Research Article

The Potential of Synbiotics Supplementation using Gembili Inulin as a Prebiotic in Improving Fermentability and Productivity of Lambs Fed Diet of Different Fibre Content

Hanung Dhidhik Arifin1,2, Edy Rianto1*, Endang Purbowati1, Anis Muktiani1

1Department of Animal Sciece, Faculty of Animal and Agricultural Sciences, Diponegoro University, Semarang, Indonesia; 2Animal Husbandry Study Program, Faculty of Agriculture, University of Muhammadiyah Purworejo, Purworejo, Indonesia.

Received | March 19, 2025; Accepted | May 11, 2025; Published | June 05, 2025

*Correspondence | Edy Rianto, Department of Animal Sciece, Faculty of Animal and Agricultural Sciences, Diponegoro University, Semarang, Indonesia; Email: [email protected]

Citation | Arifin HD, Rianto E, Purbowati E, Muktiani A (2025). The potential of synbiotics supplementation using gembili inulin as a prebiotic in improving fermentability and productivity of lambs fed diet of different fibre content. Adv. Anim. Vet. Sci. 13(7): 1502-1516.

DOI | https://dx.doi.org/10.17582/journal.aavs/2025/13.7.1502.1516

ISSN (Online) | 2307-8316; ISSN (Print) | 2309-3331

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

Fibrous feed plays an important role ins ruminant production. In addition to maintain the life of microorganisms in the rumen, feed with high fibre content has consequences in decreasing digestibility and indirectly have negative impact in livestock productivity and resulted in high methane production (Galyean and Hubbert, 2014; Luthfi et al., 2023). Several studies found that the ability to digest fibre in feed and livestock productivity can be optimized by administering probiotics, prebiotics or a combination of both. Probiotics and prebiotics can increase the number of microbes, the number of enzymes and rumen microbial activity (Riswandi et al., 2015; Hartono et al., 2016). The combination of probiotics with prebiotics are called synbiotics which have great potential to improve conditions in the rumen (Markowiak and Śliżewska, 2018). Increasing the rumen microbial population will increase microbial activity, secretion and concentration of cellulolytic enzymes, thereby increasing the digestibility of fibre in feed and protein and VFA (Irwanto et al., 2019; Muhtarudin et al., 2020).

Commonly, buffalo rumen fluid was used as a source of probiotic. Buffalo rumen fluid contains bacteria, fungi, yeast and cellulolytic protozoa with the highest ability to degrade fibre in feed. Adult buffalo rumen fluid contains a total of 18.45 x 108 CFU/mL bacteria, total cellulolytic bacteria 6.86 x 108- 3.3 x 109 CFU/mL, cellulolytic activity 43.2%/day, VFA production rate is faster and higher than cow rumen fluid (Aminah et al., 2020). On the other hand, a combination of probiotic and prebiotic sources is required to form synbiotic. Prebiotics with high fibre are fermented by rumen bacteria to be used as an energy source (Samanta et al., 2013). One of the substances that functions as a prebiotic is inulin. Inulin is a compound that plays an indirect role as a prebiotic by forming short-chain fatty acids in the large intestine after fermentation (Tarini and Wolever, 2010; Ríos-Covián et al., 2016) and can increase the number of Bifidobacterium and Lactobacillus in the large intestine and increase their activity (Samanta et al., 2012). Inulin can be easily found in Gembili (Dioscorea esculenta L.). The previous study showed that Gembili contains inulin ranging from 10.53% to 14.80%; (Bahlawan et al., 2020; Hilman et al., 2021). Gembili is also a source of carbohydrate (Bahlawan et al., 2020). Gembili contains 59.06–60.52% carbohydrates, including amylose of 9.22–11.4% and amylopectin of 49.84–50.24% (Afoakwa et al., 2013; Retnowati et al., 2019; Riley et al., 2014). The production of Gembili in Indonesia is not recorded. However, this stuff is found abundant in many places in Indonesia, such as Papua (60 – 70 ton/ha/year; Sabda et al., 2019), Moluccas (43,02 – 67,56 ton/ha/year; Pesireron et al., 2021), Java (6,1-7,1 ton/ha/year; Fitria et al., 2024).

Studies of the potential synbiotic supplementation that use Gembili inulin as a source of prebiotic in the fibrous diet of lambs are still limited. It is necessary a study to examine the effect of synbiotic derived from the combination of Gembili inulin as a source of prebiotic to stimulate fermentability and digestibility in fibrous diet of lambs. This study was aimed to determine the potential of Gembili inulin as prebiotic source in synbiotic supplementation on nutrient utilization, fermentability and productivity of lambs fed different fibrous diet.

MATERIALS AND METHODS

There were 2 experiments in this study. The first was an in vitro experiment that consisted of two stages (In vitro 1 and 2). The experiment 1 was to examine the inulin levels of Gembili as a source of prebiotic in forming synbiotics supplementation in the next study. The flow chart of protocol of experiment 1 is presented in Figure 1. The experiment II was conducted in vivo to evaluate the nutrient utilization, fermentability and productivity of lambs. The flow chart of protocol of experiment 1 is presented in Figure 1.

 

Experiment 1

In vitro 1: Experiment I, in vitro 1 and 2 used a Completely Randomized Design (CRD) with 4 treatments and 4 replications. The first step aimed to determine the best

 

Table 1: Nutritional content of the in vitro diet.

Feed stuff	Moisture (%)	DM (%)	OM (%)	Nutrient Contents (%)					TDN (%)
				Ash	CP	EE	CF	NFE
				100% DM
Napier Grass	28.80	71.20	51.20	20.00	9.34	1.26	40.52	28.88	43.99
Concentrate	13.12	86.88	78.30	8.57	18.25	1.44	11.14	60.60	67.89
Total	24.10	75.90	59.33	16.57	12.01	31.71	1.32	38.39	51.16

 

Abbreviations: DM: Dry Matter; OM: Organic Matter; CP: Crude Protein; EE: Ether Extract; CF: Crude Fibre; NFE: Nitrogen Free Extract. TDN was calculated based on the equation proposed by Harris et al. (1972), as presented in Hartadi et al. (1980).

 

Table 2: Nutritional content of the in vivo diet.

