## 2 Materials and Methods
### Evaluation of Nutritional Assets  
Standard physical, chemical, enzymatic, volumetric, and gravimetric techniques were applied to measure the core nutrients, minerals, and digestibility of S1, S2, and S3. Subsamples were withdrawn from each original sample. The subsamples were used to measure each nutrient separately according to standard recommended techniques. Crude protein (CP) value is an important indicator for growth and performance. CP was calculated as a % of the dry matter (DM) after measuring nitrogen (N) content by GAFTA and ISO methods [33; 34]. Soluble protein (SP) is measured as a % of the CP and determines how much protein is water soluble. CP solubility helps to determine the available protein. Available CP is also measured as a % of the CP and determines how much of the total CP is available for digestion. Available CP is calculated by subtracting the acid detergent fiber (ADF) bound protein. Plant ADF content is a measure of the cellulose and lignin in plant DM. Plant ADF is a good indicator of digestibility. In addition, it defines the amount of fiber needed by milk-producing mammals. The acid detergent insoluble CP (ADICP) measures the tightly bound protein that is not available for digestion. ADICP is defined as an ADF bound protein according to ISO 13906. It is expressed as a % of the DM [35]. Neutral detergent in soluble CP (NDICP) measures the protein that is bound to the neutral detergent fiber fraction (NDF). It is measured according to ISO 16,472 and is expressed as a % of the DM [36]. NDF is a measure of the cell wall content of plant-based feeds and limits total feed intake in diets with high plant content. Plant ADF is part of the NDF. NDF and ADF contents vary with plant phenotype and plant age. NDF and ADF were measured according to ISO 16,472 [36] and ISO 13,906 [35], respectively. The NDF corrected for ash content (aNDF) is also expressed as a % of DM. High aNDF values generally occur in plant species with low CP values [37]. Lignin is a woody fiber linkage. Lignin is not digestible by humans or ruminants. The lignin content is measured using sulfuric acid reagent grade (Biopack, Buenos Aires, Argentina) according to an adapted method based on ISO 13,906, and it is expressed as a % of DM [35]. The fiber digestibility (NDFD) is expressed as a % of NDF. NDFD is important for nutritionists to estimate how much fiber can be digested. Fiber digestion times of 24, 30,48, 72, 120 and 240 h were selected to define the extent of total fiber digestion. The NDFD was determined using the \"traditional\" [PERSON] method (tNDFD) [38] and the standardized Combs-Goeser method (sNDFD) [39]. The part of the fiber that has not been digested after a certain time (uNDF t) was expressed as a % of DM. In particular, uNDF 240 is, by definition, the fraction of fiber that can never be digested and is equated with the indigestible NDF (iNDF). Non-fibrous carbohydrates (NFCs) are the sugar-related carbohydrates and starches found inside the cell walls of plant tissues. In general, starch is the most important fraction of the NFC. These compounds are nearly 100% digestible. NFCs are necessary in the intestine for bacterial growth and for the production of high-quality bacterial proteins. However, an excess of NFC could lead to clinical acidosis [40]. The starch content is expressed as a % of DM and was measured by cleaving the starch molecules into individual glucose molecules and then reading the resulting glucose content. The ash content is expressed as a % of DM and was measured by burning the sample according to GAFTA [41] and ISO [42] methods. Ash stems from soil contamination and from minerals within the plant. Fat is a high energy nutrient. The content of fat-like compounds (EE) is expressed as a % of DM and was measured using an ether extraction method according to GAFTA [43] and ISO [44] methods. The total minerals were determined by an Inductively Coupled Plasma Mass Spectrometer (ICP-MS) (NexION 2000 ICP Mass Spectrometer, Perking Elmer, Boston, USA).