Influence of Dietary Clove oil Supplementation on Proximate Constituents,in vitro Ruminal Fermentation Efficiency and Microbial Composition

Abstract

Objectives

This study sought to determine the effect of clove oil (CL) as a replacement to flavomycin antibiotic growth promoter (AGP) in a goat basal diet on proximate components, in vitro ruminal fermentation characteristics, and microbiome profile.

Methods

Six treatment groups were formulated using a goat basal diet without AGP and CL (NEGCON), diet with flavomycin AGP (POSCON), and AGP-diet supplemented with 0.5% (CL5), 1.0% (CL10), 1.5% (CL15), and 2.0% v/w (CL20) of clove oil. Nine independent replicate samples from each treatment group were homogenized, oven-dried, milled and analysed for proximate components. Each replicate sample (1 g) was inoculated with donor goats’ ruminal fluid in a pre-mixed phosphate buffer solution (pH 6.8) and incubated (39 °C) to record in vitro ruminal gas production, fermentation kinetics, volatile fatty acids (VFA), and detect archaeal and bacterial diversity using 16 s rRNA gene sequencing.

Results

Supplementation with CL in place of AGP linearly increased the organic matter content of the diets. Replacing the AGP with CL significantly decreased VFA production but had no effects on the in vitro fermentation characteristics. The abundance of Anaerovibrio spp. was higher at 1% CL supplementation but lower for NEGCON. Lactobacillus mucosae abundance was highest at 2% CL followed by 1.5% CL supplementation, while both control groups caused the lowest abundance.

Conclusion

It was concluded that CL can modulate rumen microbial communities by enhancing microbial diversity and promoting beneficial probiotic populations. However, the associated reduction in VFA production at higher supplementation levels suggests that CL could negatively affect the energy status of ruminants. Future in vivo studies are needed to validate these findings and determine the optimal inclusion level of CL that could enhance fermentation efficiency in goats.

Keywords

Antibiotic growth promoter Essential oils Rumen microbiota Small ruminants Volatile fatty acids Goat nutrition

Introduction

Goat production holds a significant role in the socio-economy and food security of many countries.¹ However, uncontrolled disease outbreaks and low productivity in goat production have led goat meat producers to use antibiotic growth promoter (AGP) such as flavomycin to relieve the burden of infectious diseases and maximise profit.² The overuse of AGP has burdened the human healthcare sector with increased morbidity and mortality due to the emergence of antimicrobial-resistant diseases globally.³ These bacteria undergo mutation and does not respond to antibiotic treatments.⁴ Opportunistically, these bacteria spread to humans through antibiotic residues in meat products resulting from the misuse of in-feed antibiotics in livestock production.⁵ This has increased worldwide concerns and led to the ban of AGP in many countries.⁶ Alternatively, natural alternatives to AGP including herbal plants, seeds or essential oils have gained global research interests.⁷ These natural additives contain terpenoids, phenylpropanoids and other phytochemicals that possess antimicrobial and antioxidant effects that are detrimental to pathogenic bacteria⁸ but beneficial to animal performance.⁹

Clove essential oil (CL) contains at least 50% eugenol and about 10% to 40% of its content comprise of β-caryophillene, eugenyl acetate, α-caryophyllene, and α-copaene⁹ and has been shown to enhance nutrient utilization and modulate rumen fermentation in small ruminants.¹⁰ However, the optimal dose that CL can effectively replace AGP has not been investigated in vitro. Therefore, the purpose of the study was to evaluate the effect of supplementing a basal goat diet with varying levels of clove oil replacing AGP on proximate composition, in vitro ruminal fermentation, volatile fatty acids, and microbial profile. It was hypothesized that CL supplementation in place of flavomycin will enhance fermentation efficiency by promoting beneficial ruminal microbial populations.

Materials and Methods

Ethics Statement

The in vitro study was conducted at the Animal Science Laboratory of the North-West University Farm, Molelwane Research Farm in Mafikeng (North West, South Africa) for 96 h. The study protocol was approved by the Animal Production Research Ethics Committee of North-West University (Ethics Approval Number: NWU-00816-23-A5). All procedures adhered to the ARRIVE 2.0 guidelines for the reporting of animal research.¹¹

Sources and Treatment Formulation

The feed ingredients and synthetic antibiotic growth promoter (flavomycin) used for this experiment were sourced from Simplegrow Agric Services (Pty) Ltd in Centurion (Gauteng, South Africa). The CL used in this study was purchased from Dychem Industrial (PTY) LTD (Kempton park, South Africa). A basal goat diet was formulated using Spesfeed software to be isocaloric and isonitrogenous and to meet the growing requirements of meat-type goats¹² as follows: POSCON = a basal diet with flavomycin (0.025 g/kg); NEGCON = a basal diet without flavomycin; CL5 = basal diet with 0.5% CL; CL10 = basal diet with 1% CL; CL15 = basal diet with 1.5% CL; and CL20 = basal diet with 2.0% (v/w) CL as shown in Table 1. Each treatment had 9 independent replicates and the samples were ground (1 mm) and homogenised using a KINEMATICA Polymix grinder (model PX-MFC 90 D 230 V/ EU, Eschbach, Germany).

