Effects of dietary saponin and polyphenol supplementation in broiler chickens exposed to multiple mild stressors of cyclic elevated ambient temperature, feed withdrawal, and coccidiosis infection

Laney E Froebel; Michael J Rincker; Ryan N Dilger

doi:10.1016/j.psj.2026.106816

. 2026 Mar 18;105(6):106816. doi: 10.1016/j.psj.2026.106816

Effects of dietary saponin and polyphenol supplementation in broiler chickens exposed to multiple mild stressors of cyclic elevated ambient temperature, feed withdrawal, and coccidiosis infection

Laney E Froebel ^a, Michael J Rincker ^b, Ryan N Dilger ^a,^⁎

PMCID: PMC13022671 PMID: 41865655

Abstract

Broilers are commonly exposed to numerous environmental and immunological stressors in a production setting. In this study, two botanical feed additives were evaluated for their effects on growth performance, antioxidant defenses, and intestinal health in stressed broilers. To achieve a multiple mild stressors challenge, birds underwent a 12-h feed withdrawal prior to a coccidiosis vaccine challenge on d 15 of the study, which was designated as 0 d post-inoculation (DPI). Additionally, birds were exposed to cyclic elevated ambient brooder temperatures during the grower phase. A total of 420 male Ross 708 chicks at 2 d post-hatch were assigned to 1 of 5 treatment groups with 7 birds allotted to each of 12 replicate cages. The 42-d study (starter d 0-14, grower d 14-28, finisher d 28-42) was conducted with treatments including: 1) control diet + birds not exposed to stressors (NC), 2) control diet + birds exposed to stressors (PC), 3) PC + 500 mg of Micro-Shield/kg of diet in the starter phase only (MS), 4) PC + 500 mg of Micro-Shield/kg diet in the starter, grower, and finisher phases (MSall), and 5) PC + 500 mg of Micro-Shield/kg diet in the starter phase + 250 mg of Micro-Aid/kg diet in the grower and finisher phases (MS+MA). All data were subjected to a 1-way ANOVA using the MIXED procedure of SAS with results considered significant when P < 0.05. Birds exposed to stressors had impaired growth performance during the grower phase compared with birds in the NC group (P < 0.05). On 6 DPI, birds in the MS+MA treatment had the highest (P < 0.05) levels of catalase in blood serum, potentially indicating higher antioxidant capacity. While birds in the NC treatment had the highest gene expression of zonula occludens-1 (ZO-1) in jejunal mucosa tissue on 6 DPI (P < 0.05), MS+MA birds had similar ZO-1 expression compared with NC birds. These results suggest botanicals may confer health benefits in broilers experiencing environmental and immunological stressors.

Keywords: Broiler, Eimeria, Environmental stress, Polyphenol, Saponin

Introduction

Broilers typically experience a combination of environmental and immunological stressors in commercial production settings. Avian coccidiosis is a prevalent parasitic disease caused by the Eimeria protozoan that is ubiquitous in the poultry-rearing environment (Williams, 1998). Coccidiosis is extremely costly to the poultry industry, with estimates reporting coccidiosis cost the global poultry industry approximately $14.5 billion in 2016 (Blake et al., 2020). These losses are associated with diminished growth performance as well as costs for prophylaxis and treatment. Previously, technologies including antibiotics, chemicals, and vaccines had been successfully utilized to control diseases such as coccidiosis (Adhikari et al., 2020). However, the prevalence of drug-resistant strains of Eimeria as well as consumers driving the production of poultry products raised without the usage of antibiotics has created the need for alternative strategies to mitigate coccidiosis (Diarra and Malouin, 2014; Cervantes and McDougald, 2023).

In addition to immunological challenges such as coccidiosis, broilers are becoming increasingly threatened by increasing volatility in environmental temperatures. When the amount of heat produced by the host surpasses its ability to dissipate extra body heat to their surrounding environment, heat stress can occur (Farag and Alagawany, 2018). While broilers reared in tropical and subtropical climates are more threatened with heat stress conditions, birds are physiologically predisposed to experience heat stress due to their feathering and lack of sweat glands (Richards, 1970; Nichelmann et al., 1986). The most recent estimate of annual financial losses associated with heat stress in the U.S. poultry industry is $128-165 million (St-Pierre et al., 2003). These losses are associated with reduced growth performance as well as impaired digestive system functionality and permeability (Rostagno, 2020; Rocchi et al., 2022; Teyssier et al., 2022). In addition to the environmental stressor of heat stress, birds can experience off-feed events in commercial production systems, which can further alter intestinal structure, induce a stress response in broilers, and impair feed efficiency (Yamauchi et al., 1996; Thompson and Applegate, 2006; Najafi et al., 2018; Froebel et al., 2026).

In recent years, phytogenic compounds, including saponins and polyphenols, have been investigated as alternatives to improve broiler growth performance and health (El-Shall et al., 2022). Saponins are a class of botanical compounds with soap-like properties that can disrupt the membranes of Eimeria oocysts and sporozoites, leading to parasitic cell lysis and ultimately, cell death (Francis et al., 2002; Nazarro et al., 2013). This mechanism of action diminishes the ability of Eimeria to invade intestinal epithelial cells, thus reducing overall parasite load and severity of infection. Additionally, saponins have been previously reported to have antioxidant and immunomodulatory effects in broilers (Oelschlager et al., 2019; Dai et al., 2023). Polyphenols are an additional class of botanical compounds that have been evaluated for their ability to support broiler performance and health. Because of their chemical structure, polyphenols can alter membrane permeability of pathogens like Eimeria to influence the cationic transfer of hydrogen and potassium ions, ultimately leading to pathogen death (Idris et al., 2016). Further, polyphenols have been previously reported to increase antioxidant capacity and improve intestinal barrier function (Lee et al., 2020; Madkour et al., 2024).

Coccidiosis, heat stress, and acute feed withdrawal periods often occur concurrently in commercial broiler production settings. However, their combined effects remain poorly characterized in the literature. As such, the objective of this study was to evaluate if dietary supplementation of commercially available feed additives containing saponins and polyphenols influence the growth and immune responses of broilers during a multiple mild stressors challenge of coccidiosis, elevated brooder temperatures, and an acute off-feed event.

Materials and methods

All animal care and experimental procedures were approved by the University of Illinois Institutional Animal Care and Use Committee prior to initiation of experiment.

Bird husbandry and experimental design

A total of 420 Ross 708 male broilers were obtained from a commercial hatchery (Hoover’s Hatchery, Rudd, IA) 2 d post-hatch and placed in thermostatically controlled battery cages (99 cm × 34 cm floor space; model SB5T; Alternative Design Manufacturing, Siloam Springs, AR) with raised wire flooring in an environmentally controlled room. Birds were exposed to 1 h of darkness for the first 4 d of the trial and then underwent a step-down lighting program to reach 4 h of darkness on d 7, which remained constant for the remainder of the trial. Upon arrival, birds were individually weighed, selected, wing-banded, and assigned to 1 of 5 treatment groups with 7 birds allotted to each of the 12 replicate cages per treatment. On d 28 of the trial, 4 birds/cage (48 birds/treatment) were moved into floor pens (6 pens/treatment) with fresh pine shavings. Standard corn-soybean meal-based diets (Table 1) were formulated to meet or exceed broiler nutritional requirements (NRC, 1994) for starter (d 0-14), grower (d 14-28), and finisher (28-42) phases. Proximate analyses of diets were analyzed using a native calibration package B-FEED-M program of TANGO utilizing a Fourier transform near-infrared spectrometer (i.e., FT-NIR; Bruker Optics, Billerica, MA) and are reported in Table 2.

Table 1.

Ingredient and calculated nutrient composition of experimental basal diets.

