predictions of animal performance. Finally, we explored variations in the sheep rumen microbiome in response to the diet treatments utilized in Experiment 2. We observed minimal impact of protein source on the microbiome, but numerous microbial responses were evident when fiber source was varied. These responses included decreases of fiber-degrading bacterial populations and increases in pectin-degrading populations when beet pulp was fed compared to timothy hay. Taken together, these experiments help to provide us with a more comprehensive picture of the numerous factors involved in the process of converting feed to a usable form of energy for ruminant livestock.

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.

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

Short chain fatty acids (SCFA) synthesized in the rumen from carbohydrate fermentation are an essential energy source for ruminants. Current literature supports that SCFA are absorbed across the rumen epithelium via passive diffusion or protein-mediated transport, however, the rate and degree to which these pathways adapt to a change in diet fermentability is unknown. Furthermore, Na+ flux is partially determined by SCFA absorption, and thus is a key indicator of functional changes in the rumen epithelium. The objectives of this study were to determine the time required for a change in SCFA and Na+ absorption across the bovine rumen epithelium and to evaluate the rate and degree to which absorption pathways adapt to an increase in diet fermentability relative to changes in surface area. Twenty-five weaned Holstein steer calves were blocked by body weight and randomly assigned to either the control diet (CON; 91.5% hay and 8.5% vitamin/mineral supplement) or a moderately fermentable diet (50% hay; 41.5% barley grain, and 8.5% vitamin/mineral supplement) fed for 3 (G3), 7 (G7), 14 (G14), or 21 d (G21). All calves were fed at 2.25% BW at 0800 h. Reticular pH was recorded every 5 min for 48 h prior to killing (1000 h). Ruminal tissue was collected for Ussing chamber, barrier function, surface area measurements, and gene expression. Net 22Na+ flux (JNET-Na; 80 kBq/15 mL), the rate and pathway of mucosal to serosal 3H-acetate (JMS-acetate; 37 kBq/15 mL) and 14C-butyrate (JMS-butyrate; 74 kBq/15 mL) flux, and serosal to mucosal flux of 3H-mannitol (JSM-mannitol; 74 KBq/15 mL) and tissue conductance were measured. Half of the chambers assigned to measure JMS-acetate and JMS-butyrate were further assigned to 1 of 2 acetate and butyrate concentration treatments: 10 mM (Low) and 50 mM (High). Furthermore, JSM-mannitol flux was also measured during an acidotic and hyperosmotic challenge (CHAL) and recovery (REC) to measure barrier function of ruminal tissue. Mean reticular pH, which was positively correlated with ruminal pH (R2 = 0.5477), decreased from 6.90 for CON to 6.59 for G7 then increased. Net Na+ flux increased 125% within 7 d. Total JMS-acetate and JMS-butyrate increased from CON to G21, where passive diffusion was the primary SCFA absorption pathway. Total JMS-acetate and JMS-butyrate were greater when incubated in High vs. Low. Effective surface area of the ruminal epithelium was not affected by dietary treatment. Increased JSM-mannitol, tissue conductance, and increased expression of IL-1β and TLR2 (tendencies) with increased days fed the moderate grain diet indicated reduced rumen epithelium barrier function. Furthermore, the CHAL treatment reduced barrier function, which was not reversible during REC. This study indicates that a moderate increase in diet fermentability increases rumen epithelium absorptive function in the absence of increased SA, but reduces barrier function. Data from this study also suggests that absorption and barrier function follow different timelines, posing a challenge for ruminant diet adaptation to moderately to highly fermentable diets.

