Phenolic plant extracts are additive in their effects against in vitro ruminal methane and ammonia formation.

OBJECTIVE: The methane mitigating potential of various plant-based polyphenol sources is known, but effects of combinations have rarely been tested. The aim of the present study was to determine whether binary and 3-way combinations of such phenol sources affect ruminal fermentation less, similar or more intensively than separate applications. METHODS: The extracts used were from Acacia mearnsii bark (acacia), Vitis vinifera (grape) seed, Camellia sinensis leaves (green tea), Uncaria gambir leaves (gambier), Vaccinium macrocarpon berries (cranberry), Fagopyrum esculentum seed (buckwheat), and Ginkgo biloba leaves (ginkgo). All extracts were tested using the Hohenheim gas test. This was done alone at 5% of dry matter (DM). Acacia was also combined with all other single extracts at 5% of DM each, and with two other phenol sources (all possible combinations) at 2.5%+2.5% of DM. RESULTS: Methane formation was reduced by 7% to 9% by acacia, grape seed and green tea and, in addition, by most extract combinations with acacia. Grape seed and green tea alone and in combination with acacia also reduced methane proportion of total gas to the same degree. The extracts of buckwheat and gingko were poor in phenols and promoted ruminal fermentation. All treatments except green tea alone lowered ammonia concentration by up to 23%, and the binary combinations were more effective as acacia alone. With three extracts, linear effects were found with total gas and methane formation, while with ammonia and other traits linear effects were rare. CONCLUSION: The study identified methane and ammonia mitigating potential of various phenolic plant extracts and showed a number of additive and some non-linear effects of combinations of extracts. Further studies, especially in live animals, should concentrate on combinations of extracts from grape seed, green tea leaves Land acacia bark and determine the ideal dosages of such combinations for the purpose of methane mitigation.

The requirements for rumen-degradable protein per unit of fermentable organic matter differ between fibrous feed sources.

Ruminant feed evaluation systems use constant minimum requirements of rumen-degradable protein (RDP) and often relate this to apparently degradable organic matter (OM). However, studies with tropical forages indicate that RDP: apparently degraded OM might not be constant across high-fiber diets. This was tested with semi-continuous ruminal cultures (Rusitec) using dried contrasting low-protein fiber sources: brachiaria hay (high in fiber, medium lignified), apple pomace (medium in fiber, highly lignified), and sugar beet pulp (medium in fiber and lignification). Each feed was incubated at 14 g dry matter day(-1) with 0, 0.85, 1.7, 3.4, 6.8, 13.6, or 27.2 mg g(-1) urea. The amount of urea needed to reach a similar basal concentration of ammonia in the incubation fluid was tested for each feed in advance. Apparent fiber and OM degradability were determined after 48 h of incubation. Data was evaluated by regressions and analysis of variance. The response curve of incubation fluid ammonia to urea supplementation was similar in slope in all feeds. Plateaus in apparent OM degradability in relation to ammonia concentration were determined. The ammonia concentration where apparent OM and fiber degradability reached 95% of maximum was approached in the order of pomace < pulp < hay. With regard to fiber degradability, a plateau was reached at ≥ 80 g kg(-1) crude protein only with hay and pomace, whilst a linear relationship existed between RDP and OM degradation for pulp. In hay the ratio RDP: OM degraded was equal to 1.6 but was only 1.0 in the other feeds. There was no obvious lack of branched short-chain fatty acids at low RDP. Thus, the hypothesis was confirmed but the demand for RDP seems even higher in tropical forage compared to food industrial byproducts. The efficiency of urea to promote apparent OM and fiber degradation was also variable. Thus, it seems that minimum thresholds of either RDP or ruminal ammonia concentration may not be reflected appropriately by constants.

In vitro methane formation and carbohydrate fermentation by rumen microbes as influenced by selected rumen ciliate species.