Item	Starter	Grower	Finisher
Ingredient, g/kg
Corn	525.8	529.7	575.4
Soybean meal	390.0	378.0	325.0
Soybean oil	35.0	50.0	57.0
Sodium chloride	4.0	4.0	4.0
Limestone	12.0	11.0	11.0
Dicalcium phosphate	21.0	18.0	18.0
Vitamin premix¹	2.0	2.0	2.0
Mineral premix²	1.5	1.5	1.5
Choline chloride	3.2	3.2	3.2
L-Lys HCl	1.4	0.0	0.3
DL-Met	3.2	2.3	2.3
L-Thr	0.9	0.3	0.3
Calculated composition
ME, kcal/kg	3,046	3,162	3,248
Protein, g/kg	229.3	223.9	202.3
Ca, g/kg	10.6	9.5	9.3
Total P, g/kg	8.0	7.4	7.1
Non-phytate P, g/kg	5.3	4.7	4.6
Ca:tP	1.3	1.3	1.3
Ca:nPP	2.0	2.0	2.0
Na, g/kg	2.0	2.0	2.0
Digestible AA, g/kg
Arg	14.3	14.0	12.4
His	5.8	5.7	5.1
Ile	8.8	8.6	7.7
Leu	17.3	17.0	15.7
Lys	12.7	11.3	10.2
Met	6.4	5.5	5.2
Met + Cys	9.5	8.5	8.0
Phe	10.3	10.0	9.1
Phe + Tyr	16.3	15.9	14.3
Thr	8.3	7.5	6.8
Trp	2.5	2.5	2.2
Val	9.5	9.3	8.4

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Provided per kilogram of complete diet: retinyl acetate, 4,400 IU; cholecalciferol, 25 µg; dl-α-tocopheryl acetate, 11 IU; vitamin B12, 0.01 mg; riboflavin, 4.41 mg; d-Ca-pantothenate, 10 mg; niacin, 22 mg; and menadione sodium bisulfite, 2.33 mg.

Provided per kilogram of complete diet: Mn, 75 mg from MnO; Fe, 75 mg from FeSO₄•7H₂O; Zn, 75 mg from ZnO; Cu, 5 mg from CuSO₄•5H₂O; I, 0.75 mg from ethylene diamine dihydroiodide; and Se, 0.1 mg from Na₂SeO₃.

Table 2.

Analyzed composition of experimental diets¹.

	Starter		Grower			Finisher
Item² , % as-is	NC, PC	MS, MSall, MS+MA	NC, PC, MS	MSall	MS+MA	NC, PC, MS	MSall	MS+MA
Moisture	11.74	11.57	11.75	11.78	11.66	11.60	11.55	11.64
Fat	4.63	4.56	6.12	6.00	5.93	6.73	6.72	6.63
Protein	22.00	22.07	21.85	21.76	22.09	19.95	19.71	20.12
Fiber	3.16	3.20	3.36	3.27	3.20	3.42	3.45	3.49
Ash	6.00	6.02	6.04	6.05	5.89	5.89	5.91	5.85
Starch	36.26	36.03	35.01	35.09	35.35	37.04	37.31	36.84

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Dietary phases were provided as follows: d 0-14 starter diet, d 14-28 grower diet, and d 28-42 finisher diet. The following diet names have been assigned to a respective experimental treatment group: NC, control diet + not exposed to multiple mild stressors; PC, control diet + exposed to multiple mild stressors; MS, 500 mg/kg Micro-Shield in the starter diet + exposed to multiple mild stressors; MSall, 500 mg/kg of Micro-Shield in the starter diet + 500 mg/kg of Micro-Shield in the grower diet + 500 mg/kg of Micro-Shield in the finisher diet + exposed to multiple mild stressors; MS+MA, 500 mg/kg of Micro-Shield in the starter diet + 250 mg/kg of Micro-Aid in the grower diet + 250 mg/kg of Micro-Aid in the finisher diet + exposed to multiple mild stressors.

Compostion of diets measured using a native calibration package B-FEED-M program of TANGO on a Bruker Fourier transform near-infrared spectrometer (i.e., FT-NIRS).

Commercially available feed additives (Micro-Aid and Micro-Shield; DPI Global, Porterville, CA) were supplemented on top of the formulation (i.e., no space reserved in the formulation) as their contribution to the overall nutritive value of the diet was considered negligible. Micro-Aid contains Yucca shidigera-derived saponins, whereas Micro-Shield is a blend of bioactive ingredients (i.e. rosemary, fenugreek seed, Quillaia, oregano) that contribute both saponins and polyphenols to ingredient composition. The following treatment names were assigned to the experimental treatment groups: 1) control diet + not exposed to multiple mild stressors (NC), 2) control diet + exposed to multiple mild stressors (PC), 3) 500 mg/kg of Micro-Shield in the starter diet + exposed to multiple mild stressors (MS), 4) 500 mg/kg of Micro-Shield in the starter diet + 500 mg/kg of Micro-Shield in the grower diet + 500 mg/kg of Micro-Shield in the finisher diet + exposed to multiple mild stressors (MSall), and 5) 500 mg/kg of Micro-Shield in the starter diet + 250 mg/kg of Micro-Aid in the grower diet + 250 mg/kg of Micro-Aid in the finisher diet (MS+MA). Broilers had ad libitum access to feed and water for the duration of the trial apart from some birds experiencing a 12-h feed withdrawal prior to a coccidiosis inoculation.

Birds and feeders were weighed on d 0, 14, 28, and 42 to calculate body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR) to evaluate growth performance. Birds were checked twice daily to record mortality and culls, which were used to adjust FI and FCR. Study mortality is reported in Supplementary Table 1. Because some birds had variable feed intake on d 14 due to the 12-h feed withdrawal intervention, feed intake for this day was not included in the data analysis. On d 21 and 28 [i.e., 6 and 13 d post-inoculation (DPI)], one bird per battery cage (12 birds/treatment) was randomly selected and euthanized via CO₂ asphyxiation to permit the collection of blood via cardiac puncture and tissue samples. On d 42 (27 DPI), two birds per floor pen (12 birds/treatment) were euthanized via CO₂ asphyxiation prior to collecting blood and tissue samples. Bird and liver weights were recorded from sample birds on d 6, 13, and 27 DPI.

Multiple mild stressor challenge

A previously identified multiple mild stressors challenge was utilized to impose a combination of environmental and immunological stressors in broilers (Froebel et al., 2026). To achieve a multiple mild stressors challenge, birds exposed to stressors underwent a 12 h feed withdrawal on d 14 prior to a coccidiosis vaccine challenge. A validated vaccine challenge was utilized to induce a coccidiosis infection of d 15, and the day of challenge served as DPI 0. Chicks were orally gavaged with a vaccinator gun fitted with an oral gavage needle to administer 1 mL of either tap water (NC treatment group) or Eimeria oocysts (PC, MS, MSall, and MS+MA treatment groups). The commercial vaccine (Coccivac-B52; Merck Animal Health, Madison, NJ) contained live oocysts of E. acervulina, E. maxima, E. mivati, and E. tenella species.

On d 14, the average body weight of all birds was calculated to determine the dose for vaccine inoculation similar to how previously described (Froebel et al., 2026). The dosage was administered at 5 × the manufacturer’s recommendation (i.e. 1 dose per 40 g of body weight) using the average body weight on d 14. The average d 14 body weight was 472 g, and therefore, the required doses to achieve a 5 × challenge was 59 doses/bird. Additionally, birds in treatment groups experiencing multiple mild stressors were exposed to cyclically elevated brooder temperatures (daily routine of 34°C for 8 h then 24°C for 16 h) during the entire grower phase (d 14-28). Birds assigned to the NC treatment group were housed in thermoneutral conditions (24°C, 24-h) during this period. All birds were housed at 24°C after study d 28 and until study conclusion.

