Evaluation of dietary supplementation of frankincense oil on broiler chicken growth performance, hepatic histomorphology, antioxidant activity, blood biochemical parameters, and inflammatory responses

Abstract

This investigation aimed to assess the potential impact of frankincense oil (FKO) from Boswellia serrata on broiler chicken growth performance parameters, including body weight (BW), body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR) during the starter, grower, and finisher phases. We also evaluated the hepatic histology, serum hepatorenal function tests, antioxidant activity, and inflammatory responses. A total of 400 three-day-old male chicks (Ross 308 broiler) (100.40 ± 0.13 g) were randomly assigned to four treatment groups (10 replicates/group, ten chicks/replicate). The birds were fed basal diet (FKO0, control group) or basal diet supplemented with 200, 400, and 600 FKO/kg of diet (FKO200, FKO400, FKO600, respectively). The experiment lasted 35 days. Gas chromatography-mass spectrometry analysis revealed that FKO contained 36 constituents, with the dominant compounds being 1,6,10-DODECATRIEN-3-OL, 3,7,11-TRIMETHYL-, [S-(Z)]- (12.42%), ç-Elemene (12.42%), à-Farnesene (12.42%), PHENOL and 2,4-BIS(1,1-DIMETHYLETHYL)- (7.15%). Distinctive FKO levels (200-600 mg/kg) showed greater BW without influencing total feed intake compared to the FKO0 treatment. The FCR was improved by the addition of FKO to the diets. The blood concentrations of alanine aminotransferase and creatinine increased (p < 0.05) in the FKO600 treatment. Dietary FKO linearly lowered serum malondialdehyde levels and enhanced blood total antioxidant capacity, catalase, superoxide dismutase, and pro-inflammatory cytokines (interleukine-1 β and interferon γ) compared to the control group. Broilers fed FKO at levels 200–600 mg kg⁻¹ diet also exhibited lower serum total cholesterol, triglyceride, and low-density lipoprotein cholesterol levels. Furthermore, the FKO400 and FKO600 treatments showed an increase in high-density lipoprotein cholesterol (p < 0.01). Histomorphological analysis of the liver indicated no significant differences between the FKO-supplemented groups and the control group. However, the immunoexpression of the pro-inflammatory cytokine (transforming growth factor β) was considerably increased in the liver and spleen tissues of birds fed FKO in a level-dependent manner. In conclusion, dietary supplementation of FKO at levels up to 600 mg/kg can serve as a natural growth promoter in broiler chickens, leading to enhanced growth, hypolipidemic properties, antioxidant status, and immune responses.

HIGHLIGHTS

• Dietary FKO up to 600 mg Kg⁻¹ improved the growth performance of broiler chickens without affecting the total feed intake.

• FKO at levels 200–600 mg Kg ⁻¹ had no adverse effects on hepatic transaminases or renal parameters. FKO works as a hypolipidemic agent at all tested levels.

• Dietary FKO 200–600 boosted the antioxidant capacity.

• FKO addition boosted the inflammatory reactions in broiler chickens, as evidenced by higher serum levels of IL-1β and IFN-γ, and higher TGF-β immunoexpression in the liver and spleen tissues.

Keywords:

• Broilers chickens
• frankincense oil
• antioxidants
• lipid profile
• liver histoarchitecture
• immunohistochemistry

Introduction

In the absence of antibiotics for promoting animal growth, nutritional immunomodulators serve as important alternatives that can enhance farm animal productivity and immune system health (Munyaka et al. 2012; Landy et al. 2020; Amer, Attia, et al. 2022; Amer, Farahat, et al. 2023; Landy and Kheiri 2023,). In poultry farming, there is an ongoing exploration of new feeding programs aimed at improving production levels while ensuring the well-being of poultry throughout the feeding period (Tufan et al. 2023). As customers become more informed about the advantages of using natural feed ingredients, nutritionists are looking for phytonutrients to replace synthetic ones (Selim et al. 2021). These phytochemicals offer significant benefits, including a reduction in the incidence of metabolic diseases, increased stability of meat during storage, and improvements in food security and quality (Turcu et al. 2021; Amer, Abdel-Wareth, et al. 2022; Amer, Al-Khalaifah, et al. 2022).

Boswellia is an aromatic substance derived from the resin of tree trunks belonging to the genus Boswellia (Burseraceae family) (Al-Yasiry et al. 2016). It is also known by various other names, including frankincense, olibanum, salai guggul, loban, and kundur (Afsharypuor and Rahmany 2005). Boswellia serrata (BS) resin is among the approved food supplements recommended for inclusion in broiler production under Regulatory frameworks (EC) No 1831/2003, as listed in the European Union Registration of Feed Additives (Al-Yasiry, Kiczorowska and Samolińska 2017).

Oil (60%) is the most crucial component of frankincense resin (Kiczorowska et al. 2016). For instance, Hosny et al. (2020) reported that frankincense oil (FKO) comprises carvone (2.96%), octanol (10.1%), thymol (16.69%), and octyl acetate (67.44%). Wang et al. (1993) reported p-cymene (8.7%), octanol (12.7%), and octyl acetate (60.0%). In addition, Mohamed et al. (2015) demonstrated that the principal components were verticiol (14.48%), followed by isobutylcyclopentane (12.25%), n-octyl acetate (9.20%), and 9-oxabicyclo-[6.1.0]- non-3-yne (9.12%). Chemical properties and yield vary based on variables such as the tree’s geographic area, wood surface, collection season, collection procedure, tree maturity level, and mobilization. (Almeida-da-Silva et al. 2022; Vuddanda et al. 2016).

