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Oregano essential oil enhanced body weight and well-being by modulating the HPA axis and 23-nordeoxycholic acid of cecal microbiota in Holstein steers under cold stress

Animal Microbiome volume 7, Article number: 34 (2025) Cite this article

Abstract

Background

Prolonged exposure to cold stress in cattle increases basal energy consumption and impedes optimal production. Consequently, herds require adequate attention during cold, extended winters to alleviate cold stress and maintain profitability. This study investigated the effects of oregano essential oil (EO) on body weight (BW), well-being, blood parameters, and cecal microbiota. Eighteen steers were randomly divided into two groups (n = 9) and fed either a basal diet (CK) or the same diet supplemented with 20 g/(d·head) EO for 270 days.

Results

EO increased BW by increasing cecal microbial abundance and carbohydrate metabolism CAZymes, leading to elevated the total volatile fatty acids (VFA) levels. Cold stress activated the HPA axis, and mitigated stress by reducing serum levels of cortisol (COR), corticosterone (CORT), adrenocorticotropic hormone (ACTH), and dopamine (DA). EO increased well-being by decreasing viral species without apparent contribution to drug or antibiotic resistance development, and cecal metabolites were primarily enriched in growth, carbohydrate metabolism, and amino acid metabolism pathways. Specifically, tryptophan metabolism (2-picolinic acid, quinolinic acid, and oxindole) enhanced steer well-being by increasing antioxidants (superoxide dismutase (SOD), peroxidases (POD), and glutathione (GSH)) and reducing inflammatory factors (interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α)) following EO treatment. Notably, low-abundance microorganisms (s_Streptomyces_gardneri, s_Paenibacillus_sp._S09, and s_Nocardia_sp._Root136) may play a significant role in growth and immunity.

Conclusion

These findings provide fundamental insights into how EO alleviates cold stress by modulating the HPA axis, promotes growth and well-being of steers under cold stress by influencing mediates tryptophan metabolism of cecal microbiota in Holstein steers.

Background

Livestock welfare has long been a foundation for maximizing production potential and remains a key societal and political concern globally. More than 68% of dairy farms in China are located in the northern region, where cows are exposed to cold stress, preventing optimal production during long and cold winters [1]. Cold stress is a dysfunctional and defensive response exhibited by cattle in cold environments [2]. This stress signaling responds across the brain-gut axis, using blood as a junction to regulate the body metabolism to regain physiological homeostasis. The gut-brain axis involves bidirectional and continuous regulation of molecular, cellular, and host states [3]. On one hand, stressors are integrated into the central nervous system and trigger the hypothalamic-pituitary-adrenal (HPA) axis to release glucocorticoids from the adrenal gland, which restore homeostasis by modulating enteric volatile fatty acids (VFA), gut function, and microbial composition [4]. On the other hand, microbiota deficiency and disorders leading to gut or microbiota-derived factors may compromise gut barriers, allowing these factors to enter the bloodstream. This not only exacerbates HPA activity in response to stressors during homeostasis but also induces the release of proinflammatory cytokines [5, 6]. These processes might lead to traumatic injury, disruption of nutrition-metabolism balance, altered hormone secretion levels, compromised immune competence, and changes in intestinal flora, affecting animal health, product quality, and disease risk, potentially causing death and hidden economic losses [7,8,9]. Therefore, cattle require extra attention and care to help alleviate cold stress and improve production during winter.

Oregano essential oil (EO) is extracted from Origanumvulgare, a plant widely distributed across Asia, using steam distillation. Research indicates that the primary components of EO are terpenes (Carvacrol and Thymol, ≥ 75 ~ 85%), which exhibit various biological functions including antimutagenic, antioxidant, antihyperglycemic, antifungal, antiviral, anti-inflammatory, and antibacterial effects. These properties make EO valuable in the food, pharmaceutical, and cosmetics industries [10, 11]. Studies have also reported the use of EO or Origanumvulgare to enhance health and performance in livestock [12,13,14]. However, limited research exists on the application of EO to alleviate cold stress and improve the well-being of Holstein steers during winter. While rumen microbes are well-characterized, knowledge about cecum microorganisms in ruminants remains limited. Researchers have highlighted the significant physiological role of cecum microorganisms in production, noting that the cecum is more susceptible than the rumen to high-concentrate feeding conditions in beef cattle. Previous research has found the predominant phyla in the rumen were Bacteroidetes and Firmicutes collectively accounting for 91% of the ASV; but the primary phylum in the cecum was Firmicutes followed by Bacteroidetes, and they differ in the evolutionary branches of function. Revotella ruminicola and Ruminococcus flavefaciens were the most abundant bacterial species in the rumen microbiome, while Clostridiales bacterium and Eubacterium rectale were predominant bacterial species in the cecum microbiome [15, 16]. Furthermore, the rumen and cecum morphometrics may be different, rumen tissue structure tends to ferment, absorption and promote growth, while cecum tissues undergo secondary fermentation and are more inclined to host health, even local inflammation in the cecum may significantly promote systemic inflammation [17, 18]. When potentially digestible fibers reach the cecum, they provide 8.6% energy through VFA after secondary fermentation and absorption, contributing to improved growth performance in steers [19]. A pertinent question is whether EO can reach the steers’ cecum. Recent research indicates that EO can indeed reach the colon and modulate colonic inflammation, microbiome homeostasis and intestinal barrier function in fattening bulls [20]. Consequently, EO treatment may represent an effective approach to enhance the well-being and growth of cattle by modulating cecum microbiota and metabolites.

This study integrates metagenomic and metabolomic approaches to analyze blood biochemical parameters and inflammatory factors, cecal fermentation parameters and microorganisms, blood and cecum metabolites in Holstein steers subjected to cold stress and treated with EO. The research aims to elucidate the host response to cold stress, identify cold stress biomarkers, and evaluate the potential of EO to alleviate cold stress in Holstein steers. The findings are intended to provide data support and fundamental insights for winter welfare strategies and the selection of supplements to mitigate cold stress in the cattle industry.

