The objective of this study was to determine the effects of replacing grass silage (GS) with corn silage (CS) in dairy cow diets on enteric methane (CH4) production, rumen volatile fatty acid concentrations, and milk fatty acid (FA) composition. A completely randomized block design experiment was conducted with 32 multiparous lactating Holstein-Friesian cows. Four dietary treatments were used, all having a roughage-to-concentrate ratio of 80:20 based on dry matter (DM). The roughage consisted of either 100% GS, 67% GS and 33% CS, 33% GS and 67% CS, or 100% CS (all DM basis). Feed intake was restricted (95% of ad libitum DM intake) to avoid confounding effects of DM intake on CH4 production. Nutrient intake, apparent digestibility, milk production and composition, nitrogen (N) and energy balance, and CH4 production were measured during a 5-d period in climate respiration chambers after adaptation to the diet for 12 d. Increasing CS proportion linearly decreased neutral detergent fiber and crude protein intake and linearly increased starch intake. Milk production and milk fat content (on average 23.4 kg/d and 4.68%, respectively) were not affected by increasing CS inclusion, whereas milk protein content increased quadratically. Rumen variables were unaffected by increasing CS inclusion, except the molar proportion of butyrate, which increased linearly. Methane production (expressed as grams per day, grams per kilogram of fat- and protein-corrected milk, and as a percent of gross energy intake) decreased quadratically with increasing CS inclusion, and decreased linearly when expressed as grams of CH4 per kilogram of DM intake. In comparison with 100% GS, CH4 production was 11 and 8% reduced for the 100% CS diet when expressed per unit of DM intake and per unit fat- and protein-corrected milk, respectively. Nitrogen efficiency increased linearly with increased inclusion of CS. The concentration of trans C18:1 FA, C18:1 cis-12, and total CLA increased quadratically, and iso C16:0, C18:1 cis-13, and C18:2n-6 increased linearly, whereas the concentration of C15:0, iso C15:0, C17:0, and C18:3n-3 decreased linearly with increasing inclusion of CS. No differences were found in short- and medium-straight, even-chain FA concentrations, with the exception of C4:0 which increased linearly with increased inclusion of CS. Replacing GS with CS in a common forage-based diet for dairy cattle offers an effective strategy to decrease enteric CH4 production without negatively affecting dairy cow performance, although a critical level of starch in the diet seems to be needed.

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3-Nitrooxypropanol (NOP) is a promising methane (CH) inhibitor. Recent studies have shown major reductions in CH emissions from beef and dairy cattle when using NOP but with large variation in response. The objective of this study was to quantitatively evaluate the factors that explain heterogeneity in response to NOP using meta-analytical approaches. Data from 11 experiments and 38 treatment means were used. Factors considered were cattle type (dairy or beef), measurement technique (GreenFeed technique, C-Lock Inc., Rapid City, SD; sulfur hexafluoride tracer technique; and respiration chamber technique), dry matter (DM) intake, body weight, NOP dose, roughage proportion, dietary crude protein content, and dietary neutral detergent fiber (NDF) content. The mean difference (MD) in CH production (g/d) and CH yield (g/kg of DM intake) was calculated by subtracting the mean of CH emission for the control group from that of the NOP-supplemented group. Forest plots of standardized MD indicated variable effect sizes of NOP across studies. Compared with beef cattle, dairy cattle had a much larger feed intake (22.3 ± 4.13 vs. 7.3 ± 0.97 kg of DM/d; mean ± standard deviation) and CH production (351 ± 94.1 vs. 124 ± 44.8 g/d). Therefore, in further analyses across dairy and beef cattle studies, MD was expressed as a proportion (%) of observed control mean. The final mixed-effect model for relative MD in CH production included cattle type, NOP dose, and NDF content. When adjusted for NOP dose and NDF content, the CH-mitigating effect of NOP was less in beef cattle (-22.2 ± 3.33%) than in dairy cattle (-39.0 ± 5.40%). An increase of 10 mg/kg of DM in NOP dose from its mean (123 mg/kg of DM) enhanced the NOP effect on CH production decline by 2.56 ± 0.550%. However, a greater dietary NDF content impaired the NOP effect on CH production by 1.64 ± 0.330% per 10 g/kg DM increase in NDF content from its mean (331 g of NDF/kg of DM). The factors included in the final mixed-effect model for CH yield were -17.1 ± 4.23% (beef cattle) and -38.8 ± 5.49% (dairy cattle), -2.48 ± 0.734% per 10 mg/kg DM increase in NOP dose from its mean, and 1.52 ± 0.406% per 10 g/kg DM increase in NDF content from its mean. In conclusion, the present meta-analysis indicates that a greater NOP dose enhances the NOP effect on CH emission, whereas an increased dietary fiber content decreases its effect. 3-Nitrooxypropanol has stronger antimethanogenic effects in dairy cattle than in beef cattle.