Diet	DM (%)	OM (%)	Nutritional content					TDN (%)	TDN/CP	Energy (kal/g)
			Ash	CP	EE	Cfi	NFE
			------------------100%-------------------
D1S0	90.05	77.00	13.05	6.29	0.97	28.95	50.74	49.43	7.86	3900.29
D1S1	92.61	79.49	13.11	6.33	1.00	28.97	53.58	52.06	8.22	4018.01
D2S0	90.35	79.34	11.01	9.68	2.81	25.87	50.62	59.30	6.12	4022.70
D2S1	92.91	81.83	11.08	9.73	2.84	25.89	53.47	61.93	6.37	4140.42
D3S0	90.75	82.45	8.30	14.21	5.27	21.76	50.47	72.45	5.10	4185.91
D3S1	93.31	84.95	8.36	14.25	5.29	21.78	53.31	75.08	5.27	4303.63

 

Legend: DM: Dry Matter; OM: Organic Matter; CP: Crude Protein; Cfi: Crude Fibre; EE: extra ether, NFE: Nitrogen free extra; TDN: Total digestible nutrients.

 

Gembili level to make synbiotic flour. The treatment applied in experiment 1, in vitro 1 was the level of Gembili inulin (GI) in 50 mL of fermenter (10 mL of buffalo rumen fluid + 40 mL of McDougall + 0.556 g of diet), namely:

G1 = 0 mg GI + 50 mL fermenter

G2 = 5 mg GI + 50 mL fermenter

G3 = 10 mg GI + 50 mL fermenter

G4 = 15 mg GI + 50 mL fermenter

The concentrate contained 67.89% TDN and 18.25% CP. The Napier grass contained 43.99% TDN and 9.34% CP. The nutritional content of diet is presented in Table 1.

Each treatment was conducted in a 100 mL capacity of fermenter tube consisting of a 100-mL fermenter tube filled with 0.556 g of diet + levels of GI + fermenter (40 mL of McDougall’s solution + 10 mL of rumen fluid), then supplied with CO gas for 30 seconds and closed with a ventilated rubber cover. The tube was put into a shaker water bath at 39°C, then incubated for 3 hours for volatile fatty acids (VFAs), rumen ammonia, microbial protein; 6 hours for dry matter digestibility (DMD), organic matter digestibility (OMD), crude fibre digestibility (CFD), neutral detergent fibre digestibility (NDFD), and acid detergent fibre digestibility (ADFD). Those incubation were centrifuged to obtain the supernatant and the solid residue. The supernatant liquid was used for testing rumen ammonia, rumen VFAs concentration, DMD, OMD, CFD, NDFD, and ADFD. The solid residue was used for testing rumen microbial protein (Aminah et al., 2020; Nuswantara et al., 2020).

Synbiotic test (In vitro 2): The best results from in vitro 1 were then made into synbiotic flour and continued with the test stage. The treatment applied in experiment 1, in vitro 2 study was Synbiotic (Sin) supplementation in 0.556 g of diet and 50 mL of fermenter (10 mL of sheep rumen fluid (SRF) + 40 mL of McDougall) (Salman et al, 2017; Tian et al., 2019), namely:

S1 = 0% Sin + 50 mL fermenter

S2 = 1% Sin + 50 mL fermenter

S3 = 2% Sin + 50 mL fermenter

S4 = 3% Sin + 50 mL fermenter

Making of symbiotic: Cassava flour was sterilized by autoclaving at a temperature of 121°C, a pressure of 1 ATM for 15 minutes. Liquid synbiotics were mixed with cassava flour as much as 1.5 times the weight of the liquid synbiotic until homogeneous and smooth. The dough was placed on a sterile tray and ovened at a temperature of 40°C for 24-48 hours. The synbiotics were then ground, sieved, and tested for the total number of bacteria (Total Plate Count method). A 100 mL capacity fermenter tube was filled with 0.556 g of ration (70% Napier Grass + 30% Concentrate), 0-3% synbiotic flour from the ration and 50 mL of fermenter (10 mL CRF + 40 mL McDougall’s solution) (Salman et al., 2017; Tian et al., 2019), then flowed with CO gas for 30 seconds and closed with a ventilated rubber cover. The tube was inserted into an shaker water bath at a temperature of 39°C, then incubated for 3 hours (VFA, rumen ammonia, microbial protein); 6 hours (DMD, OMD, CfiD, NDFD, ADFD) and incubated for 6 days to measure cellulolytic enzyme activity (Aminah et al., 2020; Nuswantara et al., 2020).

Fermentability and digestibility measurements: The parameters observed in experiment I were rumen ammonia concentration, microbial protein concentration, rumen volatile fatty acid (VFA) concentration, dry matter digestibility (DMD), organic matter digestibility (OMD), and crude fibre digestibility (CfiD). Rumen ammonia concentration and microbial protein concentration were analysed using the Conway method (Putri et al., 2021); rumen VFA concentration (acetic acid, propionic acid, and butyric acid) was analysed using chromatography method (Jiang et al., 2013); dry matter digestibility (DMD), organic matter digestibility (OMD), and crude fibre digestibility (CFD) were analysed using methods employed by Evitayani et al. (2023) and Saleem et al. (2019), neutral detergent fibre digestibility (NDFD), and acid detergent fibre digestibility (ADFD) were analysed using AOAC method (AOAC, 2016; method 2002.04) as used by Ran et al. (2021).