Submitted by DANIELA DUTRA ESTEVAM null (daniela_estevam@yahoo.com.br) on 2016-02-01T14:08:30Z No. of bitstreams: 1 DISSERTAÇÃO DANIELA.pdf: 1615348 bytes, checksum: 18226c859c846688ff9fa236460a54de (MD5) / Approved for entry into archive by Sandra Manzano de Almeida (smanzano@marilia.unesp.br) on 2016-02-01T18:01:26Z (GMT) No. of bitstreams: 1 estevam_dd_me_bot.pdf: 1615348 bytes, checksum: 18226c859c846688ff9fa236460a54de (MD5) / Made available in DSpace on 2016-02-01T18:01:26Z (GMT). No. of bitstreams: 1 estevam_dd_me_bot.pdf: 1615348 bytes, checksum: 18226c859c846688ff9fa236460a54de (MD5) Previous issue date: 2016-01-22 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O objetivo deste estudo foi avaliar os efeitos dos períodos de 6, 9, 14 e 21 dias de adaptação de bovinos da raça Nelore confinados a dietas de alto teor de concentrado em relação ao desempenho, variação na ingestão de massa seca, energia líquida, perfil metabólico sanguíneo, comportamento ingestivo, saúde ruminal e características de carcaça. Os animais foram alimentados durante 88 dias, independentemente do período de adaptação adotado e o delineamento utilizado foi em blocos casualizados, com seis repetições cada, em que 96 bovinos da raça Nelore não castrados (391,1 ± 30,9 kg) foram alimentados em 24 baias (4 animais/baia), de acordo com os diferentes períodos de adaptação adotados: 6, 9, 14 e 21 dias. O programa de adaptação utilizado foi em escadas, com três dietas ao longo de períodos de adaptação com seguintes níveis de concentrado: 70, 75, 80,5 e 86% da massa seca na dieta. Contrastes ortogonais foram utilizados para avaliar a relação linear, quadrática e cúbica entre os dias de adaptação e a variável dependente, além da interação de tratamento e fase (P≤0,10). Os animais adaptados por 14 dias apresentaram resultados superiores em relação ao ganho de peso diário, peso vivo final e eficiência alimentar, bem como peso de carcaça quente (P=0,04) e área de olho de lombo final (P=0,01). A adaptação por 14 dias também proporcionou aos animais melhor desenvolvimento do epitélio ruminal, pois apresentaram maior área de superfície absortiva (P=0,02) e largura de papilas (P=0,06). Além disso, esses animais demonstraram que estavam adequadamente adaptados, pois a renovação celular do epitélio ruminal demonstrou-se estabilizada no índice de proliferação celular (P=0,003) e nos núcleos em morte celular (P=0,0004). Com base nesses resultados, recomenda-se adaptar bovinos da raça Nelore a dietas de alto concentrado com o protocolo em escadas por 14 dias, pois este proporcionou maior desempenho produtivo e desenvolvimento do epitélio ruminal. / This study was designed to determine the effects of adaptation periods of 6, 9, 14 and 21 days on feedlot performance, dry matter intake fluctuations, energy gain, blood metabolic profile, feeding behavior, rumen health and carcass traits of Nellore cattle. Cattle were fed for 88-d regardless of adaptation period adopted and the experiment was designed as a completely randomized block, replicated 6 times, in which ninety-six 20-mo-old yearling Nellore bulls (391.1 ± 30.9 kg) were fed in 24 pens (4 animals/pen) according to the different adaptation periods adopted: 6, 9, 14, and 21 days. The adaptation program consisted of ad libitum feeding of 3 diets over adaptation periods with concentrate level increasing from 70, 75, 80.5 and 86% of diet dry matter. Orthogonal contrasts were used to evaluate linear, quadratic, and cubic relationship between adaptation periods and the dependent variable, moreover the interaction the treatment and phase (P≤0.10). Cattle adapted for 14 days had significantly greater for average daily gain, final body weight, G:F ratio, hot carcass weight (P=0,04) and rib-eye area (P=0,01). The adaptation for 14 days the animals also provided better development of the rumen epithelium for a greater absorptive surface area (P=0.02) and width papillae (P=0.06). Moreover, these cattle showed that were properly adapted because the cell epithelium renovation proved to be stable in cell proliferation index (P=0.003) and cell death (P=0.0004). Thus, based on the results of this study, yearling Nellore bulls should be adapted for the step protocol for 14 days, because these provided greater performance and development of the rumen epithelium. / FAPESP: 2013/25403-0

No description available.

