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. 2026 Feb 12;105(5):106630. doi: 10.1016/j.psj.2026.106630

Effects of chitosan–nanoparticles on hematological indices, renal and liver function, lipid metabolism, abdominal adipose deposition, carcass traits, and growth performance of broiler chickens

Razagh Kazem Badr a, Mokhtar Fathi b,⁎, Kianoosh Zarrinkavyani a,⁎, Zahra Biranvand c
PMCID: PMC12934293  PMID: 41713090

Abstract

This study evaluated chitosan nanoparticles (ChNPs) as a feed additive for broilers. A total of 400 d-old male Ross 308 broilers were randomly allocated into four treatments with five replication pens, each pen had 20 broilers. The experimental diets were as follows: First group was fed a basal diet only (control); 2nd, 3rd, and 4th groups received a basal diet supplemented with 50 (ChNPs-50), 100 (ChNPs-100), and 150 (ChNPs-150) mg ChNPs /kg of feed, respectively. Results showed that dietary supplementation with ChNPs reduced mortality and feed conversion ratio, while weight gain and feed intake showed non-significant trends toward improvement. Abdominal fat decreased significantly, whereas breast weight increased, with the highest values in the 100 and 150 mg/kg groups. Dressing percentage improved significantly, while liver weight remained unaffected. Linear trends confirmed dose-dependent improvements in abdominal fat, breast yield, and dressing percentage. Regarding liver function, ChNPs significantly reduced alanine aminotransferase activity. In renal function, creatinine decreased significantly in the highest ChNPs group, while uric acid and urea showed non-significant declines. ChNPs markedly improved lipid profiles. Low-density lipoprotein and total cholesterol decreased significantly, while high-density lipoprotein increased in the 150 mg/kg group. Triglycerides also declined significantly at the highest supplementation. Linear effects for all lipid parameters indicated dose-dependent improvements. Heterophils decreased and lymphocytes increased in ChNPs-supplemented birds, resulting in a significantly lower HET/LYM ratio. Overall, dietary ChNPs supplementation enhanced growth performance, carcass quality, liver and kidney function, lipid metabolism, and immune cell distribution in broilers, with dose-dependent effects evident across multiple physiological and biochemical parameters.

Keywords: Abdominal adipose deposition, Broiler, Carcass traits, Chitosan nanoparticles, Performance

Introduction

Feed additives play a crucial role in modern poultry production, where they are incorporated into diets to support animal health, optimize growth, and improve the quality of meat and eggs (Gerber et al., 2007; Mahasneh et al., 2024). For many years, antibiotic growth promoters (AGPs) were routinely included in poultry rations to enhance growth rates and suppress harmful microorganisms (Shao et al., 2021; Swaggerty et al., 2022).

However, the long-term and often uncontrolled use of AGPs has contributed to the development of antibiotic-resistant bacteria, the presence of drug residues in poultry products, and the possible transmission of resistant strains to humans (Anusuya et al., 2012; Zhang et al., 2022). Growing concerns regarding public health, antimicrobial resistance, and cross-contamination led the European Union to prohibit antibiotics as growth enhancers in 2006. This ban accelerated scientific efforts to identify natural, safe replacements—such as organic acids, probiotics, prebiotics, herbal extracts, and essential oils—to sustain poultry performance without relying on antibiotics (Arslan and Tufan, 2018).

Among the wide range of natural alternatives, chitosan stands out as a relatively underutilized yet promising biopolymer.

As the second most abundant biodegradable compound after cellulose, it is obtained through the deacetylation of chitin. Chitosan is considered environmentally safe, non-toxic, and beneficial in improving animal productivity and health. It can be extracted from crustacean shells, fungi, and insect exoskeletons (Jacaúna et al., 2021; Algothmi et al., 2024). Research has demonstrated that chitosan exhibits antimicrobial, antioxidant, immunoregulatory, antitumor, anticoagulant, and cholesterol-lowering activities (Fathi et al., 2023). When added to animal diets, it has been shown to enhance growth performance, immune status, inflammatory response, and antioxidant activity (Fathi et al., 2023).

