Inhibitory Effect Mediated by Deoxynivalenol on Rumen Fermentation under High-Forage Substrate

: Deoxynivalenol (DON) is a type B trichothecene mycotoxin produced by Fusarium fungi. To investigate its ruminal degradability and its effect on rumen fermentation, a 2 × 5 factorial experiment was conducted in vitro with two feed substrates with different forage levels (high forage (HF), forage-to-concentrate = 4:1; low forage (LF), forage-to-concentrate = 1:4) and ﬁve DON additions per substrate (0, 5, 10, 15, and 20 mg/kg of dry matter). After 48 h incubation, the DON degradability in the HF group was higher than in the LF group ( p < 0.01), and it decreased along with the increase in DON concentrations ( p < 0.01), which varied from 57.18% to 29.01% at 48 h. In addition, the gas production rate, total VFA production and microbial crude protein decreased linearly against the increase in DON additions ( p < 0.05). Meanwhile, the proportion of CH 4 in the fermentation gas end-products increased linearly, especially in the HF group ( p < 0.01). In brief, rumen microorganisms presented 29–57% of the DON degradation ability and were particularly signiﬁcant under a high-forage substrate. Along with the increasing DON addition, the toxin degradability decreased, showing a dose-dependent response. However, DON inhibited rumen fermentation and increased methane production when it exceeded 5 mg/kg of dry matter.


Introduction
Mycotoxins have contaminated a large number of cereal grains during growth, harvest, transportation, processing or storage, causing yield losses and economic losses of up to billions of dollars every year all over the world [1].Deoxynivalenol (DON) is a type B trichothecene mycotoxin produced by the Fusarium species of fungi, whose formula for 3,7,15-trihydroxy-12, 13-epoxytrichothec-9-en-8-one is a ubiquitous contaminant of cereal grains worldwide [2,3].As reported in previous studies, DON exerts its toxic effects via the epoxy group sites of C12 and C13 [4] to bind to the peptidyl transferase site of the ribosome and disrupt eukaryotic protein synthesis and trigger immunostimulation and even immunosuppression and apoptosis via activating mitogen-activated protein kinases [5,6].
The excessive dietary intake of DON in livestock leads to anorexia, vomiting, diarrhea, and some severe diseases [7].In ruminants, the toxic risk of dietary DON was thought relatively insensitive compared to other livestock [8], which might be attributed to the degradation by rumen microorganisms.It was reported that rumen microorganisms transformed DON to DOM-1-an epoxide metabolite with less toxicity (1/55 of DON) [9].An earlier in vitro study reported that the fermentation substrate of cellulose instead of corn starch, as a carbon source, presented higher DON degradability [10].The Food and Drug Administration had stipulated the advised upper limit of DON, which is 5 ppm for swine, 10 ppm for chickens, and 30 ppm for ruminants, and is assumed on an 88% dry matter basis [11].
Although rumen microbes had been demonstrated to be somewhat capable of DON detoxification, it was difficult to elucidate the specific impact of different concentrations of DON on rumen fermentation in vivo.Hildebrand et al. [12], in a previous study, noted the feed contaminated by DON at 4.4 mg/kg dry matter (DM) inhibited rumen fermentation in cows, as well as the study of Jeong et al. [10], with 40 mg/kg of purified DON in vitro.However, the dietary DON concentrations at 6 [13], 12 [14], and 64.9 [15] mg/kg DM did not present an inhibitory effect on rumen fermentation, and the studies on the effect of rumen fermentation by adding different concentrations of DON is still unclear.Thus, the objective of the present in vitro study attempted to investigate the effect of increasing DON concentrations on rumen fermentation and DON degradability under different forage levels.

Chemicals
The DON used for the quantitative analysis in this experiment was purchased from the Beijing Clovertech Limited Company (Beijing, China).The DON standard is 99% pure and exists in white needle-like crystal form.Acetonitrile was purchased from Thermo Fisher Scientific (Waltham, MA, USA).The other reagents used in the experiment were all purchased from Sinopharm (Beijing, China).

