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Chemical fractionation of carbohydrate and protein composition of corn silages fertilized with increasing doses of nitrogen/ Composicao quimica fracional de carboidratos e de proteinas de silagens de milho fertilizadas com doses crescentes de nitrogenio.

INTRODUCTION

Silage nutritional quality is closely related to the fertilizer support offered to the crop, since the nutrient content the plant extracts from the soil during its cycle will influence not only productively but also directly the bromatological composition of the plant and the resulting silage (MALAVOLTA et al., 1997). Therefore, it is necessary to ensure a nutrient supply to replace the quantity extracted by the plant to maximize productivity, especially in crops for whole plant corn silage production, which generates nutrient exportation without soil recomposition and that could cause its rapid impoverishment (UENO et al., 2011).

Among all nutrients, nitrogen is the most frequently exported, from the soil to the plant, in silage production (NEUMANN et al., 2005); however, approximately 75% of this nitrogen is transferred directly to grains. In addition, nitrogen has a great relevance in plant health and productivity as it is a nutrient active in several metabolic routes, participating in the synthesis of protein, nucleic acids, membranes, hormones, and other cellular constituents (TAIZ & ZEIGER, 2002).

In another perspective, ingested nitrogen has several functions in animals, the most common of which is amino acid synthesis. This ingested nitrogen may be in the form of proteins or peptides, or also in its free form (non-protein nitrogen); based on that, the degradation of these compounds in the ruminal environment is modulated, being subdivided into fractions according to its degradation rate (NRC, 1996).

In addition, proteolysis and fixation of these compounds by ruminal microorganisms is energy dependent; thus, higher nitrogen concentrations may imply higher energy expenditure-a fact that leads to alteration of carbohydrate fractions (SNIFFEN et al., 1992). Therefore, there must be an ideal balance between carbohydrates and protein fractions in the diet of these animals.

Fractionation of these nutrients is essential for nutrition of precision ruminants. Through this fractionation, it is possible to promote dietary changes aiming to generate greater nutrient utilization and synchronism, inducing better rumen health, longevity, reduction of reproductive problems, and reduced environmental pollution by reducing excretion of nutrients, such as nitrogen (CNCPS, 2002).

Thus, the objective of this study was to evaluate the effect of increasing levels of nitrogen fertilization on the chemical composition of carbohydrates and proteins in the resulting corn silage (Zea mays L.).

MATERIALS AND METHODS

This study was conducted in the Animal Production Center (Nupran, Nucleo de Producao Animal) from the Department of Agrarian and Environmental Sciences of Universidade Estadual do Centro-Oeste (Unicentro), located in the city of Guarapuava, state of Parana, Brazil. The soil of the study area is classified as typical haplohumox (POTT et al., 2007), and presented the following chemical characteristics on its 0 to 20cm profile, before planting: pH Ca[Cl.sub.2] 0.01M: 4.7; P: 1.1mg [dm.sup.3]; [K.sup.+]: 0.2[cmol.sub.c] [dm.sup.-3]; Mo: 2.62%; [Al.sup.3+]: 0.0[cmol.sub.c] [dm.sup.-3]; [H.sup.+] + [Al.sup.3+]: 5.2[cmol.sub.c] [dm.sup.-3]; [Ca.sup.2+]: 5.0[cmol.sub.c] [dm.sup.3]; [Mg.sup.2+]: 5.0[cmol.sub.c] [dm.sup.-3] and base saturation: 67.3%.

The corn crop (Zea mays L.) was created on November 4th through direct planting system. The experimental material was hybrid Dow 766, in early cycle, planted observing 80cm spacing, sowing depth of 4cm, and density of five plants per linear meter, according to the recommendations from the breeding company. Corn planting was performed in plots of total area of 28.8[m.sup.2] (4.8m x 6.0m), from which 16[m.sup.2] (3.2m x 5.0m) in the central area was used for analysis. Base fertilization consisted of 350kg [ha.sup.1] of 08-30-20 (N-[P.sub.2][O.sub.5]-[K.sub.2]O) formulation, according to the Fertilization and Liming Guidelines for the states of Rio Grande do Sul and Santa Catarina (2004). Phytosanitary management was conducted through crop technical reports. Thirtyfive days after planting, in V5 crop development stage, cover fertilization was applied in the form of urea (4500-00) in different dosage according to the following treatments: [T.sub.1]: 0kg N [ha.sup.-1]; [T.sub.2]: 45kg N [ha.sup.-1]; [T.sub.3]: 90kg N [ha.sup.-1]; and [T.sub.4] 135kg N [ha.sup.-1]. These fertilization dosages were based on those used by local farmers.