Results

Temperature fluctuations in the fattening period of steers

The daily highest, lowest, and average temperatures were recorded at a commercial ranch during a 270-day experiment, based on data published by the China Meteorological Administration (https://www.cma.gov.cn/). The average temperature fell below 5 ℃ starting on November 16, 2020 and remained at or below this temperature throughout experimental stages V to IX. Additionally, the body weights (BW) from 120 to 270 days, and average daily gain (ADG) from 90 to 120 and 120 to 150 days increased significantly following EO treatment (P < 0.05, Fig. 1).

Fig. 1
figure 1

Changes in temperature (A), body weight (B), and average daily gain (C) during the entire fattening stage. CK, a basal diet; EO, the same diet supplemented with 20 g per steer/day oregano essential oil; * indicates P < 0.05

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Effects of EO treatment on hypothalamic and pituitary hormone levels

The level of TRH (P = 0.026) was significantly elevated in the EO group compared to the CK group. Conversely, GHRH (P = 0.001) and GH (P < 0.001) were significantly lower in the EO group than in the CK group. However, TSH (P = 0.094) did not exhibit significant differences following EO treatment in steers subjected to cold stress (Fig. 2).

Fig. 2
figure 2

Effects of EO treatment on hypothalamic and pituitary hormones (n = 6). CK, a basal diet and EO, the same diet supplemented with 20 g per steer/day oregano essential oil; TRH: thyrotropin releasing hormone, GHRH: growth hormone releasing hormone, GH: growth hormone, and TSH: thyroid stimulating hormone; * indicates P ≤ 0.05 and ** indicates P ≤ 0.01

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Effects of EO treatment on hematological parameters of steers under cold stress

The EO group exhibited significant increases in RBC count (P = 0.018), HGB (P = 0.002), and HCT (P = 0.003) in blood cells, as well as SOD (P = 0.029), POD (P = 0.009), and GSH (P = 0.034) in serum antioxidants. Conversely, the EO group showed significant decreases in serum hormones SS (P < 0.001), COR (P = 0.001), CORT (P = 0.001), ACTH (P < 0.001), and DA (P = 0.008), as well as serum inflammatory factors IL-1β (P < 0.001), IL-6 (P < 0.001), and TNF-α (P = 0.017) compared to the CK group. Other indicators remained unaffected (P > 0.05, Table 1).

Table 1 Comparative analysis of hematological parameters after EO treatment
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Comparative analysis of cecal VFA following EO administration

The concentrations of the total VFA (P = 0.041), acetate (P = 0.013) and propionate (P = 0.029) were significantly higher in the EO group compared to the CK group, but butyrate (P = 0.006) and valerate (P = 0.004) were significantly higher in the CK group compared to the EO group (Table 2).

Table 2 Comparative analysis of VFA after EO treatment (n = 6)
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Effects of EO treatment on cecal microorganisms

Compositions and differences of cecal microorganisms

Metagenome sequencing was conducted to elucidate the impact of EO treatment on the cecal microbiome. The CK and EO groups yielded an average of 67,170,198 and 69,743,534 raw reads, respectively. After eliminating low-quality and n-containing reads, 66,218,282 and 68,698,358 clean reads were obtained, constituting 98.58% and 98.50% of the raw reads in CK and EO, respectively. Following the removal of host genome sequences, 52,889,808 and 53,016,775 optimized reads were obtained for subsequent analysis, representing 78.74% and 76.02% of the raw reads in CK and EO, respectively. The sequence reads were deemed reliable for further analysis (Table S1). A Venn diagram of cecum microbial species revealed 5,622 shared species, with 1,352 and 321 unique species in the CK and EO groups, respectively. Analysis of similarities (ANOSIM, P = 0.003) and principal co-ordinates analysis (PCoA) demonstrated significant clustering of microbial species between the two groups.

Fig. 3
figure 3

Cecal microbiological compositions and differences. A. Cecal microbial Venn diagram; B. Cecal microbial ANOSIM and PCoA analysis; C. Genus-level differences between CK and EO groups; D. Dominant species in CK and EO groups; CK, basal diet; EO, basal diet supplemented with 20 g oregano essential oil per steer/day

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The dominant microorganisms in the cecum were Firmicutes (CK: 42.27%, EO: 76.77%) and Bacteroidetes (CK: 47.42%, EO: 15.25%). Of the 227 phyla identified, 18 (16 increasing and 2 decreasing) showed significant differences in abundance (P < 0.05, Table S2). Among 522 genera with significant variations, 417 increased and 105 decreased significantly after EO treatment (P < 0.05). Out of 26,624 identified microorganisms at the species level, 2,823 exhibited significantly different abundances. The dominant bacteria were Clostridiales_bacterium (CK: 8.44%, EO: 17.89%), Bacteroidales_bacterium (CK: 1.75%, EO: 4.91%), Phocaeicola_coprophilus (CK: 5.19%, EO: 0.33%), and Firmicutes_bacterium_CAG:110 (CK: 1.60%, EO: 3.55%) (Fig. 3). Differential analysis of archaea revealed significant differences in 34 species between the two groups, with 10 species lower and 24 higher in the EO group compared to the CK group (P < 0.05). For bacteria, 2,712 species showed significant differences, with 525 species lower and 2,187 higher in abundance in the EO group (P < 0.05). Among eukaryotes, 37 species differed significantly, with 5 lower and 32 higher in the EO group (P < 0.05). For viruses, 39 species showed significant differences, with 30 less abundant and 9 more abundant in the EO group (P < 0.05). The abundance of unclassified: uncultured_organism (P = 0.005) was significantly reduced after EO treatment (Table S3). These results suggest that EO effectively increases the abundance of cecum microbiome while decreasing the abundance of viruses.

