Effect of phloretin on antioxidant status, inflammatory cytokines, blood and biochemical indices, cecal microbial load, and growth performance in broiler chickens subjected to pulmonary arterial hypertension

Abstract

This experiment was conducted to investigate the effects of dietary phloretin on broiler chickens under pulmonary arterial hypertension (PAH) conditions. A total of 500 one-day-old male broiler chickens were randomly allocated into five treatments with five replicate pens containing 20 birds per pen. The negative control group was reared at normal temperature and fed with basal diet while the PAH-treatments (positive control, F-200, F-400, and F-600) were exposed to low temperatures and received drinking water supplemented with excess salt to induce PAH and fed with basal diet containing 0, 200, 400, and 600 mg of phloretin per kilogram, respectively. Blood samples were collected from broilers at 42 days of age. Results showed that PAH decreased body weight gain (BWG), antioxidant capacity, immunoglobulin G (IgG), hematological indices, and cecal lactic acid bacteria population (CLBP), but increased feed intake (FI), PAH-related mortality, feed conversion ratio (FCR), PAH index, and pro-inflammatory cytokines, alanine transaminase (ALT), aspartate transaminase (AST), triglyceride (TG), and total cholesterol (TC) contents in serum (P<0.001). Among the PAH treatments, supplementary phloretin improved growth performance indices and reduced PAH index, and PAH-related mortality (P < 0.01). The activities of superoxide dismutase, catalase, and glutathione peroxidase, and IgG, and IgM in serum were increased, and malondialdehyde was reduced by phloretin supplementation (P <0.01). The PAH-induced effect on contents of pro-inflammatory cytokines were reduced by dietary phloretin supplementation (P <0.01). Moreover, supplementation of diets with phloretin alleviated the adverse effect of PAH as reflected by a reduction in ALT, AST, TG, and TC (P <0.01). The red blood cell count, hemoglobin, hematocrit, and heterophil levels reduced by phloretin supplementation. Phloretin increased the population of CLBP. In conclusion, phloretin supplementation during PAH may mitigate PAH-associated physiological and biochemical alterations in broiler chickens.

Introduction

Fast-growing meat-type broiler chickens (Gallus gallus) can spontaneously develop pulmonary arterial hypertension (PAH) with an estimated incidence of approximately 3% in all broiler chickens. Cold stress, hypobaric hypoxia (high altitude) or sodium chloride toxicity predisposes the birds to developing PAH (Shahir et al., 2012). Cold environmental temperatures tend to increase blood triiodothyronine (T₃) levels, required for the generation of additional metabolic heat to maintain body temperature in colder environments. The subsequent increase in basal metabolic rate results in an increase in oxygen demand and the heart attempts to maintain oxygen supply to the organs and muscles, ultimately leading to pulmonary arterial hypertension (PAH), right ventricular hypertrophy, ascites or water belly, and eventually death (Gupta, 2011).

According to Fathi et al. (2023a) the main causes of PAH etiology can be classified as: pulmonary hypertension, cardiac pathologies, and cellular damage due to oxidative stress caused by increased reactive oxygen species (ROS) production. They confirmed that chickens with ascites suffered from high oxidative stress. Mitochondrial electron leak and ROS production are increased during hypoxia and ascites (Iqbal et al., 2002). There is high electron leakage in the heart mitochondria of broilers developing ascites. There are also some reports indicating that following the occurrence of oxidative stress, severe inflammatory reactions occur in the involved cells, which lead to more severe tissue damage and can activate tissue apoptosis (Jiang et al. 2018; Fathi et al., 2023a). The increase in lipid peroxidation and the activities of aspartate aminotransferase and lactate dehydrogenase in serum are an indicator of severe liver cell damage which is a result of high production of free radicals under the influence of cold stress (Fathi et al., 2016).

In addition, oxidative stress status also releases pro-inflammatory cytokines, including interleukin (IL-6), tumor necrosis factor α (TNF-a), and IL-1β, to enhance the inflammatory response and injure cells or tissues (Jiang et al. 2018; Fathi et al., 2023a, b). Therefore, inhibiting the release of these inflammatory cytokines and mediators by macrophages may suppress or decrease tissue injury during the inflammatory process. As a result of oxidative stress, major antioxidants are depleted in the liver and the lungs of broilers (Fathi et al., 2023a). Therefore, antioxidants that relieve oxidative damage can improve oxidative stress and inflammation stress in broilers (Fathi et al., 2023a, b).

