Potential of Kersen Leaf Extract (Muntingia calabura L) and Basil Leaf Extract (Ocimum basilicum L.
) as Natural Antibacterial Candidates against Salmonella typhi Ae Weni et al.
Effectiveness of Diets Incorporating Nano-Zeolite and UreaImpregnated Nano-Zeolite on Fermentation Efficiency in Rumen Fluid In Vitro Dede Kardaya1.
Pia Nurmala Dewi1.
Deden Sudrajat1.
Dewi Wahyuni1.
Ikhsan Qodri Pramartaa1.
Ruslan Abdul Gopar2.
Windu Negara2.
Raden Siti Nurlaela3 Volume 6 Issue 3 (December 2.
e-ISSN 2722-6395 doi: 10.
30997/ijar.
ARTICLE INFO
Article history:
Received: 07-28-1025 Revised version received: 12-12-2025 Accepted: 12-15-2025 Available online: 12-30-2025 Keywords:
Ammonia.
in vitro rumen fermentation.
nano-zeolite.
volatile fatty acids.
VFA/NH3 ratio.
How to Cite:
Kardaya.
Dewi.
Sudrajat.
Wahyuni.
Pramartaa.
Gopar, .
A Nurlaela.
Effectiveness of Diets Incorporating Nano-Zeolite and Urea-Impregnated Nano-Zeolite on Fermentation Efficiency in Rumen Fluid In Vitro.
Indonesian Journal of Applied Department of Animal Science.
Universitas Djuanda.
Indonesia National Research and Innovation Agency (BRIN).
Indonesia Department of Halal Food Science.
Universitas Djuanda.
Indonesia
ABSTRACT
Zeolite had been widely investigated in animal feed for its ability to improve feed efficiency and reduce ammonia emissions, and nano-zeolite offered greater adsorption capacity due to its expanded surface area.
This study evaluated the effects of nano-zeolite supplementation on ammonia (NHCE), volatile fatty acids (VFA), the VFA/NHCE ratio, and pH during in vitro rumen fermentation.
A two-factor randomized block design .
with four replicates was used.
Incubation time .
, 4, 8, 12, 24, and 48 .
served as the first factor, and six dietary treatments served as the second factor: control, urea-supplemented concentrate, inactivated nano-zeolite, activated nano-zeolite, urea-impregnated inactivated nanozeolite, and urea-impregnated activated nano-zeolite.
Nanozeolite, whether active or urea-impregnated, did not significantly improve rumen fermentation efficiency based on NHCE.
VFA, or VFA/NHCE ratio.
All nano-zeolite diets performed similarly to the control and urea-only diets.
Fermentation efficiency peaked at 4 and 8 hours of incubation, indicating that nano-zeolite combined with urea supported stable fermentation, particularly during early to midincubation.
Research (IJAR), 6.
, 209Ae223.
https://doi.
org/10.
30997/ijar.
Corresponding Author:
Dede Kardaya kardaya@unida.
Indonesian Journal of Applied Research (IJAR), volume 6 issue 3 Ae December 2025 Potential of Kersen Leaf Extract (Muntingia calabura L) and Basil Leaf Extract (Ocimum basilicum L.
) as Natural Antibacterial Candidates against Salmonella typhi Ae Weni et al.
Available online at https://iojs.
id/index.
php/IJAR Copyright .
2025 by Indonesian Journal of Applied Research (IJAR) Introduction Tropical forages are widely characterized by high structural fiber content and low soluble carbohydrate and nitrogen fractions, which collectively constrain microbial degradation in the rumen (Van Soest, 2018.
Tedeschi et al.
, 2.
Under such conditions, the rate of carbohydrate fermentation slows, and microbial protein synthesis becomes limited by asynchronous availability of fermentable energy and nitrogen (Haque et al.
, 2.
Synchronizing the release of nitrogen with carbohydrate degradation is therefore essential to improve fermentation efficiency and reduce nitrogen losses (Kardaya et al.
, 2.
When nitrogen becomes available too rapidlyAisuch as from conventional ureaAirumen microbes cannot capture it efficiently, leading to excess ammonia accumulation and reduced feed utilization (Patra, 2.
Zeolite has been widely studied as a feed additive capable of modifying rumen fermentation by adsorbing ammonia, buffering pH, and improving nutrient utilization (Wang et al.
, 2025.
KozCowski et al.
, 2.
Kardaya et al.
first advanced the concept of ureaimpregnated zeolite (UZ) as a slow-release urea (SRU) agent.
This approach enabled a controlled release of ammonia during in vitro fermentation, reducing nitrogen losses and stabilizing pH.
Subsequent vivo research further confirmed that UZ supplementation in sheep improved fermentation patterns by lowering ammonia and acetate concentrations while enhancing propionate production and maintaining normal plasma urea levels (Kardaya et al.
Nguyen & Dang, 2.
Recent advances in nanotechnology have opened new possibilities for enhancing the functional properties of mineral-based feed additives.
