The objective of this study was to investigate the effects of essential oil mixture (EOM) supplementation on rumen fermentation characteristics and microbial changes in an in vitro. Three experimental treatments were used: control (CON, no additive), EOM 0.1 (supplementation of 1 g EOM/kg of substrate), and EOM 0.2 (supplementation of 2 g EOM/kg of substrate). An in vitro fermentation experiment was carried out using strained rumen fluid for 12 and 24 h incubation periods. At each time point, in vitro dry matter digestibility (IVDMD), neutral detergent fiber digestibility (IVNDFD), pH, ammonia nitrogen (NH3-N), and volatile fatty acid (VFA) concentrations, and relative microbial diversity were estimated. After 24 h incubation, treatments involving EOM supplementation led to significantly higher IVDMD (treatments and quadratic effect; p = 0.019 and 0.008) and IVNDFD (linear effect; p = 0.068) than did the CON treatment. The EOM 0.2 supplementation group had the highest NH3-N concentration (treatments; p = 0.032). Both EOM supplementations did not affect total VFA concentration and the proportion of individual VFAs; however, total VFA tended to increase in EOM supplementation groups, after 12 h incubation (linear; p = 0.071). Relative protozoa abundance significantly increased following EOM supplementation (treatments, p<0.001). Selenomonas ruminantium and Ruminococcus albus (treatments; p<0.001 and p = 0.005), abundance was higher in the EOM 0.1 treatment group than in CON. The abundance of Butyrivibrio fibrisolvens, fungi and Ruminococcus flavefaciens (treatments; p< 0.001, p<0.001, and p = 0.005) was higher following EOM 0.2 treatment. The addition of newly developed EOM increased IVDMD, IVNDFD, and tended to increase total VFA indicating that it may be used as a feed additive to improve rumen fermentation by modulating rumen microbial communities. Further studies would be required to investigate the detailed metabolic mechanism underlying the effects of EOM supplementation.

Streptococcus bovis (S. bovis) is one of the critical initiators of acute acidosis in ruminants. Therefore, we aimed to develop and characterize the endolysin LyJH307, which can lyse ruminal S. bovis. We tested the bactericidal activity of recombinant LyJH307 against S. bovis JB1 under a range of pH, temperature, NaCl, and metal ion concentrations. In silico analyses showed that LyJH307 has a modular design with a distinct, enzymatically active domain of the NLPC/P60 superfamily at the N-terminal and a cell wall binding domain of the Zoocin A target recognition domain (Zoocin A_TRD) superfamily at the C-terminal. The lytic activity of LyJH307 against S. bovis JB1 was the highest at pH 5.5, and relatively higher under acidic, than under alkaline conditions. LyJH307 activity was also the highest at 39 °C, but was maintained between 25°C and 55°C. LyJH307 bactericidal action was retained under 0-500 mM NaCl. While the activity of LyJH307 significantly decreased on treatment with ethylenediaminetetraacetic acid (EDTA), it was only restored with supplementation of 10 mM Ca²⁺. Analyses of antimicrobial spectra showed that LyJH307 lysed Lancefield groups D (S. bovis group and Enterococcus faecalis) and H (S. sanguinis) bacteria. Thus, LyJH307 might help to prevent acute ruminal acidosis.

