Mixed Ruminal Microorganisms Research Articles

Grazing cotton crop residue to reduce winter supplementation cost in late-gestation beef cows and assessment of the negative effects of gossypol on fermentation of mixed ruminal microorganisms

This research aimed to evaluate the use of standing cotton crop residue as a winter supplementation source for gestating beef cows and the effects of gossypol, a naturally occurring pigment with potential anti-quality characteristics, on in vitro mixed ruminal microorganism fermentations. Experiment 1 used a 3-yr completely randomized study with 108 Angus cross cows (BW 559 ± 7.9 kg) enrolled in a 30-d grazing trial. Cows were weighed, stratified by weight, and randomly assigned to 1 of 2 treatments: dormant bermudagrass ( Cynodon dactylon ) pasture + bermudagrass hay (GR) or cotton ( Gossypium ) crop residue + bermudagrass hay (CT). Experiment 2 used a completely randomized in vitro mixed ruminal microorganism fermentation with 3 × 5 factorial arrangements of treatments. This included 3 substrates: corn ( Zea mays ; CC), average relative forage quality (RFQ 100) coastal bermudagrass (ABG), and high relative forage quality (RFQ 120) coastal bermudagrass (HBG), along with 5 concentrations of gossypol: 0, 0.0615, 0.123, 0.1845, or 0.246 mg of gossypol/mL. Total hay offered and hay per head per day were greater ( P ≤ 0.02) in the GR group compared with the CT group. Economic analysis indicated that total hay cost, cost per head per day, and cost of gain were all significantly decreased for cows in the CT treatment compared with cows in the GR treatment ( P < 0.002). In vitro DM digestibility and total VFA production were lower ( P < 0.002) as gossypol concentration reached the highest concentration. Results suggest that grazing cotton crop residue is a partial replacement for hay supplementation during the winter months. Increased levels of gossypol have negative effects on DM disappearance and VFA production. The gossypol amount found in crop residue is approximately the same as the lowest concentration evaluated in the in vitro experiment. These results indicate that grazing cotton crop residue should not influence ruminal fermentation.

Impact of camphor on the in vitro mixed ruminal microorganism fermentation from goats selected for consumption of low and high levels of Juniperus spp. 1.

Encroaching plant species in West Texas continues to significantly reduce livestock production capacity and cause an imbalance between plant and livestock ecology. Juniperus ashei and Juniperus pinchotii are encroaching species rarely used by browsing animals, mainly due to phytochemical defenses. Juniperus spp. contain large concentrations of monoterpenes in their essential oil profiles to deter herbivory. Since 2003, two divergent bloodlines of meat goats have been selected to consume low or high (LJC or HJC) amounts of juniper foliage, through screening fecal samples with near-infrared reflectance spectroscopy. However, it remains unclear whether HJC goats expressed a physiological ability to consume a greater amount of juniper or if they were colonized by a ruminal microbial population that could detoxify juniper phytochemicals. Therefore, this study aimed to investigate the impact of 0.00 and 1.97 mM of camphor on the mixed ruminal microorganism fermentation after 0, 1, 2, 4, 12, and 24 h of incubation. Five LJC and five HJC goats were fed a juniper-free diet (n = 10) and five LJC and five HJC goats (n = 10), were fed a diet containing 30% fresh J. ashei leaves for 21 days prior to ruminal fluid collection. In vitro fermentations used LJC and HJC, ruminal fluid inoculum was added (33% v/v) to anoxic media in sealed Balch tubes. Total short-chain fatty acid (SCFA) production and acetate to propionate ratio were increased (P < 0.05), but there was no effect on pH (6.56 ± 0.09). Goats that received the juniper-free diet had higher (P < 0.05) SCFA production than juniper-containing diets. There was no consistent difference in LJC and HJC microbial fermentation end products caused by the addition of 1.97 mM of camphor, and goats receiving a juniper-free diet consistently generated more SCFAs in the presence of 1.97 mM of camphor. Furthermore, bloodline differences in juniper consumption were likely related to physiological adaption capacities within the animal and not a ruminal microbial detoxification advantage.

