Production of ammonia N, a-amino N, and peptide N was investigated following in vitro ruminal incubation of solvent soybean meal (SBM), dehydrated alfalfa, corn gluten feed, fish meal, distillers dried grains with solubles (DDG), cotton seed meal, brewers fried grains, meat and bone meal, blood meal, prolac, and casein (CAS). The influence of milling procedures on the production of ammonia N, a-amino N, and peptide N was also evaluated using different batches of soybean meals and distillers dried grains with solubles. The concentrations of peptide N and ammonia N measured in the cell free media at 0, 2, 4, 6, and 8 h were increased linearly (P < 0.001) with time. The mean concentrations of a-amino N were lower (P < 0.05) than the mean concentrations of peptide N and ammonia N. Production of peptide N, a-amino N, and ammonia N were varied (P < 0.05, time x protein) between proteins and between batches. Irrespective of the protein used, the amino acid composition of peptides (<3,000 MW) that appeared at 8 h had specific patterns suggesting differential utilization of peptides by ruminal microorganisms. Cell-free supernatants obtained following incubation (8 h) of SBM, CAS, and DDG were used as mucosal substrates in parabiotic chambers to quantify absorption of free and peptide-bound amino acids via ruminal and omasal epithelia of sheep. Serosal appearance of amino acids in peptide form was nearly three times higher (P < 0.001) than free amino acids. On tissue dry weight basis, serosal appearance of amino acids was greater (P < 0.01) across omasal than via ruminal tissues. There was a greater serosal appearance of amino acids from CAS than from SBM. Total, total essential (EAA), total nonessential (NEAA), and individual amino acid appearance in serosal fluids varied (P < 0.05, amino acid form x protein source) among SBM, CAS, and DDG. Collectively, these results indicate that the forestomach epithelia of sheep possess the potential to absorb ruminally derived peptides (relatively large amounts) and free amino acids (relatively small amounts). Also, the ruminal microbial degradation of dietary proteins may influence the amounts and types of free and peptide-bound amino acids absorbed via forestomach. / Ph. D.

Colony counts of proteolytic ruminal bacteria in the order of 1 x 10⁹ organisms per gram of whole rumen contents and total colony counts in the order of 2 to 3 x 10⁹ organisms per gram were obtained from rumen contents of cattle fed a maintenance ration of hay and grain. The proteolytic counts averaged 381 of the total counts. An anaerobic, differential medium characterizing proteolytic colonies by clear zones in an opaque skim milk suspension was utilized. Proteolytic isolates were assigned to the following taxa; Butyrivibrio fibrisolvens, Succinivibrio dextrinosolvens, Selenomonas ruminantium var. lactilyticas, Borrelia, Bacteroides (butyric acid-producing R•2 group of Bryant), and selenomonad-like organisms similar to B-385 group of Bryant. 1 A portion of the Ph.D. dissertation by the senior author. 2 Present address: Division of Natural Sciences, Susquehanna University, Selinsgrove, Pennsylvania. Proteolysis in the ruminal fermentation may benefit the host animal if the resulting products are later synthesized to digestible microbial proteins of higher biological value than the feed protein, or conversely this activity may be detrimental because of net protein loss. In spite of the nutritional significance of this activity the proteolytic ruminal bacteria have received relatively little attention as a physiological group. Studies have largely been restricted to observations of the degradation of gelatin or casein incidental to other studies of ruminal organisms. Gelatin proteolysis was reported among isolates from the rumen by Bryant (1951), Bryant and Burkley (1953a, 1953b, 1953c), Bryant and Small (1956a), Bryant et al. (1958a), Hamlin and Hungate (1956), Buhtanen and Gall (1953), Hungate (1957), and Hann et al. (1954). Bryant and Doetsch -- (1954) also reported isolates which attacked casein but not gelatin and Bryant (1956) reported a strain of Selenomonas ruminantium which digested casein but not gelatin. Bryant (1959) revealed the paucity of information on the proteolytic flora of the rumen in his excellent review of the bacteriology of the rumen. A casein medium designed for the isolation of proteolytic ruminal bacteria was described by Appleby (1955). Blackbum and Hobson (1960a) found proteolytic activity in all fractions of rumen contents (protozoa, large bacteria and small bacteria) and they initiated isolation of proteolytic bacteria from the ovine rumen (Blackburn and Hobson, 