Review: Reducing enteric methane emissions improves energy metabolism in livestock: is the tenet right?

The production of enteric methane in the gastrointestinal tract of livestock is considered as an energy loss in the equations for estimating energy metabolism in feeding systems. Therefore, the spared energy resulting from specific inhibition of methane emissions should be re-equilibrated with other factors of the equation. And, it is commonly assumed that net energy from feeds increases, thus benefitting production functions, particularly in ruminants due to the important production of methane in the rumen. Notwithstanding, we confirm in this work that inhibition of emissions in ruminants does not transpose into consistent improvements in production. Theoretical calculations of energy flows using experimental data show that the expected improvement in net energy for production is small and difficult to detect under the prevailing, moderate inhibition of methane production (≈25%) obtained using feed additives inhibiting methanogenesis. Importantly, the calculation of energy partitioning using canonical models might not be adequate when methanogenesis is inhibited. There is a lack of information on various parameters that play a role in energy partitioning and that may be affected under provoked abatement of methane. The formula used to calculate heat production based on respiratory exchanges should be validated when methanogenesis is inhibited. Also, a better understanding is needed of the effects of inhibition on fermentation products, fermentation heat, and microbial biomass. Inhibition induces the accumulation of H2, the main substrate used to produce methane, that has no energetic value for the host, and it is not extensively used by the majority of rumen microbes. Currently, the fate of this excess of H2 and its consequences on the microbiota and the host are not well known. All this additional information will provide a better account of energy transactions in ruminants when enteric methanogenesis is inhibited. Based on the available information, it is concluded that the claim that enteric methane inhibition will translate into more feed-efficient animals is not warranted.

Evaluating the effect of phenolic compounds as hydrogen acceptors when ruminal methanogenesis is inhibited in vitro - Part 1. Dairy cows.

Some antimethanogenic feed additives for ruminants promote rumen dihydrogen (H2) accumulation potentially affecting the optimal fermentation of diets. We hypothesised that combining an H2 acceptor with a methanogenesis inhibitor can decrease rumen H2 build-up and improve the production of metabolites that can be useful for the host ruminant. We performed three in vitro incubation experiments using rumen fluid from lactating Holstein cows: Experiment 1 examined the effect of phenolic compounds (phenol, catechol, resorcinol, hydroquinone, pyrogallol, phloroglucinol, and gallic acid) at 0, 2, 4, and 6 mM on ruminal fermentation for 24 h; Experiment 2 examined the combined effect of each phenolic compound from Experiment 1 at 6 mM with two different methanogenesis inhibitors (Asparagopsis taxiformis or 2-bromoethanesulfonate (BES)) for 24 h incubation; Experiment 3 examined the effect of a selected phenolic compound, phloroglucinol, with or without BES over a longer term using sequential incubations for seven days. Results from Experiment 1 showed that phenolic compounds, independently of the dose, did not negatively affect rumen fermentation, whereas results from Experiment 2 showed that phenolic compounds did not decrease H2 accumulation or modify CH4 production when methanogenesis was decreased by up to 75% by inhibitors. In Experiment 3, after three sequential incubations, phloroglucinol combined with BES decreased H2 accumulation by 72% and further inhibited CH4 production, compared to BES alone. Interestingly, supplementation with phloroglucinol (alone or in combination with the CH4 inhibitor) decreased CH4 production by 99% and the abundance of methanogenic archaea, with just a nominal increase in H2 accumulation. Supplementation of phloroglucinol also increased total volatile fatty acid (VFA), acetate, butyrate, and total gas production, and decreased ammonia concentration. This study indicates that some phenolic compounds, particularly phloroglucinol, which are naturally found in plants, could improve VFA production, decrease H2 accumulation and synergistically decrease CH4 production in the presence of antimethanogenic compounds.

Evaluating the effect of phenolic compounds as hydrogen acceptors when ruminal methanogenesis is inhibited in vitro - Part 2. Dairy goats.