Animal and clinical studies have indicated that some plant-based flavonoids may attenuate conditions mediated by the inflammatory response, including asthma, rheumatoid arthritis, and cardiovascular disease (Chang et al., 2012). Phloretin, extracted from fruits (including apples, pears, and peaches), leaves, trees, and various vegetables, is a type of bioactive flavonoid (Wang et al., 2018). Phloretin has many biological activities, including antioxidant, hypoglycemic, vascular protective, improving immunity, and antitumor functions (Chang et al., 2012; Barreca et al., 2014).

In particular, it can neutralize the active free radicals in the cytoplasm and improve the overall ability of cells to resist oxidative stress (Mendes et al., 2018). Recent research has shown that phloretin can enhance the antioxidant activity of the body by regulating cells in various pathways (Behzad et al., 2017). As a new, natural, and high-efficient antioxidant, phloretin has been approved for use in several fields such as medicine, cosmetics, and food processing (Behzad et al., 2017; Wang et al., 2020). However, few studies have explored the use of natural sources of phloretin as a new type of green antioxidant feed additive in broiler production. In addition, treatment with phloretin can also inhibit the expression of IL-8, and TNF-a mRNAs in LPS-stimulated human acute monocytic leukemia-derived cell line (MonoMac 6) (Jung et al., 2009) and it has demonstrated anti-inflammatory effect that reduces contents of pro-inflammatory cytokines and mediators in RAW264.7 cells (Chang et al., 2012). The objective of the present study was to evaluate the effect of supplementary phloretin on growth performance, serum biochemical parameters, inflammation, and antioxidant profiles in broilers reared in a PAH.

Materials and methods

Ethics statement

All experimental procedures involving animals were approved by the Animal Care and Use Committee of the Department of Animal Science at Payame Noor University (Approval No. 02.10.1402). All experiments were conducted in accordance with the national regulations of the Iranian Ministry of Science, Research and Technology for the care and use of laboratory animals. Every effort was made to minimize animal discomfort and reduce the number of animals used.

Experimental birds and diet

A total of 500 one-day-old male broiler chickens (Ross 308) were randomly allocated into five treatments with five replicate pens (with 20 broiler chickens per pen). The negative control group was reared at normal temperature and fed with basal diet while the PAH-treatments (positive control, F-200, F-400, and F-600) were subjected to cool temperature with excessive salt in their drinking water to induce PAH and fed with basal diet containing 0, 200, 400, and 600 mg phloretin per kilogram, respectively until day 42 of age (for a 28-day trial period).

A continuous exposure of lighting program was set at 23 hours of Light: 1 hour of Dark throughout the duration of experiments. For each cage under experiment, spherical pen feeders and drinkers were used; each feeder and drinker was sized 150 × 200 cm. Broiler chickens were vaccinated against New Castle disease and other infectious diseases regularly. A diet based on corn and soybean meal was formulated according to Ross 308 nutrient recommendations, this diet was used for the starter (1 to10 days), grower (11 to 24 days), and finisher (25 to 42 days) periods (Table 1). Diets and freshwater were provided ad libitum. The phloretin (purity: ≥ 98%) used in this research was purchased from Aladdin Reagent Co., Ltd., (Shanghai, China).

Table 1.

The ingredients and composition of the basal diet.

	Starter (0 to10 days)	Grower (11 to 24 days)	Finisher (25 to 42 days)
Ingredients (%)
Mize, 8% CP	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, 21%Ca	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
Methionine, %	0.71	0.64	0.58
Methionine‏cysteine, %	1.07	0.89	0.89
Lysine, %	1.46	1.30	1.17
Arginine, %	1.56	1.45	1.30
Threonine, %	0.96	0.87	0.78
Tryptophan, %	0.35	0.32	0.29

^b Vitamin concentrations per kilogram of diet: 4,500 IU vitamin A; 4000 IU vitamin D3; 3000 IU vitamin E; 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.

