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.