Effect of Dietary Supplementation with Methyl Salicylate on Performance, Blood Biochemistry, Intestinal Morphology, and Meat Quality of White-Feather Broilers

Abstract

This study aimed to enhance meat quality and intestinal health of white-feathered broilers by utilizing methyl salicylate (MS), a fragrant plant-derived essential oil, for its anti-inflammatory, antibacterial, and antiplatelet properties. Specifically, we investigated the effects of dietary MS supplementation on the growth performance, carcass characteristics, intestinal morphology, and blood biochemistry and breast meat quality. A total of 270 one-day-old white-feather broilers were randomly assigned to three dietary treatments: a basal diet (CON), a basal diet supplemented with methyl salicylate at a low dosage of 0.25 g/kg (MSL), or at high dosage of 0.5 g/kg (MSH). Dietary MS treatment did not significantly affect the growth performance of broilers. However, dietary methyl salicylate supplementation significantly increased the blood levels of high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), total cholesterol (Tchol), and total antioxidant capacity (T-AOC) (P < 0.05). High-dose methyl salicylate supplementation also increased villus height in the jejunum (P < 0.05). Furthermore, methyl salicylate supplementation improved the a* value (redness) of breast muscle (P < 0.05). Additionally, the addition of methyl salicylate supplementation significantly retarded the increase in thiobarbituric acid (TBA) and total volatile basic nitrogen (TVB-N) levels in breast muscle (P < 0.05), potentially benefiting long-term meat preservation. Moreover, methyl salicylate increased the content of monounsaturated fatty acids in breast meat (P < 0.05). Consequently, our findings indicate that dietary supplementation with MS enhances meat quality and may extend the shelf life of white-feathered broilers by improving blood lipid parameters and modifying muscle fatty acid profiles.

Introduction

China is the world’s second-largest producer of broiler meat, with an annual output exceeding 20 million tons. Poultry meat, particularly breast meat, is widely favored by Chinese households because of its affordability, low-fat content, and high protein value[1]. However, the emergence of antimicrobial resistance has restricted the use of antibiotic growth promoters in animal production, creating a critical need for effective alternatives[2]. Consequently, maintaining animal health, improving feed efficiency, and reducing mortality without the use of antibiotics has become urgent challenges for the livestock and poultry industries.

Plant extracts obtained from botanical sources through physical and chemical methods represent important reservoirs of natural bioactive compounds. Their biological activities are primarily attributed to secondary metabolites, such as polyphenols, flavonoids, and terpenoids[3], which exert antioxidant, anti-inflammatory, antimicrobial, and cell-signaling modulatory effects[4]. Woody plants are an abundant and sustainable local resource in China. Their bioactive extracts have demonstrated considerable potential as effective alternatives to in-feed antibiotics for enhancing livestock productivity and health[5].

Wintergreen oil, distilled from the leaves of Gaultheria species, exhibits inhibitory effects against Gram-negative bacteria and modulates the expression of inflammatory cytokines such as IL-6 and TNF-α, thereby exerting anti-inflammatory activity[6,7]. Methyl salicylate (MS), the primary component of wintergreen oil, accounts for approximately 98% of its composition[8]. Importantly, MS can now be chemically synthesized at low cost, suggesting its potential as a practical alternative to natural wintergreen oil. After ingestion, MS, aspirin, and sodium salicylate are metabolized into salicylic acid in the intestine. Notably, aspirin reduces the incidence of wooden-breast myopathy in broilers, which is associated with vascular inflammation and hypoxia induced by rapid growth[9]. Furthermore, both aspirin and salicylates inhibit the proliferation of pathogenic bacteria in a dose-dependent manner[10] and function as antioxidants via single-electron transfer mechanisms[11]. Sodium salicylate also acts as a chemical trap for hydroxyl radicals and alleviates hypoxia/reoxygenation

(H/R) injury in various tissues, directly demonstrating its ability to mitigate oxidative damage[12].

The antioxidant capacity of MS is critically related to meat quality. In muscle tissues, postmortem oxidative stress is a primary driver of quality deterioration, primarily through lipid peroxidation and protein oxidation[13]. These biochemical changes directly compromise key meat attributes, including color stability, water-holding capacity, and flavor profile[6]. By mitigating oxidative damage in vivo, MS may help preserve cellular integrity during the pre-slaughter phase, which could translate into improved postmortem meat quality[7]. Therefore, through its potential to modulate systemic oxidative status, MS supplementation is hypothesized to positively influence downstream meat quality parameters and sensory characteristics.

Building on this evidence, our previous research indicated that dietary supplementation with methyl salicylate at 40–50 mg/kg body weight improved growth performance, antioxidant capacity, and intestinal health in weaned pigs[14]. These findings suggest that MS exerts similar antimicrobial and antioxidant effects in broilers. This study aims to evaluate the effects of methyl salicylate supplementation on broiler health, feed utilization, and production performance, providing insights into its potential as a natural alternative to antibiotic growth promoters.

