Direct-fed Microbials Research Articles

Effect of Gestational Direct-Fed Microbials Supplementation on the Metabolic Profile in Periparturient Dairy Cows.

This study was conducted at a commercial dairy farm in Queensland, Australia to evaluate the effects of feeding a lactic acid bacteria-based direct-fed microbial (DFM) during gestation on the metabolic profile of periparturient dairy cows and its effects on milk production and body weight. A total of 150 multiparous Holstein cows were randomly selected based on parity (2.3) and days in milk (130 DIM) and divided into two groups of 75 cows each (control and DFM). The control cows were assigned to a basal diet consisting of a silage-based partial mixed ration (PMR), concentrate fed in the dairy twice a day, and ad libitum pasture. The DFM group received the same basal diet supplemented with three strains of Lactobacillus top-dressed in the feed. The DFM supplementation continued during both the dry period and the subsequent lactation. A subset of 82 cows (40 control and 42 DFM) were monitored during the calving season (March to July 2022) to assess the metabolic profile and postpartum performance. Blood samples were collected during the periparturient period (-4 to -2 w prepartum, around calving, and at weeks 1, 3, and 6 postpartum) to measure the levels of metabolites, enzymes, and minerals. Overall, the serum glucose, NEFA, and chloride levels were higher, while protein and urea were lower in cows supplemented with the DFM (p < 0.005). The pre-calving levels of glucose were higher and the total bilirubin, urea, and BHB were lower in cows supplemented with DFM than in the control (p < 0.05). The post-calving levels of glucose and Mg were also higher in the DFM cows than in the control cows (p < 0.05). Average milk production at 110 DIM was significantly higher in the DFM cows compared to control cows (p = 0.03). Although the total milk production over 305 days was numerically greater in the DFM cows, the difference was not statistically significant (p = 0.3), whereas the milk protein percentage was higher in the control cows (p = 0.03). The body weight of the DFM cows was greater during the periparturient period (p = 0.001) than that of the control cows. In the DFM cows, glucose levels had a positive correlation (r = 0.16) with milk yield, at 110 DIM, while serum total protein had a positive correlation with body weight (r = 0.32) (p < 0.05). In conclusion, feeding Lactobacillus-based DFM during gestation can positively influence the metabolic profile of periparturient cows, which, in turn, may affect the milk production and body weight of postpartum dairy cows.

Short communication: A multispecies bacterial-based direct-fed microbial alleviates Salmonella invasion and supports in vitro epithelial integrity.

Managing bacterial infections is of great importance in livestock production, particularly those caused by Salmonella enterica serovars Typhimurium or Dublin, which can impact both animal health and performance, as well as human food safety. Direct-fed microbials (DFM) can support gastrointestinal function and alleviate the potential negative effects of bacterial infections. In the present study, the capacity of a multispecies bacterial-based DFM containing Ligilactobacillus (formerly Lactobacillus) animalis 506, Propionibacterium freudenreichii 507, Bacillus licheniformis 809, and B. subtilis 597 to reduce S. Typhimurium ATCC14028 invasion was investigated using a co-incubation model with the HT29-MTX-E12 cell line (Exp. 1). Next, a possible antagonistic effect of the DFM against S. Dublin ATCC 41286 was evaluated using an in vitro agar well diffusion method following a co-incubation of 48h (Exp. 2). At last, a series of experiments were performed to evaluate how different doses (6.25 × 106, 2.50 × 107, or 1.00 × 108 CFU/well) of the DFM would support the integrity of intestinal epithelial cells challenged or not with S. Typhimurium ATCC14028 or hydrogen peroxide under a transepithelial electrical resistance (TEER) assay with Caco-2 cells (Exp. 3 and 4). In Exp. 1, BDP significantly (P < 0.001) reduced by 90.8% the invasion of S. Typhimurium into HT29-MTX-E12 cells, whereas viability of the potentially harmful bacteria was reduced by 21.0% (P < 0.0001). In Exp. 2, the antagonistic properties of BDP towards S. Dublin were confirmed by the detection of a clear inhibition zone (size = 8.6mm). Lastly, without challenge, the lowest dose of the DFM (6.25 × 106 CFU) provided the greatest support on the cells (treatment × hour; P < 0.0001). However, when the cells were challenged with S. Typhimurium, all doses alleviated the loss of integrity caused by the pathogen (treatment × hour; P < 0.0001). In cells challenged with hydrogen peroxide, the greater dose (1.00 × 108 CFU) supported the cells for a longer period of time (treatment × hour; P < 0.0001). These in vitro findings set the stage for exploring the potential benefits of using a novel DFM as a promising tool and strategy to mitigate Salmonella enterica infections in ruminants and improve animal health, food safety, and public health. Further in vivo confirmation needs to be developed to validate these preliminary in vitro results.

The effect of dietary supplementation of a Bacillus-based direct fed-microbial on Pekin duck subjected to heat stress challenge

Limited research investigating the use of direct-fed microbials (DFM) to mitigate the effects of heat stress in Pekin ducks exists. This study evaluated the use of a DFM at different inclusion rates. Treatments included commercial-type basal diet (CON), or CON supplemented with 125, 250, 500, or 750 ppm DFM. On d 21–35, ducks were subjected to a cyclic heat stress challenge for 12 h/d. Performance, gait scores, footpad lesions, complete cell blood counts, total plasma corticosterone concentrations, fear testing, bone health, litter quality, and gut health were measured. Body weight on d 35 was lower for CON (p < 0.05) than all other treatments. Feed conversion (d 15–35) was higher (p > 0.05) in CON than all other treatments. Total plasma corticosterone concentrations and heterophil to lymphocyte ratios were greater for CON (p < 0.05) compared to all other treatments, indicating lower stress susceptibility in DFM treatments. The CON had lower villus height (p < 0.05) than the 125 and 250 ppm DFM treatments. There was a quadratic response observed with 500 ppm as the peak in performance and stress measures indicating it may be the optimum. These results indicate that DFM supplementation during heat stress can improve duck performance and welfare.

