Effect of ergot alkaloids and a mycotoxin deactivating product on in vitro ruminal fermentation using the Rumen simulation technique (RUSITEC)

Abstract

The rumen simulation technique (RUSITEC) was used to investigate the effect of ergot alkaloids (EA) and a mycotoxin deactivating product (Biomin AA; MDP) on nutrient digestion, ruminal fermentation parameters, total gas, methane, and microbial nitrogen production. Ruminal fermentation vessels received a feedlot finishing diet of 90:10 concentrate:barley silage (DM basis). Using a randomized complete block design, treatments were assigned (n = 4 vessels/treatment) within two RUSITEC apparatuses in a 2 × 2 factorial arrangement. Treatments included: (1) control (CON) diet (no EA and no MDP); (2) CON diet + 1 g/d MDP; (3) CON diet + 20 mg/kg EA; and (4) CON diet + 20 mg/kg EA + 1 g/d MDP. The study was conducted over 14 d with 7 d of adaptation and 7 d of sample collection. Data were analyzed in SAS using PROC MIXED including fixed effects of EA, MDP, and the EA×MDP interaction. Random effects included RUSITEC apparatus and cow rumen inoculum (n = 4). Ergot alkaloids decreased dry matter (DMD) (P = 0.01; 87.9 vs. 87.2%) and organic matter disappearance (OMD) (P = 0.02; 88.8 vs. 88.4%). Inclusion of MDP increased OMD (P = 0.01; 88.3 vs. 88.9%). Neutral detergent fiber disappearance (NDFD) was improved with MDP; however, an EA×MDP interaction was observed with MDP increasing (P < 0.001) NDFD more with EA diet compared to CON. Acetate proportion decreased (P = 0.01) and isovalerate increased (P = 0.03) with EA. Consequently, acetate:propionate was reduced (P = 0.03) with EA. Inclusion of MDP increased total volatile fatty acid (VFA) production (P < 0.001), and proportions of acetate (P = 0.03) and propionate (P = 0.03), and decreased valerate (P < 0.001), isovalerate (P = 0.04), and caproate (P = 0.002). Treatments did not affect (P ≥ 0.17) ammonia, total gas, or methane production (mg/d or mg/g of organic matter fermented). The inclusion of MDP reduced (P < 0.001) microbial nitrogen (MN) production in the effluent and increased (P = 0.01) feed particle-bound MN. Consequently, total MN decreased (P = 0.001) with MDP. In all treatments, the dominant microbial phyla were Firmicutes, Bacteroidota, and Proteobacteria, and the major microbial genus was Prevotella. Inclusion of MDP further increased the abundance of Bacteroidota (P = 0.04) as it increased both Prevotella (P = 0.04) and Prevotellaceae_UCG-003 (P = 0.001). In conclusion, EA reduced OMD and acetate production due to impaired rumen function, these responses were successfully reversed by the addition of MDP.

Ergot alkaloids can affect ruminal metabolism of cattle, which can have large impacts on production, welfare, and health. Mycotoxin deactivators can alleviate some of the negative impacts that dietary ergot has on cattle.

Introduction

The metabolism and impact of ergot alkaloids within the rumen are not well described. Over the last two decades in Western Canada, ergot bodies produced by the parasitic fungus Claviceps purpurea, have been more prevalent in the rye, wheat, barley, and oats (Tittlemier et al., 2015; Coufal-Majewski et al., 2017). The ergot body is comprised of secondary metabolites, known as ergot alkaloids (EA) which include ergometrine, ergotamine, ergosine, ergocristine, ergocryptine, ergocornine, and their corresponding S-epimers (ergo-inines) (Krska et al., 2008). Some research trials have attempted to characterize the impact of these alkaloids on ruminal function (Hill et al., 2001; Matthews et al., 2005; Schumann et al., 2008; Foote et al., 2014). Schumann et al. (2008) observed changes in ruminal fermentation when cattle were fed an EA-contaminated diet and suggested that it impacted rumen microorganisms as evidenced by a shift from acetate to propionate formation, and increased isovalerate and ammonia nitrogen in the ruminal fluid. Furthermore, there is conflicting evidence on the impact of EA on fiber digestion (Coufal-Majewski et al., 2017; Stanford et al., 2018).

To reduce concentrations of ergot bodies, contaminated grain can be separated based on size, weight, or color, but these procedures fail to completely eliminate EA residues from cattle feed (European Food Safety Authority, 2012). Chronic exposure to EA even at low concentrations can be deleterious to cattle. This is primarily due to the structural similarity of EA to the biogenic amine neurotransmitters norepinephrine, epinephrine, and serotonin, with consequent impacts on feed intake and the circulatory system (Klotz, 2015). Consequently, feed additives have been studied that exhibit mycotoxin deactivating properties through adsorption, decreasing bioavailability, and uptake of the toxin (Jard et al., 2011). Mycotoxin deactivators can be made from clay or yeast derivatives, although these products may also reduce digestion and metabolism of diet nutrients (Vila-Donat et al., 2018). Further studies on EA and the use of mycotoxin deactivators may allow researchers to better understand their effect on ruminal fermentation, allowing producers to minimize adverse impacts.

The objective of this study was to determine the effect of EA on a semi-continuous ruminal fermentation system using a diet without and with EA (20 mg/kg) in the presence and absence of a mycotoxin deactivator. It was hypothesized that EA would reduce volatile fatty acid (VFA) and total gas production and lower microbial nitrogen (MN), thus reducing microbial protein synthesis (MPS) with MDP preventing these deleterious effects.

Materials and Methods

The use of animals in this study was approved by the University of Saskatchewan Animal Research Ethics Board (protocol 20190120) with care and management of cows following the guidelines of the Canadian Council on Animal Care (CCAC, 2009).