Animal Diet and Preparation of Rumen Fluid
Three rumen-cannulated lactating Holstein cows (average body weight: 517 ± 25 kg, 125 days in milk, and 36 kg/d of milk yield at the beginning of the study) were chosen as donor animals.The cows were fed at 05: 00 and 17: 00 and had free access to water.The rations are shown in Table 1.The rumen fluids were collected 1 h before the morning feeding through a rumen fistula and strained through four layers of cheesecloth.Then, the rumen fluids were kept in pre-warmed vacuum flasks with an anaerobic environment filled with carbon dioxide.All animal procedures in the present study were approved by the Animal Ethics Committee of China Agricultural University.The sampling procedures followed the Guidelines on the Ethical Treatment of Experimental Animals (2022) No. 398, established by the Ministry of Science and Technology, China.

Experimental Design and In Vitro Fermentation
The composition and nutrient concentrations of two substrate treatments (low forage (LF), forage:concentrate = 1:4; high forage (HF), forage:concentrate = 4:1) used in the present experiment are presented in Table 2. Based on the Association of Official Analytical Chemists [16], the substrate samples were analyzed for crude protein (CP, as 6.25 × N), ether extract (EE) and ash.Based on Van Soest et al. [17], the neutral detergent fiber (NDF) was analyzed with a heat-stable α-amylase and sodium sulfite addition, and the acid detergent fiber (ADF) was determined and expressed inclusive of residual ash.Following the method of Tilley and Terry [18], 500 mg of each substrate sample was incubated at 39  O per liter) and 25 mL of filtered rumen liquor in a fermentation bottle.A stock solution of DON was prepared in Acetonitrile at a concentration of 500 µg/mL.The stock solution of DON was added to each bottle, resulting in 0, 5, 10, 15, and 20 mg/kg of DON/DM in the culture fluid.The entire batch culture was conducted and repeated in five runs on the same days.The bottles were purged with CO 2 for 5 s and sealed with butyl rubber stoppers and Hungate's screw caps, then connected to the gas inlets of a Chinese-patented Automated Trace Gas Recording System (AGRS-III, China Agricultural University, Beijing, China) and incubated at 39 • C as described by Zhang and Yang [19].A 3.0 mL calibrated cumulative gas production for each vent was automatically recorded with a differential pressure switch (pressure range: 20 to 300 Pa, Huba Control Inc., Zurich, Switzerland) when the pressure inside the bottle reached 100 Pa.The cumulative gas production in each bottle was recorded against the time of incubation.The fermentation gas samples were collected from the treated bottles (three bottles/treatment/incubation time) by connecting them to pre-emptied airbags, which were then removed at 3, 6, 12, 24 and 48 h of incubation.A 1.0 mL gas sample was taken from the airbags, and the CH 4 , CO 2 and H 2 contents in the fermentation gas samples were determined using a gas chromatographic method [19].

Samples Collection and Analysis
An amount of 1.5 mL of fermentation liquid at 3, 6, 12, 24, and 48 h was drawn with a 2 mL syringe, which was used to determine the DON content using an ELISA kit (SEKSM-0007; Solarbio, Beijing, China).After 48 h of fermentation, the bottles were removed, and the contents of the bottles were fully transferred into a Nylon bag (pore size 48 µm) that was preweighed, and the filtrate was collected.The nylon bags were washed with the contents to clear and were then dried at 65 • C for less than 48 h to determine the in vitro dry matter disappearance (IVDMD).The value of pH in the culture fluid was measured immediately when the bottle was taken from the system, and a 1.0 mL sample of the culture fluid was mixed with 0.3 mL of 25% (w/v) meta-phosphoric acid solution, then centrifuged at 15,000× g (TG20M high-speed centrifuge, PingFan Instrumentation Co., Ltd., Changsha, China) for 15 min at 4 • C after 30 min.The supernatants were kept at −20 • C for subsequent determination.The measurement of volatile fatty acid (VFA) was conducted as described by Yang et al. [20] on a gas chromatograph (GC1120; Wufeng Instruments, Shanghai, China).The eight microliter sample was mixed with a 0.5 mL phenol chromogenic solution and 0.4 mL hypochlorite solution, and it was heated in a 37 • C water bath for 30 min.Then, the absorbance value was read at 630 nm with a microplate reader to determine the ammonia N (NH 3 -N) (Bio-Rad, Hercules, CA, USA) [21].After the protozoa and feed particles were removed by 430× g for 5 min, the supernatant was washed with distilled water and alkaline hydrolysis with 0.1 m sodium hydroxide.After centrifugation for 30 min at 16,110× g, the supernatant was collected, and the absorbance was measured at 540 nm by adding 50 µL of the supernatant and 150 µL of coomash bright blue chromogenic solution on a 96-well plate to determine the microbial protein (MCP) [22].For the determination of the gas composition, a 1-mL gas sample was removed from the airbags for the measurement of H 2 , CH 4 and CO 2, following the method described by Zhang and Yang [19].The gas sample (1 mL) was injected into a gas chromatograph (GC522, Wufeng Instruments, Shanghai, China) packed with porous carbon beads (TDX-1) in a 2 m stainless steel column (2.0 mm inner diameter).

