*Article* **Antioxidant Status, Blood Constituents and Immune Response of Broiler Chickens Fed Two Types of Diets with or without Different Concentrations of Active Yeast**

**Youssef A. Attia 1,\* , Hanan Al-Khalaifah 2,\* , Hatem S. Abd El-Hamid <sup>3</sup> , Mohammed A. Al-Harthi <sup>1</sup> , Salem R. Alyileili <sup>4</sup> and Ali A. El-Shafey <sup>5</sup>**


**Simple Summary:** Rations for broilers can be safely supplemented with probiotics such as active *Saccharomyces cerevisiae* (SC) yeast to stimulate oxidative reactions and immune response against stress and infectious agents. The current study suggested that SC yeast enhanced antioxidant capacity, growth rate, immune organ weights, immune response and the survival rate of broilers after Avian Influenza virus challenge at 38 days of age.

**Abstract:** Probiotics, such as active yeasts, are widely used to enhance poultry production and reduce feeding costs. This study aimed to investigate the antioxidant and immune responses of broilers to different concentrations of active *Saccharomyces cerevisiae* (SC) when supplemented to two types of diets. A total of 216 1-day-old Arbor Acres unsexed chicks were used in a factorial design, involving two feeds (regular- versus low-density diet) and three concentrations of SC (0%, 0.02% and 0.04%). The results revealed that the low-density diet reduced the body weight and production index of broilers. The addition of SC improved the production index more than the control diet. Total antioxidant capacity (TAC), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and eosinophils were significantly higher in response to the regular-density diet than the low-density diet; however, phagocytic activity (PA), lymphocyte and lysozyme activity (LYS) were lower. *Saccharomyces cerevisiae* reduced ALT, AST, malondialdehyde (MAD) and TAC more than the standard set, but improved packed cell volume (PCV), hemoglobin (Hgb), red blood cells (RBCs), lymphocytes, monocytes, heterophils, phagocytic index (PI) and the immune response to Newcastle disease virus (NDV) and avian influenza (AI). In conclusion, supplementation of a regular- or low-density diet with SC at a concentration of 0.02% or 0.04% improved the antioxidant parameters, immune status and production index of broilers against stress and infectious agents.

**Keywords:** active yeast; antioxidant status; broilers; nutrient density; immune response

#### **1. Introduction**

There is a growing interest in the innovative biofortification of poultry feed rations through the use of functional ingredients to improve feed utilization and enhance production performance and the immune status of the flocks [1–8].

**Citation:** Attia, Y.A.; Al-Khalaifah, H.; Abd El-Hamid, H.S.; Al-Harthi, M.A.; Alyileili, S.R.; El-Shafey, A.A. Antioxidant Status, Blood Constituents and Immune Response of Broiler Chickens Fed Two Types of Diets with or without Different Concentrations of Active Yeast. *Animals* **2022**, *12*, 453. https://doi.org/10.3390/ ani12040453

Academic Editor: Rosalia Crupi

Received: 9 November 2021 Accepted: 9 February 2022 Published: 12 February 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

*Saccharomyces cerevisiae* (SC) has been used in poultry feed rations to enhance resistance to aflatoxicosis in poultry [9]. The antioxidant status and capacity of poultry have been shown to be significantly enhanced by supplementing poultry feed rations with SC, either alone or in combination with probiotics [10,11]. The weights of the primary and secondary immune organs in broilers have been shown to be increased after dietary supplementation with active yeast, potentially indicating immunocompetence in broilers [12]. In addition, Kiarie et al. [13] revealed that when added with feed enzymes, yeast derivatives can modulate cellular- and humoral-mediated immunity in broilers against intestinal coccidial infections. Zhou et al. [14] investigated the ability of yeast fractions to prevent pullorum disease and fowl typhoid in breeders. The results of the study revealed that dietary fortification of yeast fractions significantly reduced disease infection in the challenged breeders. In the same study, the positive culling rate of the pullets and their body weight were significantly reduced because of the addition of yeast fractions to the feed of birds challenged with *Salmonella* infection [14]. The intestinal microflora balance has also been shown to be improved in birds with dietary supplementation of yeast, due to the presence of mannan-oligosaccharides and fructo-oligosaccharides in the cell wall of yeast [15–19]. However, debate remains regarding the effects of active yeast on the production performance and immune status of chickens, particularly when they are fed diets containing different nutrient profiles or are placed under environmental stress [20–23]. Hayat et al. [24] suggested that this could be due to genetic differences, or differences in species, age, or environmental conditions. Thus, this study was carried out to fill this knowledge gap.

The current study aimed to fill the gap in the literature about the response of broilers fed two types of diets, with or without different concentrations of SC (active yeast) with respect to antioxidant status, blood constituents and immune status. Although previous studies investigated the effect of SC on the productive performance parameters in broiler chickens, there are relatively limited data in the literature on the direct effect of SC probiotic on the antioxidative status, blood constituents and immune status in broiler chickens fed low-density diet. The current literature does not yet adequately address the approaches of nutrient manipulation in broiler feed rations to shed light on the relationship between the effect of SC in low-density diets and the immune response of broiler chickens. Accordingly, this study was executed to elaborate on this vital relationship.

#### **2. Materials and Methods**

#### *2.1. Birds, Dietary Treatments, Experimental Plan and Housing*

This research work was approved by the Deanship of Scientific Research, King Abdulaziz University, Saudi Arabia, under protocol no: (FP-217-42 H). The protocol recommends general humane treatment of animals that did not cause animal (s) pain, suffering, distress, or lasting harm, according to the Royal Decree number M59 in 14/9/1431H.

A total of 216, 1-day-old Arbor Acres broilers (mixed sexes) were acquired. The chicks were marked randomly by way of wing-banding and were housed in 36 pens with 6 birds per pen (replicate). Each treatment involved 6 replicates. The body weight (BW) of all chicks was similar at the start of the experiment.

The chickens were provided mash feeds ad libitum, along with freely accessed waterers. During the first 7 days, 23 h of light were used, followed by 20 h of light until the end of the experiment.

A factorial design (2 × 3) was applied to the experiment using two diets (a regular versus a low-density diet containing 10% fewer nutrients than the regular diet) and three levels of SC (an unfortified standard, 0.02%, or 0.04% SC). The broiler feed rations were formulated based on the Arbor Acres broilers guide [25]. There were six replications in each treatment and each replicate involved six birds. The SC yeast was purchased from China Way Co-operation, Taiwan, and had 12,000,000,000 active yeast per gram. The optimal dosage of SC was 200 to 400 g per ton of feed. Table 1 shows the composition of the dietary treatments fed to the chickens.


**Table 1.** Basal chemical composition of the experimental diets.

<sup>1</sup> Vit + Min mix. contains the following nutrients. Values are per kilogram of the diet: Vit. A, 12,000 IU; Vit. E (dl-α-tocopheryl acetate), 20 mg; menadione, 2.3 mg; Vit. D3, 2200 ICU; riboflavin, 5.5 mg; calcium pantothenate, 12 mg; nicotinic acid, 50 mg; Choline, 250 mg; Vit. B12, 10 µg; Vit. B6, 3 mg; thiamine 3 mg; folic acid, 1 mg; d-biotin, 0.05 mg; Trace minerals (mg/kg of diet): Mn, 80; Zn, 60; Fe, 35; Cu, 8; Selenium, 0.1 mg, <sup>2</sup> Calculated analyses, <sup>3</sup> Determined analyses.

#### *2.2. Data Gathering*

Average pen body weight (g) was recorded at 1, 21 and 38 days of age and used to calculate the body weight gain (BWG, g/bird). The average pen feed intake (g/bird) was recorded and used to calculate the feed conversion rate (FCR, g feed/g gain) and survival rate (100–mortality rate) during the following periods: 1–21 days, 22–38 days and 1–38 days of age. The production index was calculated as follows: BW (kg) × survival rate Production index = ×100 production period in days × FCR.

#### *2.3. Blood Sampling*

Blood was collected from each treatment group (*n* = 6) before vaccination and again at 8 days post-vaccination. The serum was harvested by centrifuging the blood at 1500× *g* for 15 min.

#### *2.4. Antioxidant Status and Biochemical Traits*

Serum total antioxidant capacity (TAC) and malondialdehyde (MAD) were assayed as described in Erel [26] and Wyatt et al. [27], respectively. They were determined using commercial kits produced by Diamond Diagnostics (23 EL-Montazah St. Heliopolis, Cairo, Egypt, http://www.diamonddiagnostics.com (accessed on 1 February 2022). Total plasma protein and albumin concentrations were measured using the methods outlined in Armstrong and Carr [28] and Doumas and Peters [29], respectively. Subtracting albumin concentration from serum total protein gives an estimate of the globulin concentration, as described in Giangiacomo et al. [30]. Various kinds of globulin (α-, β- and γ-globulin) were determined based on methods described in Elias [31]. The activities of alanine aminotransferase (ALT, U/L) and aspartate aminotransferase (AST, U/L) were determined using techniques described in Reitman and Frankel [32]. Alkaline phosphatase (ALKP) enzyme action was measured in plasma, as described by Kim and Wyckoff [33].

#### *2.5. Hematological Parameters*

Wintrobe hematocrit tubes were used to measure the packed cell volume (PCV, %) by centrifugation for 20 min. at 2000× *g*. Hemoglobin (Hgb) level was estimated using the technique described in Eilers [34]. The mean corpuscular volume (MCV, µm 3), mean corpuscular hemoglobin (MCH, Pg) and mean corpuscular hemoglobin concentration (MCHC, g/dL) were measured using the equations described in Jain [35].

#### *2.6. Immune Indices*

The phagocytic activity (PA, % of phagocytic cells engulfing yeast cells) and phagocytic index (PI, number of yeast cell phagocytized/number of phagocytic cells) were determined as described in Kawahara et al. [36].

Broiler chickens were vaccinated according to the following schedule: inactivated avian influenza (AI) H5N2 at 10 days of age. Chickens were vaccinated with clone 30 eye drop on day 8 for Newcastle disease (NDV) and bivalent NDV vaccine was administered underneath the neck membrane, simultaneously with clone 30, at 8 days of age. The Gumboro intermediate vaccine and clone 30 were administered at 12 and 21 days of age, respectively (Nobilis, Intervet, Boxmeer, The Netherlands).

Blood samples (*n* = 6 per group) were taken just before vaccination (0 days postvaccination) and again on 8th day post-vaccination. The samples were centrifuged at 1500× *g* for 15 min for serum separation, to determine antibody titers against NDV via the hemagglutination inhibition test (HI) test. This test was done using hemagglutination inhibition (HI) test according to OIE [37]. The geometric mean titer was calculated as recommended by the World Organization for Animal Health (OIE) [38].

Antibody responses were determined by the HI test, according to Seal et al. [39]. The assay measures antibodies attached to influenza antigen-coated plates [40]. Hemagglutination inhibition for NDV and AI were measured as described in Takatsy and Hamar [41].

A lymphocyte transformation test was performed, as described in Balhaa et al. [42]. Lympholyte-H (Cedarlane Laboratories Ltd., Burlington, ON, Canada) was used to layer the collected heparinized blood. After centrifugation, the lymphocytes in the interface layer were collected, washed and suspended in culture medium.

Serum bactericidal activity to the *Aeromonas hydrophila* strain was conducted following the protocols described in Rainger and Rowley [43]. The turbidimetric method was used to measure serum lysozyme activity [44]. The results were reflected as one unit of lysozyme activity as a reduction in absorbency of 0.001/min Lysozyme activity = (A0 − A)/A.