Functions and differences of cecal microorganism

To investigate the impact of EO on cecum microbial function, KEGG map and genes encoding carbohydrate-active enzymes (CAZyme) were analyzed. KEGG analysis revealed six pathways annotated at the first level: metabolism (MB), genetic information processing (GP), environmental information processing (EP), cellular processes (CP), human diseases (HD), and organizational systems (OS). Among these, CP in the EO group was significantly higher than in the CK group (P = 0.031). At the second level, 45 pathways were observed: 5-EP, 3-GP, 4-GP, 11-HD, 12-MB, and 10-OS. Ten differential pathways were identified, with five pathways enriched in each group. The CK group showed enrichment in metabolism of cofactors and vitamins, glycan biosynthesis and metabolism, metabolism of other amino acids, drug resistance: antimicrobial, and transport and catabolism. The EO group exhibited enrichment in nucleotide metabolism, endocrine and metabolic disease, infectious disease: viral, membrane transport, and cellular community – prokaryotes (Fig. 4A). At the third level, 56 out of 320 pathways were significantly enriched. The top 5 enriched pathways in the EO group were microbial metabolism in diverse environments, carbon metabolism, pentose phosphate pathway, methane metabolism, and glucagon signaling pathway. The top 5 enriched in the CK group were glycosphingolipid biosynthesis - ganglio series, glycosaminoglycan degradation, cationic antimicrobial peptide (CAMP) resistance, lysosome, and other glycan degradation, respectively (Table S4). Furthermore, Comprehensive Antibiotic Resistance Database (CARD) analysis revealed a significant increase in antibiotic inactivation and a significant decrease in reduced permeability to antibiotics after EO treatment (P ≤ 0.05, Fig. 4B). CAZyme mapping identified 116 significant genes encoding CAZyme out of 541 total, including 5 accessory activities (AA), 8 carbohydrate-binding modules (CBMs), 6 carbohydrate esterase (CE), 67 glycoside hydrolases (GH), 22 glycosyltransferases (GT), and 8 polysaccharide lyases (PL). In the EO group, 55 genes were enriched (4 AA, 6 CBM, 5 CE, 37 GH, and 3 PL), while 38 genes were enriched in the CK group (1 AA, 2 CBM, 1 CE, 30 GH, and 5 PL) for CAZyme of carbohydrate breakdown. For CAZyme of carbohydrate synthesis, 8 and 14 GTs were enriched in the EO and CK groups, respectively (Fig. 4C & Table S5). These findings suggest that EO reduces drug and antibiotic resistance while enhancing basal carbohydrate degradation.

Fig. 4
figure 4

Cecal microbiological functions and differences. A. Composition and difference of pathway at first and second level; B. Differences of CARD between CK and EO; C. Differences of genes encoding CAZyme between CK and EO; CP, cellular processes; EP, environmental information processing; GP, genetic information processing; HD, human diseases; MB, metabolism and OS, organizational systems; CK, basal diet; EO, the same diet supplemented with 20 g per steer/day oregano essential oil

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Effects of EO treatment on cecal metabolites

To comprehensively assess the impact of the cecum microbiome, we conducted a cecal metabolite profiling of bulls following EO treatment using untargeted liquid chromatography-mass spectrometry (LC-MS). The analysis identified 798 compounds in the cecum metabolites. OPLS-DA revealed a clear separation between clusters (R2X = 0.451, R2Y = 0.875, Q2 = 0.334), indicating differential metabolites in the cecum contents between the two groups and confirming the reliability of the OPLS-DA models for further analysis (Fig. 5A & B). Screening with FC (FC ≥ 2 & FC ≤ 0.5) and VIP (VIP ≥ 1) criteria identified 174 significantly different metabolites, comprising 71 upregulated and 103 downregulated compounds. These included 38 derived from amino acids and their metabolites, 34 from organic acids and derivatives, 20 from fatty acyls, 17 from glycerophospholipids, and 65 from other sources (Fig. 5C & Table S6). KEGG pathway analysis revealed that these 174 metabolites were significantly enriched in 146 pathways, primarily associated with tryptophan metabolism, lysine biosynthesis, amino acid biosynthesis, taurine and hypotaurine metabolism, glucagon signaling pathway, tricarboxylic acid cycle (TCA), and 2-oxocarboxylic acid metabolism (Fig. 5D). Notably, six metabolites related to growth, amino acid, and carbohydrate metabolism were selected based on multiple enrichments in the aforementioned metabolic pathways and the top 50 significantly different metabolites. 2-picolinic acid and quinolinic acid were downregulated, while oxindole was upregulated in the tryptophan metabolism pathway. 23-nordeoxycholic acid, enriched in the amino acid metabolism pathway, was upregulated. Cyclic AMP was upregulated, and 1-methyl-hydantoin was downregulated, both enriched in the glucagon signaling and growth hormone synthesis, secretion, and action pathways. These findings suggest that EO treatment may exert significant regulatory effects on growth, amino acid, and carbohydrate metabolism in the cecum metabolites of steers, with particular emphasis on tryptophan metabolism.

Fig. 5
figure 5

Cecal metabolite composition and function. A. OPLS-DA model chart; B. OPLS-DA analysis; C. Cecal metabolite volcano plot; D. KEGG functional enrichment analysis of cecal metabolites; E. Violin diagram of key metabolites; CK, a basal diet; EO, the same diet supplemented with 20 g per steer/day of oregano essential oil

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Effects of EO treatment on serum metabolites

Fig. 6
figure 6

Serum metabolite composition and function. A. OPLS-DA model chart; B. PCA analysis; C. Serum metabolite volcano plot; D. KEGG functional enrichment analysis of serum metabolites; E. Heatmap of cecum and serum correlation with tryptophan metabolites; CK, a basal diet; EO, the same diet supplemented with 20 g per steer/day of oregano essential oil

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To assess the impact of the cecum microbiome on serum metabolism, we conducted a serum metabolite profiling of bulls following EO treatment using untargeted LC-MS. The analysis identified 728 compounds in the serum metabolites. PCA revealed a clear separation between clusters, R2X = 0.4, R2Y = 0.999, and Q2 = 0.484 indicating differential metabolites in the serum contents between the two groups and confirming the reliability of the OPLS-DA models for further analysis (Fig. 6A & B). Screening with FC (FC ≥ 2 & FC ≤ 0.5) and VIP (VIP ≥ 1) criteria identified 15 significantly different metabolites, comprising 12 upregulated and 3 downregulated compounds (Fig. 6C & Table S7). KEGG pathway analysis revealed that these 15 metabolites were significantly enriched in purine metabolism, metabolic pathways, ABC transporters, arachidonic acid metabolism, serotonergic synapse, bile secretion, inositol phosphate metabolism, cysteine and methionine metabolism, cortisol synthesis and secretion, steroid hormone biosynthesis (Fig. 6D). To further explore the contribution of cecal microbiome mediated tryptophan metabolism to serum metabolites, we performed Pearson correlation analysis. The results showed that there was no significant difference between serum tryptophan and its metabolites, and the correlation showed that oxindole in serum was positively correlated with 23-nordeoxycholic acid in cecum (r = 2.470, P = 0.040, Fig. 6E). These results suggest that cecal microbiome-mediated 23-nordeoxycholic acid influence serum oxindole, however, there was no significant contribution to other tryptophan and its metabolites.