Gas Production and Curve Fitting
The data for the cumulative gas production can be obtained directly from the automated gas production recording system because they were fitted to the exponential equation as described by Ørskov and McDonald [23].
The cumulative gas production values (GP, mL/g dry matter) exported from the automated gas production recording system were fitted with time (t) to the exponential model as Equation (1) [24]: where A is the estimated asymptotic gas production (mL/g DM); c is the fractional gas production rate (/h); t is the gas recording time, and L is the lag time phase before GP commenced.The parameters A, c and lag were estimated by an iterative least-squares procedure using the NLIN procedure from the Statistical Software Package for Windows (version 9.02, 1999; SAS Institute Inc., Cary, NC, USA) [25].The average gas production rate (AGPR, mL/h) was calculated to obtain the rate between the start of the incubation and the time at which the cumulative gas production was half of its asymptotic value with Equation (2) [26]: where AGPR is the gas production rate of 1/2 of the maximum gas production (mL/h).
The fermentation efficiency (FE) of energy from carbohydrates to VFAs was estimated using Equation ( 5):

Statistical Analysis
Data were subjected to the analysis of variance using the general linear model procedure of SAS (1999), in which the ration type and DON dosage were treated as fixed effects [25].The least-square means were separated using a multiple comparison test (Tukey).The significance was declared at p < 0.05 and p < 0.01.

The DON Degradability
The DON degradability in the HF group was higher than that in the LF group at all-time points, as shown in Table 3 (p < 0.01).In addition, increasing the DON concentration linearly also decreased the DON degradability in both substrates (p < 0.01).The 48 h DON degradability at 5, 10, 15, and 20 mg/kg of DON/DM was 57.18%, 42.59%, 34.83%, and 29.58% in the HF group and 52.08%, 42.25%, 32.81%, and 29.01% in the LF group, respectively.DON degradability increased gradually over time when the DON concentration was 15 and 20 mg/kg, but increased rapidly when the DON concentration was 5 and 10 mg/kg (Figure 1).Furthermore, the degree of decrease in DON's degradability was higher in the HF group than in the LF group no matter the time, when the DON concentration ranged from 5 to 20 mg/kg DM (p < 0.01).It decreased by 29.38% in the HF group compared to 12.69% in the LF group at 6 h.

In vitro Dry Matter Disappearance and Kinetic Gas Production
The in vitro dry matter digestibility (IVDMD), cumulative gas production at 48 h (GP48), and the asymptotic gas production (A) in the LF group were significantly higher than that in the HF group in Table 4 (p < 0.01), and such a situation was observed in the fractional gas production rate (c), the average gas production rate between the start of the incubation and the time when half of A occurred (AGPR), and the time when half of A occurred (T1/2) (p < 0.01).The gas production rate decreased significantly with the increased DON concentration (p = 0.031), while the other indicators were not affected by increasing the DON concentration.Figure 2 showed that the maximum gas production in the LF group was higher than the HF group (p < 0.01), while the DON concentration did not affect the gas production.a,b Means within a row without a common superscript letter differ at p < 0.05; 1 IVDMD48, in vitro dry matter disappearance of 48 h (%); GP48, cumulative gas production at 48 h (mL/g DM); A, the asymptotic gas production (mL/g DM); c, the fractional gas production rate (/h); T1/2, the time when

