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.

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.

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.

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.

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.

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.

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.

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.

Methanogens and methanogenesis in the rumens and ceca of lambs fed two different high-grain-content diets.

The amount and nature of dietary starch are known to influence the extent and site of feed digestion in ruminants. However, how starch degradability may affect methanogenesis and methanogens along the ruminant's digestive tract is poorly understood. This study examined the diversity and metabolic activity of methanogens in the rumen and cecum of lambs receiving wheat or corn high-grain-content diets. Methane production in vivo and ex situ was also monitored. In vivo daily methane emissions (CH(4) g/day) were 36% (P < 0.05) lower in corn-fed lambs than in wheat-fed lambs. Ex situ methane production (µmol/h) was 4-fold higher for ruminal contents than for cecal contents (P < 0.01), while methanogens were 10-fold higher in the rumen than in the cecum (mcrA copy numbers; P < 0.01). Clone library analysis indicated that Methanobrevibacter was the dominant genus in both sites. Diet induced changes at the species level, as the Methanobrevibacter millerae-M. gottschalkii-M. smithii clade represented 78% of the sequences from the rumen of wheat-fed lambs and just about 52% of the sequences from the rumen of the corn-fed lambs. Diet did not affect mcrA expression in the rumen. In the cecum, however, expression was 4-fold and 2-fold lower than in the rumen for wheat- and corn-fed lambs, respectively. Though we had no direct evidence for compensation of reduced rumen methane production with higher cecum methanogenesis, the ecology of methanogens in the cecum should be better considered.

Short communication: Toxicokinetics of ochratoxin A in dairy ewes and carryover to milk following a single or long-term ingestion of contaminated feed.

Ruminal microbes have the capacity to inactivate ochratoxins, rendering ruminants less sensitive to this fungal contaminant found in cereal feeds. However, ochratoxin A has been reported in milk surveys. The objective of this study was to assess the toxicokinetics, excretion, and transmission into milk of ochratoxin A using doses similar to those of naturally occurring field contaminations. Six Lacaune dairy ewes in late lactation were separated into 2 groups that received a single dose of contaminated wheat containing 5 or 30 µg of ochratoxin A/kg of body weight. After administration, toxicokinetics and excretion were monitored for 48 h. Subsequently, ewes were administered the corresponding toxin dose daily for 24 d followed by a second toxicokinetics and excretion monitoring period for this long-term exposure. The doses used did not affect production or health of ewes. After a single dose, ochratoxin A and its main metabolite, ochratoxin α, were found in blood 1h postexposure. The maximum blood concentrations of ochratoxin A and α, respectively, were dose dependent and were observed, on average, 6 and 8h after exposure. Long-term exposure increased the maximum concentration of ochratoxin A detected in blood, whereas ochratoxin α was not affected. In contrast, the time to reach the maximum concentration was reduced to 3h for both molecules. Ochratoxins, essentially ochratoxin α, were mainly excreted in feces. Ochratoxin A and α were detected in milk at concentrations that were dose dependent but with a low carryover rate (<0.02%). Chronic administration did not increase the concentration of toxin in milk. Even though ochratoxin A can escape ruminal degradation and traces were found in milk of experimentally exposed ewes, the low carryover of ochratoxin A in milk minimizes the risk to consumers.

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.

Effectiveness of modified yeast cell wall extracts to reduce aflatoxin B1 absorption in dairy ewes.

This study investigated the effect of a modified yeast cell wall extract preparation (YCW) on the excretion of aflatoxin B1 (AFB1) and aflatoxin M1 (AFM1) in feces, urine, and milk of dairy ewes fed an aflatoxin-contaminated diet. Sixteen ewes in mid-lactation were assigned to 4 treatment groups: control, AF (60 µg of AFB1/kg of feed), YCW (2 g/kg of feed), and AF+YCW. The trial consisted of a short-term (3-d) exposure period followed by a long-term (21-d) exposure period. At the end of each exposure period, milk, urine, and feces were collected over 72 h. The treatments did not affect feed intake, milk production, milk composition, or body weight. The presence of AFM1 was detected in all matrices, whereas AFB1 was only present in feces. Daily excretion was higher following long-term exposure and reached 26.9 µg of AFB1/d in feces, 37.2 µg of AFM1/d in feces, and 10.7 µg of AFM1/d in urine. Supplementation with YCW was effective in increasing aflatoxin excretion in feces in the long-term exposure (up to 156% increase). The effect was accompanied by a trend of decreasing urinary excretion of AFM1. In contrast, the addition of YCW to the contaminated diet did not affect the transfer of aflatoxins from feed to milk under the present experimental conditions with low-producing ewes. The transfer rates of AFM1 in milk ranged from 0.24 to 0.54%. In conclusion, feed supplementation with YCW reduced the absorption of AFB1 and increased the elimination of AFB1 and AFM1 in ewe feces. Yeast cell wall extract could be used to protect ruminants from chronic exposure to aflatoxins present in feeds.

