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Published in Agron. J. 97:399-407 (2005).
© American Society of Agronomy
677 S. Segoe Rd., Madison, WI 53711 USA

Waste Management

Long-Term Evaluation of Poultry Litter as a Source of Nitrogen for Cotton and Corn

Charles C. Mitchella,* and Shuxin Tub

a Dep. Agron. & Soils, Auburn Univ., Auburn, AL 36849
b Soil and Plant Nutrition Dep., Central China Agric. Univ., Wuhan 430070, China

* Corresponding author (mitchc1{at}auburn.edu)

Received for publication December 5, 2003.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Land application of poultry broiler litter (BL) in agricultural production is a widely used practice. However, cotton (Gossypium hirsutum L.) growers in poultry-producing regions of the southern USA have been reluctant to use BL as a crop nutrient source. Field experiments were conducted for 13 yr to study the effect of BL application to cotton and corn (Zea mays L.) under conventional and conservation tillage systems. Nitrogen rates from 0 to 269 kg ha–1 were applied annually at two locations to compare the effect of BL and ammonium nitrate (AN) as N sources. The relationship between the total N rates (N) applied and the relative N availability (y) based on the crop yield by application of BL and AN is described by linear equation: y = 71.58 + 0.15N (r = 0.66). In most years, there were no differences in relative yields from total N applied as BL or AN. The amplitude of yield increase based on N source varied with rainfall during the growing season. The residual effect of BL in the second year after application resulted in 30 to 50% of the cotton lint yield and 25 to 65% of the corn grain yield that resulted from a standard N fertilization rate. General observations suggest that N availability from BL is similar whether surface-applied as in conservation tillage systems or incorporated as in conventionally tilled systems.

Abbreviations: AN, ammonium nitrate • BL, poultry broiler litter • CP, Coastal Plain • PSNT, presidedress soil nitrate test • TV, Tennessee Valley


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
POULTRY BL IS A MIXTURE of manure with wood shavings, peanut (Arachis hypogaea L.) hulls, rice (Oryza sativa L.) hulls, or some other bedding materials. About 7.6 x 109 broilers and about 11.4 million tonnes of BL (1.5 kg litter/broiler) are produced every year in the USA (Georgia Agric. Stat. Serv., 1997; Moore, 1998). Alabama ranks third in broiler production among U.S. states and produces almost 12% of all broilers produced in the United States (Alabama Agric. Stat. Serv., 2001). Compared with other animal manures, BL contains relatively high concentrations of N, P, K, Ca, micronutrients, and organic matter. Although excessive land application of BL has resulted in water quality problems in some areas, BL remains an important source of plant nutrients for crop and forage production in poultry-producing regions (Mitchell and Mask, 1992; Mitchell and Donald, 1999; Huang and Lu, 2000; Stevenson et al., 1990).

Because BL use as a source of nutrients has been so widely accepted, much of the basis for its use has come from decades of on-farm tests and demonstrations by county extension agents and others and has never been formally published in scientific journals. Most BL in the southeastern USA is applied to pastures and hayfields. Although cotton is the number one row crop in Alabama, Georgia, Mississippi, and Arkansas in terms of acreage and value, very little BL is used as a source of nutrient on this crop. Cotton producers in the southern USA have traditionally avoided the use of animal manure for several reasons:

  1. Because of excessive applications in the past, there is a perception among cotton producers that manure N sources would produce excessive vegetative growth and late maturity of cotton.
  2. There is a suspicion that all animal manures may introduce weed seed into prime cotton land.
  3. Most cotton land is often remote from the smaller farms where poultry are produced (e.g., the Mississippi Delta cotton farms vs. the Arkansas Ozark Mountain poultry farms).
  4. Availability of BL may not coincide with optimum time of fertilizing cotton at planting in the spring.
  5. There is a reluctance of cotton producers to change successful production practices.
  6. There is a lack of extensive published, applied research with manures on cotton.