Pulmonary arterial hypertension (PAH) induction protocol

On day16, birds were exposed to cool environmental temperatures in combination with excess salt in their drinking water to induce PAH as we have previously described (Tan et al., 2005). Briefly, starting on day 16, the brooding temperature was gradually decreased by 1°C per day until a final temperature of 17°C was reached. Along with the exposure to cold stress, sodium chloride (0.3%, w/v) was given in the drinking water to further accelerate the development of PAH. Bird mortality was recorded daily and necropsies were performed to identify PAH-related death from d 16 onward. Diagnosis of ascites generally depends on observation of the following symptoms: cardiac muscle laxation; Swollen and stiff liver; Clear, yellowish, colloidal fluid in the abdominal cavity (Fathi et al., 2016).

Broiler chicken's growth performance analysis

The broiler chickens were individually weighed at days 14 and 42, and at the end of the experiment body weight gain (BWG) and feed intake (FI) were measured and FCR was calculated for a 28-day trial period. The broiler chickens were inspected daily and the dates of any deaths were recorded. The FCR was calculated as FI/BWG and the body weight of the dead broiler chickens was taken into consideration (Bahrampour et al., 2021).

Blood Sampling for hematological and biochemistry indices

On day 42 of age, 10 birds per treatment (2 birds from each replicate) were selected and slaughtered after overnight fasting in the present study. The birds were killed by exsanguination. The blood sample of broiler was collected. Individual serum sample was separated by centrifuged at 3500 rpm for 12 min under 4°C condition and then stored at −20°C for detecting serum biochemical parameters, oxidative state, and pro-inflammation cytokines contents.

The purpose of this work was measuring hematological indices and analyzing them using an automatic blood analyzer (Sysmex KX-21N, Japan). According of leukocyte differential counts were done by methodology that was described Rasha et al. (2017). Preparation of serum was done by centrifugation at 2,500 × g for 10 min at room temperature and then stored at –20°C for later biochemical analysis (Fathi et al., 2022). Serum contents of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC) and triglyceride (TG) were evaluated by an autoanalyzer (Abbott alcyon 300, US) by laboratory kits (Pars Azmoon, Tehran, Iran). Determination of immunoglobulin G (IgG), and immunoglobulin M (IgM) were measured using commercial diagnostic kits according to the protocol provided by the manufacturer (DIASORIN S.P.A. Italia) by using an autoanalyser (Technicon RA1000, Bayer Diagnostics, Puteaux, France).

Cecal bacterial population

The microbial population analysis in broiler cecum was estimated as follows: In a screw bottle, broiler (from the same slaughtered birds, ten birds per replicate) cecal samples were collected and transferred quickly to the microbiological laboratory. An amount of 10 g from broiler cecal samples were separately collected by gently squeezing into a tube containing 4.5 ml of sterile saline and then 1 g from each sample was homogenized in 9 mL sterile water by a vortex shaker for 30 s and stored at−20°C for the enumeration of microbial population. Enumeration of target bacterial treatments was performed using selective agar media (Merck, Darmstadt, Germany): Lactobacillus spp. (MRS agar); and coliforms (MacConkey agar medium) (Abolfathi et al., 2019). The number of colony-forming units was expressed as log10 CFU/ g of, fresh sample.

Right ventricle / total ventricle (RV/TV) index and mortality because of PAH

Mortalities in the group of chickens were recorded daily, and autopsy were performed to specify related death of mortalities from d 14 onward. Broiler chickens that died with an RV to TV ratio above 0.25 or observation of fluid in the ventricular or pericardium of the heart were included in the PAH mortality (Table 2) (Shao et al., 2022). On the last day of the assessment, day 42, two of ten broiler chickens that were under treatment were dissected. During these dissections, hearts of chickens were weighed, and the atria, pericardium, major vessels, and fat were trimmed off. Separation of left and right ventricles was done, so, their weights were measured on an analytical balance (Scaltec SBA41, Goettingen, Germany; precision 10⁻³ g), and the RV to TV ratio was calculated (Fathi et al., 2022).

Table 2.

Effect of dietary supplementation of phloretin on performance in broiler exposed to pulmonary arterial hypertension (PAH) at day 42 of age.