PSV-5 Characterizing the activity of bovine Lactobacillus strains against the top 7 Shiga-toxin Producing Escherichia coli strains

Abstract Shiga toxigenic Escherichia coli (STEC), specifically serogroups O26, O45, O111, O103, O121, O145, and O157 (Top 7), pose significant health risks. Cattle are the primary reservoir of STEC, and effective intervention remains elusive. The identification of Lactobacillus spp. with antimicrobial activity as direct-fed microbials (DFMs) presents a potential strategy for pathogen reduction. These strains are not only capable of favorably modulating the gut microbiota but also possess the innate ability to produce bacteriocins that can target specific pathogens including STEC. Selection of viable anti-STEC Lactobacillus strains requires assessment of the presence or absence of prophage that may revert from the lysogenic to lytic phase and affect their efficacy. This study aimed to characterize anti-STEC potential of 14 Lactobacillus strains isolated from cattle and to identify their associated bacteriocins and prophages. Antimicrobial activity of 14 Lactobacillus strains against the top 7 STEC strains was determined using live culture spot assays and cell-free supernatants (CFS) well-diffusion assays. To further assess their anti-STEC efficacy, Lactobacillus strains with the strongest activity were selected and further evaluated in MRS broth culture and rumen fluid. The ability of Lactobacillus strains to inhibit STEC attachment to Caco-2 epithelial cells was also assessed. Prokka 1.14.6, and Bagel5 were utilized to identify genes encoding bacteriocins. Additionally, PHASTEST 3.0 was utilized to identify prophages. All 14 strains demonstrated antimicrobial activity against the top 7 STEC serogroups. Notably, L. agilis strains L3 and L6, with genome sizes of 2,063,576 bp and 2,071,091 bp respectively, and L. amylovorus L7 (883,016 bp) exhibited the greatest anti-STEC activities, targeting O157, O26, O45, and O103 for L3 and L6, and O157, O45, and O103 for L7. The CFS from these strains showed anti-STEC activity at pH 4.4-4.6, but not at 7, suggesting that organic acids played a role in inhibition. In MRS, DFM reduced STEC O157:H7 by 5 to 7.4 logs after 24 h. However, in rumen fluid, no notable reduction in STEC O157:H7 was observed. Bioinformatic analysis revealed that the L7 genome encoded Helveticin-J, a Class III bacteriocin. Prophage identified included 1 intact (51,818 bp) and 1 non-intact (17,267 bp) in L3, and 2 intact (46,916 bp, 20,295 bp) and 1 non-intact (13,939 bp) in L6, while no prophage was identified in L7. This study identified novel DFMs with activity against STEC, but future studies with cattle are required to confirm their ability to inhibit STEC.

127 Effects of maternal pre- and post-partum supplementation of a Bacillus-based DFM on plasma metabolome profile of Bos indicus-influenced heifers and their offspring

Abstract This study evaluated the effects of maternal supplementation during pre- and postpartum of a Bacillus-based direct-fed microbial (DFM) on plasma metabolome of Bos indicus-influenced cow-calf pairs. On d 0, 72 pregnant Brangus crossbred beef heifers (20 to 22 mo of age) were stratified by their initial body weight (BW; 431 ± 31 kg) and body condition score (BCS; 6.0 ± 0.36), and randomly allocated into 1 of 12 bahiagrass pastures (1 ha and 6 heifers/pasture). Treatments were randomly assigned to pastures (6 pastures/treatment) and consisted of heifers supplemented with 1 kg/d of soybean hulls dry matter added (BAC) or not (CON) with a Bacillus-based DFM mixture (Bovacillus; 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 12 drylot pens and limit-fed the same concentrate at 3.25% of BW until day 319. On d 271 and 287, calves were vaccinated against pathogens associated with bovine respiratory disease and Clostridium. Blood samples were collected from all heifers on d 0 and 63 and from all calves on d 271, 274, and 287 to determine the plasma concentration of metabolites using liquid chromatography. The PLS-DA revealed a clear separation of plasma metabolome profile of BAC and CON heifers and offspring. For the heifer metabolome, 66.5% of the treatment separation on d 63 was explained using 3 components (R2 = 0.96), and BAC heifers had increased (P ≤ 0.05) plasma concentration of 17 metabolites, including phosphatidylcholines and amino acids related classes, but decreased (P ≤ 0.05) plasma concentration of 4 metabolites from triglycerides class compared with CON heifers. For the calf metabolome, 45.6 (R2 = 0.95), 56.7 (R2 = 0.95), and 61.3% (R2 = 0.99) of the treatment separation on d 271, 274, and 287, respectively, were explained using 3 components. Plasma concentrations of 9, 10, and 28 metabolites associated with protein and fatty acid metabolism increased (P ≤ 0.05) in BAC vs. CON calves on d 271, 274, and 287, respectively. Compared with CON, BAC calves had increased (P ≤ 0.05) concentration of metabolites from phosphatidylcholines class all days, from amino acids related class on d 274 and 287, and from triglycerides class on d 287. Plasma concentrations of 3 and 6 metabolites from triglycerides class decreased (P ≤ 0.05) in BAC vs. CON calves on d 271 and 274, respectively. Thus, maternal supplementation of Bacillus-based DFM altered the plasma metabolome of heifers and their calves, leading to increased plasma concentrations of phosphatidylcholine and amino acids related classes in heifers and calves, but fluctuating effects on triglycerides class concentrations in calves.

