Reductive acetogenesis via the acetyl-CoA pathway is a hydrogenotrophic pathway that has the potential to reduce methanogenesis from ruminant livestock. However our understanding of the organisms capable of this transformation (acetogens) is hindered by a lack of specific molecular tools for this group. In the present thesis, a PCR primer set specific for a wide range of acetogens was developed, targeting the acetyl-CoA synthase (ACS) gene which is unique to the acetyl-CoA pathway. ACS was found to be useful marker for potential acetogens and ACS sequences could be used to infer family-level phylogeny for many acetogens. ACS gene specific primers were used in combination with existing molecular tools targeting the gene encoding formyltetrahydrofolate synthetase (FTHFS, present in the acetyl-CoA pathway but not unique to it) and 16S rRNA genes, as well as cultivation techniques, to investigate acetogen diversity in the rumen and two analogous gut systems where microbial hydrogenotrophy differs: the forestomach of a native Australian marsupial, the tammar wallaby Macropus eugenii; and the developing rumen of young lambs. Novel potential acetogens present naturally in the rumen of pasture fed and grain fed cattle affiliated with the Ruminococcaceae/Blautia group and distantly with the Lachnospiraceae. A large diversity of potential acetogens with functional genes affiliating broadly between the Lachnospiraceae and Clostridiaceae though without a close sequence from a cultured relative were also detected. Rumen acetogen enrichment cultures revealed the presence of a known acetogen, Eubacterium limosum, in grain fed cattle, as well as novel acetogens affiliating with the Lachnospiraceae and Ruminococcaceae/Blautia group. The novel potential acetogen population detected in this study may represent an important hydrogenotrophic group in the rumen that we understand very little about and that requires further investigation. The tammar wallaby, which exhibits foregut fermentation analogous to that of the rumen but resulting in lower methane emissions, housed a different acetogen population to that of the bovine rumen (LIBSHUFF, p <0.0001) though novel potential acetogens in the tammar wallaby forestomach affiliated broadly in the same family groups (Blautia group, Lachnospiraceae and between Lachnospiraceae and Clostridiaceae without a close cultured isolate). Acetogen enrichment cultures from the tammar wallaby forestomach facilitated isolation of a novel acetogen, which was closely related to potent reductive acetogens from kangaroos. The differences between the acetogen population of the tammar wallaby forestomach and the bovine rumen may be a factor in explaining lower methane emissions and methanogen numbers in tammar wallabies relative to ruminants. Using a gnotobiotically reared lamb model, the unique acetogen population present in the developing rumen was identified and it’s response to methanogen colonisation examined. The acetogen E. limosum and potential acetogen Ruminococcus obeum were identified as well as a small diversity of novel potential acetogens affiliating with the Blautia group and the Lachnospiraceae. A small but diverse population of naturally resident methanogens were also identified in gnotobiotically reared lambs that had been isolated at 17 hours of age. After inoculation with Methanobrevibacter sp. 87.7, methanogen numbers in gnotobiotically reared lambs significantly increased but acetogen diversity was not altered, indicating that this population is resilient to methanogen colonisation to some degree. The potential acetogen population in gnotobiotically reared lambs was significantly different (LIBSHUFF, p < 0.0001) to that in conventionally reared sheep, which indicates that factors other than methanogen establishment alone, probably relating to other microbes and associated hydrogen concentrations in the rumen, affect acetogens during rumen development.

Bibliography: leaves 226-261. Investigates nitrogen utilization in some species of rumen bacteria with the object of understanding the role of ammonia versus exogenous amino acids in relation to microbial growth.

