Nutritional composition and aerobic stability of wheat and corn silages stored under different environmental conditions

Composição nutricional e estabilidade aeróbia de silagens de trigo e milho armazenadas em diferentes ambientes

Ensiling aims to preserve the nutritional quality which characterizes a fodder at harvest. Accordingly, ensiled material must be kept in an anaerobic environment in order to create appropriate conditions for the development of lactic acid bacteria (LAB), which will convert soluble carbohydrates into organic acids. These acids have the ability to reduce the pH, thereby halting enzymatic activities and limiting the growth of deleterious microorganisms; thus, leading to the preservation of the forage for long periods (JOBIM; NUSSIO, 2013).

Another important issue relates to the type of forage for ensiling, which may influence the fermentation profile experienced by silage. Whole crop corn (Zea mays) is the standard silage plant, since it combines high productivity per hectare with high nutritional quality. Additionally, its characteristics promote ideal conditions for fermentation by LAB, such as dry matter (DM) content between 30% and 35% at the time of harvest, a low buffer capacity and a high soluble carbohydrate content (JOBIM; NUSSIO, 2013).

On the other hand, in order to optimize crop performance, particularly to increase plant health, corn hybrids containing the Bt gene have been widely used. However, some differences in chemical composition and fermentation can be observed in comparison with conventional hybrids (AULRICH et al., 2001; SAXENA; STOTZKY, 2001; BALIEIRO et al., 2011; KAMOTA et al., 2011).

Another cereal that is gaining acceptance for silage production is wheat (Triticum aestivum), as an alternative fodder during off-season periods. Wheat is an interesting option due to its good productivity and nutritional quality (MEINERZ et al., 2011). However, due to the high buffer capacity and a lower concentration of soluble carbohydrates presented by wheat, undesirable fermentations may occur in this type of material (MCDONALD et al., 1991). Therefore, the use of inoculants containing homolactic bacteria may be useful, as they transform the carbohydrates consumed into lactic acid with a more efficient use of energy (PAHLOW et al., 2003).

Nevertheless, during exposure to oxygens, spoilage microorganisms development, especially yeasts, implies the consumption of soluble substrates (such as carbohydrates, proteins, and fermentation products), which could potentially be used by cattle (WILKINSON; DAVIES, 2013). Thus, it is essential to evaluate this parameter in order to develop measures to control deterioration during the period of unavailability of the silo. It is important to emphasize that an ambient temperature above 10°C may have an influence on the evaluation of the aerobic stability of silages (ASHBELL et al., 2002). Therefore, the evaluation of the aerobic stability in more than one environment is important for decision-making in the attempt to control forage deterioration.

Therefore, the objective of this study was to evaluate the nutritional quality, fermentation profile and aerobic stability of conventional and transgenic corn silages, as well as wheat silage, treated with homolactic inoculant under different environments.

Experiments were carried out in the animal production sector of the State University of the Center-West (UNICENTRO) and in the ruminant nutrition sector of the State University of Maringá, in 2012. Experimental treatments included wheat silage (WS), wheat silage with bacterial inoculant (WSI), conventional corn silage (CCS), and transgenic corn silage (TCS).

Conventional and transgenic corn crops were planted in the second half of October 2010, under a No-tillage system, in succession with a forage mixture of black oats (Avena strigosa) and ryegrass (Lolium multiflorum), using 80 cm row spacing and seeding depth of 4 cm. Five seeds were planted per linear meter. The conventional hybrid AG 8088 and its isogenic counterpart carrying the Bt gene were used in an area of 1 hectare for each hybrid.

Basic fertilization of the corn crop consisted in application of 350 kg ha^-1 of NPK fertilizer 08-30-20 (N-P₂O₅-K₂O). Coverage fertilization consisted in 120 kg ha^-1 of N in the form of urea (46-00-00) applied when the plants had four expanded leaves, as recommended for a corn crop for grain production by the Soil Fertility Commission of Santa Catarina and Rio Grande do Sul (CQFS-RS/SC, 2004). Corn plants were harvested at physiological maturity during formation of chalky to hard corn grain, 121 days after emergence.

In the first half of June 2011, wheat was planted in succession, with a row spacing of 0.17 cm, at a depth of 2 cm and at a seed density of 220 seeds/m². The experimental material was wheat ‘BRS Umbu’ of a non-ridge, double-purpose genotype, in an area of 3 ha.

For base fertilization, 300 kg ha^-1 of NPK fertilizer 08-30-20 (N-P₂O₅-K₂O) were applied according to recommendations of the Soil Fertility Commission of Santa Catarina and Rio Grande do Sul (CQFS-RS/SC, 2004). Coverage fertilization consisted in a single dose of 118 kg ha^-1 of N in the form of urea (46-00-00), applied at tillering stage, 30 days after emergence. Wheat plants were harvested at physiological maturity, at the chalky corn ear stage, 144 days after emergence. Bacterial inoculant used for the WSI treatment was composed of Lactobacillus plantarum and Pediococcus acidilactici (1.0 × 10⁵ CFU g^-1).

