Structural features of lignin macromolecules extracted with ionic liquid from poplar wood

1-Ethyl-3-methylimidazolium acetate ([Emim][CH?COO]) was used for the extraction of lignin from poplar wood (Populus albaglandulosa), which was called to ionic liquid lignin (ILL) and structural features of ILL were compared with the corresponding milled wood lignin (MWL). Yields of ILL and MWL were 5.8±0.3% and 4.4±0.4%, respectively. The maximum decomposition rate (V(M)) and temperature (T(M)) corresponding to V(M) were 0.25%/ °C and 308.2 °C for ILL and 0.30%/ °C and 381.3 °C for MWL. The amounts of functional groups (OMe and phenolic OH) appeared to be similar for both lignins; approximately 15.5% and 6.7% for ILL and 14.4% and 6.3% for MWL. However, the weight average molecular weight (M(w)) of ILL (6347 Da) was determined to be 2/3-fold of that of MWL (10,002 Da) and polydispersity index (PDI: M(w)/M(n)) suggested that the lignin fragments were more uniform in the ILL (PDI 1.62) than in the MWL (PDI 2.64).     

Degradation of natural lignins and lignocellulosic substrates by soil-inhabiting fungi imperfecti

Abstract: The most powerful lignin-degraders among the 82 microbial strains isolated during a screening of ligninolytic microorganisms from forest soil were identified as Penicillium chrysogenum, Fusarium oxysporum and Fusarium solani . These fungi imperfecti mineralized 27.4%, 23.5% and 22.6% of ¹⁴C-labelled milled wood lignin (MWL) from wheat straw after 28 days of incubation in liquid media. Degradation of MWL from pine by P. chrysogenum was 8% and 19% when it was evaluated by spectrophotometry and Klason lignin, respectively, but this substrate was hardly mineralized. All fungi were able to attack the hemicellulosic, cellulosic and also lignin fractions of wheat straw during solid-state fermentation, F. solani being capable of degrading about 25% of both carbohydrates and lignin. When the selected fungi were tested for dye decolourization, they all readily attacked the polymeric dye Remazol brilliant blue R (RBBR) and also poly R-478 to a minor extent.     

Expression of conditioned preference for low-quality food in sheep is modulated by foraging costs

Past positive experiences can increase herbivores’ motivation to eat low-quality foods. However, this is not always translated into a higher preference for low-quality foods in choice tests among foods of higher nutritional quality. Foraging behavior is also affected by properties of the feeding context because the quality and abundance of foods in nature change in time and space. We hypothesized that in a choice situation, the expression of a past positive experience with a low-quality food is modulated by the costs associated with selecting a high-quality food option. A total of 24 sheep were randomly assigned into two groups (n=12). During conditioning phase, one group (CS+; i.e., conditioned group) was fed with oat hay (a low-quality food) for 20 min and immediately after a ration of soybean meal (a nutritious food), whereas the other group was also fed with oat hay but the offer of soybean meal was delayed 5 h (CS−; i.e., control group). After conditioning, we assessed sheep motivation to eat the oat hay in an experimental arena in which accessibility to alfalfa hay (a high-quality food) was increasingly restricted. When alfalfa hay was readily accessible, CS+ and CS− sheep almost exclusively selected this food, showing a small and similar preference for oat hay. However, when accessibility to alfalfa hay decreased, intake and selection of oat hay was greater in the CS+ sheep than in the CS− sheep. The latter was a consequence of differential changes in behavior between groups; for example, sheep in CS+ spent more time foraging oat hay and were more likely to switch to oat hay if they had previously been eating alfalfa hay than sheep in CS−. Our results show that behavioral expression of the conditioned preference for a low-quality food depends on parameters of the feeding context (e.g., availability). We suggest that this can be the link between learning models and optimal foraging models of diet selection.     

Specificity of Rumen Ciliate Protozoa in Cattle and Sheep*

SYNOPSIS. Six protozoa-free sheep, 3 fed alfalfa hay and 3 fed a concentrate diet, were inoculated with rumen contents from a steer fed the same alfalfa hay. All 24 species of protozoa in the inoculum became established in the sheep fed alfalfa hay, while only 9 species established in the sheep fed concentrate. Percentage species composition in the alfalfa-fed sheep was fairly similar to that of the inoculum. Rumen volumes of the alfalfa hay-fed sheep were significantly higher than those of the concentrate-fed sheep; however, fluid turnover rates were similar. Total protozoan numbers per ml of rumen contents were significantly higher in the concentrate-fed sheep, but after adjustment for rumen volume, there was no significant difference in the total number of protozoa in the rumen.     

