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CROP QUALITY & UTILIZATION

Protein Degradation and Fermentation Characteristics of Red Clover and Alfalfa Silage Harvested with Varying Levels of Total Nonstructural Carbohydrates

^a Plant Science Dep., South Dakota State Univ. Box 2207A, Brookings, SD 57007 USA
^b Dep. of Agronomy, 1575 Linden Dr., Univ. of Wisconsin-Madison, Madison, WI 53706 USA
^c US Dairy Forage Research Center, 1925 Linden Dr., Madison, WI 53706 USA

owensv{at}ces.sdstate.edu

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion Conclusions REFERENCES

 
Extensive degradation of protein during fermentation of high-protein crops reduces efficiency of dietary N utilization in ruminants. Evidence suggests that enhanced levels of fermentable carbohydrates can reduce proteolysis. Our objective was to evaluate whether delaying daily cutting time, to allow total nonstructural carbohydrates (TNC) to accumulate, would inhibit protein degradation by way of greater acid production in the silo. Red clover (Trifolium pratense L.) and alfalfa (Medicago sativa L.) were harvested at 0600, 1000, 1400, and 1800 h in 1993, 1994, and 1995 and wilted to a dry matter (DM) content of 350 g kg^-1 before ensiling. The level of TNC in fresh forage of both species increased throughout the day. Starch accounted for most of the daily change in TNC in fresh alfalfa, whereas in red clover, quantitative increases in sugar and starch impacted TNC similarly. Level of TNC at initiation of ensiling did not consistently affect protein degradation during fermentation as confirmed by generally insignificant correlation coefficients. The extent of proteolysis in the silo was consistently greater in alfalfa than red clover. Silage pH typically decreased and starch increased as cutting time was delayed from 0600 to 1800 h. While the extent of proteolysis was largely unaffected by inherent increases in TNC, lower silage pH and higher starch concentrations indicate that silage from the afternoon cuttings may be better preserved and higher in quality.

Abbreviations: NPN, nonprotein nitrogen • PN, protein nitrogen • TN, total nitrogen • TNC, total nonstructural carbohydrates

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion Conclusions REFERENCES

 
A KEY OBJECTIVE

of forage preservation as hay or silage is to minimize quality and dry matter (DM) losses. A major disadvantage to preserving forage as hay is the risk of exposure to adverse weather conditions since several days are usually required for drying. Much of the forage grown in humid regions of the USA, where the climate is not conducive to hay making, is preserved as silage. Harvest and quality losses associated with inclement weather are usually lower in silage because drying time is typically reduced by at least 50% compared with making hay. Quality reductions due to detrimental microbial activity are minimized in silage by excluding oxygen from the silo and rapidly attaining a pH of 3.8 to 5.0 (Pitt, 1990).

Preservation of high quality legumes as silage is limited by extensive protein degradation that occurs in many forage species during fermentation (McKersie, 1985; Charmley and Thomas, 1987; Albrecht and Muck, 1991). Of the total N (TN) in fresh forages, 75 to 90% is protein N (PN), and the remainder is nonprotein N (NPN) (Oshima and McDonald, 1978). After ensiling, NPN may account for as much as 80% of TN (Papadopoulos and McKersie, 1983; Albrecht and Muck, 1991). Waldo (1985) concluded that extensive protein degradation in the silo may result in lower dry matter intake by ruminants and reduce the efficiency with which N is utilized. Inefficient forage N utilization by ruminants also poses potential environmental problems since excess N is excreted in urine (Tamminga, 1992).

Protein degradation in the silo is influenced by several factors including forage species (Papadopoulos and McKersie, 1983; Albrecht and Muck, 1991), pH (Brady, 1961; Finley et al., 1980; Scalet et al., 1984; McKersie, 1985), DM content of the crop at ensiling (Carpintero et al., 1979; Muck, 1987), and temperature (Brady, 1961; Muck and Dickerson, 1988). Albrecht and Muck (1991) evaluated several legumes and detected a significant negative relationship between tannin concentration and NPN in the silo. Red clover and cicer milkvetch (Astragalus cicer L.) consistently underwent less protein degradation than alfalfa during fermentation although neither of these species contains measurable levels of tannins. Other researchers have also noted that the increase in NPN during ensiling is lower in red clover than alfalfa (Papadopoulos and McKersie, 1983; McKersie, 1985; Jones et al., 1995c).

