The study on the impact of indoleacetic acid on enhancing the ability of the rumen’s original microecology to degrade aflatoxin B1

The study on the impact of indoleacetic acid on enhancing the ability of the rumen's original microecology to degrade aflatoxin B1. 1 Jiajin Sun 1 ✝, Zhonghao Wang 1 ✝, Xinyu Yan 1, Yuqi Zhao 1, Li Tan 1, Xuning Miao 1, Rong 2 Zhao 1, Wenjie Huo 1, Lei Chen 1, Qinghong Li 1, Qiang Liu 1, Cong Wang 1, Gang Guo 1* 3 1College of Animal Science, Shanxi Agricultural University, Taigu, 030800 4 * Correspondence: Gang Guo 5 ✝ First authorship: These authors share firstauthorship 6 Corresponding Author 7 [email protected] 8 Abstract: 9 Aflatoxin B1 (AFB1) poses a serious threat to the health of cattle and is strictly monitored in their 10 diets. Rumen microorganisms in cattle have the ability to degrade AFB1, but this capability is 11 limited. Enhancing the degradation capacity of the original rumen microecology for AFB1 is a novel 12 approach. From a molecular perspective,indoleacetic-3-acid (IAA) promotes the expression of 13 cytochromes, which can improve the degradation of AFB1. Therefore, this study aims to investigate 14 the impact of different concentrations of IAA on the degradation of AFB1 by the rumen 15 microecology. Experiments used rumen fluid from three adult cows as donors, and the cows were all 16 fed the same total mixed ration. Rumen fluid was collected from these three cannulated cows before 17 morning feeding to prepare in vitro fermentation fluid. The experiments used a completely .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 3, 2024. ; https://doi.org/10.1101/2024.08.02.606440doi: bioRxiv preprint 2 18 randomized design, with each treatment repeated four times. The results showed that as the 19 fermentation time increased, the content of AFB1 gradually decreased, with a degradation rate of up 20 to 75.73% after 24 h. AFB1 altered the rumen fermentation pattern, with a significant reduction in 21 the content of acetic acid (P<0.05) and a significant decrease in the acetic acid to propionic acid ratio 22 (P<0.05). It also affected the rumen microecology, causing a significant reduction in the abundance 23 of Ruminococcus amylophilus, Prevotella ruminicola, and Fusobacterium succinogenes (P<0.05). In 24 addition, this study found that with the increase in the amount of IAA added, the content of AFB1 in 25 the rumen gradually decreased. IAA enhances the degradation capacity of the original rumen 26 microecology for AFB1, and the addition of IAA alleviates the impact of AFB1 on Ruminococcus 27 amylophilus, Prevotella ruminicola, and Fusobacterium succinogenes in the rumen (P<0.05). 28 Moreover, the addition of IAA can promote the stability of the rumen microecology, with 29 significantly higher acetic acid and acetic acid to propionic acid ratios in the fermentation fluid 30 compared to the non-added group (P<0.05). In summary, the addition of IAA can improve the 31 degradation capacity of the rumen microecology for AFB1, providing a new solution for alleviating 32 the impact of AFB1 on animal health. 33 Keywords: Indole-3-acetic acid; Aflatoxin B1; Rumen fermentation;Rumen microorganisms; 34 Degradation rate 35 36 1 Introduction 37 Mycotoxins are toxic secondary metabolites produced by molds during their growth process and are 38 widely present in grains and animal feed [1]. Among them, aflatoxins are considered to be important 39 class I carcinogens, posing a serious threat to the health of humans and animals [2,3]. Therefore, 40 controlling the content of aflatoxin B1 (AFB1) in animal diets is particularly important. In recent .