In Vitro Dry Matter Disappearance and Kinetic Gas Production
The in vitro dry matter digestibility (IVDMD), cumulative gas production at 48 h (GP48), and the asymptotic gas production (A) in the LF group were significantly higher than that in the HF group in Table 4 (p < 0.01), and such a situation was observed in the fractional gas production rate (c), the average gas production rate between the start of the incubation and the time when half of A occurred (AGPR), and the time when half of A occurred (T 1/2 ) (p < 0.01).The gas production rate decreased significantly with the increased DON concentration (p = 0.031), while the other indicators were not affected by increasing the DON concentration.Figure 2 showed that the maximum gas production in the LF group was higher than the HF group (p < 0.01), while the DON concentration did not affect the gas production.A, the asymptotic gas production (mL/g DM); c, the fractional gas production rate (/h); T 1/2 , the time when half of A occurred (h); AGPR, the average gas production rate (mL/h) between the start of the incubation and the time when half of A occurred. 2 L, linear effect of DON addition; Q, quadratic effect of DON addition; I, interaction effect between diet and DON addition.
half of A occurred (h); AGPR, the average gas production rate (mL/h) between the start of the incubation and the time when half of A occurred. 2 L, linear effect of DON addition; Q, quadratic effect of DON addition; I, interaction effect between diet and DON addition.

Fermentation Characteristics in Culture Fluids
As shown in Table 5, the pH in the LF group was significantly lower than in the HF group (p < 0.01), but the opposite situation was observed in NH3-N, MCP and tVFA (p < 0.05).Regarding the rumen fermentation pattern, the molar acetate proportion in the HF group tended to be numerically higher than that in the LF group (p = 0.066), while the LF group significantly increased butyrate, valerate, and isovalerate in comparison with the HF group (p < 0.01).The increasing DON concentration linearly decreased tVFA and MCP (p < 0.01), but it linearly increased NGR (p < 0.05).Moreover, the DON concentration interacted with the substrates on MCP and butyrate (p < 0.05).Along with the increase in DON concentration, MCP decreased in the HF group, but no significant decrease occurred in the LF group.In addition, both the substrate and increasing DON concentration had no effect on FE.

Fermentation Characteristics in Culture Fluids
As shown in Table 5, the pH in the LF group was significantly lower than in the HF group (p < 0.01), but the opposite situation was observed in NH 3 -N, MCP and tVFA (p < 0.05).Regarding the rumen fermentation pattern, the molar acetate proportion in the HF group tended to be numerically higher than that in the LF group (p = 0.066), while the LF group significantly increased butyrate, valerate, and isovalerate in comparison with the HF group (p < 0.01).The increasing DON concentration linearly decreased tVFA and MCP (p < 0.01), but it linearly increased NGR (p < 0.05).Moreover, the DON concentration interacted with the substrates on MCP and butyrate (p < 0.05).Along with the increase in DON concentration, MCP decreased in the HF group, but no significant decrease occurred in the LF group.In addition, both the substrate and increasing DON concentration had no effect on FE. a,b Means within a row without a common superscript letter differ at p < 0.05; 1 NH 3 -N, ammonia nitrogen (mg/dL); MCP, microbial crude protein (mg/mL); tVFA, total volatile fatty acids (mM); NGR, ratio of nonglucogenic to glucogenic acids; FE, fermentation efficiency; 2 L, linear effect of DON addition; Q, quadratic effect of DON addition; I, interaction effect between diet and DON addition.

Fermentation Gas Composition
As shown in Table 6, the proportion of H 2 and CH 4 in the HF group was significantly higher than that in the LF group, but it did not affect the proportion of CO 2 (p < 0.01).The increasing DON concentration linearly increased the proportion of H 2 and CH 4 (p < 0.05), while CO 2 showed a quadratic decreasing trend (p = 0.051).Moreover, there was an interaction between the DON supplemental concentration and forage-to-concentrate ratio on H 2 , CH 4 and CO 2 production (p < 0.01).

Discussion
The in vitro rumen fermentation technology mimicking the rumen environment can estimate feed digestibility, rapidly evaluate the interaction effect between the additives and feed, and provide gas production kinetics data based on the IVDMD and gas production [29].In the present study, this method was used to explore the degradability characteristics of DON in rumen fluid and its effect on in vitro rumen fermentation under high and low proportions of forage in feed substrates.

