Abstract: Background: Intercropping involves growing two or more crops on the same piece of land to produce rations for livestock, particularly ruminants. In this study, the silage quality of in situ rations produced from Sorghum intercropped with Indigofera was evaluated to determine which Sorghum variety produced the best silage. Methodology: The pH, Neutral Detergent Fiber (NDF), Acid Detergent Fiber (ADF), ammonia-N (N-NH3), Volatile Fatty Acids (VFA) and total bacteria in silage for use in situ rations were verified. Experiments were conducted using a Completely Randomized Design (CRD) with three replications of three factors: (1) Sorghum variety (Patir-37 and Citayam-33), (2) Indigofera composition (30, 40 and 50% Indigofera ) and (3) Microbial inoculant (Lactobacillus plantarum, Lactobacillus casei and non-microbial inoculant). Data were analyzed using analysis of variance and HSD test. Results: For all rations tested, the pH and N-NH3 values indicated good ensilage. Rations that included the Sorghum variety Citayam-33 had lower pH and N-NH3 production relative to those with Patir-37. In whole crop silages, the inoculants did not significantly affect fermentation. Meanwhile, rations with higher amounts of Indigofera (up to 50%) had lower NDF and ADF. Conclusion: Together the results show that in situ rations made from intercropped Sorghum and Indigofera ensilage well and different compositions can be obtained directly from intercropped fields to produce rations that improve ruminant performance.
INTRODUCTION
Inconsistent supplies of reliable, high-quality forage, especially during the dry season are a major constraint to improving ruminant performance. About 70% of forages used by farmers originate from local grasses that have low protein content (7-9%), high amounts of crude fiber and low digestibility. Rations that have crude protein contents below 6-7% depress microbial activity in the rumen due to a lack of N1. As such, farmers often add nutritional concentrates to ruminant rations to improve feed quality and nutrient intake. However, these concentrates are associated with regional increases in the price of raw feed materials and also have varying qualities. One alternative to these concentrates for complementing the low protein content of grass-based rations is legume forage that has high contents of crude protein, vitamins and specific minerals2 such as P and Ca.
Sorghum (Sorghum bicolor L.) is a cereal plant of the Gramineae family that has great potential for supplementing fodder resources because of its wide adaptation, rapid growth and high green and dry fodder yields, as well as its high ratoon capacity. As such, Sorghum can be grown on poorer quality land to produce ruminant forage. Citayam and brown midrib (BMR) strains are genetically mutated Sorghum that have superior agronomic traits. One important Sorghum cultivar is the BMR mutant line like Patir-37, which has higher dry matter production and lower lignin content and is comparable to the BMR mutant3 line Patir-3.1.
Legume forage has a high crude protein content (20-30%)4 and is frequently used in ruminant feed. Indigofera legumes such as Indigofera zollingeriana have good growth with high production and nutritive value5-7. Moreover, incorporation of Indigofera zollingeriana increased the protein content, dry matter degradability and Volatile Fatty Acid (VFA) values of rations in an in vitro rumen model8.
Legume and Sorghum crops are often grown separately and mixed at the time of feeding. Yet these crops can be grown simultaneously using intercropping, which is one of the most common cultivation practices used in sustainable agricultural systems and plays an important role in increasing land productivity and yield stability9. Furthermore, intercropping can conserve soil water by providing shade, buffering winds and increasing infiltration with mulch layers that improve soil structure. The enhanced productivity of multi-species agroecosystems through intercropping relative to that of monospecific agroecosystems (i.e., each species is grown alone) can be explained by complementarity and facilitation processes that result in improved resource use9.
Tropical forages are abundant during the wet season but usually are unavailable during the dry season. If forage is not harvested and consumed during the wet season, it will continue to grow and the nutritive value will decrease as the plants become more fibrous and lignified. Excess tropical forage available during the wet season can be conserved as silage for use during the dry period through ensiling10. The aim of this study was to determine the effect of ensiling on silage quality of rations produced from crops grown using intercropping of Sorghum and the legume Indigofera, as well as determine the composition of rations that optimizes nutritional value.