Items	Negative control1	PAH treatments2				SEM	P- value Linear3	Quadratic3
		Positive control	F-200	F-400	F-600
Feed Intake [g]	4475c	5116a	4780b	4769b	4650bc	75.12	0.012	0.381
Body weight gain [g]	2695a	2460b	2490b	2510b	2500b	49.10	0.014	0.192
Feed conversion ratio [g/g]	1.66d	2.08a	1.92b	1.90bc	1.86cd	0.02	0.001	0.421
RV/TV	0.22d	0.35a	0.28bc	0.29bc	0.25cd	0.02	0.001	0.612
Mortality (%)	4.00c	15.00a	9.00b	7.00b	4.00c	1.52	0.001	0.513

^a,b,c Mean values in the same row with different superscript letters were significantly (N=10)

Negative control: Chicken in control treatment were kept in the normal temperature environment and fed a basal diet. ² PAH treatments (Positive control, F-200, F-400 and F-600): birds were exposed to cool temperature with excessive salt in their drinking water to induce pulmonary arterial hypertension and fed a basal diet supplemented with 0, 200, 400 and 600 mg/kg phloretin.

³The linear and quadratic effects of phloretin were detected by orthogonal polynomials. Abbreviations: RV/TV, Right ventricle/total ventricle.

Antioxidant indices

Determination of Glutathione peroxidase (GPx) activity was done using an enzyme kit supplied by Ransel, RANDOX/RS-504 that supplied by Randox Laboratories, Crumlin, UK. Catalase (CAT) and superoxide dismutase (SOD) activity was assayed using the commercially enzyme kit supplied by Ransod, RANDOX/SD-125 that supplied by Randox Laboratories. The autoanalyzer (Alcyon 300, US) that was used in these research was according to the manufacturers’ protocols. The content of malondialdehyde (MDA) in serum and liver tissue, as an indicator of lipid peroxidation, was performed according to the methodology described by Fathi et al. (2022). Evaluation of oxidative stress in this method was based on measurement of MDA, So MDA is the final product of lipidolysis caused by oxidative stress. Pro-Inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), concentrations in serum and liver tissue were determined by using ELISA kits supplied by Pars Azmoon, Tehran, Iran, according to the manufacturer’s instructions.

Statistical Analysis

The SAS v 11 (SPSS Inc., Chicago, IL, US) and one-way ANOVA test were used to analyze the replicated data using GLM procedures. The statistical model used was: Yij = µ + Ti + eij. Where Y ij = observed value; µ = overall mean; Ti = treatment effect (control, and 1–4); and eij = random error. The differences between means were compared by Tukey’s test at 5% of probability. The SEM and mean values were reported. The linear and quadratic effects of phloretin were detected by orthogonal polynomials. The differences between treatments are considered significant at P < 0.05. Mortality rate was transformed to log₁₀ to normalize the data distribution before running the statistical analysis.

Result

Growth performance, RV/TV index, and mortality because of PAH

As shown in Table 2, PAH induction increased the RV/TV ratio and mortality associated with PAH (P < 0.01). Dietary supplementation with phloretin significantly reduced both the RV/TV ratio and PAH-related mortality (P < 0.01). Additionally, PAH induction significantly decreased body weight gain while increasing feed intake and feed conversion ratio (FCR) (P < 0.01). Although phloretin supplementation did not significantly affect body weight gain, it significantly reduced feed intake and improved FCR (P < 0.01).

Hematological indices

Hematological indices are shown in Table 3. As shown, PAH induction significantly increased the values of white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), mean corpuscular volume (MCV), and neutrophils (HET), while it decreased lymphocyte (LYM) counts (P <0.01). Supplementary phloretin significantly improved hematological indices (P < 0.01).

Table 3.

Effect of dietary supplementation of phloretin on hematological parameters of broiler exposed to pulmonary arterial hypertension (PAH) at day 42 of age.

Items	Negative control1	PAH treatments2				SEM	P- value Linear3	Quadratic3
		Positive control	F-200	F-400	F-600
RBCs count (× 106/ μl)	2.58b	2.98a	2.59b	2.27c	2.23c	0.02	0.006	0.513
HGB (g/dl)	11.10c	14.40a	14.30a	13.20bc	11.70c	0.45	0.003	0.710
MCV (%)	27.81c	36.30a	34.20a	29.30bc	29.41bc	0.95	0.002	0.432
WBCs count (× 103/ μl)	16.60b	19.06a	18.24a	18.10a	15.18bc	0.39	0.005	0.621
HET (%)	9.02c	17.20a	14.56b	12.14bc	10.83c	1.45	0.001	0.292
LYM (%)	23.10a	15.18c	15.90b	18.41b	19.66b	1.58	0.002	0.391
HET / LYM Ratio	0.393e	1.13a	0.915b	0.659c	0.550d	0.05	0.001	0.481

^a,b,c Mean values in the same row with different superscript letters were significantly. (N=10)

Negative control: Chicken in control treatment were kept in the normal temperature environment and fed a basal diet. ² PAH treatments (Positive control, F-200, F-400 and F-600): birds were exposed to cool temperature with excessive salt in their drinking water to induce pulmonary arterial hypertension and fed a basal diet supplemented with 0, 200, 400 and 600 mg/kg phloretin.