Experimental design and treatments

The level of six EA (ergocristine, ergocornine, ergocryptine, ergosine, ergotamine, and ergometrine) was determined in a sample of infected rye screenings using the method outlined in Grusie et al. (2017) for an EA panel completed at Prairie Diagnostic Services (Saskatoon, Saskatchewan). Based on the estimate of 496 mg of total alkaloids/kg rye, EA-infected rye was added to the treatment diets to achieve a concentration of 20 mg of total ergot alkaloids/kg of diet DM. A mycotoxin deactivator (MDP) was supplied by BIOMIN Holding GmbH (Getzersdorf, Austria). The MDP was a combination of diatomaceous earth and dried yeast, both presenting adsorbent activities.

Two RUSITEC apparatuses containing 8 vessels each were used in a complete randomized block design. Four treatments were applied as follows: (1) control (CON) diet (no EA and no MDP); (2) CON diet + 1 g/d MDP; (3) CON diet + 20 mg/kg EA; and (4) CON diet + 20 mg/kg EA + 1 g/d MDP. The CON diet consisted of 84.8% barley grain, 10% barley silage, and 5.2% mineral/vitamin supplement (DM basis) (Table 1). The concentration of EA for each diet treatment is outlined in Table 2. Diets containing MDP had an additional 1g/d of MDP added to the 10g/d of diet DM substrate to maintain the same amount of fermentable organic matter from diet in the incubated bags. The weight of MDP added was calculated considering the volume of the fermenters in relation to an average rumen volume (1 liter vessel: 100 liters rumen) rather than the feed:binder ratio. Treatments were replicated within 4 vessels, each replicate had rumen inoculum from a different cow. Diets were ground to pass a 2-mm screen and incubated (10.0–11.0 g DM) in Ankom nylon bags (pore size = 50 μm; Ankom Technol. Corp., Macedon, NY). The EA composition of the diets is shown in Table 1.

Table 1.

Ingredients and chemical composition of the experimental diets

Item	Control	EA1
Diet ingredient, % of DM
Barley grain	84.7	80.8
Barleysilage	10.0	10.0
Mineral2	5.2	5.2
Ergot contaminated rye screenings3	0.0	4.0
Chemical composition
Dry matter, %	94.13	93.96
Organic matter, % of DM	95.23	95.34
Crude protein, % of DM	11.20	11.50
Neutral detergent fiber, % of DM	16.91	17.15
Starch, % of DM	55.24	52.96
NEg, Mcal/kg of DM4	1.33	1.31

EA, ergot alkaloids.

Mineral contained (per kg): 457.73 g of ground wheat, 272.95 g of limestone, 91.38 g of dried molasses, 70.30 g of urea, 40.94 g of magnesium carbonate, 27.30 g of white salt, 13.72 g of tallow, 13.65 g of dicalcium phosphate, 4.27 g of Rumensin (Elanco Animal Health, Greenfield, IN), 2.60 g of MGA 100 premix (Zoetis Canada Inc., Kirkland, QC, Canada), 2.05 g of vitamin E (500,000 IU/kg), 0.91 of vitamin A (12,500,000 IU/kg), 0.47 g of zinc oxide, 0.41 g of copper oxide, and 0.08 g of manganese oxide.

Ergot concentration, 496 mg/kg.

NEg were estimated according to NASEM (2016).

Table 2.

Concentrations (mg/kg) of alkaloid R and S epimers in experimental diets fed to fermentation vessels with or without a mycotoxin deactivating product (MDP)

Alkaloid (R + S Epimer)	Control		EA3
	-MDP1	+MDP2	-MDP	+MDP
Ergocornine/inine	0.00	0.00	0.86	1.61
Ergocristine/inine	0.02	0.02	11.08	13.33
Ergocryptine/inine	0.01	0.00	1.60	1.98
Ergometrine/inine	0.00	0.00	2.27	2.42
Ergosine/inine	0.00	0.00	0.70	0.67
Ergotamine/inine	0.01	0.00	4.46	5.35
Total Ergot alkaloids	0.05	0.04	20.95	25.34

-MDP, diet without mycotoxin deactivating product.

+MDP, diet with mycotoxin deactivating product: 1g of MDP added to 10 g of diet DM, containing mineral adsorbents and dried yeast, fed at 1g/vessel per day.

EA, ergot alkaloids.

The experiment was conducted over 14 d with 7 d for adaptation within the vessel and 7 d for sample collection. Following the 7 d of adaptation, samples were collected daily and analyzed for nutrient disappearance, volatile fatty acids (VFA), ammonia (NH₃-N), microbial nitrogen (MN), total gas, and methane (CH₄) production as described by Ribeiro et al. (2018). In addition, feed residue after incubation was analyzed for total EA by HPLC (Grusie et al. 2017).

RUSITEC start-up, procedure, and design

The ruminal inoculum was collected from 4 ruminally cannulated Holstein cows (770 kg) fed a 50% barley silage:50% barley-based concentrate diet (DM basis). The inoculum was collected 2 h after feeding from 4 distinct locations within the rumen. Rumen fluid was strained through 4 layers of cheesecloth, rumen solids were retained and placed in a plastic bag, and both were immediately transported to the laboratory in preheated insulated thermos containers. Each vessel was filled with 200 mL of prewarmed artificial saliva (pH = 8.2; McDougall, 1948) modified to contain 0.3 g/l of (NH₄)₂SO₄, and 700 mL of filtered ruminal fluid. One bag with solid rumen digesta (20 g wet basis), and one bag containing the assigned diet treatment were added to each fermentation vessel. Vessels were placed into the RUSITEC and maintained at 39°C. Bags were continuously agitated within each vessel at 3 cycles/min. After 24-h, the rumen digesta bag was replaced with one bag containing the assigned diet. Thereafter, nylon feed bags were replaced daily in a manner that ensured that each bag remained in the vessel for 48 h.