Impact of concentrations of camphor on the in vitro mixed ruminal microorganism fermentation from goats selected for consumption of low and high levels of Juniperus spp.-2.

The microbial population in the gastrointestinal tract of ruminant animals aids in the utilization of forages with high levels of secondary plant compounds. Two divergent bloodlines of meat goats have been selected by screening fecal samples with near-infrared reflectance spectroscopy to assess the goat’s consumption of high or low levels of Juniperus sp. leaves containing several monoterpenes, including camphor. The mechanism by which these goats can consume greater concentrations of Juniperus spp. leaves than their counterparts is unclear, and therefore, this study was designed to determine if differences existed between the ruminal microbial populations of the low and high juniper-consuming bloodlines (LJC vs. HJC) by analyzing their ruminal microbiota and fermentation end products. In the present study, concentrations (0.00, 0.5, 0.99, 1.97, or 5.91 mM) of camphor were added to mixed ruminal microorganism fermentation. Five LJC and five HJC goats were fed a juniper-free diet (n = 10), and five LJC and five HJC goats (n = 10) were fed a diet that contained 30% fresh Juniperus ashei leaves for 21 d prior to ruminal fluid collection. In vitro fermentations used LJC and HJC, ruminal fluid inoculum added (33% v/v) to anoxic media in sealed Balch tubes. Camphor increased (P < 0.05) total short-chain fatty acid (SCFA) concentrations for all but one experimental group. Between the main dietary and bloodline goat effects, the diet was significant for all SCFA results except butyrate. In contrast, bloodline was only significant for acetate and butyrate molar proportions. Rumen fluid from juniper-free-fed goats exhibited greater concentrations of Ruminococcaceae, whereas juniper-fed goats contained more Coriobacteriaceae. Results demonstrated that mixed ruminal microorganisms fermentations from HJC goats did not produce greater concentrations of SCFAs or have the ability to degrade camphor at a higher rate than did that from LJC goats. Results suggest that camphor tolerance from J. ashei, was related to hepatic catabolic mechanisms instead of ruminal microbial degradation; however, further in vivo work is warranted.

Biogas production from sunn hemp

Sunn hemp at a cutting age of 50 days was investigated for biogas production by studying the anaerobic digestion of sunn hemp alone and the co-digestion of sunn hemp with cow dung at a concentration of 20% by fresh weight but with various proportions. The single-stage, semi-continuous reactors were operated at a hydraulic retention time of 40 days. Mixed ruminal microorganisms from cows were used as inoculum. The results indicated that the CH4 content from the digestion of sunn hemp alone was 47.57% and the CH4 produced per area of cultivation was 9,231 m3/hectare.year. Co-digestion could increase the CH4 content to greater than 50%. Proportion of sunn hemp:cow dung:water of 10:10:80 by fresh weight was optimal for co-digestion. At this recommended ratio, the CH4 yield reached 412 L/kg total volatiles solids degraded. Sunn hemp could be a very good renewable energy source for biogas production.

Biogas production in semi-continuous-flow reactors using fresh water hyacinth from the Chao Phraya River

Biogas production was studied from the digestion of water hyacinth (WH), co-digestion of WH with cow dung and co-digestion of pre-treated WH with dilute sodium hydroxide (NaOH). The reactors were fed with 20% by fresh weight of the substrate mixture of various ratios in semi-continuous-flow reactors at 30°C. Cow mixed ruminal microorganisms were used as the inoculum. The results indicated that the co-digestion of WH to cow dung to water with a ratio of 10:10:80 was optimal for biogas production. The average methane contents obtained from digestion of WH, co-digestion of WH with cow dung and co-digestion of the pre-treated WH at the recommended ratio were 48.56%, 51.14% and 53.62%, respectively. In co-digestion of WH with cow dung and that of the pre-treated WH at the optimum ratio, the methane yield increased from 144 to 170 L/kg total volatile solids or 115 L to 137 L/kg total solids added in the reactor.