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.

The aim was to obtain data regarding the effects of 4 freestall bedding materials (i.e., box compost, sand, horse manure, and foam mattresses) on cow comfort and risks for lameness and mastitis. The comfort of freestalls was measured by analyzing the way cows entered the stalls, the duration and smoothness of the descent movement, and the duration of the lying bout. The cleanliness of the cows was evaluated on 3 different body parts: (1) udder, (2) flank, and (3) lower rear legs, and the bacteriological counts of the bedding materials were determined. The combination of the cleanliness of the cows and the bacteriological count of the bedding material provided an estimate of the risk to which dairy cows are exposed in terms of intramammary infections. The results of the hock assessment revealed that the percentage of cows with healthy hocks was lower (20.5 ± 6.7), the percentage of cows with both damaged and swollen hocks was higher (26.8 ± 3.2), and the severity of the damaged hock was higher (2.32 ± 0.17) on farms using foam mattresses compared with deep litter materials [i.e., box compost (64.0 ± 10.4, 3.5 ± 4.7, 1.85 ± 0.23, respectively), sand (54.6 ± 8.2, 2.0 ± 2.8, 1.91 ± 0.09, respectively), and horse manure (54.6 ± 4.5, 5.5 ± 5.4, 1.85 ± 0.17, respectively)]. In addition, cows needed more time to lie down (140.2 ± 84.2s) on farms using foam mattresses compared with the deep litter materials sand and horse manure (sand: 50.1 ± 31.6s, horse manure: 32.9 ± 0.8s). Furthermore, the duration of the lying bout was shorter (47.9 ± 7.4 min) on farms using foam mattresses compared to sand (92.0 ± 12.9 min). These results indicate that deep litter materials provide a more comfortable lying surface compared with foam mattresses. The 3 deep litter bedding materials differed in relation to each other in terms of comfort and their estimate of risk to which cows were exposed in terms of intramammary infections [box compost: 17.8 cfu (1.0(4)) ± 19.4/g; sand: 1.2 cfu (1.0(4)) ± 1.6/g; horse manure: 110.5 cfu (1.0(4)) ± 86.3/g]. Box compost had a low gram-negative bacterial count compared with horse manure, and was associated with less hock injury compared with foam mattresses, but did not improve lying behavior (lying descent duration: 75.6 ± 38.8s, lying bout duration: 46.1 ± 18.5 min). Overall, sand provided the best results, with a comfortable lying surface and a low bacterial count.

