In order to study the effect of delayed ensiling and application of some organic acid-based additives on fermentation of corn silage, chopped whole plant corn mixed with 4 different additives consist propionic acid in addition to control treatment without any additive. Additives were: 1-Propionic acid 2-Propionic acid (85%) + Formic acid (15%) 3-Calcium propionate 4-Propionic acid (80%) + Formic acid (15%) + Ammonia (5%). 10 g kg-1 dry matter of each additives mixed with chopped corn forage in 2 different times (0.48 h). Silages were assessed using method of appearance evaluation and method of DM, pH evaluation CP, NDF, ADF, TVFA, WSC, aerobic stability and DM degradation each of treatments were determined after 60 days. Silages that exposed to air 48 h, before ensiling had better appearance quality in Filg`s method and whole additives in this experiment had good effect on appearance quality compared with control group. All of silages containing buffered propionic acid-based additive, in the method DM, pH evaluation, were good and very good. These silages had lower pH than control (p<0.05). Amount of dry matter of control silage which ensiled immediately was lower than other treated silages. NDF in the silages (with Propionic acid (85%) + Formic acid (15%)) was lower than the other silages and content of CP and TVFA increased with addition of the additives especially additive containing Propionic acid (85%) + Formic acid (15%) (p<0.05). WSC in treated silage with additive containing propionic acid (85%) + Formic acid (15%) that wilted 48 h before ensiling was more than other samples (p<0.05). Buffered propionic acid-based additives caused to increase aerobic stability in treated silages compared with untreated silages. Degradation of DM (in situ) in untreated silages that delayed ensiled was lower. Application of the additive containing propionic acid (80%) + Formic acid (15%) +Ammonia (5%) resulted in highest degradation dry matter among experimental additives. Generally, treating with propionic acid-based additives prevented degradation DM decrease (in vitro).
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Ensiling is a preservation method for moist forage crops. Many chemical additives have been used to alter silage fermentation. For example, ammonia has been used to treat high moisture corn and corn silage. Moderate concentrations of ammonia have increased the concentrations of lactic and acetic acids, decreased proteolysis, improved DM recovery and improved the aerobic stability of corn silage. Unbuffered propionic acid-based preservatives have also been used to improve the aerobic stability of corn silages. In recent years, marked changes have been made to the formulations and recommended application rates of additives containing propionic acid. Current recommendations for use of buffered propionic acid additives are considerably lower (0.1 to 0.2% of fresh forage weight) than classical recommendations for use of the unbuffered acid (0.75 to 1.5%). Data are lacking that directly compare the effects of buffered propionic acid based preservatives and ammonia on the aerobic stability of corn silage. A direct comparison is warranted to allow producers to make educated decisions on their relative usefulness (Kung et al., 2000). Moisture content at the time of harvest (too wet or too dry) may, also, be a problem if the proper maturity is not matched to the area. When, the effects of grain content and stover digestibility are combined, very significant differences in corn silage quality can result due to hybrid differences. Maturity at harvest may affect corn silage quality because it influences grain and moisture content as well as stover digestibility. A good fermentation process is not only dependent on the type and quality of the forage crop, but also on the harvesting and ensiling technique. Attention to details such as speed of harvesting, moisture content, length of chop, silage distribution and compaction can greatly influence the fermentation process and storage losses. Efficient fermentation ensures a more palatable and digestible feed, which encourages optimal dry matter intake that translates into improved animal performance. Making consistent, high-quality silage requires sound management decisions and attention to details quality silage is achieved when lactic acid is the predominant acid produced, as it is the most efficient fermentation acid and will drop the pH of the silage the fastest. The faster the fermentation is completed, the more nutrients will be retained in the silage (Johnson et al., 2002, 2003; Tapia et al., 2005). Chopping forages too long makes compaction difficult and air will remain trapped in the silage resulting in heating and spoilage (Pauly and Lingvall, 1999). The crop should be harvested and the silo filled as rapidly as possible. Filling delays will result in excessive respiration and increased silage losses. Packing should begin immediately when storing silage in bunker silos (Johnson et al., 2002, 2003; Tapia et al., 2005). Fermentation inhibitors could in theory be used for all types of forages. However, in practice, they are generally only used in wet crops with a low water-soluble carbohydrate content and/or high buffer capacity (McDonald et al., 1991). In the Netherlands salts from acids have become the most popular fermentation inhibitors (Hoogkamp, 1999). An advantage of these salts is that they are easier and safer to handle than their corresponding acids. Silage additives inhibiting silage fermentation can reduce clostridial spore counts. In wilted grass, silages a decrease in spore counts by a factor 5 to 20 has been observed. A similar decrease in spore counts could be obtained by adding molasses, a fermentation stimulant.
