Short communication: a novel multispecies bacteria-based direct-fed microbial supports in vitro gut barrier integrity challenged with a pathogen or pro-inflammatory cytokines.

We conducted two experiments to evaluate the effects of a novel bacterial-based direct-fed microbial (DFM) on intestinal barrier integrity using the in vitro transepithelial electrical resistance (TEER) assay. In experiment 1, human-derived Caco-2 cells received or not (CON) a DFM containing Ligilactobacillus (formerly Lactobacillus) animalis 506, Propionibacterium freudenreichii 507, Bacillus paralicheniformis 809, and B. subtilis 597 (BDP; BOVAMINE DEFEND® Plus) at a rate of 1 × 108 CFU/transwell. Concurrently with treatment application (CON or BDP), a pathogenic challenge of Clostridium perfringens type A was added alone (PAT) or with BDP (PAT + BDP) at a rate of 2.8 × 107 CFU/transwell in a 2 × 2 factorial arrangement. In experiment 2, Caco-2 cells were also assigned in a 2 × 2 factorial design to CON or BDP and then, 2 h post-treatment administration (CON and BDP), a mixture of tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) was added alone (CYT) or with BDP (CYT + BDP) at a 10:1 ratio, respectively. In both experiments, TEER was measured for 18 h. In experiment 1, a DFM × pathogen × hour interaction was observed for TEER (P < 0.0001). Adding the PAT alone initially tended to increase TEER vs. CON from 1.1 to 2.2 h (P ≤ 0.09), increased TEER at 3.2 h (P < 0.01), but reduced TEER from 5.4 to the end of the experimental period at 18.4 h (P ≤ 0.01). On the other hand, adding DFM, with or without the pathogenic challenge, yielded greater TEER vs. CON-CON and CON-PAT for most of the experimental period (P ≤ 0.04). A similar interaction was detected and reported in experiment 2 (P < 0.0001). The CYT challenge reduced mean TEER compared with all other treatments from 3.2 h to the remainder of the study (P ≤ 0.03). On the other hand, BDP-CYT was able to maintain the integrity of the epithelial cells when compared with CON-CON throughout the experimental period (P ≤ 0.03), the exception being at 3.2 h (P = 0.20). Moreover, BDP-CON increased (P ≤ 0.04) TEER when compared with CON-CON from 3.2 to 18.4 h, but also in comparison with BDP-CYT from 4.3 to 18.4 h post-DFM and challenge administration into the cells. In summary, C. perfringens type A and a pro-inflammatory cytokine cocktail compromised the integrity of intestinal epithelial cell monolayers in vitro, whereas adding a multispecies bacteria-based DFM counteracted these damaging effects.

Two experiments were designed to evaluate the effects of adding a bacterial-based direct-fed microbial (DFM) containing Lactobacillus animalis 506, Propionibacterium freudenreichii 507, Bacillus paralicheniformis 809, and Bacillus subtilis 597 on the integrity of intestinal epithelial cells challenged with Clostridium perfringens type A or a pro-inflammatory cytokine cocktail. Regardless of the challenge, the addition of the DFM maintained the integrity of the intestinal epithelial cells in vitro. These results help to elucidate the potential beneficial effects that the bacterial-based DFM containing L. animalis 506, P. freudenreichii 507, B. paralicheniformis 809, and B. subtilis 597 may bring to livestock species.

Bacillus paralicheniformis 809 and Bacillus subtilis 810 support in vitro intestinal integrity under hydrogen peroxide and deoxynivalenol challenges.

We designed and conducted two in vitro experiments to evaluate the effects of two Bacillus spp. probiotics on gut barrier integrity using the transepithelial electrical resistance (TEER) assay under two different challenge models. In Exp. 1, intestinal epithelial cells received or not (CON) B. paralicheniformis 809 (BLI) or B. subtilis 810 (BSU) at a rate of 1 × 108 colony forming units (CFU)/transwell. Two hours after treatment application (CON, BLI, or BSU), 5 mM of the reactive oxygen species hydrogen peroxide, mimicking mucosal oxidative stress, was added alone (HYP) or with each of the Bacillus spp. (HYP + BLI or HYP + BSU). In Exp. 2, cells were assigned to the same treatments as in Exp. 1 (CON, BLI, and BSU), or mycotoxin deoxynivalenol (DON), which was added alone or in combination with BLI or BSU, resulting in another two treatments (DON + BLI and DON + BSU). Transepithelial electrical resistance was measured for 14 h postchallenge. In Exp. 1, a treatment × hour interaction was observed for TEER (P < 0.0001). Adding BLI and BSU resulted in greater TEER values vs. CON for most of the experimental period (P < 0.02), whereas HYP reduced mean TEER and area under the curve (AUC), while increasing the amount of sugar that translocated through the monolayer cells (P < 0.001). A treatment × hour interaction was also observed in Exp. 2 (P < 0.0001), as DON led to an immediate and acute drop in TEER that lasted until the end of the experimental period (P < 0.0001). Both BLI and BSU alleviated the DON-induced damaging effects on the integrity of intestinal epithelial cells, whereas both Bacillus spp. alleviated the damage caused by DON alone and the proportion of sugar that translocated through the monolayer cells was not different between CON and DON + BLI (P = 0.14) and DON + BLI and DON + BSU (P = 0.62). In summary, both Bacillus spp. strains (B. paralicheniformis 809 and B. subtilis 810) were able to counteract the damaging effects of the challenge agents, hydrogen peroxide and deoxynivalenol, on gut barrier integrity.