Utilization by sheep of dried pig faeces containing a high concentration of copper

Seven groups of three sheep were given pelleted diets as follows: A, 97% hay + 3% molasses; B, C, D, 82% hay + 3% molasses + 15% dried pig faeces; E, F, G, 67% hay + 3% molasses + 30% dried pig faeces. In an attempt to counteract potential toxic effects of copper in the pig faeces (613 mg/kg dry matter), molybdenum was added to diets C and F at the rate of 90 mg/kg diet, and to diets D and G at the rate of 175 mg/kg diet. Sulphate was included in all diets at the rate of 1.08% of diet. Measurements were made of digestibility, copper retention, and plasma glutamic oxaloacetic transaminase (GOT) values during a 70-day feeding period, after which the animals were slaughtered.Dry matter digestibility coefficients were : diet A, 49.2; diets B, C, D, 44.4; diets E, F, G, 42.5, from which the coefficient for pig faeces alone is calculated to be < 30. Mean copper retention over the 70 days ranged from 210 mg for diet A to 1225 mg for diet G (excluding diet E which showed an aberrant retention of 53 ± 185 mg). Plasma GOT values fluctuated between diets and between sheep with a maximum value of 182 units/ml. Mean liver copper concentrations ranged from 718 mg/kg dry matter for diet A to 1740 mg/kg for diet E. Necrotic lesions occurred in some livers. Kidneys were apparently normal. There were no clear differences in copper status of animals receiving 15% versus 30% pig faeces except for a tendency for liver damage to be greater in the latter. There were no apparent effects of additions of molybdenum.The pig faeces were poorly utilized and, because of the high copper content, potentially hazardous to the health of the sheep.     

A comparative study of the effect of two-stage olive cake added to alfalfa on digestion and nitrogen losses in sheep and goats

This work was conducted to evaluate the effect of tannins, contained in a by-product derived from olive oil extraction (two-stage olive cake), on nutrient digestibility, nitrogen (N) losses and liver and kidney functions in goats and wethers. Six Segureña wethers and six Granadina goats were fed three experimental diets: alfalfa hay (AH); alfalfa hay and a concentrate (formulated with two-stage olive cake, barley and a mineral-vitamin mixture) without (AHCO) or with polyethylene glycol (PEG) addition (AHCOP). The inclusion of two-stage olive cake increased condensed tannins content of the diet and led to a decrease ( P < 0.001) in dry matter, organic matter, neutral-detergent fibre, acid-detergent fibre and crude protein (CP) digestibility. PEG supply increased ( P < 0.001) CP digestibility and N losses in urine and faeces only in wethers. Concentrations of creatinine and alkaline phosphatase activity, measured in the serum of animals, were not modified either by olive cake inclusion or by PEG supply. Results from this work indicate a higher sensitivity of sheep to diets containing condensed tannins, compared with goats, and reveal no toxic effect of tannins from two-stage olive cake at the concentration used in the experimental diets.     

Oxidation of milled wood lignin with laccase, tyrosinase and horseradish peroxidase

In this paper the oxidation of milled wood lignin (MWL), catalysed by three enzymes, i.e. laccase, tyrosinase and horseradish peroxidase (HRP) was studied. The oxidation was followed by measuring the consumption of O₂ during laccase and tyrosinase treatment and of H₂O₂ during HRP treatment. Both laccase and HRP were found to oxidise lignin effectively, whereas the effect of tyrosinase was negligible. The changes in MWL molecular-weight distributions caused in the reactions were analysed by gel permeation chromatography. Both laccase and HRP treatments were found to polymerise MWL. Peroxidase treatment was found to decrease the amount of phenolic hydroxyls in MWL, whereas no such effect could be detected in the laccase-treated sample. Both laccase and HRP treatments were, however, found to increase the amount of conjugated structures in MWL. The formation of phenoxy radicals during the treatments was studied by electron paramagnetic resonance spectroscopy. Phenoxy radicals were detected in both laccase and HRP-treated samples. The amount of the formed phenoxy radicals was found to be essentially constant during the detected time (i.e. 20–120 min after the addition of enzyme).     