Materials and Methods

Ethical statement

All animal experiments were conducted in accordance with the Measures for the Administration of Experimental Animal Use in Zhejiang Province. This study was approved by the Animal Care and Use Committee of the Zhejiang University (ethics permit no. ZJU20250213).

Broilers and Diets

A total of 270 white-feather boilers were raised in stainless steel cages (120×120×120cm= width × length × height), naturally ventilated, free to eat and drink, the temperature was maintained at 34–36°C (1-7days), and gradually decreased to 24°C at a rate of 3°C per week until the end of the end of experiment. All boilers with similar body weight were randomly divided into 3 dietary treatments with six replicate groups (fifteen broilers were fed in each cage): a basal diet (CON), a basal diet supplemented with methyl salicylate at low dosage of 0.25 g/kg (MSL) or at high dosage of 0.5 g/kg (MSH).

The trial lasted 42 days and indicators such as feed intake, average daily gain (ADG), average daily feed intake (ADFI), and feed/gain ratio (FCR) were recorded every three days for each cage. The body weight of each bird was measured separately after a 12-h fast on the 21st day and 41th day. The basal diet formulation met the nutrient requirements (National Research Council, 1994; Ministry of Agriculture of China, 2006) and was applied during the starter period (0–21 d) and grower period (21–42 d) periods. The composition and nutritional levels of the basal diet are shown in Table 1. After 42 days of the experiment, eight broilers were randomly selected from each group, euthanized by cervical dislocation, and necropsied immediately to collect tissue samples from the breast meat, blood, and other organs.

Table 1.  Basic composition of diets and nutritional levels during starter and grower phases.

Ingredients, g/kg	Starter (1 to 21d)	Grower (22 to 42d)
Corn	600	620
Soybean meal	255	220
Rice bran	35	40
Corn gluten meal	35	30
Extruded soybean	25	-
By-product citric acid residue	-	20
Soy oil	5	27
Calcium hydrogen phosphate	11	10
Calcium carbonate	14	13
Wheat middlings	6.0	6.2
Salt	3.2	3.0
Choline chloride, 50%	1.0	1.0
Lysine hydrochloride, 98%	4.0	4.0
Methionine, 98%	2.0	2.0
Threonine, 98%	1.0	1.0
Antioxidant	0.2	0.2
Enzyme complex	0.3	0.3
Premix1	2.3	2.3
Nutrient level2
Digestive energy(kcal/kg)	2920	3080
Crude protein	20.50	18.50
Crude fat	4.20	6.20
Calcium	0.95	0.90
Total phosphorus	0.60	0.60
Fiber	2.70	2.90
Lysine	1.23	1.05
Methionine	0.55	0.50
Threonine	0.86	0.75

¹ Provided per kilogram of diet: Vitamin A, 12,000 IU; Vitamin D₃, 3000 IU; Vitamin E, 27 mg; Vitamin K₃, 4.5 mg; Vitamin B₁, 3 mg; Vitamin B₂, 9 mg; Vitamin B₆, 5.4 mg; Vitamin B₁₂, 30 μg, Niacin, 54 mg; Calcium pantothenate, 15 mg; Folate, 1.5 mg; Biotin, 0.6 mg; Mn, 100 mg; Zn 80 mg; Cu, 6 mg; Fe, 90 mg; Se 0.4 mg; I 0.6 mg.

² Digestible energy and metabolizable energy are calculated values, while the levels of other nutrients are measured.

Morphological Analysis

After removing the intestinal contents with pre-cooled saline, one-centimeter segments from the middle of the duodenum, jejunum, and ileum were collected and fixed in 10% neutral buffered formalin. Fixed tissues were subjected to graded ethanol dehydration, followed by xylene clearing and paraffin embedding. Subsequently, 3–5 μm-thick sections were cut using a microtome. For H&E staining, the sections were deparaffinized in xylene, gradually rehydrated by passing through decreasing concentrations of ethanol (100%, 95%, and 75%), and stained according to standard procedures.

After removing the intestinal contents with pre-cooled saline, one-centimeter segments from the middle of the duodenum, jejunum, and ileum was collected, immersed in 10% neutral buffered formalin, and embedded in paraffin. The waxed tissue was manually cut into 3μm-thick sections, and subjected to deparaffinization and dehydration. Sections were deparaffinized in xylene, rehydrated using a graded alcohol series (100%, 95%, and 75%), and stained using standard procedures. An optical microscope system (Olympus Corporation, Tokyo, Japan) was used to obtain micrographs at a magnification of 100×, and ImageJ software (National Institutes of Health, Bethesda, MD, USA) was employed for analysis. The villus height and crypt depth were measured using a previously described method[15]. The ratio of villus height to crypt depth (VH/CD) was also calculated.