The pH of well fermented silage should rapidly fall to 5 or less to maintain forage quality and limit the extent of protein degradation. While not completely prevented, the activity of plant proteinases decreases linearly between pH 6 and 4 (McKersie, 1985; Jones et al., 1995a). Management practices used to promote a rapid pH decline during ensiling include rapid filling, good packing, and good sealing (Muck, 1988). In addition, it is critical that an anaerobic environment be achieved as quickly as possible and that sufficient substrate be available for fermentation by lactic acid bacteria. The amount of substrate necessary for successful fermentation depends primarily on buffering capacity (defined as the amount of acid needed to reduce forage pH from 6 to 4 per unit DM) and DM content of the crop at ensiling (Muck, 1988). The buffering capacity of legumes is typically higher than that of grasses (Pitt, 1990), and among legumes, alfalfa is usually more highly buffered (McDonald et al., 1991, p. 31) and has less fermentable substrate (Raguse and Smith, 1966) than red clover. Consequently, pH values tend to be lower in red clover than alfalfa silage.

The amount of fermentable substrate affects the extent of pH decline, which in turn may affect proteolysis in the silo. Naturally available substrate for fermentation varies diurnally. Total nonstructural carbohydrates (TNC), including sugar and starch, are lowest early in the morning and increase through mid to late afternoon (Holt and Hilst, 1969; Lechtenberg et al., 1971). We hypothesized that forage legumes harvested later in the day would contain greater levels of available substrate for fermentation, thus reducing the extent of protein degradation in the silo. Therefore, our objective was to determine whether delaying cutting time during the day, to enhance TNC accumulation, would inhibit protein degradation as a result of greater acid production in the silo.

	Materials and methods

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion Conclusions REFERENCES

 
Plant material used in this research was harvested in 1993, 1994, and 1995 at the University of Wisconsin Arlington Agricultural Research Station, Arlington, WI (43°18'N, 89°21'W). `Marathon' red clover and `Dart' alfalfa were established in the spring of 1993 on a well drained to moderately well drained Plano silt-loam soil (fine-silty, mixed, mesic, Typic Argiudoll). Soil nutrients and pH were maintained at recommended levels for alfalfa throughout the experiment. Eptam (S-ethyl dipropylcarbamothioate) was applied pre-plant incorporated to control weeds during establishment, and malathion (O,O-dimethyldithiophosphate of diethyl mercaptosuccinate) was used as needed to control alfalfa weevil (Hypera postica Gyll.) and potato leafhopper (Empoasca fabae Harris).

Harvesting and Sample Preparation
Wilted silage was made from forage harvested on 24 Aug. 1993 (growth cycle succeeding new seeding growth), 27 May 1994 (first growth cycle), and 26 June 1995 (second growth cycle). Plants were clipped to a 5-cm stubble height at 0600, 1000, 1400, and 1800 h in 1993 and 1994. In 1995, forage was harvested at 0600, 1000, and 1400 h on 26 June, and the 1800-h cutting was taken the next day (27 June) because of cloudy and rainy weather the afternoon of 26 June. Fresh forage from each species and cutting time was bulked, conditioned by crimping the stem at approximately 2.5-cm intervals, and thoroughly mixed for sampling. Four samples in 1993 and 1994 and three samples in 1995 (each representing a replicate for statistical analyses) were randomly taken from the bulk forage of each species at each cutting time. Each of the fresh forage samples was further divided into two portions: (i) approximately 900 g (wet weight) was immediately placed on wire screens elevated approximately 4 cm off the ground and allowed to wilt in the field to a DM content of 350 g kg^-1; and (ii) 500 g (wet weight) was used for analysis of fresh herbage characteristics. A microwave oven was used to determine rapidly the DM content of 100-g fresh forage in order to calculate the amount of water loss required to reach the targeted DM level.