Table 1.

Ingredient composition of the experimental treatments.

Ingredients (g/kg as is basis)	¹Treatments
Ingredients (g/kg as is basis)	NEGCON	POSCON	CL5	CL10	CL15	CL20
Yellow maize (8.0% CP)	434.6	432.6	402.0	349.7	307.0	287.5
Wheat bran (15% CP)	−	−	27.61	88.08	131.7	134.6
Molasses	80.00	80.00	80.00	80.00	80.00	80.00
Clove oil	−	−	5.000	10.00	15.00	20.00
Flavomycin	−	2.500	−	−	−	−
Soya oilcake (47% CP)	20.90	20.90	−	−	−	−
Sunflower oilcake (38% CP)	130.0	130.0	151.6	138.7	130.5	133.4
Eragrostis hay	150.0	150.0	150.0	150.0	150.0	150.0
Lucerne meal	150.0	150.0	150.0	150.0	150.0	150.0
Acid buffer	6.000	6.000	6.000	6.000	6.000	6.000
Ammonium chloride	10.00	10.00	10.00	10.00	10.00	10.00
Ammonium sulphate	3.000	3.000	3.000	3.000	3.000	3.000
Monodicalcium phosphate (21% phosphorus)	0.450	0.450	−	−	−	−
Limestone powder	9.090	9.090	9.170	9.060	11.39	19.99
Magnesium oxide (51% Magnesium)	0.420	0.420	0.150	−	−	−
Salt course (2 mm)	5.000	5.000	5.000	5.000	5.000	5.000
¹Melis P	0.160	0.160	0.160	0.160	0.160	0.160
²Ruminant premix	0.330	0.330	0.330	0.330	0.330	0.330
Total	1000	1000	1000	1000	1000	1000

Treatments = POSCON: basal diet with flavomycin (0.025 g/kg), NEGCON: basal diet without flavomycin or clove essential oil, CL5: basal diet containing clove essential oil at 0.5% inclusion level, CL10: basal diet containing clove essential oil at 1.0% inclusion level, CL15: basal diet with clove essential oil at 1.5% inclusion level, CL20: basal diet containing clove essential oil at 2.0% inclusion level. ¹Melis P: a taste enhancer containing natural and artificial sweeteners (Agribution Canada Ltd, Steinbach, Manitoba, Canada). ²Ruminant premix = vitamin A (7 million IU/kg), manganese (60 g/kg), zinc (125 g/kg), copper (15 g/kg), cobalt (0.45 g/kg), iodine (15 g/kg), selenium (0.3 g/kg), magnesium (80 g/kg).

Chemical Composition

Proximate composition of the experimental treatments was analysed according to Association of Official Analytical Chemists¹³ guidelines while the detergent methods described by¹⁴ were used to determine neutral detergent fibre (NDF) and acid detergent fibre (ADF) using an ANKOM²⁰⁰⁰ Fibre Analyzer designed by ANKOM Technology (New York, USA).¹⁴ Acid detergent lignin (ADL) was analysed by submerging the ADF residue into 72% sulphuric acid for 3 h while agitating every 30 min. The samples were washed thoroughly with water, submerged into acetone, air-dried, and then oven-dried (105 °C) before being weighed. Hemicellulose was calculated as the difference between NDF and ADF, whereas cellulose was calculated as the difference between ADF and ADL.

In Vitro Ruminal Fermentation Experiment

The in vitro experiment was conducted by firstly feeding three donor goats with blue buffalo grass and a concentrate at a ratio of 30:70 for 2 weeks. To avoid invasive procedure on live goats, the goats were slaughtered at an abattoir and the rumen fluid was harvested immediately into pre-warmed insulated flasks under continuous CO₂ flushing to maintain anaerobic conditions and prevent oxidation. Equal volumes of rumen fluid from the three donors were pooled to create a single inoculum batch for the experiment. The pooled rumen fluid was strained with a 4-layer muslin cloth (1 mm pore size) into an Erlenmeyer flask that was previously warmed (39 °C) while flushing with CO₂ to maintain anaerobic conditions.¹⁵ The flask was tightly sealed and transported to the Animal Science Laboratory at the North-West University Research Farm.