Most mitigation strategies to reduce enteric methane (CH4) production in the rumen induce an excess of rumen dihydrogen (H2) that is expelled and consequently not redirected to the synthesis of metabolites that can be utilised by the ruminant. We hypothesised that phenolic compounds can be potential H2 acceptors when added to the diet, as they can be degraded to compounds that may be beneficial for the animal, using part of the H2 available when ruminal methanogenesis is inhibited. We performed four in vitro incubation experiments using rumen inoculum from Murciano-Granadina adult goats: Experiment 1 examined the inhibitory potential of Asparagopsis taxiformis (AT) at different concentrations (0, 1, 2, 3, 4 and 5% of the substrate on a DM basis) in 24 h incubations; Experiment 2 investigated the effect of a wide range of phenolic compounds (phenol, catechol, resorcinol, hydroquinone, pyrogallol, phloroglucinol, gallic acid and formic acid) at different doses (0, 2, 4, and 6 mM) on rumen fermentation for 24 h; Experiment 3 evaluated the combined effect of each phenolic compound at 6 mM with AT at 2% DM in sequential batch cultures for 5 days; and Experiment 4 examined the dose-response effect of phloroglucinol at different concentrations (0, 6, 16, 26 and 36 mM) combined with AT in sequential batch cultures for 5 days. Results from Experiment 1 confirmed that AT at 2% DM substantially inhibited CH4 production while significantly increasing H2 accumulation and decreasing the acetate:propionate ratio. Results from Experiment 2 showed that phenolic compounds did not negatively affect rumen fermentation at any dose. In Experiment 3, each phenolic compound at 6 mM combined with AT at 2% DM inhibited CH4 production. Phloroglucinol numerically decreased H2 accumulation and significantly increased total gas production (TGP), volatile fatty acid (VFA) production and the acetate:propionate ratio. In Experiment 4, phloroglucinol at increasing doses supplemented with AT at 2% DM significantly decreased H2 accumulation and the abundances of archaea, protozoa and fungi abundances, and increased TGP, total VFA production and the acetate:propionate ratio in a dose-dependent way. In conclusion, combined treatment with AT and phloroglucinol was successful to mitigate CH4 production while preventing the accumulation of H2, leading to an increase in acetate and total VFA production and therefore an improvement in rumen fermentation in goats.

Microbial colonisation of tannin-rich tropical plants: Interplay between degradability, methane production and tannin disappearance in the rumen.

Condensed tannins in plants are found free and attached to protein and fibre but it is not known whether these fractions influence rumen degradation and microbial colonisation. This study explored the rumen degradation of tropical tannin-rich plants and the relationship between the disappearance of free and bound condensed tannin fractions and microbial communities colonising plant particles using in situ and in vitro experiments. Leaves from Calliandra calothyrsus, Gliricidia sepium, and Leucaena leucocephala, pods from Acacia nilotica and the leaves of two agricultural by-products: Manihot esculenta and Musa spp. were incubated in situ in the rumen of three dairy cows to determine their degradability for up to 96 h. Tannin disappearance was determined at 24 h of incubation, and adherent microbial communities were examined at 3 and 12 h of incubation using a metataxonomic approach. An in vitro approach was also used to assess the effects of these plants on rumen fermentation parameters. All plants contained more than 100 g/kg of condensed tannins with a large proportion (32-61%) bound to proteins. Calliandra calothyrsus had the highest concentration of condensed tannins at 361 g/kg, whereas Acacia nilotica was particularly rich in hydrolysable tannins (350 g/kg). Free condensed tannins from all plants completely disappeared after 24-h incubation in the rumen. Disappearance of protein-bound condensed tannins was variable with values ranging from 93% for Gliricidia sepium to 21% for Acacia nilotica. In contrast, fibre-bound condensed tannin disappearance averaged âˆ¼ 82% and did not vary between plants. Disappearance of bound fractions of condensed tannins was not associated with the degradability of plant fractions. The presence of tannins interfered with the microbial colonisation of plants. Each plant had distinct bacterial and archaeal communities after 3 and 12 h of incubation in the rumen and distinct protozoal communities at 3 h. Adherent communities in tannin-rich plants had a lower relative abundance of fibrolytic microbes, notably Fibrobacter spp. whereas, archaea diversity was reduced in high-tannin-containing Calliandra calothyrsus and Acacia nilotica at 12 h of incubation. Concurrently, in vitro methane production was lower for Calliandra calothyrsus, Acacia nilotica and Leucaena leucocephala although for the latter total volatile fatty acids production was not affected and was similar to control. Here, we show that the total amount of hydrolysable and condensed tannins contained in a plant govern the interaction with rumen microbes affecting degradability and fermentation. The effect of protein- and fibre-bound condensed tannins on degradability is less important.

Spot urine collection: A valid alternative to total urine collection for metabolomic studies in dairy cattle.

Urine is a highly suitable biological matrix for metabolomics studies. Total collection for 24-h periods is the gold standard as it ensures the presence of all metabolites excreted throughout the day. However, in animal studies, it presents limitations related to animal welfare and also due to alterations of the metabolome originating from the use of acid for preventing microbial growth or microbial contamination. In this study, we investigated whether spot urine collection is a practical alternative to total collection for metabolomic studies in lactating cows. For this purpose, we collected urine samples from 4 lactating Holstein cows fed 4 diets in a 4 × 4 Latin square design. Urine was collected for 24 h using a collecting device (i.e., total collection) or collected once per day 4 h after the morning feeding (i.e., spot urine collection). Dietary treatments differed by the amount of nitrogen content (high vs. low) and by the nature of the energy (starch vs. fiber). Urine metabolome was analyzed by 2 untargeted complementary methods, nuclear magnetic resonance and hydrophilic-interaction liquid chromatography (HILIC) coupled to a time-of-flight mass spectrometer, and by 1 targeted method, HILIC-tandem mass spectrometry. Although sampling technique had an effect on the abundance of metabolites detected, spot urine samples were equally capable of showing differences in urine metabolome than samples from total collection. When considering nitrogen levels in the diet, the robustness and precision for discriminating high- and low-nitrogen diets was equally achieved with both sampling techniques. A total of 22 discriminant metabolites associated with the N level of diets were identified from untargeted HILIC coupled to a time-of-flight mass spectrometer (n = 9) and nuclear magnetic resonance (n = 11), and 2 from targeted HILIC-tandem mass spectrometry. Alternatively, starch or fiber in the diet induced less changes in the metabolome that were not clearly discriminated independently of the sampling technique. We concluded that spot urine collection can successfully reveal differences in the urine metabolome elicited by dietary N levels and be used as a substitute of total urinary 24-h collection for metabolomic studies.

