The adverse effects of fungal infestation of silage include allergic airway diseases due to spore inhalation and reduced palatability due to a ‘mouldy’ scent caused by the production of volatile organic compounds . Moreover, some fungi can produce mycotoxins and are then called toxigenic. In the current silage-making practices, it is difficult if not impossible to avoid mycotoxin contamination of forage crops [34, 35]. Hence, ensiled forages may contain a mixture of mycotoxins, originating from pre-harvest contamination [36, 37] and or from postharvest contamination with toxigenic moulds that are common in silage . In consequence, in the market exist a large number of silage additives that aim to control silage fermentation and spoilage processes . On one hand, biological additives (homo- and hetero-fermentative strains) which inhibited the growth of spoilage moulds and improve the shelf life of silage at feedout. On the another hand, chemical silage additives with stronger antifungal and antibacterial properties . However, the positive effects of silage additives were not observed in this study in the orchardgrass silage within the same cut for each variety. The nature and intensity of the effect of silage additives may differ across plant species , suggesting that either biological or chemical additives no reduced the moulds presence in orchardgrass silage, as consequence were not differences in ERG and phenolic compounds content. Therefore, the use of the silage additives should never be regarded as a substitute for good silage-making practices .
ERG is the primary sterol present in the cell wall (membrane) of filamentous fungi [41, 42] used to determine the quantity of moulds . As it is can be seen in Table 2, the augmentative tendency of ERG content found in the fresh forage samples of orchardgrass from 2012 to 2013 was related at the higher rainy conditions in the second year, contributing to the occurrence and development of fungi. The ERG increase at high relative humidity and lower temperature was previously corroborated by Kalač . In addition, the high ERG content is related to the delayed harvest date  and the higher frequency of rains  at the timing of harvest. This explains why the ERG content was higher in the second cuts of each year than in the first ones. In 2012, 47 mm and 0.5 °C less of precipitation and temperature, respectively, were measured than in 2013 (Fig. 1). These parameters contributed to the higher incidence of fungi in the cuts of 2013, resulting in higher ERG content in fresh forage and silage than cuts of 2012.
This study showed that ERG content in silage increased considerably in comparison to the fresh forage of orchardgrass, thus, our findings are in good agreement with Skládanka et al.  which evidenced that ensilage process does not decrease the number of moulds, and hence a higher risk of mycotoxins production in the forage. Therefore, a high fungal infestation by moulds explain the significant correlations of ERG concentration in silage . Fungal growth leads to a loss or reduction in nutrients and dry matter, and a lowering of palatability, with consumption generating losses in animal performance . The importance of ensiled forage crops as sources of mycotoxins in the ruminant diet has been confirmed by numerous authors . Hence, fungal spoilage and mycotoxin contamination are one of the greatest risks in silage.
The ergosterol content found in our study in the first cut of 2012 in the silage treated with biological additives was consistent with previous studies, where the ERG degradation was visible by the inoculation with bacterial additives . While the use of chemical silage additives, based on organic acids and salts widely recommended in silage crops to limit the growth of fungi  have not a positive effect. Due to that a high ERG content evidenced that growth of fungi by chemical additives was not prevented in our orchardgrass silages. Hence, it can be suggested that the effect of silage additives in preventing growth of moulds and their metabolites is not always efficient, not even in (in vitro) experiments.
ERG concentrations in forage grasses can vary from 20 to 400 mg kg-1 DM depending on the grass species (see review by Kalač ), as well as the variety within the species, as observed in this review for Festulolium . Besides, Opitz von Boberfeld and Banzhaf proved  the lower the production of ERG content, the higher the quality of silage Until today, it has not been established a safe limit for ERG content in silages, due to all depends on the mould species which are contaminating the silage, the mycotoxins that it produces and on the ensiled forage species. For example, 110 mg kg-1 DM of ERG in Festulolium forage was considered low and this grass as being resistant to mildew infestation in comparison to 139.6 mg kg-1 DM of ERG in Arrhenatherum elatius forage that was infected with a high content of zearalenone . Furthermore, Skládanka et al.  suggested that ERG amounts (ranging from 3.8 to 190.8 mg kg-1 DM) in different harvest dates (summer and winter) indicated high content of mycotoxins [e.g. deoxynivalenol (DON) and zearalenone (ZEN)] in summer period. As a result, this could indicate that the ERG amounts recorded in this study (ranging from 42.7 to 75.7 mg kg-1 DM in 2013) cannot be considered safe for silage. Previous studies claim that in the case of toxin-forming fungi that occur in the environment, the results of analyses of ergosterol content in cereal grain usually demonstrate a significant correlation with mycotoxin concentrations found in grain crops [50, 51]. For example Cegielska-Radziejewska et al.  showed a statistically significant correlation between deoxynivalenol/ERG and total trichothecenes/ERG in poultry feeds. Consequently, high fungal infestation in mouldy parts may explain the high ERG concentration obtained in silage sub-samples . In addition, Pietri et al.  reported that the quality of maize is acceptable if the level of ERG content is less than 3 mg kg-1. There is a high possibility of fungal invasion and mycotoxin contamination if the level of ERG content exceeds more than 3 mg kg-1. Because there are no specific regulations on mycotoxins in silage (e.g. grass silage, only for maize-based product guidance value is available), currently recommended levels for animal feed could also be considered as guidelines for silage . Regarding silage the presence of DON and ZEN is recommended not to exceed 12 mg kg-1 and 3 mg kg-1, respectively . Thus, our findings suggest that to determine the ERG content as a biochemical indicator is relevant to assess silage safety but does not allow the establishment of safe limits for ruminants.
Regarding silages safety, high phenolic compound concentration gives us a hint of mould presence as many plant tissues accumulate phenolic compounds on their cell walls on interactions with fungal pathogens. Therefore, the accumulation process constitutes a protective mechanism against cell wall degradation, similar to the barrier provided by lignins, limiting the spread of pathogens . Nevertheless, their presence up to a certain threshold is actually considered to be positive, due to their antioxidant activity, ability to chelate metals, inhibit lipoxygenase and scavenge free radicals . Besides, phenolic compounds play an important role in the synthesis of the biological mimic cell wall, where they may also inhibit the diffusion of extracellular enzymes and toxins, protecting it from degradation .
The concentration of total phenolic compounds in orchardgrass silage models ranged from 24.0 to 77.5 g GAE kg-1 DM, which proved a safety issue with the silage, as the minimum phenolic compounds [condensed tannins (CT)] concentration needed to make forages bloat-safe has been proposed to be 5 g kg-1 . Consequently, high CT concentrations (> 55 g kg-1 DM) reduce forage intake and digestibility and depress rates of body and wool growth in ruminants . Moreover, elevated doses of tannins can impair fibre digestion causing toxicosis in sheep . Plants tend to produce complex mixtures of tannins and not all tannins have the same effects on feeding. The study of polyphenols (tannins) in animal production has primarily focused on CT, and little information is available on the effects of hydrolysable tannins (HT) in livestock production. Therefore, further research is required on the concentration of HT in silage grasses to provide a stronger basis and to prevent intoxications in animals ill-adapted to HT consumption .
Similar to ERG, the phenolic compounds concentration can vary between genotypes of the different varieties ensiled and to the experienced environmental . In addition, the concentration of phenolic compounds depends on the type of analysed sources .
Nowadays there are better tools which can help us to understand which microorganisms and secondary plant metabolism are involved in the ensiling process over beyond of the fermentation. Despite this, the total concentration of phenolic compounds in orchardgrass has not been reported yet, therefore, further studies are required in these issues to determine the safe limits of phenolic compounds for ruminants in silages.