Regulatory Effects of Cinnamon–Pepper–Chili Essential Oil Complex on Growth Performance, Immune Function, Complete Blood Count, and Intestinal Microbiota in Simmental CrossBred Cattle During the Late Fattening Stage

Abstract

This trial aimed to investigate the effects of compound essential oils (EO) on the fattening performance, blood physiological–biochemical indices, and intestinal microbiota in late-fattening Simmental crossbred bulls. Twenty healthy Simmental crossbred bulls (Simmental × Charolais × Angus) with similar initial body weights of 442 (±72.49) kg were randomly divided into two groups: a control group (basal diet, CON group) and a compound essential oil group (basal diet + 16 g/head/day, EO group). The trial included a 14-day pre-feeding period and a 42-day experimental period, totaling 56 days. The results showed the following: (1) The EO group exhibited a significantly higher average daily gain (ADG), immunoglobulin A (IgA), immunoglobulin G (IgG), total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-PX), glucose (GLU), dopamine (DA), basophil count (Baso), mean corpuscular hemoglobin (MCH), and platelet distribution width (PDW) compared to the CON group (p < 0.05), while the mean corpuscular volume (MCV) was significantly lower (p < 0.05). (2) Although the compound essential oil supplementation did not alter the relative abundance of major intestinal microbial taxa, it significantly improved the intestinal microbiota structure (p < 0.05), increased fiberdegrading microbiota, and promoted short-chain fatty acid production. (3) The relative abundance of the intestinal microbiota norank_f__UCG-010 showed significant positive correlations with ADG, GSH-PX, IgG, DA, T-SOD, GLU, IgA, and Baso (p < 0.05), while Christensenellaceae_R-7_group abundance was positively correlated with ADG, IgA, and Baso (p < 0.05). In conclusion, the compound essential oil enhances healthy and efficient fattening in beef cattle by improving the intestinal microbial structure, increasing beneficial bacteria, regulating the nutrient metabolism through key bacterial genera, and enhancing the immune function, antioxidant capacity, and energy metabolism levels.

1. Introduction

According to the 2022 FAO revenue and expenditure table data, the global average meat consumption is 122.05 g per day, with beef accounting for one-fifth of this total [1]. Projections from the comprehensive economic model integrating the grain demand and livestock supply indicate that by 2050, the per capita meat demand in East Asia will increase by 82%, while Africa’s meat demand is expected to double, reaching 22 kg per person [2]. Given that fattening beef cattle remains one of the critical sources of meat supply and current production cannot meet the growing demand for beef, enhancing the fattening efficiency, increasing the beef output, and ensuring food security while maintaining cattle health have become vital challenges in the development of animal husbandry.

Essential oils (EO), typically extracted from various parts of plants such as the leaves, flowers, stems, seeds, roots, and epidermis [3], exhibit a wide range of biological activities [4]. They have been proven effective in inhibiting multiple microorganisms, including bacteria [5], protozoa [6], fungi [7], and archaea [8], while also regulating physiological functions and promoting growth in animals [9]. Due to their natural, safe, and non-toxic properties, EOs have been widely adopted in livestock and poultry farming. Among them, cinnamon, pepper, chili, and oregano essential oils are the most extensively studied in animal husbandry. Experimental studies demonstrate that EOs improve the growth performance and regulate physiological functions in livestock. For instance, adding a composite of cinnamon, oregano, and eucalyptus essential oils to a milk replacer for suckling calves can enhance their immune and antioxidant capabilities while improving the feed utilization efficiency [10]. In ruminants, adding chili essential oil (200 mg/kg) to Holstein bulls’ diets notably increases their daily weight gain and serum antioxidant indices [11]. When lamb diets are supplemented with 10 mL/kg of pepper essential oil, the rumen microbial abundance and digestive enzyme activity are effectively elevated [12]. Notably, synergistic effects exist among EOs: supplementing fattening cattle diets with a composite EO (containing chili oleoresin, clove EO, and garlic EO, 500 mg/head/day) improves the average daily gain, feed conversion rate, and immune parameters [13].

As indicated by the aforementioned research, the addition of essential oils such as cinnamon, chili, and pepper to feed can enhance the production performance, improve health indicators, and promote the digestion and absorption of feed nutrients. Moreover, mixtures of multiple plant essential oils have demonstrated synergistic effects in livestock production. However, there is limited research on the combined use of these three specific oils. Therefore, this study hypothesizes that a composite of cinnamon, prickly ash, and chili essential oils exhibits positive synergistic effects capable of improving the growth performance, maintaining physiological health, ensuring the health status of fattening cattle, and enhancing the fattening efficiency. Using Simmental crossbred cattle as the subjects, this experiment investigates the effects of a three-plant essential oil mixture in diets on the growth performance, blood routine parameters, immune and antioxidant functions, and gut microbiota in late-fattening-stage Simmental crossbred cattle. The results aim to provide scientific evidence for applying composite plant essential oils in beef cattle fattening practices.

