Effects of direct-fed microbial supplement on ruminal and plasma metabolome of early-lactation dairy cows: Untargeted metabolomics approach.

We examined the effects of 2 multispecies direct-fed microbial (DFM) supplements on ruminal and plasma metabolome of early-lactation dairy cows using a high-coverage untargeted metabolomics approach. A total of 45 multiparous Holstein cows (41 ± 7 DIM) were enrolled for the 14-d pre-experimental and 91-d experimental period and were a subset from a lactation performance study, which used 114 cows. Cows were blocked using pre-experimental energy-corrected milk yield and randomly assigned within each block to 1 of 3 treatments: (1) corn silage-based diet with no DFM supplement (control; CON), (2) basal diet top-dressed with a mixture of Lactobacillus animalis and Propionibacterium freudenreichii at 3 × 109 cfu/d (PRO-A), or (3) basal diet top-dressed with a mixture of L. animalis, P. freudenreichii, Bacillus subtilis, and Bacillus licheniformis at 11.8 × 109 cfu/d (PRO-B). The basal diet was fed ad libitum daily as a TMR at 0600 and 1200 h for a duration of 91 d. Rumen fluid and blood samples were taken on d -3, 28, 49, 70, and 91 and immediately stored at -80°C. Before analysis, ruminal and plasma samples from d 28, 49, 70, and 91 were composited. An in-depth, untargeted metabolome profile of the composite rumen and plasma samples and the d -3 samples was developed by using a chemical isotope labeling/liquid chromatography-mass spectrometry (LC-MS)-based technique. Differentially abundant metabolites (taking into account fold change [FC] values and false discovery rates [FDR]) were identified with a volcano plot. In the rumen, compared with the CON diet, supplemental PRO-A increased (FC ≥1.2; FDR ≤0.05) the relative concentrations of 9 metabolites, including 2-hydroxy-2,4-pentadienoic acid, glutaric acid, quinolinic acid, and shikimic acid, and PRO-B increased relative concentrations of 16 metabolites, including 2-hydroxy-2,4-pentadienoic acid, glutaric acid, 16-hydroxypalmitic acid, and 2 propionate precursors (succinic and methylsuccinic acids). Relative to PRO-A, supplemental PRO-B increased (FC ≥1.2; FDR ≤0.05) relative rumen concentrations of 3 metabolites, 16-hydroxypalmitic acid, indole-3-carboxylic acid, and 5-aminopentanoic acid, but reduced relative rumen concentrations of 13 metabolites, including carnitine, threonic acid, and shikimic acid. Compared with the CON diet, relative concentrations of 13 plasma metabolites, including myxochelin A and glyceraldehyde, were increased (FC ≥1.2; FDR ≤0.05) by PRO-A supplementation, whereas those of 9 plasma metabolites, including 4-(2-aminophenyl)-2,4-dioxobutanoic acid, N-acetylornithine, and S-norlaudanosolin, were reduced (FC ≤0.83; FDR ≤0.05). Supplemental PRO-B increased (FC ≥1.2; FDR ≤0.05) relative concentrations of 9 plasma metabolites, including trans-o-hydroxybenzylidenepyruvic acid and 3-methylsalicylaldehyde, and reduced relative concentrations of 4 plasma metabolites, including ß-ethynylserine and kynurenine. Pathway analysis of the differentially abundant metabolites in both rumen and plasma revealed that these metabolites are involved in AA and fatty acid metabolism and have antimicrobial and immune-stimulating properties. The results of this study demonstrated that dietary supplementation with either PRO-A or PRO-B altered the plasma and ruminal metabolome. Notably, ruminal and plasma metabolites involved in the metabolism of AA and fatty acids and those with immunomodulatory properties were altered by either or both of the 2 microbial additives.

Effects of direct-fed microbial supplementation on performance and immune parameters of lactating dairy cows.

We evaluated the effects of supplementing bacterial direct-fed microbial (DFM) on performance, apparent total-tract digestibility, rumen fermentation, and immune parameters of lactating dairy cows. One hundred fourteen multiparous Holstein cows (41 ± 7 DIM) were used in a randomized complete block design with an experiment comprising 14 d of a covariate (pre-experimental sample and data collection) and 91 d of an experimental period. Cows were blocked based on energy-corrected milk (ECM) yield during the covariate period and the following treatments were randomly assigned within each block: (1) control (CON), corn silage-based total mixed ration without DFM; (2) PRO-A, basal diet top-dressed with a mixture of Lactobacillus animalis and Propionibacterium freudenreichii at 3 × 109 cfu/d; and 3) PRO-B, basal diet top-dressed with a mixture of L. animalis, P. freudenreichii, Bacillus subtilis, and Bacillus licheniformis at 11.8 × 109 cfu/d. Milk yield, dry matter intake (DMI), and body weight were measured daily, while milk samples for component analysis were taken on 2 consecutive days of each week of data collection. Feces, urine, rumen, and blood samples were taken during the covariate period, wk 4, 7, 10, and 13 for estimation of digestibility, N-partitioning, rumen fermentation, plasma nutrient status and immune parameters. Treatments had no effect on DMI and milk yield. Fat-corrected milk (3.5% FCM) and milk fat yield were improved with PRO-B, while milk fat percent and feed efficiency (ECM/DMI) tended to increase with PRO-B compared with PRO-A and CON. Crude fat digestibility was greater with PRO-B compared with CON. Feeding CON and PRO-A resulted in higher total volatile fatty acid concentration relative to PRO-B. Percentage of neutrophils tended to be reduced with PRO-A compared with CON and PRO-B. The mean fluorescence intensity (MFI) of anti-CD44 antibody on granulocytes tended to be higher in PRO-B compared with CON. The MFI of anti-CD62L antibody on CD8+ T cells was lower in PRO-A than PRO-B, with PRO-A also showing a tendency to be lower than CON. This study indicates the potential of DFM to improve fat digestibility with consequential improvement in fat corrected milk yield, feed efficiency and milk fat yield by lactating dairy cows. The study findings also indicate that dietary supplementation with DFM may augment immune parameters or activation of immune cells, including granulocytes and T cells; however, the overall effects on immune parameters are inconclusive.

