Assessment of Lavandula angustifolia L. essential oil as a natural feed additive on broiler chicken’s growth, blood physiological markers, immunological status, intestinal histomorphology, and immunoexpression of CD3 and CD20

Abstract

Lavender essential oil (LVEO) (Lavandula angustifolia L) was investigated for its impacts on broiler chicken growth, intestinal histomorphology, blood biomarkers, and immunological status. Four hundred 3-days-old male chicks (ROSS 308 broilers) (91.47 ± 0.15 g) were randomised to receive four test groups and nourished on basal diets having four levels of LVEO: 0, 200, 400, and 600 mg Kg⁻¹ over 35 days. Dietary addition of LVEO linearly increased the body weight and weight gain during the grower period, while its addition did not affect the overall growth performance. Dietary LVEO increased the villous height, and the higher villous height was detected in the LVEO600 group. Dietary LVEO increased cluster of differentiation (CD3 and CD20) immunoexpression within the spleens of birds given LVEO400 and LVEO600 diets. Serum triiodothyronine and thyroxine hormone levels were the highest in the LVEO400 and LVEO600 groups. LVEO supplementation raised serum growth hormone levels in a dosage-dependent manner. The serum concentrations of glucose and leptin hormone did not vary significantly between the LVEO-supplemented and LVEO0 groups. All levels of supplemental LVEO diminished serum cholesterol, triglyceride, and low-density lipoprotein cholesterol levels, while raising the high-density lipoprotein cholesterol concentration. Total protein values were augmented in all LVEO groups, with the highest concentrations of albumin and globulin in the LVEO600 group, followed by the LVEO400 group. Supplemental LVEO increased serum concentration of complement 3, interleukin-10, and lysozyme activity in a level-dependent manner. Modifications in the birds’ physiological activities, blood biochemical, intestinal morphological characteristics, and immunological status support the conclusion that dietary LVEO supplementation had numerous positive effects. However, it didn’t influence the birds’ growth.

HIGHLIGHTS

• LVEO supplementation linearly increased the body weight during the grower stage, while its addition did not affect the overall growth performance.

• LVEO supplementation had a hypolipidemic effect, and the LVEO400 and LVEO600 groups showed the highest serum triiodothyronine and thyroxine hormone levels.

• LVEO supplementation (200-600 mg Kg⁻¹ diet) improved the immune status of birds, indicated by increased serum concentration of complement 3, interleukin-10, and lysozymes activity with increased CD3 and CD20 immunoexpression in the spleens of LVEO400 and LVEO600 groups.

Keywords:

• Broilers
• lavender essential oil
• growth
• intestinal histomorphology
• immune status
• clusters of differentiation

Introduction

According to the Organisation of Food and Agriculture (mFAO 2021), global poultry meat production in 2022 is more than 138.8 million tonnes. Regarding human nutrition, the poultry industry offers healthier and less expensive substitutes for red meat alongside additional protein sources (Obaid Ul et al. 2022).

Even though several nations are attempting to limit the utilisation of antibiotic growth promoters (AGP) in feed ingredients, they are still ubiquitously employed in the world poultry industry to boost gut healthiness and growth performance (Hashem et al. 2021). Continuous and excessive AGP use is thought to reduce AGP efficacy and put public health at risk by attempting to spread antibiotic-resistant microbes and gene mutations in the poultry sector (Yang et al. 2015). So, there is a great need in the poultry industry for an effective AGP replacement.

Herbal extracts and essential oils (EOs) are increasingly marketed as immune and digestive enhancers in animal feeds. Animal nutrition applications have been neglected for a long time, but they have recently regained popularity because of the prohibition of antibiotics in livestock diets (Abdelhadi and Abd El-Wahab 2022). Supplementation with EOs has been researched and implemented to boost animal gut wellness and growth performance (Choi et al. 2020). EOs have many biological impacts, including hypercholesterolaemia, flavour enhancement, digestion stimulation, antiviral, antimycotic, antiparasitic properties, odour inhibition, and ammonia control (Abdelhadi and Abd El-Wahab 2022).

Lavandula angustifolia, a blooming plant species within the Lamiaceae family broadly developed within the Mediterranean locale, is commonly used to create lavender essential oil (LVEO). The monoterpenoids linalool, camphor, linalyl-acetate, terpinen-4-ol, and 1,8-cineole are abundant in LVEO, extracted via steam distillation from the plant’s flowers and leaves (Sandner et al. 2020; Amer et al. 2022). It also has a high concentration of polyphenolic compounds, for example, such as flavonoids, which are capable of a wide variety of biological functions, including destroying free radicals (Rabiei et al. 2014; Amer et al. 2022).

Because of its bioactivities and appealing odour, LVEO is frequently employed in the food trade (Danh et al. 2013). Among the most crucial aspects of LVEO is its antimicrobial properties that incorporate antibacterial, nematicidal, antifungal, anti-mite, and antiviral activity (Kot et al. 2018; Adaszyńska-Skwirzyńska et al. 2021). LVEO even has therapeutic activities such as antioxidative, anti-depressant, anti-inflammatory, analgesic, tranquilising, hypnotic, and anti-carcinogenic activity, which has led to its use in drugs and cosmetics (Ouedrhiri et al. 2017).

This aromatic plant seems to have a long and storied history of being used in herbal medicine as a natural remedy to treat inflammatory conditions. LVEO governs the inflammatory reaction by blocking macrophage production of several bacterial-induced proinflammatory mediators, particularly interleukin-1α (IL-1α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) (Sandner et al. 2020). Aromatherapy massage with lavender, cypress, and sweet marjoram oil blend significantly increased blood lymphocytes in normal individuals, particularly the increment in a cluster of differentiation 8 (CD8) T cells and a cluster of differentiation 16 (CD16) cells. These findings imply that LVEO therapies can improve immune function and lower stress (Kuriyama et al. 2005).

Still, there has been minimal research on the impact of LVEO supplementation on broiler chicken performance and immune status. Few studies have been performed to investigate the action mechanism and paths through which LVEO may exert its health benefits in broiler chickens. As a result, this research aimed to see how LVEO implementation to the basal diet affected broiler growth performance, intestinal architecture, physiological blood markers, and immunological status.

