Degradability in the rumen of several protein sources was determined by suspending from 12 to 13 g of feedstuff in dacron bags into the rumen for 0, 2, 6, 12, 24, 36, 48, and 72 h. Rumen cannulated lactating Holstein cows consuming a diet of corn silage, alfalfa, soybean, and high moisture corn were used. Degradability of protein varied from 18.6% for corn gluten meal to 72.3% for soybean meal. Gel electrophoresis was used to monitor rates of degradation in the rumen of fractions of corn gluten (CGM), CORN, cottonseed (CSM), peanut (PM), and soybean meal (SBM) protein fractions. Fractional degradation rates in the rumen were determined from densitometric analysis of stained polypeptides bands on SDS-PAGE gels. Acidic subunits of soybean glycinin were degraded at a faster rate than basic subunits (.144 vs .104 h⁻¹). Rates of degradation of zein in corn and corn gluten meal were .026 and .015 h⁻¹, respectively. Protein degradability estimated by using B subfractional components did not differ from degradability measured using total B fractions. Lag phase associated with dacron bags suspension technique did not change effective degradability. Protein solubility in SDS-PAGE sample buffer was highly correlated (R²=.958) with in situ protein degradability of CORN, CSM, DBG, FM, PM, and SBM. Different rates of degradation of each fraction may directly influence protein and amino acid contribution to the animal. / Master of Science

Three commercially prepared rumen cultures and fresh rumen liquor were compared to determine their effect on the rate of gain and efficiency of feed utilization of a) twenty-four steer calves, b) twenty-four two-year old steers in pasture, and c) twelve weanling lambs. Two of the cultures were prepared from the rumen contents of fast and slow gaining steers from experimental feeding tests, the other was a commercial product marketed under the trade name of Ruzyme. / Master of Science

Forage may undergo extensive proteolysis during fermentation. The objectives of this study were to determine if treatment of forage with heat can reduce proteolysis during subsequent fermentation. In Experiment 1, direct-cut barley and alfalfa were either microwaved or steamed then ensiled in laboratory silos as were untreated and wilted forage. Silages of microwaved or steamed forage showed marked increase in NDIN and recovery of hot water insoluble N; however, alfalfa silages also had high pH and butyric acid. In Experiment 2, steaming was compared to formic acid and anhydrous ammonia treatments for their ability to prevent proteolysis in alfalfa silages. Steamed and ensiled alfalfa also was evaluated with addition of microbial inoculant or formic acid. Silage of steamed alfalfa had greater NDIN and recovery of precipitable N than controls, formic acid, or ammonia treated silage. There was no difference in precipitable N between formic acid and ammonia treatments. Silage of steam treatment had lower pH than wilted or direct-cut controls, and additives to steamed forage favored a more homolactic fermentation. Additives to steamed forage also increased aerobic stability of the silage. Steamed silage had less aerobic stability than direct-cut silage. Rumen degradability of silage CP and OM from both experiments were evaluated. In Experiment 1, CP degradability of microwaved or steamed silages was 8 to 26% less than unheated silages, but all had similar undegraded CP after incubation for 72 h. In Experiment 2, wilting, steam, formic acid and ammonia treatments had similar, but decreased CP degradability when compared to direct-cut silage. Longer duration heat in Experiment 1 resulted in greater silage protein preservation, and greater decrease in rumen degradability of CP than Experiment 2. / Master of Science

Dairy cows are extremely inefficient at converting dietary nitrogen (N) to productive N. Approximately 25-30% of dietary N is used for milk protein while the remaining N is lost to the environment. According to National Research Council (NRC, 2001) recommendations, dairy cow rations are formulated in terms of metabolizable protein (MP) which often causes many amino acids (AA) to be fed in excess. A better understanding of protein and AA requirements could help to improve the nitrogen efficiency of dairy cows. The objective of this work was to examine the effects of feeding a low protein diet supplemented with rumen protected (RP) AA on production and N efficiency of dairy cows. Twenty-four Holstein and 24 Holstein x Jersey crossbred cows were used in a Youden square design consisting of 3 periods. Cows were randomly assigned to one of 8 treatments: 1) a standard diet containing 17% crude protein (+Con), 2) a 15% crude protein diet (-Con), 3) â Con plus RP methionine (+M, 16g/d), 4) â Con plus RP lysine (+K, 47g/d), 5) â Con plus RP leucine (+L, 181g/d), 6) â Con plus RP methionine and lysine (+MK), 7) â Con plus RP methionine and leucine (+ML), and 8) â Con plus RP methionine, lysine, and leucine (+MKL). Cows fed the â Con as well as the +MKL diet experienced a reduction in milk production and milk protein yield (P < 0.05). Dry matter intake decreased only for those animals on the +ML diet (P < 0.05). Milk urea N (MUN) decreased for all diets when compared to the +Con treatment (P < 0.05). In accordance with the decrease in MUN, N efficiency was numerically increased in the diets supplemented with RP AA, but this improvement was not significant. Phosphorylation of signaling proteins important for protein synthesis were also examined. Animals fed the +MK treatment increased phosphorylated and total forms of eukaryotic elongation factor 2 (eEF2) when compared to the +Con and â Con (P < 0.05), but this increase in abundance did not affect the ratio of phosphorylated to total abundance. Feeding dairy cows a low protein diet supplemented with RP AA has the ability to alleviate the loss in milk production associated with feeding a low protein diet as well as to increase nitrogen efficiency. / Master of Science