2. Materials and Methods

2.1. Test Animals

This trial was conducted at the Pinghu Lake Breeding Farm in Ganzhou District, Zhangye City, China, in accordance with the Standards for the Use and Care of Research Animals in China [14]. The experimental design, procedures, and methods followed a protocol approved by the Animal Care and Use Committee of Gansu Agricultural University under the guidelines of the Experimental Animal Affairs Administration of the Ministry of Science and Technology of the People’s Republic of China (Approval No. GSAU-Eth-AST-2023-036). Twenty healthy Simmental × Charolais × Angus crossbred bulls with similar body weights of 442 ± 72.49 kg were randomly divided into two groups: a control group (basal diet, CON group) and a compound essential oils group (basal diet + 16 g/head/day, EO group). The trial included a 14-day adaptation period followed by a 42-day experimental period, totaling 56 days. The compound essential oil is provided by the New Rural Development Research Institute of Northwest Normal University and consists of Sichuan pepper essential oil, cinnamon essential oil, chili essential oil (primarily composed of micronized particles with a particle size < 5 μm, uniform in size), and carriers (attapulgite and calcium silicate). The specific composition includes Sichuan pepper (10%), cinnamon (10%), chili (10%), attapulgite (60%), and calcium silicate (10%).

2.2. Feeding Procedure

According to the body weight and fattening objectives of the trial cattle, total mixed diets were formulated in phases based on the target daily weight gain, nutritional standards for minimum cost and optimal performance specified in the NRC (2016) (Nutrient Requirements of Beef Cattle). The composition and nutritional levels of the basal diets for each phase are shown in

Table 1. Composition and nutritional levels of the basal diet (dry matter basis, %).

Ingredient Composition	Content	Nutrient Level	Content
Corn	33.0	DM	63.28
Soybean meal	7.30	CP	13.30
Rapeseed meal	4.30	EE	3.20
Wheat bran	3.10	NDF	34.40
Silage corn	20.80	ADF	21.30
Corn stover	13.40	Ca	0.80
Wheat straw	8.90	P	0.41
Alfalfa hay	4.50
NaHCO3	0.30
NaCl	0.30
5%Premix 1	0.60

¹ 5% Premix: Cu: 500 mg/kg; Fe: 1200 mg/kg; Mn: 1000 mg/kg; Zn: 1800 mg/kg; I: 35 mg/kg; Se: 9 mg/kg; Co: 20 mg/kg; Vit A: 180 KIU/kg; Vit D3: 75 KIU/kg; Vit E: 1400 IU/kg.

.

2.3. Sample Collection and Measurement

2.3.1. Growth Performance Measurement

The experimental cattle were weighed on an empty stomach at the beginning and end of the trial. After the experiment concluded, the average daily gain (ADG) for each group was calculated based on the measurement data.

2.3.2. Measurement of Blood Parameters

Before the commencement of the trial and on the final day of the experiment, 10 mL of fasting blood samples were collected from the jugular vein prior to morning feeding. The blood samples were allowed to clot at room temperature for 30 min and then centrifuged at 3000× g for 10 min to separate the serum. The serum was transferred into cryovials and frozen at −20 °C for storage. For complete blood count (CBC) analysis, blood was collected into standard blood collection tubes using a similar protocol. The measurements of CBC parameters, serum antioxidant markers, and immune indices were performed according to the methodology described in Reference [15]. The main blood routine test indicators include White Blood Cell Count (WBC), Neutrophil Count (NEU), Lymphocyte Count (LYM), Monocyte Count (MONO), Eosinophil Count (EOS), Basophil Count (Baso), Neutrophil Percentage (NEU%), Lymphocyte Percentage (LYM%), Monocyte Percentage (MONO%), Eosinophil Percentage (EOS%), Basophil Percentage (Baso%), Red Blood Cell Count (RBC), Hemoglobin Concentration (HGB), Hematocrit (HCT), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Red Cell Distribution Width—Coefficient of Variation (RDW-CV), Red Cell Distribution Width—Standard Deviation (RDW-SD), Platelet Count (PLT), Mean Platelet Volume (MPV), Platelet Distribution Width (PDW), and Plateletcrit (PCT). Serum antioxidant biomarkers, immune index, and other indicators include immunoglobulin (IgA, IgG, IgM), Interleukin-6 (IL-6), Interferon-gamma (IFN-γ), Tumor Necrosis Factor-alpha (TNF-α), Total Superoxide Dismutase (T-SOD), Total Antioxidant Capacity (T-AOC), Glutathione Peroxidase (GSH-PX), Malondialdehyde (MDA), Blood Urea Nitrogen (BUN), Glucose (GLU), Total Cholesterol (CHO), Triglycerides (TG), Adrenocorticotropic Hormone (ACTH), Growth Hormone (GH), Insulin-like Growth Factor-1 (IGF-1), Gastrin (Gas), Cholecystokinin (CCK), and Dopamine (DA).

