Direct-fed Microbials Research Articles

PSIV-19 Effects of Flavolac feed top dress in lactation on sow body weight change and litter performance

Abstract A total of 271 mixed parity sows (PIC 1050) were used to evaluate the effect of feeding Flavolac in lactation diets on sow farrowing performance and litter growth performance during summer conditions. Flavolac (Agroceres Multimix; Rio Claro, Sao Paulo, Brazil) is a commercial product containing several compounds, including amino acids, antioxidants, botanicals, direct fed-microbials, and vitamin-like compounds, that individually have previously been shown to improve litter weaning weight or preweaning mortality. However, there are no published data on the benefit when these compounds are used in combination. The experiment was conducted at a commercial sow farm located in northwest Texas. At approximately d 112 to 114 of gestation, sows were moved to the farrowing house, and randomly allotted to 1 of 2 treatments based on parity and caliper score. Treatments consisted of a control diet (no Flavolac) or the control diet with an added Flavolac top-dress (80 g/d in a single feeding). Sows were weighed before entering the farrowing house and at weaning. Sows were provided approximately 1.8 kg per day of a common lactation diet without Flavolac pre-farrowing. After farrowing, sows were provided ad libitum access to lactation feed. Application of treatments began after cross fostering, approximately 24 h post-farrowing. The weaning age averaged approximately 21.3 d. There was no evidence of differences (P > 0.10) in sow body weight (BW) at entry, weaning, or overall BW change (Table). There was no evidence of differences (P > 0.10) for sow entry caliper, weaning caliper score, caliper score change, or average daily lactation feed disappearance. In addition, there was no evidence of differences (P > 0.10) in total litter or piglet birth weight. However, administration of Flavolac tended (P < 0.10) to increase litter weaning weight, total litter weight gain, and litter average daily gain. The inclusion of Flavolac led to a tendency (P = 0.091) for a reduction in pre-weaning mortality and therefore, an improvement in percentage of pigs weaned relative to those on d 2. In summary, inclusion of Flavolac during the lactation period resulted in a tendency for reduced pre-weaning mortality and tended to improve total litter weight gain, and thus litter weight at weaning.

150 Development of a leaky gut model: Meta-analysis of plasma and fecal markers of leaky gut

Abstract Intestinal integrity can be compromised in livestock and poultry species during stress events, including off-feed events. To develop a leaky gut model, weanling barrows 26 to 28 d old [n = 307; Duroc terminal line Fast genetics, initial body weight (BW) = 5.90 ± 0.47 kg), were used in a total of 6 randomized complete block experiments at the NutriQuest modeling center (Fergus Falls, MN). All trials included positive control (PC) and negative control (NC) treatments. The other treatments evaluated (n = 8) were various strains and levels of direct-fed microbial (DFM) products. With the exception of PC, all treatments had complete 24-h feed restriction (FR) approximately 18 d after arrival. Pigs assigned to PC had ad libitum access to feed. Pigs were housed 3 pigs per pen in 1 of 30 pens, and all pigs were fed a common high-quality basal diet. For 3 trials, pigs received Excede (Zoetis; Parsipanny, NJ) IM upon arrival and were provided gentamycin via water for 3 d post-arrival. For 3 trials, no prophylactic antibiotics were administered. Eighteen days post-arrival, fecal and plasma samples were collected from all pigs and feed was removed from FR pens. After 24 h, plasma and fecal samples were collected from all pigs, and one-half of the pigs per trial were harvested for tissue collection. Four days after FR, samples were collected similarly, and the remaining pigs were harvested. Plasma samples were analyzed for LPS binding protein concentrations (LBP), and fecal samples were analyzed for myeloperoxidase (MPO) concentrations. Data from the PC (n = 90) and NC (n = 92) treatments only were analyzed as Individual Participant Data meta-analysis using PROC MIXED of SAS. Data from all pigs (n = 307) were analyzed using PROC REG of SAS with forward step-wise selection to determine relationships between response variables. Plasma LBP concentrations did not differ between PC and NC 24 h after FR (P = 0.14). However, change in LBP concentrations and the ratio of LBP immediately before and after FR, differed between PC and NC (P < 0.001). For NC, LBP concentrations decreased, and for PC, LBP concentrations increased (Table 1). There was no effect of treatment on fecal MPO concentrations. Using the broader dataset (n = 307), variables that were predictive of plasma LBP concentrations immediately following FR were trial (P < 0.001), treatment (P < 0.001), and fecal MPO concentrations (P < 0.05). Variables that were predictive of change in LBP concentrations before and after FR were trial (P < 0.001), treatment (P < 0.001), and initial BW (P < 0.01). These data, taken with data presented in a companion abstract on ileal tight junction gene expression, suggest that plasma LBP concentrations immediately following a FR event are a useful marker of leaky gut. Fecal MPO concentrations do not consistently respond to FR.

