Research Article

Rumen Metrics in Sheep Fed Diets Enriched with Urea-Impregnated Nano-Zeolites

Dede Kardaya*, Deden Sudrajat, Dewi Wahyuni, Ruslan Abdul Gopar, Ikhsan Qodri Pramartaa

Department of Animal Husbandry, Faculty of Agriculture Universitas Djuanda, Jalan Tol Ciawi No.1, Ciawi, Bogor, Indonesia.

Received | January 04, 2025; Accepted | February 24, 2025; Published | March 27, 2025

*Correspondence | Dede Kardaya, Department of Animal Husbandry, Faculty of Agriculture Universitas Djuanda, Jalan Tol Ciawi No.1, Ciawi, Bogor, Indonesia; Email: dede.kardaya@unida.ac.id

Citation | Kardaya D, Sudrajat D, Wahyuni D, Gopar RA, Pramartaa IQ (2025). Rumen metrics in sheep fed diets enriched with urea-impregnated nano-zeolites. J. Anim. Health Prod. 13(2): 223-234.

DOI | https://dx.doi.org/10.17582/journal.jahp/2025/13.2.223.234

ISSN (Online) | 2308-2801

Copyright © 2025 Kumar et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Copyright: 2025 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Ruminants, mainly sheep, play a crucial role in the agricultural sector due to their ability to convert fibrous plant materials into high-quality protein. This unique digestive capability is primarily facilitated by the rumen, a specialized stomach compartment where microbial fermentation occurs (Perez et al., 2024; Qi et al., 2024). The rumen hosts a complex microbial ecosystem that is essential for breaking down cellulose and fermenting carbohydrates, thus facilitating nutrient absorption (Pressman and Kebreab 2024; Weimer 2022). The efficiency of this fermentation process is significantly influenced by the diet provided to these animals, which often includes a combination of forage and concentrated feeds (Alizadeh et al., 2021; Khattab et al., 2023; Wang et al., 2024). However, achieving optimal nutrient utilization remains a challenge, particularly regarding nitrogen management, as the rapid degradation of non-protein nitrogen sources can lead to inefficient nitrogen use and environmental pollution (Manju et al., 2022; Netto et al., 2021; Niazifar et al., 2024).

Urea has been widely used as a non-protein nitrogen source in ruminant diets to enhance protein synthesis in the rumen. It is a cost-effective alternative that can improve the nitrogen balance in sheep diets, mainly when high-quality forage is scarce (Hristov et al., 2019; Ma and Faciola, 2024; Mynhardt et al., 2016). However, its rapid degradation in the rumen can result in ammonia toxicity and poor nitrogen utilization (Kardaya et al., 2018; Saro et al., 2023). Researchers have explored various strategies to mitigate these issues, including using slow-release urea formulations (Estrada-Angulo et al., 2016; Heidari et al., 2022; Kardaya et al., 2023; Saro et al., 2023).

Recent research has focused on applying slow-release urea agents in ruminant diets, with numerous studies highlighting their potential to enhance nitrogen utilization. For instance, studies have demonstrated that incorporating slow-release urea can improve microbial protein synthesis in the rumen, ultimately enhancing animal performance (Alizadeh et al., 2021; de Lucena et al., 2024; Niazifar et al., 2024; Saro et al., 2023). Furthermore, zeolites’ use as urea carriers has significantly reduced ammonia concentrations in the rumen, indicating a more controlled release of nitrogen (Ghoneem et al., 2022; Kardaya et al., 2023; Kardaya et al., 2018; Roque-Jiménez et al., 2019; Toprak et al., 2016).

Using nanotechnology in animal nutrition is gaining traction as it offers potential benefits such as improved nutrient delivery and reduced waste production (Gelaye 2024; Hameed 2021; Kumar et al., 2023; Reddy et al., 2023; Sertova, 2020). One of the applications of nanotechnology in livestock nutrition is the use of nano-zeolite as a carrier for urea or ammonia in livestock diets. Nano-zeolites, with their unique properties, have emerged as promising carriers for urea and have shown potential in improving nutrient absorption and modulating rumen fermentation patterns (El-Nile et al., 2023; Valpotić et al., 2017). These materials can improve urea’s stability and release profile in the rumen, potentially leading to better nitrogen utilization and overall animal performance (El-Nile et al., 2023; El-Nile et al., 2021; Soltan et al., 2021; Zhang et al., 2020).

One innovative approach to using nano-zeolites is to prepare urea-impregnated nano-zeolite preparations as a slow-release urea medium in ruminant diets. Nano-zeolite has a larger surface area and porosity than conventional zeolite. Hence, it is more capable of carrying and holding urea, and its release in the rumen becomes more controlled. Integrating urea-impregnated nano-zeolite into the sheep diet is an innovative approach that can address nutritional and environmental issues associated with conventional feeding strategies.

Recent studies have highlighted the importance of rumen fluid metrics in assessing the health and efficiency of ruminant digestion. Parameters such as pH, volatile fatty acids (VFAs), and ammonia nitrogen levels are critical indicators of ruminal fermentation and microbial activity (Jiang et al., 2023; Qi et al., 2024; Wang et al., 2024). Despite the advancements in understanding the role of slow-release urea agents, there remains a gap in the literature regarding the specific metrics of rumen fluid in sheep fed diets enriched with urea-impregnated nano-zeolites. Investigating these metrics is essential for assessing the efficacy of this feeding strategy and its potential impact on sheep health and productivity (Alizadeh et al., 2021; Heidari et al., 2022; de Lucena et al., 2024; Mahdavirad et al., 2021). Therefore, this research article aims to investigate the effects of diets enriched with urea-impregnated nano-zeolites on sheep rumen metrics.

MATERIALS AND METHODS

All procedures in implementing this research were approved and authorized by the Animal Maintenance and Use Ethics Commission of the National Research and Innovation Agency concerning Research Ethics Clearance in the Field of Livestock and Utilization, Number 203/KE.02/SK/08/2024.

Experimental Animals and Housing

A total of 24 male sheep, with an average initial weight of 20.82 ± 1.09 kg, were included in the study. The animals were housed in individual pens that measured 90 cm x 120 cm, which allowed for adequate movement and comfort. Each pen was fitted with separate feeding and drinking facilities to eliminate the risk of diet contamination. The pen’s floor is made of wooden battens with bars 1.5 cm apart to ensure that dirt can escape falling under the cage’s floor. The floor materials were cleaned, and proper ventilation was maintained to reduce stress and promote animal comfort.

Basal Diet Preparation

The basal diet consisted of dwarf elephant grass (Pennisetum purpureum cv. Mott) and concentrate, prepared on a dry matter (DM) basis with a ratio of 55:45. The dwarf elephant grass was harvested at 40 days post-regrowth, chopped into 3–5 cm lengths, and sun-dried to achieve uniform moisture levels. The basal concentrate (control diet) was formulated from pollard, corn kernels (coarsely milled), soybean meal, coconut meal, and cassava pulp.

 

Table 1: Composition of feed ingredient and nutrients of experimental diets.

Feed Ingredients (%)	Diets (DM basis)
	R1	R2	R3	R4	R5	R6
Field grass	55	55	55	55	55	55
Pollard	4	11	2	2	6.6	6.6
Yellow corn	14	15	15	15	16	16
Soybean meal	16.5	10.2	17	17	14	14
Coconut meal	9.5	5	9	9	4.8	4.8
Molasses	-	1	-	-	1	1
Nonactivated nano zeolite	-	-	1	-	-	-
Thermal activated nano zeolite	-	-	-	1	-	-
Urea	-	0.8	-	-	-	-
Urea-impregnated nonactivated nano zeolite	0	0	0	0	1.6	-
Urea-impregnated thermal activated nano zeolite	-	-	-	-	-	1.6
Tapioca dregs	1	2	1	1	1	1
Total	100	100	100	100	100	100
Nutrients (%)
Dry matter (DM)	88.97	84.49	90.27	90.66	88.61	87.61
Crude protein (CP)	16.43	16.64	16.29	16.29	16.66	16.66
Total digestible nutrient (TDN)	68.03	66.55	67.47	67.47	66.53	66.53

 

R1: control diet with natural protein; R2: urea-based diets; R3 = inactivated nano-zeolite diets; R4: thermally activated nano-zeolite diets; R5: urea-impregnated inactivated nano-zeolite diets; R6: urea-impregnated thermally activated nano-zeolite diets.

