In vitro gas and methane production in rumen fluid from dairy cows fed grass silages differing in plant maturity,compared to in vivo data

The relationship between in vitro rumen CH₄ production of grass silages, using the gas production technique, and in vivo data obtained with the same cows and rations in respiration chambers was investigated. Silages were made from grass harvested in 2013 on May 6th, May 25th, July 1st and July 8th. The grass silages were used to formulate four different rations which were fed to 24 cows in early and late lactation, resulting in a slightly different dry matter intake (DMI; 16.5 kg/day vs. 15.4 kg/day). The experimental rations consisted of 70% grass silage, 10% maize silage, and 20% concentrates on a dry matter basis. Cows were adapted to the rations for 17 days before rumen fluid was collected via oesophageal tubing, and in vitro gas and CH₄ production were analysed. In vitro total gas and CH₄ production of the (ensiled) grass expressed as ml/g OM decreased with advancing maturity of the grass. The in vitro CH₄ production after 48 hr of incubation expressed in ml/g OM did not correlate with the in vivo CH₄ production expressed in g/kg organic matter intake or g/kg DMI (R² = .00–.18, p ≥ .287). The differences in CH₄ emission per unit of intake observed in vivo were rather small between the different rations, which also contributed to the observed poor relationship. Utilizing stepwise multiple regression improved the correlation only slightly. In vitro gas and CH₄ production varied based on whether donor cows were previously adapted to the respective ration or not, suggesting that careful adaption to the experimental diet should be envisaged in in vitro gas and CH₄ production experiments.     

Effects of illite supplementation on in vitro and in vivo rumen fermentation,microbial population and methane emission of Hanwoo steers fed high concentrate diets

This study was conducted to evaluate the effects of feeding supplemental illite to Hanwoo steers on methane (CH₄) emission and rumen fermentation parameters. An in vitro ruminal fermentation technique was conducted using a commercial concentrate as substrate and illite was added at different concentrations as treatments: 0%, 0.5%, 1.0%, and 2.0% illite. Total volatile fatty acids (VFA) were different (P < 0.05) at 24 h of incubation where the highest total VFA was observed at 1.0% of illite. Conversely, lowest CH₄ production (P < 0.01) was found at 1.0% of illite. In the in vivo experiment, two diets were provided, without illite and with addition of 1% illite. An automated head chamber (GreenFeed) system was used to measure enteric CH₄ production. Cattle received illite supplemented feed increased (P < 0.05) total VFA concentrations in the rumen compared with those fed control. Feeding illite numerically decreased CH₄ production (g/day) and yield (g/kg dry matter intake). Rumen microbial population analysis indicated that the population of total bacteria, protozoa and methanogens were lower (P < 0.05) for illite compared with the control. Accordingly, overall results suggested that feeding a diet supplemented with 1% illite can have positive effects on feed fermentation in the rumen and enteric CH₄ mitigation in beef cattle.     

Effects of pure plant secondary metabolites on methane production,rumen fermentation and rumen bacteria populations in vitro

In this study, the effects of seven pure plant secondary metabolites (PSM s) on rumen fermentation, methane (CH₄) production and rumen bacterial community composition were determined. Two in vitro trials were conducted. In trial 1, nine concentrations of 8‐hydroxyquinoline, α‐ terpineol, camphor, bornyl acetate, α‐ pinene, thymoquinone and thymol were incubated on separate days using in vitro 24‐hr batch incubations. All compounds tested demonstrated the ability to alter rumen fermentation parameters and decrease CH₄ production. However, effective concentrations differed among individual PSM s. The lowest concentrations that reduced (p  <  .05) CH₄ production were as follows: 8 mg/L of 8‐hydroxyquinoline, 120 mg/L of thymoquinone, 240 mg/L of thymol and 480 mg/L of α‐ terpineol, camphor, bornyl acetate and α‐ pinene. These concentrations were selected for use in trial 2. In trial 2, PSM s were incubated in one run. Methane was decreased (p  <  .05) by all PSM s at selected concentrations. However, only 8‐hydroxyquinoline, bornyl acetate and thymoquinone decreased (p  <  .05) CH₄ relative to volatile fatty acids (VFA s). Based on denaturing gradient gel electrophoresis analysis, different PSM s changed the composition of bacterial communities to different extents. As revealed by Ion Torrent sequencing, the effects of PSM s on relative abundance were most pronounced in the predominant families, especially in Lachnospiraceae , Succinivibrionaceae , Prevotellaceae , unclassified Clostridiales and Ruminococcaceae . The CH ₄ production was correlated negatively (?.72; p  <  .05) with relative abundance of Succinivibrionaceae and positively with relative abundance of Ruminococcaceae (.86; p  <  .05). In summary, this study identified three pure PSM s (8hydroxyquinoline, bornyl acetate and thymoquinone) with potentially promising effects on rumen CH₄ production. The PSM s tested in this study demonstrated considerable impact on rumen bacterial communities even at the lowest concentrations that decreased CH₄ production. The findings from this study may help to elucidate how PSM s affect rumen bacterial fermentation.     