In broiler chickens, supplementation with chitosan can reduce colonization by foodborne pathogens such as Salmonella typhimurium (Kong et al., 2010; Menconi et al., 2014). It also exerts strong immunostimulatory effects by elevating serum levels of immunoglobulins (IgG, IgM, IgA) (Li et al., 2016; Fathi et al., 2023), increasing total leukocyte counts (Nuengjamnong and Angkanaporn, 2017), and promoting cytokine and chemokine secretion. Additionally, chitosan has been linked to cholesterol reduction, possibly through modulation of circulating adipocytokines and decreased fat deposition in muscle tissues (Xu et al., 2014).

In recent years, nanomaterials have been explored as a strategy to improve poultry growth performance and production efficiency (Fathi et al., 2023, 2025a, b). Chitosan nanoparticles (CNPs), due to their extremely small size (<100 nm), demonstrate even stronger antimicrobial, antioxidant, and immune-enhancing effects, while also supporting growth and maintaining healthy gut microbiota (Divya and Jisha, 2018; Xu et al., 2018; Adil et al., 2024). The present study hypothesized that ChNPs may positively affect broilers’ growth criteria and health status. Therefore, the current investigation was intended to estimate the efficacy of chitosan nanoparticles (ChNPs), as feed additives in broiler chicken on performance, serum biochemistry, and carcass traits.

Materials and methods

Ethics statement

All animal-related experimental protocols were reviewed and approved by the Animal Ethics and Welfare Committee of the Department of Animal Science at Payame Noor University (Approval Code: IR.PNU.REC.1404.148). The entire study was conducted in strict compliance with the national ethical guidelines for the care and use of laboratory animals, as issued by the Iranian Ministry of Science, Research, and Technology. All necessary measures were undertaken to minimize animal suffering and to ensure the use of the minimum number of animals required to achieve statistical validity.

Birds, diets and experimental design

A total of 400 one-day-old male Ross 308 broiler chicks were obtained from a local commercial hatchery. Upon arrival, chicks were weighed and randomly allocated to four dietary treatments in a completely randomized design (CRD). Each treatment consisted of five replicate pens (20 birds per pen), resulting in 100 birds per treatment. The experimental diets were as follows: the first group received a basal diet without supplementation (control); the second, third, and fourth groups received the basal diet supplemented with 50 (ChNPs-50), 100 (ChNPs-100), and 150 (ChNPs-150) mg of chitosan nanoparticles per kg of feed, respectively. The dosages were selected based on previous studies in broilers (e.g., Adil et al., 2024; Fathi et al., 2023b) and confirmed by an internal pilot trial. All birds were housed in an environmentally controlled house with uniform conditions: temperature was maintained at 32–34 °C during the first week and gradually reduced to 20–22 °C by the end of the experiment; relative humidity was kept at 50–65 %; and a 23 h light:1 h dark lighting schedule was applied throughout the trial. Pens were evenly distributed within the house to avoid any positional bias. Feed and water were provided ad libitum

The birds were kept in floor pens, each measuring 150 × 200 cm per replicate, and furnished with circular feeders and drinkers. All broilers received vaccinations following standard guidelines for Newcastle disease and other prevalent poultry infections. Diets were formulated using corn and soybean meal to meet the nutritional requirements recommended for Ross 308 broilers. The feeding program consisted of three phases: a starter diet from days 1 to 10, a grower diet from days 11 to 24, and a finisher diet from days 25 to 42, as shown in Table 1.

Table 1.

The ingredient composition and nutrient content of the basal diets.

