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Article

Effects of a Defined Blend of Phytochemicals on Growth Performance, Rumen Fermentation, Bacterial Diversity, and Blood Biochemical and Physiological Parameters in Altay Sheep

by
Mingyue Di
1,2,
Mengjian Liu
1,2,
Wenshuai Zeng
1,2,
Mei Xu
1,2,
Zhanlin Ma
3,
Dong Xu
4 and
Yong Chen
1,2,*
1
Research Center for Biofeed and Animal Gut Health, College of Animal Science, Xinjiang Agricultural University, Urumqi 830052, China
2
Xinjiang Herbivore Nutrition Laboratory for Meat & Milk, Xinjiang Agricultural University, Urumqi 830052, China
3
Fuhai County Daweiyang Co., Ltd., Fuhai 836400, China
4
Agricultural Development Service Center of Aerda Township, Fuhai 836400, China
*
Author to whom correspondence should be addressed.
Fermentation 2026, 12(5), 241; https://doi.org/10.3390/fermentation12050241
Submission received: 7 April 2026 / Revised: 1 May 2026 / Accepted: 5 May 2026 / Published: 15 May 2026
(This article belongs to the Section Animal and Feed Fermentation)
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Abstract

This study aimed to evaluate the effects of dietary supplementation with a defined blend of phytochemicals (DBP) composed of carvacrol, thymol, and cinnamaldehyde on the growth performance, slaughter performance, rumen fermentation, microbial diversity, and blood physiological and biochemical parameters of sheep. Twenty-four healthy male Altay lambs, aged six months, were randomly assigned to three groups: (1) fed a basal diet (CON), (2) basal diet with 400 mg/kg DM of DBP (DBP1), and (3) basal diet with 800 mg/kg DM of DBP (DBP2). Results show that DBP supplementation had no significant effect on growth or slaughter performance (p > 0.05). The molar proportion of acetate and the acetate-to-propionate ratio increased linearly, and the molar proportions of propionate and valerate decreased linearly (p < 0.05). DBP supplementation had no significant effect on rumen bacterial α-diversity; however, in the DBP1 group, the relative abundances of Succinivibrionaceae UCG-002, Prevotellaceae UCG-004, Sphaerochaeta, Monoglobus, and Moryella were significantly increased, whereas in the DBP2 group, the relative abundances of Coprococcus and U29-B03 were significantly increased (p < 0.05). DBP exhibited a significant quadratic effect on interleukin-2 and superoxide dismutase activity (p < 0.05). In conclusion, although the DBP altered the rumen microbial community structure and rumen fermentation pattern in sheep to some extent, it showed minimal efficacy in improving growth performance, slaughter performance, immune function, and antioxidant status. Further large-scale studies are warranted to determine the optimal inclusion level and timing of this phytochemical blend in sheep diets.

1. Introduction

Rumen microorganisms and their composition play important roles in feed digestion, health maintenance, and productivity in ruminants [1]. Short-chain fatty acids (SCFAs), produced by rumen microbial fermentation, serve not only as important energy sources and precursors for fat synthesis in the host but also play critical roles in regulating immune function and alleviating inflammation [2]. Therefore, targeted regulation of the rumen microbiota holds promise for improving host health status and growth performance. For instance, dietary supplementation with red clover isoflavones enhances feed utilization by modulating the rumen microbiota [3]; probiotics improve nutrient digestion and absorption, participate in immune regulation, increase milk yield, and enhance reproductive performance by optimizing microbial balance, stabilizing the rumen environment, and promoting beneficial fermentation [4].
Phytochemicals are non-nutritive bioactive secondary metabolites derived from plants, including polyphenols, saponins, organosulfur compounds, and essential oils (EOs) [5,6]. Numerous studies have demonstrated that phytochemicals possess various biological functions, including anti-inflammatory, antioxidant, antimicrobial, antiparasitic, immunomodulatory, and flavor-enhancing properties. They also enhance feed efficiency and animal growth performance by regulating gut microbial balance [7,8]. In ruminants, phytochemicals also exhibit significant regulatory effects on rumen fermentation. Their active components can modulate rumen microbial community structure, promote the proliferation of beneficial bacteria, inhibit methanogens, reduce methane emissions, and increase volatile fatty acid (VFA) production [9,10]. In addition, phytochemicals can promote rumen papilla development, increase absorptive surface area, enhance intestinal barrier function, and improve nutrient absorption efficiency [9,11]. Among the various active components of phytochemicals, thymol, carvacrol, eugenol, cinnamaldehyde, and their blends have garnered considerable attention due to their potential to improve animal growth performance and maintain health. For example, supplementation of corn- or barley-based diets with 0.20 g/kg of carvacrol or cinnamaldehyde significantly reduced rumen fluid pH and increased total VFA concentration in lambs, with a tendency to increase liver weight, but had no significant effects on feed intake, average daily gain (ADG), or sensory characteristics of the sirloins [12]. Dietary supplementation with 240 mg/kg thymol in a hay- and alfalfa cube-based diet significantly increased the molar proportion of acetate in the rumen fluid, while significantly decreasing that of propionate; furthermore, thymol treatment increased the abundance of several lactic acid bacteria, ammonia-producing bacteria, and archaea [13]. Given that cinnamaldehyde, thymol, and carvacrol act on microorganisms via different mechanisms, their combination may produce unexpected modulatory effects on the rumen microbial community and its ecological environment. Previous study showed that dietary supplementation in lambs with 30–120 mg/kg of a phytochemical blend rich in cinnamaldehyde and carvacrol linearly reduced dry matter (DM) and neutral detergent fiber (NDF) digestibility, total VFA concentration, acetate molar proportion, and the acetate-to-propionate ratio, while it significantly increased final body weight (BW) and ADG. The 120 mg/kg supplementation also reduced the relative abundance of Firmicutes in the rumen and promoted the deposition of polyunsaturated fatty acids in the longissimus lumborum muscle [11]. Supplementation with 0.60 mg/kg of a defined blend of phytochemicals (DBP; containing 11% cinnamaldehyde, 6% thymol, and 3% carvacrol) had no significant effect on rumen VFA concentrations or nutrient digestibility in Hu sheep but improved growth performance and rumen ammonia nitrogen (NH3-N) levels [14]. Despite numerous studies confirming the positive effects of phytochemicals in ruminants, a meta-analysis indicated that their effects on rumen fermentation and overall production performance remain uncertain, with some studies reporting contradictory results. Future randomized trials are needed to more comprehensively evaluate their efficacy and underlying mechanisms in rumen fermentation, antioxidant activity, and production enhancement [15].
The Altay sheep, a renowned indigenous breed in Xinjiang, are characterized by high roughage adaptability, cold resistance, and strong environmental adaptability. However, during autumn and winter, herders often adopt roughage-based diets to control production costs, resulting in insufficient fermentable substrates in the rumen, limited fermentation efficiency, and restricted energy supply, which compromises fattening performance and economic returns. Regulating rumen fermentation to enhance fermentation efficiency is an effective approach to improving the production performance of fattening sheep. We hypothesized that phytochemicals could improve feed digestibility and growth performance in sheep by modulating the rumen microbiota, thereby altering rumen fermentation and antioxidant status. In this study, a DBP containing carvacrol, thymol, and cinnamaldehyde was added to a roughage-based diet to investigate its effects on growth performance, rumen microbial diversity, and blood biochemical parameters in fattening Altay sheep. The findings provide a theoretical basis and practical reference for elucidating the mechanisms by which phytochemicals regulate rumen fermentation and for improving fattening efficiency in sheep.

2. Materials and Methods

2.1. Experimental Design, Animals, and Management

The feeding trial was conducted from October to November 2024 at the sheep farm of Altay Fuhai County Big Tail Sheep Co., Ltd. (Fuhai, Xinjiang, China). A total of twenty-four healthy male Altay lambs during the fattening period, aged six months and weighing approximately 45 kg, were randomly assigned using a random number table to three groups with eight replicates per group and one lamb per replicate: (1) a control diet without DBP supplementation (CON); (2) DBP supplemented at 400 mg/kg of dietary DM (DBP1); (3) DBP supplemented at 800 mg/kg of dietary DM (DBP2). All lambs were housed in a free-stall barn with natural ventilation and natural lighting, and a temperature range of 10–25 °C during the trial period. Each group was kept in an earthen-floor pen measuring 6.2 m × 6.2 m, and the flooring was regularly maintained to ensure dryness and animal comfort. The DBP product (Mude Biotechnology Co., Ltd., Qingdao, China) contained 10% carvacrol, 5% thymol, and 5% cinnamaldehyde, with the remainder consisting of carriers. The supplementation levels were selected based on the manufacturer’s recommendation. The basal diet (concentrate-to-roughage ratio of 37.02:62.98) was formulated in accordance with the Nutrient Requirements of Meat-type Sheep and Goat (NY/T 816-2021) [16]. The diet composition and nutrient levels are presented in Table 1 [17]. Before the start of the experiment, the sheep house and equipment were thoroughly cleaned and disinfected. Deworming, vaccination, and management procedures were performed in accordance with the Technical Specification for Feeding and Management of Stall Feeding Meat-producing Sheep and Goats (NY/T 3052-2016) [18]. The experiment lasted 45 days, and the sheep were fed twice daily at 09:00 and 21:00, with free access to water. The DBP was first thoroughly mixed with the concentrate supplement and then combined with roughage to form a total mixed ration (TMR). The feed amount was adjusted daily to maintain 5–10% orts, and feed intake was recorded daily.

