Moulds and mycotoxins in wrapped forages for horses: Can we not only analyze the hygienic quality but also prevent the presence of moulds and mycotoxins?

Take home messages

The presence of moulds and mycotoxins in forages is a sign of impaired hygienic quality for two main reasons; the presence of mould spores, and the potential risk of mycotoxin production. Mould spores may cause airway diseases in horses and mycotoxins can cause different diseases that could be fatal (Scudamore & Livesey, 1998; Tell, 2005; Gallo et al., 2015). Moulds in feeds are often classified into two main groups: the field fungi, and the storage fungi. The field fungi is present on the standing crop and usually consist of genera such as Cladosporium, Alternaria and Fusarium, but other species can also be present. Although these species are often regarded as a problem primarily for plant growth, especially in cereals, they may also be present on grass crops for forage production and they may pose a threat to equine health. The storage fungi develops during improper storage of feeds and commonly includes species belonging to Aspergillus, Fusarium and Penicillium in forages, but other species may also be present (Scudamore & Livesey, 1998; Samson et al., 2010; Gallo et al., 2015).

Forage is commonly conserved as hay, haylage and silage, where haylage and silage are counted as wrapped forages (Müller, 2018; Schenck, 2019). It is important to know the major conservation principles of these forages for the understanding of why moulds may proliferate in them, and which actions should be taken to inhibit it. Hay is conserved through removal of water to a sufficiently low dry matter content (at least 840 g per kg) and water activity (maximum 0.70) where microbes can no longer grow. Hay can therefore be stored aerobically, if it is kept dry during the entire storage period. Moulds are aerobic and only need moisture to be able to grow in hay. Silage has a dry matter concentration up to 500 g per kg and is conserved through lactic acid fermentation (e.g., Müller, 2005). The lactic acid lowers pH and together with the anaerobic environment created by the wrapping mould growth is inhibited. If the anaerobic seal is broken, the low pH and presence of lactic acid may postpone growth of fungi but it cannot hinder it completely. Therefore, anaerobiosis is required for inhibition of mould growth in silage. Haylage can be considered to be “in between” hay and silage, with a dry matter concentration over 500 and up to 840 g per kg, and the conservation relying mainly on air-tight storage for avoidance of mould growth (e.g., Müller, 2018; Schenck, 2019). Some lactic acid can be produced in haylage with dry matter contents up to about 600 g per kg, but in very small amounts and it is unlikely that it has any major conservation effects. Haylage can therefore, compared to silage, be regarded as even more sensitive to mould growth, if the wrappings are not air-tight. Moulds only need oxygen to be able to grow in haylage. It is important to understand that haylage is not the same thing as silage, or hay, as biochemical and microbial composition differ between them. Results from studies on lactic acid fermented silage can therefore not be used straight-off for haylage, as it can result in incorrect conclusions.

Both field and storage fungi may produce mycotoxins (e.g. Wambacq et al., 2016). Field fungi can produce mycotoxins for example when crops are stressed due to cold or dry weather, and storage fungi can produce mycotoxins above freezing temperatures in the presence of oxygen and dry matter contents above 20 % (Scudamore and Livesey, 1998). Fungi that are potential mycotoxin producers do not always produce mycotoxins, but if environmental factors are optimal for mycotoxin production, it could occur. Due to the possible detrimental health effects of moulds and mycotoxins, their presence in feeds in the European Union is regulated to some extent (https:// www.efsa.europa.eu/en/topics/topic/mycotoxins). There is however a lack of knowledge for many mould species and mycotoxins.