Exploring a novel direct-fed microbial for ruminants to mitigate in vitro Salmonella invasion and infection

Abstract Managing bacterial infections is of great importance in cattle production, particularly those caused by Salmonella enterica serovars Typhimurium or Dublin, which can impact both animal health and human food safety. Direct-fed microbial (DFM) can support gastrointestinal health and alleviate potential bacterial infections. In the present study, the capacity of a bacteria-based DFM product [BOVAMINE DEFEND Plus; BDP; Novonesis, Denmark (BDP)] containing Lactobacillus animalis, Propionibacterium freudenreichii, Bacillus licheniformis, and B. subtilis to reduce Salmonella Typhimurium ATCC14028 invasion was investigated using the HT29-MTX cell line (Exp. 1) and also to evaluate the possible antagonistic effect against S. Dublin using an in vitro agar well diffusion method (Exp. 2). Briefly, in Exp. 1, after a 30 min pre-incubation with 1.0 ×108 CFU of BDP and/or 2.5 × 106 CFU of S. Typhimurium were added to each transwell. The quantity of S. Typhimurium invading the cells was measured after 90 min of co-incubation using CFU counts. As a control, the viability of the total S. Typhimurium was assessed to confirm the mode of action of BDP targets the pathogen invasion properties. In Exp. 2, the antagonistic efficacy of BDP against S. Dublin CHCC41286, isolated from cattle, was tested by adding BDP to wells in CLED agar plates casted with the pathogen above, zones of inhibition measured after 48 h incubation. In Exp. 1, BDP significantly (P &amp;lt; 0.05) reduced by 80.6 % the invasion of S. typhimurium into HT-29-MTX cells as well as from 16 % the viability of the pathogen. The antagonistic properties of BDP towards S. Dublin were confirmed by clear inhibition zones (8.6 mm). These two in vitro findings set the stage for exploring the potential benefits of using a novel DFM as a promising tool and strategy to mitigate Salmonella infections in ruminants and improve animal health, food safety, and public health. Further in vivo confirmation needs to be developed to validate these preliminary in vitro results.

46 Feeding the weaned pig gut and beyond with non-nutritional additives

Abstract Weaning imposes simultaneous stress, resulting in reduced growth performance, increased susceptibility to diseases, and increased morbidity and mortality in weaned pigs. Weaning stress can induce negative changes in the intestine, including damaged intestinal integrity, disturbed digestive and absorptive capacity, increased intestinal oxidative stress, and microbial dysbiosis. Improving intestinal development and health is critically important for enhancing disease resistance and overall health of weaned pigs. This, in turn, can increase their survival rate during this vulnerable stage and improve their productive performance in later stages. A healthy gut may include, but is not limited to, several important features: a healthy proliferation of intestinal epithelial cells, an integrated gut barrier function, a preferable or balanced gut microbiota, and well-developed intestinal mucosa immunity. Burgeoning evidence suggests that many non-nutritional additives can enhance overall intestinal health and beyond by targeting one or several functions of the gut. However, the exact protective mechanisms may vary and are still not completely understood. For example, direct-fed microbials may confer health benefits on the host by producing antimicrobial products, regulating gut microbial profile or immunity, and/or enhancing gut barrier function. Non-digestible carbohydrates and prebiotics exhibit striking impacts on reshaping the composition of gut microbiota in the host by boosting the production of health-promoting bacteria. Phytochemicals, a large variety of secondary plant metabolites, have been known for their broad and promising biological properties, including anti-microbial, antioxidant, and immune-modulating activities. Several short chain fatty acids derivatives aim to target the upper gastrointestinal tract, where the major nutrients are digested and absorbed, to promote intestinal health of pigs. Moreover, butyrate and valerate glycerides exhibit strong in vitro antimicrobial effects against enterotoxigenic Escherichia coli F18, one of the dominant pathogens causing post-weaning diarrhea globally. There are more candidates in the list that have been used in weaned pigs and shown promising results on growth and health promotion. Therefore, increasing our current understanding of non-nutritional additives and their influences on intestinal function during weaning will facilitate the development of more dietary strategies that increase stress resilience of weaned pigs.

Effects of a Bacillus-based direct-fed microbial on performance, blood parameters, fecal characteristics, rumen morphometrics, and intestinal gene expression in finishing beef bulls.

