Effect of monensin withdrawal on rumen fermentation,methanogenesis and microbial populations in cattle

This study was designed to obtain information on the residual influence of dietary monensin on ruminant fermentation, methanogenesis and bacterial population. Three ruminally cannulated crossbreed heifers (14 months old, 363 ± 11 kg) were fed Italian ryegrass straw and concentrate supplemented with monensin for 21 days before sampling. Rumen fluid samples were collected for analysis of short chain fatty acid (SCFA) profiles, monensin concentration, methanogens and rumen bacterial density. Post‐feeding rumen fluid was also collected to determine in vitro gas production. Monensin was eliminated from the rumen fluid within 3 days. The composition of SCFA varied after elimination of monensin, while total production of SCFA was 1.78 times higher than on the first day. Methane production increased 7 days after monensin administration ceased, whereas hydrogen production decreased. The methanogens and rumen bacterial copy numbers were unaffected by the withdrawal of monensin.     

Effects of dietary supplementing tannic acid in the ration of beef cattle on rumen fermentation,methane emission,microbial flora and nutrient digestibility

Four adult Simmental male cattle (376 ± 9.0 kg initial BW), fitted with permanent rumen cannulas, were used in a 4 × 4 Latin square design to investigate the effects of dietary supplementing tannic acid (TA) on rumen fermentation, methane (CH₄) production, rumen microbes, nutrient digestibility and plasma biochemical parameters. Four levels of TA, that is 0, 6.5, 13.0 or 26.0 g/kg dry matter (DM), were added to the basal ration (composed of corn silage and concentrate mixture) as experimental treatments respectively. Each experimental period consisted of a 12‐day adaptation phase followed by a 3‐day sampling phase. The results showed that supplementing TA at 26.0 g/kg DM decreased the relative abundance of protozoa, methanogens and Ruminococcus albus to the total ruminal bacterial 16S rDNA in beef cattle (p < 0.05). The results also showed that supplementing TA at 6.5, 13.0 or 26.0 g/kg DM decreased (p < 0.01) the CH₄ production (l/kg DM intake) by 11.1%, 14.7% and 33.6% respectively. Supplementing TA at 13.0 or 26.0 g/kg DM decreased the ratio of acetate to propionate and ammonia nitrogen (NH₃–N) (p < 0.05) and tended to decrease the total volatile fatty acid (VFA) concentration of rumen fluid (p = 0.07). Supplementing TA at 26.0 g/kg DM decreased DM and organic matter (OM) digestibility (p < 0.05), supplementing TA at 6.5, 13.0 or 26.0 g/kg DM decreased (p < 0.01) crude protein (CP) digestibility by 5.0%, 8.6% and 15.7%, respectively, and supplementing TA at 6.5, 13.0 or 26.0 g/kg DM increased (p < 0.05) the plasma total antioxidant capability. It was concluded that supplementing TA in the ration of beef cattle decreased the CH₄ production and digestibility of CP of beef cattle. Supplementing TA could be an effective option to mitigate CH₄ emission form cattle, further research is necessary to study the effects of TA on the performance of cattle.     

Dietary supplementation with mulberry leaf flavonoids inhibits methanogenesis in sheep

The effects of flavonoids on methanogenesis and microbial flora in Dorper × thin‐tailed Han crossbred ewes were evaluated in two experiments. To investigate the effects of flavonoids on nutrient digestibility and nitrogen balance, 18 ewes (60.0 ± 1.73 kg body weight (BW)) were allotted to two dietary treatments in experiment one, a control diet and the control diet supplemented with flavonoids (2 g/head/day). In experiment two, the effects of supplementary flavonoids on ruminal fermentation and microbial flora were investigated using quantitative polymerase chain reaction with six ewes (67.2 ± 0.79 kg BW) with ruminal cannula assigned to the identical dietary treatments used in experiment one. Supplementary flavonoids improved the apparent digestibility of nitrogen (N, P < 0.001) and neutral detergent fibre (NDF, P = 0.024) and decreased daily CH₄ output (P < 0.001). The ruminal pH (P = 0.638) and ammonia (P = 0.690) were not affected by supplementary flavonoids, whereas the total volatile fatty acid (VFA) content increased (P = 0.037). Supplementary flavonoids decreased ruminal populations of protozoans (P = 0.002) and methanogens (P < 0.001) and increased the populations of Fibrobacter succinogenes (P = 0.016). In conclusion, flavonoids improved the digestibility of organic matter and reduced CH₄ output by inhibiting the populations of microbes involved in methanogenesis.     