During daily feeding (bag exchange) vessels were flushed with carbon dioxide (CO₂) to maintain anaerobiosis. Using a peristaltic pump (model 205S, Watson Marlow, Falmouth, Cornwall, UK), artificial saliva (McDougall, 1948) was infused into each vessel at a continuous rate of 2.9%/h, replacing 70% of the vessel volume daily. The effluent output port was connected to a 1-liter Erlenmeyer flask which had a 2-liter reusable vinyl bag (Curity, Covidien Ltd., Mansfield, MA) connected for total gas collection.

Digestibility, fermentation gas, and pH

After 48-h of incubation, bags were removed from the vessels, rinsed, and dried at 55°C (days 8–10) or freeze-dried (days 11–13). Pooled bag residues from days 8 to 10 and from days 11 to 13 for each vessel were analyzed for dry matter (AOAC, 2006; method 930.15), and ash (AOAC, 2006; method 942.05). The neutral detergent fiber (NDF) was analyzed (Van Soest et al. 1991) using the ANKOM200 Fiber Analyzer (ANKOM Technology Corp., Macedon, NY, USA; Vogel et al., 1999). During NDF determination, sodium sulfite (S430-500 sodium sulfite anhydrous, Fisher Scientific, Italy) and α-amylase (ANKOM Technology Corp., 2052, O’Neil Road, Macedon, NY, USA) were included. Diet and incubation residue samples were also analyzed for starch (Megazyme, 2014; modified from AOAC method 996.22 and 76-13.01), nitrogen (N; AOAC, 2006; method 990.03.), and total EA (Grusie et al., 2017).

Daily total gas production was measured on days 1–14 using a gas meter (model DM3A, Alexander-Wright, London, UK). High gas-producing vessels that exceeded the holding capacity of the bag were measured twice daily. On days 8–10, a 20-mL syringe with a 26-gauge needle was used to sample 20 mL of gas sample from the septum of each collection bag and transferred it to an evacuated 5.9 mL exetainer vial (Labco Ltd., Wycombe, Bucks, UK) for CH₄ analysis. Methane concentration was determined using a Scion 456-Gas Chromatograph (Goes, the Netherlands, EU) with hydrogen (6 mL/min) as the carrier gas and a packed column of 3.4 m filled with Hayesep N.

Fermentation fluid pH was measured daily (Denver Instrument Company model 250, Arvada, Colorado, USA) during feed bag exchange.

Volatile fatty acids and ammonia concentrations

A day prior to sampling for VFA and NH₃-N, 3 mL of 20% (wt/vol) sodium azide solution was added to effluent flasks to arrest microbial fermentation. On days 8–10, two 1.5-mL subsamples were collected directly from the effluent flask, with one being mixed with 0.3 mL of metaphosphoric acid (25%; w/w) for VFA analysis and the other with 0.3 mL of H₂SO₄ (1%; w/w) for NH₃-N analysis. Samples were frozen at −20°C until analysis. Concentrations of VFA were analyzed by gas chromatography (Agilent 6890, Mississauga, ON, Canada) as described by Khorasani et al. (1996) and NH₃-N was analyzed using a colorimetric method as described by Fawcett and Scott (1960).

Microbial nitrogen production

On d 9-13, the McDougall’s buffer was modified, replacing (NH₄)₂SO₄ (1.0 g/l) with ¹⁵N-enriched (NH₄)₂SO₄ (1.0 g/l) (Cambridge Isotope Laboratories, Inc., Andover, MA, USA; minimum ¹⁵N enrichment 10%). Prior to sampling, 3 mL of sodium azide solution (20%; wt/vol) was added to the effluent flask to arrest microbial growth. On days 11–13, the 48-h bags removed from the vessels were processed to obtain the feed particle-bound (FPB) microbial fraction. Bags were removed from the vessel, gently squeezed, then placed in a plastic bag with 20 mL of McDougall’s buffer and manually agitated to dislodge loosely associated microbiota from feed particles. The processed liquid was exuded and retained in the effluent flask. Feed residues were washed twice with 10 mL of McDougall’s buffer in each wash. The wash buffer was retained and pooled with the initially expressed fluid to obtain the liquid-associated microbial fraction. Washed solid feed residues were considered to represent FPB. A 35 mL sample of effluent was then collected as the liquid microbial fraction.

The liquid samples were centrifuged (500 × g, 10 min, 4°C) and the supernatant was retained. The supernatant was then centrifuged (20,000 × g, 30 min, 4°C), and the resulting pellet was washed with distilled water. This process was repeated three more times to isolate the microbial pellet. The microbial pellet was then re-suspended in distilled water and stored at −20ºC until freeze-dried. Additionally, to determine the FPB microbial fraction, solid residue from feed bags was freeze-dried and ball ground. The effluent microbial pellets and FPB fractions were subsampled, encapsulated, and analyzed for total N and ¹⁵N using a Costech ECS4010 elemental analyzer (Costech Analytical Technologies Inc, Valencia, California) coupled to a Delta V mass spectrometer with a Conflo IV interface (Thermo Scientific, Bremen, Germany).

Analysis of bacteria and archaeal diversity

The freeze-dried microbial pellet, as described above, was then analyzed for bacteria and archaeal diversity. Microbial DNA was extracted from ~0.1 g of the freeze-dried, ground material using the Zymobiomics DNA extraction kit as per the manufacturer’s instructions (Zymo Research, Irvine, CA). The concentration and purity of the extracted metagenomic DNA were determined by measuring the ratios of absorbance at 260/280 and 260/230 using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Mississauga, ON, Canada).