Untersuchungen über die Höhe der Karotinverluste bei der Lagerung von Trockengrünfutter

Cannulated beef cattle (four cows: 556 kg initial weight; four steers: 504 kg initial weight) were used in an experiment with two simultaneous Latin squares to determine effects of substituting alfalfa and(or) corn for vegetative bermudagrass (BER; 77% neutral detergent fibre and 5.5% acid detergent lignin) or mature bromegrass (BRO; 70% neutral detergent fibre and 6.6% acid detergent lignin) hay on digestion characteristics. For Controls, BER or BRO was fed at 1.32 or 1.54% body weight of cows and steers, respectively; other treatments entailed substitution for hay DM of alfalfa cubes (17%) or ground corn (33%). A protein supplement was given to all animals. In vitro neutral detergent fibre (NDF) digestion was slightly greater for BER than BRO. Supplement treatments did not affect the concentration of total volatile fatty acids or the molar proportion of propionate in ruminai fluid. True ruminai organic matter (OM) digestion was similar among diets; greater duodenal microbial OM flow and postruminal NDF digestion for BRO than BER diets were responsible for higher (P < 0.05) postruminal OM digestion for BRO diets. Supplement treatment did not affect duodenal microbial nitrogen flow or efficiency of microbial growth. Corn supplementation increased total tract OM digestion (P < 0.05). Alfalfa addition depressed total tract OM and NDF digestibilities more when added to BER than BRO; depressions in total tract NDF digestion with alfalfa and corn substitutions were additive. With constant DM intake, slightly less than ad libitum, alfalfa or corn substituted alone or together for hay did not improve characteristics of digestion by cattle consuming vegetative bermudagrass or mature bromegrass other than increased postruminal and total tract OM digestiblities with corn.     

Solubility and flow of calcium,magnesium and phosphorus in the digestive tract of sheep given maize or alfalfa silages

Four sheep, each fitted with a rumen cannula and a re-entrant cannula in the proximal duodenum and terminal ileum, were used for collection of rumen, duodenal and ileal digesta. Four normal sheep were used for collection of faeces. They were given maize silage, maize silage ensiled with urea, wilted alfalfa silage or formic acid-treated alfalfa silage. Although the solubility of the calcium (Ca) in the duodenal digesta was higher with sheep on maize silages than with those on alfalfa silages, sheep fed on maize silage showed negative apparent absorption (?32.7 and ?16.5%) owing to the low concentration of Ca in whole-plant maize. The apparent absorption of magnesium (Mg) was 40.6–42.3% for maize silages and 24.2–26.8% for alfalfa silages. The differences between silages with respect to apparent absorption of phosphorus (P) were not significant and ranged between 1.5 and 10.9%. There were no appreciable differences in soluble proportions, flow and apparent absorption of Ca, Mg and P between the two maize silages or the two alfalfa silages. There was a net absorption of Ca and Mg and a net secretion of P in the stomach of animals on all silages.     

Monolignol acylation and lignin structure in some nonwoody plants: a 2D NMR study

Lignins from three nonwoody angiosperms were analyzed by 2D NMR revealing important differences in their molecular structures. The Musatextilis milled-wood-lignin (MWL), with a syringyl-to-guaiacyl (S/G) ratio of 9, was strongly acylated (near 85% of side-chains) at the γ-carbon by both acetates and p-coumarates, as estimated from ¹H-¹³C correlations in C_γ-esterified and C_γ-OH units. The p-coumarate H_3,5-C_3,5 correlation signal was completely displaced by acetylation, and disappeared after alkali treatment, indicating that p-coumaric acid was esterified maintaining its free phenolic group. By contrast, the Cannabissativa MWL (S/G ∼0.8) was free of acylating groups, and the Agavesisalana MWL (S/G ∼4) showed high acylation degree (near 80%) but exclusively with acetates. Extensive C_γ-acylation results in the absence (in M.textilis lignin) or low abundance (4% in A.sisalana lignin) of β-β′ resinol linkages, which require free C_γ-OH to form the double tetrahydrofuran ring. However, minor signals revealed unusual acylated β-β′ structures confirming that acylation is produced at the monolignol level, in agreement with chromatographic identification of γ-acetylated sinapyl alcohol among the plant extractives. In contrast, resinol substructures involved 22% side-chains in the C.sativa MWL. The ratio between β-β′ and β-O-4′ side-chains in these and other MWL varied from 0.32 in C.sativa MWL to 0.02 in M.textilis MWL, and was inversely correlated with the degree of acylation. The opposite was observed for the S/G ratio that was directly correlated with the acylation degree. Monolignol acylation is discussed as a mechanism potentially involved in the control of lignin structure.     