Corn and wheat crops were monitored according to agronomic technical reports. Thus, 4 silos were filled, one for each treatment. The silos used were trench type, 3.5 m x 11 m and 1.0 m in height; completely sealed and protected with three layers of polyethylene lining (200 µ). Opening of the storage silos was in the first half of 2012.

Samples of wheat and corn silages were collected directly from the storage silo panel for seven consecutive weeks in feed use; they were weighed immediately and then pre-dried in a forced air oven at 55°C for 72 hours for determination of partially dry matter.

Dry matter in the pre-dried samples (DM), mineral matter (MM), crude protein (CP) and crude fat (CF) were estimated at 105°C according to the techniques described in Cunniff (1995). Concentration of neutral detergent fiber (NDF) with thermostable γ-amylase, acid detergent fiber (ADF), and lignin were obtained according to the method of Van Soest et al. (1991).

Total carbohydrates (TC), non-fibrous carbohydrates (NFC) and total digestible nutrients (TDN) were calculated according to the equations of Sniffen et al. (1992). The in vitro digestibility of DM (IVDMD) and the digestibility of NDF (DNDF) were determined after Holden (1999), and Hall and Mertens (2008), respectively.

Determination of lactic acid, acetic acid, propionic and butyric acids and alcohol, were performed by gas chromatography according to Erwin et al. (1961).

Assessment of aerobic stability was done separately on the four treatments under evaluation. Silage samples (3.5 kg) were deposited in buckets (10 L capacity) without compaction and placed in two different environments; one without temperature control (environment 1), and the other at a constant temperature of 24°C (environment 2), where they remained for seven consecutive days, for a total of 168 hours.

Silage temperature was measured twice a day, at 8:00 and again at 16:00 h, using a long stem Gulterm 1001 digital thermometer inserted in the center of the silage mass. After temperature measurement, these buckets were weighed for further DM loss calculation. The pH values were determined daily at 17 h according to Silva and Queiroz (2002), on a second set of containers, as well as 25 g of sample were removed for calculation of DM, as described by Jobim et al. (2007), in order to assess DM loss.

The variables analyzed in the aerobic stability test included: aerobic stability (h), maximum temperature recorded (°C); sum of observed temperatures (°C); mean of observed temperatures (°C), maximum observed pH; mean pH, time to reach maximum pH and dry matter loss.

Variables related to chemical composition and fermentation profile were analyzed in a completely randomized design, according to the following model: $Y_{\mathrm{ij}}=\mu +T_i+e_{\mathrm{ij}}$. Where: µ= overall average; T_i = effect of silage (i = 1 to 4); e_ij = error associated with observation Y_ij. Data for chemical composition and fermentation profile were submitted to ANOVA by the SAS Program (2012), and means were compared by Tukey’s test at a probability level of 5%.

Aerobic stability was tested in a completely randomized design with nine replicates, in a factorial scheme with 2 silages (wheat silage × wheat silage with inoculant, conventional corn silage × transgenic corn silage) and 2 environments under evaluation (ambient temperature × controlled temperature at 24°C), according to the following model: $Y_{\mathrm{ijk}}=\mu +T_i+A_j+{\mathrm{SA}}_{\mathrm{ij}}+e_{\mathrm{ijk}}$. Where: µ = overall mean; T_i = effect of silage (i = 1 to 2); A_j = effect of the environment (j = 1 to 2); TA_ij = interaction between silage and environment; e_ijk = error associated with observation Y_ijk. Data collected for each variable were submitted to ANOVA using the PROC GLM option of the SAS Program (2012), while means were analyzed by the F-test at 5% significance.

As expected, corn silage yielded better results in nutritional composition, compared to wheat silage (Table 1). The intrinsic characteristics of each crop, such as physiology, morphological and chemical composition before ensiling and productivity, all of which underlie the difference in fermentation dynamics, were undoubtedly the reason for the difference in final nutritional composition of silage in each case.

Table 1
Chemical composition of silages of conventional or transgenic corn and wheat silages with and without inoculant.

Means within the same row followed by different letter differ from each other (P <0.05) by the Tukey test.a WS: wheat silage;b WSI: wheat silage inoculant;c CCS: conventional corn silage;d TCS: transgenic corn silage;f NM: natural matter.1 SEM: standard error of the mean.

Corn silage, in comparison to wheat silage, presented higher organic matter and starch content, as well as lower NDF (NDF being more digestible) and ADF values. These results led to an increase of the estimated NFC and TDN concentrations in corn silage. Due to the better nutritional composition, corn silage had higher digestibility. However, corn silage showed lower CP concentration than wheat silage; probably because wheat is a temperate C3 grass; thus, it has a higher proportion of the Rubisco enzyme, which increases the amount of nitrogen in the plant leaves in comparison to C4 grasses (TAIZ et al., 2006).

