Year of Publication


Degree Name

Master of Science (MS)

Document Type

Master's Thesis


Agriculture, Food and Environment


Animal and Food Sciences

First Advisor

Dr. Rachel R. Schendel

Second Advisor

Dr. Youling Xiong


Grass cell walls are rich in cellulose, hemicellulosic arabinoxylan (AX) polysaccharides, and lignin. AX structural differences such as degree and pattern of branching and the ester-linked phenolic acid content could affect plants’ digestibility when used as forage for livestock. However, there is little information about how these structural elements change over the growing season in the vegetative tissue of cool-season perennial grasses. Enhanced information about the cell wall composition and carbohydrate structure of forage material will provide a foundation for expanding our knowledge of how forage cell wall carbohydrate structures are utilized by ruminants. The objectives of this study were to investigate changes in the cell wall composition of five cool-season grasses grown in central Kentucky (perennial ryegrass (Lolium perenne L.), orchardgrass (Dactylis glomerata L.), tall fescue (Schedonorus arundinaceus (Schreb.) Dumort), Kentucky bluegrass (Poa pratensis L.), and timothy (Phleum pratense L.)) over the growing season and between growing years and also to explore structural carbohydrate changes in cool season forage material following fermentation with pure cultures of fibrolytic bacteria. Forages were planted in September 2019 in a randomized block design, and vegetative material was harvested in April, June, August, and October of 2020 & 2021. The collected samples were lyophilized; milled (< 0.5mm); defatted; and destarched to isolate insoluble cell wall material. The AX enzymatic fingerprint was determined by digesting cell wall material with endoxylanases and then separating and quantifying the released oligosaccharides with high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Phenolic acids were released via alkaline hydrolysis; extracted with diethyl ether following acidification; and separated, detected, and quantified using high-performance liquid chromatography coupled with a diode array detector (HPLC-DAD). The monosaccharide profile of the cell wall polysaccharides was determined via Saeman hydrolysis followed by HPAEC-PAD separation and detection. Lignin content was determined using the Acetyl Bromide Soluble Lignin (ABSL) method. Forage material was also incubated with pure bacterial cultures (Fibrobacter succinogenes (S85) and Acetivibrio thermocellus (27405)), and carbohydrate structural changes during fermentation were analyzed. Statistical significance was analyzed via one-way ANOVA, and Tukey-Kramer post-hoc testing was used to reveal significant pairwise differences. Incubation with selected pure cultures of fibrolytic bacteria revealed a tendency for the microorganisms to selectively remove arabinose units from the AX polysaccharides and a preference for utilization of cellulose. Reproducible seasonal changes were observed in the forage material, such as a general increase in lignin content over the growing season in both years, and an increase in ester-linked coumaric acid from the spring to summer sampling points. However other seasonal compositional changes were observed only in one sampling year, such as a significant increase in the arabinose/xylose (A/X) ratio for most species in 2020, but not 2021, which likely reflects the different weather conditions in the two growing seasons. Other compositional elements, such as ester-linked ferulic acid, showed clear differences between species at selected sampling points, but these differences were not reproduced in the second sampling year. Taken together, these data illustrate the importance of screening multiple forage species at multiple sampling points to observe the full range of cell wall structural composition possibilities for cool-season forages and to avoid incorrect generalizations.

Digital Object Identifier (DOI)

Funding Information

This research was funded through the United States Department of Agriculture-Animal Research Service (USDA-ARS) Forage Animal Production Research Unit (FAPRU) NP 215 Project: Sustainable Forage Production Systems for the Mid-South Transition Zone in 2021.