 

Feeding Management and Experimental Setup

The research employed a completely randomized design (CRD) with six dietary treatments. R1 was the control diet with natural protein, R2 used urea-based diets, R3 featured inactivated nano-zeolite diets, R4 included thermally activated nano-zeolite diets, R5 had urea-impregnated inactivated nano-zeolite diets, and R6 comprised urea-impregnated thermally activated nano-zeolite diets. Each treatment group consisted of four sheep (n = 4), receiving diets at 2.5% of their body weight twice daily at 8:00 am and 4:00 pm. Water was available ad libitum. The diets were formulated in iso-protein (16.29 – 16.66%) and iso-energy (66.53 – 68.03%) as presented in Table 1.

Rumen Fluid Collection and Analysis

Rumen fluid collection: Rumen fluid collection was carried out through a carefully regulated method utilizing a stomach tube attached to a vacuum pump, which improved the efficiency and effectiveness of the sampling process. Samples were taken four hours after feeding to align with the peak fermentation times. Each session yielded about 75 mL of rumen fluid from each sheep, with the initial 15 mL discarded to preserve sample integrity and prevent saliva contamination. After collection, the rumen fluid was filtered through four layers of cheesecloth to remove larger feed particles and debris that could interfere with further analyses. The filtered samples were then transferred into pre-labeled containers to avoid mix-ups and facilitate easy identification during analysis.

Furthermore, the rumen fluid in each container was promptly assessed for pH and securely sealed. All samples were kept on ice to maintain sample stability and prevent degradation of the components of interest during transport to the laboratory. For the analysis of ammonia nitrogen (NH₃-N) and volatile fatty acids (VFAs), aliquots were treated with 1 mL of 25% orthophosphoric acid to inhibit microbial activity, prevent any changes in the samples and subsequently stored at -20°C until further analysis.

Rumen fluid analysis: Rumen fluid analysis involves several critical assessments to evaluate the rumen’s microbial environment. The pH of the rumen fluid was determined immediately following collection with a calibrated digital pH meter (Accumet Basic pH Meter, Fisher Scientific, Pittsburgh, PA), adhering to the standards set by AOAC (2006). Before each measurement, the pH meter was calibrated using buffer solutions at pH values of 4.0 and 7.0, ensuring accuracy in the readings. The ammonia content of the rumen fluid was determined colorimetrically at a wavelength of 630 nm using a spectrophotometer (Beckman DU 64, Beckman Instruments, Fullerton, CA) as described by Broderick and Kang (1980). Individual VFA levels were determined by gas chromatography (Model 5890, Hewlett-Packard, Avondale, PA) utilizing a Flame Ionization Detector (FID) to identify individual acids, including acetic, propionic, butyric, and valeric acids as described by Vanzant and Cochran (1994).

Methane production is intricately linked to the specific volatile fatty acids (VFAs) generated within the rumen, as each type of VFA influences hydrogen dynamics differently during their synthesis. The equation for estimating methane is expressed as;

CH4 (mmol) = 0.5 ×Acetate - 0.25 × Propionate + 0.5 × Butyrate

which is based on stoichiometric analyses conducted by Moss et al. (2000).

The non-glucogenic ratio (NGR) and the conversion efficiency hexose to VFA were calculated using the formula outlined in reference (Orskov 1975; Wang et al., 2023):

The numerator (C2 + 2C4 + C5) represents the non-glucogenic VFAs. The denominator (C3 + C5) represents the glucogenic VFAs, with propionate (C3) being the principal glucogenic VFA. The interpretation of the NGR value reveals that a higher ratio indicates a tendency towards producing non-glucogenic VFAs, while a lower ratio suggests an increase in glucogenic VFA production.

The calculation of the efficiency with which hexose, a six-carbon sugar, is transformed into volatile fatty acids (VFAs) can be expressed through the following equation:

In this equation, A represents acetic acid’s molar percentage, P denotes propionic acid’s molar percentage, and B indicates the molar percentage of butyric acid. This formula considers the energy contributions from each VFA generated during the fermentation process. The coefficients, 0.622 for acetic acid, 1.092 for propionic acid, and 1.560 for butyric acid, reflect the relative energy yields of these acids compared to the initial hexose molecule.

Statistical Analysis

An experimental design employing a completely randomized design (CRD) involving six dietary treatments, including a control group and five experimental groups, was tested on 24 male sheep, evenly distributed across the groups. The assessment focused on various metrics, including rumen pH, ammonia concentrations, volatile fatty acids (VFAs), methane emissions, and energy conversion efficiency. Specific ratios such as VFA/NH3, the acetic-to-propionic acid ratio (A/P), and the non-glucogenic ratio (NGR) were also calculated. The impact of the dietary treatments on the rumen metrics was evaluated using analysis of variance (ANOVA), followed by Duncan’s multiple range test to pinpoint specific differences among treatments, denoted by superscripts in the tables. A significance level of p < 0.05 was established to ascertain statistical significance. The results were reported as means ± standard deviations, with superscripts indicating groups that exhibited statistically significant differences. The statistical analysis was conducted using IBM SPSS 26 software.

RESULTS AND DISCUSSION

Table 2 presents the experimental diet’s effect on rumen variables, including pH, ammonia, volatile fatty acids (VFA), methane gas, and A/P, VFA/NH3, NGR, and e-VFA ratios.

The pH value across the treatments ranged from 6.5 to 6.7, with no significant difference (p>0.05) between treatments. All treatments maintain acidity levels conducive to the rumen’s microbial activity. The resulting pH value of some researchers ranged from 6.3 – 7.00 (Belanche et al., 2021; Liang et al., 2024). In this study, inactive and active nano-zeolites, both non-and urea-impregnated, produced a stable rumen pH value (p>0.05). Several researchers have also reported the pH stability of the rumen due to the use of zeolite urea and other types of loose urea in sheep diets (Ali and Ahmed Alqutbi 2023; Netto et al., 2021; Silva et al., 2023). The pH stability of this rumen is crucial because any decrease in pH value of 0.5 units significantly changes the structure of the microbial community, especially in the liquid fraction of the rumen content, leading to a decrease in the proportion of certain phylum bacteria and protozoa (Li et al., 2021). Low rumen pH values can reduce bacterial diversity while increasing rumen fungal diversity (Belanche et al., 2021).

The concentration of acetic acid (C2) showed significant variation (p<0.05), with the R6 diet having the lowest value (41.1 ±4.0 mM) and R4 having the highest value (60.2 ±3.2 mM). The data showed that treatment with heat-activated nano-zeolites significantly increased acetic acid production compared to other treatments. The use of urea (R2) and inactivated nano-zeolite (R3) produced the same acetate levels as the control ratio (R1). However, the use of heat-activated nano-zeolite (R4) and urea-impregnated inactivated nano-zeolite (R5) resulted in higher acetate levels (p<0.05) than the R1 and R2 diets.

