In vitro Inoculation of Fresh or Frozen Rumen Fluid Distinguishes Contrasting Microbial Communities and Fermentation Induced by Increasing Forage to Concentrate Ratio

In vitro rumen batch culture is a technology to simulate rumen fermentation by inoculating microorganisms from rumen fluids. Although inocula (INO) are commonly derived from fresh rumen fluids, frozen rumen fluids are also employed for the advantages of storing, transporting, and preserving rumen microorganisms. The effects of frozen INO on microbial fermentation and community may be interfered with by substrate type, which has not been reported. This study was designed to test whether rumen fluid treatments (i.e., fresh and frozen) could interact with incubated substrates. A complete block design with fractional arrangement treatment was used to investigate the effects of INO (fresh or frozen rumen fluids) and concentrate-to-forage ratios (C/F, 1:4 or 1:1) on rumen fermentation and microbial community. The effects of increasing C/F were typical, including increased dry matter (DM) degradation and total volatile fatty acids (VFA) concentration (P < 0.001), and decreased acetate to propionate ratio (P = 0.01) and bacterial diversity of richness and evenness (P ≤ 0.005) with especially higher fermentative bacteria such as genus Rikenellaceae_RC, F082, Prevotella, Bacteroidales_BS11, Muribaculaceaege, and Christensenellaceae_R-7 (P ≤ 0.04). Although frozen INO decreased (P < 0.001) DM degradation and altered rumen fermentation with lower (P ≤ 0.01) acetate to propionate ratio and molar proportion of butyrate than fresh INO, typical effects of C/F were independent of INO, as indicated by insignificant INO × C/F interaction on substrate degradation, VFA profile and bacterial community (P ≥ 0.20). In summary, the effect of C/F on fermentation and bacterial diversity is not interfered with by INO type, and frozen INO can be used to distinguish the effect of starch content.

DHA-Rich Aurantiochytrium Biomass, a Novel Dietary Supplement, Resists Degradation by Rumen Microbiota without Disrupting Microbial Activity

We first sought to evaluate the effect of dietary supplementation with the docosahexaenoic acid (DHA)-rich microalgae, Aurantiochytrium limacinum (AURA), on rumen fermentation and the resistance of DHA to degradation and biohydrogenation by rumen microbes through ex vivo fermentation experiments. Subsequently, we sought to quantify the diet-derived DHA content of milk and the impact of AURA on microbial composition and metabolism in a pilot feeding trial with rumen-cannulated dairy cows. To achieve our aims, rumen fluid from cannulated cows was used as inoculum, and the effect of AURA inclusion on fermentation ex vivo was examined. At doses corresponding to the amount of AURA recommended for commercial production animals, only ~10% of DHA was degraded or biohydrogenated by rumen microorganisms. The results show that feeding with AURA had no effect on either total bacterial density or short-chain fatty acid production. Real-time quantitative PCR analysis of the rumen fluid samples collected during a seven-week in vivo trial revealed that microbes related to lactic acid metabolism and methanogenesis were significantly suppressed by the AURA-supplemented diet. The DHA concentration in milk increased over 25-fold with the AURA-supplemented diet and dropped by 30–40% within one week of washout. The addition of A. limacinum biomass to dairy cow diets resulted in positive effects on rumen microbial composition with no adverse effect on fermentation activity. AURA-derived DHA was stable, with only modest degradation in the rumen, and was successfully deposited in milk. This is the first study to investigate the effect of supplementing the diet of dairy cows with a protist-based biomass, namely, on important rumen fermentation parameters and on DHA deposition in milk, using a combination of ex vivo and in vivo approaches.