This study evaluated degradable nutrient supply effects on VFA concentrations, fluid flux and pool sizes, rumen epithelial metabolic and absorptive genes, and post-absorptive muscle and blood responses. Six ruminally cannulated Holstein heifers (BW=330 ± 11.3 kg) were used in a partially replicated Latin Square experiment with four treatments consisting of beet pulp or timothy hay and barley or corn grain. Periods were18 d with 3 d diet adaptation and 15 d of treatment. During each period, d 10 to 14 was used for in situ nutrient degradation assessment, d 16 to 18 was used for rumen fluid sampling, and d 18 was used for rumen papillae and skeletal muscle biopsies and blood sampling. In situ ruminal starch disappearance rate (barley 7.61 to 10.5 %/h vs corn 7.30 to 8.72%/h; P = 0.05) and extent of fiber disappearance (timothy hay 22.2 to 33.4 % DM vs beet pulp 34.4 to 38.7 % DM P=0.0007) differed significantly among diets. Acetate (P = 0.02) and isovalerate (P = 0.008) molar percentages (% mol) were increased by timothy hay, but propionate (P = 0.06) and valerate (P = 0.10) molar percentages were decreased. Corn increased propionate (P = 0.02) and valerate (P = 0.049) molar percentage, but decreased butyrate (P = 0.04) molar proportion. Fluid volume and fluid passage rate, and individual VFA pool sizes were not influenced by diet (P > 0.05). Four epithelial genes, two metabolic and two absorptive, had increased expression on timothy hay diets (P < 0.15). Blood acetate concentration was influenced by treatment (P = 0.067) but no other blood metabolites were. Skeletal muscle metabolic rate was significantly increased on corn diets (P = 0.023). The results of this study provide a whole-system snapshot of how the rumen environment changes on diets differing in nutrient degradability and how the post-absorptive system adapts in response. / Master of Science / Over the last 50 years, dairy cattle have been bred to optimize milk production to meet growing population demands for milk and dairy products. The world population continues to grow and is projected to reach 9.7 billion people by 2050. Because of this growing population, there is an overwhelming need for dairy nutritionists to optimize the conversion of human inedible fibers into human edible food. The ruminant animal accomplishes this conversion through microbial fermentation of feedstuffs into volatile fatty acids (VFA), which account for approximately 70% of total energy available for meat, milk, and fiber production. Because rumen fermentation is a complex biochemical system, it is influenced by myriad factors including the substrate provided, the pH of the environment, and the absorptive and metabolic capacity of the rumen wall, among others. Although we understand how diet influences individual aspects of rumen fermentation, few studies have concurrently evaluated how diet influences the rumen chemical environment, the epithelium, and the resulting shifts in postabsorptive metabolism. Our study sought to understand the impacts of feedstuffs with different expected ruminally available starch and fiber supplies on these aspects of ruminant physiology. Six ruminally cannulated Holstein heifers were fed four different diets which used either beet pulp (low fiber ingredient) or timothy hay (high fiber ingredient), and ground corn (low starch ingredient) or ground barley (high starch ingredient). Heifers were fed each diet for a period of 18 days. From day 10 to day 14 of the period, nutrient degradability was assessed by incubating bags of feed in the rumen and conducting feed analysis after removed from the rumen. During the last four days of each period, rumen fluid samples, blood samples, muscle biopsies, and rumen papillae biopsies were collected. Feed analysis indicated that the starch sources differed in degradation rates (i.e. the speed of degradation) and fiber sources different in extent of rumen degradation (i.e. the percentage of feed degraded). Timothy hay caused greater concentrations of Total VFA, Total branched-chain VFA, acetate isobutyrate, and isovalerate. Timothy hay caused greater molar proportions of acetate and isovalerate. Corn caused greater molar proportions of propionate and valerate when barley caused greater molar proportions of butyrate. Rumen papillae biopsies were used to evaluate gene expression. Out of 14 genes, four were impacted by diet. Two rumen transporters responsible for the absorption of VFA had greater expression when animals were fed timothy hay diets versus beet pulp diets. Two metabolic genes also had greater expression due to timothy hay. The changes of both absorptive genes and metabolic genes is likely connected to the increased presence of VFA in the rumen. Lastly, blood acetate was increased, but there was not a specific ingredient or combination that caused the change. These results provide an overall snapshot of rumen fermentation characteristics and how changes in the rumen affect other biology.

Einleitung: Kurzkettige Fettsäuren (SCFA) stellen das hauptsächliche Energiesubstrat für Wiederkäuer dar. In Anbetracht des - bedingt durch höhere Milch-, Mast und Reproduktionsleistung - steigenden Energiebedarfs von Hauswiederkäuern wie Milchkuh und Mastbulle ist es von zentraler Bedeutung, die Mechanismen zur Resorption dieser Energielieferanten bzw. Ansatzpunkte für die Beeinflussung dieser Transportprozesse genau zu kennen. Dieses Wissen kann möglicherweise dabei helfen, zukünftig die Energieaufnahme der Tiere zu unterstützen bzw. sogar effizienter zu gestalten. Ziele der Untersuchungen: Deshalb war es Ziel der vorliegenden Arbeit, die Mechanismen zur Resorption von SCFA zu charakterisieren, wobei der Schwerpunkt auf den Transport aus den Pansenepithelzellen ins Blut gelegt wurde, da hierzu im Gegensatz zu ihrer Aufnahme aus dem Pansenlumen in die Epithelzellen noch sehr wenig bekannt war. In einem zweiten Schritt sollte untersucht werden, inwiefern die nachgewiesenen Mechanismen einer Regulation unterliegen und über welche Signalwege diese vermittelt werden könnte. Materialien und Methoden: Zur Charakterisierung der beteiligten Resorptionsmechanismen wurden Epithelstücke aus dem ventralen Pansensack von Schafen in Ussing-Kammern