2.2. Sample Collection and Preservation

A 1.0 kg feed sample was collected, air-dried, ground to pass through a 40-mesh sieve, and stored at room temperature for subsequent analysis. On the first day after the end of the experiment, 10 mL of blood was collected from the jugular vein of each lamb before the morning feeding. The blood samples were allowed to stand at 4 °C for 30 min and then centrifuged at 2500× g for 10 min. The serum was stored at −20 °C for subsequent analysis. Prior to slaughter, the lambs were fasted for 24 h, with water withheld during the final 12 h. From each group, six lambs were randomly selected and humanely slaughtered at a commercial abattoir (Fuhai, China) in accordance with the Code of Practice for Livestock and Poultry Meat Fabrication—Sheep and Goat meat (GB/T 43562-2023) [19]. After slaughter, the rumen was removed, and the rumen contents were thoroughly mixed. A 100 mL sample of rumen fluid was collected, and the pH was immediately measured using a pH meter (FE20; METTLER TOLEDO, Shanghai, China). The rumen fluid was then filtered through four layers of gauze, and the filtrate was stored at −20 °C for subsequent analysis.

2.3. Feed Nutrients

The contents of DM, ash, crude protein (CP), ether extract (EE), calcium (Ca), and phosphorus (P) in the feed were determined following AOAC official methods [20]. NDF and acid detergent fiber (ADF) were assayed according to the method reported by Van Soest et al. [21]. The metabolizable energy was calculated according to the basal diet composition and nutritive values of feedstuffs in China [22].

2.4. Growth and Slaughter Performance

The BW was recorded before the morning feeding on days 1 and 46 of the experiment, and ADG, average daily feed intake (ADFI), and feed-to-gain ratio (F/G) were calculated. Pre-slaughter live weight, carcass weight, dressing rate, and backfat thickness were determined according to the Technical Specification for the Performance Test of Goat and Sheep Stud (NY/T 1236-2023) [23]. Liver weight and heart weight were also recorded.
Dressing rate (%) = (carcass weight (kg)/pre-slaughter live weight (kg)) × 100%.

2.5. Rumen Fermentation Parameters

The concentrations of NH3-N and VFAs in rumen fluid were determined according to the method described by Wei et al. [24], and the molar proportions of individual VFA were calculated. Lactate concentration in rumen fluid was measured using a lactate analyzer (LM5, Analox, Stourbridge, UK) following the manufacturer’s instructions.

2.6. 16S rDNA Amplicon Sequencing

A 1.0 mL aliquot of rumen fluid was collected, and microbial genomic DNA was extracted using a modified cetyltrimethylammonium bromide (CTAB) method [25]. The V3–V4 region of the bacterial 16S rRNA gene was amplified by PCR using primers 341F (5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′-GGACTACNNGGGTATCTAAT-3′) [26]. The amplified products were purified using magnetic beads, and sequencing libraries were constructed. The qualified libraries were subjected to paired-end sequencing on the Illumina NovaSeq 6000 platform at Novogene Technology Co., Ltd. (Beijing, China).
After removing barcodes and primer sequences from the raw sequencing data, paired-end reads were merged using FLASH software (v1.2.11) to obtain raw tags [27]. The raw tags were filtered, and chimeric sequences were removed to obtain effective tags [28]. Operational taxonomic units (OTUs) were clustered from the effective tags using the UPARSE algorithm (v7.0) with a 97% sequence identity threshold. The sequence with the highest abundance in each OTU was selected as the representative sequence, and species annotation was performed using the SILVA database (v138.1) to obtain the composition and relative abundance distribution of each taxonomic unit.
At the OTU level, a Venn diagram was constructed using the VennDiagram function in R software (v4.0.3). Alpha diversity indices, including observed features, Chao1, Shannon, and Simpson, were calculated using QIIME 2 (v2024.2), and significance tests were performed on the Persona Gene Cloud online platform (https://www.genescloud.cn; accessed on 23 November 2025). Principal coordinate analysis (PCoA) based on weighted UniFrac and Bray–Curtis distances was performed using the ade4 and ggplot2 packages in R software (v4.0.3). Analysis of similarities (ANOSIM) was performed using the OmicShare Cloud platform (Gene Denovo) (https://www.omicshare.com; accessed on 23 November 2025). Stacked bar charts were constructed for the top 10 taxa at the phylum and genus levels based on relative abundance, and unweighted pair-group method with arithmetic means (UPGMA) cluster analysis was performed using QIIME (v1.9.1) based on weighted UniFrac distances. Linear discriminant analysis effect size (LEfSe) analysis was performed to identify biomarkers among groups (linear discriminant analysis [LDA] score > 2.5, p < 0.05).

2.7. Serum Biochemical Parameters

The concentrations of total protein (TP), albumin, total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), blood urea nitrogen (BUN), glucose, non-esterified fatty acids (NEFA), lactate, malondialdehyde (MDA), catalase and total antioxidant capacity (T-AOC), as well as the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) in serum, were determined using colorimetric methods. Globulin concentration was calculated as total protein minus albumin. The concentrations of leptin, growth hormone (GH), insulin, adiponectin, fatty acid synthase (FAS), immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), interferon-γ (IFN-γ), and interleukin-2 (IL-2) in serum were determined using enzyme-linked immunosorbent assay (ELISA) kits. All kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjiang, China), and the serum biochemical parameters were determined according to the manufacturer’s instructions.

2.8. Statistical Analysis

The Shapiro–Wilk test in IBM SPSS software (v26.0) was used to test the normality of data distribution, and Levene’s test was used to test homogeneity of variances. When homogeneity of variances was assumed, one-way analysis of variance (ANOVA) was performed. When treatment effects were significant, multiple comparisons were performed using Duncan’s multiple range test. When homogeneity of variances was not assumed, Welch’s ANOVA was performed, and when treatment effects were significant, multiple comparisons were performed using the Games–Howell test. Polynomial contrasts were used to test linear and quadratic effects of DBP supplementation. Results are presented as means and standard error of the mean (SEM). Statistical significance was declared at p < 0.05, and tendencies were considered at 0.05 < p < 0.10.

3. Results

3.1. Effects of DBP Supplementation on Growth Performance of Altay Sheep

The effects of DBP supplementation on the growth performance of Altay sheep are presented in Table 2. Compared with the CON group, dietary DBP supplementation had no significant effects on final BW, ADG, ADFI, or F/G (p > 0.05).

3.2. Effects of DBP Supplementation on Slaughter Performance of Altay Sheep

The effects of DBP supplementation on the slaughter performance of Altay sheep are presented in Table 3. Compared with the CON group, dietary DBP supplementation had no significant effects on slaughter performance, organ weights, or backfat thickness (p > 0.05). However, carcass weight tended to increase linearly with increasing DBP supplementation levels (p = 0.085).

3.3. Effects of DBP Supplementation on Rumen Fermentation in Altay Sheep

The effects of DBP supplementation on rumen fermentation parameters in Altay sheep are presented in Table 4. Dietary DBP supplementation had no significant effects on rumen fluid pH, the molar proportions of butyrate, isobutyrate, and isovalerate, or the concentrations of NH3-N and lactate (p > 0.05); however, total VFA concentration tended to increase linearly (p = 0.066). Compared with the CON group, the DBP2 group showed a significant increase in the molar proportion of acetate and the A/P (p < 0.05), whereas the molar proportions of propionate and valerate were significantly decreased (p < 0.05).