Early life dietary intervention in dairy calves results in a long-term reduction in methane emissions.

Recent evidence suggests that changes in microbial colonization of the rumen prior to weaning may imprint the rumen microbiome and impact phenotypes later in life. We investigated how dietary manipulation from birth influences growth, methane production, and gastrointestinal microbial ecology. At birth, 18 female Holstein and Montbéliarde calves were randomly assigned to either treatment or control (CONT). Treatment was 3-nitrooxypropanol (3-NOP), an investigational anti-methanogenic compound that was administered daily from birth until three weeks post-weaning (week 14). Samples of rumen fluid and faecal content were collected at weeks 1, 4, 11, 14, 23, and 60 of life. Calves were tested for methane emissions using the GreenFeed system during the post-weaning period (week 11-23 and week 56-60 of life). Calf physiological parameters (BW, ADG and individual VFA) were similar across groups throughout the trial. Treated calves showed a persistent reduction in methane emissions (g CH4/d) throughout the post-weaning period up to at least 1 year of life, despite treatment ceasing three weeks post-weaning. Similarly, despite variability in the abundance of individual taxa across weeks, the rumen bacterial, archaeal and fungal structure differed between CONT and 3-NOP calves across all weeks, as visualised using sparse-PLS-DA. Similar separation was also observed in the faecal bacterial community. Interestingly, despite modest modifications to the abundance of rumen microbes, the reductive effect of 3-NOP on methane production persisted following cessation of the treatment period, perhaps indicating a differentiation of the ruminal microbial ecosystem or a host response triggered by the treatment in the early development phase.

Review: The application of omics to rumen microbiota function.

Rumen microbiome profiling uses 16S rRNA (18S rRNA, internal transcribed spacer) gene sequencing, a method that usually sequences a small portion of a single gene and is often biased and varies between different laboratories. Functional information can be inferred from this data, but only for those that are closely related to known annotated species, and even then may not truly reflect the function performed within the environment being studied. Genome sequencing of isolates and metagenome-assembled genomes has now reached a stage where representation of the majority of rumen bacterial genera are covered, but this still only represents a portion of rumen microbial species. The creation of a microbial genome (bins) database with associated functional annotations will provide a consistent reference to allow mapping of RNA-Seq reads for functional gene analysis from within the rumen microbiome. The integration of multiple omic analytics is linking functional gene activity, metabolic pathways and rumen metabolites with the responsible microbiota, supporting our biological understanding of the rumen system. The application of these techniques has advanced our understanding of the major microbial populations and functional pathways that are used in relation to lower methane emissions, higher feed efficiencies and responses to different feeding regimes. Continued and more precise use of these tools will lead to a detailed and comprehensive understanding of compositional and functional capacity and design of techniques for the directed intervention and manipulation of the rumen microbiota towards a desired state.

Invited review: Large-scale indirect measurements for enteric methane emissions in dairy cattle: A review of proxies and their potential for use in management and breeding decisions.

Efforts to reduce the carbon footprint of milk production through selection and management of low-emitting cows require accurate and large-scale measurements of methane (CH4) emissions from individual cows. Several techniques have been developed to measure CH4 in a research setting but most are not suitable for large-scale recording on farm. Several groups have explored proxies (i.e., indicators or indirect traits) for CH4; ideally these should be accurate, inexpensive, and amenable to being recorded individually on a large scale. This review (1) systematically describes the biological basis of current potential CH4 proxies for dairy cattle; (2) assesses the accuracy and predictive power of single proxies and determines the added value of combining proxies; (3) provides a critical evaluation of the relative merit of the main proxies in terms of their simplicity, cost, accuracy, invasiveness, and throughput; and (4) discusses their suitability as selection traits. The proxies range from simple and low-cost measurements such as body weight and high-throughput milk mid-infrared spectroscopy (MIR) to more challenging measures such as rumen morphology, rumen metabolites, or microbiome profiling. Proxies based on rumen samples are generally poor to moderately accurate predictors of CH4, and are costly and difficult to measure routinely on-farm. Proxies related to body weight or milk yield and composition, on the other hand, are relatively simple, inexpensive, and high throughput, and are easier to implement in practice. In particular, milk MIR, along with covariates such as lactation stage, are a promising option for prediction of CH4 emission in dairy cows. No single proxy was found to accurately predict CH4, and combinations of 2 or more proxies are likely to be a better solution. Combining proxies can increase the accuracy of predictions by 15 to 35%, mainly because different proxies describe independent sources of variation in CH4 and one proxy can correct for shortcomings in the other(s). The most important applications of CH4 proxies are in dairy cattle management and breeding for lower environmental impact. When breeding for traits of lower environmental impact, single or multiple proxies can be used as indirect criteria for the breeding objective, but care should be taken to avoid unfavorable correlated responses. Finally, although combinations of proxies appear to provide the most accurate estimates of CH4, the greatest limitation today is the lack of robustness in their general applicability. Future efforts should therefore be directed toward developing combinations of proxies that are robust and applicable across diverse production systems and environments.

