Direct-fed microbial products (DFM) are probiotics that can be used advantageously in ruminant production. The in vitro gas production technique (IVGPT) is a method to simulate rumen fermentation and can be used to measure degradation, gas production, and products of fermentation of such additives. However, inter-laboratory differences have been reported. Therefore, tests using the same material were used to validate laboratory reproducibility. The objective of this study was to assess the effect of adding two DFM formulations on fermentation kinetics, methane (CH₄) production, and feed degradation in two different basal feeds while validating a newly established IVGPT laboratory. Six treatments, with three replicates each, were tested simultaneously at the established IVGPT lab at the University of Copenhagen, and the new IVGPT lab at Chr. Hansen Laboratories. Maize silage (MS) and grass silage (GS) were fermented with and without the following DFM: P1: Ligilactobacillus animalis and Propionibacterium freudenreichii (total 1.5 × 10⁷ CFU/mL), P2: P1 with added Bacillus subtilis and B. licheniformis (total 5.9 × 10⁷ CFU/mL). The DFM were anaerobically incubated in rumen fluid and buffer with freeze-dried silage samples for 48 h. Total gas production (TGP: mL at Standard Temperature and Pressure/gram of organic matter), pH, organic matter degradability (dOM), CH₄concentration (MC) and yield (MY), and volatile fatty acid (VFA) production and profiles were measured after fermentation. No significant differences between the laboratories were detected for any response variables. The dOM of MS (78.3%) was significantly less than GS (81.4%), regardless of the DFM added (P1 and P2). There were no significant differences between the effects of the DFM within the feed type. MS produced significantly more gas than GS after 48 h, but GS with DFM produced significantly more gas at 3 and 9 h and a similar gas volume at 12 h. Both DFM increased TGP significantly in GS at 48 h. There was no difference in total VFA production. However, GS with and without probiotics produced significantly more propionic acid and less butyric acid than MS with and without probiotics. Adding P2 numerically reduced the total methane yield by 4–6% in both MS and GS. The fermentation duration of 48 h, used to determine maximum potential dOM, may give misleading results. This study showed that it is possible to standardize the methodology to achieve reproducibility of IVGPT results. Furthermore, the results suggest that the P2 DFM may have the potential to reduce CH₄ production without affecting organic matter degradation.

This study investigated the impact of L. animalis 506 on gut barrier integrity and regulation of inflammation in vitro using intestinal epithelial cell lines. Caco-2 or HT29 cell monolayers were challenged with enterotoxigenic E. coli (ETEC) or a ruminant isolate of Salmonella Heidelberg in the presence or absence of one of six probiotic Lactobacillus spp. strains. Among these, L. animalis 506 excelled at exerting protective effects by significantly mitigating the decreased transepithelial electrical resistance (TEER) as assessed using area under the curve (AUC) (p < 0.0001) and increased apical-to-basolateral fluorescein isothiocyanate (FITC) dextran translocation (p < 0.0001) across Caco-2 cell monolayers caused by S. Heidelberg or ETEC, respectively. Similarly, L. animalis 506 and other probiotic strains significantly attenuated the S. Heidelberg- and ETEC-induced increase in IL-8 from HT29 cells (p < 0.0001). Moreover, L. animalis 506 significantly counteracted the TEER decrease (p < 0.0001) and FITC dextran translocation (p < 0.0001) upon challenge with Clostridium perfringens. Finally, L. animalis 506 significantly attenuated DON-induced TEER decrease (p < 0.01) and FITC dextran translocation (p < 0.05) and mitigated occludin and zona occludens (ZO)-1 redistribution in Caco-2 cells caused by the mycotoxin. Collectively, these results demonstrate the ability of L. animalis 506 to confer protective effects on the intestinal epithelium in vitro upon challenge with enteric pathogens and DON known to be of particular concern in farm animals.

Few data are available evaluating the stability of direct-fed microbials (DFM) following their inclusion in different feed matrices. Therefore, six Exp. evaluated the recovery of bacilli spores (BOVACILLUS^TM; Exp. 1 to 3) and an Enterococcus faecium DFM (LACTIFERM^®; Exp. 4 to 6) when included in different feed preparations. The Bacillus-based DFM was included into pelleted feed prepared in different temperatures (75 to 95 °C), whereas both DFM were assessed in premix and milk replacer preparations. Bacillus spores and E. faecium recovery was evaluated through standard methodologies and data were reported as log10 colony forming units/gram of feed. The recovery of Bacillus spores was within the expected range and was not impacted by the temperature of pellet preparation (Exp. 1). Bacilli recovery was also stable up to 12 months in the premix and was not impacted by the temperature of milk replacer preparation. Regarding the Exp. with E. faecium (Exp. 4 to 6), its recoveries in the mineral premix and milk powder did not differ from T0 and were not impacted by the conditions of milk replacer preparation. These data are novel and demonstrate the stability of a Bacillus-based and an E. faecium-based DFM when included in different feed matrices often used in dairy production.

Two experiments were conducted to evaluate the in vitro and in vivo effects of a novel direct-fed microbial (DFM) containing Lactobacillus animalis LA-51, Propionibacterium freudenreichii PF-24, Bacillus licheniformis CH-200, and Bacillus subtilis King (BOVAMINE DEFEND^® Plus) against Clostridium perfringens pathogenic strains. In Experiment 1 (in vitro), an agar diffusion assay was performed to qualitatively evaluate the in vitro inhibitory effects of the DFM against C. perfringens types A and C. Including the DFM in the tested yielded inhibition zones with greater than three ring diameters in a 96-well plate. In Experiment 2 (in vivo), twenty 1-day-old beef calves were allocated to control (n = 10) or DFM (n = 10) for 21 days. All calves were orally challenged with 1.0 × 10⁸ colony forming units of C. perfringens type A strain S-107 per head. The procedures such as general health scores, body weight, and fecal sample collections were performed following the C. perfringens challenge. Daily feeding of DFM significantly reduced the incidence of diarrhea while improving general impression and appearance scores of calves. Overall, these results highlight the ability of the DFM containing L. animalis LA-51, P. freudenreichii PF-24, B. licheniformis CH-200, and B. subtilis (BOVAMINE DEFEND^® Plus) to inhibit C. perfringens types A and C under different experimental settings.