CASE STUDY: A Snapshot in Time of Fatty Acids Composition of Grass Herbage as Affected by Time of Day

INTRODUCTION

Increases in the amount of functional fatty acids in foods have positive effects on human health, reducing the severity several chronic diseases, including cancers, atherosclerosis, obesity, and diabetes (McGuire and McGuire, 2000). Manipulation of functional PUFA in ruminant products depends on rumen biohydrogenation rates and on the net amount and composition of fatty acid ingested (Bauman et al., 2003). Despite the relatively low concentration of fatty acids in forage plants, the ability to supply de novo C18:3n-3 (the building block of PUFA; Givens et al., 2001; Dewhurst et al., 2003) makes grazed herbage an excellent source of PUFA precursors for nonsupplemented grazing ruminants. The concentration of PUFA in herbage changes with season (Dewhurst et al., 2001), developmental stage of the plant (Hawke 1963, 1973; Gray et al., 1967; Dewhurst et al, 2001, 2003), and cutting interval (Dewhurst et al., 2001, 2003; Elgersma et al., 2003, 2004). At the plant cellular level, fatty acids are produced in cell plastids (Ohlrogge and Jaworski, 1997), with the greatest concentrations occurring within the thylakoid membranes of the chloroplast (Harwood, 1980). Thus, fatty acids are related to plant photosynthetic activity (Erwin and Bloch, 1963; O'Brien and Benson 1964; Belury, 2002). Under environmentally controlled conditions, plant physiology research has shown that fatty acid synthesis varied diurnally in small discs of spinach (Spinacia oleracea) leaves and recently emerged maize (Zea mays) leaf blades (Browse et al., 1981). Several field studies have shown significant increases in photosynthate concentrations of herbage during the course of a day (Mayland et al., 2003, 2005; Griggs et al., 2005); however, there is still a lack of information regarding diurnal fluctuation of fatty acids in grass herbage. The knowledge of these potential changes would help improve grazing management aimed at increasing functional fatty acids in ruminant products. From these works emerges the hypothesis that the concentration of fatty acids in herbage varies during the day. The objective of this experiment was to partially test this hypothesis by quantifying herbage fatty acids at different times of day in 2 grass species commonly found in temperate cool-season pastures of the northeastern United States.

MATERIALS AND METHODS

Site and Experimental Setup

The experiment was conducted at the USDA-ARS Pasture Systems and Watershed Management Research Unit, University Park, Pennsylvania. In January 2007, monoculture microswards (Orr et al., 2005) of meadow fescue (Festuca pratensis Hud.) and orchardgrass (Dactylic glomerata L.) were established in 44 plastic boxes (79 × 47 × 11.5 cm). All boxes had 6-mm drainage holes drilled in the bottom and spaced at 10 cm in 4 rows of 7 (28 total holes). Each box was filled with 40 to 45 kg of a potting medium (Scott's Sierra Horticultural Products Co., Marysville, OH). Seeding rate was 500 seeds/m^sup 2^, based on a germination rate previously tested. The micro-swards were established and grown in the greenhouse for 14 wk and then were placed outdoors in May 2007. Micro-swards were cut (6-cm stubble height) approximately every 21 d to maintain vegetative (3 fully expanded leaves) tillers. Microswards were watered regularly to maintain soil moisture at field capacity and fertilized after each 21-d cutting interval with N, P, and K at 1.79, 3.58, and 1.79 g/m^sup 2^, respectively.

Micro-sward Sampling, Chemical Composition Analyses, and Weather Condition Measurements

Sampling was conducted in 2 consecutive days (on July 2 and 3 for orchardgrass and on September 12 and 13 for meadow fescue) using one set of 11 micro-swards per day. Micro-swards were sampled 4 times/d: sunrise (0537 or 0650 h); morning (1037 or 1110 h); afternoon (1537 or 1530 h); and sunset (2037 or 1930 h) for orchardgrass and meadow fescue, respectively. Micro-swards were gridded and samples were taken from randomly selected grid points within each micro-sward. At each time of day, approximately 10 g (DM basis) of herbage was harvested from each micro-sward. Herbage was harvested at a 6-cm stubble height.