The 500-g fresh plant portions were immediately placed on ice and taken into the lab where they were chopped into 1-cm pieces with a paper cutter and then thoroughly mixed. Approximately 100-g fresh forage from each sample was placed in a forced air oven for 48 h at 60°C for DM determination. Two 50-g subsamples were placed in separate 530-mL sterile sampling bags and immediately placed on dry ice. One of these subsamples was later used for determination of TN and NPN and the other was used for sugar (primarily glucose, fructose, and sucrose [Lechtenberg et al., 1971]) and starch analysis. The subsample used to determine N fractions was kept at -70°C, and the subsample used for determination of sugar and starch was lyophilized and stored at -20°C until analyses could be completed. Wilted forage samples were taken into the lab, chopped into 1-cm pieces, and thoroughly mixed. Two 50-g subsamples (one for TN and NPN and one for sugar and starch) were treated in the same manner as fresh forage. A separate 100-g subsample was dried in a forced-air oven for 48 h at 60°C for DM determination.

Ensiling
Duplicate 50-g subsamples of wilted forage were ensiled using methods described previously (Owens et al., 1999). Briefly, forage was ensiled in 100-mL polypropylene centrifuge tubes after inoculating with appropriate lactic acid bacteria. Silos, with gas traps attached, were placed in a 30°C water bath for 35 d and then stored at -20°C until further chemical analyses were performed.

Characterization of Fresh Forage, Wilted Forage, and Wilted Silage
Ten grams of the frozen fresh forage, wilted forage, and wilted silage were diluted to 100 g with distilled water and macerated for 30 s at high speed in a Waring blender. The pH of the homogenate was measured immediately after blending, then the homogenate was prepared for analysis of NPN and fermentation products (Owens et al., 1999). Two 20-mL aliquots were dispensed into separate 50-mL polypropylene centrifuge tubes. Five milliliters of 25% (w/v) trichloroacetic acid (TCA) was added to one of the tubes to precipitate protein from the solution. Tubes with and without TCA were centrifuged and the supernatant decanted into 20-mL scintillation vials and stored at -20°C. The solution to which TCA had been added (TCA extract) was evaluated for NPN concentration using the micro-Kjeldahl procedure of Bremner and Breitenbeck (1983). The solution to which TCA had not been added (water extract) was used for determination of fermentation products by high pressure liquid chromatography according to the method of Muck and Dickerson (1988).

The remaining unmacerated material was weighed and dried in a forced-air oven at 60°C for DM determinations in 1993. In 1994 and 1995, unmacerated fresh forage, wilted forage, and wilted silage were lyophilized for DM determination due to concerns with the loss or transformation of carbohydrate or N components during drying at 60°C (Raguse and Smith, 1965). We suspected that some NH₃ was lost from several of the silage samples during drying at 60°C in 1993. To correct the data from these samples, values for TN used in statistical analyses were calculated by combining the measurements for TN (measured from oven-dried silage) and NH₃–N (determined from the aqueous extract). In 1994 and 1995, this problem was avoided by lyophilizing the remainder of the frozen material. Oven-dried samples from 1993 and lyophilized samples from 1994 and 1995 were ground to pass a 0.85-mm (20 mesh) screen with an intermediate Thomas-Wiley mill. Total N was measured on these samples by the micro-Kjeldahl method of Bremner and Breitenbeck (1983). The oven-dried or lyophilized silage samples were also used for sugar and starch analysis. Sugar and starch concentrations in fresh forage were determined from the subsamples that had been lyophilized and stored at -20°C.

TNC Analysis
Sugar Extraction and Measurement
. Sugar and starch were determined by the methods described by Rong et al. (1996). Approximately 45-mg lyophilized and ground tissue was rinsed four times with 80% (v/v) ethanol (1.0 mL ethanol per rinse), centrifuged, and the supernatant decanted into a graduated test tube. Anthrone reagent was added to an aliquot containing up to 200-µg free sugar, boiled for 8 min, and then cooled to room temperature in a cold water bath. Absorbance at 625 nm was determined with a Shimadzu UV-1201 spectrophotometer attached to a ASC-5 auto sample changer (Shimadzu Corporation, Kyoto, Japan). Total sugars were expressed as glucose equivalents based on standards containing 0, 5, 10, 50, 100, 150, and 200 µg glucose mL^-1 80% ethanol.