The in vitro gas production experiment followed the method of¹⁶ wherein replicate samples (1 g) were added in 125 mL airtight serum bottles, filled with 90 mL phosphate buffer (pH 6.8) and 25 mL rumen fluid, flushed with CO₂, and incubated for 96 h. Gas pressure was recorded using a pressure transducer (PX4200-015GI, Omega Engineering, QC, Canada) with a 23-gauge needle through the rubber stoppers. Measurements were taken from 2 to 96 h and corrected using blank bottles.¹⁷ Gas pressure (psi) was converted to volume (mL) using the formula:

y = 0.034 x^{2} + 6.2325 x + 1.8143

Where y = gas volume (mL) and x = measured gas pressure (psi).

Cumulative gas data were fitted to the¹⁸ model:

y = a + b (1 - e^{- c (t - l t)})

Where, y = gas production per period “t”; a = gas produced from the rapid fermentable portion (mL/g OM); b = emitted gas from the slow fermentable portion (mL/g OM); c = the constant for the gas production rate in the insoluble portion b (%/hrs); t = period of incubation time in hours; and lt = lag time (hrs). The potential gas (Pgas) produced was calculated by adding a and b.

At 24 h post-incubation, three bottles per treatment were randomly selected, and 2 mL of the medium was centrifuged (Cryste S4100G, Varispin 4, 4000 RPM, Korea) for VFA analysis. Samples were stored at 4 °C and analysed for acetic, propionic, butyric, valeric, iso-butyric, and iso-valeric acids via gas chromatography (GC-MS Laboratory, Stellenbosch University, South Africa).

Measures of Fermentation Efficiency

After the 96 h of incubation period, the residues were decanted by pouring the fermentation mixture from the serum bottles into ANKOM F57 filter bags to filter the solid residues. The bags were gently squeezed to remove the liquid before being transferred into pre-weighed crucibles and oven-dried at 105 °C for 12 h. The dry residues were weighed and incinerated to determine ash residue content at 600 °C for 6 h, which ensured complete oxidation of the residue.¹⁹ The organic matter was calculated as the difference between dry residue and ash residue. The result was used to calculate in vitro organic matter degradability (ivOMD) as follows:

ivOMD (g / kg) = \frac{OM content of incubated sample - OM content of residues}{OM content of incubated sample} \times 1000

Partition factor (PF96) at 96 h post-incubation was calculated as a ratio of the cumulative gas and in vitro organic matter degradability.

\begin{aligned} Partition factor (mL / mg OM) = \frac{96 h cumulative gas production}{96 h OMD} \end{aligned}

Characterization of the Ruminal Microbiome

Three replicate rumen fermentation samples per treatment were randomly collected and preserved with DNA/RNA shield for genomic DNA extraction. 16S rRNA (V4 region) sequencing targeting both bacteria and archaea was conducted at Inqaba Biotec™ using Illumina NextSeq with primers 515F–806R. Sequence data were processed using USEARCH and classified via the Ribosomal Database Project. OTUs representing less than 1% of total reads were excluded. Microbial abundance was assessed at the kingdom, phylum, genus, and species levels.

Statistical Analysis

Proximate composition, in vitro fermentation parameters, and VFA data (apart for the POSCON data) were assessed for linear and quadratic coefficients using the procedure of response surface regression (PROC RSREG) in SAS version 9.4.²⁰ A non-linear model was used to determine the level of CLO inclusion that maximized and minimized the response variables.

y = a x^{2} + b x + c

Where: y = dependent variable, a and b are the coefficients of the quadratic equation, and c is

dietary CL levels. The x value that minimized or maximized the response variables was determined as: $\frac{- b}{2 a}$ . Treatment effects on proximate composition, in vitro fermentation parameters, VFA and microbial abundance were evaluated using PROC GLM, and means were separated using Tukey HSD test, where p < .05.

Linear or quadratic trends were disregarded if PROC GLM results were not significant. The control groups (POSCON and NEGCON) were further contrasted using pre-planned orthogonal contrasts.

Alpha diversity indices were calculated using the vegan package in R and reported as mean ± SD. Beta diversity was assessed via PERMANOVA (Adonis2, 999 permutations) on Bray-Curtis dissimilarity and visualized using principal coordinate analysis (PCoA). Significance was set at p < .05.

Results

Table 2 shows that OM linearly increased [R² = 0.492; P = .0004] in response to CL inclusion. All CL levels increased the organic matter (OM) values than NEGCON and POSCON, however, there were no significant differences on dry matter (DM), crude fat (CF) and crude protein (CP), fibre, and lignin values.

Table 2.

Chemical composition of a basal goat diet supplemented with varying levels of clove essential oil.