eingespannt und mit Hilfe radioaktiv markierten Azetats, Butyrats und L-Laktats der Transport dieser Substrate unter verschiedenen Bedingungen sowie verschiedenen Hemmstoffeinflüssen untersucht. Zur Charakterisierung regulativer Einflüsse wurden die Epithelstücke über sechs bzw. 24 Stunden mit Butyrat inkubiert und anschließend RNA bzw. Totalprotein extrahiert. Hiermit konnten Veränderungen in mRNA- und Proteinexpression mittels quantitativer Echtzeit-PCR bzw. Western Blot nachgewiesen werden. Ergebnisse: Die Untersuchungen der vorliegenden Arbeit konnten zeigen, dass der Transport von SCFA über die basolaterale Membran des Pansenepithels hauptsächlich proteinvermittelt erfolgt. Eine signifikante Beteiligung lipophiler Diffusion, d.h. ein passiver Transport, kann weitgehend ausgeschlossen werden. Der aktive Transport wies eine bikarbonatabhängige und eine bikarbonatunabhängige Komponente auf. Der Einsatz von Hemmstoffen verschiedener Transportproteine ergab deutliche Hinweise darauf, dass der Monocarboxylattransporter (MCT) 1 eine Rolle beim bikarbonatgekoppelten Transport von Azetat bzw. allgemein unmetabolisierten SCFA spielt. Diese Hinweise wurden untersetzt durch die Beobachtung, dass MCT 1, aber auch der apikal bzw. intrazellulär lokalisierte MCT 4 durch langfristige Inkubation des Epithels mit Butyrat sowohl auf mRNA- als auch auf Proteinebene signifikant erhöht exprimiert wurden, was als Anpassungsreaktion an eine Substratakkumulation interpretiert werden kann. Außerdem wurde auch die mRNA-Expression des Putativen Anionentransporters (PAT) 1 durch Inkubation mit Butyrat erhöht, was für eine Beteiligung auch dieses Transportproteins am SCFA-Transport über das Pansenepithel spricht. Allerdings ist im Gegensatz zu MCT 1 die Lokalisation des PAT 1 in der basolateralen Membran noch fraglich. Die Expressionssteigerung von Zielgenen des Nukleären Faktors ĸB und des Peroxisomenproliferator-aktivierten Rezeptors α sowie des Hypoxie-induzierbaren Faktors selbst deuten weiterhin darauf hin, dass die Steigerung der Transportkapazitäten von MCT 1 und 4 und auch PAT 1 über diese Signalwege vermittelt wird. Schlussfolgerungen: Zusammenfassend konnte in dieser Arbeit erstmals der Transport von SCFA über die basolaterale Membran des Pansenepithels näher charakterisiert werden, sodass es nun möglich ist, zusammen mit den bereits vorliegenden Befunden für die apikale Membran ein komplettes Modell dafür zu erstellen. Auch wurden Erkenntnisse zu regulativen Einflüssen auf diesen Transport gewonnen, die es zukünftig ermöglichen könnten, die Resorption der SCFA aus dem Pansen nutritiv oder eventuell pharmakologisch zu beeinflussen. / Introduction: The main energy source for ruminants are short chain fatty acids (SCFA). Considering the ever increasing energy requirements of cattle due to increasing milk yield and meat production, it is crucial to identify the mechanisms for the resorption of these energy sources as well as possibilities to influence these transport mechanisms. This knowledge could help support the animals’ energy uptake or even making it more efficient. Aim: Thus, the aim of the present study was to characterise mechanisms for the resorption of SCFA focusing on their transport from the epithelial cells into the blood. In particular, since – compared to the research findings on the uptake of SCFA from ruminal lumen into the cells – so far only very little was known regarding this side of the epithelium. In a second step, the study aimed to elucidate whether the mechanisms observed are subject to regulatory processes and which signalling pathways are involved. Materials and methods: To characterise the transport mechanisms involved, epithelial pieces from the ventral sac of ovine rumen were mounted in Ussing chambers. Using radioactively labelled acetate, butyrate and L-lactate, the transport of these substrates was investigated under different conditions and by applying different inhibitors for potential SCFA transport proteins. To characterise regulatory influences, epithelial pieces were incubated with butyrate for six and 24 hours, respectively. Subsequently, total RNA and protein were extracted to detect changes in mRNA and protein expression using quantitative real time PCR and western blot, respectively. Results: The present study could show that transport of SCFA across the basolateral membrane of rumen epithelium is mainly realised by protein-mediated mechanisms. A significant participation of lipophilic diffusion, i.e. a passive transport, can almost entirely be excluded. The active transport could be divided into a bicarbonate-dependent and a bicarbonate-independent part. The experiments with inhibitors of different transport proteins showed clear evidence of an involvement of monocarboxylate transporter (MCT) 1 in the bicarbonate-dependent transport of acetate and non-metabolised SCFA in general. This evidence was supported by the finding that the expression of MCT 1 but also of the apically and intracellularly localised MCT 4 was increased significantly on both mRNA- and protein-level after long-term incubation of the epithelium with butyrate. This can be interpreted as an adaptation to a substrate accumulation. Additionally, butyrate incubation led to an increased mRNA expression of putative anion transporter (PAT) 1, which makes an involvement of this transport protein in SCFA transport across ruminal epithelium likely as well. However, in contrast to MCT 1 the localisation of PAT 1 in the basolateral membrane is still questionable. The increased expression of target genes of nuclear factor ĸB and peroxisome-proliferator activated receptor α as well as of hypoxia inducible factor strongly point to an involvement of these pathways in the increased expression of MCT 1 and 4 as well as PAT 1. Conclusions: In summary, this study could characterise the transport of SCFA across the basolateral membrane of ruminal epithelium in detail for the first time. This enables us to draw a complete model of ruminal SCFA transport. Also, evidence for regulatory influence on this transport processes was found, perhaps making it possible to influence resorption of SCFA from rumen by nutritive or pharmacological means in the future.

