Rapid Communication: Evaluation of methane inhibitor 3-nitrooxypropanol and monensin in a high-grain diet using the rumen simulation technique (Rusitec).

The objective of this study was to evaluate the effects of 3-nitrooxypropanol (NOP), a known methane (CH) inhibitor; the ionophore monensin (MON); and their combination on in vitro CH production in a high-grain diet (85% barley grain, 10% barley silage, and 5% vitamin-mineral supplement; DM basis) using a rumen simulation technique (Rusitec). Sixteen fermentation vessels in 2 Rusitec apparatuses (blocks) were used in a completely randomized block design with 4 treatments: Control, NOP (200 µg/g DM), MON (200 µg/g DM), and the combination of 200 µg NOP/g DM and 200 µg MON/g DM (NOP + MON). Two fermenters within each apparatus were randomly assigned to a treatment. Treatments were mixed with 10 g of substrate and supplied on a daily basis. The study included an 8-d adaptation period without treatment supplementation and a 6-d period for addition of treatments. Dry matter disappearance, pH, and total VFA were not affected by treatment ( ≥ 0.34). Acetate proportion was decreased by 8.3% and 14.9% with NOP and NOP + MON ( < 0.01), respectively; however, propionate proportion was not affected by treatment ( = 0.44). The acetate to propionate ratio was lowered by 21.1% with the combination of NOP and MON ( = 0.02), whereas ammonia-N concentration was not affected by treatment ( = 0.50). Total gas production was unaffected ( = 0.50), but CH production decreased by 77.7% and 75.95% ( < 0.01) with NOP and NOP + MON addition, respectively. Concurrently, H gas production increased by 131.3% and 185.6% ( = 0.01) with NOP and NOP + MON treatments, respectively. The copy number of methanogens was decreased in both solid and liquid phases ( < 0.01) with NOP and NOP + MON treatments. Despite the combination of NOP + MON showing the greatest decrease in acetate molar proportion and acetate to propionate ratio, it did not further inhibit CH beyond the effect of NOP alone. The decrease in CH emissions with treatments that included NOP occurred along with a decrease in the copy number of methanogens associated with the solid and liquid phases, confirming the inhibitory effects of NOP on these microorganisms. In conclusion, the combined effects of NOP and MON on CH mitigation did not exceed the effect of NOP alone when using a high-grain diet in vitro.

Effects of sustained reduction of enteric methane emissions with dietary supplementation of 3-nitrooxypropanol on growth performance of growing and finishing beef cattle.

The study objective was to evaluate the effects of sustained reduction of enteric methane (CH) emissions with dietary supplementation of the inhibitor 3-nitrooxypropanol (NOP) on growth rate and feed conversion efficiency of growing and finishing beef cattle. Eighty-four crossbred steers were used in a 238-d feeding study and fed a backgrounding diet for the first 105 d (backgrounding phase) and transition diets for 28 d followed by a finishing diet for 105 d (finishing phase) with 3 doses of NOP (0, 100, and 200 mg/kg DM). The experiment was a completely randomized design using 21 pens (4 cattle/pen) with 7 pens per treatment. When cattle were fed the backgrounding diet, pen DMI was reduced ( < 0.01) whereas G:F tended to improve ( = 0.06) with increasing dose of NOP supplementation. During the finishing phase, DMI ( = 0.06) and ADG ( = 0.07) tended to decrease with increasing dose of NOP supplementation. Although both levels of NOP were effective in reducing CH emissions from the backgrounding diet ( < 0.01), only NOP supplemented at the highest dose was effective in reducing total CH emissions from the finishing diet ( < 0.01). Methane yield (g/kg DMI) was reduced whereas hydrogen emissions were increased at the highest dose of NOP supplementation with both backgrounding and finishing diets ( < 0.01). Overall, these results demonstrate efficacy of NOP in reducing enteric CH emissions from cattle fed backgrounding and finishing diets, and these effects were negated once NOP supplementation was discontinued.

Sustained reduction in methane production from long-term addition of 3-nitrooxypropanol to a beef cattle diet.