It is clear that to inhibit aerobic spoilage organisms, in particularly the once causing the onset of deterioration (i.e., yeasts and acetic acid bacteria) have to be inhibited in their activity and growth. Some additives which have proven to be effective in this respect include chemical additives based on volatile fatty acids such as propionic and acetic acid and biological additives based bacteriocin producing micro-organisms such as lactobacilli and bacilli (Woolford, 1975; McDonald et al., 1991; Phillip and Fellner, 1992; Weinberg and Muck, 1996). The effect of formic acid as a silage, additive for grass crops is well documented (McDonald et al., 1991). When, added to crops that are difficult to ensile, a sufficient dose of formic acid will increase the lactic acid fermentation, reduce the fermentation to acetic, propionic and butyric acids, reduce proteolysis and reduce silage pH compared with untreated silage. In order to achieve the major goal in silage making that is to preserve silage material with minimum nutrient loss, formic acid is widely used. Addition of formic acid to silage material has been reported to have generally positive effects on fermentation (Haigh, 1988; Snyman and Joubert, 1996). Formic acid as silage additive has anti-bacterial effect on many bacteria species, including lactic acid bacteria; thus, addition of formic acid into silage results in limited fermentation and reduction in organic acid content of silage. This type of silage contains a greater amount of water soluble carbohydrate, which is a better source of energy for rumen microbe than lactic acid (Baytok et al., 2005). In some cases, a moderate dose of formic acid may inhibit lactic acid bacteria to a greater extent than yeasts and entrobacteria which are undesirable organisms in silage. Yeasts are highly tolerant to formic acid and, when grown under anaerobic conditions, dependent on high sugar concentrations, which are usually present in formic acid-treated silage (McDonald et al., 1991; Driehuis and Wikeselaar, 2000). Propionic acid-based additives have been used to inhibit yeasts that assimilate lactic acid when silages are exposed to air and thus, they improve aerobic stability (Woolford, 1975). After chopping, the presence of air in forage mass delays the onset of fermentation and encourages the growth of undesirable microbes, which results in negative effects on the ensuing fermentation (McDonald et al., 1991). To eliminate air from the forage mass, forages should be packed rapidly and packed tightly into silos. However, poor management decisions, such as leaving chopped forage in wagons or piles, can result in substantial delays in filling silos; thus, forages may be exposed to air for prolonged periods of time. In addition, large bunk or trench silos sometimes require weeks to fill, which expose forages to excessive amounts of air. Air is also detrimental during storage because it stimulates the growth of yeasts that metabolize lactic acid, which results in loss of nutrients (Woolford, 1990). High moisture grain silages are prone to spoil rapidly when exposed to air (McAllister et al., 1995; Woolford, 1985). To improve the aerobic stability of these grains, methods to increase the concentration of propionic acid (because of its antifungal properties) in silages have been studied (Woolford, 1975). The direct method of adding propionic acid to silages has resulted in more consistent improvements in aerobic stability (Kung et al., 1998, 2000), but the effects of adding this acid to forage that is or has been exposed to excessive amounts of air before ensiling has not been well studied. The objective of this study was to compare the effects of delayed ensiling on silage fermentation. Effects of application of buffered propionic acid-based additives on fermentation, aerobic stability and nutritive value of these silages were also investigated.
