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.

Symposium review: Technologies for improving fiber utilization.

The forage lignocellulosic complex is one of the greatest limitations to utilization of the nutrients and energy in fiber. Consequently, several technologies have been developed to increase forage fiber utilization by dairy cows. Physical or mechanical processing techniques reduce forage particle size and gut fill and thereby increase intake. Such techniques increase the surface area for microbial colonization and may increase fiber utilization. Genetic technologies such as brown midrib mutants (BMR) with less lignin have been among the most repeatable and practical strategies to increase fiber utilization. Newer BMR corn hybrids are better yielding than the early hybrids and recent brachytic dwarf BMR sorghum hybrids avoid lodging problems of early hybrids. Several alkalis have been effective at increasing fiber digestibility. Among these, ammoniation has the added benefit of increasing the nitrogen concentration of the forage. However, few of these have been widely adopted due to the cost and the caustic nature of the chemicals. Urea treatment is more benign but requires sufficient urease and moisture for efficacy. Ammonia-fiber expansion technology uses high temperature, moisture, and pressure to degrade lignocellulose to a greater extent than ammoniation alone, but it occurs in reactors and is therefore not currently usable on farms. Biological technologies for increasing fiber utilization such as application of exogenous fibrolytic enzymes, live yeasts, and yeast culture have had equivocal effects on forage fiber digestion in individual studies, but recent meta-analyses indicate that their overall effects are positive. Nonhydrolytic expansin-like proteins act in synergy with fibrolytic enzymes to increase fiber digestion beyond that achieved by the enzyme alone due to their ability to expand cellulose microfibrils allowing greater enzyme penetration of the cell wall matrix. White-rot fungi are perhaps the biological agents with the greatest potential for lignocellulose deconstruction, but they require aerobic conditions and several strains degrade easily digestible carbohydrates. Less ruminant nutrition research has been conducted on brown rot fungi that deconstruct lignocellulose by generating highly destructive hydroxyl radicals via the Fenton reaction. More research is needed to increase the repeatability, efficacy, cost effectiveness, and on-farm applicability of technologies for increasing fiber utilization.

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.