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 3, 2024. ; https://doi.org/10.1101/2024.08.02.606440doi: bioRxiv preprint 3 41 years, silage, corn, and its by-products have been widely used as the main feed materials for cattle, 42 but frequent occurrences of AFB1 exceeding standards have led to serious waste of feed resources 43 and economic losses [4,5]. Studies have shown that rumen microorganisms have the ability to 44 degrade AFB1, but this ability is limited. This is mainly achieved through the following mechanisms: 45 Biotransformation: Certain rumen microorganisms can convert AFB1 into metabolites with lower 46 toxicity or non-toxic. Adsorption: Metabolites produced by some microorganisms or cell surface 47 structures may have the ability to adsorb AFB1, thereby reducing its absorption in the body. 48 Competitive inhibition: Rumen microorganisms may compete with AFB1 for absorption sites, 49 reducing its absorption in the intestine. Enzymatic reactions: Specific microbial enzymes may 50 catalyze the breakdown of AFB1, producing harmless or low-toxic metabolites [6,7]. However, the 51 ability of rumen microorganisms to degrade AFB1 is limited, which may be affected by the following 52 factors: Types and quantities of microorganisms: Different types of rumen microorganisms have 53 different degradation capabilities for AFB1, and the composition and quantity of microbial 54 communities can affect the efficiency of degradation. Feed components: Some components in the 55 feed may affect the activity of microorganisms or their interaction with AFB1. Therefore, finding a 56 plant extract additive to enhance the degradation capacity of the original rumen microecology for 57 AFB1 is a new approach for degradation. 58 Fresh green forage contains some extracts and other features that are non-toxic and environmentally 59 friendly, which will become a potential resource library for screening degradative toxins. 60 Indoleacetic-3-acid (IAA), as a regulatory factor in plants, has various special functions in microbial 61 metabolism [8,9], and has a certain regulatory effect on the synthesis of tryptophan and cytochromes 62 by rumen microorganisms [10]. At the same time, IAA, as an activator of the cellular aryl 63 hydrocarbon receptor (AhR), activates the expression of cytochrome genes such as CYP1A1, 64 CYP1A2, and CYP1B1 in cells, playing a key role in the physiological functions and immune .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 3, 2024. ; https://doi.org/10.1101/2024.08.02.606440doi: bioRxiv preprint 4 65 responses of cells. In addition, cytochrome P450 enzymes have been found in different organisms 66 and are involved in the biotransformation of AFB1 [11]. CYP450 enzymes are a group of 67 mixed-function oxidases involved in the biotransformation of endogenous compounds (such as bile 68 acids, prostaglandins, steroids, fatty acids, etc.) and exogenous compounds (drugs, carcinogens, 69 pro-mutagens), and are present in most organisms. In addition, IAA is involved in the metabolism of 70 various beneficial bacteria [12,13] and promotes the growth of probiotics [14]. Probiotics have 71 shown significant potential in degrading AFB1, with diverse mechanisms of action, including 72 biodegradation, adsorption, competitive exclusion, regulation of the intestinal environment, 73 enhancement of host immune response, gene regulation, and synergistic action with other 74 detoxification enzymes [15,16]. These mechanisms work together to reduce the toxicity of AFB1, 75 decrease its absorption in the body, and improve the host's clearance ability [17,18]. However, there 76 are few reports on the alleviation of the negative impact of AFB1 on rumen fermentation by adding 77 IAA. Therefore, the coordination of rumen microorganisms by IAA to achieve the degradation of 78 AFB1 in the rumen requires further study. 79 In summary, as a potential rumen regulatory substance, whether IAA can promote the degradation of 80 AFB1 in the rumen is not yet clear. Therefore, this study investigates the impact of IAA on the 81 degradation of AFB1 in the rumen by adding IAA, elucidates the impact of IAA on the degradation 82 of AFB1 in the rumen, and promotes the research on the synergistic degradation of AFB1 by 83 harmless chemicals and intestinal microorganisms. This work is of great significance for the 84 degradation of mycotoxins in feed and raw materials. It can not only improve the safety of animal 85 feed but also reduce the waste of grain, which has an important impact on the long-term and stable 86 development of China's animal husbandry and agriculture. 87 2 Materials and Methods .