3.4. Effects of DBP Supplementation on Rumen Bacterial Diversity in Altay Sheep

After sequencing, assembly, quality control, and chimera removal, an average of 83,420 effective tags were obtained per sample, with an average sequence length of 415.70 bp. The average Q20 value was 98.88% (minimum 98.75%), and the average Q30 value was 95.99% (minimum 95.56%).
As shown in Figure 1A, based on the OTU level (97% sequence identity), a number of unique OTUs were present in each group, with 180 unique OTUs in the CON group, 203 in the DBP1 group, and 250 in the DBP2 group, suggesting that the DBP2 group had higher species richness. As shown in Figure 1B, compared with the CON group, there were no significant differences in α diversity indices (observed species, Shannon, Simpson, Chao1, and ACE) among the groups (p > 0.05).
The PCoA analysis based on weighted UniFrac and Bray–Curtis distances revealed different patterns of explained variance. Based on weighted UniFrac distance, PCoA1 and PCoA2 accounted for 32.38% and 24.17% of the variation, respectively, cumulatively explaining 56.55% of the total variation (Figure 1C). However, based on Bray–Curtis distance, PCoA1 and PCoA2 accounted for only 10.69% and 10.65% of the variation, respectively, cumulatively explaining 21.34% of the total variation (Figure 1D). Under both distance metrics, sample points from different treatment groups largely overlapped and did not form clear separations, indicating that the effect of DBP supplementation on the overall structure of the rumen bacterial community was negligible. Analysis of similarities (ANOSIM) results showed that differences among groups were not statistically significant (p > 0.05) (Figure 1E), although intra-group distance distribution in the DBP1 group was relatively more dispersed. Collectively, these results suggest that under the conditions of this experiment, DBP supplementation did not significantly reshape the overall structure of the rumen bacterial community, and its effects may be reflected in changes in specific taxa.
At the phylum level, the rumen microbiota in all three groups was dominated by Bacteroidota and Firmicutes, followed by Euryarchaeota (Figure 1F). Compared with the CON group, the phylum-level composition remained largely consistent across DBP-treated groups, although the DBP2 group showed a slightly higher relative abundance of Euryarchaeota and a relatively lower relative abundance of Firmicutes. At the genus level, the dominant genera in each group mainly included Prevotella, Christensenellaceae R-7 group, Rikenellaceae RC9 gut group, Methanobrevibacter, and NK4A214 group (Figure 1G). Compared with the CON group, the relative abundances of these dominant genera exhibited certain changes following DBP supplementation, with the DBP2 group showing a higher relative abundance of Rikenellaceae RC9 gut group. UPGMA clustering based on weighted UniFrac distance showed that, at both the phylum and genus levels, the DBP1 and DBP2 groups clustered together and were separated from the CON group, suggesting that the community compositions of the two treatment groups were more similar to each other following DBP supplementation.
The LEfSe analysis revealed that at the genus level, the relative abundances of NK4A214 group, Tyzzerella, Lachnospiraceae UCG-006, Marvinbryantia, Lachnospira, Eubacterium cellulosolvens group, and Oribacterium were significantly higher in the CON group than in the treatment groups. The biomarkers in the DBP1 group were Succinivibrionaceae UCG-002, Prevotellaceae UCG-004, Sphaerochaeta, Monoglobus, and Moryella, whereas those in the DBP2 group were Coprococcus and U29-B03 (Figure 2A). These results indicate that supplementation with the DBP induced remodeling of several rumen bacterial taxa across multiple phylogenetic branches, leading to significant differentiation of biomarkers among the treatment groups.
As illustrated in Figure 2B, dietary supplementation with the DBP led to differential alterations in the rumen bacterial community of Altay sheep along specific branches of the phylogenetic tree. Specifically, the CON group exhibited significant enrichment in multiple branches within RF39, Lachnospirales, and Oscillospirales. In contrast, the DBP1 group showed significant enrichment in branches associated with Izemoplasmatales and Monoglobales. The DBP2 group displayed differential enrichment primarily in a few terminal branches on the phylogenetic tree, with the significantly enriched taxa concentrated at the genus level, mainly corresponding to the phylogenetic branches of Coprococcus and U29-B03. This pattern contrasted with the clustered enrichment observed in the CON and DBP1 groups across multiple higher-order taxonomic units. Collectively, these findings suggest that DBP supplementation can, to some extent, influence the abundance and composition of certain rumen bacterial taxa, thereby driving structural changes in the rumen microecology.

3.5. Effects of DBP Supplementation on Protein and Lipid Metabolism in Altay Sheep

The effects of DBP supplementation on protein and lipid metabolism in Altay sheep are presented in Table 5. Compared with the CON group, dietary DBP supplementation had no significant effects on the concentrations of TP, albumin, globulin, BUN, ALT, TC, TG, HDL-C, or LDL-C (p > 0.05); however, AST activity tended to increase linearly (p = 0.063).

3.6. Effects of DBP Supplementation on Energy Metabolism and Hormone in Altay Sheep

The effects of DBP supplementation on blood glucose and hormones in Altay sheep are presented in Table 6. Compared with the CON group, dietary DBP supplementation had no significant effects on the concentrations of glucose, GH, insulin, leptin, adiponectin, or FAS (p > 0.05). Serum lactate concentration in the DBP1 group was significantly decreased, and showing a quadratic effect (p < 0.05).

3.7. Effects of DBP Supplementation on Serum Immune Parameters in Altay Sheep

The effects of DBP supplementation on serum immune parameters in Altay sheep are presented in Table 7. Dietary DBP supplementation resulted in a quadratic effect on serum interleukin-2 (IL-2) levels, with values initially increasing and then decreasing (p < 0.05). In addition, with increasing DBP supplementation levels, interferon-γ (IFN-γ) levels showed a linear decrease (p = 0.049), although no significant differences were observed among groups (p > 0.05). Dietary DBP supplementation had no significant effects on serum immunoglobulins (p > 0.05).

3.8. Effects of DBP Supplementation on Serum Antioxidant Parameters in Altay Sheep

The effects of DBP supplementation on serum antioxidant parameters in Altay sheep are presented in Table 8. Compared with the CON group, SOD activity in the DBP1 group was significantly decreased (p < 0.05). Dietary DBP supplementation had no significant effects on glutathione peroxidase (GSH-Px), catalase, malondialdehyde (MDA), or total antioxidant capacity (T-AOC) (p > 0.05).