DON Degradability
Dietary components play an essential role in ruminants regarding growth performance, rumen fermentation and meat yield.The forage-to-concentrate ratio in a diet might shift the rumen fermentation pattern and influence the rumen microorganisms reflected in the population and the composition [30].In the present study, the DON degradability was higher in the high-forage group instead of the low-forage group, consistent with the result of the previous study, which pointed out that the DON degradation rate tended to be higher with cellulose as the carbon source instead of corn starch [10].Additionally, previous evidence suggested that the degradability of DON was inhibited when the pH declined below 5.2 [31], implying a poor DON degradability in low-forage diets, which reduces the rumen pH.The authors in the present study speculated that cellulolytic rumen microbes in a high-forage ration presented a greater capacity for the transformation of DON to a less toxic metabolite (DOM-1), in accordance with the results obtained in previous studies [9,32,33].
In previous studies, little evidence has been reported on DON concentrations and their corresponding degradability during rumen fermentation.It was observed that the transformation ranged from 89% to 37% within 48 h when the DON concentration increased from 10 ppm to 100 ppm in rumen fluid [34], suggesting a reduced ability of rumen fluid to degrade DON as its concentration increased.In the present study, as the concentration of DON increased, a decrease in DON degradability at all incubation time points, regardless of the substrate types, was noted.A similar DON degradability of 89% at a 2 mg/L DON concentration [35], and 56.6% at 20 mg/L, has been reported [36].The results obtained in the current study confirmed that there was an inhibition effect with a high dose of DON on the microbial activity and population in the high-forage diet but not in the low-forage diet.A similar correlation between DON degradability and microbial activity has been reported before [13].More interestingly, the decrease in DON degradability could be associated with a lowered microbial activity in response to a greater DON addition.
In the present study, the DON degradability was 29.01-57.18%at 48 h within the DON concentration of 5-20 mg/kg, while it reached 70-90% at 8 mg/kg DON concentration in some in vivo studies [37,38].Previous studies have speculated that the lack of protozoa in vitro might cause a lower DON degradability [39,40] due to the toxin's degrading capacity of protozoa [41].However, whether protozoa are the main factor causing the difference between the in vivo and in vitro studies remains to be further explored.Consistent with our results, DON cannot completely be degraded by rumen microbes, which is also according to a previous study [42].Even the cultured and purified anaerobic strains incubated in vitro had limits on toxins degradability [43][44][45].As a result, residual DON in rumen fluid might negatively affect rumen fermentation.

Rumen Fermentation
Generally, the gas production extent and rate are pivotal indexes that determine kinetic rumen fermentation.A previous in vitro study with 4.4 mg/kg of DON showed that DON reduced the gas production rate of slowly fermentable components (/h) in a low-forage diet [12], which was in accordance with c (/h) in our study.According to the results of Jeong et al. [10], 40 mg/kg of DON significantly reduced gas production, regardless of the carbon source (corn starch or cellulose).However, DON had no effect on the gas production in the present study, which was consistent with the findings of Razzazi et al. [46].This might be a result of the lower concentrations of DON additions in the present experiment, which was only 1/2 of the Jeong et al. study.As for the gas component, the present study is the first to explore the effect of DON on methane production.The increase in DON additions promoted the production of H 2 and CH 4 , especially in the high-forage substrates, which reflected the interaction between the DON dose and substrates.In the rumen, H 2 and CO 2 were well believed to be used to synthesize CH 4 , which indirectly explained the reason why CO 2 decreased in the present study.In brief, although DON had no significant effect on cumulative gas production, it decelerated the gas production speed in the HF substrate and promoted methanogenesis in the rumen via a reduction in CO 2 with hydrogen.
The VFAs, including acetate, propionate and butyrate, are vital short-chained acids yielded in rumen fermentation, and they provide most of the energy for host ruminants [47].Thus, the level of VFA production can indirectly reflect the efficiency of microbial digestion and the metabolism of nutrients in diets.In the present study, total VFA production was inhibited by an increased DON dose, especially in the LF group, and this was consistent with the results of Jeong et al. [10] and Razzazi et al. [46].Some studies found no significant difference between the DON group and the control group [14,38,48,49], and this might be due to the lower DON level applied in these studies.In the present study, it appeared that increasing the DON addition promoted the production of acetate slightly and inhibited the propionate production, though the stats were wonky.The authors in the present study speculated that the DON addition could change the activity of clostridia that decomposes cellulose to produce acetate and altered rumen fermentation via the selective antibacterial effect, as noted in the previous study [13].Moreover, increasing the DON dosage reduced the NGR, representing the ratio of non-glycogenic VFAs (e.g., acetate) to glycogenic VFAs (e.g., propionate) [50].Taken together, the results obtained in the present study implicated that the inclusion of DON could shift rumen fermentation towards non-glycogenic acid production.Glycogenic VFAs are the precursor of gluconeogenesis, and they are of great importance for supplying available energy to host ruminants.Thus, the presence of DON might, to some extent, decrease rumen fermentation efficiency.
Rumen microbial protein entering the small intestine represents a major source of amino acids to ruminant animals [51], and the level of MCP in the rumen also indirectly reflects microbial growth activity.In the present study, the presence of DON remarkably inhibited the MCP in culture fluids, especially in the high-forage group, and was in accordance with the study by Hildebrand et al. [12].Some previous studies pointed out that DON had adverse effects on protein degradation in the rumen [13,38,52].Feeding DONcontaminated diets to cows caused a greater negative N balance, resulting in decreased milk protein and urea contents [53].A decrease in the MCP content was attributed to compromised rumen fermentation by reducing protein digestion.The lower MCP in the present study implicated that the DON addition presented a detrimental effect on rumen microbial activity, and this also explained why the total VFA production was decreased, as aforementioned.

