The influence of plant essential oil/palygorskite composite on growth performance, blood parameters and intestinal morphology of broiler chickens

Abstract The purpose of this experiment was to use a single-factor design to investigate the effects of different supplementation levels of plant essential oil/palygorskite composite (EPO-Pal) on growth performance, blood parameters, and intestinal morphology of broiler chicken. A total of 960 one-day-old chicks were randomly assigned into 4 treatments with 6 replicates of 40 chicks each. The broilers were fed the basal diet supplemented with 0, 500, 750, and 1000 mg/kg EPO-Pal for 42 days, respectively. The results showed that the 500 or 750 mg/kg groups significantly increased the body weight, average daily feed intake and average daily gain of the broilers at 28 and 42 days (p < 0.05). Additionally, as compared to the control group, the 1000 mg/kg EPO-Pal group exhibited higher levels of immunoglobulins-A, immunoglobulins-G, immunoglobulins-M, and high-density lipoprotein cholesterol while having lower levels of tumour necrosis factor-α (p < 0.05). The content of interleukin-2 and the villus height-to-crypt depth ratio were significantly increased in the 750 mg/kg EPO-Pal group (p < 0.05). Moreover, the activity of total antioxidant capacity and superoxide dismutase were significantly enhanced in the 500 mg/kg EPO-Pal group (p < 0.05). The results are very promising, and EPO-Pal can be considered as an additive to promote the growth by enhancing immunity, antioxidant activity, and improving the intestinal morphology of broiler chickens. HIGHLIGHTS Dietary supplementation of EPO-Pal improved the growth performance by increased average daily feed intake of broiler chickens. Dietary supplementation of EPO-Pal enhanced plasma antioxidation and immune response of broiler chickens. Dietary supplementation of EPO-Pal can postively affect the intestinal morphology of broiler chickens.


Introduction
Antibiotic growth promoters or in-feed antibiotics have been widely used in animal production since 1946 when they were discovered to promote animal growth (Moore et al. 1946).However, issues such as antibiotic residues and drug resistance have garnered increased attention from consumers.In the interest of food safety, the European Union (EU) banned the use of antibiotics as early as 2006, and China also implemented a ban on antibiotics since 2020 (http://www.moa.gov.cn/gk/,accessed on January 6th, 2020).Consequently, there has been a growing demand for safe, efficient, and residue-free growth-promoting feed additives in animal husbandry (Landy et al. 2020;Landy and Kheiri 2023).
Plant essential oil (PEO) refers to the extraction of plant secondary metabolites from plant tissues and organs, which contain a large number of aldehydes, ketones, esters, terpenes, phenols and phenylpropanoids (Dhifi et al. 2016;Perumal et al. 2022).PEO have been widely used because of its effects promoting growth (Tiihonen et al. 2010), anti-inflammatory (Luna et al. 2019), antioxidant activity (Dhifi et al. 2016), and antibacterial properties (Bora et al. 2020).As green and natural additives, numerous studies have proven the safety and efficiency of PEO in livestock production, including pigs (Long et al. 2021), poultry (Ding et al. 2022), ruminants (Palhares et al. 2021), among others.However, PEO cannot be used as a feed additive for production because of its volatility, instability, and difficulty preservation (Sharma et al. 2019;Shlosman et al. 2022).Therefore, there is a need for effective protective materials to facilitate its application.
Palygorskite (Pal) is a hydrous magnesium-rich aluminium silicate clay mineral, which has been approved by the EU as a carrier of feed additive since 2003 due to its characteristics of good thermal stability, acid resistance, and adsorptive capacity (Gal� an 1996; Murray 2000).By modifying its charge, altering surfactant groups, and employing other processes to load PEO onto Pal, PEO can be effectively preserved and utilised (Lei et al. 2017;Ghrab et al. 2018).Our previous research has shown that supplementing 750 mg/ kg PEO-Pal improved egg production by enhancing antioxidative and immune functions and altering the intestinal morphology of laying hens (Cheng et al. 2022).We speculate that similar effects may be observered in broiler chickens, such as improving the growth performance thereby enhancing economic benefits of broilers.
'Yunwu No.1' is a high-quality broiler chicken breed produced by crossbreeding Xuefeng black-bone chickens with fast-growing broilers.It combines the flavour characteristics of traditional Chinese chicken breeds with the rapid growth rates commonly seen in commercial broilers.This makes it of significant economic and research importance.Consequently, this study was designed to investigate the effects of different levels of PEO-Pal on the growth performance, blood parameters, and intestinal morphology of broiler chickens.