4. Discussion

The DBP used in this study contained three active components: carvacrol, thymol, and cinnamaldehyde. Carvacrol and thymol are monoterpenic phenols and structural isomers commonly found in aromatic plants such as Origanum and Thymus [29,30]. Cinnamaldehyde is an aldehyde organic compound abundantly present in plants of the genus Cinnamomum [31]. The three compounds are the active ingredients of plant EOs and exhibit antioxidant, antimicrobial, anti-inflammatory, and anticancer properties [30,32].
Phytochemicals have been reported to exert varying effects on the growth and slaughter performance of ruminants [6]. Some studies have found that phytochemicals improve animal production performance and carcass characteristics by promoting feed intake and digestion, whereas others report no significant effects on feed intake and growth performance, and some have even observed growth inhibition [6,33,34]. Supplementation with an EO blend in dairy cows increased milk yield, whereas supplementation with carvacrol or thymol alone had no significant effect on milk yield [35,36]. In a study using Suffolk × Small-tailed Han F1 male lambs (approximately 3 months of age), supplementation with 4 or 7 g/d of an oregano EO and cobalt lactate blend (EOC) showed that the 7 g/d EOC group had significantly higher body weight on days 48 and 72 compared with the control group, along with improved overall average daily gain and feed conversion efficiency [33]. It has been suggested that phytochemicals can increase feed intake and digestibility by improving appetite and enhancing digestive enzyme activity [37,38]. However, other studies reported that supplementation with phytochemicals had no significant effects on BW, weight gain, loin eye area, backfat thickness, or dressing percentage [34,39]. In the present study, no positive effects of the DBP on growth or slaughter performance were observed in fattening sheep. In contrast to some findings, supplementation with a plant EO blend (containing carvacrol, eugenol, thymol, and capsaicin) in dairy cow diets resulted in significantly reduced dietary DM intake and BW [40]. These findings suggest that the effects of phytochemicals on production performance are influenced by multiple factors, including ingredients, supplementation dose, diet type, and feeding duration.
Thymol, carvacrol, and cinnamaldehyde exhibit favorable antimicrobial activity against both Gram-positive and Gram-negative bacteria. Thymol has been shown to have a higher minimum inhibitory concentration (MIC) against Staphylococcus aureus than against Escherichia coli, whereas carvacrol displays similar antimicrobial activity against both Gram-positive and Gram-negative bacteria. In contrast, cinnamaldehyde exhibits greater antimicrobial activity against Gram-negative bacteria than against Gram-positive bacteria [41,42]. These three compounds may target different bacterial populations, potentially broadening the antimicrobial spectrum and reducing the risk of microbial adaptation. When these three compounds are added to the diets of ruminants, they may exert positive modulatory effects on rumen fermentation [34,43]; however, their antimicrobial effects can also perturb the rumen microecology and microbial community structure, which may account for reduced feed intake, decreased in vitro gas production, reduced VFA production, and even impaired growth performance [11,40,44]. These discrepancies may be closely related to the composition of active ingredients and the dosage used. In vitro studies have shown that medium to high doses of thymol (100–500 mg/L) modulate rumen fermentation parameters, whereas low doses (50 mg/L) exert no significant effects [43]. Therefore, further in vivo studies are needed to determine the optimal dosage that harnesses the beneficial effects of phytochemicals while avoiding inhibition of feed digestion [45]. In this study, the addition of DBP shifted the rumen fermentation pattern, characterized by an increase in acetate and a decrease in propionate. These findings may be attributed to the differential antimicrobial activities of thymol, carvacrol, and cinnamaldehyde. High doses of thymol have been reported to significantly reduce the relative abundances of Bacteroidota and Prevotella 1 in the rumen while increasing the relative abundances of Firmicutes, Succinivibrio, Streptococcus, and Pseudobutyrivibrio [46]. Thyme EO, which is rich in carvacrol, reduced fecal Escherichia coli counts and increased Lactobacillus and Bifidobacterium in lamb [47]. In the present study, 400 mg/kg DBP increased the relative abundances of Sphaerochaeta and Moryella, whereas 800 mg/kg DBP significantly increased that of Coprococcus. Sphaerochaeta utilizes carbohydrates to produce acetate, ethanol, and hydrogen [48]. Moryella ferments dietary polysaccharides to produce SCFAs such as lactate and butyrate, which are crucial for maintaining gastrointestinal barrier integrity and regulating inflammatory responses [49,50]. Coprococcus is a typical butyrate-producing genus that also produces some acetate; together with Faecalibacterium and Roseburia, it plays a vital role in limiting the production of pro-inflammatory cytokines, releasing anti-inflammatory factors, and maintaining intestinal health [51]. Although thymol and carvacrol possess antimicrobial activity, they exhibit relatively weak inhibitory effects on major fibrolytic bacteria (e.g., Ruminococcus, Fibrobacter) in the rumen [52,53], thereby indirectly promoting fiber degradation and acetate production [53]. Following DBP supplementation, the increased relative abundances of these bacteria contributed to enhanced acetate and butyrate production, which may be beneficial for maintaining intestinal health. We acknowledge that the specific contribution of cinnamaldehyde to the observed effects cannot be isolated from the blend. Given the limited in vivo evidence for cinnamaldehyde alone [12], its inclusion represents a potential limitation. The observed fermentation changes may be primarily driven by carvacrol and thymol, with cinnamaldehyde playing a minor or synergistic role. Future studies using individual compounds and their combinations in dose–response designs are warranted to dissect their respective contributions.
Unlike ionophores (e.g., monensin), which typically increase the proportion of propionate to enhance feed efficiency [54], our supplementation with 800 mg/kg DBP unexpectedly increased acetate proportion while reducing that of propionate. Propionate is the primary gluconeogenic precursor in ruminants, supplying up to 70% of glucose requirements [55]. A reduction in propionate might, theoretically, compromise glucose status. However, our results showed no negative effects on growth performance or blood glucose levels (Table 6), suggesting the presence of compensatory mechanisms. One possibility is enhanced gluconeogenesis from alternative substrates, such as amino acids, as suggested by a tendency for a linear increase in AST activity (p = 0.063, Table 5). Another possibility is that the numerically lower blood lactate concentration in the DBP1 group (7.37 vs. 10.27 mmol/L, p < 0.05) may reflect a redirection of lactate toward gluconeogenesis. Acetate, the proportion of which was increased, is primarily utilized for lipogenesis. Notably, the DBP2 group showed a tendency for linearly increased carcass weight (p = 0.085) and a numerically greater backfat thickness (9.24 vs. 8.66 mm), although these differences were not statistically significant. This suggests that the acetate shift may have directed nutrients toward fat deposition rather than affecting glucose-dependent growth. Future studies should incorporate glucose kinetics and lipogenic gene expression to fully understand this divergence.
The phenolic hydroxyl groups of carvacrol and thymol, as well as the α, β-unsaturated aldehyde group of cinnamaldehyde, confer antioxidant and anti-inflammatory activities to these compounds. Carvacrol exerts potent anti-inflammatory effects by increasing the levels of antioxidant enzymes such as SOD, GSH-Px, GR, and catalase to prevent polyunsaturated fatty acid peroxidation, while reducing the levels of pro-inflammatory cytokines such as TNF-α, IL-6, IL-8, and IL-10 [29]. Studies in weaned lambs have confirmed that carvacrol enhances antioxidant capacity and immunity by alleviating oxidative stress and inflammatory responses [44]. The antioxidant mechanisms of thymol and carvacrol primarily involve two aspects: first, the phenolic hydroxyl group directly scavenges oxygen free radicals (e.g., superoxide anion, hydroxyl radical), thereby blocking lipid peroxidation [56]; second, they upregulate the expression of antioxidant enzyme genes via the Nrf2/ARE signaling pathway, alleviating H2O2-induced oxidative damage [57]. Due to differences in the chemical structures of monoterpenic phenols and aldehydes, their antioxidant and anti-inflammatory mechanisms may vary, suggesting that the combination of carvacrol, thymol, and cinnamaldehyde may exert synergistic effects. Dietary supplementation with a blend of essential oils (carvacrol, eugenol, cinnamaldehyde, and capsaicin) in early-lactation Jersey cows produced multiple physiological effects [58]. Regarding inflammatory and immune responses, it reduced blood leukocyte and lymphocyte counts and C-reactive protein levels, while increasing IgA and immunoglobulin heavy chain. With respect to oxidative status, it decreased lipid peroxidation and enhanced TAC, as well as glutathione S-transferase and GSH-Px activities [58]. These findings indicate that dietary supplementation with phytochemicals positively affects cow health, manifesting as immunostimulatory, antioxidant, and anti-inflammatory effects [58]. Numerous studies have demonstrated that cinnamaldehyde can reduce the expression of pro-inflammatory cytokines and chemokines (e.g., IL-1β, IL-6, and TNF-α), promote the production of anti-inflammatory cytokines [59,60], and inhibit the activation of the nuclear factor kappa-B (NF-κB) pathway [61]. Additionally, cinnamaldehyde has been shown to alleviate oxidative stress by activating the NRF2/HO-1 pathway and inhibiting the production of reactive oxygen species (ROS) [62]. In the present study, 400 mg/kg DBP reduced SOD activity, whereas 800 mg/kg DBP significantly increased SOD activity. This suggests that the DBP may exert a bidirectional regulatory effect on SOD activity: at low doses, the active components may directly scavenge free radicals, resulting in lower oxidative stress levels and thus reducing the need for induced SOD synthesis, leading to relatively decreased basal SOD activity. At high doses, higher concentrations of active components may activate the Nrf2/ARE pathway, significantly upregulating SOD gene expression and thereby increasing its activity [57].
Phytochemicals also exhibit positive effects on immune regulation. Supplementation with oregano EO in sheep and calf diets has been reported to increase blood levels of IgA, IgG, and IgM [63,64]. In the present study, DBP supplementation did not exert positive effects on immunoglobulin indices in Altay sheep. However, blood levels of IL-2 and IFN-γ were reduced. This phenomenon may be related to the inhibition of the NF-κB signaling pathway, which plays a critical role in regulating immune responses, including the production of cytokines such as IL-2 and IFN-γ. NF-κB is a transcription factor that regulates gene expression involved in inflammation, immune responses, and cell survival. Several studies have confirmed that inhibition of NF-κB reduces the production of IL-2 and IFN-γ. For example, thymol and carvacrol have been shown to reduce the production of IL-2 and IFN-γ in Jurkat T cells by inhibiting the transcription factors NFAT-2 and c-Fos, which are involved in the regulation of these cytokines [65]. Furthermore, cinnamaldehyde has been demonstrated to inhibit NF-κB activation in macrophages, thereby reducing the production of IL-2 and IFN-γ [66]. Collectively, under the conditions of this study, DBP supplementation did not significantly affect humoral immune parameters (IgG, IgA, IgM) in Altay sheep but modulated the levels of the cytokines IL-2 and IFN-γ, suggesting that the DBP may primarily influence immune function through regulation of cellular immune responses rather than humoral immunity. The underlying mechanisms warrant further investigation.

5. Conclusions

Dietary supplementation with 400–800 mg/kg of the DBP composed of carvacrol, thymol, and cinnamaldehyde had only limited direct effects on growth performance, slaughter performance, blood antioxidant status, and immune function in fattening Altay sheep. Rather, DBP primarily influenced rumen fermentation patterns by modulating rumen microbial composition, exerting a mild modulatory effect on the rumen microecology. Further large-scale studies are needed to determine the efficacy, optimal inclusion level, and timing of DBP supplementation in sheep.

Author Contributions

Conceptualization, Y.C. and M.L.; methodology, M.D., W.Z. and M.X.; software, M.D. and Y.C.; validation, W.Z., M.X. and Z.M.; formal analysis, M.D.; investigation, M.D., W.Z., M.X. and D.X.; resources, Z.M. and D.X.; data curation, M.D. and M.L.; writing—original draft preparation, M.D. and M.L.; writing—review and editing, Y.C.; visualization, M.D. and M.X.; supervision, Y.C. and D.X.; project administration, M.L.; funding acquisition, Y.C. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the Program for Science and Technology Innovation Talents (2022TSYCLJ0014) and the Xinjiang Key Research and Development Program of China (2023B02015).