Chemical and nutritional characteristics of Cannabis sativa L. co-products

Cannabis sativa L. is an annual herbaceous plant. It was used for centuries to obtain different products. In the last century, hemp cultivation was forbidden due to the psychoactive effects of tetrahydrocannabinol acid (THCA). In the last years, new strains, characterized by high cannabidiolic acid (CBDA) and low THCA level, were developed renewing the interest in hemp cultivation to obtain food or to extract essential oils from flowers. All these processes produce many residues with different chemical–physical characteristics. In order to evaluate their potential use also in animal nutrition, some hemp co-products were evaluated. Two different co-products of seed processes (flour and oil) and two co-products obtained trimming the flowers, differing in granulometry were used. The samples were analysed for chemical composition and evaluated in vitro using the gas production technique with buffaloes' ruminal inoculum. All hemp co-products showed interesting nutritional characteristics, such as crude protein content always higher than 20% on a dry matter basis, and high neutral detergent fibre concentration partially lignified. The in vitro gas production parameters at 120 h of incubation showed quite low fermentability testified by the low organic matter degradability and cumulative gas volume (OMD from 28.09 to 45.64% and OMCV from 110 to 164 ml/g, respectively). Also, the methane produced after 24 h of incubation was particularly low (from 1.78 to 11.73 ml/g dOM). These results could be due to the high lipid and ash amounts or to the CBDA content that probably affected the CH₄ formation processes. According to preliminary results obtained by this study, it is possible to hypothesize that these co-products could be useful to mitigate the methane production into the rumen. Further studies are necessary in order to evaluate the correct inclusion into the diet for ruminants.     

Reducing methane production by supplementation of Terminalia chebula RETZ. containing tannins and saponins

This study investigates the effects of Terminalia chebula Retz. meal supplementation on rumen fermentation and methane (CH₄) production by using an in vitro gas technique. The experimental design was a completely randomized design (CRD) and the dietary treatments were T. chebula supplementation at 0, 4, 8, 12, 16 and 20 mg with 0.5 g of roughage and concentrate ratio at 60:40. The results revealed that cumulative gas production (96 h of incubation) were higher (P < 0.01) with T. chebula supplementation at 12, 16 and 20 mg than other treatments. However, in vitro dry matter degradability (IVDMD) and in vitro organic matter digestibility (IVOMD) were not significantly different among treatments (P > 0.05). The NH₃‐N concentrations tended to quadratically increase with increasing levels of T. chebula in the diet. In addition, total volatile fatty acids (VFA) and propionate concentrations were increased (P < 0.01), while acetate concentration, acetate‐to‐propionate ratio, CH₄ production and protozoal populations were decreased (P < 0.01) when supplemented with T. chebula at 8, 12 and 16 mg, respectively. Based on this study, it could be concluded that supplementation of T. chebula at 12 mg could improve rumen fermentation by reducing CH₄ production and protozoa populations, thus improving in vitro gas production and VFA profiles.     

