Ergosterol and polyphenols: Potential biochemical indicators of silage safety

Background: Silage, one of the most important feed sources for cattle, is vulnerable to contamination by spoilage moulds and mycotoxin production because ensilage forage crops are excellent substrates for fungal growth. Whereas there are many factors involved in mycotoxins contamination such as biological factors, harvesting, storage, and processing conditions, the climate is the most important factor. Silage additives can improve the safety of silage and inhibit moulds and other detrimental silage microorganisms; however, their ecacy varies with the type and level of toxigenic fungi contamination. Several studies provided evidence showing that the presence of various mycotoxins was correlated with the presence of ergosterol in cereals and feedstuffs. Therefore, ergosterol ( ERG ) has been suggested as a potential indicator of fungal contamination with polyphenols concentration analysis allowing accurate prediction on silage safety. The main goal of this study was to use the ERG and polyphenols content as potential biochemical indicators to predict the silage safety in ensiled grasses. The study was carried out using ten orchardgrass varieties (untreated and treated with biological and chemical additives). The determination of the samples was performed on high-performance liquid chromatography using UV detection (ERG) and spectrophotometer UV/VIS (polyphenols). Results: Interestingly, in the silage ‘Bepro’ was the unique variety that no presented content of ERG (0.0 mg kg -1 dry matter) in comparison with other varieties in the rst cut in 2012. The biological additives reduced the content of ERG in both cuts in 2012 compared with untreated silage. In contrast, the chemical additives in silage grass had a negative effect ( P < 0.05) by increasing the ERG and polyphenols content in comparison with untreated silage. Conclusions: These results of this study provide indisputable evidence that silage contaminated with fungi increase the concentration of polyphenols, which prove a progressive deterioration of silage quality. In consequence, ERG and polyphenols seem to be relevant as a rapid method for screening silage safety.


Plant materials
Ten orchardgrass varieties from breeding programmes in different European countries ('Greenly' and 'Starly' from France; 'Otello' from Italy; 'Sw-Luxor' from Sweden; 'Amera', 'Dika', and 'Bepro' from Poland; 'Husar' from Germany; and 'Dana' and 'Vega' from the Czech Republic) were used. The  form. The rst and second growth were harvested at the heading phase (vegetation stage when in orescence is emerging, but before shedding pollen). The biomass was wilted on the plot for 14 h to reduce the water content after mowing. Afterwards, the forage samples (10 kg per treatment) were taken to the laboratory and chopped with a conventional forage harvester to a particle length of 40-60 mm.
At the end of the ensiling period (90 days), the silos were opened, and samples were taken for chemical analysis. The pre-drying of subsamples (fresh forage and silage) was performed in a speci c drying oven at 60 °C for 48 hours to determine the silage quality. Subsequently, the forage samples for analysis were ground in a mill and then ltered through a 1 mm sieve. The fresh forage and silage samples were analysed to determine the concentration of ERG and phenolic compounds.

Ergosterol determination
In order to determine ERG in fresh grass and silage, a total of 250 mg of the sample and 2 mL of the 10% solution of potassium hydroxide in methanol were weighed using analytical scales into 4 mL glass vials with a screw closure. The vials were then closed with lids with a te on antiseptic sealing. The content was intensively mixed for 30 s in a mixer (MS2 Minishaker IKA, USA) and the vial was kept in a thermostat (Evaterm, Labicom, CZ) for a period of 90 min at 80 °C. Volume of 0.5 mL of distilled water and 1 mL of hexane were added after cooling to laboratory temperature and the vial content was mixed for 30 s. After thorough separation of aqueous and organic phases, the content was centrifuged (Universal 32R, Hettich, Germany) for 5 min at 4 000 rpm. The upper organic layer was poured into a 1.8 mL vial and evaporated under nitrogen ow. The remaining aqueous phase was added to 1 mL of hexane and the whole extraction process was repeated twice to achieve a quantitative ergosterol yield.
The joint extracts were evaporated to the dry phase. The evaporation residue was nally dissolved in 400 µL mixture of methanol/toluene (75:25, v/v) and analysed with the use of liquid chromatography.
The actual determination of ergosterol took place in a HPLC reverse phase using a Zorbax SB-C18 column of size 4.6 × 30 mm at a particle size of 1.8 µm (Agilent Technologies, USA). The separation was carried out at laboratory temperature using isocratic elution -mobile phase with the composition of methanol/water of 97.5:2.5 (v/v) at a volumetric velocity of 0.6 mL min − 1 . Ergosterol was detected in the ultraviolet zone at 282 nm. The dosed extract volume was 2 µL. To measure the calibration curve, a method of standard addition was used. Various amounts of a standard ergosterol solution were poured into seven vials, corresponding after conversion to a range of 0.1 to 1.000 µg g − 1 forage. The vial content was then evaporated by nitrogen ow. A forage amount of 250 mg was inserted into each vial. The eighth vial was lled with forage without the added standard. The following procedure was the same as in the The concentration of original solid plant samples was calculated using the formula: where m = weight of phenolic compounds in 100 g of original dry plant sample (g); c g = concentration of phenolic acid in measured sample received from calculation from calibration line (g mL − 1 ); V c = volume of calibration solutions used for reaction (1 mL); m r = weight of reference sample used for calculation (100 g); V ex = volume of extraction solution (10 mL); m s = weight of sample used for extraction; V p = volume of extracted sample used for reaction (1 mL). The total content of phenolic compounds was expressed as g gallic acid (GAE) kg − 1 DM.