Background: Endolysins, the bacteriophage-originated peptidoglycan hydrolases, are a promising replacement for antibiotics due to immediate lytic activity and no antibiotic resistance. The objectives of this study were to investigate the lytic activity of endolysin LyJH307 against S. bovis and to explore changes in rumen fermentation and microbiota in an in vitro system. Two treatments were used: 1) control, corn grain without LyJH307; and 2) LyJH307, corn grain with LyJH307 (4 U/mL). An in vitro fermentation experiment was performed using mixture of rumen fluid collected from two cannulated Holstein steers (450 ± 30 kg) and artificial saliva buffer mixed as 1:3 ratio for 12 h incubation time. In vitro dry matter digestibility, pH, volatile fatty acids, and lactate concentration were estimated at 12 h, and the gas production was measured at 6, 9, and 12 h. The rumen bacterial community was analyzed using 16S rRNA amplicon sequencing. Results: LyJH307 supplementation at 6 h incubation markedly decreased the absolute abundance of S. bovis (approximately 70% compared to control, P = 0.0289) and increased ruminal pH (P = 0.0335) at the 12 h incubation. The acetate proportion (P = 0.0362) was significantly increased after LyJH307 addition, whereas propionate (P = 0.0379) was decreased. LyJH307 supplementation increased D-lactate (P = 0.0340) without any change in L-lactate concentration (P > 0.10). There were no significant differences in Shannon’s index, Simpson’s index, Chao1 estimates, and evenness (P > 0.10). Based on Bray-Curtis dissimilarity matrices, the LyJH307 affected the overall shift in microbiota (P = 0.097). LyJH307 supplementation induced an increase of 11 genera containing Lachnoclostridium, WCHB1–41, unclassified genus Selenomonadaceae, Paraprevotella, vadinBE97, Ruminococcus gauvreauii group, Lactobacillus, Anaerorhabdus furcosa group, Victivallaceae, Desulfuromonadaceae, and Sediminispirochaeta. The predicted functional features represented by the Kyoto Encyclopedia of Genes and Genomes pathways were changed by LyJH307 toward a decrease of carbohydrate metabolism. Conclusions: LyJH307 caused a reduction of S. bovis and an increase of pH with shifts in minor microbiota and its metabolic pathways related to carbohydrate metabolism. This study provides the first insight into the availability of endolysin as a specific modulator for rumen and shows the possibility of endolysin degradation by rumen microbiota.

Objective: The objective of this study was to evaluate the effects of lysophospholipids (LPL) supplementation on rumen fermentation, degradability, and microbial diversity in forage with high oil diet in an in vitro system.Methods: Four experimental treatments were used: i) annual ryegrass (CON), ii) 93% annual ryegrass +7% corn oil on a dry matter (DM) basis (OiL), iii) OiL with a low level (0.08% of dietary DM) of LPL (LLPL), and iv) OiL with a high level (0.16% of dietary DM) of LPL (HLPL). An in vitro fermentation experiment was performed using strained rumen fluid for 48 h incubations. In vitro DM degradability (IVDMD), in vitro neutral detergent fiber degradability, pH, ammonia nitrogen (NH3-N), volatile fatty acid (VFA), and microbial diversity were estimated.Results: There was no significant change in IVDMD, pH, NH3-N, and total VFA production among treatments. The LPL supplementation significantly increased the proportion of butyrate and valerate (Linear effect [Lin], p = 0.004 and <0.001, respectively). The LPL supplementation tended to increase the total bacteria in a linear manner (p = 0.089). There were significant decreases in the relative proportions of cellulolytic (Fibrobacter succinogenes and Ruminococcus albus) and lipolytic (Anaerovibrio lipolytica and Butyrivibrio proteoclasticus) bacteria with increasing levels of LPL supplementation (Lin, p = 0.028, 0.006, 0.003, and 0.003, respectively).Conclusion: The LPL supplementation had antimicrobial effects on several cellulolytic and lipolytic bacteria, with no significant difference in nutrient degradability (DM and neutral detergent fiber) and general bacterial counts, suggesting that LPL supplementation might increase the enzymatic activity of rumen bacteria. Therefore, LPL supplementation may be more effective as an antimicrobial agent rather than as an emulsifier in the rumen.

Saponin possessed antiprotozoal activity; therefore, it is generally used to mitigate enteric methane (CH₄) emission in ruminant nutrition. The objective of this study was to evaluate the effects of Aloe saponaria (AS) supplementation on rumen fermentation, CH₄, and microbial diversity in an in vitro experiment over 48 h incubation. Five treatments were used: (1) no additives (CON), 1% and 2% dosage of heat-dried AS (HAS), and freeze-dried AS (FAS), and the experiments were performed three times. After the incubation, volatile fatty acids (VFAs), CH₄ proportion in total gas, and microbial diversity were evaluated. Gas production was assessed at 3, 6, 9, 12, 24, 36, and 48 h incubation. FAS addition significantly increased gas production compared to CON except for 6 and 48 h, whereas HAS addition did not significantly affect the gas production at all observed time points. AS addition significantly increased total VFA and the absolute abundances of fungi and Ruminococcus albus regardless of drying method. There was no significant change in the CH₄ proportion at 48 h, although the reduction in ciliate protozoa was detected in FAS-treated groups. In conclusion, FAS may be an effective feed additive to improve ruminal fermentation via enhancing feed utilization by rumen microbes.