Transgenic corn silage had a higher DM content, in detriment of NDF, when compared to CCS. However, according to some reports, the transgene can negatively alter fiber quality, as there may be a greater deposition of lignified tissue by the plant, which provides higher resistance to diseases, while at the same time, it would result in increased NDF content compared to non-transgenic plants (SAXENA; STOTZKY, 2001; POERSCHMANN et al., 2005; KAMOTA et al., 2011). However, this was not observed in the present study.

Reduced levels of DM, OM and total carbohydrates were observed for WS. In this way, the consumption of soluble substrate led to an increasing of CP level in WS, by a dilution effect. The type of fermentation occurred in each silage (Table 2) played a key role, as higher concentration of acetic and butyric acids have been observed in WS. Heterolactic fermentation consumes a greater amount of soluble carbohydrates compared to homolactic fermentation, since more oxidized compounds and CO₂ are formed, which are lost to the gas phase (AXELSSON, 2004). The presence of butyric acid is related to metabolism of energy-rich compounds (such as pyruvate and lactic acid) by microorganisms of the genus Clostridium, with production of CO₂, heat and increase in energy losses. These results indicate that inoculation of wheat silage provided better preservation of the material.

Table 2
Fermentation products of conventional and transgenic corn silages and wheat silages with no bacterial inoculant.

Means within the same row followed by different letter differ from each other (P <0.05) by the Tukey test.a WS: wheat silage;b WSI: wheat silage inoculant;c CCS: conventional corn silage;d TCS: transgenic corn silage.1 SEM: standard error of the mean.

Both wheat silages displayed higher levels of ammonia compared to corn silage, which is related to the higher concentration of CP and buffer capacity of the former, which favors proteolysis during storage.

In our experiments, corn silage displayed lower mean lactic acid levels than those described by Kung and Shaver (2001) for this type of silage (from 6% to 10% of DM). Furthermore, TCS had the lowest content of lactic acid (0.797%). TCS also displayed lower levels of acetic acid, propionic acid, alcohol, and NH₃/NT. However, the concentration of butyric acid was higher for TCS, compared to CCS; thus, denoting lower activity of heterolactic microorganisms. This behavior contrasts that described by Balieiro et al. (2011), who reported a higher concentration of propionic and acetic acids in Bt corn silage, with no difference in relation to butyric acid.

Inoculated wheat silage had a lower aerobic stability than WS, as shown by the significant difference (P <0.05) observed for stability, maximum temperature, sum, and mean temperature (Table 3). WSI silage presented lower acetic acid and butyric acid, accelerating deterioration, which was also demonstrated by Li et al. (2015). Acetic acid has the capacity to inhibit yeast and fungi development, which are responsible for starting the process of aerobic deterioration (DANNER et al., 2003). Although undesirable, butyric acid has the same effect. In addition, the higher amount of substrate in WSI silage due to the higher OM content in this fodder-likely increased microbiological growth, which in turn caused lower stability and high DM loss, compared to the uninoculated silage.

Table 3
Aerobic stability of wheat silage with and without bacterial inoculant.

Means within the same rows followed by different letter differ from each other (P <0.05) by the Tukey test. ¹WS: wheat silage and WSI: wheat silage with inoculant.2 N: ambient temperature; C: temperature kept at 24ºC.3 SEM: standard error or the mean.

When ambient temperature was maintained at 24°C, the loss of OM was higher. This is likely because this temperature is near the optimum temperature for the development of lactate-consuming organisms, as reported by Ashbell et al. (2002). These authors observed a rapid breakdown of the aerobic stability of corn and wheat silages at 30°C, compared to higher (40°C) or lower temperatures (10°C). On the other hand, temperature fluctuation outside the controlled environment may retard microbiological development (RATKOWSKY et al., 1982), as there is an exchange of heat between the mass of silage and the environment, thus, raising the aerobic stability of silages.

The comparison between CCS and TCS silages did not reveal any significant differences regarding the stability (Table 4). The maximum temperature in hours was higher for TCS, which could be associated with the lower concentration of acetic acid in this silage.

Table 4
Aerobic stability of conventional and transgenic corn silages.

Means within the same row followed by different letter differ from each other (P <0.05) by the Tukey test.1 CCS: Conventional corn silage and TCS: Transgenic corn silage.2 N: ambient temperature; C: temperature kept at 24 ºC.3 SEM: Standard error of the mean.

With respect to different environments, the results were similar to those observed for wheat silages; i.e., the stability of silages that remained in the controlled environment was negatively affected, which eventually caused greater DM losses.

The use of inoculant on wheat protected organic matter and soluble carbohydrates from deterioration. However, there was a reduction in the aerobic stability of wheat silages compared to conventional corn silage. The transgene did not interfere with the quality of corn silage, although it did affect the fermentation process in it. Environmental control interfered negatively with aerobic stability of wheat and corn silages and increased the losses of dry matter in both cases.