2.3.3. Determination of Gut Microbiota

On the final day of the trial, fresh fecal samples (5 g) were collected via rectal sampling and immediately transferred into cryogenic storage tubes. The samples were flash-frozen in a liquid nitrogen tank for preservation. The gut microbiota analysis was conducted following the methodology described in Reference [16].

2.3.4. Statistical Analysis

The experimental data were analyzed using the SPSS 26.0 software for independent samples t-test, with results presented as “mean ± standard deviation”. A significance threshold of p < 0.05 indicated statistically significant differences, while p < 0.01 denoted highly significant differences. α-diversity indices (Chao, Shannon, Ace, and Simpson) were calculated using Mothur software (version 1.30) and intergroup differences in α-diversity were assessed via the Wilcoxon rank sum test. Principal Coordinate Analysis (PCoA) based on the Bray–Curtis distance algorithm was employed to evaluate microbial community structure similarity between samples, with the Adonis non-parametric test used to determine significant differences in microbial community structure across groups. Linear discriminant analysis (LDA) effect size (LDA > 3, p < 0.05) was applied to identify bacterial taxa with significantly different abundances from phylum to genus levels between groups. Functional abundance profiles of microbial sequences were predicted using PICRUSt2 (v2.2.0-b).

3. Results

3.1. The Effect of Compound Essential Oils on the Growth Performance of Simmental Crossbred Cattle

As shown in

Table 2. Effect of compound essential oils on growth performance (kg).

Index	Treatment		Content
	CON	EO
Initial weight	442.00 ± 73.10	443.70 ± 75.83	0.960
Final weight	515.10 ± 73.38	538.70 ± 90.51	0.530
Average daily gain	1.74 ± 0.27 b	2.26 ± 0.55 a	0.020

Different lowercase letters as superscripts within the same row indicate significant differences (p < 0.05). No superscript letters indicate no significant difference (p > 0.05). The same notation applies to the tables below.

, compared with the CON group, the EO group exhibited a significantly higher ADG (p < 0.05). However, there were no significant differences in the initial body weight and final body weight of Simmental crossbred cattle between the EO group and the CON group (p > 0.05).

3.2. Effect of Compound Essential Oil on Hematological Parameters in Simmental Crossbred Cattle

According to

Table 3. Effect of complex essential oils on blood counts.