Blood and Serum Biochemical Indices

Blood samples were collected in two tubes. Blood in K₂EDTA-containing anticoagulant tubes was kept at 4 °C prior to plasma separation. For serum preparation, blood in procoagulant tubes was allowed to clot at room temperature for 1 h and then centrifuged at 2,000 × g for 10 min at room temperature. The harvested serum samples were immediately aliquoted and frozen at −80 °C for subsequent analysis. Serum biochemical indices (ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALB, albumin; GLOB, globulin; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; TC, total cholesterol; TG, triglycerides; UA, uric acid; Urea; Ca, calcium; P, phosphorus; TP, total protein) were determined using an automatic XN-2000-A1 blood cell analyzer (SYSMEX, Kobe, Japan). Antioxidant parameters, including total antioxidant capacity (T-AOC), malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px), were determined following the manufacturer’s instructions using commercial kits. T-AOC (no. BC1315), SOD (no. BC5165), and MDA (no. BC0025) kits were obtained from Beijing Solarbio Science & Technology Co., Ltd., and GSH-PX (no. A005-1-2) kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

The red blood cell (RBC) count, hematocrit (HCT) value, white blood cell (WBC) count, platelet (PLT) count, thrombocytocrit (PCT) value, neutrophil (NEU) count, monocyte (MONO) count, eosinophil (EOS) count, basophil (BASO) count, and lymphocyte (LYM) count in the collected blood were determined using the ProCyte Dx™ hematology analyzer (IDEXX Laboratories, Westbrook, Maine, USA).

Measurement of Meat pH, Color and Drip Loss

Measurements of pH levels, meat color, drip loss, cooking loss, and sensory attributes were conducted on the right breast muscle stored at 4 °C for 1, 4, and 7 days of storage. Ground broiler samples were homogenized in 90 ml of distilled water and shaken at room temperature for 30 min. The mixture was filtered, and the pH was measured three times using an FE20 pH meter (Mettler Toledo, Zurich, Switzerland).

Meat samples were cut into uniformly sized pieces, wrapped in plastic film, and oxygenated at 1–2°C for 30 min. The colorimeter was calibrated using a white standard board before measurement. Six different areas of each sample were randomly selected, and the L*, a*, and b* values were determined three times using a CR-400 chromaticity meter (Hangzhou Ke Sheng Instrument Co., Ltd., Hangzhou, China).

For drip loss determination, meat samples were removed from storage and the surface moisture was wiped off. The initial weight (W₁) and the weight at different storage intervals (W₂) were recorded. Drip loss (%) was calculated as: Drip loss (%) = [(W₁₋W₂)/W₁ ×100%]

Cooking loss was determined by weighing a meat sample (W3) of uniform size, placing it in a cooking bag, and heating in a water bath for 15 min. The samples were then cooled to room temperature to record the final weight (W4) after removing surface moisture. Cooking loss (%) was calculated as follows: cooking loss (%) = (W3−W4) / W3 × 100%.

Meat sensory evaluation

Sensory evaluation of chicken breast samples was conducted on days one, four, and seven after sampling. A panel of ten trained researchers assessed the samples based on five attributes: color, water loss, stickiness, elasticity, and odor. The sensory evaluation results were recorded on a 20-point scale. The overall sensory score was derived from the composite assessment. Sensory evaluation of the chicken breast tissue was performed according to reported literature[16].

Determination of TVB-N and TBA

According to the Chinese Food Safety Standard (GB 166 5009.228–2016), the total volatile basic nitrogen (TVB-N) was determined using the steam distillation method. Ground broiler meat (10 g) was homogenized in 90 mL of distilled water and stirred for 30 min. The mixture was passed through filter paper, and the filtrate was collected. The filtrate was then treated using a FOSS Kjeldahl 8400 automatic nitrogen analyzer (FOSS, Denmark) to perform steam distillation.

The thiobarbituric acid reactive substance (TBARS) detection method was used to evaluate the degree of lipid oxidation in the broiler meat samples. A 10 g sample was homogenized with 50 mL of 7.5% (w/v) trichloroacetic acid (TCA) solution, shaken at 50 °C for 30 min, and centrifuged at 4000 ×g for 15 min. The supernatant was mixed with 0.02 M thiobarbituric acid (TBA) (1:1, v/v) and incubated in a water bath at 95 °C for 30 min. After cooling, the absorbance was measured at 532 nm, and TBARS values were expressed as milligrams of malondialdehyde (MDA) equivalents per kilogram of meat (mg MDA·kg⁻¹).