Ciliate protozoa contribute to ruminal digestion and emission of the greenhouse gas methane. Individual species of ciliates co-cultured with mixed prokaryote populations were hypothesized to utilize carbohydrate types differently. In an in vitro batch culture experiment, 0.6 g of pure cellulose or xylan was incubated for 24 h in 40-mL cultures of Entodinium caudatum, Epidinium ecaudatum, and Eudiplodinium maggii with accompanying prokaryotes. Irrespective of ciliate species, gas formation (mL) and short-chain fatty acids (SCFA) concentrations (mmol L(-1)) were higher with xylan (71; 156) than with cellulose (52; 105). Methane did not differ (7.9% of total gas). The SCFA profiles resulting from fermentation of the carbohydrates were similar before and after removing the ciliates from the mixed microbial population. However, absolute methane production (mL 24 h(-1)) was lower by 50% on average after removing E. caudatum and E. maggii. Methanogen copies were less without E. maggii, but not without E. ecaudatum. Within 3 weeks part of this difference was compensated. Butyrate proportion was higher in cultures with E. maggii and E. ecaudatum than with E. caudatum and only when fermenting xylan. In conclusion, the three ciliate species partly differed in their response to carbohydrate type and in supporting methane formation.

Effect of the rumen ciliates Entodinium caudatum, Epidinium ecaudatum and Eudiplodinium maggii, and combinations thereof, on ruminal fermentation and total tract digestion in sheep.

The quantitative importance of individual ciliate species and their interaction in the rumen is still unclear. The present study was performed to test whether there are species differences in the influence on ruminal fermentation in vivo and if combinations of ciliates act additive in that respect. Six adult wethers fed a hay-concentrate diet were defaunated, then refaunated either with Entodinium caudatum (EC), Epidinium ecaudatum (EE) or Eudiplodinium maggii (EM) alone, then progressively with all possible species combinations. Feed, faeces, urine, ruminal fluid and gas were sampled for eight days always after at least 21 days of adaptation. With a linear mixed model, accounting for the 2 x 2 x 2 full factorial study design, mean marginal effect sizes, i.e., the magnitude of change in variables as caused by the presence of each ciliate species or of combinations of them, were estimated. The apparent digestibility of organic matter and neutral detergent fibre remained unaffected. The apparent N digestibility increased by 0.054 with EM (0.716 with defaunation). Ruminal ammonia increased by 1.6, 4.0 and 8.7 mmol/l in the presence of EM, EC and EE, respectively, compared to defaunation (6.9 mmol/l). In the EM + EE combination, ruminal ammonia was lower than would have been expected from an additive effect. With EE, total short-chain fatty acids increased by 23 mmol/l (100 mmol/l with defaunation), but not when EE was combined with EM. The acetate-to-propionate ratio decreased by 0.73 units in the presence of EE (4.0 with defaunation), but only when EE was the sole ciliate species in the rumen. In the presence of any ciliate species, the 16S rDNA copies of total Bacteria and major fibrolytic species decreased to 0.52- and 0.22-fold values, respectively of that found without protozoa. Total Archaea were unaffected; however, Methanobacteriales copies increased 1.44-fold with EC. The CH4-to-CO2 ratio of ruminal gas decreased by 0.036 with EM and 0.051 with EE (0.454 with defaunation). In conclusion, individual ciliates affected ruminal fermentation differently and, when different species were combined, sometimes in a non-additive manner. From the ciliates investigated, EE affected ruminal fermentation most and might play a dominant role in mixed ciliate populations.

Stable carbon isotope composition of c9,t11-conjugated linoleic acid in cow's milk as related to dietary fatty acids.