Index		Treatment		p-Value
		CON	EO
WBC (109/L)	0 d	7.43 ± 0.86	7.27 ± 0.96	0.762
	42 d	7.76 ± 1.04	7.76 ± 0.94	0.998
NEU (109/L)	0 d	3.25 ± 0.65	3.23 ± 0.52	0.970
	42 d	3.05 ± 0.62	3.89 ± 1.12	0.135
LYM (109/L)	0 d	4.02 ± 0.58	3.98 ± 0.58	0.908
	42 d	4.56 ± 1.10	4.41 ± 1.18	0.832
MONO (109/L)	0 d	0.44 ± 0.09	0.44 ± 0.11	0.978
	42 d	0.27 ± 0.04	0.30 ± 0.06	0.396
EOS (109/L)	0 d	0.41 ± 0.10	0.43 ± 0.12	0.766
	42 d	0.15 ± 0.03	0.19 ± 0.06	0.187
BASO (109/L)	0 d	0.07 ± 0.02	0.06 ± 0.04	0.858
	42 d	0.06 ± 0.02 b	0.10 ± 0.02 a	0.009
NEU%	0 d	38.60 ± 8.23	39.01 ± 5.81	0.923
	42 d	39.24 ± 7.03	40.50 ± 8.09	0.779
LYM%	0 d	48.16 ± 4.57	49.70 ± 9.72	0.732
	42 d	54.91 ± 7.39	55.03 ± 15.80	0.987
MONO%	0 d	2.77 ± 0.82	2.94 ± 0.40	0.659
	42 d	3.54 ± 0.60	3.20 ± 0.65	0.371
EOS%	0 d	3.16 ± 0.36	2.82 ± 0.75	0.340
	42 d	1.88 ± 0.51	3.22 ± 1.52	0.067
BASO%	0 d	0.69 ± 0.16	0.70 ± 0.20	0.915
	42 d	0.74 ± 0.24	1.05 ± 0.42	0.145
RBC (109/L)	0 d	7.81 ± 1.07	7.25 ± 0.82	0.336
	42 d	7.47 ± 0.82	8.02 ± 1.07	0.339
HGB (g/L)	0 d	135.87 ± 13.33	138.21 ± 11.44	0.751
	42 d	141.44 ± 11.42	138.67 ± 14.64	0.722
HCT (%)	0 d	35.94 ± 3.74	37.13 ± 4.30	0.618
	42 d	31.31 ± 2.05	30.02 ± 3.00	0.405
MCV (fL)	0 d	40.56 ± 1.89	43.51 ± 3.59	0.105
	42 d	42.30 ± 3.39 a	37.58 ± 2.05 b	0.015
MCH (pg)	0 d	18.59 ± 1.44	17.86 ± 1.00	0.335
	42 d	17.35 ± 1.09 b	19.06 ± 1.49 a	0.047
MCHC (g/L)	0 d	460.94 ± 5.73	458.46 ± 8.40	0.563
	42 d	451.22 ± 7.35	462.17 ± 9.77	0.053
RDW-CV (%)	0 d	20.73 ± 0.71	21.15 ± 0.77	0.350
	42 d	20.60 ± 0.58	20.27 ± 0.64	0.367
RDW-SD (fL)	0 d	29.81 ± 3.38	31.80 ± 2.01	0.245
	42 d	30.63 ± 2.25	28.65 ± 2.69	0.197
PLT (109/L)	0 d	385.13 ± 48.39	419.21 ± 47.14	0.245
	42 d	405.56 ± 53.88	367.83 ± 52.04	0.246
MPV (fL)	0 d	6.08 ± 0.04	6.25 ± 0.27	0.149
	42 d	5.76 ± 0.24	5.58 ± 0.20	0.204
PDW (fL)	0 d	15.51 ± 0.29	15.23 ± 0.30	0.130
	42 d	14.73 ± 0.22 b	15.02 ± 0.20 a	0.039
PCT (%)	0 d	0.97 ± 0.19	1.22 ± 0.30	0.123
	42 d	0.23 ± 0.03	0.21 ± 0.04	0.146

, the Simmental hybrid cattle in the EO group exhibited significantly higher Baso, MCH, and PDW compared to the CON group (p < 0.05), while showing a significantly lower MCV than the CON group (p < 0.05). No significant differences were observed in other routine blood parameters between the two groups (p > 0.05).

3.3. The Effect of Compound Essential Oils on Serum Indices in Simmental Crossbred Cattle

According to

Table 4. The effect of compound essential oils on serum indicators.

Index		Treatment		p-Value
		CON	EO
Blood immune parameters
IgA (μg/mL)	0 d	1117.06 ± 13.31	1123.52 ± 13.78	0.428
	42 d	980.95 ± 142.18 b	1296.01 ± 306.46 a	0.045
IgG (mg/mL)	0 d	1.24 ± 0.29	1.24 ± 0.23	0.974
	42 d	2.32 ± 0.48 b	3.61 ± 1.19 a	0.033
IgM (μg/mL)	0 d	708.76 ± 190.68	693.63 ± 81.41	0.862
	42 d	753.17 ± 373.75	782.59 ± 384.60	0.896
IL-6 (pg/mL)	0 d	99.63 ± 9.55	87.90 ± 17.40	0.178
	42 d	78.51 ± 26.49	74.79 ± 32.13	0.831
IFN-γ (pg/mL)	0 d	301.66 ± 24.90	291.42 ± 37.16	0.785
	42 d	394.01 ± 139.98	395.32 ± 166.71	0.989
TNF-α (pg/mL)	0 d	81.23 ± 8.82	77.41 ± 18.15	0.652
	42 d	88.08 ± 20.83	67.62 ± 27.91	0.180
Blood antioxidant indicators
T-SOD (U/mL)	0 d	190.25 ± 11.04	189.57 ± 19.86	0.943
	42 d	162.17 ± 19.97 b	187.22 ± 15.33 a	0.035
T-AOC (U/mL)	0 d	5.53 ± 2.18	4.98 ± 1.45	0.618
	42 d	4.63 ± 3.23	5.18 ± 3.49	0.781
GSH-PX (U/mL)	0 d	108.30 ± 23.66	112.45 ± 17.67	0.738
	42 d	111.41 ± 9.02 b	128.54 ± 15.03 a	0.038
MDA (nmol/mL)	0 d	2.51 ± 0.46	2.73 ± 0.27	0.323
	42 d	3.41 ± 0.30	3.11 ± 0.21	0.083
Biochemical index
BUN (mmol/L)	0 d	2.77 ± 1.00	2.13 ± 0.64	0.213
	42 d	3.16 ± 0.74	3.04 ± 0.68	0.768
GLU (mmol/L)	0 d	3.64 ± 0.39	3.98 ± 0.59	0.275
	42 d	2.18 ± 0.48 B	3.82 ± 0.67 A	0.001
CHO (mmol/L)	0 d	2.53 ± 0.17	2.73 ± 0.65	0.503
	42 d	2.90 ± 0.74	2.69 ± 0.34	0.542
TG (mmol/L)	0 d	0.16 ± 0.05	0.14 ± 0.04	0.542
	42 d	0.16 ± 0.04	0.15 ± 0.04	0.654
ACTH (pg/mL)	0 d	28.19 ± 12.12	29.68 ± 11.08	0.828
	42 d	16.05 ± 4.07	27.46 ± 19.80	0.197
GH (ng/mL)	0 d	5.13 ± 1.70	4.39 ± 2.04	0.511
	42 d	6.16 ± 2.46	8.25 ± 3.19	0.232
IGF-1 (ng/mL)	0 d	112.16 ± 39.72	105.75 ± 25.84	0.747
	42 d	71.76 ± 26.45	107.73 ± 57.27	0.193
Gas (pg/mL)	0 d	102.29 ± 40.74	106.77 ± 25.59	0.824
	42 d	120.57 ± 34.21	122.63 ± 63.15	0.954
CCK (ng/mL)	0 d	1366.14 ± 207.48	1378.08 ± 192.49	0.920
	42 d	1241.35 ± 481.71	1420.18 ± 443.91	0.519
DA (pg/mL)	0 d	240.73 ± 25.94	258.62 ± 10.18	0.147
	42 d	212.97 ± 109.41 b	374.23 ± 93.65 a	0.021