Effects of a xylanase-rich enzyme on intake, milk production, and digestibility of dairy cows fed a diet containing a high proportion of bermudagrass silage.

We previously reported that milk production in dairy cows was increased by adding a specific xylanase-rich exogenous fibrolytic enzyme (XYL) to a total mixed ration (TMR) containing 10% bermudagrass silage (BMD). Two follow-up experiments were conducted to examine whether adding XYL would increase the performance of dairy cows consuming a TMR containing a higher (20%) proportion of BMD (Experiment 1) and to evaluate the effects of XYL on in vitro fermentation and degradability of the corn silage, BMD, and TMR (Experiment 2). In Experiment 1, 40 lactating Holstein cows in early lactation (16 multiparous and 24 primiparous; 21 ± 3 d in milk; 589 ± 73 kg of body weight) were blocked by milk yield and parity and randomly assigned to the Control and XYL treatments. The TMR contained 20% BMD, 25% corn silage, 8% wet brewer's grain, and 47% concentrate mixture in the dry matter (DM). Cows were fed the XYL-treated or untreated experimental TMR twice per day for 10 wk after a 9-d covariate period. In Experiment 2, ruminal fluid was collected from 3 cannulated lactating Holstein cows fed a diet containing 20% bermudagrass haylage, 25% corn silage and 55% concentrate. In Experiment 1, compared with Control, application of XYL did not affect DM intake (24.0 vs. 23.7 kg/d), milk yield (35.1 vs. 36.2 kg/d), fat-corrected milk yield (36.1 vs. 36.9 kg/d), or yields of milk fat (1.29 vs. 1.31 kg/d) or protein (1.07 vs. 1.08 kg/d). However, intake of neutral detergent fiber (4.67 vs. 4.41 kg/d) tended to increase with XYL; consequently, milk protein concentration was increased by XYL (3.02 vs. 2.95%). Feed efficiency tended to be lower in cows fed XYL (1.57 vs. 1.52 kg of fat-corrected milk/kg of DM intake) compared with Control. In Experiment 2, XYL tended to increase the rate of gas production in the TMR, the molar proportion of propionate for corn silage, and that of valerate for the TMR. In addition, XYL increased in vitro DM, neutral detergent fiber, and acid detergent fiber degradability of BMD and corn silage. Application of XYL to a diet with a relatively high proportion of BMD tended to increase digestible neutral detergent fiber intake, increased milk protein concentration, and in vitro degradability of DM, neutral detergent fiber, and acid detergent fiber. However, XYL did not affect milk production and tended to decrease feed efficiency in early lactation cows.

Short communication: Effects of a physiologically relevant concentration of aflatoxin B1 with or without sequestering agents on in vitro rumen fermentation of a dairy cow diet.

Aflatoxin is a potent carcinogen commonly found in animal feeds that can impair rumen fermentation at high concentrations; however, its effects at physiologically relevant concentrations are unknown. This study examined the effects of aflatoxin B1 (AFB1), with or without bentonite clay (CL) and Saccharomyces cerevisiae fermentation product (SCFP)-based sequestering agents on in vitro rumen fermentation and digestibility of a dairy cow TMR. Corn silage-based TMR (0.5 g, 17.3% crude protein and 1.67 Mcal/kg of net energy for lactation) was incubated in a rumen fluid-buffer inoculum (1:2 ratio; 50 mL) with the following treatments: (1) no additives (control); (2) control + 0.75 µg/L AFB1 (T); (3) T + 80 mg/L sodium bentonite clay (CL; Astra-Ben-20, Prince Agri Products Inc., Quincy, IL); or (4) CL + 14 mg/L SCFP (CL+SCFP; Diamond V, Cedar Rapids, IA). Ruminal fluid was collected 3 h after the morning feeding from 3 cannulated cows fed the same TMR, and rumen fluid from individual cows was used to prepare separate inocula. Each treatment was incubated in duplicate at 39°C for 0, 4, 8, 16, and 24 h in each of 3 runs. Adding T reduced total volatile fatty acid (VFA) concentration after 4 and 8 h and molar proportion of propionate after 4 and 24 h of incubation relative to control. Adding sequestering agents (CL and CL+SCFP) with T did not affect total VFA concentration after 4 or 8 h, but increased total VFA after 16 h and tended to increase molar proportion of propionate after 24 h compared with T. At 24 h, T had lower DM digestibility and higher NH3-N concentration compared with the control. Thus, AFB1, even at very low concentration (0.75 µg/L), had detrimental effects on rumen fermentation and subsequently DM digestibility of the TMR. Adding sequestering agents did not prevent negative effects of T on rumen fermentation within 8 h of incubation; however, sequestering agents were effective after 16 h of incubation.