Analysis of combined metagenome, metabolome and phenotype

The interrelations among cecum microbiota, metabolome, and different phenotypes were quantified by calculating Pearson’s correlation coefficients. The top 20 microorganisms and metabolites with the largest linkage effects were identified using O2PLS analysis (Fig. 7A). Among the top 20 microorganisms, 7 upregulated species (s_Streptomyces_globisporus, s_Mesonia_phycicola, s_Streptomyces_gardneri, s_Paenibacillus_sp._S09, s_Thalassospira_xiamenensis, s_Nocardia_sp._Root136, s_Methylocystis_sp._ATCC_49242) were screened (effect size = meanCK - meanEO < − 0.0001) for subsequent analyses. Among the top 20 metabolites, 5 upregulated metabolites (coproporphyrin III, biliverdin, methyl L-tyrosinate, azelaic acid and L-pipecolic acid) were screened with FC > 2.5 for subsequent analyses. Additionally, 2-picolinic acid, quinolinic acid, oxindole, 23-nordeoxycholic acid, cyclic AMP and 1-methyl-hydantoin from the previous KEGG pathway were also used for subsequent analyses. Furthermore, correlation analysis revealed that the 7 upregulated species positively correlated with coproporphyrin III, biliverdin, methyl L-tyrosinate, azelaic acid and L-pipecolic acid (Fig. 7B). To further explore the relationship between phenotype and omics, network correlation analysis was conducted. The results showed that acetate was negatively correlated with 1-methyl-hydantoin (r = -0.602, P = 0.038). ACTH was positively correlated with 1-methyl-hydantoin (r = 0.838, P = 0.001), and negatively correlated with s_Streptomyces_gardneri (r = -0.597, P = 0.040) and s_Nocardia_sp._Root136 (r = -0.593, P = 0.042). COR was positively correlated with 1-methyl-hydantoin (r = 0.702, P = 0.011), and negatively correlated with L-pipecolic acid (r = -0.623, P = 0.031). DA was positively correlated with 2-picolinic acid (r = 0.805, P = 0.002) and quinolinic acid (r = 0.789, P = 0.002). GH was positively correlated with 2-picolinic acid (r = 0.697, P = 0.012), quinolinic acid (r = 0.664, P = 0.019) and 1-methyl-hydantoin (r = 0.697, P = 0.012). GHRH was positively correlated with 1-methyl-hydantoin (r = 0.824, P = 0.001), and negatively correlated with oxindole (r = -0.622, P = 0.031) and cyclic AMP (r = -0.583, P = 0.047). HCT was positively correlated with 23-nordeoxycholic acid (r = 0.627, P = 0.029). HGB was negatively correlated with 1-methyl-hydantoin (r = -0.741, P = 0.006). RBC was positively correlated with coproporphyrin III (r = 0.625, P = 0.030), L-pipecolic acid (r = 0.694, P = 0.013), s_Paenibacillus_sp._S09 (r = 0.586, P = 0.045) and s_Nocardia_sp._Root136 (r = 0.708, P = 0.010), and negatively correlated with 1-methyl-hydantoin (r = -0.741, P = 0.006). SOD was negatively correlated with 2-picolinic Acid (r = -0.839, P = 0.001) and quinolinic acid (r = -0.834, P = 0.001). SS was positively correlated with 2-picolinic acid (r = 0.577, P = 0.049) and 1-methyl-hydantoin (r = 0.638, P = 0.026). Additionally, BW (r = -0.515, P = 0.086) and ADG (r = -0.555, P = 0.061) were negatively correlated with 1-methyl-hydantoin, while ADG was positively correlated with oxindole (r = 0.363, P = 0.245, Fig. 7C).

Fig. 7
figure 7

Combined analysis of metagenome, metabolome and phenotype. A. O2PLS analysis of the metagenome and metabolome; B. Correlation analysis of target metabolite and microorganism; C. Network diagram of target metabolite, microorganism and different phenotype; → indicates a causal relationship between the two omics; CK, a basal diet; EO, the same diet supplemented with 20 g of oregano essential oil per steer daily

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Discussion

The thermal neutral zone for dairy cattle ranges from approximately 5 to 20 °C. When temperatures fall below 5 °C, cattle adapt to chronic cold stress conditions by increasing thermal insulation and basal metabolic intensity [21]. Moreover, under extreme cold conditions, cattle experience a negative energy balance, redirecting energy from productive purposes to heat production, resulting in economic losses [22]. In this study, the average temperature fell below 5 ℃ starting on November 16, 2020, and remained at or below this temperature until the experiment’s conclusion, indicating that the steers were experiencing cold stress. Additionally, Fig. 1 B&C and a previously published study [23, 24] demonstrated that feeding EO improved the BW and ADG of steers based on equivalent dry matter intake (CK: 10.45 ± 0.25 kg, EO: 10.45 ± 0.23 kg, P = 0.990), suggesting that EO supplementation contributed to enhanced BW and ADG. Notably, the steers’ ADG decreased significantly as temperatures dropped, particularly from 180 to 240 days, indicating that the animals expended more energy to withstand the cold, thereby reducing their growth rate under cold stress conditions.