Effect of incremental proportions of Desmanthus spp. in isonitrogenous forage diets on growth performance, rumen fermentation and plasma metabolites of pen-fed growing Brahman, Charbray and Droughtmaster crossbred beef steers

Desmanthus (Desmanthus spp.), a tropically adapted pasture legume, is highly productive and has the potential to reduce methane emissions in beef cattle. However, liveweight gain response to desmanthus supplementation has been inconclusive in ruminants. This study aimed to evaluate weight gain, rumen fermentation and plasma metabolites of Australian tropical beef cattle in response to supplementation with incremental levels of desmanthus forage legume in isonitrogenous diets. Forty-eight Brahman, Charbray and Droughtmaster crossbred beef steers were pen-housed and fed a basal diet of Rhodes grass (Chloris gayana) hay supplemented with 0, 15, 30 or 45% freshly chopped desmanthus forage on dry matter basis, for 140 days. Varying levels of lucerne (Medicago sativa) hay were added in the 0, 15 and 30% diets to ensure that all diets were isonitrogenous with the 45% desmanthus diet. Data were analyzed using the Mixed Model procedures of SAS software. Results showed that the proportion of desmanthus in the diet had no significant effect on steer liveweight, rumen volatile fatty acids molar proportions and plasma metabolites (P ≥ 0.067). Total bilirubin ranged between 3.0 and 3.6 μmol/L for all the diet treatments (P = 0.67). All plasma metabolites measured were within the expected normal range reported for beef cattle. Rumen ammonia nitrogen content was above the 10 mg/dl threshold required to maintain effective rumen microbial activity and maximize voluntary feed intake in cattle fed low-quality tropical forages. The average daily weight gains averaged 0.5 to 0.6 kg/day (P = 0.13) and were within the range required to meet the target slaughter weight for prime beef markets within 2.5 years of age. These results indicate that desmanthus alone or mixed with other high-quality legume forages can be used to supplement grass-based diets to improve tropical beef cattle production in northern Australia with no adverse effect on cattle health.

Effect of Oregano Oil and Cobalt Lactate on Sheep In Vitro Digestibility, Fermentation Characteristics and Rumen Microbial Community

The objective of this experiment was to evaluate the effect of different EOC (0.1425% cobalt lactate + 1.13% oregano essential oil + 98.7275% carrier) levels on in vitro rumen fermentation and microbial changes. Six EOC levels (treatments: 0 mg·L−1, CON; 50 mg·L−1, EOC1; 100 mg·L−1, EOC2; 400 mg·L−1, EOC3; 800 mg·L−1, EOC4 and 1500 mg·L−1, EOC5) were selected to be used to in vitro incubation. The in vitro dry matter digestibility (IVDMD), in vitro neutral detergent fiber digestibility (IVNDFD), in vitro acid detergent fiber digestibility (IVNDFD), pH, ammonia-nitrogen (NH3-N) concentration, total volatile fatty acid (TVFA) concentration and microbial protein (MCP) concentration were measured after 48 h incubation, after which the groups with significant nutrient digestibility and fermentation parameters were subjected to 16S rRNA sequencing. The results showed that the total gas production (GP) of the EOC5 group was higher than that of the other groups after 12 h of in vitro incubation. TVFA, NH3-N and MCP concentrations were also shown to be higher in group EOC5 than those in other groups (p < 0.05), while NH3-N and MCP concentrations in the EOC2 group were lower than those in other groups significantly (p < 0.05). The molar ratio of acetic acid decreased while the molar ratio of propionic acid increased after the addition of EOC. 16S rRNA sequencing revealed that the rumen microbiota was altered in response to adding EOC, especially for the EOC5 treatment, with firmicutes shown to be the most abundant (43.1%). The relative abundance of Rikenellaceae_RC9_gut_group was significantly lower, while the relative abundance of uncultured_bacterium_f_Muribaculaceae and Succiniclasticum was significantly higher in the EOC5 group than those in other groups (p < 0.05). Comprehensive analysis showed that EOC (1500 mg·L−1) could significantly increase gas production, alter sheep rumen fermentation parameters and microbiota composition.