#### *2.7. Challenge Test*

The challenge test was conducted to study the impact of the diet on the survival rate of chicks [45,46] between 38 and 48 days of age. Six broiler chickens per treatment were randomly selected at 38 days of to represent all treatment replications. The chickens were vaccinated with inactivated avian influenza (AI) H5N2 at 10 days of age and then challenged with H5N1 at 38 days via the oculo-nasal route with 0.2 mL/bird (106/dose). The H5N1 was from research laboratory of Poultry Disease, Fac. Vet. Med., Damanhour University, where the challenge test was carried out following the regulations for animal welfare approved by the authorized ethics committee of the Egyptian Ministry of Agriculture according to Decree No. 27, 1967. The mortality was recorded daily during 38–48 days of age.

#### *2.8. Histopathological Study*

On day 38, 6 chickens from each treatment replicate were randomly selected and euthanized under anesthesia via an intravenous injection of sodium pentobarbital (50 mg/kg;

CAMEO chemicals, Tampa, FL, USA). Necropsies were performed for sample collection. Lymphoid organs (bursa of Fabricius, thymus and spleen) were weighed, and the body weight ratios of organs were calculated.

In addition, intestine, bursa of Fabricious, thymus and spleen specimens were collected from randomly collected broilers (*n* = 6 per treatment) at 38 days of age. Tissue specimens were prepared as previously described by Culling [47].

#### *2.9. Morphometrical Study*

An Optika binocular microscope, with an Optika imaging analyzer, was used to examine the morphological appearance of intestinal villi, determine the absorption surface and measure the longitudinal axis of the large follicle of the bursa. Five segments from each bird were used for this examination. In addition, quantitative measurements of the thymus cortical: medullary ratio were performed and the hyperplasia of the lymphoblastic cells was assayed by examining the spleen. The scale used was as follows: (−) for weak hyperplasia; (+) for moderate hyperplasia; (++) for severe hyperplasia.

#### *2.10. Statistical Analyses*

The data were analyzed using general linear models in SAS (SAS Institute, Cary, NC, USA [48]). A two-way factorial design (two kinds of diets × three concentrations of SC) was used to analyze the effects of the treatments on most of the parameters. An exception was survival rate in the challenge study, where age was included as a main effect only. The replicate was the experimental unit. Data were arcsine transformed prior to analysis to improve normality. Student–Newman–Keuls (SNK) post hoc tests were applied to evaluate differences between factor levels in the model. Differences were considered significant if *p* ≤ 0.05.

#### **3. Results**

#### *3.1. Growth Performance*

Table 2 shows the impact of yeast concentrations on body weight and the European Production Efficiency Index (EPEI) of broilers fed regular- and low-density diets. During the experiment, the low-density diet was found to decrease the final body weight and EPEI, reaching 5% over the duration of the study. Diets supplemented with 0.02% and 0.04% SC resulted in a production index that was significantly enhanced relative to the control diet. However, the addition of SC in the diet at a concentration of 0.04% had a more substantial effect on the growth of 38-day-old chickens than the 0.02% level (Table 2). There was no significant relationship between the amount of SC and diet on the growth of broilers or EPEI between 1 and 38 days of age.

**Table 2.** Impact of different concentrations of *Saccharomyces cerevisiae* on body weight, survival rate, European Production Efficiency Index and blood hematological parameters of broilers fed regular- or low-density diets from days 1 to 38 of age.



**Table 2.** *Cont.*

a,b,c Means within a column with different superscripts are significantly different based on Student–Newman– Keuls (SNK) post hoc tests. MCV = Mean corpuscular volume; MCH = Mean corpuscular hemoglobin; MCHC = Mean corpuscular hemoglobin concentration; Number of observations was 6 replicates per interaction cell. ND = Not done.

#### *3.2. Antioxidant Status and Biochemical Traits*

Tables 3 and 4 show the impact of different yeast concentrations on the liver enzyme index, peroxidation index and blood serum biochemical constituents of broilers fed regular and low-density diets from 1 to 38 of age.

**Table 3.** Impact of different concentrations of *Saccharomyces cerevisiae* on liver enzymes and malondialdehyde (MDA) of broilers fed regular- and low-density diets from days 1 to 38 of age.


a,b,c Means within a column with different superscripts are significantly different based on Student– Newman–Keuls (SNK) post hoc test. ALT = Alanine aminotransferase; AST = Aspartate aminotransferase; AST/ALT = Aspartate aminotransferase to alanine aminotransferase ratio; MAD = Malondialdehyde. Number of observations was 6 per interaction cell.


**Table 4.** Impact of different concentrations of *Saccharomyces cerevisiae* on the biochemical constituents of blood serum of broilers fed regular- and low-density diets from days 1 to 38 of age.

a,b Means within a column with different superscripts are significantly different based on Student–Newman–Keuls (SNK) post hoc tests. GAR = Globulin to albumin ratio. Number of observations was 6 chicks per interaction cell.

Data for the biochemical components of blood serum show no significant impact of diet type on serum biochemical constituents (total protein, albumin, α-, β-globulin, globulin and globulin/albumin ratio). However, γ-globulin, AST and ALT were significantly lower in birds fed a low-density diet than a regular-density diet (Tables 3 and 4). Supplementation of the diet with SC at 0.02 g/kg and 0.04% significantly lowered serum AST, ALT and MAD relative to the standard. Additionally, total serum protein and β- globulin were significantly greater in the groups that received SC supplementation compared with control groups without SC. There was no interaction effect between SC level and dietary treatment on blood biochemical constituents (ALT/AST and alkaline phosphatase, Table 3; and total protein, albumin, α, β and δ- globulin, G/A ratio, Table 4). However, there was a significant impact of the interaction on serum AST, ALT and MAD. Supplementation with SC at both concentrations significantly decreased serum AST and ALT of broilers on the low-density diet compared to the regular-density diet. Additionally, 0.02% of SC decreased MAD in broilers fed a low-density diet compared to a regular density diet.

#### *3.3. Hematology of Blood*

Table 5 shows the effects of different concentrations of yeast on white blood cells and its subpopulations of broilers fed regular- and low-density diets from 1 to 38 days of age. The results showed that lymphocytes of broilers fed a low-density diet were significantly higher than that of broilers fed a regular-density diet, but eosinophils were lower. The addition of SC to the diet greatly enhanced PCV, lymphocytes and monocytes; and 0.02% SC significantly increased Hgb and RBCs, but decreased heterophils and the H/L ratio, relative to control diet (Table 5). However, the addition of SC to the diet at 0.04% significantly increased heterophils.


**Table 5.** Impact of different concentrations of *Saccharomyces cerevisiae* on white blood cells (WBC) and its subpopulations in broilers fed regular- and low-density diets from 1 to 38 days of age.

a,b Means within a column with different superscripts are significantly different based on Student–Newman–Keuls (SNK) post hoc tests. H/L = Heterophil to lymphocyte ratio. Number of observations was 6 per interaction cell.

There were significant effects of interaction between dietary treatments and SC concentrations on PCV, Hgb, MCV, MCH, white blood cells (WBCs) and the H/L ratio. Results showed that SC supplementation increased PCV and Hgb for birds fed a regular-density diet, but 0.4 g/kg SC significantly decreased Hgb in birds fed a low-density diet. In addition, MCV and MCH were increased dramatically by the addition of 0.04% SC to the regular-density diet, but they were reduced in birds fed the low-density diet. The results showed that using 0.02% and 0.04% of SC increased WBCs in birds fed a regular-density diet, but 0.04% of SC significantly decreased WBCs in those fed a low-density diet. On the other hand, 0.02% of SC significantly decreased H/L in birds fed the low-density diet only (Table 5).

#### *3.4. Lymph Organs and Immune Response*

Table 6 shows the effects of the experimental treatments on the lymphoid organs of broilers. The results showed no significant effect of diet density on lymph structures like the spleen, absolute weight of thymus and bursa of Fabricius. However, the percentage of thymus was significantly greater in birds fed a low-density diet than that in birds fed a regular-density diet. Immune responses to NDV and AI, as measured by HI, were not influenced by diet type. These organs, as well as the immune response to NDV and AI, were significantly greater in broilers fed a diet supplemented with 0.02% or 0.04% of SC than those fed a diet without SC supplementation. Moreover, the effects on the thymus, bursa of Fabricius and NDV and AI showed stepwise increases. There were significant effects of interactions between diet type and SC on the percentage of spleen weight, bursa weight and immune response to NDV. The results indicated that the absolute weights of the spleen and bursa of Fabricius significantly decreased in the group fed the regular-density diet supplemented with 0.04% of SC. Still, both levels of SC significantly increased immune response to NDV. On the other hand, both concentrations of SC significantly increased the absolute weights of the spleen and bursa of Fabricius in birds fed the low-density diet, but the response to NDV was stepwise (Table 6).


**Table 6.** Impact of different concentrations of *Saccharomyces cerevisiae* on immune organs and HI titer (log2) in response to avian influenza and Newcastle disease virus in 38-day-old broilers fed either a regular- or low-density diet from days 1 to 38 of age.

a,b,c,d Means within a column with different superscripts are significantly different based on Student–Newman– Keuls (SNK) post hoc tests. HI = Hemagglutination inhibition test; NDV = Newcastle disease virus; I = Influenza antigen. Number of observations was 6 chicks per interaction cell.

#### *3.5. Immune Indices*

Tables 7 and 8 show the impacts of the different experimental treatments on the immune parameters and survival rate of broilers, respectively. Diet type significantly influenced LYS, TAC and PA, revealing an increasing effect of a low-density diet on immune parameters, but lower TAC (Table 7).

Supplementation of the diet with SC significantly decreased TAC, but enhanced PI relative to the standard diet and had no impact on other traits related to the immunity, including serum LTT, BACT, LYS and PA (Table 7). In addition, the survival rate of broiler chickens fed a diet fortified with 0.04% of SC was significantly greater than that of chickens fed a dietary supplement of 0.02% SC, or a diet without SC (Table 8).

The interaction between SC concentration and diet type did not influence the LTT, BACT, LYS, TAC, PI, PA, or survival rate during the challenge experiment (Tables 7 and 8).

**Table 7.** Impact of different concentrations of *Saccharomyces cerevisiae* on immune indices of broilers fed a regular- or low-density diet from 1 to 38 days of age.



**Table 7.** *Cont.*

a,b Means within a column with different superscripts are significantly different based on Student–Newman–Keuls (SNK) post hoc tests. LTT = Lymphocyte transformation test; BACT = Bactericidal activity; LYS = Lysozyme activity; TAC = Total antioxidant capacity; PI = Phagocytic index; PA = Phagocytic activity. Number of observations was 6 per interaction cell.

**Table 8.** Impact of different concentrations of *Saccharomyces cerevisiae* on the survival rate of broiler chickens between 38 and 48 days of age that were fed a regular- or low-density diet and infected with HPAIV H5N1 at 38 days of age.


a,b,c Means within a column with different superscripts are significantly different based on Student–Newman– Keuls (SNK) test. Number of observations was 6 broilers of 38-day-old per interaction cell.

#### *3.6. Histology Study* testinal villi (Table 9) and the diameter of the large bursal follicle (Table 9 and Figure 1).

ND = Not done.

Table 9 shows the impact of diet type and SC concentration on the morphology of the intestines and bursa of Fabricius and spleen, as shown in Figures 1–3. Diet type did not influence the length of intestinal villi or diameter of the large follicle of the bursa of Fabricius (Table 9). No changes were seen in the spleen and thymus due to diet type, SC concentration, or the interaction between these terms (Table 9; Figures 2 and 3). The intestinal villi and bursa of Fabricious were enhanced by 29.8% and 22.9%, respectively. Both traits were significantly enhanced with the supplementation of SC at a concentration of 0.02%. The large bursal follicle's intestinal villi length and diameter were not affected by the interaction between diet type and SC concentration.

a,b Means within a column with different superscripts are significantly different based on Student– Newman–Keuls (SNK) post hoc tests. Number of observations was 6 per interaction cell per age.

