Original ResearchEffects of Sampling Time, Cultivar, and Methodology on Water- and Ethanol-Soluble Carbohydrate Profiles of Three Cool-Season Grasses in Central Kentucky
Introduction
Cool-season grasses (CSGs) accumulate water-soluble carbohydrates (WSCs), consisting of monosaccharides, sucrose, and fructans [1]. Other carbohydrates, such as raffinose, may be present as well [2]. Fructans are fructose polymers which often have a terminal sucrose unit [3] but may consist only of fructose [4]. Cool-season grass fructan may consist of fructose units joined by beta-1,2 or beta-2,6 linkages [5]. The chain length or degree of polymerization (DP) varies with CSG genotype and season [6], sometimes exceeding 200 [7].
Consuming large quantities of WSC may exacerbate the risk of equine pasture-associated laminitis (PAL) [8], but it is not clear which component of the WSC is of most concern. Feeding studies indicate that consuming glucose and fructose is correlated with insulin resistance, which can exacerbate the risk of PAL [9]. Consuming large amounts of chicory fructan (inulin), either in a single dose [10] or in more moderate doses over consecutive days [11], may induce laminitis. Although chicory fructan and CSG fructan are not identical in structure [12], these observations suggest that both the monosaccharides and the fructans in CSG could be problematic to at-risk horses. In addition, other soluble carbohydrates, such as sucrose, occur in grasses [1], [13], [14], [15], [16], which may also be important to consider for at-risk horses. More detailed information on the composition of WSC of CSG may help in understanding the roles of different carbohydrates in exacerbating the risk of PAL.
Individual carbohydrates in WSC can be quantified by high-performance liquid chromatography (HPLC) [17], [18], [19]. Concentrations of the quantified compounds can be summed to give a value for total WSC concentrations [19]. Total WSC can also be quantified colorimetrically [19], [20]. Fructan, in addition to being quantified colorimetrically [14], [18] or chromatographically, is sometimes estimated as the difference between colorimetrically determined WSC and ethanol-soluble carbohydrates (ESCs) [21]. Ethanol (80%) was previously thought to extract only glucose, fructose, and sucrose from forages [1], although it is now known to extract fructans of short chain length as well [7], [22]. The solubility, and hence the maximum chain length, of fructan polymers increases as the ethanol-to-water ratio decreases [7]. Differences in fructan solubility in ESC extracts from different CSG have received little attention, although these have been studied in ornamental bulb plants, which also accumulate fructans [23].
Accumulation of WSC in CSG is influenced by light [14], [24], temperature [13], [25], rainfall [26], cultivar [25], [27], and defoliation regime [28]. Consequently, WSC concentrations and profiles vary with geographic region. Studies of diurnal variation of forage WSC or ESC have been conducted in Australia [14], the United States Midwest [16], and the U.S. Piedmont region [15], but not in the central Kentucky region, which produces a large proportion of the U.S. annual foal crop. The goal of this study was to compare concentrations and profiles of water and ethanol extracts of several CSG cultivars collected in the morning and afternoon on two harvest dates, in order to obtain more accurate information about diurnal and cultivar variation of different soluble carbohydrates in CSG Kentucky pastures. A second goal was to gain more understanding of the effects of different extraction and quantitation methods on detected concentrations and types of WSC or ESC found in CSG.
Section snippets
Forage Sampling
Perennial ryegrass (Lolium perenne cvs. “Linn” and “Calibra”) and endophyte-free tall fescue (Lolium arundinaceum cv. “Cajun”) were planted on September 5, 2013, at the Spindletop Research Farm of the University of Kentucky (Lexington, KY). Kentucky bluegrass (Poa pratensis cv. “Ginger”) was planted at the same farm on September 12, 2012. Three replicate plots (1.5 m × 4.5 m) were sampled in late April, early May, and late May 2014, between 8 AM and 11 AM, and between 2 PM and 4 PM. Due to
Harvest Date, Diurnal, and Solvent Effects on Soluble Carbohydrate Concentrations and Fructan Chain Length
Concentrations of individual and total soluble carbohydrates, averaged across results obtained from both water and ethanol extracts, were higher on harvest 1 in late April 2014 than on harvest 2 in early May 2014 (Table 3). Concentrations of sucrose and reducing sugars (glucose and fructose) have been found to decrease during early growth periods, due to the incorporation of photosynthate into cellulosic tissue [37]. Similarly, fructan concentrations have been found to decrease with the onset
Conclusions
The results of this study indicate that methodology may affect concentrations and types of soluble carbohydrates observed in CSG. Colorimetrically determined concentrations of total WSC or ESC may exceed chromatographically determined results. Glucose and fructose may be abundant in WSC extracts due to hydrolysis of sucrose, unless degradative enzymes are inactivated at the start of extraction.
Fructans and fructan fermentation have been considered key risk factors for laminitis [10]. In this
Acknowledgments
The authors thank Gene Olson, Department Plant and Soil Sciences, University of Kentucky, for providing access to plots of field variety trials. The authors also thank Dr Jong-Duk Kim, Cheonan Yonam College, South Korea, for helpful discussions; and Andrea Crum, Susan Hayes, and numerous student helpers for assistance with sample harvesting. The authors also thank Tracy Hamilton, USDA-ARS, for laboratory assistance. This project was funded by the United States Department of Agriculture. K.J.P.
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Animal welfare/ethical statement: Authors have adhered to ethical guidelines outlined in https://www.elsevier.com/authors/journal-authors/policies-and-ethics.
Conflict of interest statement: The authors declare no conflicts of interest.
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