Cutting interval of Napier grass affecting methane yield in anaerobic digestion

Digestion of Napier grass at cutting intervals of 30, 45, 60 and 90 days was investigated in two-stage anaerobic reactors. Four sets of reactors with working volumes of 4 and 5 L for acidogenic and methanogenic reactors were operated at 30°C. Acidogenic reactors were fed slurry of Napier grass at 200 ml/day. Hydraulic retention times were 20 and 25 days for the acidogenic and methanogenic reactors, respectively. Mixed ruminal microorganisms from cow were used as inoculum. The highest methane yield was obtained from Napier grass with a cutting interval of 60 days producing 6,500 m3/ha.year.

Cutting interval of Napier grass affecting methane yield in anaerobic digestion

Digestion of Napier grass at cutting intervals of 30, 45, 60 and 90 days was investigated in two-stage anaerobic reactors. Four sets of reactors with working volumes of 4 and 5 L for acidogenic and methanogenic reactors were operated at 30°C. Acidogenic reactors were fed slurry of Napier grass at 200 ml/day. Hydraulic retention times were 20 and 25 days for the acidogenic and methanogenic reactors, respectively. Mixed ruminal microorganisms from cow were used as inoculum. The highest methane yield was obtained from Napier grass with a cutting interval of 60 days producing 6,500 m3/ha.year.

The effect of nitrate and monensin on in vitro ruminal fermentation.

Two experiments evaluated the effect of calcium ammonium nitrate decahydrate (calcium nitrate [NIT]) and monensin sodium (MON) on in vitro fermentation parameters of 2 contrasting diets (100:0 and 10:90 forage-to-concentrate ratios). Diet addition of NIT (0, 1.25, and 2.5 g/100 g DM) and MON (0, 3, and 6 mg/L) were tested alone and combined (9 treatments total; 5 bottles per treatment). Mixed ruminal microorganisms were incubated in anaerobic media containing 0.5 g of substrate diet, 1 of 9 treatments, and 40 mL buffer solution. Incubations were performed in batch cultures for 48 h at 39°C. Headspace gas volume was measured and sampled at 4, 8, 12, 24, and 48 h, and the VFA profile was assessed at the end of the experiment. Total gas production was reduced by NIT (87.9 vs. 94.6 mL; < 0.01) and MON (78.6 vs. 94.6 mL; < 0.01) and, in Exp. 2, further reduced by NIT+MON when the additives were combined (161.1 vs. 196.9 mL; < 0.01). Methane production from control in Exp. 1 and Exp. 2 averaged 9.1 and 15.3 mL, respectively, and was decreased by NIT (3.4 and 8.3 mL in Exp. 1 and Exp. 2, respectively; P < 0.01), MON (4.1 and 7.7 mL; in Exp. 1 and Exp. 2, respectively; < 0.01) and NIT+MON (1.1 and 1.5 mL; in Exp. 1 and Exp. 2, respectively; < 0.01). Both experiments demonstrated a significant increase in nitrous oxide (NO; < 0.01) when NIT was added. Compared to the control treatment, IVDMD was reduced when NIT+MON was added at the higher doses in EXP1 (31.7 vs. 37.4%; < 0.01) and EXP2 (76.6 vs. 79.9 %; < 0.01). Net VFA production was not affected by treatments ( > 0.10), but molar proportions of acetate and butyrate were reduced by MON ( < 0.01). Propionate molar proportion was increased in both experiments by MON ( < 0.01) and further increased in Exp. 2 when the additives were combined at lower doses ( < 0.01). Compared to the control treatment, the acetate:propionate (A:P) ratio was reduced by MON in Exp. 1(1.2 vs. 2.8; < 0.01) and Exp. 2 (1.0 vs. 2.3; < 0.01). Fermentation efficiency (%) was increased by MON (81.7 vs. 73.7%; < 0.01) and further increased in Exp. 2 when the additives were combined at lower doses (87.2 vs. 76.6%; < 0.01). The combination of NIT and MON in 2 contrasting diets proved beneficial by altering fermentation products toward lower CH and more propionate; however, the addition of NIT consistently increased NO production. Negative effects of the additives on IVDMD were found only when the additives were combined at higher doses.