Supplementation of the diet with 0.04% SC significantly enhanced the length of in-

*Animals* **2022**, *12*, x 12 of 19

**Figure 1.** Micrograph of bursa of Fabricious of broiler at day 38 of age stained with HandE (X40) to investigate the follicle diameter in different groups; the distance between two follicular polar as presented all groups by lines: (**A**) Broilers supplemented with 0.02% *Saccharomyces cerevisiae*;(**B**) broilers supplemented with 0.04% *Saccharomyces cerevisiae*. Moderate enhancement in the follicular diameter was detected in broilers supplemented with 0.04% *Saccharomyces cerevisiae* (**B**). **Figure 1.** Micrograph of bursa of Fabricious of broiler at day 38 of age stained with HandE (X40) to investigate the follicle diameter in different groups; the distance between two follicular polar as presented all groups by lines: (**A**) Broilers supplemented with 0.02% *Saccharomyces cerevisiae*;(**B**) broilers supplemented with 0.04% *Saccharomyces cerevisiae*. Moderate enhancement in the follicular diameter was detected in broilers supplemented with 0.04% *Saccharomyces cerevisiae* (**B**).


**Table 9.** Impact of different concentrations of *Saccharomyces cerevisiae* on the morphology of the intestine, bursa of Fabricius and follicular cortical:medullary ratio of the thymus in 38-day-old broilers fed regular- or low-density diet.

a,b Means within a column with different superscripts are significantly different based on Student–Newman–Keuls (SNK) post hoc tests. Number of observations was 6 per interaction cell per age. ND = Not done.

Supplementation of the diet with 0.04% SC significantly enhanced the length of intestinal villi (Table 9) and the diameter of the large bursal follicle (Table 9 and Figure 1). The intestinal villi and bursa of Fabricious were enhanced by 29.8% and 22.9%, respectively. Both traits were significantly enhanced with the supplementation of SC at a concentration of 0.02%. The large bursal follicle's intestinal villi length and diameter were not affected by the interaction between diet type and SC concentration. *Animals* **2022**, *12*, x 13 of 19

**Figure 2.** Micrograph of the thymus stained with HandE (X40) to explore the thymic cortical: medullary ratio. **Figure 2.** Micrograph of the thymus stained with HandE (X40) to explore the thymic cortical: medullary ratio.

**Figure 3.** Micrograph of the spleen stained with HandE (X40) of regular density diet presented normal splenic histology featuring splenic arteriole (thin arrow) with white and red pulp (thick arrow).

The present work was conducted to fill the gap in knowledge regarding the impact of active SC yeast in relation to dietary composition on the antioxidant status, blood constituents and immune response of broiler chickens. Adding an SC product to the feed at concentrations of 0.02% or 0.04% improved the performance of broilers from 1 to 38 days

To our knowledge, this is the first study investigating the interactive relationship between using yeast in low-density diets and its effect on immune response in broiler chickens. The percentage of thymus was significantly greater in birds fed a low-density diet than that of birds fed a regular-density diet. The immune response to NDV and AI were significantly greater in broilers fed a diet supplemented with 0.02% or 0.04% of SC than

All groups presented the normal splenic histology as the control.

**4. Discussion** 

of age.

**Figure 2.** Micrograph of the thymus stained with HandE (X40) to explore the thymic cortical: me-

**Figure 3.** Micrograph of the spleen stained with HandE (X40) of regular density diet presented normal splenic histology featuring splenic arteriole (thin arrow) with white and red pulp (thick arrow). All groups presented the normal splenic histology as the control. **Figure 3.** Micrograph of the spleen stained with HandE (X40) of regular density diet presented normal splenic histology featuring splenic arteriole (thin arrow) with white and red pulp (thick arrow). All groups presented the normal splenic histology as the control.

#### **4. Discussion 4. Discussion**

dullary ratio.

The present work was conducted to fill the gap in knowledge regarding the impact of active SC yeast in relation to dietary composition on the antioxidant status, blood constituents and immune response of broiler chickens. Adding an SC product to the feed at concentrations of 0.02% or 0.04% improved the performance of broilers from 1 to 38 days of age. The present work was conducted to fill the gap in knowledge regarding the impact of active SC yeast in relation to dietary composition on the antioxidant status, blood constituents and immune response of broiler chickens. Adding an SC product to the feed at concentrations of 0.02% or 0.04% improved the performance of broilers from 1 to 38 days of age.

To our knowledge, this is the first study investigating the interactive relationship between using yeast in low-density diets and its effect on immune response in broiler chickens. The percentage of thymus was significantly greater in birds fed a low-density diet than that of birds fed a regular-density diet. The immune response to NDV and AI were significantly greater in broilers fed a diet supplemented with 0.02% or 0.04% of SC than To our knowledge, this is the first study investigating the interactive relationship between using yeast in low-density diets and its effect on immune response in broiler chickens. The percentage of thymus was significantly greater in birds fed a low-density diet than that of birds fed a regular-density diet. The immune response to NDV and AI were significantly greater in broilers fed a diet supplemented with 0.02% or 0.04% of SC than those fed a diet without SC supplementation. In addition, there were significant interactions between diet type and SC on the percentage of spleen weight, bursa weight and immune response to NDV. The results indicated that the absolute weights of the spleen and bursa of Fabricious were significantly high in the group fed the low-density diet supplemented with 0.04% of SC. Still, both levels of SC significantly increased immune response to NDV. Interestingly, both concentrations of SC significantly increased the absolute weights of the spleen and bursa of Fabricious in birds fed the low-density diet.

The increase in feed cost accompanied by the reduction in the availability of corn as a main feed ingredient will affect the production efficiency of poultry on the global level, especially during global pandemics such as the current coronavirus crisis. Nutritional manipulation by using the low-density diet supplemented with yeast could provide a great opportunity to improve the economic outcome by reducing the feed cost, which constitutes approximately 60–70% of the total poultry operation cost. Using low densitydiet in broiler rations will provide a positive alternative to reduce feed cost. The other side of the coin is that using yeast in these diets compensated for the low-density contents of the diets by improving the antioxidant status and immune response of the broiler chickens. Any improvement in nutrition management and feed cost will have a direct impact on profitability and efficiency of poultry industry [49,50].

In addition, the group treated with 0.04% of SC had a significantly higher immune response than the other groups. These results contribute to the poultry industry important information that will improve production efficiency.

In addition, 0.02% of SC decreased MAD of broilers fed a low-density diet compared with a regular-density diet. These results indicate that SC enhances the oxidative status of broilers. Interestingly, Czech et al. [11] revealed that using 3% of *Yarrowia lipolytica* or SC yeast, in combination with *Bacillus* sp. probiotic, in the feed of turkeys from 7 to 112 days of age improved the antioxidant status of birds by preventing lipid peroxidation. This effect enhances the ability of poultry to handle stress and infectious agents. In another study [10], the authors investigated the mechanism by which SC enhances the oxidative status of broiler chickens. The authors included SC in either the feed or drinking water of stressed broilers and measured CYP1A2 and melanocortin-2 receptor (MC2R) gene expression in the adrenal glands and IL10 and AvBD1 in the spleen. The authors concluded that using SC in broilers' feed or drinking water for 40 days decreased stress and MC2R gene expression. They also showed that supplementation of SC fermentate in the feed was marginally more effective than adding it to drinking water in stimulating oxidative status and reducing stress in broiler chickens [10] and in detoxifying nitrate (21, 22) and aflatoxin (22, 23).

There has been an interest in using a low-density diet to feed broilers, to lower the growing pressure on the skeletal system of the bird and decrease skeletal diseases, the cost of feed and environmental pollution [51,52].

The body weight and EPEI were significantly decreased by 7.8% and 5.4%, respectively, over the study period for birds on the low-density diet compared to the regular-density diet. Indeed, the main effect of regular diet under the three SC levels was 2099 g while the mean body weight of the control diet without SC supplementations (1946 + 1784) was 1965 g. These findings indicate that the negative impact of diet structure persisted during the experimental period from 1 to 38 days of age [53].

The outcomes showed that the low-density diet improved liver function and increased the percentage of thymus and lymphocytes and PA, but decreased γ-globulin, eosinophils, TAC, ALT and AST. The current study revealed that supplementation of the feed with SC at 0.02 g/kg and 0.04% significantly lowered serum AST, ALT and MAD relative to the standard diet. Gheisari and Kholeghipour [9] showed that using live yeast had no significant impact on hematological indices such as RBCs, WBCs and PCV. On the other hand, in another study, there was a positive association between supplementation of feed with SC and hematological traits of chickens, such as RBCs, WBCs and PCV [51]. In the same study, probiotics had no effect (*p* > 0.05) on hemoglobin and WBCs at the finisher phase. Yet, a significant effect (*p* < 0.05) was observed for RBCs and packed cell volume.

Gut morphology was modulated because of the addition of 10% wheat bran in the lowdensity diet. The results indicated that the diets had no impact on the length of intestinal villi. Previous studies have shown that dietary cereal with a high nonstarch polysaccharides (NSP) level could enhance the dimension of the gastrointestinal tract [54]. Steenfeldt [55] observed that the arabinoxylan level in wheat is significantly and positively correlated to the relative masses of the duodenum, jejunum and ileum. It has been stated that dietary supplementation may modulate the morphology of the intestinal mucosa. Accordingly, NSP in the diet can also impact the morphology of the gastrointestinal tract [56]. Iji [57] showed that the crypt deepness of the jejunum and ileum was significantly enhanced by dietary addition of guar gum and xanthin gum. This finding demonstrated that NSP can improve cell turnover in the gastrointestinal tract. Enhanced crypt deepness indicates enhanced villus cell proliferation and in turn improved utilization of the nutrients by the gastrointestinal tract. This suggests that these cereals impact the mass of the gastrointestinaltract and morphology of the intestine [58].

These results suggest that the dilution of nutrients in feed via adding 10% wheat bran improves the immune response and production index of broilers 1 to 38 days of age. These outcomes agree with earlier studies by Abudabos [53] and Attia [54]. Wheat bran polysaccharides have been shown to act as antioxidants and immunostimulators and have anti-inflammatory, antitussive, anticancerous and antimutagenic properties [59–62]. Furthermore, wheat bran arabinoxylans have been shown to to enhance macrophage phagocytosis in animals [63]. They are immunostimulants of the antibody response in

chickens by enhancing the total IgG and IgM anti-SRBC antibody titers on 7th and 14th day post primary antibody response (PPI) and post-secondary inoculation (PSI) of sheep red blood cells (SRBCs) compared to the control. Additionally, Korte et al. [64] stated that supplementing feed with arabinoxylans significantly induced anti-SRBC antibody titers, representing enhanced humoral immunity in chickens.

The results indicated that supplementing feed with SC at either 0.02% or 0.04% significantly affected growth and EPEI relative to the control dietary treatment. The effect persisted throughout all tested periods. Furthermore, supplementing the low-density diet with 0.02% or 0.04% of SC resulted in an enhanced production index compared with the regular-density diet lacking the addition.

These results agree with other studies that have investigated similar effects of SC on growth performance [65–68]. In addition, the positive effect of a higher dose of SC are in line with results reported by Valdivie [69], who found that the growth performance of broilers significantly improved with an increased supplementation dose of SC. The improved gastrointestinal health and growth performance of broilers supplemented with SC may be due to the presence of effective ingredients in SC such as Vitamin B, cellulostic enzymes, phytase, monooligosaccharides (MOS) and glucomannan [70].

A diet supplemented with 0.04% SC increased the length of the villi and SC supplementation enhanced the production index and the body weight of broiler from 1 to 38 days of age in a dose-dependent manner.