Die vorliegende Arbeit befasst sich mit der Einflussnahme von konjugierten Linolsäuren (CLA) und strukturverwandten Fettsäuren auf Pansenepithelzellen (PEZ) in Kultur. Die in-vitro Untersuchungen mit primär kultivierten PEZ vom Schaf haben gezeigt, dass eine 48-stündige Inkubation mit CLA C18:2 cis-9, trans-11 (c9t11) und CLA C18:2 trans-10, cis-12 (t10c12) sowie Linolsäure, Ölsäure und trans-Vaccensäure (TVA) zu Veränderungen im Fettsäuremuster der Zellen sowie in der Expression der mRNA verschiedener Proteine führte. Aus den Veränderungen im Fettsäuremuster wurde die Aufnahme sowie die Metabolisierung der supplementierten Fettsäuren abgeleitet. Als wesentlicher Metabolit der TVA wurde die c9t11 identifiziert. Mit dem Nachweis der Stearoyl-CoA-Desaturase (SCD) -mRNA in den PEZ sowie im nativen Gewebe konnte so die endogene CLA-Synthese in den PEZ bestätigt werden. Analysiert wurden außerdem mittels quantitativer rt-PCR die Expression der SCD-mRNA und die mRNA von zwei Transportproteinen, den Monocarboxylat-Transportern (MCT) 1 und 4. Von beiden ist bekannt, dass sie in den Transport kurzkettiger Fettsäuren (SCFA) involviert sind. Die Inkubation mit den einzelnen Fettsäuren führte einheitlich zur Abnahme der SCD-mRNA Expression sowie zu einem verminderten Gehalt der Hauptprodukte der SCD, der C16:1 cis-9 und der C18:1 cis-9. Bezüglich der MCT1 und 4 mRNA-Expression wurde in der Regel eine Heraufregulierung nach Zugabe der Fettsäuren beobachtet. Des Weiteren wurden regulative Einflüsse von PPARα auf die MCTs und die SCD und von PPARγ auf den MCT1 und die SCD nach c9t11-Inkubation mit entsprechenden Antagonisten-Versuchen festgestellt. Die Studien beleuchten somit grundlegende Mechanismen des Stoffwechsels von langkettigen ungesättigten Fettsäuren in PEZ und deren Einflussnahme auf Transkriptionsfaktoren und auf spezifische, für den Wiederkäuer bedeutende Transportproteine für SCFA. Die funktionelle Relevanz dieser Ergebnisse für den Pansen und den Wiederkäuer aber auch für andere Gewebe und Species muss in weiteren Studien geklärt werden.:Inhaltsverzeichnis Abkürzungsverzeichnis 3 1 Einleitung 5 Allgemein 5 1.1 CLA-Nomenklatur 6 1.2 Bildung von CLAs und deren Vorkommen 7 1.3 CLA-Supplementierung beim Rind 9 1.4 Zu CLAs strukturverwandte Fettsäuren im Pansen 10 1.5 Potenzielle Wirkungen von CLAs und strukturverwandten Fettsäuren auf das Pansenepithel 11 1.5.1 Aufbau und Bedeutung des Pansenepithels 11 1.5.2 Resorption von langkettigen Fettsäuren über das Pansenepithel 11 1.5.3 Veränderungen der Fettsäurezusammensetzung nach Supplementierung 11 1.5.3.1 Metabolisierung 12 1.5.3.2 SCD und die endogene Synthese von CLA c9t11 13 1.5.4 CLA-Targets im Pansenepithel 15 1.5.4.1 SCD 15 1.5.4.2 Monocarboxylattransporter 16 1.5.4.3 PPAR als Transkriptionsfaktor für die SCD und die MCTs 19 2 Zielstellung 21 3 Originalarbeit 22 4 Ergänzung zur Originalarbeit 43 4.1 Nachweis von SCD-mRNA im nativen Pansenepithel des Schafes 43 Zusammenfassung der Arbeit 44 Literaturverzeichnis 49 Anhang (Supplemental Material) 59 Erklärung zum Eigenanteil der Dissertationsschrift 67 Erklärung über die eigenständige Abfassung der Arbeit 69 Publikationen und Vorträge im Rahmen der Dissertation 70