Effect of sequestering agents based on a Saccharomyces cerevisiae fermentation product and clay on the ruminal bacterial community of lactating dairy cows challenged with dietary aflatoxin B1.

This study was conducted to examine the effects of clay (CL) and Saccharomyces cerevisiae fermentation product (SCFP) on the ruminal bacterial community of Holstein dairy cows challenged with aflatoxin B1 (AFB1). A second objective was to examine correlations between bacterial abundance and performance measures. Eight lactating dairy cows stratified by milk yield and parity were randomly assigned to 4 treatments in a 4 × 4 Latin square design with 2 replicate squares, four 33-d periods, and a 5-d washout between periods. The treatments included (1) control (basal diet, no additive); (2) T (control + 63.4 µg/kg AFB1, oral dose); (3) CL (T + 200 g/head per day of sodium bentonite clay, top-dress); and (4) CL+SCFP [CL + 19 g/head per day Diamond V NutriTek (Diamond V Inc., Cedar Rapids, IA) + 16 g/head per day MetaShield (Diamond V Inc.), top-dress]. Cows were adapted to diets containing no AFB1 from d 1 to 25 (predosing period). From d 26 to 30 (dosing period), AFB1 was orally dosed and then withdrawn for d 31 to 33 (withdrawal period). During the predosing period, compared with the control, feeding CL and CL+SCFP increased the relative abundance of the most dominant phylum, Bacteroidetes (55.1 and 55.8 vs. 50.6%, respectively), and feeding CL+SCFP increased Prevotella abundance (43.3 and 43.6 vs. 40.0%, respectively). During the dosing period, feeding AFB1 did not affect the ruminal bacterial community, but the relative abundance of Fibrobacteraceae increased with CL+SCFP compared with T (1.45 vs. 0.97%); Fibrobacter abundance also tended to increase with CL+SCFP compared with T and control, respectively (1.45 vs. 0.97 and 1.05%, respectively). Feeding AFB1 with or without CL or CL+SCFP did not affect ruminal pH or concentrations of NH3-N, total volatile fatty acids, or individual volatile fatty acids. Milk yield and milk component yields were positively correlated with the relative abundance of unclassified Succinivibrionaceae, unclassified YS2, or Coprococcus. Feed efficiency was positively correlated (r ≥ 0.30) with the relative abundance of unclassified YS2, Coprococcus, or Treponema. Feeding aflatoxin at 63 µg/kg, a common contamination level on farms, did not affect the abundance of dominant bacteria or rumen fermentation. When aflatoxin was fed, CL+SCFP increased the abundance of Fibrobacter, a major fibrolytic bacteria genus. Milk yield and DMI were positively correlated with abundance of Succinivibrionaceae and Coprococcus. Feed efficiency was positively correlated with abundance of Coprococcus, Treponema, and YS2. Future studies should speciate culture and determine the functions of the bacteria to elucidate their roles in the rumen and potential contribution to increasing the performance of dairy cows.

Exogenous fibrolytic enzymes and recombinant bacterial expansins synergistically improve hydrolysis and in vitro digestibility of bermudagrass haylage.

