Methylation-GC-MS/FID-Based Glycosidic Linkage Analysis of Unfractionated Polysaccharides in Red Seaweeds.

Glycosidic linkage analysis was conducted on the unfractionated polysaccharides in alcohol-insoluble residues (AIRs) prepared from six red seaweeds (Gracilariopsis sp., Prionitis sp., Mastocarpus papillatus, Callophyllis sp., Mazzaella splendens, and Palmaria palmata) using GC-MS/FID analysis of partially methylated alditol acetates (PMAAs). The cell walls of P. palmata primarily contained mixed-linkage xylans and small amounts of sulfated galactans and cellulose. In contrast, the unfractionated polysaccharides of the other five species were rich in galactans displaying diverse 3,6-anhydro-galactose and galactose linkages with varied sulfation patterns. Different levels of cellulose were also observed. This glycosidic linkage method offers advantages for cellulose analysis over traditional monosaccharide analysis that is known for underrepresenting glucose in crystalline cellulose. Relative linkage compositions calculated from GC-MS and GC-FID measurements showed that anhydro sugar linkages generated more responses in the latter detection method. This improved linkage workflow presents a useful tool for studying polysaccharide structural variations across red seaweed species. Furthermore, for the first time, relative linkage compositions from GC-MS and GC-FID measurements, along with normalized FID and total ion current (TIC) chromatograms without peak assignments, were analyzed using principal component analysis (PCA) as a proof-of-concept demonstration of the technique's potential to differentiate various red seaweed species.

Evaluation of Rumen Fermentation and Microbial Adaptation to Three Red Seaweeds Using the Rumen Simulation Technique.

Several red seaweeds have been shown to inhibit enteric CH4 production; however, the adaptation of fermentation parameters to their presence is not well understood. The objective of this study was to examine the effect of three red seaweeds (Asparargopsis taxiformis, Mazzaella japonica, and Palmaria mollis) on in vitro fermentation, CH4 production, and adaptation using the rumen simulation technique (RUSITEC). The experiment was conducted as a completely randomized design with four treatments, duplicated in two identical RUSITEC apparatus equipped with eight fermenter vessels each. The four treatments included the control and the three red seaweeds added to the control diet at 2% diet DM. The experimental period was divided into four phases including a baseline phase (d 0-7; no seaweed included), an adaptation phase (d 8-11; seaweed included in treatment vessels), an intermediate phase (d 12-16), and a stable phase (d 17-21). The degradability of organic matter (p = 0.04) and neutral detergent fibre (p = 0.05) was decreased by A. taxiformis during the adaptation phase, but returned to control levels in the stable phase. A. taxiformis supplementation resulted in a decrease (p < 0.001) in the molar proportions of acetate, propionate, and total volatile fatty acid (VFA) production, with an increase in the molar proportions of butyrate, caproate, and valerate; the other seaweeds had no effect (p > 0.05) on the molar proportions or production of individual VFA. A. taxiformis was the only seaweed to suppress CH4 production (p < 0.001), with the suppressive effect increasing (p < 0.001) across phases. Similarly, A. taxiformis increased (p < 0.001) the production of hydrogen (H2, %, mL/d) across the adaptation, intermediate, and stable phases, with the intermediate and stable phases having greater H2 production than the adaptation phase. In conclusion, M. japonica and P. mollis did not impact rumen fermentation or inhibit CH4 production within the RUSITEC. In contrast, we conclude that A. taxiformis is an effective CH4 inhibitor and its introduction to the ruminal environment requires a period of adaptation; however, the large magnitude of CH4 suppression by A. taxiformis inhibits VFA synthesis, which may restrict the production performance in vivo.

Comparative analysis of macroalgae supplementation on the rumen microbial community: Asparagopsis taxiformis inhibits major ruminal methanogenic, fibrolytic, and volatile fatty acid-producing microbes in vitro.