Nanomaterials possess extremely large surface areas, high porosity, and enhanced ion-exchange capacity, which can alter chemical reactivity and adsorption characteristics within the rumen environment (Duhan et al.
, 2.
In livestock systems, nanotechnology applicationsAioften referred to as agronanotechnologyAihave shown promise for improving nutrient delivery, reducing environmental emissions, and enhancing feed efficiency (El-Nile et al.
, 2021.
El-Saadony et , 2.
When zeolite is processed to the nanoscale, its adsorption capacity for cations, including ammonium, increases markedly compared to the conventional form (Kardaya et al.
Despite the known benefits of zeolite, studies specifically evaluating nano-zeolite in rumen fermentation remain limited.
El-Nile et al.
reported that nano-zeolite improved the rumen fermentation pattern and nutrient digestibility in ruminants.
however, results have varied across diet types and experimental conditions.
The nanoscale structure enables more efficient adsorption of urea and ammonia, raising the possibility that nano-zeolite could serve as an improved alternative to conventional slow-release urea technologies (Mousa et al.
Moreover, nano-zeolite may influence fermentation by supporting microbial growth, stabilizing rumen pH, and reducing nitrogen waste through enhanced sequestration of free ammonia (Arshad et al.
, 2.
Given the diverse capabilities of nano-zeolites, there is an expectation that incorporating them into diets could enhance the efficiency of rumen fermentation while mitigating its adverse effects.
Consequently, it is essential to perform in vitro experiments to assess ammonia (NH.
Volatile Fatty Acid (VFA) concentrations, and pH levels, which are crucial indicators of microbial fermentation efficiency in the rumen.
This research seeks to evaluate how nano-zeolites in diets influence the production of ammonia (NH.
VFA, and the pH of rumen fluid in vitro.
Indonesian Journal of Applied Research (IJAR), volume 6 issue 3 Ae December 2025 Potential of Kersen Leaf Extract (Muntingia calabura L) and Basil Leaf Extract (Ocimum basilicum L.
) as Natural Antibacterial Candidates against Salmonella typhi Ae Weni et al.
Methods The National Research and Innovation Agency's Ethics Commission for Animal Maintenance and Use has granted its approval and authorization for all procedures in this study, following the Research Ethics Clearance in the Field of Livestock and Utilization, with reference number 203/KE.
02/SK/08/2024.
The ingredients used consisted of six types of experimental diets, rumen liquid.
CO2 gas, and saturated HgCl2.
NaHCO3 9.
80 g/L.
Anhydrous Na2HPO4 3.
71 g/L.
KCl 0.
57 g/L.
NaCl 47 g/L.
Heptahydrate MgSO4 0.
12 g/L.
Dihydrate CaCl2 0.
05 g/L, and urea .
eagent grad.
90 g/L, supernatant.
Na2CO3 saturated.
HCl 0.
01 N, mixed indicator solution, boric acid solution.
H2SO4 15%, supernatants.
NaOH 0.
5 N, aquadest.
PP indicator 1%, and HCl 0.
5 N.
Table 1 displays the composition of the feed ingredients included in the diet, while Table 2 outlines the nutrient content.
Table 1 Feed ingredients that make up the diet and nutrient content of the proximate analysis Ingredients Field Grass Pollard Yellow Corn Soybean Meal Coconut Meal Molasses Inactive nano-zeolite Activated nano-zeolite Urea Urea-impregnated inactive nano-zeolite Urea-impregnated nano-zeolite Squirt Total Dry matter basis (%) Table 2 Nutrient contents of diets based on proximal analysis Nutritional Content Dry Matter (%) Crude Protein (%) Crude Fat (%) Crude Fiber (%) Ash (%) NFE (%) * DE (Kcal/k.
* ME (Kcal/k.
* R1= Control diet without supplementation.
R2= Urea-supplemented concentrate diet.
R3= Inactivated nanozeolite supplemented concentrate diet.
R4= Active nano-zeolite supplemented concentrate diet.
R5= Ureaimpregnated inactive nano-zeolite supplemented concentrate diet.
R6= Urea-impregnated active nano-zeolite supplemented concentrate diet.
*) Calculation results: NFE (Nitrogen Free Extrac.
= %BK Ae (%PK %LK %SK .
DE (Digestible Energ.
ME (Metabolizable Energ.
= 0.
82 x DE (NRC 2.
Indonesian Journal of Applied Research (IJAR), volume 6 issue 3 Ae December 2025 Potential of Kersen Leaf Extract (Muntingia calabura L) and Basil Leaf Extract (Ocimum basilicum L.
) as Natural Antibacterial Candidates against Salmonella typhi Ae Weni et al.
Research Methods This research employed a 2-factor Randomized Block Design (RBD) structured as 6 x 6 with 4 repetitions.
The first factor involved the incubation duration, which had 6 different time periods .
, 4, 8, 12, 24, and 48 hour.
, while the second factor was the experimental diet, also comprising 6 variations, resulting in a total of 168 experimental units.
The experimental diet treatments included 6 types: a control diet without any supplementation (R.