Samples were immediately frozen in liquid N for further freeze-drying (Ultra 35 Super ES, Virtis, Gardiner, NY) and chemical analysis. Dry samples were ground to pass a 1-mm screen and analyzed for NDF, ADF, CP (N × 6.25), total nonstructural carbohydrates (TNC), and PUFA. The NDF and ADF concentrations were determined using the batch procedures outlined by Ankom Technology Corp. (Ankom Fiber Analyzer, Ankom, Fairport, NY). Concentrations of N were determined by total Kjeldahl N (AOAC, 2000, official method 976.06; using 75-mL calibrated tubes and CuSO^sub 4^-K^sub 2^SO^sub 4^ catalyst) and analysis on Quickchem 8000 ion analyzer (Lachat Instruments, Milwaukee, WI). The TNC content was determined following the procedures described by Burns et al. (2006). Fatty acid methyl esters were prepared from the samples by the method outlined in Murrieta et al. (2003). Fatty acids were separated using a Varian CP 3800 gas Chromatograph (Varian Inc., Palo Alto, CA) equipped with an Omegawax 320 capillary column (30 m × 0.32 mm i.d., 0.25-µm film thickness, Supelco, Bellefonte, PA). Injector and detector temperatures were 275°C. The oven temperature was held at 175°C for 40 min and was then increased to 240°C in increments of 10°C/min. Helium was the carrier gas with a split ratio of 50:1 and 0.8 mL/min column flow. A reference standard for peak identification and the calibration standards for quantification of the fatty acids of interest were made from the following fatty acid methyl esters (all obtained from Sigma-Aldrich, St. Louis, MO): methyl palmitate (C16:0), methyl oleate (cis-9 C18:1), methyl linoleate (cis-9,12 C18:2), and methyl linolenate (cis-9,12,15 C18:3). Methyl undecanoate (C11:0, Nu-Check Prep, Elysian, MN) was added to each sample tube as an internal standard.

Photosynthetically active radiation (LI-COR, LI190SB Quantum Sensor, Logan, UT), air temperature, and relative humidity (HMP35C, Vaisala, Logan, UT) were measured every 5 min each day with an automated weather station.

Experimental Design, Treatments, and Statistical Analyses

The data from each grass species were analyzed as a completely randomized design with micro-sward as experimental unit. The 4 herbage sampling times (sunrise, morning, afternoon, and sunset) were considered treatments. Dependent variables were analyzed by ANOVA using PROC MIXED (SAS Inst. Inc., Cary, NC). The model included the effects of day, micro-sward, treatment, and the interaction day × treatment. The random effect was micro-sward within treatment, specified in the RANDOM statement. Least squares means were separated using the PDIFF function of SAS. A value of P < 0.05 was considered significant.

RESULTS AND DISCUSSION

There was no treatment × day interaction (P > 0.01) for any variable analyzed. Therefore, averages over both sampling days are presented and variables are discussed in terms of treatment.

The predominant fatty acids in grasses are myristic, palmitic, palmitoleic, stearic, oleic, linoleic, and a-linolenic acids (Givens et al., 2001; Clapham et al., 2005). We quantified the content of palmitic, oleic, linoleic, and a-linolenic acids, which make up about 90% of the total fatty acids of forage plants (Dewhurst et al., 2003; Clapham et al., 2005). Among these fatty acids, a-linolenic acid emerges as the most important because of its nutritional-functional value and relative concentration (Harwood, 1988; Bauman et al., 2003). Despite the differences in weather conditions (Figure 1) and species and season in which herbage was sampled, the total and profile of fatty acids analyzed were similar (Table 1). These results are supported by Clapham et al. (2005), who reported uniform fatty acid composition of herbage from traditional and novel grass species subjected to differential cut interval. The harvesting protocol in the present study followed a developmental stage criterion of cutting (3 fully expanded leaves blades) instead of a fixed interval of time. Thus, at harvesting time both species were sampled at the same developmental stage. Consequently, the similarity in total and profile of fatty acids between the species in this study suggests that developmental stage might be more relevant as a determining factor of total and profile of herbage fatty acids than species or even season.

