Effects of ensiling density on nutritive value of maize and sorghum silages

Studies were conducted to determine the effects of different ensiling densities on fermentation, aerobic stability, and nutritive value of maize and sorghum silages. Maize and sorghum were harvested at dough (363 g/kg) and milk stages (275 g/kg), respectively. Herbages were chopped approximately 1.5 cm after harvest and then ensiled in mini silos at high and low-bulk densities for 8.5 weeks. Different bulk densities were achieved by ensiling different weights of herbage in the fixed-volume mini silos (1.5-L anaerobic jars, Weck, Germany). The obtained dry matter (DM) densities were 168 and 216 kg of DM/m3 for maize forage and 132 and 178 kg of DM/m3 for sorghum forage. Fermentation kinetics, the rate of aerobic deterioration upon aerobic exposure, and nutrient digestibility were followed during the periods of ensiling (on days 2, 4, 8, 15, and 60). In all cases, increased packing density resulted in silages with lower acetate content, ammonium N levels, and fermentation losses, but lactate content did not differ. Butyrate was detected in appreciable amounts only in sorghum silage. Propionate was not detected in any silage. Tightly packed silages remained stable upon exposure to air. Tight packing increases the digestibly of nutrients and improves the energy content of silages. These data show that high density limits air infiltration and reduces the oxidation loss during storage and feed-out. As a consequence, more dry matter is recovered and more energy


Introduction
In the last decade, the cost of animal production has increased, while the income from milk and meat products have not increased as quickly.This has created the need for animal production to find methods that are more efficient and profitable.One of the most effective methods is producing high-quality forages and using more forage in the diet of animals.Maize and sorghum are the main silages for feeding cattle (Wilkinson and Toivonen, 2003).The most important factor that affects the quality of silage is the ability to pack the silage to control and maintain anaerobic conditions (McDonald et al., 1991).This is achieved by packing silage at a higher density.Packing silage tightly minimizes excessive air, which can result in aerobic respiration and heat (Muck et al., 2004;Savoie et al., 2004).Heat can cause the loss of digestible nutrients and can increase the production of mold spores and mycotoxins (McDonald et al., 1991;Charley, 2008).This was similar to the research of Kung Jr (2010), who stated that lower packing density slowed down ensiling fermentation and aided the level of yeasts produced in the silage, which were found upon opening.It has been suggested that air infusion during storage and feed-out in commercial scale maize silage can contribute to clostridial growth in the peripheral areas of the silo (Borreani and Tabacco, 2009).Ruppel et al. (1995) found that the packing density negatively affected storage losses.Their findings included using a model for a six-month period and they found that losses decreased from 20 to 10% when packing was increased from 160 to 320 kg DM/m 3 .Another important factor affected by density is the maintenance and preservation of nutrients between the time of opening the silo and the time of using the silage as feed (Johnson et al., 2002;Wilkinson and Davies, 2013).Deteriorated silages cause a serious risk to the quality and safety of animal products and to animal health (Driehuis and Oude Elferink, 2000).
Under our conditions within the Mediterranean climate, two effects would be of greatest interest: a reduction of in-silo losses by an optimized fermentation process and a reduction of losses after the opening of the silo by improving the aerobic stability.Both types of losses are closely correlated to the concentration of oxygen present in the silo, which promotes unwanted microbes.There is little published information about the initial packing density effects on silage quality.Therefore, the objective of this study was to investigate the effects of different ensiling densities on fermentation, aerobic stability, and nutritive value of maize and sorghum silages.