This study explores the potential use of stable carbon isotope ratios (δ(13)C) of single fatty acids (FA) as tracers for the transformation of FA from diet to milk, with focus on the metabolic origin of c9,t11-18:2. For this purpose, dairy cows were fed diets based exclusively on C(3) and C(4) plants. The FA in milk and feed were fractionated by silver-ion thin-layer chromatography and analyzed for their δ(13)C values. Mean δ(13)C values of FA from C(3) milk were lower compared to those from C(4) milk (-30.1‰ vs. -24.9‰, respectively). In both groups the most negative δ(13)C values of all FA analyzed were measured for c9,t11-18:2 (C(3) milk = -37.0 ± 2.7‰; C(4) milk -31.4 ± 1.4‰). Compared to the dietary precursors 18:2n-6 and 18:3n-3, no significant (13)C-depletion was measured in t11-18:1. This suggests that the δ(13)C-change in c9,t11-18:2 did not originate from the microbial biohydrogenation in the rumen, but most probably from endogenous desaturation of t11-18:1. It appears that the natural δ(13)C differences in some dietary FA are at least partly preserved in milk FA. Therefore, carbon isotope analyses of individual FA could be useful for studying metabolic transformation processes in ruminants.

Diallyl disulphide and lovastatin: effects on energy and protein utilisation in, as well as methane emission from, sheep.

Currently research on feed supplementation with natural compounds to improve energy and protein utilisation and to mitigate the greenhouse gas methane in ruminants is intensively pursued. Two compounds, diallyl disulphide (DADS), an important component of garlic oil, and lovastatin, an inhibitor of a key enzyme of methanogenic Archaea, were selected on the basis of their in vitro anti-methanogenic potential. In three 23-day experimental runs, six sheep received hay and concentrate in a duplicate 3 x 3 Latin square design. The concentrate was either not supplemented or supplemented with either 4 g DADS or 80 mg lovastatin per kg of total dietary dry matter. There were no refusals of concentrate for any treatment. Respiratory measurements were conducted on experimental days 7/8 (Period 1) and days 17/18 (Period 2). Relative to the control, digestibility of neutral detergent fibre (NDF) tended to increase (p = 0.09) with DADS by 14%. This was associated with an increased (p = 0.07) body energy retention of the animals. Effects on nitrogen balance and ruminal fermentation traits were never significant. No influence of supplements or period was found on total daily CH4 production which averaged at 28.6 g per sheep. However, across both periods the amount of CH4 produced per kg NDF digested was lower (-8%; p = 0.02) with DADS than without supplementation, and the lovastatin treatment ranged in between. In conclusion, the study demonstrated a certain potential of DADS to improve fibre digestion and body energy retention and to limit CH4 formation in relation to digestible fibre intake, while lovastatin remained ineffective.

Effects of monolaurin on ruminal methanogens and selected bacterial species from cattle, as determined with the rumen simulation technique.

Before being able to implement effective ruminal methane mitigation strategies via feed supplementation, the assessment of side effects on ruminal fermentation and rumen microbial populations is indispensable. In this respect we investigated the effects of monolaurin, a methane-mitigating lipid, on methanogens and important carbohydrate-degrading bacteria present in ruminal fluid of dairy cattle in continuous culture employing the rumen simulation technique. In six experimental runs, each lasting for 10 days, four diets with different carbohydrate composition, based on hay, maize, wheat and a maize-wheat mixture, either remained non-supplemented or were supplemented with monolaurin and incubated in a ruminal-fluid buffer mixture. Incubation liquid samples from days 6 to 10 of incubation were analyzed with relative quantitative polymerase chain reaction (qPCR) of 16S rRNA genes to assess monolaurin-induced shifts in specific rumen microbial populations in relation to the corresponding non-supplemented diets. Monolaurin completely inhibited Fibrobacter succinogenes in all diets while the response of the other cellulolytic bacteria varied in dependence of the diet. Megasphaera elsdenii remained unaffected by monolaurin in the two diets containing maize, but was slightly stimulated by monolaurin with the wheat and largely with the hay diet. The supply of monolaurin suppressed Methanomicrobiales below the detection limit with all diets, whereas relative 16S rRNA gene copy numbers of Methanobacteriales increased by 7-fold with monolaurin in case of the hay diet. Total Archaea were decreased by up to over 90%, but this was significant only for the wheat containing diets. Thus, monolaurin exerted variable effects mediated by unknown mechanisms on important ruminal microbes involved in carbohydrate degradation, along with its suppression of methane formation. The applicability of monolaurin for methane mitigation in ruminants thus depends on the extent to which adverse effects on carbohydrate-degrading bacteria actually impair the supply of digested carbohydrates to the animal.

