Oral administration of Pinus koraiensis cone essential oil reduces rumen methane emission by altering the rumen microbial composition and functions in Korean native goat (Capra hircus coreanae).

This study aimed to investigate Pinus koraiensis cone essential oil (PEO) as a methane (CH4) inhibitor and determine its impact on the taxonomic and functional characteristics of the rumen microbiota in goats. A total of 10 growing Korean native goats (Capra hircus coreanae, 29.9 ± 1.58 kg, male) were assigned to different dietary treatments: control (CON; basal diet without additive) and PEO (basal diet +1 g/d of PEO) by a 2 × 2 crossover design. Methane measurements were conducted every 4 consecutive days for 17-20 days using a laser CH4 detector. Samples of rumen fluid and feces were collected during each experimental period to evaluate the biological effects and dry matter (DM) digestibility after PEO oral administration. The rumen microbiota was analyzed via 16S rRNA gene amplicon sequencing. The PEO oral administration resulted in reduced CH4 emission (eructation CH4/body weight0.75, p = 0.079) without affecting DM intake; however, it lowered the total volatile fatty acids (p = 0.041), molar proportion of propionate (p = 0.075), and ammonia nitrogen (p = 0.087) in the rumen. Blood metabolites (i.e., albumin, alanine transaminase/serum glutamic pyruvate transaminase, creatinine, and triglyceride) were significantly affected (p < 0.05) by PEO oral administration. The absolute fungal abundance (p = 0.009) was reduced by PEO oral administration, whereas ciliate protozoa, total bacteria, and methanogen abundance were not affected. The composition of rumen prokaryotic microbiota was altered by PEO oral administration with lower evenness (p = 0.054) observed for the PEO group than the CON group. Moreover, PICRUSt2 analysis revealed that the metabolic pathways of prokaryotic bacteria, such as pyruvate metabolism, were enriched in the PEO group. We also identified the Rikenellaceae RC9 gut group as the taxa potentially contributing to the enriched KEGG modules for histidine biosynthesis and pyruvate oxidation in the rumen of the PEO group using the FishTaco analysis. The entire co-occurrence networks showed that more nodes and edges were detected in the PEO group. Overall, these findings provide an understanding of how PEO oral administration affects CH4 emission and rumen prokaryotic microbiota composition and function. This study may help develop potential manipulation strategies to find new essential oils to mitigate enteric CH4 emissions from ruminants.

Blood and milk metabolites of Holstein dairy cattle for the development of objective indicators of a subacute ruminal acidosis.

OBJECTIVE: The purpose of this study was to perform a comparative analysis of metabolite levels in serum and milk obtained from cows fed on different concentrate to forage feed ratios. METHODS: Eight lactating Holstein cows were divided into two groups: a high forage ratio diet (HF; 80% Italian ryegrass and 20% concentrate of daily intake of dry matter) group and a high concentrate diet (HC; 20% Italian ryegrass and 80% concentrate) group. Blood was collected from the jugular vein, and milk was sampled using a milking machine. Metabolite levels in serum and milk were estimated using proton nuclear magnetic resonance and subjected to qualitative and quantitative analyses performed using Chenomx 8.4. For statistical analysis, Student's t-test and multivariate analysis were performed using Metaboanalyst 4.0. RESULTS: In the principal component analysis, a clear distinction between the two groups regarding milk metabolites while serum metabolites were shown in similar. In serum, 95 metabolites were identified, and 13 metabolites (include leucine, lactulose, glucose, betaine, etc.) showed significant differences between the two groups. In milk, 122 metabolites were identified, and 20 metabolites (include urea, carnitine, acetate, butyrate, arabinitol, etc.) showed significant differences. CONCLUSION: Our results show that different concentrate to forage feed ratios impact the metabolite levels in the serum and milk of lactating Holstein cows. A higher number of metabolites in milk, including those associated with milk fat synthesis and the presence of Escherichia coli in the rumen, differed between the two groups compared to that in the serum. The results of this study provide a useful insight into the metabolites associated with different concentrate to forge feed ratios in cows and may aid in the search for potential biomarkers for subacute ruminal acidosis.

Comparison of metabolites in rumen fluid, urine, and feces of dairy cow from subacute ruminal acidosis model measured by proton nuclear magnetic resonance spectroscopy.