Sequencing was performed at Genome Quebec Innovation Center (Montreal, Canada) using the Illumina MiSeq Reagent Kit v2 (500 cycles) following the manufacturer’s guidelines. The primers 515F (5ʹ-GTGCCAGCMGCCGCGGTAA-3ʹ) and 806R (5ʹ-GGACTACHVGGGTWTCTAAT-3ʹ) targeting the V4 region of the 16S rRNA gene were used to examine both bacterial and archaeal diversity (Caporaso et al., 2012). A 33-cycle PCR using 1 μl of a 1 in 10 dilution of genomic DNA and the Fast Start High Fidelity PCR System (Roche, Montreal, PQ) was conducted with the following conditions: 94°C for 2 min, followed by 33 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s, with a final elongation step at 72°C for 7 min. Fluidigm Corporation (San Francisco, CA, USA) barcodes were incorporated in a second PCR reaction using the FastStart High Fidelity PCR System under the following conditions: 95°C for 10 min, followed by 15 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 1 min, followed by a final elongation step at 72°C for 3 min. After amplification, PCR products were assessed in a 2% agarose gel to confirm adequate amplification. All samples were quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies, Carlsbad, CA) and were pooled in equal proportions. Pooled samples were then purified using calibrated Ampure XP beads (Beckman Coulter, Mississauga, ON). The pooled samples (library) were quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies, Carlsbad, CA) and the Kapa Illumina GA with Revised Primers-SYBR Fast Universal kit (Kapa Biosystems, Wilmington, MA). The average fragment size was determined using a LabChip GX (PerkinElmer, Waltham, MA, USA) instrument.

Raw fastq files were imported into Qiime2 for sequence analysis (Bolyen et al., 2019). Primer and adapter sequences were removed from sequence files with the plugin ‘cutadapt’ (Martin, 2011). Following the removal of primer and adapter sequences, the program DADA2 (Katoh and Standley, 2013; Callahan et al., 2016) was used for quality control, and removal of chimeric sequences and assignment of amplicon sequence variants (ASVs) (>99.9 % id). The Mafft program (Katoh and Standley, 2013) was used to perform a multiple sequence alignment and to mask highly variable regions. A phylogenetic tree was generated with FastTree (Price et al., 2010) and taxonomy was assigned to sequences using a Naïve-Bayes classifier trained with the Silva 128 reference database and the ‘feature-classifier’ (Bokulich et al., 2018). Samples were rarefied to the lowest number of sequences found in all samples to ensure that α-diversity analysis used the same number of sequences per sample. The plugin, ‘core-diversity-metrics’ was used to assess α-diversity using Shannon’s and Faith’s Phylogenetic Diversity indices. Sequences were deposited to the Small Reads Archive (NCBI) with accession number PRJNA828192.

Ergot alkaloids

To determine EA disappearance, the feed residue from 3 individual days was compiled (days 8–10, 11-13) by vessel. Feed residues and diets were sent to Prairie Diagnostic Services (Saskatoon, Saskatchewan) for EA analyses. As per Grusie et al. (2017) samples were extracted with solvents and analyzed using liquid chromatography-mass spectrometry.

Statistical analysis

Data were analyzed using the MIXED model of SAS (SAS Institute Inc.) with individual vessels considered as the experimental unit. Fixed effects included dietary EA, MDP, EA × MDP interaction, and random effects included RUSITEC system (1 to 2) and inoculum (cow 1 to 4). Day of sampling and the interaction of day and treatments were included in the model as fixed effects and treated as a repeated measure for gas, methane, pH, VFA, and ammonia-N parameters. Various covariance structures were tested with the final structure chosen based on the minimum Akaike’s information criteria value (Wang and Goonewardene, 2004). Significance was declared at P ≤ 0.05.

Results

pH, total gas production, and methane production

Ergot alkaloids and MDP did not affect fermenter pH or total gas production (P ≥ 0.30; Table 3). An EA × MDP interaction (P = 0.02) was observed for % of CH₄ where inclusion of EA with MDP reduced CH₄ relative to CON with MDP while the other treatments were intermediate and not different. However, there was no effect on CH₄ when expressed in mL/d (P > 0.31), mg/d (P > 0.31), or mg/g of organic matter (OM) fermented (P > 0.17).

Table 3.

Effect of ergot alkaloids (EA) and a mycotoxin deactivating product (MDP) on pH, total gas, and methane (CH₄) production when fed a high grain diet in the RUSITEC

Item	Control		EA		SEM	P-value
	-MDP1	+MDP2	-MDP	+MDP		EA	MDP	EA × MDP
pH	6.62	6.61	6.61	6.60	0.014	0.35	0.30	0.91
Total gas, litersd	2.99	3.01	3.00	3.18	0.292	0.57	0.51	0.59
CH4, %3	4.46ab	4.80a	4.54ab	3.70b	0.454	0.04	0.30	0.02
CH4, mL/d	129	141	138	121	14.3	0.66	0.85	0.31
CH4, mg/d	85.6	93.2	90.9	80.0	9.45	0.66	0.85	0.31
CH4, mg/g of OM fermented	10.14	11.65	10.84	9.25	1.14	0.45	0.97	0.17

Within a row, means without a common superscript difference (P < 0.05).

-MDP, diet without mycotoxin deactivating product.

+MDP, diet with mycotoxin deactivating product: 1g of MDP added to 10 g of diet DM, containing mineral adsorbents and dried yeast, fed at 1g/vessel per day.

Percentage of total gas

Nutrient and EA disappearance

Feed disappearance was influenced by EA and MDP (Table 4). Dietary EA decreased DM (P = 0.01), OM (P = 0.02), and starch disappearance (P = 0.04). Furthermore, the inclusion of MDP increased OMD (P = 0.01) and total EA disappearance (P = 0.01). Additionally, interaction of EA × MDP was observed for NDF disappearance (P < 0.001). The addition of MDP increased NDF disappearance for both diets, but the impact of MDP was greater when provided with EA.

Table 4.

