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Abstract The objective of this study was to determine the influence of a total-mixed ration including unsalable carrots at 45% DM on the rumen microbiome; and the plasma, rumen and liver metabolomes. Carrots discarded at processing were investigated as an energy-dense substitute for barley grain in a conventional feedlot diet, and improved feed conversion efficiency by 25%. Here, rumen fluid was collected from 34 Merino lambs at slaughter (n = 16 control; n = 18 carrot) after a feeding period of 11-weeks. The V4 region of the 16S rRNA gene was sequenced to profile archaeal and bacterial microbe communities. Further, a comprehensive, targeted profile of known metabolites was constructed for blood plasma, rumen fluid and biopsied liver metabolites using a gas chromatography mass spectrometry (GC–MS) metabolomics approach. An in vitro batch culture was used to characterise ruminal fermentation including gas and methane (CH4) production. In vivo rumen microbial community structure of carrot fed lambs was dissimilar (P

Despite considerable effort to develop and optimise additives to reduce methane emissions from cattle, little information on additive effectiveness exists for cattle under grazing scenarios. As the majority of Australian cattle production occurs on grazing land it is pertinent to report on the use of additives under simulated conditions. The current study evaluated the addition of nine additives to Rhodes grass hay under in vitro conditions, to estimate their impact on methane (CH4), gas production, and rumen fermentation parameters (volatile fatty acids, rumen pH and in vitro dry matter digestibility [IVDMD]). Citral extract at 0.1% of rumen media decreased all CH4 production parameters, but reduced gas production and digestibility, compared to a 100% hay control. Similarly, Sandalwood essential oil decreased CH4 production at 48 h, IVDMD and gas production, compared to the control. Biochar + nitrates at 5 and 8% DM, and Biochar + Asparagopsis at 5% DM decreased cumulative CH4 production (15.6%, 25.9%, 23.8%, respectively; P < 0.01), compared to the control. No changes in IVDMD and gas production were observed. As such, the biochar additives were considered the most promising additives from those evaluated with a substrate designed to replicate Australian grazing systems.

This study characterized the nutritive and microbial profiles and the fermentation characteristics of silage with the following compositions on a dry matter (DM) basis: (1) 100% sorghum, (2) 70% sorghum + 30% carrot or pumpkin, and (3) 40% sorghum + 60% carrot or pumpkin. The treatments were further divided based on the addition or no addition of a probiotic inoculant. After 70 days of ensiling, the silage was incubated for 48 h using the in vitro batch culture technique. Crude protein and non-fiber carbohydrates in the silage increased (P ≤ 0.01) by 5.7 percent point (pp) and 9.6 pp, respectively, with pumpkin at 60% DM. The V4 region of the 16S rRNA gene was sequenced to profile pre-ensiled and ensiled archeal and bacterial communities. Silages containing carrot or pumpkin strongly influenced the microbial structure (PERMANOVA: R2 = 0.75; P < 0.001), despite the ensiled treatments being dominated by Lactobacillus spp., except for the control, which was dominated by Weissella and Pediococcus spp. (P < 0.01). Linear discriminant analysis indicated that carrot and pumpkin silages were responsible for the increased relative abundance of Lactobacillus and Acinetobacter spp. (log LDA score ≥ 2), respectively. After 48 h of incubation, carrot and pumpkin inclusion increased (P < 0.01) the in vitro DM digestibility by 22.5 and 31.3%, increased the total volatile fatty acids (VFAs) by 16 and 20.6% (P < 0.01), respectively, and showed a tendency (P = 0.07) to increase the gas production. Therefore, this study supports the use of carrot or pumpkin in sorghum silages to maximize feed digestibility and total VFA concentrations.