The objective was to evaluate whether long-term addition of 3-nitrooxypropanol (NOP) to a beef cattle diet results in a sustained reduction in enteric CH4 emissions in beef cattle. Eight ruminally cannulated heifers (637 ± 16.2 kg BW) were used in a completely randomized design with 2 treatments: Control (0 g/d of NOP) and NOP (2 g/d of NOP). Treatments were mixed by hand into the total mixed ration (60% forage, DM basis) at feeding time. Feed offered was restricted to 65% of ad libitum DMI (slightly over maintenance energy intake) and provided once per day. The duration of the experiment was 146 d, including an initial 18-d covariate period without NOP use; a 112-d treatment period with NOP addition to the diet, divided into four 28-d time intervals (d 1 to 28, 29 to 56, 57 to 84, and 85 to 112); and a final 16-d recovery period without NOP use. During the covariate period and at the end of each interval and the end of the recovery period, CH4 was measured for 3 d using whole animal metabolic chambers. The concentration of VFA was measured in rumen fluid samples collected 0, 3, and 6 h after feeding, and the microbial population was evaluated using rumen samples collected 3 h after feeding on d 12 of the covariate period, d 22 of each interval within the treatment period, and d 8 of the recovery period. Average DMI for the experiment was 7.04 ± 0.27 kg. Methane emissions were reduced by 59.2% when NOP was used (9.16 vs. 22.46 g/kg DMI; P < 0.01). Total VFA concentrations were not affected (P = 0.12); however, molar proportion of acetate was reduced and that for propionate increased when NOP was added (P < 0.01), which reduced the acetate to propionate ratio (3.0 vs. 4.0; P < 0.01). The total copy number of the 16S rRNA gene of total bacteria was not affected (P = 0.50) by NOP, but the copy number of the 16S rRNA gene of methanogens was reduced (P < 0.01) and the copy number of the 18S rRNA gene of protozoa was increased (P = 0.03). The residual effect of NOP for most of the variables studied was not observed or was minimal during the recovery period. These results demonstrated that the addition of NOP to a diet for beef cattle caused a sustained decrease of methanogenesis, with no sign of adaptation, and that these effects were reversed once NOP addition was discontinued

The potential of 3-nitrooxypropanol to lower enteric methane emissions from beef cattle.

This study evaluated if 3-nitrooxypropanol reduces enteric methane (CH4) emissions when added to the diet of beef cattle. The effects of 3-nitrooxypropanol on related variables including diet digestibility, ruminal fermentation, and ruminal microorganisms were also investigated. Eight ruminally cannulated Angus heifers (549 ± 64.3 kg [mean BW ± SD]) were fed a high forage diet (backgrounding diet) supplemented with 4 levels of 3-nitrooxypropanol (0, 0.75, 2.25 and 4.50 mg/kg BW). The experiment was designed as a duplicated 4 × 4 Latin square with 2 groups of heifers and four 28-d periods. Methane emissions were measured during 3 consecutive days using metabolic chambers. Up to a 5.8% reduction in ad libitum DMI was observed when 2.5 mg/kg BW of 3-nitrooxypropanol was fed (P = 0.03). Increasing level of 3-nitrooxypropanol linearly (P < 0.001) reduced CH4, with 33% less CH4 (corrected for DMI) at the highest level of supplementation compared with the control. Feed energy lost as CH4 was also reduced when 3-nitrooxypropanol was supplemented (P < 0.001). Molar proportion of acetate was reduced (P < 0.001) and that for propionate increased (P < 0.001) with increasing dose of 3-nitrooxypropanol, which in turn led to a reduction in the acetate to propionate ratio (P < 0.001). Total copy numbers of 16S ribosomal RNA (rRNA) genes for bacteria, methanogens, and 18S rRNA genes for protozoa in ruminal contents were not affected by 3-nitrooxypropanol supplementation (P ≥ 0.31). There was no effect of 3-nitrooxypropanol on DM (P = 0.1) digestibility in the total tract. The use of 4.5 mg/kg BW of 3-nitrooxypropanol in beef cattle consuming a backgrounding diet was effective in reducing enteric CH4 emissions without negatively affecting diet digestibility.

Effect of a blend of essential oil compounds on the colonization of starch-rich substrates by bacteria in the rumen.

AIMS: To investigate the mode of action of a blend of essential oil compounds on the colonization of starch-rich substrates by rumen bacteria. METHODS AND RESULTS: Starch-rich substrates were incubated, in nylon bags, in the rumen of sheep organized in a 4 x 4 latin square design and receiving a 60:40 silage : concentrate diet. The concentrate was either high or low in crude protein, and the diet was supplemented or not with a commercial blend of essential oil compounds (110 mg per day). The total genomic DNA was extracted from the residues in the bags. The total eubacterial DNA was quantified by real-time PCR and the proportion of Ruminobacter amylophilus, Streptococcus bovis and Prevotella bryantii was determined. Neither the supplementation with essential oil compounds nor the amount of crude protein affected the colonization of the substrates by the bacteria quantified. However, colonization was significantly affected by the substrate colonized. CONCLUSIONS: The effect of essential oils on the colonization of starch-rich substrates is not mediated through the selective inhibition of R. amylophilus. SIGNIFICANCE AND IMPACT OF THE STUDY: This study enhances our understanding of the colonization of starch-rich substrates, as well as of the mode of action of the essential oils as rumen manipulating agents.