Four experiments were conducted to examine the effects of a recombinant bacterial expansin-like protein (BsEXLX1) from Bacillus subtilis and a commercial exogenous fibrolytic enzyme (EFE) preparation for ruminants on hydrolysis of pure substrates (cellulose and xylan) and in vitro digestibility of bermudagrass haylage (BMH). Recombinant Escherichia coli BL21 strain was used to express BsEXLX1; the protein was purified using an affinity column. In experiment 1, carboxymethylcellulose, Whatman #1 filter paper (General Electric, Boston, MA) and oat-spelt xylan substrates were subjected to 4 treatments (1) sodium citrate buffer (control), (2) BsEXLX1 (162 µg/g of substrate), (3) EFE (2.3 mg/g of substrate), and (4) EFE + BsELX1 in 3 independent runs. Samples were incubated at optimal conditions for both additives (pH 5 and 50°C) or at ruminal (pH 6 and 39°C) or ambient (pH 6 and 25°C) conditions for 24 h and sugar release was measured. In experiment 2, digestibility in vitro of BMH was examined after treatment with the following: (1) control (buffer only), (2) BsEXLX1 (162 µg/g of dry matter), (3) EFE (2.2 mg/g of dry matter), and (4) EFE + BsEXLX1 in 3 independent runs at 39°C for 24 h. Experiment 3 examined effects of EFE and BsEXLX1 on simulated preingestive hydrolysis and profile of released sugars from BMH after samples were suspended in deionized water with sodium azide at 25°C for 24 h in 2 independent runs. In experiment 4, the sequence of the BsEXLX1 purified protein was compared with 447 ruminal bacterial genomes to identify similar proteins from the rumen. In experiment 1, compared with EFE alone, EFE and BsEXLX1 synergistically increased sugar release from carboxymethylcellulose and Whatman #1 filter paper under all simulated conditions; however, hydrolysis of xylan was not improved. In experiment 2, compared with EFE alone, treatment with EFE and BsEXLX1 increased neutral detergent fiber and acid detergent fiber digestibility of bermudagrass haylage (by 5.5 and 15%, respectively) and total volatile fatty acid concentrations, and decreased acetate-propionate ratio. In experiment 3, compared with EFE alone. The EFE and BsEXLX1 synergistically reduced concentrations of neutral detergent fiber and acid detergent fiber and increased release of sugars by 9.3%, particularly cellobiose (72.5%). In experiment 4, a similar sequence to that of BsEXLX1 was identified in Bacillus licheniformis, and similar hypothetical protein sequences were identified in Ruminococcus flavefaciens strains along with different protein structures in E. xylanophilum and Lachnospiraceae. This study showed that an expansin-like protein synergistically increased the hydrolysis of pure cellulose substrates and the hydrolysis and digestibility in vitro of BMH.

Silage review: Mycotoxins in silage: Occurrence, effects, prevention, and mitigation.

Ensiled forage, particularly corn silage, is an important component of dairy cow diets worldwide. Forages can be contaminated with several mycotoxins in the field pre-harvest, during storage, or after ensiling during feed-out. Exposure to dietary mycotoxins adversely affects the performance and health of livestock and can compromise human health. Several studies and surveys indicate that ruminants are often exposed to mycotoxins such as aflatoxins, trichothecenes, ochratoxin A, fumonisins, zearalenone, and many other fungal secondary metabolites, via the silage they ingest. Problems associated with mycotoxins in silage can be minimized by preventing fungal growth before and after ensiling. Proper silage management is essential to reduce mycotoxin contamination of dairy cow feeds, and certain mold-inhibiting chemical additives or microbial inoculants can also reduce the contamination levels. Several sequestering agents also can be added to diets to reduce mycotoxin levels, but their efficacy varies with the type and level of mycotoxin contamination. This article gives an overview of the types, prevalence, and levels of mycotoxin contamination in ensiled forages in different countries, and describes their adverse effects on health of ruminants, and effective prevention and mitigation strategies for dairy cow diets. Future research priorities discussed include research efforts to develop silage additives or rumen microbial innocula that degrade mycotoxins.

Silage review: Foodborne pathogens in silage and their mitigation by silage additives.

Silage is one of the main ingredients in dairy cattle diets and it is an important source of nutrients, particularly energy and digestible fiber. Unlike properly made and managed silage, poorly made or contaminated silage can also be a source of pathogenic bacteria that may decrease dairy cow performance, reduce the safety and quality dairy products, and compromise animal and human health. Some of the pathogenic bacteria that are frequently or occasionally associated with silage are enterobacteria, Listeria, Bacillus spp., Clostridium spp., and Salmonella. The symptoms caused by these bacteria in dairy cows vary from mild diarrhea and reduced feed intake by Clostridium spp. to death and abortion by Listeria. Contamination of food products with pathogenic bacteria can cause losses of millions of dollars due to recalls of unsafe foods and decreases in the shelf life of dairy products. The presence of pathogenic bacteria in silage is usually due to contamination or poor management during the fermentation, aerobic exposure, or feed-out stages. Silage additives and inoculants can improve the safety of silage as well as the fermentation, nutrient recovery, quality, and shelf life. This review summarizes the literature on the main foodborne pathogens that occasionally infest silage and how additives can improve silage safety.

Effect of adding clay with or without a Saccharomyces cerevisiae fermentation product on the health and performance of lactating dairy cows challenged with dietary aflatoxin B1.