1960b). Fulghum (1958) described the development of two anaerobic, differential media for the isolation and enumeration of proteolytic ruminal bacteria, these media were developed through modification of the media of Hamlin and Hungate (1956) and ling and Smith (1955), and of the medium used by Donovan and Vincent (1955) for studying proteolytic organisms from milk. Fulghum (1958) found the optimum level of clarified rumen fluid added to these media to be 401. Colonies of proteolytic organisms in these media were characterized by clear zones in opaque skim milk or plant protein suspensions in the media. Plant proteins failed to maintain a uniform opacity and were therefore of limited value in delineating the proteolytic segment of the flora even though the plant proteins stimulated total counts by a factor of from three to five. Earlier Fulghum et al. (1958) reported that dispersion of whole rumen contents in anaerobic diluting fluid in a blendor increased total counts by a factor of four when compared with total counts obtained from rumen fluid samples which were diluted by shaking in anaerobic diluting fluid. Proteolytic counts were the same from both inocula. In later studies (Fulghum, 1958), proteolytic counts were also found to be increased by a factor of four when dispersed whole contents were compared with shake dilutions of rumen fluid. Proteolytic and total counts were found to be slightly higher in the dorsal sac of the rumen than in the ventral sac, although this phenomenon was variable with regard to time of sampling following feeding of animals. Similarly, the ratio of proteolytic to total counts varied at different times following feeding. The proteolytic flora remained constant while the total counts varied. The sequence of fluctuation was different in each individual animal. / Ph. D.

Volatile fatty acids (VFA) are important products of ruminal fermentation. The VFA are not only the major source of energy to the ruminant animals but also influence methane production in the rumen. Therefore it is important to understand mechanism controlling VFA production and to depict VFA production in a model. This will allow us to devise strategies to enhance energy utilization and reduce methane production in ruminant livestock. An evaluation of a mechanistic model in predicting VFA production was conducted and equations were introduced into the model to improve the predictions. Later a continuous culture experiment was conducted to test the hypothesis on which those equations were based on. A mechanistic model -" Molly, was evaluated using a dataset with reported VFA production rates. The results of residual error analysis indicated that the root mean square prediction errors (RMSPE) were 63, 63, and 49% for acetate, propionate and butyrate, respectively. An assessment from two studies reporting VFA production revealed a potential of reducing errors of prediction by representing interconversion among VFA. In the second study, equations based on thermodynamics influence of pH and VFA concentration were introduced in the model to represent interconversion among VFA. The parameters for de novo VFA production and VFA absorption were re derived with (VFAInt) and without (BASE) the new interconversion equations. There were some improvements in the VFA concentration predictions but the improvements were both in VFAInt and BASE models. The RMSPE of VFA production were still above 50% for acetate, propionate and butyrate. The larger errors of predictions were attributed to measurement variation in VFA production literature, or possible incorrect rate constants for interconversion equations. Finally, a third study was conducted to assess the effect of pH, and VFA concentration on VFA and methane production in continuous culture. The treatments consisted of control, 20 mmol/d acetate infusion (INFAC), 7 mmol/d propionate infusion (INFPR), and low pH (LOWPH). Individual isotopes of acetate, propionate and butyrate were infused in the fermenters to estimate interconversions among VFA. With LOWPH treatment methane emission was reduced whereas production of propionate was increased. Hydrogen production was higher in INFAC indicating that some of the acetate could have been degraded to CO2 and H2. It was estimated that around 3 % of de novo acetate was converted to propionate and 9 % to butyrate. Exchange between propionate and butyrate was insignificant and below 1% of de novo production of either VFA. However, treatments did not affect interconversion rates among VFA. These results indicated that pH and VFA concentration do not have thermodynamic influence on VFA interconversion as hypothesized. / Ph. D.

