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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.

Call number: LD2668 .T4 1984 A43 / Master of Science

Includes bibliographical references (leaves [259]-290) Investigates nitrogen assimilation and metabolism in rumen bacteria with the object of understanding the basic process and their controls.

Includes bibliographical references (leaves 122-138). / xvii, 138 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Investigates the use of microbial inoculants to prevent the onset of acidosis in acutely grain fed animals; and, the most effective combination of virginiamycin and lactic acid utilising bacteria (selenomonas ruminantium subsp. lactilytica and Megasphaera elsdenii) in controlling lactic acid accumulations in vitro. / Thesis (Ph.D.)--University of Adelaide, Dept. of Animal Science, 1995

Includes bibliographical references (leaves [259]-290) / xxviii, 290 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Investigates nitrogen assimilation and metabolism in rumen bacteria with the object of understanding the basic process and their controls. / Thesis (Ph.D.)--University of Adelaide, Dept. of Animal Sciences, 1991

Bibliography: leaves 122-138. Investigates the use of microbial inoculants to prevent the onset of acidosis in acutely grain fed animals; and, the most effective combination of virginiamycin and lactic acid utilising bacteria (selenomonas ruminantium subsp. lactilytica and Megasphaera elsdenii) in controlling lactic acid accumulations in vitro.

The yeast expression system Pichia pastoris was investigated as an encapsulation technology capable of serving as a rumen escape vehicle. Cellularly encapsulated protein is protected from the ruminal environment so long as the cell membrane, which surrounds and isolates the intracellular protein is physically intact. Intracellular expression of Green Fluorescent Protein (GFP) allows for the monitoring of cellular integrity as necessary for the protection of encapsulated protein from ruminal proteases. Upon cellular lysis GFP is exposed to extracellular proteases which result in both the proteolytic degradation of the protein-based GFP chromophore and its associated fluorescence. Visualization of rumen fluid under epifluorescent microscopy revealed a high level of background autofluorescence owing to the fluorescent plant particles, microbes, and fluorescent compounds therein. Visualization of GFP in rumen fluid can be optimized through GFP variant selection, filter set design, and light source selection based on bulb emission spectra. Incubation of intracellular GFP expressing P. pastoris in batch culture ruminal in vitro simulations demonstrated that 93%, 97%, and 25% of the P. pastoris inoculum maintained cellular integrity in clarified rumen fluid, bacterial fraction of rumen fluid, and whole rumen fluid, respectively, when incubated over 36 to 48 h. Continuous fermentation in vitro rumen simulations (Rusitec) demonstrated a P. pastoris escape rate of 19% when added daily to fully adapted Rusitec vessels having a dilution rate of 0.75d-1. Abomasal in vitro simulations demonstrated that 84% of the P. pastoris inoculum was lysed within 12 h, as necessary for the release of encapsulated protein. P.pastoris may be an effective post-fuminal delivery vehicle, provided that similar results are obtained in vivo. / xiv, 120 leaves : ill. ; 28 cm.

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

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