Effects of supplementing direct-fed microbials on health and growth of preweaning Gyr × Holstein dairy calves

This study aimed to evaluate the effects of direct-fed microbials (DFM) on health and growth responses of pre-weaning Bos indicus × B. taurus (Gyr × Holstein) crossbred calves. Ninety newborn heifer calves [initial body weight (BW) 35 ± 4.0 kg] were used. At birth, calves were ranked by initial BW and parity of the dam and assigned to: 1) whole milk without DFM supplementation (CON; n = 30), 2) whole milk with the addition of 1.0 g/calf per day of a Bacillus-based DFM (BAC; n = 30), or 3) whole milk with the addition of 1.0 g/calf per day of BAC and 1.2 g/calf per day of Enterococcus faecium 669 (MIX; n = 30). Milk was fed individually during the study (77 d) and the BAC and MIX treatments were offered daily throughout the 77-d pre-weaning period. All calves were offered a starter supplement and corn silage starting on d 1 and 60 of age, respectively. Milk and starter supplement intake were evaluated daily, and BW was recorded on d 0 and at weaning (d 77). Diarrhea and pneumonia were assessed daily, and fecal samples were collected on d 0, 7, 14, 21, and at weaning (d 77) for assessment of the presence of bacterial and protozoal pathogens via qPCR. All data were analyzed using SAS (v. 9.4) with calf as the experimental unit and using single-df orthogonal contrasts (BAC + MIX vs. CON; BAC vs. MIX). Daily feeding of DFM, regardless of type, improved weaning BW. Odds ratio for occurrence of pneumonia was lower for DFM-supplemented calves, but occurrence of both did not differ between BAC and MIX calves. No Salmonella spp. or E. coli F41 were detected in any of the calves. The proportion of calves positive for E. coli F17 was greater for DFM calves on d 7 (92 and 96% vs. 81% for BAC, MIX, and CON, respectively), 21 (13 and 26% vs. 7% for BAC, MIX, and CON, respectively), and at weaning (48 and 35% vs. 22% for BAC, MIX, and CON, respectively). For C. difficile, more DFM calves were positive on d 7 (65 and 30% vs. 35% for BAC, MIX, and CON, respectively) and 14 (20 and 28% vs. 7% for BAC, MIX, and CON, respectively), but also greater for BAC vs. MIX on d 7. More CON calves were positive for C. perfringens on d 14 (14% vs. 3 and 8% for CON, BAC, and MIX, respectively) compared with DFM-fed calves. Incidence of calves positive for C. perfringens was greater in BAC vs. MIX on d 7 (50 vs. 18%), and greater for MIX vs. BAC at weaning (9 vs. 0%). For protozoa occurrence, a lower proportion of DFM calves were positive for Cryptosporidium spp. on d 7 (58 and 48% vs. 76% for BAC, MIX, and CON, respectively), but opposite results were observed on d 21 for Cryptosporidium spp. (3 and 11% vs. 0% for BAC, MIX, and CON, respectively) and Eimeria spp. on d 14 (7 and 8% vs. 0% for BAC, MIX, and CON, respectively) and 21 (50 and 59% vs. 38% for BAC, MIX, and CON, respectively). In summary, DFM feeding alleviated the occurrence of pneumonia, improved growth rates, while also modulating the prevalence of bacteria and protozoa in pre-weaning Gyr × Holstein calves.

The Effect of a Direct Fed Microbial on Liveweight and Milk Production in Dairy Cattle.

This longitudinal study aimed to quantify the effects of dietary supplementation of a direct-fed microbial (DFM) consisting of three lactobacilli isolates on milk yield, milk fat and protein yields, somatic cell count (SCC), and liveweight in a single dairy herd in Australia. A total of 150 dairy cows were randomly selected based on parity and days in milk and divided into two groups: control (n = 75) and DFM treatment (n = 75). Throughout the study, the two groups of cows were housed separately in a dry lot yard, and each group had their own feeding area. For the DFM treatment group, selected cows in mid-lactation were supplemented with 10 mL/cow/day of the DFM via top dressing of the feed for the remainder of the lactation and through the dry period, extending into subsequent lactation. The control group had no supplementation. The milk yield and liveweight were recorded daily. Milk samples were collected every two months for milk component analysis (fat, protein, and somatic cell count [SCC]). The DFM-treated cows gained more liveweight across the study (19.40 kg, 95% CI 0.44 kg; 38.30 kg, p = 0.05) compared to the control cows. In the second production year, the DFM-treated cows mobilized more liveweight (-6.06 kg, 95% CI -10.49 kg; -1.61 kg, p = 0.01) and produced more milk (0.39 L/d 95% CI 0.10; 0.89, p = 0.05). Over a full lactation, DFM cows yielded at least 258 L (95% CI 252 L; 265 L) more milk than controls. No significant differences were found in fat and protein yield or SCC. This study suggests that consistent and ongoing supplementation with a Lacticaseibacillus- and Lentilactobacillus-based DFM could have a positive effect on milk production, but further research is needed to understand the underlying mechanism.

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, 2h 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 18h. 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 2.2h (P ≤ 0.09), increased TEER at 3.2h (P < 0.01), but reduced TEER from 5.4 to the end of the experimental period at 18.4h (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 3.2h 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 3.2h (P = 0.20). Moreover, BDP-CON increased (P ≤ 0.04) TEER when compared with CON-CON from 3.2 to 18.4h, but also in comparison with BDP-CYT from 4.3 to 18.4h 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.

Influence of BOVAMINE DEFEND Plus on growth performance, carcass characteristics, estimated dry matter digestibility, rumen fermentation characteristics, and immune function in finishing beef steers.