Increased acetate levels of rumen liquid in sheep fed with nano-zeolite diets or urea-impregnated nano-zeolites can be associated with modulation of the rumen fermentation process. Nano-zeolites, a natural aluminosilicate mineral, function as an ammonia adsorbent and release it back into the rumen liquid to improve the efficiency of using ammonia as a nitrogen source instead of protein by rumen microbes. Nano-zeolites impregnated with urea act as a medium that can hold and release urea slowly so that it does not readily dissolve in the rumen so that the fermentation of urea into ammonia by urease enzymes is controlled so that the efficiency of ammonia used by rumen microbes is optimal. This combination increases volatile fatty acids (VFAs), primarily acetate, the primary source of energy for ruminants. This increase in acetate is due to the increased fermentation of fibrous carbohydrates supported by adequate amounts of non-protein nitrogen sources for acetate-producing bacteria. This explanation aligns with the opinion (Ali and Ahmed Alqutbi, 2023) that increased nitrogen availability favored the growth of acetate-producing bacteria. The acetate level of sheep rumen fluid increased due to adding zeolite or other buffers, which was also reported (Mahdavirad et al., 2021; Pikhtirova et al., 2024).

The level of propionic acid (C3) was also influenced by the treatment of diets (p<0.05). In this case, R1 showed the highest concentration (25.0 ±3.6 mM) while R6 and R2 had the lowest concentration range (14.9 ±1.1 mM and 16.7±1.3 mM). Urea-impregnated inactivated nano-zeolite (R5) diets produced higher propionate (p<0.05) compared to conventional urea diets (R2) and urea-impregnated active nano-zeolite diets (R6). These results show that using urea-impregnated inactivated nano-zeolites is better in producing propionate than urea-impregnated active nano-zeolites. The effectiveness of nano-zeolites is comparable to that of a control diet without conventional urea and nano-zeolite supplements in influencing the levels of propionic acid in rumen fluid. However, the use of thermally activated nano-zeolites.

Using non-activated nano-zeolites impregnated with urea in sheep diets has produced higher rumen propionate than conventional urea-containing diets. This difference is mainly due to the properties of zeolite that can release ammonia slowly and controllably, which modulates the availability of ammonia and affects the fermentation process of rumen. Zeolite acts as a buffering agent, stabilizes the pH of the rumen, and improves the efficiency of nitrogen utilization, which in turn affects the production of volatile fatty acids (VFAs) such as propionates. The propionate levels of sheep rumen liquid in the control diet without the addition of zeolite or other buffer were higher (p<0.05) than those of the zeolite or other buffer diets, also disclosed by (Mahdavirad et al., 2021).

For iso-butyric acid (iC4), the R2 diet showed a much lower concentration compared to R1 (p<0.05), while the other diet produced the same iso-butyrate level as the R1 diet. These data show that treating urea without nano-zeolite in the diet can reduce the level of iso-butyric acid in the sheep rumen. The levels of n-butyric acid (nC4) also varied significantly (p<0.05). The lowest value was seen in R2 (7.4 ±1.0 mM), while R1 had the highest value (12.1 ±1.1 mM), while the other diets produced the same iso-butyrate levels as R1 diets. These data show that treating urea without nano-zeolite in the diet can reduce the level of iso-butyric acid in the sheep rumen. Butyric acid (C4) also showed the same significance pattern (p<0.05) as iso-butyrate and n-butyrate. The R1 diet produced the highest level of butyric acid (13.9±1.2 mM), but statistically, the value was the same as the R4 and R5 diets. The R2 and R3 diets were in the lowest range of butyric acid levels (8.7±1.1 mM and 9.7±0.5 mM). The R1, R4, and R5 diets produced higher levels of butyric acid (p<0.05) than the R2 and R3 diets. These data showed that the active and inactivated nano-zeolite diets impregnated with urea showed the same level of effectiveness as the control diet in increasing butyric acid levels, while the use of conventional urea and inactivated nano-zeolites in the diet could reduce the butyric acid levels in the sheep rumen.

Conventional urea is rapidly hydrolyzed in rumens, rapidly releasing ammonia, resulting in inefficient nitrogen utilization and lower production of volatile fatty acids (VFAs) such as butyric acid. This condition is supported by an explanation (Chanjula et al., 2021) that rapid release can cause ammonia to escape the rumen before it is thoroughly utilized by microbes, thereby reducing VFA production. Nano-zeolites that are not heat-activated, although they have a large surface area, are less effective in capturing and releasing ammonia out of sync with the rate of fermentation of carbohydrates into VFA, including butyric acid, resulting in low levels of butyric acid. According to Manikandan and Subramanian (2014), An inactivated zeolite does not provide the benefit of a slow-release system and, therefore, cannot increase microbial activity and VFA production.

Heat-activated nano-zeolites increase the surface area and adsorption capacity of nano-zeolites, allowing for a more controlled release of nitrogen, thus supporting more stable and sustained microbial activity in the rumen. On the other hand, both structural carbohydrates supplied by forage and nonstructural carbohydrates contained in concentrates are fermented by rumen microbes so that there is a synchronization between ammonia release and carbohydrate fermentation, which has an impact on increasing the production of VFA, including butyric acid. Although nano-zeolites are not heat-activated, if they are impregnated with urea, this impregnation process can change the urea release pattern, providing a more gradual release of nitrogen so that it can control the hydrolysis of urea into ammonia. It can optimize the activity of rumen microbes and increase butyric acid production compared to conventional urea. Previous research (Kardaya et al., 2012) using urea-impregnated micro-zeolites can increase rumen microbial activity due to the synchronization between ammonia release and VFA production in the rumen.

 

Table 2: Effect of the experimental diet on sheep rumen metrics.

Rumen metrics	R1	R2	R3	R4	R5	R6
pH	6.5 ±0.1	6.7 ±0.2	6.6 ±0.1	6.7± 0.2	6.7± 0.2	6.7± 0.2
C2 (mM)	53.2 ±2.0b	49.7 ±4.9b	50.3 ±5.9bc	60.2 ±3.2d	57.2 ±2.3c	41.1± 4.0a
C3 (mM)	25.0 ±3.6d	16.7 ±1.3ab	18.1 ±1.9bc	19.2 ±0.7bc	21.0 ±1.2 c	14.9± 1.1a
iC4 (mM)	1.8 ±0.1b	1.3 ±0.3a	1.5± 0.1ab	1.5± 0.2b	1.7± 0.2b	1.4± 0.1ab
nC4 (mM)	12.1 ±1.1d	7.4 ±1.0a	8.2± 0.5ab	10.3± 0.7cd	10.2± 2.4bcd	8.7± 1.3abc
C4 (mM)	13.9 ±1.2c	8.7 ±1.1a	9.7± 0.5a	11.9± 0.9bc	11.9± 2.4bc	10.2± 1.2ab
iC5 (mM)	2.4 ±0.4b	1.6 ±0.6 a	1.6± 0.6 a	2.0± 0.3 ab	2.2± 0.7 ab	1.8± 0.4 ab
nC5 (mM)	1.8 ±0.3	1.1 ±0.2	1.3± 0.4	1.4± 0.3	1.6± 0.3	1.4± 0.1
C5 (mM)	4.2 ±0.7b	2.8 ±0.8a	2.9± 1.0 a	3.4± 0.5 ab	3.8± 1.0 ab	3.2± 0.5 ab
VFA (mM)	103.2 ±5c	83.0 ±6.5ab	86.3 ±6.7b	100.8 ±3.8c	100.5 ±8.0c	75.7 ±6.0a
NH3 (mM)	11.6 ±1.0c	8.8 ±0.8a	8.1± 1.3 a	9.2± 0.8 ab	10.7± 1.7bc	10.8 ±0.8bc
Methan (mmol)	27.3 ±0.8bc	25.0 ±3.1ab	25.5 ±2.8ab	31.2± 1.5d	29.3± 2.4cd	21.9 ±2.4a
VFA/NH3	9.01 ±1.18b	9.48 ±0.60bc	10.81 ±1.72bc	11.02 ±1.23c	9.50± 1.28bc	7.01± 0.58a
C2/C3 (A/P)	2.1± 0.37a	3.0± 0.44b	2.8± 0.27b	3.1± 0.11b	2.7± 0.22b	2.8± 0.26b
NGR	2.94 ±0.35a	3.60± 0.41b	3.48± 0.39ab	3.87± 0.09b	3.42± 0.30ab	3.57± 0.40b

 

R1: basal diets; R2: basal diets + urea; R3: basal diets + non-activated nano-zeolite; R4: basal diets + thermal activated nano-zeolite; R5: basal diets + urea impregnated non-activated nano-zeolite; R6: basal diets + urea impregnated pre-thermal activated nano-zeolite. NGR: Non-Glucogenic Ratio. Different superscripts on the same row indicate significant differences (p<0.05).