The PEO-Pal
Plant Essential oil/palygorskite composite (PEO-Pal) was supplied by Jiangsu Sinitic Biological Technology Co., Ltd (Jiangsu, China).The preparation method for PEO-Pal was consistent with the procedure previously described in our study (Cheng et al. 2022).The main components of PEO-Pal consistent of 11% PEO (including carvacrol and thymol) and 89% palygorskite.

Animals, experimental design, and management
All the birds and the experimental protocols were approved by the Institutional Animal Care and Use Committee of Hunan Agricultural University, Hunan, China.The 'Yunwu No.1' broiler chicken were sourced from Hunan Yunfeifeng Agricultural Commercial Company (Hunan, China).This experiment utilised a single-factor experimental design.A total of 960 oneday-old healthy female broiler chicks, each with a similar weight (33.80 ± 0.21), were randomly and evenly divided into 4 treatment groups, with 6 replicates consistenting of 40 chicks in each.The dietary treatments were as follows: (1) basal diet; (2) basal diet þ 500 mg/kg PEO-Pal; (3) basal diet þ 750 mg/kg PEO-Pal; (4) basal diet þ 1000 mg/kg PEO-Pal.The PEO-Pal was incorporated into the feed through gradual mixing, and the composition and ingredients of the diets are detailed in Table 1.The experimental diets were formulated to meet the nutrient requirements of the birds according to the China Agricultural Standard (NY/T 33-2004).
Each replicate consisted of 8 cages, with 5 chicks raised in each cage (50 � 50 � 36 cm; length-� width � height).All the chicks were fed their respective experimental diets for a duration of 42 days.The chicks were fed twice a day (06:30 am and 2:30 pm) and given ad libitum access to water throughout the experiment.The temperature in the chicken coop was 33 � C for the first three days, and then gradually decreased 1 � C every three days until the final temperature reaches 20 � C. The lighting regimen used was 24 h light throughout the entire experimental period.Immunisation procedures are carried out according to company standards.

Sample collection
At the 22 d and 43 d of the experiment, 12 chicks (2 chicks per replicate) were randomly selected from each treatment after fasting for 12 h.Blood samples (3 mL) were obtained via the jugular vein using the vacutainer tube.Blood samples were placed at room temperature for 1 h, subsequently centrifuged at 3000 � g for 10 min to separate plasma, and plasma were stored in 1.5 mL Eppendorf tube at -20 � C for further analysis.After collected blood samples, the chicks were exsanguinated and dissected to obtain immune organ (include spleen and bursa of Fabricius).And immune organ index is obtained by the weight of the immune organ divided by live weight of the bird.And the duodenum, jejunum and ileum were taken 2 cm, washed with normal saline and fixed in 4% paraformaldehyde for intestinal morphology measurements.

Growth performance
The chicks were weighed on 1 d, 15 d, 29 d, and 43 d respectively, and recorded the feed intake at the same time.To ensure data credibility, mortality and health status were visually recorded daily.The growth performance index including body weight (BW), average daily feed intake (ADFI), average daily gain (ADG), and feed/gain ratio (F/G ratio).

Intestinal morphology
The intestinal tissue fixed samples were embedded in paraffin, sectioned (4 mm), followed by hematoxylineosin staining.Morphological measurements were performed on 10 villi chosen from each segment by CaseViewer Image analysis software, and measurements of the villus height (VH) and crypt depth(CD) were taken and the villus height-to-crypt depth ratio (VH/CD) were calculated subsequently.

Statistical analysis
All data were analysed by one-way analysis of variance (ANOVA) using SPSS 22.0 statistical software (SPSS Institute Inc., Chicago, Illinois), and the statistical differences among the groups were determined by Duncan's multiple range test, and the replicate was used as the experimental unit.The results are expressed as arithmetic mean ± standard deviation, p < 0.05 was considered statistically significant.

Growth performance
As presented in Table 2, the BW of 500 and 750 mg/ kg PEO-Pal groups showed a significant increase compared with the control group at 28 and 42 days, respectively (p < 0.05).The ADFI of the PEO-Pal groups and the ADG of 500 mg/kg PEO-Pal group were significantly improved compared to the control group during the day15-28 (p < 0.05).At 29-42 days, the ADFI of 750 mg/kg PEO-Pal group and the ADG of 500 and 750 mg/kg PEO-Pal groups were significantly increase compared with the control group (p < 0.05).And throughout the entire experimental period, the ADFI of the PEO-Pal groups and the ADG of 500 and 750 mg/kg PEO-Pal groups were significantly increase compared with the control group (p < 0.05).