Fatty Acids Analysis

Muscle samples were freeze-dried and stored under nitrogen at low temperature. Approximately 0.20 g of the freeze-dried sample was homogenized with 10 mL of extraction solvent (isopropanol:n-hexane, 3:2, v/v) and vortexed for 1 min. Subsequently, 4 mL of a sodium sulfate solution (66.8 g·L⁻¹) was added, and the upper hexane layer was collected. The extraction was repeated once, and the combined organic phases were evaporated under nitrogen to obtain total lipids. For fatty acid methyl ester (FAME) derivatization, the extracted lipids were reacted with 3 mL of sodium hydroxide–methanol solution (0.5 mol·L⁻¹) and 100 μL of internal standard (C19:0 methyl ester) at 55°C for 30 min under nitrogen atmosphere. Next, 2 mL of 14% boron trifluoride–methanol solution was added, and the mixture was incubated at 55°C for another 30 min. After cooling to room temperature, 2 mL water and 2 mL n-hexane were added. The mixture was vortexed for 1 min and the organic layer was collected. Hexane extraction was repeated, and the combined organic layers were evaporated under nitrogen. The residue was reconstituted in 500 μL of n-hexane, filtered, and analyzed using a gas chromatograph (Agilent 7890B GC system, Agilent Technologies, Santa Clara, Ca, USA).

Statistical Analysis

Analyses were performed using the GraphPad Prism (version 9.5.1, GraphPad Software, Boston, MA, USA). A one-way analysis of variance (ANOVA) was performed, followed by Tukey’s multiple comparison test for normally distributed and homogeneous data. In cases of non-normal distribution or heterogeneous variances, the Kruskal–Wallis test with Dunn’s multiple comparison test was used. The data were expressed as mean ± standard error (SEM). Statistical significance was defined as P < 0.05, while P values between 0.05 and 0.1 were considered indicative of a trend.

Results

Growth Performance and Organ Index

The effects of methyl salicylate on broiler growth are shown in Table 2. During the starter period, birds with low-dose methyl salicylate supplementation tended to have increased feed conversion ratio (FCR) compared to the CON group (P =0.07). Throughout the trial, there were no significant differences (P > 0.05) among the three groups. There was no significant difference in the mortality rate of the broilers among the groups (P > 0.05). Compared to the CON group, the MSH group had a significantly increased heart index percentage (P < 0.05; Table 3). Different doses of MS supplementation in the diet did not affect other organ indices compared to the CON group (P > 0.05).

Table 2.  Effects of different doses of methyl salicylate on growth performance of broilers.

Items1	CON	MSL	MSH	SEM2	P-Value
Initial BW(g)	42.5	42.5	42.6	0.11	0.819
Final BW(g)	2406.5	2361.6	2397.3	34.87	0.353
Starter (1 to 21D)
ADFI (g)	58.6	60.1	58.8	0.89	0.658
ADG (g)	38.0	37.1	37.3	0.71	0.754
FCR (g/g)	1.545	1.621	1.58	0.02	0.072
Grower (22 to 42D)
ADFI(g)	146.3	147.5	146.2	1.63	0.604
ADG(g)	74.3	73.1	74.6	1.45	0.574
FCR(g/g)	1.974	2.023	1.961	0.04	0.427
Overall (1 to 42D)
ADFI (g)	103.5	104.9	103.5	1.10	0.620
ADG (g)	57.7	56.6	57.4	0.85	0.516
FCR (g/g)	1.799	1.856	1.804	0.03	0.207
Mortality rate (%)	2.22	2.22	4.45	1.678	0.585

^a–c Means within a row with different superscripts are significantly different at P < 0.05.

¹ BW: body weight; ADFI, average daily feed intake; ADG, average daily gain; FCR, feed conversion ratio.

² SEM: standard error of the mean.

Table 3.  Effects of different doses of methyl salicylate on the organ index of broilers.

Items	CON	MSL	MSH	SEM1	P-Value
Liver index	2.67	2.74	2.61	0.101	0.647
Spleen index	0.11	0.11	0.12	0.009	0.885
Heart index	0.45b	0.46b	0.53a	0.012	<0.001
Bursa of Fabricius index	0.17	0.19	0.17	0.015	0.625

^a–c Means within a row with different superscripts are significantly different at P < 0.05.

¹ SEM: standard error of the mean.

Serum Biochemical Indicators Level

Serum biochemical indicators of broilers are shown in Table 4. Compared to the CON group, methyl salicylate at both high and low doses increased the serum levels of high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and total cholesterol (TC) (P < 0.05). The ALT levels in the MSH group were significantly higher than those in the MSL group (P < 0.05). Dietary high-dose MS supplementation significantly decreased serum urea concentration compared to the CON group (P < 0.05), while serum phosphorus levels showed a decreasing trend without statistical significance (P = 0.09).

Table 4.  Effects of methyl salicylate supplementation on serum biochemical indicators levels of white feather broilers.