Suitability of different media for in vitro cultivation of the ruminal protozoa species Entodinium caudatum, Eudiplodinium maggii, and Epidinium ecaudatum.

Three protozoal cultivation media were tested to determine the medium which best facilitated growth and viability of key B-type ciliates isolated from the sheep rumen. Entodinium caudatum and Eudiplodinium maggii were grown anaerobically in 50-ml flasks for 32 days in Caudatum-type (C), Kisidayova (K) or Dehority (M) medium. On day 32, in media K and M, E. caudatum cell counts were high with 5.6×10(3) and 7.8×10(3) mL(-1), respectively, and the proportion of dead cells was low with 0.6 and 1.4%, respectively. E. maggii concentrations when grown in medium M and C were 2.7×10(3) and 2.4×10(3) mL(-1), respectively, with 3.9 and 14.1% dead cells. Medium M, which favoured growth of both protozoa species, was tested again and Epidinium ecaudatum was included. Protozoa were grown for a 4-month period and samples were taken in the last two months on days 1, 7, 35 and 57. Average cell concentrations were 10.0, 0.8 and 0.5×10(3) mL(-1) for E. caudatum, E. maggii, and E. ecaudatum, respectively. In conclusion, medium M would appear to be the best choice for cultivating these three species in one medium.

Ruminal methane inhibition potential of various pure compounds in comparison with garlic oil as determined with a rumen simulation technique (Rusitec).

Ruminants represent an important source of methane (CH(4)) emissions; therefore, CH(4) mitigation by diet supplementation is a major goal in the current ruminant research. The objective of the present study was to use a rumen simulation technique to evaluate the CH(4)-mitigating potential of pure compounds in comparison with that achieved with garlic oil, a known anti-methanogenic supplement. A basal diet (15 g DM/d) consisting of ryegrass hay, barley and soyabean meal (1:0·7:0·3) was incubated with the following additives: none (negative control); garlic oil (300 mg/l incubation liquid; positive control); allyl isothiocyanate (75 mg/l); lovastatin (150 mg/l); chenodeoxycholic acid (150 mg/l); 3-azido-propionic acid ethyl ester (APEE, 150 mg/l); levulinic acid (300 mg/l); 4-[(pyridin-2-ylmethyl)-amino]-benzoic acid (PABA, 300 mg/l). Fermentation profiles (SCFA, microbial counts and N turnover) and H(2) and CH(4) formation were determined. Garlic oil, allyl isothiocyanate, lovastatin and the synthetic compound APEE decreased the absolute daily CH(4) formation by 91, 59, 42 and 98 %, respectively. The corresponding declines in CH(4) emitted per mmol of SCFA were 87, 32, 40 and 99 %, respectively, compared with the negative control; the total SCFA concentration was unaffected. Garlic oil decreased protozoal numbers and increased bacterial counts, while chenodeoxycholic acid completely defaunated the incubation liquid. In vitro, neutral-detergent fibre disappearance was lower following chenodeoxycholic acid and PABA treatments (- 26 and - 18 %, respectively). In conclusion, garlic oil and APEE were extremely efficient at mitigating CH(4) without noticeably impairing microbial nutrient fermentation. Other promising substances were allyl isothiocyanate and lovastatin.

Methane output of tortoises: its contribution to energy loss related to herbivore body mass.

