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This study aimed (1) to provide estimates of total mean retention times of milk replacer (MR), concentrates, and roughage in veal calves fed a mixed diet; (2) to determine the effect of level and type of solid feed (SF) on passage kinetics of MR, concentrates, and roughages in veal calves; and (3) to compare passage kinetics in veal calves using the fecal excretion curves of indigestible markers and a noninvasive 13C tracer breath test approach to determine whether the latter technique can serve as an alternative. At the start of the trial, 48 Holstein-Friesian calves (6 wk of age; 68 ± 7.7 kg of body weight; BW) were assigned to 1 of 4 dietary treatments (for statistical analysis, only 39 calf observations were used). Three treatments contained chopped wheat straw as roughage in the SF mixture in a concentrate:roughage ratio of 90:10 (dry matter basis). The SF level was 20 g/kg of metabolic BW per day (low straw), 30 g/kg of metabolic BW per day (middle straw), or 40 g/kg of metabolic BW per day (high straw). The fourth treatment (high hay) contained long perennial ryegrass hay as roughage in the SF mixture in a concentrate:roughage ratio of 70:30 (dry matter basis, at 40 g/kg of metabolic BW per day). The quantity of MR was fixed for the high straw treatment, whereas the amount of MR for the other treatments during the adaptation period was adjusted based on a pair gain strategy (i.e., exchanging ration components but keeping similar net energy). At the end of the adaptation period, calves ranged from 12 to 15 wk of age with an average BW of 123 ± 8.6 kg. Passage kinetics of concentrates were estimated by measuring 13C enrichment excess of CO2 in breath from a pulsed-dose of [1-13C]octanoate. Passage kinetics of roughage, concentrates, and MR were also estimated using fecal excretion curves obtained after ingestion of chromium-mordanted roughage, Yb2O3, and Co-EDTA, respectively. We conclude that [1-13C]octanoate cannot serve as a measure for oro-duodenal transit of concentrates because of unrealistic estimates. Based on the fecal excretion curves, we concluded that the total mean retention time of MR (i.e., time to peak; the moment that the excretion curve reaches peak concentration) was, on average, 12.4 h, and that the passage kinetics of MR was not affected by the level or type of SF. The mean retention time of concentrates was shorter (21.4 h) than that of both straw (59.1 h) and hay (36.8 h), and was not affected by the level or type of SF. Also, the mean retention time of the slowest compartment (i.e., the rumen) was shorter for concentrates (39.6 h) than that of straw (110.0 h) and hay (59.2 h). Contrary, the passage of roughage was affected by level and type of SF. Long hay increased time to peak by 22.3 h and decreased ruminal mean retention time by 50.8 h relative to chopped straw, indicating that the passage rate of long hay is faster than that of chopped straw. We conclude that the level and type of SF only affects the passage kinetics of roughage and not that of MR and concentrates.