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.     

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.     

Rumen microbial response in production of CLA and methane to safflower oil in association with fish oil or/and fumarate

Supplementation effect of fish oil and/or fumarate on production of conjugated linoleic acid (CLA) and methane by rumen microbes was examined when incubated with safflower oil. One hundred and twenty milligrams of safflower oil (SO), safflower oil with 24 mg fish oil (SOFO), safflower oil with 24 mmol/L fumarate (SOFA), or safflower oil with 24 mg fish oil and 24 mmol/L fumarate (SOFOFA) were added to the 90 mL culture solution. The culture solution was also made without any supplements (control). The SOFA and SOFOFA increased pH and propionate (C3) compared to other treatments from 3 h incubation time. An accumulated amount of total methane (CH₄) for 12 h incubation was decreased by all the supplements compared to control. The concentrations of c9,t11CLA for all the incubation times were increased in the treatments of SOFO, SOFA and SOFOFA compared to SO. The highest concentration of c9,t11CLA was observed from SOFOFA among all the treatments at all incubation times. Overall data indicate that supplementation of combined fumarate and/or fish oil when incubated with safflower oil could depress CH₄ generation and increase production of C₃ and CLA under the condition of current in vitro study.     

Nutritional evaluation of the legume Macrotyloma axillare using in vitro and in vivo bioassays in sheep

This study consisted of two experiments with the following objectives: to evaluate the effects of tannins from the tropical legume macrotiloma (Macrotyloma axillare) on total gas and methane (CH₄) production, as well as on ruminal fermentation parameters by performing an in vitro bioassay, with samples incubated with and without polyethylene glycol (PEG) in a semi‐automatic system; and secondly in a 17 day in vivo experiment, to determine apparent total tract digestibility (ATTD) of dietary nutrients and ruminal fermentation parameters of 12 intact 8‐ to 9‐month‐old Santa Inês (averaging 24.95 ± 1.8 kg body weight) ewes fed tropical grass hay supplemented with macrotiloma hay. The ewes were divided into two treatment groups depending on their diet: chopped aruana grass hay (Panicum maximum cv. Aruana) (control—CON); and aruana grass hay supplemented with chopped macrotiloma hay (macrotiloma—MAC). The animals were kept for 5 consecutive days in metabolic cages for the ATTD assay, and at the end of this period, samples of rumen fluid were collected from each ewe to determine ammoniacal nitrogen (NH₃‐N) and short‐chain fatty acid (SCFA) production, and protozoa count. For the in vitro assay, a decrease in total gas and CH₄ production was observed for samples incubated without PEG (p < .05). No differences were observed for the other parameters evaluated (p > .05). In the in vivo experiment, increased intake and ATTD of crude protein were observed for the animals fed MAC when compared to CON (p < .05). For rumen fermentation parameters, increased NH₃‐N, total SCFA and isobutyrate concentrations, as well as reduced protozoa count were observed for MAC when compared to CON (p < .05). The results observed here indicated the potential of macrotiloma for use as a ruminant feed, and antimethanogenic potential of this plant was noted.     

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.     

Effects of rare earth element lanthanum on rumen methane and volatile fatty acid production and microbial flora in vitro

The objectives of the trial were to study the effects of rare earth element (REE) lanthanum (La) on the in vitro rumen methane (CH₄) and volatile fatty acid (VFA) production and the microbial flora of feeds. Four feed mixtures with different levels of neutral detergent fibre (NDF), that is 20.0% (I), 31.0% (II), 41.9% (III) and 52.7% (IV), were formulated as substrates. Five levels of LaCl₃, that is 0, 0.4, 0.6, 0.8 and 1.0 mmol/kg dry matter (DM), were added to the feed mixtures, respectively, as experimental treatments in a two‐factor 5 × 4 randomized design. The in vitro incubation lasted for 24 h. The results showed that supplementing LaCl₃ increased the total gas (p < 0.001) production and tended to increase the total VFA production (p = 0.072) and decreased the CH₄ production (p = 0.001) and the ratios of acetate/propionate (p = 0.019) and CH₄/total VFA (p < 0.001). Interactions between LaCl₃ and NDF were significant in total gas production (p = 0.030) and tended to be significant in CH₄ production (p = 0.071). Supplementing LaCl₃ at the level of 0.8 mmol/g DM decreased the relative abundance of methanogens and protozoa in the total bacterial 16S rDNA analysed using the real‐time PCR (p < 0.0001), increased F. succinogenes (p = 0.0003) and decreased R. flavefaciens (p < 0.0001) whereas did not affect R. albus and anaerobic fungi (p > 0.05). It was concluded that LaCl₃ decreased the CH₄ production without negatively affecting feed digestion through manipulating rumen microbial flora when feed mixtures with different levels of NDF were used as substrates.     