We evaluated the effects of supplementing direct-fed microbials (DFM), containing Bacillus licheniformis and Bacillus subtilis, on performance, rumen morphometrics, intestinal gene expression, and blood and fecal parameters in finishing bulls. Nellore × Angus bulls (n = 144; initial BW = 401 ± 45.5kg) were distributed at random in 36 pens (4 bulls/pen and 18 pens/treatment), following a completely randomized design. A ground corn-based finishing diet was offered for ad libitum intake twice a day for 84 d, containing the following treatments: 1) control (without DFM); 2) DFM (B. licheniformis and B. subtilis) at 6.4 × 109 CFU (2g) per animal. The data were analyzed using the MIXED procedure of SAS, with a pen representing an experimental unit, the fixed effect of the treatment, and the random effect of pen nested within the treatment. For fecal parameters (two collections made), the collection effect and its interaction with the treatment were included in the model. Bulls that received the DFM had a decreased dry matter intake (P ≤ 0.01), did not differ in average daily gain (2.05kg; P = 0.39), and had a 6% improvement in gain:feed (P = 0.05). The other performance variables, final BW, hot carcass weight, and hot carcass yield, did not differ (P > 0.10). Plasma urea-N concentration decreased by 6.2% (P = 0.02) in the bulls that received DFM. Glucose, haptoglobin, and lipopolysaccharides were not different between treatments (P > 0.10). Ruminal morphometrics were not affected by the treatment (P > 0.10). The use of DFM tended to reduce fecal starch (P = 0.10). At slaughter, bulls fed DFM had an increased duodenal gene expression of tryptophan hydroxylase-1 (P = 0.02) and of superoxide dismutase-1 (P = 0.03). Overall, supplementation with DFM based on B. licheniformis and B. subtilis to Nellore × Angus bulls in the finishing phase decreased dry matter intake, did not influence ADG, improved gain:feed, and increased the expression of genes important for duodenal function.

Male Holstein calves fed a milk replacer and pelleted calf starter containing a botanical extract or a direct fed microbial (DFM) alone or in combination

Botanical extracts (BE; Apex, Adisseo, North America, Atlanta, GA) are known to enhance DMI and gut health, while direct fed microbials (DFM), such as a lactobacillus acidophilus fermentation product (EX: Excel; Pacer Technology, Inc., Murtaugh, ID), has demonstrated improved gut health and growth performance when fed to growing neonatal Holstein calves. The hypothesis was this combination may be synergistic to neonatal calf growth performance and intestinal health. Eighty 2-5-d old Holstein bull calves were blocked by BW and randomly assigned to 1 of 4 treatments arranged using a randomized complete block design. Treatments were: 1) Control: no additives; 2) BE added at 496 mg/kg to the calf starter (CS); 3) EX added to the CS at 2.50 g/kg with EX added to the milk replacer (MR) at 5 g/d; and 4) BE&EX: BE and EX added to CS at same rates and added EX to MR. Calves received 0.283 kg MR in 1.9 L fed 2 x/d for the first 14 d, then increased to 0.42 kg in 2.84 L fed 2x/d through d 35, followed by 0.42 kg MR in 2.84 L fed 1x/d through d 42, followed by weaning. The CS was a 25% CP DM basis mini-pellet and the MR was a 22:20 (CP:fat) fed 2x/d at 0630 and 1800 h along with free choice water. Weaning occurred after d 49 of the 70-d experiment. Calves fed CS EX alone demonstrated greater BW gain compared with calves fed BE&EX with calves fed Control and BE being intermediate and similar (63.9, 63.5, 65.0, and 59.7 kg for Control, BE, EX, and BE&EX, respectively). Total DMI (MR+CS) was lower for calves fed BE&EX compared with calves fed the remaining treatments (116.5, 114.2, 116.4, and 104.9 kg). The feeding of a BE in combination with EX (DFM) to neonatal calves reduced BW during wk 8, 9, and 10 and reduced 0-70 d ADG (874.7, 870.0, 889.7, and 817.6 g/d) compared with calves fed the remaining treatments. Calves fed BE&EX demonstrated the lowest calf starter intake during wk 4 through 10 compared with calves fed the other treatments. The study average calf starter intake for calves fed Control and EX was greater compared with calves fed BE&EX with calves fed BE being similar and intermediate. Calves fed EX and BE&EX demonstrated greater total d of fecal scour score = 1 (5.4, 5.7, 8.0 and 8.3 d) compared with calves fed Control and BE. Gains in frame measurements of hip height, hip width, withers height, and body length were similar while calves fed BE&EX demonstrated lower heart girth gains (19.2, 18.3, 19.7 and 17.6 cm) during the milk feeding phase (0 - 7 wk) compared with calves fed Control and EX, with calves fed BE being similar and intermediate. It is not known why this study demonstrated an antagonism between BE and EX in growth performance and feed intake but further research is needed to identify the mechanism of action.

Impact of Lactobacillus- and Bifidobacterium-Based Direct-Fed Microbials on the Performance, Intestinal Morphology, and Fecal Bacterial Populations of Nursery Pigs.