 

The highest isovalerate level (2.4±0.4 mM) was obtained from the R1 diet, and the lowest (1.6±0.6 mM) was obtained from the R2 and R3 diets. Diets containing active nano-zeolite (R4), urea-impregnated inactivated nano-zeolite (R5) and urea-impregnated active nano-zeolite (R6) showed isovalerate levels equivalent to (p>0.05) with R1 diets. These data show that R4, R5, and R6 diets can help maintain high isovaleric acid levels comparable to R1 diets. The n-valerate content showed insignificant variation (p>0.05) between treatments, although the R1 diet showed the highest n-valerate level numerically, and R2 still showed the lowest n-valerate level. The levels of valeric acid show the same pattern as the levels of isovalerates. The highest valerate acid content (4.2±0.7 mM) was obtained from the R1 diet, and the lowest range (2.8±0.8 mM - 2.9±1.0 mM) was obtained from the R2 and R3 diets. Diets containing active nano-zeolite (R4), urea-impregnated inactive nano-zeolite (R5) and urea-impregnated active nano-zeolite (R6) showed isovalerate levels equivalent to (p>0.05) R1 diets. These data suggest that R4, R5, and R6 diets can help maintain high isovaleric acid levels comparable to R1 diets higher than conventional urea diets (2).

Lower levels of ruminal valeric acid in sheep fed conventional urea diets and inactive zeolite diets compared to control diets can be attributed to the effects of urea and zeolite on rumen fermentation dynamics. When used as a nitrogen source, urea can alter the rumen environment by affecting ammonia levels and the balance of volatile fatty acids (VFAs), including valeric acid. Nano-zeolites, minerals with ion-exchange properties, greater surface area, and porosity, can further influence this fermentation process by modulating the release of ammonia and other nutrients. Urea supplementation increases the concentration of rumen ammonia nitrogen, which can affect the production of VFA, including valeric acid. High ammonia levels can cause an imbalance with VFA synthesis, resulting in less optimal microbial activity and potentially reducing the production of certain VFAs (Kardaya et al., 2012; Lira-Casas et al., 2019).

Heat-activated nano-zeolites, urea-impregnated inactivated nano-zeolites, and urea-impregnated heat-activated nano-zeolites have the same potential as control diets in influencing valeric acid production. It is thought that the three diets (R4, R5, and R6) act as slow-release agents for ammonia, which can stabilize the pH of the rumen (Table 2) and the production of certain VFAs, including valeric acid. This stabilization can lead to a more controlled fermentation, potentially increasing valeric acid levels. As conveyed by (Lira-Casas et al., 2019), slow-release urea may lead to a more gradual and sustained release of ammonia, which may not support the same level of valeric acid production as seen in a control diet without this supplement. The results of this study are also supported by (Silva et al., 2023), which states that zeolite, with its cation exchange capacity, can affect the availability of nutrients and microbial activity in the rumen, which further affects the production of VFAs.

Total VFA levels were significantly affected (p<0.05) by diet treatment. The R6 diet showed the lowest total VFA (75.7 ±6.0 mM), while R4 had a higher concentration (100.8 ±3.8 mM). The R1 diet showed the highest concentration of VFA among all diets, with significant differences from R2, R3, and R6. The R2 diet had the second lowest level of VFA, significantly different from R1, R4, and R5, suggesting that adding urea alone did not increase VFA production as effectively as other treatments. The R3 diet is in the middle, with a concentration statistically similar to R2 but significantly different from R4 and R5. R4 an d R5 are similar to R1, each showing no significant differences. However, they differ significantly (p<0.05) from R2, R3, and R6, suggesting that nano-zeolite activation or urea impregnation positively impacts VFA production. The R6 diet had the lowest level of VFA, significantly different (p<0.05) from all other diets except R2 (p>0.05), suggesting that while urea impregnation may have some benefits, the process of thermal activation of urea-impregnated nano-zeolite was not as effective as using urea-impregnated inactivated nano-zeolites in producing VFA in sheep rumen fluid.

This VFA production pattern resembles the production pattern of other short-chain fatty acids, except acetic acid. As previously explained, conventional urea is rapidly hydrolyzed in rumens, leading to rapid ammonia release, which can result in inefficient nitrogen utilization and lower production of volatile fatty acids (VFAs). This condition is supported by (Chanjula et al., 2021) that rapid release can cause ammonia to escape the rumen before it is thoroughly utilized by microbes, thereby reducing VFA production. Non-heat-activated nano-zeolites, although they have a large surface area, are less effective in capturing and releasing ammonia out of sync with the rate of fermentation of carbohydrates into VFA, resulting in low VFA levels. According to Manikandan and Subramanian (2014), inactivated zeolite does not provide the benefit of a slow-release system and, therefore, cannot increase microbial activity and VFA production. The slow release of nitrogen from this system allows microbes to utilize ammonia more efficiently and increases VFA production. This efficiency is less dominant in conventional urea and inactive nano-zeolites, which do not provide the same controlled release (Kardaya et al., 2012; Silviani et al., 2024). A study that aligned with this study comparing conventional urea with SRU found that SRU increased total VFA production (Alipour et al., 2021).

Ammonia levels also varied significantly across treatments. The R1, R5, and R6 diets showed a high range of ammonia levels (11.6±1.0 mM, 10.8 ±0.8 mM, and 10.7±1.7 mM), suggesting that the R1, R5, and R6 diets could maintain higher ammonia levels, which could be beneficial for microbial protein synthesis. High ammonia levels at R1 indicate a high level of protein degradation. In contrast, high ammonia levels at R5 and R6 indicate the ability of urea-impregnated nano-zeolites to maintain high enough ammonia levels to supply the microbe’s need for nitrogen instead of protein to synthesize their cell proteins. The highest ammonia levels obtained in this study (R1, R5, and R6) were slightly below the ammonia levels (13 – 14.9 mM) of rumen liquid to support maximum fermentation activity (Vidya et al., 2024).

The R2, R3, and R4 diets produced a low range of ammonia levels (8.8 ±0.8 mM, 8.1±1.3 mM, and 9.2±0.8 mM). The low ammonia level in conventional urea diets (R2) indicates that although urea is easily soluble and quickly fermented into ammonia in rumen liquid, the amount is quickly depleted due to the absorption of rumen ammonia through the rumen wall to the liver so that the rumen liquid ammonia level is low. Meanwhile, the low ammonia levels of rumen liquid produced by inactivated nano-zeolite (R3) and heat-activated nano-zeolite (R4) diets are due to their ability to capture, maintain, and release ammonia in a controlled and sustainable manner so that the utilization of ammonia by rumen microbes becomes more optimal.

Thus, diets without supplementation (R1) can produce higher levels of ammonia due to effective protein degradation from natural sources equivalent to rumen liquid ammonia levels produced by inactivated urea-impregnated diets (R5) and heat-activated and urea-impregnated nano-zeolites (R6). These data indicate that urea-impregnated nano-zeolites can replace natural protein nitrogen sources in supplying ammonia for rumen microbes. On the other hand, diets with conventional urea (R2) do not maintain high enough ammonia levels due to faster absorption of ammonia through the rumen wall. In other words, the slow-release formulation of urea in urea-impregnated inactivated nano-zeolites (R5) and urea-impregnated heat-activated nano-zeolite (R6) provides a more sustainable source of ammonia, leading to higher levels compared to conventional urea (R2) and non-urea nano-zeolites, both non-activated (R3) and heat-activated (R4) which both produce low levels of ammonia.