Quantitative analysis of ruminal methanogenic microbial populations in beef cattle divergent in phenotypic residual feed intake (RFI) offered contrasting diets

Background

Methane (CH₄) emissions in cattle are an undesirable end product of rumen methanogenic fermentative activity as they are associated not only with negative environmental impacts but also with reduced host feed efficiency. The aim of this study was to quantify total and specific rumen microbial methanogenic populations in beef cattle divergently selected for residual feed intake (RFI) while offered (i) a low energy high forage (HF) diet followed by (ii) a high energy low forage (LF) diet. Ruminal fluid was collected from 14 high (H) and 14 low (L) RFI animals across both dietary periods. Quantitative real time PCR (qRT-PCR) analysis was conducted to quantify the abundance of total and specific rumen methanogenic microbes. Spearman correlation analysis was used to investigate the association between the relative abundance of methanogens and animal performance, rumen fermentation variables and diet digestibility.

Results

Abundance of methanogens, did not differ between RFI phenotypes. However, relative abundance of total and specific methanogen species was affected (P < 0.05) by diet type, with greater abundance observed while animals were offered the LF compared to the HF diet.

Conclusions

These findings suggest that differences in abundance of specific rumen methanogen species may not contribute to variation in CH₄ emissions between efficient and inefficient animals, however dietary manipulation can influence the abundance of total and specific methanogen species.     

Feeding of tropical trees and shrub foliages as a strategy to reduce ruminal methanogenesis: studies conducted in Cuba

The aim of this paper was to present the main results obtained in Cuba on the effects of feeding tropical trees and shrubs on rumen methanogenesis in animals fed with low quality fibrous diets. More than 20 tree and shrub foliages were screened for phytochemicals and analyzed for chemical constituents. From these samples, seven promising plants (Samanea saman, Albizia lebbeck, Tithonia diversifolia, Leucaena leucocephala, Trichantera gigantea, Sapindus saponaria, and Morus alba) were evaluated for methane reduction using an in vitro rumen fermentation system. Results indicated that the inclusion levels of 25% of Sapindo, Morus, or Trichantera foliages in the foliages/grass mixtures (grass being Pennisetum purpureum) reduced (P < 0.01) methane production in vitro when compared to Pennisetum alone (17.0, 19.1, and 18.0 versus 26.2 mL CH₄/g fermented dry matter, respectively). It was demonstrated that S. saman, A. lebbeck, or T. diversifolia accession 23 foliages when mixed at the rate of 30% in Cynodon nlemfuensis grass produced lower methane compared to the grass alone. Inclusion levels of 15% and 25% of a ruminal activator supplement containing 29% of L. leucocehala foliage meal reduced methane by 37% and 42% when compared to the treatment without supplementation. In vivo experiment with sheep showed that inclusion of 27% of L. leucocephala in the diet increased the DM intake but did not show significant difference in methane production compared to control diet without this foliage. The results of these experiments suggest that the feeding of tropical tree and shrub foliages could be an attractive strategy for reduction of ruminal methanogenesis from animals fed with low-quality forage diets and for improving their productivity.     

Dose–response effect of a pine bark extract on in vitro ruminal ammonia and methane formation kinetics

ABSTRACT

This study assessed the potential of a pine bark extract (PBE) to decrease methane (CH₄) and ammonia nitrogen (NH₃-N) production in vitro. Dietary substrates, mixed hay, soybean meal and corn grain, were supplemented 0, 2, 4 and 6% of PBE and incubated in an in vitro batch culture for 24-h. Incubations were run three times. Total gas production (GP) was determined at 6, 12 and 24-h and gas samples were analysed for CH₄. Samples were collected for volatile fatty acids (VFA) and NH₃-N analysis. Treatments were compared by polynomial contrasts for PBE concentration. Increasing PBE caused linear decreases in NH₃-N, microbial biomass production and digestibility, whereas the degradation rate was quadratically reduced. Total VFA were decreased but total GP and CH₄ production and kinetics were unaffected. The inclusion of 2% PBE in ruminant feed has the capability to reduce NH₃-N concentration by 50%, without affecting diet digestibility or CH₄ production.     