Effects of bacterial direct-fed microbials on ruminal characteristics, methane emission, and milk fatty acid composition in cows fed high- or low-starch diets.

This study investigated the effects of bacterial direct-fed microbials (DFM) on ruminal fermentation and microbial characteristics, methane (CH4) emission, diet digestibility, and milk fatty acid (FA) composition in dairy cows fed diets formulated to induce different ruminal volatile fatty acid (VFA) profiles. Eight ruminally cannulated dairy cows were divided into 2 groups based on parity, days in milk, milk production, and body weight. Cows in each group were fed either a high-starch (38%, HS) or a low-starch (2%, LS) diet in a 55:45 forage-to-concentrate ratio on a dry matter (DM) basis. For each diet, cows were randomly assigned to 1 of 4 treatments in a Latin square design of (1) control (CON); (2) Propionibacterium P63 (P63); (3) P63 plus Lactobacillus plantarum 115 (P63+Lp); (4) P63 plus Lactobacillus rhamnosus 32 (P63+Lr). Strains of DFM were administered at 1010 cfu/d. Methane emission (using the sulfur hexafluoride tracer technique), total-tract digestibility, dry matter intake, and milk production and composition were quantified in wk 3. Ruminal fermentation and microbial characteristics were measured in wk 4. Data were analyzed using the mixed procedure of SAS (SAS Institute Inc., Cary, NC). The 2 diets induced different ruminal VFA profiles, with a greater proportion of propionate at the expense of acetate and butyrate for the HS diet. Greater concentrations of total bacteria and selected bacterial species of methanogenic Archaea were reported for the HS diet, whereas the protozoa concentration in HS decreased. For both diets, bacterial DFM supplementation raised ruminal pH (+0.18 pH units, on average) compared with CON. Irrespective of diet, P63+Lp and P63+Lr increased ruminal cellulase activity (3.8-fold, on average) compared with CON, but this effect was not associated with variations in ruminal microbial numbers. Irrespective of diet, no effect of bacterial DFM on ruminal VFA was observed. For the LS diet, supplementing cows with P63+Lr tended to decrease CH4 emission (26.5%, on average, when expressed per kilogram of milk or 4% fat-corrected milk). Only P63 supplementation to cows fed the HS diet affected the concentration of some milk FA, such as cis isomers of 18:1 and intermediates of ruminal biohydrogenation of polyunsaturated FA. Overall, bacterial DFM could be useful to stabilize ruminal pH. Their effects on CH4 production mitigation and milk FA profile depended on DFM strain and diet and should be confirmed under a greater variation of dietary conditions.

Tea saponin reduced methanogenesis in vitro but increased methane yield in lactating dairy cows.

The effect of tea saponin supplementation in the ruminant diet on methane emissions, rumen fermentation, and digestive processes is still under debate. The objective of this study was to assess the effect of this plant extract on methanogenesis, total-tract digestibility, and lactating performances of dairy cows. The work included 2 independent and successive experiments. First, the effect of 7 tea saponin doses (from 0 to 0.50 g/L) on methane emissions and protozoa concentrations was tested in 2 repeated in vitro batch culture incubations using bovine rumen contents as inoculum and a cereal mixture as substrate. After 18 h of incubation, total gas production and composition as well as rumen fermentation parameters and protozoa concentration were analyzed. Increasing dosage of the plant extract reduced methane production and protozoa concentration, with a maximum reduction of 29% for CH4 (mL/g of substrate) and 51% for protozoa (105/mL). Tea saponin did not affect volatile fatty acids concentration, but marginally decreased total gas production by 5% at the highest dose. Second, a 2-period crossover design experiment was carried out with 8 lactating dairy cows fed a basal diet (54% corn silage, 6% hay, and 40% pelleted concentrates on a dry matter basis) without (control) or with 0.52% tea saponin (TSP). Each experimental period lasted 5 wk. Animals were fed ad libitum during the first 3 wk of the period (wk 1, 2, and 3) and restricted (95% of ad libitum intake) during the last 2 wk (wk 4 and 5). Intake and milk production were recorded daily. Methane emissions were quantified using open chambers (2 d, wk 4). Total-tract digestibility and nitrogen balance were determined from total feces and urine collected separately (5 d, wk 5). Rumen fermentation parameters and protozoa concentration were analyzed from samples taken after morning feeding (1 d, wk 5). Milk production, dry matter intake, and feed efficiency were reduced with TSP (-18, -12, and -8%, respectively). As daily methane production (g/d) was not affected, methane emissions (g/kg of dry matter intake) increased by 14% with TSP. Total-tract digestibility and nitrogen balance were similar between diets, except for acid detergent fiber digestibility, which tended to be improved with TSP (+4 percentage units). Rumen fermentation parameters and protozoa concentration were relatively unchanged by diets. Under the conditions of this experiment, tea saponin is not efficient to reduce methane emissions from dairy cows.