Starch Hydrolysis and Measurement
The tube containing the ethanol-insoluble pellet was placed in a 55°C oven for 24 h to evaporate any residual ethanol before starch hydrolysis. Distilled deionized water (0.5 mL) was added to each tube and starch gelatinized by boiling samples for 10 min. After cooling to room temperature, 1 U amyloglucosidase (Sigma Chemical product A3514) and 40 U -amylase (Sigma Chemical product A2643) in 200 mM acetate buffer (pH 5.0) were added to each tube and incubated at 55°C for 24 h. At the conclusion of the incubation period the samples were centrifuged at 16000 x g for 10 min. Glucose released from starch hydrolysis was determined with glucose Trinder reagent (Sigma Chemical Diagnostic kit No. 315). Absorbance was read at 505 nm and the results compared to glucose standards containing 0, 5, 10, 20, 40, 60, and 80 µg glucose mL^-1 distilled deionized water. Starch was calculated by multiplying the glucose concentration by 0.9.

Statistical Analysis
When duplicate samples were evaluated, the average of the two values was used for statistical analysis. The experiment was conducted in a completely randomized design with four replicates in 1993 and 1994 and three replicates in 1995. Numerous year x treatment interactions and other higher order interactions were detected when data were analyzed across years. Some of these interactions resulted in changes in order of treatments; therefore, data were analyzed by individual years. Analysis of variance was used to test statistical significance of species, cutting time of day (CT), and species x CT interaction using the general linear model (GLM) of SAS (SAS Institute, Cary, NC). Species and cutting time were treated as fixed effects in the analysis of variance model. Means were separated by Fisher's protected least significant difference when F-tests were determined to be significant. The CORR procedure of SAS (SAS Institute, Cary, NC) was used to generate Pearson correlation coefficients to evaluate the relationship of sugar and TNC concentrations of wilted forage with silage NPN levels and silage pH values, and silage pH values with silage NPN concentrations.

	Results and discussion

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion Conclusions REFERENCES

 
General Characteristics of Fresh Forage, Wilted Forage, and Wilted Silage
Forage was cut when the stage of maturity was typical of what would be harvested and preserved as silage in the North Central USA. Red clover was harvested at about 33% bloom stage in 1993, at early bud stage in 1994, and at 50% bloom stage in 1995. Alfalfa was harvested at early-bud stage in 1993, at late-bud stage in 1994, and at 10% bloom stage in 1995. Total N for fresh red clover ranged from 27 to 35 g kg^-1 DM and for alfalfa ranged from 29 to 49 g kg^-1 DM, and was lowest in 1995 because both species were harvested at more advanced stages of maturity (Tables 1 to 3). The concentration of TN in both species tended to decrease slightly during the day, in agreement with findings by Youngberg et al. (1972) with alfalfa. The decrease in TN is likely attributed to dilution of N compounds during daytime fixation of carbon into nonstructural carbohydrates. Loss of TNC during wilting caused the level of TN in wilted forage to be more similar across cutting times within each harvest, although a trend of decreasing TN with later cutting times was still present (Tables 1 to 3) . Dry matter losses associated with respiration and fermentation resulted in slightly higher levels of TN in silage. The observed changes are comparable to those reported by Albrecht and Muck (1991) for unwilted red clover and alfalfa silage.

Nonprotein N in Fresh Forage, Wilted Forage, and Wilted Silage
The NPN (on a TN basis) concentration of fresh red clover ranged from 110 to 194 g kg^-1 TN and fresh alfalfa 130 to 171 g kg^-1 TN. The level of NPN in fresh forage was not consistently affected by species or cutting time. In 1993, NPN levels were similar between species (Table 1), in 1994, red clover was higher in NPN than alfalfa (Table 2), and in 1995, alfalfa contained more NPN than red clover (Table 3).

Protein hydrolysis by plant enzymes (Kemble, 1956; Oshima and McDonald, 1978) generally resulted in greater levels of NPN in wilted than in fresh forage (Tables 1 to 3). The increase in NPN during wilting was usually greater in alfalfa than in red clover, indicating that protein in red clover is protected from degradation soon after harvest. These results concur with Jones et al. (1995b) who found that red clover extracts browned (an indication of the reaction believed to be responsible for the inhibition of proteolysis) very quickly.