MATERIALS AND METHODS
Whole-plant corn was harvested one half milk line and chopped to a theoretical length of 1-2 cm with a forage harvester. Forage treated with nothing (control), or with 0.1% of a buffered propionic acid-based additives (propionic acid) (1), Propionic acid (85%)+formic acid (15%) (2), Calcium propionate (3), Propionic acid (80%)+Formic acid (15%)+Ammonia (5%) (4) and ensiled immediately in quadruplicate 10 L micro silos. Portions of the chopped forage, untreated and treated, were left in loose piles on a clean concrete floor in a barn for 48 h before being packed into silos. Empty and full weights of silos were recorded and silos were stored in the dark at ambient temperatures ranging between 18 and 25°C. All of the silos for each treatment were opened after 60 day of ensiling. The DM content of fresh forage and silage samples was determined by drying (70 to 75 g) in a forced-air oven at 60°C for 48 h. After drying, samples were ground. Ten gram samples of silage from each treatment were diluted 100 mL sterile deionized water and blended for 2 min. Silage pH determined immediately. ADF by was measured the procedure of Robertson and Van Soest (1981). Crad Protein was calculated by multiplying total nitrogen by 6.25. Fresh forage (25 g) was added to a dilution bottle that contained 225 mL of sterile quarter-strength Ringer`s solution (Oxoid BR54; Unipath, Basingstoke, UK) and homogenized in a blender for 1 min. After blending, water extracts were filtered through Whatman 54 filter paper (Whatman Inc., Clifton, NJ), acidified with 50% sulfuric acid and frozen before analysis of Water-Soluble Carbohydrates (WSC) (Nelson, 1944). The pH of silage extracts was determined within 20 min of homogenization. After 60 day of ensiling, when silos were opened, silage was mixed well and a 2 kg sample was returned to its respective silo. No physical packing of the silage took place. A thermometer was placed in the geometric center of each silage mass and temperatures were recorded every 3 h. A double layer of cheesecloth was placed on the top of each silo to prevent contamination but allowed for penetration of air because silos were incubated between 21 and 22°C. Aerobic stability was defined as the number of hours before a 2°C increase in temperature of the silage mass relative to ambient temperature. The temperature and pH of silages were recorded for 144 h, each 24 h once. For the determination of in situ ruminal DM, dry corn silage samples were ground to pass through a 2 mm screen and 0.5 g of silage was weighed into Dacron bags (5x10 cm, 50 μm pore size; Ankom, Fairport, NY) (Van Soest and Wine, 1967). Duplicate bags were placed in the rumen of a fistulated sheep fed a diet that was 82.4% alfalfa hay/grass hay, 8.8% soybean meal and 8.8% dry rolled barely. Bags were incubated for 0, 4, 8, 16, 24, 48 and 72 h. After removal from the sheep, bags were washed with water until effluent was clear and then were dried at 55°C for 48 h. Dried bags were weighed and DM disappearance was determined. The chemical composition of forages and silages was analyzed as a factorial in a completely randomized design by the general linear models procedure of SAS/STAT (1996). Factors were %ADF, %NDF, %CP, %WSC, TVFA (Mmol kg-1), %DDM, DE (Mcal kg-1), ME (Mcal kg-1), %TDN Duncan test at 0.05 probability level. Means were separated by Duncan test. The kinetics of ruminal DM disappearance in situ was estimated by the Naway software.
In this study, each of the factors including like to smell, color and structure (appearance characters) based on Filg`s method evaluation has given a number (Nikpur et al., 1981). Treated forage with the additive that exposured to air for 48 h before ensiling, had better appearance than others. Control group had lower appearance quality than the other treatments in all of sealing times.
The results of appearance evaluation based on dry matter and pH Filg`s method is presented in Table 1. In this evaluation method, the maximum of pH for stating in very good degree silage with 35% DM is 4.5. In the present study, average DM was 35% and pH ranged from 4.4 to 4.96, therefore all of silages were stated in good and very good class (Table 1).
Untreated silage (control group) that was ensiled either immediately or after 48 h, had higher pH than treated silages (p<0.05). The control group that was ensiled immediately, had lower DM content compared with treated silages (p<0.05). The chemical composition of corn silage after 60 day of ensiling is presented in Table 2 and 3. Timing of treatment with buffered propionic acid either at chopping or after exposure to air made no difference in ADF content , but amount of NDF in treated silage with additive 2 (Propionic acid (85%)+Formic acid (15%)) was lower than the other treatments and control groups (p<0.05). Significant interaction was between for concentration of Water-Soluble Carbohydrates (WSC). The WSC concentration (Table 3) of either immediating sealing and delayed ensiling silages without any additive (control) were lower than the other treatments. However, for additive 2* Immediating ensilage (15.8 g kg-1) amount of WSC were higher than additives 3, 4 (p<0.05). For type of additive *48 h delayed ensiling, silage with additive 2 had higher concentration of WSC compared with the other treatments (p<0.05).