Different lowercase letters as superscripts within the same row indicate significant differences (p < 0.05), while different uppercase letters denote extremely significant differences (p < 0.01). No su-perscript letters indicate no significant difference (p > 0.05).

, compared with the CON group, the EO group exhibited significantly higher levels of IgA and IgG in immune indicators (p < 0.05), while no significant differences were observed in the IgM, IL-6, IFN-γ, or TNF-α levels between the two groups (p > 0.05). For the antioxidant indicators, T-SOD and GSH-PX levels in the EO group were significantly higher than those in the CON group (p < 0.05), whereas T-AOC and MDA levels showed no significant intergroup differences (p > 0.05). In the blood biochemical indices, GLU and DA levels were significantly elevated in the EO group compared to the CON group (p < 0.05). No significant differences were detected between the groups for the BUN, CHO, TG, ACTH, GH, IGF1, Gas, or CCK levels (p > 0.05).

3.4. The Effect of Compound Essential Oils on the Intestinal Microbiota of Simmental Crossbred Cattle

3.4.1. Dilution Curve and OTUs Analysis

Following the rarefaction of sequencing data to the minimum sample size sequence, the species dilution curves for both groups plateaued at a value of 10,000, indicating the adequacy of the sequencing data volume. The Venn diagram revealed a total of 1528 OTUs in the CON group and 1577 OTUs in the EO group, with 1170 shared OTUs between the groups (see Figure 1).

3.4.2. Alpha Diversity Analysis

According to the ACE index, Chao index, Shannon index, and Simpson index shown in the figure, there were no significant differences in the species abundance and diversity between the groups (p > 0.05) (see Figure 2).

3.4.3. Beta Diversity Analysis

As shown in Figure 3, principal coordinate analysis (PCoA) based on the Bray–Curtis distance revealed that PC1 and PC2 contributed 28.5% and 18.17% to intergroup variations, respectively. Adonis analysis testing differences between the CON and EO groups demonstrated an R² value of 0.337 and a Pr (>F) value of 0.034 (

Table 5. Adonis test.

#	Df	SumsOfSqs	MeanSqs	F.Model	R2	Pr (>F)
group_factor	1	0.02772	0.02772	4.06921	0.33715	0.034
Residuals	8	0.05450	0.00681	-	0.66284	-
Total	9	0.08223	-	-	1	-

Df: Degrees of Freedom; SumsOfSqs: Sum of Squares; MeanSqs: Mean Squares; F.Model: F-statistic value; R²: Proportion of variance explained by groups and residuals relative to the total variance, respectively; Pr (>F): p-value obtained via permutation test.

), indicating significant structural differences in gut microbial communities within the multivariate space between the two groups, with the EO group exhibiting better clustering than the CON group.

3.4.4. Analysis of Microbial Composition at the Phylum and Genus Levels

A total of 12 bacterial phyla were identified, as illustrated in the figure, with the combined relative abundance of the dominant phyla Firmicutes and Bacteroidota exceeding 70%(

Table 6. Relative abundance of gut microorganisms (%).