²Parameters (g/kg)	¹Treatments						SEM	P values
²Parameters (g/kg)	POSCON	NEGCON	CL5	CL10	CL15	CL20	SEM	Overall	Linear	Quadratic	PvsN
Dry matter	921	919	920	916	926	927	3.300	0.151	0.124	0.144	0.665
Organic matter	834^b	832^b	859^a	850^a	858^a	860^a	3.800	<.0001	0.0004	0.1123	0.444
Crude protein	135	136	135	136	138	139	0.222	0.647	0.270	0.476	0.073
Crude fat	33.7	32.0	39.1	38.7	36.2	32.9	3.720	0.661	0.706	0.211	0.900
Neutral detergent fibre	311	322	305	284	289	300	8.600	0.055	0.036	0.053	0.415
Acid detergent fibre	129	127	133	116	124	132	3.900	0.060	0.922	0.159	0.397
Acid detergent lignin	33.9	32.4	28.5	35.3	32.0	39.5	7.490	0.940	0.673	0.627	0.903
Cellulose	183	195	172	169	166	167	7.150	0.060	0.004	0.055	0.397
Hemicellulose	94.7	94.8	104.0	80.3	91.8	92.4	5.700	0.158	0.505	0.523	0.999

^{a, b, c}

Means with the same superscripts did not vary (P > .05).

Treatments = POSCON: Control diet containing flavomycin, NEGCON: Control diet containing zero level of clove oil and flavomycin, CL5: basal diet containing clove essential oil at 0.5% inclusion level, CL10: basal diet containing clove essential oil at 1.0% inclusion level, CL15: basal diet with clove essential oil at 1.5% inclusion level, CL20: basal diet containing clove essential oil at 2.0% inclusion level.

PvsN: preplanned orthogonal contrasts for NEGCON and POSCON.

Table 3 shows that in vitro ruminal fermentation parameters were not (P > .05) affected by CL supplementation, with no significant linear, quadratic, or orthogonal contrast effects observed.

Table 3.

Effect of clove essential oil on in vitro ruminal fermentation parameters.

²Parameters	¹Treatments						P values
	POSCON	NEGCON	CL5	CL10	CL15	CL20	Overall	Linear	Quadratic	PvsN
a (mL/g OM)	45.2 ± 7.240	49.0 ± 6.610	58.2 0 ± 11.45	70.7 ± 7.240	50.5 ± 8.100	51.8 ± 7.241	0.214	0.704	0.262	0.638
b (mL/g OM)	660 ± 64.99	744 ± 59.32	762 ± 102.8	778 ± 64.99	756 ± 72.66	744 ± 64.99	0.853	0.694	0.926	0.470
c (%/hrs)	0.05 ± 0.008	0.03 ± 0.007	0.03 ± 0.007	0.03 ± 0.008	0.03 ± 0.007	0.04 ± 0.008	0.617	0.133	0.381	0.302
Lag time (hrs)	1.54 ± 0.178	1.39 ± 0.163	1.24 ± 0.283	1.05 ± 0.179	1.51 ± 0.200	1.40 ± 0.179	0.458	0.817	0.238	0.556
Pgas (mL/g OM)	705 ± 70.70	793 ± 64.53	820 ± 111.8	849 ± 70.70	806 ± 79.04	795 ± 70.70	0.812	0.691	0.803	0.474
CUMG96 (mL/g OM)	717 ± 65.04	722 ± 72.73	841 ± 110.2	794 ± 89.96	754 ± 89.96	786 ± 77.92	0.108	0.627	0.171	0.121
ivOMD96 (g/kg)	633 ± 69.88	458 ± 60.52	540 ± 85.58	563 ± 60.52	618 ± 69.88	539 ± 60.51	0.479	0.776	0.382	0.964
PF96 (mL/mg OM)	1.17 ± 0.159	1.70 ± 0.138	1.56 ± 0.194	1.62 ± 0.138	1.61 ± 0.159	1.61 ± 0.138	0.261	0.626	0.811	0.291

^{a, b, c}

Means with the same superscripts did not vary (P > .05).

Treatments = POSCON: control diet containing flavomycin, NEGCON: control diet containing zero level of clove oil and flavomycin, CL5: basal diet containing clove essential oil at 0.5% inclusion level, CL10: basal diet containing clove essential oil at 1.0% inclusion level, CL15: basal diet with clove essential oil at 1.5% inclusion level, CL20: basal diet containing clove essential oil at 2.0% inclusion level. ²Parameters = a: the gas production from the immediately fermentable fraction (mL), b: the gas production from the slowly fermentable fraction (mL), c: the gas production rate constant for the insoluble fraction, CUMG96: Cumulative gas at 96 h of incubation, Pgas: potential gas production, PF96: partition factor at 96 h post-incubation, ivOMD96: In vitro organic matter digestibility at 96 h post-incubation. PvsN: preplanned orthogonal contrasts for NEGCON and POSCON.

The inclusion of CL reduced (P < .05) VFA production with CL20 recording the least compared to POSCON, which showed no significant variation with NEGCON as seen on table 4. Table 5 shows that acetic, propionic, isobutyric, isovaleric, valeric and butyric acids recorded a positive quadratic response (P < .05) to clove essential oil supplementation.