Plasma immune response
The effects of dietary PEO-Pal on plasma immune response results are reported in Table 4.At day 21, the concentration of IgA and IgM in 1000 mg/kg PEO-Pal group was significantly higher than that in the control group (p < 0.05), while the concentration of TNF-a was significantly lower than that in the control group (p < 0.05).At day 42, compared with the control group, the concentration of IgA and IgG in 1000 mg/ kg PEO-Pal group was significantly increased (p < 0.05) and the concentration of TNF-a in 1000 mg/kg PEO-Pal group was significantly decreased (p < 0.05),   additionally, the content of IL-2 in 750 mg/kg PEO-Pal group was significantly improved (p < 0.05).

Plasma antioxidant capacity
The results of plasma antioxidant capacity are presented in Table 5.There was no significant difference in plasma antioxidant capacity among the 4 groups at 21 days (p > 0.05).However, during day 42, the content of T-AOC and SOD in 500 mg/kg PEO-Pal group was significantly higher than the control group (p < 0.05).

Plasma biochemical indexes
The results of plasma biochemical indexes in broiler chick are presented in Table 6.Compared with the control group, the content of HDL-C was significantly higher in the 1000 mg/kg PEO-Pal group at 42 days (p < 0.05).

Intestinal morphology
The results of broiler chick intestinal morphology are presented in Table 7.The VH/CD of the jejunum in 750 mg/kg PEO-Pal group was significantly higher than that in the other groups (p < 0.05).There were no significant differences in VH and CD of all intestinal segments among all the groups (p > 0.05).moreover, the VH/CD of the duodenum and ileum did not show significant difference among the 4 groups (p > 0.05).