Effect of triacylglycerol structure on absorption and metabolism of isotope-labeled palmitic and linoleic acids by humans.

The effect of dietary TAG structure and fatty acid acyl TAG position on palmitic and linoleic acid metabolism was investigated in four middle-aged male subjects. The study design consisted of feeding diets containing 61 g/d of native lard (NL) or randomized lard (RL) for 28 d. Subjects then received an oral dose of either 1,3-tetradeuteriopalmitoyl-2-dideuteriolinoleoyl-rac-glycerol or a mixture of 1,3-dideuteriolinoleoyl-2-tetradeuteriopalmitoyl-rac-glycerol and 1,3-hexadeuteriopalmitoyl-2-tetradeuteriolinoleoyl-rac-glycerol. Methyl esters of plasma lipids isolated from blood samples drawn over a 2-d period were analyzed by GC-MS. Results showed that absorption of the 2H-fatty acids (2H-FA) was not influenced by TAG position. The 2H-FA at the 2-acyl TAG position were 85+/-4.6% retained after absorption. Substantial migration of 2H-16:0 (31.2+/-8.6%) from the sn-2 TAG position to the sn-1,3 position and 2H-18:2n-6 (52.8+/-6.4%) from the sn-1,3 position to the sn-2 position of chylomicron TAG occurred after initial absorption and indicates the presence of a previously unrecognized isomerization mechanism. Incorporation and turnover of the 2H-FA in chylomicron TAG, plasma TAG, and plasma cholesterol esters were not influenced by TAG acyl position. Accretion of 2H-16:0 from the sn-2 TAG position in 1-acylphosphatidylcholine was 1.7 times higher than 2H-16:0 from the sn-1,3 TAG positions. Acyl TAG position did not influence 2H-18:2n-6 incorporation in PC. The concentration of 2H-18:2n-6-derived 2H-20:4n-6 in plasma PC from subjects fed the RL diet was 1.5 times higher than for subjects fed the NL diet, and this result suggests that diets containing 16:0 located at the sn-2 TAG position may inhibit 20:4n-6 synthesis. The overall conclusion is that selective rearrangement of chylomicron TAG structures diminishes but does not totally eliminate the metabolic and physiological effects of dietary TAG structure.

Bioactive crude plant seed extracts from the NCAUR oilseed repository.

Over four-hundred crude extracts from 202 plant species distributed among 131 plant families were evaluated for their bioactivity against brine shrimp (Artemia salina). Activity was determined for both the organic (CH2Cl2:MeOH) and aqueous extracts against A. salina in a 96 well-plate assay. Of the greater than four-hundred extracts tested, 21 organic and 6 aqueous extracts demonstrated potent cytotoxic activity (LC50 = < 100 microg/ml). Three of these organic extracts (Crateva religiosa, Diospyros dichrophylla, and Olax subscorpioidea) were chosen for chemical investigations due to their high activity and a lack of prior investigations. Chemical analysis of these extracts resulted in the isolation of oleanolic acid (1) and 4-epi-hederagenin (2) from C. religiosa, isodiospyrin (3) from D. dichrophylla, and santalbic acid (4) from O. subscorpioidea.

Effect of dietary docosahexaenoic acid on desaturation and uptake in vivo of isotope-labeled oleic, linoleic, and linolenic acids by male subjects.