Weaning is a critical stage in the swine production cycle, as young pigs need to adjust to sudden and dramatic changes in their diet and environment. Among the various organ systems affected, the gastrointestinal tract is one of the more severely impacted during this transition. Traditionally, challenges at weaning have been managed by prophylactic use of antibiotics, which not only provides protection against diarrhea and other gut dysfunction but also has growth-promoting effects. With banning or major restrictions on the use of antibiotics for this purpose, various alternative products have been developed as potential replacements, including direct-fed microbials (DFMs) such as probiotics and postbiotics. As their efficiency needs to be improved, a continued effort to gain a deeper understanding of their mechanism of action is necessary. In this context, this report presents a study on the impact of a Lactobacillus-based probiotic (LPr) and a Bifidobacterium-based postbiotic (BPo) when added to the diet during the nursery phase. For animal performance, an effect was observed in the early stages (Day 0 to Day 10), as pigs fed diets supplemented with either DFMs were found to have higher average daily feed intake (ADFI) compared to pigs fed the control diet (p < 0.05). Histological analysis of intestinal morphology on D10 revealed that the ileum of supplemented pigs had a higher villus height/crypt depth ratio (p < 0.05) compared to controls, indicating a benefit of the DFMs for gut health. In an effort to further explore potential mechanisms of action, the effects of the DFMs on gut microbial composition were investigated using fecal microbial communities as a non-invasive representative approach. At the bacterial family level, Lactobacillaceae were found in higher abundance in pigs fed either LPr (D10; p < 0.05) or BPo (D47; p < 0.05). At the Operational Taxonomic Unit (OTU) level, which can be used as a proxy to assess species composition, Ssd-00950 and Ssd-01187 were found in higher abundance in DFM-supplemented pigs on D47 (p < 0.05). Using nucleotide sequence identity, these OTUs were predicted to be putative strains of Congobacterium massiliense and Absicoccus porci, respectively. In contrast, OTU Ssd-00039, which was predicted to be a strain of Streptococcus alactolyticus, was in lower abundance in BPo-supplemented pigs on D47 (p < 0.05). Together, these results indicate that the DFMs tested in this study can impact various aspects of gut function.

Immune modulation and cecal microbiome changes in broilers fed with fenugreek seeds and Bacillus-based probiotics

Intensive broiler production systems face challenges like enteric diseases, impacting global food security. Strategies to enhance broiler immunity and gut health, particularly amidst antibiotic growth promoter restrictions, are crucial. The present study investigated the combined effects of fenugreek seeds (FS) and Bacillus-based direct-fed microbials (DFM) on immune-related gene expression in the ileum and alteration of microbial population in the cecum of broiler. The study involved 160 Ross 308 broiler chicks, which were divided into four groups consisting of 5 replicates, each containing eight birds. The chicks were grown for a period of 42 d, during which they had ad libitum access to feed and water. Dietary treatments were: Control (basal diet), FS5 (basal + 5g/kg fenugreek seeds), FS5DFM (basal + 5g/kg fenugreek seeds + 0.1g/kg Bacillus-based DFM), and DFM (basal + 0.1g/kg Bacillus-based DFM). Ileum tissue and cecal contents were collected on d 42 for gene expression and gut microbiome analysis. Ileal gene expression analysis revealed the downregulation of IL-6, IL-8L2, CASP6, PTGS2, and IRF7 in both FSs and DFMs groups compared to the control, suggesting individual immunomodulatory effects. However, avian β-defensin genes exhibited complex regulation, highlighting the need for further investigation. Cecal microbiome diversity remained stable, with subtle shifts in specific taxa influenced by FSs and DFMs. Interestingly, the combination of the FSs and DFMs uniquely impacted specific taxa, including Clostridiales vadin BB60. These findings suggest that both FSs and DFMs demonstrated potential for improving broiler immunity through inflammation reduction. The combination of FSs and DFMs offers a synergistic effect in immune modulation and specific microbial modulation, warranting further investigation with pathogen challenge models for comprehensive understanding.

Dietary fiber source and direct-fed microbial supplementation effects on lactation performance and feeding behavior of high-producing dairy cows

The objective of this study was to evaluate the effects of dietary fiber source and direct-fed microbial supplementation on lactation performance and feeding behavior of high-producing dairy cows. Sixty-four multiparous Holstein cows (3.5 ± 1.6 lactations; 76 ± 22 DIM and 735 ± 67 kg of BW at covariate period initiation) and 32 gate feeders were enrolled in a study with a completely randomized design and a 2 × 2 factorial arrangement. Cows and gate feeders were randomly assigned to treatments (16 cows and 8 gate feeds per treatment). Cows were allowed 1 week to acclimate to feeding gates followed by a 2-week covariate period. During the acclimation and covariate periods, all cows were fed the same diet to meet or exceed the nutrient requirements. Following the covariate period, cows were enrolled in a 8-week treatment period during which cows were randomly assigned to 1 of 4 treatments consisting of forage inclusion in the diet, either 45.8% (LF) or 56.7% (HF) of DM, and the supplementation of 75 mL/hd/d of a direct-fed microbial (DFM) containing herbal extracts (mallow, mint, and sage), L. plantarum, L. buchneri, Saccharomyces cerevisiae, and sugar cane molasses (Valibiom Mix, Valibiotics, Traiskirchen, Austria) or without supplementation (CON). The average covariate period value of each variable was used as a covariate. Three-way interactions were observed for DMI and feed efficiency. Dry matter intake was 2 to 3 kg greater for LF-DFM than HF-CON and HF-DFM during wk 2, 3, 5, and 8 of the treatment period. Milk production was 2.1 kg/d greater for LF than HF diets. Both milk fat and MUN concentrations were greater for HF than LF diets. Conversely, milk protein concentration was lower for HF than LF diets. The respiration rate measured in the morning was lower with DFM supplementation than CON. Rectal temperature measured in the morning and averaged for the day were greater for LF than HF diets. Under the conditions of the present study, feeding high-forage diets may be an alternative for producers to reduce feeding costs depending on the price of purchased feeds. However, non-forage fiber sources (i.e., soy hulls) must be considered when producers are challenged by either forage shortages or forage with a lower nutritive value. Additionally, DFM supplementation reduced respiration rate in the morning and affected meal behavior of lactating cows.