Effects of ergot alkaloids (EA) and a mycotoxin deactivating product (MDP) on the disappearance of a high grain diet in the RUSITEC

Item	Control		EA		SEM	P-value
	-MDP1	+MDP2	-MDP	+MDP		EA	MDP	EA × MDP
Disappearance
DM, %	87.8	88.0	86.8	87.5	0.38	0.01	0.09	0.33
OM, % of DM	88.7	89.0	87.9	88.8	0.30	0.02	0.01	0.18
Starch, % of DM	99.6	99.7	99.4	99.6	0.07	0.04	0.28	0.49
N, % of DM	96.7	96.6	96.3	95.9	0.35	0.07	0.34	0.56
NDF, % of DM	44.1c	52.7b	41.8c	58.2a	0.83	0.06	<0.001	<0.001
Total EA,%	-	-	73.7	82.4	2.32	-	0.01	-

-MDP, diet without mycotoxin deactivating product.

+MDP, diet with mycotoxin deactivating product: 1 g of MDP added to 10 g of diet DM, containing mineral adsorbents and dried yeast, fed at 1 g/vessel per day.

Ammonia and volatile fatty acids

Ergot alkaloids did not affect daily NH₃-N production (P = 0.90; Table 5). EA inclusion did not affect total daily VFA production, although EA decreased the proportion of acetate (P = 0.01), resulting in a reduced acetate:propionate ratio (P = 0.03). Additionally, EA increased the proportion of isovalerate (P = 0.03). Total daily VFA production and the proportions of acetate and propionate increased with MDP (P < 0.03). Conversely, MDP decreased valerate (P < 0.001), isovalerate (P = 0.04) and caproate (P = 0.002) proportions in the effluent.

Table 5.

Effects of ergot alkaloids (EA) and a mycotoxin deactivating product (MDP) on ammonia and volatile fatty acid production in RUSITEC fermenters fed a high grain diet

Item	Control		EA		SEM	P-value
	-MDP1	+MDP2	-MDP	+MDP		EA	MDP	EA × MDP
NH3-N3, mg/d	67.9	72.7	65.9	73.6	5.10	0.90	0.15	0.72
Total VFA4, mmol/d	63.2	67.4	60.6	67.3	2.70	0.32	<0.001	0.35
VFA proportions, mol/100 mol
Acetate (C2)	47.7	48.4	45.6	47.4	0.97	0.01	0.03	0.29
Propionate (C3)	25.6	25.9	24.7	27.0	1.12	0.85	0.03	0.09
Butyrate	15.6	16.0	17.3	15.8	1.23	0.13	0.30	0.06
Valerate	5.45	4.53	6.17	4.72	0.574	0.09	<0.001	0.32
Isovalerate	4.60	4.05	5.19	4.60	0.393	0.03	0.04	0.95
Isobutyrate	0.78	0.81	0.75	0.79	0.263	0.33	0.08	0.88
Caproate	0.32	0.16	0.27	0.14	0.058	0.41	0.002	0.75
C2/C35	1.89	1.86	1.81	1.75	0.071	0.03	0.28	0.77

-MDP, diet without mycotoxin deactivating product.

+MDP, diet with mycotoxin deactivating product: 1g of MDP added to 10 g of diet DM, containing mineral adsorbents and dried yeast, fed at 1g/vessel per day.

NH₃-N, ammonia nitrogen.

VFA, volatile fatty acid.

C2/C3, acetate:propionate ratio.

Microbial nitrogen (N) production, microbial diversity, phylum and genus abundancies

Ergot alkaloids did not impact MN production (P > 0.19); however, MDP increased production of MN (mg/d) in the feed particlebound fraction (P = 0.01), while decreasing MN in the effluent (P < 0.001) consequently, MDP decreased (P = 0.001) total MN production (Table 6).

Table 6.

Effects of ergot alkaloids (EA) and a mycotoxin deactivating product (MDP) on microbial nitrogen production in RUSITEC fermenters fed a high grain diet

Item	Control		EA		SEM	P-value
	-MDP1	+MDP2	-MDP	+MDP		EA	MDP	EA × MDP
Production of microbial nitrogen, mg/d
FPA1	61.0	47.4	57.0	48.2	3.30	0.48	<0.001	0.30
FPB2	4.8	6.1	5.5	6.4	0.39	0.19	0.01	0.65
Total	65.8	53.5	62.4	54.6	3.28	0.62	0.001	0.33

-MDP, diet without mycotoxin deactivating product.

+MDP, diet with mycotoxin deactivating product: 1g of MDP added to 10 g of diet DM, containing mineral adsorbents and dried yeast, fed at 1g/vessel per day.

FPA, feed particle-associated fraction (from liquid effluent).

FPB, feed particle-bound fraction (from residue of forage bags).

The Shannon Diversity Index (Table 7) indicated a decrease in diversity associated with EA diets (P = 0.05). Compared to the CON, there was no influence (P = 0.82) of MDP on microbial diversity. The ruminal microbiota was dominated by bacteria (>97.6%), while archaea accounted for 2.3–2.4% of the composition. Phylogenetic analysis identified 15 bacterial phyla within the effluent microbial pellet, with eight (Firmicutes, Bacteroidota, Proteobacteria, Actinobacteriota, Spirochaetota, Synergistota, Verrucomicrobiota, and Planctomycetota) being at an abundance >0.5%. The three most abundant phyla include Firmicutes, Bacteroidota, and Proteobacteria. EA increased the relative abundance of Planctomycetota (P = 0.05), whilst decreasing Verrucomicrobiota (P = 0.05), but it did not influence other phyla. The relative abundance of Bacteroidota (P = 0.04), Actinobacteriota (P = 0.04), and Planctomycetota (P = 0.01) increased in vessels with MDP as compared to those that did not receive MDP. Archaea were not impacted (P > 0.85) by either EA or MDP.

Table 7.