Intermittent shedding of thermophilic campylobacters by sheep at pasture

The rates at which sheep on different types of pasture shed campylobacters in their faeces were measured over 12 months. Overall, shedding of campylobacters at pasture was between a third and a half of the carriage rate (92%) of the intestines of sheep at slaughter. Shedding was highest during saltmarsh grazing, followed by upland fell and farm grazing. The rate of shedding varied at different times of the year, with the highest rates (100%) coinciding with lambing, weaning, and movement onto new pasture. The lowest rates (0%) occurred when sheep were fed on hay and silage. On the farm, low rates occurred during the whole of gestation, both when the sheep were indoors and outdoors. Campylobacter jejuni was the main species isolated and survived for up to 4 d in sheep faeces. Lambs became colonized by Campylobacter within 1-5 d of being born. Ewes, which were not shedding campylobacters prior to lambing, started to shed after lambing, and ewes which were shedding low numbers of Campylobacter before lambing, increased the numbers of bacteria being shed after lambing.     

Influence of tannic acid application on alfalfa hay: in vitro rumen fermentation, serum metabolites and nitrogen balance in sheep

Alfalfa protein is poorly utilised by ruminants due to its rapid degradation in rumen. The objective of the study was to assess the influence of spraying tannic acid (TA) on chopped alfalfa hay on in vitro rumen fermentation and nitrogen (N) retention by sheep. Alfalfa hay with and without TA was fed to sheep to determine nutrient digestibility and N balance. TA was sprayed on chopped alfalfa at three concentrations to determine its effect on in vitro fermentation of dry matter (DM) and N balance in sheep. Final TA concentrations were 0, 30, 60 and 90 g TA per kg DM. The control was sprayed with the same amount of water but without TA. In vitro DM degradation and the production of gas, ammonium-N (NH4-N) and short-chain fatty acid (SCFA) were measured. TA-sprayed alfalfa and the control were fed to sheep to determine nutrient digestibility and N retention. Addition of TA had no influence on the extent and rate of gas production but significantly decreased NH4-N concentration at 30 (P < 0.05), 60 and 90 (P < 0.0001) g/kg DM. Addition of polyethylene glycol (PEG) to TA-sprayed alfalfa increased NH4-N to a level comparable to non-TA-sprayed alfalfa. Spraying of alfalfa with TA significantly decreased (P < 0.05) isovalerate but did not affect the total and individual SCFA acid production. Tannic acid significantly (P < 0.05) reduced in vitro true degradability of DM (IVTD) after 24 h incubation at levels of 60 and 90 g TA per kg DM. Neutral-detergent fibre digestibility (dNDF) after 24 h (P < 0.01), 60 and 90 (P < 0.0001) g TA per kg DM. The effect of TA on either IVTD or dNDF was not significant (P > 0.05) after 48 h of incubation. There was a strong linear relationship between percentage increase in gas production due to PEG and protein precipitation capacity (R2 = 0.94). N digestibility was significantly reduced with all three levels of TA additions. However, the proportion of urine-N to total N output was reduced by adding 60 g (P < 0.05) and 90 g (P < 0.01) TA per kg DM. Serum metabolites and liver enzymes were not affected by TA (P > 0.05). Higher faecal N as the TA level increased indicates incomplete dissociation of tannin-protein complexes post ruminally. Factors affecting dissociation of tannin-protein complexes need further study.     