Einleitung: Kurzkettige Fettsäuren (SCFA) stellen das hauptsächliche Energiesubstrat für Wiederkäuer dar. In Anbetracht des - bedingt durch höhere Milch-, Mast und Reproduktionsleistung - steigenden Energiebedarfs von Hauswiederkäuern wie Milchkuh und Mastbulle ist es von zentraler Bedeutung, die Mechanismen zur Resorption dieser Energielieferanten bzw. Ansatzpunkte für die Beeinflussung dieser Transportprozesse genau zu kennen. Dieses Wissen kann möglicherweise dabei helfen, zukünftig die Energieaufnahme der Tiere zu unterstützen bzw. sogar effizienter zu gestalten. Ziele der Untersuchungen: Deshalb war es Ziel der vorliegenden Arbeit, die Mechanismen zur Resorption von SCFA zu charakterisieren, wobei der Schwerpunkt auf den Transport aus den Pansenepithelzellen ins Blut gelegt wurde, da hierzu im Gegensatz zu ihrer Aufnahme aus dem Pansenlumen in die Epithelzellen noch sehr wenig bekannt war. In einem zweiten Schritt sollte untersucht werden, inwiefern die nachgewiesenen Mechanismen einer Regulation unterliegen und über welche Signalwege diese vermittelt werden könnte. Materialien und Methoden: Zur Charakterisierung der beteiligten Resorptionsmechanismen wurden Epithelstücke aus dem ventralen Pansensack von Schafen in Ussing-Kammern eingespannt und mit Hilfe radioaktiv markierten Azetats, Butyrats und L-Laktats der Transport dieser Substrate unter verschiedenen Bedingungen sowie verschiedenen Hemmstoffeinflüssen untersucht. Zur Charakterisierung regulativer Einflüsse wurden die Epithelstücke über sechs bzw. 24 Stunden mit Butyrat inkubiert und anschließend RNA bzw. Totalprotein extrahiert. Hiermit konnten Veränderungen in mRNA- und Proteinexpression mittels quantitativer Echtzeit-PCR bzw. Western Blot nachgewiesen werden. Ergebnisse: Die Untersuchungen der vorliegenden Arbeit konnten zeigen, dass der Transport von SCFA über die basolaterale Membran des Pansenepithels hauptsächlich proteinvermittelt erfolgt. Eine signifikante Beteiligung lipophiler Diffusion, d.h. ein passiver Transport, kann weitgehend ausgeschlossen werden. Der aktive Transport wies eine bikarbonatabhängige und eine bikarbonatunabhängige Komponente auf. Der Einsatz von Hemmstoffen verschiedener Transportproteine ergab deutliche Hinweise darauf, dass der Monocarboxylattransporter (MCT) 1 eine Rolle beim bikarbonatgekoppelten Transport von Azetat bzw. allgemein unmetabolisierten SCFA spielt. Diese Hinweise wurden untersetzt durch die Beobachtung, dass MCT 1, aber auch der apikal bzw. intrazellulär lokalisierte MCT 4 durch langfristige Inkubation des Epithels mit Butyrat sowohl auf mRNA- als auch auf Proteinebene signifikant erhöht exprimiert wurden, was als Anpassungsreaktion an eine Substratakkumulation interpretiert werden kann. Außerdem wurde auch die mRNA-Expression des Putativen Anionentransporters (PAT) 1 durch Inkubation mit Butyrat erhöht, was für eine Beteiligung auch dieses Transportproteins am SCFA-Transport über das Pansenepithel spricht. Allerdings ist im Gegensatz zu MCT 1 die Lokalisation des PAT 1 in der basolateralen Membran noch fraglich. Die Expressionssteigerung von Zielgenen des Nukleären Faktors ĸB und des Peroxisomenproliferator-aktivierten Rezeptors α sowie des Hypoxie-induzierbaren Faktors selbst deuten weiterhin darauf hin, dass die Steigerung der Transportkapazitäten von MCT 1 und 4 und auch PAT 1 über diese Signalwege vermittelt wird. Schlussfolgerungen: Zusammenfassend konnte in dieser Arbeit erstmals der Transport von SCFA über die basolaterale Membran des Pansenepithels näher charakterisiert werden, sodass es nun möglich ist, zusammen mit den bereits vorliegenden Befunden für die apikale Membran ein komplettes Modell dafür zu erstellen. Auch wurden Erkenntnisse zu regulativen Einflüssen auf diesen Transport gewonnen, die es zukünftig ermöglichen könnten, die Resorption der SCFA aus dem Pansen nutritiv oder eventuell pharmakologisch zu beeinflussen.:Inhaltsverzeichnis 1 Einleitung 1 2 Literaturübersicht 3 2.1 Bedeutung kurzkettiger Fettsäuren für Wiederkäuer 3 2.2 Metabolismus von SCFA im Pansenepithel 4 2.2.1 Aufrechterhaltung des Konzentrationsgradienten vom Pansenlumen ins Epithel 4 2.2.2 Produktion von HCO3- aus CO2 durch die Carboanhydrase 5 2.2.3 Bereitstellung von Energie für die Epithelzellen 