Effect of ergot alkaloids (EA) and a mycotoxin deactivating product (MDP) on microbial diversity indexes, the abundance of archaea and bacteria, and phylum-level taxonomic composition of the most abundant bacterial communities (%) in the RUSITEC when fed a high grain diet (n = 4 per treatment)

Item	Control		EA		SEM	P-value
	-MDP1	+MDP2	-MDP	+MDP		EA	MDP	EA × MDP
Shannon Diversity Index	6.5	6.6	6.4	6.4	0.07	0.05	0.09	0.60
Phylogenetic Diversity	26.3	27.8	27.1	27.3	0.68	0.82	0.25	0.40
Archaea	2.3	2.3	2.3	2.4	0.99	0.85	0.89	0.91
Bacteria	97.7	97.7	97.7	97.6	0.99	0.85	0.89	0.91
Firmicutes	50.4	42.0	49.7	47.0	3.74	0.56	0.15	0.45
Bacteroidota	24.8	31.6	26.2	29.5	3.45	0.88	0.04	0.42
Proteobacteria	8.6	7.8	9.5	9.3	2.03	0.54	0.79	0.87
Actinobacteriota	3.6	5.5	2.7	4.0	0.72	0.09	0.04	0.65
Spirochaetota	4.0	3.6	3.5	2.3	1.70	0.20	0.27	0.57
Synergistota	3.8	2.7	3.0	2.6	0.76	0.40	0.12	0.49
Verrucomicrobiota	2.1	2.2	1.4	1.4	0.46	0.05	0.87	0.87
Planctomycetota	0.9	1.6	1.4	2.0	0.20	0.05	0.01	0.92
Others (<0.5%)	1.9	2.5	1.9	2.1	0.5617	0.68	0.56	0.61

-MDP, diet without mycotoxin deactivating product.

+MDP, diet with mycotoxin deactivating product: 1g of MDP added to 10 g of diet DM, containing mineral adsorbents and dried yeast, fed at 1g/vessel per day.

A total of 28 genera with relative abundancies >0.5% were identified (Table 8) with Prevotella, Succiniclasticum and uncultured Selenomonadaceae being the most abundant. EA addition increased (P = 0.02) the relative abundancies of Prevotellaceae_UCG-003 (P = 0.01) and uncultured Selenomonadaceae, while decreasing WCHB1-41 (P = 0.01). An EA × MDP interaction was observed for Treponema (P = 0.03), with its relative abundance decreased by MDP in the EA diet only. Compared to the CON treatment, MDP increased the relative abundancies of Prevotella (P = 0.04), Prevotellaceae_UCG-003 (P = 0.001), Bifidobacterium (P = 0.002) and Pirellulaceae_p-1088-a5_gut_group (P = 0.05), whilst it decreased Succiniclasticum (P < 0.01), Lachnospiraceae_NK3A20_group (P = 0.04), and Oribacterium (P = 0.003).

Table 8.

Effect of ergot alkaloids (EA) and a mycotoxin deactivating product (MDP) on genus-level taxonomic composition of the most abundant bacterial communities (%) in the RUSITEC when fed a high grain diet (n = 4 samples per treatment)

Phylum Genus	Control		EA		SEM	P-value
	-MDP1	+MDP2	-MDP	+MDP		EA	MDP	EA × MDP
Bacteroidota
Prevotella	18.61	24.71	19.60	20.81	2.503	0.37	0.04	0.14
Bacteroidales uncultured	1.96	2.84	2.04	2.23	0.312	0.40	0.11	0.29
Bacteroidales;f__F082;g__F082	1.25	1.88	1.51	2.76	0.617	0.24	0.07	0.51
Rikenellaceae_RC9_gut_group	1.35	1.46	0.98	1.48	0.258	0.25	0.06	0.21
Prevotellaceae_YAB2003_group	0.89	0.85	1.17	0.79	0.203	0.58	0.30	0.41
Prevotellaceae_UCG-003	0.26	0.67	0.57	1.11	0.112	0.01	0.001	0.56
Firmicutes
Succiniclasticum	11.02	5.36	11.03	6.46	1.139	0.63	<0.01	0.64
Selenomonadaceae uncultured	7.63	6.84	11.91	8.72	1.227	0.02	0.09	0.28
Lactobacillus	4.31	4.97	5.46	2.83	2.148	0.72	0.48	0.25
Acetitomaculum	2.35	2.43	2.14	2.86	1.833	0.78	0.36	0.42
Megasphaera	4.49	6.33	6.90	4.25	1.563	0.92	0.80	0.18
Streptococcus	1.86	1.51	1.32	1.23	0.549	0.47	0.69	0.81
Lachnospiraceae_NK3A20_group	2.23	0.83	1.47	1.12	0.547	0.52	0.04	0.17
Schwartzia	0.65	0.69	0.73	1.02	0.157	0.14	0.21	0.34
Erysipelotrichaceae_UCG-002	0.77	1.04	0.69	0.82	0.320	0.55	0.42	0.76
Christensenellaceae_R-7_group	0.58	1.03	0.54	0.67	0.229	0.17	0.06	0.27
Selenomonas	0.90	0.60	0.60	0.51	0.157	0.21	0.21	0.48
Oribacterium	1.01	0.31	0.83	0.38	0.149	0.72	0.003	0.43
Proteobacteria
Ruminobacter	4.13	3.64	4.09	5.12	1.490	0.64	0.86	0.62
Succinivibrionaceae_UCG-002	2.04	2.22	2.63	1.52	0.421	0.90	0.28	0.15
Succinivibrio	1.51	1.51	1.84	1.92	0.387	0.35	0.92	0.92
Spirochaetota
Treponema	3.94a	4.30a	4.23a	2.32b	1.832	0.08	0.10	0.03
Actinobacteriota
Olsenella	3.03	3.54	2.82	2.88	0.748	0.57	0.71	0.76
Bifidobacterium	0.44	0.86	0.39	0.82	0.113	0.65	0.002	0.96
Synergistota
Pyramidobacter	3.22	2.30	2.60	2.14	0.704	0.35	0.12	0.57
Planctomycetota
Pirellulaceae_p-1088-a5_gut_group	0.73	1.27	1.04	1.26	0.172	0.41	0.05	0.37
Verrucomicrobiota
WCHB1-41	1.01	1.47	0.62	0.83	0.175	0.01	0.08	0.48
Pedosphaeraceae_DEV114	0.54	0.71	0.54	0.41	0.325	0.15	0.85	0.15
Others (<0.5%)	10.19	14.01	10.48	11.60	1.715	0.38	0.06	0.27

-MDP, diet without mycotoxin deactivating product.