Seaweeds have received a great deal of attention recently for their potential as methane-suppressing feed additives in ruminants. To date, Asparagopsis taxiformis has proven a potent enteric methane inhibitor, but it is a priority to identify local seaweed varieties that hold similar properties. It is essential that any methane inhibitor does not compromise the function of the rumen microbiome. In this study, we conducted an in vitro experiment using the RUSITEC system to evaluate the impact of three red seaweeds, A. taxiformis, Palmaria mollis, and Mazzaella japonica, on rumen prokaryotic communities. 16S rRNA sequencing showed that A. taxiformis had a profound effect on the microbiome, particularly on methanogens. Weighted Unifrac distances showed significant separation of A. taxiformis samples from the control and other seaweeds (p < 0.05). Neither P. mollis nor M. japonica had a substantial effect on the microbiome (p > 0.05). A. taxiformis reduced the abundance of all major archaeal species (p < 0.05), leading to an almost total disappearance of the methanogens. Prominent fiber-degrading and volatile fatty acid (VFA)-producing bacteria including Fibrobacter and Ruminococcus were also inhibited by A. taxiformis (p < 0.05), as were other genera involved in propionate production. The relative abundance of several other bacteria including Prevotella, Bifidobacterium, Succinivibrio, Ruminobacter, and unclassified Lachnospiraceae were increased by A. taxiformis suggesting that the rumen microbiome adapted to an initial perturbation. Our study provides baseline knowledge of microbial dynamics in response to seaweed feeding over an extended period and suggests that feeding A. taxiformis to cattle to reduce methane may directly, or indirectly, inhibit important fiber-degrading and VFA-producing bacteria.

Effects of feeding a pine-based biochar to beef cattle on subsequent manure nutrients, organic matter composition and greenhouse gas emissions.

Biochar in ruminant diets is being assessed as a method for simultaneously improving animal production and reducing enteric CH4 emissions, but little is known about subsequent biochar-manure interactions post-excretion. We examined chemical properties, greenhouse gas (GHG) emissions and organic matter (OM) composition during farm scale stockpiling (SP) or composting (CP) of manure from cattle that either received a pine-based biochar in their diet (BM) or did not (RM). Manure piles were monitored hourly for temperature and weekly for top surface CO2, N2O and CH4 fluxes over 90 d in a semiarid location near Lethbridge, AB, Canada. Results indicate that cumulative CO2, N2O and CH4 emissions were not affected by biochar, implying that BM was as labile as RM. The pH, total C (TC), NO3-N and Olsen P were also not influenced by biochar, although it was observed that NH4-N and OM extractability were both 13% lower in BM than RM. Solid-state 13C nuclear magnetic resonance (NMR) showed that biochar increased stockpile/compost aromaticity, yet it did not alter the bulk C speciation of manure OM. Further analysis by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) revealed that dissolved OM was enriched by strongly reduced chemical constituents, with BM providing more humic-like OM precursors than RM. Inclusion of a pine-based biochar in cattle diets to generate BM is consistent with current trends in the circular economy, "closing the loop" in agricultural supply chains by returning C-rich organic amendments to croplands. Stockpiling/composting the resulting BM, however, may not provide a clear advantage over directly mixing low levels of biochar with manure. Further research is required to validate BM as a tool to reduce the C footprint of livestock waste management.

Molecular speciation and aromaticity of biochar-manure: Insights from elemental, stable isotope and solid-state DPMAS 13C NMR analyses.

The use of biochar (BC) in feedlot cattle diets has recently been explored as an approach to simultaneously improving animal production and reducing enteric methane (CH4) emissions. This study examines the impact of BC on manure properties and whether BC affects manure composition and carbon (C) and nitrogen (N) outputs from feedlot steers offered a barley-based diet with BC at 0.0, 0.5, 1.0 and 2.0% (BC0, BC0.5, BC1 and BC2) of diet dry matter. Manure was sampled three times over a 235 day feeding trial conducted in southern Alberta, Canada. Results showed that BC2 increased total C and the C/N ratio by 5.7 and 6.6% relative to BC0, respectively (P < 0.05), while total N exhibited a quadratic response from BC0 to BC2 (P = 0.005). Manure 15δN signatures, ranging from +3.83 to +7.34‰, were not affected (P > 0.05) by BC treatment. DPMAS 13C NMR revealed similar structural features among BC0 and BC2; indigestible BC had a minor impact on the bulk-C speciation of manure organic matter (OM). Compositional changes were limited to the aromatic-C region of the 13C NMR spectra. Fused-ring domains, mainly pyrogenic-C, were increased by 1.56-fold at BC2 relative to BC0. Overall, results demonstrated that BC stabilizes recalcitrant-C in manure OM, potentially sequestering soil-C when applied to croplands. This approach provides an added value to its use in ruminant diets, mainly from a nutrient cycling perspective. However, whole-farm studies are further required to validate the incorporation of BC into beef production systems.

Effect of a pine enhanced biochar on growth performance, carcass quality, and feeding behavior of feedlot steers.