Items1	CON	MSL	MSH	SEM2	P-Value
ALT	3.50ab	3.00b	4.13a	0.248	0.018
AST	371.1	377.4	342.4	41.557	0.847
HDL-C	1.67b	1.98a	1.98a	0.065	0.003
LDL-C	1.10b	1.43a	1.38a	0.060	0.005
TC	2.69b	3.30a	3.21a	0.106	0.002
TG	1.12	1.03	0.99	0.092	0.602
TP	32.31	30.95	31.21	1.035	0.647
ALB	12.14	11.66	12.01	0.397	0.708
GLOB	20.18	19.29	19.20	0.740	0.621
Urea	1.13 a	1.03ab	0.81b	0.076	0.031
UA	238.0	235.8	268.6	21.159	0.472
Ca	2.58	2.68	2.50	0.060	0.364
P	2.67	2.47	2.39	0.085	0.078

^a–c Means within a row with different superscripts are significantly different at P < 0.05.

¹ ALT: alanine aminotransferase; AST, aspartate aminotransferase; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; TC, total cholesterol; TG, triglycerides; TP, total protein; ALB, albumin; GLOB, globulin; Urea: blood urea nitrogen; UA, uric acid; Ca, calcium; P, phosphorus.

² SEM: standard error of the mean.

Serum Antioxidant Capacity and Complete Blood Count Levels

Total antioxidant capacity (T-AOC) levels were significantly higher in the MSL group than in the CON group (P < 0.05; Table 5). Serum malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) levels were not significantly different between the groups (P > 0.05). The complete blood counts of the broilers are shown in Table 6. Different doses of MS supplementation in the diet did not affect red blood cell (RBC) count, platelet (PLT) count, or related indicators (P > 0.05). Compared with the CON group, dietary supplementation with low-dose MS tended to increase the WBC count, but the differences were not statistically significant (P =0.07). Specifically, the MONO and GRANS counts were higher in the MSL and MSH groups than in the CON group (P > 0.05).

Table 5.   Effects of methyl salicylate supplementation on antioxidant capacity in breast muscle of white feather broilers.

Items1	CON	MSL	MSH	SEM2	P-Value
T-AOC	1.516a	2.509b	2.198ab	0.166	0.011
SOD	12.01	17.12	22.66	4.775	0.324
MDA	5.46	4.95	5.01	0.693	0.854
GSH-Px	2663	2546	2784	108.12	0.345

^a–c Means within a row with different superscripts are significantly different at P < 0.05.

¹ T-AOC, total antioxidant capacity; SOD, superoxide dismutase; MDA, malondialdehyde; GSH-Px, glutathione peroxidase.

² SEM: standard error of the mean.

Table 6.  Effects of methyl salicylate supplementation on complete blood count levels of white-feather broilers.

Items1	CON	MSL	MSH	SEM2	P-Value
RBC	2.433	2.560	2.363	0.062	0.119
HGB	12.85	13.58	12.72	0.279	0.113
HCT	33.97	35.72	33.38	0.801	0.162
MCV	140.0	139.9	141.5	1.548	0.731
MCH	52.83	53.08	53.77	0.227	0.725
PLT	642.7	630.5	596.3	27.040	0.473
MPV	5.483	5.517	5.633	0.114	0.639
PCT	0.355	0.348	0.336	0.022	0.835
WBC	265.7	272.8	267.3	20.060	0.067
LYM	236.6	240.0	235.0	2.057	0.237
MONO	6.500	7.867	7.533	0.477	0.147
GRANS	22.60	24.92	24.80	1.019	0.231

^a–c Means within a row with different superscripts are significantly different at P < 0.05.

¹ RBC: red blood cell count; HGB, hemoglobin; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; PLT, platelet count; MPV, mean platelet volume; PCT, plateletcrit; WBC, white blood cell count; LYM, lymphocyte count; MONO, monocyte count; GRANS, granulocyte count.

² SEM: standard error of the mean.

Intestinal Morphology

The VH/CD ratio in the jejunum of chickens was enhanced by MS supplementation (P < 0.05; Table 7). Compared to the control group, the MSL and MSH groups showed significantly improvement in in villus height (VH) in the jejunum (P < 0.05). However, broilers fed different levels of MS exhibited no differences in the ileum compared to those fed the basal diet (P > 0.05).

Table 7.  Effects of methyl salicylate supplementation on intestinal morphology of white-feather broilers.

Items1	CON	MSL	MSH	SEM2	P-Value
Jejunum
VH	863.7a	1036b	1017b	48.810	0.019
CD	153.8	144.0	145.4	8.052	0.429
VH:CD	5.66	7.36	7.17	0.467	0.067
Ileum
VH	647.3	701.9	723.8	43.921	0.791
CD	136.0	137.1	155.5	6.099	0.287
VH:CD	4.75	5.14	4.68	0.248	0.562

^a–c Means within a row with different superscripts are significantly different at P < 0.05.

¹ VH, villus height; CD, crypt depth; VH:CD, villus height-crypt-depth ratio.

² SEM: standard error of the mean.