Materials and methods

Gas chromatography-mass spectrometry (GC-MS) analysis of LVEO

To assess and evaluate the chemical composition of LVEO, a Trace GC1310-ISQ mass spectrometer (Thermo Scientific, Austin, TX, USA) with a specific capillary column TG-5MS (30 m 0.25 mm 0.25 m film thickness) was used. The column temperature controller was maintained at 50 °C, then continued to increase at a rate of 5 °C per min to 230 °C for 2 min, then continued to increase at a rate of 30 °C per min to 290 °C, and then decided to hold for 2 min. The injector and MS transfer line temperatures were held at 250 and 260 °C, respectively, and helium was employed as a carrier gas with a constant flow rate of 1 mL/min. The eluent wait time was 3 min, and 1 L diluted samples were implanted instantly using an Autosampler AS1300 in split mode coupled with GC. EI mass spectra spanning m/z 40–1000 were obtained in full scan mode at 70 eV ionisation energy voltages. The heat of the ion source was fixed to 200 °C. The elements were recognised by comparing their retention times and mass spectra to those of the WILEY 09 and NIST 11 mass spectral databases, as previously reported by Amer et al. (2022).

Birds, diet preparation, and experimental protocol

This study occurred in a poultry research centre at Egypt’s Zagazig University’s Veterinary Medicine Faculty. All experiment operations were certified by the ZU-IACUC committee (approval number ZU-IACUC/2/F/152/2022). A total of 400 one-day-old male Ross 308 broiler chicks were bought from a commercial hatchery (Dakahlia Poultry, Mansoura, Egypt). Before the experiment, the birds underwent a 3-day acclimation period, gaining a typical body weight of 91.47 ± 0.15 g. They were then randomly placed into one of four experimental groups, each with ten replicates (10 chicks per replicate). The birds consumed basal diets containing LVEO (lavender essential oil, ORGANIC EGYPT, Cairo, Egypt) at four different levels: 0, 200, 400, and 600 mg Kg⁻¹ (Supplementary Figure 1). LVEO was mechanically mixed with other feed ingredients. The experimental diets were fed to the birds throughout three feeding periods: starter (4^th–10^th day), grower (11^th–23^rd day), and finisher (24^th–35^th day) periods. The roughly elemental nutritional makeup of the main diet is listed in Table 1. Dietary trials and management concerns were performed in conformity with the nutrition-specific requirements for the Ross 308 broiler (AVIAGEN 2014). Chicks were raised in a well-ventilated open home along with sawdust bedding (7 birds/m²). The investigation continued for 35 days. The construction site temperature was kept at 34 °C for the first week and steadily dropped until it achieved 25 °C after the trial. The illumination regime was set to 23:1 h light/dark, then 20:4 h light/dark until the end of the experiment.

Table 1. The proximate chemical constituents of the basal diet as fed (g Kg⁻¹).

Ingredients	Starter	Grower	Finisher
Corn 7.5% CP	560	593	620
Soybean meal 47% CP	333.3	281	238.2
Corn gluten meal 60% CP	39.2	52.7	60.7
Oil (soya) - e76	22	30	40
Dicalcium phosphate 18%	15	14	13
Calcium carbonate	12	12	11
Sodium bicarbonate	2.5	2.5	2.5
Dl methionine 99%	4	3	3.3
Broiler premix*	3	3	3
L-LYSINE HCL 98%	4.6	4.5	4
Salt	1.5	1.1	1.5
Antimycotoxin	1	1	1
Choline 60 veg	0.7	0.7	0.7
L-Threonine 98.5%	1	1	1
Enzyme Phytase	0.05	0.05	0.05
Chemical analysis (%)
Moisture	11.15	11.16	11.09
ME poultry (kcal/kg)	3004	3100	3201
Crude protein	23.03	21.53	20.16
Lysine	1.46	1.31	1.16
Methionine	0.72	0.61	0.62
Calcium	0.94	0.90	0.83
Av. Phosphorus	0.48	0.44	0.42

* Premix per kg of diet: vitamin A (1 500 IU), vitamin D3 (200 IU), vitamin E (10 mg), niacin (35 mg), pantothenic acid (10 mg), riboflavin (3.6 mg), pyridoxine (3.5 mg), thiamine (1.8 mg), vitamin K3 (0.5 mg), folic acid (0.55 mg), cobalamin (0.01 mg), Fe (80 mg), Mn (60 mg), Zn (40 mg), Cu (8 mg), I (0.35 mg), Se (0.15 mg), biotin (0.15 mg). CP: crude protein.

Growth performance

Weighing the birds was performed on the 4th day of life to identify the average preliminary body weight; body weights were then recorded at 10, 23, and 35 days to assess the mean body weight of the birds in each cluster.

Equation (1)(1) $$\text{BWG }=\text{ W2 }-\text{ W1}$$(1) was employed to compute the body weight gain (BWG); (1) $$\text{BWG }=\text{ W2 }-\text{ W1}$$(1)

W2 is the intended period’s final body weight, and W1 is the initial body weight.

The average feed intake per bird was derived by subtracting the weight of the feed provided from the residues left in each replicate and dividing the result by the number of birds.

Equation (2)(2) $$\mathit{FCR}=FI\left(g\right)/\mathit{BWG}\left(g\right)$$(2) was involved in evaluating the feed conversion ratio (FCR) (2) $$\mathit{FCR}=FI\left(g\right)/\mathit{BWG}\left(g\right)$$(2)

Equation (3)(3) $$\mathit{RGR}=W2-W1/0.5\left(W1+W2\right)\times 100$$(3) was required to estimate the relative growth rate (RGR) (Brody 1945). (3) $$\mathit{RGR}=W2-W1/0.5\left(W1+W2\right)\times 100$$(3)

W1 is the preliminary live weight (g), and W2 is the live weight at the end of the time-conferred period (g).