Material and Methods
Silages were prepared from maize (Zea mays L.) and sorghum (Sorghum bicolor L.) and grown in the same years at the Agricultural Experimental Station (40 o 14' N, 28 o 50' E).Whole-crop maize and sorghum were cut at the dough (363 g/kg) and milk stages (275 g/kg), respectively.The forages were chopped (1.5 cm) with a laboratory chopper (Fimaks, Turkey).Three representative samples of fresh chopped forages were collected and frozen for subsequent analysis.Different bulk densities were achieved by ensiling different weight of herbage in the fixed-volume mini silos (1.5-L anaerobic jars, Weck, Germany).To achieve two different bulk densities for maize forage, jars were filled with 1050 and 1350 g (wet weight) of chopped maize, without a headspace.The obtained dry matter (DM) densities were 168 and 216 kg of DM/m 3 for maize forage.To achieve two different bulk densities for sorghum forage, jars were filled with 1100 and 1450 g (wet weight) of chopped sorghum, without a headspace.The obtained DM densities were 132 and 178 kg of DM/m 3 for sorghum forage.The openings of the mini silos were performed in a five-day temporal series on days 2, 4, 8, 15, and 60 after filling.There were 60 jars (two forages × five days × two ensiling density × three parallels) and they were stored at an ambient temperature of 25-28 °C.Ensiled forages (on days after ensiling, three jars per treatment for each time) were sampled for further analysis.
Chemical analyses of fresh forage and silages were performed in triplicate and presented on DM basis.The silage pH was measured directly from the silage juice using a pH meter (Sartorius PB-20, Germany).Fresh forages and silage samples were dried at 60 °C for DM determination (AOAC, 1990).Dry matter content of the silages was corrected (DM cor ) for the loss of volatile substances during drying through the following equation (Weißbach, 2009): DM cor = DM + 0.95 × sum of fatty acids (C2-C6) + 0.08 × lactic acid + 0.77 × 1,2 propanediol + 1.00 × other alcohols (C2-C6 including butanediol) [g/kg].Fresh forage and silages were analyzed for crude protein (CP) and ash according to AOAC (1990).The neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined using the sequential analysis scheme of Van Soest et al. (1991).Wet samples stored at -20 °C were extracted for 3 min in a blender in water or in ethyl acetate (1:9) for watersoluble carbohydrates (WSC) and analysis of fermentation products.The WSC were determined as described by Dubois et al. (1956).Lactic acid was determined by the procedures of Barker and Summerson (1941).The volatile fatty acids (VFA) and alcohol concentrations were analyzed using a gas chromatograph with a capillary column (over a temperature range of 45-230 °C).Ammonia-N was determined using a Kjeltech auto analyzer (Gerhardt, Germany).Microbiological analyses of fresh forage and silages were presented on fresh and wet silage basis.Microbiological evaluation included enumeration of lactobacilli on pourplate rogosa agar (Oxoid CM627, Oxoid, Basingstoke, U.K.) and yeast and mold on spread-plate malt extract agar (Difco, Detroit, MI), acidified with lactic acid to pH 4.0.Plates were incubated for 3 d at 30 °C.All microbiological data were transformed into log 10 .
At the end of the ensiling period (d 60), the silages were subjected to an aerobic stability test at room temperature (22 °C), which lasted 5 d, in a "polyethylene terephthalate (P.E.T.) bottle" system developed by Ashbell et al. (1991).The system was constructed from recycled soft drink bottles (polyethylene terephthalate) in two parts: the upper part (1-L) was filled with 250 g (wet weight) of loosely packed silage and the lower part with 100 mL of 20% KOH.Gas was exchanged through 1-cm holes in the lid of the upper part to the lower part.The CO 2 produced during aerobic exposure was absorbed in the base and determined by titration with 1 N HCl.In addition, silage pH was measured and yeast and mold analyses were performed as the indicators of aerobic spoilage as well.The pH and yeast and mold analyses were determined by the previously explained analysis methods.Analyses were carried out in the silage samples after 5 d of exposure to air.
The data obtained from silage quality were analyzed as a completely randomized design with three replications and subjected to analysis of variance by the GLM procedure of SAS (Statistical Analysis System, version 6.0).Differences among means were tested using Tukey's test and significance was declared at P<0.05.R. Bras.Zootec., 45(10):596-603, 2016