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 3, 2024. ; https://doi.org/10.1101/2024.08.02.606440doi: bioRxiv preprint 5 88 2.1. Experimental design 89 (1) Alfalfa silage and starch, each 0.2 g, were weighed and mixed in nylon bags and placed in 90 fermentation bottles in advance. 60 mL of rumen fluid and 40 μL of AFB1 standard solution (10 91 μg/mL) were added to the bottles, setting the AFB1 concentration to 1 mg/kg. The rumen fluid 92 fermentation bottles containing 1 mg/kg AFB1 were divided into 8 groups, with 4 replicates each, 93 and placed in a 39°C constant temperature shaker for in vitro fermentation at 0, 1, 2, 3, 4, 5, 24, and 94 48 h. After the fermentation, pre-column derivatization [19] was carried out to detect AFB1 content 95 and rumen fermentation indicators. 96 (2) Alfalfa silage and starch, each 0.2 g, were weighed and mixed in nylon bags and placed in 97 fermentation bottles in advance. 60 mL of rumen fluid and 40 μL of AFB1 standard solution (10 98 μg/mL) were added to the bottles, setting the AFB1 concentration to 1 mg/kg. The rumen fluid 99 fermentation bottles containing 1 mg/kg AFB1 were supplemented with IAA standard solution (600 100 μg/mL) in amounts of 0, 10 μL, 100 μL, 1 mL, and 5 mL, setting the IAA concentrations in the 5 101 fermentation bottles to 0, 15 mg/kg, 150 mg/kg, 1500 mg/kg, and 7500 mg/kg, respectively. Each 102 group had 4 replicates and was placed in a 39°C constant temperature shaker for in vitro fermentation 103 for 24 h. After pre-column derivatization [19], the AFB1 content and rumen fermentation indicators 104 were detected. 105 2.2 Experimental materials and animals 106 In this experiment, three healthy adult cows with similar body conditions and ages were selected as 107 rumen fluid donors. IAA (Shanghai Macklin Biochemical CO, LTD), Aflatoxin B1 (Stanford 108 Analytical Chemicals Inc). 109 2.3 Collection and in vitro cultivation method of rumen fluid .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 3, 2024. ; https://doi.org/10.1101/2024.08.02.606440doi: bioRxiv preprint 6 110 Before sampling, the fermentation bottles were preheated in a 39°C constant temperature incubator. 111 Before the morning feeding (07:00), rumen fluid was collected from the three Holstein cows through 112 the oral cavity. The mixed rumen fluid was filtered through four layers of gauze and temporarily 113 stored in a thermos preheated to 39°C and supplied with carbon dioxide (CO 2), maintaining an 114 anaerobic environment. The rumen fluid was mixed with artificial saliva in a 1:1 ratio as described by 115 Longland et al. [20] and continuously supplied with CO 2. Alfalfa silage and starch were mixed in a 116 1:1 ratio and placed in nylon bags. The reaction mixtures and collected rumen fluid were evenly 117 mixed, and each 60 mL was divided into individual fermentation bottles, sealed, and placed in a 39°C 118 shaker for cultivation [21] (speed of 120 r/min). When the fermentation time reached the set value, it 119 was immediately removed, stopped in an ice bath, and samples were taken for detection of various 120 indicators. 121 2.4 Determination items and methods 122 After 24 h of in vitro fermentation, the fermentation fluid was cooled and the contents of AFB1, 123 ammonia nitrogen (NH 3-N), volatile fatty acids (VFA), and four types of cellulase were measured. 124 The content of AFB1 was determined using an Agilent 1260 Infinity II high-performance liquid 125 chromatograph (HPLC) with a C18 column, 4.6×250 nm, 5 μm. The mobile phase was 126 acetonitrile-water (20:80); column temperature: 40°C; mobile phase flow rate: 1.0 mL/min; injection 127 volume: 20 μL; excitation wavelength 360 nm, emission wavelength 440 nm; detection time 20 min. 128 The degradation rate of AFB1 = (AFB1 content in the blank control - AFB1 content in the 129 experimental group) / AFB1 content in the blank control × 100%. 130 The content of ammonia nitrogen was determined using the phenol-hypochlorous acid colorimetric 131 method [22]. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 3, 2024. ; https://doi.org/10.1101/2024.08.02.606440doi: bioRxiv preprint 7 132 pH was measured using a pH meter (LE438, Mettler Toledo Instruments Co., Ltd.). 