Analysis of presence of moulds and mycotoxins in forage can be performed. Moulds can be analysed both qualitatively and quantitatively by culturing on selective substrates and/or by molecular methods (Samson et al., 2010). Multi-mycotoxin chromatographic assays are available (e.g. Andersen et al., 2020). However, forages are very heterogenous and mould growth in forages is not evenly distributed within for example a bale. Representative sampling can therefore be very difficult, and different sampling methods can influence the analytical result for both moulds and mycotoxins. In a Swedish-Norwegian study on 124 horse farms where three sampling methods were used on all farms, results showed that the method influenced the possibility to detect different mould species or genera (Schenck et al., 2019a). Visible fungi on bale surfaces was not a useful indicator of moulds in core samples, while direct plating of core samples overestimated fungi growing with hyphae (e.g. Arthrinium spp.) and dilution plating of cores samples overestimated spore-producing fungi (Schenck et al., 2019a). One hundred of the core samples in the same study were analysed for mycotoxins, where Fusarium toxins Enniatin B and Deoxynivalenol were the most common and present in 14 and 12 % of the samples, respectively (Schenck et al., 2019b).

Most of the detected mycotoxins were present in low concentrations and in fewer than 10 % of the samples. As shown in previous studies, visual appearance of moulds in forages was not associated with presence of mycotoxins, and mycotoxins could be present in forage samples with no visible moulds (Schenck et al., 2019b). Considering the difficulties involved in sampling, analysis and interpretation of analytical data for risk assessment of moulds and mycotoxins in forages, and the fact that a batch of wrapped bales infected with fungi cannot be cured in any way in practice, it is highly important to have a preventive approach in forage production. To be able to prevent mould growth in wrapped forages, knowledge of which factors that are associated with mould growth in such feeds is required. Forage management and production factors were analysed for their association with the presence of moulds and mycotoxins through correlation and multivariate regression analyses in the previously mentioned study (Schenck et al., 2019a,b). Increasing dry matter content and pH, less than 8 layers of stretch film, lower seal integrity, wilting the crop in windrows instead of wide-spread in the field, and higher compared to lower latitude all increased the risk of finding moulds in the forage. Increasing dry matter content also increased the risk of finding Fusarium mycotoxins in the forage in the same study. It was also found that as forage dry matter concentration increased, the preventive effect of a higher number of stretch film layers on inhibition of mould presence disappeared, but at about 50 % dry matter the risk of finding moulds in the forage was halfed by using more than 8 layers of stretch film (Schenck, 2019).

The effect of increased number of stretch film layers on preventing mould growth has been reported previously in several studies where airtightness of bales, carbon dioxide concentration within the bale gas volume and extent of bale surface moulds has been measured (Paillat & Gaillard, 2001; Keles et al., 2009; O’Brien et al., 2007, 2008; Spörndly et al., 2017). These results all point in the same direction, that if haylage is going to be conserved with low risk of mould growth, the wrappings have to be air-tight.

Future challenges within the area include among others increased knowledge of if and which health problems in horses that can be expected from different moulds and mycotoxins in forage. Some secondary metabolites may prove to not be toxic to horses, while others may explain conditions or diseases where we currently do not have a good understanding of the causes. Another challenge is which effect climate change will have on the field fungal flora in grass, and especially on endophytic fungi. These are fungi growing inside the plants and they may produce toxins in the field. One example is Epichloë spp. which produces alkaloids acting as neurotoxins and causing reproductive problems in horses (Anas et al., 1998). It is known that endophytic fungi are present in many grass species, but less is known about when these fungi produce toxins and if it is a problem that will increase with a warmer climate. Yet another challenge are the environmental aspects of using large amounts of polyethylene stretch film for forage conservation. It is required for restricting mould growth in wrapped forages, but could be questioned from a sustainability perspective.