Methane production from napier grass by two-stage anaerobic digestion

Digestion of Napier grass was investigated in two-stage anaerobic reactors. The reactor volume was 4 and 5 litres for the acidogenic and methanogenic reactors, respectively. The acidogenic reactors were fed once daily at 100, 150, 200, 250, and 300 mL/day with a slurry of Napier grass. The organic loading rates (OLRs) were 0.60, 0.90, 1.20, 1.50 and 1.80 kg chemical oxygen demand (COD)/m3.day. Mixed ruminal microorganisms from cows were used as the inoculum. The pH was adjusted to 7.5 for all methanogenic reactors at the start-up period. The reactors were operated at 30°C. The maximum methane (CH4) yield was observed at an OLR of 1.20 kg COD/m3.day. The average pH in the acidogenic and methanogenic reactors was 5.3 and 7.4, respectively. At the maximum CH4 yield OLR, the CH4 production was 148 litres at STP/kg dry Napier grass added to the reactor, which indicated that 6.76 kg of dry Napier grass is needed to produce 1 m3 of pure CH4.

Production of medium-chain volatile fatty acids by mixed ruminal microorganisms is enhanced by ethanol in co-culture with Clostridium kluyveri

Mixed bacterial communities from the rumen ferment cellulosic biomass primarily to C2–C4 volatile fatty acids, and perform only limited chain extension to produce C5 (valeric) and C6 (caproic) acids. The aim of this study was to increase production of caproate and valerate in short-term in vitro incubations. Co-culture of mixed ruminal microbes with a rumen-derived strain of the bacterium Clostridium kluyveri converted cellulosic biomass (alfalfa stems or switchgrass herbage) plus ethanol to VFA mixtures that include valeric and caproic acids as the major fermentation products over a 48–72h run time. Concentrations of caproate reached 6.1gL−1, similar to or greater than those reported in most conventional carboxylate fermentations that employ substantially longer run times.

Effects of Cordyceps militaris on the growth of rumen microorganisms and in vitro rumen fermentation with respect to methane emissions

This experiment was designed to investigate the effects of different concentrations (0.00, 0.10, 0.15, 0.20, 0.25, and 0.30g/L) of dried Cordyceps militaris mushroom on in vitro anaerobic ruminal microbe fermentation and methane production using soluble starch as a substrate. Ruminal fluids were collected from Korean native cattle, mixed with phosphate buffer (1:2), and incubated anaerobically at 38°C for 3, 6, 9, 12, 24, 36, 48, and 72h. The addition of C. militaris significantly increased total volatile fatty acid and total gas production. The molar proportion of acetate was decreased and that of propionate was increased, with a corresponding decrease in the acetate:propionate ratio. As the concentration of C. militaris increased from 0.10 to 0.30g/L, methane and hydrogen production decreased. The decrease in methane accumulation relative to the control was 14.1, 22.0, 24.9, 39.7, and 40.9% for the 0.10, 0.15, 0.20, 0.25, and 0.30g/L treatments, respectively. Ammonia-N concentration and numbers of live protozoa decreased linearly with increasing concentrations of C. militaris. The pH of the medium significantly decreased at the highest level of C. militaris compared with the control. In conclusion, C. militaris stimulated mixed ruminal microorganism fermentation and inhibited methane production in vitro. Therefore, C. militaris could be developed as a novel compound for antimethanogenesis.

Expression of a fungal ferulic acid esterase in alfalfa modifies cell wall digestibility

BackgroundAlfalfa (Medicago sativa) is an important forage crop in North America owing to its high biomass production, perennial nature and ability to fix nitrogen. Feruloyl esterase (EC 3.1.1.73) hydrolyzes ester linkages in plant cell walls and has the potential to further improve alfalfa as biomass for biofuel production.ResultsIn this study, faeB [GenBank:AJ309807] was synthesized at GenScript and sub-cloned into a novel pEACH vector containing different signaling peptides to target type B ferulic acid esterase (FAEB) proteins to the apoplast, chloroplast, endoplasmic reticulum and vacuole. Four constructs harboring faeB were transiently expressed in Nicotiana leaves, with FAEB accumulating at high levels in all target sites, except chloroplast. Stable transformed lines of alfalfa were subsequently obtained using Agrobacterium tumefaciens (LBA4404). Out of 136 transgenic plants regenerated, 18 independent lines exhibited FAEB activity. Subsequent in vitro digestibility and Fourier transformed infrared spectroscopy (FTIR) analysis of FAEB-expressing lines showed that they possessed modified cell wall morphology and composition with a reduction in ester linkages and elevated lignin content. Consequently, they were more recalcitrant to digestion by mixed ruminal microorganisms. Interestingly, delignification by alkaline peroxide treatment followed by exposure to a commercial cellulase mixture resulted in higher glucose release from transgenic lines as compared to the control line.ConclusionModifying cell wall crosslinking has the potential to lower recalcitrance of holocellulose, but also exhibited unintended consequences on alfalfa cell wall digestibility due to elevated lignin content. The combination of efficient delignification treatment (alkaline peroxide) and transgenic esterase activity complement each other towards efficient and effective digestion of transgenic lines.