Table 4.

Effect of supplementing a basal goat diet with varying levels of clove essential oil on in vitro ruminal volatile fatty acids.

Parameter (mg/L)	¹Treatments						P-value	Contrast
Parameter (mg/L)	POSCON	NEGCON	CL5	CL10	CL15	CL20	P-value	PvsN
Acetic	1413 ± 54.60^a	1370 ± 54.60^ab	1141 ± 54.60^bc	1070 ± 63.00^c	1035 ± 54.60^c	1039 ± 63.0° ^c	0.008	0.589
Propionic	698.7 ± 21.22^a	701.8 ± 21.22^a	526.7 ± 21.22^b	503.1 ± 24.50^b	529.4 ± 21.22^b	486.3 ± 24.50^b	0.0004	0.918
Isobutyric	77.30 ± 3.340^a	83.20 ± 3.340^a	46.40 ± 3.340^b	41.40 ± 3.850^b	43.80 ± 3.340^b	38.60 ± 3.850^b	<.0001	0.384
Isovaleric	68.60 ± 2.420^a	73.10 ± 2.420^a	45.30 ± 2.420^b	39.80 ± 2.800^b	42.70 ± 2.420^b	40.00 ± 2.800^b	<.0001	0.064
Valeric	96.20 ± 3.210^a	104.8 ± 3.200^a	62.80 ± 3.210^b	58.80 ± 3.710^b	67.00 ± 3.210^b	58.10 ± 3.710^b	<.0001	0.327
Butyric	461.6 ± 12.63^a	502.8 ± 12.63^a	354.4 ± 12.63^b	320.2 ± 14.58^b	337.3 ± 12.63^b	318.5 ± 14.58^b	<.0001	0.162

a, b, c

Means with the same superscripts did not vary (P > 0.05).

Parameters = PvsN: preplanned orthogonal contrasts for NEGCON and POSCON.

Table 5.

Regression equations for volatile fatty acids in response to varying levels of clove essential oil in a goat basal diet.

volatile fatty acids	Equations	Levels that Minimized VFAs	P-linear	P-quadratic	Overall R²
Acetic	y = 206 (±43.12) $x$ ² - 523 (±84.13) $x$ + 1365 (±32.83)	1.27%	<.0001	0.0003	0.795
Butyric	y = 106 (±18.25) $x$ ² - 277 (±35.61) $x$ + 491 (±13.89)	1.31%	<.0001	<.0001	0.866
Propionic	y = 123 (±24.97) $x$ ² −309 (±48.73) $x$ + 685 (±19.01)	1.26%	<.0001	0.0002	0.796
Isobutyric	y = 24.3 (±4.790) $x$ ² - 64.2 (±9.346) $x$ + 79.7 (±3.65)	1.32%	<.0001	0.0002	0.838
Isovaleric	y = 19.8 (±3.343) $x$ ² - 51.4 (±6.523) $x$ + 70.8 (±2.545)	1.30%	0.0001	0.0001	0.866
Valeric	y = 27.7 (±5.925) $x$ ² - 69.7 (±11.56) $x$ + 100 (±4.510)	1.25%	<.0001	0.0004	0.780

Supplementary Table 1 shows that the alpha diversity metrics (Shannon, Simpson, Fisher Alpha and Richness) tended to increase with increasing levels of CL inclusion.

Supplementary Figure 1 shows that CL significantly affected the Bray Cutis dissimilarity index of the microbiome. The treatments are clustered together in pairs, with POSCON and NEGCON, CL5 and CL10, and CL15 and CL20 promoting similar microbes. The dissimilarity index (R² = 0.932; P = .001) significantly varied among the treatments when accessed with PERMANOVA and the pairwise results were not significant.

The Bacterial and Archaeal populations were not (P > .05) affected by the inclusion of CL in the goat basal diet as shown in supplementary Figure 2.

Supplementary Figure 3a shows the microbes identified at the bacterial phylum level with 17 major phyla identified in all samples. Supplementary Figure 3 (b - d) shows that a high (P < .05) abundance of Firmicutes and Proteobacteria were recorded for CL5 and CL10 while other treatments recorded similar values. CL10 had the highest (P < .05) Chloroflexi microbes compared to other treatment groups, which do not differ.

There were 44 genera identified as shown in supplementary Figure 4. The genera Adlercreutzia recorded the highest (P < .05) abundance for CL20 followed by CL15. Treatment CL10 increased (P < .05) the abundance of Anaevibrio, followed by POSCON, while NEGCON had the least value. Clostridium was more (P < .05) abundant in CL10 and CL20 groups than in POSCON and NEGCON. CL10 increased (P < .05) the abundance of Moryella followed by CL15 and CL20, which had no treatment variation with POSCON and NEGCON. CL5 resulted in the lowest (P < .05) Prevotella abundance and did not (P > .05) differ from POSCON or the other treatments. The SHD and Sphingomonas had higher (P < .05) abundance for CL10 while other treatments did not vary.