The antibacterial and antifungal properties of FKO have been documented in previous studies (Di Stefano et al. 2020; Almeida-da-Silva et al. 2022). These studies primarily focused on the resin’s anti-inflammatory, immunomodulatory, and anti-leukotriene activity, particularly its main component, boswellic acid derivatives (Mikhaeil et al. 2003). Frankincense exerts its effects by modulating immune cells derived from the innate and acquired immune systems by hindering leukotriene formation, cyclooxygenase and lipoxygenase, and the generation of reactive oxygen species. Clinical research has demonstrated that frankincense and its phytochemicals can help treat osteoporosis, multiple sclerosis, and asthma. Moreover, frankincense has shown efficacy in managing oedema associated with a brain tumour, leading to significant therapeutic implications (Efferth and Oesch 2022).

Suppliers of animal-derived foods are increasingly drawn to frankincense as a herbal supplement to maximize yield production and promote livestock health by providing them with a nutritious diet (Al-Yasiry and Kiczorowska 2016b). A previous study has demonstrated the antioxidant properties of BS gum resin extracts (Sharma et al. 2011). Pandey et al. (2005) found that the extracts of BS gum resin reduced total serum cholesterol (TC) while increasing high-density lipoprotein-cholesterol (HDL-C) in rats. However, there is limited research focusing on the potential application of FKO in poultry production (Amer, Gouda, et al. 2023). The current study aimed to investigate the effects of FKO, a natural antioxidant in broiler diets, on growth performance, blood biochemistry, liver histoarchitecture, and immune expression of transforming growth factor beta (TGF-β) in the liver and spleen of broiler chicken.

Materials and methods

Gas chromatography-mass spectrometry (GC-MS) analysis of frankincense oil (FKO)

FKO (Boswellia serrata) was obtained from ORGANIC EGYPT, Cairo, Egypt. A temperature-dependent Scientific Detect GC1310-ISQ mass spectrum analyzer (Austin, TX, USA) equipped with an immediate capillary column TG-5MS (30 m × 0.25 mm × 0.25 µm film thickness) was applied to assess the chemical composition of FKO (Thermo Scientific, Austin, TX, USA). The column temperature was initially set to 50 °C and then increased to 230 °C at the rate of 5 °C per minute, which was maintained for 2 min. Subsequently, the temperature was increased at the rate of 30 °C per minute to a temperature close to 290 °C, where it was held for 2 min. The injector and MS transfer lines were retained at temperatures of 250 and 260 °C, respectively, with helium acting as a carrier gas with a steady flow rate of 1 mL/min. The solvent time limit was 3 min, and diluted samples of 1 µL were inoculated instantly utilizing an Autosampler AS1300 combined with a GC in splitless mode. EI mass spectra were acquired in full scan mode at 70 eV ionization voltages over the m/z 40–1000 range. The ion source temperature was initially set to 200 °C. The elements were recognized by comparing their retention times and spectral data with the WILEY 09 and NIST 11 mass spectral database systems.

Birds, diets preparation and experimental design

This investigation was carried out in a poultry research centre at Egypt’s Zagazig University’s Faculty of Veterinary Medicine. All experimental procedures were approved by the ZU-IACUC committee (approval number ZU-IACUC/2/F/152/2022). All protocols used in this study were carried out following the relevant institutional guidelines; all experiments involving animal models were performed following the ARRIVE guidelines.

Four hundred one-day-old male Ross 308 broiler chickens were readily accessible from an industrial broodstock (Dakahlia Poultry, Mansoura, Egypt). Chicks were reared in an air-conditioned open house with wood chips bedding (7 birds/m²). The investigation lasted for 35 days. The ambient temperature during construction was maintained at 34 °C for the first week and gradually decreased until it reached 25 °C on the final day of the experiment. The illumination system was set to 23:1 h light/dark for the initial hour, and then 20:4 h light/dark until the experiment ended. Before the commencement of the experiment, the chickens were acclimated for 3 days, acquiring a typical body weight (BW) of 100.40 ± 0.13 g. They were then randomly assigned to one of four test groups, each with ten replicates (10 chicks per replicate). FKO was fed to the birds at four doses: 0, 200, 400, and 600 mg kg⁻¹ diet over three feeding intervals: starter (4^th–10^th day), grower (11^th–23^rd day), and finisher phases (24^th–35^th day). FKO was thoroughly mixed with other feed ingredients using mechanical means. Table 1 shows the approximate elemental analysis of the basal diet. The experimental diets and management practices adhered to the nutritional demands of the Ross 308 broiler (AVIAGEN 2014). Standard vaccination protocols were implemented to protect against Newcastle (on days 4 and 14) and Gumboro diseases (on days 7 and 22). The birds were monitored daily for any signs of disease or mortalities. No mortalities were recorded during the experimental period.

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

Ingredients	Starter period (4–10 d)	Grower period (11–23 d)	Finisher period (24–35 d)
Yellow corn 7.5% CP	559	593	620
Corn gluten, 60% CP	39.35	52.75	60.7
Soybean meal, 48% CP	334.2	281	238
Soybean oil	22	30	40
Calcium dibasic phosphate	15	14	13
Calcium carbonate	12	12	11
Common salt	1.5	1.5	1.5
DL-methionine, 98%	4	3	3.3
Premix*	3	3	3
Lysine HCl, 78%	4.7	4.5	4
Phytase	0.05	0.05	0.05
Threonine	1	1	1
Choline	0.7	0.7	0.7
Antimycotoxin	1	1	1
Na2Co3	2.5	2.5	2.5
Chemical composition (g kg^-1)
Crude protein	230.5	215.2	201.5
ME (Kcal/kg)	3004	3101	3201
Calcium	9.41	9.04	8.32
Available phosphorus	4.81	4.48	4.17
Lysine	14.6	13.1	11.6
Methionine	7.21	6.10	6.26
Threonine	8.24	7.65	7.13

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

ME: metabolizable energy.