Effects of Branched-chain Amino Acids on In vitro Ruminal Fermentation of Wheat Straw.

This study investigates the effects of three branched-chain amino acids (BCAA; valine, leucine, and isoleucine) on the in vitro ruminal fermentation of wheat straw using batch cultures of mixed ruminal microorganisms. BCAA were added to the buffered ruminal fluid at a concentration of 0, 2, 4, 7, or 10 mmol/L. After 72 h of anaerobic incubation, pH, volatile fatty acids (VFA), and ammonia nitrogen (NH3-N) in the ruminal fluid were determined. Dry matter (DM) and neutral detergent fiber (NDF) degradability were calculated after determining the DM and NDF in the original material and in the residue after incubation. The addition of valine, leucine, or isoleucine increased the total VFA yields (p≤0.001). However, the total VFA yields did not increase with the increase of BCAA supplement level. Total branched-chain VFA yields linearly increased as the supplemental amount of BCAA increased (p<0.001). The molar proportions of acetate and propionate decreased, whereas that of butyrate increased with the addition of valine and isoleucine (p<0.05). Moreover, the proportions of propionate and butyrate decreased (p<0.01) with the addition of leucine. Meanwhile, the molar proportions of isobutyrate were increased and linearly decreased (p<0.001) by valine and leucine, respectively. The addition of leucine or isoleucine resulted in a linear (p<0.001) increase in the molar proportions of isovalerate. The degradability of NDF achieved the maximum when valine or isoleucine was added at 2 mmol/L. The results suggest that low concentrations of BCAA (2 mmol/L) allow more efficient regulation of ruminal fermentation in vitro, as indicated by higher VFA yield and NDF degradability. Therefore, the optimum initial dose of BCAA for in vitro ruminal fermentation is 2 mmol/L.

Addition of high concentration of inorganic selenium in orchardgrass (Dactylis glomerata L.) hay diet does not interfere with microbial fermentation in mixed ruminal microorganisms in continuous cultures

ABSTRACT The current literature lacks information on ruminal microbial metabolism in response to high selenium (Se) concentration in the diet. The current study was performed to investigate changes in ruminal fermentation when high concentration of Se was administered in mixed ruminal cultures in fermentors. Two mature beef cows, fitted with a rumen cannula, were grazed on tall-fescue pasture and used as donor animals for ruminal contents. Filtered ruminal contents were allowed 11 d of adaptation to diets followed by 3 d of data collection. A dual-flow continuous culture system was used in a completely randomized design (n = 4) to test 2 dietary treatments: control (no Se addition) and 50 mg/kg of Se addition. Orchardgrass hay (20 g of DM/d) containing 0.12 mg/kg of Se was added to the fermentors in 2 equal portions at 0800 and 1700 h. Selenium (sodium selenate) was added to the Se-addition treatment by gradually increasing the concentration from 2 to 50 mg/kg over the 11-d adaptation period. Culture pH averaged 6.04 and was not affected by treatment. Total VFA concentration averaged 52.2 and 55.5 mM in the control and Se treatment cultures, respectively, and addition of Se did not affect the total VFA concentration. Molar proportions of acetate and propionate did not differ because of Se addition, resulting in a similar acetate-to-propionate ratio. However, addition of Se tended to increase (P = 0.09) ammonia-N concentration and methane production (P = 0.10). Selenate-respiring microorganisms were detected by the most probable number enumeration technique in 3 of the 4 replications receiving Se. Addition of 50 mg/kg of Se in orchardgrass-hay diet had no negative effects on ruminal fermentation, as was observed in similar culture pH and VFA concentration. However, addition of Se affected microbial metabolism in the mixed cultures by increasing ammonia-N concentration and methane production. Overall data in this study suggest that the addition of Se up to 50 mg/kg would not interfere with in vitro ruminal metabolism by microbiota and that some adaptation of Se-reducing microbes may occur.