Discussion
While numerous studies have demonstrated the growth-promoting properties and safety of plant essential oils (PEO), research results often vary due to differences in the type and dosage of PEO, as well as the breed and age of animals.China, in particular, boasts a rich variety of resources, and there may be different effects when supplementing the diet with PEO in different breeds.Our previous research has also indicated that PEO is suitable for Chinese local chicken breeds (Liu et al. 2020), but there are no report in 'Yunwu No.1' broiler chicken.Based on this, we hypothesise that PEO-Pal could effectively improve broiler chicken production performance, but the specific mechanism is not very clear.So we conducted further investigations to understand the functional mechanisms of PEO-Pal by evaluating its effects on immune response, antioxidant capacity, and intestinal morphology.
Previous studies have demonstrated that plant essential oils primarily enhance the growth performance of animals through several mechanisms.Firstly, they can improve the palatability of feed, thereby increasing daily feed intake in animals (Clouard and Val-Laillet 2014).Secondly, plant essential oils can enhance nutrient utilisation by modulating the intestinal microflora and, consequently, improve digestive capabilities (Windisch et al. 2008;Bravo et al. 2014).Lastly, plant essential oils can regulate the secretion of growth-related hormones, thereby promoting animal growth (Ariza-Nieto et al. 2011;Li et al. 2012).In our study, we observed significant improvements in body weight, average daily gain, and average daily feed intake due to dietary supplementation with PEO-Pal.However, it's important to note that the results of our study differ from some previous research.For instance, Xue et al. (2020) reported that dietary supplementation with 100 mg/kg of mixed plant essential oil had no influence on the growth performance of broilers.On the other hand, Ding et al. (2022) found that supplementing the diet with 400 mg/kg of essential oils improved average daily gain and body weight while reducing the feed conversion ratio in chickens.Park and Kim (2018) also observed increased body weight gain and reduced feed conversion ratio in broiler chicks fed a diet containing 0.03% essential oils.These variations in results may be attributed to differences in dosage and the specific varieties of essential oils used in the studies The spleen and bursa of Fabricius are important immune organs in poultry, and they serve as indicators of the body's immune capability (Rivas and Fabricant 1988).The bursa of Fabricius plays a unique role in avian immune function, primarily contributing to humoral immunity.The spleen, being the largest peripheral immune organ in poultry, also plays a crucial role in immune responses.The weight of these immune organs is closely linked to their cell proliferation rates and their overall contribution to the body's immune function (Hassan et al. 2010).Several previous studies have reported that dietary supplementation with plant essential oils has no significant impact on the immune organs of animals (Kim et al. 2016;Wang et al. 2019;Oladokun et al. 2021).Our findings in this study align with these previous research results.Specifically, we observed no significant differences in immune organ indices among all experimental groups at both 21 and 42 days of age.
However, the results of the plasma immune response tests indicated that PEO-Pal can enhance the immune competence of chickens.Immunoglobulins play a crucial role in the immune system as they regulate the inflammatory response, and their levels serve as important indicators of immune function (Magnad� ottir 2006;Elghandour et al. 2020).Among the key immunoglobulins, IgA primarily contributes to mucosal immunity, IgG plays a central defensive role throughout the inflammatory response due to its longer half-life, and IgM exhibits the fastest response and plays a pivotal role in early inflammation (Elghandour et al. 2020;Kim et al. 2022).
Our study indicated that dietary supplementation with 1000 mg/kg of PEO-Pal had a positive impact on the concentrations of IgA, IgG, and IgM.Furthermore, in comparison to the control group, the 1000 mg/kg PEO-Pal group showed a significant reduction in TNFa levels, while the 750 mg/kg PEO-Pal group exhibited a remarkable increase in IL-2 levels.Cytokines encompass both pro-inflammatory factors, such as TNF-a, and anti-inflammatory factors, like IL-2.As intercellular signalling molecules, the primary role of cytokines in the immune system is to mediate and regulate immune responses (Hou et al. 2015).Previous studies have demonstrated that PEO can enhance an animal's immune response by increasing immunoglobulin levels and the presence of anti-inflammatory factors, while inhibiting the activity of pro-inflammatory factors (Liu et al. 2019;Sivandzade et al. 2019;Su et al. 2021).Our research also proves this point.
Oxidative stress refers to an increase in the generation of free radicals in the body or a decrease in its ability to scavenge them, which caused accumulation of free radicals and then result in oxidative damage (Sohal and Allen 1990).Oxidative stress is also a significant factor contributing to the reduction in animal growth performance and the quality of animal products.In this paper, we observed that the group supplemented with 500 mg/kg PEO-Pal showed an increase in the activity of T-AOC and SOD in the plasma of broiler chickens compared to the control group.T-AOC represents the total antioxidant capacity of the body when exposed to external stimuli (Siegel and Latimer 1984).SOD and GSH-Px are the primary antioxidant enzymes.SOD activity reflects the body's ability to scavenge free radicals, and its primary function is to catalyse the disproportionation reaction of superoxide anions and free radicals, thereby protecting cells and tissues from damage (Giardino et al. 2002).GSH-Px, an enzyme that catalyses the decomposition of hydrogen peroxide, helps maintain the integrity of cell membrane structure and function (Surai et al. 2018).Previous studies have shown that dietary supplementation with essential oils can significantly increase T-AOC, SOD, and GSH-Px activities in the plasma, jejuna, and ileal mucosa of chickens (Ruan et al. 2021).Other studies by Zhang et al. (2021) and Su et al. (2018) have also demonstrated that dietary essential oils can enhance the activity of T-AOC, SOD, and GSH-Px in chicken plasma.PEO enhance the antioxidant function of plasma through two aspects, on the one hand, the phenolic hydroxyl group of PEO can bind to peroxy radicals, thereby alleviating oxidative stress (Brenes and Roura 2010;Asghari et al. 2021), on the other hand, PEO can influence the activity of antioxidant enzymes by regulating nuclear factor erythroid-2 related factor2 (Meeran and Prince 2012; Kang et al. 2015).
Plasma biochemical indices are important markers for assessing the health status and physiological metabolism of an organism, and they can be influenced by a variety of factors (Vicari et al. 2008).Hesabi et al. (2019) found that a blend of essential oils significantly altered the levels of cholesterol, aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase, while not affecting glucose, triglycerides, HDL, and LDL.Ibrahim et al. (2019) demonstrated that essential oils improved dyslipidemia, leading to significant reductions in total cholesterol, triacylglycerol, and low-density lipoproteincholesterol, along with an increase in high-density lipoprotein-cholesterol.And Maral et al. ( 2022) also demonstrated that essential oils significantly increased the content of HDL-C and decreased the content of LDL-C in athlete plasma.In our paper, the addition of 1000 mg/kg PEO-Pal to the diet significantly increased the plasma concentration of HDL-C in broiler chickens.The differences in our results compared to previous studies may be attributed to factors such as the chicken breed, nutritional composition of the diet, dosage of the additive, or other variables.Previous research has suggested that thymol and carvacrol, two components of essential oils, can promote adipocyte metabolism, reduce sterol regulatory element-binding protein-1c levels, and inhibit lipid accumulation (Saravanan and Pari 2016;Choi et al. 2017).HDL-C is considered a beneficial form of cholesterol due to its ability to reduce overall cholesterol levels and its antiinflammatory, antioxidant, and anti-apoptotic properties (Rosenson et al. 2016).
Intestinal morphology plays a crucial role in the process of digestion and absorption, and indicators such as villus height (VH), crypt depth (CD), and the VH/CD ratio are commonly used to assess intestinal structure.The VH/CD ratio, in particular, is positively correlated with the digestive and absorptive functions of the intestine (Dubrovsky and Dunn 2018;Mazzoni et al. 2022).Du et al. (2016) reported that dietary supplementation with thymol and carvacrol improved the villus height and VH/CD ratio in the ileum of broilers.Hesabi et al. (2019) demonstrated that diets containing 150 or 200 mg/kg of essential oils could increase the ileal villus height and VH/CD ratio in broilers.Furthermore, our previous study showed that the addition of 750 mg/kg PEO-Pal to the diet improved the VH/CD ratio in the jejunum of laying hens (Cheng et al. 2022).In this study, we observed that the VH/CD ratio in the jejunum of the 750 mg/kg PEO-Pal group was significantly higher than in the other groups.Numerous studies have provided evidence that PEO can improve the intestinal health of animals.PEO helps maintain a balanced intestinal microbiota, preventing the destructive effects of pathogenic bacteria on the intestine, and preserving the architecture of intestinal epithelial cells.Meanwhile, its antioxidant function can promote the growth of the intestinal villi (Castillo et al. 2006;Jang et al. 2007;Yang et al. 2014).
In conclusion, dietary supplementation with PEO-Pal can improve growth performance by boosting immunity and antioxidant activity while also positively affecting intestinal morphology in broiler chicken.Our findings suggest that dietary supplement with 500 mg/kg PEO-Pal during days 1-28 and increase the dosage of PEO-Pal to 750 mg/kg on days 29-42, which may obtain optimal growth performance in broiler chickens.This study provides a theoretical basis for the scientific use of PEO-Pal.