There were significant improvements in blood serum biochemistry and liver function due to SC supplementation. Consistent with these results, Paryad and Mahmoudi [71] and Hosseini [72] showed that SC at 1.5% significantly enhanced total plasma protein, albumin and globulin and WBCs and decreased the H/L ratio. Furthermore, Zhang et al. [64] revealed that SC supplementation to broiler chickens significantly lowered the 2-thiobarbituric acid-reactive substances (TBARS) in the breast and drumstick meats and increased villus height, compared to the control group.

Supplementation of SC significantly enhanced the spleen, thymus, bursa of Fabricious and HI in response to NDV and AI, with a positive concentration-dependent impact of SC on the thymus, bursa of Fabricious and immune response to NDV and AI. The diameter of the bursal follicle significantly enhanced at 0.04% SC, indicating an improvement in the number of B-lymphoblasts, leading to an increase in the B-lymphocytes responsible of humoral immunity stimulation through antibody production. Further evidence of this effect was reflected by the increased survival rate of broilers challenged with AI at 38 d of age. In addition, SC supplementation was associated with improvements in β-globulin and hematological traits such as PCV, Hgb, RBCs, lymphocytes, monocytes and PI. These data provide more evidence for an improved health status of broilers fed a diet supplemented with SC. The effect of SC on the relative weights of the thymus and bursa and immune response to NDV and AI, was dose-dependent. Similarly, Newman [73], Spring et al. [74] and Zhang et al. [70] showed that SC supplementation of the diet enhanced production performance by improving the immune status, intestinal lumen health and digestion and nutrient utilization of birds. In addition, Gheisari and Kholeghipour [9] found that broiler chickens fed SC at a concentration of 0.02% had higher antibody titers against NDV than the control at 38 d of age, but it did not affect AI titers. The positive effect of SC on immune response could be attributed to its cell wall constituents, including chitin, mannan and glucan, which have immunostimulant effects [2,71,75–78].

#### **5. Conclusions**

The antioxidant status and total antioxidant capacity of broiler chickens were improved by supplementation of the diet with SC. Supplementation of either a regular-density diet or a low-density diet with SC at either 0.02% or 0.04% enhanced the BWG and EPEI of broilers during 1 to 38 days of age. Additionally, broilers fed a low-density diet supplemented with either 0.02% or 0.04% SC had a greater body weight and EPEI than birds fed the control diet with no supplementary SC. However, fortification of the diet with 0.04% resulted in significantly enhanced immune organs and a higher immune response.

**Author Contributions:** Y.A.A., H.S.A.E.-H. and A.A.E.-S. conducted the experimental design and first draft of the manuscript; H.S.A.E.-H. and A.A.E.-S. carried out the experiment, data collection and contribution to the experimental set-up and laboratory work; H.A.-K. and S.R.A. carried out the statistical analyses and revision; Y.A.A., M.A.A.-H. and H.S.A.E.-H. proofread the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors would like to thank the Kuwait Institute for Scientific Research, Kuwait, for providing the publications fees (APC).

**Institutional Review Board Statement:** The Animal Care and Use Committee (ACUC) at King Fahd Medical Research Center has approved the referenced protocol used in the current study, numbered ACUC-22-1-2, dated 1-2-2022.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data were presented in the manuscript.

**Acknowledgments:** The Deanship of Scientific Research (DSR) at King Abdulaziz University, Jeddah, Saudi Arabia, has funded this project, under grant no. (FP-217-43H). The authors acknowledged the administrative, technical and financial support by DSR, King Abdulaziz University, Jeddah, Saudi Arabia.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Article* **Effects of** *Eimeria tenella* **Infection on Key Parameters for Feed Efficiency in Broiler Chickens**

**Janghan Choi <sup>1</sup> , Hanseo Ko <sup>1</sup> , Yuguo Hou Tompkins <sup>1</sup> , Po-Yun Teng <sup>1</sup> , Jeferson M. Lourenco <sup>2</sup> , Todd R. Callaway <sup>2</sup> and Woo Kyun Kim 1,\***


**Simple Summary:** Coccidiosis, which can be induced by *Eimeria* spp., causes tremendous economic losses in the poultry production. *Eimeria tenella* (*E. tenella*) is one of the poultry *Eimeria* spp. that damage cecal tissue. Broilers infected with *E. tenella* can have reduced body weight, feed efficiency, and gut health because ceca are the main site for producing volatile fatty acids (VFA; important energy sources) and ceca accommodate diverse pathogens. To find appropriate strategies to cope with *E. tenella* infection, modes of actions of *E. tenella* infection on broiler growth and health should be investigated, and experimental infection model should be established. In the study, different levels of sporulated *E. tenella* oocysts were inoculated to the broilers, and the inoculation dosages induced mild infection in the ceca of broilers. The current study showed that *E. tenella* infection damaged feed efficiency and small intestinal health in broilers, mainly by reducing cecal volatile fatty acids (VFA) production. Different inoculation levels modulated the tendency of fecal moisture content and fecal oocyst shedding at different time points. Based on the results, energy supplementation and/or modulation of cecal microbiota potentially ameliorates negative effects of *E. tenella* infection in broilers.

**Abstract:** The purpose of the study was to investigate effects of different inoculation dosages of *E. tenella* on growth performance, gastrointestinal permeability, oocyst shedding, intestinal morphology, fecal consistency, ileal apparent digestibility, antioxidant capacity, and cecal VFA profile in broiler chickens. Five different dosages (T0: 0, T1: 6250, T2: 12,500, T3: 25,000, and T4: 50,000) of *E. tenella* oocysts were inoculated via oral gavage to fourteen-day-old broilers. Inoculation of *E. tenella* linearly increased FCR (*p* < 0.05), and feed intake was quadratically increased on 6 days post-infection (dpi; *p* = 0.08) and 7 dpi (*p* = 0.09). Cecal lesion score of each treatment was T0: 0; T1: 0.39 ± 0.14; T2: 0.93 ± 0.21; T3: 1.25 ± 0.16; and T4: 1.58 ± 0.2. Cecal total VFA production was linearly reduced due to *E. tenella* infection on 6 dpi (*p* < 0.01). *E. tenella* infection deepened cecal crypts depth on 6 dpi (CD; *p* < 0.05). Gastrointestinal permeability tended to be linearly increased (*p* = 0.07). *E. tenella* infection tended to linearly reduce duodenal VH (*p* = 0.1) and jejunal VH on 9 dpi (*p* = 0.09). Different dosages of *E. tenella* modulated the tendency of fecal moisture content and oocyst shedding. Therefore, *E. tenella* infection impaired feed efficiency and small intestinal health mainly by reducing cecal VFA production and deepening cecal CD in broilers.

**Keywords:** *Eimeria tenella*; broiler chickens; oocyst shedding; volatile fatty acids; feed efficiency; cecal health

#### **1. Introduction**

Coccidiosis causes tremendous economic losses in broiler production by impairing gut health and depressing growth performance and feed efficiency of broiler chickens, and expensive anti-coccidial treatments also increase the overall production cost [1,2].

**Citation:** Choi, J.; Ko, H.; Tompkins, Y.H.; Teng, P.-Y.; Lourenco, J.M.; Callaway, T.R.; Kim, W.K. Effects of *Eimeria tenella* Infection on Key Parameters for Feed Efficiency in Broiler Chickens. *Animals* **2021**, *11*, 3428. https://doi.org/10.3390/ ani11123428

Academic Editor: Raffaella Rossi

Received: 8 November 2021 Accepted: 29 November 2021 Published: 1 December 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Avian coccidiosis are induced by *Eimeria* spp., which are protozoan parasites, and there are 7 known *Eimeria* spp. that can infect chickens: *Eimeria acervulina*, *E. maxima*, *E. tenella*, *E. brunetti*, *E. necatrix*, *E. mitis,* and *E. praecox* [3]. Each species resides at the different section of the intestinal tract of broiler chickens, and thereby it has different modes of actions to affect growth performance and gut health of broilers [4]. Teng et al. [5] reported that *E. maxima* decreased digestibility of crude proteins and amino acids in broilers. *Eimeria* spp. can be transmitted via the fecal–oral route. The infection is initiated by ingestion of sporulated (infectious) oocysts, and after the asexual and sexual replications, un-sporulated oocysts are excreted with feces [6]. In an appropriate environment, the oocysts can be sporulated and become infectious, and this life cycle can be repeated with poultry growth cycle.

*Eimeria tenella* (*E. tenella*) resides in the mucus membrane of ceca, and during its replications, epithelial cells in ceca are damaged, resulting in hemorrhagic diarrhea and impaired growth performance and intestinal health in broilers [7]. The ceca, the main intestinal compartment for bacterial fermentation, can be reservoirs for pathogenic bacteria and their toxins that can cause oxidative stress after entering the blood stream of broilers [8]. However, ceca also play crucial roles in producing beneficial bacterial metabolites including vitamins, volatile fatty acids (VFA), lactic acid, and antimicrobial compounds via bacterial fermentation [9]. The VFA are not only inhibit the growth of pathogenic bacteria, but also are energy substrates for the host and induce gut development of chickens by accelerating gut epithelial cell proliferation [10]. Moreover, VFA interact with fat metabolism via mitogen-activated protein kinase (MAPK) pathway [11]. These suggest that VFA are closely associated with feed efficiency by providing extra energy to the host or influencing metabolism of chickens.

To cope with *Eimeria* spp. infection in broiler production, anti-coccidial drugs, and vaccination has been used in the broiler industry. However, the use of anti-coccidial drugs has been restricted by inhibiting the use of old anti-coccidial drugs and requiring Veterinary Feed Directives (VFD) registrations because of the spread of resistant *Eimeria* strain and consumer pressure [12,13]. Furthermore, vaccination is expensive and can prevent spread of *Eimeria* spp. [14]. Recently, a lot of attention has been paid to find nutritional interventions to control *Eimeria* spp. infection in broilers. Diverse bioactive compounds, including essential oils [15], probiotics [16], sodium butyrate [17], and plant extracts [18], were studied to control or to ameliorate negative effects of *E. tenella* infection and in broilers. The modes of actions of those bioactive compounds may include damaging cell wall of *E. tenella*, modulating cecal microbiota, and/or enhancing the immunity of broilers. To find suitable nutritional interventions, it is important to understand mode of actions of *E. tenella* on the growth of chickens and to set up appropriate experimental infection models to test novel nutritional interventions. Therefore, the hypothesis of this study was that impaired cecal health due to *E. tenella* infection may result in reduced growth performance and impaired intestinal health because of reduced VFA production and increased oxidative stress. The purpose of the study was to investigate the effects of different inoculation dosages of *E. tenella* on growth performance, gastrointestinal permeability, oocyst shedding, fecal consistency, intestinal morphology, ileal apparent digestibility, antioxidant capacity, and cecal VFA in broiler chickens.

#### **2. Materials and Methods**

#### *2.1. Experimental Design, Diets, and Growth Performance*

This study was approved by the Institutional Animal Care and Use committee of the University of Georgia, and this experiment was conducted at the Poultry Research Center, University of Georgia, Athens, GA. A total of 360 fourteen-day-old male Cobb500 broiler chickens were distributed to 5 treatments with 6 replicates (12 birds per battery cage) in a completely randomized design. The experimental treatments were (1) treatments 0 (T0): administration with 1 mL of phosphate-buffered solution (PBS) as a sham-challenged group; (2) treatments 1 (T1): administration with 6250 sporulated oocysts of *E. tenella*; treatment 2 (T2): administration with 12,500 sporulated oocysts of *E. tenella*; treatments (T3): administration with 25,000 sporulated oocysts of *E. tenella*; and treatment (T4), administration

with 50,000 sporulated oocysts of *E. tenella*. The sham-challenged groups were placed on the top of the cages to minimize the cross-infection. The *E. tenella* used in the study was a wild-type strain. To each bird, 1 mL of inoculum was administrated by oral gavage. As shown in Table 1, the experimental diet (D 14 to 23) was formulated to meet or exceed Cobb 500 nutrient requirements (2018) and included 3 g/kg of titanium (IV) oxide (Acros Organics, Morris Plains, NJ, USA) as an indigestible marker to determine the apparent ileal digestibility (AID). In-feed anticoccidials were not included in the experimental diet (D 14 to 23) and in the pre-experimental diet (D 0 to 14).