Differential blood cell counts

At 6 and 13 DPI, approximately 4 mL of blood from 1 bird per cage was submitted to the Veterinary Clinical Pathology Laboratory at the University of Illinois Urbana-Champaign for measuring total and differential blood cell counts. Because of the unreliability of automated blood counting machines associated with avian blood cells, counts were done manually.

Intestinal permeability

On 6 and 13 DPI, fluorescein isothiocyanate-dextran (FITC-d, CAS 60842-46-8; MW 4000; Sigma-Aldrich, St. Louis, MO) was used to evaluate intestinal permeability. One bird per cage was orally gavaged with 1 mL of 2.2 mg FITC-d/mL as reconstituted in water. Two hours after being orally gavaged, birds were euthanized and blood was collected via cardiac puncture into evacuated serum tubes, allowed to clot at room temperature, and centrifuged to obtain serum, which was stored at −20°C until analysis. The amount of FITC-d in serum was measured at an excitation wavelength of 485 nm and an emission wavelength of 528 nm using a multi-mode microplate reader (Synergy HT; BioTek Instruments, Inc., Winooski, VT).

Jejunal histomorphology and histopathology

Upper jejunum samples were collected on 6 and 13 DPI for obtaining histomorphological measurements and histopathological lesion scores. Jejunal samples were collected in 10% neutral buffered formalin from 1 bird per cage and stored at room temperature until further analysis. Samples were examined by a board-certified histopathologist (Veterinary Diagnostic Pathology, LLC, Fort Valley, VA) who was blinded to treatment identification. Intestinal samples were scored on a 0–5-point scale as previously described (Abdul Rasheed et al., 2020).

Antioxidative defenses and oxidative stress markers

Serum samples were collected on 6 and 13 DPI from 1 bird per battery cage as previously described and stored at −80°C for the evaluation of catalase (CAT; Cat. No.: 707002), glutathione peroxidase (GPx; Cat. No.: 703102), nitrate (NO₃^-; Cat. No.: 780001), nitrite (NO₂^-; Cat. No.: 780001) and superoxide dismutase (SOD; Cat. No.: 706002) using commercially available kits (Cayman Chemical, Ann Arbor, MI). All assays were measured using a multi-mode microplate reader (Synergy HT; BioTek Instruments, Inc.) according to individual kit manufacturer’s instructions. Additionally, serum, liver, and breast tissue were collected from one bird per battery cage (12 birds/treatment) on 6 and 13 DPI and two birds per floor pen (12 birds/treatment) on 27 DPI for the measurement of malondialdehyde (MDA) using a commercially available kit (Cat. No.: 700870; Cayman Chemical).

Gene expression

Representative samples from the liver and jejunum (tissue separate from mucosal scrapings) were collected from 1 bird per cage on 6 and 13 DPI to evaluate gene expression. Approximately 50-100 mg of tissue were homogenized in 1 mL of Trizol (Cat No.: 15596018; Invitrogen, Carlsbad, CA) for 2 minutes at 30 Hz (TissueLyser II; Qiagen, Valencia, CA). RNA extraction from tissue samples was conducted according to manufacturer instructions. Extracted RNA was evaluated for quantity and purity using a spectrophotometer (NanoDrop ND-100; Nano-Drop Technologies, Wilmington, DE) and reverse-transcribed to obtain complementary DNA (cDNA) using a high-capacity reverse transcription kit (Cat No.: 4368814; Thermo Fisher Scientific Inc., Waltham, MA). Sample RNA was put into a thermocycler (Bio-Rad, Hercules, CA) for 10 min at 25°C, 120 min at 37°C, and 5 min at 85°C before being cooled to 4°C. Synthesized cDNA was stored at −20°C until plating.

The TaqMan Gene Expression Assay (Thermo Fisher Scientific Inc., Waltham, MA) was used to evaluate relative gene expression of heat shock protein-70 (HSP70) and heat shock protein-90 (HSP90) in liver tissue, interleukin-1 beta (IL-1β) and interleukin-10 (IL-10) in jejunal tissue, and occludin (OCLN) and zonula occludens-1 (ZO-1) in jejunal mucosa (Thermo Fisher Scientific Inc.). Gene details and National Center for Biotechnology Information reference numbers are shown in Table 3. Amplification by real-time, quantitative PCR was achieved for target (HSP70, HSP90, IL-1β, IL-10, OCLN, ZO-1) and reference (glyceraldehyde-3-phosphate dehydrogenase; GAPDH; NM_204305.1; Thermo Fisher Inc.) genes. Synthesized cDNA was amplified with Taqman (Cat No.: 4304437; Thermo Fisher Scientific Inc) oligonucleotide probes containing 5′ fluorescent reporter dye and 3′ non-fluorescent quencher dye. Generation of fluorescence was determined using the QuantStudio™ 7 Flex Real-Time PCR System (Applied Biosystems, Forest City, CA) set to a maximum of 40 cycles. Gene expression of target genes was normalized through parallel amplification of endogenous threshold cycle method (Livak and Schmittgen, 2001), and results were expressed as fold-change relative to birds in the NC treatment group.

Table 3.

Assays used for amplification of target and reference genes.

Gene	Assay ID	NCBI Reference	Amplicon length (bp)
Cytokines
IL-1β	Gg03347157_g1	NM_204524.1	92
IL-10	Gg03358689_m1	NM_001004414.2	56
Heat Shock Proteins
HSP70	Gg03370143_s1	NM_001006685.1	102
HSP90	Gg03362127_m1	NM_001109785.1	79
Tight Junction Proteins
OCLN	Gg07157847_m1	NM_205128.1	81
ZO-1	Gg07169480_m1	XM_015278975.2	75
Reference Gene
GAPDH	Gg03346982_m1	NM_204305.1	107

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Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HSP70, heat shock protein-70; HSP90, heat shock protein-90; IL-1β, interleukin-1 beta; IL-10, interleukin-10; NCBI, National Center for Biotechnology Information; OCLN, occludin; ZO-1, zonula occludens-1.

Statistical analyses

All data were initially subjected to a 1-way ANOVA using the MIXED procedure of SAS (version 9.4; SAS Institute, Cary, NC) and results were considered significant when P < 0.05. Means separation was conducted if the overall model was significant, and least-square means and pooled SEM estimates were reported. Outliers were identified as having an absolute Studentized residual value of 3 or greater. Histopathological scores were evaluated as previously described in Sommer (2022). Briefly, histopathological outcomes were scored with discrete and ordinal values (0-5), and cumulative probabilities for each outcome were obtained using the PLM procedure of SAS (version 9.4; SAS Institute, Cary, NC) via a Tukey-Kramer test. The probabilities of a specific score within each treatment were calculated, and the sum of all probabilities equaled 1 within a treatment. Average weighted scores were calculated for each treatment to account for the probability of each score occurring. Each score was multiplied by its respective proportion, and the sum of all scores for each treatment was calculated.

Results

Growth performance

Growth performance results are reported in Table 4. In the starter phase (d 0-14), there were no differences (P > 0.05) in any growth performance parameters. In the grower phase, the NC treatment group had higher BWG (P < 0.001), higher FI (P < 0.001), and better FCR (i.e., lower; P < 0.001) compared with the infected treatment groups. There were no differences (P > 0.05) between treatment groups during the finisher phase (d 28-42) for any growth performance outcomes.

Table 4.

Growth performance for broilers fed different botanical supplements while experiencing environmental and immunological stressors¹.