Conclusions
With the increased DON levels, from 5 to 20 mg/kg, the present in vitro rumen fermentation study confirmed that a high-forage substrate compared with a low-forage substrate was more conducive to DON degradability in the rumen in vitro.Moreover, as the concentration of DON increased, a decrease in DON degradability was noted.Although the DON addition did not affect the in vitro cumulative gas production, it remarkably decelerated the fermentation process, inhibited microbial fermentation in terms of decreased VFAs and MCP, and shifted the fermentation pattern to a more non-glycogenic acid and methane production.

Figure 1 .
Figure 1.Degradability of DON over time in the presence of HF substrate (a) and LF substrate (b).

Figure 1 .
Figure 1.Degradability of DON over time in the presence of HF substrate (a) and LF substrate (b).

Figure 2 .
Figure 2. Cumulative gas production profiles of in vitro fermentation of substrates with LF (a) and HF (b) in response to increasing DON concentration expressed in µ g/mL of culture fluid.

Figure 2 .
Figure 2. Cumulative gas production profiles of in vitro fermentation of substrates with LF (a) and HF (b) in response to increasing DON concentration expressed in µg/mL of culture fluid.

Table 2 .
Fermentation substrates composition and nutrient concentrations of experiment fermentation substrates (g/kg DM).

Table 3 .
Effect of DON and substrate levels on ratio of DON degradability (%) in in vitro fermentation.

Table 4 .
Effect of DON concentration (mg/kg) in culture fluids on kinetic gas production during 48 h incubation.

Table 4 .
Effect of DON concentration (mg/kg) in culture fluids on kinetic gas production during 48 h incubation.

1 Diet DON Concentration (mg/kg) SEM p-Value 2
a,b Means within a row without a common superscript letter differ at p < 0.05; 1 IVDMD 48 , in vitro dry matter disappearance of 48 h (%); GP 48 , cumulative gas production at 48 h (mL/g DM);

Table 5 .
Effect of DON concentration (mg/kg DM) on pH, ammonia nitrogen (NH3-N), microbial crude protein (MCP) and volatile fatty acid (VFA) production in fermentation fluids after 48 h incubation.

Table 5 .
Effect of DON concentration (mg/kg DM) on pH, ammonia nitrogen (NH 3 -N), microbial crude protein (MCP) and volatile fatty acid (VFA) production in fermentation fluids after 48 h incubation.

Table 6 .
Effect of DON concentration (mg/kg) in culture fluids on fermentation gas composition during 48 h incubation.Means within a row without a common superscript letter differ at p < 0.05; 1 H 2 , hydrogen gas; CO 2 , carbon dioxide; CH 4 , methane; 2 L, linear effect of DON addition; Q, quadratic effect of DON addition; I, interaction effect between diet and DON addition.