The objective of this study was to investigate whether the CH4 mitigation potential of 3-nitrooxypropanol (3-NOP) in dairy cattle was affected by basal diet (BD) composition. The experiment involved 64 Holstein-Friesian dairy cows (146 ± 45 d in milk at the start of trial; mean ± SD) in 2 overlapping crossover trials, each consisting of 2 measurement periods. Cows were blocked according to parity, d in milk, and milk yield, and randomly allocated to 1 of 3 diets: a grass silage-based diet (GS) consisting of 30% concentrates and 70% grass silage (DM basis), a grass silage- and corn silage-mixed diet (GSCS) consisting of 30% concentrates, 42% grass silage, and 28% corn silage (DM basis), or a corn silage-based diet (CS) consisting of 30% concentrates, 14% grass silage, and 56% corn silage (DM basis). Two types of concentrates were formulated, viz. a concentrate for the GS diet and a concentrate for the CS diet, to meet the energy and protein requirements for maintenance and milk production. The concentrate for the GSCS diet consisted of a 50:50 mixture of both concentrates. Subsequently, the cows within each type of BD received 2 treatments in a crossover design: either 60 mg of 3-NOP/kg of DM (NOP60) and a placebo with 0 mg of 3-NOP/kg of DM (NOP0) in one crossover or 80 mg of 3-NOP/kg of DM (NOP80) and NOP0 in the other crossover. Diets were provided as total mixed ration in feed bins, which automatically recorded feed intake. Additional concentrate was fed in the GreenFeed system that was used to measure emissions of CH4 and H2. The CS diets resulted in a reduced CH4 yield (g/kg DMI) and CH4 intensity (g/kg milk). Feeding 3-NOP resulted in a decreased DMI. Milk production and composition did not differ between NOP60 and NOP0, whereas milk yield and the yield of major components decreased for NOP80 compared with NOP0. Feed efficiency was not affected by feeding 3-NOP. Interactions between BD and supplementation of 3-NOP were observed for the production (g/d) and yield (g/kg DMI) of both CH4 and H2, indicating that the mitigating effect of 3-NOP depended on the composition of the BD. Emissions of CH4 decreased upon 3-NOP supplementation for all BD, but the decrease in CH4 emissions was smaller for GS (−26.2% for NOP60 and −28.4% for NOP80 in CH4 yield) compared with both GSCS (−35.1% for NOP60 and −37.9% for NOP80 for CH4 yield) and CS (−34.8% for NOP60 and −41.6% for NOP80 for CH4 yield), with no difference between the latter 2 BD. Emissions of H2 increased upon 3-NOP supplementation for all BD, but the H2 yield (g/kg DMI) increased 3.16 and 3.30-fold, respectively, when NOP60 and NOP80 were supplemented to GS, and 4.70 and 4.96 fold, respectively, when NOP60 and NOP80 were supplemented to CS. In conclusion, 3-NOP can effectively decrease CH4 emissions in dairy cows across diets, but the level of CH4 mitigation is greater when supplemented in a corn silage-based diet compared with a grass silage-based diet.

Next to rumen acidosis, other forms of acidosis may also affect lactational performance of cows. Therefore, the effects of hindgut acidosis, induced via abomasal infusion of ground corn, and metabolic acidosis, induced via abomasal infusion of NH4Cl, were studied in cows in early lactation. Observations were made on intake and digestibility of nutrients, lactation performance, energy and N partitioning, blood acid-base status, and rumen and hindgut fermentation characteristics. In a 6 × 6 Latin square design, 6 rumen-fistulated, second-lactation Holstein-Friesian dairy cows (48 ± 17 d in milk) were subjected to 5 d of continuous abomasal infusions of water as control, or solutions of 2.5 mol of NH4Cl/d, 5.0 mol of NH4Cl/d, 3.0 kg of ground corn/d, or the combination of ground corn with either of the 2 NH4Cl levels, followed by 2 d of rest. Treatment solutions were administered via peristaltic pumps through infusion lines attached to the rumen cannula plug and an abomasal infusion line with a flexible disk (equipped with holes to allow digesta passage) to secure its placement through the sulcus omasi. A total mixed ration consisting of 70% grass silage and 30% concentrate (on dry matter basis) was fed at 95% of ad libitum intake of individual cows. The experiment was conducted in climate respiration chambers to determine feed intake, lactation performance, and energy and N balance. Abomasal infusion of NH4Cl affected the acid-base status of the cows, but more strongly when in combination with abomasal infusion of ground corn. Metabolic acidosis (defined as a blood pH < 7.40, blood HCO3 concentration < 25.0 mmol/L, and a negative base excess) was observed with 5.0 mol of NH4Cl/d, 3.0 kg of ground corn/d + 2.5 mol of NH4Cl/d, and 3.0 kg of ground corn/d + 5.0 mol of NH4Cl/d. Metabolic acidosis was associated with decreased milk lactose content, metabolic body weight, energy retained as protein, and fecal N excretion, and increased urine N excretion, and tended to decrease intake of nutrients. Digestibility of several nutrients increased with 5.0 mol of NH4Cl/d, likely as a result of decreased intake. Abomasal ground corn infusion resulted in hindgut acidosis, where fecal pH decreased from 6.86 without ground corn to 6.00 with ground corn, regardless of NH4Cl level. The decrease in fecal pH was likely the result of increased hindgut fermentation, evidenced by increased fecal volatile fatty acid concentrations. Hindgut acidosis was associated with decreased digestibility of nutrients, except for starch, which increased, and crude fat, which was not affected. No systemic inflammatory response was observed, suggesting that the hindgut epithelium was not severely affected by the more acidic conditions or barrier damage. Abomasal infusion of ground corn increased milk yield, milk protein and lactose yield, fecal N excretion, N use efficiency, and total energy retained as well as energy retained in fat, and reduced milk fat content and urine N excretion.