Abstract Background Effects of Saccharomyces cerevisiae fermentation products (SCFP) on rumen microbiota were determined in vitro and in vivo under a high and a depressed pH. The in vitro trial determined the effects of Original XPC and NutriTek (Diamond V, Cedar Rapids, IA) at doses of 1.67 and 2.33 g/L, respectively, on the abundances of rumen bacteria under a high pH (> 6.3) and a depressed pH (5.8–6.0) using quantitative PCR (qPCR). In the in vivo trial eight rumen-cannulated lactating dairy cows were used in a cross-over design. Cows were randomly assigned to SCFP treatments (Original XPC, Diamond V, Cedar Rapids, IA) or control (No SCFP) before two 5-week experimental periods. During the second period, SCFP treatments were reversed. Cows on the SCFP treatment were supplemented with 14 g/d of SCFP and 126 g/d of ground corn. Other cows received 140 g/d ground corn. During the first 4 wk. of each period, cows received a basal diet containing 153 g/kg of starch. During week 5 of both periods, the rumen pH was depressed by a SARA challenge. This included replacing 208 g/kg of the basal diet with pellets of ground wheat and barley, resulting in a diet that contained 222 g/kg DM of starch. Microbial communities in rumen liquid digesta were examined by pyrosequencing, qPCR, and shotgun metagenomics. Results During the in vitro experiment, XPC and NutriTek increased the relative abundances of Ruminococcus flavefaciens, and Fibrobacter succinogenes determined at both the high and the depressed pH, with NutriTek having the largest effect. The relative abundances of Prevotella brevis, R. flavefaciens, ciliate protozoa, and Bifidobacterium spp. were increased by XPC in vivo. Adverse impacts of the in vivo SARA challenge included reductions of the richness and diversity of the rumen microbial community, the abundances of Bacteroidetes and ciliate protozoa in the rumen as determined by pyrosequencing, and the predicted functionality of rumen microbiota as determined by shotgun metagenomics. These reductions were attenuated by XPC supplementation. Conclusions The negative effects of grain-based SARA challenges on the composition and predicted functionality of rumen microbiota are attenuated by supplementation with SCFP.

This study evaluated the fermentation quality, nutritive profile, in vitro fermentation, and microbial communities colonising sorghum ensiled with an unsalable vegetable mixture (chopped beans, carrot, and onion (1:1:1) ) including: (1)−100% sorghum; (2)−80% sorghum + 20% vegetable mix or (3)−60% sorghum + 40% vegetable mix, on a dry matter (DM) basis, with or without a probiotic inoculant. Samples were obtained across 0, 1, 3, 5,7, and 101 days ensiling and after 14 d aerobic exposure. The V4 region of the 16S rRNA gene and the ITS1 region were sequenced to profile bacterial, archaeal, and fungal communities. Compared to the 0% DM, ethanol increased (p < 0.01) from 8.42 to 20.4 ± 1.32 mM with 40% DM vegetable mix inclusion, while lactate decreased from 5.93 to 2.24 ± 0.26 mM. Linear discriminant analysis revealed that relative abundances of 12 bacterial taxa were influenced by silage treatments (log LDA score ≥ 4.02; p ≤ 0.03), while predicted functional pathways of alternative carbohydrate metabolism (hexitol, sulfoquinovose and glycerol degradation; N-acetyl glucosamine biosynthesis; log LDA score ≥ 2.04; p ≤ 0.02) were similarly enriched. This study indicated that carbohydrate metabolism by heterofermentative lactic acid bacteria can increase the feed value of sorghum when ensiled with an unsalable vegetable mixture at 40%DM, without requiring a high quantity of lactate.

Background: A foal undergoes considerable growth and development from birth to weaning, progressing from a milk-based diet to complete herbivory. The symbiotic relationships between bacteria, archaea and fungi substantiate this energy demand by colonising the hindgut and remaining flexible throughout the diet transitions. Methods: A total of 70 faecal samples were collected from 14 mares and their foals across five studs in NSW as they aged from 0 to 5 months old. DNA was extracted from faecal samples and underwent amplification and sequencing of the 16S rRNA gene V4 hypervariable region of archaea and bacteria, and the fungal internal transcribed spacer-1 (ITS1) region. The fungal and bacterial community structure was assessed using Bray-Curtis dissimilarities, and the effect of age at sampling and location was determined using PERMANOVA. Results: Age at sampling had a substantial effect on the foal’s archaeal and bacterial faecal microbiota (PERMANOVA: R2 = 0.16; p < 0.01), while the effect of geographical location was smaller but still significant (PERMANOVA: R2 = 0.07; p < 0.01). The overall abundance, diversity and richness of bacterial and archaeal populations increased (p < 0.01) as foals aged, most noticeably rising between foals 1 to 2 and 2 to 3 months of age. The 15 most relatively abundant fungal species were all environmental saprophytes, most strongly affected by geographical location (p < 0.01) rather than age at sampling. There was an effect of location on Preussia Africana (p = 0.02) and a location × age interaction for fungal species Preussia persica (p < 0.01), Acremonium furcatum (p = 0.04), and Podospora pseudocomata (p = 0.01). There was no effect of age, location, or location × age interaction on the relative abundance of the remaining fungal species. Conclusions: The faecal microbiome appeared to stabilise for most bacterial and archaeal genera by 2 to 3 months of age, resembling an adult mare. Bacterial genera isolated from faecal samples belonged mainly to the Firmicutes phylum. Age at sampling more strongly affected the archaeal and bacterial faecal microbiota than the effect of the geographical location where the horse was sampled. The lack of effect of location on microbe populations suggests that although environmental factors may influence population structure, there are distinct differences at each stage of foal maturation.