An increase in body mass (M) is traditionally considered advantageous for herbivores in terms of digestive efficiency. However, recently increasing methane losses with increasing M were described in mammals. To test this pattern in non-mammal herbivores, we conducted feeding trails with 24 tortoises of various species (M range 0.52-180 kg) fed a diet of grass hay ad libitum and salad. Mean daily dry matter and gross energy intake measured over 30 consecutive days scaled to M(0.75 (95%CI 0.64-0.87)) and M(0.77 (95%CI 0.66-0.88)), respectively. Methane production was measured over two consecutive days in respiration chambers and scaled to M(1.03 (95%CI 0.84-1.22)). When expressed as energy loss per gross energy intake, methane losses scaled to 0.70 (95%CI 0.47-1.05) M(0.29 (95%CI 0.14-0.45)). This scaling overlaps in its confidence intervals to that calculated for nonruminant mammals 0.79 (95%CI 0.63-0.99) M(0.15 (95%CI 0.09-0.20)), but is lower than that for ruminants. The similarity between nonruminant mammals and tortoises suggest a common evolution of the gut fauna in ectotherms and endotherms, and that the increase in energetic losses due to methane production with increasing body mass is a general allometric principle in herbivores. These findings add evidence to the view that large body size itself does not necessarily convey a digestive advantage.

Methane output of rabbits (Oryctolagus cuniculus) and guinea pigs (Cavia porcellus) fed a hay-only diet: implications for the scaling of methane production with body mass in non-ruminant mammalian herbivores.

It is assumed that small herbivores produce negligible amounts of methane, but it is unclear whether this is a physiological peculiarity or simply a scaling effect. A respiratory chamber experiment was conducted with six rabbits (Oryctolagus cuniculus, 1.57±0.31 kg body mass) and six guinea pigs (Cavia porcellus, 0.79±0.07 kg) offered grass hay ad libitum. Daily dry matter (DM) intake and DM digestibility were 50±6 g kg⁻°·75 d⁻¹ and 55±6% in rabbits and 59±11 g kg⁻°·75 d⁻¹ and 61±3% in guinea pigs, respectively. Methane production was similar for both species (0.20±0.10 L d⁻¹ and 0.22±0.08L d⁻¹ and represented 0.69±0.32 and 1.03±0.29% of gross energy intake in rabbits and guinea pigs, respectively. In relation to body mass (BM) guinea pigs produced significantly more methane. The data on methane per unit of BM obtained in this study and from the literature on the methane output of elephant, wallabies and hyraxes all lay close to a regression line derived from roughage-fed horses, showing an increase in methane output with BM. The regression, including all data, was nearly identical to that based on the horse data only (methane production in horses [L d⁻¹]=0.18 BM [kg]°·97(95%CI °·9²â»¹·°²)) and indicates linear scaling. Because feed intake typically scales to BM°·75, linear scaling of methane output translates into increasing energetic losses at increasing BM. Accordingly, the data collection indicates that an increasing proportion of ingested gross energy is lost because relative methane production increases with BM. Different from ruminants, such losses (1%-2% of gross energy) appear too small in non-ruminant herbivores to represent a physiologic constraint on body size. Nevertheless, this relationship may represent a physiological disadvantage with increasing herbivore body size.

Methods of emulsifying linoleic acid in biohydrogenation studies in vitro may bias the resulting fatty acid profiles.

The effects of three emulsifying methods on ruminal fatty acid biohydrogenation (BH) in vitro were compared. Using a static in-vitro gas test system, four replicates of each treatment were incubated in buffered ruminal fluid. Hemicellulose (300 mg dry matter) was supplemented either with or without linoleic acid (9c12c-18:2, 5% in diet dry matter) and incubated for 4 and 24 h. Three methods of emulsifying 9c12c-18:2 were tested: (1) ethanol, (2) Tween 80, and (3) sonication. The products were then compared to non-emulsified 9c12c-18:2. Out of the three emulsifying methods tested, ethanol and sonication resulted in stable 9c12c-18:2 emulsions, indicating good 9c12c-18:2 distribution, while the Tween 80 emulsion was less stable. BH was strongly inhibited by treating 9c12c-18:2 with ethanol and sonication at different steps of the BH-pathway, resulting in changed concentrations of certain BH intermediates. The fatty acid profile generated from the major BH-pathways of 9c12c-18:2 with Tween 80 was comparable to that without emulsification after 24 h of incubation. We conclude that it is not recommended to emulsify lipids before incubating them in vitro when investigating fatty acid BH. If emulsification of 9c12c-18:2 is necessary, Tween 80 seems to be the method that interferes least with BH.