L'objectif principal de cette thèse est d'étudier in vitro et in vivo, le métabolisme de certains terpènes caractéristiques de régimes d'hiver-printemps ingérés par les chèvres laitières dans la région du Basilicate (Italie). Nous avons cherché à quantifier dans une première étape le métabolisme des terpènes en milieu fermentaire in vitro, et de caractériser les facteurs majeurs de variation de leur métabolisme. Nous avons également effectué un essai in vivo sur 4 terpènes particuliers (-pinène, -pinène, p-cymène, et linalool, les terpènes principaux ingérés par les chèvres en Basilicate) dans le but de valider les résultats obtenus in vitro, de caractériser leur devenir dans le rumen, et leur flux au niveau duodénal et sanguin. En parallèle nous avons étudié l'impact de ces terpènes sur le métabolisme ruminal des parois et des protéines, ainsi que leurs effets sur les performances des chèvres laitières. La partie expérimentale est composée de trois séries d'études. Les études méthodologiques, les études de métabolisme des terpènes in vitro et celles in vivo. Les études méthodologiques de mise au point d'une méthode d'extraction et d'analyse (SPME «Solid Phase Microextraction») ont été réalisées afin d'obtenir les conditions optimales pour l'extraction des terpènes en milieu fermentaire et dans la matrice sanguine. Le premier essai in vitro a conduit à une discrimination des terpènes testés en termes de leur disparition dans le jus de rumen, qui a été associée au type chimique du terpène considéré. Les terpènes hydrocarbones ont montré en effet une disparition beaucoup plus marquée par rapport aux terpènes oxygénés. Cette différence tient principalement à leurs différences de propriétés physico-chimiques, qui sépare en partie leur voie métabolique. La disparition de terpènes semble avoir deux causes principales, une intervention biologique d'origine microbienne et une interaction avec la matrice. Une propriété moins toxique et plus hydrophobe semble renforcer le métabolisme (dégradation) microbien et l'intervention de la matrice sur les terpènes hydrate de carbones par rapport à ceux oxygénés. Dans le deuxième essai in vitro, l'effet de trois facteurs de variation, le type d'inoculum (riche en fibre vs. riche en amidon), le type des bactéries (Gram + vs Gram -, sélectionnées par l'apport d'antibiotique) et le potentiel redox ont été étudiés sur le métabolisme des terpènes en milieu fermentaire. Les résultats obtenus ont montré que l'inoculum riche en fibre, théoriquement favorisant les bactéries cellulolytiques accroît la dégradation apparente de terpènes. L'absence d'un effet de l'ajout de l'antibiotique éliminant des bactéries Gram + a montré que ce sont les bactéries Gram - qui contribuent au métabolisme des terpènes. Enfin, l'augmentation du potentiel redox a augmenté la disparition de certains des terpènes testés, notamment dans l'inoculum riche en amidon. Les résultats obtenus dans les études in vitro concernant le métabolisme des terpènes, ont été confirmé par ceux observés in vivo. Toutefois une disparition supérieure observée in vivo a suggéré l'intervention d'autres facteurs de variation qui peuvent être attribué à l'absorption dans le rumen et une perte causée par rumination et éructation. L'impact de terpènes sur le métabolisme du rumen et la performance des chèvres laitières a été étudié par l'incorporation d'un mélange de quatre terpènes (mentionnées ci-dessus) à deux doses (0.05 et 0.5 ml/kg MSI). L'ingestion de terpènes n'a provoqué aucun effet sur le métabolisme ruminal des fibres, et des protéines. Les performances de production des chèvres n'ont pas été affectées par les terpènes; cependant une modification du taux protéique (chute du Taux protéique à dose faible) observée, peut être considérée comme un effet secondaire (effet de dilution) lié à une augmentation numérique de la production du lait à cette dose

Two experiments were conducted to evaluate the effects of dietary inclusion level of a low lignin hull, high-oil groat (CDC SO-I) oat on the performance, carcass characteristics and rumen degradation characteristics of feedlot cattle. In the first trial, 200 crossbred steers (average weight of 427.3 ± 22.4 kg) were allocated to 20 pens. Five treatments, formulated by replacing barley with increasing levels of CDC SO-I oat (Barley grain:CDC SO-I oat ratios of 100:0; 75:25; 50:50; 25:75 and 0:100 ; DM basis) were used. Four pens were randomly allocated to each treatment diet. Over the entire study there was a linear decrease (P< 0.01) in DMI and ADG with increasing inclusion level of CDC SO-I oat, whereas feed efficiency (gain:feed) decreased (P= 0.03) quadratically. Days on feed also increased (P= 0.03) quadratically for the steers fed the higher levels of CDC SO-I oat. Increasing the inclusion level of CDC SO-I oat in the diet also decreased (P< 0.01) carcass weight, dressing percentage and grade fat linearly. However, there was no effect of treatment on rib eye area and lean yield percentage. There was no significant effect of treatment on marbling score. While the results of this trial point to a negative effect of CDC SO-I oat on finishing performance, there were minimal differences between cattle fed 100% barley as the concentrate versus those fed 75% barley: 25% oat blend.<p> Trial 2 involved a metabolism trial to determine the effect of CDC SO-I oat inclusion level on rumen fermentation parameters of 5 fistulated heifers fed the same diets used in Trial 1. A 5 × 5 Latin square experiment design was used. Rumen degradation parameters (rumen pH, VFA, osmolality and ammonia nitrogen levels) and feeding behavior (time spent eating, ruminating, chewing and drinking) were measured. Mean rumen pH for the barley-based diet was 5.88 which was not different (P> 0.05) than the mean pH of 5.5 for the oat-fed cattle. Treatment did not affect (P> 0.05) time spent below pH cutoff values of 5.8, 5.5 and 5.2. No effect of oat inclusion level (P> 0.05) was observed on total VFA levels, molar proportion of individual fatty acids and osmolality while isobutyrate (P= 0.05) and ruminal ammonia nitrogen concentrations decreased linearly (P= 0.02) with the higher inclusion of CDC SO-I oat. Time spent eating was linearly (P< 0.01) increased with higher inclusion level of CDC SO-I oat.<p> Over all, the results of this study indicate that the replacement of barley by CDC SO-I oat in finishing diets decreases dry matter intake and as a result leads to reduced ADG, increased days on feed and lower slaughter and carcass weights. The reduced performance might be the result of higher fat content, high hull and/or faster degradation rate of oat starch leading to subacute ruminal acidosis in cattle fed higher levels of oat. However, replacing barley with CDC SO-I oat does not significantly change the rumen environment. The results of this study indicate that CDC SO-I oat can be successfully included up to a maximum level of 25% without any adverse effect on performance and carcass characteristics in the diets of finishing cattle.