, a concentrated diet with added urea (R.
, a concentrated diet with inactivated nano-zeolite (R.
, a concentrated diet with active nano-zeolite (R.
, a concentrated diet with inactivated nanozeolite combined with urea (R.
, and a concentrated diet with active nano-zeolite combined with urea (R.
The study focused on observing variables such as ammonia production (NH.
VFA, the VFA:NH3 ratio, and pH levels.
The collected data were subjected to multivariate General Linear Model (GLM) analysis using the IBM SPSS 27 software on the variables studied.
If a significant difference was detected (P<0.
, a subsequent analysis was performed using the Duncan Multiple Range Test.
Research Procedure Preparation of Nano-zeolite and Urea Impregnated Nano-zeolite The process of creating urea-impregnated nano-zeolite begins with the preparation of natural zeolite, which is ground into a fine powder with a mesh size of 200.
Zeolites are divided into two categories: inactive and those activated at 600 AC.
To make urea-impregnated zeolite, 310 grams of urea are dissolved in distilled water at temperatures between 80 and 119 AC.
Once the urea is dissolved, zeolite is added to the mixture and stirred thoroughly until it cools, after which it is ground again.
After separating the zeolite based on treatment, its size is further reduced using nanotechnology.
These processed nano-zeolites are then incorporated into the Sample Preparation The sample was composed of a blend of 55 g of forage and 45 g of concentrate, measured on a dry matter basis, which were then mashed together.
A quantity of 0.
5 g of these samples was placed into a 100 ml glass bottle, labeled according to the test and sample treatment.
Intake of Rumen Fluid Rumen fluid is obtained from fistulated cattle housed at the BRIN Cibinong cattle barn.
To begin, a thermos is preheated with water at 39AC, which is then discarded before the thermos is filled with rumen fluid to maintain microbial viability.
The collection process involves sampling from different areas within the rumen, capturing both solid and liquid parts The thermos containing the rumen fluid is then taken to the laboratory, where CO2 is promptly introduced.
CO2 gas is infused into the rumen liquid inside the thermos, and the contents are mixed to ensure the solid and liquid rumen inoculum are uniform, followed by filtration through two layers of gauze.
CO2 gas is continuously added for 30 seconds throughout the procedure (Camacho et al.
, 2.
Preparation of McDougall Buffer Solution McDougall's buffer solution is composed of sodium bicarbonate, which acts as a McDougall buffers were selected due to their ability to create a more favorable environment for fiber digestion (Camacho et al.
, 2.
The materials for the buffer solution are measured and placed into an Erlenmeyer flask.
Then, 1.
5 L of distilled water is added, and the mixture is homogenized using a magnetic stirrer.
The CaCl2.
2H2O is introduced at the end, once all other components are fully dissolved.
After thorough mixing.
CO2 gas is passed through the solution until the pH reaches 6.
Indonesian Journal of Applied Research (IJAR), volume 6 issue 3 Ae December 2025 Potential of Kersen Leaf Extract (Muntingia calabura L) and Basil Leaf Extract (Ocimum basilicum L.
) as Natural Antibacterial Candidates against Salmonella typhi Ae Weni et al.
Following the methods described by Menke .
, the incubation media solution is created by mixing one portion of the filtered rumen fluid with two portions of McDougall's Table 3 outlines a timetable for incubation periods along with the respective amounts of the incubation media solution mixture.
Table 3 Incubation time and amount of incubation media solution mixture Incubation period Sample .
R,4U) Total Rumen fluid Buffer .
L) .
L) Ratio 1:2 Total rumen fluid buffer .
L) In each glass bottle, 0.
5 g of the sample was combined with 50 ml of incubation media solution, followed by a 30-second infusion of CO2 gas.
The bottles were then sealed with a rubber stopper and an aluminum seal using a vial crimper.
These bottles were placed in a shaking water bath at 39AC for incubation periods of 2 (I.
, 4 (I.
, 8 (I.
, 12 (I.
, 24 (I.
, and 48 (I.
Following each incubation period, the sample in the tube is mixed thoroughly using a vortex, its pH is measured, and 2 to 3 drops of saturated HgCl 2 are added to halt the fermentation process.
Subsequently, it is placed in the freezer for VFA and NH3 Ammonia Analysis (NH.
Ammonia (NH.
was examined using the Conway microdiffusion technique.
In this process, 1 ml of a saturated sodium carbonate (Na2CO.
solution is placed on one side of the cup's partition, while 1 ml of the incubation supernatant is added to the opposite side.
The center of the cup is filled with 1 ml of boric acid, which contains the indicators bromocresol green and methyl red (BCG: MR).
The cup is then securely sealed with a lid coated with The saturated Na2CO3solution and the supernatant are combined by shaking the cup, and the mixture is left undisturbed for 24 hours at room temperature.
During this period, the boric acid solution's color shifts from red to a purple-bluish hue.
After 24 hours, the sample undergoes titration with a 0.
01 N HCl solution until the solution turns bright red.
The NH3 concentration is determined using the formula:
(VaOeV.
x N HCL x 1000 N-NH3 .