Institutional Review Board Statement

The animal study protocol was approved by the Animal Welfare Ethics Committee of Xinjiang Agricultural University (protocol no. 2023055), with the approval date of 11 April 2023.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

Author Zhanlin Ma was employed by the company Fuhai County Daweiyang Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Abbreviations

The following abbreviations are used in this manuscript:
DBPDefined blend of phytochemicals
EOsEssential oils
TMRTotal mixed ration
UPGMAUnweighted pair-group method with arithmetic means
ANOSIMAnalysis of similarities

References

  1. Chuang, S.T.; Ho, S.T.; Tu, P.W.; Li, K.Y.; Kuo, Y.L.; Shiu, J.S.; Wang, S.Y.; Chen, M.J. The rumen specific bacteriome in dry dairy cows and its possible relationship with phenotypes. Animals 2020, 10, 1791. [Google Scholar] [CrossRef]
  2. Yang, J.; Li, Y.; Sun, M.; Zhang, Y.; Guo, S.; Zhou, D.; Lin, P.; Wang, A.; Jin, Y. Associations of rumen and rectum bacteria with the sustained productive performance of dairy cows. Front. Microbiol. 2025, 16, 1565034. [Google Scholar] [CrossRef] [PubMed]
  3. Bu, Y.; Zhang, X.; Xiong, Z.; Li, K.; Zhang, S.; Lin, M.; Zhao, G.; Zheng, N.; Wang, J.; Zhao, S. Effect of red clover isoflavones on ruminal microbial composition and fermentation in dairy cows. Appl. Microbiol. Biotechnol. 2025, 109, 107. [Google Scholar] [CrossRef]
  4. Kulkarni, N.A.; Chethan, H.S.; Srivastava, R.; Gabbur, A.B. Role of probiotics in ruminant nutrition as natural modulators of health and productivity of animals in tropical countries: An overview. Trop. Anim. Health Prod. 2022, 54, 110. [Google Scholar] [CrossRef] [PubMed]
  5. Popovic, L.; Rijkers, G.T. Phytochemicals: Principles and practice. Biology 2026, 15, 18. [Google Scholar] [CrossRef] [PubMed]
  6. Priyashantha, H.; Jayathissa, I.S.; Vidanarachchi, J.K.; Jayarathna, S.; Mapiye, C.; Maggiolino, A.; Ponnampalam, E.N. Phytochemicals in ruminant diets: Mechanistic insights, product quality enhancement, and pathways to sustainable milk and meat production—Invited review. Animals 2026, 16, 425. [Google Scholar] [CrossRef]
  7. Valdivieso-Ugarte, M.; Gomez-Llorente, C.; Plaza-Díaz, J.; Gil, Á. Antimicrobial, antioxidant, and immunomodulatory properties of essential oils: A systematic review. Nutrients 2019, 11, 2786. [Google Scholar] [CrossRef]
  8. Wang, J.; Deng, L.; Chen, M.; Che, Y.; Li, L.; Zhu, L.; Chen, G.; Feng, T. Phytogenic feed additives as natural antibiotic alternatives in animal health and production: A review of the literature of the last decade. Anim. Nutr. 2024, 17, 244–264. [Google Scholar] [CrossRef]
  9. Nhara, R.B.; Baloyi, J.J. Complementary effects of essential oils and organic acids on rumen physiology as alternatives to antibiotic feed additives. Animals 2025, 15, 2910. [Google Scholar] [CrossRef]
  10. Nasir, M.; Rodríguez-Prado, M.; Simoni, M.; Martín-Orúe, S.M.; Pérez, J.F.; Calsamiglia, S. Optimizing essential oil mixtures: Synergistic effects on cattle rumen fermentation and methane emission. Animals 2025, 15, 2105. [Google Scholar] [CrossRef]
  11. Ma, Z.; Meng, Y.; Li, F.; Ungerfeld, E.; Lv, J.; Liu, B.; Li, S.; Wang, X. Effects of an essential oil blend rich in cinnamaldehyde and carvacrol on rumen biohydrogenation and fatty acid profile in the longissimus lumborum of growing lambs. J. Sci. Food Agric. 2024, 104, 9581–9591. [Google Scholar] [CrossRef] [PubMed]
  12. Chaves, A.V.; Stanford, K.; Gibson, L.L.; McAllister, T.A.; Benchaar, C. Effects of carvacrol and cinnamaldehyde on intake, rumen fermentation, growth performance, and carcass characteristics of growing lambs. Anim. Feed Sci. Technol. 2008, 145, 396–408. [Google Scholar] [CrossRef]
  13. Fukuda, E.; Lu, Y.; Jessup, R.; Drewery, M.L. Effect of thymol on forage utilization, rumen fermentation, and rumen microorganisms in beef steers. J. Anim. Sci. 2024, 102, 487–488. [Google Scholar] [CrossRef]
  14. Wang, G.; Geng, W.; Yang, H.; Zhang, X.; Zhang, H.; Shen, B.; Liu, C. Effect of essential oil on growth performance, rumen fermentation and nutrient digestibility of Hu sheep. Indian J. Anim. Res. 2025, 59, 1370–1375. [Google Scholar] [CrossRef]
  15. Li, W.; Wang, F.; Han, Y.; Sun, F.; Liu, C.; Zhu, Y.; Zhong, P. Effects of essential oils on calf growth, ruminal fermentation, and antioxidative status: A meta-analysis. Front. Vet. Sci. 2025, 12, 1573846. [Google Scholar] [CrossRef]
  16. NY/T 816-2021; Nutrient Requirements of Meat-Type Sheep and Goat. China Agriculture Press: Beijing, China, 2021.
  17. Xu, M.; Di, M.; Zeng, W.; Li, X.; Xu, D.; Ma, Z.; Wang, Y.; Liu, M.; Chen, Y. Effects of arginine supplementation on growth performance, serum parameters, and rumen microbial diversity in fattening Altay sheep fed a forage-based diet. Agriculture 2026, 16, 932. [Google Scholar] [CrossRef]
  18. NY/T 3052-2016; Technical Specification for Feeding and Management of Stall Feeding Meat-Producing Sheep and Goats. China Agriculture Press: Beijing, China, 2016.
  19. GB/T 43562-2023; Code of Practice for Livestock and Poultry Slaughtering Operation-Sheep and Goat. China Standards Press: Beijing, China, 2023.
  20. AOAC International. Official Methods of Analysis, 18th ed.; Academy Press: Washington, DC, USA, 2005. [Google Scholar]
  21. Van Soest, P.J.; Robertson, J.B.; Lewis, B.A. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 1991, 74, 3583–3597. [Google Scholar] [CrossRef]
  22. Institute of Animal Sciences; Chinese Academy of Agricultural Sciences. State Key Laboratory of Animal Nutrition and Feeding; China Feed Database Information Network Center; National Agriculture Science Data Center. Tables of feed composition and nutritive values in China (thirty-fifth edition). China Feed 2024, 182–197. [Google Scholar] [CrossRef]
  23. NY/T 1236-2023; Technical Specification for the Performance Test of Goat and Sheep Stud. China Agriculture Press: Beijing, China, 2023.
  24. Wei, H.; Liu, J.; Liu, M.; Zhang, H.; Chen, Y. Rumen fermentation and microbial diversity of sheep fed a high-concentrate diet supplemented with hydroethanolic extract of walnut green husks. Anim. Biosci. 2024, 37, 655–667. [Google Scholar] [CrossRef] [PubMed]
  25. Aphale, D.; Kulkarni, A. Modifications and optimization of manual methods for polymerase chain reaction and 16S rRNA gene sequencing quality community DNA extraction from goat rumen digesta. Vet. World 2018, 11, 990–1000. [Google Scholar] [CrossRef] [PubMed]
  26. Zhao, X.; Zhang, Z.; Hu, B.; Huang, W.; Yuan, C.; Zou, L. Response of gut microbiota to metabolite changes induced by endurance exercise. Front. Microbiol. 2018, 9, 765. [Google Scholar] [CrossRef]
  27. Magoc, T.; Salzberg, S.L. FLASH: Fast length adjustment of short reads to improve genome assemblies. Bioinformatics 2011, 27, 2957–2963. [Google Scholar] [CrossRef]
  28. Bokulich, N.A.; Subramanian, S.; Faith, J.J.; Gevers, D.; Gordon, J.I.; Knight, R.; Mills, D.A.; Caporaso, J.G. Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing. Nat. Methods 2013, 10, 57–59. [Google Scholar] [CrossRef]
  29. Mączka, W.; Twardawska, M.; Grabarczyk, M.; Wińska, K. Carvacrol—A natural phenolic compound with antimicrobial properties. Antibiotics 2023, 12, 824. [Google Scholar] [CrossRef]
  30. Peter, S.; Sotondoshe, N.; Aderibigbe, B.A. Carvacrol and thymol hybrids: Potential anticancer and antibacterial therapeutics. Molecules 2024, 29, 2277. [Google Scholar] [CrossRef]
  31. Nora, L.; Marcon, C.; Deolindo, G.L.; Signor, M.H.; Muniz, A.L.; Bajay, M.M.; Copetti, P.M.; Bissacotti, B.F.; Morsch, V.M.; da Silva, A.S. The effects of a blend of essential oils in the milk of suckling calves on performance, immune and antioxidant systems, and intestinal microbiota. Animals 2024, 14, 3555. [Google Scholar] [CrossRef]
  32. Weng, X.L.; Ho, C.T.; Lu, M.W. Biological fate, functional properties, and design strategies for oral delivery systems for cinnamaldehyde. Food Funct. 2024, 15, 6217–6231. [Google Scholar] [CrossRef]
  33. Wu, J.P.; Zhou, R.; Liu, L.S.; Casper, D.P.; Lang, X.; Wang, C.L.; Zhang, L.P.; Wei, S.; Liu, H.B. Growth performance, nutrient digestibility, blood parameters, and carcass characteristics by lambs fed an oregano and cobalt blend. Animal 2021, 15, 100365. [Google Scholar] [CrossRef]
  34. Biricik, H.; Oral, H.H.; Taluğ, A.M.; Cengiz, Ş.Ş.; Koyuncu, M.; Dikmen, S. The effects of carvacrol and/or thymol on the performance, blood and rumen parameters, and carcass traits of Merino sheep. Turk. J. Vet. Anim. Sci. 2016, 40, 651–659. [Google Scholar] [CrossRef]
  35. Benchaar, C. Feeding oregano oil and its main component carvacrol does not affect ruminal fermentation, nutrient utilization, methane emissions, milk production, or milk fatty acids composition of dairy cows. J. Dairy Sci. 2020, 103, 1516–1527. [Google Scholar] [CrossRef]
  36. Benchaar, C. Diet supplementation with thyme oil and its main component thymol failed to favorably alter rumen fermentation, improve nutrient utilization, or enhance milk production in dairy cows. J. Dairy Sci. 2021, 104, 324–336. [Google Scholar] [CrossRef]
  37. Basmacıoğlu Malayoğlu, H.; Baysal, S.; Misirlioğlu, Z.; Polat, M.; Yilmaz, H.; Turan, N. Effects of oregano essential oil with or without feed enzymes on growth performance, digestive enzyme, nutrient digestibility, lipid metabolism and immune response of broilers fed on wheat-soybean meal diets. Br. Poult. Sci. 2010, 51, 67–80. [Google Scholar] [CrossRef]