Equation (4)(4) $$\text{PER }=\text{ Live weight gain }\left(\mathrm{g}\right)/\text{protein intake }\left(\mathrm{g}\right)$$(4) was utilised to analyse the protein efficiency ratio (PER) (McDonald et al. 1973). (4) $$\text{PER }=\text{ Live weight gain }\left(\mathrm{g}\right)/\text{protein intake }\left(\mathrm{g}\right)$$(4)

Sample collection

Blood samples were drawn randomly after the slaughter at the finale of the conferred feeding time (two birds per replicate, 20 birds per group). The euthanasia method applied to the chicks was cervical dislocation, as endorsed by the American Veterinary Medical Association (Association AVM 2013). The blood was taken without the addition of an anticoagulant, forced to allow coagulation to occur at room temperature, followed by centrifugation for fifteen min at 3000 rpm for serum separation before being deep frozen at −20 C until physicochemical analysis. Histomorphology tests were done on small intestine samples taken from various locations. On spleen tissue sections, CD3 and CD20 immunohistochemistry was conducted.

Blood physiochemical analysis

My Biosource Co., San Diego, CA, USA, chicken ELISA kits with CAT. NO., MBS265796, MBS269454, MBS266317, and MBS025331 were utilised to evaluate thyroxine (T4), triiodothyronine (T3), growth hormones (GH), and leptin, respectively, agreeing with the directions in the pamphlets.

An automated biochemical detector (Robotnik Prietest ECO Ambernath (W), Thane, India) was required to estimate the serum glucose concentrations (Trinder 1969).

Serum lipid and protein profiles

Spectrum-bioscience colorimetric diagnostic kits (Egyptian Company for Biotechnology, Cairo, Egypt) were involved in estimating serum total cholesterol (TC), triglycerides (TG), and high-density lipoprotein cholesterol (HDL-C), as previously described by Allain et al. (1974), McGowan et al. (1983), and Vassault et al. (1986), respectively. The low-density lipoprotein cholesterol (LDL-C) level was computed agreeing to the Iranian formula LDL-C = TC/1.19 + TG/1.9 – HDL-C/1.1 (Ahmadi et al. 2008). Griffin and Whitehead’s turbidimetric method measured very low-density lipoprotein cholesterol (VLDL-C) (Griffin and Whitehead 1982).

Total protein serum concentration was assessed consistent with Grant (1987). Serum albumin content was measured, agreeing with Doumas et al. (1981). Serum globulin levels were theoretically approximated by subtracting albumin values from total proteins (Doumas et al. 1972).

Immune indices

The level of serum interleukin-10 (IL-10) was assessed using MyBioSource Co. CAT.NO. MBS701683 chicken ELISA kits. Likewise, the serum complement 3 (C3) level was checked using an ELISA kit of CAT. NO. LS-F9287 (Life Span Biosciences, Inc., Seattle, WA, USA). The serum activity of lysozyme was computed using (Lie et al. 1986).

Histology and morphometry of the small intestine

Two-centimeter samples were isolated from each section of the small intestine (the duodenum, jejunum, and ileum) and conserved in 10% neutral buffered formaldehyde (NBF) for 72 h before being dehydrated, cleared, and inserted in wax. The histological examination was carried out on 5-um dense oblique sections (cut with a microtome), which were placed on slides and stained with haematoxylin and eosin (H&E) (Suvarna et al. 2013). The length of the villous (from the edge of the crypt to the apex of the villous), the width of the villous, the number of villi in each section (NVIS), the muscular thickness (MTh), and the number of goblet cells (G cell) were all tested. All parameters were estimated using an OLYMPUS TH4-200 camera and computer-assisted digital-image pro plus (IPP) analysis software (Image-Pro Plus 4.5, Media Cybernetics, Silver Spring, MD, USA).

Immunohistochemical procedures

The avidin-biotin-peroxidase complex (ABC) procedure was designated by Hsu et al. (1981) for immunohistochemical staining for CD3 and CD20. In brief, xylene was used to deparaffinized paraffin-embedded intestinal sections and then rehydrated in ethyl alcohol. Endogenous peroxidase blocking reagent comprised of hydrogen peroxide and sodium azide was applied to tissue sections (DAKO peroxidase blocking reagent, Cat. No.S 2001). The sections were treated with one to two drops of the supersensitive mouse anti-Chicken CD3, clone CT-3 (Bio-Rad Lab., Dubai, UAE), and CD20 (ThermoFisher Scientific, Waltham, MA, USA). Haematoxylin was used to counterstain the sections, and the slides were inspected via the microscope. According to Rizzardi et al. (2012), the percentages of immunostaining positive areas were calculated using ImageJ software in 5 sections from every group under high magnification.

Data analysis

A randomised design and the SAS 9.2 general linear model (GLM) protocol were applied to the statistical analysis. Pens served as experimental units all through the research. The linear and quadratic influences of vastly increased tested concentrations were evaluated by orthogonal polynomial contrasts. Tukey’s test compared mean differences with a 5% chance of success. The data’s variation was affirmed as combined SEM, with the significance value set at p < 0.05.

Results

GC-MS analysis of LVEO

The bioactive compounds of LVEO were previously reported and published (Amer et al. 2022). The foremost bioactive composites recognized were α-pinene, acetic acid linalool ester, terpineol, α-linalool, eucalyptol, phellandral, geranyl vinyl ether, nerolidyl acetate, and acetic acid. The fatty acids recognized in LVEO were 9,12,15-octadecatrienoic acid (α-linolenic acid), arachidonic acid, 3,7,11-tridecatrienoic acid, 5,10-undecadienoic acid, z-8-methyl-9- tetradecenoic acid, trans-2-undecenoic acid, 13-docosenoic acid, and 11-octadecenoic acid.

Growth performance

The effect of supplemental LVEO on broiler chicken growth parameters is shown in Table 2. During the starting period, the FCR was reduced in the LVEO600 group likened to the LVEO0 group (p < 0.05), but the BW and BWG did not fluctuate abruptly (p > 0.05). During the grower period, the LVEO-treated groups had a linear rise in the BW and BWG (p < 0.05), but the FI and FCR remained insignificantly different at (p > 0.05). A dietary combination of LVEO did not affect bird growth parameters during the finisher period or overall performance (p > 0.05).

Table 2. The impacts of dietary addition of Lavender essential oil (LVEO) on broiler chicken growth over 35 days.