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.

Cell wall component and mycotoxin moieties involved in the binding of fumonisin B1 and B2 by lactic acid bacteria.

AIMS: The ability of lactic acid bacteria (LAB) to bind fumonisins B1 and B2 (FB1, FB2) in fermented foods and feeds and in the gastrointestinal tract could contribute to decrease their bioavailability and toxic effects on farm animals and humans. The aim of this work was to identify the bacterial cell wall component(s) and the functional group(s) of FB involved in the LAB-FB interaction. METHODS AND RESULTS: The effect of physicochemical, enzymatic and genetic treatments of bacteria and the removal/inactivation of the functional groups of FB on toxin binding were evaluated. Treatments affecting the bacterial wall polysaccharides, lipids and proteins increased binding, while those degrading peptidoglycan (PG) partially decreased it. In addition, purified PG from Gram-positive bacteria bound FB in a manner analogue to that of intact LAB. For FB, tricarballylic acid (TCA) chains play a significant role in binding as hydrolysed FB had less affinity for LAB. CONCLUSIONS: Peptidoglycan and TCA are important components of LAB and FB, respectively, involved in the binding interaction. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactic acid bacteria binding efficiency seems related to the peptide moiety structure of the PG. This information can be used to select probiotics with increased FB binding efficiency.

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.

Aflatoxin M1 and ochratoxin A in raw bulk milk from French dairy herds.

Mycotoxins in milk are a public health concern and have to be regularly monitored. A survey on the presence of aflatoxin M1 (AFM1) and ochratoxin A (OTA) in raw bulk milk was conducted in 2003 in the northwest of France, the main French milk-producing basin. Randomly selected farms (n = 132) were characterized by a diet based on corn silage and containing a large proportion of on-farm produced cereals, feeding sources that are frequently contaminated by mycotoxins. Farms were surveyed twice in winter and in summer. At each sampling time, a trained surveyor completed a questionnaire recording farm management procedures and production traits. The AFM1 was found in 3 out of 264 samples but at levels (26 ng/L or less) that are below the European legislation limit of 50 ng/L. Traces of AFM1 (less than 8 ng/L) were also found in 6 other samples. The OTA was detected in 3 samples also at low levels, 5 to 8 ng/L. Farms that tested positive to the presence of mycotoxins, 12 in total including 6 farms that had traces of AFM1, differed from negative farms by a more extensive use of total mixed rations, 58 vs. 27%. In addition, the positive farms tended to have lower milk yields. Although the incidence of milk contamination with AFM1 and OTA at the farm level was low during the period studied, production and management data from the surveyed farms suggest a link between feeding management practices and mycotoxin contamination.

Development and validation of a HPLC method for the quantitation of ochratoxins in plasma and raw milk.

A HPLC method with improved sensitivity for the determination of ochratoxins (OT) A, B and alpha in plasma and milk was developed. Plasma analysis involved a simple liquid-liquid extraction with chloroform; while for milk, an additional immunoaffinity clean-up step was necessary. The method showed a good linearity (r(2)>0.999). The limit of quantification (LOQ) of OTA was 5 and 200 ng/l for milk and plasma, respectively. Average recovery was 89% in both matrices, except for OTalpha in milk that was only 18% due to poor immunoaffinity binding. OT remained stable in -20 degrees C stored samples; OTA concentration in plasma and milk did not change after 8 and 18 months of storage, respectively. The developed method has been applied to contaminated plasma and milk samples obtained from dairy ewes fed with ochratoxin-contaminated feed.