The study was conducted to examine the effect of supplementing bentonite clay with or without a Saccharomyces cerevisiae fermentation product (SCFP; 19 g of NutriTek + 16 g of MetaShield, both from Diamond V, Cedar Rapids, IA) on the performance and health of dairy cows challenged with aflatoxin B1 (AFB1). Twenty-four lactating Holstein cows (64 ± 11 d in milk) were stratified by parity and milk production and randomly assigned to 1 of 4 treatment sequences. The experiment had a balanced 4 × 4 Latin square design with 6 replicate squares, four 33-d periods, and a 5-d washout interval between periods. Cows were fed a total mixed ration containing 36.1% corn silage, 8.3% alfalfa hay, and 55.6% concentrate (dry matter basis). Treatments were (1) control (no additives), (2) toxin (T; 1,725 µg of AFB1/head per day), (3) T + clay (CL; 200 g/head per day; top-dressed), and (4) CL+SCFP (CL+SCFP; 35 g/head per day; top-dressed). Cows were adapted to diets from d 1 to 25 (predosing period) and then orally dosed with AFB1 from d 26 to 30 (dosing period), and AFB1 was withdrawn from d 31 to 33 (withdrawal period). Milk samples were collected twice daily from d 21 to 33, and plasma was sampled on d 25 and 30 before the morning feeding. Transfer of ingested AFB1 into milk aflatoxin M1 (AFM1) was greater in T than in CL or CL+SCFP (1.65 vs. 1.01 and 0.94%, respectively) from d 26 to 30. The CL and CL+SCFP treatments reduced milk AFM1 concentration compared with T (0.45 and 0.40 vs. 0.75 µg/kg, respectively), and, unlike T, both CL and CL+SCFP lowered AFM1 concentrations below the US Food and Drug Administration action level (0.5 µg/kg). Milk yield tended to be greater during the dosing period in cows fed CL+SCFP compared with T (39.7 vs. 37.7 kg/d). Compared with that for T, plasma glutamic oxaloacetic transaminase concentration, indicative of aflatoxicosis and liver damage, was reduced by CL (85.9 vs. 95.2 U/L) and numerically reduced by CL+SCFP (87.9 vs. 95.2 U/L). Dietary CL and CL+SCFP reduced transfer of dietary AFB1 to milk and milk AFM1 concentration. Only CL prevented the increase in glutamic oxaloacetic transaminase concentration, and only CL+SCFP prevented the decrease in milk yield caused by AFB1 ingestion.

Bacterial diversity and composition of alfalfa silage as analyzed by Illumina MiSeq sequencing: Effects of Escherichia coli O157:H7 and silage additives.

The first objective of this study was to examine effects of adding Escherichia coli O157:H7 with or without chemical or microbial additives on the bacterial diversity and composition of alfalfa silage. The second objective was to examine associations between the relative abundance of known and unknown bacterial species and indices of silage fermentation quality. Alfalfa forage was harvested at 54% dry matter, chopped to a theoretical length of cut of 19 mm, and ensiled in quadruplicate in laboratory silos for 100 d after the following treatments were applied: (1) distilled water (control); (2) 1 × 105 cfu/g of E. coli O157:H7 (EC); (3) EC and 1 × 106 cfu/g of Lactobacillus plantarum (EC+LP); (4) EC and 1 × 106 cfu/g of Lactobacillus buchneri (EC+LB); and (5) EC and 0.22% propionic acid (EC+PA). After 100 d of ensiling, the silage samples were analyzed for bacterial diversity and composition via the Illumina MiSeq platform (Illumina Inc., San Diego, CA) and chemically characterized. Overall, Firmicutes (74.1 ± 4.86%) was the most predominant phylum followed by Proteobacteria (20.4 ± 3.80%). Relative to the control, adding E. coli O157:H7 alone at ensiling did not affect bacterial diversity or composition but adding EC+LP or EC+LB reduced the Shannon index, a measure of diversity (3.21 vs. 2.63 or 2.80, respectively). The relative abundance of Firmicutes (69.2 and 68.8%) was reduced, whereas that of Proteobacteria (24.0 and 24.9%) was increased by EC+LP and EC+PA treatments, relative to those of the control (79.5 and 16.5%) and EC+LB (77.4 and 18.5%) silages, respectively. Compared with the control, treatment with EC+LP increased the relative abundance of Lactobacillus, Sphingomonas, Pantoea, Pseudomonas, and Erwinia by 426, 157, 200, 194, and 163%, respectively, but reduced those of Pediococcus, Weissella, and Methylobacterium by 5,436, 763, and 250%, respectively. Relative abundance of Weissella (9.19%) and Methylobacterium (0.94%) were also reduced in the EC+LB silage compared with the control (29.7 and 1.50%, respectively). Application of propionic acid did not affect the relative abundance of Lactobacillus, Weissella, or Pediococcus. Lactate concentration correlated positively (r = 0.56) with relative abundance of Lactobacillus and negatively (r = -0.41) with relative abundance of Pediococcus. Negative correlations were detected between ammonia-N concentration and relative abundance of Sphingomonas (r = -0.51), Pantoea (r = -0.46), Pseudomonas (r = -0.45), and Stenotrophomonas (r = -0.38). Silage pH was negatively correlated with relative abundance of Lactobacillus (r = -0.59), Sphingomonas (r = -0.66), Pantoea (r = -0.69), Pseudomonas (r = -0.69), and Stenotrophomonas (r = -0.50). Future studies should aim to speciate, culture, and determine the functions of the unknown bacteria detected in this study to elucidate their roles in silage fermentation.

Effects of the dose and viability of Saccharomyces cerevisiae. 2. Ruminal fermentation, performance of lactating dairy cows, and correlations between ruminal bacteria abundance and performance measures.