Variables	LVEO0	LVEO200	LVEO400	LVEO600	SEM	P values
						ANOVA	Linear	Quadratic
IBW (g)	90.85	91.33	91.67	92	0.21	0.263	0.16	0.85
Starter period
BW(g)	315.56	321	328	333.33	3.90	0.44	0.12	0.99
BWG(g)	224.70	229	236.33	241.33	3.80	0.48	0.13	0.97
FI (g)	260.67	257	262	261.67	2.23	0.89	0.73	0.75
FCR	1.16	1.12	1.11	1.09	0.01	0.114	0.02	0.68
Grower period
BW(g)	1103.06	1144. 67	1167	1211	17.68	0.18	0.04	0.97
BWG(g)	787 .50	823.67	839.33	878	15.21	0.21	0.05	0.97
FI (g)	1134.91	1123.33	1141	1114.67	18.21	0.97	0.83	0.87
FCR	1.44	1.37	1.36	1.27	0.03	0.29	0.08	0.94
Finisher period
BW(g)	1991.11	1912.33	2009.33	2077.33	33.97	0.44	0.28	0.31
BWG(g)	888.06	767.67	842.33	866	31.39	0.62	0.98	0.31
FI(g)	1480.07	1539.33	1447.33	1415.33	36.03	0.72	0.44	0.58
FCR	1.70	2.03	1.72	1.63	0.07	0.22	0.41	0.14
Overall performance
BW(g)	1991.11	1912.33	2009.33	2077.33	33.97	0.44	0.28	0.31
BWG(g)	1900.26	1820.67	1917.67	1985.67	33.93	0.44	0.28	0.31
FI (g)	2875.65	2919.67	2850	2791	49.93	0.87	0.54	0.66
FCR	1.52	1.61	1.49	1.40	0.03	0.11	0.07	0.12
PER	3.16	2.98	3.21	3.41	0.06	0.12	0.07	0.12
RGR	182.50	181.67	182.33	183	0.26	0.37	0.36	0.17

p < 0.05 was used to control whether the regressions were significant. The data variation was expressed as combined SEM. LVEO0: basal diet without LVEO, LVEO200: basal diet with 200 mg LVEO Kg⁻¹ diet, LVEO400: basal diet with 400 mg LVEO Kg⁻¹ diet, and LVEO600: basal diets with 600 mg LVEO Kg⁻¹ diet. IBW: initial body weight, BW: body weight, BWG: body weight gain, FCR: feed conversion ratio, PER: protein efficiency ratio, RGR: relative growth rate.

Serum physiochemical parameters

As illustrated in Table 3, the measured serum values of glucose and leptin did not vary significantly among groups (p > 0.05). The blood level of GH elevated linearly (p < 0.01) and quadratically (p = 0.05) in a concentration-dependent manner, with the highest level observed in the LVEO400 and LVEO600 groups (p < 0.05). Linear enhancement in serum T3 and T4 levels was observed in the LVEO400 and LVEO600 groups (p < 0.01).

Table 3. The impacts of dietary addition of Lavender essential oil (LVEO) on the serum physiochemical parameters of broiler chickens.

Variables	LVEO0	LVEO200	LVEO400	LVEO600	SEM	P values
						ANOVA	Linear	Quadratic
Glucose (mg/dL)	338	341	342.33	341.33	1.18	0.665	0.345	0.452
GH (ng/mL)	2.62 ^c	3.85 ^b	5.01 ^a	5.25 ^a	0.33	0.000	0.000	0.045
Leptin (ng/mL)	2.05	2.05	2.09	2.05	0.02	0.951	0.88	0.78
T3 (ng/mL)	3.41 ^b	4.24 ^ab	4.80 ^a	5.05 ^a	0.21	0.002	0.000	0.200
T4 (ng/mL)	19.43 ^b	21.26 ^b	23.30 ^a	24.96 ^a	0.66	0.000	0.000	0.853

p < 0.05 was used to control whether the regressions were significant. The data variation was expressed as combined SEM. Means with different superscripts ^“a,b,c” in the same row vary significantly at (p < 0.05). LVEO0: basal diet without LVEO, LVEO200: basal diet with 200 mg LVEO Kg⁻¹ diet, LVEO400: basal diet with 400 mg LVEO Kg⁻¹ diet, and LVEO600: basal diets with 600 mg LVEO Kg⁻¹ diet. GH: growth hormone, T3: triiodothyronine, T4: thyroxine.

Lipid profile

A quadratic decline in serum TC was reported with LVEO supplementation (p = 0.02). In all LVEO groups, serum TG and LDL-C concentrations were lowered linearly and quadratically compared to the LVEO0 group (p < 0.05). The serum level of HDL-C linearly enhanced in LVEO-supplemented groups in a concentration-dependent approach, with the top levels observed in the LVEO600 group (p < 0.01). The VLDL-C levels did not differ substantially among groups (p > 0.05) (Table 4).

Table 4. The impacts of dietary addition of Lavender essential oil (LVEO) on the serum lipid profile of broiler chickens.

Variables	LVEO0	LVEO200	LVEO400	LVEO600	SEM	P values
						ANOVA	Linear	Quadratic
TC (mmol/L)	3.55 ^a	3.37 ^b	3.41 ^ab	3.44 ^ab	0.02	0.025	0.070	0.015
TG (mmol/L)	1.33 ^a	1.21 ^b	1.19 ^bc	1.15 ^c	0.02	0.000	0.000	0.004
LDL-C (mmol/L)	1.33 ^a	1.06 ^b	1.04 ^b	1.02 ^b	0.04	0.005	0.002	0.030
HDL-C (mmol/L)	1.99 ^b	2.09 ^ab	2.13 ^ab	2.17 ^a	0.02	0.018	0.003	0.400
VLDL-C (mmol/L)	0.23	0.23	0.23	0.24	0.01	0.780	0.406	0.591

p < 0.05 was used to control whether the regressions were significant. The data variation was expressed as combined SEM. Means with different superscripts ^“a,b,c,” in the same row vary significantly at (p < 0.05). LVEO0: basal diet without LVEO, LVEO200: basal diet with 200 mg LVEO Kg⁻¹ diet, LVEO400: basal diet with 400 mg LVEO Kg⁻¹ diet, and LVEO600: basal diets with 600 mg LVEO Kg⁻¹ diet. TC: total cholesterol, TG: triglycerides, HDL-C: high-density lipoprotein cholesterol, LDL-C: low-density lipoprotein cholesterol, VLDL-C: very low-density lipoprotein cholesterol.