Two experiments were conducted to evaluate the effects of dietary inclusion level of a low lignin hull, high-oil groat (CDC SO-I) oat on the performance, carcass characteristics and rumen degradation characteristics of feedlot cattle. In the first trial, 200 crossbred steers (average weight of 427.3 ± 22.4 kg) were allocated to 20 pens. Five treatments, formulated by replacing barley with increasing levels of CDC SO-I oat (Barley grain:CDC SO-I oat ratios of 100:0; 75:25; 50:50; 25:75 and 0:100 ; DM basis) were used. Four pens were randomly allocated to each treatment diet. Over the entire study there was a linear decrease (P< 0.01) in DMI and ADG with increasing inclusion level of CDC SO-I oat, whereas feed efficiency (gain:feed) decreased (P= 0.03) quadratically. Days on feed also increased (P= 0.03) quadratically for the steers fed the higher levels of CDC SO-I oat. Increasing the inclusion level of CDC SO-I oat in the diet also decreased (P< 0.01) carcass weight, dressing percentage and grade fat linearly. However, there was no effect of treatment on rib eye area and lean yield percentage. There was no significant effect of treatment on marbling score. While the results of this trial point to a negative effect of CDC SO-I oat on finishing performance, there were minimal differences between cattle fed 100% barley as the concentrate versus those fed 75% barley: 25% oat blend.<p> Trial 2 involved a metabolism trial to determine the effect of CDC SO-I oat inclusion level on rumen fermentation parameters of 5 fistulated heifers fed the same diets used in Trial 1. A 5 × 5 Latin square experiment design was used. Rumen degradation parameters (rumen pH, VFA, osmolality and ammonia nitrogen levels) and feeding behavior (time spent eating, ruminating, chewing and drinking) were measured. Mean rumen pH for the barley-based diet was 5.88 which was not different (P> 0.05) than the mean pH of 5.5 for the oat-fed cattle. Treatment did not affect (P> 0.05) time spent below pH cutoff values of 5.8, 5.5 and 5.2. No effect of oat inclusion level (P> 0.05) was observed on total VFA levels, molar proportion of individual fatty acids and osmolality while isobutyrate (P= 0.05) and ruminal ammonia nitrogen concentrations decreased linearly (P= 0.02) with the higher inclusion of CDC SO-I oat. Time spent eating was linearly (P< 0.01) increased with higher inclusion level of CDC SO-I oat.<p> Over all, the results of this study indicate that the replacement of barley by CDC SO-I oat in finishing diets decreases dry matter intake and as a result leads to reduced ADG, increased days on feed and lower slaughter and carcass weights. The reduced performance might be the result of higher fat content, high hull and/or faster degradation rate of oat starch leading to subacute ruminal acidosis in cattle fed higher levels of oat. However, replacing barley with CDC SO-I oat does not significantly change the rumen environment. The results of this study indicate that CDC SO-I oat can be successfully included up to a maximum level of 25% without any adverse effect on performance and carcass characteristics in the diets of finishing cattle.