This study examined effects of the dose and viability of supplemented Saccharomyces cerevisiae yeast strain YE1496 on ruminal fermentation and performance of lactating dairy cows. A second objective was to examine correlations between ruminal bacteria abundance and performance measures. Four ruminally cannulated lactating cows (284 ± 18 days in milk) were assigned randomly to 1 of 4 treatment sequences in a 4 × 4 Latin square experimental design using four 21-d experimental periods. Cows were fed a nonacidotic total mixed ration comprising 22.5% starch (minimum ruminal pH >5.8), 41.7% corn silage, 7.60% wet brewers grain, and 50.7% concentrate on a dry matter (DM) basis. The diet was supplemented with no yeast (control), a low (5.7 × 107 cfu/d; LLY) or high (6.0 × 108 cfu/d; HLY) dose of live yeast, or a high dose of killed yeast (6.0 × 108 cfu/d; killed by heating at 80°C for 1.5 h; HDY). Milk production and composition were measured twice daily from d 11 to 21 of each period, and rumen fluid samples were collected on d 21. In vivo digestibility was measured using chromic oxide as a marker. Pearson correlation analysis was used to assess whether animal performance parameters were correlated with relative abundance (RA) of ruminal bacteria. Supplemental LLY increased yields (kg/d) of milk (29.6 vs. 31.7) and milk protein (0.95 vs. 1.03), tended to increase milk fat yield (1.10 vs. 1.17) and ruminal acetate:propionate ratio (1.92 vs. 2.21), and increased in vivo apparent digestibility (%) of DM (64.5 vs. 69.1), neutral detergent fiber (NDF; 45.0 vs. 54.5), and ADF (53.1 vs. 60.9) compared with the control. Feeding HLY had no effects on milk yield compared with the control (30.0 vs. 29.6 kg/d). Feeding HDY tended to increase in vivo digestibility (%) of NDF (45.0 vs. 50.7), ADF (53.1 vs. 57.7), and the ruminal concentration of lactate (0.78 vs. 2.82 mM) but did not affect milk yield compared with the control. Dry matter and NDF digestibility correlated negatively with RA of unclassified Lachnospiraceae in both solid (r = -0.50 and -0.52, respectively) and liquid (r = -0.56 and -0.57, respectively) fractions, whereas milk yield correlated positively with RA of Lachnospiraceae [Ruminococcus] (an incompletely classified genus; r = 0.43) in the solid ruminal fraction. Supplemental LLY, HLY, or HDY increased or tended to increase DM, NDF, and ADF digestibility, but only LLY increased yields of milk, milk fat, and milk protein.

Fate of Escherichia coli O157:H7 and bacterial diversity in corn silage contaminated with the pathogen and treated with chemical or microbial additives.

Inhibiting the growth of Escherichia coli O157:H7 (EC) in feeds may prevent the transmission or cycling of the pathogen on farms. The first objective of this study was to examine if addition of propionic acid or microbial inoculants would inhibit the growth of EC during ensiling, at silo opening, or after aerobic exposure. The second objective was to examine how additives affected the bacterial community composition in corn silage. Corn forage was harvested at approximately 35% dry matter, chopped to a theoretical length of cut of 10 mm, and ensiled after treatment with one of the following: (1) distilled water (control); (2) 1 × 105 cfu/g of EC (ECCH); (3) EC and 1 × 106 cfu/g of Lactobacillus plantarum (ECLP); (4) EC and 1 × 106 cfu/g of Lactobacillus buchneri (ECLB); and (5) EC and 2.2 g/kg (fresh weight basis) of propionic acid, containing 99.5% of the acid (ECA). Each treatment was ensiled in quadruplicate in laboratory silos for 0, 3, 7, and 120 d and analyzed for EC, pH, and organic acids. Samples from d 0 and 120 were also analyzed for chemical composition. Furthermore, samples from d 120 were analyzed for ammonia N, yeasts and molds, lactic acid bacteria, bacterial community composition, and aerobic stability. The pH of silages from all treatments decreased below 4 within 3 d of ensiling. Escherichia coli O157:H7 counts were below the detection limit in all silages after 7 d of ensiling. Treatment with L. buchneri and propionic acid resulted in fewer yeasts and greater aerobic stability compared with control, ECCH, and ECLP silages. Compared with the control, the diversity analysis revealed a less diverse bacterial community in the ECLP silage and greater abundance of Lactobacillus in the ECLP and ECA silages. The ECLB silage also contained greater abundance of Acinetobacter and Weissella than other silages. Subsamples of silages were reinoculated with 5 × 105 cfu/g of EC either immediately after silo opening or after 168 h of aerobic exposure, and EC were enumerated after 6 or 24 h, respectively. All silages reinoculated with EC immediately after silo opening (120 h) had similar low pH values (<4.0) and EC counts were below the detection limit. The ECCH and ECLP silages reinoculated with EC after 168 h of aerobic exposure had relatively high pH values (>5.0) and EC counts (5.39 and 5.30 log cfu/g, respectively) 24 h later. However, those treated with L. buchneri or propionic acid had lower pH values (4.24 or 3.96, respectively) and lower EC counts (1.32 log cfu/g or none, respectively). During ensiling, EC was eliminated from all silages at pH below 4.0. During aerobic exposure, the growth of EC was reduced or prevented in silages that had been treated with L. buchneri or propionic acid at ensiling, respectively.