Fifty-one species were identified as shown in supplementary Figure 5. The abundance of Anaerovibrio spp. was increased by all CL inclusion levels compared to NEGCON with the highest (P < .05) value recorded for CL10, however, the CL inclusion levels did not vary with POSCON. CL20 promoted the highest (P < .05) value for CF231 spp. while POSCON had the least value. CL10 caused the highest (P < .05) Desulfovibrio spp. abundance while CL5, CL15 and CL20 did not vary (P > .05) with POSCON and NEGCON. Lactobacillus mucosae had the highest (P < .05) abundance in CL20, followed by CL15, whereas POSCON and NEGCON promoted comparably the lowest abundance values. CL10 and CL20 had the highest (P < .05) abundance of RFN_20 spp. when compared to CL5 and CL15.

Discussion

Nutrient composition is essential in goat production as it enhances the growth of rumen microbes for efficient fermentation,²¹ which is key to nutrient metabolism and absorption. Dietary crude protein is essential to ruminal microbes to allow the synthesis of microbial proteins, while the fibre content maintains balance in the rumen and promote microbial fermentation processes that break down complex carbohydrates.²² The products of microbial fermentation become precursors for volatile fatty acid production, which is an energy source for goats. Hence, a balanced nutrient profile is essential to maintain healthy rumen microbiota and enhance the overall productivity of goats.²³ The results of this study showed that CL increased organic matter, however there were no effects on other proximate components of the diets. This suggests that CL could improve the availability of dietary organic components, without altering the overall nutritional composition of the feed. Neutral detergent fiber (NDF) and cellulose tended to decrease, particularly at higher inclusion levels, while hemicellulose values fluctuated across treatments. Park et al,²⁴ dry matter intake (DMI) is positively correlated with total volatile fatty acids (TVFA) and NDF intake with acetate production. This relationship suggests that reduced effective fiber in this study may have lowered substrate availability for acetate-producing microbes, thereby contributing to the reduction in VFA yields observed at higher CL inclusion levels. Clove oil contains 63.94% eugenol and 33.97% caryophyllene, these compounds have antioxidant and antimicrobial properties²⁵ which makes them able to modulate microbial populations and activity in the rumen. In this study, it was expected that CL inclusion would enhance the energy efficiency of the fermentation process by reducing excessive gas production, which is considered a form of energy loss. The in vitro ruminal fermentation parameters were not statistically different, however, CUMG96 and ivOMD96 increased numerically at CL10 but declined at higher doses suggesting that CL inclusion is dose-dependent. The partitioning factor (PF96), which reflects microbial efficiency, remained similar across treatments, suggesting that CL did not compromise the efficiency of microbial protein synthesis.

The inclusion of CL caused positive quadratic responses from the volatile fatty acids (VFA) and their production was minimized at 1.27, 1.31, 1.26, 1.32, 1.30 and 1.25% for acetic, propionic, isobutyric, isovaleric, valeric and butyric acids, respectively. In support of these findings, VFA concentrations decreased with increasing inclusion of oregano essential oil (0, 13, 52, 91 and 130 mg/L) when incubated with 150 ml of buffered sheep rumen fluid with 1200 mg of substrate for 24 h.²⁶ This result suggests that the efficiency of essential oils in modulating the rumen fermentation dynamics is highly-dose dependent and might compromise VFA production. This is not a desirable effect as VFAs are energy precursors that the animal uses for productivity.²⁷

In this study, the alpha, Shannon and Simpson diversity metrics showed a numerical increase with CL inclusion levels and was notable at CL15 and CL20, showing an increase in the abundance and variability of species and suggesting a healthier ruminal environment.²⁸ Alpha diversity reflects microbial diversity within each treatment, while Shannon and Simpson indices assess species abundance and evenness.²⁹ Their increase suggests more diverse and evenly distributed microbial populations. In this study, the richness of the microbiome environment was measured using the species richness and fisher alpha's index. The species’ richness only considers the number of unique species, while Fisher Alpha accounts for the number of unique species and their distribution. Similar to the current findings,³⁰ reported that there was no effect of phytogenics on rumen alpha diversity in bovines fed concentrate diets.

Beta diversity, which measures differences in species across environments,²⁹ revealed significant group variations, indicating that CL altered microbial community structure. To explore this further, microbial abundance was assessed at kingdom, phylum, genus, and species levels.