The aim of the present study was to investigate, firstly, whether the substitution of dietary starch by rumen-protected fat results in visible changes in the protein metabolism by increasing the urea flux and decreasing of AA levels in blood plasma as indicators for a change of AA oxidation, secondly, whether the fat-fed cows use predominantly long chain FA in the processes of milk fat synthesis or as an energy source in oxidation processes, and thirdly, whether the ratio of glucogenic to lipogenic nutrients could affect the supply of glucose in the metabolism of dairy cows. Two experiments were conducted. In either experiments, about 1.8 kg of starch in the ration of the control group were substituted by about 0.7 kg fat as protected fat (Ca salts of palm, soybean and sunflower oils in addition to protected soybeans) in the ration of the fat group. The first experiment was carried out on 32 multiparous high yielding dairy cows (16 Holstein-Friesian cows in each group) during the first 100 d of lactation. The second experiment was carried out in four periods in a cross-over design. Two cows were used in each period during the 6th to the 10th lactation weeks. The cows were infused intravenously with D-[U-13C6]-Glucose. The substitution of starch by protected fat tended to increase the milk production and milk lactose output and to decrease the microbial protein synthesis in the rumen and plasma glucose level. Also, the levels of ß-HBA and NEFA in plasma, the milk urea content and the total urea-flux were increased (P<0.05). Milk protein content but not yield and plasma levels of insulin, Met, Ser and His decreased (P<0.05) but the branched chain amino acids in plasma increased (P<0.05). The oxidation rate of FA was lower in comparison to other sub`strates. In the second experiment, the enrichment of milk fat and blood CO2 by 13C decreased but the recovery of 13C in milk lactose increased (P<0.065) due to high fat intake. The results indicate that the substitution of starch by protected fat can save glucose in the intermediary metabolism for lactose synthesis in the mammary gland and the cows used fatty acids predominantly for milk fat synthesis and not for oxidation.

Three duodenally cannulated lactating Holstein cows fed cotton-seed meal (CSM), corn gluten meal (CGM) or blood meal (BM) as protein supplement were used in a 3 x 3 Latin Square experiment to determine microbial crude protein (MCP) in duodenal digesta. Diets, formulated to contain 15% crude protein (CP) on a dry matter basis, consited of 60% concentrate, 31% corn silage and 9% alfalfa hay. Chromium oxide was employed as flow marker. Microbial protein fraction of digesta CP (MCP/DCP) was estimated by three microbial markers: ¹⁵N, diaminopimelic acid (DAP) and ribonucleic acid (RNA). The isotopic method gave the most reliable results. Variability was higher with DAP and RNA. Results from RNA were lower (P < .01) and unreasonable. Based on ¹⁵N, MCP/DCP differed among treatments (P < .10) with means of 61.5, 59.4, and 50.0% for CSM, CGM, and BM, respectively, but differences were not significant for absolute amounts of total CP and MCP in duodenal digesta.

Six ruminally fistulated steers (average BW 317 kg) were used in a 5 X 5 + 1 Latin Square design to determine effects of humic/fulvic acid (HFA) on serum urea nitrogen (SUN), rumen pH, rumen ammonia nitrogen (RAN), and rumen VFA production in Holstein steers. Treatments included a control (0%), 0.5, 1.0 or 1.5% HFA, and a diet containing monensin (33 mg/kg). A quadratic feed intake response was observed; with intake lowest for 0.5 and 1.0 and highest for 1.5 relative to control steers. No treatment x time interaction was observed for RAN, pH, SUN, total VFA or individual VFA. No treatment differences were observed for total VFA concentrations (mM), or for molar proportion of acetate, propionate, butyrate or valerate. A quadratic response was observed for butyrate and isovalerate. However, HFA does not alter rumen fermentation in the same mode of action as monensin.

Health and productivity of grown-up animals highly depends on technology of breeding of calf’s. It‘s very important, that calf’s aged 3-4 months shouldn‘t fix diseases of digestive and airways systems. The main aim of this work was to determine influence of composite start feed „Milaflo“ to the intensity of breeding, development of internal organs and wellness of calf’s. Summing-up the result conclusions are provided as follows: organism of heifers develops faster, when during the period of feeding with milk combined start feed was used; composite start feed „Milaflo“ meets all zootechnical requirements and grants intensive growth of calf’s during feeding with natural milk and other crops without any negative reaction to wellness of calf’s; during test period in ŽŪB „ Pauliukai“, daily gain of calf’s which were feeded with „Milaflo“ contained 778,3 g/ per day, while ones feeded with standard composite feed - 713,9 g; during test period in Center of Practical Education of LVA daily gain of control group calf’s contained 708,9 g, heifers, which were feeded with additive „Milaflo“ and powder of conditioned milk – 752,2 g, heifers which were feeded with additive „Milaflo“ and natural milk – 966,7 g; use of composite start feed „Milaflo“ has no negative reaction to morphological composition of blood; highest efficiency of composite start feed concerning the development of great prestomach was noted during week 3 - 4.

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.