133 The content of volatile fatty acids (VFA) was determined using a high-performance gas 134 chromatograph (GC-TRACE 1300, column model 30 m×0.25 mm×0.25 μm) [23]. 135 The determination of the four types of cellulase was carried out according to the method described by 136 Agarwal et al. [24]. 137 Microbial DNA was extracted using the bead-CTAB method, and real-time fluorescent quantitative 138 PCR (ABI StepOnePlus, USA) was performed for RT-PCR. [25] (Primers are in the attachment) 139 2.5 Data processing and statistical analysis 140 Experimental data were recorded using Excel, and data analysis was performed using SPSS software, 141 with paired sample T-tests and repeated measures models. The level of statistical significance was set 142 at P<0.05, and the level of highly significant difference was set at P<0.001. Figures were drawn 143 using KingDraw, Prism 9, and Adobe Illustrator 2022 for graphic illustration. 144 3. Results 145 3.1. The impact of different fermentation times on the degradation rate of AFB1 in vitro 146 fermentation fluid 147 Figure 1(a) reveals that during the in vitro fermentation process of alfalfa silage, the degradation 148 efficiency of AFB1 shows a stable and significant increasing trend as the reaction time is extended. 149 In the 0-24 hour reaction, the degradation rate rises rapidly; between 24-48 h, the rate of increase in 150 degradation slows down. When the reaction reaches 48 h, the fermentation fluid's degradation rate of 151 AFB1 can reach 80.09%. Taking all factors into consideration, this experiment selects 24 h as the 152 final fermentation time. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 3, 2024. ; https://doi.org/10.1101/2024.08.02.606440doi: bioRxiv preprint 8 153 154 Fig.1. ( a ) The effect of different fermentation time on the degradation rate of AFB1 in in vitro 155 fermentation broth. ( b ) volatile fatty acids, ( c ) Acetic acid/propionic acid ratio, ( d ) pH, ( e ) 156 ammonia nitrogen production. Consistent with * means P < 0.05, * * means P < 0.001. 157 3.2 The impact of AFB1 on the production of volatile fatty acids, pH, and ammonia nitrogen in 158 vitro fermentation fluid 159 Data from Figures 1(b, c, d, e) indicate that in the alfalfa silage in vitro fermentation fluid, compared 160 to the control group, the addition of 1 mg/kg AFB1 significantly reduced the content of acetic acid 161 (P<0.05); meanwhile, the content of propionic acid, isobutyric acid, valeric acid, and isovaleric acid 162 significantly increased (P<0.05). Further analysis shows that the addition of 1 mg/kg AFB1 to the in 163 vitro fermentation fluid led to a significant decrease in the acetic acid to propionic acid ratio (P<0.05). 164 In addition, the addition of AFB1 did not significantly affect the pH value and ammonia nitrogen 165 content of the in vitro fermentation fluid. 166 3.3 The impact of AFB1 on the activity of major fiber-degrading enzymes in vitro fermentation 167 fluid 168 The impact of adding 1 mg/kg AFB1 on the activity of major cellulose-degrading enzymes in the 169 rumen fermentation fluid is shown in Figure 2. It can be seen from Figure 2 that the addition of 170 AFB1 has a highly significant effect on reducing the activity of xylanase after in vitro fermentation 171 of alfalfa silage ( P<0.05), while it does not significantly affect the activity of pectinase, 172 carboxymethyl cellulase sodium, and β-glucosidase. 173 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 3, 2024. ; https://doi.org/10.1101/2024.08.02.606440doi: bioRxiv preprint 9 174 Fig.2.Effect of AFB1 on the activity of major fiber degrading enzymes in vitro fermentation broth, * 175 indicates P < 0.05, * * indicates P < 0.001. 176 3.4 The impact of AFB1 on the microbial flora in vitro fermentation fluid 177 Data from Figure 3 reveal that after the addition of 1 mg/kg AFB1, the content of Prevotella 178 ruminicola and Fusobacterium succinogenes significantly decreased (P<0.05), indicating that the 179 presence of AFB1 inhibits the growth of these two types of bacteria. This result has potential 180 implications for the balance of the microbial community within the rumen. 181 182 Fig.3.Effect of AFB1 on microbial flora in in vitro fermentation broth, * indicates P < 0.05, * * 183 indicates P < 0.001. 