M) = Va = Volume of sample titration Vb = volume of blank titration Vc = Sample volume N HCl = Normality of HCL VFA Analysis Volatile fatty acids (VFA) can be measured using a modified version of the steam distillation method outlined in the General Laboratory Procedure .
The distillation equipment.
Omni Lab Food Alyt, is used, and the D3000 is activated to allow the steam generator to heat up.
A 5 ml supernatant sample is placed into a VFA distillation tube, followed by the addition of 1 ml of 15% H2SO4.
The distillation tube is securely connected to the The distillate is collected in a 250 ml Erlenmeyer flask that already contains 5 ml of 5 N NaOH solution.
The distillation is halted once the distillate volume reaches 100 ml.
Indonesian Journal of Applied Research (IJAR), volume 6 issue 3 Ae December 2025 Potential of Kersen Leaf Extract (Muntingia calabura L) and Basil Leaf Extract (Ocimum basilicum L.
) as Natural Antibacterial Candidates against Salmonella typhi Ae Weni et al.
Two drops of a 1% phenolphthalein (PP) indicator are added to the Erlenmeyer flask with the Titration is performed using a 0.
5 N HCl solution until the solution changes from pink to colorless.
The same procedure is followed for blanks as in VFA measurements, but without using supernatants from the experimental sample.
The VFA concentration is determined using the formula:
titrant blankOev.
titrant sampl.
y N HCl y 1000 Total VFA .
M) N-HCl = Normality of HCl solution titrant blank = Quantity of HCl needed to neutralize 5 ml of NaOH .
titrant sample = Quantity of HCl titters required to condense the distillation outcome Results and Discussion Results Ammonia Production Ammonia generation serves as a useful measure for evaluating the fermentability of feed, which is linked to the digestibility of feed protein, as well as the activity and population of rumen microbes.
Monitoring the ammonia concentration in the rumen is crucial, as it significantly influences the optimal growth of rumen microorganisms.
Approximately 80% of these microbes can utilize ammonia as a nitrogen source for their development.
Table 4 below presents the ammonia production levels across different dietary treatments during the incubation period.
Table 4 Concentrations of ammonia produced under different dietary treatments during the incubation phase Hour Diets Means 37A2.
92A2.
70A2.
25A2.
25A3.
25A1.
45A2.
20A2.
67A4.
95A2.
57A2.
42A5.
00A5.
80A3.
02A3.
07A4.
12A4.
02A2.
37A2.
47A3.
85A3.
73A0.
83A1.
13A0.
03A0.
90A0.
33A0.
32A0.
42A4.
67A2.
05A2.
55A3.
42A2.
92A1.
84A2.
32A2.
82A1.
87A3.
70A5.
45A3.
80A0.
82A3.
R1= Control diet without supplementation.
R2= Urea-supplemented concentrate diet.
R3= Inactivated nano-zeolite supplemented concentrate diet.
R4= Active nano-zeolite supplemented concentrate diet.
R5= Urea-impregnated inactive nano-zeolite supplemented concentrate diet.
R6= Urea-impregnated active nano-zeolite supplemented concentrate diet.
Different superscripts in the same column show a marked difference (P<0.
based on the results of Duncan's analysis.
Production of Volatile Fatty Acids (VFA) The in vitro VFA analysis results (Table .
indicated differences in concentration depending on the incubation times and diet treatments.
Table 5 VFA Concentration .
M) Across Different Diet Treatments Over Incubation Period Hour Diets Means 79A23.
57A29.
25A37.
57A20.
83A19.
81A21.
30A24.
14A18.
78A13.
75A18.
77A16.
04A13.
95A19.
40A15.
83A18.
73A17.
68A19.
17A21.
07A12.
57A16.
34A16.
95A8.
99A30.
95A27.
40A36.
56A28.
18A28.
51A25.
78A13.
94A13.
22A9.
06A6.
48A7.
50A10.
16A10.
Indonesian Journal of Applied Research (IJAR), volume 6 issue 3 Ae December 2025 Potential of Kersen Leaf Extract (Muntingia calabura L) and Basil Leaf Extract (Ocimum basilicum L.
) as Natural Antibacterial Candidates against Salmonella typhi Ae Weni et al.
33A22.
69A17.
77A24.
52A24.
81A18.
00A19.
35A19.
Remark: R1= Control diet without supplementation.
R2= Urea-supplemented concentrate diet.
R3= Inactivated nano-zeolite supplemented concentrate diet.
R4= Active nano-zeolite supplemented concentrate diet.
R5= Ureaimpregnated inactive nano-zeolite supplemented concentrate diet.
R6= Urea-impregnated active nano-zeolite supplemented concentrate diet.
The different superscripts in the same column show a marked difference (P<0.
based on the results of DuncanAos analysis Ratio VFA/NH3 Table 6 below displays the data concerning the ratio of volatile fatty acids (VFA) to ammonia (NH.
derived from in vitro fermentation outcomes across different feed treatments.