Recent research with poultry BL focused on the environmental implications of excessive nutrients (Kingery et al., 1994; Ritter, 2000; Cabrera and Sims, 2000). In the 1980s and early 1990s, N monitoring techniques such as the chlorophyll meter and the presidedress soil nitrate test (PSNT) for corn offered new techniques to monitor N fertilization especially where organic sources of N were being used (Fox et al., 1989; Magdoff et al., 1984; Meisinger et al., 1992).

Because of an ongoing need to continually evaluate crop production with BL fertilization, evaluate new soil and crop monitoring techniques such as the PSNT, and document soil and environmental changes from long-term BL applications, BL experiments were begun in the early 1990s at outlying units of the Alabama Agricultural Experiment Station. These experiments addressed only the soil fertility issues listed above. The initial objectives of these studies were to (i) evaluate BL as a source of N for cotton, (ii) determine the availability of N in BL compared with AN fertilizer, (iii) determine if plant growth regulators would be needed to control excessive vegetative growth of cotton fertilized with BL, and (iv) demonstrate to area cotton producers the practicality of using BL as an alternative fertilizer for cotton. After 4 yr of conventional cotton production, an experiment in a central Alabama Coastal Plain (CP) soil was modified to (i) evaluate surface-applied BL as a source of N for conservation-tilled corn, (ii) evaluate the PSNT for corn on a CP soil in the southern USA, and (iii) determine the residual effects of BL on corn production and soil properties. Cotton production practices in the South changed rapidly during the decade of the 1990s. By 1998, many producers had switched to conservation tillage. The boll weevil had been effectively eliminated as a production problem in the southeastern CP, and genetically modified cotton varieties were available that reduced losses to boll worms and offered simplified weed control with glyphosate [N-(phosphonomethyl)glycine] herbicide (e.g., Roundup). With conservation tillage practices, surface BL application with no incorporation became necessary for producers. The CP experiment in central Alabama was modified again to (i) evaluate surface-applied BL as a source of N for reduced-tilled cotton and (ii) determine the residual effects of BL and fertilizer N on reduced-tilled cotton.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Broiler litter experiments were conducted for 3 yr at the Tennessee Valley (TV) Research and Extension Center near Huntsville, AL, and for 12 yr in the CP of Central Alabama at the E.V. Smith Research Center.

The experiment at the TV site began in 1990 with conventionally tilled cotton in a Decatur silty clay loam (clayey, kaolinitic, thermic, Rhodic Paleudults). Tillage included moldboard plowing in the fall followed by disking and cultivation before and after planting in the spring. The experiment used a randomized, complete block design with 11 treatments and 4 blocks (Table 1). The same experiment was begun in 1991 on a Norfolk fine sandy loam (fine-loamy, siliceous, thermic Typic Kandiudults) in the CP of central Alabama. Plot size at both sites was 7.6 m long and 7.3 m wide, which accommodated eight 0.91-m (36-inch) rows. The TV experiment was conducted for 3 yr and terminated. The CP experiment was continued for 12 yr with modifications in 1995 and again in 1998. Both experiments were planted with conventionally tilled cotton from 1990 through 1994 with three rates of N as AN (0, 67, and 134 kg N ha–1) and three rates of BL to supply total N rates of 134, 202, and 269 kg N ha–1. Broiler litter was not applied at the 67 kg N ha –1 rate because application at this low rate was not practical in 1990 with the spreader technology available at that time. The standard N recommendation for cotton in Alabama is 67 kg N ha–1 for the finer-textured soils of the TV and 134 kg N ha–1 for the sandier soils of the CP (Adams et al., 1994). Variable N treatments with and without Pix [mepiquat chloride (1,1-dimethylpiperidium chloride)] were used to control vegetative growth. Pix was applied at early bloom at a rate of 230 g ha–1. Ammonium nitrate was applied in two equal, split applications at planting and at early squaring. Broiler litter was applied by hand just before planting and incorporated into the soil with final seedbed preparation. The untreated control and the AN treatments received 30 kg P ha–1 yr–1 as concentrated superphosphate (46% P2O5) and 83 kg K ha–1 yr–1 as KCl (60% K2O) at planting.


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  		Table 1. Treatments used on cotton and corn at the Tennessee Valley (1990–1992) and Coastal Plain (1991–2002) sites.