Experiment 2

The best liquid synbiotic (polynomial orthogonal test results) was made into synbiotic flour using the method used by Tampangallo et al. (2018) which was modified. Orthogonal polynomial analysis showed the highest value in the cubic phase (R = 0.906) with a regression equation of Y = 0.816X³ - 4.507X² + 9.230X + 61.485, resulting in an optimal digestibility value of 69.06% at a synbiotic level of 2.46%, then the result was rounded to 3%. The second experiment was conducted based on the ethical feasibility of the study protocol with No. 58-09/A-9/KEP-FPP. This study used 24 lambs aged 3 months and weighed 12.10 ± 0,67 kg. The lambs were divided into 6 groups of treatments and reared for 3 months. The grouping of lambs based on Completely Randomized Design (CRD) 3x2 Factorial with 4 replications. The first factor was Pakchong grass (PG): concentrate ratio (C), namely 100% PG and 0% C (D1); 70% PG and 30% C (D2); 30% PG and 70% C (D3). The second factor was synbiotic supplementation (S) namely without synbiotic supplementation (S0); synbiotic supplementation 3% of the diet (S1). The combination of the diet and synbiotic supplementation were as follows:

D1S0 = 100% PG: 0% C + 0% synbiotic

D1S1 = 100% PG: 0% C + 3% synbiotic

D2S0 = 70% PG: 30% C + 0% synbiotic

D2S1 = 70% PG: 30% C + 3% synbiotic

D3S0 = 30% PG: 70% C + 0% synbiotic

D3S1 = 30% PG: 70% C + 3% synbiotic

The diet was given in the form of mash. Lambs were fed and watered ad libitum. The parameters observed in Experiment 2 were: feed intake, digestibility, fermentability in rumen and lambs productivity. Feed intake was recorded daily from the calculation of the provision minus the remaining rations every morning. The lambs were weighed every week to obtain body weight data as a basis for feeding (measured ad libitum).

A measurement of feed digestibility and fibre fractions and estimation of body protein production through purine derivatives were carried out through total collection of feces and urine accordance by Luthfi et al. (2024). Total collection was carried out in the 3rd week for 7x24 hours, with rations given as much as 4% of body weight. Analysis of dry matter of fresh feces and urine was carried out every day for a week. Feces and urine were collected using a metabolic cage connected to a jerrycan filled with 20% H2SO4 as much as 20 mL to keep the nitrogen in the urine from evaporating. The results of the 24-hour feces collection were then weighed every morning and samples of 10% of the total collection were taken for analysis of feces DM levels. The remaining feces samples were then mixed until homogeneous and dried, then ground. The feces samples were then analysed proximately (DM, ash, CP, EE, Cfi, and NFE), energy and Van Soest fibre fraction analysis (NDF ADF, hemicellulose, cellulose and lignin). The results of the 24-hour urine collection were then weighed every morning and samples of 10% of the total collection were taken for analysis of CP levels, and energy and purine derivatives.

Measurement of feed fermentability was carried out by taking rumen fluid at 0; 3 and 6 hours after morning feeding in the 12th week. Rumen fluid was taken by inserting a plastic tube into the lamb’s mouth, until it passed through the esophagus into the rumen cavity. The next step was to connect the plastic tube to a stemmed Erlenmeyer flask connected to a vacuum pump and then the vacuum pump was turned on to suck out the rumen fluid until it entered the Erlenmeyer flask as much as ± 50 mL (Luthfi et al., 2024). The rumen fluid that had been placed in the Erlenmeyer flask was then filtered using gauze and its pH was measured with a pH meter and H2SO4 was added to a pH value of ± 3 then put into a plastic bottle so that could be stored in the freezer and then taken to the laboratory to be analysed for VFA, NH3 and rumen microbial protein. Measurement of VFA production was carried out using the Gas Chromatography method according to Susilo et al. (2019). Measurement of NH3 production was carried out using the Conway microdiffusion method as used by Suharti et al. (2018). Calculation of CH4 production was carried out based on the concentration of acetate, propionate and butyrate, according to the method used by Susilo et al. (2019). The flowchart of experiment 2 is presented in Figure 2.

 

Parameters in experiments 2 were measured as follows:

a	Nutrients intake	:	Feed nutrients provided – Remaining feed nutrients
b	Nutrient digestibility (%)	:
Fermentability
a	Rumen pH Value	:	pH meter
b	Total VFA of Rumen fluid	:
c	A partial VFA (mM)	:	* molecular weight of acetate, propionate, and butyrate
d	Ammonia (NH3) of Rumen fluid	:
e	Methane production (mM)	:	0,5 A - 0,25 P + 0,5 B A: mol acetate; P: mol propionate; B: mol butirate
Feed Utilization
a	Retained Protein (g)	:	Protein intake - Fecal Protein - Urine Protein
b	Retained Energy (MJ)		Energy intake - Fecal energy - Urine Energy
c	Derivate purine	:	Allantoin, uric acid, xanthine-hypoxanthine levels (mmol/L) Allantoin, uric acid, xanthine-hypoxanthine volume (mmol/day) Percentage of allantoin, uric acid, xanthine-hypoxanthine 9%)
d	Microbial protein production	:	Y = 0,84X+(0,15BW0,75 e-0,25X); BW = Body Weight = 0,727 × X
Lambs Productivity
e	Average Daily Gain (ADG;g)	:
f	Feed conversion ratio (FCR)

 

Data Analysis

All data obtained from experiment 1 and 2 were analysed using analysis of variance (F test) with 5% accuracy to determine the effect of treatment. If there was a significant effect. The duncan test was carried out to determine the effect of synbiotic supplementation.

 

Table 3: Fibre fraction content of the diet in vivo.

Diet	NDF	ADF	Hemicellulose
	.....................................%...........................................
D1S0	65.45	42.38	23.07
D1S1	65.92	42.47	23.46
D2S0	58.24	35.55	22.68
D2S1	58.71	35.64	23.07
D3S0	48.62	26.45	22.17
D3S1	49.09	26.54	22.55

 

RESULTS AND DISCUSSIONS

Experiment 1

In vitro 1: The Gembili inulin significantly increased ruminal ammonia, microbial protein production, propionic, butyric acid and acetic/propionic ratio (P<0.05) while acetic acid concentration was significantly decreased (P<0.05). However, total VFA and methane energy were similar among the treatments (P>0.05). The fermentability profile of the diet is presented in Table 4.

The treatment G3 resulted in the highest ruminal ammonia production, while G0 was the lowest one (P<0.05). This study showed that higher inulin supplementation led to higher ruminal ammonia production. This study also showed treatment G3 resulted in the highest microbial protein production (P<0.05). It indicated that the supplementation of inulin encourage the microbial synthesize so that can improve nitrogen utilization. Salman et al. (2017) stated that inulin had the potential to improve nitrogen utilization. Wang et al. (2020) claimed that inulin fermentation improved nitrogen metabolism in the rumen. Rumen ammonia concentration is determined by protein breakdown by rumen microbes. Inulin provides extra energy for the growth of rumen microorganisms and balances the ratio of carbohydrates to nitrogen. which can contribute to microbial protein synthesis in the rumen.