³The linear and quadratic effects of phloretin were detected by orthogonal polynomials. Abbreviations: RBCs, Red blood cells; HGB, Hemoglobin; MCV; Mean corpuscular volume, Hematocrit; WBCs, White blood cells; HET, Heterophil; LYM, Lymphocyte,

Biochemical indices

PAH induction significantly increased the serum levels of ALT, AST, triglycerides (TG), and total cholesterol (TC) (Table 4) (P <0.01). Supplementation with 400 and 600 mg/kg of phloretin significantly reduced these parameters (P < 0.01). In addition, PAH induction decreased the serum levels of glutathione peroxidase (GPx) and superoxide dismutase (SOD), and increased malondialdehyde (MDA) concentrations (Table 4) (P<0.01). Supplementation with 400 and 600 mg/kg of phloretin significantly enhanced GPx and SOD levels and decreased MDA concentrations in serum (P < 0.01). As shown in Table 4, PAH induction significantly reduced serum concentrations of immunoglobulin G (IgG) and immunoglobulin M (IgM) (P < 0.01). Inclusion of 400 and 600 mg/kg of phloretin tended to increase IgG and IgM concentrations (P < 0.01).

Table 4.

Effect of dietary supplementation of phloretin on biochemical parameters of broiler exposed to pulmonary arterial hypertension (PAH) at day 42 of age.

Items	Negative control1	PAH treatments2				SEM	P- value Linear3	Quadratic3
		Positive control	F-200	F-400	F-600
TG (mg/dL)	79.58c	133.53a	122.46a	96.01b	70.15c	4.45	0.002	0.781
TC (mg/dL)	97.05c	158.07a	109.47b	107.33b	98.53c	3.75	0.001	0.591
ALT (U/L)	8.27c	17.28a	16.54ab	14.30b	9.92c	1.05	0.021	0.642
AST (U/L)	122.85c	169.43a	162.69a	155.38ab	131.19bc	3.95	0.010	0.490
GSH-Px (U/mL)	594.50a	340.35c	391.05c	474.11b	556.40a	35.10	0.001	0.592
SOD (U/mL)	151.66a	115.18cd	129.20c	134.5bc	148.60a	11.25	0.011	0.620
MDA (nm/mL)	7.61b	9.47a	9.46a	8.38ab	7.06b	0.16	0.002	0.494
IgM (U/ml)	3.47a	2.84c	2.89c	3.27b	3.37b	0.35	0.001	0. 312
IgG (U/ml)	5.47b	4.13c	4.49c	6.42a	6.12a	0.41	0.001	0.490
IL-1β (ug/mL)	2.10d	15.10a	14.28ab	6.20c	5.48c	0.51	0.002	0.481
TNF-α (ug/mL)	5.20d	15.20a	13.24b	9.52c	8.57c	0.40	0.001	0.521

^a,b,c Mean values in the same row with different superscript letters were significantly. (N=10)

Negative control: Chicken in control treatment were kept in the normal temperature environment and fed a basal diet. ² PAH treatments (Positive control, F-200, F-400 and F-600): birds were exposed to cool temperature with excessive salt in their drinking water to induce pulmonary arterial hypertension and fed a basal diet supplemented with 0, 200, 400 and 600 mg/kg phloretin.

³The linear and quadratic effects of phloretin were detected by orthogonal polynomials. Abbreviations:TG, triglyceride; TC, total cholesterol; ALT, alanine transaminase; AST, aspartate aminotransferase. GPx, glutathione peroxidase; SOD, superoxide dismutase; MDA, malondialdehyde. IgG: Immunoglobulin G, IgM: Immunoglobulin M. IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-α.