The variability in calf management can change the physiological state of the calf as they are weaned or attain puberty. It is up to the producer to ensure that the calves develop properly to meet their expected needs on the farm. While there are guidelines from the NRC in place, there is a substantial range in the amount of protein and fat that a calf can be fed. This physiological state can be reflected in the proteins produced in tissues, the expression of gene regulatory pathways, or even the microbes present in the gut. The purpose of this study was to examine how an increase in dietary energy in milk replacer of pre-weaned Holstein heifers impacts the microbial profile of the rumen as well as the transcriptome in tissues related to growth and metabolism. Our hypothesis was that pre-weaned Holstein heifers on milk replacer diets with lower dietary energy will have a different rumen microbiome composition and a different transcriptome in growth related tissues. Holstein heifer calves (n = 36) were assigned randomly to 1 of 2 milk replacer diets: restricted (R; 20.9% CP, 19.8% Fat; n = 18) or enhanced (E; 28.9% CP, 26.2% Fat; n = 18). Calves were euthanized and rumen fluid was collected at pre-weaning (8 wks; n = 6) or post-weaning (10 wks; n = 6). Liver (L), adipose (A), and longissimus dorsi (LD) tissues were collected at pre-weaning (8 wks; n = 12). Average daily gain (ADG) and gain-to-feed ratio (G:F) were calculated for each calf. Analysis of ADG and G:F was performed using a PROC GLM in SAS with diet as the main effect; E calves had increased ADG and G:F compared to R calves. For rumen samples, libraries were constructed from extracted DNA and DNASeq was conducted using a paired-end analysis at 100 bp using Illumina HiSeq 2500. Operational taxonomic unit (OTU) clustering analysis was conducted using the 16s rRNA Greengenes reference. A PERMANOVA analysis was conducted in R to determine OTU populations for age and treatment. There was no difference in microbiome composition between pre-weaning and post-weaning calves (P = 0.761). Microbiome composition differed between E and R calves (P < 0.001). Bacteroidetes and Firmicutes represented the most abundant phyla for both E and R calves. Enhanced calves had 49.4% (5141 reads) Bacteriodetes and 36.4% (3789 reads) Firmicutes; whereas, R calves had 31.6% (2491 reads) Bacteriodetes and 41.1% (3236 reads) Firmicutes. For L, A, and LD samples, libraries were constructed from extracted RNA for RNA-Seq analyses. RNA-Seq analysis was performed using CLC Genomics Workbench and the Robinson and Smith Exact Test was used to identify differentially expressed genes between diets. There were 238 differentially expressed genes in A, 227 in LD, and 40 in L. Of the differentially expressed genes, 10 appeared in at least 2 tissues. PANTHER was used to identify functional categories of differentially expressed genes. The majority of genes were associated with metabolic processes (A = 112, 26.7%; L = 16, 32.0%; LD = 81, 34.0%) or cellular processes (A = 93, 22.1%; L = 13, 26.0%; LD = 73, 30.7%). In E calves, upregulated genes included those regulating NADH dehydrogenation (LD = 17, A = 5; i.e. ND1, ND4), gluconeogenesis (LD = 2, A = 6; i.e. ALDOB, PCK2), and cell proliferation (LD = 2, A = 3; i.e. GADD45A, CDKN1A). There was a difference in both the transcriptome and rumen microbiome of calves fed differing levels of dietary energy. The calves on the R diet had a rumen microbial composition more similar to a younger calf, while the composition of E calves was more similar to a mature calf. The change in regulation of genes involved in the cell cycle and ATP synthesis in response to dietary energy could explain the change in ADG between diets. Because the R calves appeared to have stunted development of their microbiomes and an expression profile similar to oxidative stress, it is possible that the R diet did not meet the nutritional requirements of that calves. / Master of Science / Changes in the way a calf is raised from birth can affect the biological processes that occur when they change from liquid to solid feed or reach reproductive maturity. While there are guidelines in place in how much a calf should be fed, there is still a large range in the amount of protein and fat in the liquid feed. The change in nutrition levels changes the biological processes occurring in the calf, which are reflect by changes in expression of genes in different parts of the calf as well the levels of microbes in the gut. The purpose of this study was to examine how the change in protein and fat in the liquid feed of female calves affects the microbes in the first section of the stomach, the rumen, as well as the genes expressed in parts of the calf associated with growth. Our hypothesis was that female calves fed liquid diets with lower protein and fat will have different rumen microbes and a different level of gene expression in growth related tissues. Female calves (n = 36) were randomly assigned 1 of 2 diets at birth: restricted (R; 20.9% Crude Protein, 19.8% Fat; n = 18) or enhanced (E; 28.9% Crude Protein, 26.2% Fat; n = 18). Calves were euthanized and rumen contents were collected at removal of the liquid feed (8 wks; n = 6) or 2 wks after calves were switched to an all dry feed diet (10 wks; n = 6). Liver (L), adipose (A), and longissimus dorsi (LD) tissues were collected at removal of the liquid feed (8 wks; n = 12). Bacterial DNA was extracted from the rumen samples and RNA was extracted from L, A, and LD samples. DNA and RNA were sequenced at the University of Missouri DNA Core Lab. Microbiome composition differed between E and R calves (P < 0.001). Enhanced calves had 49.4% Bacteriodetes and 36.4% Firmicutes; whereas, R calves had 31.6% Bacteriodetes and 41.1% Firmicutes. There were 238 differentially expressed genes in A, 227 in LD, and 40 in L. Of the differentially expressed genes, 10 appeared in at least 2 tissues. In E calves, upregulated genes included those regulating NADH dehydrogenation (LD = 17, A = 5; i.e. ND1, ND4), gluconeogenesis (LD = 2, A = 6; i.e. ALDOB, PCK2), and cell growth (LD = 2, A = 3; i.e. GADD45A, CDKN1A). There was a difference in both the gene expression and rumen microbiome of calves fed differing levels of protein and fat. The calves on the R diet had a rumen microbial composition more similar to a younger calf, while the composition of E calves was more similar to a mature calf. Because the R calves appeared to have stunted development of their microbiomes and an expression profile similar to oxidative stress, it is possible that the R diet did not meet the nutritional requirements of that calves.