One hundred and eighty crossbred beef steers (406.0 ± 2.2kg) were used to determine the impact of a novel direct-fed microbial (DFM) on growth performance, carcass characteristics, rumen fermentation characteristics, and immune response in finishing beef cattle. Steers were blocked by body weight (BW) and randomly assigned, within block, to 1 of 2 treatments (3 replicates/treatment: 30 steers/replicate). Treatments included: (1) no DFM (control) and (2) DFM supplementation at 50 mg ∙ animal-1 ∙ d-1 (BOVAMINE DEFEND Plus). All steers were fed a high-concentrate finishing diet and individual feed intake was recorded daily via the GrowSafe system. BWs were collected every 28 d. On day 55, 10 steers per pen were injected with ovalbumin (OVA). Jugular blood samples were collected from each steer on days 0, 7, 14, and 21 post injection. On day 112, the same steers were injected again with OVA and intramuscularly with a pig red blood cell solution. Jugular blood samples were collected from each steer on days 0, 7, 14, and 21 post injection. On day 124, rumen fluid was collected from 3 steers per treatment and used to estimate in vitro rumen fermentation characteristics. Equal numbers of steers per treatment were transported to a commercial abattoir on days 145, 167, and 185 of the experiment, harvested, and carcass data were collected. Initial BW was similar across treatments. On days 28 and 55, steers receiving DFM had heavier BW (P < 0.01) compared to controls. The average daily gain was greater in DFM-supplemented steers from days 0 to 28 (P < 0.01) and days 0 to 55 (P < 0.01) of the experiment compared to controls. Overall dry matter intake (DMI) was greater (P < 0.04) and overall feed efficiency was similar in DFM-supplemented steers compared to controls. Dressing percentage (P < 0.02) was greater in steers receiving DFM compared to controls. Antibody titers to injected antigens were similar across treatments. However, red blood cell superoxide dismutase activity was greater (P < 0.05) in DFM-supplemented steers compared to controls. In vitro molar proportions of isobutyric and butyric acid were greater (P < 0.01) and dry matter (DM) digestibility tended (P < 0.07) to be greater in rumen fluid obtained from steers supplemented with DFM. These data suggest that BOVAMINE DEFEND Plus supplementation improves growth performance during the initial period of the finishing phase, increases overall DMI and dressing percentage, and may impact antioxidant status in beef cattle.

104 Maternal pre- and post-partum supplementation of Bacillus-based DFM enhanced cow and calf performance

Abstract This study evaluated the effects of maternal supplementation of Bacillus-based direct-fed microbials (DFM) on physiology and growth performance of Bos indicus-influenced cow-calf pairs. On d 0 (~139 d before expected calving date), 72 fall-calving, Brangus crossbred beef heifers (25 to 37.5% Bos indicus; 20 to 22 mo of age) pregnant with their first offspring were stratified by their initial body weight (BW; 431 ± 31 kg) and body condition score (BCS; 6.0 ± 0.36; scale 1 to 9), and randomly allocated into 1 of 12 bahiagrass pastures (1 ha and 6 heifers per pasture). Treatments were randomly assigned to pastures (6 pastures per treatment) and consisted of heifers supplemented with 1 kg/d of soybean hulls dry matter (DM) added (BAC) or not (CON) with a Bacillus-based DFM mixture (BovacillusTM; Chr. Hansen A/S, Hørsholm, Denmark) from d 0 to 242 (139 ± 4 d prepartum to 104 ± 4 d postpartum). Calves were weaned on d 242 (96 ± 30 d of age) and then allocated into 1 of 16 drylot pens and limit-fed the same concentrate at 3.25% of BW until d 319. Maternal treatment effects were not detected (P ≥ 0.29) for herbage allowance, forage nutritive value, and heifer activity on pasture. Heifer BCS on d 39 and 63 tended (P ≤ 0.09) to be greater, whereas heifer BCS on d 91 was greater (P = 0.01) for BAC vs. CON heifers and did not differ (P ≥ 0.20) between treatments on d 179 and 242. Plasma concentrations of glucose did not differ from d 0 to 63 (P ≥ 0.14) but were greater (P = 0.005) on d 179 and tended (P = 0.09) to be greater on d 242 for BAC vs. CON heifers. First offspring BW at birth, ADG from birth to early weaning, and BW weaning did not differ (P ≥ 0.19), but BW at drylot exit (d 319) was greater (P = 0.05) for BAC vs. CON calves. Maternal treatment effects were not detected (P ≥ 0.42) for calf serum concentrations of IgG at birth, and post-vaccination plasma concentrations of glucose, cortisol and haptoglobin. However, serum titers against bovine respiratory syncytial virus (BRSV) were greater (P = 0.04) for BAC vs. CON calves on d 287, whereas seroconversion against parainfluenza 3 virus (PI-3) was greater (P &amp;lt; 0.01) for BAC vs. CON calves on d 271. Thus, maternal supplementation of Bacillus-based DFM increased prepartum BCS gain and postpartum plasma concentrations of glucose of heifers and led to positive carryover effects on post-weaning BW gain and some indicators of humoral immune response to vaccination in their first offspring.