Identification of rumen microbial biomarkers linked to methane emission in Holstein dairy cows

Mitigation of greenhouse gas emissions is relevant for reducing the environmental impact of ruminant production. In this study, the rumen microbiome from Holstein cows was characterized through a combination of 16S rRNA gene and shotgun metagenomic sequencing. Methane production (CH₄) and dry matter intake (DMI) were individually measured over 4–6 weeks to calculate the CH₄ yield (CH₄y = CH₄/DMI) per cow. We implemented a combination of clustering, multivariate and mixed model analyses to identify a set of operational taxonomic unit (OTU) jointly associated with CH₄y and the structure of ruminal microbial communities. Three ruminotype clusters (R1, R2 and R3) were identified, and R2 was associated with higher CH₄y. The taxonomic composition on R2 had lower abundance of Succinivibrionaceae and Methanosphaera, and higher abundance of Ruminococcaceae, Christensenellaceae and Lachnospiraceae. Metagenomic data confirmed the lower abundance of Succinivibrionaceae and Methanosphaera in R2 and identified genera (Fibrobacter and unclassified Bacteroidales) not highlighted by metataxonomic analysis. In addition, the functional metagenomic analysis revealed that samples classified in cluster R2 were overrepresented by genes coding for KEGG modules associated with methanogenesis, including a significant relative abundance of the methyl-coenzyme M reductase enzyme. Based on the cluster assignment, we applied a sparse partial least-squares discriminant analysis at the taxonomic and functional levels. In addition, we implemented a sPLS regression model using the phenotypic variation of CH₄y. By combining these two approaches, we identified 86 discriminant bacterial OTUs, notably including families linked to CH₄ emission such as Succinivibrionaceae, Ruminococcaceae, Christensenellaceae, Lachnospiraceae and Rikenellaceae. These selected OTUs explained 24% of the CH₄y phenotypic variance, whereas the host genome contribution was ~14%. In summary, we identified rumen microbial biomarkers associated with the methane production of dairy cows; these biomarkers could be used for targeted methane-reduction selection programmes in the dairy cattle industry provided they are heritable.     

Effect of methanol on methanogenesis and fermentation in the rumen simulation technique (RUSITEC)

Introduction Major methanol sources in ruminant feeds are pectins esterified with methoxyl groups. Methanol can be released by pectin esterase activity of rumen bacteria (R exova -B enkova and M arkovic 1976). It has been detected in rumen fluid of cows in vivo and in vitro (V antcheva et al. 1970, 1972). It is, however, not likely to accumulate in the rumen fluid since it can be readily used by methylotrophic organisms. Methanogenic archaebacteria such as Methanosarcina barkeri (H utten et al. 1980; M& uuml ; ller et al. 1986) are able to use methanol as energy and carbon source. Two equations have been reported for this conversion: 4CH₄ → 3CH₄ + CO₂ + 2 H₂O (1) CH₃OH + H₂ → CH₄ + H₂O (2) Methanogenesis from methanol has been shown to occur during in vitro fermentations with rumen fluid as inoculum (C zerkawski and B reckenridge 1972; P ol and D emeyer 1988). However, acidogenic micro-organisms, such as Eubacterium limosum or Butyribacterium methylotrophicum, may also utilize methanol. In rumen fluid of sheep fed a molasses-rich diet, Eubacterium limosum was the predominant methanol-utilizing bacterium (G enthner et al. 1981). Major products of these acidogenic methylotrophs are acetate and butyrate. These different fermentation end-products, methane versus fatty acids, will affect the metabolizable energy content of feedstuffs that are rich in highly methoxylated pectins or other methyl group-containing compounds. The purpose of the present study was to investigate whether the rumen simulation technique (RUSITEC) would be a suitable model to evaluate the methanogenic or acidogenic potential of methanol during ruminal fermentation. The study focused on methane production, fermentation characteristics and methanol turnover. Fermentation traits included pH, redox potential, ammonia and volatile fatty acid production, and degradation of feed constituents.     

