Antibodies targeting specific bacterial species could allow for modification of the rumen microbial population to enhance rumen fermentation. However, there is limited knowledge of targeted antibody effects on rumen bacteria. Therefore, our objective was to develop efficacious polyclonal antibodies to inhibit the growth of targeted cellulolytic bacteria from the rumen. Egg-derived, polyclonal antibodies were developed against pure cultures of Ruminococcus albus 7 (anti-RA7), Ruminococcus albus 8 (anti-RA8), and Fibrobacter succinogenes S85 (anti-FS85). Antibodies were added to a cellobiose-containing growth medium for each of the three targeted species. Antibody efficacy was determined via inoculation time (0 h and 4 h) and dose response. Antibody doses included: 0 (CON), 1.3 × 10−4 (LO), 0.013 (MD), and 1.3 (HI) mg antibody per ml of medium. Each targeted species inoculated at 0 h with HI of their respective antibody had decreased (P < 0.01) final optical density and total acetate concentration after a 52 h growth period when compared with CON or LO. Live/dead stains of R. albus 7 and F. succinogenes S85 dosed at 0 h with HI of their respective antibody indicated a decrease (≥ 96%; P < 0.05) in live bacterial cells during the mid-log phase compared with CON or LO. Addition of HI of anti-FS85 at 0 h in F. succinogenes S85 cultures reduced (P < 0.01) total substrate disappearance over 52 h by at least 48% when compared with CON or LO. Cross-reactivity was assessed by adding HI at 0 h to non-targeted bacterial species. Addition of anti-RA8 or anti-RA7 to F. succinogenes S85 cultures did not affect (P ≥ 0.45) total acetate accumulation after 52 h incubation, indicating that antibodies have less of an inhibitory effect on non-target strains. Addition of anti-FS85 to non-cellulolytic strains did not affect (P ≥ 0.89) OD, substrate disappearance, or total VFA concentrations, providing further evidence of specificity against fiber-degrading bacteria. Western blotting with anti-FS85 indicated selective binding to F. succinogenes S85 proteins. Identification by LC-MS/MS of 8 selected protein spots indicated 7 were outer membrane proteins. Overall, polyclonal antibodies were more efficacious at inhibiting the growth of targeted cellulolytic bacteria than non-targeted bacteria. Validated polyclonal antibodies could serve as an effective approach to modify rumen bacterial populations.

The objective was to determine if a 7-day lactic acid adaptation in cannulated steers affected ruminal fermentation during an acidosis challenge. Steers (n=18; 790 ± 68 kg) were assigned to one of two treatments: control (CON) 500 mL of H2O or 1 mM DL-Lactic acid solution per kg steer BW (LAC). Steers were dosed with treatments via the cannula prior to feeding for seven days during the adaptation period. The adaptation diet consisted of 25% corn silage, 30% grass hay, 15% dry distillers grains, 25% dry rolled corn, 5% molasses and 10% supplement. To induce acidosis, steers were fasted for 24 hours and subsequently fed a high concentrate diet consisting of 15% corn silage, 15% dry distillers grains, 55% high moisture corn, 5% molasses, 10% supplement. Rumen fluid was collected throughout the acidosis challenge to measure fermentation characteristics including rumen pH, redox, ammonia, volatile fatty acids, and lactic acid. Data were analyzed using the MIXED procedure of SAS 9.4 with a repeated measures analysis. Dry matter intake was not affected (P ≥ 0.65) by treatment before or after the acidosis challenge. A treatment × time interaction was not observed (P = 0.83) for ruminal pH, but pH was affected (P < 0.01) by time and remained between 5.6 and 5.0 for about 14 hours during the challenge. A treatment × hour interaction tended (P = 0.08) to be observed for lactate; LAC had a greater concentration than CON at hour 14 during the challenge. However, a treatment × hour interaction was not detected (P ≥ 0.38) for ammonia, volatile fatty acids, fecal pH and redox potential. In conclusion, these results indicate that a 7-day lactic acid adaption at 1 mM per kg BW did not affect ruminal fermentation characteristics during an acidosis challenge.