Starter diet Grower diet Finisher diet
Ingredients (%)
Corn 47.53 51.63 57.56
Soybean meal, 44 %CP 42.35 37.99 32.35
Soybean oil, 9000 kcal/kg 5.54 6.24 6.29
Limestone, 38 % Ca 1.20 1.12 1.05
Di-calcium phosphate 1.79 1.56 1.34
Vitamin premixb 0.25 0.25 0.25
Mineral premixc 0.25 0.25 0.25
NaCl 0.40 0.40 0.40
DL-Methionine, 99 % 0.37 0.32 0.28
Lysine, 78 % 0.28 0.22 0.22
Threonine, 98.5 % 0.05 0.02 0.00
Calculated values d
Metabolizable energy, kCal/kg 2990 3082 3218
Crude protein, % 23 21.3 19.3
Calcium (Ca), % 0.96 0.87 0.79
Available phosphorus, % 0.456 0.409 0.361
Sodium (Na), % 0.16 0.16 0.16
Met, % 0.71 0.64 0.58
Met + Cys, % 1.07 0.89 0.89
Lys, % 1.46 1.30 1.17
Arg, % 1.56 1.45 1.30
Thr, % 0.96 0.87 0.78
Try, % 0.35 0.32 0.29
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b Vitamin concentrations per kilogram of diet: retinol, 13.50 mg; cholecalciferol, 4.15 mg; tocopherol acetate, 32.00 mg; vitamin K3, 2 mg; thiamin, 2 mg; riboflavin, 6.00 mg; biotin, 0.1 mg; cobalamin, 0.015 mg; pyroxidine, 3 mg; niacin, 11.00 mg; d-pantothenic acid, 25.0; menadione sodium bisulphate, 1.10; folic acid, 1.02; choline chloride, 250 mg; nicotinamide, 5 mg;.

CMineral concentrations per kilogram of diet:calcium pantothenate, 25 mg; Fe (from ferrous sulphate), 35 mg; Cu (from copper sulphate), 3.5 mg; Mn (from manganese sulphate), 60 mg; Zn (from zinc sulphate), 35 mg; I (from calcium iodate), 0.6 mg; Se (from sodium selenite), 0.3 mg.

Chitosan nanoparticles (ChNPs) preparation

Chitosan used in this study was sourced from Zhejiang Golden-Shell Biochemical Co. Ltd. (Zhejiang, China) and had a degree of deacetylation of about 90 % and a molecular weight of approximately 150 kDa. Pentasodium tripolyphosphate (TPP) and chlortetracycline were purchased from Sigma Chemical Co. (St. Louis, MO). Chitosan nanoparticles (ChNPs) were prepared following the ionic gelation method described by Ali et al. (2023) and Adil et al. (2024), which is based on electrostatic interactions between chitosan’s amine groups and negatively charged TPP (Fig. 1) . For nanoparticle synthesis, chitosan was dissolved in 1 % (v/v) acetic acid to create a 0.2 % (w/v) solution, after which a 0.06 % (w/v) TPP solution was added dropwise under vigorous stirring. The formed nanoparticles were collected by centrifugation at 12,000 g for 30 min and resuspended in deionized water. In an additional preparation, 0.25 g of low–molecular-weight chitosan (93 % deacetylated) was dissolved in water and acetic acid, the pH was adjusted to 4.6–4.8, and TPP was added to induce nanoparticle formation, indicated by a white precipitate. Nanoparticles were purified by centrifugation at 9000 rpm for 30 min and repeatedly washed with distilled water to remove residual NaOH. The morphology and size of the final ChNPs were evaluated using transmission electron microscopy (TEM) (Fig. 2).

Fig. 1.

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Schematic diagram illustrating the formation of chitosan nanoparticles (ChNPs).

Fig. 2.

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Electron microscope image of a chitosan–nanoparticles (ChNPs) at different magnifications.

Productive performance and carcass traits

In this study, growth performance parameters—including weight gain (WG), body weight (BW), and feed conversion ratio (FCR)—were evaluated. Birds were individually weighed at weekly intervals beginning in the third week of age and continuing through the seventh week. Each week, in the early morning and prior to feeding, both the birds and the remaining feed were weighed. Weekly weight gain was calculated by subtracting a bird’s BW in a given week from its BW in the subsequent week. Feed conversion ratio was determined by dividing the total feed intake (g) during each week by the corresponding WG (g) for that same period.

At six weeks of age, following an 8-hour fast, two birds per replication pen were randomly selected for sampling. Blood was collected and centrifuged at 3,500 × g and 4 °C for 10 min to obtain serum, which was stored at −20 °C. Birds were then euthanized by cervical dislocation, and liver and abdominal fat samples were harvested. Slaughter traits, including semi-eviscerated, eviscerated, and dressed weights, along with percentages of breast, liver, and abdominal fat, were recorded. Relative weights were calculated as a percentage of body weight (Lan et al., 2025).

Biochemical indices

Serum concentration of aspartate amino transferase (AST), alanine amino transferase (ALT), total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), urea, uric acid, and creatinine were determined using an autoanalyser (Abbott alcyon 300, USA) by laboratory kits (Pars Azmoon, Tehran, Iran).