**Table 1.** Diet composition and calculated analysis of the broiler diet (g/kg, as fed basis).

<sup>1</sup> Vitamin mix provided the following in mg/100 g diet: thiamine-HCl, 1.5; riboflavin 1.5; nicotinic acid amide 15; folic acid 7.5; pyridoxine-HCl, 1.2; d-biotin 3; vitamin B-12 (source concentration, 0.1%) 2; d-calcium pantothenate 4; menadione sodium bisulfite, 1.98; α-tocopherol acetate (source 500,000 IU/g), 22.8; cholecalciferol (source 5,000,000 IU/g) 0.09; retinyl palmitate (source 500,000 IU/g), 2.8; ethoxyquin, 13.34; I-inositol, 2.5; dextrose, 762.2; <sup>2</sup> Mineral mix provided the following in g/100 g diet: Ca(H2PO4)2·H2O, 3.62; CaCO3, 1.48; KH2PO4, 1.00; Na2SeO4, 0.0002; MnSO4·H2O, 0.035; FeSO4·7H2O, 0.05; MgSO4·7H2O, 0.62; KIO3, 0.001; NaCl, 0.60; CuSO4·5H2O, 0.008; ZnCO3, 0.015; CoCl2·6H2O, 0.00032; NaMoO4·2H2O, 0.0011; KCl, 0.10; dextrose, 0.40; <sup>3</sup> SID: standard ileal digestible amino acid.

During the entire experiment period, birds had free access to water and feed, and temperature was controlled according to the recommendation of Cobb Broiler Management Guide. Body weight (BW) of the birds per cage were recorded on 6 days post-infection (dpi) and 9 dpi, and feed disappearance were recorded daily to calculate average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR). The acute phase was 0 to 6 dpi, and recovery phase was 6 to 9 dpi.

#### *2.2. Gastrointestinal Permeability, Oocyst Shedding, and Fecal Consistency*

Gastrointestinal permeability was measured according to Teng et al. [19] with minor modifications. On 5, 6, and 7 dpi, one bird per cage was administrated with 1 mL of 2.2 mg/mL of fluorescein isothiocyanatedextran 4 kDa (FITC-D4; Sigma-Aldrich Co., St. Louis, MO, USA) dissolved in PBS. After 2 h, birds were euthanized and blood from the heart was collected into heparin-free vacutainer tubes. The tubes stood in a dark container at room temperature for 1 h for clotting and centrifuged at 1000× *g* for 15 min to recover serum. The collected serum samples were transferred to a 96 black well plate (Greiner Bio-

one, Monroe, NC, USA) in duplicate, and the fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 528 nm by using a ICTOR3 Multilabel Plate Reader (Perkin Elmer, Waltham, MA, USA). The FITC-D4 concentration in the serum was quantified by prepared standard solution using pooled serum from birds that were not inoculated with FITC-D4 (not part of the study) and expressed relative to the T0 group.

Fresh fecal samples were collected on 5 to 6 dpi, 6 to 7 dpi, and 7 to 8 dpi to measure fecal moisture content and fecal oocyst shedding. Fecal samples were put in a 60 ◦C oven until constant weight, and the weights before and after drying were recorded to calculate moisture content. Oocysts in fecal samples were counted using a McMaster chamber to calculate oocyst shedding per gram of feces. Briefly, 3 to 5 g of fecal samples were put in 50 mL tubes and mixed thoroughly with 25 mL of distilled water to ensure a uniform suspension. Afterwards, 1 mL of the suspension were mixed with 9 mL of the saturated salt solutions. The mixed solution was loaded into a McMaster chamber, and number of oocysts were counted using a microscope. Oocyst shedding were expressed as log<sup>10</sup> (oocysts/g feces).

#### *2.3. Sample Collection and Lesion Score*

On 6 and 9 dpi, 4 birds per cage were euthanized by the cervical dislocation method for sample collection and lesion scoring (6 dpi). Cecal lesion scoring from 4 birds per cage was conducted in a blind fashion according to the 4-score scale method [20]. Around 3 to 5 cm of intestinal sections of mid-duodenum, mid-jejunum, mid-ileum, and mid-ceca were collected, and then rinsed with PBS to remove digesta, and stored in 10% neutralbuffered formalin for further steps. From 4 birds per cage, ileal digesta (from the Meckel's diverticulum to 15 cm upper from the ileo-cecal-colic junction) were collected and dried in a 60 ◦C oven until constant weight to determine ileal moisture content and for digestibility analysis. Liver and ceca samples were collected and snap-frozen and stored at −80 ◦C.

#### *2.4. Intestinal Morphology*

The fixed tissues in 10% neutral-buffered formalin were embedded in paraffin and cut into 4 µm, and hematoxylin and eosin (H&E) staining was conducted. The H&E-stained slides were read using a microscope (BZ-X810; Keyence, Osaka, Japan). The villus height (VH) and crypts depth (CD) of five well-oriented villi per section and their corresponding crypts for the five villi were measured for duodenum, jejunum, and ileum samples, and CD was measured for ceca samples by using ImageJ (National Institutes of Health, Bethesda, MD, USA). The ratios of VH to CD were calculated for each villus and crypt.

#### *2.5. Apparent Ileal Digestibility*

The concentrations of titanium dioxide in oven-dried samples (0.3 g for ileal digesta samples and 0.5 g for the feed sample) were analyzed according to Short et al. [21]. Dry matter (DM), organic matter (OM), and ash apparent ileal digestibility were determined according to Lin and Olukosi [22].

#### *2.6. Liver Total Antioxidant Capacity (TAC)*

Total antioxidant capacity (TAC) in the liver was measured using a commercial kit (QuantiCromAntioxidant Assay Kit; DTAC-100) (BioAssay Systems, Hayward, CA, USA). Approximately 100 mg of frozen liver samples were homogenized in 1 mL of PBS for 45 s using a beads beater and centrifuged at 10,000× *g* for 10 min. Aliquots of supernatants were taken for the analyses of protein content using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Cleveland, OH, USA) after 20 times dilution. Afterwards, TAC was measured according to the manufacture's protocol without further dilutions. The absorbance was measured using SpectraMax® ABS Plus microplate reader (Molecular devices, San Jose, CA, USA). The TAC values were expressed as nM trolox quivalents/mg protein.

#### *2.7. VFA Concentrations in Cecal Digesta*

Concentrations of VFA in cecal digesta were analyzed according to Lourenco et al. [23]. Cecal samples were collected from birds on 6 and 9 dpi, and the samples were snap-frozen in liquid nitrogen and stored at −80 ◦C for further analyzes. Once thawed, samples were diluted and homogenized by placing 0.5 g into 3 mL of distilled water. The samples were vigorously homogenized for 1 min and subsequently frozen at −20 ◦C. After the samples were thawed, these were centrifuged at 10,000× *g* for 10 min, and 850 µL of supernatant were collected and mixed with 170 µL of the fresh 25% (wt/vol) meta-phosphoric acid solution, and immediately frozen at −20 ◦C overnight. The samples were centrifuged at 10,000× *g* for 10 min, and 800 µL of supernatant was collected and mixed with 1600 µL ethyl acetate. Samples were vigorously homogenized for 10 s and allowed to settle for 5 min. The top layer was transferred to a screw-thread vial and analyzed in a gas chromatograph (Shimadzu GC-2010 plus; Shimadzu Corporation, Kyoto, Japan) equipped with an autoinjector (AOC-20i; Shimadzu Corporation, Kyoto, Japan). A capillary column (Zebron ZB-FFAP; 30 m × 0.32 mm × 0.25 µm; Phenomenex Inc., Torrance, CA, USA) was used for the separation of the VFA. The sample injection volume was set at 1 mL, and helium was used as the carrier gas. The column temperature was initially set at 110 ◦C, and gradually increased to 200 ◦C over the course of 6 min. The flame ionization detector was set at 350 ◦C.

#### *2.8. Statistical Analyses*

Statistical analyses were performed using SAS (version 9.4; SAS Inst. Inc., Cary, NC, USA). Data normality was checked using proc univariate except for lesion score data. All groups were compared using proc mixed in a completely randomized design followed by Tukey's comparison test. Kruskal–Wallis test followed by the Dwass, Steel, Critchlow-Fligner post hoc test was used to analyze lesion score data. Orthogonal polynomial contrasts were utilized to evaluate the significance of linear or quadratic effects of different *E. tenella* inoculation dosages, and the inoculation dosages of *E. tenella* were normalized by using the base 2 logarithm of the number of sporulated *E. tenella* number for orthogonal polynomial contrasts [19]. Statistical significance was set at *p* < 0.05, and trends (0.05 ≤ *p* ≤ 0.1) were also presented.

#### **3. Results**

#### *3.1. Growth Performance and Lesion Score*

As shown in Table 2, no significant differences were observed in BW, ADG, and ADFI in the acute phase (*p >* 0.1) among the treatments. However, daily feed intake tended to quadratically increase on 6 dpi (*p* = 0.08) and 7 dpi (*p* = 0.09) due to *E. tenella* infection (Figure 1). In the acute phase, FCR was linearly increased due to the inoculation of *E. tenella* (*p* < 0.05). There were no significant differences in growth performance among the treatments in the recovery phase.

**Table 2.** Growth performance parameters including body weight (BW; g), average daily gain (ADG; g/d), average daily feed intake (ADFI; g/d), and feed conversion ratio (FCR; g/g) of broiler chickens infected with different dosages of *Eimeria tenella* during the acute phase [0 to 6 days post-infection (dpi)] and recovery phase (6 to 9 dpi) <sup>1</sup> .



**Table 2.** *Cont.*

<sup>1</sup> T0: treatment 0 (Sham-challenged with phosphate-buffered saline); T1, treatment 1 (challenged with 6250 sporulated oocysts of *Eimeria tenella*); T2, treatment 2 (challenged with 12,500 sporulated oocysts of *Eimeria tenella*); T3, treatment 3 (challenged with 25,000 sporulated oocysts of *Eimeria tenella*); T4, treatment 4 (challenged with 50,000 sporulated oocysts of *Eimeria tenella*). At each time point, orthogonal polynomial contrasts analysis was conducted to see linear pattern (L) and quadratic pattern (Q) among the treatments. *tenella* (*p* < 0.05). There were no significant differences in growth performance among the treatments in the recovery phase.

**Figure 1.** Daily feed intake of broiler chickens infected with different dosages of *Eimeria tenella*. Daily feed intake was measured in the T0 (treatment 0; Sham-challenged with phosphate-buffered saline); T1 (treatment 1; challenged with 6250 sporulated oocysts of *E. tenella*); T2 (treatment 2; challenged with 12,500 sporulated oocysts of *E. tenella*); T3 (treatment 3; challenged with 25,000 sporulated oocysts of *E. tenella*); T4 (treatment 4; challenged with 50,000 sporulated oocysts of *E. tenella*) groups during 1 to 9 days post-infection. At each time point, orthogonal polynomial contrasts analysis was conducted to see linear pattern (L) and quadratic pattern (Q) among the treatments. **Figure 1.** Daily feed intake of broiler chickens infected with different dosages of *Eimeria tenella*. Daily feed intake was measured in the T0 (treatment 0; Sham-challenged with phosphate-buffered saline); T1 (treatment 1; challenged with 6250 sporulated oocysts of *E. tenella*); T2 (treatment 2; challenged with 12,500 sporulated oocysts of *E. tenella*); T3 (treatment 3; challenged with 25,000 sporulated oocysts of *E. tenella*); T4 (treatment 4; challenged with 50,000 sporulated oocysts of *E. tenella*) groups during 1 to 9 days post-infection. At each time point, orthogonal polynomial contrasts analysis was conducted to see linear pattern (L) and quadratic pattern (Q) among the treatments.