|Table 1:||Appearance evaluation based on dry matter and pH Filg`s method|
|1Delaying time of sealing = 0 h, 48 h, 2Control = Without any additive. Additive 1 = propionic acid. Additive 2 = Propionic acid (85%) +formic acid (15%), Additive 3 = Calcium propionate. Additive 4 = Propionic acid (80%) +Formic acid (15%) +Ammonia (5%)|
|Table 2:||Chemical composition (DM based) corn silage after 60 day of ensiling ( sealeing time is fixed)|
|1Control = Without any additive. Additive 1 = Propionic acid. Additive 2 = Propionic acid (85%) +formic acid (15%), Additive 3 = Calcium propionate. Additive 4 = Propionic acid (80%) + Formic acid (15%) + Ammonia (5%). 2Digestable Dry Matter = 88.9-0.779 (ADF), 3Digestable Energy = 0.027+0.0427(%DDM), 4Metabolizable Energy = DE *0.821. 5Total digestible nutrition = DE/0.04409 (Khalil et al., 1986), Unlike superscript in a row differ significantly (p<0.05)|
|Table 3:||Significant interaction between delaying time and additive type for concentration of water-soluble carbohydrates (WSC) in corn silage after 60 day of ensiling|
|1Delaying time of sealing = 0 h, 48 h. 2Control = Without any additive. Additive 1 = propionic acid. Additive 2 = Propionic acid (85%) + formic acid (15%), Additive 3 = Calcium propionate. Additive 4 = Propionic acid (80%) +Formic acid (15%) +Ammonia (5%). a, ab, bc, cd, d: Bars with unlike letter(s) differ (p<0.05)|
|Table 4:||Apparent in situ DM digestion of corn silages (sealing time is fixed)|
|1Control = Without any additive. Additive 1 = Propionic acid. Additive 2 = Propionic acid (85%) + formic acid (15%), Additive 3 = Calcium propionate. Additive 4 = Propionic acid (80%) + Formic acid (15%) + Ammonia (5%). 2Delaying time of sealing = 0 h, 48 h. 3Kd = Rate of digestion. Unlike superscript in a row differ significantly (p<0.05)|
Totally the WSC concentrations of either immediating sealing or delayed ensiling silages with additive 2 were higher than the other treatments. The TVFA concentration of treatment 2 (1364.0 Mmol kg-1) were significantly higher than other treatments and control group (p<0.05). The content of CP increased with addition of the additives especially the additive 2. The CP concentration of the additive 2 (108.1 g kg-1) were significantly higher than the treatments 1 and 3(p<0.05). For the concentration of DDM, DE, ME, TDN in the silages no significant main effects were found for both the type of additive and for the sealing time.
In situ Ruminal DM Digestibility
Apparent in situ ruminal DM digestibility is presented in Table 4. Addition of the additives 1 and 4 to silages effected on in situ ruminal DM digestibility of treated silages. So that, in situ ruminal DM digestion of control group (340.8 g kg-1) was lower than treatment 1 (430.5 g kg-1) and 4 (462.7 g kg-1), significantly (p<0.05). Sealing time had no effect on in situ ruminal DM digestibility of silages.
|Fig. 1:||The aerobic stability of corn silage. Control = Without any additive. Additive 1 = Propionic acid. Additive 2 = Propionic acid (85%) +Formic acid (15%), Additive 3 = Calcium propionate. Additive 4 = Propionic acid (80%) +Formic acid (15%) +Ammonia (5%). Delaying time of sealing = 0 h, 48 h. SE = 10. a,b,ab,c,d Bars with unlike letters differ (p<0.05)|
Significant interactions between additives and sealing time were found for pH and temperature (p<0.05). The temperature of the control silages in all of the sealing time after 48 h exposuring to air 2°C increased and after 144 h received to own peak of temperature (immediately = 33.95°C, 48 h delaying = 25.80°C). The acidity of the control silages in all of the sealing time after 48 h exposuring increased too (Fig. 1).
Applications of organic acid-based additives improve quality of fermentation corn Silage (Knicky, 2005).
Untreated silage had higher pH than treated silages. This is similar the results of Kung et al. (2000, 2004) and Randby (2000). Addition of the buffered propionic acid-based additive decrease pH, which suggests that these additives partially reduced the metabolism of some aerobic microorganisms (Kung et al., 1998).
Observations about of DM were similar to those of Florek et al. (2004) and Kung et al. (2004) that the reason for this finding related to the limiting fermentation power of formic acid and increasing DM content power of propionic acid with unknown reason. Addition and blending some additives at time of ensiling improve fermentation and increase DM of silages (Harrison and Blauwiekel, 1994; Givens et al., 1993).