Transfer of linoleic and linolenic acid from feed to milk in cows fed isoenergetic diets differing in proportion and origin of concentrates and roughages.

The transfer of ingested alpha-linolenic acid (ALA) and linoleic acid (LA) determines the nutritional quality of milk, but the factors determining this transfer are unclear. The present experiment investigated the influence of roughage to concentrate proportions and the effect of concentrate types on milk fat composition. Respectively, six lactating dairy cows were fed one of three isoenergetic (5.4+/-0.05 MJ net energy for lactation/kg dry matter; DM) and isonitrogenous (215+/-3.5 g crude protein/kg DM) diets, consisting of ryegrass hay only (33 g fatty acids/kg DM; ALA-rich, no concentrate), maize (straw, whole maize pellets and gluten; 36 g fatty acids/kg DM; LA-rich; 560 g concentrate/kg DM), or barley (straw and grain plus soybean meal; 19 g fatty acids/kg DM; LA-rich; 540 g concentrate/kg DM). The fatty acid composition of feeds and resulting milk fat were determined by gas chromatography. The ALA concentration in milk fat was highest (P<0.001) with the hay-diet, but the proportionate transfer of ALA from diet to milk was lower (P<0.001) than with the maize- or barley-diets. The LA concentration in milk fat was highest with the maize-diet (P<0.05, compared with hay) but relative transfer rate was lower (P=0.01). The transfer rates of ALA and LA were reciprocal to the intake of individual fatty acids which thus contributed more to milk fat composition than did roughage to concentrate proportions. The amount of trans-11 18:1 in milk fat was lowest with the barley-diet (P<0.001) and depended on the sum of ALA and LA consumed. The milk fat concentration of cis-9, trans-11 18:2 (rumenic acid) was more effectively promoted by increasing dietary LA (maize) than ALA (hay). Amounts of 18:0 secreted in milk were four (maize) to seven (hay) times higher than the amounts ingested. This was suggestive of a partial inhibition of biohydrogenation in the maize-diet, possibly caused by the high dietary LA level.

Efficiency of monolaurin in mitigating ruminal methanogenesis and modifying C-isotope fractionation when incubating diets composed of either C3 or C4 plants in a rumen simulation technique (Rusitec) system.

Mitigation of methanogenesis in ruminants has been an important goal for several decades. Free lauric acid, known to suppress ruminal methanogenesis, has a low palatability; therefore, in the present study the aim was to evaluate the mitigation efficacy of its esterified form (monolaurin). Further, 13C-isotope abundance (delta13C) and 13C-12C fractionation during methanogenesis and fermentation were determined to evaluate possible microbial C-isotope preferences. Using the rumen simulation technique, four basal diets, characterised either by the C3 plants grass (hay) and wheat (straw and grain), or the C4 plant (13C excess compared with C3 plants) maize (straw and grain), and a mixture of the latter two, were incubated with and without monolaurin (50 g/kg dietary DM). Added to hay, monolaurin did not significantly affect methanogenesis. When added to the other diets (P < 0.05 for the wheat-based diet) methane formation was lowered. Monolaurin decreased fibre disappearance (least effect with the hay diet), acetate:propionate ratio, and protozoal counts. Feed residues and SCFA showed the same delta13C as the diets. Methane was depleted in 13C while CO2 was enriched in 13C compared with the diets. Monolaurin addition resulted in 13C depletion of CO2 and enrichment in CH4 (the latter only in the hay diet). In conclusion, monolaurin proved to effectively decrease methanogenesis in the straw-grain diets although this effect might partly be explained by the concomitantly reduced fibre disappearance. The influence on 13C-isotope abundance and fractionation supports the hypothesis that ruminal microbes seem to differentiate to some extent between C-isotopes during methanogenesis and fermentation.