Quality of Breast Muscle

Compared to the control group, the overall sensory score of the MSH group was significantly higher (P < 0.05; Fig 1). The pH and cooking loss levels of broiler breast muscle at the three time points (1, 4, and 7 d) showed no significant differences among the groups (P > 0.05, Fig 2A, B). On the first day, the drip loss in the CON group was significantly higher than in the other groups (P < 0.05; Fig 2C). As time increased, all three groups displayed identical traits; the a-value gradually decreased, while the L-value and b-value gradually increased. On the first day, the MSH group had the highest a value among the three groups, which was significantly higher than that of the other groups (P < 0.05, Fig 2D, E, F). Meanwhile, the b-value was significantly lower in the MSH group than in the MSL group (P < 0.05). On the fourth day, there was no difference among the three groups in terms of flesh color (P > 0.05). On the seventh day, The MSH group had the lowest L-value compared to CON and MSL groups (P < 0.05). Moreover, compared to the CON group, the MSH group showed a significant decrease in the b-value (P < 0.05) and the numerically highest a-value.

Fig. 1.

Effects of methyl salicylate supplementation on sensory evaluation of broiler meat.

(A) Overall sensory scores of broiler meat samples on days 1, 4, and 7. (B) Five-dimensional radar plots of sensory attributes for meat samples assessed on days 1, 4, and 7. * Differences were considered significant at P < 0.05 (** P < 0.001, *** P < 0.0001, **** P < 0.00001).

Fig. 2.

Effects of methyl salicylate supplementation on meat quality of white-feather broilers. (A) pH; (B) Cooking loss; (C) Drip loss; (D) L value, (lightness); (E) a value, (redness); (F) b value, (yellowness); (G) thiobarbituric acid reactive substances (TBA); (H) total volatile basic nitrogen (TVB-N). Error bars represent standard error of the mean. * Differences were considered significant at P < 0.05 (** P < 0.001, *** P < 0.0001, **** P < 0.00001).

Oxidative Stability and Protein Degradation

During the three distinct periods, a significant reduction in thiobarbituric acid (TBA) levels was observed in both the MSL and MSH groups compared with the control group. Notably, TBA levels in the MSH group were significantly lower than those in the CON group after the first day (P < 0.05, Fig 2G). On the fourth day, the TBA level in the MSL group was significantly lower than that in the CON group (P < 0.05). Prior to the seventh day, there were no significant differences in total volatile basic nitrogen (TVB-N) levels among the three groups. However, on the seventh day, the TVB-N level in the MSH group was significantly lower than that in the CON group (P < 0.05; Fig 2 H).

Fatty Acid Profile of Breast Muscle and Correlation Analysis

Seventeen fatty acids were detected in the breast muscle of broilers. Compared to the control group, dietary supplementation with MS resulted in significant differences in the contents of the five fatty acids (P < 0.05; Table 8). Palmitic acid (C16:0) and stearic acid (C18:0) levels were lower in the MSL-fed group than in the control group. However, no significant differences were observed in other saturated fatty acids (SFA) between the groups (P > 0.05). Among the monounsaturated fatty acids (MUFA), the oleic acid (C18:1n9c) content was higher (P < 0.05) in the MSL group, whereas the content of palmitoleic acid (C16:1n7) was lower (P < 0.05). Among polyunsaturated fatty acids (PUFA), the essential fatty acid alpha-linolenic acid (C18:3n3) content was lower (P < 0.05) after MS supplementation than in the control group. Compared with the CON group, the MSL group had a significantly lower SFA content and significantly higher MUFA content (P < 0.05). Mantel and chord-graph correlations were used to analyze the correlation between meat quality and fatty acid composition of the breast muscle in broilers. The results indicated a correlation between the changes in pH and TBA (Fig. 3A) and the fatty acid composition on day 1 (P < 0.05). Furthermore, changes in a*, TVB-N, and cooking loss (Fig. 3B) correlated with fatty acid composition on day 7 (P < 0.05).

Table 8.  Effects of methyl salicylate supplementation on fatty acid composition in breast muscle of white-feather broilers (%).