+MDP, diet with mycotoxin deactivating product: 1 g of MDP added to 10 g of diet DM, containing mineral adsorbents and dried yeast, fed at 1 g/vessel per day.

Discussion

Limits of total ergot bodies in cereal grains destined for livestock feed have been established in the European Union, United States, and Canada and are less than 0.10, 0.30, and 0.10-0.33 mg/kg, respectively (Scott, 2009; Coufal-Majewski et al., 2016). The Canadian Food Inspection Agency (CFIA) recommends maximum levels of total EA in beef cattle diets to be no greater than 2-3 mg/kg (CFIA, 2017). No action levels for total EA in cattle diets were found for the United States, but Evans et al. (2004) recommend less than 1 mg/kg. Quantifying an exact maximum allowable limit for EA is not simple due to factors like the absolute concentration of an individual EA, alkaloid profile, and presence of other mycotoxins that may play an additive or synergistic role in toxicity (Coufal-Majewski et al., 2016). Furthermore, EA may undergo epimerization, resulting in interconversion between R and S alkaloid epimers (European Food Safety Authority, 2012) that may influence toxicity. Moreover, the current CFIA guidelines do not consider feed additives that may deactivate mycotoxins. Feed additives that deactivate mycotoxins via binding, have allowed animals to consume concentrations of mycotoxins that are higher than recommended (Debevere et al., 2020), with most of this research being done using monogastric animals (Debevere et al., 2020). The MDP used in the present study includes mineral adsorbents and dried yeast. Other adsorbent materials used for mycotoxin deactivation include aluminosilicates, bentonites, zeolites, and various types of clay (Huwig et al., 2001).

Previous research has shown that adsorbents reduce the toxicity of aflatoxin (Ramos et al., 1996), with their affinity for other mycotoxins being much more variable (Stoev, 2013). To our knowledge, there is very limited research on the impact of mycotoxin deactivators on the toxicity of EA or their impact on rumen fermentation or microbial profiles.

Impact on fermenter pH and gas production

Ergot alkaloids and MDP had no impact on fermenter pH. This was expected, as there was a constant volume of the strong buffer being pumped into each vessel and the numerical variation in pH among treatments was small. Gas production from the fermentation of the substrate was abundant in the fermenters signifying effective anaerobic digestion, but it was not affected by EA or MDP. The reduction in the % of CH₄ in the total gas produced by EA diets when MDP was added is hard to explain as there were no differences in CH₄ when expressed as mg/d or mg/g of OM fermented.

Impact of EA on nutrient disappearance

The differences between CON diet and EA diet on ruminal fermentation parameters are assumed to be due to the toxic effect of EA, but it is important to note that the inclusion of EA is through rye screenings, and therefore may have interference due to a small inclusion of rye in a barley-based diet. EA appeared to have a clear impact on the degradation of nutrients as we observed reduced dry matter disappearance (DMD), an observation confirmed by previous studies (Matthews et al., 2005), along with decreased OMD, and starch degradation. Coupled with a decrease in the proportion of acetate, inhibited digestion may be due to a shift in the microbial population. Using the Shannon Diversity Index, we observed a decrease in microbial diversity associated with EA treatments. Certain microbial species may be more susceptible to EA, resulting in a reduced population critical for efficient nutrient degradation. When analyzing bacterial abundance, the phyla Verrucomicrobiota decreased with EA diets, as Verrucomicrobiota has been shown to be associated with lignocellulose degradation (Gharechahi et al., 2021) this may play a role in suppressed DM or OM digestion.

Impact of MDP on nutrient disappearance

The addition of MDP to the diet promoted pronounced ruminal OM, EA, and NDF disappearance. Although MDP promoted neutral detergent fiber disappearance (NDFD), the effect was greater with the EA diet, suggesting that MDP may reverse the adverse effects on NDFD from EA inclusion. The influence on OMD is likely driven by the amplified NDFD as there was no change in starch or N disappearance. Improvements in nutrient disappearance may be due to components within MDP, including mineral adsorbents and dried yeast. Methods to amplify digestion within the rumen include the addition of enzymes, bacterial attachment, and colonization of feed particles (Wang and McAllister, 2002). The inclusion of the yeast derivatives may allow for an improvement in the rumen ecosystem through enzymatic activity amplifying digestion (Ghazanfar et al., 2017). Additionally, as MDP promoted an increase in feed particle-bound MN, this may signify a greater attachment of microbes to fermentable material amplifying OMD, NDFD, and EA degradation. Microbial adhesion accelerates nutrient breakdown as it increases the duration and proximity of feed particles to microbial enzymes (Wang and McAllister, 2002). Due to the wide array of ingredients in MDP, it is uncertain which constituent of the product is responsible for this effect or perhaps a synergistic effect may have occurred influencing digestion through increased microbial attachment or possible increased enzyme activity. In previous studies, when ingredients of the MDP used in the present study have been fed independently, the effects were minimal (Grochowska et al., 2012; Belanche et al., 2016; Ortiz et al., 2016); however, the concentration of 1 g/d to a 10 g/d diet, offers a ratio much higher than those used in in vivo studies. Although the amount of MDP compared to diet was high, the volume of binder to the volume in the vessel was calculated to be similar to that in the rumen.

