*Article* **Dietary Cinnamon Bark Affects Growth Performance, Carcass Characteristics, and Breast Meat Quality in Broiler Infected with** *Eimeria tenella* **Oocysts**

**Mohammed M. Qaid \* , Saud I. Al-Mufarrej , Mahmoud M. Azzam , Maged A. Al-Garadi , Abdulmohsen H. Alqhtani \* , Abdulaziz A. Al-abdullatif , Elsayed O. Hussein and Gamaleldin M. Suliman**

> Animal Production Department, College of Food and Agriculture Sciences, King Saud University, Riyadh 11451, Saudi Arabia; salmufarrej@ksu.edu.sa (S.I.A.-M.); mazzam@ksu.edu.sa (M.M.A.); malgaradi@ksu.edu.sa (M.A.A.-G.); aalabdullatif@ksu.edu.sa (A.A.A.-a.); shessin@ksu.edu.sa (E.O.H.); gsuliman@ksu.edu.sa (G.M.S.)

**\*** Correspondence: mqaid@ksu.edu.sa (M.M.Q.); ahalqahtani@ksu.edu.sa (A.H.A.); Tel.: +966-509-844-024 (M.M.Q.); Tel: +966-540-000-232 (A.H.A.)

**Simple Summary:** Antimicrobial tolerance problems have culminated in an increased focus on raising broiler chickens without using any antibiotics, and an increasing interest has developed in nonantibiotic feed supplements with potential productivity and health benefits. Previous studies have shown beneficial results linked to the addition of cinnamon to broiler diets under health conditions without induced coccidiosis. In this study, different amounts of dietary cinnamon, a phytogenic of the Lauraceae family, were evaluated for their effects on carcass characteristics and meat quality in broilers infected with oocysts of *Eimeria tenella*. Overall, emeriosis negatively affects bird slaughter weight, carcass yield, and most carcass traits. Compared with the positive control, cinnamon increased slaughter weight, carcass yield, and the percentage weights of heart, proventriculus, gizzard, breast, and pancreas. In addition, cinnamon at 2 g/kg diet improved performance, cocking loss, and meat tenderness among cinnamon groups. The ionophore salinomycin (Sacox®) group had the highest slaughter yield, myofibril fragmentation index (MFI), and texture profile analysis (TPA) of meat. The current research offers equivalent and unbiased findings from a study of substitutes for commercial coccidiostats in a consistent experimental paradigm that applies well to commercial conditions.

**Abstract:** A total of 150 broiler chicks were used to determine the impact of dietary cinnamon bark powder (CBP; *Cinnamomum verum*) on breast meat quality, growth performance, and carcass characteristics of birds under coccidiosis, as one of the protozoan parasitic diseases. A total of 5 replicates of birds received 1 of the following 6 groups for 34 days: control groups (1 and 2) received a basal diet without the addition of CBP or salinomycin; group 3 received a basal diet with 0.066 g salinomycin; groups 4–6 were given a basal diet supplemented with 2, 4, and 6 g CBP/kg feed, respectively. On day 21, 4 <sup>×</sup> <sup>10</sup>4/100 <sup>µ</sup>L of *Eimeria tenella* oocysts/bird were challenged, except for the negative control (NC). At the end of the experimental trial, five birds/group were sampled for carcass characteristics and breast attributes. Overall, emeriosis negatively affects slaughter body mass, carcass yield, and the majority of carcass characteristics in birds, and cinnamon can mitigate these effects. Cinnamon groups, particularly at the 2 g level, alleviated the negative effect on performance caused by coccidia infestation to the same or greater extent as the negative control and salinomycin treatment groups. Furthermore, when compared with the other experimental groups, the addition of cinnamon improved some physicochemical properties with some affecting meat quality, such as decreasing MFI and increasing toughness in cinnamon-treated groups. In summary, it can be concluded that CBP can enhance the shelf life, carcass, and quality of birds' meat by maximizing the productive performance efficiency and breast meat productivity of birds under coccidiosis infestation. Further research is required to investigate the use of cinnamon to optimize the quality of meat and the productivity of both healthy and diseased broilers.

**Citation:** Qaid, M.M.; Al-Mufarrej, S.I.; Azzam, M.M.; Al-Garadi, M.A.; Alqhtani, A.H.; Al-abdullatif, A.A.; Hussein, E.O.; Suliman, G.M. Dietary Cinnamon Bark Affects Growth Performance, Carcass Characteristics, and Breast Meat Quality in Broiler Infected with *Eimeria tenella* Oocysts. *Animals* **2022**, *12*, 166. https:// doi.org/10.3390/ani12020166

Academic Editors: Natalie Morgan and Youssef A. Attia

Received: 27 October 2021 Accepted: 5 January 2022 Published: 11 January 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/).

**Keywords:** breast; broiler; carcass traits; cinnamon bark; meat quality

#### **1. Introduction**

Total imports of Saudi Arabian chicken meat were reduced from 618,000 to 520,000 tons in 2020 and 2021 as local production of Saudi Arabian chicken meat increased. Globally, increases in broiler meat production were also expected, with ~100.827 million tons produced in 2020 and projections for 2022 of ~100.9 million tons [1]. These increases in production are related to the high demand for poultry meat, which exceeds that of other animals, reflecting its rich nutritional value and favorable profit margin. As a result of the sharp increase in the production and popularity of broiler meat, the demand for improving the palatability and quality of meat has increased [2]. Important criteria for assessing the slaughter value of poultry carcasses include the percentage of dressing, the percentage of culinary, the tenderness of the meat, and the muscle content of the carcass. According to [3], broilers have a greater dressing percentage, reaching ~75%. This indicator is considered to be essential for evaluating the quantity of produce. Likewise, the composition of the carcass is essential because a high muscle content, particularly of the breast muscle, and a low fat content stimulate the consumer's appetite. While the quality of the meat comprises a sequence of attributes, the consumer is most concerned about the tenderness, color, juiciness, and palatability of the meat [4–6]. Meat color is a simple and quick criterion for evaluation and seems to be an important indicator to consumers. Fresh chicken meat should be light red in color. Thus, at first glance by a consumer, any change in color indicates declining nutritional value and deterioration in quality. The capacity of bird meat to retain its natural and added moisture during processing, storage, and when subjected to an external force is termed its water-holding capacity (WHC). Measuring WHC is one of the easiest ways to detect the quality of meat, and it has a direct impact on yield and appearance [7].