The inclusion of CL in goat basal diet had no effect on the bacteria and archaeal communities. At the phylum level, 17 groups were identified and the Actinobacteria (6.55% – 10.12%), Bacteriodates (39.04 – 46.36%), Firmicutes (32.6% – 38.2%), and Unknown (5.51% – 7.32%) dominated the rumen microbiota. Similarly, the phyla Bacteroidetes, Firmicutes, and Proteobacteria were the dominant ruminal microbes reported by.²⁶ The lower doses (CL5 and CL10) increased the population of Firmicutes but had no effect on Bacteriodates, while the higher doses (CL15 and CL20) reduced the abundance of Firmicutes and promoted comparable values to POSCON and NEGCON. Likewise,³¹ reported that origanum oil, garlic oil, and peppermint oil (0.50 g/L each) caused a decline in ruminal Firmicutes abundance while origanum and peppermint oils increased Bacteriodates. This confirms the ability of CL to selectively impact the growth of ruminal microbes. This is consistent with the report of⁹ which states that eugenol selectively distrupts bacterial cell membranes.

Higher abundance of Chloroflexi and Proteobacteria were recorded for CL10 when compared to POSCON and NEGCON. Chloroflexi is known to assist in the digestion of complex organic matter to support microbial growth.³² Although the function and effects of Chloroflexi in the rumen has not been fully explored, As reported by,³³ 80 mg/kg blend of essential oils (5.5% cinnamaldehyde, 9.5% eugenol, and 3.5% Capsicum oleoresin.) included in high concentrate diet for lambs reduced Chloroflexi. The reduction of Chloroflexi observed at the higher CL levels is consistent with findings where a cinnamaldehyde–eugenol–capsicum blend similarly decreased the relative abundance of Chloroflexi in lambs fed high-concentrate diets.³³ Some previous studies have associated Proteobacteria with low methane emitting cows,^34,35 meaning that its abundance in the ruminal microbiota at 1.0% CL may indicate lower methane production.

Although the role of Adlercreutzia has not been fully documented, a report suggested it is inversely correlated with oxidative stress and inflammatory cytokines meaning it may have health benefits³⁶ and it is also associated with weak absorption and weight loss in a Crohn disease (CD) rat model.³⁷ Genera CF231 and Prevotella, belonging to the phylum Bacteriodates are fibre degraders,³⁸ and Prevotella is also involved in protein and amino acid metabolism.³⁹ The response of these microbes tended to be dose dependent as the highest abundance of CF231 and Prevotella was recorded at CL20 and POSCON when compared to other inclusion levels. This is indicative of the impact of CL to selectively suppress other bacterial growth to favour the growth of some microbes. To corroborate this finding,²⁶reported that the abundance of Prevotella was increased linearly by increasing doses of oregano essential oil (0, 13, 52, 91 and 130 mg/L) incubated with sheep rumen fluid. The abundance of Bifidobacterium and Lactobacillus were higher in CL treatments with the highest abundance recorded at CL20 compared to POSCON and NEGCON. These two genera are probiotic bacteria that prevent ruminal acidosis and manage gut health.⁴⁰ These increases are consistent with reports showing that eugenol-containing blends modulate rumen bacterial communities, improving epithelial health and contributing to a more balanced microbial environment.³³

Sphingomonas, which is also a probiotic bacterium, was detected in only three treatments (POSCON, CL5 and CL10) but were significantly higher in CL10. The increase recorded for these genera suggests that CL inclusion enhanced probiotic bacteria and may have positive effects on gut health and host immune systems.⁴¹ CL10 increased Anaevibrio compared to NEGCON while other treatments were comparable to POSCON. This result is similar to that of⁴² who found that 1% tucuma oil on hay concentrate mixture enriched the abundance of Anaevibrio. Although, the abundance of Clostridium was similar for all treatments, CL10 and CL20 were higher. Clostridium is a hyper ammonia producing bacteria that is considered a harmful microbe because of its potential to initiate acidosis.⁴⁰ However, the values recorded in this study are similar to NEGCON and POSCON and do not suggest toxicity.

The abundance of Adlercreutzia spp., CF231 spp., Desulfovibrio spp., Lactobacillus mucosae, RFN_20 spp., and Treponema increased with CL increasing inclusions. Although, there are few studies that have discussed the functional effects of most of these species in the rumen, Desulfovibrio spp. is from the phylum Preoteobacteria and they are known for their nitrate and sulphate reduction abilities. They use lactate and pyruvate as organic substrates in the rumen.⁴³ This might explain the slight reduction of the Lactobacillus mucosae recorded for CL10, where Desulfovibrio spp. had the highest abundance.