The effect of dietary docosahexaenoic acid (22:6n-3, DHA) on the metabolism of oleic, linoleic, and linolenic acids was investigated in male subjects (n = 6) confined to a metabolic unit and fed diets containing 6.5 or <0.1 g/d of DHA for 90 d. At the end of the diet period, the subjects were fed a mixture of deuterated triglycerides containing 18:1n-9[d6], 18:2n-6[d2], and 18:3n-3[d4]. Blood samples were drawn at 0, 2, 4, 6, 8, 12, 24, 48, and 72 h. Methyl esters of plasma total lipids, triglycerides, phospholipids, and cholesterol esters were analyzed by gas chromatography-mass spectrometry. Chylomicron triglyceride results show that the deuterated fatty acids were equally well absorbed and diet did not influence absorption. Compared to the low-DHA diet (LO-DHA), clearance of the labeled fatty acids from chylomicron triglycerides was modestly higher for subjects fed the high DHA diet (HI-DHA). DHA supplementation significantly reduced the concentrations of most n-6[d2] and n-3[d4] long-chain fatty acid (LCFA) metabolites in plasma lipids. Accumulation of 20:5n-3[d4] and 22:6n-3[d4] was depressed by 76 and 88%, respectively. Accumulations of 20:3n-6[d2] and 20:4n-6[d2] were both decreased by 72%. No effect of diet was observed on acyltransferase selectivity or on uptake and clearance of 18:1n-9[d6], 18:2n-6[d2], and 18:3n-3[d4]. The results indicate that accumulation of n-3 LCFA metabolites synthesized from 18:3n-3 in typical U.S. diets would be reduced from about 120 to 30 mg/d by supplementation with 6.5 g/d of DHA. Accumulation of n-6 LCFA metabolites synthesized from 18:2n-6 in U.S. diets is estimated to be reduced from about 800 to 180 mg/d. This decrease is two to three times the amount of n-6 LCFA in a typical U.S. diet. These results support the hypothesis that health benefits associated with DHA supplementation are the combined result of reduced accretion of n-6 LCFA metabolites and an increase in n-3 LCFA levels in tissue lipids.

Effect of dietary arachidonic acid on metabolism of deuterated linoleic acid by adult male subjects.

The influence of dietary supplementation with 20:4n-6 on uptake and turnover of deuterium-labeled linoleic acid (18:2n-6[d2]) in human plasma lipids and the synthesis of desaturated and elongated n-6 fatty acids from 18:2n-6[d21 were investigated in six adult male subjects. The subjects were fed either a high-arachidonic acid (HIAA) diet containing 1.7 g/d or a low-AA (LOAA) diet containing 0.21 g/d of AA for 50 d. Each subject was then dosed with about 3.5 g of 18:2n-6[d2] as the triglyceride (TG) at 8:00 A.M., 12:00, and 5:00 P.M. The total 18:2n-6[d21] fed to each subject was about 10.4 g and is approximately equal to one-half of the daily intake of 18:2n-6 in a typical U.S. male diet. Nine blood samples were drawn over a 96-h period. Methyl esters of plasma total lipid (TL), TG, phospholipid, and cholesterol ester were analyzed by gas chromatography-mass spectroscopy. Dietary 20:4n-6 supplementation did not affect uptake of 18:2n-6[d2] in plasma lipid classes over the 4-d study period nor the estimated half-life of 24-36 h for 18:2n-6[d2]. The percentages of major deuterium-labeled desaturation and elongation products in plasma TL, as a percentage of total deuterated fatty acids, were 1.35 and 1.34% 18:3n-6[d2]; 0.53 and 0.50% 20:2n-6[d2]; 1.80 and 0.92% 20:3n-6[d2] and 3.13 and 1.51% 20:4n-6[d2] for the LOAA and HIAA diet groups, respectively. Trace amounts (<0.1%) of the 22:4n-6[d2] and 22:5n-6[d2] metabolites were present. Plasma TL concentration data for both 20:3n-6[d2] and 20:4n-6[d2] were 48% lower (P < 0.05) in samples from the HIAA diet group than in samples from the LOAA diet group. For a normal adult male consuming a typical U.S. diet, the estimated accumulation in plasma TL of 20:4n-6 synthesized from 20 g/d (68 mmole) of 18:2n-6 is 677 mg/d (2.13 mmole). Dietary supplementation with 1.5 g/d of 20:4n-6 reduced accumulation of 20:4n-6 synthesized from 20 g/d of 18:2n-6 to about 326 mg/d (1.03 mmole).

Influence of dietary arachidonic acid on metabolism in vivo of 8cis,11cis,14-eicosatrienoic acid in humans.