184 3.5 The impact of different concentrations of IAA on the degradation rate of AFB1 in 24-hour 185 in vitro fermentation fluid 186 Figure 4 illustrates that compared to the control group with only 1 mg/kg AFB1 added, there is a 187 noticeable upward trend in the degradation rate of AFB1 in the alfalfa silage in vitro fermentation 188 fluid as the amount of IAA increases. It can be observed that there is a certain positive correlation 189 between the amount of IAA added and the degradation rate of AFB1 by the rumen fermentation fluid. 190 As the addition of IAA gradually increases, the degradation effect on AFB1 by the rumen 191 fermentation fluid is enhanced. When the IAA addition reaches 7500 mg/kg, the degradation rate of 192 AFB1 by the fermentation fluid peaks at 75.1%. 193 194 Fig.4.Effects of different concentrations of IAA on the degradation rate of AFB1 in 24 h .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 3, 2024. ; https://doi.org/10.1101/2024.08.02.606440doi: bioRxiv preprint 10 195 fermentation broth in vitro. P L represents a linear response, PQ represents a quadratic linear response. 196 3.6 The impact of different concentrations of IAA on the production of volatile fatty acids, pH, 197 and ammonia nitrogen in 24-hour in vitro fermentation fluid 198 Figure 5 indicates that compared to the control group with only 1 mg/kg AFB1 added, the addition of 199 low and medium concentrations of IAA shows a positive correlation between the amount of IAA and 200 the content of volatile acids in the fermentation fluid. Specifically, there is a significant increasing 201 trend in the content of acetic acid, propionic acid, butyric acid, and total volatile fatty acids (TVFA) 202 (linear effect, P=0.007, 0.028, 0.026, 0.006; quadratic effect, P=0.028, 0.021). Additionally, when 203 the IAA addition is 150 mg/kg, there is also a significant increase in isovaleric acid in the 204 fermentation fluid (P<0.05). Furthermore, when 1500 mg/kg IAA is added, the content of acetic acid, 205 valeric acid, the acetic acid to propionic acid ratio, and TVFA in the fermentation fluid all 206 significantly increase (P<0.05), while the content of propionic acid, isobutyric acid, butyric acid, and 207 isovaleric acid significantly decrease (P<0.05). The addition of IAA did not significantly affect the 208 pH and ammonia nitrogen content in the fermentation fluid. 209 210 Fig.5.Effects of different concentrations of indole acetic acid on the production of volatile fatty acids, 211 pH and ammonia nitrogen in 24 h in vitro fermentation broth. P L represents a linear response, P Q 212 represents a quadratic linear response. 213 3.7 The impact of different concentrations of IAA on the activity of major fiber-degrading 214 enzymes in 24-hour in vitro fermentation fluid 215 The impact of adding different concentrations of IAA on the activity of major fiber-degrading 216 enzymes in the 24-hour in vitro fermentation fluid is shown in Figure 6. It can be seen that, compared .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 3, 2024. ; https://doi.org/10.1101/2024.08.02.606440doi: bioRxiv preprint 11 217 to the control group with only 1 mg/kg AFB1 added, the addition of 15 and 150 mg/kg IAA shows an 218 enhancing trend in the activity of pectinase, xylanase, and carboxymethyl cellulase sodium after the 219 in vitro fermentation of alfalfa silage (where pectinase activity shows a linear effect, P=0.002; 220 xylanase and carboxymethyl cellulase sodium activities show a quadratic effect, P=0.027, 0.005). In 221 contrast, high concentrations of IAA exhibit a certain inhibitory effect on the activity of pectinase, 222 xylanase, and carboxymethyl cellulase sodium. 223 224 Fig.6.Effects of different concentrations of IAA on the activity of major fiber degrading enzymes in 225 24 h in vitro fermentation broth. PL represents a linear response, and PQ represents a quadratic linear 226 response. 