The increase of inulin stimulated the microbial growth and lead the microbes to digest fibre of the diet to be better. This study agrees with Zhao et al. (2016) that ammonia concentration was determined by protein breakdown of rumen microorganisms. Inulin was more effective in promoting bacterial growth when rumen ammonia was sufficient. This finding was also in parallel with the study of Wang et al. (2021) that inulin increased the diversity and richness of rumen microbes, the elevated synthesis of microbial protein in rumen.

This study showed that the supplementation of GI up to 15mg (G4) in the fibrous diet increased the proportion of propionic and butyric while decreased the concentration of acetic (P<0.05). Therefore, the acetic/propionic ratio in this

 

Table 4: Fermentability and digestibility with Gembili inulin (In Vitro 1) .

In Vitro 1	Level of Gembili Inulin				P Value
	G1	G2	G3	G4
Rumen Fluid Profile
Ammonia (mg/100mL)	9.27c	11.62b	12.35ab	12.79a	0.00
Microbial Protein Production (mg/100mL)	19.99bc	22.64ab	18.88c	24.27a	0.02
VFA (mMol)	10.49	9.35	12.04	13.15	0.64
Acetic (%)	75.83a	75.44a	73.82b	73.77b	0.00
Propionic (%)	16.87c	17.36bc	18.11a	18.02ab	0.01
Butyric (%)	7.30b	7.20b	8.08a	8.21a	0.00
Acetic/Propionic Ratio	4.50a	4.35a	4.08b	4.10b	0.01
Methane energy (MJ)	0.06	0.05	0.06	0.07	0.35
Digestibility
DM	39.87	43.23	40.27	40.76	0.29
OM	47.36	51.35	48.34	48.48	0.21
Cfi	43.81	48.65	44.89	47.27	0.07
NDF	37.39	38.00	39.29	40.63	0.93
ADF	37.51	37.11	40.26	43.57	0.47

 

Abbreviations: VFA: Volatile Fatty Acid; DM: Dry Matter; OM: Organic matter; CF: Crude Fibre; NDF: Neutral Detrgent Fibre; ADF: Acid Detergent Fibre; a, b, c) different superscripts on the same line shows significant differences (P<0.05).

 

study decreased, parallel with the increase of GI. In feedlot system, this result was highly expected as indicator of deposition of protein in the body tissue. This was due to inulin can alter the fermentation pattern of rumen microbes without changing the total VFA production. It also indicated that the activity of ruminal microbes lead to a more efficient fermentation pathway for producing propionic and butyric acids, which has the potential to enhance energy efficiency in digestion. Changes in the diversity of rumen microbes or fermentation substrates (fibre and carbohydrates) can alter the composition of VFA without affecting the total amount of VFA produced. Zhao et al. (2016) and Zhang et al. (2021) stated that properties of inulin as a prebiotic containing fructose, which is easily and quickly fermented by rumen microbes, producing propionic and butyric acids. Inulin supports the growth of non-cellulolytic bacteria (Prevotella spp), where the production of propionate through the succinate pathway efficiently utilizes carbon and hydrogen, thereby reducing the availability of hydrogen for methanogenesis. Additionally, the increase in rumen ammonia concentration, which supports microbial protein synthesis, can enhance fermentation efficiency towards propionate production (Misra et al., 2022).

Methane energy in this study was similar among the treatments (averaged 0.06 MJ; P>0.05). It indicated that there was no significant change in fermentation efficiency. When the total VFA remains stable (Table 6), there was not enough change in the energy flow produced by fermentation to affect methane production or methane energy. Although there were changes in the proportion of propionate, these changes were not significant enough to reduce methane production. The change of concentration of acetate, propionate, and butyrate was not significant enough to drastically change hydrogen flow or methanogen activity. Therefore, even though there were changes in the VFA composition, the effects on methane emissions and methane energy were not visible.

The digestibility of fibrous diet supplemented GI is presented in Table 5. The supplementation of GI in fibrous diet did not change the DMD, OMD, NDFD, and ADFD of the fibrous diet (P>0.05).

The average of DMD was 41.03%; OMD was 48.88%; CfiD was 46.16%, NDFD was 38.83%; and ADFD was 39.61%. This indicated that inulin in first stage only worked in the rumen fermentation, but did not change the digesta absorption in the digestive tract after the rumen. Inulin was more appropriate to the fermentation substrate composition to promote more efficient alterations in rumen microorganisms than digestion. The DMD and OMD values in this study were lower than the finding of Salman et al. (2017), which showed that the inclusion inulin up to 100 mg/l in the rumen fluid fermenter of Karayaka sheep resulted in 66.69-73.21% DMD, 68.55-75.32% OMD, and 20.06-35.70% NDFD. According to Jayanegara et al. (2019), the in vitro digestibility value for DMD was 34.0% and OMD was 30.9%.

 

Table 5: Fermentability and digestibility of the diet supplemented synbiotic in vitro 2.

In Vitro 2	Synbiotic
	S1	S2	S3	S4	P value
Rumen Fluid Profile
Ammonia (mg/100mL)	17,20	18,05	18,21	18,41	0,61
Microbial Protein Production (mg/100mL)	25,96	26,15	28,46	30,42	0,31
VFA (mMol)	12,22	12,14	11,18	8,81	0,26
Acetic (%)	75,23	71,97	71,07	67,73	0,06
Propionic (%)	16,16	15,55	15,31	14,65	0,15
Butyric (%)	8,60c	12,48bc	13,63ab	17,63a	0,01
Acetic/Propionic Ratio	4,67	4,64	4,64	4,63	0,99
Methane energy (MJ)	0,07	0,07	0,06	0,05	0,39
Digestibility
DM	58,81c	63,99b	65,38ab	67,90a	0,00
OM	64,16c	70,05b	71,43ab	73,37a	0,00
Cfi	61,48c	67,02b	68,41ab	70,63a	0,00
NDF	40,45	42,69	44,13	44,21	0,09
ADF	21,17	21,23	23,66	24,01	0,52

 

Abbreviations: VFA: Volatile Fatty Acid; DM: Dry Matter; OM: Organic matter; CF: Crude Fibre; NDF: Neutral Detrgent Fibre; ADF: Acid Detergent Fibre; a, b, c) different superscripts on the same line shows significant differences (P<0.05).