Table 6 Ratio of VFA to NH3 Across Different Dietary Treatments Over Incubation Period Hour Diets 49A2.
15A3.
53A3.
75A0.
40A2.
60A0.
09A3.
33A4.
32A2.
38A4.
86A2.
98A3.
35A4.
48A8.
77A3.
09A3.
61A2.
47A2.
45A1.
63A1.
23A2.
68A0.
69A0.
31A1.
Means 76A3.
88A2.
98A2.
65A9.
19A9.
37A5.
36A2.
45A4.
16A4.
78A1.
04A2.
62A2.
65A1.
51A1.
81A1.
05A1.
93A1.
04A1.
Description: R1= Control diet without supplementation.
R2= Urea-supplemented concentrate diet.
R3= Inactivated nano-zeolite-supplemented concentrate diet.
R4= Active nano-zeolite-supplemented concentrate diet.
R5= Urea-impregnated inactive nano-zeolite supplemented concentrate diet.
R6= Urea-impregnated active nanozeolite supplemented concentrate diet.
The different superscripts in the same column show a marked difference (P<0.
based on the results of Duncan's analysis pH Level Table 7 displays the pH assessment data for fermentation media across different dietary treatments (R1 to R.
during the incubation periods I2.
I4.
I8.
I12.
I24, and I48.
Table 7 Rumen Fluid pH Levels Across Different Dietary Treatments During the Incubation Phase Hour 09A0.
06A0.
00A0.
95A0.
86A0.
83A0.
13A0.
06A0.
98A0.
98A0.
86A0.
85A0.
13A0.
04A0.
01A0.
96A0.
88A0.
84A0.
Diets 13A0.
08A0.
01A0.
97A0.
88A0.
86A0.
13A0.
09A0.
02A0.
99A0.
88A0.
82A0.
14A0.
10A0.
00A0.
98A0.
92A0.
86A0.
Means 13A0.
07A0.
00A0.
97A0.
88A0.
84A0.
Description: R1= Control diet without supplementation.
R2= Urea-supplemented concentrate diet.
R3= Inactivated nano-zeolite-supplemented concentrate diet.
R4= Active nano-zeolite-supplemented concentrate diet.
R5= Urea-impregnated inactive nano-zeolite supplemented concentrate diet.
R6= Urea-impregnated active nanozeolite supplemented concentrate diet.
The different superscripts in the same column show a marked difference (P<0.
based on the results of Duncan's analysis Discussion Ammonia Production Overall, ammonia levels for each treatment diet increased from incubation I2 to I48, with values ranging from 11.
7 mM in R3 to 19.
8 mM in R6.
The ammonia concentrations reported by Kardaya et al.
ranged from 7.
5 mM to 22.
45 mM, which closely align with Indonesian Journal of Applied Research (IJAR), volume 6 issue 3 Ae December 2025 Potential of Kersen Leaf Extract (Muntingia calabura L) and Basil Leaf Extract (Ocimum basilicum L.
) as Natural Antibacterial Candidates against Salmonella typhi Ae Weni et al.
the findings of this study.
Similarly.
Kardaya et al.
observed ammonia concentrations 11 Ae 11.
6 mM.
The GLM multivariate full factorial analysis indicated no interaction between incubation duration .
and diet treatment.
While diet treatment did not affect.
Table 8 Ratio of VFA to NH3 Across Different Dietary Treatments Over Incubation Period Hour Diets 49A2.
15A3.
53A3.
75A0.
40A2.
60A0.
09A3.
33A4.
32A2.
38A4.
86A2.
98A3.
35A4.
48A8.
77A3.
09A3.
61A2.
47A2.
45A1.
63A1.
23A2.
68A0.
69A0.
31A1.
Means 76A3.
88A2.
98A2.
65A9.
19A9.
37A5.
36A2.
45A4.
16A4.
78A1.
04A2.
62A2.
65A1.
51A1.
81A1.
05A1.
93A1.
04A1.
Description: R1= Control diet without supplementation.
R2= Urea-supplemented concentrate diet.
R3= Inactivated nano-zeolite-supplemented concentrate diet.
R4= Active nano-zeolite-supplemented concentrate diet.
R5= Urea-impregnated inactive nano-zeolite supplemented concentrate diet.
R6= Urea-impregnated active nanozeolite supplemented concentrate diet.
The different superscripts in the same column show a marked difference (P<0.
based on the results of Duncan's analysis pH Level Table 9 displays the pH assessment data for fermentation media across different dietary treatments (R1 to R.
during the incubation periods I2.
I4.
I8.
I12.
I24, and I48.
Table 9 Rumen Fluid pH Levels Across Different Dietary Treatments During the Incubation Phase Hour 09A0.
06A0.
00A0.
95A0.
86A0.
83A0.
13A0.
06A0.
98A0.
98A0.
86A0.
85A0.
13A0.
04A0.
01A0.
96A0.
88A0.
84A0.
Diets 13A0.
08A0.
01A0.
97A0.
88A0.
86A0.
13A0.
09A0.
02A0.
