INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNO V A T ION (IJRSI)

ISSN No. 2321-2705 | DOI: 10.51244/IJRSI |V o lume XII Issue XI November 2025

Effect of the Ratio Between Corn Forage and Red Calliandra on Ph,

Lactic Acid, Ammonia, and Organic Matter Changes in Silage

Putri Ayu Winenggar¹, Iin Susilawati², Ujang Hidayat Tanuwiria²

¹Student at the Faculty of Animal Husbandry, Department of Animal Nutrition and Feed Technology,

Padjadjaran University

²Faculty of Animal Husbandry, Department of Animal Nutrition and Feed Technology, Padjadjaran

University, Sumedang, Indonesia

DOI: https://dx.doi.org/10.51244/IJRSI.2025.12110091

Received: 21 November 2025; Accepted: 29 November 2025; Published: 09 December 2025

ABSTRACT

The objective of this study was to determine the effect of corn stover and red calliandra ratios on pH, lactic acid,

ammonia, and organic matter (OM) change of silage. A Completely Randomized Design (CRD) was used with

four treatments and five replications, consisting of P1 (90% corn stover + 10% red calliandra), P2 (80% corn

stover + 20% red calliandra), P3 (70% corn stover + 30% red calliandra), P4 (60% corn stover + 40% red

calliandra). All data were analyzed using analysis of varians (ANOVA) followed by Duncan’s multiple range

test. The observed variables included pH, lactic acid, ammonia, and changes in silage organic matter. The results

showed that the ratios of corn stover and red calliandra had no significant effect on pH, lactic acid, ammonia or

changes in silage organic matter (p > 0,05). The best treatment was obtained in P1 (90% corn stover + 10% red

calliandra), which produced the lowest pH (4,07), the lowest ammonia level (4,68%), and the smallest organic

matter loss (-0,35%), indicating slightly more efficient fermentation. It can be concluded that the combination

of corn stover and red calliandra produces silage with relatively uniform chemical quality, while the ratio of 90%

corn stover and 10% red calliandra tends to provide the most optimal fermentation outcome.

Keywords: Corn stover silage, Red calliandra, Fermentation quality, Ammonia concentration, Organic matter loss.

INTRODUCTION

The availability of reliable and nutritious forage is a key factor influencing the productivity of ruminant animals.

Seasonal fluctuations, particularly during dry periods, often cause a reduction in forage quality and quantity.

These conditions create challenges for farmers, making preservation methods such as ensiling an important

option to maintain feed value over extended storage periods [1]. Ensiling depends on an anaerobic fermentation

process in which lactic acid bacteria utilize water-soluble carbohydrates to form lactic acid, reduce pH, and limit

the growth of undesirable microorganisms [2]. The efficiency of this process varies according to the

characteristics of the material being ensiled, including dry matter content, carbohydrate supply, and the stability

of anaerobic conditions throughout fermentation. Key chemical indicators such as pH, lactic acid, ammonia, and

organic matter change provide a comprehensive evaluation of these fermentation processes and are widely used

to assess overall silage quality.

Corn stover (Zea mays) is widely used as a silage material because it is available in large quantities and contains

fermentable carbohydrates that support lactic acid bacteria activity [3]. Despite this advantage, its crude protein

level is relatively low, making it less ideal as a standalone substrate. Red calliandra (Calliandra calothyrsus), on

the other hand, is a leguminous plant with higher protein content and rumen-resistant protein fractions [4]. Its

main limitation comes from its tannin content, which can suppress microbial activity and potentially slow

fermentation. Combining these two materials may help balance their strengths and weaknesses, resulting in more

stable fermentation.

Page 988

ww w .rsisinternational.org

INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNO V A T ION (IJRSI)

ISSN No. 2321-2705 | DOI: 10.51244/IJRSI |V o lume XII Issue XI November 2025

Based on these considerations, evaluating silage formulated from corn stover and red calliandra is necessary.

This study investigates how different ratios of the two forages affect silage pH, lactic acid content, ammonia

concentration, and changes in organic matter during fermentation.

MATERIAL AND METHODS

This research was conducted at the Ruminant Nutrition and Animal Feed Chemistry Laboratory, Faculty of

Animal Husbandry, Padjadjaran University, Indonesia for 1 months.