 

Table 6: Nutrient intake of lambs fed different Pakcong grass: concentrate ratio and different synbiotic levels .

Intake	Synbiotics	Diet			average	P value
		R1	R2	R3
DM (g/d)	S0	509,27	623,70	739,23	624,07	Diet	0,00
	S1	499,84	693,08	799,83	664,25	Synbiotics	0,16
	average	504,56c	658,39b	769,53a	644,16	Interaction	0,45
OM (g/d)	S0	392,14	494,83	609,52	498,83y	Diet	0,00
	S1	397,34	567,15	679,42	547,97x	Synbiotics	0,04
	Average	394,74c	530,99b	644,47a	541,36	Interaction	0,38
CP (g/d)	S0	32,03	60,40	105,04	65,82	Diet	0,00
	S1	31,65	67,41	113,99	71,02	Synbiotics	0,07
	Average	31,842c	63,91b	109,52a	68,42	Interaction	0,34
Cfi (g/d)	S0	147,44	161,33	160,84	156,53	Diet	0,03
	S1	144,82	179,44	174,20	166,15	Synbiotics	0,20
	Average	146,13b	170,39a	167,52a	161,34	Interaction	0,48
TDN (g/d)	S0	251,75	369,84	536,54	385,71y	Diet	0,00
	S1	260,23	429,20	600,49	429,97x	Synbiotics	0,01
	Average	255,99c	399,52b	568,01a	407,84	Interaction	0,32
Energy (MJ/d)	S0	8,31	10,50	12,95	10,59y	Diet	0,00
	S1	8,40	12,01	14,40	11,60x	Synbiotics	0,04
	Average	8,36c	11,25b	13,68a	10,60	Interaction	0,38

 

Explanation: a,b,c Different superscripts in the same row indicate significantly different feed ratio factors (P<0.05; Duncan’s Test); x,y Different superscripts in the same column indicates significant differences between synbiotic supplementation levels (P<0.05).

 

In vitro 2: Based on the first stage, the results of the Gembili inulin level from the previous stages were then made into synbiotic flour and continued with stage 2. Fermentability of fibrous diet is presented in Table 6. The study showed that there was significant effect on butyrate (P<0.05).

 

Table 7: Nutrient digestibility of lambs fed different Pakcong grass: Concentrate ratio and different synbiotic levels.

Digestibility (%)	Synbiotic	Diet			Average	P value
		R1	R2	R3
DM	S0	54,20	61,03	66,93	60,72	Diet	0,00
	S1	52,45	68,11	69,18	60,79	Synbiotics	0,45
	Average	53,32c	60,28b	68,85a	60,78	Interaction	0,77
OM	S0	54,19	61,03	66,93	60,72	Diet	0,00
	S1	54,18	62,32	63,80	60,79	Synbiotics	0,25
	Average	54,18c	61,48b	65,37a	60,40	Interaction	0,67
CP	S0	54,40	66,31	66,47	68,71	Diet	0,00
	S1	58,84	67,36	68,35	76,87	Synbiotics	0,45
	Average	56,62c	66,83b	67,41a	72,79	Interaction	0,98
Cfi	S0	34,09	43,00	42,09	39,73	Diet	0,33
	S1	43,88	43,10	50,52	45,83	Synbiotics	0,14
	Average	39,98	43,05	46,30	42,78	Interaction	0,56
Retained
Digested protein (g/d)	S0	14,04	33,09	71,67	39,60	Diet	0,00
	S1	11,89	34,51	75,65	40,68	Synbiotic	0,65
	Average	12,96c	33,80b	73,66a	40,18	Interaction	0,58
Retained Protein (g/d)	S0	6,90	27,35	57,61	30,62	Diet	0,00
	S1	7,84	27,00	65,82	33,54	Synbiotic	0,20
	Average	7,37c	27,16b	61,723a	32,08	Interaction	0,25
Digested energy (MJ/d)	S0	3,34	5,16	7,80	5,55	Diet	0,00
	S1	3,67	6,18	9,38	6,41	Synbiotic	0,10
	Average	3,50c	5,85b	8,59a	5,98	Interaction	0,57
Retained energy (MJ/d)	S0	2,29	4,83	6,48	4,53	Diet	0,00
	S1	3,13	4,78	8,44	5,45	Synbiotic	0,10
	Average	2,71c	4,81b	7,46a	5,49	Interaction	0,20

 

Explanation: a,b,c Different superscripts on the same row indicate significantly different feed ratio factors (P<0.05); x,y,z Different superscripts in the same column indicate significantly different synbiotic supplementation factors (P<0.05).

 

The higher supplementation synbiotic up to 3% increased concentration of butyric acid. It indicated that the increase in butyrate was also supported by synbiotic, which aids the growth of butyrate-producing bacteria (Butyrivibrio fibrisolvens), and provided a substrate that is easily and quickly fermented through the butyrogenic pathway, especially under conditions of high rumen ammonia concentration. This study was similar to the finding of Zhao et al. (2016) that the inclusion of inulin in the low crude protein (CP) 8% and high crude protein (CP) 16% in the diet decreased the concentration of acetic acid, AP ratio, and methane concentration and increased the concentration of propionate and butyrate. Zhao et al. (2016) claimed that inulin was likely to suppress rumen microbes involved in fibre fermentation, resulting in a reduction in acetic production. The percentage of acetic, propionic, and butyric acids was higher than the finding of Umucallilar et al. (2010), which showed that inulin supplementation of 0–4% in rations with concentrate forage ratios of 20/80, 40/60, and 60/40 analysis in vitro using rumen fluid of male Holstein sires resulted in acetic acid percentages of 52.84–55.67%, propionic acid 18.27–20.07%, and butyric acid 25.05–28.54%.

The digestibility of fibrous diet supplemented synbiotic is presented in Table 7. The supplementation of synbiotic in fibrous diet had significant effect in nutrient digestibility (P<0.05).