The study of⁴⁴ associated the genus Oscillospira with increased butyrate formation when they administered Pinus koraiensis cone essential oil orally at 1 g/day in a basal diet of native Korean goats. The RFN_20 spp. which is from the phylum Tenericutes was also associated with VFA production in the rumen.⁴⁵ However, due to their low abundance, they did not increase VFA production. Pyramidobacter spp. are from the phylum Synergistetes and are known to be fibre degraders associated with high production of acetic acid and methane.⁴² However, this study did not follow the expected trend, as CL10 had the highest abundance of Pyramidobacter spp. but was associated with reduced acetic acid production. Given the positive relationship between acetate formation and methane output,²⁴ this finding suggests that an increase in Pyramidobacter abundance may not necessarily enhance methane production. This agrees with study of⁴² who reported that 1.0% tucumã oil increased the genus Pyramidobacter and decreased acetic acid production. Consistently,⁴⁵ found that genus Pyramidobacter is negatively correlated to acetate and methane production. This suggests that the eugenol and caryophyllene content of clove can suppress hydrogen-producing microbes and potentially redirect hydrogen toward more efficient fermentation pathways.

Treponema spp. which has been associated with acetate production, increased with dietary CL inclusion levels implying symbiotic interaction between microbes where Clostridium aminophilum produces ammonia.³⁸ And this ammonia is further reduced by Desulfovibrio spp. Both Desulfovibrio spp. and Pyramidobacter spp. seem to be using acetate as substrate^43,44 in the medium as Treponema spp. drives acetate production.⁴⁶ These shifts may reflect broader community adjustments driven by the phenolic activity of eugenol and caryophyllene, contributing to a more balanced rumen ecosystem even though overall VFA production was not enhanced. Anaerovibrio spp. increased at all CL levels, peaking at CL10, suggesting higher doses may inhibit its growth. The study of⁴² linked Anaerovibrio abundance to propionate production, but this was not observed here, likely due to its lower abundance (0.12%) compared to 5.16% in their study. This may reflect differences in oil types or microbial sensitivity to CL. POSCON and all CL treatments also showed elevated levels of unknown microbes, consistent with findings by.^47,48

Conclusions

This study found that incremental levels of CL had no significant effect on dry matter, crude fat, crude protein, or fibre content however, it improved organic matter. The inclusion of clove essential oil did not enhance fermentation parameters and decreased the production of volatile fatty acids. Inclusion of CL led to distinct differences in microbial composition. The abundance of Anaerovibrio, Desulvibrio and Sphingomonas were highest at CL5 and CL10, whereas Bifidobacterium and Lactobacillus were notably higher for CL15 and CL20. These findings suggest that including clove essential oil may have positive impact on ruminal health. The higher abundance of unknown microbes recorded for CL15 and CL20 indicates the emergence of novel microbial species that require further investigation. It is recommended that further studies identify and characterize unknown taxa to better understand their functions and overall effect on nutrient absorption and productivity.

Limitations of the Study

This study was conducted in vitro, and may not reflect long-term microbial adaptation.

The minor adjustments of treatments during formulation might have its effect.

The dose range of clove essential oil (CL) may not have captured its full biological potential.

Microbial analysis was based on 16S rRNA gene sequencing, which provides taxonomic but not functional insights, limiting interpretation of microbial activity and functional conclusions.

Although microbial shifts suggested potential impacts on ammonia dynamics and methane-related microbes, ammonia concentration and methane production were not measured, making it difficult to assess nitrogen utilization or energy loss.

Lastly, the chemical composition of the specific clove essential oil used was not independently verified, relying instead on literature values, which may vary due to extraction method, source, and storage.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X251410059 - Supplemental material for Influence of Dietary Clove oil Supplementation on Proximate Constituents, in vitro Ruminal Fermentation Efficiency and Microbial Composition

Supplemental material, sj-docx-1-npx-10.1177_1934578X251410059 for Influence of Dietary Clove oil Supplementation on Proximate Constituents, in vitro Ruminal Fermentation Efficiency and Microbial Composition by Adeola P. Idowu, Lebogang E. Motsei, Chidozie F. Egbu and Caven M. Mnisi in Natural Product Communications

Footnotes

Acknowledgements

The authors are appreciative to the Department of Animal Science in North-West University (South Africa) for the financial support.

ORCID iDs

Adeola P. Idowu

Chidozie F. Egbu

Caven M. Mnisi

Ethical Approval

This study was approved (NWU-0081623A5) by the Animal Production Research Ethics Committee of North-West University, South Africa

Author's contributions

Adeola P. Idowu: Conceptualization, Investigation, Data curation and writing of the original draft.

Lebogang E. Motsei: Conceptualization, Methodology funding acquisition, Project administration, and Supervision.

Chidozie F. Egbu: Project administration, Supervision, Methodology.

Caven M. Mnisi: Methodology, Data Validation, Supervision, Resources, Review & Editing.

Funding

The authors received no external funding for the research, authorship, and/or publication of this article.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data Availability Statement

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

Statement of Human and Animal Rights

All procedures in this study were conducted in accordance with the North-West University Animal Production Sciences Research Ethics Committee (NWU-AnimProdREC) (NWU-0081623A5)* approved protocols.

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

Supplemental Material

Supplemental material for this article is available online.

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