This result is the same as that of a study in goats, in which natural zeolite and nano-zeolite both produce lower ammonia levels (El-Nile et al., 2021). The addition of conventional urea yielded 32 mM ammonia nitrogen content in rumen liquid in vitro studies (Shen et al., 2023). One of the limitations of in vitro is that fermentation residues, including ammonia, will accumulate in the fermenter tubes, while the number of microbes decreases as the incubation time increases. Sheep fed a diet of pastural grass with sorghum seeds produced an ammonia level of 37.15 mg/100 ml of rumen fluid or the equivalent of 26.5 mM (Aguerre et al., 2009). The study’s results on sheep fed solid diets based on agricultural waste produced ammonia levels ranging from 18.25 – 20.82 mg/100 ml of rumen liquid (Vidya et al., 2024), equivalent to 13 – 14.9 mM. These data reveal that the optimal ammonia content of sheep rumen liquid for rumen microbial activity cannot be generalized, depending on the source of nitrogen and carbohydrates provided in the diet and the alignment of the fermentation process of those nitrogen and carbohydrates in the liquid.

The highest methane content (p<0.05) was obtained from the R4 diet (31.2 ±1.5 mM), and the lowest was obtained from the R6 diet (21.9 ±2.4 mM), which was statistically comparable (p>0.05) to the R2 and R3 diets. The R2, R3, and R5 diets produced methane levels equivalent to the control diets. especially between R4 and R6, where R4 causes an increase in methane emissions while R6 results in a reduction in methane gas emissions (p<0.05). These data imply that heat-activated nano-zeolites (R4) produce higher methane emissions than non-activated nano-zeolites. These results differ from those obtained (El-Nile et al., 2021) in that nano-zeolite causes a reduction in methane (CH4) production in goats. This difference is more due to the heat activation process in the nano-zeolites used in the R4 diet. This heat activation can increase urea’s adsorption, retention, and release in a controlled manner. It can supply a non-protein nitrogen source in an amount proportional to the rate of structural carbohydrate degradation, thereby changing the VFA production pattern and leading to an increase in acetic acid production (Table 2). The increase in acetic acid production increases the production of protons (hydrogen), the raw material for methane production, so methane production increases in the R4 diet. On the other hand, heat-activated nano-zeolites impregnated with urea produce the lowest acetic acid (Table 2), so hydrogen gas production is low, which has an impact on decreasing methane gas production. There are indications that heat-activated and urea-impregnated (R6) nano-zeolites are more effective in inhibiting methanogenic activity than urea-impregnated inactivated nano-zeolites.

The production of methane gas from goats fed rice straw diets treated with a mixture of urea, nitrate, and corn oil supplements can reduce methane gas emissions, with the number of emissions ranging from 8.92 – 9.91 g of methane gas per day (Zhang et al., 2019) or equivalent to 55.75 – 61.94 mmol per day. The production of methane gas in goats fed unconventional diets derived from bio-fermentation and supplementation can reduce emissions, ranging from 18.23 – 28.76 mM of methane gas (Partama and Mudita, 2019). Urea-nitrate supplementation with a zeolite layer produced the same methane levels as the control treatment, ranging from 3.25 – 4.92 mmol/mL or equivalent to 32.5 – 49.2 mM (Silviani et al., 2024). The difference in methane gas production reported by the researchers is suspected to be caused by several factors, such as the level of consumption and quality of diets, the weight of livestock, the ambient temperature, and the way rumen liquid is sampled. These factors were also conveyed in the review article (Broucek, 2014), that many factors affect the production of CH4 in ruminants, including intake levels, feed type and quality, energy consumption, animal size, growth rate, production rate, and environmental temperature.

The VFA/NH3 ratio is an important indicator of fermentation efficiency and nitrogen utilization. The highest ratio was found in R4 (11.02 ±1.23), comparable to the ratio obtained from R2, R3, and R5 diets, while R6 had the lowest ratio (7.01±0.58). There are indications that in this study, diets that produce a higher VFA/NH3 ratio show better utilization of nitrogen and carbohydrates so that the fermentation process becomes more efficient. In vitro studies using diets supplemented with microminerals in sheep also produced a high VFA/NH3 ratio, ranging from 13.15 to 18.38, with no significant difference in the statistical analysis results (Fathul and Sitti Wajizah, 2010) However, as previously explained, one limitation of in vitro is that fermentation residues, including VFA and ammonia, will accumulate in the fermenter tube. At the same time, the number of microbes decreases as the incubation time increases, thus reflecting less of the actual conditions in the rumen.

A study on the use of nano-zeolites in goat diets that focused on rumen fermentation and digestibility of nutrients found that diets containing nano-zeolites resulted in higher total VFA and a more favorable VFA/NH3 ratio with a range of 6.05 - 8.07 compared to control diets or diets containing natural zeolite (El-Nile et al., 2021). The lowest VFA/NH3 ratio value (R6) in this study indicates that heat-activated nano-zeolite impregnated with urea is less supportive in the fermentation process of carbohydrates into VFA than other diets.

The acetic acid to propionic acid (A/P) ratio ranged from 2.1±0.37 in the R1 diet to 3.1±0.11 in the R4 diet. The acetate-to-propionate ratio is almost the same as the A/P (2.36 – 2.94) results of in vitro studies using lipogenic and glucogenic food substances (Hua et al., 2021). The control diet (R1) results in a lower C2/C3 ratio (p<0.05) than the other diets. However, after butyric acid was included with acetic acid, which is both non-glucogenic fatty acids, it turned out that the control diet produced the same non-glucogenic ratio (NGR) as the R3 and R5 diets.

The non-glucogenic ratio (NGR) in sheep rumen fluid refers to the ratio of non-glucogenic volatile fatty acids (VFA) to glucogenic VFA. This ratio is an important indicator of the type of fermentation that occurs in the rumen, which can be affected by the composition of the food, especially the balance of structural and nonstructural carbohydrates. The range of NGR values in sheep rumen fluid can vary depending on several factors, including the type of food and the degradability of the carbohydrates and proteins consumed. Acetic and butyric acids are lipogenic and support energy for fat synthesis in livestock but do not contribute to glucose synthesis (non-glucogenic). Propionic acid is classified as glucogenic because it contributes to glucose synthesis through gluconeogenesis. The NGR value ranged from 2.94±0.35 in the R1 diet and 3.87±0.09 in the R4 diet. This value is not far from the NGR value of PE goat rumen liquid fed with forage diets and concentrates with different ratios, ranging from 3.06 – 3.47, with no significant variation (Suryani et al., 2014). The NGR value of the control diet (R1) was balanced (p>0.05) with the R3 and R5 diets but lower (p<0.05) than the R2, R4, and R6 diets. These data revealed that diets containing inactivated nano-zeolites, both non- and urea-impregnated, tended to match control diets in improving the fermentation efficiency of structural and nonstructural carbohydrates in rumen fluids. When the ratio of structural carbohydrates to nonstructural carbohydrates increases, the NGR tends to increase due to the higher proportion of non-glucogenic VFAs such as acetate and butyrate. Structural carbohydrates are more likely to be fermented into non-glucogenic VFA (Xia and Meng, 2007). In summary, the NGR value explains the balance between glucogenic and non-glucogenic VFAs in the rumen, providing insights into energy utilization efficiency, the type of fermentation that occurs, and potential health implications for livestock because extreme reductions in NGR can lead to metabolic disorders such as acidosis.