Palatability in sheep and in vitro nutritional value of dried and ensiled sainfoin (Onobrychis viciifolia) birdsfoot trefoil (Lotus corniculatus), and chicory (Cichorium intybus).

Three temperate forages, sainfoin, birdsfoot trefoil, and chicory, characterized by elevated contents of plant secondary compounds, were compared to a ryegrass-clover mixture (control) in dried (Experiment 1) and ensiled form (Experiment 2) in their palatability and nutritional value. Palatability was measured in adult wethers (n = 6) allowed to choose between the familiar control forage and one of the three test plants. Palatability index was calculated from differences in intake of control and test plants measured after given times. Generally at first contact, palatability of the unfamiliar plants was low. Lag time until palatability index approached or exceeded a value of 100 was 2-5 d, but could not be related to the content of condensed tannins. Sainfoin had a high palatability, the highest content of condensed tannins (77.4 +/- 10.23 g/kg DM), a high content of duodenally utilisable crude protein (94.7 +/- 16.87 g/100 g CP), and a high content of metabolizable energy (9.5 +/- 0.38 MJ ME/kg DM), making this plant most promising for various purposes including anthelmintic action.

Rumen simulation technique study on the interactions of dietary lauric and myristic acid supplementation in suppressing ruminal methanogenesis.

The interactions of lauric (C12) and myristic acid (C14) in suppressing ruminal methanogenesis and methanogens were investigated with the rumen simulation technique (Rusitec) using bovine ruminal fluid. The fatty acids were added to basal substrates (grass hay:concentrate, 1:1.5) at a level of 48 g/kg DM, provided in C12:C14 ratios of 5:0, 4:1, 3:2, 2.5:2.5, 2:3, 1:4 and 0:5. Additionally, an unsupplemented control consisting of the basal substrates only was employed. Incubation periods lasted for 15 (n 4) and 25 (n 2) d. CH4 formation was depressed by any fatty acid mixture containing at least 40 % C12, and effects persisted over the complete incubation periods. The greatest depression (70 % relative to control) occurred with a C12:C14 ratio of 4:1, whereas the second most effective treatment in suppressing CH4 production (60 % relative to control) was found with a ratio of 3:2. Total methanogenic counts were decreased by those mixtures of C12 and C14 also successful in suppressing methanogenesis, the 4:1 treatment being most efficient (60 % decline). With this treatment in particular, the composition of the methanogenic population was altered in such a way that the proportion of Methanococcales increased and Methanobacteriales decreased. Initially, CH4 suppression was associated with a decreased fibre degradation, which, however, was reversed after 10 d of incubation. The present study demonstrated a clear synergistic effect of mixtures of C12 and C14 in suppressing methanogenesis, mediated probably by direct inhibitory effects of the fatty acids on the methanogens.

Myristic acid supports the immediate inhibitory effect of lauric acid on ruminal methanogens and methane release.

Two in vitro experiments were carried out with the Hohenheim gas test (HGT) apparatus in order to investigate dose-dependent effects and interactions of non-esterified lauric acid (C(12)) and myristic acid (C(14)) given either individually or in mixture on ruminal methanogens and methanogenesis. Special emphasis was also put on the relationship between effects on methane formation and methanogenic counts. The in vitro incubations were conducted in 10mL ruminal fluid and 20mL buffer solution and lasted for 24h. In the first experiment, 14 levels of C(12), C(14) and stearic acid (C(18); control) were supplied each in increasing steps of 2.5mg covering the range from 0 to 32.5mg. In the second experiment, dosages ranging from 2.5 to 30mg C(12) were supplemented in steps of 2.5mg either without or with 10, 20 or 30mg of C(14). Counts of total Archaea and individual methanogenic orders were determined by the fluorescence in situ hybridization technique using 16S rRNA oligonucleotide probes. In experiment 1, a methane-suppressing effect of more than 80% was achieved with a supply of 30mg C(12), whereas C(14) and C(18) had no effect. Incubation liquid counts of total Archaea and individual methanogenic orders (Methanococcales, Methanosarcinales, Methanomicrobiales and Methanobacteriales) exponentially decreased as a response to C(12) and C(14) to about the same degree (up to 90%) and, to a lesser extent, by C(18). The proportions of the orders of total methanogenic population were not altered by any of the fatty acids. In experiment 2, an additional supply of 10 or 20mg of C(14) supported the suppression of methanogenesis and methanogens by C(12) synergistically. Supplementing 30mg instead of 20mg of C(14) did not further increase the efficacy of C(12) in suppressing methane formation and methanogens. The study illustrated the advantage of using mixtures of C(12) and C(14) in ruminant nutrition to suppress methane emission since mixtures will reduce the amounts of the less palatable C(12) required in feed.