In vitro microbial protein synthesis,ruminal degradation and post‐ruminal digestibility of crude protein of dairy rations containing Quebracho tannin extract

This study evaluated the effects of Quebracho tannin extract (QTE) on in vitro ruminal fermentation, chemical composition of rumen microbes, ruminal degradation and intestinal digestibility of crude protein (iCPd). Three treatments were tested, the control (basal diet without QTE), the basal diet with 15 g QTE/kg dry matter (DM) and the basal diet with 30 g QTE/kg DM. The basal diet contained (g/kg DM): 339 grass silage, 317 maize silage and 344 concentrate. In vitro gas production kinetic was determined using the Hohenheim gas test (Experiment 1). The Ankom RF technique, a batch system with automatic gas pressure recordings, was used to determine in vitro production of short‐chain fatty acids (SCFA) and ammonia–nitrogen concentration (NH₃‐N), as well as nitrogen and purine bases content in liquid‐associated microbes (LAM) and in a residue of undegraded feed and solid‐associated microbes (Feed+SAM) (Experiment 2). Ruminal degradation and iCPd were determined using the nylon bag technique and the mobile nylon bag technique, respectively (Experiment 3). Gas production (Experiment 1), total SCFA and NH₃‐N (Experiment 2) decreased with increasing QTE levels. Microbial mass and composition of LAM were not affected by QTE, but total mass of Feed+SAM linearly increased, likely due to decreased substrate degradation with increasing QTE levels. The total amount of N in microbial mass and undegraded feed after the in vitro incubation increased with increasing QTE levels, suggesting a potential greater N flow from the rumen to the duodenum. In contrast to in vivo studies with the same QTE, no effects were detected on ruminal effective degradability and iCPd, when using the nylon bag techniques. Based on the in vitro procedures, QTE increased the supply of N post‐rumen; however, some evidence of a decreased fibre degradation were also observed. Therefore, the benefit of adding QTE to diets of cattle is still questionable.     

Effects of the ruminal comminution rate and microbial contamination of particles on accuracy of in situ estimates of ruminal degradability and intestinal digestibility of feedstuffs

Effects of considering the comminution rate (k_c) and the correction of microbial contamination (using ¹⁵N techniques) of particles in the rumen on estimates of ruminally undegraded fractions and their intestinal digestibility were examined generating composite samples (from rumen‐incubated residues) representative of the undegraded feed rumen outflow. The study used sunflower meal (SFM) and Italian ryegrass hay (RGH) and three rumen and duodenum cannulated wethers fed with a 40:60 RGH to concentrate diet (75 g DM/kgBW^0.75). Transit studies up to the duodenum with Yb‐SFM and Eu‐RGH marked samples showed higher k_c values (/h) in SFM than in RGH (0.577 vs. 0.0892, p = 0.034), whereas similar values occurred for the rumen passage rate (k_p). Estimates of ruminally undegraded and intestinal digestibility of all tested fractions decreased when k_c was considered and also applying microbial correction. Thus, microbial uncorrected k_p‐based proportions of intestinal digested undegraded crude protein overestimated those corrected and k_c?k_p‐based by 39% in SFM (0.146 vs. 0.105) and 761% in RGH (0.373 vs. 0.0433). Results show that both k_c and microbial contamination correction should be considered to obtain accurate in situ estimates in grasses, whereas in protein concentrates not considering k_c is an important source of error.     

Influence of condensed tannin on intake, digestibility, and efficiency of protein utilization in beef steers fed high concentrate diet