 
In 1995, the CP experiment was converted from conventionally tilled cotton to conservation-tilled corn. Tillage practices involved in-row subsoiling to a depth of 35 cm and planting of corn into a herbicide-killed residue of winter rye (Secale cereale L.). Ammonium nitrate treatments were modified (Table 1). From 1995 through 2002, half of the BL treatments were not applied. These untreated, residual BL treatments alternated such that each year there were residual BL treatments and treatments receiving litter the current year.

The experiment at CP was planted with conservation-tilled cotton in 1998 after 3 yr of consecutive conservation-tilled corn. The same tillage practices used for the corn crop were also used for cotton. The fertility treatments remained the same from 1995 through 2002. All fertilizer and broiler litter were broadcast by hand on the surface and not incorporated when the experiment was in conservation tillage.

Analyses of BL applied during selected years are given in Table 2. Broiler litter used came from houses using peanut hull bedding with at least six flocks of birds produced on the same bedding. Broiler litter was dry-stacked before spreading. All crops were nonirrigated.


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  		Table 2. Moisture and nutrient concentrations of broiler litter on a dry matter basis for 11 of the 13 yr, 1990 through 2002.

 
Cotton leaf blade samples were collected from the uppermost mature leaves at first bloom during each year of the TV experiment and in 1991, 1992, and 1993 of the CP experiment. Corn leaf samples were taken at V8 growth stage by collecting the uppermost, fully expanded leaf from at least 20 plants in each plot. Plant samples were dried in a forced-air oven at 60°C, ground to pass a 0.5-mm screen, and analyzed for total N by combustion (Campbell, 1992) and for other nutrients using inductively coupled argon plasma emission spectroscopy on a dry-ashed sample (Donohue and Aho, 1992). Seed cotton was harvested by mechanically picking the center two rows in each plot after defoliation. Yield was adjusted for percentage lint each year by ginning a composite sample from each treatment. The two center rows of corn in each plot were mechanically harvested using a plot combine and the grain yield adjusted for 15.5% moisture.

Data were analyzed using ANOVA and GLM procedures (SAS Inst., 1987). To determine the effect of total N rate on yields, analysis of variance was performed for each experiment using mixed model analysis with years, N rate, and years x N rate as fixed effects and blocks within years as random effects. Because the year x N rate interaction was highly significant (P < 0.001), further analysis was based on least squares year x N rate means. Yield at a given rate of applied N, either AN or BL, was converted to a percentage yield relative to the no-N control treatment. This relative response was modeled by stepwise regression analysis with P < 0.15 as the condition for an additional term to be included in the model. Independent variables were N rate (linear and quadratic) and dummy variables representing year, source of N (AN or BL), or Pix application for cotton only from 1990 to 1994 and their interactions. Because yield responses were relative to the no-N control, the fitted models did not contain an intercept term. The final decision on which terms to retain in the model was based on the main objective, i.e., estimate the response to applied N as precisely as possible. A quadratic term or additional independent variables were added to the model if it resulted in a significant improvement in the coefficient of determination as suggested by Draper and Smith (1998). For the models chosen, Mallow's Cp statistic was close to the number of predictors in the model, indicating that a best subset of predictor variables had been chosen (Draper and Smith, 1998).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Leaf Nutrient Concentration
Application of BL increased leaf blade concentrations of N, P, K, Ca, Mg, Cu, and B of cotton in conventional till compared with the no-N check plot (Table 3). With increasing rates of BL, concentrations of N, P, K, Ca, Mg, Cu, and B increased proportionally. With the range of BL N application from 134 to 269 kg ha–1, the percentage increase of nutrient concentrations in leaves over years and locations was as follows, N = 6 to 39%, K = 4 to 72%, Ca = 1 to 24%, Mg = 3 to 38%, Cu = 9 to 143%, and B = 1 to 82%. Application of AN significantly increased the concentration only of N and sometimes Mg. When rates of total N as AN are compared with total N as BL, application of BL increased K and B concentrations significantly. A slight increase in leaf blade N in the no-N check treatment from 1991 to 1993 at the CP site was likely due to normal variation in atmospheric N fixation and soil mineralization of N due to year-to-year variation in weather. Soil nitrate N was monitored in May in 1995, 1996, and 1997 to a depth of 60 cm. In 1995 and 1996, soil nitrate N in the no-N treatment was less than 5 mg NO3–N kg–1. However, in 1997, it was 16 mg NO3–N kg–1 for no apparent reason. Like leaf blade N, soil nitrate N in the upper 15 cm increased slightly with increasing N rates (data not shown). Rainfall was highly variable, which is typical for Gulf CP sites. These soils have very low soil organic C (<5 g organic C kg–1). Jackson (1998) observed no measurable differences in surface nitrate levels over a 2-yr period in similar soils. Corn leaf analysis during 1995–1997 had similar trends as the cotton leaf analysis (data not shown).