Hematological indices

Whole blood samples were collected by venipuncture into EDTA-K3 anticoagulation tubes for measuring hematological parameters including white blood cells count (WBCc), red blood cells count (RBCc), hemoglobin (HGB), hematocrit (HCT), heterophils (HET) and lymphocytes (LYM) in whole blood were analyzed using an automatic blood analyzer (Sysmex KX-21N Automatic blood analyzer, Japan). For differential leukocyte counts (H/L ratio), a drop of blood was smeared on a glass slide, left to dry and then stained with Giemsa stain. One hundred leucocytes were counted on each slide including heterophils and lymphocytes. The H/L ratio was calculated by dividing the number of heterophils by the number of lymphocytes. Two slides were counted and the means were calculated for each bird (Al Wakeel et al., 2017).

Statistical Analysis

All data were analyzed using the general linear model (GLM) procedure of SPSS (version 16.0; SPSS Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) was performed to determine the overall treatment effects. When a significant F-test was observed (P < 0.05), means were separated by Duncan’s multiple-range test. The assumption of normality was checked with the Shapiro–Wilk test, and homogeneity of variances was verified using Levene’s test; no violations were detected. Orthogonal polynomial contrasts were used to evaluate linear and quadratic trends with increasing levels of ChNPs supplementation. Data are presented as means of five replicates with the standard error of the mean (SEM). For readers interested in interval estimates, the SEM provided can be used to approximate confidence intervals.

Results

Growth performance traits and mortality

Dietary supplementation with chitosan nanoparticles (ChNPs) significantly affected the growth performance and mortality of broiler chickens (Table 2). Mortality was markedly reduced (P < 0.01) in all ChNPs-supplemented groups compared with the control, which recorded a 4 % mortality rate. No mortality occurred in birds fed diets containing 50, 100, or 150 mg/kg ChNPs. Feed conversion ratio (FCR) was significantly improved (P < 0.01) with increasing levels of ChNPs. Birds receiving 100 and 150 mg/kg ChNPs showed lower FCR values (1.89 and 1.87, respectively) than the control group (2.07). Average daily gain (ADG) tended to increase with ChNPs supplementation, reaching the highest value (61.20 g) in the ChNPs-150 group, although this change was not statistically significant (P = 0.320). Similarly, average daily feed intake (ADFI) was not significantly influenced by dietary treatments (P = 0.211).

Table 2.

Effects of dietary chitosan–nanoparticles (ChNPs) on growth performance and mortality of broiler chickens.

Treatments ADFI (g) ADG (g) FCR Mortality (%)
Control 114.68 55.42 2.07a 4.00a
ChNPs-50 110.33 55.95 1.97a 0.00b
ChNPs-100 112.71 59.52 1.89b 0.00b
ChNPs-150 114.56 61.20 1.87b 0.00b
SEM 2.01 0.61 0.04 0.02
P value Linear 0.134 0.291 0.000 0.000
0.541 Quadratic 0.531 0.451 0.651 0.541
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a, b, c Mean values in the same column with different superscript letters were significantly different (P < 0.05).

ChNPs-50, 100, 150: basal diet with 50, 100, or 150 mg/kg chitosan–nanoparticles (ChNPs), respectively; ADFI: Average daily feed intake; ADG: Average daily gain; FCR: feed conversion ratio.

Carcass traits

Dietary supplementation with chitosan nanoparticles (ChNPs) influenced carcass traits of broiler chickens (Table 3). The relative weight of abdominal adipose tissue decreased significantly with increasing ChNPs levels, from 2.52 % in the control to 2.01 % in the 150 mg/kg group (P < 0.01). In contrast, breast weight increased significantly, with the highest values observed in the 100 and 150 mg/kg ChNPs groups (33.45 % and 34.60 %, respectively; P < 0.01). Dressing percentage improved significantly with ChNPs supplementation, reaching 83.15 % in the 150 mg/kg group (P < 0.01). Liver relative weight was not significantly affected by dietary treatments (P > 0.05).

Table 3.

Effects of dietary chitosan–nanoparticles (ChNPs) on carcass attributes of broiler chickens.