**Table 2.** Growth performance parameters including body weight (BW; g), average daily gain (ADG; g/d), average daily feed intake (ADFI; g/d), and feed conversion ratio (FCR; g/g) of broiler chickens infected with different dosages of *Eimeria tenella* during the acute phase [0 to 6 days post-infection (dpi)] and recovery phase (6 to 9 dpi) 1. Cecal lesion due to *E. tenella* infection was not detected in the T0 group on 6 dpi (Figure 2). The T4 group had a higher lesion score compared to the T1 group (*p* < 0.05); lesion scores > 0 and <2 were also obtained in T1, T2, and T3 groups.

#### *3.2. Oocyst Shedding and Fecal/ileal Moisture Content*

**Items** *Eimeria tenella***-Challenged Polynomial Contrast T0 T1 T2 T3 T4 SEM** *p* **Value Lin. Quad.**  Initial BW 359.4 360.3 356 359.5 358 3.72 0.38 0 to 6 dpi BW 703.4 705.5 697 699.5 680.77 27.28 0.55 0.16 0.43 As shown in Figure 3, *E. tenella* was not detected in the feces of the sham-challenged (T0) group at all time points. On 5 to 6 dpi, the T4 group had significantly higher oocyst shedding compared to T1 group, and *E. tenella* infection linearly increased oocyst shedding. However, there were no significant differences in oocyst shedding among the treatments on 6 to 7 dpi and 7 to 8 dpi.

ADG 57.33 57.53 57.01 56.54 53.80 4.32 0.56 0.16 0.37 ADFI 84.92 87.24 88.63 86.05 84.31 4.61 0.55 0.69 0.11

BW 881.8 859 876.9 885.73 889.19 79.63 0.97 0.69 0.72 ADG 65.81 57.40 61.68 63.83 69.70 13.74 0.62 0.43 0.22 ADFI 115.4 120.9 123.77 123.1 123.1 14.86 0.87 0.4 0.55 FCR 1.9 2.17 2.03 1.97 1.77 0.39 0.50 0.38 0.16

ADG 60.16 57.49 58.83 58.97 59.10 6.47 0.97 0.94 0.66 ADFI 95.09 98.47 100.6 98.16 97.24 6.81 0.72 0.65 0.22 FCR 1.62 1.74 1.71 1.67 1.64 0.14 0.54 0.85 0.15 1 T0: treatment 0 (Sham-challenged with phosphate-buffered saline); T1, treatment 1 (challenged with 6250 sporulated oocysts of *Eimeria tenella*); T2, treatment 2 (challenged with 12,500 sporulated

6 to 9 dpi

0 to 9 dpi

scores > 0 and <2 were also obtained in T1, T2, and T3 groups.

ratic pattern (Q) among the treatments.

**Figure 2.** Cecal lesion score of broiler chickens infected with different dosages of *Eimeria tenella* on 6 days post-infection. Cecal lesion score was measured in the T0 (treatment 0; Sham-challenged with phosphate-buffered saline); T1 (treatment 1; challenged with 6250 sporulated oocysts of *E. tenella*); T2 (treatment 2; challenged with 12,500 sporulated oocysts of *E. tenella*); T3 (treatment 3; challenged with 25,000 sporulated oocysts of *E. tenella*); T4 (treatment 4; challenged with 50,000 sporulated oocysts of *E. tenella*) groups on 6 days post-infection. Different letters at the same time point represent significantly different (*p* < 0.05) by utilizing the Kruskal–Wallis test followed by the Dwass, Steel, Critchlow-Fligner post hoc test. **Figure 2.** Cecal lesion score of broiler chickens infected with different dosages of *Eimeria tenella* on 6 days post-infection. Cecal lesion score was measured in the T0 (treatment 0; Sham-challenged with phosphate-buffered saline); T1 (treatment 1; challenged with 6250 sporulated oocysts of *E. tenella*); T2 (treatment 2; challenged with 12,500 sporulated oocysts of *E. tenella*); T3 (treatment 3; challenged with 25,000 sporulated oocysts of *E. tenella*); T4 (treatment 4; challenged with 50,000 sporulated oocysts of *E. tenella*) groups on 6 days post-infection. Different letters at the same time point represent significantly different (*p* < 0.05) by utilizing the Kruskal–Wallis test followed by the Dwass, Steel, Critchlow-Fligner post hoc test. *Animals* **2021**, *11*, x FOR PEER REVIEW 8 of 17

oocysts of *Eimeria tenella*); T3, treatment 3 (challenged with 25,000 sporulated oocysts of *Eimeria tenella*); T4, treatment 4 (challenged with 50,000 sporulated oocysts of *Eimeria tenella*). At each time point, orthogonal polynomial contrasts analysis was conducted to see linear pattern (L) and quad-

Cecal lesion due to *E. tenella* infection was not detected in the T0 group on 6 dpi (Figure 2). The T4 group had a higher lesion score compared to the T1 group (*p* < 0.05); lesion

**Figure 3.** Oocyst shedding of broiler chickens infected with different dosages of *Eimeria tenella*. Fecal *oocyst shedding* was measured using McMaster chamber in the T0 (treatment 0; Sham-challenged with phosphate-buffered saline); T1 (treatment 1; challenged with 6250 sporulated oocysts of *E. tenella*); T2 (treatment 2; challenged with 12,500 sporulated oocysts of *E. tenella*); T3 (treatment 3; challenged with 25,000 sporulated oocysts of *E. tenella*); T4 (treatment 4; challenged with 50,000 sporulated oocysts of *E. tenella*) groups on 5 to 6, 6 to 7, and 7 to 8 days post-infection. Number of *E. tenella* oocysts is shown as log10 (oocysts/g feces). At each time point, orthogonal polynomial contrasts analysis was conducted to see linear pattern (L) and quadratic pattern (Q) among the treatments. Different letters at the same time point represent significantly different (*p* < 0.05) by proc mixed followed by the Tukey's multiple comparison test among the treatment groups. **Figure 3.** Oocyst shedding of broiler chickens infected with different dosages of *Eimeria tenella*. Fecal *oocyst shedding* was measured using McMaster chamber in the T0 (treatment 0; Sham-challenged with phosphate-buffered saline); T1 (treatment 1; challenged with 6250 sporulated oocysts of *E. tenella*); T2 (treatment 2; challenged with 12,500 sporulated oocysts of *E. tenella*); T3 (treatment 3; challenged with 25,000 sporulated oocysts of *E. tenella*); T4 (treatment 4; challenged with 50,000 sporulated oocysts of *E. tenella*) groups on 5 to 6, 6 to 7, and 7 to 8 days post-infection. Number of *E. tenella* oocysts is shown as log<sup>10</sup> (oocysts/g feces). At each time point, orthogonal polynomial contrasts analysis was conducted to see linear pattern (L) and quadratic pattern (Q) among the treatments. Different letters at the same time point represent significantly different (*p* < 0.05) by proc mixed followed by the Tukey's multiple comparison test among the treatment groups.

As shown in Table 3, Fecal moisture content was modulated due to *E. tenella* infection on 7 to 8 dpi (*p* < 0.05). Fecal moisture content was quadratically decreased on 5 to 6 dpi (*p* < 0.05), and quadratically increased on 6 to 7 dpi (*p* < 0.05) due to *E. tenella* infection. *E. tenella* infection linearly increased fecal moisture content on 7 to 8 dpi (*p* < 0.05). On 6 dpi, As shown in Table 3, Fecal moisture content was modulated due to *E. tenella* infection on 7 to 8 dpi (*p* < 0.05). Fecal moisture content was quadratically decreased on 5 to 6 dpi (*p* < 0.05), and quadratically increased on 6 to 7 dpi (*p* < 0.05) due to *E. tenella* infection. *E. tenella* infection linearly increased fecal moisture content on 7 to 8 dpi (*p* < 0.05). On

*E. tenella* infection altered ileal moisture content, and ileal moisture content was linearly

**Table 3.** Fecal moisture content (fecal consistency) and ileal moisture content of broiler chickens infected with different dosages of *Eimeria tenella on* 5 to 6 days post-infection (dpi), 6 to 7 dpi, and 7

**Items** *Eimeria tenella***-Challenged Polynomial** 

5 to 6 dpi 78.23 76.76 75.91 76.56 76.99 1.66 0.22 0.22 0.04 6 to 7 dpi 68.14 69.68 73.38 74.64 68.66 4.87 0.12 0.35 0.03 7 to 8 dpi 70.81 71.87 73.09 75.07 74.86 2.52 0.03 <0.01 0.65

6 dpi 81.75 81.83 80.6 80.94 80.81 0.76 0.03 0.01 0.33 9 dpi 80.34 80.1 81.24 80.62 79.93 1.22 0.42 0.9 0.18 1 T0: treatment 0 (Sham-challenged with phosphate-buffered saline); T1, treatment 1 (challenged with 6250 sporulated oocysts of *E. tenella*); T2, treatment 2 (challenged with 12,500 sporulated oocysts of *E. tenella*); T3, treatment 3 (challenged with 25,000 sporulated oocysts of *E. tenella*); T4, treatment 4 (challenged with 50,000 sporulated oocysts of *E. tenella*). At each time point, orthogonal polynomial contrasts analysis was conducted to see linear pattern (L) and quadratic pattern

**T0 T1 T2 T3 T4 SEM** *p* **Value Lin. Quad.** 

**Contrast** 

### *3.2. Oocyst Shedding and Fecal/ileal Moisture Content*

Feces

Ileum

(Q) among the treatments.

*3.3. Gastrointestinal Permeability* 

to 8 dpi (fecal moisture content), and at 6 and 9 dpi (ileal moisture content) 1.

6 dpi, *E. tenella* infection altered ileal moisture content, and ileal moisture content was linearly decreased on 6 dpi due to *E. tenella* infection (*p* < 0.05).

**Table 3.** Fecal moisture content (fecal consistency) and ileal moisture content of broiler chickens infected with different dosages of *Eimeria tenella on* 5 to 6 days post-infection (dpi), 6 to 7 dpi, and 7 to 8 dpi (fecal moisture content), and at 6 and 9 dpi (ileal moisture content) <sup>1</sup> .


<sup>1</sup> T0: treatment 0 (Sham-challenged with phosphate-buffered saline); T1, treatment 1 (challenged with 6250 sporulated oocysts of *E. tenella*); T2, treatment 2 (challenged with 12,500 sporulated oocysts of *E. tenella*); T3, treatment 3 (challenged with 25,000 sporulated oocysts of *E. tenella*); T4, treatment 4 (challenged with 50,000 sporulated oocysts of *E. tenella*). At each time point, orthogonal polynomial contrasts analysis was conducted to see linear pattern (L) and quadratic pattern (Q) among the treatments. *Animals* **2021**, *11*, x FOR PEER REVIEW 9 of 17

#### *3.3. Gastrointestinal Permeability* No significant differences in gastrointestinal permeability were observed on 5 dpi

No significant differences in gastrointestinal permeability were observed on 5 dpi and 6 dpi (Figure 4). However, gastrointestinal permeability tended to be linearly increased (*p* = 0.07). However, the numerically highest group, T4, had only 1.16 folds FITC fluorescence compared to the T0 group. and 6 dpi (Figure 4). However, gastrointestinal permeability tended to be linearly increased (*p* = 0.07). However, the numerically highest group, T4, had only 1.16 folds FITC fluorescence compared to the T0 group.