Growth performance

The birds were weighed on the fourth day of life to find the initial BW, and subsequently, the BWs and feed intake (FI) were noted down at 10, 23, and 35 days. The growth parameters were calculated as follows: $$\text{Body weight gain }\left(\text{BWG}\right) \left(g\right)\text{ = W2 }\mathrm{–}\text{ W1}$$

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

The average FI/bird (g) = feed offered (g) - feed residue (g)/No. of birds in each replicate

Feed conversion ratio (FCR) = FI (g)/BWG (g)

The relative growth rate (RGR) was determined (Brody 1945). $$\text{RGR = W2 }\mathrm{–}\text{ W1/0.5}\left(\text{W1 + W2}\right)\mathrm{\times }\text{ 100}$$

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

The protein efficiency ratio (PER) = live weight gain (g)/protein intake (g) (McDonald et al. 1973).

Protein intake (g) = feed intake (g) × crude protein %

Blood biochemical assay

After the euthanization of birds through cervical displacement, blood samples (n = 10) were harvested from the carotid artery as per the protocol recommended by the American Veterinary Medical Association (Association AVM 2013). The serum was obtained by vortexing blood at 3000 rpm for 20 min and stored at −20 °C until used in the biochemical assays.

Following the manufacturer’s instructions, colorimetric diagnostic kits were used to test liver enzymes, including aspartate transaminase (AST) and alanine transaminases (ALT), as well as renal function like creatinine and uric acid (Biodiagnostic Co., Giza, Egypt).

Spectrum-bioscience colorimetric diagnostic kits (Egyptian Company for Biotechnology, Cairo, Egypt) were used to estimate 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 calculated using 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 was used to measure the very low-density lipoprotein cholesterol (VLDL-C) (Griffin and Whitehead 1982).

Inflammatory and antioxidant indices assay

Interferon γ (INF-γ) and interleukin 1β (IL-1β) levels were measured using ELISA assay kits (MyBioSource, San Diego, CA, USA) (Cat. No. MBS700243 and MBS2024496, respectively). Malondialdehyde (MDA), total antioxidant capacity (TAC) levels, catalase (CAT), and superoxide dismutase (SOD) activities were assessed using MyBioSource ELISA Kits (Cat. Nos. MBS2700234, MBS038818, and MBS705758, respectively). The techniques described by (Mcdonald and Hultin 1987), (Rice-Evans and Miller 1994), (Aebi 1984), and (Nishikimi et al. 1972), were employed for the respective measurements.

Histopathological investigations

Ten liver specimens per group were collected from the chickens and fixed in neutral buffered formalin 10% for analysis. The tissue samples were dehydrated in a series of gradually increasing ethanol concentrations (75–100%). Subsequently, they were submerged in xylol I and II before being embedded in paraffin. Cross-sectional and longitudinal sections were cut into 4 µm using a microtome (Leica RM 2155, England). These sections were stained with hematoxylin and eosin (H&E) (Suvarna et al. 2013). Each animal in the group was photographed under high-power magnification (×100 and ×400 magnification) using AmScope 5.0 MP microscope digital camera (25 images for each group). Stained sections were examined for vascular disruptions, inflammatory processes, degenerative changes, cell death, necrosis, and other liver damage.

Immunohistochemical estimation

The liver and spleen leucocytic populations in broiler chickens were examined for inflammatory mediators, specifically (TGF-β), in response to the dietary addition of FKO. After the investigation, liver and spleen samples (ten samples per group) were collected to evaluate TGF-β immunoexpression (Saber et al. 2019). Endogenous peroxidase blocking reagent containing hydrogen peroxide and sodium azide was applied to the tissue sections (DAKO peroxidase blocking reagent, Cat. No. S 2001). Next, one to two drops of the supersensitive primary monoclonal antibody against TGF-β (Cat. BAF240, Novus Biologicals, Briarwood Avenue, Centennial, CO, USA) was added to these sections; the slides were then stained with hematoxylin and observed under a microscope. Morphometric analysis was performed using Image J software (bundled with 64-bit Java 1.8.0_172, National Institutes of Health, Maryland, US) to accurately measure various immune-positive cells and their proportions in the liver and spleen of tested chicken groups in three high-power fields (Rizzardi et al. 2012).

Data evaluation

A randomized design and the SAS 9.2 general linear model (GLM) protocol were employed for statistical analysis in this study. Throughout the study, pens served as experimental units. The effects of the graded levels of FKO were evaluated for linear and quadratic trends using orthogonal polynomial contrasts. Tukey’s test was used to compare mean differences. The data variation was confirmed based on the pooled standard error of the mean (SEM), with p < 0.05 as the level of significance.

Results

GC-MS analysis of FKO

Our recent publication reported the main constituents of FKO obtained using GC-MS analysis (Amer et al. 2023). The main bioactive compounds in FKO included 1,6,10-DODECATRIEN-3-OL, 3,7,11-TRIMETHYL-, [S-(Z)]- (12.42%), ç-Elemene (12.42%), à-Farnesene (12.42%), PHENOL, 2,4-BIS(1,1-DIMETHYLETHYL)- (7.15%), 10-Undecyn-1-ol (3.50%), 2-Furanmethanol, 5-ethenyltetrahydro-à,à,5-trimethyl-, cis- (2.97%), Dodecanoic acid, 2,3-bis(acetyloxy)propyl ester (2.44%), 1,3-Butanediol, 2TMS derivative (2.36%), (4-ISOPROPENYL-1-CYCLOHEX EN-1-YL) METHANOL (2.08%), and d-Mannose, 2,3,4,5,6-pentakis-O-(trimethylsilyl)-o-methyloxyme, (1E)- (2.06%) (Table 2).