Level of NPN in wilted material was affected by cutting time in 1994 (Table 2), and there was a species x cutting time interaction for NPN in wilted forage in 1993 (Table 1). This interaction was the result of the low level of NPN found in wilted alfalfa from the 1800-h cutting time compared with all other alfalfa cutting times. In contrast, NPN levels in wilted red clover were similar across cutting times in 1993. In 1994, wilted alfalfa cut at 0600 and 1800 h and red clover cut at 1800 h contained less NPN than the other cutting times (Table 2). It is likely that differences in wilting time were the cause of variation in NPN in these samples since they reached the targeted DM content 3 to 12.5 h sooner than the other forage samples (Table 4). In 1995, fresh herbage from all cutting times required similar wilting periods, resulting in comparable NPN levels in wilted forage (Table 3).

Averaged over years and time of day at harvest, proteolysis in the silo caused NPN levels to increase by 136% in red clover and 232% in alfalfa. Most of the protein is degraded in 1 to 2 d (Oshima and McDonald, 1978), and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the most abundant protein in the plant, is particularly susceptible to degradation (Fairbairn et al., 1988). The lower level of NPN in red clover compared with alfalfa silage may be a direct or indirect result of the action of polyphenol oxidase on plant proteinases in red clover (Jones et al., 1995b).

Increasing the level of TNC at ensiling by harvesting later in the day did not significantly decrease final silage NPN concentrations in either species. In 1994, there was a significant effect of cutting time, but the concentration of NPN was actually higher in silage from the late afternoon harvest. Furthermore, there was no difference in NPN of silage harvested at 0600, 1000, and 1400 h (Table 2). The absence of a strong correlation of silage NPN with wilted sugar and TNC levels provides further evidence that protein degradation in wilted silage is not greatly affected by cutting time (Table 5) . McKersie (1985) stated that the amount of protein hydrolyzed during ensiling is largely dependent on the rate of pH decline and the "proteolytic potential," i.e., the total proteinase activity and the availability and susceptibility of protein to degradation. We speculated that a higher rate of pH decline would have occurred in forage harvested later in the day (by increasing the level of TNC), thus inhibiting protein degradation in the silo. Since delaying cutting time did not result in large increases in sugar, we did not eliminate the possibility that addition of a rapidly fermentable substrate could further increase the rate of pH decline and reduce protein hydrolysis during ensiling. McKersie (1985) also proposed that proteolytic potential might be different between years, cuts, harvest dates, and cultivars. Our data support this hypothesis since substantial variability for NPN exists over the three harvest years, which included different growth cycles, harvest dates, and species.

Year-to-year variation in sugar concentration was greater than cutting time differences, although sugar levels tended to increase throughout the day of each harvest. Starch had a greater effect than sugar on daytime changes in TNC in fresh alfalfa, whereas quantitative increases in sugar and starch concentrations affected TNC similarly in fresh red clover (Tables 1 to 3). These results for alfalfa concur with those of Lechtenberg et al. (1971) who reported that starch was responsible for 65% of the daily increase in TNC in first-growth alfalfa. It is apparent from these data and those of Lechtenberg et al. (1971) that excess photosynthate production late in the day results in starch synthesis and storage in leaves.

Wilted red clover contained 2 to 32% and wilted alfalfa 16 to 43% less TNC than fresh forage. As a result of these changes, the effect of cutting time on TNC concentration was generally less pronounced in wilted forage (Tables 1 to 3). Close examination of the data in Tables 1 to 3 reveals that a greater proportion of starch than sugar was lost during wilting. In fact, the concentration of sugar in wilted forage was comparable to that found in fresh herbage. Loss of original sugar from fresh forage may have been masked by the release of glucose from starch hydrolysis. In 1993 and 1994 starch decreased by 1.7 to 15.0 g kg^-1 DM in red clover and 1.2 to 34.5 g kg^-1 DM in alfalfa during wilting. Nonetheless, wilted forage from the afternoon cuttings continued to maintain a higher concentration of starch than wilted herbage from the morning harvests. In 1995, the starch concentration of wilted forage was similar across cutting times because of extensive starch hydrolysis during wilting of all samples.