Treatments Dressinga (%) Relative weight of livera, % Breast weight (%) Relative weight of abdominal adiposea ,%
Control 79.20b 2.50 30.10b 2.52a
ChNPs-50 81.01ab 2.48 30.91b 2.17b
ChNPs-100 82.01a 2.45 33.45a 2.04c
ChNPs-150 83.15a 2.47 34.60a 2.01c
SEM 0.621 0.05 0.815 0.005
P value Linear 0.001 0.326 0.000 0.001
Quadratic 0.651 0.629 0.651 0.391
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a, b, c Mean values in the same column with different superscript letters were significantly different (P < 0.05).

ChNPs-50, 100, 150: basal diet with 50, 100, or 150 mg/kg chitosan–nanoparticles (ChNPs), respectively;.

aCalculated as relative to body weight.

Lipid metabolism

Dietary supplementation with chitosan nanoparticles (ChNPs) markedly affected serum lipid-metabolism parameters in broiler chickens (Table 4). Low-density lipoprotein (LDL-C) concentrations decreased significantly with ChNPs inclusion, from 106.44 mg/dL in the control group to values ranging between 94.72 and 98.92 mg/dL in all supplemented groups (P < 0.05). In contrast, high-density lipoprotein (HDL-C) levels increased significantly in birds receiving ChNPs, reaching the highest value (65.23 mg/dL) in the 150 mg/kg group compared with 56.95 mg/dL in the control (P < 0.05). Total cholesterol concentrations decreased substantially and significantly with increasing ChNPs levels, from 157.46 mg/dL in the control to 130.56 mg/dL in the 150 mg/kg treatment (P < 0.05). A similar pattern was observed for triglycerides (TG), which declined from 229.45 mg/dL in the control to 168.54 mg/dL at the highest supplementation level (P < 0.05).

Table 4.

Effects of dietary chitosan–nanoparticles (ChNPs) supplementation on lipid metabolism related parameters in serum of broiler chickens.

Treatments TG (mg/dL) TC (mg/dL) HDL-C (mg/dL) LDL-C (mg/dL)
Control 229.45a 157.46a 56.95b 106.44a
ChNPs-50 205.48b 141.16b 62.16a 98.92b
ChNPs-100 173.04c 133.36c 64.28a 94.72b
ChNPs-150 168.54c 130.56c 65.23a 95.21b
SEM 2.03 1.32 1.25 1.02
P value Linear 0.001 0.002 0.012 0.001
Quadratic 0.402 0.591 0.365 0.561
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a, b, c Mean values in the same column with different superscript letters were significantly different (P < 0.05).

ChNPs-50, 100, 150: basal diet with 50, 100, or 150 mg/kg chitosan–nanoparticles (ChNPs), respectively. TG, triacylglycerol; TC, total cholesterol; HDL-C, high density lipoprotein cholesterol; LDL-C, low density lipoprotein cholesterol;.

Kidney and liver functions

Dietary supplementation with chitosan nanoparticles (ChNPs) influenced renal and liver function indices in broiler chickens (Table 5). Uric acid concentrations decreased progressively with increasing ChNPs levels, from 7.53 mg/dL in the control group to 4.03 mg/dL in the 150 mg/kg group; however, these reductions were not statistically significant (P > 0.05). Similarly, urea levels showed a numerical decline in birds receiving 100 and 150 mg/kg ChNPs, although the differences did not reach significance. In contrast, creatinine levels were significantly affected by dietary treatments (P < 0.05), decreasing markedly from 0.195 mg/dL in the control group to 0.075 mg/dL in the highest ChNPs group. Broilers fed 100 and 150 mg/kg ChNPs exhibited significantly lower creatinine concentrations compared with the control group.

Table 5.

Effects of dietary chitosan–nanoparticles (ChNPs) on renal and liver function of broiler chickens.

Treatments Creatinine (mg/dL) Urea (mg/dL) Uric acid (mg/dL) ALT (U/L) AST (U/L)
Control 0.195a 27.30a 7.53a 26.91a 179.35
ChNPs-50 0.185a 27.51a 5.46b 25.58a 171.48
ChNPs-100 0.080b 19.44b 4.39c 14.02b 159.65
ChNPs-150 0.075b 18.25b 4.03c 10.75b 155.45
SEM 0.011 1.01 0.15 0.41 8.20
P value Linear 0.001 0.012 0.011 0.010 0.360
Quadratic 0.621 0.463 0.391 0.294 0.751
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a, b, c Mean values in the same column with different superscript letters were significantly different (P < 0.05).