Table 1 .
Composition and nutrient levels of the experimental diets (dry matter basis).

Table 2 .
Effects of Plant Essential oil/palygorskite composite (PEO-Pal) on growth performance of broiler chicken 1 .Data represent the means of 6 replicates per group with 40 chick per replicate.Values are expressed as means ± SD.Means in the same row assigned different lowercase letters are significantly different (p < 0.05). 2 Body weight (BW); Average daily feed intake (ADFI); Average daily gain (ADG); Feed/Gain ratio (F/G ratio).

Table 3 .
Effects of Plant Essential oil/palygorskite composite (PEO-Pal) on immune organ index of broiler chicken 1 .Data represent the means of 6 replicates per group with 2 chick per replicate.Values are expressed as means ± SD.Means in the same row assigned different lowercase letters are significantly different (p < 0.05).

Table 4 .
Effects of Plant Essential oil/palygorskite composite (PEO-Pal) on plasma immune indexes of broiler chicken 1 .Data represent the means of 6 replicates per group with 2 chick per replicate.Values are expressed as means ± SD.Means in the same row assigned different lowercase letters are significantly different (p < 0.05).

Table 5 .
Effects of Plant Essential oil/palygorskite composite (PEO-Pal) on plasma antioxidant capacity of broiler chicken 1 .Data represent the means of 6 replicates per group with 2 chick per replicate.Values are expressed as means ± SD.Means in the same row assigned different lowercase letters are significantly different (p < 0.05).2T-AOC: total antioxidant capacity; SOD: superoxide dismutase; GSH-Px: glutathione peroxidase.

Table 6 .
Effects of Plant Essential oil/palygorskite composite (PEO-Pal) on plasma biochemical indexes of broiler chicken at 42 day 1 .Data represent the means of 6 replicates per group with 2 chick per replicate.Values are expressed as means ± SD.Means in the same row assigned different lowercase letters are significantly different (p < 0.05).

Table 7 .
Effects of Plant Essential oil/palygorskite composite (PEO-Pal) on intestinal morphology of broiler chicken at 42 day 1 .Data represent the means of 6 replicates per group with 2 chick per replicate.Values are expressed as means ± SD.Means in the same row assigned different lowercase letters are significantly different (p < 0.05).