Hematological parameters directly reflect the immune and anti-inflammatory function, as well as the well-being of cattle. Cold stress activates the HPA axis to release glucocorticoids, facilitating the organism’s adaptation to environmental changes [4]. The TRH, a tripeptide hormone (pGlu–His–Pro-NH2), initiates the hypothalamo–pituitary–thyroid (HPT) axis, a signaling cascade essential for metabolic homeostasis and vertebrate development. TRH stimulates the pituitary to release TSH, which then acts on the thyroid to stimulate T3 and T4 production. T4 and T3 regulate TRH and TSH secretion through negative feedback to maintain physiological levels of the main HPT axis hormones [25]. In this study, except for TRH, no differences were observed in other axis hormones, potentially due to negative feedback. GHRH, a 44-amino acid peptide released by the hypothalamus, induces GH secretion and production through cyclic adenosine monophosphate (cAMP)-dependent pathways [26]. SS, produced by neuroendocrine neurons of the hypothalamic ventromedial nucleus, inhibits GH secretion upon binding to G protein-coupled receptors [27]. GH secretory bursts vary in frequency and amplitude according to factors such as sleep, exercise, diet, starvation, hypoxia, glucocorticoids, and stress. These factors likely influence somatotroph secretion by affecting CNS neurotransmitters, other hormone concentrations (cortisol, corticosterone, adrenocorticotropic hormone, and dopamine), and circulating metabolic fuel levels [28]. The elevated levels of GHRH, GH, SS, cortisol, corticosterone, ACTH, and dopamine in the CK group may be attributed to accelerated metabolic fuel levels to overcome cold stress, fasting, and pre-slaughter fear. Notably, cortisol, corticosterone, and ACTH levels decreased after EO treatment. Researchers suggest that the secretion of these hormones positively correlates with stressors, and they serve as stress markers due to their role in neutralizing oxidants to prevent oxidative stress intensification [29]. An imbalance between oxidants and antioxidants can exacerbate oxidative stress, leading to inflammation and compromised health. RBCs, which lack nuclei and mitochondria, are the primary oxygen transporters in vertebrate blood. They continuously produce energy through hemoglobin auto-oxidation and anaerobic glycolysis [30]. Due to long-term exposure to endogenous (from hemoglobin auto-oxidation) and exogenous (from neutrophils and macrophages) reactive oxygen species (ROS), RBCs possess a wide range of antioxidant systems to minimize ROS-mediated lipid and protein damage [31]. SOD, POD, and GSH are primary cellular antioxidant enzymes crucial for scavenging ROS. This study observed increased SOD, POD, and GSH levels, and decreased IL-1β, IL-6, and TNF-α levels in steer serum after EO treatment. These findings suggest that cold stress primarily elicits a response through the HPA axis; EO improves BW by increasing GH levels, decreasing SS levels, and reducing energy loss; and enhances well-being by boosting antioxidants and reducing inflammation in steers under cold stress.

Cecum microbes and their metabolism significantly impact digestion, growth performance, and health of steers. In this study, cecum microbial richness increased significantly, while the number of viruses decreased after EO treatment. Previous research has reported that effective reduction in viruses may occur upon EO supplementation, as EO is a hydrophobic molecule that is permeable through the cell membrane and causes expansion of the cellular content [32]. Besides, these compounds (Carvacrol and Thymol) exert their antimicrobial effects by disrupting cell membrane permeability and inhibiting metabolites and key enzymes in the tricarboxylic acid cycle pathway, ultimately resulting in bacterial cell death [33]. Here, death of the viruses may occur through the drainage of crucial molecules and ions from the bacterial cell, and resulting bacteria in a dominant niche for increasing abundance. Increased biodiversity in the cecum creates a functional buffer against changing environmental conditions and contributes to growth and feed efficiency [34, 35]. The primary phyla in the cecum were Firmicutes and Bacteroidetes. The Firmicutes to Bacteroidetes ratio is often used to assess host BW because these phyla play an important role in hydrolyzing indigestible dietary polysaccharides to VFA, such as components of plant cell walls and undigested starch [16, 36]. Furthermore, a high F/B ratio may lead to host inflammation [37]. In this study, the decreasing F/B ratio indicated that EO could reduce steer inflammation by regulating cecum microflora, although this analysis at the phylum level has low resolution from a taxonomic perspective. Additionally, CAZymes, sourced from a vast array of microbes, represent the most widespread and structurally diverse set of enzymes involved in the breakdown, biosynthesis, or modification of lignocellulose found in living organisms [38]. CAZyme degrades diet structural polysaccharides to provide nutrient substances for absorption by epithelium [39]. The enrichment of genes encoding CAZyme, which are more involved at AA, CBM, CE, GH and PL in the cecum microbiota of EO steers, further demonstrated that the EO provided steers with a greater ability to degrade complex substrates. In this study, genes encoding β-glucosidase, β-xylosidase, and cellulose were highly enriched in the EO group, further demonstrating that the steers had a high ability to degrade complex substrates to VFA after EO treatment, corresponding to increasing the total VFA, acetate and propionate levels in the EO group. Interestingly, KEGG and CARD analyses showed decreases in drug resistance: antimicrobial and transport and catabolism, while antibiotic inactivation significantly increased and permeability to antibiotics reduced after EO treatment. KEGG analysis revealed that differential metabolites were mainly enriched in amino acid metabolism, particularly tryptophan metabolism in steer cecum metabolite analysis. Tryptophan metabolism primarily involves three metabolic pathways: kynurenine (Kyn, 95%), 5-hydroxytryptamine (HT, 1 ~ 2%), and indole pathways (2 ~ 3%) [40, 41]. Intestinal microorganisms convert tryptophan to tryptamine and indole pyruvic acid, and indole pyruvic acid to indole, indole acetaldehyde, and indole lactate. Indole acetaldehyde can be converted to indole acetic acid and tryptophol, and the former can then be converted to skatole. Indole lactate may be converted to indole acrylic acid and subsequently to indole propionic acid [42]. Researchers have advocated that tryptophan and its metabolism play an important role in neuronal function, inflammatory responses, oxidative stress, immune responses, and intestinal homeostasis. For example, they improve the mitochondrial respiration and glycolysis of T cells to alleviate intestinal inflammation and inhibit intestinal inflammation via the activation of Aryl hydrocarbon receptor and enhancement of Treg cell function under high abundance of Bacteroides thetaiotaomicron [43]. Furthermore, 2-picolinic acid and quinolinic acid, end products of tryptophan, are implicated as major biomarkers of inflammatory disorders [44, 45], and oxindole regulates host biological processes, such as maintenance of epithelial barrier integrity, immune response, protection against pathogens, inflammation, and metabolic disorders [46]. The high concentration of 23-nordeoxycholic acid restrained host inflammation and enhanced antioxidant activity, and it is related to the abundance of Enterococcus faecalis [47]. With increasing levels of cyclic AMP, there may be direct control of IL-10, IL-12, and TNF-α expression early [48]. In this research, 2-picolinic acid, quinolinic acid, and cyclic AMP were downregulated, while oxindole and 23-nordeoxycholic acid were upregulated, and the abundance of Enterococcus faecalis increased, which showed improved steer immunity and antioxidant activity (SOD, Fig. 7C) after EO treatment. The above results indicated that EO increased the abundance of fiber-degrading bacteria and converted fiber into VFA to provide energy for the host to increase BW and resist cold, while improving the steers’ immunity and antioxidant activity by intervening in tryptophan and its metabolites. Notably, EO may not contribute to the development of resistance and antibiotic residues.