Table 2. The retention time (RT) and peak area (%) of the chemical constituents contained in frankincense oil (FKO), as evidenced by GC-MS.

Compound	RT (min)	Peak area %	Molecular formula	Molecular weight
1,6,10-dodecatrien-3-OL, 3,7,11-Trimethyl-, [S-(Z)]-	43.53	12.42	C15H26O	222
à-Farnesene	43.53	12.42	C15H24	204
Phenol,2,4-bis(1,1-dimethylethyl)-	17.78	7.15	C14H22O	206
2,4-Di-tert-butylphenol	17.78	7.15	C14H22O	206
10-Undecyn-1-ol	30.56	3.50	C11H20O	168
2-Furanmethanol,5-ethenyltetrahydro-à,à,5-trimethyl-,cis-	29.97	2.97	C10H18O2	170
Dodecanoic acid,2,3-bis(acetyloxy)propyl ester	27.91	2.44	C19H34O6	358
1,3-Butanediol, 2TMS derivative	33.07	2.36	C10H26O2Si2	234
(4-Isopropenyl-1-Cyclohexen-1-Yl)methanol	27.48	2.08	C10H16O	152
d-Mannose, 2,3,4,5,6-pentakis-O-(trimethylsilyl)-, o-methyloxyme, (1E)-	26.13	2.06	C22H55NO6Si5	569
Silane, (butoxymethyl)trimethyl-	26.13	2.06	C8H20OSi	160
2,3-Butanediol	33.55	1.81	C4H10O2	90
4′-Hydroxy-4-methoxy-2′-methylchalcone	36.37	1.59	C17H16O3	268
Carbitol, TBDMS derivative	25.95	1.57	C12H28O3Si	248
Benzeneethanamine, N,à-dimethyl-	34.11	1.54	C10H15N	149
Thiocyanic acid	34.11	1.54	CHNS	59
Octadecanoic acid,trimethylsilyl ester	37.43	1.51	C21H44O2Si	356
Guanosine 3′,5′-cyclicmonophosphate	37.43	1.51	C10H12N5O7P	345
Furazolidone	35.73	1.51	C8H7N3O5	225
Propanoic acid, ethyl ester	36.56	1.51	C5H10O2	102
4-Methylcinnamic acid	37.14	1.50	C10H10O2	162
GLYCINE	37.71	1.41	C2H5NO2	75
3-Nonyn-1-ol	30.35	1.30	C9H16O	140
Silane, trimethyl(1-phenylethyl)-	48.50	1.18	C11H18Si	178
L-THREONINE	38.44	1.17	C4H9NO3	119
Idebenone	18.63	1.16	C19H30O5	338
Z-THUJENOL	18.63	1.16	C10H16O	152
2′,4′-Dihydroxy-4-methoxychalcone	38.04	1.14	C16H14O4	270
6,9,12-Octadecatrienoic acid,phenylmethyl ester, (Z,Z,Z)-	30.65	1.08	C25H36O2	368
L-(-)-Fucose	38.22	1.06	C6H12O5	164
1,3-Butanediol, 2TMS derivative	48.40	1.04	C10H26O2Si2	234
S-Methyl-L-cysteine	34.38	1.04	C4H9NO2S	135
Arachidonyl trifluoromethyl ketone	35.47	0.94	C21H31F3O	356
Sedoheptulosan	34.17	0.84	C7H12O6	192
Heptadecanoic acid, 16-methyl-,methyl ester	34.53	0.63	C19H38O2	298

Growth performance

Table 3 displays the growth parameter data. The various levels of FKO had minimal impact on BW, BWG, FCR, or FI during the starter period compared to the FKO0 treatment (p > 0.05). During the grower period, BW and BWG increased in birds given FKO200, followed by FKO600 (p ≤ 0.01) during the grower period. FCR and FI showed non-significant change among different FKO treatments during the grower period compared to FKO0 (p > 0.05). FKO600 treatment increased BW and decreased the FCR (p < 0.05) during the finisher period compared to FKO0. All FKO levels did not affect the average BWG, and FI did not differ significantly across all FKO levels compared to FKO0 (p > 0.05). However, the different FKO levels linearly increased the BW and BWG (p < 0.05) without affecting the total FI over the entire period. Overall, FCR was reduced in the FKO600 group (linear p ≤ 0.01). Compared to the FKO0 group, FKO supplementation at 600 mg Kg ⁻¹ enhanced the PER and RGR (linear, p < 0.05).

Table 3. The impact of dietary frankincense oil (FKO) supplementation on the growth of broiler chickens.