Holstein calves fed a milk replacer with a direct-fed microbial and a starter containing a botanical extract or a direct-fed microbial alone or in combination

Botanical extracts (BE; Apex, Adisseo, North America) have demonstrated enhanced DMI and improved gut health, while direct fed microbials (DFM), such as Lactobacillus acidophilus fermentation product (EX: Excell, Pacer Technology, Inc.), has demonstrated improved gut health and growth performance of growing Holstein calves. The hypothesis was this combination may be synergistic to neonatal calf growth performance and intestinal health. Eighty, 2-5-d old Holstein bull calves were blocked by BW and randomly assigned to one of 8 treatments arranged in a 2 × 4 factorial using a randomized complete block design. The main factors were milk replacer (MR) without (Control) and with EX added at 5 g/d fed and calf starter (CS). The CS containing no additives (Control); CS containing BE at 496 mg/kg; CS containing EX at 2.50 g/kg; and CS containing BE and EX at the same inclusion rates. The MR were fed 2x/d at 0630 and 1800 h along with free choice CS (amounts and orts weighed d) and water. Weaning occurred after d 42 for the 56-d experiment. No MR by CS main effects interactions were detected for BW, ADG, CS intake, total DMI, feed efficiency or body frame gain parameters. The BW gain (38.0 and 39.3 kg for control and EX, respectively) for MR main effect was similar for calves fed both MR, while CS main effects (38.7, 39.7, 39.2, and 37.2 kg for control, BE, EX, and BE&EX, respectively) was similar among all CS. Gains in body length (10.6 and 10.8 cm), hip width (4.5 and 4.5 cm), withers height, (10.5 and 10.6 cm) heart girth (18.6 and 19.9 cm) and body length (9.1 and 7.9 cm) were similar for calves fed both MR, while CS main effects for hip height (10.5, 10.2, 10.3, and 10.9 cm), hip width (4.7, 4.6, 4.4, and 4.3 cm) withers height (10.7, 10.9, 10.3 and 10.6 cm), heart girth (19.9, 18.9, 18.9, and 19.4 cm), and body length (11.7, 9.1, 8.3, and 8.4 cm) were similar. Total days of a fecal score = 0 was greater for calves fed Control MR and BE CS compared with calves fed Contol MR and the combination of BE&EX with calves fed the remaining treatments being intermediated and similar. This study demonstrated little calf growth performance and health benefits when feeding a BE or EX alone or in combination compared with calves fed control.

Effect of a Multi-Species Direct-Fed Microbial on Growth Performance, Nutrient Digestibility, Intestinal Morphology and Colonic Volatile Fatty Acids in Weanling Pigs

The potentials of ABO replacer of ENZ and DFM on growth performance, AID, colonic VFAs, gut morphology, fecal score and diarrhea incidence were evaluated. We randomly assigned 120 piglets to four experimental diets that included: (1) control diet (CON), fed the basal ration; (2) ABO was added at 250 ppm of in-feed ABO; (3) ENZ was added at a rate of 3 kg/ton feed; (4) DFM was added with 50 × 106 cfu/g of Bacillus subtilis and 2 × 106 cfu/g of Lactobacillus spp. at a rate of 1.2 kg/ton feed. A complete randomized design used six pens per treatment with five pigs per pen. Pigs had ad libitum access to feed and water throughout the 6-week trial. Feed intake and BW were recorded on weeks 0, 2, 4 and 6, as well as fecal scores and diarrhea incidences (visually recorded and calculated). At weeks 2 and 4, a sub-sample of pigs (n = 6) was sacrificed for intestinal morphology, enzyme activity and VFAs. The results of the study demonstrated that DFM piglets showed increased final BW (3 kg) (p &lt; 0.001) vs. CON. Likewise, ADG was positively affected by the incorporation of ABO, ENZ and DFM in the diets, with an average increase of 8 to 17% on ADG compared with CON (p &lt; 0.001). The AID of gross energy, organic matter, CP and EAAs in piglets fed ENZ and DFM were significantly higher (p &lt; 0.05) than those of CON and ABO at weeks 2 and 4. Inclusion of DFM increased intestinal morphology, enzymatic activities and propionic and butyric acid more than in pigs fed CON, ABO and ENZ (p &lt; 0.05). The fecal score and diarrhea incidence generally decreased over time in pigs fed DFM (p &lt; 0.05). These findings indicate that dietary supplementation with DFM has better effects at any period on growth performance, CP and AA digestibility and beneficially altered the intestinal health in weanling piglets.

Enrichment and Characterization of Sheep Caecum Mixed Lactic Acid Bacteria as Potential Direct-fed Microbials (DFMs) in Livestock

Abstract Lactic Acid Bacteria (LAB) have been widely used as feed supplements in the form of live bacteria or Direct-fed microbials (DFMs) which are expected to have a positive impact on livestock productivity. However, each LAB strain has different characteristics so its ability to influence its host livestock will also be different. This research aims to characterize the LAB enrichment process obtained from the caecum of sheep. The sheep caecum LAB underwent re-inoculated while being enriched in MRS broth medium. After obtaining the LAB isolates, characterization was carried out in the form of pH measurements, total plate count, bacteriocin production, and bacterial kinetic growth. The results showed that LAB could be grown from the digesta of sheep caecum as seen from the decrease in the average pH of the growth medium reaching pH 4.14 with a bacterial colony count of 6,01 log CFU/ml. The grown LAB had a Ks value of 0.24 μM glucose and a μmax value of 2.69 OD. Apart from that, the clear zone test showed a positive result. In conclusion, Enrichment of mixed LAB cultures from sheep caecum has decreased environmental pH by 33%. Sheep caecum mixed LAB culture has easy growth characteristics and antimicrobial activity so it can be a suitable alternative DMF candidate. Further research focusing on the application of this LAB strain is needed to be able to determine the effect of bacteria on livestock.