  38. Nguyen, N.P.K.; Tran, K.N.; Nguyen, L.T.H.; Shin, H.M.; Yang, I.J. Effects of essential oils and fragrant compounds on appetite: A systematic review. Int. J. Mol. Sci. 2023, 24, 7962. [Google Scholar] [CrossRef]
  39. Muñoz-Cuautle, A.; Ortega-Cerrilla, M.E.; Herrera-Haro, J.G.; Nava-Cuellar, C.; Gutiérrez-Olvera, C.; Ramírez-Bribiesca, J.E.; Zetina-Córdoba, P. Effect of oregano (Lippia graveolens) essential oil as a phytogenic feed additive on productive performance, ruminal fermentation, and antioxidant activity in lamb meat. Agriculture 2022, 12, 973. [Google Scholar] [CrossRef]
  40. Diepersloot, E.C.; Pupo, M.R.; Heinzen, C., Jr.; Souza, M.S.; Ferraretto, L.F. Effects of monensin and essential oil blend supplementation on lactation performance and feeding behavior in dairy cows. J. Dairy Sci. 2025, 108, 2517–2526. [Google Scholar] [CrossRef]
  41. Mith, H.; Duré, R.; Delcenserie, V.; Zhiri, A.; Daube, G.; Clinquart, A. Antimicrobial activities of commercial essential oils and their components against food-borne pathogens and food spoilage bacteria. Food Sci. Nutr. 2014, 2, 403–416. [Google Scholar] [CrossRef]
  42. Radocchia, G.; Giammarino, A.; Barberini, S.; Verdolini, L.; De Angelis, M.; Simonetti, G.; Pantanella, F.; Schippa, S.; Angiolella, L. Carvacrol and thymol, a synergistic antimicrobial activity against bacterial and Candida species. Microbiologyopen 2025, 14, e70089. [Google Scholar] [CrossRef]
  43. Ma, J.; Li, T.; Lin, L.; Lu, Y.; Chen, X.; Li, S.; Du, C.; Wei, C.; Yin, F.; Gan, S. Effects of grape seed extract supplementation on the growth performance, nutrients digestion and immunity of weaned lambs. Front. Vet. Sci. 2024, 11, 1402637. [Google Scholar] [CrossRef]
  44. Ahmadibonakdar, Y.; Vakili, A.; Javadmanesh, A.; Seradj, A.R.; Rajaei-Sharifabadi, H. Thymol modulates rumen barrier function and inflammation in feed-restricted lambs. Anim. Nutr. 2026, 25, 19–29. [Google Scholar] [CrossRef]
  45. Benchaar, C.; Hassanat, F. Assessing the effects of high-carvacrol oregano oil on rumen microbial fermentation, gas production, and methane production in vitro. Can. J. Anim. Sci. 2025, 105, 1–6. [Google Scholar] [CrossRef]
  46. Yu, J.; Cai, L.; Zhang, J.; Yang, A.; Wang, Y.; Zhang, L.; Guan, L.L.; Qi, D. Effects of thymol supplementation on goat rumen fermentation and rumen microbiota in vitro. Microorganisms 2020, 8, 1160. [Google Scholar] [CrossRef]
  47. Cetin, I.; Cetin, E.; Karakcı, D.; Ercetin, E. Thyme essential oil supplementation in growing merino lambs: Effects on growth performance, blood metabolites, and fecal microbiology. Anim. Sci. J. 2025, 96, e70051. [Google Scholar] [CrossRef]
  48. Lu, H.; Wang, H.; Li, B.; Lv, Z.; Li, S.; Xia, Y.; Wang, L. Effects of soybean meal replacement on growth performance, rumen fermentation, rumen microorganisms, and metabolites in Dumont lambs. Animals 2025, 15, 3096. [Google Scholar] [CrossRef]
  49. Du, D.; Jiang, W.; Feng, L.; Zhang, Y.; Chen, P.; Wang, C.; Hu, Z. Effect of Saccharomyces cerevisiae culture mitigates heat stress-related damage in dairy cows by multi-omics. Front. Microbiol. 2022, 13, 935004. [Google Scholar] [CrossRef]
  50. Wang, C.Z.; Dong, J.N.; Sun, W.W.; Qu, K.; Lin, Z.C.; Aschalew, N.D.; Zhao, Y.; Sun, Z.; Ta, N.; Zhao, Z.K.; et al. Myo-inositol affects the health, metabolome, and lactation performance of peripartum dairy cows. Front. Immunol. 2025, 16, 1605244. [Google Scholar] [CrossRef]
  51. Singh, V.; Lee, G.; Son, H.; Koh, H.; Kim, E.S.; Unno, T.; Shin, J.H. Butyrate producers, “the sentinel of gut”: Their intestinal significance with and beyond butyrate, and prospective use as microbial therapeutics. Front. Microbiol. 2023, 13, 1103836. [Google Scholar] [CrossRef]
  52. Calsamiglia, S.; Busquet, M.; Cardozo, P.W.; Castillejos, L.; Ferret, A. Invited review: Essential oils as modifiers of rumen microbial fermentation. J. Dairy Sci. 2007, 90, 2580–2595. [Google Scholar] [CrossRef]
  53. Cobellis, G.; Trabalza-Marinucci, M.; Yu, Z. Critical evaluation of essential oils as rumen modifiers in ruminant nutrition: A review. Sci. Total Environ. 2016, 545–546, 556–568. [Google Scholar] [CrossRef]
  54. Rezaei Ahvanooei, M.R.; Norouzian, M.A.; Piray, A.H.; Vahmani, P.; Ghaffari, M.H. Effects of monensin supplementation on rumen fermentation, methane emissions, nitrogen balance, and metabolic responses of dairy cows: A systematic review and dose-response meta-analysis. J. Dairy Sci. 2024, 107, 607–624. [Google Scholar] [CrossRef]
  55. Wang, G.Y.; Qin, S.L.; Zheng, Y.N.; Geng, H.J.; Chen, L.; Yao, J.H.; Deng, L. Propionate promotes gluconeogenesis by regulating mechanistic target of rapamycin (mTOR) pathway in calf hepatocytes. Anim. Nutr. 2023, 15, 88–98. [Google Scholar] [CrossRef]
  56. Chen, X.; Shang, S.; Yan, F.; Jiang, H.; Zhao, G.; Tian, S.; Chen, R.; Chen, D.; Dang, Y. Antioxidant activities of essential oils and their major components in scavenging free radicals, inhibiting lipid oxidation and reducing cellular oxidative stress. Molecules 2023, 28, 4559. [Google Scholar] [CrossRef]
  57. Zou, Y.; Wang, J.; Peng, J.; Wei, H. Oregano essential oil induces SOD1 and GSH expression through NRF2 activation and alleviates hydrogen peroxide-induced oxidative damage in IPEC-J2 cells. Oxidative Med. Cell. Longev. 2016, 2016, 5987183. [Google Scholar] [CrossRef]
  58. Leal, K.W.; Leal, M.L.R.; Breancini, M.; Signor, M.H.; Vitt, M.G.; Silva, L.E.L.; Wagner, R.; Jung, C.T.K.; Kozloski, G.V.; de Araujo, R.C.; et al. Essential oils and capsaicin in the diet of Jersey cows at early lactation and their positive impact on anti-inflammatory, antioxidant and immunological responses. Trop. Anim. Health Prod. 2024, 56, 247. [Google Scholar] [CrossRef]
  59. Yin, L.; Hussain, S.; Tang, T.; Gou, Y.; He, C.; Liang, X.; Yin, Z.; Shu, G.; Zou, Y.; Fu, H.; et al. Protective effects of cinnamaldehyde on the oxidative stress, inflammatory response, and apoptosis in the hepatocytes of Salmonella gallinarum-challenged young chicks. Oxidative Med. Cell. Longev. 2022, 2022, 2459212. [Google Scholar] [CrossRef]
  60. Wang, R.; Li, S.; Jia, H.; Si, X.; Lei, Y.; Lyu, J.; Dai, Z.; Wu, Z. Protective effects of cinnamaldehyde on the inflammatory response, oxidative stress, and apoptosis in liver of Salmonella typhimurium-challenged mice. Molecules 2021, 26, 2309. [Google Scholar] [CrossRef]
  61. Guo, J.; Yan, S.; Jiang, X.; Su, Z.; Zhang, F.; Xie, J.; Hao, E.; Yao, C. Advances in pharmacological effects and mechanism of action of cinnamaldehyde. Front. Pharmacol. 2024, 15, 1365949. [Google Scholar] [CrossRef]
  62. Thiruvengadam, M.; Venkidasamy, B.; Subramanian, U.; Samynathan, R.; Ali Shariati, M.; Rebezov, M.; Girish, S.; Thangavel, S.; Dhanapal, A.R.; Fedoseeva, N.; et al. Bioactive compounds in oxidative stress-mediated diseases: Targeting the NRF2/ARE signaling pathway and epigenetic regulation. Antioxidants 2021, 10, 1859. [Google Scholar] [CrossRef] [PubMed]
  63. Chen, H.; Liu, T.; Wu, J.P.; Wu, N.; He, B.; Liu, L.S. Effects of oregano essential oil on growth and hematogenic immunity of newborn calves. Pratacult. Sci. 2017, 11, 2141–2148. [Google Scholar] [CrossRef]
  64. Jia, L.; Wu, J.; Lei, Y.; Kong, F.; Zhang, R.; Sun, J.; Wang, L.; Li, Z.; Shi, J.; Wang, Y.; et al. Oregano essential oils mediated intestinal microbiota and metabolites and improved growth performance and intestinal barrier function in sheep. Front. Immunol. 2022, 13, 908015. [Google Scholar] [CrossRef]
  65. Gholijani, N.; Gharagozloo, M.; Kalantar, F.; Ramezani, A.; Amirghofran, Z. Modulation of cytokine production and transcription factors activities in human Jurkat T cells by thymol and carvacrol. Adv. Pharm. Bull. 2015, 5, 653–660. [Google Scholar] [CrossRef]
  66. Cui, H.; Zhang, C.; Su, K.; Fan, T.; Chen, L.; Yang, Z.; Zhang, M.; Li, J.; Zhang, Y.; Liu, J. Oregano essential oil in livestock and veterinary medicine. Animals 2024, 14, 1532. [Google Scholar] [CrossRef]
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Figure 1. Venn diagram and α- and β-diversity analyses of rumen bacteria. (A) Venn diagram of OTUs from different groups; (B) boxplot of α-diversity index; (C) PCoA based on weighted Unifrac distance; (D) PCoA based on Bray–Curtis distance; (E) ANOSIM analysis; (F) UPGMA clustering tree based on weighted Unifrac distance at phylum level; (G) UPGMA clustering tree based on weighted Unifrac distance at genus level. OUTs, operational taxonomic units; PCoA, principal coordinate analysis; ANOSIM, analysis of similarities; UPGMA, unweighted pair-group method with arithmetic means; CON, the control group; DBP1, DBP supplemented at 400 mg/kg; DBP2, DBP supplemented at 800 mg/kg.
Figure 1. Venn diagram and α- and β-diversity analyses of rumen bacteria. (A) Venn diagram of OTUs from different groups; (B) boxplot of α-diversity index; (C) PCoA based on weighted Unifrac distance; (D) PCoA based on Bray–Curtis distance; (E) ANOSIM analysis; (F) UPGMA clustering tree based on weighted Unifrac distance at phylum level; (G) UPGMA clustering tree based on weighted Unifrac distance at genus level. OUTs, operational taxonomic units; PCoA, principal coordinate analysis; ANOSIM, analysis of similarities; UPGMA, unweighted pair-group method with arithmetic means; CON, the control group; DBP1, DBP supplemented at 400 mg/kg; DBP2, DBP supplemented at 800 mg/kg.
