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Effects of additives on fermentation characteristics and chemical composition of ensiled Chromolaena odorata leaves

Muhammad Rusdy

Department of Forage Crops and Grassland Management, Faculty of Animal Science Hasanuddin University, Indonesia


An experiment was carried out to determine the effects of readily fermented carbohydrates (RFC) sources on fermentation characteristics and chemical composition of ensiled Chromolaena odorata leaves.The experiment used cassava tuber flour, maize meal, rice bran and cane molasses as sources of RFC and nutrients. The additives were mixed with unwilted Chromolaena odorata leaves and ensiled in polvinyl chloride (PVC) laboratory silo measuring 16 cm in diameter and 20 cm length at the rate of 5% (w/w fresh basis) and ensiled for 30 days. The experiment was assigned in a completeley randomized design with four additives mixed with Chromolaena leaves plus control (no additives) as treatments with three replications.

The results from experiment showed that additives improved fermentation quality by reducing final pH, NH3-N and increasing lactic acid content of post-ensiled Chromolaena odorata. Compared to pre-ensiled Chromolaena odorata, ensiling reduced dry matter, crude protein and nitrate, but increased NDF and ADF contents of silage. Additives increased dry matter, crude protein, in vitro dry matter digestibility and reduced NDF, ADF contents of ensiled Chromolaena odorata with molasses was the best additive. Ensiling could not reduce nitrate content of Chomolaena odorata leaves to very safe levels. It can be concluded that locally available additives such as molasses and cassava could be utilized to improve fermentation characteristics and nutritive value of ensiled Chromolaena odorata.

Keywords: ensiling, fermentation quality, nutritive value, readily fermentable carbohydrate sources, siam weed


A major constrain to animal production in developing countries including Indonesia is the scarcity of available feed and fluctuating the quantity and quality of year around feed supply. During the rainy season, there is abundant of feed supply without followed by increasing of animals number reared, however, in the dry season, natural pastures and crop residues available for animals after crop harvest are usually lacks of most essential nutrients required for good performance of animal. This is manifested in loss of weight conditions, reduced reproduction capacity and increased mortality rate.

With increasing demand for livestock products as results of rapid growth population and economies and shrinking of land area, the future hope of feeding of millions of people will depend on better utilization of non-conventional feed resources (Makkar 2002).

Since 40 years ago, a large areas of plantation and pasture in Indonesia, have been invaded by Chromolaena odorata (here after is called Chromolaena) that reduced availability of forage supply. This weed can grow rapidly and form infestation that can suppress pasture growth by competing nutrients and water and their overshading and allelophathic effects lowering productivity of desirable forage species with a concomitant loss of livestock production. In Maiwa ranch of South Sulawesi Indonesia, more than 50% of pasture areas have been covered by this weed.

The threat of Chromolaena to agriculture has been a global concern such that many studies have been carried out all over the world to control the weed. However, an evaluation made by Ambika and Jayachandra (2014) revealed that, after several years of research efforts, the control problem of Chromolaena odorata has remained unsolved. Because of its difficulty to control, some researches recommended to use this weed for some useful purposes such as animal feed (Aro et al 2009; Bamikole et al 2004). Assessment of nutritive value of the weed showed that it has a good potential for feeding livestock due to its high crude protein content, low fibre and low extractable phenolic content (Apori et al 2000). Morever, this weed is evergreen and can be used as potential source of forage for animals year around, including dry season. However, in the fresh state, this plant is avoided by livestock because it has exceptionally high nitrate content (Sajise et al1974) and presence of antipedants (odor) in the leaves (Apori et al 2000). High levels of nitrate in forages is toxic to animals due to it can interfere with the ability of animal to transport oxygen in the blood (Stalling, 2006). The practical way to reduce nitrate content of forage is by ensiling, because enterobacteria in ensiling process are usually effective at degrading nitrates, McDonald et al 1991.

For successful ensiling, the ensilability of plants must be considered. To ensure a fast development of naturally occurring lactobacillus bacteria, adequately levels of RFC are required. The use of additive as a source of RFC and nutrients may be a suitable way to preserve the nutrients in Chromolaena leaves.The present study aims to determine the effects ensiling on nitrate content and the use of additives on fermentation characteristics and chemical composition of ensiled Chromolaena leaves.