Effect of Citrus Byproducts on Survival of O157:H7 and Non-O157Escherichia coliSerogroups withinIn VitroBovine Ruminal Microbial Fermentations

Citrus byproducts (CBPs) are utilized as a low cost nutritional supplement to the diets of cattle and have been suggested to inhibit the growth of both Escherichia coli O157:H7 and Salmonella. The objective of this study was to examine the effects in vitro that varying concentrations of CBP in the powdered or pelleted variety have on the survival of Shiga-toxin Escherichia coli (STEC) serotypes O26:H11, O103:H8, O111:H8, O145:H28, and O157:H7 in bovine ruminal microorganism media. The O26:H11, O111:H8, O145:H28, and O157:H7 serotypes did not exhibit a change in populations in media supplemented with CBP with either variety. The O103:H8 serotype displayed a general trend for an approximate 1log10 reduction in 5% powdered CBP and 20% pelleted CBP over 6 h. There was a trend for reductions in populations of a variant form of O157:H7 mutated in the stx1 and stx2 genes in higher concentrations of CBP. These results suggest that variations exist in the survival of these serotypes of STEC within mixed ruminal microorganism fluid media when supplemented with CBP. Further research is needed to determine why CBPs affect STEC serotypes differently.

The influence of diet type (dairy versus intensive fattening) on the effectiveness of garlic oil and cinnamaldehyde to manipulate in vitro ruminal fermentation and methane production

The objective of this study was to evaluate the effects of increasing doses [0 (control: CON), 20, 60, 180 and 540 mg/L incubation medium] of garlic oil (GO) and cinnamaldehyde (CIN) on in vitro ruminal fermentation of two diets. Batch cultures of mixed ruminal microorganisms were inoculated with ruminal fluid from four sheep fed a medium-concentrate diet (MC; 50 : 50 alfalfa hay : concentrate) or four sheep fed a high-concentrate diet (HC; 15 : 85 barley straw : concentrate). Diets MC and HC were representative of those fed to dairy and fattening ruminants, respectively. Samples of each diet were used as incubation substrates for the corresponding inoculum, and the incubation was repeated on 4 different days (four replicates per experimental treatment). There were GO × diet-type and CIN × diet-type interactions (P &lt; 0.001–0.05) for many of the parameters determined, indicating different effects of both oils depending on the diet type. In general, effects of GO were more pronounced for MC compared with HC diet. Supplementation of GO did not affect (P &gt; 0.05) total volatile fatty acid (VFA) production at any dose. For MC diet, GO at 60, 180 and 540 mg/L decreased (P &lt; 0.05) molar proportion of acetate (608, 569 and 547 mmol/mol total VFA, respectively), and increased (P &lt; 0.05) propionate proportion (233, 256 and 268 mmol/mol total VFA, respectively), compared with CON values (629 and 215 mmol/mol total VFA for acetate and propionate, respectively). A minimum dose of 180 mg of GO/L was required to produce similar modifications in acetate and propionate proportions with HC diet, but no effects (P &gt; 0.05) on butyrate proportion were detected. Methane/VFA ratio was reduced (P &lt; 0.05) by GO at 60, 180 and 540 mg/L for MC diet (0.23, 0.16 and 0.10 mol/mol, respectively), and by GO at 20, 60, 180 and 540 mg/L for HC diet (0.19, 0.19, 0.16 and 0.08 mol/mol, respectively), compared with CON (0.26 and 0.21 mol/mol for MC and HC diets, respectively). No effects (P = 0.16–0.85) of GO on final pH and concentrations of NH3-N and lactate were detected. For both diet types, the highest CIN dose decreased (P &lt; 0.05) production of total VFA, gas and methane, which would indicate an inhibition of fermentation. Compared with CON, CIN at 180 mg/L increased (P &lt; 0.05) acetate proportion for the MC (629 and 644 mmol/mol total VFA for CON and CIN, respectively) and HC (525 and 540 mmol/mol total VFA, respectively) diets, without affecting the proportions of any other VFA or total VFA production. Whereas for MC diet CIN at 60 and 180 mg/L decreased (P &lt; 0.05) NH3-N concentrations compared with CON, only a trend (P &lt; 0.10) was observed for CIN at 180 mg/L with the HC diet. Supplementation of CIN up to 180 mg/L did not affect (P = 0.18–0.99) lactate concentrations and production of gas and methane for any diet. The results show that effectiveness of GO and CIN to modify ruminal fermentation may depend on diet type, which would have practical implications if they are confirmed in vivo.