It indicated that the digestibility of fibrous diet can be optimized by supplementation of synbiotics. This was due to synbiotics up to 3% increased the population of fermentative microbes in the rumen, which supports feed degradation, especially through the role of inulin as a prebiotic that stimulates the growth of Fibrobacter succinogenes and Ruminococcus albus, the main bacteria in the breakdown of crude fibre (Amin and Mao, 2021). Buffalo rumen fluid in synbiotics increased the population of more adaptive microbes in the sheep rumen, thereby increasing the activity of cellulase and protease enzymes, accelerating fibre hydrolysis, increasing nutrient availability, and optimizing fermentation efficiency and feed utilization (Zhao et al., 2022).

Experiment 2

Feed intake: Based on the results of Experiment I, the study continued to in vivo treatments. The nutrient intake of lambs is presented in Table 6. The study showed that nutrient intakes had significantly different among treatments (P<0.05). Although there were significant impacts on feed intakes in each treatment factor, there were no interaction between synbiotics and different diet quality (P>0.05). Lambs fed R3 (high concentrate) had highest nutrient intake than the others. The highest DMI intakes was in line with OM, CP, Cfi, TDN and Energy intakes of lambs fed R3. The higher the DM intake, the higher the intakes of OM, CP, Cfi, TDN and energy. This was parallel with the studies of Luthfi et al. (2023) and Luthfi et al. (2024) that the nutrient intake was influenced by the amount of dry matter intake; the higher the dry matter consumed, the higher the nutrient intake.

The highest nutrient intake in R3 was due to high fibre sources feed such as R1 and R2 have bulky feed characteristics. Increasing concentrate and decreasing grass in feed has consequences for decreasing NDF. The higher the grass used in this study, the bulkier the feed. Bulky feed easily fills the rumen cavity of sheep, so that it physically pushes on the rumen wall and causes faster physical satiety. Increasing concentrate and decreasing grass in feed has consequences for decreasing NDF. Karimazadeh et al. (2017) claimed that feed with low volume and high density of nutrient content increases voluntary intake of animals. This study was similar to the finding by Quang et al. (2015) and Truong (2021), which found that feed intake enhanced as the amount of concentrate consumed increased in Brahman cattle. On the other hand, supplementation of Synbiotics had significant effects on OM, TDN and energy intakes (P<0.05) but did not on DM, CP and Cfi (P>0.05). Lambs fed S1 had higher intakes than those of S0. It indicated that feed supplemented by Synbiotics improve the organic consumption and total energy.

Digestibility and utilization: The feed digestibility is presented in Table 7. The study showed that the different nutrient quality of feed have significant effect on nutrient digestibility (P<0.05). However, synbiotic supplementation up to 3% in the diet did not change the digestibility (P>0.05). There were no interaction among treatments in nutrient digestibility. Lambs fed R3 had highest digestibility than those of R1 and R2 (P>0.05).

This study indicated that the lower grass and the higher concentrate composition in the diet caused the low fibre content in the diet (Table 3) and it increase the nutrient digestibility. Mirzaei-Alamouti et al. (2021) found that the lower NDF resulted in high digestibility. The concentration of NDF was corelated to the cell wall of fibre sources and has the highest impact on feed intake and thus on nutrient intake between diet containing different fibre (Tedeschi et al., 2019; McDonald et al., 2021).

Supplementation of synbiotic up to 3% in the diet have no effect on the digestibility of DM, OM, CP and Cfi. The digestibility of DM, OM, CP and Cfi was 43.56%, 45.86%, 53.05% and 42.78%, respectively. This study indicated that for ruminants, the addition of synbiotics in the diet of lambs was not strong enough to improve overall digestibility. In this study, there was no influence of synbiotic supplementation on the digestibility value of sheep feed, resulting in no difference in protein and energy utilization in lambs (P>0.05). This finding was in line with the study by Hamasalim (2016), that the concept of synbiotics in supplementation was to change the composition of the gut microbiota with beneficial organisms and non-absorbable organism substrates. The non-significant results on the synbiotic effects of nutrient utilization in sheep were due to improved feed quality, thus improving microbiota and digestibility values. the previous study by Arowolo and He (2018), found that the high digestibility of feed supplemented by synbiotic- mannooligosaccharides (similar to inulin) was largely due to the high digestibility of NDF on the diet. This is thought to be due to the basal diet (different fibre content) may also be a contributing factor and the type and level of probiotics/prebiotics used in synbiotic supplementation on fibre digestion was not yet large enough. This finding was similar to the study by Shrama et al. (2023) that supplementation of synbiotic did not affect DM digestibility in the diet of calve. The study by Kazemi-Bonchenar, (2013) dry matter digestibility, crude protein and organic matter were not affected by the use of synbiotics.

The digestion and retention of feed were presented in Table 8. As the results of feed intake and digestibility, this study showed that lowering fibre content in the diet improved the digested and retained nutrients (P<0.05). There was no significant impact on digested and retained nutrient of lambs fed the diet supplemented synbiotic levels (P>0.05) and also there was no interaction among factor treatments (P<0.05). The higher quality diet, the higher the digested and retained nutrient intake of lambs. Lambs fed R3 had the highest digested-retained protein and energy than those of the others. It was due to the significant and the highest nutrient intake of Lambs fed R3 with high digestibility. Therefore, a high feed intakes resulted in high nutrients being digested and metabolized. Luthfi et al. (2022) and Luthfi et al. (2024) found that digested nutrient of feed was highly affected the degradability and the amount of feed intake. This is in line with this study, where

 

Table 8: Rumen fluid profile of lambs fed different Pakchong grass: concentrate ratio and different synbiotic levels.