227 3.8 The impact of different concentrations of IAA on the microbial flora in 24-hour in vitro 228 fermentation fluid 229 230 Figure 7 illustrates that when 15 and 150 mg/kg of IAA were added to the fermentation fluid, there 231 was an increasing trend in the content of total bacteria and Prevotella ruminicola, showing 232 significant linear effects with corresponding p-values of 0.013 and 0.033, respectively. Further 233 research found that when the concentration of IAA was increased to 15, 150, and 1500 mg/kg, the 234 content of Butyrivibrio fibrisolvens, Megasphaera elsdenii, Ruminococcus amylophilus, and total 235 methanogenic archaea in the fermentation fluid also showed an upward trend. Similarly, this trend 236 exhibited significant linear effects with corresponding p-values of 0.05, 0.009, 0.008, and 0.002, 237 respectively. It is worth noting that Ruminococcus amylophilus not only showed a linear effect under 238 the influence of changes in IAA concentration but also a quadratic effect, with the corresponding .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 3, 2024. ; https://doi.org/10.1101/2024.08.02.606440doi: bioRxiv preprint 12 239 p-value being 0.078. This finding suggests that the variation in the content of Ruminococcus 240 amylophilus may be influenced by a combination of factors, and its regulatory mechanism may be 241 more complex. 242 243 Fig.7.Effects of different concentrations of indole acetic acid on the microbial flora in the 24 h in 244 vitro fermentation broth. P L represents a linear response, and P Q represents a quadratic linear 245 response. 246 4. Discussion 247 Ruminant animals have a complex microbial community in their rumen that has been formed through 248 long-term evolution. These microorganisms are interdependent and mutually restrictive. They are 249 closely connected with the host's immune system, nutritional metabolism, and interactions between 250 organisms. Studies have found that certain microorganisms in the rumen have the ability to degrade 251 AFB1. [26] The research by Upadhaya et al. [27] also confirmed that the microorganisms in the 252 bovine rumen have the ability to degrade AFB1. This phenomenon can be attributed to several key 253 factors: first, microorganisms reduce the toxicity of AFB1 through adsorption; second, enzymes 254 secreted by rumen microorganisms can break down the structure of AFB1, converting it into other 255 substances, further reducing its toxicity. During in vitro fermentation, 1 mg/kg of AFB1 was added to 256 the fermentation fluid, and as the fermentation time increased, the concentration of AFB1 gradually 257 decreased, reaching a stable state after a period of time. Especially when the fermentation time 258 reached 48 h, the degradation efficiency of AFB1 can reach about 80%. In addition, research has 259 shown that AFB1, as a harmful secondary metabolite widely present in feed and its raw materials, 260 can disrupt the balance of microorganisms in the rumen when ingested by ruminants and enter the 261 rumen, leading to changes in the protein content and pH value of the rumen microorganisms, thereby .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 3, 2024. ; https://doi.org/10.1101/2024.08.02.606440doi: bioRxiv preprint 13 262 affecting the normal fermentation process of the rumen. [28, 29] Volatile fatty acids are the main 263 source of energy for ruminants to obtain from feed [30], but the presence of AFB1 has a significant 264 inhibitory effect on the rumen microbial community, leading to a decrease in the total production of 265 volatile acids and a change in the fermentation type. In vitro fermentation experiments show that 266 after exposure to AFB1, the abundance of core microorganisms in the rumen such as Prevotella and 267 Fusobacterium decreased, affecting the balance and function of the microbial community. In addition, 268 AFB1 also reduced the activity of xylanase, consistent with the changes of cellulolytic bacteria, while 269 the activity of other enzymes such as pectinase did not change significantly, which may be related to 270 the amount of AFB1 added. The rumen environment, especially the pH value, is crucial to the 271 microbial balance and the health of the rumen [31]. The metabolic process in the rumen includes the 272 degradation of cellulose and hemicellulose, the metabolism of soluble sugars, and the fermentation to 273 produce organic acids and short-chain fatty acids. These findings indicate that AFB1 not only 274 changes the fermentation pattern of the rumen but may also affect the nutritional absorption and 275 health of ruminants. 