OBJECTIVE: In this study, metabolites that changed in the rumen fluid, urine and feces of dairy cows fed different feed ratios were investigated. METHODS: Eight Holstein cows were used in this study. Rumen fluid, urine, and feces were collected from the normal concentrate diet (NCD) (Italian ryegrass 80%: concentrate 20% in the total feed) and high concentrate diet (HCD) groups (20%: 80%) of dairy cows. Metabolite analysis was performed using proton nuclear magnetic resonance (NMR) identification, and statistical analysis was performed using Chenomx NMR software 8.4 and Metaboanalyst 4.0. RESULTS: The two groups of rumen fluid and urine samples were separated, and samples from the same group were aggregated together. On the other hand, the feces samples were not separated and showed similar tendencies between the two groups. In total, 160, 177, and 188 metabolites were identified in the rumen fluid, urine, and feces, respectively. The differential metabolites with low and high concentrations were 15 and 49, 14 and 16, and 2 and 2 in the rumen fluid, urine, and feces samples, in the NCD group. CONCLUSION: As HCD is related to rumen microbial changes, research on different metabolites such as glucuronate, acetylsalicylate, histidine, and O-Acetylcarnitine, which are related to bacterial degradation and metabolism, will need to be carried out in future studies along with microbial analysis. In urine, the identified metabolites, such as gallate, syringate, and vanillate can provide insight into microbial, metabolic, and feed parameters that cause changes depending on the feed rate. Additionally, it is thought that they can be used as potential biomarkers for further research on subacute ruminal acidosis.

Red seaweed extracts reduce methane production by altering rumen fermentation and microbial composition in vitro.

A series of in vitro batch culture incubations were carried out to investigate changes in rumen fermentation characteristics, methane (CH4) production, and microbial composition in response to supplementation with five different red seaweed species (Amphiroa anceps, AANC; Asparagopsis taxiformis, ATAX; Chondracanthus tenellus, CTEN; Grateloupia elliptica, GELL; and Gracilaria parvispora, GPAR). Prior to the incubations, the total flavonoid and polyphenol content of the red seaweed extracts was quantified. The incubated substrate consisted of timothy hay and corn grain [60:40 dry matter (DM) basis]. Treatments were substrate mixtures without seaweed extract (CON) or substrate mixtures supplemented with 0.25 mg/mL of red seaweed extract. Samples were incubated for 6, 12, 24, 36, and 48 h. Each sample was incubated in triplicates in three separate runs. In vitro DM degradability, fermentation parameters (i.e., pH, volatile fatty acids, and ammonia nitrogen), total gas production, and CH4 production were analyzed for all time points. Microbial composition was analyzed using 16S rRNA amplicon sequencing after 24 h of incubation. The highest CH4 reduction (mL/g DM, mL/g digested DM, and % of total gas production) was observed in ATAX (51.3, 50.1, and 51.5%, respectively, compared to CON; P < 0.001) after 12 h of incubation. The other red seaweed extracts reduced the CH4 production (mL/g DM; P < 0.001) in the range of 4.6-35.0% compared to CON after 24 h of incubation. After 24 h of incubation, supplementation with red seaweed extracts tended to increase the molar proportion of propionate (P = 0.057) and decreased the acetate to propionate ratio (P = 0.033) compared to the CON. Abundances of the genus Methanobrevibacter and total methanogens were reduced (P = 0.050 and P = 0.016) by red seaweed extract supplementation. The linear discriminant analysis effect size (P < 0.05, LDA ≥ 2.0) showed that UG Succinivibrionaceae, Anaeroplasma, and UG Ruminococcaceae, which are associated with higher propionate production, starch degradation, and amylase activity were relatively more abundant in red seaweed extracts than in the CON. Our results suggest that supplementation with red seaweed extracts altered the microbiota, leading to the acceleration of propionate production and reduction in CH4 production.

Dose-Response Effects of Bamboo Leaves on Rumen Methane Production, Fermentation Characteristics, and Microbial Abundance In Vitro.