The objective of this study was to evaluate the effect of enhanced biochar (EB) on growth performance, carcass quality, and feeding behavior of feedlot steers fed high-forage and high-grain diets. A total of 160 crossbred steers (initial 286 ± 26 kg body weight [BW]) were blocked by BW and randomly assigned to 16 pens (10 steers per pen), 8 of which were equipped with the GrowSafe system for monitoring feeding behavior. Treatments were EB included in the diet at 0% (control), 0.5%, 1.0%, or 2.0% (dry matter [DM] basis) with four pens per treatment. The backgrounding phase (84 d) was divided into four 21-d periods, and the finishing phase (112 d) was divided into four 28-d periods, with a 28-d transition period for dietary adaptation. Pen was the experimental unit for all parameters except for feeding behavior, where steer was considered the experimental unit. Treatment was included as a fixed effect, and period was considered a repeated measure. Total weight gain and overall average daily gain (ADG) tended to decrease (P = 0.06) with 2.0% EB. There was no effect (P ≥ 0.13) of EB on dry matter intake (DMI), gain-to-feed ratio (G:F), net energy for gain, ADG, or final BW for the backgrounding or finishing phases. There was a treatment × period effect (P < 0.05) of EB on DMI, ADG, and G:F for both backgrounding and finishing phases. Hot carcass weight, dressing %, back fat, rib-eye area, and meat yield were not affected (P ≥ 0.26) by EB. Lean meat yield was increased (P = 0.03) by 2.0% EB compared to all other treatments. Compared to the control, 2.0% EB increased (P = 0.02) the number of carcasses that achieved Canada 1 grade. More (P = 0.05) carcasses from control steers were graded as Canada 3 as compared to those fed 0.5% or 2.0% EB. Quality grade and incidences of liver abscesses were not affected (P ≥ 0.44) by EB. Enhanced biochar had no effect (P ≥ 0.11) on feeding behavior during backgrounding or finishing phases. In conclusion, EB did not result in changes in growth rate, feed efficiency, or feeding behavior in feedlot cattle, but 2.0% EB increased lean carcass yield grade.

Pretreatment of crop residues by ammonia fiber expansion (AFEX) alters the temporal colonization of feed in the rumen by rumen microbes.

This study examines the colonization of barley straw (BS) and corn stover (CS) by rumen bacteria and how this is impacted by ammonia fiber expansion (AFEX) pre-treatment. A total of four ruminally cannulated beef heifers were used to investigate in situ microbial colonization in a factorial design with two crop residues, pre-treated with or without AFEX. Crop residues were incubated in the rumen for 0, 2, 4, 8 and 48 h and the colonizing profile was determined using 16 s rRNA gene sequencing. The surface colonizing community clustered based on incubation time and pre-treatment. Fibrobacter, unclassified Bacteroidales, and unclassified Ruminococcaceae were enriched during late stages of colonization. Prevotella and unclassified Lachnospiraceae were enriched in the early stages of colonization. The microbial community colonizing BS-AFEX and CS was less diverse than the community colonizing BS and CS-AFEX. Prevotella, Coprococcus and Clostridium were enriched in both AFEX crop residues, while untreated crop residues were enriched with Methanobrevibacter. Several pathways associated with simple carbohydrate metabolism were enriched in the primary colonizing community of AFEX crop residues. This study suggests that AFEX improves the degradability of crop residues by increasing the accessibility of polysaccharides that can be metabolized by the dominant taxa responsible for primary colonization.

A Pine Enhanced Biochar Does Not Decrease Enteric CH4 Emissions, but Alters the Rumen Microbiota.

The objective of this study was to examine the effect of a pine enhanced biochar (EB) on rumen fermentation, apparent total tract digestibility, methane (CH4) emissions, and the rumen and fecal microbiome of Angus × Hereford heifers fed a barley silage-based diet. The experiment was a replicated 4 × 4 Latin square using 8 ruminally cannulated heifers (565 ± 35 kg initial BW). The basal diet contained 60% barley silage, 35% barley grain and 5% mineral supplement with EB added at 0% (control), 0.5, 1.0, or 2.0% (DM basis). Each period lasted 28 days, consisting of 14 days adaptation and 14 days of measurements. Samples for profiling of the microbiome in rumen liquid, solids and feces were collected on d 15 before feeding. Rumen samples for fermentation characterization were taken at 0, 3, 6, and 12 h post feeding. Total collection of urine and feces was conducted from days 18 to 22. Heifers were housed in open-circuit respiratory chambers on days 26-28 to estimate CH4 emissions. Ruminal pH was recorded at 1-min intervals during CH4 measurements using indwelling pH loggers. Data were analyzed with the fixed effects of dietary treatment and random effects of square, heifer within square and period. Dry matter intake was similar across treatments (P = 0.21). Ammonia N concentration and protozoa counts responded quadratically (P = 0.01) to EB in which both were decreased by EB included at 0.5 and 1.0%, compared to the control and 2.0% EB. Minimum pH was increased (P = 0.04), and variation of pH was decreased (P = 0.03) by 2.0% EB. Total tract digestibility, N balance and CH4 production were not affected (P ≥ 0.17) by EB. Enhanced biochar decreased the relative abundance of Fibrobacter (P = 0.05) and Tenericutes (P = 0.01), and increased the relative abundance of Spirochaetaes (P = 0.01), Verrucomicrobia (P = 0.02), and Elusimicrobia (P = 0.02). Results suggest that at the examined concentrations, EB was ineffective at decreasing enteric CH4 emissions, but did alter specific rumen microbiota.