The dairy industry today is beginning to dedicate more focus on the growth of the calf from birth to first breeding to better improve the milk production as well as the overall performance of the individual cows. While the development of the rumen is one of the most vital contributors to the performance of the calf, it remains unknown what molecular mechanisms are responsible for the development of the rumen, and more specifically the proliferation of rumen epithelial cells. The objectives of this study were to investigate the existing data on rumen development through meta-analysis and to explore the effects of sodium butyrate and lipopolysaccharide (LPS) on rumen development in calves through experiment. In the first study a meta-analysis was performed to summarize the literature on calf performance and derive equations that relate rumen (e.g., rumen pH, reticulorumen weight, papillae area) and non-rumen factors (e.g., feed composition, form of feed, housing) to animal performance (e.g., intake of milk replacer (MR), starter, and forage; average daily gain (ADG); and feed efficiency). We looked at four different relationships to further investigate the connections between rumen, non-rumen, and performance factors. In the first and second relationships of interest, the effect of dietary and environmental variables on rumen variables and performance variables were examined, respectively. The third relationship of interest was how rumen variables influenced performance variables. The final relationship of interest was investigating the additive effects of the rumen, dietary, and environmental variables on the performance variables. Forward selection, multiple regression was used to derive equations to select variables that explained variation in the response variable in each model. Results showed that the variation in calf ADG was explained by daily forage intake, calves that were weaned, total starter intake, and total MR intake (concordance correlation coefficient (CCC) = 0.976). The variation in feed to gain ratio was explained by the weight of the ruminal contents, daily forage, MR, and starter intakes, percent of starter in the diet, and total starter intake (CCC = 0.992). The variation in daily forage intake was explained by the percent of the diet that was starter or MR (CCC = 0.998). The variation in daily starter intake was explained by the percent of acid detergent fiber in the starter, a pelleted starter (versus a texturized), diets including starter and forage (versus a milk replacer only diet), and the percent of the diet that was MR (CCC = 0.998). The variation in daily MR intake was explained by the percent of the diet that was starter, final body weight, ruminal propionate concentration, and daily starter intake (CCC = 0.918). Based on these analyses, although dietary and environmental factors are closely associated with calf performance, ruminal factors such as volatile fatty acid (VFA) concentration and ruminal contents appear to have additional, additive influences on calf performance. In the second study, 24 Holstein bull calves were challenged with oral doses of LPS and sodium butyrate. The hypothesis here was that LPS and sodium butyrate would instigate rumen cell proliferation independently and additively. Calves were assigned to one of four treatments: control (CON; n=5), butyrate (BUTY; n=5), LPS only (LPS-O) (n=6), or LPS plus butyrate (LPSB; n=6). All treatments were administered orally twice daily consisting of either: 0.9% saline (CON); 11 mM sodium butyrate (BUTY); LPS ranging from 2.5 to 40 µg/kg metabolic body weight (BW0.75, LPS), or both butyrate and LPS (LPSB). Calves were fed milk replacer (22% CP, 20% fat, as-fed) and starter (20% CP, 3% fat, as-fed) based on metabolic BW, or about 12% BW of MR and 3% BW of starter. Feed intake, fecal and respiratory scores, and rectal temperature were recorded daily. Calf BW, hip height, jugular blood samples, and rumen content samples (via oroesophageal tube) were collected weekly. Calves were weaned at 6 wk of age and euthanized at 8 wk of age, whereupon ruminal weights and ruminal samples for papillae area and epithelial thickness were collected. Blood and rumen samples were analyzed for concentrations of beta-hydroxybutyrate, glucose, LPS-binding protein, and VFA. Data were analyzed as a 2x2 factorial with the repeated effect of week. Three non-orthogonal contrasts (CON versus the average of all other treatments; LPS-O versus LPSB, and LPSB versus BUTY) were investigated. Feed intake, health measures, and blood metabolites did not differ by treatment. Calf BW increased by week (P < 0.0001). Irrespective of week, LPS calves weighed more and had higher ADG than BUTY calves (P = 0.020). Irrespective of week, withers height was