Item	NC	PC	MS	MSall	MS+MA	Pooled SEM	P-value
BWG, g/bird
d 0-14	443	431	428	425	429	7.2	0.42
d 14-28	1,003^a	824^b	815^b	797^b	784^b	17.8	<0.001
d 28-42	1,601	1,687	1,671	1,699	1,583	35.2	0.09
FI², g/bird
d 0-14	494	499	496	488	496	6.8	0.80
d 14-28	1,315^a	1,188^b	1,165^b	1,155^b	1,167^b	19.9	<0.001
d 28-42	2,558	2,632	2,621	2,669	2,481	47.3	0.07
FCR², g:g
d 0-14	1.12	1.15	1.14	1.15	1.16	0.013	0.18
d 14-28	1.32^c	1.45^ab	1.43^b	1.45^ab	1.49^a	0.018	<0.001
d 28-42	1.60	1.56	1.57	1.57	1.57	0.014	0.45

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^a-cMeans without a common superscript letter differ within a row (P < 0.05).

Values are least-square means derived from 12 replicate pens per treatment allotted with 7 chicks at study initiation for d 0-28 growth performance parameters. On d 28, 4 birds/cage of median weight (48 birds/treatment) were moved into floor pens with fresh pine shavings and pooled based on treatment group (6 pens/treatment). The following diet names have been assigned to a respective experimental treatment group: NC, control diet + not exposed to multiple mild stressors; PC, control diet + exposed to multiple mild stressors; MS, 500 mg/kg Micro-Shield in the starter diet + exposed to multiple mild stressors; MSall, 500 mg/kg of Micro-Shield in the starter diet + 500 mg/kg of Micro-Shield in the grower diet + 500 mg/kg of Micro-Shield in the finisher diet + exposed to multiple mild stressors; MS+MA, 500 mg/kg of Micro-Shield in the starter diet + 250 mg/kg of Micro-Aid in the grower diet + 250 mg/kg of Micro-Aid in the finisher diet + exposed to multiple mild stressors. Abbreviations: BWG, body weight gain; FCR, feed conversion ratio; FI, feed intake; SEM, standard error of the mean.

Outcomes were corrected for mortality.

Liver weights

Results for liver weights are found in Table 5. There were no differences (P > 0.05) in absolute liver weight on 6 DPI. However, the NC treatment group had lower (P = 0.015) relative liver weight compared with the infected treatment groups on 6 DPI. On 13 DPI, the MSall treatment group had the lowest (P = 0.039) absolute liver weight. Additionally, on 13 DPI the NC treatment group had the lowest (P = 0.004) relative liver weight compared with all other treatments. On 27 DPI, there were no differences (P > 0.05) in absolute or relative liver weights.

Table 5.

Absolute and relative liver weights of broilers fed different botanical supplements while experiencing environmental and immunological stressors¹.

Item	NC	PC	MS	MSall	MS+MA	Pooled SEM	P-value
6 DPI
Abs. Wt., g²	23.4	23.5	23.5	22.3	23.7	0.83	0.76
Rel. Wt., %³	2.55^b	2.99^a	2.94^a	2.90^a	2.91^a	0.10	0.015
13 DPI
Abs. Wt., g²	33.0^a	32.6^a	34.6^a	28.6^b	31.1^ab	1.37	0.039
Rel. Wt., %³	2.19^c	2.45^ab	2.57^a	2.36^bc	2.36^bc	0.07	0.004
27 DPI
Abs. Wt., g²	64.0	69.6	68.9	65.3	68.0	2.45	0.43
Rel. Wt., %³	2.05	2.29	2.23	2.15	2.26	0.07	0.14

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^a-cMeans without a common superscript letter differ within a row (P < 0.05).

Values are least-square means derived from 1 sample bird per battery cage (12 birds/treatment) on 6 and 13 DPI and from 2 sample birds per floor pen (12 birds/treatment) on 27 DPI. The following diet names have been assigned to a respective experimental treatment group: NC, control diet + not exposed to multiple mild stressors; PC, control diet + exposed to multiple mild stressors; MS, 500 mg/kg Micro-Shield in the starter diet + exposed to multiple mild stressors; MSall, 500 mg/kg of Micro-Shield in the starter diet + 500 mg/kg of Micro-Shield in the grower diet + 500 mg/kg of Micro-Shield in the finisher diet + exposed to multiple mild stressors; MS+MA, 500 mg/kg of Micro-Shield in the starter diet + 250 mg/kg of Micro-Aid in the grower diet + 250 mg/kg of Micro-Aid in the finisher diet + exposed to multiple mild stressors. Abbreviations: DPI, days post-inoculation; SEM, standard error of the mean.

Absolute liver weight.

Relative liver weight; calculated using the equation: (liver weight/bird weight) × 100.

Differential blood cell counts

Blood hematology results are found in Table 6. On 6 DPI, the NC treatment group had higher (P < 0.001) blood protein concentration compared with all other treatment groups. There were no differences (P > 0.05) in hematocrit values or white blood cell populations between any of the treatment groups on 6 DPI. On 13 DPI, MS and MSall treatment groups had the highest (P = 0.042) hematocrit values. Further, the NC treatment group had the highest (P = 0.007) percentage of heterophils on 13 DPI compared with all other treatments. Conversely, the NC treatment had the lowest percentage of eosinophils (P = 0.036) and basophils (P = 0.003) on 13 DPI. There were no differences (P > 0.05) in populations of band heterophils, lymphocytes, mono/azuro granules, or the heterophil:lymphocyte ratio on 13 DPI.

Table 6.

Blood clinical hematology in broilers fed different botanical supplements while experiencing environmental and immunological stressors¹.

Item	NC	PC	MS	MSall	MS+MA	Pooled SEM	P-value
Hematocrit, %
6 DPI	30.2	30.7	30.9	30.7	30.8	0.97	0.98
13 DPI	30.2^b	30.3^b	32.3^a	32.5^a	31.8^ab	0.69	0.042
Protein, g/dL
6 DPI	3.1^a	2.7^b	2.6^b	2.6^b	2.8^b	0.09	<0.001
13 DPI	2.9	2.9	2.9	2.9	2.9	0.08	0.97
Hetero, % of WBC
6 DPI	37.5	27.8	27.6	28.3	24.4	4.77	0.34
13 DPI	51.1^a	35.7^b	39.8^b	35.7^b	39.7^b	3.29	0.007
Band, % of WBC
6 DPI	0.9	0.8	1.3	1.2	0.8	0.42	0.88
13 DPI	2.6	2.8	3.6	2.3	3.0	0.49	0.44
Lymph, % of WBC
6 DPI	52.7	60.8	60.3	60.5	64.8	5.44	0.59
13 DPI	39.2	43.9	42.7	48.3	44.5	3.35	0.43
Mono, % of WBC
6 DPI	3.4	6.4	5.7	6.3	6.3	1.18	0.29
13 DPI	2.9	3.8	3.9	5.2	4.8	0.88	0.36
Eos, % of WBC
6 DPI	1.7	1.5	1.1	1.3	1.2	0.31	0.62
13 DPI	2.5^c	4.8^a	4.7^ab	3.1^bc	3.8^abc	0.60	0.036
Baso, % of WBC
6 DPI	3.8	2.7	3.6	2.4	2.7	0.75	0.53
13 DPI	1.8^b	5.8^a	4.5^a	5.4^a	4.3^a	0.73	0.003
H:L
6 DPI	1.0	0.5	0.5	0.5	0.5	0.16	0.09
13 DPI	1.4	0.8	1.1	0.8	1.1	0.18	0.08

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^a-cMeans without a common superscript letter differ within a row (P < 0.05).