Figure 1. Chromolaena odorata plant

Materials and Methods

The experiment was conducted at Faculty od Animal Science Hasanuddin University, Indonesia between August and October 2013. Chromolaena leaves were obtained from the campus of Hasanuddin University. Chromolaena leaves were harvested at early bloom stage of growth and ensiled without wilting. Cassava tuber flour, maize meal, rice bran, and cane molasses, each at the rate of 5% of Chromolaena leaves (w/w) were used as nutrients and RFC sources. Before ensiling, Chromolaena was analyzed for dry matter, crude protein, neutral detergent fibre (NDF), acid detergent fibre (ADF), readily fermented carbohydrates (RFC) and nitrate contents. The additives were mixed withChromolaena leaves and ensiled in polyvinil chloride (PVC) pots silo measuring 16 cm in diameter and 20 cm length for 30 days. Each silo was filled with 1.41 kg of Chromolaena – additives mixture that had been shaken vigorously to make a good mixture and pressed to make the mixture airtight.

The experiment was assigned ina completely randomized designwith the four additives mixed with Chromolaena leaves plus control (Chromolaena leaves only) as treatments with three replications. After filling, the open end of each silo was sealed by polyethylene sheet held securely by a strong rubber. The silos were stored in laboratory at room temperature of around 28 - 32°C. After 30 days of fermentation, the siloswere opened for determination of nitrate, pH, lactic acid, ammonia – nitrogen, dry matter, crude protein,neutral detergent fibre (NDF) , acid detergent fibre (ADF) contents and in vitro dry matter digestibility (IVDMD). Nitrate content was measured for free additives Chromolaena silage only. Dry matter contents weredetermined by drying it in oven at 60°C for 72 hours. The pH of each sample was determined by using approximately 25 g wet silage added to 100 ml of distilled water. After hydration for 10 minutes using blender, the pH was determined using digital pH meter. Protein contents of silages were detemined following of Association of Official Analytical Chemist (AOAC) (1990). NDF and ADF were analyzed according to the method of Van Soest and Robeston (1979). RFC contents were determined by the anthrone method (Yemm and Willis, 1954). One gram of freeze-dried sample was boiled 150 ml of distilled water with a cold-finger condenser for two hours and filtered through Toyo paper. Two ml of 29.5 N of sulfuric acid containing 1 mg of thio-urea and 1 mg of anthrone were added into 2 ml of filtrates and the mixed solutions was boiled again for 20 minutes. The solution was photometrically measured at 625 nm against glucose standard solution. In vitro dry matter digestibiity used the method proposed by Goto and Minson (1977). Spectrophotometer was used to determine nitrate-content. The silages were analyzed for lactic acid content according to Playne (1985) and NH3-N according to Anonymous (1986).

Statistical analysis

The changes in chemical composition of Chromolaena as result of ensiling were calculated by comparing values of chemical composition ofpre-ensiled and post ensiled plant, All fermentation quality and chemical composition data from post-ensiled Chromolaenawere subjected to analysis of variance using General Linear Model procedures of SPSS version 16. Least Significant Difference (LSD) for all statistically analyzed data were used to record the difference between treatment means with significance of 5%.

Results and Discussion

Chemical composition of silage ingredients

The chemical compositions of fresh Chromolaena leaves and additives are shown in Table 1.

Table 1. Chemical composition of fresh Chromolaena laeaves and additives (dry matter basis).
Parameters Chromolaena Cassava tuber flour Corn meal Rice bran Molasses
Dry matter (%) 19.3 87.9 87.8 87.7 59.8
Crude protein (%) 25.9 0.51 8.46 9.85 4.61
NDF (%) 51.5 20.0 24.2 24.9 0.00
ADF (%) 33.6 0.16 5.77 12.3 0.00
RFC (%) 3.67 62.1 2.28 4.12 72.1
Nitrate 0.90        

The dry matter content of plant prior to ensiling plays an important role in determining the silage fermentation characteristics. The dry matter of Chromolaena in this study was lower than recommended for desirable silage preservation (30 – 40%) (Regan 2000). The RFC content in Chromolaena leaves was 3.67% DM; this value was lower than 6 – 7% DM level recommended to achieve well preserved silage conservation (Oshima et al 1996). This indicated that in order to obtain a good quality silage, Chromolaena leaves need addition of high RFC sources.

In this study, fresh Chromolaena leaves contain 0.90% nitrate that pottentially very toxic to livestock (Olson et al 2014). The tendency of plants to accumulate nitrate to poisonous level had also been reported in tropical grasses such as kikuyu grass (Pennisetum clandestinum) (Williams et al 1991) and Pennisetum purpureum (Bharathidhasan et al 2008).