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  		Table 3. Effect of a standard rate of ammonium nitrate (AN) and rates of broiler litter (BL) on nutrient content of cotton leaf blades at early bloom for selected years at the Tennessee Valley (TV) and Coastal Plain (CP) sites.

 
Plant Height
Application of both BL and AN resulted in no height differences among the treatments on cotton early in the season (21 June) (Fig. 1). However, differences in height were observed by 27 August. At the same rate of total N (134 kg ha–1), cotton plants with AN were taller than those with BL, and the growth regulator was effective at limiting the height regardless of source of N.



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  		Fig. 1. Effect of total N rates as broiler litter (BL) and ammonium nitrate (AN) and a growth regulator (Pix) on the height of cotton plants at Tennessee Valley Research and Extension Center in 1990. Error bar is standard error of the means.

 
Nitrogen Effect on Yield
Over the 13-yr period, BL and AN resulted in similar yield increases when applied based on total N in both conventional till and conservation till cropping systems involving cotton and corn (Fig. 2). As expected with any crop and especially nonirrigated crops in the southern USA, there were yield differences by year and significant N rate x year interactions. Generally, as rates of BL N increased from 134 to 269 kg ha–1, nonirrigated cotton yield in conventional tillage reached 860 to 1340 kg lint ha–1. Broiler litter increased lint yields from 16 to 116% over the no-N control. At the TV location, cotton yield response to added N was linear and consistent for all 3 yr, but the magnitude of the increase was much higher in 1992 (Fig. 2, Curves 1A and 1B; Table 4). The growth regulator was effective at increasing cotton yields only at the TV location at the highest rate of applied N as BL (data not shown). Linear yield increases to added N were also observed at the CP site under conventional tillage in 1991 through 1994, but each year resulted in a significantly different response (Fig. 2, Curves 2A, 2B, 3, and 4; Table 4). Total N in both BL and AN resulted in similar yield increases in 1991 and 1993, but AN resulted in larger increases relative to the no-N treatment in 1994. Conservation till cotton at the CP site in 1998 through 2002 averaged a maximum annual yield of 870 to 1150 kg lint ha–1 with an average percentage yield increase of 43 to 140% over the no-N control. Yield response was best described by quadratic models with 95% of maximum yield occurring at an N rate between 175 and 193 kg N ha–1 (Fig. 2, Curves 6 and 7; Table 4). This is much more N than is currently recommended (Adams et al., 1994). Nonirrigated corn grain yield reached 3.4 to 9.6 Mg ha–1 (53 to 153 bushels per acre) with an average percentage yield increase of 90 to 167% over the no-N control. There was no significant difference in N source during the 3 yr that corn grain was produced at the CP site. The yield increase for corn by application of BL was consistent during the 3-yr experiment in spite of a wide range in maximum yield. There was no year x N rate interaction with corn (Fig. 2, Curve 5; Table 4). The N rate for 95% of maximum yield was 222 kg N ha–1, which is much larger than is currently recommended for nonirrigated corn.



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  		Fig. 2. Effect of total N rate applied either as ammonium nitrate or broiler litter on crop yield increases above the no-N treatment in experiments at a Tennessee Valley and a Coastal Plain site from 1990 to 2002. Stepwise regression was performed on least squares year x treatment means to arrive at the best-fitted model based on the coefficient of determination. Because the response variable was yield increase above the zero N treatment, all regressions were modeled without an intercept term. Numbers to the right of regression lines refer to the corresponding equation given in Table 4.