Listeriosis in Sheep

Listeriosis in sheep. Listeria monocytogenes excretion and immunological state in healthy sheep. Acta vet. scand. 1979, 20, 168–179. — The excretion of Listeria monocytogenes (Lm) in the faeces and milk, and humoral and cell mediated immunity against Lm, were examined in a sheep flock where no cases of listeriosis had occurred during the last 3 years. The investigation was carried out during the indoor season. During the first part of the season 2 of the 10 pregnant, 8 months old lambs excreted Lm in the faeces, but none of the 106 ewes, 2–10 years old. At lambing the organism was isolated from the faeces of 6 of the 10 1 year old lambs and from 64% of the ewes, and from the milk of 1 of the lambs and 41% of the ewes. Nearly all the isolates (98.5%) belonged to serotype 1. Antibody titres against Lm were found in sera and whey by an indirect haemagglutination method. The titres were higher for the ewes than for the hoggs and seemed to be influenced by the number of foetuses the animals carried. Cell mediated immunity was determined by a skin test where delayed hypersensitivity against an antigen prepared from Lm, was measured. Animals fed grass silage had a stronger reaction than animals fed hay, and a stronger reaction was found in animals with ≥ 3 foetuses than in the remainder. The investigation indicates that even in a healthy sheep flock all the animals may be exposed to Lm, and the majority may be latent carriers and excrete this organism in the faeces and milk during periods of stress.     

Down-regulation of cinnamyl alcohol dehydrogenase in transgenic alfalfa (Medicago sativa L.) and the effect on lignin composition and digestibility

To improve the digestibility of the forage crop alfalfa (Medicago sativa L.), cinnamyl alcohol dehydrogenase (CAD), which catalyses the last step in the biosynthesis of the lignin monomers, was down-regulated by using an antisense approach. A subset of six transgenic lines with reduced CAD activity and control lines were analysed when grown in the greenhouse and in the field. The down-regulation of the CAD enzyme was associated with a red coloration of the stem. The lignin quantity remained unchanged, but the lignin composition, as determined by thioacidolysis, was altered. The highest reduction of CAD activity was associated with a lower syringyl/guaiacyl (S/G) ratio and a lower S+G yield, mainly because of a decreased amount of S units. An increase in in situ disappearance of dry matter and of cell wall residue was detected in one of the transgenic lines grown in the greenhouse, and for two of the lines grown in the field the rate of disappearance of dry matter slightly improved. Furthermore, these two lines had a higher solubility in alkali as shown by the lower yield of saponified residue. This study opens perspectives for improving forage crop digestibility by the modulation of enzymes involved in lignin biosynthesis.     

Influence of hay particle size at different concentrate and feeding levels on digestive processes and feed intake in ruminants. 2. passage,digestibility and feed intake

In order to study the main effects of particle size, three ruminally fistulated cows (550 to 580 kg BW) were fed a constant low concentrate level (3.56 kg DM/d, 20% of total DMI) and a fibre‐rich hay (approximately 60% NDF in DM) in long (28.7 mm), chopped (9.2 mm) and fine ground (2.9 mm) form in a 3 x 3 Latin square design. In another three factorial experiment with 8 wethers (4 animals were ruminally fistulated, mean BW = 68 kg) the main effects and interactions of the above mentioned hay particle sizes at two concentrate levels (10.4 to 13.3% and 29.5 to 40.1% of DMI, resp.) and two intake levels (restricted and ad libitum) were investigated. In comparison to long hay (28.7 mm), feeding of chopped hay (9.2 mm) at low concentrate levels, increased not only the hay intake (7% in dairy cows and 13% in sheep) but also the intake of digestible organic matter (12% in dairy cows and 32% in sheep), due to an increase in the apparent digestibility of OM by 3.8% in dairy cows and 8.2% in sheep. Ad libitum feeding of fine ground hay in combination with low concentrate amount in the ration increased the passage rate in the hindgut and consequently the hay intake, but not the intake of DOM, due to a significant depression of digestibility, especially of fibre fractions (4 to 7% in dairy cows and 4.5 to 14% in sheep), in comparison to 28.7 and 9.2 mm hay particle sizes. The digestibility decreased significantly with restricted feeding of fine ground hay in sheep only in comparison to 9.2 mm particle size. A threefold increase of concentrate amount levelled out all effects of the particle size reduction. The effect of particle size was more pronounced in sheep than in dairy cows.     