Tools and Materials

The tools and materials used in this study included 90-day-old corn stover, red calliandra, molasses, distilled

water, 10-liter plastic jars, 40×60 cm plastic bags, an 8×6 m tarpaulin, a vacuum sealer, a machete, a Five Goats

scale, a pH meter, a beaker glass, titration equipment, a drop pipette, Conway dishes, Erlenmeyer flasks, an

electric oven, a muffle furnace, an analytical balance, aluminum cups, porcelain crucibles, a desiccator, and

tongs.

Silage Production

The moisture content of the corn stover was reduced through wilting for approximately 2 hours, while red

calliandra leaves were wilted for about 24 hours to achieve an appropriate dry matter level for the ensiling

process. Both materials were then chopped using a machete into 2–4 cm pieces, weighed, and mixed according

to the treatments. Molasses diluted with water at a 1:1 ratio was added at 4% of the total fresh weight for each

treatment, and the mixture was stirred until homogeneous. The mixed materials were placed into plastic-lined

jars and compacted to remove air. The jars were tightly sealed and fermented at room temperature for 22 days.

After fermentation, the silage was opened and evaluated for quality by measuring pH, lactic acid concentration,

ammonia (NH₃) content, and changes in organic matter as indicators of fermentation success.

Research Methods

The study was conducted using a Completely Randomized Design (CRD) with 4 treatments and 5 replicates,

resulting in a total of 20 experimental units. The treatments carried out included:

P1 = 90% Corn stover + 10% red Calliandra

P2 = 80% Corn stover + 20% red Calliandra

P3 = 70% Corn stover + 30% red Calliandra

P4 = 60% Corn stover + 40% red Calliandra

Observed Variables:

Silage pH

Silage pH was measured to determine the acidity level as a primary indicator of fermentation quality. The pH

was recorded directly inside the fermentation jars using a digital pH meter calibrated with pH 4.0 and pH 7.0

buffer solutions. Measuring pH under actual in-jar conditions provides an accurate representation of the

fermentation environment and reflects the extent of lactic acid–driven fermentation and overall silage stability.

Lactic Acid

Lactic acid concentration was determined to evaluate the intensity of fermentation by lactic acid bacteria (LAB).

The analysis followed an alkali titration method based on Cappuccino and Sherman (1991). A10 g silage sample

was homogenized with 10 mL distilled water, heated to remove dissolved CO₂, and subsequently cooled. A few

Page 989

ww w .rsisinternational.org

INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNO V A T ION (IJRSI)

ISSN No. 2321-2705 | DOI: 10.51244/IJRSI |V o lume XII Issue XI November 2025

drops of phenolphthalein indicator were added before titration with 0.1N NaOH until a stable pink endpoint was

achieved. Final titration volume was used to calculate lactic acid concentration.

Ammonia (NH₃)

Ammonia content was analyzed as an indicator of protein deamination during fermentation. The Conway micro-

diffusion technique (Conway, 1957) was employed. Silage extract (supernatant) and saturated Na₂CO₃ were

placed in the outer compartments of a Conway dish, while boric acid solution containing mixed indicators was

positioned in the central well. The sealed dish was incubated for 24 hours to allow volatilized NH₃ to be absorbed

by the boric acid solution, which was subsequently titrated with standardized H₂SO₄ (e.g., 0.005 N) until a

distinct endpoint color change occurred.

Measurement of Organic Matter (OM) Change

Organic matter (OM) change was assessed to quantify the loss of organic fraction during ensiling, representing

the extent of substrate utilization by microorganisms. OM determination followed the ash method, in which

samples were combusted at 550°C until constant weight. OM content was calculated as 100% minus ash content.

The change in OM between pre-ensiling and post-fermentation samples was computed using the formula

commonly applied in mixed-forage silage studies [4]:

(

−

)

( )

% =

ℎ

× 100

This parameter serves as an indicator of nutrient conservation efficiency throughout the fermentation process.

Statistical Analysis

Statistical analysis was tested with analysis of variance (ANOVA). If the results obtained were significantly

different (P<0.05) between treatments, a further test was conducted using Duncan's multiple range test at a

significance level of 5% to determine the differences between treatments. Data processing was analyzed using

the SPSS program.