Impact of MDP on EA disappearance

Adding MDP to EA diet increased total ergot disappearance. We cannot differentiate if this was a result of MDP binding some of the EA and reducing its quantification by the laboratory analysis method, or if MDP promoted ruminal conditions that increased microbial activity and consequent EA degradation. We saw a reduction in Treponema by MDP with EA diets, and this may have played a role in increasing NDFD or ergot disappearance. Treponema saccharophilum is described as a sugar fermenter, increasing in abundance with high levels of nonstructural carbohydrates (Liu et al., 2014). Overall, we observed limited interaction effects suggesting that EA and MDP have independent effects contrary to what was hypothesized.

VFA production

The increase in total VFA production reflects the greater digestion of nutrients as a result of the addition of MDP to the diet and supports the greater ruminal nutrient disappearance. This increase in VFA production with MDP could increase the energy supply for maintenance and production/growth of ruminants. The proportions of the total VFA production were partitioned towards more acetate and propionate synthesis, therefore, valerate, isovalerate, and caproate made up a smaller proportion of the total VFA concentration. In a study with dairy cows using a similar mycotoxin binder (MycoFix), Kiyothong et al. (2012) observed a similar increase in VFA concentrations and molar proportions of propionate, but a decrease in molar proportions of acetate and the acetate:propionate ratio. The higher proportion of acetate by adding MDP in the present study likely reflects the increased digestion of NDF, as acetate is a prominent byproduct of fiber digestion (Russell, 1998). The reduction in the proportion of acetate with EA diets compared to CON diets is related to the greater concentrations of valerate and isovalerate observed. This change in the VFA profile with EA diets may be a result of the slight increase in crude protein content due to the rye screenings inclusion.

Ammonia, MN, and microbiota abundancy

There is no question that the microbial community has a major influence on digestion within the rumen, thereby affecting animal performance and health (Hernández et al., 2022). Further understanding the complex dynamics of the microbiota is vital to improving our understanding of their function within the rumen ecosystem. Although MDP in the diet had a positive influence on the degradation of OM and NDF, it promoted a decrease in total MN production. In addition, we saw a tendency for MDP to decrease the diversity of the microbial population, which may help explain the greater OM and NDF degradation as has been seen in more feed-efficient cattle (Shabat et al., 2016a, b; Li and Guan, 2017). A decrease in total MN with MDP addition may be due to MDP binding to important growth factors, which may be vital for certain species of microbes to have efficient reproduction, resulting in impacted microbial growth

In all treatments, the dominant microbial phyla were Firmicutes, Bacteroidota and Proteobacteria. The addition of MDP increased Bacteroidota, Actinobacteriota, and Planctomycetota phylum-level abundance reflecting the important role it played in shifting the microbial population. This is likely due to differences in behavior and the preferred substrate of microbial communities. The phylum Bacteroidota or Bacteroidetes usually predominates in the microbial community attached to the feed particles in the rumen of cattle fed high grain diets (Li et al., 2012). Therefore, the increase in Bacteroidota abundance with MDP may reflect the increase in the MN observed in the FPB fraction with MDP diets. Increased Bacteroidota abundance is often associated with improved hydrolysis of the plant cell wall, protein degradation (Hernández et al., 2022) and fermentation of organic matter (Li et al., 2012). As acetate and propionate are common end-products of Bacteroidota fermentation, this increased growth is reflected in the reported VFA composition and digestibility of organic matter in the present study. In addition, Planctomycetota and Verrucomicrobiota phyla are also feed particle associated (Li et al., 2012). As MDP increased the abundance of Planctomycetota, this may further contribute to the increased FPB MN. EA increased Planctomycetota to a lesser extent, although decreased the abundance of Verrucomicrobiota. The results from this study suggest that MDP may promote increased ruminal feed digestion by stimulating microbial attachment to feed particles.

The most dominant genus for all treatments was Prevotella, which is known for its nutritional versatility to support its microbial growth (Stevenson and Weimer, 2007). Prevotella is considered an acetate producer (Zhou et al., 2021), which is in agreement with an increase in acetate in MDP treatments. Additionally, MDP promoted an increase in Bifidobacterium, a genus that has been associated with increased gut health when probiotics are fed to calves (Malmuthuge et al., 2015), and that has been classified as a starch digestor (Xia et al., 2015). This increase in Bifidobacterium with MDP may be associated with the presence of dried yeast as a component of the MDP but there is limited data to support this.

Conclusion

By using an in vitro rumen simulation technique, we were able to isolate the impact of EA and MDP on ruminal fermentation parameters including digestibility, VFA production, NH₃-N, and MN production, and composition. Dietary EA seemed to negatively affect ruminal digestion, whereas MDP positively influenced digestion. Furthermore, dietary EA had suppressive effects on OMD and ruminal acetate proportions but the addition of MDP counteracted these deleterious effects. The impact of MDP on ruminal fermentation seems to be at least in part associated with changes in the ruminal bacterial population.

Glossary

Abbreviations

ADF

acid detergent fiber

CON

control

DM

dry matter

DMD

dry matter disappearance

EA

ergot alkaloids

FPB

feed particle bound

MDP

mycotoxin deactivating product

MPS

microbial protein synthesis

MN

microbial nitrogen

NDF

neutral detergent fiber

NDFD

neutral detergent fiber disappearance

OM

organic matter

OMD

organic matter disappearance

RUSITEC

rumen simulated technique

VFA

volatile fatty acids

Conflict of Interest Statement

BIOMIN Holding GmbH (now part of DSM) provided the mycotoxin deactivating product used in the study and advised on selecting the adequate dosage for testing the product. They were not involved in the study conception, design, data collection, and analysis, or writing of the manuscript. The authors declare no other real or perceived conflicts of interest.