Effects of the dose and viability of Saccharomyces cerevisiae. 1. Diversity of ruminal microbes as analyzed by Illumina MiSeq sequencing and quantitative PCR.

This study was conducted to examine effects of the dose and viability of supplemental Saccharomyces cerevisiae on the ruminal fermentation and bacteria population and the performance of lactating dairy cows. Four ruminally cannulated lactating cows averaging 284±18d in milk were assigned to 4 treatments arranged in a 4×4 Latin square design with four 21-d periods. Cows were fed a total mixed ration containing 41.7% corn silage, 12.1% brewer's grains, and 46.2% concentrate on a dry matter basis. The diet was supplemented with no yeast (control) or with a low dose of live yeast (5.7×107 cfu/cow per day; LLY), a high dose of live yeast (6.0×108 cfu/cow per day; HLY), or a high dose of killed yeast (6.0×108 cfu/cow per day; HDY). Microbial diversity was examined by high-throughput Illumina MiSeq sequencing (Illumina Inc., San Diego, CA) of the V4 region of the 16S rRNA gene. The relative abundance of select ruminal bacteria was also quantified by quantitative PCR (qPCR). Adding LLY to the diet increased the relative abundance of some ruminal cellulolytic bacteria (Ruminococcus and Fibrobacter succinogenes) and amylolytic bacteria (Ruminobacter, Bifidobacterium, and Selenomonas ruminantium). Adding live instead of killed yeast increased the relative abundance of Ruminococcus and F. succinogenes; adding HDY increased the relative abundance of Ruminobacter, Bifidobacterium, Streptococcus bovis, and Selenomonas ruminantium. The most dominant (≥1% of total sequences) bacteria that responded to LLY addition whose functions are among the least understood in relation to the mode of action of yeast include Paraprevotellaceae, CF231, Treponema, and Lachnospiraceae. Future studies should aim to speciate, culture, and examine the function of these bacteria to better understand their roles in the mode of action of yeast. A relatively precise relationship was detected between the relative abundance of F. succinogenes (R2=0.67) from qPCR and MiSeq sequencing, but weak relationships were detected for Megasphaera elsdenii, Ruminococcus flavefaciens, and S. ruminantium (R2≤0.19).

Effects of 3 sequestering agents on milk aflatoxin M1 concentration and the performance and immune status of dairy cows fed diets artificially contaminated with aflatoxin B1.

This study examined whether adding 3 mycotoxin-sequestering agents to diets contaminated with aflatoxin B1 (AFB1) would reduce milk aflatoxin M1 (AFM1) concentration, and improve the performance and alter immune status of dairy cows. Fifteen lactating dairy cows were used in an experiment with an incomplete crossover design including four 28-d periods. Treatments included a control diet (C), a toxin diet (T; 1,725µg of AFB1/head per day; 75µg/kg), and diets containing the toxin and 20g/head per day of a proprietary mixture of Saccharomyces cerevisiae fermentation product containing a low (SEQ1) or high (SEQ2) dose of a chlorophyll-based additive, or a low dose of the chlorophyll-based additive and sodium bentonite clay (SEQ3). Sequestering agents were top-dressed on the total mixed ration (TMR) daily in each period, and AFB1 was dosed orally in gelatin capsules before the TMR was fed on d 21 to 25. Milk was sampled twice daily on d 20 to 28 and plasma was sampled on d 20 and 25. Sequestering agents did not affect milk AFM1 concentration during the toxin-dosing period. However, after AFB1 was withdrawn, the sequestering agents reduced the time required (24 vs. 48h) to reduce the milk AFM1 concentration below the Food and Drug Administration action level of 0.5µg/kg. Feeding T instead of C tended to reduce milk and fat-corrected milk yields, but feeding SEQ1 prevented these effects. Red blood cell count and hemoglobin concentration were reduced by feeding T instead of C, but not by feeding SEQ1, SEQ2, or SEQ3. The mean fluorescence intensity of antibody staining for 2 leukocyte adhesion molecules, L-selectin (CD62L) and ß-integrin (CD18), tended to be greatest when SEQ1 and SEQ3 were fed. Plasma acid-soluble protein concentration was decreased by feeding SEQ1, SEQ2, and SEQ3 instead of T. Sequestering agents had no effect on milk AFM1 concentration, but they reduced the time required to reduce milk AFM1 concentration to a safe level after withdrawal of AFB1 from the diet. Only SEQ1 prevented the adverse effects of AFB1 on milk and fat-corrected milk yields.

An in vitro model to study interactions between Escherichia coli and lactic acid bacterial inoculants for silage in rumen fluid.