This trial was conducted to evaluate if the effect of condensed tannin (CT) is associated with a true protein source on intake, ruminal and total digestibility, ruminal digestion rate, protein efficiency, and microbial efficiency in beef steers fed high concentrate diet (87% of DM). Four Bos indicus steers (407 ± 12 kg of BW) fitted with rumen cannula were assigned to a 4 × 4 Latin square design, arranged in a 2 × 2 factorial arrangement. Treatments consisted of either inclusion (0.4% of DM) or exclusion of condensed tannin (CT) from quebracho extract (76% of CT) with or without the use of soybean meal (SBM) as source of true protein. The level of inclusion was calculated to provide a daily intake of 1 g/10 kg of BW of CT. Intake of DM and nutrients was not affected (P > 0.10) by CT inclusion. However, there was an effect (P < 0.10) of CT inclusion on ether extract digestibility. An interaction (P < 0.10) was observed between CT and SBM on ruminal digestibility and digestion rate of crude protein (CP): when mixed with soybean meal, CT decreased the ruminal digestibility and, consequently, reduced the digestion rate of CP. Intake of CP increased (P < 0.10) with the inclusion of SBM. No differences in DM passage rate were observed (P > 0.10) among treatments. Effects of the interaction (P < 0.10) between CT and SBM were observed on flux of rumen undegradable protein (RUP), metabolizable protein (MP), and on the ratio MP:CP. In the presence of soybean meal, the addition of CT increased (P < 0.10) the flux of RUP and MP, and improved the ratio MP:CP. The yield of microbial protein on the abomasum and the microbial efficiency did not differ among treatments (P > 0.10). There was no difference (P > 0.10) on the pH, VFA, and ruminal ammonia (N-NH₃) with the addition of condensed tannin. The N-NH₃ increased and the ruminal pH decreased with the inclusion of soybean meal (P < 0.10). The utilization of condensed tannin as an additive in beef cattle diets with high level of concentrate and soybean meal as a source of true protein implies positive effects on crude protein utilization, decreasing digestion rate and ruminal digestibility of crude protein and consequently increasing the levels of metabolizable protein, with no changes in the ruminal fermentation parameters.     

Microbial and chemical composition of liquid‐associated bacteria in goats' rumen and fermenters

This study was undertaken to investigate the relationship between chemical composition and microbial profile of rumen liquid‐associated bacteria (LAB) in vivo (Murciano‐Granadina goats) and in a rumen simulation system (single‐flow continuous‐culture fermenters). To achieve this aim, analyses of purine bases along with some molecular techniques (quantitative PCR to assess abundance and DGGE to identify biodiversity and bacterial profile) were carried out. A control diet (AHC) based on alfalfa hay (AH) and concentrate (C) in a 1:1 ratio and two experimental diets (AHCBI and AHCBII), in which concentrate was partially replaced with multinutrient blocks, were used. Diets AHCBI and AHCBII included multinutrient blocks differing in the relative amount of two‐stage olive cake and the source of protein (sunflower meal vs. fava beans). We aimed to investigate the effect of these blocks on rumen microbiota to evaluate their potential as safe substitutes of cereal‐based concentrates. Similar patterns of response to diet were found for chemical composition, microbial abundances and diversity in LAB isolated from goat's rumen and fermenters. Whereas bacterial density (log₁₀ gene copies/g FM: 11.6 and 9.4 for bacteria and methanogens, respectively, in rumen) and diversity indexes (Shannon index: 3.6) were not affected by diet, DGGE analyses showed that bacterial community profile was affected. The cluster analysis suggested differences in bacterial profile between LAB pellets isolated from the rumen of goat and fermenters. A relationship between chemical composition and bacterial community composition in LAB pellets seems to exist. Changes in the former were reflected in the bacterial community profile. Further research is needed to clarify the relationship between chemical and microbial composition of ruminal bacterial pellets with diets of different quality.     

Rumen fermentation and degradability in buffalo and cattle using the in vitro gas production technique

An in vitro trial was conducted to investigate the effect of different inoculum sources (buffalo vs. cattle) on rumen fermentation and degradability. Incubations were carried out using rumen fluid obtained from buffalo or cattle fed the same diet [60% grass hay and 40% concentrate; 18 kg dry matter (DM)/day]. The fermentation kinetics of eight feeds commonly used in ruminant nutrition (alfalfa hay, barley meal, beet pulp, corn meal and silage, ryegrass hay and silage and soya bean meal s.e.) were studied with the in vitro gas production technique and rumen fermentation parameters (substrate disappearance, pH and volatile fatty acids production) were determined after 120 h of incubation. The linear relationship indicates that the microbial metabolic pathways of the two inocula for all the substrates were qualitatively similar, albeit often quantitatively different. In this in vitro study, a significant influence of rumen inoculum (buffalo vs. cow) on fermentation and degradability of the examined substrates was found. The differences in buffalo and cattle rumen fermentation can be explained with a different microbial activity of the two ruminant species, because of different amount of microbial population or microbial population constituted by different species of bacteria and protozoa.     