Effects of feeding an inoculated corn silage with or without a direct-fed microbial on dry matter intake, milk production, and nutrient digestibility of high-producing lactating Holstein cows.

This study evaluated the effects of inoculating corn silage and/or feeding a direct-fed microbial (PRO) on performance and nutrient digestibility of lactating dairy cows. At harvesting, corn silage was treated either with water (culated or not [CON]) or Lactococcus lactis and Lentilactobacillus buchneri (INC; SiloSolve FC) at 1.5 × 105 cfu/g of corn silage. Ten mini silos and one farm-scale silo bunker per treatment were prepared for the laboratory and the lactating dairy cow trial, respectively. Five mini silos per treatment were opened on days 2 or 90 post-ensiling for pH measurement, as well as chemical analysis and aerobic stability, respectively. The farm-scale silo bunkers were opened 77 d post-ensiling for the beginning of the lactating cow trial. Eighty lactating Holstein cows were assigned in a 2 × 2 factorial design to: (1) CON silage without PRO (CON-CON; n = 20), (2) CON silage with PRO at 14 g/head/d (CON-PRO; n = 20), (3) INC silage without PRO (INC-CON; n = 20), and (4) INC silage with PRO at 14 g/head/d (INC-PRO; n = 20). Concurrently with the feeding trial, eight cows per treatment were chosen for nutrient digestibility. The pH of the corn silage was not affected by the silage inoculant (P ≥ 0.29), but INC yielded greater concentration of acetic acid and longer aerobic stability (P < 0.01). Dairy cows fed INC had a lower mean total dry matter intake (DMI), milk protein content, and somatic cell counts vs. CON (P ≤ 0.02). On the other hand, milk and fat- and protein-corrected milk (FPCM) production efficiency, milk urea-N, DM, crude protein, and starch digestibility were greater for INC-fed cows (P ≤ 0.03). Feeding direct-fed microbials (DFM) improved mean body weight, milk yield, and FPCM, as well as milk protein and lactose yield (P ≤ 0.05), but reduced milk fat and protein content (P = 0.02). A silage inoculant × DFM interaction was observed for milk production efficiency, milk protein and lactose content, and somatic cell count (P ≤ 0.05). Dairy cows fed INC-CON had a greater milk production efficiency and milk lactose content (P ≤ 0.04), but INC-PRO had lower milk protein content and SCC (P ≤ 0.03). In summary, inoculating L. lactis and L. buchneri increased acetic acid content and aerobic stability of corn silage, reduced DMI, but improved milk production efficiency and nutrient digestibility of lactating Holstein dairy cows. On the other hand, feeding PRO improved milk, protein, and lactose yield. Additionally, combining the feeding of an inoculated corn silage with PRO reduced milk somatic cell count.

Ligilactobacillus animalis 506 Protects the Intestinal Barrier from the Damaging Effects of Enteric Pathogens and Deoxynivalenol.

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.

Stability of Bacillus and Enterococcus faecium 669 Probiotic Strains When Added to Different Feed Matrices Used in Dairy Production.

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 (BOVACILLUSTM; 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.

Supplementation of direct-fed microbial Enterococcus faecium 669 affects performance of preweaning dairy calves.