Parameters	Hour	Synbiotic	Diet			average	P value
			R1	R2	R3
pH	0	S0	7,55	7,71	7,76	7,67	Interaction	0,83
		S1	7,36	7,68	7,73	7,59	Diet	0,14
		Average	7,46	7,70	7,74	7,63	Synbiotic	0,50
	3	S0	7,57	7,59	7,37	7,51	Interaction	0,99
		S1	7,56	7,56	7,31	7,47	Diet	0,46
		Average	7,56	7,57	7,34	7,49	Synbiotic	0,84
Acetic acid (mmol/L)	0	S0	13,90	19,80	14,78	16,16	Interaction	0,77
		S1	15,40	20,63	22,08	19,37	Diet	0,53
		Average	14,65	20,21	18,43	17,76	Synbiotic	0,43
	3	S0	40,26	16,34	19,98	25,52	Interaction	0,02
		S1	11,04	18,13	26,39	18,52	Diet	0,18
		Average	25,65	17,23	23,18	22,02	Synbiotic	0,07
Propionic acid (mmol/L)	0	S0	2,49	3,68	2,82	3,00	Interaction	0,97
		S1	4,31	5,60	5,36	5,09	Diet	0,71
		Average	3,40	4,64	4,09	4,04	Synbiotic	0,11
	3	S0	8,76	3,72	4,61	5,70	Interaction	0,00
		S1	2,35	3,37	7,64	4,45	Diet	0,04
		Average	5,56a	3,55b	6,12a	5,08	Synbiotic	0,13
Butyric acid (mmol/L)	0	S0	0,79	1,28	1,40	1,15	Interaction	0,91
		S1	1,54	2,44	1,99	1,99	Diet	0,58
		Average	1,16	1,86	1,69	1,57	Synbiotic	0,15
	3	S0	2,66	1,20	1,80	1,89	Interaction	0,02
		S1	0,72	1,19	3,23	1,71	Diet	0,08
		Average	1,69	1,19	2,51	1,80	Synbiotic	0,71
Acetic/Propionic Ratio	0	S0	5,46	5,67	5,30	5,48	Interaction	0,42
		S1	3,73	4,24	4,82	4,26	Diet	0,61
		Average	4,60	4,95	5,06	4,87	Synbiotic	0,12
	3	S0	4,61	4,38	4,42	4,47	Interaction	0,18
		S1	5,26	5,17	3,54	4,65	Diet	0,13
		Average	4,93	4,77	3,98	4,56	Synbiotic	0,63
VFA (mmol/L)	0	S0	17,18	24,75	18,99	20,31	Interaction	0,87
		S1	21,24	28,67	29,43	26,45	Diet	0,55
		Average	19,21	26,71	24,21	23,38	Synbiotic	0,29
	3	S0	51,67	21,26	26,39	33,10	Interaction	0,00
		S1	14,11	22,69	37,26	24,69	Diet	0,14
		Average	32,89	21,97	31,82	28,89	Synbiotic	0,09
Methane (mmol/L)	0	S0	6,73	9,62	7,38	7,91	Interaction	0,81
		S1	7,39	10,14	10,70	9,41	Diet	0,50
		Average	7,06	9,88	9,04	8,66	Synbiotic	0,45
	3	S0	19,26	7,84	9,74	12,28	Interaction	0,00
		S1	5,30	8,82	12,90	9,00	Diet	0,21
		Average	12,28	8,33	11,32	10,64	Synbiotic	0,09
NH3 (mg/100 mL)	0	S0	6,42	6,72	6,15	6,43	Interaction	0,88
		S1	6,35	6,59	6,22	6,38	Diet	0,07
		Average	6,38	6,66	6,18	6,41	Synbiotic	0,77
	3	S0	6,63	6,71	6,75	6,70	Interaction	0,91
		S1	6,60	6,88	6,82	6,77	Diet	0,66
		Average	6,61	6,79	6,79	6,74	Synbiotic	0,70

 

Table 9: Protein microbial production and efficiency of microbial protein synthesize of lambs.

Microbial Nitrogen Production (g/d)	S0	0,71	0,91	1,19	0,93	Interaction	0,50
	S1	0,68	1,05	1,36	1,03	Diet	0,00
	Average	0,69c	0,98b	1,27a	0,98	Synbiotic	0,20
Microbial protein production (g/d)	S0	4,41	5,67	7,42	5,83	Interaction	0,50
	S1	4,25	6,55	8,48	6,43	Diet	0,00
	Average	4,33c	6,11b	7,95a	6.13	Synbiotic	0,20
OMD (g/d)	S0	82,78	149,39	218,87	150,35	Interaction	0,54
	S1	96,97	167,54	270,31	178,27	Diet	0,00
	Average	88,88c	158,46b	244,59a	164,31	Synbiotic	0,08
Efficiency of microbial protein synthesize (g/kg OMD)	S0	59,36	39,81	34,18	44,45	Interaction	0,27
	S1	45,44	39,19	31,34	38,66	Diet	0,00
	Average	52,40a	39,50b	32,76b	41,56	Synbiotic	0,11

 

Explanation: a,b,c Different superscripts on the same row indicate significantly different feed ratio factors (P<0.05); x,y,z Different superscripts in the same column indicate significantly different synbiotic supplementation factors (P<0.05).

 

probiotic/prebiotic supplementation did not affect NDF digestion (Jin et al., 2014; Nunez-Benitez et al., 2021). Mc Donald et al. (2021) found that low fibrous diet (low NDF) and high dry matter intake resulted in high nutrient digestibility and promote high composition of nutrient that can be metabolized.

Rumen fluid profile: The effect of Synbiotic supplementation in the different quality feed is presented in Table 8. There were no different significant effect on pH rumen fluid among factor treatments and interaction (P>0.05). The average pH value at 0 h and 3 h after feeding was 7.34 and 7.49, respectively. It indicated that pH rumen fluid of lambs was stable even they fed different quality of diet and supplemented different synbiotics.