Potential of tannin-rich plants, Leucaena leucocephala, Glyricidia sepium and Manihot esculenta, to reduce enteric methane emissions in sheep.

An in vivo trial was conducted in sheep to investigate the effect of three tropical tannin-rich plants (TRP) on methane emission, intake and digestibility. The TRP used were leaves of Glyricidia sepium, Leucaena leucocephala and Manihot esculenta that contained, respectively, 39, 75 and 92 g condensed tannins/kg DM. Methane was determined with the sulphur hexafluoride tracer technique. Eight rumen-cannulated sheep of two breeds (four Texel, four Blackbelly) were used in two 4 × 4 Latin square designs. Four experimental diets were tested. They consisted in a tropical natural grassland hay based on Dichanthium spp. fed alone (C) or in association with G. sepium (G), L. leucocephala (L) or M. esculenta (M) given as pellets at 44% of the daily ration. Daily organic matter intake was higher in TRP diets (686, 984, 1054 and 1186 g/day for C, G, L and M respectively; p < 0.05) while apparent organic matter total tract digestibility was not affected (69.9%, 62.8%, 65.3% and 64.7% for C, G, L and M respectively; p > 0.05). Methane emission was 47.1, 44.9, 33.3 and 33.5 g/kg digestible organic matter intake for C, G, L and M, respectively, and was significantly lower (p < 0.05) for L and M than for G and C. Our results confirm the potential of some TRP to reduce methane production. The strong decrease in methane and the increase in intake with TRPs may be due to their presentation as pellets.

Bioavailability of aflatoxin B1 and ochratoxin A, but not fumonisin B1 or deoxynivalenol, is increased in starch-induced low ruminal pH in nonlactating dairy cows.

High-production dairy and beef systems require diets rich in starch. This practice may induce ruminal acidosis and also increase exposure to mycotoxins because starches in starch-rich diets are the main vehicles of mycotoxin contamination. The aim of this study was to investigate the effects of low ruminal pH on the bioavailability of 4 major mycotoxins [i.e., aflatoxin B1 (AFB1), ochratoxin A (OTA), deoxynivalenol (DON), and fumonisin B1 (FB1)]. Eight nonlactating dairy cows fitted with rumen cannulas were used in a double crossover experiment. The trial was divided into 4 periods with 2 periods per crossover. Cows were divided into 2 groups receiving a low (15% dry matter basis) and high-starch diet (30.8%) with and without live yeast supplementation (1×1010 cfu per cow) in the first and second crossover, respectively. At the end of each period, cows received a single dose of mycotoxin-contaminated feed containing 0.05, 0.2, 0.24, and 0.56mg of AFB1, OTA, DON, and FB1 per kg of feed, respectively. The fecal and urinary excretion of mycotoxins and their metabolites was monitored for up to 48h postdosing. As expected, ruminal pH decreased in cows fed the high-starch diet. The high-starch diet increased the bioavailability of OTA and AFB1. Urinary excretion of OTA 24h after mycotoxin administration increased 3-fold in the high-starch diet, correlated with lower fecal excretion. Similarly, a decrease in fecal excretion of AFB1 was accompanied by an increase in urinary excretion of its major metabolite, aflatoxin M1, 48h after mycotoxin administration. In contrast to AFB1 and OTA, the bioavailability of DON and FB1 remained unchanged. Yeast supplementation had no effect on the excretion balance of these 2 mycotoxins. In conclusion, these results show that high-starch diets increased the bioavailability of OTA and AFB1, most probably through the lowering effect on ruminal pH. This greater bioavailability potentially increases the toxic effects of these mycotoxins.

Screening of bacterial direct-fed microbials for their antimethanogenic potential in vitro and assessment of their effect on ruminal fermentation and microbial profiles in sheep.