Index	Treatment		p-Value
	CON	EO
gate level
p__Firmicutes	49.120 ± 6.830	58.490 ± 6.684	0.144
p__Bacteroidota	45.080 ± 5.088 a	35.760 ± 4.946 b	0.037
p__Spirochaetota	3.960 ± 6.318	3.259 ± 5.995	0.531
p__Patescibacteria	1.174 ± 0.353	1.701 ± 0.538	0.144
p__Verrucomicrobiota	0.186 ± 0.111	0.363 ± 0.417	0.531
p__Proteobacteria	0.235 ± 0.215	0.196 ± 0.145	0.999
p__Cyanobacteria	0.086 ± 0.068	0.117 ± 0.061	0.463
p__unclassified_k__norank_d__Bacteria	0.082 ± 0.033	0.069 ± 0.031	0.674
p__Actinobacteriota	0.070 ± 0.065	0.049 ± 0.034	0.600
p__Deinococcota	0.003 ± 0.005	0.000 ± 0.000	0.180
genus level
g__Rikenellaceae_RC9_gut_group	18.440 ± 8.036	18.220 ± 6.025	0.835
g__UCG-005	15.460 ± 5.113	17.520 ± 2.661	0.531
g__unclassified_f__Lachnospiraceae	7.276 ± 2.412	8.531 ± 5.884	0.835
g__Bacteroides	5.915 ± 6.554	4.078 ± 1.154	0.835
g__norank_f__UCG-010	2.803 ± 0.716 b	4.613 ± 1.134 a	0.012
g__Treponema	3.960 ± 6.318	3.259 ± 5.995	0.531
g__Alistipes	4.098 ± 1.315	3.041 ± 1.111	0.404
g__Prevotellaceae_UCG-003	4.913 ± 5.919	1.486 ± 0.955	0.403
g__norank_f__Muribaculaceae	3.096 ± 1.060	3.213 ± 1.144	0.834
g__norank_f__Eubacterium_coprostanoligenes_group	2.799 ± 1.832	3.487 ± 1.901	0.676

). Additionally, 220 bacterial genera were detected, as shown in the figure (Figure 4), with the dominant genera primarily including Rikenellaceae_RC9_gut_group, UCG-005, unclassified_f__Lachnospiraceae, Bacteroides, and norank_f__UCG-010.

3.4.5. Species Difference Analysis

Using the LEfSe (Linear Discriminant Analysis Effect Size) for multi-level differential species discriminant analysis revealed significantly differential taxa between the groups. In the CON group, the prominent differentially abundant taxa included seven species: p__Bacteroidota, c__Bacteroidia, o__Bacteroidales, g__Psychrobacillus, g__Salinimicrobium, c__Actinobacteria, and g__norank_f__Cyclobacteriaceae. In the EO group, 14 species showed significant differences: c__Clostridia, o__Oscillospirales, f__UCG-010, g__norank_f__UCG-010, g__Butyrivibrio, g__norank_f__Erysipelotrichaceae, g__norank_f__Clostridium_methylpentosum_group, f__Clostridium_methylpentosum_group, g__Eisenbergiella, g__Dorea, g__Family_XIII_UCG-001, g__Cellulosilyticum, f__Defluviitaleaceae, and g__Defluviitaleaceae_UCG-011 (see Figure 5).

3.4.6. Functional Abundance Analysis of Gut Microbiota via KEGG Pathways

The functional abundance analysis of the gut microbiota KEGG pathways (at level 3) revealed that the top five pathways in terms of intestinal functional abundance were metabolic pathways, amino acid biosynthesis, microbial metabolism in diverse environments, ribosomes, and a two-component system. Among these, the amino acid biosynthesis pathway showed a significant increase in the EO group (p < 0.05) (see Figure 6).

3.4.7. Association Analysis Between Gut Microbiota and Growth/Blood Parameters

According to the correlation heatmap, the relative abundance of norank_f__UCG-010 showed significantly positive correlations with ADG, GSH-PX, IgG, DA, T-SOD, GLU, IgA, and Baso (p < 0.05). The relative abundance of Christensenellaceae_R-7_group exhibited significantly positive correlations with ADG, IgA, and Baso (p < 0.05). No significant correlations were observed between other dominant bacterial taxa and the listed indicators (p > 0.05) (see Figure 7).