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.     

In vitro gas production in rumen fluid of buffalo as affected by urea‐calcium mixture in high‐quality feed block

This study aimed to determine the effect of urea‐calcium sulphate mixture (U‐cas) levels in high‐quality feed block (HQFB) on ruminal digestibility, fermentation and gas kinetics in rumen fluid of swamp buffalo by using in vitro techniques. The treatments were seven levels of U‐cas incorporated in HQFB at 0, 3, 6, 9, 12, 15 and 18% and the experimental design was a completely randomized design. Gas production rate constants for the insoluble fraction, potential extent of gas and cumulative gas were linearly increased with increasing levels of U‐cas in HQFB. The in vitro dry matter digestibility, in vitro organic matter digestibility, true digestibility and microbial mass were altered by treatments and were greatest at 18% U‐cas supplementation. Concentrations of propionate were linearly increased with increasing levels of U‐cas and was highest with U‐cas supplementation at 18%. The NH₃–N concentration was highest when urea was added in the HQFB while NH₃–N concentration tended to be reduced with increasing level of U‐cas. The findings suggest supplementation of 18% U‐cas in HQFB improves kinetics of gas production, rumen fermentation, digestibility and microbial mass as well as controlling the rate of N degradation in the rumen of swamp buffalo.     

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.     

A note on the stoichiometrical relationship of short chain fatty acid production and gas formation in vitro in feedstuffs of widely differing quality

In vitro gas production tests are increasingly used in feedstuff research, mainly because rates of fermentation can conveniently be estimated via the accumulating gas volumes (for review see Getachew et al. 1998). In Germany, the Hohenheim in vitro gas test is widely employed in routine feed evaluation to predict in vivo digestibilities and metabolizable energy contents of feeds (Menke and Steingaβ 1988). Although they are waste products, fermentative gases (mainly CO₂ and CH₄) undoubtfully represent parts of the feeds which have been degraded. Conceptually, the use of in vitro gas tests is then only justified by a close linkage between the waste products and useful fermentation products, i.e. short chain fatty acids (SCFA) and/or microbial biomass. It was shown for straw (Blümmel and O?rskov 1993), cereal grain (Opatpatanakit et al. (1994) and hay (Makkar et al. 1995) that gas volumes in the bicarbonate buffered Hohenheim in vitro gas production test reflect SCFA production very closely. Gas volumes were produced quantitatively and qualitatively (Blümmel and O?rskov 1993) according to the stoichiometry of Wolin (1960), i.e. the amount of fermentative CO₂ and CH₄ could be accurately calculated from the amount and proportion of acetate, propionate and butyrate present in the incubation medium. As previously suggested by Menke and Steingaβ (1988) additional CO₂ was produced from buffering the SCFA and about 54% of the total gas volume was attributable to this buffering reaction (Blümmel and O?rskov 1993), assuming that 1 mmol of SCFA released 1 mmol of CO₂ from the bicarbonate buffer into the gas phase. However, the stoichiometry of Wolin (1960) considers only the fermentation of carbohydrates with glucose as a model and the presence of protein (or fat) in feeds might distort the stoichiometrical relationship between SCFA and gas production. It was for example argued by Cone (1998), that the stoichiometry does not hold good for protein rich feeds. In other words, the relationship between SCFA and gas production would be getting weaker and more variable. These conclusions were based on dilution experiments of starch and glucose with casein in an in vitro gas production test (Cone 1998). Obviously, uncoupling of SCFA and gas production would make the interpretation of gas profiles difficult, since the relationship between the useful products (SCFA) and the waste product (gases) would become obscured. It was therefore the objective of this technical note to re-examine the relationship between SCFA and gas production for 55 feeds varying largely in their protein and also fat content.     