Cutting time had a significant effect on TNC in wilted silage from each harvest. There was a species x cutting time interaction in 1993 and 1995, although from a practical perspective the low level of TNC remaining in silage made this interaction less consequential.

Sugar content of wilted silage, ranging from 3 to 34 g kg^-1 DM, was not consistently affected by cutting time (Tables 1 to 3). A species x cutting time interaction in 1993 (Table 1) and 1995 (Table 3) resulted in changes in silage sugar level rankings across cutting times. Sugars remaining in silage could be derived from the hydrolysis of structural carbohydrates (Dewar et al., 1963) or starch (Melvin, 1965; Muck, 1990).

pH and Fermentation Products of Wilted Silage
The pH of silage from red clover ranged from 4.07 to 4.43 and alfalfa from 4.19 to 5.05. A species x cutting time interaction in 1993 (Table 1) indicated a differential response to cutting time for each species. The trend of decreasing pH at each cutting time was similar (Table 1); however, a greater reduction in silage pH levels was observed in alfalfa compared with red clover between 1000 and 1400 h. Red clover consistently attained a lower pH than alfalfa during fermentation as a result of higher levels of rapidly fermentable sugars and a lower buffering capacity. Because alfalfa tends to be lower in sugar content at a given cutting time and has greater buffering capacity than red clover, delaying cutting time had a greater impact on pH decline in alfalfa in the silo, however.

The pH of wilted silage was usually higher in forage harvested in the morning than the afternoon, particularly in alfalfa. The relationship between sugar or TNC and silage pH was weak for both species, except for alfalfa in 1993 (P < 0.001) and for red clover in 1994 (P < 0.05) when significant negative relationships of sugar and TNC concentrations with silage pH were observed (Table 5). Cutting time resulted in greater differences in sugar concentration in wilted alfalfa in 1993 (Table 1) and in wilted red clover in 1994 (Table 2), and was the likely cause of higher correlation coefficients. In 1995, the pH values of alfalfa silage from the 0600 and 1800 h harvests were nearly equal because of similar levels of TNC in wilted alfalfa from these cuttings (Table 3). The pH of alfalfa silage was >4.5 in several instances (harvests taken at 0600 and 1000 h in 1993 and 0600 and 1800 h in 1995), yet a lactate-to-acetate ratio >2.0 and a favorable odor indicated that all silages were well preserved. Other workers have also reported well preserved silage with pH values >4.5 (Kung et al., 1984; Muck, 1987; Fairbairn et al., 1988).

Cutting time did not consistently affect lactate and acetate concentrations, although there was a trend of increasing lactate and decreasing acetate as time of harvest went from 0600 to 1800 h (Table 6) . This is reflected by silage pH values which were usually lower in silage made from forage harvested in the afternoon (Tables 1 to 3). A species x cutting time interaction for lactate (Table 6) was present in the first 2 yr because of greater increases in lactate production in alfalfa than red clover silage in forage harvested later in the day. In 1995, red clover silage contained more lactate than alfalfa silage; however, a cutting time effect was not detected.

	Conclusions

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion Conclusions REFERENCES

 
Delaying harvest from morning to the afternoon to produce a higher TNC forage provided no major or consistent benefit in terms of lactate production or in protein protection in the silo; however, silage pH tended to decrease and starch increase as cutting time was delayed from 0600 to 1800 h. The pH of alfalfa silage was consistently higher than red clover silage as a result of lower sugar levels for fermentation and a greater buffering capacity (McDonald et al., 1991, p. 31) in wilted alfalfa. Extensive protein degradation resulted in significantly higher NPN levels in alfalfa than in red clover regardless of cutting time. Forage harvested in the afternoon frequently required less time to dry than herbage from morning cuttings (Table 4). Consequently, while delaying harvest did not improve protein protection in the silo, forage harvested in the afternoon did not necessarily lead to delayed ensiling, and may be better preserved and of increased quality because of lower silage pH and higher starch concentrations.

	ACKNOWLEDGMENTS

 
The authors are grateful to Ed Bures for excellent technical assistance provided.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion Conclusions REFERENCES

 
This research was supported by Hatch Project 3270 and is a contribution of the Wisconsin Agric. Exp. Stn.

Received for publication December 8, 1998.

	REFERENCES

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion Conclusions REFERENCES

 

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