 

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  		Table 4. Estimates for regression coefficients, standard errors, and coefficients of determination (R2) for crop yield increases above the no-N treatment in response to total N rate.{dagger}

 
Residual Effect of Broiler Litter on Yield
As expected, there were pronounced residual effects of BL on crop yield 1 yr after application (Fig. 3). However, the highest rate of BL the previous year produced only about half as much cotton and corn as the optimum N rate applied to the current crop. Treatments receiving BL the previous year resulted in corn grain yields ranging from 1.9 to 7.7 Mg ha–1 (31 to 122 bushels per acre) and cotton lint yields ranging from 590 to 810 kg ha–1 without applying any additional N. This is about 24 to 63% of corn yield and 30 to 48% of lint yield with current BL applications.



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  		Fig. 3. Residual effect of broiler litter (BL) at three rates on corn grain yield (1995–1997) and cotton lint yield (1998–2002) at three total N rates on a Coastal Plain soil. Residual effects were measured the year after application.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
Our analysis of BL used in these experiments over a 13-yr period suggests that the average fertilizer grade would be close to a 2.6–3.5–2.5 (N–P2O5–K2O) when expressed on a wet basis rather than a dry matter basis as in Table 2. Similar values were reported in other research (Gale and Gilmour, 1986; Bitzer and Sims, 1988; Stevenson et al., 1990; Marshall et al., 1998). Results from these tests indicate that most of the total N is available even when surface-applied to conservation-tilled cotton or corn under nonirrigated conditions typical of the southeastern USA. In addition, when BL is applied as a source of N for cotton or corn based on the total N analysis, more than adequate P and K are applied. In fact, increases in soil test P and K levels are documented in all treatments receiving BL (data to be published separately). Broiler litter contains relatively high concentrations of secondary and micronutrients Ca, Mg, S, B, Zn, and Cu. In the southeastern USA, B is the only micronutrient routinely recommended for cotton, and Zn is the only routinely recommended micronutrient for corn. In Alabama, 0.34 kg B ha –1 is routinely recommended for all cotton, and 3.4 kg Zn ha –1 is recommended for corn on sandy soils (Adams et al., 1994). At the lowest rate of 134 kg BL N ha–1, about 0.23 kg B ha–1 and 1.9 kg Zn ha–1 are applied. Higher concentrations of B and Cu accumulated in tissues of cotton from BL applications compared with comparable N rates as AN (Table 3).

There is also speculation that BL may improve soil physical properties such as water-holding capacity and soil structure from the addition of organic matter and decreased seasonal transport losses of nutrients from soil during the growing seasons (Wood, 1992; Wood et al., 1999; Flynn, 1995), which may result in yield increases (Kingery et al., 1993, 1994). However, when BL was used as a source of N at the rates applied in this study, changes in soil physical properties were not documented.

The effect of BL on yield increases varied by year and cropping system as is typical of non-irrigated systems in the southeastern U.S. Rainfall patterns during the April through October growing season at CP indicated that yield differences due to treatment were small in years with less rainfall than typical especially for cotton (Fig. 4). Tillage systems did not appear to have much effect on the yield response to BL although these effects could not be statistically compared.



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  		Fig. 4. Precipitation during the growing season at the Coastal Plain site on yield relative to the no-N control treatment from 1991 to 2002. AN, ammonium nitrate; BL, broiler litter.