Variables	FKO 0 mg/kg	FKO 200 mg/kg	FKO 400 mg/kg	FKO 600 mg/kg	SEM	P-values
						ANOVA	Linear	Quadratic
IBW (g)	100.56	100.63	100.00	100.42	0.14	0.43	0.42	0.55
Starter period
BW(g)	328.33	338.54	317.08	331.04	3.12	0.08	0.56	0.71
BWG(g)	227.78	237.92	217.08	230.63	3.10	0.10	0.60	0.74
FI (g)	269.63	270.63	256.46	264.58	2.54	0.18	0.18	0.45
FCR	1.18	1.14	1.18	1.15	0.01	0.60	0.66	0.85
Grower period
BW(g)	1169.70^b	1269.67^a	1224.44^ab	1260.22^a	13.45	0.01	0.01	0.07
BWG(g)	841.37^b	931.13^a	907.36^a	929.18^a	12.39	0.01	< 0.01	0.04
FI (g)	1201.85	1212.29	1121.46	1172.08	17.77	0.29	0.26	0.56
FCR	1.43	1.30	1.24	1.26	0.03	0.08	0.03	0.15
Finisher period
BW(g)	1888.89^b	2121.73^ab	2009.56^ab	2146.67^a	38.81	0.04	0.03	0.41
BWG(g)	719.19	852.06	785.12	886.44	27.55	0.13	0.07	0.74
FI(g)	1483.33	1602.08	1447.92	1450.00	31.31	0.28	0.36	0.35
FCR	2.062^a	1.878^ab	1.849^ab	1.656^b	0.05	0.03	0.01	0.95
Overall performance
BW(g)	1888.89^b	2121.73^ab	2009.56^ab	2146.67^a	38.81	0.04	0.03	0.41
BWG(g)	1788.33^b	2021.10^ab	1909.56^ab	2046.25^a	38.83	0.04	0.03	0.41
FI (g)	2954.81	3085.00	2825.83	2886.67	47.23	0.26	0.27	0.70
FCR	1.65^a	1.53^ab	1.48^ab	1.42^b	0.03	0.02	< 0.01	0.48
PER	2.90^b	3.15^ab	3.23^ab	3.39^a	0.07	0.03	0.01	0.60
RGR	994.72^b	1111.18^ab	1054.78^ab	1123.54^a	19.39	0.04	0.03	0.41

SEM = total standard error of means (n = 10).

^a,b,c Means with different superscripts are significantly different (p < 0.05). initial body weight- (IBW); body weight- (BW); body weight gain (BWG); feed intake- (FI); feed conversion ratio- (FCR); protein efficiency ratio- (PER); relative growth rate-(RGR).

Blood biochemical markers

In comparison to the control group (FKO0), FKO had a higher blood concentration of ALT at 600 mg kg⁻¹ (53.4%) (linear p = 0.01). Additionally, creatinine levels increased linearly at FKO600 (9.01%) (p < 0.01) when compared to the FKO0 group; however, no significant changes were observed in AST and uric acid levels (Table 4).

Table 4. The impact of dietary frankincense oil (FKO) supplementation on broiler chickens’ hepatic and renal function variables.

Variables	FKO0 mg/kg	FKO 200 mg/kg	FKO400 mg/kg	FKO 600 mg/kg	SEM	P-Values
						ANOVA	Linear	Quadratic
ALT (U/L)	5.00^b	6.67^ab	7.00^ab	7.67^a	0.38	0.05	0.01	0.39
AST (U/L)	47.67	53.00	52.33	54.67	1.53	0.47	0.18	0.64
Creatinine (mg/dL)	1.11^b	1.16^ab	1.15^ab	1.21^a	0.01	< 0.01	< 0.01	1.00
Uric acid (mg/dL)	2.50	2.57	2.77	2.65	0.05	0.24	0.14	0.32

SEM total standard error of means (n = 10).

^a,bMeans with different superscripts significantly differ (p < 0.05). Aspartate aminotransferase (AST); Alanine aminotransferase (ALT).

Serum antioxidant activities and inflammatory responses

Table 5 displays data regarding serum antioxidant capacity, lipid peroxidation, and inflammatory markers in broilers. Dietary supplementation with FKO at 200, 400, and 600 mg kg⁻¹ significantly diminished the MDA content (linear and quadratic, p < 0.01) by 40.92%, 53.98%, and 50.64%, respectively, but significantly increased TAC serum enzyme activity by 12.62%, 27.69%, and 34.25%, respectively compared to the FKO0 group (linear, p < 0.01). FKO supplementation at 400 and 600 mg Kg⁻¹ significantly increased CAT levels by 73.98%, 97.49%, and SOD levels by 13.97%, and 19.27%, respectively compared to the FKO0 group (linear, p < 0.01). The FKO200 group did not significantly differ from the FKO0 group (p > 0.05). IL-1β demonstrated a linear increase (p < 0.01) with FKO supplementation at 200, 400, and 600 mg kg⁻¹ by 9.03%, 12.27%, and 18.06%, respectively, compared to the FKO0 group. Additionally, there were linear (p < 0.01) and quadratic (p = 0.05) increases in INF-γ levels in all FKO-supplemented groups (55.68%, 86.96%, and 106.59%, respectively) compared to the FKO0 group.

Table 5. The impact of dietary frankincense oil (FKO) supplementation on broiler chickens’ serum antioxidant activity and inflammatory responses.

Variables	FKO0 mg/kg	FKO 200 mg/kg	FKO 400 mg/kg	FKO 600 mg/kg	SEM	P-Values
						ANOVA	Linear	Quadratic
TAC (U/mL)	10.22^c	11.51^b	13.05^a	13.72^a	0.42	0.000	< 0.01	0.22
CAT (U/mL)	3.19^c	4.16^bc	5.55^ab	6.30^a	0.40	0.002	< 0.01	0.77
SOD (U/mL)	135^c	145^bc	154^ab	161^a	3.14	0.001	< 0.01	0.58
MDA (nmol/mL)	6.28^a	3.71^b	2.89^b	3.10^b	0.43	0.000	< 0.01	< 0.01
IL1β ug/mL	144^c	157^b	161.67^b	170^a	2.90	0.000	< 0.01	0.14
IFN- γ (pg/mL)	7.13^c	11.10^b	13.33^ab	14.73^a	0.90	0.000	< 0.01	0.05

SEM: total standard error of means (n = 10).