The effects of treatments on pro-inflammatory cytokines (tumor necrosis factor-α [TNF-α] and interleukin-1β [IL-1β]) in serum are summarized in Table 4. Broiler chickens subjected to PAH showed elevated serum concentrations of TNF-α and IL-1β compared to the control group (P < 0.01). However, birds fed diets supplemented with 400 and 600 mg/kg of phloretin showed significantly lower serum levels of TNF-α and IL-1β (P < 0.01).

Cecal microbiota

The results of the cecal microbiota analysis are presented in Table 5. While the cecal coliform population was not significantly affected by treatments, PAH induction significantly decreased the population of cecal Lactobacillus compared to the control group (P < 0.01). Supplementation with 400 and 600 mg/kg of phloretin significantly increased Lactobacillus counts in the cecum (P < 0.01)

Table 5.

Effect of dietary supplementation of phloretin on cecal bacterial population of broiler exposed to pulmonary arterial hypertension (PAH) at day 42 of age.

Microbiological Count (Log 10 CFU/g fresh digesta)	Negative control1	PAH treatments2				SEM	P-value Linear3	Quadratic3
		Positive control	F-200	F-400	F-600
Coliforms	7.41	7.35	7.32	7.29	7.26	0.04	0.130	0.273
Lactic acid bacteria	7.55a	3.14c	3.07c	4.25b	7.82a	0.19	0.001	0.561

^a,b,c Mean values in the same row with different superscript letters were significantly. (n = 10).

Negative control: Chicken in control treatment were kept in the normal temperature environment and fed a basal diet. ² PAH treatments (Positive control, F-200, F-400 and F-600): birds were exposed to cool temperature with excessive salt in their drinking water to induce pulmonary arterial hypertension and fed a basal diet supplemented with 0, 200, 400 and 600 mg/kg phloretin.

³The linear and quadratic effects of phloretin were detected by orthogonal polynomials. Abbreviations:

CFU, Colony Forming Unit.

Discussion

In this study, by experimentally inducing pulmonary arterial hypertension (PAH), we investigated the hematological, biochemical, and anatomical changes of the heart due to PAH, as well as the effects of supplementing different levels of phloretin on these changes. In this regard, 35% of the broiler chickens subjected to cold-induced PAH exhibited signs and symptoms of PAH syndrome and subsequently died. Necropsy of the deceased chickens revealed that the ratio of the right ventricle to the total ventricles was significantly higher compared to the control treatment broiler chickens. Several previous studies have reported that cold stress induces ascites syndrome and leads to an increase in the relative weight of the right ventricle, cardiac hypertrophy, and mortality (Shao et al., 2022; Fathi et al., 2016, 2022; Wang et al., 2020). It has been suggested that one of the primary causes of ascites-related mortality is oxidative stress triggered by hypoxemia during cold exposure. Therefore, antioxidant therapy has been proposed as an effective strategy to reduce mortality in broiler chickens experiencing oxidative stress due to induced PAH.

In the present study, PAH-related mortality was reduced in the phloretin-treated groups, likely due to the antioxidant effects of phloretin. Many researchers have already reported the antioxidant properties of phloretin (Huang et al., 2017; Zhang et al., 2019; Han et al., 2020; Ben-Othman et al., 2021). In the present study, supplementation of phloretin in the diet improved the growth performance of chickens under cold stress associated with PAH, which may indicate its beneficial effects in counteracting the adverse consequences of cold stress.

Several studies have shown that dietary supplementation with antioxidants such as aspirin, vitamin E, chitosan, lycopene, epigallocatechin gallate, melatonin, and curcumin to the diet can alleviate various types of stress including heat stress (Zhang et al., 2015), cold stress (Fathi et al., 2016, 2022, 2023a) and physiological stress (Fathi et al., 2023b) and help maintain broiler performance. Similarly, in the current study, phloretin was able to mitigate the negative impacts of cold stress on growth performance. Zhang et al. (2015) demonstrated that the plant-derived antioxidant can also enhance the growth performance of broilers under heat stress.