99A0.
88A0.
82A0.
14A0.
10A0.
00A0.
98A0.
92A0.
86A0.
Means 13A0.
07A0.
00A0.
97A0.
88A0.
84A0.
Description: R1= Control diet without supplementation.
R2= Urea-supplemented concentrate diet.
R3= Inactivated nano-zeolite-supplemented concentrate diet.
R4= Active nano-zeolite-supplemented concentrate diet.
R5= Urea-impregnated inactive nano-zeolite supplemented concentrate diet.
R6= Urea-impregnated active nanozeolite supplemented concentrate diet.
The different superscripts in the same column show a marked difference (P<0.
based on the results of Duncan's analysis.
Discussion Ammonia Production Overall, ammonia levels for each treatment diet increased from incubation I2 to I48, with values ranging from 11.
7 mM in R3 to 19.
8 mM in R6.
The ammonia concentrations reported by Kardaya et al.
ranged from 7.
5 mM to 22.
45 mM, which closely align with the findings of this study.
Similarly.
Kardaya et al.
observed ammonia concentrations 11 Ae 11.
6 mM.
The GLM multivariate full factorial analysis indicated no interaction between incubation duration .
and diet treatment.
While diet treatment did not affectammonia concentration, the incubation duration significantly influenced (P<0.
ammonia levels in rumen fluids.
Duncan Multiple Distance Analysis follow-up tests revealed that the lowest ammonia concentrations (P<0.
occurred at incubation periods I2.
I4, and I8 Indonesian Journal of Applied Research (IJAR), volume 6 issue 3 Ae December 2025 Potential of Kersen Leaf Extract (Muntingia calabura L) and Basil Leaf Extract (Ocimum basilicum L.
) as Natural Antibacterial Candidates against Salmonella typhi Ae Weni et al.
hours, while the highest (P<0.
was at I48.
These findings are consistent with Kardaya et al.
, who noted that a rice straw-based diet with slow-release urea and varying molasses levels increased rumen fluid ammonia concentrations at I2, decreased to a minimum at I8, and then peaked at I48.
Prommachart et al.
also found a similar increase in ammonia concentration at I8 using a diet of mangosteen, rambutan, and banana flower peel feed (MARABAC) with tung oil.
Figure 1 illustrates the average ammonia concentration across different diet treatments during the incubation period.
incubation time Figure 1 Mean concentration of ammonia production across different dietary treatments throughout the incubation period Ammonia production levels are influenced by the crude protein content in animal feed.
Protein sources that are readily broken down by rumen microbes are crucial in this process.
Once feed proteins reach the rumen, they are decomposed by microbes into amino acids, which are subsequently deaminated to produce ammonia, organic acids, and CO2.
Not all proteins undergo degradation.
The ammonia generated in the rumen combines with keto alpha acids derived from protein or carbohydrate sources to create new amino acids, which are essential for synthesizing microbial proteins (Hackman & Firkins .
Detmann et al.
also noted that as the amount of digestible organic matter rises, there is a corresponding linear increase in rumen ammonia nitrogen levels.
This trend is at least partially attributed to the positive correlation between digestible organic matter in the diet and Crude Protein.
The studyAos use of diets with relatively similar protein and energy levels is believed to contribute to the lack of significant treatment effects.
Referring to the data presented in Table 4, the R2 treatment with urea supplementation and the R6 treatment with active nano-zeolite supplementation exhibited the highest concentrations during the I2 incubation, measuring 12.
92 mM and 13.
25 mM, respectively.
reduction in NH3 concentration was observed from hours I2 to I4 across all treatments (R1 to R.
The maximum NH3 concentration in the cowAos rumen is reached in the morning and evening, approximately 1 to 2 hours and 6 to 7 hours after the cow ingests the diet.
This pattern is consistent for both 50:50 unbuffered, 50:50 buffered, 75:25 unbuffered, and 75:25 buffered diets (Khorasani and Kennelly 2001.
Fathul and Wajizah, 2.
From I4 to I8.
NH3 concentrations either continue to decrease or remain unchanged across most treatments.
Specifically, the R1 treatment saw a reduction from 12.
20 mM to 02 mM, whereas the R5 treatment experienced a slight increase from 11.
42 mM to 13.
These findings align with the research by (Kardaya et al.
, which reported values 1 and 11.
6 mM.
These range are conducive to protein metabolism in the rumen and Indonesian Journal of Applied Research (IJAR), volume 6 issue 3 Ae December 2025 Potential of Kersen Leaf Extract (Muntingia calabura L) and Basil Leaf Extract (Ocimum basilicum L.
) as Natural Antibacterial Candidates against Salmonella typhi Ae Weni et al.
can enhance microbial protein synthesis.
Incubating all treatments from I8 to I12 led to a notable rise in NH3 levels.
Specifically, the R1 treatment saw an increase from 12.
02 mM to 14.
73 mM, while the R6 treatment rose 47 mM to 14.
33 mM.