Corn plants were harvested close to its physiological maturation, in chalky grain cob formation stage (R4), according to the classification proposed by RITCHIE et al. (2003). Plants of each plot were harvested 20cm from the soil surface, chopped in stationary forage equipment and ensiled in laboratory silos of PVC tubes of 10cm diameter and 50cm length. The material was manually compacted and properly identified. Silos were stored in a covered area for 30 days and, after opening, a 500g sample of each treatment was collected, weighed and pre-dried in forced air oven at 55[degrees]C until constant weight, to determine the content of dry matter (DM), according to AOAC (1995). Then the samples were milled in a "Wiley" type mill, using a 1mm sieve mesh.

The total dry matter (TDM) was determined in an oven at 105[degrees]C; gross protein (GP) was determined by micro Kjeldahl method; and mineral matter (MM) and ethereal extract (EE) were determined according to AOAC (1995). Also, neutral detergent fiber (NDF) levels were determined using thermostable a-amylase (Termamyl 120L, Novozymes Latin America Ltda.) according to Van SOEST et al. (1991); and acid detergent fiber (ADF) content was determined according to Goering & Van SOEST (1970). Levels of neutral detergent insoluble nitrogen (NDIN) and acid detergent insoluble nitrogen (ADIN) and GP fractions were calculated according to LICITRA et al. (1996).

NDF was subsequently corrected for ashes and proteins (NDFap) by discounting the remaining protein in NDF, which was determined after multiplying the factor of 6.25 by NDIN content and the residual ash. Non-fibrous carbohydrates corrected for ashes and proteins (NFCap) were obtained through the equation proposed by SNIFFEN et al. (1992): NFCap = 100 - (NDFap + EE + GP + MM).

The pH readings were performed using a digital pH meter, according to the methodology developed by CHERNEY & CHERNEY (2003). Ammoniac nitrogen determination (NH3NT-1) was based on BOLSEN et al. (1992). Soluble sugar content (SS) was determined according to HALL (2000); and organic acids according to SILVA & QUEIROZ (2009). Starch content was determined according to the methodology described by WALTER et al. (2005). The soluble fiber (SF) contents were determined through the methodology proposed by AOAC (1995). Total digestible nutrient contents (NDT) were obtained from the equation of BOLSEN et al. (1992).

This study had a randomized block design, consisting of four treatments with three replicates each. Data collected for each parameter were submitted to analysis of variance to compare averages at a significance level of 5% by Tukey test, and also to polynomial regression analysis using the "proc reg" procedure, processed in SAS statistical software (1993).

RESULTS AND DISCUSSION

Table 1 presents the average GP content, as well as its fractions according to the different levels of nitrogen fertilization. A significant difference was observed between the treatments in both GP content and C fraction. In GP, 135kg N ha-1 fertilization was associated with the highest content (8.60%) between treatments. In contrast, the highest C fraction value was observed in the treatment without nitrogen cover fertilization (10.21%).

MENGEL (2001) states that nitrogen increases in cover fertilization generates increases in plant GP content and, consequently, increases in silage, as this nutrient accounts for approximately 16% of the constitution of these molecules. Figure 1 shows the contrasting GP and C fraction behavior, with GP showing an increasing linear behavior, incrementing its content by 0.0085% for each kg of nitrogen applied, and fraction C showing the opposite behavior, linearly decreasing by 0.0087% for each increase in kg of nitrogen applied. Increasing nitrogen dose in cover fertilization provided an increase in protein value and; in contrast, decreased the indigestible fractions. It occurs when higher cover nitrogen doses exceed the plant energetic ability for protein synthesis and formation of true protein (TAIZ & ZEIGER, 2002). This process also depends on insolation, temperature, precipitation, and air relative humidity conditions. NNP is an integral part of A fraction, or also considered as readily digestible by ruminants (CNCPS, 2002).

A significant statistical difference (P < 0.05) was observed in average carbohydrates, organic acids (part of A fraction) and starch (part of B1 fraction) values between treatments. However, there was no statistically significant difference (P > 0.05) for non-fibrous carbohydrates corrected for ash and protein, soluble fiber (fraction B1), hemicellulose and cellulose (fraction B2), neither for C fraction.