Immune status

As depicted in Table 5, increasing the level of LVEO increased total protein linearly (p < 0.01). Albumin and globulin were linearly higher in LVEO600 (p < 0.05). Serum C3 was linearly and quadratically increased across all LVEO-supplemented groups; higher levels were in the LVEO400 and LVEO600 groups (p < 0.01). A significant linear and quadratic elevation was demonstrated in IL10 level in all LVEO-supplemented groups compared to the LVEO0 group (p < 0.01). The highest linear (p < 0.01) and quadratic (p < 0.05) rise in lysozyme activity was observed in the LVEO600, followed by LVEO400 and then LVEO200 groups compared to the LVEO0 group.

Table 5. Impacts of dietary addition of Lavender essential oil (LVEO) on broiler chickens’ protein profile and immune variables.

Variables	LVEO0	LVEO200	LVEO400	LVEO600	SEM	P-values
						ANOVA	Linear	Quadratic
Total protein (g/dL)	3.54 ^c	4.24 ^b	4.57 ^ab	5.20 ^a	0.19	0.000	0.000	0.798
Albumin (g/dL)	1.23 ^b	1.33 ^b	1.63 ^ab	2.04 ^a	0.11	0.015	0.003	0.302
Globulin (g/dL)	2.31 ^b	2.91 ^ab	2.94 ^ab	3.15 ^a	0.12	0.053	0.014	0.311
C3 (mg/dL)	1.08 ^c	1.18 ^b	1.24 ^a	1.27 ^a	0.02	0.000	0.000	0.011
IL-10 (pg/mL)	1.72 ^b	3.39 ^a	3.98 ^a	4.30 ^a	0.31	0.000	0.000	0.010
Lysozymes (µg/mL)	131.17 ^c	167.00 ^b	184.83 ^ab	192.83 ^a	7.53	0.000	0.000	0.035

p < 0.05 was used to control whether the regressions were significant. The data variation was expressed as combined SEM. Means with different superscripts “^a,b,c” in the same row vary significantly at (p < 0.05). LVEO0: basal diet without LVEO, LVEO200: basal diet with 200 mg LVEO Kg⁻¹ diet, LVEO400: basal diet with 400 mg LVEO Kg⁻¹ diet, and LVEO600: basal diets with 600 mg LVEO Kg⁻¹ diet. C3: complement 3, IL10: interleukin 10.

Histological findings and morphometric measures of the small intestine

Mild villous epithelial stratification was observed in all groups, with several-layered proliferating cells with centred rounded nuclei and rich eosinophilic cytoplasm. However, remodelling and rearrangement of such cells could be observed in groups (LVEO400 and LVEO600) (Figures 1-C, D). The dimensions of the villous length, villous width, crypt of Lieberkühn length, and muscular coat thickness were recorded. The best dimensions were seen in the LVEO600 and LVEO400 groups (Figures 1-D C,), respectively, followed by LVEO200 (Figure 1B) and LVEO0 (Figure 1-A). Routine H&E counting of goblet cells revealed a low number of goblet cells in groups LVEO0 and LVEO200 (Figure 1–A, B), and the highest number were in the LVEO400 LVEO600 groups (Figure 1-C, D).

Figure 1. Representative photomicrographs of the small intestines of various tested groups (A; LVEO0), (B; LVEO200), (C; LVEO400), and (D; LVEO600) exhibiting the aspects of the villous length (green arrows), villous width (brown arrows), the crypt of Lieberkühn length (orange arrows), and muscular coat thickness (black stars). Routine H&E predicted scoring of goblet cells (yellow arrows) confirmed a low number in groups a and B and the highest proportion in groups C and D. Slight villous epithelial stratification with multi-layered proliferate cells with central rounded nuclei and profuse eosinophilic cytoplasm can be identified in all groups (blue arrows). Still, regeneration and rearrangement of such cells are seen in groups C and D. H&E × 100 and 200 for each group (A–D).

Table 6 and Figure 1 demonstrate the impact of additional LVEO on the morphological features measures (villous length, villous width, Crypt of Lieberkühn length, Muscular coat thickness) of the small intestine at 35 days of age. The villous length was linearly and quadratically rose in the LVEO400 and LVEO600 groups (p < 0.01). In contrast, the villous width was linearly and quadratically diminished in the LVEO400 and LVEO600 groups (p < 0.01) compared with the LVEO0 group. Crypt of Lieberkühn length was linearly and quadratically elevated in all LVEO-supplemented groups (p < 0.01) compared to the LVEO 0 group. The jejunal and ileal villi height were linearly and quadratically augmented in all LVEO-supplemented groups (p < 0.01) related to the LVEO0 group. A non-significant change was observed in villous width in the jejunum and ileum of the LVEO600 group compared to the LVEO0 group (p > 0.05). There was a significant reduction in jejunal Crypt of Lieberkühn length in all LVEO consumed groups (p < 0.01) compared to the LVEO0 group. The highest muscular coat thickness of the duodenum and jejunum was observed in the LVEO200 group. The highest ileum villous width was observed in the LVEO200 group, while the LVEO400 and 600 groups were not distinguished statistically from the LVEO0 group. The highest crypt of Lieberkühn length of the ileum was observed in the LVEO200 group. The ileal muscular coat thickness was significantly diminished in all LVEO groups (p < 0.05).

Table 6. The impacts of dietary addition of Lavender essential oil (LVEO) on the morphometric measures (µm) of the small intestine and the CD3 and CD20 splenic immunoexpression in broiler chickens.