Direct-fed microbials (DFM) are used to modulate ruminal function and induce beneficial effects on ruminants. The objectives of this work were to 1) screen bacterial strains for their antimethanogenic potential in vitro and 2) assess the effect of 3 selected DFM on ruminal methane (CH) emissions, fermentation parameters, and microbial profiles in sheep. Forty-five bacterial strains were preselected based on their metabolism and fermentation characteristics. These bacteria were screened for their ability to reduce ruminal methanogenesis using 24-h batch incubations and an inoculum of 10 cfu/mL of medium. The addition of bacterial strains stimulated ruminal fermentation with increases in total gas production for 41 strains ( < 0.05) without a concomitant increase in CH production (only 9 strains had higher CH than the controls without DFM; < 0.05). 53-W, D31, and D1 had the greatest difference between total gas and CH production and were selected for further in vivo testing. Twelve rumen-cannulated Texel wethers were divided into 3 groups and were treated daily for 4 wk with 6 × 10 cfu/animal for and and 3 × 10 cfu/animal for . Measures of enteric CH, ruminal fermentation, and ruminal microbial traits were performed before, at 2 and 4 wk during the treatment period, and at 2 wk after the DFM treatment stopped. Methane production was reduced by 13% ( < 0.05) with after 2 wk of DFM administration, and this effect was maintained throughout the treatment and posttreatment periods. In contrast, had no effect on CH production, and increased it by 16% ( < 0.05) after 4 wk of DFM administration. There was no effect on other fermentation parameters or on the bacterial, archaeal, and protozoal numbers monitored by quantitative PCR. However, denaturing gradient gel electrophoresis profiles indicated changes in bacterial and archaeal diversity in the and groups. Although added bacteria were unable to permanently colonize the rumen, had a greater 24-h survival rate than the others, implying that the persistence of DFM may be important for modulating ruminal traits of interest. These results suggest that bacterial DFM used in this trial were able to modify CH emissions, although correlated changes in other ruminal parameters studied were minor.

Long-term effect of linseed plus nitrate fed to dairy cows on enteric methane emission and nitrate and nitrite residuals in milk.

A previous study showed the additive methane (CH4)-mitigating effect of nitrate and linseed fed to non-lactating cows. Before practical application, the use of this new strategy in dairy cows requires further investigation in terms of persistency of methanogenesis reduction and absence of residuals in milk products. The objective of this experiment was to study the long-term effect of linseed plus nitrate on enteric CH4 emission and performance in dairy cows. We also assessed the effect of this feeding strategy on the presence of nitrate residuals in milk products, total tract digestibility, nitrogen (N) balance and rumen fermentation. A total of 16 lactating Holstein cows were allocated to two groups in a randomised design conducted in parallel for 17 weeks. Diets were on a dry matter (DM) basis: (1) control (54% maize silage, 6% hay and 40% concentrate; CON) or (2) control plus 3.5% added fat from linseed and 1.8% nitrate (LIN+NIT). Diets were equivalent in terms of CP (16%), starch (28%) and NDF (33%), and were offered twice daily. Cows were fed ad libitum, except during weeks 5, 16 and 17 in which feed was restricted to 95% of dry matter intake (DMI) to ensure complete consumption of meals during measurement periods. Milk production and DMI were measured weekly. Nitrate and nitrite concentrations in milk and milk products were determined monthly. Daily CH4 emission was quantified in open circuit respiration chambers (weeks 5 and 16). Total tract apparent digestibility, N balance and rumen fermentation parameters were determined in week 17. Daily DMI tended to be lower with LIN+NIT from week 4 to 16 (-5.1 kg/day on average). The LIN+NIT diet decreased milk production during 6 non-consecutive weeks (-2.5 kg/day on average). Nitrate or nitrite residuals were not detected in milk and associated products. The LIN+NIT diet reduced CH4 emission to a similar extent at the beginning and end of the trial (-47%, g/day; -30%, g/kg DMI; -33%, g/kg fat- and protein-corrected milk, on average). Diets did not affect N efficiency and nutrients digestibility. In the rumen, LIN+NIT did not affect protozoa number but reduced total volatile fatty acid (-12%) and propionate (-31%) concentrations. We concluded that linseed plus nitrate may have a long-term CH4-mitigating effect in dairy cows and that consuming milk products from cows fed nitrate may be safe in terms of nitrate and nitrite residuals. Further work is required to optimise the doses of linseed plus nitrate to avoid reduced cows performance.

Nitrate but not tea saponin feed additives decreased enteric methane emissions in nonlactating cows.