Values are least-square means derived from 1 sample bird per battery cage (12 birds/treatment) on 6 and 13 DPI. The following diet names have been assigned to a respective experimental treatment group: NC, control diet + not exposed to multiple mild stressors; PC, control diet + exposed to multiple mild stressors; MS, 500 mg/kg Micro-Shield in the starter diet + exposed to multiple mild stressors; MSall, 500 mg/kg of Micro-Shield in the starter diet + 500 mg/kg of Micro-Shield in the grower diet + 500 mg/kg of Micro-Shield in the finisher diet + exposed to multiple mild stressors; MS+MA, 500 mg/kg of Micro-Shield in the starter diet + 250 mg/kg of Micro-Aid in the grower diet + 250 mg/kg of Micro-Aid in the finisher diet + exposed to multiple mild stressors. Abbreviations: Band, band heterophils; Baso, basophils; DPI, days post-inoculation; Eos, eosinophils; Hetero, heterophils; H:L, heterophil-to-lymphocyte ratio; Lymph, lymphocytes; Mono, mono/azuro granules; SEM, standard error of the mean; WBC, white blood cells.

Intestinal permeability

Intestinal permeability results are reported in Table 7. There were no treatment differences (P > 0.05) in the concentration of FITC-d in blood serum on either 6 or 13 DPI.

Table 7.

FITC-d concentrations in blood serum from broilers fed different botanical supplements while experiencing environmental and immunological stressors¹.

Item	NC	PC	MS	MSall	MS+MA	Pooled SEM	P-value
6 DPI, ug/mL	0.420	0.543	0.420	0.512	0.564	0.048	0.11
13 DPI, ug/mL	0.456	0.509	0.522	0.504	0.509	0.039	0.76

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Values are least-square means derived from 1 sample bird per battery cage (12 birds/treatment) on 6 and 13 DPI. The following diet names have been assigned to a respective experimental treatment group: NC, control diet + not exposed to multiple mild stressors; PC, control diet + exposed to multiple mild stressors; MS, 500 mg/kg Micro-Shield in the starter diet + exposed to multiple mild stressors; MSall, 500 mg/kg of Micro-Shield in the starter diet + 500 mg/kg of Micro-Shield in the grower diet + 500 mg/kg of Micro-Shield in the finisher diet + exposed to multiple mild stressors; MS+MA, 500 mg/kg of Micro-Shield in the starter diet + 250 mg/kg of Micro-Aid in the grower diet + 250 mg/kg of Micro-Aid in the finisher diet + exposed to multiple mild stressors. Abbreviations: DPI, days post-inoculation; FITC-d, fluorescein isothiocyanate–dextran; SEM, standard error of the mean.

Jejunal histomorphology and histopathology

Jejunal histomorphology measurements are reported in Table 8. On 6 DPI, the NC treatment group had shorter villi (P < 0.001), shallower crypt depths (P < 0.001), and increased villi height:crypt depth ratio (P = 0.008) compared with all other treatments. However, there were no differences (P > 0.05) in histomorphology measurements on 13 DPI. Cumulative pathology and immune response scores and histopathologic weighted scores are found in Table 9 and Table 10. The NC treatment group had numerically lower cumulative pathology and immune response scores compared with all other treatment groups on both 6 and 13 DPI.

Table 8.

Histomorphology measurements in broilers fed different botanical supplements while experiencing environmental and immunological stressors¹.

Item	NC	PC	MS	MSall	MS+MA	Pooled SEM	P-value
Villi, µm
6 DPI	490^b	790^a	911^a	864^a	929^a	70.0	<0.001
13 DPI	1,410	1,521	1,467	1,556	1,590	59.6	0.24
Crypt, µm
6 DPI	129^b	299^a	331^a	345^a	313^a	19.7	<0.001
13 DPI	150^b	211^a	228^a	212^a	215^a	17.5	0.018
V:C
6 DPI	3.8^a	2.8^b	2.9^b	2.5^b	3.0^b	0.25	0.008
13 DPI	9.8	7.4	7.1	7.6	8.4	0.79	0.13

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^a-bMeans without a common superscript letter differ within a row (P < 0.05).

Table 9.

Histopathological lesion scoring on 6 DPI in broilers fed different botanical supplements while experiencing environmental and immunological stressors¹.

Item	NC	PC	MS	MSall	MS+MA
Coccidia	0.00	4.98	4.89	4.99	5.00
Lamina propria GALT	1.36	2.72	2.72	2.52	2.60
Heterophils	0.14	0.10	0.02	0.00	0.02
Bacteria	0.00	0.00	0.00	0.00	0.00
Necrotic enteritis	0.00	0.00	0.00	0.00	0.00
Cystic crypts	0.00	0.29	0.29	0.43	0.17
Intraepithelial lymphocytes	0.82	2.42	2.32	2.77	2.64
Mucus	0.00	0.00	0.00	0.00	0.00
Goblet cells	0.68	0.00	0.00	0.00	0.00
Misshaped tips	0.00	2.12	1.95	2.70	2.92
Congestion	0.00	0.00	0.00	0.00	0.00
Compressed villi	1.85	0.00	0.00	0.00	0.00
Segmented filamentous bacteria	0.00	0.00	0.00	0.00	0.00
Necrosis, exfoliation	0.00	0.02	0.00	0.40	0.11
Hemorrhage	0.00	0.00	0.00	0.00	0.00
Cumulative
Pathology²	7.33	14.92	13.67	16.25	15.75
Immune Response³	2.17	5.08	5.08	5.25	5.08

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Jejunal samples were collected post-mortem and stored in 10% neutral buffered formalin until processing. Samples were scored by a board-certified histopathologist who was blinded to treatment identity. Average weighted scores (scores were on a 6-point scale of 0-5) were calculated to account for the probability of each score occurring, specifically each score was multiplied by its respective proportion, and the sum of all amounts per treatment were calculated. The following diet names have been assigned to a respective experimental treatment group: NC, control diet + not exposed to multiple mild stressors; PC, control diet + exposed to multiple mild stressors; MS, 500 mg/kg Micro-Shield in the starter diet + exposed to multiple mild stressors; MSall, 500 mg/kg of Micro-Shield in the starter diet + 500 mg/kg of Micro-Shield in the grower diet + 500 mg/kg of Micro-Shield in the finisher diet + exposed to multiple mild stressors; MS+MA, 500 mg/kg of Micro-Shield in the starter diet + 250 mg/kg of Micro-Aid in the grower diet + 250 mg/kg of Micro-Aid in the finisher diet + exposed to multiple mild stressors. Abbreviations: DPI, days post-inoculation; GALT, gut-associated lymphoid tissue.

Average sum of all histopathological lesion scores, not weighted.

Average sum of lamina propria GALT and intraepithelial lymphocytes scores, not weighted.

Table 10.

Histopathological lesion scoring on 13 DPI in broilers fed different botanical supplements while experiencing environmental and immunological stressors¹.

Item	NC	PC	MS	MSall	MS+MA
Coccidia	0.00	0.36	0.57	0.65	0.41
Lamina propria GALT	0.40	1.77	2.01	2.03	1.83
Heterophils	0.00	0.00	0.0	0.00	0.00
Bacteria	0.00	0.00	0.00	0.00	0.00
Necrotic enteritis	0.00	0.00	0.00	0.00	0.00
Cystic crypts	0.00	0.16	0.03	0.09	0.07
Intraepithelial lymphocytes	1.91	3.06	3.13	3.31	3.32
Mucus	0.00	0.00	0.00	0.00	0.00
Goblet cells	0.00	0.00	0.00	0.00	0.00
Misshaped tips	0.00	0.00	0.00	0.00	0.00
Congestion	0.00	0.00	0.00	0.00	0.00
Compressed villi	0.00	0.00	0.00	0.00	0.00
Segmented filamentous bacteria	0.00	0.00	0.00	0.00	0.00
Necrosis, exfoliation	0.00	0.00	0.00	0.00	0.00
Hemorrhage	0.00	0.00	0.00	0.00	0.00
Cumulative
Pathology²	2.33	6.58	6.83	7.58	6.67
Immune Response³	2.33	4.83	5.17	5.42	5.17

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Average sum of all histopathological lesion scores, not weighted.