 

The efficiency of glucose energy conversion to VFA (Figure 1) in the rumen can be calculated based on the result of VFA energy produced during the fermentation of glucose to VFA by rumen microbes. The e-VFA value in this study ranged from 74.56±0.26% in the R4 diet and 77.35±1.19% in the control diet (R1). The fermentation efficiency of this result is lower than the results of in vitro studies, which obtained efficiency values ranging from 81 to 82.6% (Wahyuni et al., 2024). The control diet (R1) produced the highest efficiency level (p<0.05), outperforming other diet efficiency levels that showed the same efficiency level (p>0.05). The efficiency of hexose energy conversion to VFA is inversely related to the NGR and A/P ratio (Table 2). In other words, the lower the non-glucogenic ratio (NGR) and the A/P ratio, the more efficient the hexose fermenting into VFA. The synthesis of propionic acid from glucose is classified as energy-efficient. In contrast, the synthesis of acetic and butyric acids is classified as energy-wasteful because some energy is lost as methane gas.

CONCLUSIONS AND RECOMMENDATIONS

The heat-activated nano-zeolites and urea-impregnated inactivated nano-zeolites boosted acetate, propionate, butyrate, and total VFA production compared to conventional urea diets. These diets yielded similar isovalerate and valerate production to conventional urea diets. Urea-impregnated inactivated and preheat-activated nano-zeolite diets were more sustainable ammonia source than conventional urea. The heat-activated nano-zeolite diet increased rumen fermentation efficiency but similarly to the other diets was less effective in converting energy from hexoses to VFAs compared to the control diet. Most diets, except the urea impregnated preheat-activated nano-zeolite, showed a better VFA/NH3 ratio, indicating improved nitrogen and carbohydrate utilization. However, heat-activated nano-zeolite urea impregnated inactivated nano-zeolite increased methane emissions, while control diet, conventional urea, non-activated nano-zeolite, and urea impregnated preheat-activated nano-zeolite reduced methane emissions. Incorporating urea-impregnated nano-zeolite into sheep diets improved fermentation efficiency and nutrient utilization while maintaining sutainable ammonia supply for rumen microbes. Inactive nano-zeolites impregnated with urea proved to be the most effective for enhancing rumen metrics.

ACKNOWLEDGEMENTS

Acknowledgment is given to the Indonesian Ministry of Education, Culture, Research and Technology for their financial contribution to the 2024 Applied Research Scheme, as specified in Contract Number: 014/SP2H/RT-MONO/LL4/2024, 801/01/K-X/VI/2024.

NOVELTY STATEMENTS

The research explores the use of urea-impregnated nano-zeolites as a novel approach to improve nitrogen utilization in sheep diets. This method addresses the rapid ammonia release and potential toxicity associated with conventional non-protein nitrogen sources like urea. The research assesses the impact of these formulations on key rumen fluid parameters—pH, ammonia concentration, volatile fatty acids (VFA), and fermentation efficiency—crucial for optimizing sheep nutrition and health. The incorporation of urea-impregnated nano-zeolites offers a slow-release solution, providing a more sustainable ammonia source for rumen microbes and enhances overall fermentation efficiency compared to conventional urea. The research highlights the potential of inactive nano-zeolites impregnated with urea as the most effective method for enhancing rumen metrics, offering a significant improvement over traditional urea-based diet. The findings highlight this approach as a superior alternative to conventional urea-based diets and indicate potential reductions in methane emissions, promoting more sustainable livestock farming practices.

AUTHOR’S CONTRIBUTIONS

Dede Kardaya: Conceptualization, investigation, supervision, validation, writing – review and editing.

Deden Sudrajat: Formal analysis, metodology, investigation, writing – original draft.

Dewi Wahyuni: data curation, investigation, wrtiting – origial draft, project administration.

Ruslan Abdul Gopar: Investigation, Resources, Software, Validation.

Ikhsan Qodri Paramartaa: data curation, investigation, software, project administration.

Conflict of Interest

The authors have declared no conflict of interest.

REFERENCES

Aguerre M, Repetto JL, Pérez-Ruchel A, Mendoza A, Pinacchio G, Cajarville C (2009). Rumen pH and NH 3-N concentration of sheep fed temperate pastures supplemented with sorghum grain (Internet). S. Afr. J. Anim. Sci., 39(Supplement 1):246-250. https://doi.org/10.4314/sajas.v39i1.61157

Ali H, Ahmed Alqutbi A (2023). The Zeolite and Urea Effect on the Fodder Consumed and Productive Performance and of Awassi Lamps. Al-Qadisiyah Journal For Agriculture Sciences. Al-Qadisyiah Uni - Al-Qadisiyah J. Agric. Sci., 13(1):1–6. https://doi.org/10.33794/qjas.2022.136259.1090

Alipour D, Saleem AM, Sanderson H, Brand T, Santos LV, Mahmoudi-Abyane M (2021). Effect of combinations of feed-grade urea and slow-release urea in a finishing beef diet on fermentation in an artificial rumen system. Transl. Anim. Sci. Oxford Univ.ersity Press, 4(2):839–47. https://doi.org/10.1093/tas/txaa013

Alizadeh Z, Yansari A, Chashnidel Y, Kazemifard M, Ajarpajouh S (2021). Effect of soybean meal replacement by slow=release urea on ruminal parameter, blood metabolites, and microbial protein synthesis in Zel ram. Pasture Forage Util., 43:1–11. https://doi.org/10.4025/actascianimsci.v43i1.48684

AOAC (2006). Official method of analysis. Official method of analysis. Association of Official Analytical Chemists. . 18th edition. Washington DC USA: Assoc. Off. Anal. Chem.,

Belanche A, Martín-García I, Jiménez E, Jonsson NN, Yañez-Ruiz DR (2021). A novel ammoniation treatment of barley as a strategy to optimize rumen pH, feed degradability and microbial protein synthesis in sheep. J. Sci. Food Agric., 101(13):5541–9. https://doi.org/10.1002/jsfa.11205

Broderick GA, Kang JH (1980). Automated Simultaneous Determination of Ammonia and Total Amino Acids in Ruminal Fluid and In Vitro Media. J. Dairy Sci., 63(1):64–75. https://doi.org/10.3168/jds.S0022-0302(80)82888-8

Broucek J (2014). Production of Methane Emissions from Ruminant Husbandry: A Review. J Environ Prot (Irvine, Calif). Sci. Res. Publishing Inc, 05(15):1482–93. https://doi.org/10.4236/jep.2014.515141

Chanjula P, Suntara C, Cherdthong A (2021). The effects of oil palm fronds silage supplemented with urea-calcium hydroxide on rumen fermentation and nutrient digestibility of thai native-anglo nubian goats. Fermentation. MDPI; 7(4). https://doi.org/10.3390/fermentation7040218

de Lucena KH de OS, Mazza PHS, Oliveira RL, Barbosa AM, Perreira Filho JM, Bessa RJB (2024). Slow-releasing urea coated with low-trans vegetable lipids: Effects on lamb performance, nutrient digestibility, nitrogen balance, and blood parameters. Anim. Feed Sci. Technol., 310:115925. https://doi.org/10.1016/j.anifeedsci.2024.115925

El-Nile A, Elazab M, El-Zaiat H, El-Azrak KED, Elkomy A, Sallam S (2021). In vitro and in vivo assessment of dietary supplementation of both natural or nano-zeolite in goat diets: Effects on ruminal fermentation and nutrients digestibility. Animals, 11(8). https://doi.org/10.3390/ani11082215

El-Nile A, Elazab MA, Soltan YA, Elkomy AE, El-Zaiat HM, Sallam SMA (2023). Nano and natural zeolite feed supplements for dairy goats: Feed intake, ruminal fermentation, blood metabolites, and milk yield and fatty acids profile. Anim. Feed Sci. Technol., 295. https://doi.org/10.1016/j.anifeedsci.2022.115522

Estrada-Angulo A, López-Soto MA, Rivera-Méndez CR, Castro BI, Ríos FG, Dávila-Ramos H (2016). Effects of Combining Feed Grade Urea and a Slow-release Urea Product on Performance, Dietary Energetics and Carcass Characteristics of Feedlot Lambs Fed Finishing Diets with Different Starch to Acid Detergent Fiber Ratios. Asian-Australas. J. Anim. Sci. Asian-Australas. Assoc. Anim. Prod. Soc., 29(12):1725–33. https://doi.org/10.5713/ajas.16.0013

Fathul F, Sitti Wajizah D (2010). Penambahan Mikromineral Mn dan Cu dalam Ransum terhadap Aktivitas Biofermentasi Rumen Domba Secara In Vitro. Jurnal Ilmu Ternak dan Vet., 15(1):915.

