Original Research
Effects of Sampling Time, Cultivar, and Methodology on Water- and Ethanol-Soluble Carbohydrate Profiles of Three Cool-Season Grasses in Central Kentucky

https://doi.org/10.1016/j.jevs.2017.11.011Get rights and content

Highlights

  • Monosaccharides and disaccharides constituted the majority of total water-soluble carbohydrate (WSC) or ethanol-soluble carbohydrate (ESC) in three cultivars of different cool-season grasses collected in mid-springtime from central Kentucky plots.

  • Relative amounts of glucose, fructose, and sucrose detected depended on extraction parameters.

  • Most diurnal variation in the averaged WSC and ESC concentrations was due to sucrose, not to fructan.

  • Fructan concentration and chain length varied with cultivar.

  • In one cultivar, short-chain fructans were abundant in both WSC and ESC extracts. Therefore, using the difference between WSC and ESC concentrations to calculate fructan is not a correct practice.

Abstract

Cool-season grasses (CSGs) accumulate variable amounts of water-soluble carbohydrates (WSCs, monosaccharides and disaccharides, and fructans), depending on climate, time of day and year, and genotype. Fructan concentrations in CSG are sometimes estimated as the difference between concentrations of WSC and ethanol-soluble carbohydrates (ESCs, monosaccharides and disaccharides, and variable amounts of fructan). Characterizing both WSC and ESC may improve understanding of soluble carbohydrate profiles in pastures and inform grazing management decisions, particularly for horses at risk for laminitis. Three CSG cultivars from Kentucky pastures were collected in the morning and afternoon on two springtime dates. Water-soluble carbohydrates and ESC were extracted with water or 80% ethanol, respectively, and analyzed by high-performance liquid chromatography (HPLC) and a colorimetric assay. Method of analysis (HPLC or colorimetry), and extraction solvent, affected the composition of WSC or ESC determined in CSG samples, demonstrating the need to consider methodology when interpreting results. Total soluble carbohydrate (mean of WSC and ESC) concentrations across cultivars and harvests were generally higher in the afternoon than in the morning, based on both HPLC (P = .0023) and colorimetric (P < .0001) analysis. The diurnal variation was mainly due to sucrose (P < .0001). Among cultivars, monosaccharides and disaccharides constituted the majority of the averaged WSC and ESC concentrations. The proportions of water- and ethanol-soluble fructan (P = .0101), and fructan chain lengths detected in water and ethanol extracts (P < .0001), differed among CSG cultivars. In choosing CSG cultivars for pastures, both soluble carbohydrate composition and concentrations should be considered.

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.

References (48)

  • R. Waite et al.

    The water-soluble carbohydrates of grasses. I. Changes occurring during the normal life-cycle

    J Sci Food Agric

    (1953)
  • N.J. Chatterton et al.

    Oligosaccharides in foliage of Agropyron, Bromus, Dactylis, Festuca, Lolium and Phleum

    New Phytol

    (1990)
  • C.J. Nelson et al.

    Fructans

    Physiol Plantarum

    (1984)
  • W. Van den Ende et al.

    The metabolism of fructans in roots of Cichorium intybus during growth, storage and forcing

    New Phytol

    (1996)
  • N.J. Chatterton et al.

    Fructan oligomers in Poa ampla

    New Phytol

    (1997)
  • R.D. Grotelueschen et al.

    Carbohydrates in grasses. III. Estimations of the degree of polymerization of the fructosans in the stem bases of timothy and bromegrass near seed maturity

    Crop Sci

    (1968)
  • K.E. Borer et al.

    Effect of feeding glucose, fructose, and inulin on blood glucose and insulin concentrations in normal ponies and those predisposed to laminitis

    J Anim Sci

    (2012)
  • A.W. Van Eps et al.

    Equine laminitis induced with oligofructose

    Equine Vet J

    (2006)
  • T.A. Burns et al.

