Lab Methods
Nutritional analyses were completed at the Alaska Department of Fish and Game (ADFG) Herbivore Nutrition Laboratory, in Palmer, AK. Digestible energy and digestible protein of all forage species were quantified using the methods outlined by Spalinger et al. (2010) and McArt et al (2006). Vegetation samples collected in the field were subsequently freeze-dried and ground in a Wiley mill over a 20-mm screen (McArt et al. 2006). Before being ground, winter twig samples were segregated into three diameter segments in order to quantify the relationship between nutritional quality and diameter of the twig. The goal was to divide the stems into three diameter classes that produced approximately equal masses with enough sample for the nutritional analyses. Regressions were then used to predict nutritional quality and bite size from diameter at point of browsing for any given species.
Nitrogen concentrations were determined using a LECO TruSpec CHN and Cos-tech CHNOS Analyzer with NIST apple leaves used as quality control standard for every 10th sample (McArt et al. 2006). Digestible dry matter of each forage was determined from sequential fiber analysis using the ANKOM 200 fiber analysis system according to manufacturer’s recommended methods. Neutral detergent fiber (NDF) was determined with a neutral detergent solution made with sodium sulfite and an acid detergent solution was used to determine acid detergent fiber (ADF). NDF is an estimate of the ratio of insoluble fiber of the plant in the cell wall and the highly digestible cell contents while ADF is a measure of the cellulose + lignin/cutin concentration (Van Soest 1994, Spalinger et al. 2010). Samples were then extracted in 72% sulfuric acid to determine lignin + cutin concentration (Van Soest 1994, Spalinger et al. 2010). The sulfuric acid residue was then ashed at 500℃ for 3 hours to determine the mineral/silica concentration (Van Soest 1994, Spalinger et al. 2010, Cook et al. 2022). Because of a shift in sequential fiber values since 2012, we used a modified correction as described in Cook et al. (2022). Digestible dry matter (DDM) and energy (DE) were subsequently estimated from equations presented by Spalinger et al. (Spalinger et al. 2010).
Tannins were extracted in a 50% methanol solution under high pressure and temperature in a DionexTM Accelerated Solvent Extractor (ASE-200) (Close et al. 2003, McArt et al. 2006). The tannin extract was then diluted with 50% methanol, mixed with bovine serum albumin (BSA) and purified with Sephadex G-25. The resulting solution was mixed with Bradford Protein Reagent and read on a UV-Vis microplate spectrometer at 595 nm to estimate protein precipitating capacity (PPC, mg DSA precipitated/mg forage DM) (McArt et al. 2006). PPC was then used to calculate the digestible protein, the percent protein reduction, and digestible energy of each sample (Robbins et al. 1987, Spalinger et al. 2010).
After freeze-drying, twigs were subdivided into 3 diameter classes for subsequent nutritional analyses, following the methods outlined above. After nutritional analyses, mass-diameter and nutrient density - diameter regressions were computed for each species. From these, estimates of nutritional quality and bite size were made for field measured browsed twigs.