Items1	CON	MSL	MSH	SEM2	P-Value
C10:0	0.24	0.08	0.30	0.085	0.061
C12:0	0.20	0.21	0.26	0.084	0.897
C14:0	0.35	0.40	1.25	0.229	0.589
C15:0	0.52	0.46	1.49	0.466	0.483
C16:0	24.61a	21.41b	21.62ab	0.721	0.012
C16:1n7	1.89a	1.73ab	1.39b	0.116	0.013
C17:0	0.19	0.11	0.15	0.033	0.274
C18:0	13.64a	11.72b	12.17ab	0.428	0.038
C18:1n9c	16.16a	23.54b	22.16ab	1.658	0.039
C18:2n6c	22.63	20.45	17.80	1.110	0.052
C20:1	0.31	0.19	0.27	0.078	0.548
C18:3n3	0.91a	0.78b	0.62ab	0.058	0.003
C20:2	1.03	1.02	0.99	0.093	0.896
C20:3n6	1.13	1.20	0.96	0.112	0.440
C20:4n6	11.99	13.14	13.27	1.107	0.862
C23:0	2.69	2.55	2.30	0.219	0.483
C22:6n3	1.49ab	1.02a	2.98b	0.582	0.006
SFA	42.45b	36.93a	39.56ab	1.358	0.021
MUFA	18.05a	25.27b	23.55ab	1.661	0.048
PUFA	39.50	37.80	36.89	0.920	0.302
PUFA: SFA	0.94	1.03	0.94	0.033	0.136
Total (g/100 g)	61.31	62.87	66.75	5.523	0.981

¹ SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids.

² SEM: standard error of the mean.

Fig. 3.

Correlation analysis of meat quality

(A) Correlation between fatty acids and meat quality on day 1 (B) Correlation between fatty acids and meat quality on day 7. In the correlation matrices, red represents a positive correlation and blue or green represents a negative correlation. * Differences were considered significant at P < 0.05 (** P < 0.001, *** P < 0.0001).

Discussion

Research has indicated that salicylate compounds can alleviate oxidative damage by enhancing the antioxidant system and mitigating inflammatory responses through the inhibition of pro-inflammatory mediators and signaling pathways[17,18]. Therefore, harnessing these bioactivities is particularly important for modern broiler production, where metabolic stress is prevalent.

In this study, the addition of MS did not significantly affect the growth performance of white-feathered broilers. The rapid growth rate and intense metabolism of modern broiler breeds make them particularly susceptible to oxidative stress and associated conditions such as ascites[19]. Therefore, the primary contribution of methyl salicylate may lie not in overriding their innate growth capacity but in enhancing antioxidant defenses. This improvement in metabolic health indirectly supports growth and is pivotal for improving product quality traits such as meat stability[20].

Serum HDL-C and TC levels serve as key biomarkers of hepatic lipid metabolic homeostasis. High-dose aspirin has been shown to indirectly promote hepatic lipid accumulation and inflammation, which are closely associated with aspirin-induced reactive oxygen species (ROS) generation and alterations in the antioxidant defense system[21]. This suggests that methyl salicylate may contribute to increased hepatic lipid metabolism, possibly through its ability to enhance antioxidant capacity. Similarly, sodium salicylate has been reported to elevate serum ALT levels while simultaneously upregulating antioxidant activity[22].

Oxidative stress damages cellular structures and impairs nutrient absorption and metabolism, thereby inhibiting weight gain. Conversely, antioxidant capacity directly influences the growth rate and feed utilization efficiency of broilers[23]. T-AOC levels are clear indicators of the status of the antioxidant defense system[24]. By inducing metabolic reprogramming, sodium salicylate upregulates endogenous antioxidant defenses, principally through the nicotinate-and nicotinamide-related metabolic pathways, resulting in elevated systemic T-AOC[25]. This suggests a compensatory shift toward targeted metabolic antioxidant pathways rather than a generalized upregulation of classical enzymes.

Maintaining the characteristic intestinal morphology is essential for digestion and absorption of nutrients[26]. The villus height to crypt depth ratio (VH/CD) reflects the functional status of the small intestine. In this study, dietary MS supplementation increased VH and VH/CD ratios in the jejunum under high-density conditions. This finding aligns with that of Zhang[14], who reported that MS supplementation increased VH in broilers, confirming the positive impact of MS on intestinal morphology. However, the findings of Dang et al. indicated that improving VH is not sufficient to enhance growth performance. Instead, augmentation of intestinal barrier function through anti-inflammatory mechanisms appears to be a more critical determinant of healthy growth[27]. The jejunum serves as the primary site for nutrient absorption and is characterized by the highest metabolic activity and epithelial turnover rate within the small intestine[28]. MS is primarily absorbed in the stomach and proximal small intestine. The relatively alkaline microenvironment of the jejunum likely favors dissociation and passive diffusion of lipophilic compounds, potentially leading to higher local bioavailability[29]. Taken together, the unique physiological demands of the jejunum, combined with the physicochemical properties of MS, favor its local absorption, thereby leading to a more pronounced improvement in villus morphology in the jejunum than in other intestinal segments.