Ensiling vegetables with forage crops is a suggested method of waste diversion and can be directly utilized as a livestock feed. Carrot or pumpkin, ensiled at 0, 20, or 40% dry matter (DM) with crop sorghum, and with or without a second-generation silage inoculant were assessed for nutritive composition, organic acid profiles, aerobic stability and in vitro rumen fermentation characteristics. The study was a completely randomized design, with the fixed effects consisting of vegetable type (carrot vs. pumpkin), level (i.e., the level of vegetables), inoculant (inoculant or non-inoculant) and the interactions, and mini-silos within treatment as the random effect. The experimental unit for sorghum treatments represented by each mini-silo (5 kg capacity). Silage was sampled after 70-days ensiling for nutrient composition, 14-day aerobic stability, organic acid profiles and microbial diversity. After 24 h in vitro incubation, rumen fermentation parameters were assessed, measuring gas and methane (CH4) production, in vitro digestibility and volatile fatty acid concentrations. Sorghum ensiled with carrot or pumpkin at 20% or 40% DM increased crude fat (P ≤ 0.01) and decreased (P ≤ 0.01) silage surface temperature upon aerobic exposure compared to the control. Bacterial communities analyzed through 16S rRNA gene sequencing linearly increased (P ≤ 0.01) in diversity across both vegetables when the vegetable proportion was increased in the silage; dominated by Lactobacillus species. ITS analysis of the fungal microbiota upon silage opening and after 14 days (aerobic stability) identified increased (P ≤ 0.03) fungal diversity with increasing vegetable proportions, predominantly populated by Fusarium denticulatum, Issatchenkia orientalis, Kazachstania humilis, and Monascus purpureus. Upon assessment in vitro, there was an increase (P ≤ 0.04) in in vitro digestibility and some CH4 parameters (% CH4, and mg CH4/g DM), with no effect (P ≥ 0.17) on remaining CH4 parameters (mL CH4/g DM, mg CH4/g digested DM), gas production or pH. However, increasing vegetable amount decreased percentage of acetic acid and increased percentage of propionic acid of the total VFA, decreasing A:P ratio and total VFA concentration as a result (P ≤ 0.01). The results from this study indicate including carrot or pumpkin at 20 or 40% DM in a sorghum silage can produce a highly digestible, microbially diverse and energy-rich livestock feed.

Biochar is a novel carbonized feed additive sourced from pyrolyzed biomass. This compound is known to adsorb gasses and carbon, participate in biological redox reactions and provide habitat biofilms for desirable microbiota proliferation. Therefore, biochar holds potential to modify rumen fermentation characteristics and reduce enteric CH4 emissions. The objective of this study was to investigate the effect of hardwood biochar supplementation on fermentation parameters, methane (CH4) production and the ruminal archaeal, bacterial, and fungal microbiota using the in vitro RUSITEC (rumen simulation technique) system. Treatments consisted of a control diet (oaten pasture: maize silage: concentrate, 35:35:30 w/w) and hardwood biochar included at 400 or 800 mg per day (3.6 and 7.2% of substrate DM, respectively), over a 15-day period. Biochar supplementation had no effect (P ≥ 0.37) on pH, effluent (mL/d), total gas (mL/d), dry matter (DM) digestibility or CH4 production (mg/d). The addition of 800 mg biochar per day had the tendency (P = 0.10) to lower the % of CH4 released in fermentation compared to 400 mg/d biochar treatment. However, no effect (P ≥ 0.44) was seen on total VFA, acetate, propionate, butyric, branched-chain VFA, valerate and caproate production and the ratio of acetate to propionate. No effect (P > 0.05) was observed on bacterial, archaeal or fungal community structure. However, biochar supplementation at 800 mg/d decreased the abundance of one Methanomethylophilaceae OTU (19.8-fold, P = 0.046) and one Lactobacillus spp. OTU (31.7-fold, P < 0.01), in comparison to control treatments. Two fungal OTUs classified as Vishniacozyma victoriae (5.4 × 107 increase) and Sporobolomyces ruberrimus (5.4 × 107-fold increase) were more abundant in the 800 mg/d biochar samples. In conclusion, hardwood biochar had no effects on ruminal fermentation characteristics and may potentially lower the concentration of enteric CH4 when included at higher dosages by manipulating ruminal microbiota abundances.