Tea saponin is considered a promising natural compound for reducing enteric methane emissions in ruminants. A trial was conducted to study the effect of this plant extract fed alone or in combination with nitrate on methane emissions, total tract digestive processes, and ruminal characteristics in cattle. The experiment was conducted as a 2 × 2 factorial design with 4 ruminally cannulated nonlactating dairy cows. Feed offer was restricted to 90% of voluntary intake and diets consisted of (DM basis): 1) control (CON; 50% hay and 50% pelleted concentrates), 2) CON with 0.5% tea saponin (TEA), 3) CON with 2.3% nitrate (NIT), and 4) CON with 0.5% tea saponin and 2.3% nitrate (TEA+NIT). Tea saponin and nitrate were included in pelleted concentrates. Diets contained similar amounts of CP (12.2%), starch (26.0%), and NDF (40.1%). Experimental periods lasted 5 wk including 2 wk of measurement (wk 4 and 5), during which intake was measured daily. In wk 4, daily methane emissions were quantified for 4 d using open circuit respiratory chambers. In wk 5, total tract digestibility, N balance, and urinary excretion of purine derivatives were determined from total feces and urine collected separately for 6 d. Ruminal fermentation products and protozoa concentration were analyzed from samples taken after morning feeding for 2 nonconsecutive days in wk 5. Tea saponin and nitrate supplementation decreased feed intake ( < 0.05), with an additive effect when fed in combination. Compared with CON, tea saponin did not modify methane emissions (g/kg DMI; > 0.05), whereas nitrate-containing diets (NIT and TEA+NIT) decreased methanogenesis by 28%, on average ( < 0.001). Total tract digestibility, N balance, and urinary excretion of purine derivatives were similar among diets. Ruminal fermentation products were not affected by tea saponin, whereas nitrate-containing diets increased acetate proportion and decreased butyrate proportion and ammonia concentration ( < 0.05). Under the experimental conditions tested, we confirmed the antimethanogenic effect of nitrate, whereas tea saponin alone included in pelleted concentrates failed to decrease enteric methane emissions in nonlactating dairy cows.

Additive methane-mitigating effect between linseed oil and nitrate fed to cattle.

The objective of this study was to test the effect of linseed oil and nitrate fed alone or in combination on methane (CH4) emissions and diet digestibility in cows. The experiment was conducted as a 2 × 2 factorial design using 4 multiparous nonlactating Holstein cows (initial BW 656 ± 31 kg). Each experimental period lasted 5 wk, with measures performed in the final 3 wk (wk 3 to 5). Diets given on a DM basis were 1) control (CON; 50% natural grassland hay and 50% concentrate), 2) CON with 4% linseed oil (LIN), 3) CON with 3% calcium nitrate (NIT), and 4) CON with 4% linseed oil plus 3% calcium nitrate (LIN+NIT). Diets were offered twice daily and were formulated to deliver similar amounts (DM basis) of CP (12.2%), starch (25.5%), and NDF (39.5%). Feed offer was restricted to 90% of voluntary intake (12.4 kg DMI/d). Total tract digestibility and N balance were determined from total feces and urine collected separately for 6 d during wk 4. Daily CH4 emissions were quantified using open chambers for 4 d during wk 5. Rumen fermentation and microbial parameters were analyzed from samples taken before and 3 h after the morning feeding. Rumen concentrations of dissolved hydrogen (H2) were measured continuously up to 6 h after feeding using a H2 sensor. Compared with the CON diet linseed oil and nitrate decreased (P < 0.01) CH4 emissions (g/kg DMI) by 17 and 22%, respectively, when fed alone and by 32% when combined. The LIN diet reduced CH4 production throughout the day, increased (P = 0.02) propionate proportion, and decreased (P = 0.03) ruminal protozoa concentration compared with CON diet. The NIT diet strongly reduced CH4 production 3 h after feeding, with a simultaneous increase in rumen dissolved H2 concentration, suggesting that nitrate does not act only as an electron acceptor. As a combined effect, linseed plus nitrate also increased H2 concentrations in the rumen. Diets had no effect (P > 0.05) on total tract digestibility of nutrients, except linseed oil, which tended to reduce (P < 0.10) fiber digestibility. Nitrogen balance (% of N intake) was positive for all diets but retention was less (P = 0.03) with linseed oil. This study demonstrates an additive effect between nitrate and linseed oil for reducing methanogenesis in cows without altering diet digestibility.

Presence of aflatoxin M1 in raw, reconstituted, and powdered milk samples collected in Algeria.

Aflatoxins are potent toxic metabolites produced by Aspergillus spp. Aflatoxin M1 (AFM1) is a metabolite of aflatoxin B1 that can be present in milk, and it is a public health concern. There is scarce information on the incidence of aflatoxin M1 contamination in milk consumed in Algeria. The presence of AFM1 was investigated in raw milk samples collected between February and October 2011 from 11 dairy farms representative of Algerian production conditions and that were located around Constantine city. Reconstituted and powdered milk samples were purchased from local supermarkets. The analysis was performed by liquid chromatography-fluorescence detection after immunoaffinity purification. AFM1 was detected in 5 out of 47 samples (11 %) at levels ranging from 9 to 103 ng/L, with one sample exceeding the limit of 50 ng/L set by European regulations. Traces of AFM1 (less than 8 ng/L) were also found in 11 other samples. The incidence of AFM1 contamination was higher in imported powdered milk (29 %) than in raw milk (5 %). Although the concentration of AFM1 in contaminated samples was low, the relatively considerable prevalence found in this exploratory study justifies more detailed and continuous monitoring to reduce consumers' exposure to AFM1.

Potential of tannin-rich plants for modulating ruminal microbes and ruminal fermentation in sheep.