greater in LPS compared to CON (P = 0.006). Rumen pH and rumen VFA concentrations did not differ by treatment but did decrease and increase, respectively, with week in conjunction with increased starter intake. Total empty forestomach (P = 0.014) and reticulorumen weights (P = 0.012) were greater in LPSB compared to BUTY. Overall, LPS and sodium butyrate appeared to have synergistically affected some, but not all rumen measurements without affecting calf growth, intake, or health. Results from the meta-analysis emphasize the importance of continuing to focus on the solid feed intake of the calf from birth through weaning. Implications from the LPS study are imperative to other dairy scientists who will attempt to further study the effects of LPS on the rumen. / Master of Science in Life Sciences / Dairy calves are born with an under-developed stomach. The stomach has four compartments: the rumen, reticulum, omasum, and abomasum. The rumen is the largest component where finger-like projections called papillae grow to absorb nutrients for the calf. It is vital to the calf that the rumen develops not only the papillae to absorb nutrients but also to foster a microbe-rich environment so the microbes can act as a defense mechanism for the calf to aid in fighting disease. While it is known that things like solid feed support the development of the rumen, the mechanism behind how that is happening still remains unclear in the literature. The objective of this study was first to better understand the relationships that exist in the literature between dietary, environmental, and ruminal factors, and second to investigate the claim that certain components of the bacteria in the rumen are stimulating rumen development independently and additively with sodium butyrate. In order to investigate the relationships amongst the dietary, environmental, and ruminal parameters, a computer program called R Studio was used to analyze over 30 different models that extracted data from a database that included a collection of 36 studies from the literature. This is also known as a meta-analysis. The associations of interest that we found were: average daily gain (ADG) of the calf was associated with daily forage intake, calves that were weaned, total starter intake, and total MR intake. Feed efficiency of the calf was associated with the weight of the ruminal contents, daily forage, milk replacer (MR), and starter intakes, percent of the diet composed of starter, and total starter intake. Daily forage intake was associated with the percent of the diet that was starter or MR. Daily starter intake was associated with acid detergent fiber in the starter, a pelleted starter (versus a texturized starter), diets including starter and forage (versus a MR only diet), and the percent of the diet that was MR. Daily MR intake was associated with the percentage of the diet that was starter, final body weight (BW), ruminal propionate concentration, and daily starter intake. These relationships emphasized that although dietary and environmental factors are more closely associated with calf performance, ruminal factors such as rumen contents and volatile fatty acid concentrations appear to have additional, additive influences on calf performance. The second part of the study objective was to explore an idea that, to our knowledge, has not been published in the literature. In the second study, 24 dairy calves were challenged with oral doses of a gram-negative bacteria lipopolysaccharide (LPS), and a short-chain fatty acid sodium butyrate. The hypothesis in this study was that the LPS and sodium butyrate would trigger metabolic pathways on the rumen cell membranes to a greater extent together, versus independently, to increase the amount of cells growing. Calves were assigned to one of four treatments: control (CON), butyrate (BUTY), LPS only (LPS-O), or LPS plus butyrate (LPSB). To study this effect, each treatment group was administered their respective treatment orally as a liquid twice daily. To measure the results, the following data was collected: feed intake, fecal and respiratory scores, rectal temperature BW, hip and withers height, blood samples, rumen content and pH samples, papillae area, epithelial thickness, and organ weights. Blood and rumen samples were analyzed for blood metabolites and volatile fatty acids concentrations respectively. Data were analyzed and results showed no difference amongst feed intake, health measures, rumen pH, rumen VFA concentration, and blood metabolites by treatment. Calves on the LPS treatment weighed more and had higher ADG than BUTY treatment calves. Withers height was higher in the LPS group when compared to CON. Stomach weights were higher in the LPSB group when compared to the BUTY group.

