Dietary tea tree (Melaleuca alternifolia) oil supplementation enhances the expressions of amino acid transporters in goat ileal mucosa and improves intestinal immunity

Abstract Tea tree oil (TTO) is a plant‐derived additive with anti‐inflammatory, bactericidal, and growth‐promoting properties. However, little is known about the effects of TTO on intestinal amino acid transport and immune function in goats. Twenty‐four Ganxi goats (initial body weight of 13.5 ± 0.70 kg) were randomly allotted two treatments and fed either control (CON) or CON+TTO (0.2 ml/kg) diet. The addition of TTO to the diet significantly decreased (p < .05) tumor necrosis factor‐α content and increased (p < .05) interleukin‐2 (IL‐2) content in goat serum; significantly decreased (p < .05) IL‐12, and increased (p < .05) IL‐2 content in goat ileal mucosa; significantly increased (p < .05) secreted IgA content in the jejunal and ileal mucosa; significantly upregulated (p < .05) IL‐2 and downregulated (p < .05) IL‐12 at the mRNA level in the ileal mucosa; significantly elevated the levels of serine, arginine, and total amino acids in the ileal mucosa (p < .05); significantly upregulated (p < .05) SLC1A1 and SLC7A1 in the ileum; and significantly enhanced (p < .05) the protein expression of Claudin‐1 in the ileal mucosa. In summary, adding 0.2 ml/kg of TTO to the diet enhanced SLC1A1 and SLC7A1 mRNA expression in the ileal mucosa, and SLC1A1 and SLC7A1 could transport serine and arginine from the chyme to the ileal mucosa. Thus, increased serine and arginine content in the mucosa could improve intestinal immunity. TTO supplementation upregulated the expression of IL‐2 and Claudin‐1 in goat ileal mucosa, and enhanced immune function in the intestine.


| INTRODUC TI ON
In goat breeding, many antibiotics are used in the feed to reduce the occurrence of diseases. However, considering the residue of antibiotics in animal products and the harm to human health, China banned the use of antibiotics in animal feed in 2020 (Dong et al., 2019).
Plant essential oils are rich in natural active substances such as terpenes, esters, ketones, and alcohols (Burt, 2004). Plant essential oils have attracted the attention of animal husbandry researchers because of their antibacterial, insecticidal, and antioxidant functions (Cappelli et al., 2021;Davila-Ramirez et al., 2020;Hall et al., 2021;Su et al., 2020;Zhang et al., 2021). Thus, plant essential oil can promote animal growth and improve intestinal immunity (Puvaca et al., 2020;Zhang et al., 2021). Nehme et al. (Nehme et al., 2021) recognized the immunomodulatory molecules of essential oils as a potential therapeutic option in ruminants and monogastric husbandries.
Tea tree oil (TTO), a plant essential oil, is distilled from the fresh branches and leaves of Melaleuca alterniflora and is widely used in medicine and agricultural production (Puvaca et al., 2019). TTO treatment can lead to the loss of bacterial cytoplasmic content, disrupt the integrity of the cell membrane, and ultimately lead to bacterial death (Carson et al., 2002). TTO exerts anti-inflammatory effects by stimulating lymphocyte proliferation and inhibiting the production of proinflammatory cytokines (Brand et al., 2002).
Studies have shown that adding TTO to broiler diets can significantly increase their daily weight gain (Cui et al., 2018;Khattak et al., 2014). Improvements in growth performance are often accompanied by changes in the animal body protein metabolism of animals (Xu et al., 2016). TTO supplementation can improve intestinal immunity in piglets (Dong et al., 2019). Adding Chinese herbal medicine powder to the piglet diet could increase the concentration of amino acids in the piglet's serum and promote the absorption of amino acids by the piglet's gastrointestinal tract (Kong et al., 2009). The concentration of amino acids in the blood is affected by their absorption and transport of amino acids in the gastrointestinal tract. The transport of intestinal amino acids is also closely related to muscle protein metabolism and intestinal immunity (Kong et al., 2018;Li et al., 2007). However, to the best of our knowledge, there has been no research on the effects of TTO on intestinal amino acid transport and intestinal immunity in goats.
We hypothesized that the addition of TTO would improve intestinal mucosal immunity in goats. This study explored the effects of TTO on goat intestinal amino acid transport and immunity by adding TTO to goat diets.