107 Bacterial direct-fed microbial mixtures effects on beef cattle ruminal degradability kinetics

Abstract The effects of bacterial direct-fed microbial (DFM) mixtures on beef cattle ruminal degradability were evaluated. Six ruminally cannulated beef steers (BW = 520 ± 30 kg) were used in a replicated 3 × 3 Latin square design and offered a steam-flaked corn-based finishing diet to ad libitum intake during three, 28-d periods. Treatments were: 1) Control (no DFM, lactose carrier only); 2) Treat-A (L. animalis, P. freudenreichii; B. subtilis; and B. licheniformis), at 1:1:1:3 ratio, respectively; totaling 6 × 109 CFU (50 mg)/animal-daily minimum; and 3) Treat-B, the same DFM combination, but with doses at 1:1:3:1 ratio. Bacterial counts were approximately 30% greater than the minimum expected. Pre-dehydrated (55ºC for 72 h) diets and forage substrates were ground (2 mm) and placed into 10 × 20 cm (28 µm) nylon bags (8 and 5 g, as-is respectively). The diet-substrate included a mixture of (DM basis) steam-flaked corn (70%), alfalfa hay (10%), and wet corn gluten feed (20%). The forage-substrate was represented by ‘WW-B Dahl’ Old World bluestem (Bothriochloa bladhii). Substrate in-situ bags were placed within a nylon mesh (with weights) at the ruminal ventral sac, and reversely removed at 0, 2, 4, 8, 12, 20, 32, 48, 64, 72, and 96 h after feeding. Samples were removed, rinsed, and dehydrated for 72 h at 55oC. Residues were composited within period, steer, incubation-time, corrected for residual moisture and were used to fit a first-order kinetics model using the NLIN procedure of SAS (animal = experimental unit). Steers offered Treat-A tended to show an increase on the rate of degradation of NDF (P = 0.15) and ADF (P = 0.10) fractions; increased (P &amp;lt; 0.01) hemicellulose potentially degradable fraction (B); and increased (P = 0.02) the effective degradable fraction of NDF (tendency for ADF, P = 0.08) at pre-established rate of passages of 4, 5, and 6% of the diet-substrate compared with Control, while tending to have a reduced rate of degradation for the overall DM (P = 0.06) and OM (P = 0.08) fractions. When the in-situ bags containing the forage-substrate were incubated, steers offered Treat-A showed an increased (P &amp;lt; 0.01) lag time (T0) for hemicellulose compared with Control, while those offered Treat-B had a decreased (P = 0.02) hemicellulose potentially degradable fraction (B) compared with Control. The effective degradable fraction for hemicellulose at pre-established rate of passages of 4, 5, and 6% tended (P = 0.09) to numerically increase for steers offered Treat-A compared with Control. The combination of bacteria in Treat-A enhanced the ruminal degradation of high-quality fiber components, as indicated by the improvements observed within the more degradable fractions from ingredients used in the diet and the forage substrates. Further assessment is warranted to investigate post-ruminal nutrient disappearance.

Effect of Direct Fed Microbial (DFM) on in-vitro Methanogenesis and Dry Matter Digestibility

The in-vitro rumen fermentation study was conducted to evaluate the effect of supplementation of direct-fed microbial (DFM) in the total mixed ration (TMR) on total gas production, methanogenesis and dry matter digestibility by using rumen liquor of adult Surti buffaloes. DFM was supplemented at 0, 1, 2, 3, 4, 5, 6, 7, and 8% with TMR (65% Straw and 35% concentrate) for in-vitro gas production trials. The result of the in-vitro study revealed significantly (P&lt;0.05) higher IVDMD (57.03%) and lower CH4 production (3.20 ml CH4/100 mg DDM) at a 3% level of DFM supplementation in TMR. Based on the overall results of in-vitro studies 3% level of DFM supplementation was found to be most suitable for further in-vivo studies in adult Surti buffaloes.

Effect of a Lactobacilli-Based Direct-Fed Microbial Product on Gut Microbiota and Gastrointestinal Morphological Changes.

The calf's gastrointestinal tract (GIT) microbiome undergoes rapid shifts during early post-natal life, which can directly affect calf performance. The objectives of this study were to characterise and compare differences in the establishment and succession of GIT microbiota, GIT morphological changes, and the growth of dairy calves from birth until weaned. Forty-four newborn Holstein-Friesian calves were randomly selected and assigned to Treatment (TRT) and Control (CON) groups. The TRT group calves received a once-daily dose of a direct-fed microbial (DFM) liquid product containing Lacticaseibacillus paracasei, Lentilactobacillus buchneri, and Lacticaseibacillus casei, all formerly known as Lactobacillus. Fresh faecal samples were manually taken from the rectum of all calves, and gross necropsy was performed on the forestomachs and gastrointestinal tracts. Bacterial DNA was extracted from frozen faecal samples for 16S rRNA gene amplicon sequencing. Calves in the TRT group had greater live weights (p = 0.02) at weaning compared with calves in the CON group (mean = 69.18 kg, SD = 13.37 kg). The average daily live weight gain (ADG) and total feed intake were similar between the two groups. Calves in the TRT group had greater duodenum, abomasum, and reticulum weights (p = 0.05). Rumen and intestinal development (p < 0.05) and faecal microbial diversity (p < 0.05) were more pronounced in the TRT group. The relative abundances of eight genera differed (p < 0.001) between the groups. Supplementing calves with the LAB-based DFM increased live weight at weaning and had a more pronounced effect on the development of rumen and the gastrointestinal tract and on microbiota diversity and evenness. Future work is needed to better understand the potential association of LAB-DFM products on gut mucosa-associated microbiota.

Effects of a multistrain Bacillus-based direct-fed microbial on gastrointestinal permeability and biomarkers of inflammation during and following feed restriction in mid-lactation Holstein cows

