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2015-R3 Microbial Detoxification of DON

2018-8-14 16:24:24 Comments:0 Views:200 category:Project Introduction

6.1 About Principle Investigator

Short Curriculum Vitae Education & work experience 

Ph.D degree in Animal Nutrition and Feed Science of China Agricultural University, Beijing, PR China
Postdoctoral Fellow in Dairy Cow Nutrition and Feed Processing of Wageningen Institute of Animal Science, Wageningen Agricultural University, Netherlands
Postdoctoral Fellow in Institute of Animal Science of Chinese Academy of Agricultural Sciences, Beijing, PR. China
Associate Professor in Animal biology and Animal Physiology from 2003 to 2008 at College of Biological Sciences, China Agricultural University, Beijing, PR China
Associate professor in Animal Nutrition and Feed Science since 2008 at State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, PR China

Field of expertise
His current research interest includes feed evaluation, anaerobic methanogensis inhibition and microbial degradation of forage cell wall in the ruminant animals with aims to improve feed conversation with less environmental pollution.

Scientific publications are available at:

6.2 Objectives of the study:
Fusarium is considered to be the most important toxigenic fungi, since they produce the most prevalent mycotoxins, including secondary metabolites of deoxynivalenol (DON) and zearalenone (Seeling et al., 2006). Eriksen & Pettersson (2004) noted that dietary intake of DON could cause vomiting and animal intestinal dysfunction (e.g., diarrhea, vomiting), oral mucosa and dermal damage, decreased immunity and so on. Rodrigues & Naehrer (2012) in a three-year survey on the worldwide occurrence of mycotoxins reported that a detection rate of DON was 59%, and the average of its content was 1104 µg/kg in the feedstuffs and feed sampled from the USA, Europe and Asia. Some previous studies reported that ruminants could be relatively resistant to toxic effects of DON, since the potential of ruminal microbes might degrade DON to the less toxic metabolite of de-epoxy DON (Curtui et al., 2005; Seeling et al., 2006). The objective of this study was to determine the effect of DON at different level on in vitro fermentation by assessing rumen fermentation with two forage to concentrate ratios.

6.3 Main report of the research:
6.3.1 Materials and Methods
The experimental include two substrate treatment (HF, Chinese wildrye grass hay: concentrate = 4 : 1; LF, Chinese wildrye grass hay: concentrate = 1 : 4). Freshly prepared buffer (pH 6.85, 50 mL) and 25 mL strained rumen fluid (from three rumen-cannulated lactating Holstein cow) were dispensed into a 150-mL bottle containing 500 mg rations. The working solution of DON was added to each bottles,resulting in 0, 0.5, 1, 1.5, 2 µg DON per mL of the culture fluids and the level of 1 µg/mL was equivalent with the limits standards of the state. After the in vitro fermentation .Collected the filtrate and washed the nylon bags together with the contents to clear used distilled water. Then dried at 65 °C less than 48 h to a constant weight.

6.3.2 Gas Production and Curve Fitting

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 (France, J. et al.)[10] as Equation (1):

where A is the asymptotic gas production; c is the gas production rate; and t is the gas recording time. The parameters A, c and lag were estimated by an iterative least squares procedure using the NLIN procedure of the Statistical Software Package for Windows (version 9.02, 1999; SAS Institute Inc., Cary, NC, USA). The average gas production rate (AGPR, mL/h) [11] 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):

Where AGPR is the gas production rate of 1/2 maximum gas production (mL/h). And according to (2005) the methods of Grings etc. to calculate the time of maximum gas production of 1/2 (T1 / 2, h) with equation (3)

6.3.3 Results Effect of DON in IVDMD and gas production kinetics

In the present study, IVDMD and gas production for 48 h was greater in LF than HF(P < 0.01). Neither IVDMD nor kinetic gas production was affected by the DON addition(P > 0.05).With the increasing of DON dose, the gas production rate (c) showed a significant secondary correlation(P < 0.05), decreased first and then increased. When the dose of DON was 1.5 μg/mL, it was the lowest. Effect of DON on ammonia N, VFA concentrations and VFA pattern

NH3-N: ammonia nitrogen (mg/dL), MCP: microbial crude protein (mg/mL), tVFA: total volatile fatty acids (mM), NGR: ratio of non-glucogenic to glucogenic acids, FE: fermentation efficiency, CH4e: methane production evaluated. I: Interaction effect between diet and DON addition, L: Linear effect of DON addition, Q: Quadratic effect of DON addition, FE: fermentation efficiency, CH4e: methane production evaluated, NGR: ratio of non-glucogenic to glucogenic acids.

In the present study, ammonia-N concentration was greater in LF than HF(P<0.01).Total VFA production in LF was greater than HF(P=0.003) and the pH was lower in LF than HF(P<0.01).The molar percentage of acetate was greater in HF than LF though kinetics gas production was not statistical affected (P=0.066),but those percentages of butyrate, valerate and isovalerate were lower in HF than LF(P<0.01),and other VFAs were not affected. The methane production evaluated (CH4e) of HF group was lower than LF group (P<0.01), but the ratio of non-glucogenic (e.g., acetate, butyrate) to glucogenic (e.g. butyrate) VFAs (NGR) and fermentation efficiency (FE) were not affected significantly.
The microbial protein concentration and molar percentage of butyrate were decreased in HF group,but increased in LF group with the increase of DON addition level. Total VFA production has an interaction effect between ration and DON dosage that total VFA production tended to linearly decline with the DON addition regardless of HF or LF. Molar percentage of isovalerate has an tendency to decreased by the DON addition (P=0.067), NGR and CH4e was linearly increased. Regardless of forage to concentrate ratio in the rations, the total VFA production linearly decline with DON addition, especially DON level of 1 g/mL, but other fermentation characteristics were not affected by DON addition. Degradability of DON by rumen microorganisms.