The larger body and breast weights of birds are related to larger areas and diameters of muscle fibers (myofiber), and older birds have lower myofiber density than younger birds [8]. Consumers expect tenderness; therefore, measuring tenderness is an appropriate way of measuring consumer satisfaction in eating meat products, and it is subjectively judged to be a sense of the hardness or elasticity of tough or tender meat [9]. Texture profile analysis (TPA) is a constructive technique that mimics the bite action of the mouth through a popular double compression test to determine the textural properties of foods, and it does not require a large number of investigators to assess the texture of poultry meat [10].

Humans that consume poultry meat could be directly affected by antibiotic growth promoters (AGPs) residues in the meat, or indirectly affected by the development of antibiotic-resistant pathogens in the meat [11]. Some researchers have investigated the growth and meat quality of birds fed diets containing natural herbs or extracts of natural herbs [12–16]. Coccidiosis is a parasitic disease caused by a protozoan that causes enteritis, hemorrhagic cecal lesions, and bloody diarrhea, with significant economic losses worldwide to the poultry industry [17].

Cinnamon spice is obtained from the inner bark of *Cinnamomum verum*, which is a vigorous, evergreen, annual, and aromatic ethnomedicinal plant belonging to the Lauraceae family. Various herb extracts, including cinnamon plant oils and their bioactive constituents, such as cinnamaldehyde and eugenol, are used as dietary supplements in poultry production. These extracts have a variety of uses, including antibacterial activity against many pathogens and acceleration of the growth of good bacteria, such as lactic acid bacteria and bifidobacterial, in the poultry intestine [18,19]. Furthermore, cinnamon oil has potent hypercholesterolemia, anticandidal, antioxidant, analgesic, and antiulcer activities [20], and the health-promoting and performance-enhancing effects of dietary aromatic herbs and extracts have been shown in both healthy and diseased farm animals [21]. Dietary cinnamon supplementation improves the quality and shelf life of broiler meat and maximizes meat productivity by lowering abdominal fat, increasing the dressing

percentage and redness, and reducing drip loss and antioxidant activity of the breast meat of stressed broilers [22]. The leaves and bark of the cinnamon herb and its metabolites are receiving more attention as phytogenic feed additive substitutes for AGPs because they are natural antibiotics—readily available, non-toxic, and residue-free [20]—as well as enhancing poultry growth and improving carcass characteristics and the quality of broiler meat, and enhancing immunity and microbiological factors. However, limited studies are available on the use of cinnamon powder as a phytogenic alternative antibiotic and potential nutrition enhancer in the diets of broilers facing coccidiosis challenges.

This study hypothesized that the cinnamon herb could be used to enhance growth performance, carcass traits, meat quality, and physico-chemical properties of the breast muscle of broiler chickens experiencing coccidiosis infestation. Various levels of cinnamon bark plant were evaluated to validate this hypothesis. Therefore, this study aimed to test the ability of the cinnamon powder to be used as a dietary AGP substitute for enhancing the breast quality, carcass characteristics, and growth performance of Ross broiler chickens challenged by *Eimeria tenella*.

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

#### *2.1. Ethical Approval*

The research was carried out in accordance with the Kingdom of Saudi Arabia's ethical standards for animal use (Ethic committee King Saud University, Ethical approval number: KSU-SE-20-44).

#### *2.2. Infection with Eimeria tenella*

Our previous study [23] described the source of *Eimeria tenella* (*E. tenella*) oocysts, the sporulation of unsporulated oocysts, the identification, passage, and propagation of sporulated oocysts, and the inoculation procedure. All birds, with the exception of NC, were administered 1 mL of double distilled water containing 4 <sup>×</sup> <sup>10</sup>4/100 <sup>µ</sup>L/bird of live sporulated oocysts of *E. tenella* at 21 days of age, according to previous research [24–26]. The authors choose the 7 dpi for anticoccidial indices (data published in [23]). Thus, this study is an extension of the same broiler batch experiment in which the anticoccidial indicators of CBP evaluation, namely the number of fecal oocysts, survival rate, bloody diarrhea, and lesion scores, were included. As a result, this investigation does not address the assessment of anticoccidial indicators. We found that CBP was effective on *E. tenella.* Furthermore, salinomycin, commercially called (Sacox®; Huvepharma NV, Belgium), is a standard product that protects birds from coccidiosis. For growth performance sampling, we chose 7 and 14 dpi. Following [27], meat quality and carcass variables were measured in all treatments on the last day of the trial, at 34 days of age (14 dpi here), in order to mimic the carcass traits of commercial poultry at marketing weights and to identify meat quality.

#### *2.3. Birds and Husbandry*

The study was carried out in a controlled environment—a heated battery room at the Animal Production Department's experimental poultry research unit at King Saud University, Riyadh, Saudi Arabia. The trial operated during spring month "March–April 2019" with average temperature varying between 20.4 ◦C and 33.4 ◦C and the average relative humidity in Riyadh in April was 28% during the experimental period. A total of 150, 1-day-old, mixed-sex, commercial Ross 308 broiler birds were collected from the national commercial hatchery (Al Wadi Poultry Company "Al Khomasia," Riyadh, Saudi Arabia). At the hatchery, the birds were immunized against Newcastle and infectious bronchitis diseases. The chicks were randomly divided into 30 experimental cages with 5 replicates of 6 treatments with 5 chicks per replicate. At 1 day of age, the temperature was set at 35 ◦C and gradually decreased by 1 ◦C every 2 days until a permanent temperature of 22 ◦C was reached. Then, it was maintained until the end of the trial. Relative humidity ranged from 65–85%. Feed and water were supplied *ad libitum*, and birds were kept on a "23 h on and 1 h off" light schedule.

#### *2.4. Experimental Dietary Treatments*

Poultry diets were obtained from the Arabian Agricultural Services Company. The ingredients and chemical properties of the commercial starter (1–21 days) and finisher (22–34 days) broiler chicks' diets were analyzed, formulated, and mixed in a mashed form, based on Ross 3080 s recommendation guidelines (Supplementary Table S1). On arrival, the birds were randomly allocated to one of the following 6 treatments:

T1—negative control group given an unmedicated diet, these were unchallenged coccidiosis chicks (NC); T2—positive control group given an unmedicated diet + coccidial challenge (PC); T3—medicated diet with salinomycin sodium (66 mg salinomycin/kg diet) + coccidial challenge; T4–T6—2, 4, and 6 g CBP/kg diet, respectively, + coccidial challenge. The supplemented levels of purchased CBP or salinomycin powder were mixed with the basal broiler diet.