The aim of this study was to determine the methane (CH4) mitigation potential of 3-nitrooxypropanol and the persistency of its effect when fed to dairy cows in early lactation. Sixteen Holstein-Friesian cows (all multiparous; 11 cows in their second parity and 5 cows in their third parity) were blocked in pairs, based on actual calving date, parity, and previous lactation milk yield, and randomly allocated to 1 of 2 dietary treatments: a diet including 51 mg of 3-nitrooxypropanol/kg of dry matter (3-NOP) and a diet including a placebo at the same concentration (CON). Cows were fed a 35% grass silage, 25% corn silage, and 40% concentrate (on dry matter basis) diet from 3 d after calving up to 115 d in milk (DIM). Every 4 weeks, the cows were housed in climate respiration chambers for 5 d to measure lactation performance, feed and nutrient intake, apparent total-tract digestibility of nutrients, energy and N metabolism, and gaseous exchange (4 chamber visits per cow in total, representing 27, 55, 83, and 111 DIM). Feeding 3-NOP did not affect dry matter intake (DMI), milk yield, milk component yield, or feed efficiency. These variables were affected by stage of lactation, following the expected pattern of advanced lactation. Feeding 3-NOP did not affect CH4 production (g/d) at 27 and 83 DIM, but decreased CH4 production at 55 and 111 DIM by an average of 18.5%. This response in CH4 production is most likely due to the differences observed in feed intake across the different stages of lactation because CH4 yield (g/kg of DMI) was lower (on average 16%) at each stage of lactation upon feeding 3-NOP. On average, feeding 3-NOP increased H2 production and intensity 12-fold; with the control diet, H2 yield did not differ between the different stages of lactation, whereas with the 3-NOP treatment H2 yield decreased from 0.429 g/kg of DMI at 27 DIM to 0.387 g/kg of DMI at 111 DIM. The apparent total-tract digestibility of dry matter, organic matter, neutral detergent fiber, and gross energy was greater for the 3-NOP treatment. In comparison to the control treatment, 3-NOP did not affect energy and N balance, except for a greater metabolizable energy intake to gross energy intake ratio (65.4 and 63.7%, respectively) and a greater body weight gain (average 0.90 and 0.01% body weight change, respectively). In conclusion, feeding 3-NOP is an effective strategy to decrease CH4 emissions (while increasing H2 emission) in early lactation Holstein-Friesian cows with positive effects on apparent total-tract digestibility of nutrients.

The digestive physiology of ruminants is sufficiently different (e.g., with respect to mean retention time of digesta, digestibility of the feed offered, digestion, and fermentation characteristics)that caution is needed before extrapolating results from one type of ruminant to another. The objectives of the present study were (1)to provide an overview of some essential differences in rumen physiology between dairy cattle, beef cattle, and sheep that are related to methane (CH 4 )emission; and (2)to evaluate whether dietary strategies to mitigate CH 4 emission with various modes of action are equally effective in dairy cattle, beef cattle, and sheep. A literature search was performed using Web of Science and Scopus, and 94 studies were selected from the literature. Per study, the effect size of the dietary strategies was expressed as a proportion (%)of the control level of CH 4 emission, as this enabled a comparison across ruminant types. Evaluation of the literature indicated that the effectiveness of forage-related CH 4 mitigation strategies, including feeding more highly digestible grass (herbage or silage)or replacing different forage types with corn silage, differs across ruminant types. These strategies are most effective for dairy cattle, are effective for beef cattle to a certain extent, but seem to have minor or no effects in sheep. In general, the effectiveness of other dietary mitigation strategies, including increased concentrate feeding and feed additives (e.g., nitrate), appeared to be similar for dairy cattle, beef cattle, and sheep. We concluded that if the mode of action of a dietary CH 4 mitigation strategy is related to ruminant-specific factors, such as feed intake or rumen physiology, the effectiveness of the strategy differs across ruminant types, whereas if the mode of action is associated with methanogenesis-related fermentation pathways, the strategy is effective across ruminant types. Hence, caution is needed when translating effectiveness of dietary CH 4 mitigation strategies across different ruminant types or production systems.