^a,b,c Means with different superscripts are significantly different (p < 0.05). total antioxidant capacity-(TAC); catalase-(CAT); superoxide dismutase-(SOD); malondialdehyde-(MDA); interleukin 1 beta-(IL1β); interferon-γ- (IFN-γ).

Lipid profile

Serum TC, TG, and LDL levels declined linearly and quadratically in broilers fed diets with FKO at concentrations of 200, 400, and 600 mg kg⁻¹ (p < 0.05). Serum HDL-C augmented linearly (p < 0.01) in FKO400 (8.04%) and FKO600 (11.06%) groups compared to the FKO0 group, but no significant difference was observed between the FKO200 and FKO0 groups. FKO supplementation levels had no significant influence on VLDL-C (p > 0.05) (Table 6).

Table 6. The impact of dietary frankincense oil (FKO) supplementation on serum lipid profile.

Variables	FKO0 mg/kg	FKO 200 mg/kg	FKO 400 mg/kg	FKO 600 mg/kg	SEM	P-values
						ANOVA	Linear	Quadratic
TC (mmol/L)	3.56^a	3.39^b	3.42^b	3.41^b	0.02	0.01	0.01	0.02
TG (mmol/L)	1.31^a	1.19^b	1.18^b	1.16^b	0.02	<0.01	<0.01	0.01
LDL-C (mmol/L)	1.35^a	1.06^b	1.03^b	0.96^b	0.05	<0.01	<0.01	0.03
VLDL-C (mmol/L)	0.22	0.23	0.23	0.23	0.00	0.66	0.29	0.57
HDL-C (mmol/L)	1.99^b	2.10^ab	2.15^a	2.21^a	0.03	<0.01	<0.01	0.43

SEM: total standard error of means (n = 10).

^a,bMeans with different superscripts significantly differ (p < 0.05). Total cholesterol-(TC); Triglycerides-(TG); Low-density lipoprotein cholesterol-(LDL-C); High-density lipoprotein cholesterol-(HDL-C); Very low-density lipoprotein cholesterol-(VLDL-C).

Histopathological responses

Histological examination of liver sections from the four experimental groups, which received FKO at doses of 0, 200, 400, and 600 mg kg⁻¹, revealed normal hepatic histomorphology. The FKO600 group showed mild to medium portal lymphoplasmacytic aggregations, and biliary proliferation was observed. In the FKO200 and FKO400 groups, the presence of inflammatory cells was minimal. The accumulation of portal inflammatory cells appears to be a defensive and immunomodulatory mechanism rather than a harmful inflammatory process (Figure 1).

Figure 1. Photomicrographs of liver sections from chickens administered frankincense oil (FKO) at 0 mg kg⁻¹ (A), 200 mg kg⁻¹ (B), 400 mg kg⁻¹ (C), and 600 mg kg⁻¹ (D) exhibited typical histomorphological structures, like portal area (PA, yellow arrow), hepatocytes (HC, black arrow) which are perceived as a small masses around the Central veins (CV), a few round cells are seen as a natural immune response around the portal area (PA, arrow). mild to moderate portal lymphoplasmacytic aggregations (red arrows) and biliary proliferation (BD, yellow arrow) were seen in the FKO600 group. Each group with two magnification powers; H&E × 100, × 400.

Immunohistochemical analysis and morphometric measures

Morphometric analysis of liver sections in the different experimental groups revealed the presence of positive cells per 3 high power areas for the pro-inflammatory determinant (TGF-β) at the following proportions: 0.70%, 0.54%, 1.17%, and 1.46% in the FKO0, FKO200, FKO400, and FKO600 groups, respectively (Figure 2). Meanwhile, spleen sections showed positive cells per 3 high power areas for TGF-β as follows: 0.50%, 1.72%, 1.73%, and 2.77% in the FKO0, FKO200, FKO400, and FKO600 groups, respectively (Figure 3).

Figure 2. The liver sections were immunostained for TGF-β positive cells (shown as red arrows) in the four experimental groups: (A) frankincense oil (FKO)0, (B) FKO200, (C) FKO400, and (D) FKO600. × 400 magnification power. (E) Quantitative analysis of the morphological features is as follows: 0.70%, 0.54%, 1.17%, and 1.46% for FKO0, FKO200, FKO400, and FKO600 groups, respectively.

Figure 3. The spleen sections were immunostained for TGF-β positive cells (showed as red arrows) in the four experimental groups: (A) frankincense oil (FKO)0, (B) FKO200, (C) FKO400, and (D) FKO600. × 400 magnification power. (E) Quantitative analysis of the morphological features is as follows: 0.50%, 1.72%, 1.73%, and 2.77% for FKO0, FKO200, FKO400, and FKO600 groups, respectively.

Discussion

The principal bioactive components identified in FKO through GC-MS analysis in this study were 1,6,10-DODECATRIEN-3-OL (12.42%), 3,7,11-TRIMETHYL-, [S-(Z)]-(12.42%), ç Elemene (12.42%), à-Farnesene (12.42%), PHENOL, 2,4-BIS(1,1 DIMETHYLETHYL)- (7.15%), 10-Undecyn-1-ol (3.50%), 2-Furanmethanol, 5 ethenyltetrahydro-à,à,5-trimethyl-, cis- (2.97%), Dodecanoic acid, 2,3 bis(acetyloxy)propyl ester (2.44%), 1,3-Butanediol, 2TMS derivative (2.36%), (4 ISOPROPENYL-1-CYCLOHEX EN-1-YL)METHANOL (2.08%) and d-Mannose, 2,3,4,5,6-pentakis-O-(trimethylsilyl)- o-methyloxyme, (1E)- (2.06%). These active components are responsible for the antioxidant, anti-inflammatory, immunomodulatory, hypolipidemic, and hepatoprotective properties of FKO studied in this study.