Technical note: Stearidonic acid metabolism by mixed ruminal microorganisms in vitro1

Dietary supplementation of stearidonic acid (SDA; 18:4n-3) has been considered a possible strategy to increase n-3 unsaturated fatty acid content in ruminant products; however, little is known about its metabolism in the rumen. In vitro batch incubations were carried out with bovine ruminal digesta to investigate the metabolism of SDA and its biohydrogenation products. Incubation mixtures (4.5 mL) that contained 0 (control), 0.25, 0.50, 0.75, 1.00, 1.25, or 1.50 mg of SDA supplemented to 33 mg (DM basis) of commercial total mixed ration based on corn silage, for dairy cows, were incubated for 72 h at 39°C. The content of most fatty acids in whole freeze-dried cultures was affected by SDA supplementation. Branched-chain fatty acids decreased linearly (P < 0.01), and odd-chain fatty acids decreased quadratically (P < 0.01), particularly from 1.00 mg of SDA and above, whereas most C18 fatty acids increased linearly or quadratically (P ≤ 0.04). Stearidonic acid concentrations at 72 h of incubation were very small (<0.6% of total fatty acids and ≤0.9% of added SDA) in all treatments. The apparent biohydrogenation of SDA was extensive, but it was not affected by SDA concentration (P > 0.05). Biohydrogenation followed a pattern similar to that of other C18 unsaturated fatty acids up to 1.00 mg of SDA. Stearic acid (18:0) and vaccenic acid (18:1 trans-11) were the major fatty acids formed, with the latter increasing 9-fold in the 1.00 mg of SDA treatment. At greater inclusion rates, 18:0 and 18:1 trans isomers decreased (P ≤ 0.03), accompanied by increases in unidentified 18:3 and 18:4 isomers (P = 0.02), suggesting that the biohydrogenation pathway was inhibited. The present results clearly indicate that SDA was metabolized extensively, with numerous 18:4 and 18:3 products formed en route to further conversion to 18:2, 18:1 isomers, and 18:0.

Effects of inactivated and live cells of Saccharomyces cerevisiae on in vitro ruminal fermentation of diets with different forage:concentrate ratio

SUMMARYThe effects of yeast Saccharomyces cerevisiae, either inactivated (by osmotic pressure, designated IY) or provided as a culture containing live yeast cells (YC), on ruminal fermentation of two different diets were investigated in vitro. Total mixed rations (TMR) having forage:concentrate ratios of 0·6:0·4 (medium–high forage diet) and 0·2:0·8 (low-forage diet) were incubated in batch cultures of mixed ruminal micro-organisms to which either IY (to reach concentrations of 500 and 250 mg product/l incubation medium) or YC (at a concentration of 150 mg product/l) were added directly as powder. To evaluate the effects of the additive on ruminal microbial population, sheep used as donors of rumen fluid were allocated to three experimental groups: Control (no additive), IY and YC, that received a diet with the corresponding additive for 10 days. With both diets, YC decreased ruminal pH compared to control, whereas IY had no effect. Adding yeast products to the high-fibre diet affected total volatile fatty acid (VFA) production and VFA composition, in general with a slight increase in IY and a significantly greater increase in response to the addition of YC. Ammonia nitrogen (P=0·006), total gas production (P&lt;0·001) and in vitro dry matter disappearance (IVD) (P&lt;0·001) showed the highest values with YC. Methane production was higher than the control when the IY inoculum was used, and increased even more with the YC inoculum (P&lt;0·001). With the high-concentrate TMR, no effects on total VFA concentration were observed when yeast additives were used. Similar trends were shown for lactate and methane production and total gas production, where values tended to be higher when using the YC inoculum (P values of 0·055, &lt;0·001, 0·006 and &lt;0·001, respectively). After 144 h of incubation, differences were observed only with the high-fibre diet in the cumulative gas production at 24 h of incubation and in the average fermentation rate, which was greater with YC, although the asymptotic gas production was not affected. These results indicate that live yeasts affect ruminal fermentation slightly more than inactivated yeasts, although both products require a regular administration and some adaptation of the ruminal microbial population for the stimulatory effects to become apparent. The effects of yeasts on ruminal fermentation are diet-dependent, being more noticeable with a high-fibre substrate, and subtle with a high-concentrate diet.