Consist with our result, Low environmental temperature was associated with higher RBCs, HGB, MCV, WBCs and HET values in broiler chickens (Fathi et al., 2022). The inclusion of phloretin reduced, RBC, HGB, MCV, and HET values, while increasing WBC counts (Table 3). To evaluate chickens exposed to stressors, a wide range of parameters are assessed, including physiological indicators related to metabolism, immunity and antioxidant response, along with blood or serum stress markers (Puvadolpirod and Thaxton, 2000).The heterophil-to-lymphocyte (H/L) ratio is widely recognized as one of the most reliable indicators of stress status in birds. In stressed chickens, heterophil numbers typically increase while lymphocyte counts decrease, resulting in a higher H/L ratio (Siegel, 1995). It has been suggested that phloretin's role in reducing oxidative damage may contribute to improvements in RBC counts. One of the most important features that distinguishes phloretin from other antioxidants is its ability, through its metabolites, to effectively scavenge free radicals (Huang et al., 2017; Han et al., 2020; Hu et al., 2021).

The results of the present study showed that serum ALT, AST, TG, and TC levels increased in broilers during PAH. Phloretin supplementation reduced the levels of these biochemical parameters in PAH-induced broilers. It is assumed that dietary phloretin enhances the utilization of TC and TG, effectively inhibit the catabolism of their reserves, and thereby mitigates the effects of environmental stress on serum biochemical parameters in broilers (Hu et al., 2021). Another proposed mechanism by which antioxidant compounds reduce cholesterol is by inhibiting its biosynthesis, particularly at the initial step involving hydroxymethylglutaryl coenzyme A reductase (HMG-COA). The conversion of HMG-CoA to mevalonate is catalyzed by HMG-CoA reductase in the presence of nicotinamide adenine dinucleotide phosphate (NADPH).

Antioxidants have been shown to reduce mevalonate production by inhibiting the activity of HMG-CoA reductase. Plant phenolic compounds can reduce LDL-C oxidation and increase serum HDL-C levels (Weinbrenner et al., 2004). Furthermore, antioxidants lower triglyceride levels by enhancing their absorption and reducing hepatic triacylglycerol lipase activity (Ahmadipour et al., 2015). Studies have shown that flavonoids and anthocyanins help prevent chronic cardiovascular disease and atherosclerosis by scavenging free radicals, inhibiting LDL-C oxidation, and reducing blood levels of triglycerides and cholesterol (Ustundag and Ozdogan, 2015). Therefore, due to its rich antioxidant content, particularly phenols and flavonoids, phloretin may help prevent cardiovascular complications associated with ascites.

Meanwhile, supplementing the diet of broiler chickens with antioxidant compounds such as lycopene and aspirin can protect liver cells by exerting antioxidant effects, thereby preventing their destruction and the leakage of liver enzymes into the bloodstream (Fathi et al., 2016, 2022). Therefore, it is possible that phloretin, through its antioxidant properties, reduced the serum levels of liver enzymes in birds experiencing oxidative stress induced by PAH.

Consistent with our results, cold temperature environments increase serum lipid peroxidation in broilers, accelerate the depletion of antioxidant factors, and impair the body's antioxidant capacity (Fathi et al., 2016, 2022, 2023a). Phloretin's ability to alleviate oxidative stress is attributed to its effect on modulating the cellular redox state (Han et al., 2020). Huang et al. (2017) showed that phloretin significantly increased GSH concentration and reduced MDA content in the oxidative-stressed lungs of asthmatic mice.

Zhang et al. (2019) also suggested that phloretin improves of oxidative stress in the colon of mice with ulcerative colitis by regulating MDA, SOD, and GSH levels. Hu at al. (2021) also suggested that phloretin improves oxidative stress in broilers exposed to heat stress. Based on this, we speculated that phloretin alleviates PAH-induced stress damage by enhancing the body's antioxidant defense.

It has been shown that cold stress can affect the levels of certain immunoglobulins. It has also been reported that cold stress suppresse humoral immunity and decreased the cell-mediated immunity of chickens. However, the specific effects of cold stress on immunoglobulin levels in chickens are less well understood. In the present study cold stress combined with salt in the drinking water reduced serum IgM and IgG levels. Similar to our findings, Zhao et al. (2013) reported that the mRNA expression levels of the IgM and IgG genes initially increased and then decreased in the duodenum, jejunum, and ileum of broilers under cold stress compared with the control treatment. Borsoi et al. (2015) showed that broilers exposed to cold stress exhibited lower plasma IgG levels compared to non-stressed birds. The increase in serum immunoglobulin levels due to phloretin supplementation may be attributed to the additive effect of its antioxidant properties, which enhance the birds’ immune system (Abd El-Hack et al., 2021).