Impact of variation in structure of condensed tannins from sainfoin (Onobrychis viciifolia) on in vitro ruminal methane production and fermentation characteristics

Our study investigated the effects of condensed tannins (CT) on rumen in vitro methane (CH₄) production and fermentation characteristics by incubating lucerne in buffered rumen fluid in combination with different CT extracts at 0 (control), 40, 80 and 120 g CT/kg of substrate DM. Condensed tannins were extracted from four sainfoin accessions: Rees ‘A’, CPI63763, Cotswold Common and CPI63767. Gas production (GP) was measured using a fully automated GP apparatus with CH₄ measured at distinct time points. Condensed tannins differed substantially in terms of polymer size and varied from 13 (Rees ‘A’) to 73 (CPI63767) mean degree of polymerization, but had relatively similar characteristics in terms of CT content, procyanidin: prodelphinidin (PC: PD) and cis:trans ratios. Compared to control, addition of CT from CPI63767 and CPI63763 at 80 and 120 g CT/kg of substrate DM reduced CH₄ by 43% and 65%, and by 23% and 57%, respectively, after 24‐h incubation. Similarly, CT from Rees ‘A’ and Cotswold Common reduced CH₄ by 26% and 46%, and by 28% and 46% respectively. Addition of increasing level of CT linearly reduced the maximum rates of GP and CH₄ production, and the estimated in vitro organic matter digestibility. There was a negative linear and quadratic (p < 0.01) relation between CT concentration and total volatile fatty acid (VFA) production. Inclusion of 80 and 120 g CT/kg of substrate DM reduced (p < 0.001) branched‐chain VFA production and acetate: propionate ratio and was lowest for CPI63767. A decrease in proteolytic activity as indirectly shown by a change in VFA composition favouring a shift towards propionate and reduction in branched‐chain VFA production varied with type of CT and was highest for CPI63767. In conclusion, these results suggest that tannin polymer size is an important factor affecting in vitro CH₄ production which may be linked to the CT interaction with dietary substrate or microbial cells.     

Modulation of ruminal and intestinal fermentation by medicinal plants and zinc from different sources

Two experiments were conducted on sheep to determine the effect of dietary supplementation with zinc and a medicinal plant mixture on haematological parameters and microbial activity in the rumen and large intestine. In Experiment 1, 24 male lambs were randomly divided into four groups: One group was fed an unsupplemented basal diet (control), and three groups were fed a diet supplemented with 70 mg Zn/kg diet in the form of Zn sulphate (ZnSO₄), a Zn‐chelate of glycine hydrate (Zn‐Gly) or a Zn‐proteinate (Zn‐Pro), for five months. The ruminal content was collected separately from each lamb, and batch cultures of ruminal fluid were incubated in vitro with mixture of medicinal plants (Mix) with different roughage:concentrate ratios (800:200 and 400:600, w/w). Bioactive compounds in Mix were quantified by UPLC/MS/MS. In Experiment 2, four sheep were fed a diet consisting of meadow hay and barley grain (400:600, w/w), with Zn‐Gly (70 mg Zn/kg diet), Mix (10% replacement of meadow hay) or Zn‐Gly and Mix (Zn‐Gly‐Mix) as supplements in a Latin square design. Mix decreased total gas (p < 0.001) and methane (p < 0.01) production in vitro. In Experiment 1, caecal isobutyrate and isovalerate concentrations varied among the dietary treatments (p < 0.01). The isovalerate concentration of the zinc‐supplemented groups in the distal colon was higher (p < 0.001) compared with the control. In Experiment 2, the molar proportion of isobutyrate was the highest in the faeces of the sheep fed the diet with Zn‐Gly‐Mix (p < 0.01). The plasma zinc concentration was higher in the groups fed a diet supplemented with zinc (p < 0.001). The haematological profile and antioxidant status did not differ between the dietary groups (p > 0.05). The diets containing medicinal plants and organic zinc thus helped to modulate the characteristics of fermentation in ruminants.     