Gelaye Y (2024). Application of nanotechnology in animal nutrition: Bibliographic review. Cogent Food Agric. Informa Healthcare. https://doi.org/10.1080/23311932.2023.2290308

Ghoneem WMA, El-Tanany RR, Mahmoud AEM (2022). Effect of Natural Zeolite as a Rumen Buffer on Growth Performance and Nitrogen Utilization of Barki Lambs. Pak. J. Zool., 54(3):1199–207. https://doi.org/10.17582/journal.pjz/20191207121206

Hameed HM (2021). Physiological role of Nanotechnology in Animal and Poultrynutrition. Egypt. J. Vet. Sci., 53(3):311-317. https://doi.org/10.21608/ejvs.2021.73671.1231

Heidari M, Ghorbani GR, Hashemzadeh F, Ghasemi E, Panahi A, Rafiee H (2022). Feed intake, rumen fermentation and performance of dairy cows fed diets formulated at two starch concentrations with either conventional urea or slow-release urea. Anim. Feed Sci. Technol., 290:115366. https://doi.org/10.1016/j.anifeedsci.2022.115366

Hristov AN, Bannink A, Crompton LA, Huhtanen P, Kreuzer M, McGee M (2019). Invited review: Nitrogen in ruminant nutrition: A review of measurement techniques. J. Dairy Sci., 102(7):5811–52. https://doi.org/10.3168/jds.2018-15829

Hua D, Zhao Y, Nan X, Xue F, Wang Y, Jiang L (2021). Effect of different glucogenic to lipogenic nutrient ratios on rumen fermentation and bacterial community in vitro. J. Appl. Microbiol., 130(6):1868–82. https://doi.org/10.1111/jam.14873

Jiang M, Zhang X, Wang K, Datsomor O, Li X, Lin M (2023). Effect of Slow-Release Urea Partial Replacement of Soybean Meal on Lactation Performance, Heat Shock Signal Molecules, and Rumen Fermentation in Heat-Stressed Mid-Lactation Dairy Cows. Animals. Multi. Digital Publishing Inst., (MDPI) 13(17). https://doi.org/10.3390/ani13172771

Kardaya D, Sudrajat D, Dihansih E (2012). Efficacy of dietary urea-impregnated zeolite in improving rumen fermentation characteristics of local lamb. Media Peternakan Fakultas Peternakan Institut Pertanian Bogor, 35(3): 207-213. https://doi.org/10.5398/medpet.2012.35.3.207

Kardaya D, Sudrajat D, Wahyuni D (2023). Performance of Local Sheep Fed Diets Containing Urea-Impregnated Zeolite. J. Anim. Health Prod., 11(2):199–205. https://doi.org/10.17582/journal.jahp/2023/11.2.199.205

Kardaya D, Wiryawan KG, Parakkasi A, Winugroho HM (2018). Effects of three slow-release urea inclusions in rice straw-based diets on yearling Bali bull performances. S. Afr. J. Anim. Sci., 48(4):752–7. https://doi.org/10.4314/sajas.v48i4.17

Khattab IM, Abdel-Wahed AM, Anele UY, Sallam SM, El-Zaiat HM (2023). Comparative digestibility and rumen fermentation of camels and sheep fed different forage sources. Anim. Biotechnol., 34(3):609–18. https://doi.org/10.1080/10495398.2021.1990939

Kumar P, Singh P, Chauhan S, Swaroop MN, Bhardwaj A, Datta TK (2023). Nanotechnology for Animal Sciences-New Insights and Pitfalls: A Review. Agricultural Reviews. Agric. Res. Commun. Cent., (Of). https://doi.org/10.18805/ag.R-2620

Li MM, White RR, Guan LL, Harthan L, Hanigan MD (2021). Metatranscriptomic analyses reveal ruminal pH regulates fiber degradation and fermentation by shifting the microbial community and gene expression of carbohydrate-active enzymes. Anim. Microbiome., 3(1):32. https://doi.org/10.1186/s42523-021-00092-6

Liang J, Zhang P, Zhang R, Chang J, Chen L, Wang G (2024). Response of rumen microorganisms to pH during anaerobic hydrolysis and acidogenesis of lignocellulose biomass. Waste Manage., 174:476–86. https://doi.org/10.1016/j.wasman.2023.12.035

Lira-Casas R, Efren Ramirez-Bribiesca J, Zavaleta-Mancera HA, Hidalgo-Moreno C, Cruz-Monterrosa RG, Crosby-Galvan MM (2019). Designing and evaluation of urea microcapsules in vitro to improve nitrogen slow release availability in rumen. J. Sci. Food Agric., 99(5):2541–7. https://doi.org/10.1002/jsfa.9464

Ma SW, Faciola AP (2024). Impacts of Slow-Release Urea in Ruminant Diets: A Review. Fermentation. Multidisciplinary Digital Publishing Institute (MDPI), 10(10). https://doi.org/10.3390/fermentation10100527

Mahdavirad N, Chaji M, Bojarpour M, Dehghanbanadaky M (2021). Comparison of the effect of sodium bicarbonate, sodium sesquicarbonate, and zeolite as rumen buffers on apparent digestibility, growth performance, and rumen fermentation parameters of Arabi lambs. Trop. Anim. Health Prod., 53(5):465. https://doi.org/10.1007/s11250-021-02909-7

Manikandan A, Subramanian KS (2014). Fabrication and characterisation of nanoporous zeolite based N fertilizer. Afr. J. Agric. Res., 9(2):276–84. https://doi.org/10.5897/AJAR2013.8236

Manju GU, Nagalakshmi D, Nagabhushana V, Venkateswarlu M, Rajanna NV (2022). Nutrient Utilisation in Ram Lambs Fed with Coated Slow Release Non-protein Nitrogen Sources. Indian J. Anim. Res. Agric. Res. Commun. Cent., (Of). https://doi.org/10.18805/IJAR.B-4859

Moss AR, Jouany JPierre, Newbold JC (2000). Methane production by ruminants: its contribution to global warming. Ann. Zootechnie, 49:231–53. https://doi.org/10.1051/animres:2000119

Mynhardt H, van Niekerk WA, Erasmus LJ, Hassen A, Coertze RJ (2016). Substitution of rumen degradable nitrogen with urea in sheep fed low quality: Eragrostis curvula hay. Sci Agric. Sci. Agricola, 73(6):498–504. https://doi.org/10.1590/0103-9016-2015-0277

Netto AJ, de Azevedo Silva AM, Bezerra LR, de Barros Carvalho A, da Silva Agostini DL, Vasconcelos de Oliveira DL (2021). Lipid microspheres containing urea for slow release of non-protein N in ruminant diets. Anim. Prod. Sci., 62(2):191–200. https://doi.org/10.1071/AN20694

Niazifar M, Besharati M, Jabbar M, Ghazanfar S, Asad M, Palangi V (2024). Slow-release non-protein nitrogen sources in animal nutrition: A review. Heliyon, 10(13):e33752. https://doi.org/10.1016/j.heliyon.2024.e33752