**Figure 4.** In vivo gastrointestinal permeability of broiler chickens infected with different dosages of *Eimeria tenella*. In vivo gastrointestinal permeability was measured using fluorescein isothiocyanate dextran 4 kDa in the T0 (treatment 0; Sham-challenged with phosphate-buffered saline); T1 (treatment 1; challenged with 6250 sporulated oocysts of E. tenella); T2 (treatment 2; challenged with 12,500 sporulated oocysts of *E. tenella*); T3 (treatment 3; challenged with 25,000 sporulated oocysts of *E. tenella*); T4 (treatment 4; challenged with 50,000 sporulated oocysts of *E. tenella*) groups on 5, 6, and 7 days post-infection. At each time point, orthogonal polynomial contrasts analysis was conducted to see linear pattern (L) and quadratic pattern (Q) among the treatments. **Figure 4.** In vivo gastrointestinal permeability of broiler chickens infected with different dosages of *Eimeria tenella*. In vivo gastrointestinal permeability was measured using fluorescein isothiocyanate dextran 4 kDa in the T0 (treatment 0; Sham-challenged with phosphate-buffered saline); T1 (treatment 1; challenged with 6250 sporulated oocysts of E. tenella); T2 (treatment 2; challenged with 12,500 sporulated oocysts of *E. tenella*); T3 (treatment 3; challenged with 25,000 sporulated oocysts of *E. tenella*); T4 (treatment 4; challenged with 50,000 sporulated oocysts of *E. tenella*) groups on 5, 6, and 7 days post-infection. At each time point, orthogonal polynomial contrasts analysis was conducted to see linear pattern (L) and quadratic pattern (Q) among the treatments.

#### *3.4. Intestinal Morphology and Apparent Ileal Digestibility*

*3.4. Intestinal Morphology and Apparent Ileal Digestibility*  As shown in Table 4, *E. tenella* infection linearly (*p* < 0.05) and quadratically (*p* < 0.05) increased cecal CD, whereas the sham-challenged group had the lowest cecal CD compared to the *E. tenella* challenged groups on 6 dpi (*p* < 0.05). On 6 dpi, ileal CD showed a As shown in Table 4, *E. tenella* infection linearly (*p* < 0.05) and quadratically (*p* < 0.05) increased cecal CD, whereas the sham-challenged group had the lowest cecal CD compared to the *E. tenella* challenged groups on 6 dpi (*p* < 0.05). On 6 dpi, ileal CD showed a tendency to be linearly reduced (*p* = 0.06), and ileal VH:CD tended to be increased (*p* = 0.08) due

tendency to be linearly reduced (*p* = 0.06), and ileal VH:CD tended to be increased (*p* = 0.08) due to *E. tenella* infection. On 9 dpi, the inoculation of *E. tenella* tended to linearly

CD compared to T2, T3, and T4 groups (*p* < 0.05), and *E. tenella* infection linearly (*p* < 0.05), and quadratically (*p* < 0.05) increased cecal CD. As shown in Figure 5, cecal CD were deepened, and gametocytes and developing oocysts were observed in the ceca of broiler chick-

**Table 4.** Duodenal, jejunal, ileal, and cecal morphology [villus height (VH; µm), crypts depth (CD; µm), and VH:CD] of broiler chickens infected with different dosages of *Eimeria tenella* on 6 days

**Items** *Eimeria tenella***-Challenged Polynomial** 

VH 2459.97 2585.52 2442.42 2154.12 2485.42 300.16 0.22 0.34 0.56 CD 282.46 245.17 253.82 227.51 255.55 35.36 0.17 0.13 0.08 VH:CD 9.05 10.8 9.86 10.39 9.85 1.78 0.53 0.61 0.26

VH 1411.46 1375.62 1332.65 1321.93 1303.21 147 0.71 0.17 0.76

**T0 T1 T2 T3 T4 SEM** *p* **Value Lin. Quad.** 

**Contrast** 

ens infected *E. tenella.* 

6 dpi Duodenum

Jejunum

post-infection (dpi) and 9 dpi 1.

to *E. tenella* infection. On 9 dpi, the inoculation of *E. tenella* tended to linearly decrease duodenal VH (*p* = 0.1) and jejunal VH (*p* = 0.09). The T0 group had lower cecal CD compared to T2, T3, and T4 groups (*p* < 0.05), and *E. tenella* infection linearly (*p* < 0.05), and quadratically (*p* < 0.05) increased cecal CD. As shown in Figure 5, cecal CD were deepened, and gametocytes and developing oocysts were observed in the ceca of broiler chickens infected *E. tenella.*

**Table 4.** Duodenal, jejunal, ileal, and cecal morphology [villus height (VH; µm), crypts depth (CD; µm), and VH:CD] of broiler chickens infected with different dosages of *Eimeria tenella* on 6 days post-infection (dpi) and 9 dpi <sup>1</sup> .


<sup>1</sup> T0: treatment 0 (Sham-challenged with phosphate-buffered saline); T1, treatment 1 (challenged with 6250 sporulated oocysts of *E. tenella*); T2, treatment 2 (challenged with 12,500 sporulated oocysts of *E. tenella*); T3, treatment 3 (challenged with 25,000 sporulated oocysts of *E. tenella*); T4, treatment 4 (challenged with 50,000 sporulated oocysts of *E. tenella*). At each time point, orthogonal polynomial contrasts analysis was conducted to see linear pattern (L) and quadratic pattern (Q) among the treatments. Different letters at the same time point represent significantly different (*p* < 0.05) by proc mixed followed by the Tukey's multiple comparison test among the treatment groups.

> In broiler chickens infected *E. tenella*, crypts were deepened and gametocytes and developing oocysts were observed.

> There were no significant differences in the ileal apparent digestibility of DM, OM, and ash among treatments on 6 dpi and 9 dpi (*p* > 0.1; Table 5).

*Animals* **2021**, *11*, x FOR PEER REVIEW 11 of 17

**Figure 5.** Ceca morphology of a non-challenged bird ((**A**) 2×; (**B**) 10×) and of a *Eimeria tenella* infected bird ((**C**) 2×; (**D**) 10×) in the T1 group (challenged with 6250 sporulated oocysts of *E. tenella*) on 6 days post-infection when tissues were stained with hematoxylin and eosin. Microgametocytes, macrogametocytes, and schizonts were observed in the ceca crypts (**E**,**F**). **Figure 5.** Ceca morphology of a non-challenged bird ((**A**) 2×; (**B**) 10×) and of a *Eimeria tenella* infected bird ((**C**) 2×; (**D**) 10×) in the T1 group (challenged with 6250 sporulated oocysts of *E. tenella*) on 6 days post-infection when tissues were stained with hematoxylin and eosin. Microgametocytes, macrogametocytes, and schizonts were observed in the ceca crypts (**E**,**F**).

Ash 40.16 40 37.66 40.82 42.66 5.47 0.6 0.42 0.26

OM 75.02 72.29 74.32 74.68 75.8 2.69 0.29 0.31 0.18 Ash 33.03 36.24 29.47 33.86 33.82 9.49 0.81 0.95 0.76

In broiler chickens infected *E. tenella*, crypts were deepened and gametocytes and developing oocysts were observed. **Table 5.** Apparent ileal digestibility (%) of dry matter (DM), organic matter (OM), and ash of broiler chickens infected with different dosages of *Eimeria tenella* on 6 days post-infection (dpi) and 9 dpi.


6 dpi DM 69.16 70.31 68.35 71.53 70.19 1.96 0.09 0.21 0.96 OM 70.85 72.08 70.13 73.32 71.79 1.94 0.09 0.23 0.89 <sup>1</sup> T0: treatment 0 (Sham-challenged with phosphate-buffered saline); T1, treatment 1 (challenged with 6250 sporulated oocysts of *E. tenella*); T2, treatment 2 (challenged with 12,500 sporulated oocysts of *E. tenella*); T3, treatment 3 (challenged with 25,000 sporulated oocysts of *E. tenella*); T4, treatment 4 (challenged with 50,000 sporulated oocysts of *E. tenella*). At each time point, orthogonal polynomial contrasts analysis was conducted to see linear pattern (L) and quadratic pattern (Q) among the treatments.

9 dpi

#### *3.5. Liver Total Antioxidant Capacity (TAC)* As shown in Figure 6, liver TAC was not modulated due to *E. tenella* infection in

*3.5. Liver Total Antioxidant Capacity (TAC)* 

(Q) among the treatments.

As shown in Figure 6, liver TAC was not modulated due to *E. tenella* infection in broiler chickens (*p* > 0.1) on 6 dpi. However, different inoculation dosages of *E. tenella* quadratically increased liver TAC on 9 dpi (*p* < 0.05). broiler chickens (*p* > 0.1) on 6 dpi. However, different inoculation dosages of *E. tenella* quadratically increased liver TAC on 9 dpi (*p* < 0.05).

1 T0: treatment 0 (Sham-challenged with phosphate-buffered saline); T1, treatment 1 (challenged with 6250 sporulated oocysts of *E. tenella*); T2, treatment 2 (challenged with 12,500 sporulated oocysts of *E. tenella*); T3, treatment 3 (challenged with 25,000 sporulated oocysts of *E. tenella*); T4, treatment 4 (challenged with 50,000 sporulated oocysts of *E. tenella*). At each time point, orthogonal polynomial contrasts analysis was conducted to see linear pattern (L) and quadratic pattern

*Animals* **2021**, *11*, x FOR PEER REVIEW 12 of 17

**Figure 6.** Total antioxidant capacity in the liver of broiler chickens infected with different dosages of *Eimeria tenella*. On 6 and 9 days post-infection (dpi), total antioxidant capacity in the liver was measured in the T0 (treatment 0; Sham-challenged with phosphate-buffered saline); T1 (treatment 1; challenged with 6250 sporulated oocysts of *E. tenella*); T2 (treatment 2; challenged with 12,500 sporulated oocysts of *E. tenella*); T3 (treatment 3; challenged with 25,000 sporulated oocysts **Figure 6.** Total antioxidant capacity in the liver of broiler chickens infected with different dosages of *Eimeria tenella*. On 6 and 9 days post-infection (dpi), total antioxidant capacity in the liver was measured in the T0 (treatment 0; Sham-challenged with phosphate-buffered saline); T1 (treatment 1; challenged with 6250 sporulated oocysts of *E. tenella*); T2 (treatment 2; challenged with 12,500 sporulated oocysts of *E. tenella*); T3 (treatment 3; challenged with 25,000 sporulated oocysts of *E. tenella*); T4 (treatment 4; challenged with 50,000 sporulated oocysts of *E. tenella*) groups. At each time point, orthogonal polynomial contrasts analysis was conducted to see linear pattern (L) and quadratic pattern (Q) among the treatments.

#### of *E. tenella*); T4 (treatment 4; challenged with 50,000 sporulated oocysts of *E. tenella*) groups. At each time point, orthogonal polynomial contrasts analysis was conducted to see linear pattern (L) and quadratic pattern (Q) among the treatments. *3.6. VFA Concentrations in Cecal Digesta*

*3.6. VFA Concentrations in Cecal Digesta*  As shown in Table 6, infection of *E. tenella* linearly decreased acetate concentration in ceca contents (*p* < 0.05), and the T4 group had significantly lower acetate concentration in ceca contents compared to the T0 group. Isobutyrate concentration in ceca contents was linearly decreased (*p* < 0.05) on 6 dpi as *E. tenella* dosage increased. Total VFA were linearly decreased due to *E. tenella* infection (*p* < 0.05), and the T4 group had significantly lower total VFA compared to the T0 group on 6 dpi. On 9 dpi, *E. tenella* tended to linearly decrease butyrate (*p* = 0.09), and significantly decreased valerate concentrations (Linear; *p* As shown in Table 6, infection of *E. tenella* linearly decreased acetate concentration in ceca contents (*p* < 0.05), and the T4 group had significantly lower acetate concentration in ceca contents compared to the T0 group. Isobutyrate concentration in ceca contents was linearly decreased (*p* < 0.05) on 6 dpi as *E. tenella* dosage increased. Total VFA were linearly decreased due to *E. tenella* infection (*p* < 0.05), and the T4 group had significantly lower total VFA compared to the T0 group on 6 dpi. On 9 dpi, *E. tenella* tended to linearly decrease butyrate (*p* = 0.09), and significantly decreased valerate concentrations (Linear; *p* < 0.05) in ceca contents. The T0 group had significantly lower propionate compared to the T1 group, and T4 birds had significantly lower varelate compared to the T1 group.