4. Discussion

4.1. The Effects of Compound Essential Oils on the Growth Performance of Simmental Crossbred Cattle

The inherent components of plant essential oils can enhance the sensory stimulation of feed in animals, increase the feed intake, and thereby promote growth [9]. Fugita et al. [17] found that the addition of a compound essential oil containing oregano oil, castor seeds, and cashew nuts to the diet improved the growth performance of beef cattle. Souza et al. [18] reported that dietary supplementation with a compound essential oil of eugenol, thymol, and vanillin enhanced the daily weight gain and dry matter intake of Nelore cattle. Su’s study demonstrated that capsaicin supplementation increased the daily weight gain and feed intake of Holstein calves [19]. In this experiment, the average daily gain (ADG) of Simmental crossbred cattle in the EO group was significantly higher than that in the CON group, and the final body weight of the EO group was numerically greater than that of the CON group, consistent with the aforementioned findings. The possible explanation is that capsaicin in the compound essential oil stimulates the secretion of digestive enzymes and improves the feed digestibility [20], thereby enhancing the appetite and feed intake in Simmental crossbred cattle. Additionally, cinnamon essential oil in the compound blend imparts a unique aromatic flavor to the feed, improving its palatability [21].

4.2. Effects of Compound Essential Oils on Hematological Parameters in Simmental Crossbred Cattle

Blood routine parameters are crucial physiological indicators for assessing the animal health status. Metrics such as the red blood cell count, white blood cell count, hemoglobin content, and platelet count can reveal an animal’s health condition and potential disease risks. This study found that Simmental hybrid cattle supplemented with compound plant essential oils in their diet exhibited significantly higher MCH and PDW compared to the CON group. Regarding the mechanism of an increased erythrocyte hemoglobin content, the existing research indicates that plant essential oils, with their strong anti-glycosylation activity, may reduce hemoglobin glycosylation modifications, thereby maintaining a normal hemoglobin function and promoting erythrocyte production [22]. Although the specific regulatory pathways require further elucidation, these findings align with the protein stability-enhancing properties of essential oils. The elevated PDW suggests that compound essential oils might enhance the coagulation function, supporting normal hemostatic and clotting processes [23]. The experimental results also showed significantly lower MCV in the EO group compared to the CON group. This difference may stem from the delayed erythrocyte senescence induced by essential oil supplementation. Previous studies have confirmed that oxidative stress is a key factor mediating erythrocyte aging [24], while terpene compounds in plant essential oils have demonstrated antioxidant properties [25].

Baso, a type of white blood cell, play a critical role in regulating immune responses and inflammatory processes. This study found that dietary supplementation with compound essential oils significantly increased basophil counts in Simmental hybrid cattle. The elevated Baso levels may directly enhance inflammatory responses through the release of bioactive substances such as histamine and heparin, while the chemokines secreted by basophils effectively recruit immune cells like neutrophils and monocytes to inflammatory sites, thereby strengthening the immune capacity [26]. Combined with previous research data from calf trials, these findings demonstrate that compound essential oils exhibit universal regulatory effects on basophils in ruminants [27]. A comprehensive analysis of blood routine indicators revealed that compound essential oils not only promote hematopoiesis and improve the coagulation function, but also enhance animal health by potentiating immune-mediated responses.

4.3. The Effect of Compound Essential Oils on Serum Parameters in Simmental Crossbred Cattle

Studies have shown that capsaicin exerts immunomodulatory effects by increasing immunoglobulin levels and inhibiting the production of pro-inflammatory cytokines in perinatal dairy cows [28], and this has been further validated in calf trials [20]. The potential mechanism may be related to capsaicin downregulating the expression of pro-inflammatory cytokines via the NF-κB signaling pathway [29]. A compound essential oil containing cinnamaldehyde, eugenol, and capsicum oleoresin has also demonstrated the ability to enhance plasma IgM and IgG levels and activate immune responses in ewe trials [26]. The findings of this study demonstrate that the composite plant essential oil significantly increased the concentrations of IgA and IgG while reducing TNF-α levels in Simmental crossbred cattle, consistent with previous research conclusions. This indicates that dietary supplementation with the composite essential oil can maintain the health of ruminants by enhancing the humoral immune function and suppressing inflammatory responses. Serum antioxidant markers serve as critical indicators of an organism’s ability to maintain an oxidative balance. In this trial, the essential oil group exhibited significantly elevated activity levels of serum T-SOD and GSH-Px, alongside a marked reduction in the content of MDA, a lipid peroxidation product. These results confirm that the composite essential oil effectively alleviates oxidative stress in Simmental crossbred cattle by enhancing the antioxidant enzyme activity, thereby providing crucial support for their healthy growth.