Statistical analyses
The data were processed using the statistical software STATISTICA ver. 12 (StatSoft, Inc.). The results were expressed as a mean ± standard error of the mean (SEM). Differences with P < 0.05 were considered signi cant and determined multifactorial ANOVA test (in particular, Scheffé's test), which was applied for mean comparison.

Silage Additives
The amounts of ERG and phenolic compounds in silages, within the same cut for each orchardgrass variety treated silages with biological and chemical additives compared to their respective controls, showed no differences. Therefore, the ERG and phenolic compounds data with additives and controls were presented in a single average, as will be described below (Tables 2 and 4).

Analysis of Ergosterol by HPLC
The ERG content in fresh forage of orchardgrass varieties gradually increased from the rst cut of 2012  (Table 1).
As it is shown in Table 2, ERG content in silage differed between orchardgrass varieties and cuts, being  Table 2).

Analysis of phenolic compounds by Folin-Ciocalteau assay
In the fresh cut the concentration of phenolic compounds was higher in the rst cut in both year (2012 and 2013), where the rst cut from 2013 showed higher concentration of phenolic compounds range from 38.1 to 63.1 g gallic acid per kg of DM (g GAE kg − 1 DM) in contrast to the others cuts ( Table 3).
The concentration of phenolic compounds was no different between silage and fresh forage within the same cultivars. Nevertheless, the total phenolic concentrations showed differences (P < 0.05) among orchardgrass varieties, except in the rst cut of 2013. Similarly, to ERG, a higher concentration of phenolic compounds was recorded in the silage group treated with chemical silage additives in comparison to control silages. However, there were no differences in phenolic compounds concentrations between the silage groups (treated with biological and chemical additives) in both years, except in the rst cut of 2013, where the highest concentrations of these secondary metabolites (77.5 g GAE kg − 1 DM) was observed in the silages treated with chemical additives (Table 4).

Discussion
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 [33]. Moreover, some fungi can produce mycotoxins and are then called toxigenic. In the current silage-making practices, it is di cult if not impossible to avoid mycotoxin contamination of forage crops [34,35]. Hence, ensiled forages may contain a mixture of mycotoxins, originating from preharvest contamination [36,37] and or from postharvest contamination with toxigenic moulds that are common in silage [35]. In consequence, in the market exist a large number of silage additives that aim to control silage fermentation and spoilage processes [38]. 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 [39]. 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 [40], 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 [38].
ERG is the primary sterol present in the cell wall (membrane) of lamentous fungi [41,42] used to determine the quantity of moulds [43]. 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 [44]. In addition, the high ERG content is related to the delayed harvest date [27] and the higher frequency of rains [45] at the timing of harvest. This explains why the ERG content was higher in the second cuts of each year than in the rst 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 ndings are in good agreement with Skládanka et al. [46] 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 signi cant correlations of ERG concentration in silage [2]. 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 [15]. The importance of ensiled forage crops as sources of mycotoxins in the ruminant diet has been con rmed by numerous authors [2]. Hence, fungal spoilage and mycotoxin contamination are one of the greatest risks in silage.
The ergosterol content found in our study in the rst 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 [46]. While the use of chemical silage additives, based on organic acids and salts widely recommended in silage crops to limit the growth of fungi [39] 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 e cient, 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 [44]), as well as the variety within the species, as observed in this review for Festulolium [47]. Besides, Opitz von Boberfeld and Banzhaf proved [47] 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 [48]. Furthermore, Skládanka et al. [49] suggested that ERG amounts (ranging 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 signi cant correlation with mycotoxin concentrations found in grain crops [50,51]. For example Cegielska-Radziejewska et al. [52] showed a statistically signi cant 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 [2]. In addition, Pietri et al. [53] 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 speci c 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 [54]. Regarding silage the presence of DON and ZEN is recommended not to exceed 12 mg kg -1 and 3 mg kg -1 , respectively [55].
Thus, our ndings 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 [56]. 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 [57]. 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 [56].
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 [58]. Consequently, high CT concentrations (> 55 g kg -1 DM) reduce forage intake and digestibility and depress rates of body and wool growth in ruminants [59]. Moreover, elevated doses of tannins can impair bre digestion causing toxicosis in sheep [60]. 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 [59].
Similar to ERG, the phenolic compounds concentration can vary between genotypes of the different varieties ensiled and to the experienced environmental [61]. In addition, the concentration of phenolic compounds depends on the type of analysed sources [62].
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.

Conclusions
Biological additives used during ensiling in both cuts of 2012 effectively inhibited epiphytic bacteria and enable successful reduction the content of ERG and consequently moulds proliferation associated with silage production. While in the both cuts of 2013 its effect was not observed. In contrast, silage treated with chemical additives did not avoid ERG production in both years. For this reason, silage additives are not always successful at improving silage hygiene quality, because better-adapted epiphytic micro ora might outcompete the additives and dominate the succeeding fermentation.
In consequence, the gradual increase of ERG in silages of orchardgrass between 2012 and 2013 evidenced that forage contamination by moulds simultaneously increased the concentration of phenolic compounds. As a result, this effect proved a progressive deterioration in silage safety, which could be harmful and adversely affect production and health of livestock. Therefore, the determination of ERG and phenolic compounds seem to be relevant as a rapid method for screening silage grass safety, since the animal production depends mainly on forage quality and silage safety.