ChNPs-50, 100, 150: basal diet with 50, 100, or 150 mg/kg chitosan–nanoparticles (ChNPs), respectively. ALT, Alanine aminotransferase, AST, Aspartate aminotransferase.

Dietary supplementation with chitosan nanoparticles (ChNPs) affected serum aminotransferase activity in broiler chickens (Table 5). Aspartate aminotransferase (AST) activity decreased numerically with increasing ChNPs levels, from 179.35 U/L in the control group to 155.45 U/L in the 150 mg/kg group; however, these differences were not statistically significant (P > 0.05). In contrast, alanine aminotransferase (ALT) activity was significantly reduced by ChNPs supplementation (P < 0.05), declining from 26.91 U/L in the control to 10.75 U/L at the highest inclusion level. Broilers receiving 100 and 150 mg/kg ChNPs exhibited markedly lower ALT values compared with the control group

Hematological parameters

Dietary supplementation with chitosan nanoparticles (ChNPs) had minimal effects on most hematological indices of broiler chickens (Table 6). Red blood cell count (RBCs), hemoglobin (HGB), hematocrit (HCT), and total white blood cells (WBCs) were not significantly affected by ChNPs supplementation (P > 0.05). However, significant changes were observed in differential leukocyte counts. The percentage of heterophils (HET) decreased significantly (P = 0.001), while lymphocyte (LYM) percentage increased (P = 0.002) in ChNPs-supplemented groups compared with the control. Consequently, the HET/LYM ratio was significantly reduced in all ChNPs groups (P = 0.001). Linear effects were significant for HET, LYM, and HET/LYM ratio (P < 0.05).

Table 6.

Effects of dietary chitosan–nanoparticles (ChNPs) on hematological values of broiler chickens.

Indices P-Value
 Groups
Control ChNPs-50 ChNPs-100 ChNPs-150 SEM Linear Quadratic
RBCs count (× 106/ μl) 3.01 3.12 2.90 2.87 0.512 0.190 0.613
HGB (g/dl) 14.80 14.80 12.70 13.21 0.480 0.273 0.520
HCT (%) 39.31 38.51 37.81 37.99 0.981 0.376 0.421
WBCs count (× 103/μl) 19.90 18.41 20.85 20.79 0.791 0.351 0.530
HET (%) 44.71a 37.70b 36.05b 35.01b 0.351 0.010 0.325
LYM (%) 52.76b 59.01a 59.13a 60.12a 0.601 0.012 0.621
HET / LYM ratio 0.84a 0.63b 0.60b 0.61b 0.012 0.010 0.721
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a, b, c Mean values in the same row with different superscript letters were significantly. (n = 10).

ChNPs-50, 100, 150: basal diet with 50, 100, or 150 mg/kg chitosan–nanoparticles (ChNPs), respectively.RBCs, Red blood cells; HGB, Hemoglobin; HCT, Hematocrit; WBCs, White blood cells; HET, Heterophil; LYM Lymphocyte.

Discussion

A variety of feed additives are used to enhance growth performance, improve feed efficiency, and support overall health in poultry. Nanotechnology offers notable advantages in this regard by increasing the bioavailability and effectiveness of nutritional compounds. In the present study, dietary supplementation with chitosan nanoparticles (ChNPs) at 100 mg/kg improved the feed efficiency of broiler chickens. Similarly, Adil et al. (2024) reported enhanced growth and feed conversion efficiency in broilers supplemented with 400 mg/kg of chitosan nanoparticles. Furthermore, the use of chitosan nanoparticles as a dietary supplement in Nile tilapia (Oreochromis niloticus) has shown beneficial effects on growth and feed utilization. These improvements were attributed to increased digestive enzyme activity, suppression of harmful intestinal microorganisms, and enhancement of certain innate immune responses (Abd El-Naby et al., 2019).