Average sum of lamina propria GALT and intraepithelial lymphocytes scores, not weighted.

Antioxidative defenses and oxidative stress markers

Results for antioxidative defenses and oxidative stress markers are found in blood serum are reported in Table 11. On 6 DPI, the MS+MA treatment had the highest (P = 0.008) serum CAT levels compared with all other treatments. Additionally, on 6 DPI, the MS and MS+MA treatments had higher (P < 0.001) NO₂^- serum levels compared with all other treatments. Conversely, the MS treatment group had the lowest (P < 0.001) NO₃^- levels compared with the other treatments. Also on 6 DPI, the PC treatment had the lowest (P = 0.002) serum MDA levels compared with all other treatments. There were no differences (P > 0.05) in GPx or SOD levels on 6 DPI. However, on 13 DPI, the PC and MS treatment groups had higher (P < 0.001) serum GPx levels compared with the other treatments. There were no differences (P > 0.05) in CAT, MDA, NO₂^-, NO₃^-, or SOD serum levels on 13 DPI. On 27 DPI, the MSall and MS+MA treatments had the lowest (P = 0.023) serum MDA levels compared with all other treatments.

Table 11.

Antioxidative defense and oxidative stress markers in blood serum of broilers fed different botanical supplements while experiencing environmental and immunological stressors¹.

Item	NC	PC	MS	MSall	MS+MA	Pooled SEM	P-value
CAT, nmol/min/mL
6 DPI	17.73^bc	14.20^c	22.66^ab	22.35^abc	25.01^a	2.351	0.008
13 DPI	25.59	24.24	25.24	20.26	20.40	3.306	0.62
GPx, nmol/min/mL
6 DPI	690.21	844.62	870.41	833.69	772.87	73.026	0.38
13 DPI	611.15^b	904.05^a	922.20^a	584.09^b	703.06^b	58.620	<0.001
MDA, µM
6 DPI	23.30^a	11.00^b	20.17^a	26.97^a	21.71^a	2.962	0.002
13 DPI	26.49	27.06	27.96	29.07	22.36	2.324	0.32
27 DPI	20.84^a	18.00^ab	19.50^a	14.57^b	14.56^b	1.679	0.023
NO₂^-, µM
6 DPI	16.84^b	16.51^b	22.06^a	15.77^b	19.93^a	1.048	<0.001
13 DPI	18.01	17.54	17.38	17.12	17.10	1.328	0.99
NO₃^-, µM
6 DPI	14.68^a	11.42^ab	6.58^c	10.15^bc	8.52^bc	1.301	<0.001
13 DPI	9.32	9.52	10.81	12.71	10.41	1.157	0.26
SOD, U/mL
6 DPI	2.42	2.12	2.64	2.73	2.44	0.322	0.68
13 DPI	2.64	2.62	2.42	2.62	2.58	0.292	0.98

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a-cMeans without a common superscript letter differ within a row (P < 0.05).

Values are least-square means derived from 1 sample bird per battery cage (12 birds/treatment) on 6 and 13 DPI and from 2 sample birds per floor pen (12 birds/treatment) on 27 DPI. The following diet names have been assigned to a respective experimental treatment group: NC, control diet + not exposed to multiple mild stressors; PC, control diet + exposed to multiple mild stressors; MS, 500 mg/kg Micro-Shield in the starter diet + exposed to multiple mild stressors; MSall, 500 mg/kg of Micro-Shield in the starter diet + 500 mg/kg of Micro-Shield in the grower diet + 500 mg/kg of Micro-Shield in the finisher diet + exposed to multiple mild stressors; MS+MA, 500 mg/kg of Micro-Shield in the starter diet + 250 mg/kg of Micro-Aid in the grower diet + 250 mg/kg of Micro-Aid in the finisher diet + exposed to multiple mild stressors. Abbreviations: CAT, catalase; DPI, days post-inoculation; GPx, glutathione peroxidase; MDA, malondialdehyde; NO₂^-, nitrite; NO₃^-, nitrate; SEM, standard error of the mean; SOD, superoxide dismutase.

Results for MDA levels in breast and liver tissue are reported in Table 12. On 6 and the 13 DPI, there were no differences (P > 0.05) in breast tissue levels of MDA. However, on 27 DPI, the MS+MA treatment had the highest (P < 0.001) MDA levels in breast tissue compared with all other treatments. On 6 DPI, there were no differences (P > 0.05) in MDA levels of liver tissue. However, on 13 DPI, the PC and MS treatments had the lowest (P = 0.001) MDA levels of liver tissue compared with all other treatments. Additionally, on 27 DPI, the MS treatment had the highest (P = 0.042) MDA levels of liver tissue compared with all other treatments.

Table 12.

Malondialdehyde levels in breast and liver tissue of broilers fed different botanical supplements while experiencing environmental and immunological stressors¹.

Item	NC	PC	MS	MSall	MS+MA	Pooled SEM	P-value
Breast, µM
6 DPI	136.34	190.29	189.59	146.60	127.09	21.193	0.11
13 DPI	151.11	124.35	140.00	159.95	128.10	15.967	0.45
27 DPI	177.21^cd	112.85^d	206.48^bc	259.43^ab	282.90^a	25.282	<0.001
Liver, µM
6 DPI	202.40	258.90	301.37	293.35	344.86	37.334	0.10
13 DPI	328.78^a	166.68^c	186.24^c	266.91^ab	203.37^bc	29.665	0.001
27 DPI	112.58^bc	153.15^abc	215.16^a	99.77^c	195.14^ab	30.787	0.042

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^a-dMeans without a common superscript letter differ within a row (P < 0.05).

Gene expression

Gene expression data are shown in Table 13 and are reported as a fold-change relative to the NC treatment group. There were no differences (P > 0.05) in liver HSP70 or HSP90 gene expression on 6 or 13 DPI. Further, there were no differences (P > 0.05) in IL-1β or IL-10 gene expression in jejunal tissue on 6 DPI. There was a difference in IL-1β gene expression on 13 DPI where the NC treatment group had the lowest (P < 0.001) IL-1β expression compared with all other treatments. Additionally on 13 DPI, there was a difference in IL-10 expression where the MSall treatment group had the highest (P < 0.001) IL-10 expression levels. ON 6 DPI, OCLN (P < 0.001) and ZO-1 (P = 0.022) gene expression in jejunal mucosa tissue was highest in the NC treatment group. There were no differences (P > 0.05) in OCLN or ZO-1 gene expression in jejunal mucosa tissue on 13 DPI.

Table 13.

Gene expression of the liver, jejunum, and jejunal mucosa of broilers fed different botanical supplements while experiencing environmental and immunological stressors¹.

Item	NC	PC	MS	MSall	MS+MA	Pooled SEM	P-value
Liver
HSP70
6 DPI	1.000	1.654	1.280	1.072	1.180	0.236	0.30
13 DPI	1.000	1.251	0.954	1.524	0.605	0.357	0.54
HSP90
6 DPI	1.000	0.818	1.054	1.074	0.720	0.124	0.17
13 DPI	1.000	0.866	1.007	0.883	0.778	0.205	0.91
Jejunum
IL-1β
6 DPI	1.000	0.912	0.970	1.509	0.862	0.286	0.46
13 DPI	1.000^c	2.729^ab	2.555^b	3.218^ab	3.989^a	0.502	<0.001
IL-10
6 DPI	1.000	1.045	0.610	1.155	0.909	0.187	0.30
13 DPI	1.000^c	17.234^bc	18.741^bc	43.044^a	32.961^ab	6.695	<0.001
Jejunal Mucosa
OCLN
6 DPI	1.000^a	0.666^bc	0.606^c	0.614^c	0.772^b	0.054	<0.001
13 DPI	1.000	1.050	0.888	1.147	1.048	0.098	0.46
ZO-1
6 DPI	1.000^a	0.715^c	0.672^c	0.755^bc	0.964^ab	0.088	0.022
13 DPI	1.000	0.803	0.722	0.818	0.793	0.100	0.30

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^a-cMeans without a common superscript letter differ within a row (P < 0.05).