Sensory evaluation revealed that MS supplementation significantly improved the overall sensory score of the broiler meat. This enhancement was manifested in two key aspects: superior color and tenderness/stickiness on the first day and markedly better retention of flavor and springiness after refrigerated storage compared with the control group. This phenomenon may be attributed to increased oleic acid content resulting from MS supplementation. Our analysis revealed that MS supplementation significantly increased the proportions of oleic acid (C18:1n9c) and total monounsaturated fatty acids (MUFAs). Oleic acid is widely recognized for its positive influence on sensory perception, contributing to a smoother, juicier mouthfeel and a more pleasant and rich flavor profile[30,31]. This directly accounts for the higher initial scores for attributes such as stickiness and overall liking in the MS group. Notably, sustained sensory quality during storage is likely attributable to the potent antioxidant properties of methyl salicylate and its metabolites[12]. Lipid oxidation is the primary cause of flavor deterioration, texture loss, and discoloration in meat products[32]. The antioxidant activity of MS is believed to effectively suppress the formation of undesirable secondary lipid oxidation products during storage, thereby helping preserve the native flavor profile of meat by delaying the onset of rancid off-flavors.

Consistent with the aspirin-induced reduction in water-holding capacity (WHC) reductions, MS supplementation decreased drip loss at 24 h[9]. In the present study, a consistent decrease in drip loss was observed at 24 h. The observed improvement in WHC of breast meat may be attributed to the antioxidant capacity of MS, which helps maintain the structural integrity of cell membranes[33]. Enhanced WHC can subsequently reduce the loss of pigments and soluble nutrients, thereby contributing to the stability of meat color and flavor[34]. A higher b* value is attributed to increased lipid content, whereas an elevated a* value is associated with increased myoglobin levels and lipid oxidation[35]. A previous study indicated that aspirin treatment led to a significant increase in a* values and a decrease in b* values, which is consistent with our results[9]. The application of MS may reduce the incidence of white striping and the yellowing index.

TBA is a major indicator of lipid oxidation in meat and meat products[36]. The results of this study showed that MS supplementation also reduced TBA levels, and this effect became more significant with extended storage time, suggesting that MS inhibits lipid oxidation[37]. This improvement may be attributed to the enhanced antioxidant capacity induced by MS, which is consistent with the observed increase in T-AOC. TVN-B content serves as a key indicator of protein hydrolysis, reflecting the extent of protein degradation in meat during storage[38]. Compared with the CON group, the MSH group showed significantly reduced TVB-N content in the breast muscles on day 7. Besides its antioxidant properties, this may be attributed to the antibacterial effect of MS, as the growth of Pseudomonas and Enterobacter in the muscle, which can utilize amino acids as growth substrates to produce sulfur-containing compounds and non-flavor amines, leads to a rapid increase in TVB-N content[39]. Our results suggest that the addition of MS improves muscle storage stability and extends shelf life.

Fatty acid content and composition are important indicators of meat quality[40]. Our study showed that dietary supplementation with MS altered the fatty acid profile of broiler muscles by reducing the SFA content and increasing the MUFA levels. MUFAs, such as oleic acid, oxidize at a slower rate than PUFAs, leading to greater oxidative stability[41]. This directly contributed to the delayed onset of rancidity and prolonged shelf life. The reduction in the SFA content minimizes the formation of short-chain aldehydes responsible for off-flavors[31]. From a cellular and nutritional standpoint, the incorporation of more MUFAs into muscle cell membranes helps to maintain membrane fluidity and integrity[42]. MUFAs such as oleic acid are known to inhibit ferroptosis, an iron-dependent form of cell death, potentially through pathways involving ACSL3. This mechanism protects cellular structures by suppressing lipid ROS accumulation, leading to improved water-holding capacity, reduced drip loss, and improved postmortem texture preservation[43]. Consequently, the MS-mediated shift in the fatty acid profile not only enhances the nutritional value of the meat by creating a healthier lipid composition but also directly underpins improvements in sensory attributes and storage stability. It is well known that an increased proportion of saturated fatty acids in meat can elevate serum triglyceride and total cholesterol levels, potentially contributing to the development of cardiovascular disease[44]. From a health perspective, meat with a relatively low SFA content may help reduce the risk of cardiovascular disease[45]. However, research on the effects of MS on broiler fatty acid composition remains scarce, highlighting the need for further molecular studies to address this knowledge gap.

Conclusions

Overall, methyl salicylate (MS) supplementation improved the antioxidant status, intestinal morphology, and meat quality of broilers, but had no direct effect on growth performance. MS enhances oxidative stability, water-holding capacity, and improves the muscle fatty acid profile by increasing MUFAs and reducing SFAs, thereby contributing to improved sensory traits and storage stability. These results indicate that MS can serve as a useful additive to support metabolic health and meat quality in modern broiler production, although further research is required to elucidate its regulatory effects on lipid metabolism.

Author Contributions

HW and HM conceived and designed the study. JL performed the trial, analyzed the data, and drafted the manuscript; YM, JM, WT, and YW provided assistance with animal feeding and laboratory analyses; HW revised the article. All authors have read and approved the final manuscript.

Grants and funding

This study was financially supported by a grant from the Key R&D Projects of Zhejiang Province (2026C02A1028).

Conflicts of Interest

The authors declare no conflicts of interest.

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