This can be attributed to the inclusion of zeolites in urea-based feeds, which enhances rumen fermentation and facilitates the absorption of NH3-N generated from the breakdown of urea by the urease enzyme in the rumen, allowing for a controlled release as required.
This process offers a consistent source for microbial protein synthesis by rumen microorganisms and helps maintain an optimal environment for their growth (Alrez et , 2.
Ammonia concentrations from I12 to I48 during incubation led to an increase in NH3 levels across all treatments.
The R2 treatment, with 18.
82 mM urea supplementation, and the R6 treatment, which included 19.
80 mM of urea-impregnated active nano-zeolite, exhibited the highest NH3 concentrations.
This rise is likely attributed to the buildup of ammonia due to the exhaustion of substrates available for microbial protein synthesis and the lack of absorption processes in the in vitro rumen system (Zhang et al.
Production of Volatile Fatty Acids (VFA) Overall, the in vitro VFA concentration for each treatment showed an increase from I2 to I48, with values ranging from 97.
57 mM at R4 to 128.
77 mM at R3.
The VFA concentration findings by Kardaya et al.
were similar to those in this study, ranging from 79.
5 to 5 mM, while Zahera et al.
reported higher results of 102.
29 mM to 126.
69 mM over a 4-hour incubation period.
Deitmers et al.
observed lower VFA concentrations, 2 and 113.
8 mM.
The GLM multivariate full factorial analysis indicated no significant interaction between incubation duration and diet treatment.
Although diet treatment did not affect VFA concentration, the incubation duration significantly influenced it (P<0.
Duncan Multiple Distance Analysis follow-up tests revealed that the lowest VFA concentrations (P<0.
occurred at incubation periods I2.
I4.
I8, and I12, while the highest (P<0.
were at I24 and I48.
These findings align with Wang et al.
, who reported that varying mixed ratios of wheat hay with rumen yak and cow increased rumen fluid VFA concentrations at I24.
I48, and I72.
Saleem et al.
also noted an increase in VFA concentrations from I24 to I48.
Figure 2 illustrates the average volatile fatty acid production concentration .
M) across different diet treatments during the incubation period.
incubation time Figure 2 Mean VFA concentration across different dietary treatments throughout the incubation period Indonesian Journal of Applied Research (IJAR), volume 6 issue 3 Ae December 2025 Potential of Kersen Leaf Extract (Muntingia calabura L) and Basil Leaf Extract (Ocimum basilicum L.
) as Natural Antibacterial Candidates against Salmonella typhi Ae Weni et al.
The marked variation in VFA concentration observed between the early incubation periods of I2 to I12 and the later stages of I24 and I48 suggests distinct fermentation processes occurring in the rumen in vitro.
During the initial I2 incubation, the R1 treatment .
ontrol die.
showed the highest VFA concentration at 114.
79 mM, closely followed by the R3 treatment .
iet with inactive nano-zeolite supplementatio.
25 mM, reflecting intense fermentation activity.
These VFA levels remain within the normal range of 70-150 mM.
The reduction in VFA concentration is attributed to rumen microbes utilizing the fermented products to maintain stable rumen fermentation activity(Zahera et al.
, 2.
These microbes use the products as sources of energy and nutrients.
The levels of fermented products in I4 to I8 either continued to decline or remained unchanged in most of the treatments.
Specifically, the R1 treatment saw a reduction from 14 mM to 105.
83 mM, and the R2 treatment decreased from 105.
78 mM to 100.
73 mM.
This reduction indicates that microbes persist in using fermented products to build their cellular components.
More significant reductions in some treatments might indicate the microbes' proficiency in breaking down the available substrates.
According to Syahrir et al.
, the quantity of microbes in the rumen influences the overall production of VFA.
increase in cellulolytic bacterial cells within the rumen fluid leads to a rise in total VFA More substantial decreases in certain treatments could signify the microbes' effectiveness in digesting the available substrates.
During the incubation stages from I8 to I12, there was a rise in VFA concentration.
Specifically, in I8, the R3.
R4, and R6 treatments showed an increase, while in I12, the R1 treatment reached 128.
95 mM before it began to decline, dropping to 127.
78 mM in I24 and further to 123.
33 mM by I48.
This indicates that fermentative activity reached its peak during I8 and I12, followed by a decrease due to the progressively limited availability of substrates.
While there was no statistically significant difference observed between the diet treatments (P>0.
, there is an inclination for VFA concentration to vary between diet treatments at each incubation period.
The treatment with active nano-zeolite (R.
supplementation recorded the lowest value of 97.
57 mM at the 2nd hour, which increased to 40 mM by the 12th hour.
In contrast, treatments involving urea impregnation (R5 and R.
displayed varied results but generally tended to be higher at certain times.
Diets enhanced with urea, nano-zeolite, and combinations of urea-nano-zeolite exhibited variations in VFA concentrations compared to control diets, suggesting that such supplementation could influence rumen fermentation activity.
A noticeable difference in rumen VFA values between treatments will become apparent if the feed consumed contains different carbohydrates, particularly crude fiber.