Timing of treatment with buffered propionic acid either at chopping or after exposure to air made no difference in ADF content , the reason for decrease amount of NDF in treated silage with additive 2 (propionic acid (85%) +Formic acid (15%)) compared with the other treatments and the control groups are unknown. Kung et al. (1998, 2000) reported that the application of the buffered propionic acid-based additives had no effect on fermentation end-products. In this study, the crud protein content of silages with additive 2 were higher than additive 3 (calcium propionate). Randby (2000) detected that application of formic acid in silages with 24 h delayed in ensiling, improve quality fermentation, increase amount of true protein and sugar and decrease content of acetic acid, propionic acid, ammonia-N and pH. Additives consisted of formic acid limit fermentation and decrease ammonia-N and increase quality of silage protein.
The WSC concentration of both immediating sealing and delayed ensiling silages without any additive (control) were lower than the other treatments and amount of WSC for additive 2* Immediating ensilage were higher than additives 3, 4 (p<0.05). For type of additive*48 h delayed ensiling, silage with additive 2 had higher concentration of WSC compared with the other treatments (p<0.05). Mills and Kung (2002) reported that content of WSC didn`t have any different between treatments consisted of the buffered propionic acid-based additive and ensiling at 0 and 24 h after chopping forage. For type of additive*48 h delayed ensiling, control silage had higher concentration of WSC compared with other treatments. In addition of preservative effect of buffered propionic acid-based additive, application of this additive resulted in little differences in end-productions of corn silage, but treating corn silages with 0.2% (wt/wt wet forage) of buffered propionic acid-based additive were increased amount of WSC (Kung et al., 2004). Florek et al. (2004) did not measure WSC raises caused by wilted corn forage that treated with formic acid-based additive before ensiling. Of course, other factors are effective in WSC content of silages, including: variation of plant, stage of growth, effect of long of day, effect of manure (McDonald et al., 1991), effect of witting (Umana, 1991) and protein content of plant (Nikpur et al., 1981).
TVFA concentrations of treatment 2 were significantly higher than others, which is similar to values reported by Kung et al. (2004) and Kung and Shaver (2001). They were mentioned that addition of buffered propionic acid-based additive to silage caused increasing Volatile Fatty Acid (VFA), of course the highest was for propionic acid content of silage. Timing of treatment with buffered propionic acid-based additive either at chopping or after exposure to air made difference in forage content of TVFA. Randby (2000) indicated that application of formic acid additive to silage improved quality of corn silage and when the sealing was delayed 24 h, additives could vanish negative effects of delaying. The results of some studies detected that additive compound of formic acid influence amount of VFA and content of propionic acid increase in treating corn silage with buffered propionic acid-based additive. Treating corn silage with 0.2% (wt/wt wet forage) of these additives caused by increasing lactic acid, acetic acid and propionic acid compared with untreated silages (Kung et al., 2004).
For the concentration of DDM, DE, ME, TDN in the silages have influenced by content of ADF, which is similar to results reported by Kung et al. (1998, 2000) that mentioned the buffered propionic acid-based additive have little effect on some nutrients (e.g., ADF, NDF and starch).
In situ Ruminal DM Digestibility
Sealing time had no effect on in situ ruminal DM digestibility of silages. Treating with the buffered propionic acid-based additive prevented of the negative effects of exposuring to air before ensiling on in situ ruminal DM digestibility (Mills and Kung, 2002). Collectively, these data showed that the buffered propionic acid-based products can improve the fermentation and increase nutritive value end aerobic stability of silages.
Temperature and pH values of silages after opening explained that wilting forage before ensiling, affected in inhibiting temperature raise of silage after exposuring to air (Harrison and Blauwiekel, 1994). Among of experimental additives in all sealing time, additive 1 was more effective than others for the inhibiting of raising temperature of silages, but generally all of the additives in all of the sealing time had significant effect on increasing aerobic stability, which is similar to results of Kung et al. (1998, 2004) and Mills and Kung (2002).
When, chopped corn forage is exposed to air for a prolonged period of time before ensiling, the number of detrimental microorganisms increases whereas the concentration of fermentable substrate decreases. The applications of a buffered propionic acid-based additive, some indices of fermentation quality were not as negatively affected by the exposure to air. Exposure to air before ensiling also reduced in situ DM digestion, but not if silage was treated with a buffered propionic acid additive regardless of timing of the application. These results show that use of a buffered propionic acid-based additive can partially, but not totally, compensate for poor silo management practices. However, rapid filling of silos and achieving adequate packing densities to exclude excessive air should still be high priorities for making excellent quality silage.
The authors would like to thank Company Part Pishtaz, Kimia Roshd and Gorgan University of Agricultural Science and Natural Resources.
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