#### *2.5. Preparation and Compositions: Cinnamon Bark Powder*

*Cinnamomum verum* bark was acquired from a nearby store in Riyadh, Saudi Arabia, for use in this study. The dried bark used was ground to a fine powder. Moreover, as described by [23], high-performance liquid chromatography (HPLC) and gas chromatography–mass spectrometry (GC–MS) were used to detect the biologically relevant compounds in the CBP extract mixture. A total of 26 different active compounds with the highest quality were detected by GC–MS in the CNB extract, particularly Cinnamaldehyde, 3-phenyl-, hexadecanoic acid, (E)-2-propenal, methyl ester, 14-methyl-,methyl ester, pentadecanoic acid, oxime-, methoxy-phenyl-, and 2-methyl-benzofuran, as previously accounted for in [28].

#### *2.6. Performance Measurements and Production Efficiency*

Body weight and feed intake (FI) of birds were recorded at 1, 7, and 14 dpi per replicate to evaluate performance. Then, for each pen, the body weight gain (BWG) and FI were recorded. The average feed conversion efficiency (FCR) was calculated by dividing FI (g) by BWG (g). The European production efficiency factor (PEF) was calculated as follows:

$$\text{PEF} = \text{(Live weight (kg)} \times \text{Livability)} / \text{(Age in days} \times \text{FCR)} \times 100\tag{1}$$

#### *2.7. Carcass Relative Weights*

A total of 30 34-day-old birds (Ross 308) of each treatment (*n* = 5, 1 male bird per cage, per treatment) were randomly selected for slaughter and subjected to feed withdrawal for 10 h. Before slaughtering, the birds were weighed. After bleeding, birds were defeathered and eviscerated (about two minutes after jugular vein incision). The feathers of the birds were plucked, and the viscera was eviscerated. The carcasses were dissected after the head, feathers, and shanks were removed. Live weight at slaughter (SW, kg) and carcass weight (CW, kg) were registered to calculate the yield of (CY%) (dressing percentage) = (CW/SW) × 100). Then, the CW were dissected into commercial parts, including the offal (heart, liver, and gizzard), neck, abdominal fat, heart, liver, proventriculus, gizzard, lymphoid organs, breast, leg, and pancreas, and weighed. Then, the relative weights of these weights were expressed in relation to the SW.

#### *2.8. Meat Quality Indices*

In addition to carcass parameters, the left and right parts of the breast meat (pectoralis major) of each selected bird (one breast per cage/treatment) were used for qualitative analyses. For the pH and color measurements, breast samples were kept at 4 ◦C; meanwhile, for the other quality measurements, they were frozen at −20 ◦C until further measurements were conducted for other meat quality assessments, and then thawed overnight in the fridge at 4 ◦C before analysis. The initial and ultimate pH and color components of dissected breast muscle were determined at 1 and 24 h.

2.8.1. Breast Meat Physicochemical Characteristics (pH, Temperature, and Color Indicators)

The breast pH was measured after processing with a microprocessor Hanna Instruments pH meter, and incisions were made in the cranial left side of the pectoral muscle. At 1 and 24 h, an average of 3 pH measurements for each sample had been taken.

A thermocouple thermometer probe from Eutech Instruments was placed deep in the center of the muscle 1 h postmortem to monitor internal core temperature values of the pectoral muscle.

Breast flesh color measurements, developed by Dr. Richard Hunter as the Hunter values—lightness (L\*), redness (a\*), and yellowness (b\*)—were set on CIELAB scales and assessed with a Chroma meter, 1 and 24 h after the slaughter on 2 different areas of the inner side of the cranial position of the breast muscles. Values for L\*, a\*, and b\* were converted to estimate the total color change (∆E), Chroma meter (saturation index), hue angle, browning index (BI), and whiteness index (WI), as described by [29–31]. According to [32], these measurements obtain a much more accurate assessment of how consumers perceive the color of meat. The averages of the two readings of the color components were taken.

#### 2.8.2. Water-Holding Capacity

The breast meat water-holding capacity (WHC) of the frozen meat samples was measured immediately after thawing overnight at 4 ◦C using the compression method outlined by [33].

#### 2.8.3. Cooking Loss

The cooking loss (CL) was measured as follows [33]: CL was determined by weighing muscle samples with a semi-analytical balance, placing them in a commercial indoor tabletop grill, cooking them until they reached an internal temperature of 75 ◦C, and reweighing them after cooking. The CL percentage = [(Initial weight − Cooked weight)/Initial weight] × 100.

#### 2.8.4. Drip Loss

The parts of the breasts used for the drop test were weighed separately, packed, and kept at 4 ± 1 ◦C for 24 h. Then, the difference in weights before (Wi) and after (Wu) storage was calculated and expressed as a drop loss proportion [34]. Drip loss (DL) (%) = [(Wi − Wu)/Wi] × 100.

#### 2.8.5. Myofibril Fragmentation Index

The procedures mentioned by [33] were used to evaluate myofibril fragmentation (MFI) as an indirect measure of calpain intracellular proteases. An amount of 4 g of muscle, minced with scissors, was homogenized in a blender for 30 s with 40 mL of cold MFI buffer at 2 ◦C. After many washes, the suspension aliquots were diluted in MFI buffer to a final concentration of 0.5 mg/mL and poured into a cuvette for immediate measurement of absorbance at 540 nm using a spectrophotometer. Each sample's MFI was calculated to be A540 nm × 200.