Unconventional Vegetable Oils for a Reduction of Methanogenesis and Modulation of Ruminal Fermentation.

The objective of this study was to evaluate the use of vegetable oils from plants grown in Brazil, first using the in vitro batch culture, and then evaluating the oil with methane (CH4) reducing potential in an in vivo experiment. The in vitro experiment was conducted as a completely randomized design using the seven contrasting oils. Treatments consisted of a control and 3 increasing concentrations (0, 1, 2, and 5% v/v) of oil added to a tifton 85 hay samples. All vegetable oils linearly decreased (P < 0.01) gas production after 24 h of incubation, with the greatest reduction when 5% of oil was included into the diet. Açaí and buriti had no effect of CH4 (% or mL/g DM incubated) however carrot, macaúba, basil, passionflower, and pequi oil all linearly decreased (P < 0.01) CH4 production with increasing inclusion rate of oil. Pequi oil resulted in the largest decrease in CH4 production (mL/g DM incubated) after 24 h of in vitro incubation. The objective of the in vivo experiment was to evaluate the effects of pequi oil on nutrient digestibility, CH4 production, and rumen fermentation parameters in wethers fed a hay-based diet. The experiment was conducted as a 2 × 2 Latin Square design using 4 Dorper wethers (63.4 ± 1.46 kg body weight). There were 2 experimental periods of 21 d each, with d 1-14 used for diet adaptation and d 15-21 for measurements and collections. The treatments consisted of a control diet and pequi oil fed at 70 g per animal per day. The addition of pequi oil to the diet had no effect on feed intake or the digestibility of nutrients, however there was a numerical decrease in the population of cellulolytic bacteria. There was a tendency (P = 0.06) for pequi oil addition to decrease CH4 production (g/d) by 17.5%. From this study, we can conclude that pequi oil may be used as a suitable oil for reducing CH4 production from ruminants, with no negative effects on intake or digestibility.

Effect of humic substances on rumen fermentation, nutrient digestibility, methane emissions, and rumen microbiota in beef heifers1.

Ruminants play an important role in food security, but there is a growing concern about the impact of cattle on the environment, particularly regarding greenhouse gas emissions. The objective of this study was to examine the effect of humic substances (HS) on rumen fermentation, nutrient digestibility, methane (CH4) emissions, and the rumen microbiome of beef heifers fed a barley silage-based diet. The experiment was designed as a replicated 4 × 4 Latin square using 8 ruminally cannulated Angus × Hereford heifers (758 ± 40.7 kg initial BW). Heifers were offered a basal diet consisting of 60% barley silage and 40% concentrate (DM basis) with either 0- (control), 100-, 200- or 300-mg granulated HS/kg BW. Each period was 28 d with 14 d of adaptation. Rumen samples were taken on day 15 at 0, 3, 6, and 12 h postfeeding. Total urine and feces were collected from days 18 to 22. Blood samples were taken on day 22 at 0 and 6 h postfeeding. Between days 26 and 28, heifers were placed in open-circuit respiratory chambers to measure CH4. Ruminal pH was recorded continuously during the periods of CH4 measurement using indwelling pH loggers. Intake was similar (P = 0.47) across treatments. Concentration of ammonia-N and counts of rumen protozoa responded quadratically (P = 0.03), where both increased at H100 and then decreased for the H300 treatments. Apparent total tract digestibility of CP (P = 0.04) was linearly increased by HS and total N retention (g/d, % N intake, g/kg BW0.75) was improved (P = 0.04) for HS when compared with the control. There was no effect of HS on CH4 production (g/d; P = 0.83); however, HS decreased the relative abundance of Proteobacteria (P = 0.04) and increased the relative abundance of Synergistetes (P = 0.01) and Euryarchaeota (P = 0.04). Results suggest that HS included at up to 300 mg/kg BW may improve N retention and CP digestibility, but there was no impact on CH4 production.