Values are least-square means derived from 1 sample bird per battery cage (12 birds/treatment) on 6 and 13 DPI. The following diet names have been assigned to a respective experimental treatment group: NC, control diet + not exposed to multiple mild stressors; PC, control diet + exposed to multiple mild stressors; MS, 500 mg/kg Micro-Shield in the starter diet + exposed to multiple mild stressors; MSall, 500 mg/kg of Micro-Shield in the starter diet + 500 mg/kg of Micro-Shield in the grower diet + 500 mg/kg of Micro-Shield in the finisher diet + exposed to multiple mild stressors; MS+MA, 500 mg/kg of Micro-Shield in the starter diet + 250 mg/kg of Micro-Aid in the grower diet + 250 mg/kg of Micro-Aid in the finisher diet + exposed to multiple mild stressors. Abbreviations: DPI, days post-inoculation; HSP70, heat shock protein-70; HSP90, heat shock protein-90; IL-1β, interleukin-1 beta; IL-10, interleukin-10; OCLN, occludin; SEM, standard error of the mean; ZO-1, zonula occludens-1.

Discussion

Broiler performance and health are often negatively impacted by environmental and immunological stressors in commercial production settings. However, as antibiotic growth promoters continue to fall out of use in the poultry industry, alternative nutritional interventions are being assessed for their ability to support broiler growth and health. Therefore, the purpose of this study was to evaluate the effect of dietary supplementation of saponins and polyphenols on broiler growth and health during exposure to immune and environmental stressors.

Previous literature reports variable responses to dietary saponin and polyphenol supplementation in terms of broiler growth performance. In Su et al. (2016) and Sultan et al. (2024), administration of Yucca schidigera-derived saponins improved growth performance, whereas Sariozkan et al. (2015) reported no significant effects. Similarly, broiler growth responses to dietary polyphenol supplementation have also been inconsistent. A study conducted by Abu Hafsa and Ibrahim (2018) reported improvements in growth performance with dietary polyphenol supplementation, whereas Liu et al. (2023) reported no effects of polyphenols on growth performance. However, all these studies were conducted in unchallenged birds. In contrast, our study utilized a multiple mild stressors model, where birds exposed to the challenge exhibited reduced growth performance compared with NC birds. However, no differences in growth performance were observed among challenged groups as a result of dietary intervention.

During periods of oxidative stress, dysregulation of the cellular environment can cause protein misfolding, unfolding, or aggregation to diminish cellular function (Nakamura and Lipton, 2009; Lévy et al., 2019). Heat shock proteins are part of a critical mechanism that works to mitigate cellular damage induced by oxidative stressors including coccidiosis and heat stress. As protein chaperones, heat shock proteins respond to oxidative damage by inducing correct protein refolding or inducing the degradation of irreparably damaged proteins (Abare et al., 2023). In the current study, no differences were observed in the gene expression of either liver HSP70 or HSP90 on 6 or 13 DPI. While there were no differences in HSP70 or HSP90 liver gene expression reported, birds in the MSall and MS+MA treatment groups had relative liver weights comparable to the NC group on 13 DPI. This increase in relative liver weight is most likely due to the increase in production of acute phase proteins, cytokines, and other immune-related molecules (Klasing and Austic, 1984; Robinson et al., 2016). Although this immunological response is essential for combatting diseases such as coccidiosis, prolonged immune system activation can negatively impact broiler health by contributing to tissue damage and diverting nutrients away from growth and instead towards immune responses (Humphrey and Klasing, 2004). Therefore, the observation that MS and MSall birds had relative liver weights similar to NC birds on 13 DPI may indicate a timely return to homeostatic conditions, thereby benefitting overall broiler health.

Blood clinical hematology is a critical outcome to evaluate in stress-challenged broilers as it provides a global view of metabolic and immunologic status. In the 1980s, the heterophil:lymphocyte ratio emerged as a sensitive measure to evaluate the stress response in broiler chickens (Gross et al., 1980; Gross and Siegel, 1983). Davison et al. (1983) previously described that corticosterone administered in the diet could induce lymphocytopenia and granulocytosis to, in turn, affect the granulocyte:lymphocyte ratio. In the current study, birds exposed to stressors had a lower percentage of heterophils compared with NC birds, which does not align with the literature where coccidiosis-infected broilers typically exhibit increased levels of heterophils (Akhtar et al., 2015; Moraes et al., 2019). During coccidiosis infections, hematocrit values are often decreased due to hemorrhaging intestinal tissue (Natt and Herrick, 1955). Therefore, the increased hematocrit observed in the MS, MSall, and MS+MA treatments compared with the PC could indicate lower systemic hemorrhaging in these birds. There were no differences in lymphocyte populations at peak coccidiosis on 6 DPI, which could indicate that the immune response to Eimeria had not been fully established (Freitas et al., 2023; Jespersen et al., 2024b). This observation is further supported by the absence of differences in jejunal cytokine gene expression on 6 DPI, which is discussed later. However, while challenged birds did have elevated eosinophil and basophil populations compared with NC birds on 13 DPI, all values fell within typical reference ranges reported in Akhtar et al. (2015) and Jespersen et al. (2024a). As such, we do not consider these differences biologically significant to broiler health. This was further supported by no differences in heterophil:lymphocyte ratios on either 6 or 13 DPI.

Maintenance of a healthy intestinal barrier function is critical in broilers during both coccidiosis and heat stress conditions, as both conditions disrupt gastrointestinal integrity and permeability (Ruff et al., 2020; Emami et al., 2021). Additionally, the continuous ingestion of nutrients supports the coordination between the gastrointestinal tract and the cardiovascular system to direct blood flow appropriately (Oberman et al., 2000), which is important for maintaining tight junction proteins between intestinal epithelial cells (Jabbar et al., 2003). In the current study, FITC-d was utilized as an indicator to evaluate intestinal permeability, but no differences were evident at either 6 or 13 DPI. However, histopathological lesion scoring of jejunal tissue indicated intestinal damage in birds exposed to the stressors model, as evidenced by increased cumulative pathology and immune response scores in all challenged treatments on 6 and 13 DPI compared with NC birds. The higher cumulative pathology and immune response scores of challenged birds were driven by the presence of coccidia, infiltration of intraepithelial lymphocytes, cystic crypts, and misshaped villi tips, all of which are expected in birds experiencing environmental and immunological stressors (Santos et al., 2015; Jespersen et al., 2024b). Alternatively, birds exposed to stressors surprisingly had higher villi heights in jejunal tissue on 6 DPI compared with the NC treatment, which is not in agreement with previous literature as exposure to the stressors used in our challenge model typically induces villus atrophy (Liu et al., 2023; Jespersen et al., 2024b). However, this reported increase in villus height may be indicative of increased epithelial cell turnover in response to intestinal epithelial damage (Fernando and McCraw, 1973). Challenged treatments also had deeper crypt depths compared to NC birds on both 6 and 13 DPI, which induced lower villi height:crypt depth ratios in challenged birds on 6 DPI. Crypt hyperplasia is commonly observed in birds exposed to stressors as a compensatory response to replace cells lost due to intestinal damage (Calik et al., 2019; Brugaletta et al., 2022). Further, at peak coccidiosis infection on 6 DPI, gene expression of OCLN and ZO-1 in jejunal tissue was lower in challenged treatments compared with NC birds. Overall, these results indicate that dietary saponin and polyphenol supplementation had minimal effects on the maintenance of intestinal barrier function in broilers exposed to stressors.