Orskov E (1975). Manipulation of rumen fermentation for maximum food utilization. World Rev. Nutr. Diet, 22:152–82. https://doi.org/10.1159/000397977

Partama IBG, Mudita IM (2019). Penurunan Emisi Polutan Ternak Kambing Melalui Aplikasi Teknologi Biofermentasi dan Suplementasi. J. Vet., 20(1):132–9. https://doi.org/10.19087/jveteriner.2019.20.1.132

Perez HG, Stevenson CK, Lourenco JM, Callaway TR (2024). Understanding Rumen Microbiology: An Overview. Encyclopedia. MDPI AG, 4(1):148–57. https://doi.org/10.3390/encyclopedia4010013

Pikhtirova A, Pecka-Kiełb E, Króliczewska B, Zachwieja A, Króliczewski J, Kupczyński R (2024). The Effect of Saponite Clay on Ruminal Fermentation Parameters during In Vitro Studies. Animals. Multi. Digital Publishing Instit. (MDPI), 14(5). https://doi.org/10.3390/ani14050738

Pressman EM, Kebreab E (2024). A review of key microbial and nutritional elements for mechanistic modeling of rumen fermentation in cattle under methane-inhibition. Front. Microbiol. Front. Media SA., 15(1488370):1-21. https://doi.org/10.3389/fmicb.2024.1488370

Qi W, Xue MY, Jia MH, Zhang S, Yan Q, Sun HZ (2024). Understanding the functionality of the rumen microbiota: searching for better opportunities for rumen microbial manipulation. Anim Biosci. Asian-Australas. Assoc. Anim. Prod. Soc., 370–84. https://doi.org/10.5713/ab.23.0308

Reddy PRK, Yasaswini D, Reddy PPR (2023). Nanotechnology in Veterinary Sector: Current Applications, Limitations and Future Perspective. Handb. Green, 97(8):1541-1567. https://doi.org/10.1007/978-3-031-16101-8_8

Roque-Jiménez JA, Pinos-Rodríguez JM, Rojo-Rub R, Mendoza GD, Vazquez A, De Jesus JAC (2019). Effect of natural zeolite on live weight changes, ruminal fermentation and nitrogen metabolism of ewe lambs. S. Afr. J. Anim. Sci. Afr. J. Online (AJOL), 48(6). https://doi.org/10.4314/sajas.v48i6.19

Saro C, Degeneffe MA, Andrés S, Mateo J, Caro I, López-Ferreras L (2023). Conventional Feed-Grade or Slow-Release Coated Urea as Sources of Dietary Nitrogen for Fattening Lambs. Animals,13(22). https://doi.org/10.3390/ani13223465

Sertova NM (2020). Contribution of nanotechnology in animal and human health care. Adv. Mater. Lett.,

Shen J, Zheng W, Xu Y, Yu Z (2023). The inhibition of high ammonia to in vitro rumen fermentation is pH dependent. Front. Vet. Sci., 10. https://doi.org/10.3389/fvets.2023.1163021

Silva A, Pereira Filho JM, Oliveira J, Lucena K, Mazza P, Silva Filho E (2023). Effect of slow-release urea on intake, ingestive behavior, digestibility, nitrogen metabolism, microbial protein production, blood and ruminal parameters of sheep. Trop. Anim. Health Prod., 55(6):414. https://doi.org/10.1007/s11250-023-03833-8

Silviani E, Yasmin N, Martin RSH, Jayanegara A (2024). Urea-nitrate coating with zeolite reduces in vitro ammonia concentration in the rumen. IOP Conf. Ser. Earth Environ. Sci. Inst. Phys., 1359(1):1-6. https://doi.org/10.1088/1755-1315/1359/1/012110

Soltan Y, Morsy A, Hashem N, Elazab M, Sultan M, Marey H (2021). Modified nano-montmorillonite and monensin modulate in vitro ruminal fermentation, nutrient degradability, and methanogenesis differently. Animals, 11(10):3005. https://doi.org/10.3390/ani11103005

Suryani NN, Budiasa IKM, Astawa IPA (2014). Rumen fermentation and microbial protein synthesis of ettawah grade (pe) goat fed various composition of forage and different level of concentrate. Majalah Ilmiah Peternakan, 17:56–60.

Toprak NN, Yilmaz A, Öztürk E, Yigit O, Cedden F (2016). Effect of micronized zeolite addition to lamb concentrate feeds on growth performance and some blood chemistry and metabolites. S. Afr. J. Anim. Sci., 46(3):313–20. https://doi.org/10.4314/sajas.v46i3.11

Valpotić H, Gračner D, Turk R, Đuričić D, Vince S, Folnožić I (2017). Zeolite clinoptilolite nanoporous feed additive for animals of veterinary importance: potentials and limitations. Period Biol. Croat. Soc. Nat. Sci., 159–72. https://doi.org/10.18054/pb.v119i3.5434

Vanzant ES, Cochran RC (1994). Performance and forage utilization by beef cattle receiving increasing amounts of alfalfa hay as a supplement to low-quality, tallgrass-prairie forage2. J. Anim. Sci., 72(4):1059–67. https://doi.org/10.2527/1994.7241059x

Vidya B, Venkateshwarlu M, Nagalakshmi D, Chandra AS, Preetham VC, Kumari NN (2024). Rumen Fermentation and Microbial Nitrogen Supply in Native Sheep Fed Crop Residue based Densified Feed Blocks Varying in Particle Size of Roughage. Asian J. Dairy Food Res. Agric. Res. Commun. Cent., 43(1):79–84.

Wahyuni S, Sunarso S, Eko Prasetiyono BWH, Satrija F, Jayanegara A (2024). Ruminal fermentation characteristics and methane emissions with the addition of Cassia spp. extract to a total mixed ration based on corn stover. J. Anim. Behav. Biometeorol., 12(1). https://doi.org/10.31893/jabb.2024006

Wang S, Tang W, Jiang T, Wang R, Zhang R, Ou J (2024). Effect of Dietary Concentrate-to-Forage Ratios During the Cold Season on Slaughter Performance, Meat Quality, Rumen Fermentation and Gut Microbiota of Tibetan Sheep. Animals, Multi. Digital Publishing Inst. (MDPI), 14(22). https://doi.org/10.3390/ani14223305

Wang YL, Zhang ZH, Wang WK, Wu QC, Zhang F, Li WJ (2023). The Effect of γ-Aminobutyric Acid Addition on In Vitro Ruminal Fermentation Characteristics and Methane Production of Diets Differing in Forage-to-Concentrate Ratio. Fermentation. MDPI, 9(2). https://doi.org/10.3390/fermentation9020105

Weimer PJ (2022). Degradation of Cellulose and Hemicellulose by Ruminal Microorganisms. Microorganisms. MDPI., 10(12): 1-30. https://doi.org/10.3390/microorganisms10122345

Xia ZG, Meng QX (2007). Effects of different proportions of dietary structural and nonstructural carbohydrates on ruminal fermentation and microbial growth effi ciency in sheep. J. Anim. Feed Sci., 16(Suppl. 2):218–23. https://doi.org/10.22358/jafs/74492/2007

Zhang W, Zhang L, Jiang X, Liu X, Li Y, Zhang Y (2020). Enhanced adsorption removal of aflatoxin B1, zearalenone and deoxynivalenol from dairy cow rumen fluid by modified nano-montmorillonite and evaluation of its mechanism. Anim. Feed Sci. Technol., 259:114366. https://doi.org/10.1016/j.anifeedsci.2019.114366

Zhang X, Medrano RF, Wang M, Beauchemin KA, Ma Z, Wang R (2019). Effects of urea plus nitrate pretreated rice straw and corn oil supplementation on fiber digestibility, nitrogen balance, rumen fermentation, microbiota and methane emissions in goats. J. Anim. Sci. Biotechnol. BioMed. Cent. Ltd., 10(1). https://doi.org/10.1186/s40104-019-0312-2