    Distribution of insulin receptor and insulin-like growth factor-1 receptor in the digital laminae of mixed-breed ponies: an immunohistochemical study

    Equine Vet J

    (2013)
  • J.C. Ince et al.

    In vitro degradation of grass fructan by equid gastrointestinal digesta

    Grass Forage Sci

    (2013)
  • T.A. Ciavarella et al.

    Diurnal changes in the concentration of water-soluble carbohydrates in Phalaris aquatica L. pasture in spring, and the effect of short-term shading

    Aust J Agric Res

    (2000)
  • V.L. Lechtenburg et al.

    Diurnal variation in nonstructural carbohydrates of Festuca arundinacea (Schreb.) with and without N fertilizer

    Agron J

    (1972)
  • A.C. Longland et al.

    Comparison of a colorimetric and a high-performance liquid chromatography method for the determination of fructan in pasture grasses for horses

    J Sci Food Agric

    (2012)
  • N. Pavis et al.

    Structure of fructans in roots and leaf tissues of Lolium perenne

    New Phytol

    (2001)
  • Cited by (13)

    • Water- and Ethanol-Soluble Carbohydrates of Temperate Grass Pastures: a Review of Factors Affecting Concentration and Composition

      2022, Journal of Equine Veterinary Science
      Citation Excerpt :

      The latter are fructose polymers, which usually have a glucose moiety [2], although fructose-only polymers have been found in selected species of the Asteraceae [3]. Ethanol-soluble carbohydrates (ESCs), when extracted with 80% ethanol, include glucose, fructose, sucrose, and short-chain fructans, which can have a degree of polymerization (DP) up to about 20 fructose units in length [4–6]. In grasses, nonstructural carbohydrates (WSCs plus starch) consist primarily of sucrose and fructan [7,8].

    • Diurnal Variation in Forage Nutrient Composition of Mixed Cool-Season Grass, Crabgrass, and Bermudagrass Pastures

      2022, Journal of Equine Veterinary Science
      Citation Excerpt :

      Conversely, starch was greatest for CRB, with marked increases in the late afternoon and evening. While diurnal variation in sugars and fructans has received much attention in the literature [2,24,26], variation in starch has also been found in both CSG [25] and WSG [4]. However, the peak starch in CRB (4%) was minimal in comparison to the peak WSC in CSG (19%), leading to greater overall NSC in CSG regardless of time of day.

    • Ethanol-Soluble Carbohydrates of Cool-Season Grasses: Prediction of Concentration by Near-Infrared Reflectance Spectroscopy (NIRS) and Evaluation of Effects of Cultivar and Management

      2021, Journal of Equine Veterinary Science
      Citation Excerpt :

      Gravity-filtered extracts were diluted 10-fold in water (final ethanol concentration of 8%) and frozen or refrigerated until analysis. Phenol-sulfuric acid assays (hazardous due to corrosive reagent) to quantify ESCs were performed as described previously [1]. Results were determined on the basis of freeze-dried weight, which, based on a subset of 90 samples analyzed in a related study [12], was 92 to 95% dry matter.

    • Effects of Harvest Date, Sampling Time, and Cultivar on Total Phenolic Concentrations, Water-Soluble Carbohydrate Concentrations, and Phenolic Profiles of Selected Cool-Season Grasses in Central Kentucky

      2019, Journal of Equine Veterinary Science
      Citation Excerpt :

      Absorbance at 490 nm was measured with a spectrophotometer (model DU-800; Beckman–Coulter, Brea, CA, USA). WSC concentrations of Ginger BG, Cajun TF, and Calibra PRG had been measured before this study and reported previously [21]. The method of Mattila and Kumpulainen [23] was used for extracting soluble phenolic compounds, with the following changes: 50 mg tissue was extracted by ultrasonication in a 15 mL conical polypropylene centrifuge tube, and extraction volume was decreased to 3.5 mL 85% methanol and 10% aqueous acetic acid (85:15, vol/vol).

    View all citing articles on Scopus

    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.

    Disclaimer: Mention of trade names or commercial products in the article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA.

    View full text