This study investigated the influence of dietary arachidonic acid (20:4n-6) on delta 5 desaturation and incorporation of deuterium-labeled 8cis,11 cis, 14-eicosatrienoic acid (20:3n-6) into human plasma lipids. Adult male subjects (n = 4) were fed diets containing either 1.7 g/d (HI20:4 diet) or 0.21 g/d (LO20:4 diet) of arachidonic acid for 50 d and then dosed with a mixture containing ethyl esters of 20:3n-6[d4] and 18:1n-9[d2]. A series of blood samples was sequentially drawn over a 72-h period, and methyl esters of plasma total lipid, triacylglycerol, phospholipids, and cholesteryl ester were analyzed by gas chromatography-mass spectrometry. Based on the concentration of 20:3n6[d4] in total plasma lipid, the estimated conversion of 20:3n-6[d4] to 20:4n-6[d4] was 17.7 +/- 0.79% (HI20:4 diet) and 2.13 +/- 1.44% (LO20:4 diet). The concentrations of 20:4n-6[d4] in total plasma lipids from subjects fed the HI20:4 and LO20:4 diets were 2.10 +/- 0.6 and 0.29 +/- 0.2 mumole/mL plasma/mmole of 20:3n-6[d4] fed/kg of body weight. These data indicate that conversion of 20:3n-6[d4] to 20:4n-6[d4] was stimulated 7-8-fold by the HI20:4 diet. Phospholipid acyltransferase was 2.5-fold more selective for 20:3n-6[d4] than 18:1n-9[d2], and lecithin:cholesterol acyltransferase was 2-fold more selective for 18:1n-9[ds] than 20:3n-6[d4]. These differences in selectivity were not significantly influenced by diet. Absorption of ethyl 20:3n-6[d4] was about 33% less than ethyl 18:1n-9[d2]. The sum of the n-6 retroconversion products from 20:3n-6[d4] in total plasma lipids was about 2% of the total deuterated fatty acids. Neither absorption nor retroconversion appears to be influenced by diet.

Alternate pathways in the desaturation and chain elongation of linolenic acid, 18:3(n-3), in cultured glioma cells.

Cultured C6 glioma cells rapidly incorporate and metabolize the essential fatty acids, 18:2(n-6) and 18:3(n-3), to 20- and 22-carbon polyunsaturated fatty acids. Using several deuterated fatty acid substrates we have obtained data that suggest alternate pathways, one possibly involving delta 8-desaturation, may exist in glioma cells for formation of 20:5(n-3) and 22:6(n-3) from 18:3(n-3). With 18:3(n-3)-6,6,7,7-d4 practically no 18:4(n-3)-6,7-d2 or 20:4(n-3)-8,9-d2 was detected whereas 20:3(n-3)-8,8,9,9-d4 accounted for 3.4% and delta 5,11,14,17-20:4-8,8,9,9-d4 for 21.1% of the total deuterated fatty acids recovered in phospholipids after a 16 h incubation; 20:5(n-3)-8,9-d2, 22:5(n-3)-10,11-d2, and 22:6(n-3)-10,11-d2 accounted for 42.4%, 13.2%, and 2.8% of deuterated acyl chains, respectively. When added exogneously, 20:3-8,8,9,9,-d4 was extensively converted to delta 5,11,14,17-20:4(n-3)-8,8,9,9-d4 (45%) and 20:5(n-3)-8,9-d2 (24%); a small amount (4%) of 18:3(n-3)-d4 also was detected. Both 20:4(n-3)-8,9-d2 and 18:4(n-3)-12,13,15,16-d4 were also converted to 20:5(n-3) and 22:6(n-3) with 8 and 0% of the respective original deuterated substrate remaining after 16 h. A possible pathway for 18:3(n-3) metabolism in glioma cells is described whereby an initial chain elongation step is followed by successive delta 5 and delta 8 desaturation reactions resulting in 20:5(n-3) formation and accounting for the ordered removal of deuterium atoms. Alternatively, extremely effective retroconversion may occur to chain shorten 20:3(n-3)-d4 to 18:3(n-3)-d4 followed by rapid conversion through the classical desaturation and chain elongation sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of the metabolic interconversion of deuterium-labeled fatty acids by gas chromatography/mass spectrometry.

An analytical method that was developed to analyze deuterium-labeled fatty acids in human blood has been extended to identify labeled fatty acids from C14 to C24 chain length which are formed by metabolic processes such as desaturation, elongation, or shortening of the labeled fatty acids fed. A new computer and a hardware adder have been utilized to assure reliable data acquisition. Relative standard deviations for the analysis of labeled fatty acids were measured at 0.02, 0.03, and 0.04 at the 5%, 1%, and 0.2% levels of the labeled fatty acid methyl esters, respectively. The method makes extensive use of standards and computer processing for accuracy and high productivity. Data from a chylomicron triacylglycerol fraction are included to demonstrate the sensitivity of detection of metabolites formed by desaturation and elongation.