276 IAA is an important biological regulatory substance, essential for the growth of forage, and is also 277 considered to be an efficient and environmentally friendly additive with the potential to regulate the 278 balance of animal intestinal microflora. Although research on IAA in rumen microorganisms is still 279 in its infancy, this experiment has confirmed that adding different concentrations of IAA to rumen 280 fluid containing AFB1 can effectively reduce the content of AFB1, especially when adding 7500 281 mg/kg IAA, the content of AFB1 significantly decreased. However, considering that high 282 concentrations of IAA may affect the balance of the rumen, according to the "Safety Use 283 Specifications for Feed Additives," the recommended amount of tryptophan (the precursor of IAA) in 284 the diet of ruminants is 0.1% (1 g/kg). In addition, some studies have shown that excessively high 285 concentrations of IAA may change the animal intestinal microbial community [32]. Therefore, this .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 3, 2024. ; https://doi.org/10.1101/2024.08.02.606440doi: bioRxiv preprint 14 286 experiment further explored the specific impact of IAA additions of 15 mg/kg, 150 mg/kg, and 1500 287 mg/kg on rumen fermentation parameters, aiming to find an appropriate level of IAA addition to 288 alleviate the impact of AFB1 on the rumen fermentation process and improve animal health. This 289 experiment studied the impact of different concentrations of IAA on VFAs, pH, and ammonia 290 nitrogen production in in vitro fermentation fluid containing 1 mg/kg AFB1, and found that medium 291 and low concentrations of IAA can significantly increase the content of total volatile fatty acids 292 (TVFA), while high concentrations of IAA tend to decrease. The addition of IAA, especially medium 293 and low concentrations, enhances the ability of the rumen microbial community to utilize carbon 294 sources and stimulates the degradation of nutrients in the rumen. Acetic acid, propionic acid, and 295 butyric acid are the main products of rumen fermentation and are crucial for energy metabolism and 296 nutrient absorption [33-35]. In the experiment, after the addition of IAA, the content of acetic acid 297 showed a linear upward trend, which is related to the activation of fiber-decomposing bacteria and 298 enzyme activity by IAA, promoting the growth of fiber-decomposing bacteria and fungi. With the 299 addition of IAA, the observed changes in the microbial community, such as Prevotella and 300 Fusobacterium, are closely related to the adjustment of VFAs composition [36, 37]. These 301 microorganisms decompose the fiber in the feed, promoting the production of acetic acid and 302 propionic acid. Propionic acid is key in the gluconeogenesis process, affecting body fat and lactose 303 synthesis, while butyric acid is easily absorbed by the rumen epithelial cells, providing energy 304 [38-41]. The concentration of IAA has a significant regulatory effect on propionic acid production, 305 with medium and low concentrations of IAA causing a linear increase in propionic acid content, 306 while high concentrations of IAA reduce the level of propionic acid. The change in butyric acid 307 content is also related to the amount of IAA added, with medium and low concentrations of IAA 308 increasing butyric acid content, while high concentrations of IAA reduce butyric acid content. In 309 addition, the regulation of the acetic acid/propionic acid ratio affects microbial protein synthesis and 310 the structure of the rumen microbial community, which in turn relates to the digestion and nutritional .