#### 2.8.6. Meat Texture Analysis

The shear force (SF) and TPA of the samples were determined using a texture analyzer (TA.HD. Stable Micro Systems, Surrey, UK) in 2 parts per replicate (1 breast/replicate/treatment). After the cooked samples had cooled at 22 ◦C, 5 round core meat slices (1.27 and 2.5 cm diameter for SF and TPA, respectively) were cut from each sample, parallel to the longitudinal direction of the muscle fibers, using a handheld coring tool. During the SF test, the maximum force (kgf) was applied vertically to the fibers using a TA.HD. Texture Analyzer, designed for a Warner-Bratzler shear blade, with a triple-slotted cutting edge. The speed of the crosshead was set at 200 mm/min. The SF values were estimated from the maximum point of the generated curve. A cylindrical piston (75 mm diameter) was used to compress the TPA sample to within 80% of its original height over two test cycles in 5 s. The texture

analyzers conditions were used to generate force–time curves of deformation. The hardness, springiness, chewiness, and cohesion parameters were measured following [35]. curves of deformation. The hardness, springiness, chewiness, and cohesion parameters were measured following [35]. estimated from the maximum point of the generated curve. A cylindrical piston (75 mm diameter) was used to compress the TPA sample to within 80% of its original height over two test cycles in 5 s. The texture analyzers conditions were used to generate force–time

The shear force (SF) and TPA of the samples were determined using a texture analyzer (TA.HD. Stable Micro Systems, Surrey, UK) in 2 parts per replicate (1 breast/replicate/treatment). After the cooked samples had cooled at 22 °C, 5 round core meat slices (1.27 and 2.5 cm diameter for SF and TPA, respectively) were cut from each sample, parallel to the longitudinal direction of the muscle fibers, using a handheld coring tool. During the SF test, the maximum force (kgf) was applied vertically to the fibers using a TA.HD. Texture Analyzer, designed for a Warner-Bratzler shear blade, with a triple-slotted cutting edge. The speed of the crosshead was set at 200 mm/min. The SF values were estimated from the maximum point of the generated curve. A cylindrical piston (75 mm diameter) was used to compress the TPA sample to within 80% of its original height over two test cycles in 5 s. The texture analyzers conditions were used to generate force–time

The shear force (SF) and TPA of the samples were determined using a texture analyzer (TA.HD. Stable Micro Systems, Surrey, UK) in 2 parts per replicate (1 breast/replicate/treatment). After the cooked samples had cooled at 22 °C, 5 round core meat slices (1.27 and 2.5 cm diameter for SF and TPA, respectively) were cut from each sample, parallel to the longitudinal direction of the muscle fibers, using a handheld coring tool. During the SF test, the maximum force (kgf) was applied vertically to the fibers using a TA.HD. Texture Analyzer, designed for a Warner-Bratzler shear blade, with a triple-slotted cutting edge. The speed of the crosshead was set at 200 mm/min. The SF values were

*Animals* **2022**, *12*, x 6 of 18

*Animals* **2022**, *12*, x 6 of 18

#### *2.9. Statistical Analysis 2.9. Statistical Analysis*  curves of deformation. The hardness, springiness, chewiness, and cohesion parameters

2.8.6. Meat Texture Analysis

2.8.6. Meat Texture Analysis

1 bird from each replicate).

In the statistical analysis system [36], a general linear model (GLM) was used to analyze slaughter characteristics and meat quality data. Six groups were arranged in five replicates in a completely randomized design. Each replicate cage represented an experimental unit. On the 34th day of age, male birds were sampled (*n* = 5 birds per treatment, 1 bird from each replicate). In the statistical analysis system [36], a general linear model (GLM) was used to analyze slaughter characteristics and meat quality data. Six groups were arranged in five replicates in a completely randomized design. Each replicate cage represented an experimental unit. On the 34th day of age, male birds were sampled (*n* = 5 birds per treatment, 1 bird from each replicate). were measured following [35]. *2.9. Statistical Analysis*  In the statistical analysis system [36], a general linear model (GLM) was used to analyze slaughter characteristics and meat quality data. Six groups were arranged in five rep-

All data were analyzed using one-way ANOVA and expressed as a statistical mean ± standard error of the mean (SE) using the following models: All data were analyzed using one-way ANOVA and expressed as a statistical mean ± standard error of the mean (SE) using the following models: licates in a completely randomized design. Each replicate cage represented an experimental unit. On the 34th day of age, male birds were sampled (*n* = 5 birds per treatment,

$$\mathbf{Y}\mathbf{i} = \mu + \mathbf{T}\mathbf{i} + \mathbf{G}\mathbf{i}$$

where Yij is the observed j parameters in the ith treatment, µ is the overall mean of the measurements, Ti is the effect of the ith treatment, and Ԑij is the random residual error. To assess significant differences between means for measurements using Duncan's multiple range test, a statistical significance level of *p* < 0.05 was used. where Yij is the observed j parameters in the ith treatment, µ is the overall mean of the measurements, Ti is the effect of the ith treatment, and ± standard error of the mean (SE) using the following models: Yij = µ + Ti + Ԑij where Yij is the observed j parameters in the ith treatment, µ is the overall mean of the measurements, Ti is the effect of the ith treatment, and Ԑij is the random residual error. To ij is the random residual error. To assess significant differences between means for measurements using Duncan's multiple range test, a statistical significance level of *p* < 0.05 was used.

#### **3. Results**  assess significant differences between means for measurements using Duncan's multiple **3. Results**

According to our previous research [23], clinical coccidiosis symptoms were observed in birds after infection with oocysts of *E. tenella*. It was found that 6 g of CBP had moderate anti-coccidial activity and could be used to treat poultry emeriosis in the field. Consequently, CBP decreased the severity of lesions and reduced oocyst excretion per gram in chickens' droppings. Moreover, based on the HPLC and GC–MS results, we found that cinnamaldehyde and other important bioactive compounds are present in the cinnamon bark extract. range test, a statistical significance level of *p* < 0.05 was used. **3. Results**  According to our previous research [23], clinical coccidiosis symptoms were observed in birds after infection with oocysts of *E. tenella*. It was found that 6 g of CBP had moderate anti-coccidial activity and could be used to treat poultry emeriosis in the field. Consequently, CBP decreased the severity of lesions and reduced oocyst excretion per According to our previous research [23], clinical coccidiosis symptoms were observed in birds after infection with oocysts of *E. tenella*. It was found that 6 g of CBP had moderate anti-coccidial activity and could be used to treat poultry emeriosis in the field. Consequently, CBP decreased the severity of lesions and reduced oocyst excretion per gram in chickens' droppings. Moreover, based on the HPLC and GC–MS results, we found that cinnamaldehyde and other important bioactive compounds are present in the cinnamon bark extract.

gram in chickens' droppings. Moreover, based on the HPLC and GC–MS results, we

#### *3.1. Performance Measurements and Production Efficiency*  found that cinnamaldehyde and other important bioactive compounds are present in the *3.1. Performance Measurements and Production Efficiency*