In general, there was no significant effect among treatments on volatile fatty acids and methane production (P<0.05). However, there was an interaction between treatment at 3 h after feeding. The study showed that increasing supplementation synbiotics in low quality diet was able to reduce acetic acid concentration, and methane production simultaneously. It can be seen that the addition up to 3% in 100% Pakchong grass was able to reduce acetic acid concentration from 40.26 mmol/L to 11.04 (decreased about 28 mmol/l) which also resulted in a decrease in methane from 19.26 mmol to 5.30 (decreased about 14 mmol/l). When the diet quality was improved, the addition of synbiotics actually increased acetic acid and methane production. On the other hand, the propionic acid increased along with the increase of diet quality. Therefore, the acetic/propionic ratio in this study was stable in 4.56. The previous study showed that the combination of prebiotics inulin and Enterococcus faecium stimulates postnatal rumen development and improves its function (Arne and Ilgaza, 2021). Commercial synbiotic supplementation in dairy cattle diet has been shown to enhance rumen fermentation and allow cattle to consume feed more efficiently without affecting blood parameters (Turkhachev et al., 2022). Other studies have shown that inulin and S. cerevisiae supplementation improved rumen and gastrointestinal development of Holstein crossbred calves (Jonova et al., 2021). Rumen VFA production is highly correlated with the rumen OM digestion rate, and in turn, the rate and extent of rumen fibre digestion. A study by Arne and Algaza (2021), showed that the addition of inulin as a synbiotic combination in the diet can significantly increase the development of rumen papillae and muscle layer thickness. It was evaluated that the growth of the Saccusventralis muscle and longer Saccusventralis papillae improved in calves fed medium and high doses of inulin and its combination with E. faecium. Inulin has increased the development of these tissues because the rumen requires a longer breakdown process for fibrous feed. On the other hand, prebiotic inulin enhances the growth of lactic acid bacteria such as Bifidobacterium and Lactobacillus, which have a positive impact on the microflora and increase the availability and absorption of nutrients (Singh et al., 2017).

The ammonia rumen fluid production is presented in Table 9. Ammonia rumen fluid in this study was not significantly different (P>0.05) and there were not interactions among

 

Table 10: Productivity and FCR of lambs fed different quality diet and supplemented Synbiotic.

Parameters	Synbiotic	Diet			Average	P value
		R1	R2	R3
Initial weight (kg)	S0	10,72	11,96	12,91	11,86	Interaction	0,98
	S1	10,96	12,48	13,56	12,33	Diet	0,14
	Average	10,84	12,22	13,24	12.10	Synbiotic	0,63
Final weight (kg)	S0	13,21	17,78	20,98	17,32	Interaction	0,33
	S1	12,30	18,65	24,03	18,32	Diet	0,00
	Average	12,75c	18,21b	22,50a	17,82	Synbiotic	0,36
ADG (g/hari)	S0	29,64	69,23	96,04	64,97	Interaction	0,18
	S1	16,00	73,45	124,56	71,34	Diet	0,00
	Average	22,82c	71,34b	110,30a	68,16	Synbiotic	0,48
FCR	S0	24,01	9,09	8,77	13,97	Interaction	0,31
	S1	34,02	9,72	6,55	16,76	Diet	0,00
	Average	29,04b	9,40a	7,66a	15.37	Synbiotic	0,41

 

the treatments (P>0.05). The ammonia rumen in this study was in 6.74 mg/100 mL. It indicated that supplementation synbiotics up to 3% kept the ammonia rumen to be stable. By increasing synbiotic supplementation in the diet, it could reducing the risk bloat in animals fed high protein - low fibre diets.

Microbial protein production and efficiency of microbial protein synthesize are presented in Table 9. Different quality diet had significant effect on protein microbial production and microbial protein synthesize (P<0.05). However, synbiotic did not change the protein microbial production, OM digested and microbial protein synthesize (P>0.05). This study also implicated that there was no interaction among factor treatments. The higher the quality diet, the higher protein microbial production and OM digested. Lambs fed R3 had highest protein microbial production and lowest efficiency protein microbial rumen compared to lambs fed R1 and R2. It indicated that increasing of concentrate and reducing the grass composition in the diet drive the high protein content in the diet. The higher protein content in the diet, the higher nitrogen source for synthesize the microbial protein in the rumen. Sharma et al. (2023) claimed the microbial protein production was highly associated with increased microbial protein synthesis. Both conditions depend on reduction of ruminal breakdown of dietary N and or increases on microbial protein synthesis. In this study, synbiotics did not significantly affect the amount of N and OM degraded. Therefore, ammonia concentration in the rumen did not change the even the diet were in high protein and low fibre.

Productivity

The productivity lambs fed different fibre content supplemented synbiotics is presented in Table 10. The different of fibre content in feed change the productivity of lambs (P<0.05). However, levelling of synbiotic did not affect productivity and there was no interaction between the treatment on the lambs productivity (P>0.05).

The lower fibre content of the diet, the better the productivity of lambs. The lambs fed R3 had the highest ADG, highest final weight, and lowest FCR (110.30 g/d; 22.50 kg; 7.66) and the lambs fed R2 had the lowest ADG, lowest final weight and highest FCR (22.82 g/d) than the others. It was due to the higher DMI and nutrient utilization of lambs fed R3. Therefore, increasing feed quality resulted in good performance. The lowest FCR of lambs fed R3, indicated that enhancing feed fibre was able to reduce DM requirements for increasing weight gain. Luthfi et al. (2022) and Luthfi et al. (2024) found that the higher nutrient intake and utilization resulted in high body weight gain and low feed conversion ratio. In this study also showed that no significant effect on lambs productivity in synbiotic supplementation was due to there were no significant effect of synbiotic addition in feed digestibility and utilization.

 

CONCLUSIONS AND RECOMMENDATIONS

Based on the results, it can be concluded that Gembili Inulin can be used to be a prebiotic source up to 15 mg. The level of synbiotic up to 3% can be supplemented into the diet. A synbiotic supplementation has a good impact on the fermentability of a diet with high fibre and low protein content. Inulin from Gembili as prebiotic source in synbiotic supplementation decreased acetic acid and methane production simultaneously. It kept the ammonia concentration to be stable and prevented the risk of bloat when protein was increased and fibre source of the diet was reduced.

ACKNOWLEDGEMENTS

The authors wish to thanks to KEMENRISTEKDIKTI for scholarship and Muhammadiah Purworejo University for the study permission.

NOVELTY STATEMENTS

The author stated that study on the topic presented in this paper is very limited.

AUTHOR’S CONTRIBUTIONS

Conceptualization: EDR, EWP, and ANM; methodology: EDR, ANM, EWP and HDA; validation: EDR.; formal analysis: HDA; investigation: HDA, and EDR. Data curation, EWP and ANM; writing—original draft preparation: HDA and EDR; writing—review and editing: EWP and ANM. All authors have read and agreed to the published version of the manuscript.

Conflict of Interest

The authors have declared no conflict of interest.

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