To further investigate the regulatory mechanisms of economic traits and well-being phenotypes, we conducted Pearson correlation analysis. In economic traits, BW and ADG were negatively correlated with 1-methyl-hydantoin (downregulated). We hypothesized that this correlation might be due to high levels of 1-methyl-hydantoin inhibiting the release of GHRH and GH, decreasing acetate levels, and promoting SS release. However, the specific pathway of 1-methyl-hydantoin requires further investigation. Additionally, ADG was positively correlated with oxindole (upregulated). Oxindole, a tryptophan derivative, exhibits various biological activities including antidiabetic, antimicrobial, antiviral, anti-inflammatory, antioxidant, anticholinesterase, antitubercular, and antimalarial properties. This derivative effectively repairs gut barrier function via interplay between intestinal aryl hydrocarbon receptor and Wnt/β-catenin signaling. Furthermore, enhancement of M2 macrophages in the intestinal lamina propria generates more IL-10 entering the bone marrow, thereby promoting osteoblastogenesis and increasing ADG [49, 50] (Fig. 7C). Regarding well-being phenotypes, much gut microbiome research has focused on the role and impact of dominant species due to their vital role in gut function. However, low-abundance species may also profoundly affect host health and growth [51]. For instance, ACTH may be influenced by s_Streptomyces_gardneri and s_Nocardia_sp._Root136, although their specific functions and pathways remain unclear. COR was negatively correlated with L-pipecolic acid, consistent with findings suggesting that L-pipecolic acid can potentially alleviate depression and anxiety via the γ-aminobutyric system [52]. DA was positively correlated with 2-picolinic acid and quinolinic acid, replicating trends observed in previous mouse studies where systemic inflammation originating from the gut induced a dopamine increase [53]. RBC count was positively correlated with coproporphyrin III, s_Paenibacillus_sp._S09, and s_Nocardia_sp._Root136. Coproporphyrin III serves as a biomarker for measuring organic anion transporting polypeptide (OATP) 1B activity, with OATPs mediating the hepatic uptake of diverse endogenous compounds and xenobiotics [54]. The protein product of β-1,3-glucanase gene PglA, cloned from Paenibacillus_sp._S09, contains three functional regions: an N-terminal domain, a GH16 catalytic domain, and a C-terminal domain with an Ig-like structure, participating in the degradation of polysaccharides utilized as a nutrient source [55]. In conclusion, EO improved growth and health of steers by mediating low abundance microorganisms and tryptophan metabolism.

Our findings provide a crucial foundation for enhancing the body weight and health of steers under cold stress through dietary EO supplementation, while also facilitating the identification of biomarkers associated with cold stress, in addition, the key metabolic pathway after EO treatment were found: EO—s_Paenibacillus_sp._S09—23-nordeoxycholic acid—Oxindole. However, this study has several limitations. Firstly, the small sample size (n = 9 or 6) may introduce significant variability. Secondly, functional and response pathways may vary considerably across different varieties and individuals. Lastly, as a cross-sectional study of gut microbiota, the interaction and causal relationship between microbiota and host require further investigation. Future validation using animal models is necessary to confirm these findings.

Conclusions

This research demonstrates that dietary supplementation with 20 g/(d·head) of EO, a natural bioactive compound, mitigated cold stress in Holstein steers by modulating the HPA axis, as evidenced by decreased levels of COR, CORT, ACTH, and DA. Furthermore, EO supplementation enhanced body weight, average daily gain, and overall well-being (increased RBC, SOD, POD and GSH, decreased IL-1β, IL-6 and TNF-α) in cold-stressed steers by influencing cecal microorganisms involved in tryptophan metabolism. Additionally, the study identified microbial and metabolic markers associated with growth, immunity, and oxidative stability, providing insights into the molecular mechanisms underlying cold stress alleviation in steers. These findings offer beef cattle practitioners an alternative dietary additive to enhance steer survival during cold winters and improve economic efficiency.

Methods

Animal management and sample collection

Eighteen healthy Holstein steers, aged 300 days (castrated at 60 days), were obtained from Huarui Ranch (38°43′34.92″N, 100°40′24.82″E; Minle County, Zhangye, Gansu, China). The steers, with similar body weights (mean ± standard error: 350.32 ± 4.41 kg), were randomly divided into two groups (n = 9) and housed in individual stalls within open-type sheds, where the indoor temperature matched the ambient temperature. The experimental group (EO) received a basal diet supplemented with 20 g/(d·head) of EO, the EO was acquired from Ralco Agriculture Inc. (Marshall, MN, USA) at a market price of 120 CNY/kg, with the 20 g/(d·head) EO as per the manufacturer’s recommendation, preparation and composition of EO have been previously reported [23]. While the control group (CK) was fed only the basal diet. Each morning, the EO was measured, thoroughly mixed with 1 kg of corn, and added to the feed; the CK group received 1 kg of corn without EO. The experiment lasted 270 days from June 4th, 2020 to February 28, 2021, all steers were weighed BW every 30 days and calculate the ADG. Every 30 days is a stage and nine stages in total. All steers were fed a total mixed ration (TMR) twice daily at 8:00 and 16:00. The TMR composition (Table S8) met the nutritional requirements for beef cattle [21], and the animals had unrestricted access to both feed and water.

