Croubels, Siska

De Baere, Siegrid

Debevere, Sandra

Fievez, Veerle

Haesaert, Geert

Rychlik, Michael

English

Ghent University Academic Bibliography

Debevere S.1,4*, De Baere S.1, Haesaert G.2, Rychlik M.3, Fievez V.4a, Croubels S.1a1Department of Pharmacology, Toxicology and Biochemistry, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium; 2Department of Applied Biosciences, Faculty of Bioscience Engineering, Ghent University, Valentin Vaerwyckweg 1, 9000 Ghent, Belgium; 3Chair of Analytical Food Chemistry, Technische Universität München, Alte Akademie 10, 85354, Freising, Germany; 4Department of Animal Production, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, 9000 Ghent, Belgium; asharedsenior authorshipINTRODUCTIONMycotoxin contaminated feed is increasingly being associated with subclinical health problems in highly productive dairy cows, reflected by vague and non-specificsymptoms and suboptimal milk production. In Belgium, the most important mycotoxins found in maize silage are deoxynivalenol (DON), nivalenol (NIV), zearalenone(ZEN), mycophenolic acid (MPA), roquefortine C (ROQ-C), citrinin (CIT) and enniatin B (ENN B). The detoxifying capacity of ruminal microbiota for the previouslymentioned mycotoxins, as well as the binding capacity of those mycotoxins to silage is not well known. To answer these questions, in vitro bovine and ovine rumenincubations with maize silage, spiked with single mycotoxins, and possible degradation of these mycotoxins after incubation was studied. Furthermore, inactive(autoclaved) rumen fluid and UHPLC water was used as inoculum to determine the binding capacity to maize silage. In addition, the impact of mycotoxins on rumenfermentation was studied, and was monitored by volatile fatty acids and CH4 production. To determine the mycotoxin concentrations in the samples, a sensitive andspecific analytical method for the quantitative determination of the mycotoxins DON, NIV, ZEN, MPA, ROQ-C, CIT and ENN B as well as their metabolites deepoxy-deoxynivalenol (DOM-1), α-zearalenol (α-ZOL), β -zearalenol (β-ZOL), zearalanone (ZAN), α-zearalanol (α-ZAL) and β-zearalanol (β-ZAL) in rumen fluid using UPLC-MS/MS was developed and validated.Spiked substrate:- 50 mg maize silage- spiked in triplicate with single mycotoxins at following concentrations:In vitro inoculum:- Fresh rumen fluid/buffer mixture (FRF):- mixture from 3 lactating cows- or mixture from 3 hamels- or from 4 individual cows (3x lactating, 1x non-lactating) added immediately to spiked substrate incubation for 1.5h at 39°C in shaking incubator- Autoclaved rumen fluid (ARF)- UHPLC water (H2O)Spiked matrix-matched control samples (n=3x3):FRF: without incubation; ARF: 0h; H2O: 0h, without substrateTable 1. Reduction of mycotoxins, expressed as % compared to control samples, whenfresh rumen fluid (FRF), autoclaved rumen fluid (ARF) or UHPLC water was added to spiked maize silage according to the design in Fig. 1.ContactSandra.Debevere@UGent.beSiska.Croubels@UGent.beVeerle.Fievez@UGent.beMytox.beMytoxsouth.orgRESULTS AND DISCUSSIONMATERIALS AND METHODSBEHAVIOR OF MYCOTOXINS IN MAIZE SILAGE IN IN VITRO RUMEN SIMULATIONSwwwCONCLUSIONSDisappearancecompared tocontrol (%) FRF ARF H2OCIT 96±4 100±0 100±0ROQ-C 57±13 76±9 54±5DON 24±9A 10±1B 4±3BMPA 21±13B 42±10A 52±3AENN B 29±7A 37±9A 10±3BZEN 25±15B 46±10A,B 51±4ANIV 3±5 5±5 0±0CIT DON NIV ENN B MPA ROQ-C ZEN0.5 µg/g 12 µg/g 12 µg/g 1 µg/g 6 µg/g 2 µg/g 3 µg/gAnalysis of samples for mycotoxins:- Sample preparation: salting-out liquid-liquid extraction (SALLE)1- Quantitative analysis (UHPLC-MS/MS1) of CIT, DON, DOM-1, NIV, ENN B, MPA, ROQ-C, ZEN, α-ZOL, β-ZOL, ZAN, α-ZAL, β-ZALadded to substrate 24h after spiking1Debevere et al. (2017). 39th Mycotoxin Workshop, Abstracts, p103ACKNOWLEDGEMENTS This research is funded by the government agency Flanders Innovation & Entrepreneurship (VLAIO-Belgium, grant n° LA 140971)Volatile fatty acids (VFA) and CH4 productions were normal during the in vitro incubations with FRF  good microbial activity, although the pH range of FRF and ARF was divergent: pH 5.36-6.55.High similiarities when FRF, ARF or H2O is used:- CIT: (almost) complete disappearance (90-100%)- ROQ-C: extended disappearance (>50%)- MPA, ENN B, ZEN: moderate disappearance (10-50%)- NIV: (almost) no disappearance (0-10%)Some differences:- DON: higher disappearance when FRF is used, which  may be caused by rumen microbiota.- MPA, ZEN: higher disappearance when (ARF or) H2O is used, which may be due to longer exposure/binding time to substrate (24h)- ENN B: higher disappearance when FRF or ARF is used, which may be caused by extra binding to particles in rumen fluid.Spiked maize silage+ FRF+ ARF+ H2O24hIncubation (1.5h)Sample extraction +quantification of mycotoxins(UHPLC-MS/MS)Figure 1. Setup of the in vitro rumen incubation study to determine the effect of rumen microbiota, inactive rumen fluid and substrate on recovery of mycotoxins.Degradation of DON by the microbiota is assumed, however, no DOM-1 was detected. In contrast, NIV showed nodegradation. There was no effect seen on the rumen fermentation parameters tested. Binding to the maize silagesubstrate of the mycotoxins CIT (complete), ROQ-C (high), MPA, ENN B and ZEN (moderate) is concluded asdisappearance of those mycotoxins in spiked substrate also occurs when no active rumen fluid is added. Future studies todetermine the stability of this binding in the gastrointestinal tract of ruminants are necessary, as bound mycotoxins couldbe released post-ruminally (e.g. pH effects) and exert their toxic effects.Partner in:Values are presented as mean ± SD. Values with a different superscript are significantly different (P<0.05). No metabolites were detected in the samples (range LOQ mycotoxins: 0.05- 1.56 ng/mL; NIV: 36 ng/mL)

Behavior of mycotoxins in maize silage in in vitro rumen simulations

https://biblio.ugent.be/publication/8531835/file/8531839

Behavior of mycotoxins in maize silage in in vitro rumen simulations

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