Nuengjamnong and Angkanaporn (2017) reported improved growth performance in chickens supplemented with 2 g/kg chitosan. Similarly, chitosan oligosaccharides have been shown to enhance body weight, weight gain, and feed conversion ratio in broilers (Li et al., 2019; Ahmed et al., 2021; Fathi et al., 2023b). These growth-promoting effects are primarily attributed to improved nutrient digestibility, enhanced feed utilization, and stimulation of growth-related hormones (Hu et al., 2020; Yang et al., 2022). In addition, chitosan oligosaccharides positively influence gut morphology and intestinal integrity, which promotes efficient nutrient absorption and overall digestive performance (Fathi et al., 2023b).

The present study revealed a significant improvement in dressed and breast meat yield of birds-fed diets added with ChNPs at 100 mg/kg diet related to control. Moreover, the abdominal fat yield was significantly decreased dose-dependent on increasing the concentration of ChNPs in the diet.

Consistent with our findings, Adil et al. (2024) reported that supplementation with chitosan nanoparticles (ChNPs) up to 300 mg/kg of diet increased eviscerated and breast meat yield while reducing abdominal fat. Similarly, dietary chitosan oligosaccharides have been shown to enhance carcass traits, decrease abdominal fat, modulate lipid metabolism by lowering serum lipid levels and hepatic lipid accumulation, inhibit adipocyte differentiation, and improve glycolipid metabolism in broilers (Arslan and Tufan, 2018; Tao et al., 2019, 2021; Lee et al., 2021; Lan et al., 2023). The reduction in abdominal fat is attributed to multiple mechanisms, including inhibition of lipid absorption in the gastrointestinal tract, suppression of hepatic lipogenesis, interference with adipocyte differentiation, and increased fecal lipid excretion (Li et al., 2016; Chiu et al., 2017; Pan et al., 2018; Lan et al., 2023). This decrease in abdominal fat not only enhances the market value of broilers but also reduces economic losses during processing, while adipocyte hyperplasia, resulting from triglyceride accumulation in lipid droplets, remains closely associated with abdominal fat deposition (Chen et al., 2014; Lan et al., 2025).

Furthermore, abdominal fat deposition is largely influenced by the uptake of circulating triglycerides (TG), which originate from hepatic lipid metabolism following the synthesis and secretion of TG-rich VLDL (Bai et al., 2021; Na et al., 2018). Chitosan oligosaccharides, which are readily absorbed and distributed to key metabolic organs, exert notable lipid-lowering effects by reducing serum TG and cholesterol levels and limiting hepatic lipid accumulation (Naveed et al., 2019; Tao et al., 2021). In this study, ChNPs supplementation resulted in lower serum TG, TC, and LDL-C levels, accompanied by an increase in HDL-C, indicating enhanced lipid catabolism through reverse cholesterol transport (Muanprasat and Chatsudthipong, 2017).

The reduction in abdominal fat alongside increased breast yield suggests that ChNPs not only limit lipid deposition through interference with intestinal fat absorption and adipogenesis but may also improve protein synthesis efficiency, possibly via modulation of insulin-like growth factors or mTOR signaling (Tao et al., 2021; Adil et al., 2024).

Liver enzymes such as ALT and AST are well-established biomarkers of hepatic integrity (Ambrosy et al., 2015). In the present study, dietary supplementation with ChNPs, particularly at levels exceeding 100 mg/kg, resulted in a marked reduction in serum ALT. These findings are consistent with earlier reports showing that chitosan and chitosan oligosaccharides lower circulating ALT and AST concentrations in broilers and other animal models (Qiao et al., 2011; Chang et al., 2020; Adil et al., 2024). The hepatoprotective action of chitosan derivatives has been attributed to their capacity to alleviate hepatic inflammation and limit liver injury, as demonstrated by reduced liver enzyme activity in both heat- and cold-stressed broilers (Fathi et al., 2023b). The observed improvements in lipid profile and liver enzymes likely stem from the ability of ChNPs to reduce oxidative stress and inflammatory signaling in hepatocytes, thereby suppressing lipogenic transcription factors (e.g., SREBP-1c) and promoting fatty-acid oxidation (Lan et al., 2023; Muanprasat and Chatsudthipong, 2017). Collectively, these results indicate that chitosan supplementation contributes to enhanced liver function in broiler chickens.