5 2.2.4 Bereitstellung von wasserlöslichen, glukosesparenden Energiesubstraten für die periphere Zirkulation 5 2.2.5 Verhinderung möglicher Schädigungen durch Butyrat 6 2.3 Transportmechanismen für kurzkettige Fettsäuren 7 2.3.1 Para- versus transzelluläre Resorption 7 2.3.2 Transzelluläre Resorption mittels lipophiler Diffusion 7 2.3.3 Proteinvermittelte SCFA-Permeation 9 2.3.4 Permeation von SCFA aus dem Epithel ins Blut 11 2.4 Beeinflussung der SCFA-Resorption auf Genexpressionsebene 17 2.4.1 Beeinflussung der Genexpression durch Butyrat 17 2.4.2 Beeinflussung der Genexpression durch Hypoxie 20 2.4.3 Mechanismen für die Regulation der Genexpression durch Butyrat (-Metaboliten) und Hypoxie 21 2.5 Fragestellungen dieser Arbeit 26 3 Ergebnisse 28 3.1 Publikation 1 28 3.2 Publikation 2 41 4 Diskussion 54 4.1 Transport von SCFA über die basolaterale Membran des Pansenepithels 54 4.1.1 Transport mittels lipophiler Diffusion 57 4.1.2 SCFA werden bevorzugt über die basolaterale Membran transportiert 58 4.1.3 SCFA(-Metaboliten) werden bikarbonatabhängig über die basolaterale Membran transportiert 59 4.1.4 SCFA(-Metaboliten) werden durch einen Anionenaustauschmechanismus ins Blut ausgeschleust 61 4.1.5 Azetat wird durch einen pHMB- und CHC-sensitiven Mechanismus transportiert 63 4.2 Der Transport von SCFA über das Pansenepithel unterliegt regulativen Einflüssen 68 4.2.1 Einfluss von Butyrat(-Metaboliten) auf die Expression von potentiellen SCFA Transportern 68 4.2.2 Mechanismen für die Regulation der Expression durch Butyrat(-Metaboliten) 72 4.3 Theoretisches Modell des SCFA-Transports und dessen Regulation auf Genexpressionsebene auf Grundlage der Ergebnisse der vorliegenden Arbeit 74 5 Zusammenfassung 76 6 Summary 78 7 Literaturverzeichnis 80 Danksagung 98 / Introduction: The main energy source for ruminants are short chain fatty acids (SCFA). Considering the ever increasing energy requirements of cattle due to increasing milk yield and meat production, it is crucial to identify the mechanisms for the resorption of these energy sources as well as possibilities to influence these transport mechanisms. This knowledge could help support the animals’ energy uptake or even making it more efficient. Aim: Thus, the aim of the present study was to characterise mechanisms for the resorption of SCFA focusing on their transport from the epithelial cells into the blood. In particular, since – compared to the research findings on the uptake of SCFA from ruminal lumen into the cells – so far only very little was known regarding this side of the epithelium. In a second step, the study aimed to elucidate whether the mechanisms observed are subject to regulatory processes and which signalling pathways are involved. Materials and methods: To characterise the transport mechanisms involved, epithelial pieces from the ventral sac of ovine rumen were mounted in Ussing chambers. Using radioactively labelled acetate, butyrate and L-lactate, the transport of these substrates was investigated under different conditions and by applying different inhibitors for potential SCFA transport proteins. To characterise regulatory influences, epithelial pieces were incubated with butyrate for six and 24 hours, respectively. Subsequently, total RNA and protein were extracted to detect changes in mRNA and protein expression using quantitative real time PCR and western blot, respectively. Results: The present study could show that transport of SCFA across the basolateral membrane of rumen epithelium is mainly realised by protein-mediated mechanisms. A significant participation of lipophilic diffusion, i.e. a passive transport, can almost entirely be excluded. The active transport could be divided into a bicarbonate-dependent