| MATERIAL AND ME THODS
The study was approved by the Institutional Animal Care Committee, and all procedures involving animals were conducted following the guidelines on animal care of the Institute of Subtropical Agriculture, Chinese Academy of Sciences.

| Animals and experimental design
Animal experiments were performed at the Institute of Subtropical Agricultural, Chinese Academy of Sciences (Changsha, China), from April to July 2019. Twenty-four Ganxi goats (initial body weight of 13.5 ± 0.70 kg) were randomly allotted two treatments and fed either a control (CON) or CON+TTO (0.2 ml/kg) diet. The trial lasted 60 days. During the experiment, the goats had free access to feed and water. Goats were fed twice at 08:00 a.m. and 16:00 p.m.
During the animal trial, all goats had access to water and feed ad libitum. The diet was formulated according to the feeding standard of meat-producing sheep and goats (NY/T816-2004

| Feed samples collection and analyses
Approximately 500 g of feed samples was collected and dried in an oven at 65 °C for 48 h. Feed samples were stored at −20 °C until further analysis. The determination of CP, NDF, ADF, Ca, and P in  Likewise, the levels of interleukin-10 (IL-10), interleukin-12 (IL-12),

| Amino acid profile of intestinal mucosa and muscle
After goat slaughter, the jejunal and ileal mucosa, and longissimus dorsi were immediately separated, and washed with precooled PBS (0.85% NaCl, 1.4 mM KH 2 PO 4 , 8 mM Na 2 HPO 4 , pH 7.4), and quickly put into liquid nitrogen and stored at −20°C until analysis. Amino acids in the intestinal mucosa and longissimus dorsi were determined as described elsewhere (Zhang et al., 2013). An L-8800 automatic amino acid analyzer (Hitachi) was used to determine the hydrolyzed amino acid content of the jejunal, ileal mucosa, and longissimus dorsi.

| Immunity markers, amino acid transporters, and barrier genes mRNA expression
After slaughter, the jejunum, ileum, and longissimus dorsi were immediately cut (5 cm), flushed with chilled PBS, and snap-frozen in liquid nitrogen. Samples were stored at −80 °C until further analysis.
RNAiso Plus (TaKaRa, Dalian, Code No. 9108/9109) was used to extract total RNA from the jejunum, ileum, and longissimus dorsi, while DNase I (Thermo Fisher Scientific) was used to eliminate genomic DNA. The purity and concentration of the total RNA were measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific).
Total RNA (1 μg total RNA was reverse transcribed to cDNA in a 20μl system using the Evo M-MLV RT Kit (Accurate Biology11706, Changsha, China) following the manufacturer's instructions.
Real-time quantitative polymerase chain reaction (qPCR) was performed using SYBR Premix Ex Taq II (Takara) on an ABI-7900HT qPCR system (Applied Biosystems) with β-actin as the housekeeping gene. All primers were synthesized by Sangon Biotech. The primer sequences of target genes are shown in Table S1. The relative expression of the target gene mRNA was calculated using the 2-ΔΔCT method (Livak & Schmittgen, 2001).

| Immunohistochemical analysis of claudin1 in the intestinal mucosa
Immunohistochemistry was performed as previously described by Tian et al (Tian et al., 2020). Specifically, the slides were dewaxed sequentially, endogenous peroxidase was removed, antigen-binding sites were exposed, and sections were exposed and permeabilized according to a previously described technique.

| Statistical analysis
All data were subjected to statistical analysis using SAS (version 9.4; SAS Inc.) with an independent-sample Student's t-test. A pvalue <.05 was considered a statistically significant difference. All visualizations were performed using GraphPad Prism 8 (GraphPad Software).