Table 1 shows that the challenge of coccidiosis had adverse effects on BWG, FCR, and PEF of the birds at 1st and 2nd week post-infection and suffered from it over the entire period compared with an unchallenged control group. During the second week after infection, the BWG, FCR, and PEF of the birds were dose-dependent, increasing as the cinnamon level decreased. Birds receiving a 2g CBP/kg diet had higher BWG, FCR, and PEF over the entire post-infection period (0–14 dpi) than those which received 4g CBP, 6g CBP, and PC (*p* < 0.05), but was similar to those receiving 66 mg salinomycin and NC. This means that birds given 2g CBP/kg gained more and converted feed more efficiently. The statistical models of FI and FCR did not differ significantly (*p* > 0.05) during the first or second weeks or during the entire period after the *Eimeria tenella* oocyst challenge. cinnamon bark extract. *3.1. Performance Measurements and Production Efficiency*  Table 1 shows that the challenge of coccidiosis had adverse effects on BWG, FCR, and PEF of the birds at 1st and 2nd week post-infection and suffered from it over the entire period compared with an unchallenged control group. During the second week after infection, the BWG, FCR, and PEF of the birds were dose-dependent, increasing as the cinnamon level decreased. Birds receiving a 2g CBP/kg diet had higher BWG, FCR, and PEF over the entire post-infection period (0–14 dpi) than those which received 4g CBP, 6g CBP, and PC (*p* < 0.05), but was similar to those receiving 66 mg salinomycin and NC. This Table 1 shows that the challenge of coccidiosis had adverse effects on BWG, FCR, and PEF of the birds at 1st and 2nd week post-infection and suffered from it over the entire period compared with an unchallenged control group. During the second week after infection, the BWG, FCR, and PEF of the birds were dose-dependent, increasing as the cinnamon level decreased. Birds receiving a 2g CBP/kg diet had higher BWG, FCR, and PEF over the entire post-infection period (0–14 dpi) than those which received 4g CBP, 6g CBP, and PC (*p* < 0.05), but was similar to those receiving 66 mg salinomycin and NC. This means that birds given 2g CBP/kg gained more and converted feed more efficiently. The statistical models of FI and FCR did not differ significantly (*p* > 0.05) during the first or second weeks or during the entire period after the *Eimeria tenella* oocyst challenge.

#### means that birds given 2g CBP/kg gained more and converted feed more efficiently. The *3.2. Carcass Characteristic Variables*

statistical models of FI and FCR did not differ significantly (*p* > 0.05) during the first or second weeks or during the entire period after the *Eimeria tenella* oocyst challenge. The effects of the CBP on carcass variables at 34 d of age (14 days post-inoculation) are shown in Table 2. Except for liver, leg, fat, and lymphoid organs (bursa, thymus, and spleen) values, there were significant differences in slaughter variables between treatments. The live weight, CW, and the carcass yield of slaughtered birds were statistically different (*p* < 0.05); this result indicates that the adverse effects of *Eimeria tenella* infection in birds were clearly observed in the PC group and were compensated for in the salinomycin and cinnamon groups. Thus, the heart, proventriculus, gizzard, breast, and pancreas % CW values did differ (*p* < 0.05) between treatments. Birds receiving cinnamon at levels of 4 g with their basal diet had higher percentage heart, proventriculus, gizzard, and pancreas weights, respectively, compared with those in other dietary groups. The lowest abdominal fat yield exhibited an insignificant decrease (*p* < 0.05) with increased CBP compared with the NC group.


**Table 1.** Live body weight (BW), average live body gain (BWG), average feed intake (FI), feed conversion ratio (FCR), and production efficiency factor (PEF) of broiler chickens given experimental diets (cinnamon), post coccidial challenge period (0–14 dpi).

<sup>1</sup> Treatments: NC—negative control, unsupplemented, unchallenged; PC—positive control, unsupplemented, challenged; Salinomycin—basal diet supplemented with coccidiostat salinomycin, challenged; Cinnamon—groups whose basal diet was supplemented with 2, 4, and 6 g cinnamon powder/kg diet, respectively, challenged. a–d Different letters indicate statistically significant differences (*<sup>p</sup>* < 0.05). <sup>2</sup> Values are presented in means <sup>±</sup> standard error (SE) (*n* = 5).

**Table 2.** Carcass variables of broiler chickens supplemented with cinnamon powder, 14 days post-infection.


<sup>1</sup> Treatments: NC—negative control, unsupplemented, unchallenged; PC—positive control, unsupplemented, challenged; Salinomycin—basal diet supplemented with coccidiostat salinomycin, challenged; Cinnamon—groups whose basal diet supplemented with 2, 4, and 6 g of cinnamon powder/kg of diet, respectively, challenged. <sup>2</sup> The data is presented as means with standard errors (*n* = 5). a–c Means in the rows with different superscripts differ significantly (*p* < 0.05), <sup>3</sup> Dressing percentage or carcass yield (CY%) = (carcass weight/Live weight) \* 100.

#### *3.3. Breast Meat Physicochemical Characteristics*

Tables 3 and 4 display the influence of CBP on the physicochemical characteristics of broiler breast samples at 1 and 24 h postmortem, respectively. The core temperature, initial and ultimate lightness, total color change, and WI of samples were different (*p* < 0.05). Otherwise, the initial and ultimate values for pH, redness, yellowness, total color change (Delta E: ∆E), hue angle, BI, and saturation index (Chroma) did not differ among experimental groups (*p* > 0.05). The core temperature values of breast muscle were significantly different (*p* < 0.05), with the broilers from the 2 g of CBP/kg treatment having the highest (26.55 ◦C) temperature values and the broilers fed the control diet and exposed to challenge with coccidia (PC) having the lowest values (24.89 ◦C). Although initial and ultimate pH values did not differ significantly (*p* > 0.05) between cinnamon-treated groups, pH decreased 24 h postmortem with increasing CBP content. Birds receiving 4 g of CBP and NC had higher initial color lightness than those receiving 66 mg of salinomycin and NC (*p* = 0.038) but were similar to those receiving 2 g of CBP, 6 g of CBP, and in the PC group. Birds that

received 66 mg of salinomycin had lower final color lightness than those in the NC group and 6 g of CBP (*p* < 0.01) but were similar to those receiving 2 g of CBP, 4 g of CBP, and in the PC group.

**Table 3.** Core temperature, pH, and color of the pectoralis major at 34 d of age in broilers fed diets containing varying amounts of cinnamon bark powder (CBP) were measured 1 h postmortem.