The current study demonstrated that FKO supplementation (FK200, FK400, and FK600 mg kg⁻¹) positively affected the final BW, BWG, and FCR of broiler chickens. However, it did not affect FI when compared to the FK0. The FKO600 group outperformed the others regarding FI, weight gain, and FCR. The presence of Boswellia in the digestive system is thought to be a stimulant, aid in gas removal, support appetite, and encourages the flow of gastric juices leading to an improvement in digestion and absorption by stimulating pancreatic enzyme secretion (Rao et al. 2001). The improvement in growth performance observed in this study may be attributed to the presence of components such as 2,4-Di-tert-butyl phenol which enhances food safety and promotes health properties (Varsha et al. 2015), and Furazolidone which is used as a feed additive with bacteriostatic and growth-promoting properties (Ahmed et al. 2012).

In addition, the observed improvement in performance may be attributed to the antimicrobial effects of FKO, which reduce microbial nutrient utilization such as protein and energy in the host, leading to enhanced growth and reduced ammonia production (Dibner and Buttin 2002; Chevrier et al. 2005). FKO's antimicrobial effects in the current study could be attributed to the presence of 1,6,10-Dodecatrien-3-ol, 3,7,11-trimethyl- (12.42%), PHENOL, 2,4-BIS(1,1-DIMETHYLETHYL)- (7.4%), Benzeneethanamine, N,à-dimethyl-(1.54%), 4-Methylcinnamic acid (1.50%), Furazolidone (1.51%), and 6,9,12-Octadecatrienoic acid, phenylmethyl ester, (Z,Z,Z)- (1.08%) (Ahmed et al. 2012; Sova 2012; Devi and Kottai Muthu 2015, Abubakar and Majinda 2016; Ren et al. 2019, Thanga Krishna and Mohan 2012). Similarly, Mohamed et al. (2021) reported that the addition of BS at 2.0–2.5% in diets increased broiler growth compared to control chickens. It had been reported that Boswellia carterii and BS improved body mass, weight gain, feed utilization, and the economic productivity indicator throughout the experiment, particularly in broilers provided with 3g BS/L in water supplies (Al-Yasiry, Jawad, Menati, Naji and Lokman 2016).

The present analyses revealed that ALT was linearly elevated in FKO-treated groups compared to the control group on a scale manner; AST failed to indicate any variations between treatments. However, the scores remain within the body’s normal range (Joshua et al. 2022; Khalesi et al. 2022). The histological examination of the liver in this study confirmed the absence of hepatotoxicity throughout the 35 days. The FKO600 group exhibits very little lymphoplasmacytic clustering (inflammatory cells) and slight portal vascular distention. The study by Hakkim et al. (2019) showed that the groups treated with frankincense essential oil exhibited minimal injury with intact cytoplasm and nucleus and appeared to have normal hepatocytes. BS extracts encourage liver regeneration and retard the onset and progression of hepatic fibrosis (Khan et al. 2014).

Uric acid is the primary byproduct of nitrogen metabolism in birds. Age, sex, and nutrition strongly influence the uric acid content of plasma in birds (Al-Yasiry, Kiczorowska, Samolińska, et al. 2017). The current study found no significant differences in uric acid levels among the groups. However, creatinine concentration increased linearly with FKO supplementation, with the highest level observed in the FKO600 group. Nevertheless, these values remained within the body’s normal range (Café et al. 2012).

The present study demonstrated a significant reduction in TC, TG, and LDL-C and a marked improvement in HDL-C in all FKO-supplemented groups compared to the unsupplemented group. Lower TC, TG, and LDL-C concentrations may reflect the hypocholesterolemic properties of FKO, which may be attributed to 6,9,12-Octadecatrienoic acid, phenylmethyl ester, (Z,Z,Z)- (Rehana and Nagarajan 2013), 4-Methylcinnamic acid (Das et al. 2006), S-Methyl-L-cysteine (G et al. 2013), and L-threonine (Jiang et al. 2017). FKO's lipid-lowering effects in the current study may be due to its inhibitory effect on the microsomal acyl-coenzyme A, a cholesterol acyltransferase enzyme that is incorporated in the acylation of cholesterol to cholesterol esters in the liver (Hakkim, et al. 2019). In addition, Tripathi et al. (2004) suggested that the intake of BS helps restore cell function for insulin secretion, which consequently aids in lowering serum lipid profiles. A previous study showed that the Boswellia water-soluble fraction reduces overall cholesterol levels (Pandey, Singh and Tripathi 2005).

Maintaining balanced redox homeostasis by preserving the equilibrium between oxidation and anti-oxidation is crucial for broiler health. In the present study, FKO diet supplementation at 200, 400, and 600 mg kg⁻¹ linearly reduced the MDA content while increasing TAC, CAT, and SOD serum enzyme activities compared to the control group. Antioxidants, radical reducing agents, glutathione content regulators, cell membrane preservatives, and cell permeation control systems are some of the cellular mechanisms of Boswellia species (Mahdian et al. 2020). SOD and TAC values levels were linearly enhanced in birds fed a diet containing 0.5, 1, and 1.5 g BS/Kg, resulting in a decrease in MDA compared to the control group (Montaser et al. 2021). Furthermore, Altmann et al. (2004) suggested that the antioxidant properties of Boswellia carterii may have a beneficial impact on cells.