The total of fatty acids was not affected by time of day for both grass species (P > 0.01). Time of day did not affect (P > 0.01) the concentration of particular fatty acids either, except for oleic acid (P < 0.01), in both grass species. Concentration of oleic acid increased (P < 0.01) from sunrise to sunset by 22 and 13% in orchardgrass and meadow fescue, respectively. This different magnitude of oleic acid concentration may be related to weather conditions at each sampling. The diurnal increase of oleic acids agrees with the results of Browse et al., (1981), who reported diurnal fluctuation of oleic acid for spinach (S. oleracea) and recently emerged maize (Z. mays) leaves. Oleic acid represents one of the most important nutraceutical fatty acids in plants, and has been related to reducing total cholesterol (Chen et al., 1995) and breast cancer risk in humans (Menendez et al., 2005). However, because of the highly reductive environment in the rumen as well as the low relative concentration and "monounsaturation" condition of oleic acid in grass herbage, oleic acid is easily biohydrogenated (saturated) in the rumen, losing its functional importance and negating any diurnal effect. The lack of a time of day effect (P > 0.01) on the concentration of linoleic and a-linolenic acids disagrees with studies showing that a-linolenic concentrations in different species re- spond to changes of light intensity in the environment. For example, Gray et al. (1967) found that barley (Secale cereale) leaf blades (7 d old) had 71.1 vs. 51.8 g/kg of total n-3 fatty acids at either a natural daylight or a dark environment, respectively. O'Brien and Benson (1964) further argued that in leaves of forage legumes like alfalfa (Medicago sativa) and red clover (Trifolium pratense), a-linolenic increases immediately after photosynthesis begins.

The lack of a time of day effect on the fatty acid composition of the herbage used in the present study may have been related to the herbage sampling protocol and leaf development. Herbage samples in the current study included portions of pseudo-stems, which might have exerted a dilution effect. The major concentration of PUFA in plants is found in leaf blades (Weenink, 1961; Sastry and Kates, 1963; O'Brien and Benson, 1964), and fatty acid metabolism and biosynthesis are greater in actively growing leaves (Hitchcock and James, 1964; Ohlrogge and Jaworski, 1997; Williams et al., 1998). In our study, most of the leaves of herbage sampled were completely expanded. Consequently, the majority of these leaves were not actively growing at high rates. Browse et al. (1981) showed a diurnal accumulation of fatty acids in maize leaves; however, these leaves were recently emerged and growing at fast rates. Therefore, if such a diurnal accumulation was occurring in young, recently emerging leaves of our study, it was diluted by the greater relative proportion of mature leaves.