In the present study, PAH induction increased the serum levels of pro-inflammatory cytokines, including TNF-α and IL-1β (Table 4). Similar to our findings, Zhao et al. (2013) reported that cold stress increased the pro- inflammatory cytokine concentrations in duodenum, jejunum, and ileum of broilers. In response to stress, pro-inflammatory cytokine levels increase, potentially leading to hemorrhage and necrosis in the liver and spleen, followed by visible signs of inflammation (Azuma et al., 2015). Several reports have shown that oxidative stress triggers severe inflammatory responses in affected cells, leading to extensive tissue damage and the activation of apoptosis

(Jiang et al. 2018). Previous studies have indicated that the inflammatory cytokines production is associated with the overproduction of ROS under oxidative and heat stress in broilers (Ruixia et al. 2020a) and rats (Ruixia et al. 2020b; Tan et al. 2005). In this study, dietary phloretin supplementation significantly decreased the serum levels of IL-1β and TNF-a. Our results indicated that PAH induced by low temperature and salt exposure—followed by oxidative stress and inflammation—was alleviated by phloretin supplementation.

Phloretin, a bioactive flavonoid, has been shown to exhibit both anti-inflammatory and antioxidant effects (Cahng et al., 2012; Wang et al., 2018). Wang et al. (2018) reported that Phloretin pretreatment significantly suppressed the mucins secretion, inflammatory cell infiltration, and cytokine release in cigarette smoke-exposed mouse lungs, and also inhibited CSE-induced expression of MUC5AC and IL-1β in NCI-H292 bronchial epithelial cells. Furthermore, western blot analysis revealed that phloretin attenuated the activation of EGFR, ERK and P38 pathways both in vivo and in vitro. Moreover, phloretin has been found to provide protective effects in pulmonary inflammatory disorders. Huang et al. (2017) demonstrated that phloretin suppressed the release of inflammatory cytokines in the lungs of ovalbumin-induced allergic mice. In-vitro studies also showed that phloretin alleviated interferon γ-induced inflammation in human lung fibroblasts (Jeon et al., 2017).

Additionally, phloretin was shown to inhibit the activation of MAPK signaling molecules and reduce downstream inflammatory cytokine production in interferon-γ-stimulated lung fibroblasts, TNF-α-stimulated macrophages, LPS-stimulated dendritic cells, and LPS-treated mice (Aliomrani et al., 2016).

Barreca et al. (2014) described the antimicrobial activity of phloretin against 14 Gram positive and Gram negative bacteria. Phloretin exhibited strong antimicrobial activity and was able to inhibit Gram-positive bacteria, particularly S. aureus ATCC 6538, L. monocytogenes ATCC 13932, methicillin-resistant S. aureus clinical strains and S. typhimurium ATCC 13311. Structure–activity relationship studies showed that the presence of a free hydroxyl group at the C20 position and the absence of glycosyl molecules are crucial for interaction with biological targets, as highlighted by the dramatic reduction in antimicrobial activity in glycosylated structures at C20 and at both C30 and C50. Furthermore, the analysis of key cytosolic enzymes in S. aureus provided insight into the antimicrobial mechanism of phloretin. In fact, phloretin disrupts the energy metabolism of S. aureus by reducing the enzymatic activity of LDH and IDH, altering the utilization of biological fuels (such as sugars, fatty acids, and amino acids), and impairing the bacterium's ability to resist oxidative damage by significantly inhibiting catalase activity.

Conclusion

According to the study’s findings, supplementing broiler diets with 600 mg/kg of phloretin improved growth performance, enhanced feed efficiency, reduced mortality, and promoted intestinal health. Additionally, the same level of phloretin supplementation enhanced the birds’ physiological performance and immune responses.

CRediT authorship contribution statement

Mokhtar Fathi: Conceptualization, Methodology, Formal analysis, Project administration, Data curation, Supervision, Investigation, Writing – original draft, Writing – review & editing, Supervision, Project administration. Kianoosh Zarrinkavyani: Conceptualization, Methodology, Formal analysis, Project administration, Data curation, Supervision, Resources, Writing – original draft, Writing – review & editing, Supervision, Project administration. Zahra Biranvand: Investigation, Visualization, Validation, Software. Morteza Aghil Al Abd: Investigation, Resources, Visualization, Validation, Software.

Disclosures

Authors declare no conflict of interest.