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.

Thymol Improves Barrier Function and Attenuates Inflammatory Responses in Porcine Intestinal Epithelial Cells during Lipopolysaccharide (LPS)-Induced Inflammation.

It is well-known that essential oil thymol exhibits antibacterial activity. The protective effects of thymol on pig intestine during inflammation is yet to be investigated. In this study, an in vitro lipopolysaccharide (LPS)-induced inflammation model using IPEC-J2 cells was established. Cells were pretreated with thymol for 1 h and then exposed to LPS for various assays. Interleukin 8 (IL-8) secretion, the mRNA abundance of cytokines, reactive oxygen species (ROS), nutrient transporters, and tight junction proteins was measured. The results showed that LPS stimulation increased IL-8 secretion, ROS production, and tumor necrosis factor alpha (TNF-α) mRNA abundance ( P < 0.05), but the mRNA abundance of sodium-dependent glucose transporter 1 (SGLT1), excitatory amino acid transporter 1 (EAAC1), and H+/peptide cotransporter 1 (PepT1) were decreased ( P < 0.05). Thymol blocked ROS production ( P < 0.05) and tended to decrease the production of LPS-induced IL-8 secretion ( P = 0.0766). The mRNA abundance of IL-8 and TNF-α was reduced by thymol pretreatment ( P < 0.05), but thymol did not improve the gene expression of nutrient transporters ( P > 0.05). The transepithelial electrical resistance (TEER) was reduced and cell permeability increased by LPS treatment ( P < 0.05), but these effects were attenuated by thymol ( P < 0.05). Moreover, thymol increased zonula occludens-1 (ZO-1) and actin staining in the cells. However, the mRNA abundance of ZO-1 and occludin-3 was not affected by either LPS or thymol treatments. These results indicated that thymol enhances barrier function and reduce ROS production and pro-inflammatory cytokine gene expression in the epithelial cells during inflammation. The regulation of barrier function by thymol and LPS may be at post-transcriptional or post-translational levels.

Development of Novel Microparticles for Effective Delivery of Thymol and Lauric Acid to Pig Intestinal Tract.

Antibiotics have been widely supplemented in feeds at subtherapeutic concentrations to prevent postweaning diarrhea and increase the overall productivity of pigs. However, the emergence of antimicrobial-resistant bacteria worldwide has made it urgent to minimize the use of in-feed antibiotics. The development of promising alternatives to in-feed antibiotics is crucial for maintaining the sustainability of swine production. Both medium-chain fatty acids (MCFA) and essential oils exhibit great potential to postweaning diarrhea; however, their direct inclusion has compromised efficacy because of several factors including low stability, poor palatability, and low availability in the lower gut. Therefore, the objective of this study was to develop a formulation of microparticles to deliver a model of essential oil (thymol) and MCFA (lauric acid). The composite microparticles were produced by the incorporation of starch and alginate through a melt-granulation process. The release of thymol and lauric acid from the microparticles was in vitro determined using simulated salivary fluid (SSF), simulated gastric fluid (SGF), and simulated intestinal fluid (SIF), consecutively. The microparticles prepared with 2% alginate solution displayed a slow release of thymol and lauric acid in the SSF (21.2 ± 2.3%; 36 ± 1.1%), SGF (73.7 ± 6.9%; 54.8 ± 1.7%), and SIF (99.1 ± 1.2%; 99.1 ± 0.6%), respectively, whereas, the microparticles without alginate showed a rapid release of thymol and lauric acid from the SSF (79.9 ± 11.8%; 84.9 ± 9.4%), SGF (92.5 ± 3.5%; 75.8 ± 5.9%), and SIF (93.3 ± 9.4%; 93.3 ± 4.6%), respectively. The thymol and lauric acid in the developed microparticles with or without alginate both exhibited excellent stabilities (>90%) during being stored at 4 °C for 12 weeks and after being stored at room temperature for 2 weeks. These results evidenced that the approach developed in the present study could be potentially employed to deliver thymol and lauric acid to the lower gut of pigs, although further in vivo investigations are necessary to validate the efficacy of the microparticles.