Optimization and support of health and performance of preweaning dairy calves is paramount to any dairy operation, and natural solutions, such as probiotics, may help to achieve such a goal. Two experiments were designed to evaluate the effects of direct-fed microbial (DFM) Enterococcus faecium 669 on performance of preweaning dairy calves. In experiment 1, twenty 4-d-old Holstein calves [initial body weight (BW) 41 ± 2.1 kg] were randomly assigned to either (1) no probiotic supplementation (CON; n = 10) or (2) supplementation with probiotic strain E. faecium 669 during the preweaning period (DFM; n = 10) at 2.0 × 1010 cfu/kg of whole milk. Full individual BW was analyzed every 20 d for average daily gain (ADG) and feed efficiency (FE) determination. In experiment 2, thirty 4-d-old Holstein calves (initial BW 40 ± 1.9 kg) were assigned to the same treatments as in experiment 1 (CON and DFM). The DFM supplementation period was divided into period I (from d 0 to 21) and II (from d 22 to 63), with weaning occurring when animals were 67 d of age. During the entire experimental period, DFM was mixed into the whole milk at a rate of 1.5 × 1010 and 2.5 × 109 cfu/kg of whole milk/calf per day for periods I and II, respectively (6-time reduction). Full individual BW was taken every 21 d. As a routine of the experiment, calves were monitored daily, and diarrhea cases were evaluated using a daily 3-point fecal score. For both experiments, all data were analyzed using calf as the experimental unit. In experiment 1, DFM-supplemented calves were heavier on d 40 (+ 4.5 kg) and 60 (+ 6.5 kg) and had a greater ADG (+ 118 g) versus CON. In experiment 2, supplementation with DFM significantly tended to reduce diarrhea occurrence. Treatment × day and treatment × week interactions were observed for BW, ADG, and gain-to-feed ratio. Dairy calves supplemented with DFM were 1.8 and 3.5 kg heavier on d 42 and at weaning, respectively, and had a greater ADG from d 21 to 42 (+ 52 g) and 42 to 63 (+ 77 g) and gain-to-feed ratio from d 42 to 63 (+ 8.6%). In summary, supplementation of E. faecium 669 to dairy calves improved preweaning performance, even when the dose of the DFM was reduced by 6- to 8-times. Additionally, initial promising results were observed on diarrhea occurrence, but further studies are warranted.

Evaluation of a Bacillus-based direct-fed microbial probiotic on in vitro rumen gas production and nutrient digestibility of different feedstuffs and total mixed rations.

We evaluated the effects of a Bacillus-based direct-fed microbial (DFM) on total in vitro gas production, dry matter (DM), neutral detergent fiber (NDF), and starch disappearance of different feedstuffs and total mixed rations (TMR) in three different experiments. In experiment 1, six single fiber-based feedstuffs were evaluated: alfalfa hay, buffalo grass, beet pulp, eragrostis hay, oat hay, and smutsvinger grass. Experimental treatments were control (with no probiotic inoculation; CON) or incubation of a probiotic mixture containing Bacillus licheniformis and B. subtilis (3.2 × 109 CFU/g; DFM). The calculation of DFM dose under in vitro conditions was based on the assumption of a rumen capacity of 70 liter and the dose of 3 g of the DFM mixture/head/d (9.6 × 109 CFU). Total in vitro gas production, DM, and NDF disappearance were evaluated at 24- and 48 h posttreatment incubation. Mean treatment effects were observed at 24- and 48 h gas production (P < 0.0001), as DFM incubation increased in vitro gas production by 5.0% and 6.5%, respectively. For nutrient digestibility, mean DM digestibility was increased at 48 h (P = 0.05), whereas mean NDF digestibility increased at both timepoints by incubating DFM in vitro (P ≤ 0.02). In experiment 2, nine commercial dairy TMR were collected and evaluated for the same variables and treatments described in experiment 1, with the additional analysis of starch digestibility at 7 h post in vitro incubation. The only difference was the concentration of the DFM included, being representative for a dosage of 8.8 × 109 CFU/head/d. In vitro gas production was increased only at 48 h due to DFM incubation (P = 0.05), whereas DM and NDF digestibility were improved at 24 and 48 h (P ≤ 0.02). No treatment effects were observed on in vitro starch digestibility (P = 0.31). In experiment 3, a combined analysis of DM and NDF digestibility was performed by using quality values (NDF and crude protein or CP) of 16 substrates. Regardless of CP and NDF levels of the substrates, DFM improved in vitro 24 and 48 h DM and NDF digestibility (P ≤ 0.03). In summary, incubating a Bacillus-based DFM (B. licheniformis and B. subtilis; BOVACILLUS) improved mean in vitro gas production, DM, and NDF digestibility of single feedstuffs and commercial dairy TMR, highlighting the potential of this combination of Bacillus spp. to improve nutrient utilization, mainly fiber.

Effects of a Bacillus-based direct-fed microbial on in vitro nutrient digestibility of forage and high-starch concentrate substrates.