Effect of urea coating by chitosan on the dynamics of ammonia concentration and rumen fermentation in vitro

The aim of this study was to determine the effect of coating urea by chitosan at graded levels on ammonia concentration and rumen fermentation in vitro. This study used Factorial Randomized Complete Block Design (RCBD) to test ammonia parameter and Randomized Complete Block Design (RCBD) for pH, microbial protein synthesis, dry matter and organic matter digestibility, and Volatile Fatty Acid (VFA). The treatments tested were: P0 = addition non coating urea 1%; P1 = coating urea by chitosan 1%; P2 = coating urea by chitosan 2%; P3 = coating urea by chitosan 3%. The data obtained were analysed by using ANOVA and continued with Tukey HSD test with SPSS version 25. The results of this study showed that the coating of urea chitosan had no significant effect on pH, dry matter and organic matter digestibility, microbial protein synthesis, and amonia concentration in the rumen. However, it significantly reduced (P <0.05) total VFA concentration. It can be concluded that the application of urea coating by chitosan does not affect on the degradation of urea in the rumen.

Mitragyna speciosa Korth Leaves Supplementation on Feed Utilization, Rumen Fermentation Efficiency, Microbial Population, and Methane Production In Vitro

The objective of the research was to evaluate the different levels of Mitragyna speciosa Korth leaves powder (MSLP) added to rations with 60:40 or 40:60 roughage to a concentrate (R:C ratio) on in vitro nutrient digestibility, rumen fermentation characteristics, microbial population, and methane (CH4) production. The treatments were arranged according to a 2 × 8 factorial arrangement in a completely randomized design. The two factors contain the R:C ratio (60:40 and 40:60) and the levels of MSLP addition (0, 1, 2, 3, 4, 5, 6, and 7% of the total substrate). There was no interaction between the R:C ratio and MSLP supplementation on gas production kinetics, ammonia nitrogen (NH3-N), and microbial populations. The gas production rate constant for the insoluble fraction (c) was increased by the R:C ratio at (40:60), whilst there was no difference obtained among treatments for cumulative gas production, whilst the gas production rate constant for the insoluble fraction (c) was increased by the R:C ratio at 40:60. The concentration of NH3-N was influenced by the R:C ratio and MSLP addition both at 4 and 8 h after incubation. In vitro dry matter degradability (IVDMD) and organic matter degradability (IVOMD) were significantly improved by the R:C ratio and supplementation of MSLP at 12 h. Increasing the R:C ratio and MSLP concentrations increased total volatile fatty acid (VFA) and propionic acid (C3) concentrations while decreasing acetic acid (C2) and butyric acid (C4) concentrations; thus, the C2:C3 ratio was reduced. MSLP addition reduced protozoa and methanogen populations (p < 0.05). The calculated CH4 production was decreased (p < 0.05) by the R:C ratios at 40:60 and supplementation of MSLP. Finally, the addition of MSLP as a phytonutrient may improve nutrient degradability and rumen fermentation properties while decreasing protozoa, methanogen population, and CH4 production.

Effects of Selenium Supplementation on Rumen Microbiota, Rumen Fermentation and Apparent Nutrient Digestibility of Ruminant Animals: A Review

Enzymes excreted by rumen microbiome facilitate the conversion of ingested plant materials into major nutrients (e.g., volatile fatty acids (VFA) and microbial proteins) required for animal growth. Diet, animal age, and health affect the structure of the rumen microbial community. Pathogenic organisms in the rumen negatively affect fermentation processes in favor of energy loss and animal deprivation of nutrients in ingested feed. Drawing from the ban on antibiotic use during the last decade, the livestock industry has been focused on increasing rumen microbial nutrient supply to ruminants through the use of natural supplements that are capable of promoting the activity of beneficial rumen microflora. Selenium (Se) is a trace mineral commonly used as a supplement to regulate animal metabolism. However, a clear understanding of its effects on rumen microbial composition and rumen fermentation is not available. This review summarized the available literature for the effects of Se on specific rumen microorganisms along with consequences for rumen fermentation and digestibility. Some positive effects on total VFA, the molar proportion of propionate, acetate to propionate ratio, ruminal NH3-N, pH, enzymatic activity, ruminal microbiome composition, and digestibility were recorded. Because Se nanoparticles (SeNPs) were more effective than other forms of Se, more studies are needed to compare the effectiveness of synthetic SeNPs and lactic acid bacteria enriched with sodium selenite as a biological source of SeNPs and probiotics. Future studies also need to evaluate the effect of dietary Se on methane emissions.