 
Residual effect of BL on crop yield is an important consideration when animal wastes are used as N sources in agriculture. However, the short-term effect of residual BL at the CP site is much less than would be expected with animal manure in the midwestern or northeastern USA. In our long-term experiment, residual N effect reached 30 to 50% for cotton lint and 25 to 65% for corn of current litter application treatments. Evers (1999) in Texas estimated that about 60% of N in BL would be available the first year. When used as a source of N for cereal crops, BL N efficiency ranged from 10 to 49% (Nicholson et al., 1999; Nyakatawa and Reddy, 2000; Nyakatawa et al., 2000). Kingery et al. (1996) reported that BL amendments had a larger labile N fraction (6.7%) than mineral fertilizer (4.1%) or no amendment (4.7%). In greenhouse research with maize, Hamilton and Sims (1995) calculated N use efficiency for BL of 48% over all N rates (percentage of added N recovered by plants, adjusted for soil N uptake by plants). At the CP site, we found that soil residual N was in the upper 5 cm in the form of organic matter (data not shown). Using the PSNT, soil nitrate N concentrations at the V8 growth state at sidedressing of corn were less than 10 mg NO3–N kg–1 to a depth of 60 cm except at the highest BL application rate the current season. The highest soil nitrate N measured at sidedressing during the 3 yr of corn was 23 mg N kg–1 in the surface 15 cm of the treatment receiving the highest rate of AN at planting. This is much less than values reported by Magdoff et al. (1984), Fox et al. (1989), and Meisinger et al. (1992) using the PSNT in the northeastern USA. These data are consistent with Jackson (1998), who found rapid leaching of N following cotton fertilization on similar CP soils in Alabama.

To further demonstrate the similarity in plant available N from AN and BL, we attempted to construct a relative N availability factor of BL based on corn and cotton yield compared with AN using the 13 yr of data from these experiments. The relative N availability factor (y) was calculated as:

Nitrogen availability in BL over the course of the 13-yr experiment is about 92 to 113% of AN under our experimental conditions (Fig. 5). Relative N availability may be greater than 100% because BL at a given rate of total N may produce greater numerical yields in some years than AN at the same total N rate. No attempt was made to evaluate N residual effect beyond the second year after application of BL. Although some carryover certainly occurred beyond the second year, this was evident only at the highest BL rate. Low rates of BL (less than 202 kg N ha–1) may have lower relative N availability and produce lower yield of cotton and corn compared with AN. However, analysis of N response curves showed that the yields with BL are similar or even higher than those from AN when the N rate is 202 kg ha–1 or higher. These results are in contrast with Arkansas research on a Sharkey clay (very fine, montmorillonitic, nonacid, thermic, Vertic Haplaquepts) where 260 kg N ha–1 as BL produced lower cotton yields and lower leaf petiole nitrate levels compared with 140 kg N ha–1 as urea AN solution (Glover et al., 1998).



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  		Fig. 5. The regression relationship between relative N availability factor and N rates. BL, broiler litter; AN, ammonium nitrate.

 
Although no statistical comparisons can be made for cotton response to surface-applied BL and AN at the CP site from 1998 through 2002 under conservation tillage and cotton response to incorporated BL and AN under conventional tillage from 1991 through 1994, we were surprised that the N rate for maximum yield increase was similar to the N rate for maximum yield increase under conventional tillage. Cotton producers are very interested in the optimum N rate as they continue to switch from conventional tillage to conservation tillage. General observations suggest that cotton response to BL and AN were similar whether incorporated under conventional tillage or surface-applied as in conservation tillage.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
Application of BL increased nutrient concentration in leaves, plant vegetative growth, and yield of cotton and corn significantly in both conventional till and no-till field experiments over a 13-yr period from 1990 through 1992. Compared with AN, relative N availability in BL was about 92 to 113% of N in AN, and the values varied positively with an increase in N rates. In only one site-year was there a significantly higher yield response to total N as BL compared with total N as AN. The residual effect of BL the second year after application resulted in 30 to 50% cotton lint yield and 25 to 65% corn grain yield compared with a standard, recommended N rate.


   ACKNOWLEDGMENTS
 
The authors gratefully acknowledge the assistance of Dr. Edzard van Santen with the statistical analyses. We also appreciate the help of Mr. Charles Burmester and the staff of the Tennessee Valley Research and Extension Center and Mr. Bobby Durbin and the staff of the E.V. Smith Research Center who helped with the collection of field data over a 13-yr period. This research was supported by the Alabama Cotton Commission; Cotton, Inc.; the Alabama Wheat and Feed Grains Committee; and the Alabama Agricultural Experiment Station.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 




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