Microwave-enhanced extraction of lignin from birch in formic acid: Structural characterization and antioxidant activity study

A rapid and mild extraction protocol for the preparation of lignin was achieved by microwave-assisted heating in formic acid at 101 °C under atmospheric pressure. In this case, birch lignin was extracted with microwave heating process (ML) in formic acid and characterized by elemental analysis, FTIR, GPC, ¹H NMR and ¹³C–¹H HSQC. In addition, the antioxidant activity of the samples was investigated. For comparative study, milled wood lignin (MWL) and lignin extracted with oil bath heating process (OL) were prepared. The results showed that the lignin yield under microwave heating was much higher than that under oil bath heating. A maximal delignification degree (89.77%) was achieved when microwave heating time was 30 min. When double time (60 min) was used under oil bath heating, the delignification degree was 66.11%. The structural characterization showed that the lignin structure of ML did not change dramatically, which is a mixture of GS-type with β-O-4′ ether bond as the major inter-unit linkage. As for antioxidant activity against DPPH, the radical scavenging index (RSI) of ML was 1.20, which was higher than that of MWL (0.53), suggesting that ML exhibited much higher antioxidant activity than MWL.     

Understanding medusahead low intake and palatability through in vitro digestibility and fermentation kinetics

The low intake of medusahead grass (Taeniatherum caput-medusae ssp. asperum) by sheep is attributed to the high silica content of the plant and its negative impact on digestibility, making this weed a successful competitor in grazed plant communities. The goals of this study were to determine the influence of (1) plant maturity stage (from late vegetative stage to beginning of senescence and thatch), (2) particle size (1, 5, 10 and 20 mm), and (3) high-quality feeds (alfalfa hay and a high-energy concentrate) on fermentation kinetics and apparent digestibility of medusahead relative to palatable feeds (alfalfa and tall fescue hays, and high-energy concentrates). In vitro gas production was estimated and apparent digestible organic matter (DOM) of the substrates was assessed after incubation. Medusahead from late vegetative to senescence stage had greater DOM (65% to 71%; P<0.05) than alfalfa hay (53%), similar to tall fescue hay (67%; P>0.05), and lower than the high-energy concentrates assayed (77% to 79%; P<0.05). Fermentation kinetics showed slow fermentation rates for medusahead relative to alfalfa (P<0.05), and a decline in fermentation rates with plant maturity (P<0.05). Fermentation rates of the substrates were reduced with particle sizes ⩾5 mm (P<0.05), and apparent DOM for medusahead declined as particle size increased, a relationship not found for alfalfa or tall fescue hays (P<0.05). No effects (P>0.05) on digestibility parameters were observed by the addition of high-quality feeds to medusahead. Slow fermentation kinetics and a significant inhibitory effect of particle size on apparent digestibility of organic matter contribute to explain the low use of medusahead by sheep. Such inhibitory effect may also underlie the lack of positive associative effects observed during the study.     

Impact of raking and baling patterns on alfalfa hay dry matter and quality losses

A field study was conducted to investigate the impact of different patterns of raking and baling operations on the dry matter (DM) and quality losses of the produced alfalfa (Medicago sativa) hay. The experimental work was carried out on a 50?ha center pivot irrigated alfalfa field in a commercial farm located in the Eastern Region of Saudi Arabia. Raking operation was performed following two patterns corresponding to the direction of mowing operation, namely, Raking-I in the same direction of mowing (Mowing→ ?and Raking→) and Raking-II opposite to the direction of mowing (Mowing→ and Raking←). The baling operation; however, was performed following four patterns corresponding to the directions of both mowing and raking operations, namely, Baling-I (Mowing→, Raking→ and Baling→), Baling-II (Mowing→, Raking→ and Baling←), Baling-III (Mowing→, Raking← and Baling→) and Baling-IV (Mowing→, Raking← and Baling←). Results showed that cumulative DM losses in alfalfa hay yield of 30.93% occurred during the harvesting operations. Out of which, raking induced the most DM losses of 985.22?kg?ha^?1 (59.66% of the total DM losses and 17.35% of the total hay yield). However, the least DM losses were observed during the baling operation and were estimated at 175.81?kg?ha^?1 (10.22% of the total DM losses and 3.10% of the total hay yield). Raking opposite to the direction of mowing reduced the DM losses by 130.17?kg?ha^?1 (7.88% of the total DM losses and 2.29% of the total hay yield) compared to that with the direction of mowing. Results also indicated that out of the 21.04% losses in the total crude protein (CP) content of the produced alfalfa hay, 10.91% occurred during the raking operation. However, the baling operation induced the least amount of CP losses (only 2.32% of the total CP). Overall, the best results in terms of alfalfa hay quality and quantity losses were achieved with Baling-III, where the lowest DM losses (2.01% of the total hay yield) and the lowest CP losses (1.44% of the total CP) were recorded.     