A short note on linkage between the β- and κ-caseins in the Churra and Manchega ovine breeds

Abstract

The aim of this study was to evaluate the effect of Mentha piperita L. (MP) dried leaves on the in vitro rumen methanogenesis and fermentation. It was found that after 24 hours of incubations, addition of 16.34 and 23.35 mg of MP to the 233.3 mg of substrate significantly decreased methane emission by 41.52 and 15.51%, respectively. Simultaneously, the MP supplementation exerted no effect on the dry matter digestibility and volatile fatty acid profile. Addition of MP to the ruminants' diet inhibits the methane production without altering the basic parameters of rumen fermentation.     

Effects of Quillaja saponaria extract alone or in combination with Yucca schidigera extract on ruminal fermentation and methanogenesis in vitro

The objective of the study was to evaluate effects of Quillaja saponaria (QS) with or without Yucca schidigera extract (YS) on in vitro ruminal fermentation and methanogenesis. The culture media consisted of 400 mL of strained rumen fluid collected from two non‐lactating Holstein cows and 400 mL of artificial saliva. The culture media was anaerobically incubated with 10 g of a mixture of concentrate and oat hay (1:1, w/w) at 39°C for 24 h. The treatments were arranged as 2 × 3 factorial design experiment consisting of three levels of QS (0, 2 or 4 mL/L) and two levels of YS (0 or 2 mL/L). Treatment interactions between QS and YS were observed for pH and ammonia N concentration. CH₄ production was not modified by addition of QS but decreased (P < 0.05) when added with YS. Addition of QS with YS increased (P < 0.05) propionate concentrations. Protozoa numbers were decreased (P < 0.05) by QS addition alone or with YS. Results show that QS and YS exhibited strong antiprotozoal effects and combinations of both plant extracts may have potential as safe manipulators of ruminal fermentation.     

Effects of quebracho tannin extract on intake,digestibility, rumen fermentation,and methane production in crossbred heifers fed low-quality tropical grass

The aim of this work was to evaluate the effect of quebracho tannins extract (QTE) on feed intake, dry matter (DM) digestibility, and methane (CH₄) emissions in cattle fed low-quality Pennisetum purpureum grass. Five heifers (Bos taurus × Bos indicus) with an average live weight (LW) of 295 ± 19 kg were allotted to five treatments (0, 1, 2, 3, and 4% QTE/kg DM) in a 5 × 5 Latin square design. Intake, digestibility, and total methane emissions (L/day) were recorded for periods of 23 h when cattle were housed in open-circuit respiration chambers. Dry matter intake (DMI), organic matter intake (OMI), dry matter digestibility (DMD), and organic matter digestibility (OMD) were different between treatments with 0 and 4% of QTE/kg DM (P < 0.05). Total volatile fatty acid and the molar proportion of acetate in the rumen was not affected (P < 0.05); however, the molar proportion of propionate increased linearly (P < 0.01) for treatments with 3 and 4% QTE. Total CH₄ production decreased linearly (P < 0.01) as QTE increased in the diet, particularly with 3 and 4% concentration. When expressed as DMI and OMI by CH₄, production (L/kg) was different between treatments with 0 vs 3 and 4% QTE (P < 0.05). It is concluded that the addition of QTE at 2 or 3% of dry matter ration can decrease methane production up to 29 and 41%, respectively, without significantly compromising feed intake and nutrients digestibility.     

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.     

Analysis of methanogenic archaeal communities of rumen fluid and rumen particles from Korean black goats

Molecular diversity of methanogens in the rumen of Korean black goats was investigated with 16S rRNA gene clone libraries using methanogen‐specific primers. The libraries were composed of rumen fluid‐associated methanogens (FAM) and rumen particle‐associated methanogens (PAM) from rumen‐fistulated Korean black goats. Among the 141 clones of the FAM library, the sequences were mostly related to two phyla, the Methanobacteriaceae family (77.3%) and the Thermoplasmatales family (22.7%); and among the 68 clones of the PAM library, sequences were also mainly clustered in the two phyla, the Thermoplasmatales family (63.24%) and the Methanobacteriaceae family (35.29%). Most of the sequenced clones in the two libraries were closely related to uncultured methanogenic archaeon. Quantitative real‐time PCR revealed that PAM (8.97 log 10) had significantly higher (P < 0.01) density of methanogens by the methanogenic 16S rRNA gene copies than FAM (7.57 log 10). The two clone libraries also showed difference in Shannon index (FAM library 1.70 and PAM library 1.59) and Chao 1 estimator (FAM library 18 and PAM library 17 operational taxonomic units). Apparent differences found in the microbial community from the two 16S rRNA gene libraries could be a result of such factors as the chemical and physical nature of the target material surface, types or component of diets, the interaction between the methanogens and other microbes, and age of the experimental goats.     