According to Van SOEST (1994), total carbohydrates are relevant, since they represent the main energy source for microbial fermentation, being converted into short-chain fatty acids. Between the treatments, the highest content was observed in silage without nitrogen fertilization (88.05%) and the lowest in silage of 135kg N [ha.sup.-1] treatment (86.11%), representing a 2.20% reduction. Generally, it implied a decreasing linear behavior of 0.0068 for each increase in kg of nitrogen per hectare (Figure 1). These findings may be justified by the fact that there was a greater consumption of these carbohydrates to turn the increased amount of available nitrogen into protein, which tends to be a natural response of the plant (TAIZ & ZEIGER, 2002).

This mechanism also has implications for starch, since its highest value was observed without nitrogen fertilization (25.59%), with a decrease of 0.0127kg for each kg of nitrogen applied. Regarding the organic acids, the highest value was observed in the treatment with 45kg N ha-1 (11.41%).

Soluble fibers linearly increased by 0.0138 for each kg of nitrogen applied. In other words, more fertilized silages had increased amounts of soluble fiber, which is the fraction of greatest potential for digestibility. The same was reported by MASOERO et al. (2011), who analyzed different levels of reduced nitrogen fertilizer in corn crops (without nitrogen fertilization, 100 and 150kg N [ha.sup.-1]) and observed increased fiber digestibility in corn silage, especially in leaf and inferior stalk areas.

However, e increase in nitrogen fertilization did not influence the nutritional parameters, as indicated in table 2. Even with decreased starch concentration, as mentioned above, the content of total digestible nutrients did not change with increasing nitrogen fertilization. These results demonstrate the recommendations of other authors for high quality silage production (BASSEGIO et al., 2015); i.e., the selection of hybrids presenting higher grain participation to increase starch proportion in the material, is not absolute true, since silages well fertilized with nitrogen may have the same energy contribution with greater use of fibrous fraction.

In addition, such findings have great relevance in the context of ruminant nutrition. Corn silages with more digestible fiber can result in higher DM intake, which may reduce costs with a lower participation of concentrate in the diet and ensure ruminal health due to greater rumination through ruminal buffer, higher fat milk production and extended animal production, providing that particle size is respected (FERRARETTO & SHAVER, 2015). Also, a lower content of ingested starch with highly digestible fiber may reduce metabolic disorders, such as ruminal acidosis, in milk and meat production systems (DE NARDI et al., 2013).

Table 2 shows a statistically significant difference (P<0.05) in pH; treatments without nitrogen, with 45 and 90kg N [ha.sup.-1] did not differ from each other, but had statistically lower pH (P<0.05) when compared to the treatment with 135kg N [ha.sup.-1]. An increase in GP content and the presence of organic ions from the silage generates an increase in buffer capacity, which in turn neutralizes pH inside the silo, and it would be considered undesirable (ROOKE & HATFIELD, 2003).

Although a GP increase was observed, even without any increase in N-NH3 and in the amount of more digestible nitrogen fraction, which contributed to linear pH increase in this study, no harmful effect was observed on conservation, since the pH remained below 4.0. Moreover, the levels of N-N[H.sub.3] were below 10%, which, according to Van SOEST (1994), suggestedoptimal storage quality of these silages, because they do not indicate proteolysis due to fermentation by clostridia.

CONCLUSION

Increase in nitrogen levels in cover fertilization of corn crops provided benefits in kinetics and chemical composition of resulting silage.

http://dx.doi.org/10.1590/0103-8478cr20160270

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Mikael Neumann (1) Jose Laerte Nornberg (2) Guilherme Fernando Mattos Leao (3) Egon Henrique Horst (4) Danubia Nogueira Figueira (5)

(1) Departamento de Medicina Veterinaria, Universidade Estadual do Centro-Oeste (UNICENTRO), Rua Simeao Camargo Varela de Sa, 03, Vila Carli, 85040-080, Guarapuava, PR, Brasil. E-mail: neumann.mikael@hotmail.com. Corresponding author.

(2) Departamento de Zootecnia, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brasil.

(3) Departamento de Zootecnia, Universidade Federal do Parana (UFPR), Curitiba, PR, Brasil.

(4) Departamento de Ciencia Animal, Universidade Estadual de Londrina (UEL), Londrina, PR, Brasil.