<sup>1</sup> Treatments: NC—negative control, unsupplemented, unchallenged; PC—positive control, unsupplemented, challenged; Salinomycin—basal diet supplemented with coccidiostat salinomycin, challenged; Cinnamon—groups whose basal diet supplemented with 2, 4, and 6 g cinnamon powder/kg diet, respectively, challenged. <sup>2</sup> Each mean is based on measurements from 5 birds per treatment. a–d Means in the same rows with different superscripts differ significantly at *p* < 0.05; L1, a1, and b1—initial lightness, redness, and yellowness, respectively; ∆E—total color change.

**Table 4.** pH, and color of the pectoralis major at 34 d of age in broilers fed diets containing varying amounts of cinnamon bark powder (CBP) were measured 24 h postmortem.


Ultimate pH and color values of the breast muscle were measured. <sup>1</sup> Treatments: NC—negative control, un challenged, not supplemented; PC—positive control, challenged, not supplemented; Salinomycin—challenged, basic diet supplemented with salinomycin; Cinnamon—groups whose basal diet supplemented with 2, 4, and 6 g CBP/kg diet, respectively, challenged. <sup>2</sup> Each mean is calculated using data from 5 birds per group with standard errors. abc Means differ significantly in the same rows with different superscripts at *p* < 0.05; abbreviations: L2, a2, and b2, final lightness; redness; and yellowness, respectively; ∆E—total color change.

The birds that received 66 mg of salinomycin had lower final color lightness than those in the NC group and fed 6 g of CBP (*p* < 0.01) but were similar to those receiving 2 g of CBP, 4 g of CBP, and in the PC group. Generally, the pH and color measurements of the samples were similar across all treatment groups tested, with the exception of the lightness and WI.

The higher initial lightness results in the control challenged group (PC) resulted in a lower color change and a higher WI compared with the control unchallenged group (NC), and vice versa at the ultimate lightness estimate. These findings may be attributed to the PC group having a higher pH decline than the NC group, indicating that the emeriosis challenge might alter the appearance of the lightness of breast samples. The birds that received cinnamon had lower final yellowness than those in the controls and salinomycin group (*p* < 0.021).

#### *3.4. Water-Holding Capacity, Dripping Loss, Cooking Loss, Myofibril Fragmentation Index, and Shear Force*

The broiler breast quality, WHC, DL, CL, MFI, and SF, of the samples at 34 d of age (14 days post-inoculation) are presented in Table 5. The CL, MFI, and SF values of the meat samples differed (*p* < 0.05) between groups, while the WHC and DL values did not differ (*p* > 0.05). The most favorable numerical values (*p* > 0.05) for WHC were obtained for the broilers fed 2 g of CBP/kg. Broilers fed 4 g of CBP/kg had the highest and worst CL value (36.64%; *p* < 0.05). In contrast, those fed 2 g of CBP/kg, the NC group, and the PC group had the lowest and the most convenient CL value (26.64%, 23.36%, and 22.46%, respectively) but were similar to those receiving 66 mg of salinomycin/kg and 6 g of CBP/kg (35.75% and 32.97%, respectively). The CL results demonstrate that breasts from chickens that received the anticoccidial (salinomycin) and 4 and 6 g of CBP shrank more when cooked than those given 2 g of CBP/kg and the control treatments (*p* < 0.001).

**Table 5.** Water-holding capacity (WHC), dripping loss (DL), cooking loss (CL), myofibril fragmentation index (MFI), and shear force (SF) in broilers fed diets containing varying amounts of cinnamon bark powder (CBP).


<sup>1</sup> Treatments: NC—negative control, unsupplemented, unchallenged; PC—positive control, unsupplemented, challenged; Salinomycin—basal diet supplemented with coccidiostat salinomycin, challenged); Cinnamon groups whose basal diet supplemented with 2, 4, and 6 g of cinnamon powder/kg of diet, respectively, challenged. <sup>2</sup> The data is presented as means of 5 birds per treatment with standard errors of each mean (*n* = 5). abc Means in the same rows with different superscripts differ significantly at *p* < 0.05.

The broilers fed CBP had the lowest MFI value (*p* < 0.05), with 2 g of CBP/kg being the most convenient compared with the PC, NC, and salinomycin groups. The CBP treatments had higher SF values (*p* < 0.05) than the salinomycin and control groups, indicating tough meat. However, the 2 g of CBP/kg group had the lowest SF (1.69 kgf) among the cinnamon groups, indicating that it was the CBP group with the most tenderness.

#### *3.5. Texture Analysis*

TPA quality of breast samples from broilers fed with CBP inoculation and exposed to *E. tenella* challenge is revealed in Table 6. There were significant differences between the experimental groups in the values for hardness, springiness, and chewiness (*p* < 0.05). However, there was a tendency for the cohesiveness value to differ (*p* = 0.04) among treatments. TPA values were found to be higher (*p* < 0.05) in the breast meat of the broiler chickens receiving 66 mg salinomycin/kg. Higher hardness values (*p* < 0.05) of 2 g CBP/kg compared with 4 g and 6 g of CBP/kg groups indicated that hardness improved in broilers fed diets containing 2 g CBP/kg. Springiness and chewiness did not differ significantly among groups with different levels of CBP (*p* > 0.05). Springiness values were lower in CBP groups compared with controls and salinomycin groups (*p* < 0.0001).


**Table 6.** Texture profile in broilers fed diets containing varying amounts of cinnamon bark powder (CBP).

<sup>1</sup> Treatments: NC—negative control, unsupplemented, unchallenged; PC—positive control, unsupplemented, challenged; Salinomycin—basal diet supplemented with *coccidiostat* salinomycin, challenged); Cinnamon—groups whose basal diet supplemented with 2, 4, and 6 g of cinnamon powder/kg of diet, respectively, challenged). <sup>2</sup> Each mean is based on measurements of 5 birds per treatment with standard errors of each mean (mean <sup>±</sup> SE) (*n* = 5) at the end of the trial. <sup>a</sup> and <sup>b</sup> Means in the same row with different superscripts differ significantly at *p* < 0.05.

In this trial, texture profiles differed significantly between treatments, with the CBP groups having lower texture scores compared with the salinomycin treatment, but not from the control groups. However, the CBP groups had lower springiness than the control groups. The NC and 6 g CBP/kg treatments exhibited the lowest values for hardness (0.51) and for chewiness (2.06), while the salinomycin treatment demonstrated the highest values for hardness (0.94) and for chewiness (3.07).