Rumen Fungi and Forage Fiber Degradation

The role of anaerobic rumen fungi in in vitro forage fiber degradation was determined in a two forage × two inoculum source × five treatment factorial design. Forages used as substrates for rumen microorganisms were Coastal bermuda grass and alfalfa; inoculum sources were rumen fluid samples from a steer fed Coastal bermuda grass hay or alfalfa hay; treatments were whole rumen fluid (WRF), WRF plus streptomycin (0.2 mg/ml of rumen fluid) and penicillin (1.25 mg/ml of fluid), WRF plus cycloheximide (0.5 mg/ml of fluid), WRF plus streptomycin, penicillin, and cycloheximide, and McDougall buffer. Populations of fungi as shown by sporangial development were greater on bermuda grass leaves than on alfalfa leaflets regardless of inoculum source. However, endogenous fungal populations were greater from the alfalfa hay inoculum. Cycloheximide inhibited the fungi, whereas streptomycin and penicillin, which inhibit bacterial populations, resulted in an increase in numbers of sporangia in the alfalfa inoculum, suggesting an interaction between bacteria and fungi. Bacteria (i.e., WRF plus cycloheximide) were equal to the total population in degrading dry matter, neutral-detergent fiber (NDF), acid-detergent fiber (ADF), and cellulose for both inocula and both forages. Degradation of dry matter, NDF, ADF, and cellulose by anaerobic fungi (i.e., WRF plus streptomycin and penicillin) was less than that due to the total population or bacteria alone. However, NDF, ADF, and cellulose digestion was 1.3, 2.4, and 7.9 percentage units higher, respectively, for bermuda grass substrate with the alfalfa versus bermuda grass inoculum, suggesting a slight benefit by rumen fungi. No substantial loss of lignin (72% H₂SO₄ method) occurred due to fungal degradation. The most active fiber-digesting population in the rumen was the bacteria, even when streptomycin and penicillin treatment resulted in an increase in rumen fungi over untreated WRF. The development of large numbers of sporangia on fiber may not indicate a substantial role as digesters of forage.     

Phylogenetic diversity and dietary association of rumen Treponema revealed using group-specific 16S rRNA gene-based analysis

Treponema spp. are a commonly detected bacterial group in the rumen that are involved in the degradation of soluble fibers. In this study, a ruminal Treponema group-specific PCR primer targeting the 16S rRNA gene was designed and used to assess the phylogenetic diversity and diet association of this group in sheep rumen. Total DNA was extracted from rumen digesta of three sheep fed a diet based on alfalfa/orchardgrass hay or concentrate. The real-time PCR quantification indicated that the relative abundance of the Treponema group in the total rumen bacteria was as high as 1.05%, while the known species Treponema bryantii accounted for only 0.02%. Fingerprints of the Treponema community determined by 16S rDNA-targeted denaturing gradient gel electrophoresis (DGGE) analysis tended to differ among the diets. Principal component analysis of the DGGE profiles distinguished those Treponema associated with either the hay or the concentrate diets. Analysis of a Treponema 16S rRNA gene clone library showed phylogenetically distinct operational taxonomic units for a specific dietary condition, and significant (P=0.001) differences in community composition were observed among clone libraries constructed from each dietary regimen. The majority of clones (75.4%) had <97% sequence similarity with known Treponema. These results suggest the predominance of uncultured Treponema that appear to have distinct members related to the digestion of either hay or concentrate diet.