UNLABELLED: Previous studies have shown that silages treated with lactic acid bacteria (LAB) inoculants enhance ruminants' performance. The objective of the current experiments was to develop an in vitro model to study interactions between LAB silage inoculants and inoculated silages and Escherichia coli (EC) in rumen fluid (RF). Our hypothesis was that some inoculants inhibit EC in RF. For that purpose buffered RF was incubated under anaerobic conditions at 39°C with commercial strains of LAB silage inoculants or with laboratory corn and wheat silages treated with these LAB, an EC strain and with various ruminant feed ingredients. The EC strain was originally isolated from cattle manure and tagged with a plasmid expressing the green fluorescence protein and kanamycin and streptomycin resistance. Results indicate that the LAB or the treated silages did not suppress EC numbers in the RF. When the pH of the RF decreased below 5·0 the EC disappeared. We conclude that both LAB inoculants for silage and EC survived in RF for several days; however, the inoculants and silages treated with such inoculants did not inhibit EC in RF in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: Forage crops, silage and hay are initial stages of the food chain for humans. Cattle harbours and sheds enterobacteria regularly, some strains of which are pathogens. These can contaminate forage crops through field fertilization with cattle manure. The objective of this study was to develop an in vitro model to test whether lactic acid bacteria, which are used in silage inoculants, alone or in treated silages can inhibit Escherichia coli in rumen fluid. This study presents safety aspects and it is also part of a broad research effort aimed at finding out how LAB silage inoculants and inoculated silages enhance ruminant performance or exert probiotic effects in ruminants.

Control of Escherichia coli O157:H7 in contaminated alfalfa silage: Effects of silage additives.

This study was conducted to examine if adding microbial inoculants or propionic acid to alfalfa silages contaminated with Escherichia coli O157:H7 would inhibit the growth of the pathogen during or after ensiling. Alfalfa forage was harvested at the early bloom stage, wilted to a dry matter concentration of 54%, chopped to 19-mm lengths, and ensiled after treatment with one of the following: (1) distilled water (control); (2) 1×10(5) cfu/g of E. coli O157:H7 (EC); (3) EC and 1×10(6) cfu/g of Lactobacillus plantarum (EC+LP); (4) EC and 1×10(6) cfu/g of Lactobacillus buchneri (EC+LB); and (5) EC and 2.2g/kg of propionic acid (EC+PA). Each treatment was ensiled in quadruplicate in laboratory silos for 0, 3, 7, 16, and 100d and analyzed for EC counts, pH, and organic acids. In addition, samples from d 100 were analyzed for chemical composition, ammonia-N, counts of yeasts and molds, and aerobic stability. Escherichia coli O157:H7 was detected in all silages until d 7, but by d 16 it was not detected in those treated with EC+LB and EC+LP, though it was still detected in EC and EC+PA silages. However, by d 100, the pathogen was not detected in any silage. The rate of pH decrease to 5.0 was fastest for the EC+LP silage (7d), followed by the EC+LB silage (16d). Nevertheless, all silages had attained a pH of or less than 5.0 by d 100. The rapid decrease in pH in EC+LP and EC+LB silages was observed due to higher lactate and acetate concentrations, respectively, relative to the other silages during the early fermentation phase (d 3-16). Propionic acid was only detected in the EC+PA silage. Yeast counts were lowest in EC+LB and EC+PA silages. Subsamples of all d-100 silages were reinoculated with 1×10(5) cfu/g of EC immediately after silo opening. When the pathogen was subsequently enumerated after 168h of aerobic exposure, it was not detected in silages treated with EC+PA, EC+LB, or EC+LP, which all had pH values less than 5.0. Whereas the EC silage had a pH value of 5.4 and 2.3 log cfu/g of the pathogen. Certain bacterial inoculants can hasten the inhibition of E. coli O157:H7 during ensiling, such as propionic acid, and they can also prevent its growth on silage contaminated with the pathogen after ensiling.

Effects of beak amputation and sex on the pecking rate damage and performance parameters of turkey.

This study was carried out to evaluate the effect of sex and beak trimming on pecking and the performance of turkeys. Five hundred and forty unsexed, day old British United Turkey poults were was divided into 3 treatments based on beak trimming at 0, 1/4, 1/3 measured from the tip of the beak inwards with 3 replicates of 60 poults each experiment 1 while 480 turkeys (240 each of male and female) were transferred and allotted to 4 treatment groups of 120 birds each and 4 replicates of 30 turkeys each in experiment 2. Data on performance response and severity of pecking were taken and subjected to one-way analysis of variance in a completely randomised design (experiment 1) and 2x2 factorial layout (factors were sex and beak trimming). Results showed that beak trimming had no significant (p>0.05) effect on all the performance parameters of turkey poults except feed intake while sex and beak trimming had significant (p<0.05) effect on performance indices of turkey. Debeaked male and female recorded higher feed intake, protein intake and feed conversion ratio. There was higher rate of aggressive pecking among the Toms than in the Hens and severity of damage was higher in undebeaked turkeys than the debeaked. Beak trimming can greatly reduce the severity of damage caused by aggressive pecking and should be done twice (6 and 14th week) at 1/4 measured from the tip of the beak.