Nitrate may lower methane production in ruminants by competing with methanogenesis for available hydrogen in the rumen. This study evaluated the effect of 4 levels of dietary nitrate addition on enteric methane production, hydrogen emission, feed intake, rumen fermentation, nutrient digestibility, microbial protein synthesis, and blood methemoglobin. In a 4×4 Latin square design 4 lactating Danish Holstein dairy cows fitted with rumen, duodenal, and ileal cannulas were assigned to 4 calcium ammonium nitrate addition levels: control, low, medium, and high [0, 5.3, 13.6, and 21.1g of nitrate/kg of dry matter (DM), respectively]. Diets were made isonitrogenous by replacing urea. Cows were fed ad libitum and, after a 6-d period of gradual introduction of nitrate, adapted to the corn-silage-based total mixed ration (forage:concentrate ratio 50:50 on DM basis) for 16d before sampling. Digesta content from duodenum, ileum, and feces, and rumen liquid were collected, after which methane production and hydrogen emissions were measured in respiration chambers. Methane production [L/kg of dry matter intake (DMI)] linearly decreased with increasing nitrate concentrations compared with the control, corresponding to a reduction of 6, 13, and 23% for the low, medium, and high diets, respectively. Methane production was lowered with apparent efficiencies (measured methane reduction relative to potential methane reduction) of 82.3, 71.9, and 79.4% for the low, medium, and high diets, respectively. Addition of nitrate increased hydrogen emissions (L/kg of DMI) quadratically by a factor of 2.5, 3.4, and 3.0 (as L/kg of DMI) for the low, medium, and high diets, respectively, compared with the control. Blood methemoglobin levels and nitrate concentrations in milk and urine increased with increasing nitrate intake, but did not constitute a threat for animal health and human food safety. Microbial crude protein synthesis and efficiency were unaffected. Total volatile fatty acid concentration and molar proportions of acetate, butyrate, and propionate were unaffected, whereas molar proportions of formate increased. Milk yield, milk composition, DMI and digestibility of DM, organic matter, crude protein, and neutral detergent fiber in rumen, small intestine, hindgut, and total tract were unaffected by addition of nitrate. In conclusion, nitrate lowered methane production linearly with minor effects on rumen fermentation and no effects on nutrient digestibility.

BackgroundThe selective breeding of cattle with high-feed efficiencies (FE) is an important goal of beef and dairy cattle producers. Global gene expression patterns in relevant tissues can be used to study the functions of genes that are potentially involved in regulating FE. In the present study, high-throughput RNA sequencing data of liver biopsies from 19 dairy cows were used to identify differentially expressed genes (DEGs) between high- and low-FE groups of cows (based on Residual Feed Intake or RFI). Subsequently, a profile of the pathways connecting the DEGs to FE was generated, and a list of candidate genes and biomarkers was derived for their potential inclusion in breeding programmes to improve FE.ResultsThe bovine RNA-Seq gene expression data from the liver was analysed to identify DEGs and, subsequently, identify the molecular mechanisms, pathways and possible candidate biomarkers of feed efficiency. On average, 57 million reads (short reads or short mRNA sequences < ~200 bases) were sequenced, 52 million reads were mapped, and 24,616 known transcripts were quantified according to the bovine reference genome. A comparison of the high- and low-RFI groups revealed 70 and 19 significantly DEGs in Holstein and Jersey cows, respectively. The interaction analysis (high vs. low RFI x control vs. high concentrate diet) showed no interaction effects in the Holstein cows, while two genes showed interaction effects in the Jersey cows. The analyses showed that DEGs act through certain pathways to affect or regulate FE, including steroid hormone biosynthesis, retinol metabolism, starch and sucrose metabolism, ether lipid metabolism, arachidonic acid metabolism and drug metabolism cytochrome P450.ConclusionWe used RNA-Seq-based liver transcriptomic profiling of high- and low-RFI dairy cows in two breeds and identified significantly DEGs, their molecular mechanisms, their interactions with other genes and functional enrichments of different molecular pathways. The DEGs that were identified were the CYP’s and GIMAP genes for the Holstein and Jersey cows, respectively, which are related to the primary immunodeficiency pathway and play a major role in feed utilization and the metabolism of lipids, sugars and proteins.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3622-9) contains supplementary material, which is available to authorized users.

Improving feed efficiency of dairy cows through breeding is expected to reduce enteric methane production per unit of milk produced. This study examined the effect of 2 forage-to-concentrate ratios on methane production, rumen fermentation, and nutrient digestibility in Holstein and Jersey dairy cows divergent in residual feed intake (RFI). Before experimental onset, RFI was estimated using a random regression model on phenotypic herd data. Ten lactating Holstein and 10 lactating Jersey cows were extracted from the herd and allocated to a high or low pre-experimental RFI group of 5 animals each within breed. Cows were fed ad libitum with total mixed rations either low (LC) or high (HC) in concentrates during 3 periods in a crossover design with a back-cross and staggered approach. Forage-to-concentrate ratio was 68:32 for LC and 39:61 for HC. Cows adapted to the diets in 12 to 24 d and feces were subsequently collected on 2 d. Afterward, gas exchange was measured in respiration chambers and rumen liquid was collected once after cows exited the chambers. Pre-experimental RFI was included in the statistical analysis as a class (low and high RFI) or continuous variable. Methane per kilogram of dry matter intake (DMI) was lower for Holsteins than Jerseys and the response to increased concentrate level was more pronounced for Holsteins than Jerseys (27.2 vs.13.8%); a similar pattern was found for the acetate:propionate ratio. However, methane production per kilogram of energy-corrected milk (ECM) was unaffected by breed. Further, total-tract digestibility of neutral detergent fiber was higher for Jerseys than Holsteins. For RFI as a class variable, DMI, methane production regardless of the expression, and digestibility were unaffected by RFI. For RFI as a continuous variable, DMI was lower and methane per kilogram of DMI was higher for cows with negative (efficient) than positive (inefficient) RFI values, and neutral detergent fiber digestibility was higher for Holsteins with negative than positive RFI values, but not for Jerseys. Daily methane production and methane per kilogram of ECM were unaffected by RFI. In conclusion, methane per kilogram of DMI of Jerseys was lowered to a smaller extent in response to the HC diet than of Holsteins. When pre-experimental RFI was used as a continuous variable, higher methane per kilogram of DMI was found for cows with negative RFI than positive RFI values, but not for methane per kilogram of ECM. These findings call for validation in larger studies.

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Identifying factors that influence the composition of the microbial population in the digestive system of dairy cattle will be key in regulating these populations to reduce greenhouse gas emissions. In this study, we analyzed rumen and fecal samples from five high residual feed intake (RFI) Holstein cows, five low RFI Holstein cows, five high RFI Jersey cows and five low RFI Jersey cows, fed either a high-concentrate diet (expected to reduce methane emission) or a high-forage diet. Bacterial communities from both the rumen and feces were profiled using Illumina sequencing on the 16S rRNA gene. Rumen archaeal communities were profiled using Terminal-Restriction Fragment Length Polymorphism (T-RFLP) targeting the mcrA gene. The rumen methanogen community was influenced by breed but not by diet or RFI. The rumen bacterial community was influenced by breed and diet but not by RFI. The fecal bacterial community was influenced by individual animal variation and, to a lesser extent, by breed and diet but not by RFI. Only the bacterial community correlated with methane production. Community differences seen in the rumen were reduced or absent in feces, except in the case of animal-to-animal variation, where differences were more pronounced. The two cattle breeds had different levels of response to the dietary intervention; therefore, it may be appropriate to individually tailor methane reduction strategies to each cattle breed.