Variables	LVEO0	LVEO200	LVEO400	LVEO600	SEM	P-values
						ANOVA	Linear	Quadratic
Duodenum
Villous length	1093.55 ^c	1051.91 ^d	1115.55 ^b	1366.02 ^a	37.11	0.000	0.000	0.000
Villous width	353.39 ^a	347.70 ^a	182.47 ^b	80.57 ^c	34.77	0.000	0.000	0.000
Crypt depth	206.34 ^d	219.40 ^c	229.81 ^b	307.43 ^a	11.89	0.000	0.000	0.000
Muscular coat thickness	112.95 ^b	128.52 ^a	116.73 ^b	63.38 ^c	7.53	0.000	0.000	0.000
Jejunum
Villous length	890.10 ^c	1024.69 ^a	924.25 ^b	1023.67 ^a	18.02	0.000	0.000	0.000
Villous width	149.87 ^a	109.24 ^c	118.46 ^b	154.60 ^a	5.91	0.000	0.002	0.000
Crypt depth	238.83 ^a	145.51 ^c	102.48 ^d	209.38 ^b	16.09	0.000	0.000	0.000
Muscular coat thickness	198.83 ^b	236.61 ^a	141.67 ^c	132.33 ^c	12.91	0.000	0.000	0.000
Ileum
Villous length	482.77 ^d	547.88 ^b	527.02 ^c	706.74 ^a	25.49	0.000	0.000	0.000
Villous width	115.16 ^b	141.88 ^a	114.20 ^b	116.34 ^b	3.54	0.000	0.005	0.000
Crypt depth	178.65 ^b	187.67 ^a	107.28 ^d	140.90 ^c	9.66	0.000	0.000	0.000
Muscular coat thickness	254.91 ^a	181.10 ^c	210.88 ^b	165.14 ^d	10.32	0.000	0.000	0.000
Splenic immunoexpression of CD3 and CD20 (%)
CD3	0.37 ^c	3.33 ^b	11.63 ^a	12.33 ^a	1.58	0.000	0.000	0.063
CD20	13.50 ^d	34.67 ^c	48.00 ^b	72.84 ^a	6.51	0.000	0.000	0.197

p < 0.05 was used to control whether the regressions were significant. The data variation was expressed as combined SEM. Means with different superscripts ^“a,b,c,d” in the same row vary significantly at (p < 0.05). LVEO0: basal diet without LVEO, LVEO200: basal diet with 200 mg LVEO Kg⁻¹ diet, LVEO400: basal diet with 400 mg LVEO Kg⁻¹ diet, and LVEO600: basal diets with 600 mg LVEO Kg⁻¹ diet.

Immunohistochemical analysis

Table 6 and Figures 2 and 3 exhibit splenic CD3 and CD20 immunoexpression. Immunohistochemical spleen sections stained with specific CD3 and CD20 antibodies revealed greater staining reactions within the white pulp in the LVEO200, LVEO400, and LVEO600 groups compared to weak expression in the LVEO0 group.

Figure 2. Positively immunostained CD3 cells (red arrows) and negative cells (black arrows) in the spleens of various supplemented groups. (A: LVEO0, B; LVEO200, C; LVEO400, D; LVEO600. × 400 magnifications.

Figure 3. Positively immunostained CD20 cells (red arrows) and negative cells (black arrows) in the spleens of various supplemented groups. (A: LVEO0, B; LVEO200, C; LVEO400, D; LVEO600. Bar 20. Magnification: × 400.

Discussion

Herbal products like LVEOs can assist with antibiotic resistance, invasive treatments, adverse reactions, and drug dependence. These attributes, combined with the emergence of drug resistance, render lavender a prevalent medicinal herb today (Kajjari et al. 2022). The existing research examined how LVEO affected broiler chicken growth, physiological blood markers, immunological response, and gut morphology. According to Amer et al. (2022), the primary active compounds recognised by GC-MS analysis in LVEO were α-pinene (32.24%), acetic acid linalool ester (32.24%), and α-linalool (16.06%) along with monoterpenoids like as cis-α- terpineol (16.06%), eucalyptol (11.74), and phellandral (10.91%).

Herbal EOs’ active principles act as a digestibility stimulant, adjusting the intestinal microbiota ecosystem and boosting the release of inherent digestive enzymes, thereby enhancing poultry growth performance (Popović et al. 2016). Yarmohammadi Barbarestani et al. (2020) revealed that giving birds 600 mg/kg of LVEO raised BWG and improved FCR, implying efficient feed usage. Antimicrobial properties of EOs on the intestinal bacteria can suppress pathogenic bacteria’s growth and enhance beneficial microorganisms’ competitiveness, thus maximising animal performance (Scherer et al. 2014). The antimicrobial property of LVEO could be mainly ascribed to its bioactive phytoconstituents as α-pinene, α-linalool, α-terpineol, eucalyptol, and 9,12,15- Octadecatrienoic acid (Oliveira and Santos 2021), (Jabir et al. 2018), (Badawy et al. 2019), (Di et al. 2022) and (Al-Gara et al. 2019), respectively.

In the current study, daily consumption with LVEO had no effect on animal growth except for a linear rise in BW and BWG during the grower period. Based on the consumed calories, quantity, form, and administering approach (water, feeds) and the bioactivities of the EOs’ ingredients, animal growth could be increased, decreased, or unaffected (Adaszynska-Skwirzyńska et al. 2021). There were no mortalities in the experimental groups. In the study of Torki et al. (2021), adding LVEO had no impact on feed intake or body weight alterations during the trial period, matching the current study. Furthermore, Mokhtari et al. (2018) discovered that adding 100-800 mg/kg of LVEO (main chemical ingredients: Linalool-44.31%; linalyl acetate-32.98%) to the diet seemed not to influence fattening production parameters. Additionally, Scherer et al. (2014) observed no notable variations in feed efficiency, weight gain, or final mass among broiler chickens with reduced feed consumption after 21 days, which might be attributed to an irritant smell in the diet.

Biochemical and hematological markers can reveal energy metabolism and the body’s physiological functions (Toghyani et al. 2012). According to existing findings, dietary LVEO showed no influence on either glucose or leptin hormone levels. In the present investigation, higher T3 and T4 levels have been found in the LVEO400 and LVEO600 groups, along with greater GH levels by LVEO addition in a concentration-dependent approach, with the most significant level found in the LVEO400 and LVEO600 groups. This is compatible with the results of Yarmohammadi Barbarestani et al. (2020) who explored no variations in glucose levels in their analysis of LVEO application in broiler chicken diets. All body cells need an energy source regularly, so sufficient amounts of glucose in plasma must be preserved (Azimi-Youvalari et al. 2022).