In vitro evaluation,in vivo quantification,and microbial diversity studies of nutritional strategies for reducing enteric methane production

The main objective of the present work was to study nutritive strategies for lessening the CH₄ formation associated to ruminant tropical diets. In vitro gas production technique was used for evaluating the effect of tannin-rich plants, essential oils, and biodiesel co-products on CH₄ formation in three individual studies and a small chamber system to measure CH₄ released by sheep for in vivo studies was developed. Microbial rumen population diversity from in vitro assays was studied using qPCR. In vitro studies with tanniniferous plants, herbal plant essential oils derived from thyme, fennel, ginger, black seed, and Eucalyptus oil (EuO) added to the basal diet and cakes of oleaginous plants (cotton, palm, castor plant, turnip, and lupine), which were included in the basal diet to replace soybean meal, presented significant differences regarding fermentation gas production and CH₄ formation. In vivo assays were performed according to the results of the in vitro assays. Mimosa caesalpineaefolia, when supplemented to a basal diet (Tifton-85 hay Cynodon sp, corn grain, soybean meal, cotton seed meal, and mineral mixture) fed to adult Santa Ines sheep reduced enteric CH₄ emission but the supplementation of the basal diet with EuO did not affect (P > 0.05) methane released. Regarding the microbial studies of rumen population diversity using qPCR with DNA samples collected from the in vitro trials, the results showed shifts in microbial communities of the tannin-rich plants in relation to control plant. This research demonstrated that tannin-rich M. caesepineapholia, essential oil from eucalyptus, and biodiesel co-products either in vitro or in vivo assays showed potential to mitigate CH₄ emission in ruminants. The microbial community study suggested that the reduction in CH₄ production may be attributed to a decrease in fermentable substrate rather than to a direct effect on methanogenesis.     

Modulation of Na+ transport across isolated rumen epithelium by short-chain fatty acids in hay- and concentrate-fed sheep

The effect of increasing concentrations of short-chain fatty acids [SCFA; mixture of the Na+ salts of acetic acid (62.5%), propionic acid (25.0%) and of butyric acid (12.5%)] on Na+ transport of sheep rumen epithelium was studied in vitro. The conventional Ussing chamber method was used for measuring Na+ transport rates (22Na+), short-circuit current (Isc) and tissue conductance (GT) of isolated rumen epithelium. SCFA in the buffer solution on the mucosal side caused a linear increase of Jnet Na+ from 1.14, to 1.22, 1.78 and 2.50 microeq/cm2/h in hay-fed sheep at 0, 15, 40 and 80 mmol/l SCFA, respectively. In a second study, the effect of higher SCFA concentrations [0 (control), 80, 100 and 120 mmol/l] was investigated with epithelia from two groups of sheep. One group was subjected to hay ad libitum, whereas the other received concentrate feed (800 g/day in equal portions at 7.00 am and 3.00 pm) and hay ad libitum. Epithelia from concentrate-fed sheep again showed a significant (p < 0.05) and linear increase in Jnet Na+ at 80, 100 and 120 mmol/l. However, in hay-fed sheep, the difference in increase among 80, 100 and 120 mmol/l SCFA was not significant, indicating that, above 80 mmol/l SCFA Jms and Jnet exhibit saturation. Moreover, Na+ fluxes (Jms and Jnet) were generally higher in concentrate-fed than in hay-fed sheep at all SCFA concentrations and significant differences were observed at 100 and 120 mmol/l SCFA. The obtained results confirm the effect of SCFA on Na+ transport and are in agreement with studies regarding feeding regimes and electrolyte transport in the rumen. The important new observation is the increase of Na+ transport in concentrate-fed sheep even at high concentrations of SCFA (100 and 120 mmol/l). The enhanced activity of the Na+/H+ exchanger at these SCFA concentrations supports the assumption that the capacity for regulating the intracellular pH by extrusion of protons is increased, suggesting an adaptation in concentrate-fed sheep. This adaptation could prevent possible disturbances of epithelial functions (transport and barrier) under conditions of increased SCFA absorption.