# Feasibility of Utilizing Biodegradable Plastic Film to Cover Corn Silage under Farm Conditions
## 2 Materials and Methods
### Sample Preparation and Analyses  
The pre-ensiled material and the silages were split into five subsamples. One sub-sample was analyzed immediately for the DM content by oven drying at 80 \({}^{\circ}\)C for 24 h. Dry matter was corrected according to ([PERSON] et al., 2018) in order to consider the volatile compound losses that can take place at 80 \({}^{\circ}\)C.  
The second subsample was oven-dried at 65 \({}^{\circ}\)C to constant weight and was air equilibrated, weighed and ground in a Cyclotec mill (Tecator, Herndon, VA, USA) to pass a 1 mm screen. The dried samples were analyzed for total nitrogen (TN), according to the [PERSON] method (method number 992.23, ([PERSON] et al., 2018)), using a Nitrogen Primacs SN (Skalar, Breda, The Netherlands), for crude protein (CP) (total N x 6.25) and for ash by ignition (method number 942.05, ([PERSON] et al., 2018)). Neutral detergent fiber (aNDF) was analyzed, using a Raw Fiber Extractor (FIWE, VELP Scientifica, Usmate Velate, IT), with the addition of heat-stable amylase (A3306, Sigma Chemical Co., St. Louis, MO, USA) and expressed on a DM basis, including residual ash, as described by [PERSON] et al. (2018). Acid detergent fiber (ADF) was analyzed and expressed on a DM basis, including residual ash ([PERSON] et al., 2018).  
A third fresh sub-sample was used to determine the water activity (a\({}_{w}\)), pH, nitrate (NO\({}_{3}\)) and ammonia nitrogen (NH\({}_{3}\)-N) contents and the buffering capacity (BC). The fresh forage was extracted for pH, NO\({}_{3}\) and NH\({}_{3}\)-N determination, using a Stomacher blender (Seward Ltd, Worthing, UK), for 4 min in distilled water at a 9:1 water-to-sample material (fresh weight) ratio. The total nitrate concentration was determined in the water extract, through semi-quantitative analysis, using Merckoquant test strips (Merck, Darmstadt, Germany; detection limit 100 mg NO\({}_{3}\)/kg). The ammonia nitrogen content and pH were determined using specific electrodes. The buffering capacity was determined in the water extract, as described by [PERSON] and [PERSON] (1993).  
A fourth sub-sample was extracted, using a Stomacher blender, for 4 min in H\({}_{2}\)SO\({}_{4}\) 0.05 mol/L at a 4:1 acid-to-sample material (fresh weight) ratio. An aliquot of 40 mL of silage acid extract was filtered with a 0.20-um syringe filter and used for quantification of the fermentation products. The lactic and monocarboxylic acids (acetic, propionic and butyric acids) were determined, by means of high performance liquid chromatography (HPLC), in the acid extract [32]. Ethanol, 1-propanol and 1,2-propanediol were determined by means of HPLC, coupled to a refractive index detector, on an Aminex HPX-87H column (Bio-Rad Laboratories, Richmond, CA, USA).  
The fifth subsample was used for microbial counts. Thirty grams of sample was transferred to a sterile homogenization bag, suspended 1:10 w/v in a peptone salt solution (1 g of bacteriological peptone and 9 g of sodium chloride per litre) and homogenized for 4 min in a laboratory Stomacher blender (Seward Ltd, London, UK). The yeast and mold numbers were determined by preparing serial dilutions and using the pour plate technique, with 40.0 g/L of Yeast Extract Glucose Chloramphenicol Agar (YGC agar, DIFCO, West Molesey, Surrey, UK). The yeast and mold colony-forming units (cfu) were enumerated separately, according to their macromorphological features, on plates that yielded 1 to 100 cfu, after incubation at 25 \({}^{\circ}\)C for 3 and 5 d for the yeasts and molds, respectively.