Methane-suppressing effect of myristic acid in sheep as affected by dietary calcium and forage proportion.

The efficiency of myristic acid (14 : 0) as a feed additive to suppress CH4 emissions of ruminants was evaluated under different dietary conditions. Six sheep were subjected to a 6 x 6 Latin square arrangement. A supplement of non-esterified 14 : 0 (50 g/kg DM) was added to two basal diets differing in their forage:concentrate values (1 : 1.5 and 1 : 0.5), which were adjusted to dietary Ca contents of 4.2 and 9.0 g/kg DM, respectively. Comparisons were made with the unsupplemented basal diets (4.2 g Ca/kg DM). The 14 : 0 supplementation decreased (P<0.001) total tract CH4 release depending on basal diet type (interaction, P<0.001) and dietary Ca level (P<0.05, post hoc test). In the concentrate-based diet, 14 : 0 suppressed CH4 emission by 58 and 47 % with 4.2 and 9.0 g Ca/kg DM, respectively. The 14 : 0 effect was lower (22 %) in the forage-based diet and became insignificant with additional Ca. Myristic acid inhibited (P<0.05) rumen archaea without significantly altering proportions of individual methanogen orders. Ciliate protozoa concentration was decreased (P<0.05, post hoc test) by 14 : 0 only in combination with 9.0 g Ca/kg DM. Rumen fluid NH3 concentration and acetate:propionate were decreased (P<0.05) and water consumption was lower (P<0.01) with 14 : 0. The use of 14 : 0 had no clear effects on total tract organic matter and fibre digestion; this further illustrates that the suppressed methanogenesis resulted from direct effects against methanogens. The present study demonstrated that 14 : 0 is a potent CH4 inhibitor but, to be effective in CH4 mitigation feeding strategies, interactions with other diet ingredients have to be considered.

Effect of coconut oil and defaunation treatment on methanogenesis in sheep.

The present study was conducted to evaluate in vivo the role of rumen ciliate protozoa with respect to the methane-suppressing effect of coconut oil. Three sheep were subjected to a 2 x 2 factorial design comprising two types of dietary lipids (50 g x kg(-1) coconut oil vs. 50 g x kg(-1) rumen-protected fat) and defaunation treatment (with vs. without). Due to the defaunation treatment, which reduced the rumen ciliate protozoa population by 94% on average, total tract fibre degradation was reduced but not the methane production. Feeding coconut oil significantly reduced daily methane release without negatively affecting the total tract nutrient digestion. Compared with the rumen-protected fat diet, coconut oil did not alter the energy retention of the animals. There was no interaction between coconut oil feeding and defaunation treatment in methane production. An interaction occurred in the concentration of methanogens in the rumen fluid, with the significantly highest values occurring when the animals received the coconut oil diet and were subjected to the defaunation treatment. Possible explanations for the apparent inconsistency between the amount of methane produced and the concentration of methane-producing microbes are discussed. Generally, the present data illustrate that a depression of the concentration of ciliate protozoa or methanogens in rumen fluid cannot be used as a reliable indicator for the success of a strategy to mitigate methane emission in vivo. The methane-suppressing effect of coconut oil seems to be mediated through a changed metabolic activity and/or composition of the rumen methanogenic population.