Vegetables regarded as unsalable at processing often undergo disposal into landfill, threatening food security and increasing emissions through decomposition. Ensiling vegetables with forage crops is a suggested method of waste reduction that could also double as a livestock feed. Carrot or pumpkin, ensiled at 0%, 20% or 40% DM with crop sorghum, and with or without a second-generation silage inoculant were assessed for nutritive composition, organic acid profiles, aerobic stability and in vitro rumen fermentation characteristics. Silage was sampled after 70-days ensiling for nutrient composition, 14-day aerobic stability, organic acid profiles and microbial diversity. Sorghum ensiled with carrot or pumpkin at 20% or 40% DM increased crude fat (P ≤ 0.01) and decreased (P ≤ 0.01) silage surface temperature upon aerobic exposure compared to the control. Bacterial communities analyzed through 16S rRNA gene sequencing linearly increased (P ≤ 0.01) in diversity as vegetable proportion increased in the silage; dominated by Lactobacillus species. Upon assessment in vitro, there was an increase (P ≤ 0.04) in in vitro digestibility and some CH4 parameters (% CH4, and mg CH4/g DM), with no effect (P ≥ 0.17) on remaining CH4 parameters (mL CH4/g DM, mg CH4/g DMD), gas production or pH. However, increasing vegetable proportion decreased acetic and increased propionic acid concentrations respectively, decreasing A:P ratio and total VFA as a result (P ≤ 0.01). Results from this study indicate including carrot or pumpkin at 20% or 40% DM in a sorghum silage can produce a highly digestible, microbially diverse and energy-rich livestock feed whilst acting as a method of waste diversion of considerable environmental benefit.

Microorganisms in the digestive tract of ruminants differ in their functionality and ability to use feed constituents. While cecal microbiota play an important role in post-rumen fermentation of residual substrates undigested in the rumen, limited knowledge exists regarding its structure and function. In this trial we investigated the effect of dietary supplementation with linseed oil and nitrate on methane emissions and on the structure of ruminal and cecal microbiota of growing bulls. Animals were allocated to either a CTL (control) or LINNIT (CTL supplemented with 1.9% linseed and 1.0% nitrates) diet. Methane emissions were measured using the GreenFeed system. Microbial diversity was assessed using amplicon sequencing of microbial genomic DNA. Additionally, total RNA was extracted from ruminal contents and functional mcrA and mtt genes were targeted in amplicon sequencing approach to explore the diversity of functional gene expression in methanogens. LINNIT had no effect on methane yield (g/kg DMI) even though it decreased methane production by 9% (g/day; P < 0.05). Methanobrevibacter- and Methanomassiliicoccaceae-related OTUs were more abundant in cecum (72 and 24%) compared to rumen (60 and 11%) irrespective of the diet (P < 0.05). Feeding LINNIT reduced the relative abundance of Methanomassiliicoccaceae mcrA cDNA reads in the rumen. Principal component analysis revealed significant differences in taxonomic composition and abundance of bacterial communities between rumen and cecum. Treatment decreased the relative abundance of a few Ruminococcaceae genera, without affecting global bacterial community structure. Our research confirms a high level of heterogeneity in species composition of microbial consortia in the main gastrointestinal compartments where feed is fermented in ruminants. There was a parallel between the lack of effect of LINNIT on ruminal and cecal microbial community structure and functions on one side and methane emission changes on the other. These results suggest that the sequencing strategy used here to study microbial diversity and function accurately reflected the absence of effect on methane phenotypes in bulls treated with linseed plus nitrate.