Physical and fermentation characteristics of post-ensiled Chromolaena

Observations show that the silage colour was changed from pale green at the start of ensiling to yellow brown one month after ensiling. The smell was good for all treatments. There was a little effluent was found attached in Chromolaena leaves silage, but no effluent was detected at the bottom of silos. There was a little spoilage moulds were found on the top of all silages. Presence of mould growth in the top layer of the silos suggests that moulds might have been stimulated by presence of small amount of oxygen. Based on colour and smell, the silages were considered to be acceptable, except for molded layer.

The results of the effects of RFC additive treatments on the fermentation characteristics of silage are shown in Table 2.

Table 2. pH, lactic acid and NH3-N concentrations of silage with and without additives
Parameters Control Cassava tuber flour Corn meal Rice bran Molasses SEM Prob.
pH 5.17b 4.73a 4.80a 4.80a 4.60a 0.00 0.001
Lactic acid (% DM) 0.77a 2.42c 1.47b 1.32b 2.71c 0.00 0.001
NH3-N (% TN) 20.1c 13.6a 16.1b 15.1b 13.1a 10.0 0.02
abcMeans in the same row sharing different letter are different at P<0.05

The rate of pH reduction of crops is the function of level of RFC and presence of epiphytic lactic acid bacteria in the plants before ensiling. If sufficient lactic acids bacteria are present but RFC content is low at ensiling, the result is a slow reduction in pH. In the present study, the pH of additives treated Chromolaena silages were significantly (P < 0.05) lower than control and molasses fermented silage resulted inthe lowest pH value followed by cassava fermented silage. The low pH values found in additives fermented silages, especially in molasses and cassava fermented silages may be attributed to the high concentration of RFC levels in the both additives. The low pH values of molasses treated silages was in in agree with Man and Wilktorsson (2002) as well as McDonald et al (1991). The pH might be lowered by rapid production of lactic acid caused by molasses and cassava tuber flour providing RFC for lactic acid bacteria.

Attainment of low pH and increased lactic acid content are the important determinant factor forfinal silage fermentation quality. In the present study, addition of molasees and cassava flour significantly (P < 0.05) increased lactic acid content of silage. RFC in additives, especially in molasses and cassava tuber flour might provide subtrat for lactic acid bacteria that increased its population density, resulting in increased lactic acid concentration and low pH. Besides by its higher RFC content, the higher value of lactic acid on the cassava flour treatment might be due to some starches in cassava flour were converted to soluble sugar and lactic acid (Chedly and Lee, 1998). The increase of lactic acid content as adding molasses had been reported by Yokota et al (1992) and cassava tuber meal by Panditharatne et al (1986).

Fermenation by lactic acid is preferred over other acids because of its lower disassociation constant, therefore it is the major organic acid responsible for decreasing silage pH. According to Catchpoole and Henzel (1971), good quality silage has a pH value of 4.2 or below, lactic acid between 3 and 13% and butyric acid concentration less than 0.2%. Based on their standard, the results presented in Table 2 show that from viewpoint of pH and lactic acid content, silage fermentation quality in this study was suboptimal.

The amount of NH3-N reflects how much of plant protein has been degraded by unwanted microbial activity. The uwanted protein degradation in silage in mainly done by proteolytic clostridia but also by enterobacteria. In this sudy, silage NH3-N concentration was significantly lower in molasses and cassava tuber flour treated Chromolaena silage compared to corn meal and rice bran treated silages. The high content of RFC in molasses and cassava tuber flour may reduced pH more rapidly that reducing proteolysis activity. McDonald et al (1991) reported that well-preseved silage is considered to be good with less than 100 g NH3-N/kg total nitrogen. In this study, protein degradation might was extensive because NH3-N concentration was above 10 %.for all treatments.

Chemical composition of pre-ensiled vs post-ensiled Chromolaena leaves

Chemical composition of pre-ensiled and post-ensiled Chromolaena leaves are shown in Table 1 and Table 3, respectively.