It is well known that serum biochemical indicators in animals can serve as important parameters for assessing their health status. In this study, the serum DA and GLU levels in the EO group were significantly higher than those in the CON group. This phenomenon may be attributed to capsaicin promoting a dopamine release by altering the neuronal cell membrane fluidity or modulating ion channel activity [30]. Notably, dopamine can activate voltage-dependent potassium channels via pancreatic β-cell D2-like receptors, shortening the action potential duration and inhibiting glucose-stimulated insulin secretion, ultimately leading to elevated blood glucose levels [31]. This suggests that the compound essential oils may regulate energy metabolism processes through the “dopamine-glucose metabolism axis”. In summary, the compound essential oils enhance the immune function, antioxidant capacity, and glucose metabolism in ruminants by increasing serum IgA and IgG levels, boosting the antioxidant enzyme activity, suppressing the pro-inflammatory cytokine TNF-α, and modulating the dopamine–glucose metabolism axis, thereby ensuring the healthy growth of Simmental crossbred cattle.

4.4. The Impact of Compound Essential Oils on the Intestinal Microbiota of Simmental Crossbred Cattle

Essential oils contain various components that can interact intricately with intestinal microbiota, and this interaction may influence animal health through multiple mechanisms [32]. In this study, 16S rRNA sequencing technology was employed to analyze the intestinal microbiota of Simmental hybrid cattle. The results revealed that the species richness of intestinal microbiota showed no significant changes induced by the addition of compound essential oils. However, the EO group demonstrated better microbial community clustering compared to the CON group, with significant structural differences observed between the two groups. This indicates that essential oil supplementation could improve the intestinal microbial structure in Simmental hybrid cattle. The Firmicutes phylum is primarily responsible for degrading cellulose, hemicellulose, starch, and oligosaccharides, while the Bacteroidetes phylum breaks down proteins and various carbohydrates in feed, including xylose, oligosaccharides, pectin, and cellulose. These two phyla collectively promote nutrient absorption in ruminants [33,34]. In this experiment, Firmicutes and Bacteroidetes were identified as the most abundant phyla in the intestinal tracts of both groups of Simmental crossbred cattle, which aligns with findings from previous studies [16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35]. In the differential species analysis, the CON group showed seven significantly differential species, including p__Bacteroidota, c__Bacteroidia, o__Bacteroidales, and others. The EO group exhibited 14 significantly differential species, such as c__Clostridia, o__Oscillospirales, and f__UCG-010. The EO group was predominantly characterized by fiber-degrading microbiota from the Clostridia class, which are typically involved in the degradation of complex carbohydrates such as cellulose and hemicellulose in the rumen of ruminants, producing short-chain fatty acids (SCFAs) [36]. Among other differential microbes, g__Dorea and g__Family_XIII_UCG-001 contribute to the host energy supply through the acetate or propionate metabolism [37,38]. The g__norank_f__Erysipelotrichaceae is associated with the host lipid metabolism and immune regulation, influencing inflammatory responses [39]. Similarly, in the correlation analysis between the gut microbiota and blood and growth indicators, the relative abundances of norank_f__UCG-010 and Christensenellaceae_R-7_group in the gut microbiota showed significant positive correlations with ADG, GSH-PX, T-SOD, IgG, IgA, Baso, GLU, and DA in crossbred cattle. Previous studies indicate that norank_f__UCG-010 promotes SCFA biosynthesis by participating in dietary fiber fermentation, which is closely linked to its role in maintaining intestinal health and regulating immune homeostasis [40]. Christensenellaceae_R-7_group, belonging to the phylum Firmicutes and family Christensenellaceae, similarly enhances SCFAs synthesis, and SCFAs can indirectly regulate immunoglobulin expression [41]. In this study, feeding Simmental crossbred cattle with compound essential oils resulted in a significant increase in the amino acid biosynthesis pathway, while other pathways such as carbon metabolism and microbial metabolism showed no notable changes. This suggests that the essential oils may promote amino acid metabolism, potentially through the regulation of enzyme activity related to amino acid synthesis in the cattle, thereby enhancing their amino acid synthesis capacity. In summary, the addition of compound essential oils to the diet optimized the intestinal microbiota structure of Simmental crossbred cattle, enriched fiber-degrading microbial communities, and increased amino acid metabolic pathways. These effects synergistically enhanced fiber substrate fermentation and beneficial metabolite production in the gut, collectively improving the host immune function and growth performance, ultimately achieving the comprehensive enhancement of intestinal health and physiological functions.

Although this study provides valuable insights into the effects, certain limitations should be acknowledged. For instance, only a single dosage was tested, and the observations were limited to short-term effects. These constraints highlight the need for future research to explore dose–response relationships and evaluate the long-term impacts on the microbiota diversity.

5. Conclusions

The Essential Oil Complex can enhance the daily weight gain of Simmental hybrid cattle and exert positive effects on related blood parameters. The analysis of the intestinal microbial community revealed that the compound essential oil improves the microbial community richness, diversity, and species composition, while modulating the abundance of certain beneficial microbial species. These changes demonstrate favorable impacts on the immune function, antioxidant levels, and metabolic parameters.