The objective was to evaluate the effects of dietary treatments designed to increase passage rate on the rumen microbiome. Factors such as particle size, feedstuff degradability, and rumen motility may drive ruminal passage rates and alter the composition of rumen microorganisms responsible for methane formation. Eight ruminal-cannulated steers were assigned to a replicated 4 × 4 Latin square design. All steers were fed an ad libitum diet of prairie grass hay (10.4% crude protein) with treatments designed to increase passage rate: no treatment (CON), pelleted (PEL), 7% Ca(OH)2 treated (ALK), and six 2 kg weights inserted into the rumen (WTS). Ruminal contents were collected on d 13 for 16S rRNA sequencing using specific bacterial and archaeal primers. Bacterial and archaeal taxa were not different (P > 0.1) between WTS and CON. At the genus level, PEL-fed steers had decreased (P = 0.01) relative abundance of Butyrivibrio 2 when compared with CON. Steers fed ALK had decreased (P < 0.01) relative abundance of Rikenellaceae RC9 gut group but increased (P < 0.01) Christensenellaceae R-7 group compared with CON. In contrast, relative abundance of Christensenellaceae R-7 group decreased (P = 0.03) in steers fed PEL compared with CON. The most abundant archaea, Methanobrevibacter, increased (P = 0.04) in steers fed ALK compared with CON (75 vs 65% of archaeal reads, respectively). Additionally, relative abundance of Methanosphaera increased (P < 0.01) in steers fed PEL compared with CON. Measures of richness including Chao1 and observational taxonomic units were decreased (P < 0.01) in ALK-treated steers but were not affected (P > 0.2) by PEL or WTS compared with CON. In conclusion, pelleting and alkali treatment of hay affected the rumen microbiome composition compared with steers fed basal prairie grass hay. Increased passage rates may alter composition of rumen bacteria and archaea communities.

The objective was to develop efficacious polyclonal antibodies to inhibit growth of targeted cellulolytic rumen bacterial species. Many rumen microorganisms have overlapping functions, so inhibiting the activity of targeted bacterial species may help elucidate their dynamic contributions to rumen fermentation. Egg-derived, polyclonal antibodies were developed against whole-cell cultures of Ruminococcus albus 7 (anti-RA7), Ruminococcus albus 8 (anti-RA8), and Fibrobacter succinogenes S85 (anti-FS85). Antibodies were added to a cellobiose-containing growth medium of pure cultures of the 3 targeted species. Antibody efficacy was determined via an inoculation time (0 and 4 h) and dose response. Antibody doses included: 0 (CON), 0.013 (LO), 1.3 (MD), 13 (HI) mg antibody per 10 mL of medium. Each targeted species inoculated at 0 h with HI of their respective antibody had decreased (P < 0.01) final optical density and total volatile fatty acid (VFA) concentration after a 52-h growth period when compared with CON or LO. Live/dead stains of R. albus 7 and F. succinogenes S85 dosed at 0 h with HI of their respective antibody indicated a decrease (≥ 96%; P < 0.05) of live bacterial cells during mid-log phase compared with CON or LO. Addition of HI of anti-FS85 at 0 h in F. succinogenes S85 cultures reduced (P < 0.01) total substrate disappearance over 52 h by at least 48% when compared with CON or LO. Cross-reactivity was assessed by adding HI at 0 h to non-targeted bacterial species. Addition of anti-RA8 or anti-RA7 to F. succinogenes S85 cultures did not affect (P ≥ 0.45) total VFA accumulation after 52 h incubation. Overall, polyclonal antibodies were more efficacious at inhibiting growth of targeted cellulolytic bacteria than non-targeted bacteria. Specific polyclonal antibodies can be used to further characterize functional contributions of bacterial species to rumen fermentation.