Ruminants produce large amounts of methane as part of their normal digestive processes. Recently, feed additives were shown to inhibit the microorganisms that produce methane in the rumen, consequently reducing methane emissions. The objective of this study was to evaluate the dose-response effect of Phyllostachys nigra var. henonis (PHN) and Sasa borealis supplementation on in vitro rumen fermentation, methane, and carbon dioxide production, and the microbial population. An in vitro batch culture system was used, incubated without bamboo leaves (control) or with bamboo leaves (0.3, 0.6, and 0.9 g/L). After 48 h, total gas, methane, and carbon dioxide production decreased linearly with an increasing dose of bamboo leaves supplementation. The total volatile fatty acid, acetate, and acetate-to-propionate ratio were affected quadratically with increasing doses of bamboo leaves supplementation. In addition, propionate decreased linearly. Butyrate was increased linearly with increasing doses of PHN supplementation. The absolute values of total bacteria and methanogenic archaea decreased linearly and quadratically with an increasing dose of PHN treatment after 48 h. These results show that bamboo leaves supplementation can reduce methane production by directly affecting methanogenic archaea, depressing the metabolism of methanogenic microbes, or transforming the composition of the methanogenic community. These results need to be validated using in vivo feeding trials before implementation.

Metabolic profiling of serum and urine in lactating dairy cows affected by subclinical ketosis using proton nuclear magnetic resonance spectroscopy.

Ketosis is associated with high milk yield during lactating or insufficient feed intake in lactating dairy cows. However, few studies have been conducted on the metabolomics of ketosis in Korean lactating dairy cows. The present study aimed to investigate the serum and urine metabolites profiling of lactating dairy cows through proton nuclear magnetic resonance (1H-NMR) spectroscopy and comparing those between healthy (CON) and subclinical ketosis (SCK) groups. Six lactating dairy cows were categorized into CON and SCK groups. All experimental Holstein cows were fed total mixed ration. Serum and urine samples were collected from the jugular vein of the neck and by hand sweeping the perineum, respectively. The metabolites in the serum and urine were determined using 1H-NMR spectroscopy. Identification and quantification of metabolites was performed by Chenomx NMR Suite 8.4 software. Metabolites statistical analysis was performed by Metaboanalyst version 5.0 program. In the serum, the acetoacetate level was significantly (p < 0.05) higher in the SCK group than in the CON group, and whereas acetate, galactose and pyruvate levels tended to be higher. CON group had significantly (p < 0.05) higher levels of 5-aminolevulinate and betaine. Indole-3-acetate, theophylline, p-cresol, 3-hydroxymandelate, gentisate, N-acetylglucosamine, N-nitrosodimethylamine, xanthine and pyridoxine levels were significantly (p < 0.05) higher in the urine of the SCK group than that in the CON group, which had higher levels of homogentisate, ribose, gluconate, ethylene glycol, maltose, 3-methyl-2-oxovalerate and glycocholate. Some significantly (p < 0.05) different metabolites in the serum and urine were associated with ketosis diseases, inflammation, energy balance and body weight. This study will be contributed useful a future ketosis metabolomics studies in Korea.

Effects of seaweed extracts on in vitro rumen fermentation characteristics, methane production, and microbial abundance.

Several seaweed extracts have been reported to have potential antimethanogenic effects in ruminants. In this study, the effect of three brown seaweed species (Undaria pinnatifida, UPIN; Sargassum fusiforme, SFUS; and Sargassum fulvellum, SFUL) on rumen fermentation characteristics, total gas, methane (CH4), carbon dioxide (CO2) production, and microbial populations were investigated using an in vitro batch culture system. Seaweed extract and its metabolites, total flavonoid and polyphenol contents were identified and compared. For the in vitro batch, 0.25 mg∙mL-1 of each seaweed extract were used in 6, 12, 24, 36 and 48 h of incubation. Seaweed extract supplementation decreased CH4 yield and its proportion to total gas production after 12, 24, and 48 h of incubation, while total gas production were not significantly different. Total volatile fatty acid and molar proportion of propionate increased with SFUS and SFUL supplementation after 24 h of incubation, whereas UPIN was not affected. Additionally, SFUS increased the absolute abundance of total bacteria, ciliate protozoa, fungi, methanogenic archaea, and Fibrobacter succinogenes. The relative proportions of Butyrivibrio fibrisolvens, Butyrivibrio proteoclasticus, and Prevotella ruminicola were lower with seaweed extract supplementation, whereas Anaerovibrio lipolytica increased. Thus, seaweed extracts can decrease CH4 production, and alter the abundance of rumen microbial populations.