Ten strains of anaerobic, gram negative, monotrichous, butyric acid-producing curved rods have been isolated from ingesta of the bovine rumen. These 10 strains of butyrivibrio represented 1/5 of all isolates at 1 x 10⁻⁸ dilutions. Morphological and physiological characteristics of the 10 strains and a strain isolated by gill and king (1958) have also been studied. No two of the isolates were identical in all reactions. Most of the organisms produced a large amount of butyric and some lactic, formic, propionic and succinic acids with the utilization of acetic acid in a rumen fluid glucose medium. The fermentation carried on by these organisms was sensitive to most tested environmental changes. Studies with buffered rumen fluid-glucose media demonstrated a shift of the fermentation products with pH. Addition of fatty acids to this medium indicated that these organisms were active in the conversion of acetate and possibly propionate to butyrate. Two strains apparently had the ability to produce propionate at the expense of lactate. The results of the fermentation tests in 98 per cent rumen fluid medium showed that the tested strains used acetic (plus formic) or lactic (plus succinic) to produce butyric or propionic acid, and produced higher concentrations of fatty acids under a carbon dioxide atmosphere than under nitrogen. When rumen fluid and acetic acid were absent all strains had the ability to produce either formic or acetic acid. / Master of Science

One or more factors which occur in bovine rumen fluid stimulate the growth of a strain of Butyrivibrio. The stimulating material is heat stable, organic in nature and non-dialyzable. It cannot be extracted from rumen fluid with lipid solvents and is retained in part on anion and cation exchange resins. It can be eluted from the resins with strong acid. It is stable to enzymatic hydrolysis by trypsin. Granular mucin or bovine saliva will partially replace the stimulatory activity. The part of the material which was not replaced by mucin did not appear to be any compound that is commonly used to stimulate bacterial growth. The presence of a possible inhibitor for the growth of a strain of Butyrivibrio was demonstrated. / Master of Science

Primary Objective: To find a medium, defined as clearly as possible in terms of its nitrogenous components, which will support growth of a cellulolytic rumen bacterium. Secondary Objectives: 1. To test an hypothesis that the optimum nitrogenous nutrient balance for a strongly heterotrophic organism is that mixture of the basic structural molecules ( amino acids, purines, and pyrimidines ) which most closely approximates the composition of the organism itself. 2. To apply this hypothesis toward the goal set in the first objective. / Master of Science

A procedure was developed for the quantitative separation of major fermentation products of uniformly labeled glucose-C¹⁴ produced by bovine rumen microorganisms in vitro. After 45 min, the fermentation mixture was fractionated into (a) one control subsample, and duplicate fractions of (b) solid matter “precipitate“, (c) ether extract, (d) “amino acid“, (e) “sugar“, (f) CO₂, and (g) CH₄. Similar fractionation of an unfermented control sample was made. A portion of the fermentation ether extract was subjected to column chromatography to resolve (a) C₁, (b) C₂, (c) C₃, (d) C₄, and (e) C₅ fatty acids, (f) succinic, and (g) lactic acids. Each fraction was analyzed in triplicate for C¹⁴ by a direct plating technique. Corrections for geometry, self absorption, and efficiency were made by direct plating additional triplicate fraction subsamples, each containing a uniformly labeled glucose-C¹⁴ internal standard. The data were expressed as per cent recovery of added C1u. The results indicated that glucose was rapidly fermented with most of the C¹⁴ found in the ether extractable fraction as acetic acid. Significant levels of C¹⁴ were found in the “precipitate“ fractions. The data were compatible with evidence that CH₄ was derived from CO₂. The results of 6 trials indicated that there was no significant difference in the distribution of products resulting from the in vitro fermentation of uniformly labeled glucose-C¹⁴ between animals, between days within animals, or between times within days. / Ph. D.