Two experiments evaluated the effects of a Bacillus-based direct-fed microbial (DFM) on in vitro dry matter (DM) and neutral detergent fiber (NDF; experiment 1) and starch (experiment 2) digestibility of a variety of ruminant feedstuffs. In experiment 1, 10 forage sources were evaluated: ryegrass, alfalfa hay, leucaena, corn silage, spinifex, buffel grass, flinders grass, Mitchell grass, Rhodes grass hay, and Queensland bluegrass. Experimental treatments were control (forages with no probiotic inoculation; CON) and forage sources inoculated with a mixture containing Bacillus licheniformis and Bacillus subtilis (3.2 × 109 CFU per g; DFM). In vitro DM and NDF digestibility were evaluated at 24- and 48-h post-treatment inoculation. Treatment × hour interactions were noted for IVDMD (in vitro dry matter digestibility) and IVNDFD (in vitro neutral detergent fibre digestibility) (P ≤ 0.05). More specifically, DFM inoculation increased (P ≤ 0.03) IVDMD at 24 h in four forages and increased 48-h IVDMD (P ≤ 0.02) in alfalfa hay, ryegrass, leucaena, and Mitchell grass hay, but opposite results were observed for Queensland bluegrass (P < 0.01). A 24- and 48-h IVNDFD increased following DFM inoculation (P ≤ 0.02) in five forage sources, but reduced for Queensland bluegrass (P < 0.01). When the forages were classified according to their quality, main treatment effects were detected for IVDMD (P ≤ 0.02) and IVNDFD (P < 0.01). In experiment 2, five common cereal grains were evaluated-high-density barley (82 g/100 mL), low-density barley (69 g/100 mL), corn, sorghum, and wheat-under the same treatments as in experiment 1. In vitro starch digestibility (IVSD) was evaluated at 6- and 12-h following treatment inoculation. Treatment × hour interactions were observed for starch digestibility in three out of five concentrate sources (P ≤ 0.001). Inoculation of DFM yielded greater 24-h starch digestibility for high-, low-density barley, and wheat (P ≤ 0.02), but also greater at 48 h in wheat (P < 0.0001). Moreover, mean starch digestibility improved for corn and sorghum inoculated with DFM (P < 0.01). Using a Bacillus-based DFM (B. licheniformis and B. subtilis) improved the mean in vitro DM and NDF digestibility of different forage sources of varying qualities (based on crude protein content). Similarly, IVSD was also greater following DFM inoculation, highlighting the potential of this probiotic to improve nutrient digestibility and utilization in the beef and dairy cattle herd.

Feeding lambs with silage mixtures of grass, sainfoin and red clover improves meat oxidative stability under high oxidative challenge.

This study investigated the oxidative stability of meat from lambs fed silages in which timothy grass was totally or partially replaced by sainfoin and/or red clover. Five groups of 8 lambs were fed the following silages: timothy grass (T), mixture (50:50) of timothy with either sainfoin (T-SF) or red clover (T-RC), mixture of timothy, sainfoin and red clover (50:25:25; T-SF-RC), or mixture (50:50) of sainfoin and red clover (SF-RC). Feeding the silages containing red clover (T-RC, T-SF-RC and SF-RC) decreased the deposition of vitamin E in muscle relative to the highly unsaturated fatty acids (P < .001), did not affect the oxidative stability of fresh meat, but reduced oxidative deterioration in cooked meat and in meat homogenates incubated with pro-oxidant catalysts (P < .001). The results of this study demonstrated that feeding lambs with silages containing sainfoin and red clover improve the oxidative stability of meat subjected to strong pro-oxidant conditions and suggest that these effects should be further clarified.

Sodium Hydroxide Enhances Extractability and Analysis of Proanthocyanidins in Ensiled Sainfoin (Onobrychis viciifolia).

Little information exists on the effects of ensiling on condensed tannins or proanthocyanidins. The acetone-butanol-HCl assay is suitable for measuring proanthocyanidin contents in a wide range of samples, silages included, but provides limited information on proanthocyanidin composition, which is of interest for deciphering the relationships between tannins and their bioactivities in terms of animal nutrition or health. Degradation with benzyl mercaptan (thiolysis) provides information on proanthocyanidin composition, but proanthocyanidins in several sainfoin silages have proved resistant to thiolysis. We now report that a pretreatment step with sodium hydroxide prior to thiolysis was needed to enable their analysis. This alkaline treatment increased their extractability from ensiled sainfoin and facilitated especially the release of larger proanthocyanidins. Ensiling reduced assayable proanthocyanidins by 29%, but the composition of the remaining proanthocyanidins in silage resembled that of the fresh plants.