Objectives were to evaluate the effects of a multistrain Bacillus-based (Bacillus subtilis and Bacillus pumilus blend) direct-fed microbial (DFM) on production, metabolism, inflammation biomarkers and gastrointestinal tract (GIT) permeability during and following feed restriction (FR) in mid-lactation Holstein cows. Multiparous cows (n = 36; 138 ± 53 DIM) were randomly assigned to 1 of 3 dietary treatments: (1) control (CON; 7.5 g/d rice hulls; n = 12), (2) DFM10 (10 g/d Bacillus DFM, 4.9 × 109 cfu/d; n = 12) or 3) DFM15 (15 g/d Bacillus DFM, 7.4 × 109 cfu/d; n = 12). Before study initiation, cows were fed their respective treatments for 32 d. Cows continued to receive treatments during the trial, which consisted of 3 experimental periods (P): P1 (5 d) served as baseline for P2 (5 d), during which all cows were restricted to 40% of P1 DMI, and P3 (5 d), a "recovery" where cows were fed ad libitum. On d 4 of P1 and on d 2 and 5 of P2, GIT permeability was evaluated in vivo using the oral paracellular marker Cr-EDTA. As anticipated, FR decreased milk production, insulin, glucagon, and BUN but increased nonesterified fatty acids. During recovery, DMI rapidly increased on d 1 then subsequently decreased (4.9 kg) on d 2 before returning to baseline, whereas milk yield slowly increased but remained decreased (13%) relative to P1. The DFM10 cows had increased DMI and milk yield relative to DFM15 during P3 (10%). Overall, milk lactose content was increased in DFM cows relative to CON (0.10 percentage units), and DFM10 cows tended to have increased lactose yield relative to CON and DFM15 during P3 (8% and 10%, respectively). No overall treatment differences were observed for other milk composition variables. Circulating glucose was quadratically increased in DFM10 cows compared with CON and DFM15 during FR and recovery. Plasma Cr area under the curve was increased in all cows on d 2 (9%) and 5 (6%) relative to P1. Circulating LPS binding protein (LBP), serum amyloid A (SAA), and haptoglobin (Hp) increased in all cows during P2 compared with baseline (31%, 100%, and 9.0-fold, respectively). Circulating Hp concentrations continued to increase during P3 (274%). Overall, circulating LBP and Hp tended to be increased in DFM15 cows relative to DFM10 (29% and 81%, respectively), but no treatment differences were observed for SAA. Following feed reintroduction during P3, fecal pH initially decreased (0.62 units), but returned to baseline levels whereas fecal starch markedly increased (2.5-fold) and remained increased (82%). Absolute quantities of a fecal Butyryl-CoA CoA transferase (but) gene associated with butyrate synthesis, collected by fecal swab were increased in DFM10 cows compared with CON and DFM15 cows. In summary, FR increased GIT permeability, caused inflammation, and decreased production. Feeding DFM10 increased some key production and metabolism variables and upregulated a molecular biomarker of microbial hindgut butyrate synthesis, while DFM15 appeared to augment immune activation.

An In Vitro Study on the Role of Cellulases and Xylanases of Bacillus subtilis in Dairy Cattle Nutrition.

The administration of Bacilli to dairy cows exerts beneficial effects on dry matter intake, lactation performance, and milk composition, but the rationale behind their efficacy is still poorly understood. In this work, we sought to establish whether cellulases and xylanases, among the enzymes secreted by B. subtilis, are involved in the positive effect exerted by Bacilli on ruminal performance. We took advantage of two isogenic B. subtilis strains, only differing in the secretion levels of those two enzymes. A multi-factorial study was conducted in which eight feed ingredients were treated in vitro, using ruminal fluid from cannulated cows, with cultures of the two strains conveniently grown in a growth medium based on inexpensive waste. Feed degradability and gas production were assessed. Fiber degradability was 10% higher (p < 0.001) in feeds treated with the enzyme-overexpressing strain than in the untreated control, while the non-overexpressing strain provided a 5% increase. The benefit of the fibrolytic enzymes was maximal for maize silage, the most recalcitrant feed. Gas production also correlated with the amount of enzymes applied (p < 0.05). Our results revealed that B. subtilis cellulases and xylanases effectively contribute to improving forage quality, justifying the use of Bacilli as direct-fed microbials to increase animal productivity.

Evaluation of direct-fed microbials on in vitro ruminal fermentation, gas production kinetic, and greenhouse gas emissions in different ruminants’ diet

IntroductionThree in vitro experiments were conducted to evaluate the effects of increasing levels of Enterococcus faecium and Saccharomyces cerevisiae (DFM1) and increasing levels of Bacillus licheniformis and Bacillus subtilis (DFM2) on in vitro ruminal fermentation parameters in three different dietary scenarios.MethodsFor Exp. 1, the basal diet consisted of 25:75 roughage:concentrate ratio (R:C) and was composed by 5 treatments: control (no additive), 2 levels of DFM1 (1X = 1.9 mg and 5X = 9.0 mg), and 2 levels of DFM2 (1X = 3.8 mg and 5X = 19 mg). The Exp. 2 consisted of a 41:59 R:C diet and was composed by 5 treatments: control (no additive) and 2 levels of DFM1 (1X = 3.8 mg and 5 X = 19 mg) and 2 levels of DFM2 (1X = 5.6 mg and 5X = 28 mg). The Exp. 3 consisted of a 100:0 R:C diet [Brachiaria (syn. Urochloa brizantha)] and was composed by the same treatments described in Exp. 1. The DFM1 contained 3.5 × 109 CFU per g of Enterococccus faecium and Saccharomyces cerevisiae, whereas the DFM2 contained Bacillus licheniformis and Bacillus subtilis at 3.2 × 109 CFU per g. In each Exp., an in vitro gas production (GP) system with 43-bottles (AnkomRF) was used in four consecutive 48 or 72-h fermentation batches to evaluate total GP (TGP), kinetics and fermentation profiles, methane (CH4), and carbon dioxide (CO2).ResultsFor Exp 1, DFM1 increased quadratically TGP at 24 and 48-h, which reflected in a greater in vitro organic matter digestibility (IVOMD). The concentrations of ammonia-N, CH4, and CO2 (mmol/g of IVOMD) reduced quadratically as DFM1 increased. For Exp. 2, DFM1 inclusion reduced butyrate concentration and acetate to propionate ratio. Regarding GHG emissions, DFM1 and DFM2 quadratically reduced CH4 and CO2 emission per IVOMD (mmol/g of IVOMD). For Exp. 3, DFM1 increased quadratically TGP at 48h with no impact on IVOMD. Otherwise, DFM2 increased linearly TGP at 24 and 48h which reflected in a greater IVOMD. The inclusion of DFM1 increased linearly iso-valerate and branched-chain volatile fatty acids (BCVFA) concentration and DFM2 addition increased BCVFA quadratically.DiscussionOverall, addition of DFM1 [Enterococccus faecium (5 × 109 CFU per g) + Saccharomyces cerevisiae (5 × 109 CFU per g)] or DFM2 [Bacillus licheniformis + Bacillus subtilis (3.2 × 109 CFU per g)] might enhance the fermentation process in the rumen and decrease greenhouse gas emissions in a dose-dependent manner, though the results are contingent on the specific type of diet.