RESULT AND DISCUSSION

Table I Result Of Effect Of Corn Stover And Red Calliandra On Ph, Lactic Acid, Ammonia And Organic Matter

Change

Parameter

Treatment

4,07 ± 0,05

1,55 ± 0,23

4,68 ± 0,54

-0,35 ± 0,81

4,22 ± 0,09

1,40 ± 0,15

5,85 ± 0,60

-1,31 ± 0,82

4,26 ± 0,13

1,81 ± 0,12

6,17 ± 1,14

-1,17 ± 0,68

4,44 ± 0,08

1,51 ± 0,26

7,12 ± 1,48

-0,74 ± 0,87

Lactic Acid

Ammonia

Organic Matter Change

Data are presented as mean ± SD. All parameters showed no significant differences among treatments based on

ANOVA (p > 0.05); therefore, superscript letters are not included.

Note: P1 = 90% Corn stover + 10% red calliandra; P2 = 80% Corn stover + 20% red calliandra; P3 = 70% Corn

stover + 30% red calliandra; P4 = 60% Corn stover + 40% red calliandra.

Page 990

ww w .rsisinternational.org

INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNO V A T ION (IJRSI)

ISSN No. 2321-2705 | DOI: 10.51244/IJRSI |V o lume XII Issue XI November 2025

The overall results showed a relatively uniform pattern across all measured parameters, including pH, lactic acid,

ammonia, and organic matter change. This uniformity indicates that the fermentation process proceeded in a

similar manner across treatments, despite the differing proportions of red calliandra. The predominance of corn

stover in the mixtures likely provided a consistent supply of fermentable carbohydrates that supported stable

lactic acid bacteria activity at all inclusion levels. The similarity among treatments also suggests that the tannin

content of red calliandra, at the proportions used in this study, was insufficient to alter the main microbial

fermentation pathways. These findings offer a general overview of the fermentation dynamics and serve as a

basis for the more detailed parameter-specific discussion presented in the following sections.

The statistical analysis showed that varying the proportions of corn stover and red calliandra did not lead to

significant changes in silage pH (p = 0.474 > 0.05). The observed pH values ranged from 4.07 to 4.44 (4.25 ±

0.03), indicating stable fermentation within the normal range for well-preserved silage, generally between 3.8

and 4.5. This stability suggests that including red calliandra up to 40% did not modify the acidification process.

This result is consistent with [5], who noted that pH tends to remain steady when sufficient water-soluble

carbohydrates are available to support lactic acid bacteria.

A similar trend has been reported in the review by [6], which states that materials with high carbohydrate content,

such as corn, typically maintain relatively consistent pH values even when mixed with other forages, as long as

anaerobic conditions are well maintained. Likewise, [7] observed that combining various forage sources with

substrates rich in water-soluble carbohydrates generally does not produce major differences in final pH because

lactic acid bacteria can still ferment the available substrate effectively. In the present study, the tannin content

of red calliandra also appeared too low to suppress LAB activity, and therefore did not result in any notable

increase in pH.

Overall, all treatments showed indications of proper fermentation. P1 recorded the lowest pH value (4.07),

reflecting a slightly stronger acidification pattern, although the differences among treatments were small and

remained within the acceptable range for good-quality silage.

Lactic Acid

The variation in corn stover and red calliandra ratios did not produce significant differences in the lactic acid

concentration of the silage (p = 0.730 > 0.05). The average values were relatively close, ranging from 1.40 to

1.81% (1.57 ± 0.07%), which shows that adding up to 40% red calliandra did not substantially modify the activity

of lactic acid bacteria during fermentation. Treatment P3 (70:30) had the highest lactic acid level, indicating a

slightly better balance between the water-soluble carbohydrates supplied by corn stover and the nitrogen

contribution from calliandra. This pattern supports the concept that lactic acid formation depends heavily on the

supply of water-soluble carbohydrates, which serve as the primary substrate for lactic acid bacteria [7].

The lack of significant differences also suggests that the calliandra proportion used in this study was not high

enough to shift the overall fermentation pattern, including any microbial inhibition potentially caused by tannins.

Similar trends have been observed in alfalfa-based silages, where changing the level of legume inclusion or

applying inoculants did not always alter lactic acid output when the availability of water-soluble carbohydrates

and other fermentation conditions remained adequate [8].