This review evaluates the effects of silage characteristics on enteric methane (CH 4 ) emission from ruminants by performing a meta-analysis. A total of 17 studies were selected from the literature, and the relationship between CH 4 yield (g/kg dry matter [DM] intake) and silage characteristics was determined using a mixed model univariate regression procedure. For grass silage, organic matter digestibility (%; R 2 =0.74) and crude protein content (g/kg DM; R 2 =0.36) were negatively associated, and neutral detergent fibre (NDF) content (g/kg DM; R 2 =0.44) was positively associated with CH 4 yield. This indicates that increased grass silage quality consistently decreases CH 4 yield and may be an effective enteric CH 4 mitigation strategy. Similarly, optimizing maize silage quality appears to be an effective enteric CH 4 mitigation strategy, because NDF content (g/kg DM; R 2 =0.60) was positively associated with CH 4 yield. Upon replacing grass silage with maize silage, dietary starch content (g/kg DM; R 2 =0.62) and silage replacement level (%; R 2 =0.33) were negatively associated, and NDF content (g/kg DM; R 2 =0.34) was positively associated with CH 4 yield. These results indicate that replacing grass silage with maize silage consistently decreases CH 4 yield and may be an effective enteric CH 4 mitigation strategy. In contrast, replacement of alfalfa silage with maize silage was not associated with CH 4 yield and does not appear to be an effective strategy to decrease CH 4 yield. In conclusion, management practices to improve silage quality are a potent mitigation strategy to reduce enteric CH 4 emission per unit of feed fed to ruminants, and implications of silage quality have to be addressed when assessing greenhouse gas emissions in ruminant production.

DNA based methods have been widely used to study the complexity of the rumen microbiota, and it is well known that the method of DNA extraction is a critical step in enabling accurate assessment of this complexity. Rumen fluid (RF) and fibrous content (FC) fractions differ substantially in terms of their physical nature and associated microorganisms. The aim of this study was therefore to assess the effect of four DNA extraction methods (RBB, PBB, FDSS, PQIAmini) differing in cell lysis and/or DNA recovery methods on the observed microbial diversity in RF and FC fractions using samples from four rumen cannulated dairy cows fed 100% grass silage (GS100), 67% GS and 33% maize silage (GS67MS33), 33% GS and 67% MS (GS33MS67), or 100% MS (MS100). An ANOVA statistical test was applied on DNA quality and yield measurements, and it was found that the DNA yield was significantly affected by extraction method (p < 0.001) and fraction (p < 0.001). The 260/280 ratio was not affected by extraction (p = 0.08) but was affected by fraction (p = 0.03). On the other hand, the 260/230 ratio was affected by extraction method (p < 0.001) but not affected by fraction (p = 0.8). However, all four extraction procedures yielded DNA suitable for further analysis of bacterial, archaeal and anaerobic fungal communities using quantitative PCR and pyrosequencing of relevant taxonomic markers. Redundancy analysis (RDA) of bacterial 16S rRNA gene sequence data at the family level showed that there was a significant effect of rumen fraction (p = 0.012), and that PBB (p = 0.012) and FDSS (p = 0.024) also significantly contributed to explaining the observed variation in bacterial community composition. Whilst the DNA extraction method affected the apparent bacterial community composition, no single extraction method could be concluded to be ineffective. No obvious effect of DNA extraction method on the anaerobic fungi or archaea was observed, although fraction effects were evident for both. In summary, the comprehensive assessment of observed communities of bacteria, archaea and anaerobic fungi described here provides insight into a rational basis for selecting an optimal methodology to obtain a representative picture of the rumen microbiota.