MATERIALS AND METHODS
Silage was produced from harvests of the Sorghum mutant lines Citayam-33 and BMR Patir-3.7 that were grown with Indigofera legumes in an intercropping system used at the research farm station of the Bogor Agricultural University Jonggol Animal Science Teaching and Research Unit of the Faculty of Animal Science. Lactobacillus plantarum and Lactobacillus casei were used as inocula (isolates from the Laboratory of PAU IPB).
Sorghum and Indigofera biomass were harvested simultaneously when 80% of the Sorghum was flowering (~90 days after planting). Sorghum plants were defoliated above the first node from the soil surface (~10 cm above ground). Indigofera plants were defoliated 100 cm above the soil surface. Whole Sorghum and Indigofera plant matter (stem, leaf and grain) was chopped into 2-3 cm lengths and then wilted for 12 h separately. Silage proportions were adjusted per 10 kg silage and active Lactic Acid Bacteria (LAB) were sprayed onto the silage at 1 mL kg–1 of forage or about 106 CFU g–1. Polyethylene garbage bags (15 kg capacity) were used as silos and covered by 3 layers bags to prevent leaks. A vacuum pump was used to remove the air from the bags to produce an anaerobic atmosphere.
The mixtures were fermented for 30 days, after which the silage qualities were assessed. Immediately upon opening the bags, the pH of 25 g of each silage mixture mixed with 100 mL distilled water was determined using a glass electrode coupled to a pH meter. Silage fiber components, including Neutral Detergent Fiber (NDF) and Acid Detergent Fiber (ADF) were determined using the methods described by van Soest. Ammonia-N (N-NH3) concentrations were determined by micro-diffusion using the Conway method. The VFA was analyzed using gas chromatography and the total bacteria were counted using the Total Plate Count (TPC) method.
Experiments were conducted at the Field Laboratory, Faculty of Animal Science, Bogor Agricultural University [Jonggol Animal Science Teaching and Research Unit (UP3J)] between November, 2014 and April, 2015. The study used a completely randomized factorial design with three factors (2×3×3) and three replicates. The first factor was Sorghum variety [Patir-37 (S1) and Citayam-33 (S2)]. The second factor was Indigofera composition [30% Indigofera (I1), 40% Indigofera (I2) and 50% Indigofera (I3)]. The third factor was microbial inoculant [L. plantarum (B1), L. casei (B2) and non-bacterium (B3)]. The data were then statistically analyzed using analysis of variance (ANOVA) by means of MINITAB (Version 16). Honestly Significant Difference (HSD) was applied to determine the difference among treatments. Differences were considered significant at p<0.05.
RESULTS
pH and N-NH3: The pH of the silage ranged between 3.80 and 4.53, whereas, the N-NH3 values were between 0.958 and 1.557 mM (Table 1). These values indicate that the ensilage of the treatments was effective. There were significant differences (p<0.01) in pH and N-NH3 among the treatments with different Sorghum varieties wherein silage composed of Citayam-33 had lower pH and N-NH3 values than did Patir-37. There were no significant differences (p>0.05) in pH and N-NH3 among compositions of Indigofera, microbial inoculant type and the interaction between these factors.
NDF and ADF: Acid Detergent Fiber (ADF) and Neutral Detergent Fiber (NDF) are frequently used as standard forage testing techniques for fiber analysis. The average NDF of the silage ranged between 58.55 and 65.23% and the average ADF ranged between 43.72 and 50.04% (Table 2). There were significant differences (p<0.01) in the NDF and ADF contents among the silage with different Indigofera amounts such that silage with 30% Indigofera had higher NDF and ADF content than did silage with 40 and 50% Indigofera. Meanwhile, there were non-significant differences (p>0.05) in NDF and ADF among the different Sorghum varieties, bacterial inocula and among all interactions between these factors.