The objective of this work was to study nutritional strategies for decreasing methane production by ruminants fed tropical diets, combining in vitro and in vivo methods. The in vitro approach was used to evaluate the dose effect of condensed tannins (CT) contained in leaves of Gliricidia sepium, Leucaena leucocephala, and Manihot esculenta (39, 75, and 92 g CT/kg DM, respectively) on methane production and ruminal fermentation characteristics. Tannin-rich plants (TRP) were incubated for 24 h alone or mixed with a natural grassland hay based on Dichanthium spp. (control plant), so that proportions of TRP were 0, 0.25, 0.5, 0.75, and 1.0. Methane production, VFA concentration, and fermented OM decreased with increased proportions of TRP. Numerical differences on methane production and VFA concentration among TRP sources may be due to differences in their CT content, with greater effects for L. leucocephala and M. esculenta than for G. sepium. Independently of TRP, the response to increasing doses of CT was linear for methane production but quadratic for VFA concentration. As a result, at moderate tannin dose, methane decreased more than VFA. The in vivo trial was conducted to investigate the effect of TRP on different ruminal microbial populations. To this end, 8 rumen-cannulated sheep from 2 breeds (Texel and Blackbelly) were used in two 4 × 4 Latin square designs. Diets were fed ad libitum and were composed of the same feeds used for the in vitro trial: control plant alone or combined with pellets made from TRP leaves at 44% of the diet DM. Compared to TRP, concentration of Ruminococcus flavefaciens was greater for the control diet and concentration of Ruminococcus albus was least for the control diet. The methanogen population was greater for Texel than for Blackbelly. By contrast, TRP-containing diets did not affect protozoa or Fibrobacter succinogenes numbers. Hence, TRP showed potential for mitigating methane production by ruminants. These findings suggest that TRP fed as pellets could be used to decrease methane production.

Influence of rumen protozoa on methane emission in ruminants: a meta-analysis approach.

A meta-analysis was conducted to evaluate the effects of protozoa concentration on methane emission from ruminants. A database was built from 59 publications reporting data from 76 in vivo experiments. The experiments included in the database recorded methane production and rumen protozoa concentration measured on the same groups of animals. Quantitative data such as diet chemical composition, rumen fermentation and microbial parameters, and qualitative information such as methane mitigation strategies were also collected. In the database, 31% of the experiments reported a concomitant reduction of both protozoa concentration and methane emission (g/kg dry matter intake). Nearly all of these experiments tested lipids as methane mitigation strategies. By contrast, 21% of the experiments reported a variation in methane emission without changes in protozoa numbers, indicating that methanogenesis is also regulated by other mechanisms not involving protozoa. Experiments that used chemical compounds as an antimethanogenic treatment belonged to this group. The relationship between methane emission and protozoa concentration was studied with a variance-covariance model, with experiment as a fixed effect. The experiments included in the analysis had a within-experiment variation of protozoa concentration higher than 5.3 log10 cells/ml corresponding to the average s.e.m. of the database for this variable. To detect potential interfering factors for the relationship, the influence of several qualitative and quantitative secondary factors was tested. This meta-analysis showed a significant linear relationship between methane emission and protozoa concentration: methane (g/kg dry matter intake)=-30.7+8.14×protozoa (log10 cells/ml) with 28 experiments (91 treatments), residual mean square error=1.94 and adjusted R 2=0.90. The proportion of butyrate in the rumen positively influenced the least square means of this relationship.

Amylase addition increases starch ruminal digestion in first-lactation cows fed high and low starch diets.

The objective of this study was to evaluate the effect of an exogenous amylase preparation on digestion of low- and high-starch diets in dairy cattle. Rumen and total-tract nutrient digestibility were measured in a 4×4 Latin square design with 28-d periods using 4 first-lactation cows cannulated at the rumen and duodenum. Corn silage-based diets had 20 or 30% starch, attained by changing the composition of concentrate, with or without addition of an exogenous amylase preparation. Effects of the enzyme additive were observed on ruminal digestibility but not at the total-tract level. Ruminal digestibility of starch increased from 75% in control to 81% with amylase supplementation. This difference in ruminal starch digestion was compensated postruminally, so that the total-tract digestibility of starch was almost complete and did not differ between treatments. The amylase supplement also increased the true ruminal digestibility of organic matter but did not affect microbial N flow to the duodenum. Amylase supplement reduced the proportion of acetate and butyrate and increased that of propionate, particularly in the high-starch diet, where it tended to increase the concentration of total volatile fatty acids in the rumen. Other effects were a higher amylase activity in the solid-associated microbial community and a tendency for lower numbers of protozoa. In contrast, we observed no changes in intake, production, dry matter and fiber (neutral detergent fiber and acid detergent fiber) digestibility, or ruminal digestion, and no or small changes on selected fibrolytic and amylolytic bacteria and on the microbial community in general. We conclude that the exogenous amylase improved starch digestion in the rumen in first-lactation cows with moderate intake and production levels.