The aim of our investigations was to evaluate the effect of the fermentation process on the microbiological and mycotoxicological status of alfalfa silage. The second cutting of alfalfa was harvested in the initial stage of blooming, field-wilted at a dry matter content of around 30%, and ensiled in 16 mini-silos (8654.63 cm³ volume). The silos were stored at an ambient temperature of 20°C for 12 weeks. The samples of green fodder and silage were collected for chemical, microbiological, and mycotoxicological analyses. The content of dry matter in the forage and silage was determined according to the Polish standard PN-ISO 6496 and that of crude protein according to PN-EN ISO 5983-1, PN-EN ISO 5983-2. In addition, basic fermentation parameters were determined in the silage: pH was measured by an N-517 pH-meter, and the content of organic acids (lactic acid, acetic acid, and butyric acid) by the HPLC method. The number of lactic acid bacteria was established according to the ISO 15214:2002 standard, the number of mesophilic aerobic bacteria according to PN-R-64791:1994, and the number of yeasts and molds according to ISO 7954:1999. The levels of aflatoxins (AF) and ochratoxin A (OTA) were determined by the HPLC method with fluorescence detection, and the contents of toxins T-2 and HT-2, nivalenol, deoxynivalenol, and zearalenon were determined using HPLC -MS/MS. In addition, the mold types identified were calculated as a percentage of their total numbers. Alfalfa forage was characterized by a high content of epiphytic lactic acid bacteria. The numbers of mesophilic aerobic bacteria, yeasts and molds exceeded allowable levels for high-quality feed material. The fermentation process increased the number of lactic acid bacteria and reduced the occurrence of mold in silage compared with green fodder, while totally eliminating yeast. Among the mycotoxins analyzed, only small amounts of deoxynivalenol (DON) were detected in forage. The fermentation process had no effect on the count of aerobic mesophilic bacteria. The results of these analyses demonstrated a good microbiological and mycotoxicological status of alfalfa silage.
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