| TTO supplementation could alter cytokine concentrations in goat serum and ileal mucosa
The addition of TTO to the diet significantly reduced (p < .05) the content of TNFα and increased (p < .05) IL-2 in goat serum ( Table 2).
The levels of IL-1β, IL-4, IgG, IgM, and IgA were not affected (p > .05) by the addition of TTO (Table 2). As illustrated in Table 3, adding TTO to the diet significantly increased (p < .05) the IL-2 content in the ileal mucosa of goats. TTO supplementation significantly decreased (p < .05) the IL-12 content in the goat ileal mucosa. TTO significantly increased (p < .05) the sIgA content in the goat jejunal and ileal mucosa. However, the levels of TNFα, IL-1β, IL-10, and IFNγ in the goat jejunal and ileal mucosa were unaffected (p > .05) by TTO supplementation.

| TTO improves intestinal immunity by increasing the expression of SLC1A1, SLC7A1, and Claudin-1 in ileal mucosa
As shown in Table S3, adding TTO to the diet had no significant effect (p > .05) on the amino acid content of the jejunal mucosa. TTO supplementation significantly increased (p < .05) Ser, Arg, and TAA content in the ileal mucosa ( Figure 1a). The jejunal amino acid sensor receptors and transporters were not affected (p > .05) by TTO supplementation (Table S4). Compared to the CON group, the addition of TTO significantly enhanced (p < .05) the expression of SLC1A1 and SLC7A1 in the ileum (Figure 1b). TTO supplementation significantly increased (p < .05) the mRNA expression of Claudin-1 in the ileal mucosa but did not affect (p > .05) the expression of TJP1 and Mucin2 (Figure 1c). Immunohistochemical staining showed that TTO significantly enhanced (p < .05) the protein expression of Claudin-1 in the ileal mucosa (Figure 1d,e). The mRNA expression of TNFα, IL-1β, IL-2, IL-10, IL-12, and IFNγ in the goat jejunum mucosa was not affected (p > .05) by TTO supplementation ( Figure S1). TTO supplementation significantly upregulated (p < .05) the mRNA expression of IL-2 and downregulated the mRNA expression of IL-12 in the ileal mucosa ( Figure 1f).