The objective of this study was to determine how the key features of bioreactor landfills; increased temperature, moisture and microbial activity, affect the biological stability of the landfill material. In the first part of the study the solubilization and degradation of lignin in paper exposed to these bioreactor landfill conditions are explored. The solubility of the lignin in paper was observed at different temperatures and over 27 weeks at 55°C and the anaerobic bioconversion of office paper, cardboard and Kraft lignin was observed in bench-scale reactors over 8 weeks. As the temperature rose, lignin solubility increased exponentially. With extended thermal treatment, the dissolution of lignin continues at a constant rate. This rate increases 15 times for paper and 1.5 times for cardboard in the presence of rumen inoculum compared to un-inoculated systems. At around 6 weeks the inter-monomeric linkages between the solubilized lignin molecules began breaking down, releasing monomers. In cardboard and Kraft lignin, a significant amount of the monomers mineralize to CO₂ and CH₄ during this time period. The results indicate that small, but significant rates of lignin solubilization and anaerobic lignin degradation are likely to occur in bioreactor landfills due to both higher temperature and microbial activity. In the second part of the study, field data from the Outer Loop Recycling and Disposal Facility in Louisville, Kentucky was evaluated to determine the effectiveness of an anaerobic-aerobic landfill bioreactor (AALB) vs. the control landfill that is managed as a traditional landfill. Moisture, temperature, elevation and the amount of time the MSW has spent in the landfills (age) were measured and compared to determine the factors that affect the biological stability of the landfill. The results showed that the MSW in the AALB is more biologically stable than the MSW in the control landfill, indicating that they are more degraded. Additionally, elevation or location of the MSW was the key factor in determining the extent of MSW stability within the AALB and temperature is the key factor in determining the biological stability of the MSW in the control landfill. Higher temperatures correlated with a more biologically stable waste. The cellulose to lignin ratio (C/L ratio) and biochemical methane potential (BMP) were the main biological stability parameters used. / Master of Science