(5) Departamento de Zootecnia, Universidade Estadual Paulista "Julio de Mesquita Filho" (UNESP), Jaboticabal, SP, Brasil.

Received 03.15.16 Approved 01.10.17 Returned by the author 02.21.17

Caption: Figure 1-Behavior of gross protein (GP) and its C fraction, and of total carbohydrates (TC), starch, soluble fiber (SF) and organic acids (OA) of corn silage grown with different cover nitrogen fertilizer treatments.
Table 1--Average levels of gross protein and total carbohydrates
(% in DM) and its fractions (% of GP and % of TC) in corn silage
grown with different cover nitrogen fertilizer treatments.

Parameter                               N levels in cover
                                  fertilization (kg [ha.sup.-1])

                                   1            45           90

                                % in DM

GP                              6.05 (D)     7.00 (C)     7.72 (B)

                                                          % of GP

A                                49.36        48.77        51.92
B1                                7.01         7.69         6.06
B2                               28.81        28.11        27.78
B3                                4.59         5.86         4.76
C                              10.21 (A)     9.55 (A)     9.46 (A)

                                 % In MS

TC                             90.22 (A)    89.46 (AB)   88.37 (BC)

                                 % of CT

Fraction A    Soluble sugars      2.83         2.39         3.12
              Organic acids    10.12 (AB)   11.75 (A)    9.80 (AB)
Fraction B1   Soluble fiber     9.70 (C)    10.55 (BC)   11.97 (B)
                  Starch       28.36 (A)    27.07 (AB)   26.06 (AB)
Fraction B2   Hemicellulose      20.77        19.72        20.89
                Cellulose        24.65        24.65        24.33
Fraction C        Lignin          3.57         3.86         3.84

Parameter         N levels       Average   Coefficient
                  in cover                 of Variation
               fertilization
              (kg [ha.sup.-1])

                    135

                                % in DM

GP                8.60 (A)        7.34         3.81

A                  53.55          50.90       10.38
B1                  5.12          6.47        40.53
B2                 27.81          28.13       16.84
B3                  5.91          5.39        40.42
C                 7.60 (B)        9.21         8.08

                                % of GP

TC               87.99 (C)        89.01        1.09

                                % of CT

Fraction A          3.94          3.07        28.93
                  7.18 (B)        9.71        15.18
Fraction B1      14.49 (A)        11.68       10.05
                 24.43 (B)        26.48        8.92
Fraction B2        21.72          20.78        9.07
                   24.32          24.49        8.72
Fraction C          3.91          3.80        18.92

Averages followed by different letters in the line
differ (P < 0.05) from each other by the Tukey test.

Table 2--Chemical composition of corn silage grown with
different cover nitrogen fertilizer treatments.

Parameter                    N levels of coverage           Average
                              (kg [ha.sup.-1])

                     1          45         90       135

Dry matter, %      30.15      27.49      30.60     30.09    29.58

                                   % of MS

Organic            96.27      96.47      96.75     96.42    96.48
  matter
Ethereal            2.50       2.48       2.69     2.74     2.60
  extract
Neutral            42.93      42.91      42.50     42.06    42.60
  detergent
  fiber
Acid detergent     26.16      28.56      25.59     25.53    26.46
  fiber
Non-fibrous        44.77      44.05      43.83     43.01    43.92
  carbohydrates
Total              67.47      67.31      67.97     67.92    67.74
  digestible
  nutrients

                                     Index

pH                3.31 (B)   3.32 (B)   3.35 (B)   3.40 (A) 3.35

                                     % of total nitrogen

N-N[H.sub.3]        3.77       3.57       3.92     3.45     3.68

Parameter         Coefficient of
                  Variation (%)

Dry matter, %          5.14

                      % of MS

Organic                0.44
  matter
Ethereal              14.76
  extract
Neutral                5.37
  detergent
  fiber
Acid detergent         7.54
  fiber
Non-fibrous            6.62
  carbohydrates
Total                  2.16
  digestible
  nutrients

                      Index

pH                     0.57

                      % of total
                      nitrogen

N-N[H.sub.3]          12.54

Averages followed by different letters in the line
differ (P<0.05) from each other by the Tukey test.
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Title Annotation:SOIL SCIENCE
Author:Neumann, Mikael; Nornberg Laerte, Jose; Leao Mattos, Guilherme Fernando; Horst, Egon Henrique; Figue
Publication:Ciencia Rural
Article Type:Ensayo
Date:May 1, 2017
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