Table 3. Chemical composition of post ensiled Chromolaena leaves as influenced by additives (dry matter basis)
Parameters Control Cassava tuber flour Corn Meal Rice bran Molasses SEM Prob.
Dry matter 16.6a 18.5b 17.6ab 17.5ab 18.0b 0.96 4.92
Crude protein 21.7a 25.1c 23.1b 23.0b 25.6c 0.67 0.00
NDF 55.7b 48.7a 52.7ab 51.4ab 49.5a 0.25 0.00
ADF 45.4b 39.3a 43,3b 43.6b 40.1a 1.76 0.00
IVDMD 48.2a 52.2b 53.2b 52.8b 54.4b 0.84 0.00
Nitrate 0.40
abcMeans in the same row sharing different letter are si gnificantly different at P < 0.05

Table 1 shows that dry matter content of Chromolaena silages averaged 19.3% before ensiling and reduced to 16.6% after 30 days fermentation period (Table 3). This reduction in dry matter may be due to fermentation activity that degraded nutrients and produced gasses, seepage and spoilage. During ensiling, some amounts of dry matter, mainly in the form of sugar and crude protein may be broken down for the metabolism of silage microorganism as well as for cell respiration, before anaerobic conditions are established (Tuah and Addai 1986). The low dry matter content of post-ensiled than pre-ensiled plants also had been reported by Baytok and Aksu (2005) in corn and by Panditharatne et al. (1986) in Panicum maximum.

Compared to pre-ensiled, ensiling Chromolaena reduced crude protein contentby 4.20%. This might due some crude proteins are breadown into small soluble compounds including peptide, amino acid and ammonia (Meiteka, 2008). Athough crude protein reduction occurred, however, the crude protein contents in pre-ensiled and post-ensiled Chromolaena were more than 8% DM, which according to (Norton1994), can provide ruminal ammonia above the minimum level required by rumen microorganism to support optimum growth.

The most common change in the chemical composition during ensiling are reduction in crude protein and increase in NDF and ADF contents (Kim et al 2009). In this study, compared to fresh Chromolaena leaves, ensiling increased NDF and ADF contents by 4.20% and 11.8%, respectively. This increased NDF and ADF contents of ensiled Chromolaena might be caused by a large reduction in soluble sugar and crude protein and a little change in fibre content during fermentation.The greater increase in ADF than NDF concentrationsof post ensiled Chromolaena in this study is indicative of a decreased in hemicellulose fraction, because hemicellulose is NDF minus ADF. This was in line with Yahaya et al ( 2001) that hemicellulose losses during fermentation was higher than cellulose losses.

Ensiling can reduce nitrate level in feed. In this study, ensiling reduced nitrate content of Chromolaena by 55%. This value was in agree with Koch (2014) that ensiling will reduce nitrate through fermentation as much as 50 – 60%, but lower than reported by Hartwig and Bernhart (2014) that ensiling process reduced nitrate by 80 -90%. Nitrate content of ensiled Chromolaena in this study was not too safe to pregnant livestock (Cash et al 2014) as the safe level is 0.15% or less (Olson et al 2014), so it is not recommended using ensiled Chromolaena as sole diet.

Chemical composition of post-ensiled Chromolaena

Dry matter contents of ensiled Chromolaena were significantly higher (P<0.05) in additives treated silages than control, and this probably partly, attibuted to the high dry matter content of additives. More DM recovery with additives may also due to addition of RFC that improved fermentation characteristics. Once silage gains stability, then there is no more fermentation and reduction in DM is prohibited. Sharp et al (1994) indicated that increased DM recovery may have been due to homolactic fermentation which decreased fermentation losses. Lactic acid production mayreduce the carbon loss which resuts in more DM recovery.

The additives treated silages had significantly (P < 0.05) higher crude protein content than control. This increased crude protein content may be due to relatively higher protein content of additivesand a rapid drop in pH that inhibited proteolytic activity and the NH3-N produced helps in getting the aerobic stability because of its fungicidal properties (Kung et al 2000). Liebensperger and Pitt (1987) also pointed out that a rapid decrease in pH inhibits clostridial fermentation and hydrolisis of plant protein by plant enzymes. Morever, due to stability of silage, bacteria that contain more than 75% true protein, become a part of silage.

The proportion of NDF and ADF declined with additon of additives, especially in molasses and cassava tuber flour treated silage. Additives may enhance cell wall degradation due to increased silage fermentation caused by the high RFC content and lower NDF and ADF in molasses and cassava tuber flour. The IVDMD of silages was also improved by adding additives. This could be attributed to reduced NDF and ADF contents in silages by addition of additives. The results are in agreement with Nayigihugu et al (1995) who observed that increasing molasses levels lowered pH, NDF, ADF and increased in vitro digestibility of grass. The results of this study indicated that, RFC additive is not only to improve fermentation of silage but may also improving proportion of silage that can be digested by animals.



The author would like to express gratitude to Direcorate General of Higher Education, Ministry of Education and Culture Republic of Indonesia for providing funds for this research.


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Received 25 October 2014; Accepted 4 March 2015; Published 1 April 2015

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