#### **4. Discussion**

This study assessed the success of different levels of cinnamon as a natural herb compared with the standard synthetic anticoccidial product (salinomycin) in broilers exposed to experimentally induced challenge of coccidiosis. Few or no studies have reported the effect on the performance, carcass traits, and breast quality of broilers supplemented with CBP under coccidial challenge. The null hypothesis states that the effects of CBP on growth performance, carcass traits, and meat quality of broiler breasts are the same as the effects of the control groups (non-CBP), which is based on the *p*-value is calculated using a probability level of α = 0.05. The alternative hypothesis is that CBP inhibits the weight loss in the breast and carcass caused by *E. tenella*, which is consistent with the results of [23] who found that CBP extract inhibits the weight loss caused by *Eimeria* infection.

Several studies have evaluated cinnamon powder supplementation at different levels (g/kg) in feed: 2 [37], 10 and 20 [38], 10, 30, and 50 [39], and 30, 50, and 70 [40]. The above studies have had significant effects of the levels of cinnamon powder evaluated in broiler chickens, such as BW, FI, and FCR. The authors in [41] reported that cinnamon at different levels (2.5, 5.0, or 7.5 g) did not show any positive impact on the performance or performance index, as opposed to the results of the current study.

As expected, the results of this study showed that BW, weight gain, and feed efficiency were most adversely affected by the positive control (coccidiosis-exposed group, not treated with any natural or synthetic drug). The effect of salinomycin sodium was similar to that of the cinnamon groups as a natural herb in the 1st week after the challenge. Luckily, cinnamon outcomes, particularly cinnamon at level 2 g per kilogram of diet, have been the best during the 2nd week and entire period after challenge since BWG, FCR, and PEF were improved compared with infected groups. The exploration of alternatives to synthetic antibacterial and anti-coccidial drugs is an interesting field of research for poultry scientists [42]. The performance of birds was recovered closer to the negative control and drug-treated groups at the 2nd week following a coccidiosis challenge, which is encouraging; fortunately, the effect was exceeded in the 2g CBP group and was equal in other CBP groups to salinomycin as the synthetic drug. The positive effects of the anticoccidial cinnamon herb have been linked to the presence of active compounds present in plants that reduce the parasitic oocyst, modulate intestinal microflora, improve immunity and antioxidant status, and reduce intestinal inflammation [13,43–45]. Herbal anticoccidial agents maintain the growth of the broilers by reducing the destructive effects of coccidiosis [46]. The harmful effect

of coccidiosis is clear in the infected group (PC), which has been improved in cinnamon powder-treated birds. Confirming our document, prior literature have also reported improved performance in response to anticoccidials and the ability of natural products to decrease the induced weight loss due to infection of birds [24,42,47]. Cinnamon bark oil up to 0.05% has a better preservative impact on the quality of lamb meat during storage it is supplemented at levels 0.01, 0.25, 0.5, and 5 g in the diet [48]. Cinnamon and/or citral supplementation in feed improved growth performance of chicken vaccinated or not vaccinated against coccidiosis to the level comparable with bacitracin and alter cecal microbiota composition [13]. The addition of cinnamon powder up to 5% appears to have no noticeable impact on growth and carcass characteristics except for the spleen and heart weight percentage [39]. However, they have discovered that it can be used to enhance cellular immune responses in broiler chicks.

Conventional coccidiostats have been associated with undesirable residues in meat in some areas since the late 1990s [49]. However, there is no credible scientific proof to boost the allegation that residues exist or have caused issues for consumers. There is no proof of residual effects from salinomycin or other coccidiostats, with the exception of diclazuril [50]. According to [50], any coccidiostat residue in poultry meat poses a low direct hazard to human health (<1%). Moreover, none of the natural ingredients have already been studied to determine whether they also produce residues. Furthermore, the impacts of CBP on meat quality, carcass characteristics, and marketing growth of broilers infected with *E. tenella* oocysts were studied at different doses and with different results. As a follow-up to a previously published study on the effects of CBP as a coccidiosis prevention product, the effects of CBP on meat quality, growth performance, and carcass characteristics of broiler chickens infected with *E. tenella* were investigated here. Thus, this study examined the efficacy of different quantities of CBP as a natural herb compared with the current synthetic anticoccidial product (salinomycin) in birds exposed to an experimentally induced coccidial challenge. Previously, there was limited research on the effects of CBP on the carcass traits and breast quality of birds facing a coccidial challenge.

The bird became infected with *E. tenella* after ingesting sporulated oocysts which penetrated and damaged the intestinal epithelium of the caecum due to endogenous and exogenous multiplication of the *E. tenella* stage, whereupon the developed oocysts were excreted in the feces [51]. In order to reduce the cost of eliminating the damage caused by *E. tenella* in poultry farms, the authors are actively developing various prevention methods for monitoring *E. tenella* [24,52]. The effects of *E. tenella* infection range from localized intestinal tissue degeneration to death in the most severe instances [53]. To minimize detrimental impacts on high-quality broiler meat production, new agents with cheap costs and minimum adverse effects against *E. tenella* are needed.

Here, the positive impact of cinnamon additives, particularly at level 4 g, was obvious on most carcass traits such as the CW, carcass yield, as well as breast, heart, proventriculus, gizzard, and pancreas percentage relative to CW, increased as compared with the PC. This result may be attributed to that cinnamon can improve the digestive system due to the presence of cinnamon's active components, such as cinnamaldehyde, which can stimulate appetite and digestion [54]. Therefore, birds who received a cinnamon diet, particularly at level 6 g, had a higher breast weight than those in the PC and similar to those received a diet supplemented with salinomycin. Our data showed that experimental treatments did not influence some of the carcass characteristics of meat, such as the relative weights of liver, leg, and fat. These findings are in agreement with [55], who found that the carcass characteristics did not change with the addition of various cinnamon oil levels and sodium butyrate except that the cholesterol level of the muscles was lowered in broilers. In part, this agrees with [56], who found that including CBP in broiler meal had no substantial effect (*p* > 0.05) on heart, breast, gizzard, cholesterol, abdominal fat, or triglycerides. However, [57] found that broilers fed CBP had a higher dressing percentage (*p* < 0.05). The findings of [58] corroborated our findings that dietary treatment with CBP

had no significant effect on lymphoid organ relative weights (*p* > 0.05). Immune organ weight was significantly higher in the 5.0% CBP group, according to [40].