Evaluation of long-term supplementation of a Bacillus subtilis direct-fed microbial and enzymatically hydrolyzed yeast cell culture product used alone or in combination on Clostridia, Clostridium perfringens, Escherichia coli, and Salmonella prevalence in beef steers.

The objective was to determine the influence of long-term supplementation (258 d) of a direct-fed microbial (DFM) and/or yeast cell wall (YCW) product on bacterial populations in beef steers. Single-sourced Charolais × Red Angus steers (n = 256; body weight = 246 ± 1.68kg) were used in a randomized complete block design and blocked by location into one of four treatments: 1) fed no DFM and no YCW (Control); 2) fed only the DFM (DFM; Certillus CP B1801 Dry,28 g/steer d-1 ); 3) fed only the YCW (YCW; Celmanax; 18 g/steer d-1 ); and 4) fed the DFM and the YCW (DFM+YCW). Steers were vaccinated for respiratory and clostridial diseases and treated for internal and external parasites at processing and individually weighed on days 1, 14, 42, 77, 105, 133, 161, 182, 230, and 258. To determine bacterial prevalence, fecal samples were collected on days 1, 14, 77, 133, 182, and 230 and environmental (pen area, feed, and water) samples were collected at the beginning of the week when cattle were weighed. No treatment × day interactions or treatment effects (P > 0.05) were observed between treatment groups at any sampling days for the bacterial populations. Samples on days 1, 133, and 182 had greater (P < 0.05) Clostridia levels compared to the other sampling points but were not different from each other. Clostridia levels were also greater (P < 0.05) on day 77 compared to days 14 and 230. Samples on days 77 and 230 had greater (P < 0.05) Clostridium perfringens levels compared to the other sampling points but were not different (P > 0.05) from each other. Samples on days 1 and 14 had lower (P < 0.05) total Escherichia coli levels compared to the other sampling points but were not different (P > 0.05) from each other. Escherichia coli levels on day 77 were higher (P < 0.05) compared to days 133, 182, and 230. Little Salmonella prevalence (1.5%) was observed throughout the study. This study had greater levels of Clostridia compared to small and large commercial feedlots in the Church and Dwight research database, but C. perfringens, total and pathogenic E. coli, and Salmonella prevalence were notably lower. Collectively, there were no appreciable treatment influences on bacterial populations. These data further indicate a low pathogenic bacterial challenge at the trial site, which could partially explain the lack of differences with DFM or YCW supplementation. The DFM and YCW used alone or in combination cannot be expected to show additional benefits when animals are relatively unstressed with a low pathogenic bacterial challenge.

Effects of bacterial direct-fed microbial combinations on beef cattle growth performance, feeding behavior, nutrient digestibility, ruminal morphology, and carcass characteristics.

The effects of the dietary inclusion of a mixture of bacterial direct-fed microbial (DFM) on feedlot beef cattle growth performance, carcass characteristics, nutrient digestibility, feeding behavior, and ruminal papillae morphology were evaluated. Crossbred-Angus steers (n = 192; initial body weight (BW) = 409 kg ± 8kg) were blocked by BW and randomly assigned into 48 pens (4 steers/pen and 16 pens/treatment) following a randomized complete block design. A steam-flaked corn-based fishing diet was offered to ad libitum intake once daily for 153 d containing the following treatments: (1) Control (no DFM, lactose carrier only); (2) treat-A (Lactobacillus animalis, Propionibacterium freudenreichii, Bacillus subtilis, and Bacillus licheniformis), at 1:1:1:3 ratio, respectively; totaling 6 × 109 CFU (50mg)/animal-daily minimum; and (3) treat-B, the same DFM combination, but with doses at 1:1:3:1 ratio. Bacterial counts were ~30% greater than the minimum expected. Data were analyzed using the GLIMMIX procedure of SAS, with pen as the experimental unit, the fixed effect of treatment, and the random effect of BW-block, while preplanned contrasts comparing Control × treat-A or treat-B were used. Steers offered treat-A had increased carcass-adjusted average daily gain (P = 0.03) by 6.7%, gain efficiency (P < 0.01) by 6%, tended (P = 0.07) to have increased carcass-adjusted final BW by 15kg, and hot carcass weight (P = 0.07) by 10kg, while treat-B did not differ (P ≥ 0.17) from control. Overall dry matter (DM) intake (P = 0.36) and other carcass traits (P ≥ 0.13) were not affected by treatments. Steers offered treat-A tended to have increased digestibility of DM (P = 0.07) by 3%, neutral detergent fiber (P = 0.10), and hemicellulose (P = 0.08) by 9% compared with control, while treat-B did not differ (P ≥ 0.10) from control. No treatment × period interactions (P ≥ 0.21) or main effects of treatment (P ≥ 0.12) were observed during 24-h feeding behavior. Steers ruminated, ate, chewed, and were more active (P ≤ 0.01) during the second behavioral assessment (day 113), while drinking behavior was not affected (P ≥ 0.88). Ruminal papillae morphology and ruminal ammonia concentration (ruminal fluid collected at slaughter facility) were not affected by treatment (P ≥ 0.39). Steers offered the DFM treat-A had improved growth performance and it positively affected carcass weight and nutrient digestion. The DFM combinations did not seem to affect feedlot cattle feeding behavior or ruminal papillae morphology.