DON disappearance rate decreased linearly with increasing DON dosage (P<0.01), and the disappearance rate in HF group was significantly higher than LF group at 3 h (P<0.01). DON was mainly degraded in the first 6 h, and the average disappearance rate at 6, 12, 24, 48 h was 30%, 33%, 38%, 41% , respectively, and HF group was 5% higher than the LF group.

6.4 Conclusion

Except forage to concentrate effect, though no statistical effect on nutrient digestibility and kinetic gas production were observed, the remarkable decline occurred for total VFA production when the DON addition level was greater than 1 mg/mL as equalent acceptable level of 5 mg DON/kg feed. The DON addition increased the ratio of non-glucogenic (e.g., acetate, butyrate) to glucogenic (e.g.  propionic acid) VFAs as well as CH4 production, suggesting that DON exhibited detrimental effect on rumen microbial metabolism.


6.5 Appendix

6.5.1 Effect of DON in IVDMD and gas production kinetics

Technology of rumen fermentation gas produce can estimate feed digestibility in animals, measure feed digestibility, evaluate interaction between additives and feed rapidly, provide gas production kinetics data. (Makkar,2010;Menke et al.,1979;Van Der Meer et al.,1988)[17, 18, 19]. It is based on IVDMD and gas production, Carbohydrates and crude protein is the main source of gas production during substrate fermentation, impact gas fermentation characteristics of substrates in vitro culture directly. IVDMD is higher and the gas production is more, indicates that a higher degree of fermentation of feed in rumen and a higher degradable rate. The results in this experiment, when the ratio of forage to concentrate is different, IVDMD and all gas production ratio of kinetic parameters showed a significant difference, which indicated that there is a great relationship between fermentation aerodynamic characteristics and components and physical chemical properties of substrate. With the increasing of DON doses, the gas production was decreasing, though it was not significantly different in statistics. In addition, it showed a significant quadratic relationship between Gas production rate and DON dose. And the minimum appeared at 1.5 μg/mL.

Trenholm et al. (1985) stated that DON concentrations increased from 1.5 mg / kg to 6.4 mg / kg for concentration feed, the consumption of fodder just have slight and temporary reduction, and the weight gain and hay consumption was not be affected [20]. And in this experiment, after adding DON, IVDMD of HF group changed slightly among 2.5 mg/kg (0.5 mg/μL) and 10 mg/kg (2 mg/μL). This is consistent with the results of previous studies.

Seeling et al. (2006) pointed out, DON cannot be completely degraded by rumen microbial [21]. The degree of degradation affected by animal species, age, sex and feeding methods, because these factors will influence the type and quantity of the rumen microflora. The results of this experiment can explain that DON will reduce the rate of digestion and metabolism of ruminants, which is consistent with the previous studies.

All those stated that DON has a negative effect on the fermentation of feed. But with the addition of DON, IVDMD and gas production characteristics were not affected. But the reason is still unclear. For now, there is not a report about the effect of DON on gas production in vitro fermentation of dairy cows at home and abroad.


6.5.2 Effect of DON on ammonia N, VFA concentrations and VFA pattern

Carbohydrate and crude protein is the main source of gas production, and the gas production is a very important indicator, usually the higher degree of fermentation, the higher gas production [22]. The MCP concentration, total VFA production and the molar percentage of butyrate, isovalerate, valerate in LF group is greater than HF group is very significant, and that means the LF group has higher proportion of concentrate than HF group and more available ingredient fermented by rumen microbes. As to the fermentation characteristics, the difference of some indicators is significant between HF group and LF group, and some not. This may be caused by the combined effect of forage to concentrate ratio and the DON addition. The difference of HF group and LF group may be removed by the addition of DON. The effect of DON on ruminants is very difficult to evaluate, because the microbial environment of the rumen and its inherent detoxification potential is often considered a first defense mechanism to prevent the harmful substances [23]. Many researches have demonstrated that DON can be metabolized as DOM-1, a less toxic metabolite [24, 25]. When ruminal pH declines below 5.2, the transformation of DON will be inhibited completely in vitro [25]. And the pH in the present study is within the normal range, so the metabolism of DON will not be influenced by pH. Dänicke et al. (2005) reported that rumen pH and VFA fed Fusarium toxin-contaminated wheat were not influenced by contamination of the wheat, but the postprandial ammonia concentration was consistently higher[26].This is in agreement with our study. However, Jeong et al.(2010) demonstrated that DON negatively impacts certain aspects of rumen fermentative capacity, such as total gas and VFA production. The result of present study in agreement with feeding trial [8], but inconsistent with in vitro trial [3], this difference may be caused by the different addition level and trial condition.


6.5.3 Degradability of DON by rumen microorganisms.

Many researchers have demonstrated that DON can be metabolized as DOM-1 by rumen microorganisms [23,25,27]. In the meantime, mycotoxin may modify the ruminal microflora and weaken detoxicating effects of rumen microorganisms [24]. In the present study, DON degradation was influenced by concentrate/forage ratio and DON dosage. Apparently, degradation tend to more efficiently in HF group than LF group, which is agreement with previous study [4], but we have a higher degradation. King et al. [23] observed that, when the concentration of DON in rumen fluid from 10 ppm to 100 ppm, the transformation rate from 89% to 37% within 48 h, which proves higher concentration, lower degradation. On the contrary, some researchers consider that higher substrate concentrations may promote the transformation of DON to DOM-1 by inducing greater microbial enzyme activity. In addition, the degradation of DON influenced by various factors, such as pH level, incubation time [25]. So, the degradation rate would be different under the different experiment conditions.




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