Fermentation 12 00241 g001
Fermentation 12 00241 g002
Figure 2. LEfSe analysis of rumen microbiota. (A) LDA score bar chart; (B) Cladogram. In the cladogram, the concentric rings from the inside to the outside represent the taxonomic hierarchy of bacteria from phylum to genus. The size of each node is proportional to the relative abundance of the corresponding taxon. Different colors indicate taxa significantly enriched in different treatment groups: red nodes are biomarkers in the CON group, green nodes are biomarkers in the DBP1 group, and blue nodes are biomarkers in the DBP2 group, whereas yellow nodes indicate taxa with no significant differences among groups. LEfSe, linear discriminant analysis effect size; LDA, linear discriminant analysis; CON, the control group; DBP1, DBP supplemented at 400 mg/kg; DBP2, DBP supplemented at 800 mg/kg.
Figure 2. LEfSe analysis of rumen microbiota. (A) LDA score bar chart; (B) Cladogram. In the cladogram, the concentric rings from the inside to the outside represent the taxonomic hierarchy of bacteria from phylum to genus. The size of each node is proportional to the relative abundance of the corresponding taxon. Different colors indicate taxa significantly enriched in different treatment groups: red nodes are biomarkers in the CON group, green nodes are biomarkers in the DBP1 group, and blue nodes are biomarkers in the DBP2 group, whereas yellow nodes indicate taxa with no significant differences among groups. LEfSe, linear discriminant analysis effect size; LDA, linear discriminant analysis; CON, the control group; DBP1, DBP supplemented at 400 mg/kg; DBP2, DBP supplemented at 800 mg/kg.
Fermentation 12 00241 g002
Table 1. Composition and nutrient levels of the basal diet (DM basis, %).
Table 1. Composition and nutrient levels of the basal diet (DM basis, %).
IngredientsContentNutrient Levels 2Content
Corn silage25.80Crude protein14.93
Cotton leaves37.18Organic matter87.92
Corn20.62Ether extract3.24
Spray-dried corn bran8.38Neutral detergent fiber28.23
Cottonseed meal3.91Acid detergent fiber14.93
NaHCO30.73Calcium0.81
Expanded urea1.23Phosphorus0.33
CaHPO40.29Metabolizable energy (MJ/kg)9.02
Limestone0.74  
Premix 11.12  
Total100.00  
1 The premix provided the following per kg of the diet: VA 10,000 IU, VD3 2500 IU, VE 40 IU, Fe 100 mg, Cu 12.5 mg, Zn 70 mg, Mn 50 mg, I 0.75 mg, Se 0.40 mg. 2 Except for ME, nutrient levels were measured values.
Table 2. Effect of DBP supplementation on the growth performance of Altay lambs.
Table 2. Effect of DBP supplementation on the growth performance of Altay lambs.
ItemsGroupsSEMp-Value
CONDBP1DBP2MLQ
Initial BW (kg)45.9045.3846.511.020.7410.6790.518
Final BW (kg)53.9354.1955.111.150.7530.4790.813
ADG (g/d)178.61195.70191.2515.740.7320.5760.583
ADFI (kg/d)1.861.731.720.080.3920.2150.572
F/G11.049.279.260.800.2170.1300.381
Note: BW, body weight; ADG, average daily gain; ADFI, average daily feed intake; F/G, feed-to-gain ratio; CON, the control group; DBP1, DBP supplemented at 400 mg/kg; DBP2, DBP supplemented at 800 mg/kg; SEM, standard error of the mean; M, main effect; L, linear effect; Q, quadratic effect.
Table 3. Effects of DBP supplementation on slaughter performance of Altay lambs.
Table 3. Effects of DBP supplementation on slaughter performance of Altay lambs.
ItemsGroupsSEMp-Value
CONDBP1DBP2MLQ
Live weight (kg)53.8354.1556.001.430.5260.3010.668
Carcass weight (kg)27.9727.9330.170.850.1350.0850.291
Dressing rate (%)51.9451.6253.880.890.1810.1410.252
Liver weight (g)228.33238.33250.0013.320.5300.2680.960
Heart weight (g)765.00761.67793.3337.260.8070.5990.707
Backfat thickness (mm)8.668.629.240.980.8840.6820.789
Note: CON, the control group; DBP1, DBP supplemented at 400 mg/kg; DBP2, DBP supplemented at 800 mg/kg; SEM, standard error of the mean; M, main effect; L, linear effect; Q, quadratic effect.
Table 4. Effects of DBP supplementation on rumen fermentation parameters in Altay lambs.
Table 4. Effects of DBP supplementation on rumen fermentation parameters in Altay lambs.
ItemsGroupsSEMp-Value
CONDBP1DBP2MLQ
pH6.306.436.250.120.5340.7570.289
Total VFAs (mmol/L)147.44166.58168.657.560.1260.0660.371
Acetate (%)68.87 b70.37 ab71.49 a0.560.0160.0050.781
Propionate (%)17.23 a15.78 ab14.31 b0.580.0100.0030.991
Isobutyrate (%)0.660.570.550.040.1220.0540.492
Butyrate (%)11.4711.7812.230.500.5610.2930.908
Isovalerate (%)0.700.710.830.060.2390.1380.417
Valerate (%)0.94 a0.80 ab0.73 b0.050.0430.0150.577
A/P4.04 b4.50 ab5.01 a0.1760.005<0.0010.894
NH3-N (mmol/L)12.9714.2715.101.0520.3770.1730.857
Lactate (µmol/L)452.44355.22536.00123.630.5960.6400.373
Note: VFAs, volatile fatty acids; A/P, acetate-to-propionate ratio; NH3-N, ammonia nitrogen; CON, the control group; DBP1, DBP supplemented at 400 mg/kg; DBP2, DBP supplemented at 800 mg/kg; SEM, standard error of the mean; M, main effect; L, linear effect; Q, quadratic effect. For data in the same row, values with the same letter or no letter indicate no significant difference (p > 0.05), while values with different superscript letters indicate a significant difference (p < 0.05).
Table 5. Effects of DBP supplementation on protein and lipid metabolism in Altay lambs.
Table 5. Effects of DBP supplementation on protein and lipid metabolism in Altay lambs.
ItemsGroupsSEMp-Value
CONDBP1DBP2MLQ
TP (g/L)57.7858.3258.131.570.9240.7740.791
Albumin (g/L)46.6146.7545.511.300.7640.5570.670
Globulin (g/L)10.8711.5712.620.820.3410.1520.866
BUN (mg/100 mL)7.678.017.340.320.3570.4780.215
AST (U/L)1.943.393.640.600.1290.0630.424
ALT (U/L)9.039.649.220.700.8200.8500.555
TC (mmol/L)1.631.761.700.110.7230.6520.521
TG (mmol/L)0.210.240.200.020.4110.6170.221
HDL-C (mmol/L)0.970.940.970.050.8601.0000.589
LDL-C (mmol/L)0.120.140.130.010.4480.5520.268
NEFA (mmol/L)0.510.490.370.060.2890.1400.607
Note: TP, total protein; BUN, blood urea nitrogen; ALT, alanine aminotransferase; AST, aspartate aminotransferase; TC, total cholesterol; TG, triglycerides; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; NEFA, non-esterified fatty acids; CON, the control group; DBP1, DBP supplemented at 400 mg/kg; DBP2, DBP supplemented at 800 mg/kg; SEM, standard error of the mean; M, main effect; L, linear effect; Q, quadratic effect.
Table 6. Effects of DBP supplementation on energy metabolism and hormone in Altay lambs.
Table 6. Effects of DBP supplementation on energy metabolism and hormone in Altay lambs.
ItemsGroupsSEMp-Value
CONDBP1DBP2MLQ
Glucose (mmol/L)4.615.065.250.290.3140.1440.722
Insulin (uIU/mL)22.5125.6226.712.850.5680.3130.776
GH (ng/mL)1.841.942.020.120.5690.2970.938
Leptin (ng/mL)3.854.133.110.390.1920.2000.189
Lactate (mmol/L)10.27 a7.37 b11.07 a0.770.0100.4760.003
Adiponectin (mg/L)7.457.757.520.610.9370.9390.729
FAS (ng/mL)8.409.328.320.560.3890.9170.178
Note: GH, growth hormone; FAS, fatty acid synthase; CON, the control group; DBP1, DBP supplemented at 400 mg/kg; DBP2, DBP supplemented at 800 mg/kg; SEM, standard error of the mean; M, main effect; L, linear effect; Q, quadratic effect. For data in the same row, values with the same letter or no letter indicate no significant difference (p > 0.05), while values with different superscript letters indicate a significant difference (p < 0.05).
Table 7. Effects of DBP supplementation on serum immunity in Altay lambs.
Table 7. Effects of DBP supplementation on serum immunity in Altay lambs.
ItemsGroupsSEMp-Value
CONDBP1DBP2MLQ
IgG (mg/mL)8.509.558.410.820.5650.9370.294
IgA (mg/mL)3.693.522.850.380.2730.1320.594
IgM (mg/mL)2.042.332.110.210.6030.8290.334
IL-2 (ng/L)48.89 ab53.79 a38.90 b3.640.0320.0710.042
IFN-γ (ng/L)101.6597.9887.424.690.1180.0490.557
Note: IgG, immunoglobulin G; IgA, immunoglobulin A; IgM, immunoglobulin M; IL-2, interleukin-2; IFN-γ, interferon-γ; CON, the control group; DBP1, DBP supplemented at 400 mg/kg; DBP2, DBP supplemented at 800 mg/kg; SEM, standard error of the mean; M, main effect; L, linear effect; Q, quadratic effect. For data in the same row, values with the same letter or no letter indicate no significant difference (p > 0.05), while values with different superscript letters indicate a significant difference (p < 0.05).
Table 8. Effects of DBP supplementation on serum antioxidant parameters in Altay lambs.
Table 8. Effects of DBP supplementation on serum antioxidant parameters in Altay lambs.
ItemsGroupsSEMp-Value
CONDBP1DBP2MLQ
SOD (U/mL)147.82 a133.22 b153.88 a4.140.0090.3170.003
GSH-Px (U/mL)117.14114.60125.0818.000.9120.7590.772
Catalase (U/mL)0.991.701.500.290.2310.2300.216
MDA (nmol/mL)6.247.186.180.730.5680.9530.296
T-AOC (U/mL)0.790.780.830.020.1640.1210.258
Note: SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; MDA, malondialdehyde; T-AOC, total antioxidant capacity; CON, the control group; DBP1, DBP supplemented at 400 mg/kg; DBP2, DBP supplemented at 800 mg/kg; SEM, standard error of the mean; M, main effect; L, linear effect; Q, quadratic effect. For data in the same row, values with the same letter or no letter indicate no significant difference (p > 0.05), while values with different superscript letters indicate a significant difference (p < 0.05).
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MDPI and ACS Style