Similarly, glucose levels in the 5% linalool-treated birds were not altered (Beier et al. 2014). The volatile ingredients in EOs operate through particular odour receptors, promoting the central nervous system to manage energy expenditure, balancing lipolysis and lipogenesis, and monitoring appetite (Shen et al. 2005; De Blasio et al. 2021). The repercussions mentioned above are triggered by the stimulating action of the sympathetic and parasympathetic nerve systems, plus the releasing hormones that involve insulin and leptin (Yamamoto et al. 2013; De Blasio et al. 2021). Leptin can diminish hunger, promote weight loss, and enhance basal metabolism (Izquierdo et al. 2019). When the fat mass is destroyed, plasma leptin levels drop, encouraging appetite while repressing energy consumption until fat mass is regained. Leptin levels rise as fat mass increases, restricting appetite until weight loss happens: the more fat mass there is, the more leptin is released (Friedman 2019). The non-significant change in leptin hormone in the current investigation could be attributed to steady feed intake or elevated T3 and T4 hormones with LVEO intake. Under physiological circumstances, leptin may also modify the thyroid axis set point (Nazifi et al. 2012). Rat in vivo and in vitro research proposed that elevated blood T3 reduces leptin mRNA expression at white adipose tissue and blood leptin concentrations (Medina-Gomez et al. 2004). The levels decline as euthyroidism is attained (Al-Hindawi Sahar 2018).

In contrast, data on the impacts of T3 and T4 hormones on serum leptin levels are conflicting, with recommendations that T3 and T4 hormones all had an inhibitory, stimulatory, or no critical influence on leptin levels (Al-Hindawi Sahar 2018). Thyroid hormones boost thermogenesis and raise basal metabolic activity, resulting in uncoupling oxidative phosphorylation (Nazifi et al. 2012). The current study’s thyroid hormone elevation could be attributed to lavender calcium influx blocking, which inhibits glutamate and norepinephrine liberation. Glutamate and norepinephrine function in the onset of anxiousness and act as anti-depressants (Suyono et al. 2020). Nonetheless, this could be an anti-stressor by triggering the thyroid gland release of T4 and T3 into broiler blood (Mustafa and Tayeb 2022).

The current study demonstrated a significant rise in GH, notably during LVEO 400 and 600 feeding. T3 and T4 can induce growth hormone mRNA transcription and manufacturing in the pituitary gland (Yen 2001). GH can activate bone and muscle cell growth and differentiation (Kühn et al. 2002). GH promotes growth by initiating cellular GH receptors or triggering insulin-like growth factor 1 (IGF1) efflux by the liver, which elicits many of GH’s growth-promoting actions in poultry (Nguyen et al. 2015; Zhang et al. 2022). Elevated GH without enhancing bird growth parameters implies that growth is complex and multifaceted and that GH alone cannot describe the chicken’s growth (Beccavin et al. 2001).

In contrast to the LVEO0 group, daily consumption with varying levels of LVEO enhanced the serum lipid profile by lessening TC, TG, and LDL-C concentrations while boosting HDL-C concentrations. Adaszyńska-Skwirzyńska et al. (2021) reported that 600 mg/kg LVEO consumption significantly lowered cholesterol levels compared to a control group. Linalool, one of LVEO’s active ingredients, has been coupled with cholesterol-lowering features (Eissa et al. 2017). Cho et al. (2011) discovered that providing mice linalool eliminates 3-hydroxy-3-methyl glutaryl-CoA reductase protein expression (as an indication for liver cholesterol synthesis; HMG-CoA), resulting in lower total cholesterol and LDL-C concentrations. In addition, α-pinene (Santos et al. 2023) diminishes cholesterol, its fractions, and triglycerides. Likewise, other studies on the hypercholesterolaemia characteristics of medicinal EOs in broilers and laying hens have implied similar approaches, such as inhibiting HMG-CoA reductase and cholesterol-7 hydroxylase fatty acid synthase and suppression of the pentose phosphate pathway by 6-amino nicotinamide (Torki et al. 2018). Furthermore, Abo Ghanima et al. (2020) noticed that laying hens fed rosemary and cinnamon oil (dosing 300 mg/kg feed) had lesser blood cholesterol levels. Herbal extracts (EOs) restrict specific enzymes such as peroxidase and dehydrogenase, causing a decline in low-density cholesterol (Iqbal et al. 2021). Furthermore, the decrement in lipid profile parameters identified in this study may be associated with increased T3 and T4 hormones. Hypothyroid rats revealed a remarkable rise in TC, TG, HDL-C, and LDL-C (Nazifi et al. 2012). The number of LDL receptors in the liver declines in obvious hypothyroidism, whereas blood levels of TC, LDL-C, and apolipoprotein B rise.

Blood proteins are critical immune system aspects that fluctuate when immunostimulants are applied (Chakraborty and Hancz 2011). In the present work, raising the concentration of LVEO reportedly increased total protein, albumin, and globulin in the LVEO 600 group, which is identical to previous findings (Yousefi et al. 2020). Significantly higher serum proteins may also be associated with enhanced broiler liver function and health as this organ is the site of several protein biosyntheses (Hoseini and Tarkhani 2013), and lavender extract has been proven to be hepatoprotective (Selmi et al. 2015). The hepatoprotective impact of LVEO could be ascribed to the antioxidant properties of active s as α-pinene, eucalyptol, α-linalool, α-terpineol, 9,12,15- Octadecatrienoic acid, and arachidonic acid (Seol and Kim 2016; Bouzenna et al. 2017; Złotek et al. 2017; Al-Gara et al. 2019; Hsouna et al. 2023). The rise in total protein levels verified that aromatic oil has a capability to boost protein absorption and digestion, precisely as revealed by Krishan and Narang (2014), enabling greater protein use in broiler chicken and thus bettering weight gain. Furthermore, Houghton et al. (1995) revealed that the rise in globulin explained the beneficial application of aromatic compounds in enhancing immune function due to its role in cell formation and protection and inhibiting non enzymatic oxidation.