Time of day affected (P < 0.01) DM, CP, TNC, ADF, and NDF concentrations in both orchardgrass (Table 2) and meadow fescue. Details of these variables for meadow fescue are presented elsewhere [P. Grego- rini, K. J. Soder, M. A. Sanderson, and G. R. Ziegler (Center for Food Manufacturing, The Pennsylvania State University), unpublished data]. Diurnal fluctuation of DM concen- tration of orchardgrass was not as marked as was expected; however, it followed a pattern similar to that reported in the literature for other grasses (Delagarde et al., 2000; Grego- rini et al., 2008). The main difference (P < 0.01) in DM concentration was found from sunrise to morning, which matched the rise of temperature and relative humidity at that time of day (Figure 1) as well as the increment of TNC. Although the DM concentra- tion of meadow fescue was compara- tively lower than for orchardgrass, it followed the same diurnal pattern, increasing from sunrise to sundown. The CP, ADF, and NDF concentra- tions of orchardgrass decreased from sunrise to sundown by 9.7, 4.2, and 10.3%, respectively; for meadow fescue, these decreases were 8.4, 10.3, and 4.7%, respectively. The TNC for both orchardgrass and meadow fescue increased by 41 and 48%, respectively, by sundown, although there was no difference between afternoon and sun- down for both species. This may be related to an increase in respiration and metabolic activity of the plants at sundown. Although there were clouds during all of d 2 (Figure 1) for both sampling periods, the general pattern of photosynthetic active radiation did not differ from d 1. Therefore, the lack of differences in TNC for orchardgrass and meadow fescue between afternoon and sundown can be explained by changes in photosynthetically active radiation and air temperature from 1500 h onward. The similarity in magnitude of changes in this study, independently of species and season, agrees with values presented by Delagarde et al. (2000) for perennial ryegrass (Lolium perenne L.) and by Gregorini et al. (2006) for Italian ryegrass (Lolium multiflorum Lam.). These authors reported that the seasonal effects did not interact with the variation in chemical composition of herbage throughout the daylight hours.

Despite the literature showing potential accumulations of fatty acids during the day in young leaves, our results suggest that ruminants grazing grass herbage are not totally exposed to diurnal fluctuation in total amount of fatty acids or in PUFA. Grazing ruminants encounter and consume an arrangement of leaves of different ages as well as some proportions of sheaths, stems, and even dead material. Once PUFA are ingested, biohydrogenation seems to be the major obstacle to increasing their flow to the intestines (Bauman et al., 2003). Increments in ruminai passage rates alter the rate and extent of biohydrogenation (Dewhurst et al., 2003). Consequently, grazing management stimulating faster ruminai passage rates may increase PUFA in animal products. Gregorini et al. (2008) reported faster liquid and solid ruminai turnover rates when beef heifers were stimulated to graze more during afternoon hours. Avondo et al. (2008) showed increments in a-linolenic acid content of milk for goats only grazing in the afternoon. Both authors suggest that the effect of time of day on chemical composition of herbage (DM, TNC, ADF, and NDF) was a key factor in determining their results. Diurnal increases of DM and TNC dilute NDF and ADF concentrations (Delagarde et al., 2000; Gregorini et al., 2006, 2008), which diminish herbage toughness from morning to afternoon (P. Gregorini et al., unpublished data) and makes herbage consumed in the afternoon easier to chew (faster particle breakdown) and faster in passing through the rumen [P. Gregorini, K. J. Soder, M. A. Sanderson, and G. R. Ziegler (Center for Food Manufacturing, The Pennsylvania State University), unpublished data] . Time of day may not affect the concentrations of PUFA in herbage; however, differences in the plant chemical composition contents during the day might enhance PUFA supply from the rumen when herbage is strategically grazed. This premise still warrants further and detailed research.

IMPLICATIONS

Concentrations of oleic acid in grass herbage increased during the day; however, PUFA and total fatty acid concentrations remain stable throughout the day. Increments in TNC from sunrise to sundown diluted NDF and ADF concentration of herbage during the afternoon and evening. Such a dilution diminishes chewing effort and increase the rate of forage particle size reduction, favoring faster ruminai passage rates. From these phenomena, interesting speculations emerge regarding strategic grazing managements to reduce the rate and extent of ruminai biohydrogenation, thereby increasing beneficial fatty acid contents in products from ruminants fed pasture. Regardless of the stable concentrations of herbage PUFA, diurnal differences in the plant chemical composition might enhance PUFA supply from the rumen when herbage is strategically grazed during the afternoon.

ACKNOWLEDGMENTS

The authors thank Melissa Rubano and Robert Stout, USDA-ARS, and Chuck Murrieta, Dept. of Animal Science, Univ. of Wyoming, for the laboratory expertise and time contributed to conducting this study.

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