Tucumã Oil Shifted Ruminal Fermentation, Reducing Methane Production and Altering the Microbiome but Decreased Substrate Digestibility Within a RUSITEC Fed a Mixed Hay - Concentrate Diet.

Tucumã oil is sourced from the fruit pulp of the tucumã tree and contains high concentrations of unsaturated fatty acids and carotenoids. Due to these properties it may have the potential to decrease enteric methane (CH4) from ruminants when included in the diet. The objective of this study was to determine the effect of oil mechanically extracted from the fruit pulp of tucumã on fermentation characteristics, CH4 production and the microbial community using the rumen stimulation technique. Treatments consisted of a control diet (forage:concentrate; 70:30), and tucumã oil included at 0.5 or 1.0% (v/v). Addition of tucumã oil linearly decreased (P < 0.01) dry matter disappearance. Total gas (mL/d) and carbon dioxide (CO2) production (mL/d, mL/g DM) were unaffected (P ≥ 0.36) to increasing addition of tucumã oil where 0.5% (v/v) of Tucumã oil numerically increased both variables. Acetate and butyrate percentages of total VFA were linearly decreased (P ≤ 0.01) and propionate and valerate percentages of total VFA were linearly increased (P < 0.01) by increasing concentrations of tucumã oil added to the substrate. The ratio of acetate to propionate was linearly decreased (P < 0.01) with increasing concentration of tucumã oil. Methane production (mL/d) was linearly decreased (P = 0.04) with increasing addition of tucumã oil to the substrate. Tucumã oil reduced the bacterial richness and diversity when included at 1.0% (v/v) in both solid- and liquid- associated microbes. The abundance of the genera Fibrobacter and Rikenellaceae RC9 gut group were decreased and Pyramidobacter, Megasphaera, Anaerovibrio, and Selenomonas were enriched by the addition of 1.0% tucumã oil. In conclusion, tucumã oil resulted in the favorable shift in fermentation products away from acetate toward propionate, decreasing the production of CH4 when tucumã oil was included at 1.0% (v/v), however, substrate digestibility was also inhibited. The rumen microbiota was also altered by the addition of tucumã oil.

Humic Substances Alter Ammonia Production and the Microbial Populations Within a RUSITEC Fed a Mixed Hay - Concentrate Diet.

Humic substances are a novel feed additive which may have the potential to mitigate enteric methane (CH4) production from ruminants as well as enhance microbial activity in the rumen. The aim of this study was to examine the effects of humic substances on fermentation characteristics and microbial communities using the rumen stimulation technique (RUSITEC). The experiment was conducted as a completely randomized design with 3 treatments duplicated in 2 runs (a 15-day period each run) with 2 replicates per run. Treatments consisted of a control diet (forage:concentrate; 60:40) without humic substances or humic substances added at either 1.5 g/d or 3.0 g/d. Dry matter disappearance, pH, fermentation parameters and gas production were measured from day 8 to 15. Samples for microbial profiling were taken on day 5, 10, and 15 using the digested feed bags for solid- associated microbes (SAM) and fermenter fluid for liquid- associated microbes (LAM). The inclusion of humic substances had no effect (P ≥ 0.19) on DM disappearance, pH or the concentrations of VFA. The production of NH3 was linearly decreased (P = 0.04) with increasing levels of humic substances in the diet. There was no effect (P ≥ 0.43) of humic substances on total gas, CO2 or CH4 production. The number of OTUs was significantly reduced in the 3.0 g/d treatment compared to the control on d 10 and 15; however, the microbial community structure was largely unaffected (P > 0.05). In the SAM samples, the genera Lachnospiraceae XPB1014 group, Succiniclasticum, and Fibrobacter were reduced in the 3.0 g/d treatment and Anaeroplasma, Olsenella, and Pseudobutyrivibrio were increased on day 5, 10, and 15. Within the LAM samples, Christensenellaceae R-7 and Succiniclasticum were the most differentially abundant genera between the control and 3.0 g/d HS treatment samples (P < 0.05). This study highlights the potential use of humic substances as a natural feed additive which may play a role in nitrogen metabolism without negatively affecting the ruminal microbiota.