Incorporating an EO into broiler chickens’ meals boosted serum immunological variables, as displayed by elevated lysozyme activity, immunoglobulin concentrations, and phagocytic indicators (Kishawy et al. 2022). The immune status values examined in this research showed enhanced responses to dietary LVEO. When compared to the LVEO0 group, dietary LVEO supplementation elevated serum IL-10, C3, and lysozyme levels in the current work. The complement system promotes both humoral and cellular immunity in various ways. C3 is primarily generated by the liver, but macrophages can also create it (Gao et al. 2020). IL-10 is an effective anti-inflammatory that serves as a "stop" message for myeloid cells. IL-10 regulates the immune system’s reaction to the host by inhibiting the manufacturing of pro-inflammatory cytokines such as interferon, tumour necrosis factor α (TNF-α), IL-1, IL-2, and IL-6 (Arendt et al. 2016). IL-10 acts in birds like in mammals (Rothwell et al. 2004). Whether in tiny amounts, the existence of these biomolecules (linalool and terpinene-4-ol, α-terpineol) and others may modify the anti-inflammatory impacts of the studied LVEOs (Pandur et al. 2021). The study by Aboutaleb et al. (2019) decided that post-LVEO intervention elevated IL-10 expressions in dose-dependent ways. The anti-inflammatory influences of α-pinene on inflammatory responses significantly reduce IL-6 and TNF-α creation (Kim et al. 2015), whereas α-terpineol prevents pro-inflammatory IL-6 cytokine generation (Held et al. 2007). Linalool restricts the nuclear factor kappa B (NFκB) and mitogen-activated protein kinase (MAPK) pathways (Huo et al. 2013). It triggers the production of IL-10 (Hsouna et al. 2023). In broiler chickens, eucalyptol has anti-inflammatory impacts, probably by retarding inflammation signalling while promoting IL-10 levels (Di et al. 2022). Rising C3 release, a component of the complement system, implies strong immunity and disease resistance (Delanghe et al. 2014). The results of Gao J et al. (2020) found that adding herbal EOs to the food of egg-laying birds could increase the content of C3 and C4 in their blood, indicating their efficacy for triggering the immune function. Lysozymes are believed to be an excellent predictor of the immune response (del Rocío Quezada-Rodríguez and Fajer-Ávila 2017). An enhancement in neutrophil count and lysozyme production could explain the boost in lysozyme activity (Costas et al. 2011).

The integrity of the gut barrier is pivotal to the physiological function of the intestine (Yarmohammadi Barbarestani et al. 2020). Changes in the architecture of the intestinal mucosa, such as an increment in villous length and villous length: crypt depth ratio, can indicate improved intestinal health and an expansion of the surface area for digestion and absorption. The current research showed that LVEO intake resulted in a significant rise in villous length, indicating increased digestive enzyme activity and, thus, better nutrient digestibility. These latest findings align with (Yarmohammadi Barbarestani et al. 2020) and (Salajegheh et al. 2018). Overall, plant-derived feed additives may enhance the proportion of villous height to crypt depth in the small intestine, boosting absorptive surface area and, digestive health and nutrient absorption efficiency (Khattak et al. 2014). The anti-inflammatory and anti-oxidation mechanisms of EOs, which guard the villi against oxidative damage, may be critical for enhancing the intestinal structure and function (Gao et al. 2019), (Du et al. 2016). α-pinene can guard and increment the viability of aspirin-induced oxidative stress in rat intestinal cell line (IEC-6) cells (Bouzenna et al. 2017). The percentage of villous height to crypt depth in the jejunum and ileum, along with the crypt depth in the ileum, were improved by eucalyptol intake (Di et al. 2022). It can protect the villi by strengthening the body’s humoral defences, which causes numerous antibodies to be transferred to the intestine’s mucosal surfaces (Tohid et al. 2010). Furthermore, the active ingredients selective antimicrobial properties may be linked to better intestinal morphological traits in broilers (Amer et al. 2022).

The presence of particular cell surface biomarkers categorises lymphocyte subsets. CD3 prompts a cascade of events that initiates helper and cytotoxic T cells in response to antigen recognition (Ryan 2010). CD20, a B-lymphocyte surface antigen, monitors B-cell function, differentiation, and propagation (Tedder and Engel 1994). Immunohistochemical findings suggest that LVEO contribution caused dose-dependent spleen immunostaining reactivity to CD3 and CD20. So far, the present results have demonstrated that LVEO has an immune-stimulating effect in broiler chickens, which agrees with previous studies (Sienkiewicz et al. 2011). The immunostimulatory effect of LVEO may be mainly ascribed to eucalyptol’s beneficial impacts on the humoral immunity of broilers (Di et al. 2022). α-pinene is fundamental for employing leukocyte cells in the inflammation sites (Santos et al. 2023).

Conclusion

The LVEO consumption had almost no impact on the bird’s growth except to increase BW and BWG during the grower period. However, its addition produced several beneficial consequences, including improving the bird’s gut architecture, particularly at 600 mg Kg⁻¹ diet. Furthermore, LVEO supplementation optimised the birds’ metabolic functions, associated with increased elevated T3, T4, and GH at level 400 and 600 mg Kg⁻¹ diet. Dietary LVEO had hypolipidemic effects. LVEO supplementation can improve the birds’ immune status by raising the serum C3, IL10, and lysozyme levels and provoking immunological immunoexpression of CD3 and CD20 in the spleen of the LVEO400 and LVEO600 groups.

Ethical approval

The experimental protocol received ethical approval from Zagazig University’s Institutional Animal Care and Use Committee (Approval No. ZU-IACUC/2/F/152/2022). All animal experiments were carried out in accordance with the instructions outlined in "The Guide for the Care and Use of Laboratory Animals in Scientific Investigations," and the study followed the relevant institutional standards. All animal experiments were carried out in accordance with the ARRIVE guidelines.

Acknowledgments

This work was supported by Researchers Supporting project number (RSPD2023R700), King Saud University, Riyadh, Saudi Arabia.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The datasets generated or analysed during the current study are not publicly available but are available upon reasonable request from the corresponding author.