Grass silage is typically fed to dairy cows in temperate regions. However, in vivo information on methane (CH 4 ) emission from grass silage of varying quality is limited. We evaluated the effect of two rates of nitrogen (N) fertilisation of grassland (low fertilisation (LF), 65 kg of N/ha; and high fertilisation (HF), 150 kg of N/ha) and of three stages of maturity of grass at cutting: early maturity (EM; 28 days of regrowth), mid maturity (MM; 41 days of regrowth) and late maturity (LM; 62 days of regrowth) on CH 4 production by lactating dairy cows. In a randomised block design, 54 lactating Holstein-Friesian dairy cows (168 ± 11 days in milk; mean ± standard error of mean) received grass silage (mainly ryegrass) and compound feed at 80 : 20 on dry matter basis. Cows were adapted to the diet for 12 days and CH 4 production was measured in climate respiration chambers for 5 days. Dry matter intake (DMI; 14.9 ± 0.56 kg/day) decreased with increasing N fertilisation and grass maturity. Production of fat-and proteincorrected milk (FPCM; 24.0 ± 1.57 kg/day) decreased with advancing grass maturity but was not affected by N fertilisation. Apparent total-tract feed digestibility decreased with advancing grass maturity but was unaffected by N fertilisation except for an increase and decrease in N and fat digestibility with increasing N fertilisation, respectively. Total CH 4 production per cow (347 ± 13.6 g/day) decreased with increasing N fertilisation by 4% and grass maturity by 6%. The smaller CH 4 production with advancing grass maturity was offset by a smaller FPCM and lower feed digestibility. As a result, with advancing grass maturity CH 4 emission intensity increased per units of FPCM (15.0 ± 1.00 g CH 4 /kg) by 31% and digestible organic matter intake (33.1 ± 0.78 g CH 4 /kg) by 15%. In addition, emission intensity increased per units of DMI (23.5 ± 0.43 g CH 4 /kg) by 7% and gross energy intake (7.0 ± 0.14% CH 4 ) by 9%, implying an increased loss of dietary energy with advancing grass maturity. Rate of N fertilisation had no effect on CH 4 emissions per units of FPCM, DMI and gross energy intake. These results suggest that despite a lower absolute daily CH 4 production with a higher N fertilisation rate, CH 4 emission intensity remains unchanged. A significant reduction of CH 4 emission intensity can be achieved by feeding dairy cows silage of grass harvested at an earlier stage of maturity.

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.

Complex interactions between rumen microbiota, cow genetics, and diet composition may exist. Therefore, the effect of linseed oil, DGAT1 K232A polymorphism (DGAT1), and the interaction between linseed oil and DGAT1 on CH and H emission, energy and N metabolism, lactation performance, ruminal fermentation, and rumen bacterial and archaeal composition was investigated. Twenty-four lactating Holstein-Friesian cows (i.e., 12 with DGAT1 KK genotype and 12 with DGAT1 AA genotype) were fed 2 diets in a crossover design: a control diet and a linseed oil diet (LSO) with a difference of 22 g/kg of dry matter (DM) in fat content between the 2 diets. Both diets consisted of 40% corn silage, 30% grass silage, and 30% concentrates (DM basis). Apparent digestibility, lactation performance, N and energy balance, and CH emission were measured in climate respiration chambers, and rumen fluid samples were collected using the oral stomach tube technique. No linseed oil by DGAT1 interactions were observed for digestibility, milk production and composition, energy and N balance, CH and H emissions, and rumen volatile fatty acid concentrations. The DGAT1 KK genotype was associated with a lower proportion of polyunsaturated fatty acids in milk fat, and with a higher milk fat and protein content, and proportion of saturated fatty acids in milk fat compared with the DGAT1 AA genotype, whereas the fat- and protein-corrected milk yield was unaffected by DGAT1. Also, DGAT1 did not affect nutrient digestibility, CH or H emission, ruminal fermentation or ruminal archaeal and bacterial concentrations. Rumen bacterial and archaeal composition was also unaffected in terms of the whole community, whereas at the genus level the relative abundances of some bacterial genera were found to be affected by DGAT1. The DGAT1 KK genotype was associated with a lower metabolizability (i.e., ratio of metabolizable to gross energy intake), and with a tendency for a lower milk N efficiency compared with the DGAT1 AA genotype. The LSO diet tended to decrease CH production (g/d) by 8%, and significantly decreased CH yield (g/kg of DM intake) by 6% and CH intensity (g/kg of fat- and protein-corrected milk) by 11%, but did not affect H emission. The LSO diet also decreased ruminal acetate molar proportion, the acetate to propionate ratio, and the archaea to bacteria ratio, whereas ruminal propionate molar proportion and milk N efficiency increased. Ruminal bacterial and archaeal composition tended to be affected by diet in terms of the whole community, with several bacterial genera found to be significantly affected by diet. These results indicate that DGAT1 does not affect enteric CH emission and production pathways, but that it does affect traits other than lactation characteristics, including metabolizability, N efficiency, and the relative abundance of Bifidobacterium. Additionally, linseed oil reduces CH emission independent of DGAT1 and affects the rumen microbiota and its fermentative activity.