Metabolic Profiling of Rumen Fluid and Milk in Lactating Dairy Cattle Influenced by Subclinical Ketosis Using Proton Nuclear Magnetic Resonance Spectroscopy.

Ketosis metabolic research on lactating dairy cattle has been conducted worldwide; however, there have been very few Korean studies. Biofluids from lactating dairy cattle are necessary to study ketosis metabolic diseases. Six Holstein cows were divided into two groups (healthy (CON) and subclinical ketosis diagnosed (SCK)). Rumen fluid and milk samples were collected using a stomach tube and a pipeline milking system, respectively. Metabolites were determined using proton nuclear magnetic resonance (NMR) spectroscopy and they were identified and quantified using the Chenomx NMR Suite 8.4 software and Metaboanalyst 5.0. In the rumen fluid of the SCK group, butyrate, sucrose, 3-hydroxybutyrate, maltose, and valerate levels were significantly higher than in the CON group, which showed higher levels of N,N-dimethylformamide, acetate, glucose, and propionate were significantly higher. Milk from the SCK group showed higher levels of maleate, 3-hydroxybutyrate, acetoacetate, galactonate, and 3-hydroxykynurenine than that from the CON group, which showed higher levels of galactitol, 1,3-dihydroxyacetone, γ-glutamylphenylalanine, 5-aminolevulinate, acetate, and methylamine. Some metabolites are associated with ketosis diseases and the quality of rumen fluid and milk. This report will serve as a future reference guide for ketosis metabolomics studies in Korea.

Effects of Olive (Olea europaea L.) Leaves with Antioxidant and Antimicrobial Activities on In Vitro Ruminal Fermentation and Methane Emission.

We evaluated whether olive leaves (OLs) are effective as feed additives and supplements for ruminants and the potential methane reduction effects during in vitro fermentation. Two Hanwoo cows (460 ± 20 kg) equipped with cannula were fed Timothy hay and corn-based feed 3% of the body weight at a ratio of 6:4 (8:30 a.m. and 5:00 p.m.). Ruminal fluid from the cows was collected and mixed before morning feeding. In vitro batch fermentation was monitored after 12 and 24 h of incubation at 39 °C, and OLs were used as supplements to achieve the concentration of 5% in the basal diet. At 12 h of fermentation, methane production decreased in the 5% OLs group compared to that in the control group, but not at 24 h. The proportion of cellulose-degrading bacteria, Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens, tended to increase in the 5% OLs group at 12 h. The amount of ammonia produced was the same as the polymerase chain reaction result for Prevotella ruminicola. At 12 h, the proportion of Prevotella ruminicola was significantly higher in the 5% OLs group. OLs may be used incorporated with protein byproducts or other methane-reducing agents in animal feed.

Effects of the Appropriate Addition of Antioxidants from Pinus densiflora and Mentha canadensis Extracts on Methane Emission and Rumen Fermentation.

This study aimed to investigate the optimal addition of terpene-based Pinus densiflora and Mentha canadensis extracts, with antioxidant and methane reduction effects, as feed supplements to ruminants. Two cannulated steers (450 ± 30 kg), consuming Timothy Hay and a commercial concentrate (60:40, w/w) twice daily (at 09:00 and 17:30) at 2% of body weight, with free access to water and a mineral block, were used as rumen fluid donors. In vitro fermentation experiments, with Timothy Hay as the substrate, were conducted with P. densiflora and M. canadensis extracts as supplements to achieve concentrations of 30, 50, and 70 mg/L on a Timothy Hay basis. Fibrobacter succinogenes decreased in proportion upon P. densiflora and M. canadensis extract supplementation at 50 mg/L, while the dry matter degradability of the feed was not significantly different (p < 0.05). Methane emission was significantly lower in the 50 and 70 mg/L treatment groups, for both extracts, at 12 h (p < 0.05). Based on methane production and antioxidant activity, our study suggests that 30 mg/L addition is the most appropriate level of supplementation.