< 0.05) in ceca contents. The T0 group had significantly lower propionate compared to the T1 group, and T4 birds had significantly lower varelate compared to the T1 group. **Table 6.** Concentrations of volatile fatty acids (mM) in ceca contents of broiler chickens infected with different dosages of *Eimeria tenella* at 6 days post-infection (dpi) and 9 dpi <sup>1</sup> .



**Table 6.** *Cont.*

<sup>1</sup> T0: treatment 0 (Sham-challenged with phosphate-buffered saline); T1, treatment 1 (challenged with 6250 sporulated oocysts of *E. tenella*); T2, treatment 2 (challenged with 12,500 sporulated oocysts of *E. tenella*); T3, treatment 3 (challenged with 25,000 sporulated oocysts of *E. tenella*); T4, treatment 4 (challenged with 50,000 sporulated oocysts of *E. tenella*). At each time point, orthogonal polynomial contrasts analysis was conducted to see linear pattern (L) and quadratic pattern (Q) among the treatments. Different letters at the same time point represent significantly different (*p* < 0.05) by proc mixed followed by the Tukey's multiple comparison test among the treatment groups.

#### **4. Discussion**

The purpose of the study was to investigate the effects of different inoculation dosages of *E. tenella* on growth performance, gastrointestinal permeability, oocyst shedding, intestinal morphology, fecal consistency, ileal apparent digestibility, antioxidant capacity, and cecal VFA profile in broiler chickens. Inoculation dosages were derived from our previous study [19], and the same strain of *E. tenella* was used. However, in the current study, milder infection (lesion score of T4: 1.6) was achieved compared to our previous study (lesion score of the T4 equivalent group: 2.7). The potential reason for milder infection would be that broilers were challenged with three different *Eimeria* spp. (*E. acervulina*, *E. maxima,* and *E. tenella*) in the previous study, which probably caused more severe infection by compensating the immune system in birds compared to the single species challenging in the current study. Mild-infection models are important to test a new bioactive compound because if the compounds do not show any beneficial effects at the mild infection model, they may not show any beneficial effects against coccidiosis at severe infection model either. In the present study, *E. tenella* infection decreased feed efficiency during the acute phase and increased feed intake on 6 and 7 dpi. These data are partially consistent with previous studies [24,25] which reported that FCR was increased along with reduced BW of broilers. In our current study, however, *E. tenella* infection increased FCR by increasing the feed intake of birds, whereas BW was also numerically (*p* = 0.16) decreased with a linear trend during the acute phase. Impaired feed efficiency in broilers infected with *E. tenella* during the acute phase in the present study would be associated with reduced acetate and total VFA production in the ceca. Acetate is the most abundant VFA, which are produced via bacterial fermentation in the ceca of broilers [26]. It is already well-established that *E. tenella* infection can negatively affect cecal microbiome, which results in depressed VFA production in broilers according to many previous studies [27,28]. Cecal VFA production affects the host's energy balance because VFA are important energy substrates for the host [29]. Gasaway [30] mentioned that VFA supply approximately 11% to 18% of the total energy production in chickens. In addition, *Eimeria* spp. compete for energy and nutrients for their asexual and sexual replications with the host [31]. Reduced available energy in the body can result in reduced BW or increased FCR by raising feed intake of the birds to decrease maintenance energy requirements or to meet the energy requirements, respectively [32]. In addition, reduced production of acetate, which can induce the secretion of satiety-stimulating hormones from the gut, could increase the feed intake of the birds [33]. Therefore, in this study, reduced VFA production in ceca due to *E. tenella* infection potentially reduced available energy levels in the body, and this increased feed intake and FCR to supply more energy to meet the energy requirements in broiler chickens infected with *E. tenella*.

This study showed that *E. tenella* infection deepened crypts depth of the ceca. The potential reasons for increased cecal crypts depth due to *E. tenella* infection are still unclear. It is proposed that *E. tenella* increased cecal CD (mucosa layer) to make their habitats in the ceca, or ceca crypts were deepened to increase VFA absorption because VFA production was restricted due to *E. tenella* infection. Nevertheless, deepened CD could inhibit the production and absorption of VFA in the ceca. Increased CD possibly turned in increased total goblet cells in the ceca and mucus secretion into the cecal contents. This possibly reduced concentration of VFA and modulated VFA production in ceca content by affecting microbiome of the broiler chickens. Ceca only have villi at the entrance of the ceca to filter large particles away and to act as an immunological detector of cecal contents [34,35], and middle and distal parts of the ceca only have smooth mucous membrane without villi [36]. Thereby, another possible reason for increased CD in broilers infected with *E. tenella* would be that birds increased CD to let crypts function like villi as a defensive mechanism in the proximal ceca because *E. tenella* infected ceca are vulnerable for further infections (e.g., bacterial infection).

We also hypothesized that impaired ceca health due to *E. tenella* infection may indirectly affect small intestinal health (the main area for nutrient digestion and absorption) by causing energy deficiency and inducing oxidative stress, and this may account for decreased feed efficiency in the current study. Nevertheless, no differences were observed in DM, OM, and ash apparent ileal digestibility among the treatments on 6 and 9 dpi in the current study. According to our previous study, *E. maxima* infection significantly decreased nutrient digestibility in broiler chickens, which indicates that different *Eimeria* spp. affect nutrient digestibility of broilers differently [5]. Liver total antioxidant capacity was same among the treatments on 6 dpi and even increased in broilers infected with *E. tenella* on 9 dpi, potentially because mild infection of *E. tenella* may allow birds to stimulate their antioxidant defensive system. Still, over-production of antioxidants (enzymatic and non-enzymatic) can result in over-use of energy and nutrients, which also may decrease available energy and nutrients for growth in chickens. However, a previous study by Georgieva et al. [25] reported that severe *E. tenella* infection model decreased antioxidant capacity of broilers. Ileal crypt depth was decreased, and ileal VH:CD was increased without affecting VH in *E. tenella* infected broilers in the current study. Probably, ileal morphology was enhanced with limited energy and nutrients sources as a compensation mechanism because cecal functionality was restricted due to *E. tenella* in the current study. There were negative effects of *E. tenella* on duodenal and jejunal morphology on 9 dpi and gastrointestinal permeability on 7 dpi. Energy and absorbed VFA play an important role in gut development in broiler chickens by stimulating gut epithelial cell proliferation [9]. In this study, reduced cecal VFA production would be the main factor that negatively affected gut health of *E. tenella* infected broilers rather than increased pathogens and toxin production because liver health was maintained in broilers infected with *E. tenella.* Whereas it cannot be concluded that impaired intestinal health caused impaired feed efficiency during the acute phase, energy deficiency due to poor VFA yield subsequently damaged intestinal health of broilers infected with *E. tenella*. Moreover, while differences were observed in gastrointestinal permeability, it was only less than two-fold difference. Our previous study [37] reported that more than 200 folds differences of gastrointestinal permeability were observed due to *E. maxima* infection. Hence, reduced VFA production in the ceca due to *E. tenella* infection caused energy deficiency in the body, which resulted in compensated gut health in broiler chickens.

In this study, different *E. tenella* inoculation dosages linearly increased oocyst shedding on 5 to 6 dpi, which implies that the higher inoculation dosages can results in higher oocyst shedding. Nonetheless, these data could be obtained in our study because our *E. tenella* strain induced mild-infection (lesion score below 2) in the ceca. Williams [38] demonstrated that higher inoculation dosage levels can linearly increase oocyst yields until reaching to the crowded dosages (e.g., maximally producing dosage), and higher dosages than the crowded dosages can decrease oocyst yields in broilers. However, no differences were observed on 6 to 7 dpi and 7 to 8 dpi in the current study. The current result also demonstrated that different challenge dosages of *E. tenella* have different peak point of shedding. The potential reason would be that higher number of *E. tenella* possibly decreased number of generation within the asexual and sexual stages of the life [35], which resulted in an earlier peak date for the highest dosage group in the study. Our study was the first to find that different *E. tenella* inoculation dosages resulted in different peak points for oocyst yields in the mildly infected broilers. Oocyst yields in severely infected (*E. tenella* lesion score 3 to 4) broilers at different time points should be investigated further.

Ileal and fecal moisture contents were modulated due to Eimeria infection in the current study. Ileal (6 dpi) and fecal moisture contents (5 to 6 dpi) were quadratically and linearly decreased, respectively. Afterwards, fecal moisture content was increased on 6 to 8 dpi. *E. tenella* are known to induce diarrhea containing mucus and blood. Although obvious bloody diarrhea was not achieved in the current study, fecal moisture content was increased after 6 dpi. Potentially, *E. tenella* damaged enterocytes in the ceca and modulated microbiota, and this caused electrolyte imbalance in the ceca which may explain the modulated ileal and fecal moisture contents in the current study [39]. Moreover, ceca play a crucial role in water absorption [40], and water absorption ability of the ceca of birds infected with *E. tenella* would be limited potentially due to thickened mucosa layer in the current study. Hypothetically, higher water loss due to *E. tenella* infection increased water intake [40] as birds increased their feed intake to compensate energy deficiency in the current study. In addition, increased fecal moisture content can result in increased litter moisture, and this can increase the incidence of food pad dermatitis in broilers [41].

This current study showed that mild infection of *E. tenella* impaired feed efficiency and gut health mainly through reducing VFA production in broilers. Thereby, supplementation of VFA or bioactive compounds that has high energy values and antimicrobial effects (e.g., medium chain fatty acids) can control mild-infection of *E. tenella* in broilers [11,42].

#### **5. Conclusions**

Orthogonal polynomial contrasts showed that *E. tenella* mild-infection reduced VFA production in the ceca, and this caused energy deficiency, which increased feed intake and impaired feed efficiency of broiler chickens. This suggests that the cecal VFA concentrations could be a key parameter to represent feed efficiency and *E. tenella* infection severity in broiler chickens. Furthermore, mild-infection of *E. tenella* modulated intestinal morphology, antioxidant capacity, and gastrointestinal permeability in the recovery phase. Different inoculation dosages of *E. tenella* changed oocyst shedding patterns and ileal/fecal moisture content. These current data showed that the mechanisms of *E. tenella* impair feed efficiency and gut health of broilers, which will be beneficial to study strategies to cope with *E. tenella* infection in broiler chickens. Based on the current study, 25,000 to 50,000 sporulated oocyst dosage range would be recommended as subclinical models for nutritional strategies.

**Author Contributions:** Conceptualization, J.C. and W.K.K.; methodology, J.C., P.-Y.T., J.M.L., T.R.C. and W.K.K.; validation, P.-Y.T., J.M.L., T.R.C. and W.K.K.; investigation, J.C., H.K. and Y.H.T.; data curation, J.C.; writing—original draft preparation, J.C.; writing—review and editing, H.K., Y.H.T., P.-Y.T., J.M.L. and W.K.K.; visualization, J.C. supervision, T.R.C. and W.K.K.; project administration, W.K.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** This study was approved by the Institutional Animal Care and Use committee of the University of Georgia (A2018 09-006, approved on 16 January 2019).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to further statistical analysis.

**Acknowledgments:** We appreciated Brett Marshall for his help with the gastrointestinal permeability measurement and Taylor Rae Krause for her help with the VFA analysis. The authors appreciate Alberta L. Fuller for providing the *E. tenella* strain for this study.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

