Effects on ruminal fermentation influenced by substrate, exogenous fibrolytic enzymes, and solventogenesis pathways

Abstract

The effects of dietary pre-treatment with exogenous fibrolytic enzymes on ruminal substrate degradation kinetics and ruminal microbiome relative abundances were evaluated. A 5 × 4 unbalanced Latin square design using ruminally cannulated steers (n = 5; BW = 520 ± 30 kg; experimental units) was performed. A 2 × 2 factorial arrangement of treatments was used, in which the first factor was a beef cattle grower diet type (high quality = HQ; and low = LQ); and the second factor being the dietary pre-treatment with exogenous fibrolytic enzymes (0 or 0.75 mL/kg of diet DM = ENZYME; AB Vista, UK). Steers were offered diets to ad libitum intake during four 21-d periods. The ruminal in situ degradation kinetics model was performed with pre-dehydrated (55ºC for 72 h) wheat hay substrate, which was ground (2 mm) and placed into 10 × 20 cm (28 µm) nylon bags (5g, as-is) in duplicates. Substrate in situ bags were placed within a nylon mesh (with weights) at the ruminal ventral sac on d 17, while reversely removed at 0, 3, 6, 12, 18, 24, 30, 48, 72, and 96 h after feeding. Upon removal, samples were rinsed and dehydrated for 72 h at 55oC (forced air oven). Degradation residues duplicates were composited and organized within period, steer, and incubation time. Samples were adjusted for residual moisture (100oC, 4 h), and analyzed for ash, NDF, and ADF content, while hemicellulose was calculated by difference (NDF - ADF). Substrate bag residues of each nutritional analysis was used to fit a first-order kinetics model using the NLIN procedure of SAS and the Glimmix procedure of SAS, in which the effective degradability of fractions among other variables were calculated. Ruminal content samples (45 mL) were collected on d 16 at 6 h after feeding from five locations within the rumen for DNA extraction and determination of microbial relative abundances. Microbiome data were sequenced by Illumnia® NovaSeq™ 6000 (16S rRNA). Data were analyzed using the GLIMMIX procedures of SAS using the fixed effects of dietary type, addition of enzyme, and the interaction between them, while steer within sequence of dietary treatments was used as random effect. ENZYME increased (P = 0.03) the substrate fraction B (potentially degradable) and decreased (P = 0.03) substrate fraction C (undegradable fraction) of HQ grower diets, while such effect was not observed for LQ diets. No diet × ENZYME interaction (P ≥ 0.14) was observed for other kinetic of degradation variables, except by tendencies (P = 0.09) in which ENZYME numerically increased rate of degradation (kd, %/h) of organic matter (OM) and the fraction B of hemicellulose of HQ diets while not affecting the LQ. Regardless of dietary type, ENZYME increased (P ≤ 0.05) the substrate effective degradability of OM, NDF, ADF; the kd (P ≤ 0.01) of DM, OM, and hemicellulose; and the fraction B of ADF (P ≤ 0.01); while decreasing (P ≤ 0.01) the fraction C of ADF, with few other tendencies (P = 0.06) following a similar positive pattern of response with the use of ENZYME. Regardless of the use of ENZYME, HQ diets had greater (P ≤ 0.01) DM kd, fraction B, while lower fraction C compared to LQ diets. Similar response was observed for OM, ADF, and hemicellulose. The ruminal microbiome relative abundance of the Class Bacilli tended (P = 0.11) to be numerically greater when ENZYME was used in LQ diets, while such indication was not observed for HQ diets. The same trend (P = 0.11) was also observed for the Order Lactobacillales, while no other interactions or main effects of ENZYME were observed (P ≥ 0.17). Regardless of ENZYME, steers offered LQ diet had greater (P ≤ 0.05) relative abundances of: Domain Bacteria; Phylum Bacteroidetes and Chloroflexi; Class Firmicutes (unclassified), Bacteroidia, Bacilli, Anaerolineae; Order Firmicutes (unclassified), Bacteroidales, and Lactobacillales; Family Firmicutes (unclassified), Prevotellaceae, and Ruminococcaceae; and Genus Firmicutes (unclassified) and Prevotella. Steers offered HQ diet had greater (P ≤ 0.05) relative abundances of: Domain Archea; Phylum Euryarchaeota; Class Clostridia, Methanobacteria, and Chloroplast; Order Clostridiales, Methanobacteriales, and Chloroplast; Clostridiales (unclassified) and Methanobacteriaceae; and Genus Clostridiales (unclassified). The pre-treatment of beef cattle grower diets with exogenous fibrolytic enzymes enhanced the ruminal effective degradable fraction of wheat hay substrate and tended to positively affect the relative abundance of ruminal microbiota within the Class Bacilli, represented by the Order Lactobacillales. A reduction in the ruminal degradation lag time does not seem to fully explain the effect of enzyme, but rather an improvement in the rate of degradation of organic matter, especially represented by hemicellulose. The effects of inoculum type and starch substrate level on in vitro ruminal fermentation gas production kinetics, volatile fatty acid (VFA) profile, ammonia-N, pH, lactate, and solvent production were evaluated. Ruminally cannulated crossbred beef steers (n = 6; BW = 550 ± 50 kg) were used in a 3 × 3 Latin square design with factors including ruminal fluid inoculum type (wheat-hay based diet [HAY]; steam-flaked corn-based grower diet [GROWER]; and steam-flaked corn-based finisher diet [FINISHER]); and corn starch substrate inclusion level (0, 6, and 12 g, as-is basis). The in vitro incubations were conducted on dry incubators for 24 h at 39°C with a constant agitation at 125 rpm. Gas kinetics were measured using 330 mL fermentation flask units equipped with pressure sensor (ANKOM system) and residual gas collection system (sealed foil sampling bag with septa and Tygon hoses) using 250 mL of inoculum with no buffer (the inoculum collected from the steer-pairs offered the same diet was mixed prior to the in vitro incubation). Fermentation units were incubated in triplicate, while results were averaged within each treatment lab-replication before being included in the statistical model. Metabolites were measured post-incubation (frozen samples [20oC] added with 1% of a 20% H2SO4 v/v solution), except by pH which was measured immediately after fermentation kill (ice bath) and room temperature stabilization. Data were analyzed using the GLIMMIX procedures of SAS using incubation batch as the experimental unit (n = 3), the fixed effects of inoculum type, starch level, and the interaction inoculum-type × starch-level, and the random effect of incubation batch (inoculum × starch). Ethanol production (mM) was not affected (P ≥ 0.24) by treatments and showed measurable levels (3.11) for HAY+12g and GROWER+12g combinations only. Maximum gas production (mL/g substrate) was greater (P < 0.01) for GROWER+0g (528) and FINISHER+0g (478) compared to other treatments (100). The greatest gas production lag time was observed for HAY+0g (5.75 h) compared to other treatments (0.97 h). The greatest (P = 0.04) CH4 production (mL/L ruminal fluid) was observed for GROWER+6g (482) while the least observed for HAY+0g (73), with other treatments being intermediate (269). The greatest (P = 0.05) molar proportion (mM/100mM of tVFA) of butyrate was observed for GROWER+0g & 6g (25.7), while the least was observed for HAY+0g and FINISHER+6g & 12 g (14.6), with other treatments being intermediate (20.7). Acetate molar proportion tended (P = 0.09) to increase (57.61) for HAY+0g compared to other treatments (42.16). Kill-time in vitro pH was the greatest (P = 0.02) for HAY+0g (6.16), and the least observed for GROWER & FINISHER+6g &12g (4.78), while other treatments were intermediate (5.21). The least (P < 0.01) ammonia-N (mg/dL) was observed for HAY+6g & 12g (2.12) compared to other treatments (25.56). No other inoculum × substrate interaction (P ≥ 0.24) was observed. Regardless of starch inclusion level, HAY produced the least (P < 0.01) total gas, CO2, total VFA, and propionate molar proportion, while the largest (P < 0.01) acetate and C4:C3 ratio. Other subtle differences (P < 0.01) were observed for less prominent short chain fatty acids. Regardless of inoculum, 6 & 12g of starch substrate showed the greatest (P ≤ 0.02) % and volume of CO2, total gas production, total VFA, propionate, and lactate; while the least C4:C3 ratio, and acetate compared to 0g. Other subtle differences (P < 0.01) were observed for less prominent short chain fatty acids. Current in vitro ruminal fermentation model was capable to ratify expected outcomes of ruminal inoculums collected from beef steers consuming hay, grower, and finisher diets, as well as raising starch levels. However, it was not able to detect meaningful differences in cumulative 24 h solvent production