Results
The fresh maize had 363 g DM/kg with a pH value of 5.92 (Table 1).The NDF and ADF contents of fresh maize were 542 and 333 g/kg DM, respectively.Pre-ensiled maize had adequate levels of WSC (66 g/kg DM), but was relatively low in protein content (70 g/kg DM).The fresh sorghum had 270 g DM/kg with a pH value of 6.05.The NDF and ADF contents of fresh sorghum were 578 and 360 g/kg DM, respectively.Pre-ensiled sorghum had high levels of WSC (33 g/kg DM), but contained low levels of protein (51 g/kg DM).The epiphytic lactobacilli and yeast counts were high in both herbages, each being 10-log cfu/g.
Both ensiling density and the day of fermentation affected (P<0.05) the chemical composition of silages (Table 2).These effects were stronger (P<0.05) at the initial stage of fermentation.The DM recovery was higher (P = 0.06) in tightly packed silages than in loosely packed silages.Loosely packed sorghum silages had higher (P<0.05)fermentation losses than maize silages.The fiber fractions and protein content of silages were unaffected (P>0.05) by the packing density.
Maize and sorghum silages appeared to have fermented normally, as indicated by the final pH levels of 3.8 and 3.6 and fermentation acid levels of 83 to 72 g/kg DM, respectively (Table 3).Both ensiling density and the day of fermentation exerted a substantial influence on the fermentation profile of silages (P<0.05).Thus, the difference between the higher packing densities was much greater for initial stages of fermentation than the latter stages of fermentation for both types of silages (P<0.05).Within the first week of fermentation, nearly 80% of the total carbohydrates available were metabolized in maize and sorghum silages (P<0.05).As fermentation progressed, the concentration of lactic acid increased and the concentration of acetic acid decreased (P<0.05).Overall, the level of packing density did not influence (P>0.05) the lactate production, but influenced the acetate and ammonium-N levels (P<0.05).Lower levels were obtained in tightly packed silages (P<0.05).The accumulation of butyric acid was not seen in maize silages.However, a very small amount was seen in sorghum silages and were lower in tightly packed silages (P<0.05).The concentrations of alcohols were decreased with the fermentation progress and lower levels were obtained in tightly packed silages (P<0.05).
Two weeks after the beginning of fermentation, the amount of lactobacilli was higher than after eight weeks of fermentation (Table 4).The yeast count after two weeks was higher, but at eight weeks it was negligible.The total number of yeasts was 50% higher in the loosely packed silages than in the tightly packed.The yeast counts were higher in the sorghum silages than in the maize silage.No mold growth was detected.
The pH and CO 2 production within five days of air exposure was higher (P<0.05) in loosely packed silages than in tightly packed silages (Table 5).The most significant improvement in aerobic stability was seen in the silages with the highest packing density because of the lower yeast activity (P<0.05).Loosely packed sorghum silages had higher CO 2 production and yeast counts than maize silages.Regardless of the packing rate, no mold growth was observed in the silages.Visible molds were low in both types of silages that were exposed to air, each being one.
The level of packing density affected (P<0.05) the content of digestible DM, NDF, and OM and the calculated ME content of silages (Table 6).These parameters were higher in the tightly packed vs. the loosely packed silages (P<0.05).Tightly packed maize silage had 21% higher OM digestibility and 31.5% higher energy content than sorghum silages.

Discussion
One of the most important factors influencing preservation characteristics and nutritive value of forages is the density of forage mass and the levels of air-filled porosity in the silo (McDonald et al., 1991).Thus, the ensiling density is important in the influence on the fermentation course and final fermentation quality as well as animal performance.
In this study, factors affecting the pattern of fermentation in the silo are considered and the effects  of ensiling density on silage quality are illustrated.As expected, ensiling maize and sorghum benefit the maintenance of silage protein content close to that of the fresh forage.The reduction in ammonia-N of tightly packed silages is indicative of lower proteolytic activity, which may have resulted in improved protein-N utilization in the rumen.This hypothesis for improvement in protein-N utilization of well-preserved silages has been proposed previously.Sharp et al. (1994) investigated the digestion of well-preserved silages with 16-month-old Jersey heifers and reported a 33% improvement in efficiency of microbial protein synthesis.The overall drop in pH values reflected the production of acidic metabolites.The decrease of acetic acid in silages in the later stages of fermentation is the result of the achievement of anaerobic conditions in both types of silages (McDonald et al., 1991).However, the final WSC and the fermentation acid concentrations were, respectively, 89 and 15% higher in the maize silages than in the sorghum silages.These differences can be explained by the maturities (Tabacco et al., 2009).Differences in yeast populations, resulting from aeration (because of the different consolidation rates), become less apparent as the fermentation progressed.Mold that was present on the fresh maize and sorghum was not similarly active on the corresponding silages.These results would suggest that the fermentation pattern was predominantly homolactic (McDonald et al., 1991;Gerlach et al., 2013) and this led to lower fermentation losses, especially in tightly packed silages (Ruppel, 1992;Losand, 2003;Savage et al., 2015).Zhang and Yu (2015) evaluated the effects of the two levels of ensiling wet densities (500 and 600 kg/m 3 ) on the silage quality of Leymus chinensis silage.They found that lactic acid content was higher while the pH value, butyric acid, ammonium-N concentration, and the coliform bacteria were lower in high-density silage (600 kg/m 3 ).Savage et al. (2015) observed that lactic acid concentration was higher in maize silage ensiled at a rate of 240 kg of DM/m 3 dry density (5.55%) than at 170 kg of DM/m 3 density (4.40%).Rota et al. (2012) demonstrated the fermentation quality of maize silage (256 g/kg) when ensiled at a rate of 246 kg DM/m 3 packing dry density for 110 days into 2-L micro silos.They reported that DM losses were 7.12%, lactic acid was 93.9 g/kg DM, acetic acid was 22 g/kg DM, and ethanol was 7.49 g/kg DM.
Test for the aerobic stability based on CO 2 production showed that the presence of air during ensiling due to low packing density gave rise to poor post-storage quality in both silages.The set of deterioration process showed an increased level in pH values above five and a rise in yeast counts by seven log units (Vissers et al., 2007).These numbers were found in loosely packed silages (McDonald et al., 1991).The data from the stability tests reconfirm the findings of Tabacco et al. (2011) that tightly packed silages (612 kg/m 3 ) is more stable than loosely packed silages (577 kg/m 3 ).In that study, the spoiled silages also tended to show thermal instability.Further investigations by Windle and Kung Jr (2013) showed that that aerobically spoiled silage-based total mixed ration (TMR) contained 7.8 log cfu yeast/g at the time of feed-out, whereas the fresh TMR contained 5 log cfu yeast/g.Ruppel et al. (1995) and Muck et al. (2003) stated that greater silage density and feed-out rate together increase the time that silage is exposed to air without spoiling before removal from the silo.
The improved fermentation and aerobic stability of both silages may mirror what is observed in animal performance (McDonald et al., 1991;Gerlach et al., 2013;Windle and Kung Jr, 2013).Indeed, we observed that tightly packed silages had higher amount of degradable OM in the rumen and increased the energy content by 21 (maize silage) and 13% (sorghum silage), which indicates a conservation of nutrients (McDonald et al., 1991).Our results are in agreement with Flynn (1988), who demonstrated that in well-preserved and poorly preserved silages for cattle feed, the DM digestibility was 73.5 and 70.7%, respectively.The effects of hygienic quality of silage on DM intake of dairy cows were previously examined (Wichert et al., 1998) and it has been seen that aerobic deterioration led to a decrease in DM intake of about 10-20%.Feeding heifers with spoiled TMR resulted in lower DM intakes when compared with fresh TMR (Windle and Kung Jr, 2013).Aerobic deterioration can jeopardize the nutritive value of maize silage-based diets and can cause a reduction in feed intake of goats by 53% (Gerlach et al., 2013) and steers by 16% (Bolsen et al., 2002).