FKO's antioxidant activity is mainly due to its composition, which includes components such as phenol-2, 4-bis (1, 1-dimethylethyl), 2,4-Di-tert-butyl phenol, 1,3-butanediol, 2-Furanmethanol, à-Farnesene, 5-ethenyltetrahydro-à,à,5-trimethyl-,cis-, L-Fucose, glycine, Hexadecanoic acid, idebenone, and Sedoheptulosan, which have been proven to possess antioxidant activity (ÇELİK et al. 2014; El-Shitany et al. 2015; Gueven et al. 2015, Altameme et al. 2016; Park et al. 2013; Pérez-Torres et al. 2017, Ren, Wang, Karthikeyan, Liu and Cai 2019; Swaminathan et al. 2021; Younis and Saleh 2021; Nilsson et al. 2022; Varsha et al. 2015).

The results of the current study demonstrated that increased levels of FKO supplementation led to higher inflammatory responses, as evidenced by elevated serum levels of pro-inflammatory mediators (IL-1β and IFN-γ) and a moderate rise in hepatic and splenic TGF-β immune expression. This suggests that FKO stimulates innate immunity in birds by promoting the generation of pro-inflammatory mediators that can evoke inflammatory reactions in birds. These findings are consistent with the histological evaluation of hepatocytes, which revealed mild lymphoplasmacytic clustering (inflammatory cells) and slight portal vascular distension at the highest FKO levels (600 mg Kg⁻¹). Tumor necrotic factor-α (TNF-α) and IL-1β are pro-inflammatory mediators crucial in the inflammatory process. Inflammatory mediators perform an essential part in the management of both acute and chronic inflammation (Shehata et al. 2011). TNF-α- and IL-1β can initiate inflammatory responses and activate other inflammatory cytokines, which directly affect critical cell incidents such as genetic expression, DNA damage, and cell growth, contributing to various inflammatory disorders (Al-Yasiry and Kiczorowska, 2016a). Inflammation is a defence mechanism involving the immune system, circulatory system, and adhesion molecules. The inflammatory process aims to eliminate the underlying causes of cellular damage, necrotic cells, and tissues destroyed by the injury and inflammation and promote tissue regeneration (Han et al. 2017). We hypothesize that these responses represent a defence mechanism and immune modulation rather than a harmful inflammatory process, as the growth of the birds was improved by FKO supplementation and not adversely affected. Our previous study confirms this hypothesis as we reported the immunomodulatory effects of FKO in a level-dependent manner (200, 400, 600 mg kg⁻¹) (Amer et al. 2023). In addition, these reactions may be influenced by the vaccination program administered to the chickens during the experimental period, as the modulated immune responses were higher in the FKO-supplemented groups than in the control group. FKO's immunostimulant effect could be attributed to biologically active compounds such as L-threonine, glycine, L-Fucose, à-Farnesene, and d-Mannose, 2,3,4,5,6-pentakis-O-(trimethylsilyl)-, o-methyloxyme, (1E)- (Pérez-Torres et al. 2017; Zhang et al. 2017; Yoo et al. 2019; Kim et al. 2022; Schepetkin et al. 2022).

While some studies suggest a pro-inflammatory role for TGF-β' in disease, it is important to note that TGF-β can also exhibit anti-inflammatory properties (Ashrafizadeh et al. 2020). Theacrine is an anti-inflammatory agent, preventing synovial enlargement and accumulating inflammatory cells in joints. This anti-inflammatory effect is mediated by TGF-β inducement and subsequent overexpression of the Smad protein (Gao et al. 2020). The anti-inflammatory effect of FKO may be attributed to various components present in it, including 1,6,10-Dodecatrien-3-ol, 3,7,11-trimethyl, 3-Nonyn-1-ol, à-Farnesene, Arachidonyl trifluoromethyl ketone, Guanosine 3′,5′-cyclic monophosphate, glycine, hexadecanoic acid, and Sedoheptulosan (Park et al. 2013; Devi and Kottai Muthu 2015; El-Shitany et al. 2015; Khan et al. 2015; Pérez-Torres et al. 2017; Tatipamula et al. 2019; Nguyen et al. 2022; Schepetkin et al. 2022).

Conclusion

Based on the findings of this study, it can be concluded that FKO can be added to broiler chicken diets as a natural growth promotor for enhancing growth up to 600 mg kg⁻¹. The addition of FKO200–600 to the diet had a hypolipidemic effect by reducing TC, TG, and LDL-C, while increasing HDL-C. FKO supplementation can enhance antioxidant capacity by elevating TAC, CAT, and SOD activity while reducing MDA concentration in a level-dependent manner. Dietary FKO (200–600 mg Kg⁻¹) linearly modulated broiler chickens’ immune responses, evidenced by elevated levels of serum IL-1β and IFN-γ and upregulated TGF-β immunoexpression. FKO addition had no harmful effect on hepatic tissues or blood biochemistry. It is recommended to add FKO at a level of 600 mg kg⁻¹ to improve the growth, antioxidant, and immune status of the birds. However, further studies are needed to investigate the effects of higher levels of FKO on broiler growth and health.

Ethical approval

The Ethical approval of the experimental protocol was obtained from the Institutional Animal Care and Use Committee of Zagazig University, Egypt (Approval No. ZU-IACUC/2/F/152/2022). All animal experiments were conducted in compliance with the recommendations outlined in “The Guide for the Care and Use of Laboratory Animals in scientific investigations”, the study adhered to the relevant institutional guidelines, and all animal experiments were performed following the ARRIVE guidelines.

Disclosure statement

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

Data availability statement

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

Additional information

Funding

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