Di, M.; Liu, M.; Zeng, W.; Xu, M.; Ma, Z.; Xu, D.; Chen, Y. Effects of a Defined Blend of Phytochemicals on Growth Performance, Rumen Fermentation, Bacterial Diversity, and Blood Biochemical and Physiological Parameters in Altay Sheep. Fermentation 2026, 12, 241. https://doi.org/10.3390/fermentation12050241

AMA Style

Di M, Liu M, Zeng W, Xu M, Ma Z, Xu D, Chen Y. Effects of a Defined Blend of Phytochemicals on Growth Performance, Rumen Fermentation, Bacterial Diversity, and Blood Biochemical and Physiological Parameters in Altay Sheep. Fermentation. 2026; 12(5):241. https://doi.org/10.3390/fermentation12050241

Chicago/Turabian Style

Di, Mingyue, Mengjian Liu, Wenshuai Zeng, Mei Xu, Zhanlin Ma, Dong Xu, and Yong Chen. 2026. "Effects of a Defined Blend of Phytochemicals on Growth Performance, Rumen Fermentation, Bacterial Diversity, and Blood Biochemical and Physiological Parameters in Altay Sheep" Fermentation 12, no. 5: 241. https://doi.org/10.3390/fermentation12050241

APA Style

Di, M., Liu, M., Zeng, W., Xu, M., Ma, Z., Xu, D., & Chen, Y. (2026). Effects of a Defined Blend of Phytochemicals on Growth Performance, Rumen Fermentation, Bacterial Diversity, and Blood Biochemical and Physiological Parameters in Altay Sheep. Fermentation, 12(5), 241. https://doi.org/10.3390/fermentation12050241

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