Conclusions
The more tightly packed silages enables better conservation of soluble carbohydrates, preserves silage proteins, and creates less change of structural carbohydrates.It increases post-storage stability aspects, which favor the acceptability and intake of silage that can enhance the animal performance.

Table 1 -
Chemical composition and epiphytic lactobacilli of pre-ensiled maize and sorghum DM -dry matter; DM cor -dry matter corrected for loss of volatiles; NDF -neutral detergent fiber; ADF -acid detergent fiber; NH 3 -N -ammonium nitrogen; TN -total nitrogen; cfu -colony-forming units.1Hemicellulosecalculated as the difference between NDF and ADF.

Table 2 -
Chemical composition of maize and sorghum silages (g/kg DM) at two ensiling densities DM -dry matter; DM cor -dry matter corrected for loss of volatiles; CP -crude protein; NDF -neutral detergent fiber; ADF -acid detergent fiber; HC -hemicellulose calculated as the difference between NDF and ADF; SEM -standard error of the mean.Means in the same column with different letters differ significantly (P<0.05).R. Bras.Zootec., 45(10):596-603, 2016

Table 3 -
Fermentation metabolites of maize and sorghum silages (g/kg DM) at two ensiling densities WSC -water soluble carbohydrates; ND -not detected; NH 3 -N -ammonium nitrogen; TN -total nitrogen; SEM -standard error of the mean.Means in the same column with different letters differ significantly (P<0.05).

Table 4 -
Microbiological composition of maize and sorghum silages (log cfu/g) at two ensiling densities cfu -colony-forming units.Microbiological analysis was performed on a single sample each time.Therefore, no statistical analyses are available.

Table 5 -
Results of aerobic stability test (5 d) for maize and sorghum silages at two ensiling densities after a 60 d storage period

Table 6 -
In vitro rumen digestible dry matter, fiber and organic matter, and calculated metabolizable energy values for maize and sorghum silages at two ensiling densities after a 60 d storage period dDM -digestible dry matter; dNDF -digestible neutral detergent fiber; dOM -digestible organic matter; ME -metabolizable energy; DM -dry matter; SEM -standard error of the mean.Means in the same column with different letters differ significantly (P<0.05).R. Bras.Zootec., 45(10):596-603, 2016