Objectives were to determine dry matter, fiber and nitrogen fractions, and in vitro and in situ degradability of forages and by-product feeds, and to compare in vitro methods of estimating rumen degradability with the in situ bag technique. Feeds analyzed with number of samples in parentheses included alfalfa as baled hay (23), alfalfa ensiled in conventional (43), and. oxygen limiting silos (39), ammonia· treated (25), and untreated corn silage from conventional (17) and bunker silos (17), rye (25), sorghum (7), wheat (6), barley (5), and orchardgrass (4) silages, orchardgrass (19) and fescue hay (3), and dried distillers grains dark colored (2) and light (1), wet brewers grains (1), and whole cottonseeds (3). Samples were analyzed for dry matter, crude protein, buffer-soluble protein, protease insoluble nitrogen, neutral and acid detergent fiber and insoluble nitrogen, and in situ degradability of nitrogen, dry matter, and fiber. Protease insoluble nitrogen, buffer-insoluble protein, and neutral detergent insoluble nitrogen were lowest for alfalfa from conventional upright silos. Oxygen limiting silo samples had greater dry matter, insoluble protein, and bound nitrogen compared to conventional upright silo samples. Oxygen limiting silos had 35.9% of samples with bound nitrogen greater than 15% of total nitrogen compared to 14% of conventional upright silo samples. Baled hay and oxygen limiting silo samples had similar protease insoluble nitrogen, however, ensiled samples had greater bound nitrogen. In situ nitrogen degradability was greatest for ensiled forages compared to hays. Ensiled forages had the greatest A fraction (rapidly solubilized), alfalfa hay the greatest B fraction (slowly degraded), and orchardgrass hay the greatest C fraction (not degraded). Degradation of dry matter and fiber followed similar patterns for each forage and by-product. Significant results were found by comparing in vitro and in situ techniques for estimating degradability. Due to differences between hay and silage, use of one technique can not be recommended at this time to predict degradability. For silage, the best measure related to in situ degradability was buffer-soluble protein; for hay, the best measure was neutral detergent insoluble nitrogen. / Ph. D. / incomplete_metadata

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

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.

La « biotransformation ruminale » est un concept qui regroupe l'ensemble des réactions se produisant dans le rumen (dégradation, synthèse et conversion). Ces réactions sont pilotées par trois forces motrices majeures : les lois de la cinétique chimique, de la thermodynamique et de la dynamique des populations microbiennes. Cette thèse a pour objectif d'étudier l'impact d'un facteur alimentaire (pourcentage de concentrés incorporés dans la ration, supplémentation en huile) sur le fonctionnement du rumen et la biotransformation ruminale des constituants alimentaires en termes de dynamique et d'intensité. Cette thèse a combiné trois types d'approches : un essai in vivo sur des chèvres en milieu de lactation, deux essais in vitro (méthode du gaz-test) et une approche de modélisation mécaniste. In vivo, les réactions de biotransformation ont été évaluées par un suivi de la dynamique postprandiale et des mesures de bilans duodénaux. De plus, nous avons étudié l'influence réciproque des phénomènes ruminaux et de l'animal-hôte (comportement d'ingestion, métabolisme, paramètres zootechniques, et qualité du lait) sur le long terme (6 semaines). Nos résultats sont en accord avec la plupart des études antérieures effectuées chez la chèvre ou la vache laitières. Les modifications du comportement d'ingestion observées après 6 semaines avec le régime riche en concentrés ont eu un effet sur les phénomènes digestifs ruminaux. Les flux duodénaux d'acides gras ont expliqué les profils en acides gras du lait. Les études in vitro ont donné des résultats très cohérents par rapport à l'in vivo lorsque les animaux donneurs recevaient les régimes incubés. Enfin, nous avons développé un modèle mécaniste de fonctionnement de rumen in vitro décrivant de manière spécifique les lois physico-chimiques expliquant les dynamiques d'évolution du pH et de formation de gaz. Ce modèle aboutit à des résultats satisfaisants et pourrait être intégré à un modèle de rumen plus complet. La modélisation semble être le meilleur moyen pour intégrer toutes les réactions de biotransformation observées lors d'expérimentations. / “Ruminal biotransformation” is a concept aggregating all the reactions occurring in the rumen (degradation, synthesis and conversion). These reactions are driven by three major driving forces: laws of chemical kinetics, thermodynamics and the dynamics of microbial populations. The principal objective of the thesis was to study how a dietary factor (percentage of concentrate in the ration, oil supplementation) can modify rumen function and the ruminal biotransformation of dietary components in terms of pattern and extent. To answer to this question, three approaches were used: an in vivo experiment on mid-lactation goats, two in vitro experiments (with the gas-test method) and the development of a mechanistic model. In vivo biotransformation reactions were assessed by post-feeding measurements or by duodenal flow determination. Further, the relationships between the rumen and the host animal (intake behaviour, metabolism, performance parameters, and milk quality) were studied over a period of 6 weeks. Our results were consistent with previous reports in goats and dairy cows. Animals adapted their intake behaviour in 6 weeks with the high-concentrate diet. As a consequence, modifications of ruminal digestion patterns were observed. Duodenal flow of fatty acids explained the milk fatty acid profile. The in vitro studies gave consistent results with the in vivo study when donor animals were fed the incubated diets. Finally, a mechanistic model of rumen functioning in in vitro devices was developed. It describes specifically the physicochemical laws explaining pH and gas production patterns. This model gives satisfying results and could be integrated to a larger mechanistic model of rumen. Modelling provides a logical framework within which to mathematically integrate all the biotransformation reactions observed in experiments.