Effects of forage legumes containing condensed tannins on methane and ammonia production in continuous cultures of mixed ruminal microorganisms

It has been reported that some condensed tannin (CT) containing forage legumes may reduce ruminal CH4 emissions as a result of direct and/or indirect inhibition of methanogens. Dual flow continuous fermentors (working volume of 700ml) were used to determine effects of CT containing legume hays in dairy total mixed rations (TMR) on ruminal fermentation, with an emphasis on CH4 and ammonia N production. Fermentors were inoculated with filtered rumen contents and allowed to adapt to experimental diets for 6d, followed by 3d of sampling and data collection. All dietary treatments were formulated for lactating dairy cows in early lactation. Five TMR were evaluated being: (1) alfalfa (AFT), (2) cicer milkvetch (CMVT), (3) Norcen birdsfoot trefoil (NBFTT), (4) Oberhaunstadter birdsfoot trefoil (OBFTT) and (5) sainfoin (SFT). The experiment was a 5×5 Latin square design. Compared to AFT, culture pH was not influenced by most forages, but was increased (P<0.01) by feeding SFT. Compared to AFT, the molar proportion of propionate increased (P=0.03) with CMVT, but decreased (P=0.05) with SFT. Feeding CMVT decreased (P<0.01) the molar proportion of butyrate relative to AFT. Acetate to propionate ratio decreased with CMVT (P<0.01) and OBFTT (1.65; P=0.01), but increased with SFT (P<0.01) compared to AFT. Apparent digestibility of dry matter or neutral detergent fiber (aNDF) was not influenced by forage source. Compared to AFT, CH4 production decreased with other treatments (P<0.01), whereas CH4 production (expressed as mM/g aNDF digested) was only reduced (P<0.05) by CMVT and OBFTT. NH3–N concentration was lower (P<0.01) with CMVT and SFT, but not affected by NBFTT or OBFTT compared to AFT. Legume forages eliciting reductions in CH4 and NH3–N production may offer advantages in lactating dairy diets to improve nutrient utilization and environmental sustainability of dairy cattle production.This paper is part of the special issue entitled: Greenhouse Gases in Animal Agriculture – Finding a Balance between Food and Emissions, Guest Edited by T.A. McAllister, Section Guest Editors: K.A. Beauchemin, X. Hao, S. McGinn and Editor for Animal Feed Science and Technology, P.H. Robinson.

Dietary sulfur concentration has no effect on In Vitro fermentative activity of ruminal mixed microorganisms

We previously reported that elevated concentrations of dietary sulfur (0.65% sulfur, dry basis) in finishing diets containing dried distillers grains with solubles decreased dry matter intake and average daily gains of feedlot cattle. Furthermore, high dietary sulfur concentrations yielded lower ruminal concentrations of volatile fatty acids, but were associated with increased ruminal ammonia concentrations and improved total tract digestibility of the diet. The objective of this study was to investigate, in culture tubes, effects of added sulfur on in vitro dry matter disappearance, volatile fatty acid profiles, and ammonia concentrations from substrates containing different sulfur concentrations when fermented by mixed ruminal microorganisms from a steer fed a diet based on corn and alfalfa.