Conversely, if the coarse fibers in the complete feed across treatments are nearly the same, the rumen VFA value will not show a significant difference (Doubt et al.
Urea, as a non-protein nitrogen source, can enhance the growth and activity of rumen microorganisms, especially those that use ammonia for microbial protein synthesis (Firsoni and Ansori, 2.
Nano-zeolite has the potential to act as a rumen pH buffer and ammonia adsorbent, which can influence the fermentation environment and VFA production (El-Nile et , 2.
Ratio VFA/NH3 Overall, the VFA/NH3 ratio for each dietary treatment decreased from incubation I2 to I48, with values ranging from 10.
33 for R3 to 5.
93 for R6.
Kardaya et al.
reported VFA/NH3 ratios between 7.
01 and 11.
02, which are quite similar to the findings of this study.
However, these results differ from those of Fathul and Wajizah .
, who reported higher ratios of 13.
15 to 18.
The GLM multivariate full factorial analysis indicated no interaction between the duration of incubation .
and the diet treatment.
While the diet treatment did not affect the VFA/NH3 ratio, the length of incubation had a significant impact (P<0.
on the VFA/NH3 ratio in rumen fluid.
Further analysis using Duncan's Multiple Range Test Indonesian Journal of Applied Research (IJAR), volume 6 issue 3 Ae December 2025 Potential of Kersen Leaf Extract (Muntingia calabura L) and Basil Leaf Extract (Ocimum basilicum L.
) as Natural Antibacterial Candidates against Salmonella typhi Ae Weni et al.
revealed that the lowest VFA/NH3 ratio (P<0.
occurred at incubation periods I48 and I12, while the highest (P<0.
was observed at I4 and I8.
These findings align with those of Kardaya et al.
, who found that a rice straw-based diet with added slow-release urea and varying molasses levels increased the VFA/NH3 ratio in rumen fluid in vitro at incubation I4, then decreased at I8, reaching the lowest values at I24 and I48.
The VFA/NH3 ratio observed in this study remains within the range indicative of effective rumen fermentation, aligning with the findings of Kardaya et al.
Despite this, no treatment shows statistically superior efficiency over others.
According to Chen et al.
, the balance between rumen-degradable starch (RDS) and rumen-degradable protein (RDP) influences rumen fermentation characteristics and microbial protein synthesis.
Achieving an optimal RDS:RDP ratio can enhance the efficiency of both rumen fermentation and microbial protein production.
pH Level Overall, the pH level of the fermentation medium for each dietary treatment declines from incubation I2 to I48, with values ranging from 7.
14 on R6 to 6.
82 on R5.
The pH values reported by Kardaya et al.
were between 6.
81 and 7.
32 which are quite similar to the findings of this study Xia et al.
reported even higher pH values, specifically between 15 and 7.
The GLM multivariate full factorial analysis indicated no interaction between the duration of incubation .
n hour.
and the dietary treatment.
While the diet treatment did not influence the pH value, the incubation period significantly affected it (P<0.
in rumen Further analysis using Duncan's Multiple Range Test revealed that the lowest pH values (P<0.
occurred at incubation periods I24 and I48, while the highest (P<0.
were at I2 and I4.
These findings align with Saleem et al.
, who observed that after 48 hours of incubation, the pH of rumen fluid tends to decrease when treating three types of wheat .
oft, medium, har.
with three processing methods .
illing, dry, and wet rollin.
Research indicates that ruminants consuming grass typically exhibit a physiological ruminal pH between 6 and 7.
According to the pH data collected, an increase in incubation time generally leads to a decrease in pH levels, attributed to the production of organic acids by microorganisms within the fermentation medium.
The marked decrease in pH at I48 and I24 compared to the initial hours I2 and I4 indicated that microbial activity in the fermentation medium intensified over time.
In this scenario, it is crucial to acknowledge that while the R1 to R6 treatment did not exhibit a notable change in pH, the differences in incubation duration significantly influenced the fermentation process as a whole.
Zahera et al.
also observed that pH fluctuations can impact digestibility and fermentability within the rumen system.
Conclusion Diets that include inactive, active, or urea-impregnated nano-zeolites offer a similar level of rumen fermentation efficiency as both control diets and those containing solely urea.
Furthermore, the peak feed fermentation efficiency is observed at the 4th and 8th hours of This indicates that using a combination of nano-zeolite and urea not only sustains optimal rumen fermentation efficiency but also enhances the fermentation process effectively during the early to mid-incubation stages.
Acknowledgments We express our gratitude to the Indonesian Ministry of Education.
Culture.
Research and Technology for their financial support of the 2024 Applied Research Scheme, as outlined in Contract Number: 014/SP2H/RT-MONO/LL4/2024, 801/01/K-X/VI/2024.
Indonesian Journal of Applied Research (IJAR), volume 6 issue 3 Ae December 2025 Potential of Kersen Leaf Extract (Muntingia calabura L) and Basil Leaf Extract (Ocimum basilicum L.
) as Natural Antibacterial Candidates against Salmonella typhi Ae Weni et al.
References