Following the experiment, six steers were randomly selected from each group based on median body weight. Blood samples (5 mL) were collected from the jugular vein in two EDTA tubes and one gel biochemistry tubes prior to slaughter. Subsequently, 1.5 mL of serum was isolated by centrifugation at 3000 rpm and 4 ℃ for 15 min in sterile tubes from the gel biochemistry tubes. All samples were stored at -20 ℃ for subsequent analysis. After slaughter hypothalamic and pituitary tissues were immediately collected in sterile tubes after opening the brain according to the anatomical site for subsequent hormone analysis. Additionally, the cecum was separated based on physiological anatomy after abdominal cavity opening, the mixed contents were collected into three 5 mL sterile tubes and stored at -80 °C for further VFA, metagenomic, and metabolomic analyses.

Analysis of hypothalamic and pituitary hormones

The hormone levels were measured using commercial Enzyme linked immunosorbent assay (ELISA) kits (Elabscience Biotechnology Co., Ltd., Wuhan, China) according to the manufacturer’s instructions, including thyrotropin releasing hormone (TRH, Kit No. E-EL-R0979) and growth hormone releasing hormone (GHRH, Kit No. E-EL-R0469) in the hypothalamus, and growth hormone (GH, Kit No. E-EL-R3003) and thyroid stimulating hormone (TSH, Kit No. E-EL-R0976) in the pituitaries. Briefly, tissue was homogenized in phosphate-buffered saline (pH = 7.0; 1:20 w/v) and stored at − 20 °C for 24 h. Subsequently, the supernatants were transferred to clean Eppendorf vials for ELISA after centrifugation at 5000 rpm for 5 min. Absorbance was measured at 450 nm using a microplate reader [56].

Analysis of hematological parameters

Blood cell count analysis was conducted utilizing an automatic hematology analyzer (BC-1800, Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, China). The examination encompassed the following parameters: white blood cell (WBC) profiles, including WBC count, lymphocyte, monocyte, and neutrophil counts; red blood cell (RBC) profiles, comprising RBC count, hemoglobin content (HGB), hematocrit (HCT), mean corpuscular volume (MCV), and mean corpuscular haemoglobin concentration (MCHC); and platelet count.

Serum antioxidant parameters were assessed using a colorimetric method (BS-420 automatic biochemical instrument, Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, China) in accordance with the commercial kit instructions (Zhongsheng Beihang Biotechnology Co., Ltd, Beijing, China). The measured parameters included malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), total antioxidant capacity (T-AOC), peroxidases (POD), glutathione (GSH), glutathione peroxidase (GSH-PX) and glutathione S-transferase (GSH-ST).

Serum hormone and inflammatory parameters were measured using commercial ELISA kits (Beijing Huaying Institute of Biotechnology, Beijing, China) and a plate reader (DR-200BS, Wuxi Huawei Delang Instrument Co., Ltd., Jiangsu, China) according to the manufacturer’s instructions. The measured parameters included creatine kinase (CK, Kit No. HY-50056), lactatedehydrogenase (LDH, Kit No. HY-N0042), triiodothyronine (T3, Kit No. HY-400), thyroxine (T4, Kit No. HY-10002), growth hormone (GH, Kit No. HY-C0018), testosterone (T, Kit No. HY-10027), somatostatin (SS, Kit No. HY-10172), cortisol (COR, HY-10062), corticosterone (CORT, Kit No. HY-10063), adrenocorticotropic hormone (ACTH, Kit No. HY-10175), dopamine (DA, Kit No. HY-1003), immunoglobulin A (IgA, Kit No. bs-0360Gs), immunoglobulin G (IgG, Kit No. bs-0293Gs), immunoglobulin M (IgM, Kit No. bs-0345Gs), interleukin-1β (IL-1β, Kit No. HY-10101), interleukin-6 (IL-6, Kit No. HY-10105) and tumor necrosis factor-α (TNF-α, Kit No. HY-10116).

Analysis of cecum volatile fatty acids

Metaphosphorylated cecum fluid was analyzed to determine VFA using gas chromatography [35]. The fluid was centrifuged at 5400 r/min for 10 min, after which the supernatant was carefully collected and filtered through a 0.45-µm syringe filter into a vial for GC. Subsequently, VFA content was measured using a GC ultra-gas chromatograph (TRACE-1300, Thermo Scientific, Milan, Italy).

Metagenome sequencing and bioinformatics analysis

The total DNA of cecum microbes was extracted using a Soil DNA Kit (MOBIO, Carlsbad, CA, USA) following established protocols [39]. DNA concentration and purity were assessed using a TBS-380 fluorometer (Turner Biosystems, Sunnyvale, CA, USA) and NanoDrop 2000 (NanoDrop Technologies, Wilmington, DE, USA), respectively. DNA was fragmented to approximately 400 bp using a Covaris M220 (Gene Company Limited, Hong Kong, China) for library construction. Sequencing was performed on the Illumina NovaSeq 6000 platform (Illumina Inc., San Diego, CA, USA), and low-quality sequencing reads (length < 50 bp, quality value < 20, or N bases) were filtered using FAST (version 0.20.0) (H. Li & Durbin, 2009). Reads were aligned to the Bos Taurus reference genome assembly using BWA (version 0.7.9a), and the data were assembled using MULTIPLE MEGAHIT (Version 1.1.2) [57]. Overlapping sequences of lengths ≥ 300 bp were selected as the final assembly result and used for further gene annotation. The best candidate open reading frames (ORFs) were predicted using Metagene [58]. Predicted ORFs with length ≥ 100 bp were retrieved. Cluster analysis of non-redundant gene catalogs with sequence homology and 90% coverage was conducted using CD-HIT (version 4.6.1). Subsequently, sequences of the non-redundant gene catalog were aligned with the NCBI NR database using BLASTP (version 2.2.28 +, best match e-value cutoff: 1e − 5) to obtain annotation results and species abundance [59]. Finally, the sequences were annotated against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database using BLAST search (version 2.2.28 +, best match e-value cutoff: 1e − 5) [60].