All treatments produced lactic acid concentrations within the 1–3% interval, which is considered normal for

high-quality silage [9]. This indicates that fermentation proceeded efficiently and without signs of butyric acid

development, which is typically associated with off-odors. Even though the differences were not statistically

significant, P3 showed a slight tendency toward more effective fermentation among the tested formulations.

Ammonia

The variance analysis showed that the different proportions of corn stover and red calliandra did not cause

significant changes in silage ammonia concentration (p = 0.818 > 0.05). The observed values ranged from 4.68%

Page 991

ww w .rsisinternational.org

INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNO V A T ION (IJRSI)

ISSN No. 2321-2705 | DOI: 10.51244/IJRSI |V o lume XII Issue XI November 2025

to 7.12% (5.96 ± 0.45%), indicating that protein breakdown during fermentation occurred at a low and fairly

consistent level. Such values fall within the normal range for well-preserved silage, as ammonia levels below

10% of total nitrogen generally reflect limited proteolysis and a stable fermentation process [6].

The absence of treatment differences suggests that increasing red calliandra inclusion up to 40% was not enough

to influence the extent of protein deamination. This outcome agrees with the findings of [5], who noted that

ammonia accumulation remains modest when water-soluble carbohydrates are available in sufficient amounts to

support the predominance of lactic acid bacteria. Although calliandra contains tannins that theoretically could

reduce proteolysis, their concentration in this study did not appear strong enough to create measurable effects.

Among the treatments, P1 produced the lowest ammonia level (4.68%), while P4 showed the highest (7.12%).

Even so, all values remained within acceptable limits and were statistically similar. The slightly lower value in

P1 indicates a minor tendency toward more efficient fermentation, yet the overall differences were too small to

be considered biologically important. These results demonstrate that the ratios of corn stover and red calliandra

tested in this study did not alter ammonia formation, and that fermentation remained largely driven by lactic acid

bacteria. This agrees with the review of [10], which emphasized that ammonia production is more strongly

influenced by anaerobic stability and the availability of fermentable substrates than by differences in forage

composition.

Organic Matter Changes

The variance analysis showed that different proportions of corn stover and red calliandra did not significantly

affect changes in silage organic matter (OM) (p = 0.310 > 0.05). The OM values ranged from –0.35% to –1.31%

(–0.89 ± 0.08), indicating that a portion of the organic fraction was used as a substrate during fermentation. The

small magnitude of the decrease suggests that the fermentation process ran steadily and without notable energy

loss. As noted in [6], a slight decline in OM generally reflects efficient fermentation because most of the energy

remains preserved in organic acids and digestible nutrients.

The lack of treatment differences indicates that the inclusion of red calliandra up to 40% was not sufficient to

alter the extent of OM reduction. The relatively high water-soluble carbohydrate (WSC) content in corn stover

likely remained the main factor supporting fermentation, regardless of calliandra addition. This interpretation is

consistent with [5], who reported that when WSC availability is adequate, OM losses tend to remain low because

lactic acid bacteria can operate effectively.

The highest OM reduction occurred in P2 (–1.31%), whereas P1 experienced the smallest loss (–0.35%).

Although the values did not differ statistically, the slightly lower reduction in P1 suggests a modest tendency

toward more efficient fermentation. Overall, the mixtures of corn stover and red calliandra produced stable

fermentation with minimal OM loss. This agrees with [11], who highlighted that successful ensiling is influenced

more by sufficient fermentable substrates and proper anaerobic conditions than by differences in forage

composition.

Although all parameters displayed relatively uniform patterns across treatments, this study has several limitations

that should be considered when interpreting the results. The range of red calliandra inclusion used (10–40%)

was relatively narrow, which may have limited the emergence of more pronounced biological differences among

treatments. In addition, the fermentation period of 22 days may not have been sufficient to capture changes that

typically appear during longer-term ensiling. Based on these limitations, future research is recommended to

evaluate red calliandra levels above 40%, extend the fermentation duration, and explore more diverse ensiling

conditions. Such approaches may provide a more comprehensive understanding of fermentation responses and

nutrient stability in silages combining corn stover and red calliandra.