References

• Anas, K., Cross, D.L., Poling, R., Redmond, L.M., Campbell, C .E. 1998. A survey concerning the equine fescue toxicosis malady. Journal of Equine Veterinary Science 18, 631-637.
• Andersen, B., Phippen, C., Frisvad, J.C., Emery, S., Eustace, R.A. 2020. Fungal and chemical diversity in hay and wrapped haylage for equine feed. Mycotoxin Research 36, 159-172.
• Gallo, A., Giuberti, G., Frisvad, J.C., Bertuzzi, T., Nielsen, K.F. 2015. Review on mycotoxin issues in ruminants: Occurrence in forages, effects of mycotoxin ingestion on health status and animal performance and practical strategies to counteract their negative effects. Toxins 7, 3057-3111.
• Keles, G., O'Kiely, P., Lenehan, J.J. & Forristal, P.D. 2009. Conservation characteristics of baled grass silages differing in duration of wilting, bale density and number of layers of plastic stretch-film. Irish Journal of Agricultural and Food Research 48, 21-34.
• Müller, C. E. 2005. Fermentation patterns of small-bale silage and haylage produced as a feed for horses. Grass and Forage Science 60, 109-118
• Müller, C.E. 2018. Silage and haylage for horses. Review paper. Grass and Forage Science 73, 815-827
• O’Brien, M., O'Kiely, P., Forristal, P.D., Fuller, H.T. 2006. The mycobiota of baled grass silage in Ireland. The Journal of Animal and Feed Sciences 15, 305-311
• O’Brien, M., O'Kiely, P., Forristal, P.D., Fuller, H. 2007. Visible fungal growth on baled grass silage during the winter feeding season in Ireland and silage characteristics associated with the occurrence of fungi. Animal Feed Science and Technology 139, 234-256
• O’Brien, M., O´Kiely, P., Forristal, P.D., Fuller, H.T. 2008. Fungal contamination of big-bale grass silage on Irish farms: predominant mould and yeast species and features of bales and silage. Grass and Forage Science 63, 121-137
• Paillat, J.M., Gaillard, F. 2001. PA, Precision Agriculture: Air-tightness of wrapped bales and resistance of polythene stretch film under tropical and temperate conditions. Journal of Agricultural Engineering Research 79, 15-22
• Samson, R.A., Houbraken, J., Frisvad, J.C., Thrane, U., Andersen, B. 2010. Food and Indoor Fungi. CBS Laboratory Manual series. Utrecht, Netherlands
• Schenck, J. 2019. Filamentous fungi in wrapped forages. Doctoral Thesis 2019:69. Swedish University of Agricultural Sciences, Uppsala, Sweden
• Schenck, J. S., Djurle, A., Jensen, D.F., Müller, C.E., O’Brien, M., Spörndly, R. 2019a. Filamentous fungi in wrapped forages determined with different sampling and culturing methods. Grass and Forage Science 74, 29-41
• Schenck, J. S., Müller, C.E., Djurle, A., Jensen, D.F., O’Brien, M., Johansen, A., Rasmussen, P., Spörndly, R. 2019b. Occurrence of filamentous fungi and mycotoxins in wrapped forages in Sweden and Norway and their relation to chemical composition and management. Grass and Forage Science 74, 613-625
• Scudamore, K.A., Livesey, C.T. 1998. Occurrence and significance of mycotoxin in forage crops and silage: a review. Journal of the Science of Food and Agriculture 77, 1-17
• Skaar, I. 1996. Mycological survey and characterization of the mycobiota of big bale grass silage in Norway. Doctoral Thesis. Oslo, Norway: Norwegian College of Veterinary Medicine
• Spörndly, R., Stennemark, V., Nylund, R. 2017. Relation between seal integrity and hygienic quality in silage bales and differences between baling techniques. In: (Eds.) Udén, P et al., Proceedings of the 8th Nordic Feed Science Conference, Uppsala, Sweden, pp. 169-172
• Tell, T.A. 2005. Aspergillosis in mammals and birds: impact on veterinary medicine. Medical Mycology Supplement 43, S71-S73
• Wambacq, E., Vanhoutte, I., Audenaert, K.,, De Gelder, L., Haesaert, G. 2016. Occurrence, prevention and remediation of toxigenic fungi and mycotoxins in silage: a review. Journal of the Science of Food and Agriculture 96, 2284-2302