The significant reduction in creatinine concentrations observed in broilers supplemented with higher levels of ChNPs suggests an improvement in renal physiological status. Previous literature indicates that chitosan and its nanoparticle forms exert renoprotective effects primarily through their antioxidant and anti-inflammatory activities, which help reduce oxidative burden on renal tissues (Naveed et al., 2019). Chitosan has also been shown to bind nitrogenous metabolites and reduce circulating levels of toxic compounds, thereby lowering renal excretory load and improving indicators such as creatinine (Qiao et al., 2011). Additionally, its capacity to modulate gut microbiota and decrease systemic endotoxin absorption may indirectly support kidney function by reducing inflammatory signaling pathways associated with renal stress (Naveed et al., 2019). Therefore, the decline in creatinine concentrations in the present study aligns with established mechanisms through which chitosan-based supplements enhance renal health.

The dose-dependent improvements in FCR, abdominal fat, lipid profile, and liver enzymes observed in the present study are consistent with earlier reports on chitosan and its nano-formulations in broilers (Adil et al., 2024; Lan et al., 2023; Fathi et al., 2023b). However, the clear linear trends (P < 0.05 for linear contrast) across multiple physiological domains provide new evidence for a predictable, dose-responsive effect of ChNPs. This reinforces the potential of ChNPs as a precision feed additive, where the level of supplementation can be adjusted according to specific production targets (e.g., maximizing breast yield or optimizing lipid metabolism)

The inclusion of ChNPs in all levels reduced, heterophile values in broiler compared to control group birds (Table 6). The reduction in heterophil percentage, along with higher lymphocyte counts and a markedly lower HET/LYM ratio, suggests improved immune status and reduced physiological stress. The HET/LYM ratio is a recognized stress biomarker in poultry, with lower values reflecting enhanced immunocompetence (Fathi et al., 2023b). Chitosan-based supplements have been shown to exert anti-inflammatory and immunomodulatory effects by modulating gut microbiota, limiting endotoxin absorption, and improving antioxidant activity (Naveed et al., 2019). The present hematological responses align with these reported benefits and indicate a dose-dependent enhancement of immune regulation in broilers receiving ChNPs.

Limitations and future directions

While this study demonstrates broad benefits of ChNPs, some limitations should be noted. The precise molecular pathways responsible for improved lipid metabolism, liver health, and immune balance were not delineated. Additionally, findings are based on one broiler strain during a 42-day period; longer-duration studies and breed-specific responses warrant attention. The numerical reduction in mortality (0 % vs. 4 % in control) supports a beneficial trend, though statistical significance was not reached, suggesting the need for further validation under defined health-challenge conditions. To build on these results, future work could focus on transcriptomic/proteomic profiling of key tissues, exploring ChNPs’ interactions with other feed additives, and testing efficacy across various poultry production systems.

Practical implications and future considerations

The findings of this study support the potential of chitosan nanoparticles as a functional feed additive in broiler production. The dose-dependent improvements in feed conversion ratio, breast yield, and metabolic indices indicate that an inclusion level of 100–150 mg/kg feed is optimal under the conditions of this trial. For practical implementation, further work is needed to ensure the economic viability of ChNPs production, their stability in feed, and homogeneity during mixing. Long-term safety, including residues in edible tissues and effects over multiple generations, should be verified. Environmental assessments regarding the fate of ChNPs in manure and soil are also recommended to guarantee ecological sustainability. Provided these aspects are addressed, ChNPs could offer a natural alternative to antibiotic growth promoters, contributing to safer and more efficient poultry production.

CRediT authorship contribution statement

Razagh Kazem Badr: Validation, Software, Resources. Mokhtar Fathi: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Kianoosh Zarrinkavyani: Validation, Supervision, Investigation, Funding acquisition. Zahra Biranvand: Visualization, Validation, Software, Resources, Methodology.

Disclosures

We declare that we have no financial and personal relationships with other people ororganizations that can inappropriately influence our work, and there is no professional or otherpersonal interest of any nature or kind in any product, service and/or company that could beconstrued as influencing the content of this paper.

Contributor Information

Mokhtar Fathi, Email: Mokhtarfathi@pnu.ac.ir.

Kianoosh Zarrinkavyani, Email: K.zarrinkavyani@ilam.ac.ir.

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