CONCLUSION

This study demonstrated that varying the ratios of corn stover and red calliandra up to 40% did not produce

significant effects on silage pH, lactic acid concentration, ammonia levels, or changes in organic matter. All

treatments yielded parameter values within the range of good-quality silage, reflecting stable fermentation

Page 992

ww w .rsisinternational.org

INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNO V A T ION (IJRSI)

ISSN No. 2321-2705 | DOI: 10.51244/IJRSI |V o lume XII Issue XI November 2025

dominated by lactic acid bacteria. Although not statistically different, the treatment with 90% corn stover and

10% red calliandra (P1) tended to produce the lowest pH (4,07), the lowest ammonia concentration (4,68%), and

the smallest loss of organic matter (-0,35%), indicating slightly more efficient fermentation. Overall, all tested

ratios of corn stover and red calliandra were suitable for producing silage with relatively uniform chemical

quality and meeting the characteristics of well-conducted fermentation.

ACKNOWLEDGEMENT

The authors gratefully acknowledge the financial support provided by Academic Leadership Grant (ALG) under

Grant Number (3208/UN6.J/PT.00/2025). This support was instrumental in enabling the successful completion

of this research project.

REFERENCES

1. Wardana, I. K., Suryani, N. N., & Winaya, I. N. A. (2019). Strategi penyediaan hijauan pakan pada musim

kemarau. Majalah Ilmiah Peternakan, 22(2), 45–54.

2. Kung, L., & Shaver, R. (2016). Interpretation and use of silage fermentation analysis reports. University

of Delaware Extension.

3. Sahid, S. A., Ayuningsih, B., & Hernaman, I. (2021). Kualitas silase tebon jagung (Zea mays) dengan

penambahan dedak fermentasi ditinjau dari kandungan lignin dan selulosa. Jurnal Ilmu Ternak Tropis

dan Ilmu Pakan, 4(2).

4. Mudhita, I. K., Putra, R. A., Rahman, M. M., Widyobroto, B. P., Agussalim, & Umami, N. (2024). The

silage quality of Pennisetum purpureum cultivar Gamma Umami mixed with Calliandra calothyrsus and

Lactiplantibacillus

plantarum.

Tropical

Animal

Science

Journal,

47(1),

112–124.

10.5398/tasj.2024.47.1.112

5. Huo, W., Zhang, Y., Zhang, L., Shen, C., Chen, L., Liu, Q., Zhang, S., Wang, C., & Guo, G. (2022). Effect

of lactobacilli inoculation on protein and carbohydrate fractions, ensiling characteristics and bacterial

community

alfalfa

silage.

Frontiers

Microbiology,

13,

1070175.

https://doi.org/10.3389/fmicb.2022.1070175

6. Kung, L., Shaver, R., Grant, R., & Schmidt, R. (2018). Silage review: Interpretation of chemical,

microbial, and organoleptic components of silages. Journal of Dairy Science, 101(5), 4020–4033.

https://doi.org/10.3168/jds.2017-13909

7. Ma, J., Fan, X., Wu, T., Zhou, J., Huang, H., Qiu, T., Xing, Z., Zhao, Z., Yin, F., & Gan, S. (2023). Lactic

Acid Bacteria and Cellulase Improve the Fermentation Characteristics, Aerobic Stability and Rumen

Degradation of Mixed Silage Prepared with Amaranth and Rice Straw. Fermentation, 9(9), 853.

https://doi.org/10.3390/fermentation 9090853 MDPI

8. Li, Y., Xu, W., Zhang, J., & Huang, J. (2024). Inoculation of exogenous lactic acid bacteria exerts limited

influence on silage fermentation and bacterial community compositions of reed canary grass straw.

Frontiers in Microbiology. OUP Academic

9. Worku, M., Alemu, B., & Tesfaye, A. (2021). Fermentation characteristics and sensory quality of maize

silage under different moisture conditions. Animal Nutrition and Feed Technology, 21(2), 267–276.

10. McDonald, P., Henderson, A. R., & Heron, S. J. E. (2020). The Biochemistry of Silage (3rd ed.).

Chalcombe Publications.

11. Aloba, T. A., Munyaka, A. W., & Mutsvangwa, T. (2022). Effects of ensiling length and storage

temperature on the nutritional quality of ensiled forages. Animal Feed Science and Technology, 284,

115241. https://doi.org/10.1016/j.anifeedsci.2021.115241

Page 993

ww w .rsisinternational.org