Fermentation Products, Microbial Protein Synthesis, and Hydrolytic Enzymes Activity of Rumen Fluids Local Beef Cattle Supplemented by Direct-Fed Microbials and Waru Leaf Flour

The aim of this study was to evaluate the effect of Waru Leaf Flour (WLF) and Direct-Fed Microbials (DFM) supplementation on volatile fatty acid (VFA) and ammonia (N-NH3) concentrations, total protozoa, microbial protein synthesis (MPS), and the specific activity of cellulase, amylase, protease in rumen fluids of local beef cattle. The study was conducted as an experimental investigation using a factorial Completely Randomized Design (FCRD). The first factor were three levels of DFM (P) mixed with rice straw (0%, 0.5%, and 1% dry matter of rice straw). The second factor consisted of three levels of WLF supplementation (D) in concentrates (0%, 0.24%, and 0.48% of dry matter concentrate). This resulted nine treatment groups, each it replicated three times.The diet composition consisted of rice straw and concentrate at 40:60 ratio (% dry matter). The interaction between DFM and WLF had a statistically significant impact (p&lt;0.01) on the rumen fluid's total protozoa, N-NH3, and protein content. However, this interaction was not statistically significant (P&gt;0.05) in relation to VFA, MPS, cellulase, amylase, and protease activity. While no significant interaction were observed for total VFA, cellulase, amylase, and protease, DFM supplementation had a statistically significant effect (P&lt;0.01) on reducing enzymes activity, and total VFA (P&lt;0.05). SDS-PAGE and zymography analyzes showed two protease molecules at all treatments with molecular weight of 144 kDa and 133 kDa, cellulase molecules at 62-67 kDa and 19-21 kDa respectively. The addition of DFM showed a new protease band, they had a measurement of 14 and 25 kDa.This study provides valuable insights into enzyme activities in rumen, particularly protease, cellulase, and amylase. The results suggest that the combination of 0.24% WLF and 0.5% DFM supplementation holds promise for optimizing rumen conditions and improving the performance of local beef cattle.

Efficacy of supplementing Aspergillus awamori in enhancing growth performance, gut microbiota, digestibility, immunity, and antioxidant activity of heat-stressed broiler chickens fed diets containing olive pulp

BackgroundGut microbes play a significant role in digestion, developing immunity, and intestinal health. Therefore, direct-fed microbials are used to modify gut microbiota, maintain a healthy digestive system, enhance immunity, and promote the broilers’ performance. In addition, it has a role in improving the utilization of unconventional feed ingredients (olive pulp, OP). This study provides the potential role of Aspergillus awamori in enhancing gut microbial content, nutrient utilization, growth performance, and antioxidative status in heat-stressed broiler chickens fed diets containing olive pulp.MethodsThree hundred chicks (Ross 308; one day old) were divided into four treatment groups (75 chick/ group) randomly, as follows; CON: chicks fed a basal diet based on corn and soybean meal, OP10: chicks fed a diet containing 10% OP, OA1: chicks fed a diet containing OP with A. awamori at 100 mg per kg, OA2: chicks fed a diet containing OP with A. awamori at 200 mg per kg.ResultsAdding A. awamori to the broiler diet that contains OP had a positive effect on productive performance via enhancing nutrition digestibility, body weight gain, feed conversion ratio, and carcass characteristics. A. awamori supplementation had a positive impact on immune responses by increasing serum immunoglobulin G and the relative weight of bursa of Fabricius (P < 0.05) compared to the other groups. Chickens fed A. awamori showed a noticeable improvement in the oxidative status through the increase in the level of serum superoxide dismutase, and glutathione peroxidase, and the decrease in the level of malondialdehyde. Feeding A. awamori also modified the intestinal microbial content by increasing the population of Lactobacillus (P < 0.05).ConclusionsOur study indicated that adding 200 mg A. awamori reduced the negative effect of heat stress by modifying the microbial content of the intestine, immune response, and enhancing feed utilization, thus improving broiler performance, as well as, improving the nutritional value of the olive pulp. Therefore, adding A. awamori to the OP diet can be effectively used in heat-stressed broiler diets.

Research Note: Water applied direct-fed microbial reduced mortality in heat stressed broilers