| Amino acid profile and the expression of genes related to protein synthesis and degradation of goat longissimus muscle
The amino acid profile of the goat muscle was not affected (p > .05) by TTO addition (Table 4). As shown in Abbreviations: IFNγ, Interferonγ; IL-10, Interleukin 10; IL-12, Interleukin 12; IL-1β, Interleukin-1β; IL-2, Interleukin 2; sIgA, Secretory immunoglobulin A; TNFα, Tumor necrosis factorα. Amino acids are the building blocks of protein synthesis and act as regulatory factors that participate in the immune response (Broer, 2008). Most amino acids are metabolized in the intestine, and intestinal epithelial cells transport dietary amino acids into epithelial cells to synthesize purines, pyrimidines, and polyamines (Dan et al., 2015). Animal intestines are highly sensitive to changes in amino acids. Sufficient amino acids are essential for intestinal maintenance of mucosal integrity and immune function. Amino acids regulate the proliferation and differentiation of intestinal epithelial cells (Dan et al., 2015) and affect the intestinal epithelium's barrier function (DeMarco et al., 2003). In this study, the addition of TTO to the diet significantly increased Ser, Arg, and total amino acids in the ileal mucosa of goats. It has been reported that Ser and Arg play a critical role in maintaining intestinal integrity and regulating intestinal immune function (Qiao et al., 2005;Zhou et al., 2018;Zhu et al., 2013).
Thus, dietary supplementation with Ser can prevent intestinal dysfunction (Zhou et al., 2018). Thus, dietary supplementation with Ser can prevent intestinal dysfunction. Hydroxymethyltransferase is present in the intestinal mucosa and converts Ser into Gly. N5-N10 methyl enetetrahydrofolate is produced in this process, essential for synthesizing pyrimidine and purine (Metcalf et al., 2018). Arg participates in various nutritional and physiological processes in animals and is widely believed to promote the production of NO, proline, and polyamines in the animal body (Wu & Meininger, 2000). Therefore, the increase in Ser and Arg contents in the intestinal mucosa may be beneficial to the immune function of the intestine (Figure 2).
Amino acids cannot freely pass through the cell membrane because of their polarity, and also because they require the corresponding transport carriers to enter the cytoplasm (Verrey et al., 2005).
Amino acid transporters in the mammalian intestine can be divided into anion-amino acid transporters (SLC1A1, SLC1A2, and SLC1A3), neutral amino acid transporters (SLC1A4, SLC1A5, SLC7A5, and SLC7A10) and cationic amino acid transporters (SLC7A1, SLC7A2, and SLC7A3) (Broer, 2008). Our research showed that the amino acid transporters mRNA expression in the jejunal was not affected by TTO supplementation, which is consistent with our jejunum mucosal amino acid profile data. However, the addition of TTO to the goat diet significantly enhanced the mRNA expression of SLC1A1 and SLC7A1 in the ileum. Glu plays a vital role in enteral nutrition, cell signal transduction, and anti-inflammatory response (Lee et al., 2007;Riazi et al., 2003;Wu, 2010). Asp and Glu are the specific substrates of SLC1A1 in the small intestine. SLC1A1 transports Asp and Glu from the intestinal lumen to the intestinal epithelial cytoplasm (Ye et al., 2016). Although our ileal mucosal amino acid profile data showed that the Asp and Glu contents in the ileal mucosa of the TTO group were only numerically higher than those of the CON  Heller et al., 2005;Kinugasa et al., 2000;Kucharzik et al., 2001). In the present study, TTO significantly increased the expression of Claudin-1 in the ileal mucosa of goats, suggesting that tea tree oil can improve the intestinal barrier and immune function.
Yong et al (Yong et al., 2022) found that TTO could enhance intestinal barrier function by increasing the expression of Claudin-1 in the intestines of mice, which is similar to that reported in the current study ( Figure 2).
The content and composition of amino acids in muscle are essential indicators for evaluating meat quality and flavor (Wood et al., 1996) . In animals, muscle protein turnover F I G U R E 2 Schematic diagram for tea tree oil improving intestinal immunity by enhancing amino acid transport in the ileal mucosa of goats. Tea tree oil upregulated the expression of SLC1A1, SLC7A1 and Claudin-1 in the ileal mucosa and increased the content of serine and arginine in the mucosa. Increased serine and arginine content elevated IL-2 and sIgA content in the ileal mucosa and improved intestinal immunity in goats (protein synthesis and decomposition) is a critical factor affecting growth performance, but the effect of TTO on protein turnover in muscle tissue is still unknown. The FOXO family is a transcriptional regulatory factor. FOXO1 and FOXO3 in muscle tissue are related to protein degradation (Goodman et al., 2011). Our study showed that the expression of mTOR, S6K1, 4EBP1, FOXO1, and FOXO3 in goat muscle was not affected by TTO supplementation. This indicates that tea tree oil had no adverse effect on goat muscle growth and protein metabolism.

| CON CLUS IONS
In summary, adding 0.2 ml/kg of TTO to goat diets can enhance the expression of SLC1A1 and SLC7A1 in goat ileal mucosa, thereby increasing the content of Ser and Arg in the ileal mucosa, which has a beneficial effect on goat intestinal immunity. TTO upregulates IL-2 and Claudin-1 in the ileal mucosa to protect intestinal health.

ACK N OWLED G EM ENTS
The

CO N FLI C T O F I NTE R E S T
The authors declare that no conflict of interest exist.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data may be provided following a request to the corresponding author.

E TH I C A L A PPROVA L
The study received the approval of the institutional Animal Care . Impacts of tea tree or lemongrass essential oils supplementation on growth, immunity, carcass traits, and blood biochemical parameters of broilers