Effects of supplementing galacto-oligosaccharides, Yucca schidigera or nisin on rumen methanogenesis, nitrogen and energy metabolism in sheep

The effects of galacto-oligosaccharides (GOS), Yucca schidigera (YS) or nisin (NS), as additives to a basal diet of grass silage and concentrate, on rumen methanogenesis, nitrogen and energy metabolism were conducted using four rumen cannulated wethers in a 4×4 Latin square design. Four treatments comprised basal diet (control), basal diet+20 g GOS, basal diet+120 ppm YS, basal diet+3 mg/kg BW^0.75 of NS. Apparent digestibility of DM, OM, CP, NDF and ADF were similar in all treatment diets. Animals fed YS had lower N losses in urine and total N losses resulting in a higher retained N. Ruminal pH values ranged from 6.33 to 6.47 and was significantly (P<0.05) altered by treatment. Sheep given YS had the lowest NH₃-N concentration, moderate in control and GOS, and the highest in NS. The GOS supplemented sheep had lower (P<0.01) proportion of acetic acid and higher (P<0.01) proportion of propionic acid compared to those fed the control diet. Microbial nitrogen supply was higher (P<0.05) in GOS supplemented sheep than those given control, YS and NS diets. Relative to control, YS and NS treatments, GOS reduced (P<0.05) methane production (l/kg BW^0.75). These results indicated that natural products have the potential to be used as manipulators of rumen fermentation.     

Effects of Lonicera japonica extract supplementation on in vitro ruminal fermentation,methane emission,and microbial population

Lonicera japonica (LJ; honeysuckle) is used in traditional folk medicine in Korea and is a rich source of ascorbic acid and phenolic components that are reported to have antioxidant and antibiotic properties. We performed an in vitro experiment to assess the effects of LJ extracts (LJE) on ruminal fermentation. Timothy hay (0.3 g dry matter [DM]) was incubated with buffer, ruminal fluid, and 0%, 3%, 5%, 7%, and 9% LJE. Batch culture fermentation was conducted separately for 12, 24, and 48 hr to determine gas production (GP), ruminal fermentation characteristics, and microbial population characteristics. The effects on GP were generally similar to those on DM degradability, with a linear decrease observed at 9% extract at 24 hr. NH₃‐N showed a linear increase with increasing extract concentrations at 12 hr, whereas a decrease was observed at 24 hr. Extract supplementation decreased methane (CH₄) production at 12, 24, and 48 hr. In addition, the abundance of fibrolytic bacteria and ciliate‐associated methanogen was reduced at all concentrations of extracts. These results indicate that LJE have the potential to serve as a ruminal fermentation modifier to suppress CH₄ production with minimal effects on nutrient digestion in the rumen.     

Energy utilization,nitrogen balance and microbial protein supply in cattle fed Pennisetum purpureum and condensed tannins

The aim of the experiment was to assess the effect of condensed tannins (CT) on feed intake, dry matter digestibility, nitrogen balance, supply of microbial protein to the small intestine and energy utilization in cattle fed a basal ration of Pennisetum purpureum grass. Five heifers (Bos taurus × Bos indicus) with an average live weight of 295 ± 19 kg were allotted to five treatments consisting of increasing levels of CT (0, 1, 2, 3 and 4% CT/kg DM) in a 5 × 5 Latin square design. Dry matter intake (DMI) was similar (p > 0.05) between treatments containing 0, 1, 2 and 3% of CT/kg DM and it was reduced (p < 0.05) to 4% CT (5.71 kg DM/day) with respect to that observed with 0% CT (6.65 kg DM/day). Nitrogen balance, purine derivatives excretion in urine, microbial protein synthesis and efficiency of synthesis of microbial nitrogen in the rumen were not affected (p ≥ 0.05) by the increase in the levels of condensed tannins in the ration. Energy loss as CH₄ was on average 2.7% of the gross energy consumed daily. Metabolizable energy intake was 49.06 MJ/day in cattle fed low‐quality tropical grass with a DMI of 6.27 kg/day. It is concluded that concentrations of CT between 2 and 3% of DM of ration reduced energy loss as CH₄ by 31.3% and 47.6%, respectively, without affecting intakes of dry and organic matter; however, digestibilities of dry and organic matter are negatively affected.