The objective of the present study was to characterize the rumen bacterial and archaeal communities in dairy cows fed different ratios of maize silage (MS) and grass silage (GS), and place the findings in the context of ruminal fermentation as well as previously reported methane (CH4) emissions. Rumen fluid from 12 rumen cannulated dairy cows was collected after 10 and 17 days of feeding one of four diets, all of which had the same roughage to concentrate ratio of 80:20 based on dry matter (DM). Roughage in the four diets (GS100, GS0, GS67, GS33) consisted of either 1000 g/kg DM GS (GS100), 1000 g/kg DM MS (GS0), or a mixture of both silages in different proportions [667 g/kg DM GS and 333 g/kg DM MS (GS67); 333 g/kg DM GS and 677 g/kg DM MS (GS33)]. Total volatile fatty acid (VFA) concentrations and the molar proportions of the ruminal VFA were not affected by diet. Only the molar proportion of isovalerate was affected by time, being lower on day 17 than on day 10. Bacterial and archaeal concentrations were not affected by diet but increased from day 10 to day 17. The bacterial community composition was affected by diet, time and diet × time, whereas the archaeal community composition was only affected by diet. Several bacterial and archaeal genus level groups were associated with diet, but not with time. Analysis indicated the increased use of hydrogen by succinate and lactate producing bacteria is likely to at least partially explain the previously reported lower CH4 emissions from MS fed dairy cows. Furthermore, time had a significant effect on both bacterial and archaeal concentrations, and also bacterial community composition. This indicates that the rumen microbiota had not stabilized after 10 days of feeding the experimental diets.

Diurnal patterns of ruminal fermentation metabolites and microbial communities are not commonly assessed when investigating variation in ruminal CH4 production. The aims of this study were to monitor diurnal patterns of: (i) gaseous and dissolved metabolite concentrations in the bovine rumen, (ii) H2 and CH4 emitted, and (iii) the rumen microbiota. Furthermore, the effect of dietary inclusion of linseed oil on these patterns was assessed. Four rumen cannulated multiparous cows were used in a cross-over design with two 17 days periods and two dietary treatments: a control diet and a linseed oil supplemented diet [40% maize silage, 30% grass silage, 30% concentrate on dry matter (DM) basis for both diets; fat contents of 33 vs. 56 g/kg of DM]. On day 11, rumen contents were sampled for 10 h after morning feeding to profile gaseous and dissolved metabolite concentrations and microbiota composition. H2 and CH4 emission (mass per unit of time) was measured in respiration chambers from day 13 to 17. A 100-fold increase in ruminal H2 partial pressure (contribution to the total pressure of rumen headspace gases) was observed at 0.5 h after feeding. This peak was followed by a decline to basal level. Qualitatively similar patterns after feeding were also observed for H2 and CH4 emission, ethanol and lactate concentrations, and propionate molar proportion, although the opposite pattern was seen for acetate molar proportion. Associated with these patterns, a temporal biphasic change in the microbial composition was observed as based on 16S ribosomal RNA with certain taxa specifically associated with each phase. Bacterial concentrations (log10 16S ribosomal RNA gene copies based) were affected by time, and were increased by linseed oil supplementation. Archaeal concentrations (log10 16S ribosomal RNA gene copies based) tended to be affected by time and were not affected by diet, despite linseed oil supplementation decreasing CH4 emission, tending to decrease the partial pressure of CH4, and tending to increase propionate molar proportion. Linseed oil supplementation affected microbiota composition, and was most associated with an uncultivated Bacteroidales taxon. In summary, our findings support the importance of diurnal dynamics for the understanding of VFA, H2, and CH4 production.