Total bacteria and VFA: Total bacteria in the silage ranged between 8.27 and 8.72 log10 CFU mL–1 and the total VFA was between 54.99 and 72.03 mM (Table 3).
| Table 1: | pH and N-NH3 in situ ration silage |
Means in the same column and species with different superscripts in uppercase highly differ significantly (p<0.01) and means in the same column and species with different superscripts in lowercase differ significantly (p<0.05), LP: Lactobacillus plantarum, LC: Lactobacillus casei, NB: Non-bacterium |
|
| Table 2: | NDF and ADF in situ ration silage |
Means in the same column and species with different superscripts in uppercase highly differ significantly (p<0.01) and means in the same column and species with different superscripts in lowercase differ significantly (p<0.05), LP: Lactobacillus plantarum, LC: Lactobacillus casei, NB: Non-bacterium | |
| Table 3: | VFA and total bacteria of in situ ration silage |
| LP: Lactobacillus plantarum, LC: Lactobacillus casei, NB: Non-bacterium | |
There were non-significant differences (p>0.05) in VFA and total bacteria content among the treatments with different Indigoflera compositions, microbial inoculants and the interaction between these factors. In whole crop silages, the finding that inoculants did not significantly affect fermentation could be due to the higher numbers of epiphytic LAB used and the good ensiling characteristics of the crop materials.
DISCUSSION
All silages examined in this study had pH<4, which is required for stability during fermentation. In well-preserved silages, general practice specifies that pH values should be <4.5. Ensiling is a preservation method for most crops that is based on natural lactic acid fermentation under anaerobic conditions, whereby Lactic Acid Bacteria (LAB) convert Water Soluble Carbohydrates (WSC) into organic acids, mainly lactic acid. The lactic acid concentration of inoculated silages typically ranges between 83 and 85.9% of total silage acid11. Successful silage production depends on anaerobic storage of material that contains adequate levels of WSC that can be fermented by LAB into lactic acid, which preserves the materials due to a rapid reduction in pH. Thus to improve LAB growth, adequate amounts of Water Soluble Carbohydrate (WSC) should be available as a fermentation substrate for LAB or aerobic bacteria during ensiling12. The pH value of silage produced from Sorghum Citayam-33 intercropped with Indigofera was lower than that for Patir-37, likely because of higher amounts of WSC in the former treatment. Meanwhile, our finding that the inoculant type did not influence the silage pH was consistent with earlier studies using maize silage13.
The N-NH3 content of the inoculated silage was between 0.29 and 0.43 mM11. The N-NH3 content in silage reflects the degree of protein degradation and is an indicator of the total amount of N degraded during ensiling. As such, the N-NH3 content of the silage can be used to determine its quality. In well-preserved silages, the N-NH3 content is typically below 100 g kg–1 total N. The best silage has NH3 <50 g N kg–1 total N whereas, good silage has NH3 contents between 50-100 g N kg–1 total N. High concentrations of ammonia arise from excessive protein breakdown in the silo that is caused by a steady decrease in pH or excessive Clostridia or enterobacteria growth. In general, silage with higher water contents has higher ammonia concentrations. The ammonia concentration of silage is also an indicator of silage crop damage, because ammonia can increase silage pH. The higher pH value we saw for Patir-37 Sorghum treatments was likely due to its higher N-NH3 content. During ensiling, plant proteases degrade proteins to peptides and free amino acids, which are in turn degraded to ammonia and non-protein nitrogenous fractions largely by Clostridia proteases. The N-NH3 is an indicator of the proportion of total N that has been completely degraded during ensiling and can be used to assess secondary fermentation. The lower content of N-NH3 of silage produced from Citayam-33 intercropped with Indigofera was likely due to its low pH value that would inhibit the growth of proteolytic Clostridia and reduce the amount of protein degradation.
The NDF approximates the total cell wall constituents including hemicellulose, whereas, ADF primarily represents cellulose and lignin. The NDF and ADF represent the fibrous portions of plant material and influence digestibility and energy availability from forage. As such, NDF and ADF can be used to predict intake potential and calculate digestibility, respectively. As the fiber content increases, the forage quality declines. The dynamics of ADF content are consistent with that of the NDF content during generative development of plants. As plants mature, the crude protein decreases concurrent with increases in starch and NDF14. Here the NDF values ranged from 54.30-61.28% and ADF ranged from 31.72-38.40%15. The NDF and ADF values were highest for the treatment with 30% Indigofera, which had a higher structural carbohydrate concentration relative to treatments with 40 and 50% Indigofera. Mature plants are typically higher in fiber and have lower Non-Structural Carbohydrate (NSC) content relative to immature plants. As such, environmental conditions that restrict growth (NSC utilization) to a greater extent than photosynthesis (NSC synthesis) would increase the amount of NSC in plant herbage. Starch, a storage carbohydrate, is present in low amounts in young vegetative tissues (tiller to flowering stages) and then increases during maturation14, the fiber fraction of plants that excludes hemicellulose from the total fiber can be expressed as Acid Detergent Fiber (ADF). Relative Feed Value (RFV) increases as the fiber (NDF or ADF) values decline. The NSC content and type depends on the plant species, plant part and development stage, as well as environmental conditions such as root and shoot temperature during growth. Other factors that affect NSC are light intensity and duration, plant nutrient availability and water status14.