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 3, 2024. ; https://doi.org/10.1101/2024.08.02.606440doi: bioRxiv preprint 15 311 metabolism of the whole body [42, 43]. In the rumen ecosystem, bacteria can degrade and utilize 312 starch and plant cell wall polysaccharides, such as xylan and pectin, but they cannot degrade 313 cellulose. These bacteria play an important role in the degradation of protein and the absorption and 314 fermentation of peptides [44-50]. Finally, the addition of IAA has a significant impact on the changes 315 in the rumen microbial community and the activity of the main fiber-degrading enzymes. Medium 316 and low concentrations of IAA significantly increased the activity of pectinase, xylanase, and 317 carboxymethyl cellulase, while high concentrations of IAA reduced the activity of these enzymes. 318 These results show that IAA alleviates the imbalance of the rumen fermentation microecology caused 319 by AFB1 by regulating the rumen microbial community and enzyme activity. In summary, the 320 appropriate addition of IAA has a positive impact on the rumen microbial community and metabolic 321 products, but high concentrations of IAA may inhibit the rumen fermentation process. These findings 322 provide important information for optimizing rumen fermentation and improving the nutritional 323 absorption of ruminants. 324 5. Conclusions 325 The addition of IAA has been proven to effectively enhance the ability of the native microbial 326 community in the rumen to degrade AFB1, providing a new strategy to mitigate the potential threat 327 of AFB1 to animal health. This study clarifies the key role of IAA in rumen fermentation, 328 particularly showing significant effects in reducing the content of AFB1 within the rumen. After a 329 comprehensive assessment of economic costs, the balance of animal gut microbiota, and health 330 impacts, a recommended dosage of 15 mg/kg IAA is suggested to be added to feed. This dosage not 331 only promotes the effective degradation of AFB1 in the rumen but also regulates the adverse effects 332 of AFB1 on the rumen fermentation process, while ensuring the health and production performance 333 of the animals. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 3, 2024. ; https://doi.org/10.1101/2024.08.02.606440doi: bioRxiv preprint 16 334 Supplementary Materials 335 The following supporting information can be downloaded. 336 CRediT authorship contribution statement 337 LQ: Conceptualization.XY: Methodology. YZ: Software. LT: Validation. JS: Formal analysis, 338 Writing - Original Draft. XM: Investigation. RZ: Resources. ZW: Data Curation. WH: Writing - 339 Review & Editing. LC: Visualization. CW: Supervision. QL: Project administration.GG: Funding 340 acquisition. 341 Funding 342 This research was funded by the National Natural Science Foundation of China (32001405). 343 Institutional Review Board Statement 344 The present study was approved by the Animal Health and Care Committee of the Shanxi 345 Agricultural University (Shanxi, China) and conducted according to the Guidelines for the 346 Experimental Animal Welfare of Ministry of Science Technology of China (approval code. 347 SXAU-EAW-2022C.RD.010025174). 348 Data Availability Statement 349 The data presented in this study are available on request from the corresponding author. 350 Acknowledgments 351 The author thanks the National Natural Science Foundation of China for financially supporting our 352 experiment. 353 Conflicts of Interest .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 3, 2024. ; https://doi.org/10.1101/2024.08.02.606440doi: bioRxiv preprint 17 354 No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all 355 authors for publication. All the authors listed have approved the manuscript that is submitted. I would 356 like to solemnly affirm that the material included here with in this manuscript has never been 357 published before, and furthermore ensured that none of the contents are currently under consideration 358 elsewhere. 359 References 360 [1] Fang Manxin, Hu Wei, Liu Ben. Protective and detoxifying effects conferred by selenium against mycotoxins 361 and livestock viruses: A review. Front Vet Sci.2022;9:956814. doi: 10.3389/fvets.2022.956814. 362 [2] Yao G, Yue Y, Fu Y, Fang Z, Xu Z and Ma G, et al. Exploration of the regulatory mechanism of secondary 363 metabolism by comparative transcriptomics in aspergillus flavus. 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