Metabolome measurement and bioinformatics analysis

Following thawing, 50 mg of cecum contents and blood were weighed into a 2-mL centrifuge tube, respectively. 500 µL of 70% methanol internal standard extract was added at 4 °C. The mixture was vibrated for 3 min and left at -20 °C for 30 min, then centrifuged at 12,000 r/min for 10 min at 4 °C. Subsequently, 250 µL of supernatant was centrifuged at 12,000 r/min for 5 min at 4 °C. Following this, 150 µL of supernatant was transferred into the corresponding injection vial for analysis. The sample was analyzed using ultra-performance liquid chromatography (UPLC, Shim-pack UFLC SHIMADZU CBM30A) and tandem mass spectrometry (MS/MS systems, QTRAP® 6500+, SCIEX, Framingham, MA, USA) devices [61]. Based on the LC/MS data, the extraction ion chromatographic peaks of all metabolites were integrated using MultiQuant software (Applied Biosystems, Foster, MA, USA) and the MetWare database (MWDB), then corrected according to the chromatographic peaks [62]. Relative concentrations of cecum metabolites were analyzed, and fold change (FC ≥ 2 and FC ≤ 0.5) and variable importance in projection (VIP ≥ 1) were used to screen for differential metabolites. The identified metabolites were annotated using the KEGG database and subsequently mapped to the KEGG pathway database.

Statistical analysis

The hematological parameters (including blood cell parameters, serum antioxidants, serum hormones, and serum inflammatory markers) and VFA data were statistically analyzed using independent samples t-tests in SPSS software (v.25.0; IBM Corp., Armonk, NY, USA). All values are presented as mean ± standard error, with statistical significance set at P ≤ 0.05. Charts were generated using GraphPad Prism 8 software (GraphPad Inc., La Jolla, CA, USA). For metagenomic analysis, Principal coordinates analysis (PCoA) based on bray-curtis distances and the analysis of similarities (ANOSIM) were performed to analyze the similarity or difference of the compositions. The Wilcoxon rank-sum test was used to identify differences in the composition and function of microorganisms between two groups. Metabolic differences between the two groups were determined using orthogonal projections to latent structures-discriminate analysis (OPLS-DA). Two-way orthogonal partial least squares (O2PLS) analysis was performed using OmicShare Tools (https://www.omicshare.com/tools/Home/Soft/o2pls). Relationship analysis was conducted using Pearson’s correlation.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

HPA:

Hypothalamic-pituitary-adrenal axis

EO:

Oregano essential oil

BW:

Body weight

ADG:

Average daily gain

TRH:

Thyrotropin releasing hormone

GHRH:

Growth hormone releasing hormone

GH:

Growth hormone

TSH:

Thyroid stimulating hormone

COR:

Cortisol

CORT:

corticosterone

ACTH:

Adrenocorticotropic hormone

DA:

Dopamine

VFA:

Volatile fatty acids

SOD:

Superoxide dismutase

POD:

Peroxidases

GSH:

Glutathione

IL-1β:

Interleukin-1β

IL-6:

Interleukin-6

TNF-α:

Tumor necrosis factor-α

TMR:

Total mixed ration

WBC:

White blood cell

RBC:

Red blood cell

HGB:

Hemoglobin content

HCT:

Hematocrit

MCV:

Mean corpuscular volume

CAZyme:

Carbohydrate-active enzymes

KEGG:

Kyoto Encyclopedia of Genes and Genomes

CAMP:

Cationic antimicrobial peptide

CARD:

Comprehensive Antibiotic Resistance Database

LC-MS:

Liquid chromatography-mass spectrometry

FC:

Fold change

VIP:

Variable importance in projection

TCA:

Tricarboxylic acid cycle

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Acknowledgements

We would like to thank TopEdit (www.topeditsci.com) for its linguistic assistance during the preparation of this manuscript.

Funding

This research received funding from the Beef Cattle Quality Fattening Project of Gansu Province (GSA-XMLZ-2021-01), the Major Science and Technology Special Project of Gansu Province (GSSLCSX-2020-1), the Industry Support Project of Gansu Province (2024CYZC-36), and the Major Science and Technology Special Project of Gansu Province (25ZDNA008).

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Authors and Affiliations

  1. College of Animal Science and Technology, Gansu Agricultural University, Lanzhou, 730070, China

    Yongliang Huang, Jinping Shi, Pengjia He, Yue Ma, Xu Zhang, Yongzhi Cao & Zhaomin Lei

  2. China Resources Ng Fung, Ltd., Shenzhen, 518000, China

    Siyu Cheng

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  1. Yongliang Huang
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Contributions

YLH and ZML was contributed to the study design and interpretation of the findings; YM, XZ, and YZC conducted the animal work; SYC, JPS, and PJH participated in lab analysis, and manuscript preparation; YLH, and ZML oversaw the development of the experiment and wrote the final version of the manuscript. The final version of the manuscript was read and approved by all authors.

Corresponding author

Correspondence to Zhaomin Lei.

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Ethical approval

The study received approval from the Animal Care Committee of Gansu Agricultural University (No. GSAU-Eth-AST-2022-035, Lanzhou, China). All procedures adhered to the rules and standards set forth by the committee.

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The authors declare no competing interests.

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Huang, Y., Cheng, S., Shi, J. et al. Oregano essential oil enhanced body weight and well-being by modulating the HPA axis and 23-nordeoxycholic acid of cecal microbiota in Holstein steers under cold stress. anim microbiome 7, 34 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s42523-025-00401-3

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Keywords

Animal Microbiome

ISSN: 2524-4671