The main source of energy for ruminants is VFA originating from microbial fermentation of carbohydrates in the rumen. The VFA are absorbed from the rumen wall into the circulation. Lower VFA values indicate a lower rate of carbohydrate degradation by microbes. In this study, the total VFA produced was still below normal levels (70-150 mM)4. The treatments did not have different effects on VFA production due to the overhaul of silage dry matter. During both fermentation and respiration, organic matter is hydrolyzed into CO2, H2O and energy. The EFSA16 reported that additives containing Saccharomyces cerevisiae, Lactobacillus plantarum and Lactobacillus casei did not improve silage production. Moreover, the total VFA for legume silage mixed with Citayam and BMR 36 Sorghum was 52.5 and 59.28 mM, respectively17.
Thomas et al.15 reported that total LAB counts for silage grass-legume mixtures ranged from 2.84×109 CFU mL–1 with legume to 1.62×109 CFU mL–1 without legume, the addition of LAB to 40% legumes resulted in lower bacterial counts (2.53×107 to 3.9×107 CFU mL–1). Legumes with 70% LAB ranged from 2.84×108 to 5.98×108 CFU mL–1 and the total bacteria was between15 5.76 and 8.76 log CFU g–1. Higher numbers of LAB can arise from high levels of WSC in the silage. The higher bacterial population could also be due in part to an increase in the numbers of amylolytic bacteria when more fermentable substrates are available. Bacterial proliferation rates increase with increasing supplies of carbohydrates that are more readily fermentable18. The count of epiphytic LAB, yeast and enterobacteria was approximately 108 CFU g–1 in the crop material. Therefore, bacteria in the inoculants would not dominate the ensiling process or affect fermentation before the pH declined to values needed for silage stability. Biochemical differences in plant tissue composition could affect the composition of microbial communities in silage19. Furthermore, the pH from lactic acid production can inhibit the growth of other microbes such as yeast, bacilli, enterobacteria and Clostridia and eventually even LAB themselves20.
CONCLUSION
Citayam-33 Sorghum had lower pH and N-NH3 production than did Patir-37 Sorghum. The silage pH ranged between 3.80 and 4.53 and the N-NH3 content was between 0.958 and 1.557 mM, indicating that the treatments ensilaged very well. In whole crop silages, inoculants did not significantly affect fermentation because of the higher numbers of epiphytic LAB and the good ensiling characteristics of this crop. Incorporation of up to 50% Indigofera resulted in lower NDF and ADF. The results also revealed that a combination of Sorghum varieties with different Indigofera compositions and inoculant bacteria in an intercropping system did not affect VFA or total bacteria levels.
SIGNIFICANCE STATEMENT
A comprehensive study of silage for use in in situ rations made from intercropped Sorghum and Indigofera will be beneficial for improving ruminant performance. Ensilage of the different treatments was good and could produce a healthy rumen environment with a pH that optimizes microbial activity. Improving microbial activity in the rumen will enhance feed intake, digestibility and feed metabolism. Together these factors can contribute to better production performance and efficiency as well as animal health. Commercial in situ rations produced from intercropped Sorghum and Indigofera could also increase ruminant industry profits and support sustainability of ruminant production.
ACKNOWLEDGMENT
The authors would like to thank Directorate General of Higher Education of Indonesia for Postgraduate Scholarship (BPPDN) and supporting this study through Doctoral Grant-Programs (Project ID DIPA No. 023.04.1.673453/2015).