Pressure to reduce the use of antibiotics in the poultry industry has intensified research on alternative solutions to support intestinal health, including but not limited to direct fed microbials (DFM). Heat stress is known to impact intestinal health and function. The aim of this study was to determine efficacy of a water applied DFM product on broiler performance during the summer period. One of two treatments were randomly allocated to 12 replicate floor pens each: a control treatment and a treatment provided daily with a dual strain DFM comprised of Lactobacillus acidophilus AG01 and Bifidobacterium animalis AG02 at 1 × 108 CFU/bird/d. Each pen contained 20 Ross 308 broilers. All birds were fed the same three-phased wheat- and soybean meal-based diets. Body weight, feed intake, feed conversion ratio, and mortality were measured at d 0, 10, 24, 35, and 42. Due to natural extreme external temperature conditions, all birds were subject to heat stress during the end of the grower phase up to and including the finisher phase. Temperature was on average 5°C higher compared to industry recommendation. No significant differences were found in growth performance between the control and DFM treatment, yet BW at d 42 in both treatments was reduced by 19% compared to the breed standard. The DFM treatment significantly reduced mortality among the birds. Overall mortality from d 1 to d 35 was reduced from 4.58% to 0.42% (P = 0.023) and overall mortality from d 1 to d 42 was reduced from 5.83 to 0.83% (P = 0.027). This was driven by the difference in heat-stress related mortality in the finisher phase from d 25 to d 42, where mortality reached only 0.44% in the DFM treatment versus 2.88% in the unsupplemented control treatment. Post-mortem analysis confirmed heat-stress related hypoxia. In conclusion, the dual strain DFM may have provided improved (intestinal) homeostasis and barrier function allowing increased resilience to heat stress in broilers.

149 Development of a leaky gut model: Impact of 24-h feed restriction on ileal expression of tight junction proteins in weaned pigs

Abstract Weanling barrows, 26 to 28 d old [n = 90; Duroc terminal line Fast genetics, initial body weight (BW)= 5.63 ± 0.43 kg] were used to evaluate the effect of 24-h complete feed restriction on growth and intestinal integrity. Upon arrival at the NutriQuest modeling center (Fergus Falls, MN), pigs were weighed, blocked by BW, and randomly assigned to 1 of 30 pens. Five treatments were randomly assigned to 6 pens: 1) Positive Control (PC), and 4 treatments that had 24-hr feed restriction (FR); 2) Negative control (NC), 3) Direct Fed Microbial (DFM) A, 4) DFM B, and 5) DFM C. Feed intake and BW were determined weekly and before and after FR. All pigs were fed a common diet. Fifteen pigs were removed prior to FR due to enteric disease. On d 18, feed was removed from pigs on treatments 2 to 5 for 24 h. After 24 h, 8 pigs per treatment were harvested for tissue collection. Feed access was restored to the remaining pigs who were harvested 3 d later. Ileal samples were collected and immediately frozen. Plasma and fecal samples were collected upon arrival, at FR, and 24 h and 4-d post-FR. Ileal expression of claudin-3, occludin, and tight-junction-1 genes was determined using QuantStudio 3 Real Time PCR system. Fold-change in gene expression compared with NC was calculated for each treatment. Plasma samples were analyzed for LPS binding protein (LBP) concentration, and fecal samples were analyzed for myeloperoxidase (MPO) concentration. Pig (n = 75) was the experimental unit for BW, LBP, MPO, and gene expression data. Pen (n = 30) was the experimental unit for feed intake. Data were analyzed using PROC GLIMMIX of SAS. Preplanned orthogonal contrasts were performed to determine the effect of PC vs. 4 FR treatments. To determine the relationship of tight junction gene expression and LBP and MPO, PROC REG of SAS was used. Data are described as PC vs. FR treatments to focus on model development. Final BW for pigs on PC was greater compared with the FR treatments (P &amp;lt; 0.05; Table 1). From 1 to 4 d post-FR, average daily gain (ADG) for FR treatments was greater than PC (P &amp;lt; 0.01). There was no effect of treatment on feed intake or feed efficiency. Plasma LBP and fecal MPO concentrations were greater 24 h post-FR for pigs on PC compared with FR treatments (P &amp;lt; 0.01). Fold change in occludin and claudin-3 expression was greater for PC compared with FR treatments (P ≤ 0.05). For pigs euthanized 24 h post-FR, there was a positive relationship between LBP concentrations and occludin expression (P &amp;lt; 0.02). Twenty-four hour FR in weaned pigs alters ileal tight junction gene expression and plasma LBP concentrations; therefore, a timed FR event may serve as a model for leaky gut.

PSV-8 Development of genetically engineered microbes as probiotics to enhance ruminant productivity

Abstract Alternatives to antibiotics (especially metaphylactic use of antibiotics in livestock) are critically needed to ensure animal and human health while meeting the increasing food demand of the growing human population. One of the proposed alternatives to antibiotic use that is gaining momentum is the use of probiotics or direct-fed microbials (DFMs), which are live microbes that have specialized functions that improve gastrointestinal health and performance. However, to date, most probiotics or DFMs tested as alternative to antibiotics to reduce pathogen colonization have shown limited effectiveness with inconsistent results. This is due to the fact that only, generally accepted as safe (GRAS) bacterial strains are being used as DFMs or probiotics in the animal industry. However, metagenome studies have clearly shown the presence of unique biosynthetic gene clusters and other beneficial genes within gut environments in livestock species in non-GRAS organisms. As such, with advances in gene editing techniques, tools are now available to strategically integrate beneficial genes found in non-GRAS approved microbial strains into GRAS approved strains to develop genetically engineered microbes with beneficial functions. As a first step towards developing genetically engineered microbes to improve animal health and performance, we engineered a biosynthetic gene cassette identified from rumen microbes into GRAS approved Bacillus strains. We utilized chasis-independent recombinase-assisted genome engineering (CRAGE) and random transposon mutagenesis to insert biosynthetic genes into GRAS approved strains. Furthermore, we performed functional screening experiments to identify strains containing the engineered gene cassette and confirmed using sequencing. This approach can be used to engineer beneficial genes and generate personalized probiotics to improve animal health and productivity.