Maternal pre- and postpartum supplementation of a Bacillus-based DFM enhanced cow and calf performance.

This study evaluated the effects of maternal supplementation of a Bacillus-based direct-fed microbial (DFM) on the physiology and growth performance of Bos indicus-influenced cow-calf pairs. On day 0 (~139 d before expected calving date), 72 fall-calving, Brangus crossbred beef heifers (20 to 22 mo of age) pregnant with first offspring were stratified by their initial body weight (BW; 431 ± 31kg) and body condition score (BCS; 6.0 ± 0.36; scale 1 to 9), and randomly allocated into 1 of 12 bahiagrass pastures (1 ha and six heifers per pasture). Treatments were randomly assigned to pastures (six pastures per treatment) and consisted of heifers supplemented with 1kg/d of soybean hulls (dry matter, DM) that was added (BAC) or not (CON) with DFM containing Bacillus subtilis and B. licheniformis (Bovacillus; Chr. Hansen A/S, Hørsholm, Denmark). Treatments were provided from days 0 to 242 (139 ± 4 d prepartum to 104 ± 4 d postpartum). Calves were weaned on day 242 (96 ± 30 d of age) and then allocated into 1 of 16 drylot pens and fed the same concentrate at 3.25% of BW (DM) until day 319. Maternal treatment effects were not detected (P ≥ 0.29) for herbage allowance and forage chemical composition. Heifer BCS on days 39 and 63 tended (P ≤ 0.09) to be greater for BAC vs. CON heifers, whereas heifer BCS on day 91 was greater (P = 0.01) for BAC vs. CON heifers. Heifer BCS did not differ (P ≥ 0.20) between treatments on days 179 and 242. Plasma glucose concentration did not differ from days 0 to 63 (P ≥ 0.14) but were greater (P < 0.01) on day 179 and tended (P = 0.09) to be greater on day 242 for BAC vs. CON heifers. Calf BW at birth, ADG from birth to weaning, and BW at weaning did not differ (P ≥ 0.19) between treatments, but calf BW at drylot exit (day 319) was greater (P = 0.05) for BAC vs. CON calves. Maternal treatment effects were not detected (P ≥ 0.42) for calf serum concentration of IgG at birth and postvaccination plasma concentrations of glucose, cortisol, and haptoglobin. Serum titers against bovine respiratory syncytial virus (BRSV) were greater (P = 0.04) for BAC vs. CON calves on day 287, whereas seroconversion against parainfluenza-3 virus (PI-3) was greater (P < 0.01) for BAC vs. CON calves on day 271. Thus, maternal supplementation of a Bacillus-based DFM increased prepartum BCS gain and postpartum plasma glucose concentration of heifers and led to positive carryover effects on postweaning BW gain and humoral immune response in their offspring.

Effects of bacterial direct-fed microbial mixtures offered to beef cattle consuming finishing diets on intake, nutrient digestibility, feeding behavior, and ruminal kinetics/fermentation profile.

Effects of bacterial direct-fed microbial (DFM) mixtures on intake, nutrient digestibility, feeding behavior, ruminal fermentation profile, and ruminal degradation kinetics of beef steers were evaluated. Crossbred Angus ruminally cannulated steers (n = 6; body weight [BW] = 520 ± 30kg) were used in a duplicated 3 × 3 Latin square design and offered a steam-flaked corn-based finisher diet to ad libitum intake for 3, 28-d periods. Treatments were 1) Control (no DFM, lactose carrier only); 2) Treat-A (Lactobacillus animalis, Propionibacterium freudenreichii, Bacillus subtilis, and Bacillus licheniformis), at 1:1:1:3 ratio, respectively; totaling 6 × 109 CFU (50mg)/animal-daily minimum; and 3) Treat-B, the same DFM combination, but doses at 1:1:3:1 ratio. Bacterial counts were ~30% greater than the minimum expected. Data were analyzed using the GLIMMIX procedure of SAS with the model including the fixed effect of treatment and the random effects of square, period, and animal (square). For repeated measure variables, the fixed effects of treatment, time, and their interaction, and the random effects of square, period, animal (square), and animal (treatment) were used. Preplanned contrasts comparing Control × Treat-A or Treat-B were performed. Intake and major feeding behavior variables were not affected (P ≥ 0.17) by treatments. Steers offered Treat-A had an increased (P = 0.04) ADF digestibility compared with Control. Steers offered Treat-A experienced daily 300min less (P = 0.04) time under ruminal pH 5.6, a greater (P = 0.04) ruminal pH average and NH3-N concentration (P = 0.05) and tended (P = 0.06) to have a lower ruminal temperature compared to Control. Ruminal VFA was not affected (P ≥ 0.38) by treatments. Steers offered Treat-A increased (P = 0.02) and tended (P = 0.08) to increase the ruminal effective degradable NDF and ADF fractions of the diet-substrate, respectively. When the forage-substrate (low quality) was incubated, steers offered Treat-A tended (P = 0.09) to increase the effective degradable hemicellulose fraction compared to Control. In this experiment, the bacterial combinations did not affect intake and feeding behavior, while the combination with a greater proportion of B. licheniformis (Treat-A) elicited an improved core-fiber digestibility and a healthier ruminal pH pattern, in which the ruminal environment showed to be more prone to induce the effective degradability of fiber fractions, while also releasing more NH3-N.

Supplementing a Bacillus-based direct-fed microbial improves feed efficiency in lactating dairy cows.