Two media were adapted to the culture of proteolytic bacteria from the bovine rumen. Modifications were made in the double indicator dairy medium of Donovan and Vincent (SRBP) and in the medium of Hungate for rumen bacteria (SRP). Modifications included use of plant protein suspensions, casein, and skim milk as the nitrogen sources of which skim milk was the most suitable, producing a uniformly opaque medium. Proteolytic colonies were characterized by clear zones in the medium. A third medium containing the artificial sheep-saliva salts mixture of McDougall was developed but was found unsatisfactory for the study of proteolytic organisms. Dilutions of rumen contents to 1 x 10⁻⁸/ml were made in anaerobic dilution fluids. Cultures were grown in roll tubes or bottles containing CO₂ atmosphere. All of the media used in this study repeatedly produced an average count of 40 colonies per tube with 10⁻⁸ dilutions of comminuted whole rumen ingesta as inocula. The average ratio of proteolytic to total colonies was found to be 1 to 5. Each of the media was compared for its ability to support the proteolytic isolates from the other. Minor differences in the specificity of the media were found to exist. Colonial and morphological studies of the proteolytic isolates were made. / Master of Science

There has been a growing interest in development of nature-inspired miniature mobile robotics, for navigating complex ground scenarios, unknown terrains, and disaster-hit areas. One application is the development of a remotely operated vehicle (ROV) for rumen monitoring to improve our understanding of microbiology, and real-time physical changes and correlations with health. This interest is being driven from the desire to improve the safety and efficiency of food production by improving precision animal agriculture, which involves understanding the digestive system of ruminant animals and responding to the biochemical and physical changes. Most miniature robotic locomotion methods have taken inspiration from insects and have focused on adopting approaches that results in improved gait performance with respect to stability, velocity, cost-of-transport, and ability to navigate uneven surface terrains. In order to operate in the rumen environment, the locomotion mechanism should have the ability to handle large frictional and viscous forces in the direction of motion performing submerged burrowing-like action. The rumen environment consists of varying stiffness content with different fluidic concentration across the layers, reaching high viscosity and densities similar to wet soil or mud. Taking inspiration from millipedes for a locomotion mechanism to function in such an environment is attractive as these organisms have evolved to be proficient burrowers in similar substrates. In this dissertation, the bio-mechanics of millipedes were investigated in-depth and modeled using analytical approaches. Multiple experiments were conducted on real animals to gain fundamental understanding of their locomotive abilities under varying environmental conditions. From this understanding, their gait behavior was emulated on a robotic platform to confirm the predicted dynamics and practically demonstrate the phenomena of modulating thrust force. The robotic models were also utilized to validate the parametric analysis and gain insight of the burrowing ability in varying gait behavior and body morphology. The primary features that govern the millipede behavior for effective burrowing were analyzed and utilized to design a locomotion mechanism for a rumen ROV. The design of the locomotion mechanism was tested in rumen-like media consisting of a wet mud mixture, where both locomotion thrust and steering ability were demonstrated. / Ph. D. / In this dissertation, the movement of millipedes utilize to traverse effectively within an environment that provides significant resistance is studied. Through various experimental observations and mathematical modeling, we are able to develop an understanding of the techniques millipedes use to be effective burrowers. To validate our model and understanding the millipede movement techniques, a robot was designed to emulate a millipede’s body structure and movement behavior. The performance of the millipede robot was found to be consistent with that of the biological creatures, indicating that we are able to emulate their behavior to achieve desirable tasks. With this developed understanding of the fundamental concepts that allow millipedes to effectively move against large resistances, we introduce the ability to design robots or devices that can achieve similar performance for various applications ranging from search and rescue to health inspection. One such application is a device that traverse within the stomach (rumen) of dairy cows to investigate its biological features and characteristics for improvement in animal agricultural efficiency. The fundamental concepts of millipede motion are translated to a rumen monitoring vehicle design, which would operate in a wet-soil-like environment, similar to millipedes. The device motion techniques are demonstrated, an indication of successfully transferring the fundamental mechanism used by millipedes for an engineering application.