On 13 DPI, birds exposed to the stressors model had higher jejunal gene expression of IL-1ß compared with NC birds. This aligns with previous findings of increased IL-1ß gene expression in birds challenged with coccidiosis (Tan et al., 2014; Oelschlager et al., 2019). While IL-1β is critical in initiating an immune response, this cytokine has been reported to induce sickness-like behavior of decreased feed intake, resulting in stunted growth (Dantzer et al., 1993). Additionally, prolonged immune responses have the costly effect of diverting nutrients away from skeletal muscle protein accretion to support the synthesis of immune molecules such as cytokines (Humphrey and Klasing, 2004). Therefore, regulation of the inflammatory response induced by IL-1β is critical in mitigating the damage associated with a prolonged immune response. In correspondence with the increased IL-1β expression, expression of immune regulatory IL-10 was highest in the MSall and MS+MA treatment groups on 13 DPI. Previously, botanicals have been reported to alter phosphorylation of the Janus kinase-signal transducers and activators of transcription pathway (Zhu et al., 2023). As signal transducer and activator of transcription 3 is a critical transcription for IL-10 production by immune cells (Hutchins et al., 2013), these data suggest that the saponins and polyphenols administered may have had some beneficial immunomodulatory or anti-inflammatory properties in challenged broilers.

Exposure to environmental and immunological stressors often induces oxidative stress in broilers, which is a physiological state marked by an overabundance of circulating reactive oxygen and nitrogen species such as NO₂^- and NO₃^- (Oke et al., 2024). While reactive oxygen and nitrogen species both play vital roles in cellular functions under homeostatic conditions, their overabundance often leads to cellular and molecular damage (Halliwell and Whiteman, 2004; Zhang et al., 2016). Broilers possess an enzymatic antioxidant defense system to combat oxidative stress in which SOD first catalyzes the dismutation of O₂^- and H₂O₂ into water and oxygen (Surai, 2016). Following this reaction, CAT converts H₂O₂ into water and oxygen molecules, removing the reactive species (Surai, 2016). Further, GPx can remove H₂O₂ and other lipid peroxides by reducing them into water and alcohol compounds (Arthur, 2000).

In the current study, CAT activity was highest in the MS+MA treatment on 6 DPI. This increase could suggest that the dietary inclusion of saponins and polyphenols enhanced the CAT-mediated removal of H₂O₂ at peak coccidiosis infection (Leff et al., 1992), potentially through activation of the nuclear factor erythroid 2-related factor (Nrf2) pathway, a key activator of the broiler endogenous antioxidant defense pathway (Surai et al., 2019). However, on 13 DPI, there were no detectable differences in CAT activity. In terms of GPx activity, there were no differences between treatments on 6 DPI. However, on 13 DPI, GPx activity was highest in the PC and MS treatment groups, indicating sustained glutathione-based detoxification mechanisms in these birds perhaps through means of increased Nrf2 activation or increased free glutathione levels in these birds (Moskaug et al., 2005; Chi et al., 2017; Zhang et al., 2018) While there were no differences in SOD activity on either 6 or 13 DPI, the modulated levels of both CAT and GPx at both peak and recovery phases of the coccidiosis infection could indicate variations in SOD activity outside of the time points when samples were collected (Szymonik-Lesiuk et al., 2003). In terms of reactive nitrogen species, NO₂^- levels were highest in the MS and MS+MA treatments on 6 DPI, which could indicate enhanced nitric oxide turnover during peak coccidiosis infection. However, these increased NO₂^- levels coincided with lower NO₃^- levels in these same treatments.

Lipid peroxidation, a major event during oxidative stress, can induce further damage in the host by altering cell membranes, proteins, and DNA to induce inflammation and cell death (Del Rio et al., 2005; Cordiano et al., 2023). A naturally formed compound during lipid peroxidation is MDA, which is commonly utilized as a biomarker to quantify oxidative stress (Cordiano et al., 2023). On 6 DPI, the PC treatment group had the lowest serum MDA, however, this did not translate to any systemic differences of MDA levels in breast or liver tissue on 6 DPI. On 13 DPI, liver tissue of PC and MS birds had the lowest levels of MDA compared with other treatments, which could indicate reduced levels of lipid peroxidation in these birds (Altan et al., 2003). On 27 DPI, the MSall and MS+MA treatments had the lowest MDA serum levels, which coincided with the MSall treatment group having the lowest MDA levels in breast tissue. Overall, the dietary supplementation of saponins and polyphenols study had marginal effects on lipid peroxidation in the current study.

Conclusions

Collectively, these results suggest that dietary supplementation of saponins and polyphenols influences the immune and oxidative stress response in broilers undergoing environmental and immunological stressors of coccidiosis, elevated ambient temperature, and an acute off-feed event. Birds exposed to the multiple mild stressors model exhibited a reduction in growth, however, no differences were observed between any of the treatments imposed with the stressors model. All stressed birds had higher relative liver weights on 6 DPI at peak coccidiosis infection but had weights comparable to unchallenged birds in MSall and MS+MA treatment groups by 13 DPI, which may be indicative of a reduced immune-related nutrient expenditure. Lymphocyte populations remained within normal ranges for all treatment groups, but the increased hematocrit levels in the botanical supplemented treatment groups could reflect reduced intestinal hemorrhaging due to coccidiosis. Although no differences in intestinal permeability measured by FITC-d were observed, gene expression of tight junction proteins in the jejunal mucosa and histopathological measurements in jejunal tissue results indicate marginal effects of botanical supplementation on intestinal barrier integrity. The supplementation of saponins and polyphenols had some modulatory effects on CAT and GPx antioxidant defenses as well as IL-β and IL-10 jejunal gene expression, indicating potential immunomodulatory effects of these interventions. Collectively, the dietary administration of saponins and polyphenols may support immune and antioxidant defenses in stressed broilers, and as such, additional research should be conducted to evaluate how their supplementation effects the broiler response throughout the duration of stressor exposure.

CRediT authorship contribution statement

Laney E. Froebel: Writing – review & editing, Writing – original draft, Visualization, Validation, Software, Resources, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Michael J. Rincker: Writing – review & editing, Resources, Funding acquisition, Conceptualization. Ryan N. Dilger: Writing – review & editing, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Disclosures

M. J. Rincker is an employee of DPI Global (Porterville, CA). No other authors declare conflicts of interest.

Acknowledgments

This research was funded in part by an unrestricted gift from DPI Global (Porterville, CA). Coccivac-B52 vaccine vials were kindly provided by Merck Animal Health (Madison, NJ).

Footnotes

Section: Metabolism and Nutrition

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.psj.2026.106816.

Appendix. Supplementary materials

mmc1.docx^{(15.8KB, docx)}

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PERMALINK

Effects of dietary saponin and polyphenol supplementation in broiler chickens exposed to multiple mild stressors of cyclic elevated ambient temperature, feed withdrawal, and coccidiosis infection

Laney E Froebel

Michael J Rincker

Ryan N Dilger

Abstract

Introduction

Materials and methods

Bird husbandry and experimental design

Table 1.

Table 2.

Multiple mild stressor challenge

Differential blood cell counts

Intestinal permeability

Jejunal histomorphology and histopathology

Antioxidative defenses and oxidative stress markers

Gene expression

Table 3.

Statistical analyses

Results

Growth performance

Table 4.

Liver weights

Table 5.

Differential blood cell counts

Table 6.

Intestinal permeability

Table 7.

Jejunal histomorphology and histopathology

Table 8.

Table 9.

Table 10.

Antioxidative defenses and oxidative stress markers

Table 11.

Table 12.

Gene expression

Table 13.

Discussion

Conclusions

CRediT authorship contribution statement

Disclosures

Acknowledgments

Footnotes

Appendix. Supplementary materials

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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