This experiment was conducted to evaluate the effects of feeding a Bacillus-based direct-fed microbial (DFM) on performance and nutrient digestibility of lactating dairy cows. Seventy-six lactating (42 ± 6 days in milk[DIM]) Holstein-Friesian primiparous and multiparous cows were enrolled to a 16-wk experiment. Cows were blocked by lactation number and DIM and within blocks, assigned to 1 of the 2 treatments: 1) basal partial-mixed ration (PMR) without DFM addition (n = 38; CON) or 2) basal PMR with the addition of 3g/head/d of a DFM containing B. licheniformis 809 and B. subtilis 810 (n = 38; BOVACILLUS, Chr. Hansen A/S, Hørsholm, Denmark; DFM). The DFM was mixed in a protein-based pellet, whereas the CON group was fed the same pellet without DFM (0.6kg/cow/d). The PMR contained (dry matter [DM] basis) 50% of forage and 48% of a concentrate feed based on corn meal, soybean meal, wheat meal, wheat middlings, and a mineral-vitamin premix, with the remaining part of the diet being represented by the pellet used as a carrier for the treatments (CON and DFM). Dry matter intake (DMI), milk yield, and production efficiency were recorded daily, whereas milk protein and fat concentrations were recorded using electronic milk meters. An additional milk sample was collected every second week of the study for milk composition. On week 15 of the study, fecal samples were collected from each cow for apparent nutrient digestibility calculation. All data were analyzed using the MIXED procedure of SAS (version 9.4; SAS Inst. Inc., Cary, NC). No treatment effects were observed on cow final body weight, daily DMI, milk yield, energy-corrected milk (ECM), ECM efficiency, milk composition (yield or content), and somatic cell count (SCC) (P ≥ 0.12). However, cows fed DFM had a greater feed and N efficiency (P ≤ 0.03) compared to cows fed CON. Moreover, DM digestibility tended to be greater for DFM-fed cows when compared to CON (P = 0.10), whereas no further nutrient digestibility differences were observed (P ≥ 0.24). In summary, supplementing a DFM containing Bacillus licheniformis and B. subtilis benefited feed efficiency of lactating dairy cows fed a PMR, while also tending to improve the digestibility of DM.

Restriction of growth and biofilm formation of ESKAPE pathogens by caprine gut-derived probiotic bacteria.

The accelerated rise in antimicrobial resistance (AMR) poses a significant global health risk, necessitating the exploration of alternative strategies to combat pathogenic infections. Biofilm-related infections that are unresponsive to standard antibiotics often require the use of higher-order antimicrobials with toxic side effects and the potential to disrupt the microbiome. Probiotic therapy, with its diverse benefits and inherent safety, is emerging as a promising approach to prevent and treat various infections, and as an alternative to antibiotic therapy. In this study, we isolated novel probiotic bacteria from the gut of domestic goats (Capra hircus) and evaluated their antimicrobial and anti-biofilm activities against the 'ESKAPE' group of pathogens. We performed comprehensive microbiological, biochemical, and molecular characterizations, including analysis of the 16S-rRNA gene V1-V3 region and the 16S-23S ISR region, on 20 caprine gut-derived lactic acid bacteria (LAB). Among these, six selected Lactobacillus isolates demonstrated substantial biofilm formation under anaerobic conditions and exhibited robust cell surface hydrophobicity and autoaggregation, and epithelial cell adhesion properties highlighting their superior enteric colonization capability. Notably, these Lactobacillus isolates exhibited broad-spectrum growth inhibitory and anti-biofilm properties against 'ESKAPE' pathogens. Additionally, the Lactobacillus isolates were susceptible to antibiotics listed by the European Food Safety Authority (EFSA) within the prescribed Minimum Inhibitory Concentration limits, suggesting their safety as feed additives. The remarkable probiotic characteristics exhibited by the caprine gut-derived Lactobacillus isolates in this study strongly endorse their potential as compelling alternatives to antibiotics and direct-fed microbial (DFM) feed supplements in the livestock industry, addressing the escalating need for antibiotic-free animal products.

Effects of direct-fed microbials on productive performance, qualitative traits of eggs, blood biochemicals and ileal microflora of laying hens fed wheat-soybean-based diet

In this study, effects of direct-fed microbial on qualitative traits of eggs and productive performance, ileal and cecal microflora and pH of hens fed wheat-based diets were investigated from 60 to 68 wk of age. The total number of 240 Hy-Line W-36 laying hens were randomly distributed between 40 cages, and the five experimental diets including (A) corn-soybean meal-based control diet, (B) wheat-based diet with no microbial additive, (C) wheat-based diet supplemented with Bacillus velezensis, (D) wheat-based diet supplemented with S. cerevisiae and (E) wheat-based diet supplemented with B. velezensis + S. cerevisiae were assigned to hens with 8 replicate cages per diet and 6 hens per each replicate. Increased egg weight and production (EP), feed intake (FI), improved feed conversion ratio (FCR) and decreased pH of ileum and caecum were observed in layers fed the diet included B. velezensis + S. cerevisiae (P&lt;0.05). Improved intestinal morphology characteristics were observed in hens fed the microbe-added diets (P&lt;0.05). Increased plasma protein, albumen and HDL in hens fed microbe-added diets (P&lt;0.05). Adding B. velezensis + S. cerevisiae to diet, modulated the ileal and caecal microflora composition by decreasing the numbers of Salmonella and increasing the numbers of Lactobacilli. Based on the results of the current study, it can be concluded that adding B. velezensis + S. cerevisiae to the wheat-based diet improve performance and intestinal morphology characteristics of laying hens, decrease plasma levels of cholesterol, triglyceride, and LDL and the intestinal pH and number of Salmonella.