and a bicarbonate-independent part. The experiments with inhibitors of different transport proteins showed clear evidence of an involvement of monocarboxylate transporter (MCT) 1 in the bicarbonate-dependent transport of acetate and non-metabolised SCFA in general. This evidence was supported by the finding that the expression of MCT 1 but also of the apically and intracellularly localised MCT 4 was increased significantly on both mRNA- and protein-level after long-term incubation of the epithelium with butyrate. This can be interpreted as an adaptation to a substrate accumulation. Additionally, butyrate incubation led to an increased mRNA expression of putative anion transporter (PAT) 1, which makes an involvement of this transport protein in SCFA transport across ruminal epithelium likely as well. However, in contrast to MCT 1 the localisation of PAT 1 in the basolateral membrane is still questionable. The increased expression of target genes of nuclear factor ĸB and peroxisome-proliferator activated receptor α as well as of hypoxia inducible factor strongly point to an involvement of these pathways in the increased expression of MCT 1 and 4 as well as PAT 1. Conclusions: In summary, this study could characterise the transport of SCFA across the basolateral membrane of ruminal epithelium in detail for the first time. This enables us to draw a complete model of ruminal SCFA transport. Also, evidence for regulatory influence on this transport processes was found, perhaps making it possible to influence resorption of SCFA from rumen by nutritive or pharmacological means in the future.:Inhaltsverzeichnis 1 Einleitung 1 2 Literaturübersicht 3 2.1 Bedeutung kurzkettiger Fettsäuren für Wiederkäuer 3 2.2 Metabolismus von SCFA im Pansenepithel 4 2.2.1 Aufrechterhaltung des Konzentrationsgradienten vom Pansenlumen ins Epithel 4 2.2.2 Produktion von HCO3- aus CO2 durch die Carboanhydrase 5 2.2.3 Bereitstellung von Energie für die Epithelzellen 5 2.2.4 Bereitstellung von wasserlöslichen, glukosesparenden Energiesubstraten für die periphere Zirkulation 5 2.2.5 Verhinderung möglicher Schädigungen durch Butyrat 6 2.3 Transportmechanismen für kurzkettige Fettsäuren 7 2.3.1 Para- versus transzelluläre Resorption 7 2.3.2 Transzelluläre Resorption mittels lipophiler Diffusion 7 2.3.3 Proteinvermittelte SCFA-Permeation 9 2.3.4 Permeation von SCFA aus dem Epithel ins Blut 11 2.4 Beeinflussung der SCFA-Resorption auf Genexpressionsebene 17 2.4.1 Beeinflussung der Genexpression durch Butyrat 17 2.4.2 Beeinflussung der Genexpression durch Hypoxie 20 2.4.3 Mechanismen für die Regulation der Genexpression durch Butyrat (-Metaboliten) und Hypoxie 21 2.5 Fragestellungen dieser Arbeit 26 3 Ergebnisse 28 3.1 Publikation 1 28 3.2 Publikation 2 41 4 Diskussion 54 4.1 Transport von SCFA über die basolaterale Membran des Pansenepithels 54 4.1.1 Transport mittels lipophiler Diffusion 57 4.1.2 SCFA werden bevorzugt über die basolaterale Membran transportiert 58 4.1.3 SCFA(-Metaboliten) werden bikarbonatabhängig über die basolaterale Membran transportiert 59 4.1.4 SCFA(-Metaboliten) werden durch einen Anionenaustauschmechanismus ins Blut ausgeschleust 61 4.1.5 Azetat wird durch einen pHMB- und CHC-sensitiven Mechanismus transportiert 63 4.2 Der Transport von SCFA über das Pansenepithel unterliegt regulativen Einflüssen 68 4.2.1 Einfluss von Butyrat(-Metaboliten) auf die Expression von potentiellen SCFA Transportern 68 4.2.2 Mechanismen für die Regulation der Expression durch Butyrat(-Metaboliten) 72 4.3 Theoretisches Modell des SCFA-Transports und dessen Regulation auf Genexpressionsebene auf Grundlage der Ergebnisse der vorliegenden Arbeit 74 5 Zusammenfassung 76 6 Summary 78 7 Literaturverzeichnis 80 Danksagung 98