As expected, the results of this study showed that slaughter weight, carcass weight, and then dressing or carcass yield were adversely affected by the positive control compared with medicated or NC groups. Our findings support those of [59,60], who were orally challenged with Eimeria-populated oocysts harmed performance indices and carcass yield. Where [59] observed *Rumex nervosus* leaves can mitigate *E. tenella* suffering and improve dressing percentage. In addition, [60] observed that the dressing percentage was significantly higher in the group receiving a mixture of all 3 herbs (*Aloe barbadensis*, *Ferulafoetida regal*, and *Tamarindus indica*) at 2 mL/L mixed with citric acid and lowest in the control group (without medicinal herbs supplementation and challenged with Eimeria-populated oocysts).

Instead of glucose under aerobic conditions, muscle glycogen is the primary metabolic fuel for anaerobic glycolysis after slaughter. When anaerobic glycolysis occurs, pyruvate is reduced to lactate. The accumulation of lactate causes a decrease in pH whenever the muscle is converted to meat [61]. When the pH reaches acidic conditions, glycolytic enzymes are probably inactivated. Thus, [62] mention that pH is one of the most important alterations that occur during rigor mortis and that it has a direct influence on the quality characteristics of the meat, such as juiciness, texture (tenderness), WHC, color, and shelf life. The meat of birds with a high pH has a higher WHC than meat with a lower pH. The pH of meat is easily determined by its color. The pH of meat is high when it is very dark and low when it is very light. The lower pH in bird meat groups with herbs may be responsible for inhibiting the integration of the deterioration of the growth of microorganisms [63]. The meat quality (PSE, DFD) of birds can be measured quickly and precisely to determine the pHi value of meat samples. The threshold pH value categories of the breast meat of broilers are 5.8 (reddish, soft, and exudative), 5.9–6.2 (standard meat quality), and 6.3 (pale, firm, and non-exudative or dark, firm, and dry) [64]. A duration of 15 min postmortem, the pH parameters were a good predictor of meat traits [65] and ranged from 5.78 to 6.59 [66]. The authors of [61] reviewed the literature and found that the highest quality commercial poultry meat products are more likely to fall within the pH range of 5.7–6.0. In this study, the initial pH ranged from 6.08 to 6.36, and the ultimate pH ranged from 5.71 to 5.83. There was acceptable color and increased wateriness (reddish, soft, and exudative) as well as pale color and good juice retention (pale, firm, and non-exudative). Although there were no significant differences between the experimental groups, the pH of chicken breast meat in the present study decreased mathematically with a rise in CBP doses. The variation in the pH could be attributed to the high antioxidant of cinnamon and its other hydroxyl derivatives attributed to the action of hydroxyl radicals (•OH) present in the phenyl ring of phenolic compounds acting as hydrogen donors [67]. Therefore, increasing CBP nutritional levels in diets increase the donation of hydroxyl groups, effectively reducing the pH value noticed in broiler breast muscle.

Meat color is influenced by many influences, such as pre-slaughter factors, stunning methods, cooling regimes, moisture content, heme pigments, protein physical status, strain, stress, and sex [4,5]. In [68], it was pointed out that raising the L\* value was desirable in terms of consumer acceptance. Here, the 4 g of CBP/kg group had a higher initial L\* value and then a higher WI and lower color change, while the ultimate L\* value and WI were increased with increasing doses of CBP. Metmyoglobin cumulating on the exterior part of the storage meat contributes to the discoloration of the meat [69], which eliminates the a\* value discrepancies. The formation of metmyoglobin and an elevation of lipid oxidation are the key components responsible for distinctions in the b\* value [70]. Differences in initial and ultimate of L\* values, total color change (∆E), and WI were found by [31] when testing CBP. Few researchers have described ∆E and BI parameters, which could assist in elucidating the L\*, a\*, and b\* behavior in bird breast meat, as impacted by CBP intake in the feed. These findings revealed that partial alterations in color variables could be due to the effects of experimental groups on the ∆E and WI. However, other authors, such as

those of [55], did not discover any effect of CBP supplemented diets on broiler meat color measurements. Thus, decreasing water retention tends to lead to less reflective surface light that reduces L\* values [71], and is associated with a decrease in the nutritional value of the meat due to the loss of some nutrients, and as a result, the breast meat becomes less tender. The experimental treatment had no effect on both WHC and DL (*p* > 0.05). WHC is a phrase used to refer to a muscle's ability to bind water under a particular set of circumstances. Commonly, the increase in muscle fat content results in higher WHC and a reduction in the percentage CL [72]. After death, oxygen deprivation causes lactic acid production, leading to a decline in pH, which causes protein denaturation, loss of protein solubility, and an overall reduction in the number of reactive groups available for water binding on muscle protein [62]. CL is a measurement of how much water is lost during cooking because of shrinkage. The degree of shrinkage that occurs during cooking is proportional to the loss of juiciness on the palate. CL was considerably lower in the study when birds were fed a diet containing 2 g of CBP/kg. In contrast, [73] did not observe any influence of 0.5 or 1 mL of cinnamon oil in broiler diets on CL.

The CBP treatment affected the MFI of the breast muscle. Myofibril fragmentation refers to the degree to which homogenization causes myofibrils to be destroyed. The authors of [74] have shown that the values of MFI are strongly correlated with other muscle measurements, such as tenderness and SF. Therefore, cinnamon supplementation could cause less fragmentation of myofibrils. On another hand, the SF in the breast muscle of birds ranged from 5.5 to 5.8 kgf/g [75] and between 2.71 and 3.31 kgf/g [76]. Therefore, the CBP groups in this trial had no effect on meat tenderness as the SF values were between 1.69 and 2.10 kgf/g, and they were almost 67% and 37% lower than the values reported by [75], respectively. However, the CBP treatments had higher SF values than the controls, and they were similar to the salinomycin group. These findings are in contrast with [55], who found that different levels of diets supplemented with cinnamon oil and sodium butyrate did not influence the meat SF value (kg force/cm<sup>2</sup> ) of the broilers.

In comparison with the salinomycin group, the CBP groups had lower levels of texture profiles. Additionally, the CBP groups had lower springiness than the control groups. Meat texture was evaluated using TPA and SF as having a myofibril structure. Recent investigations have been performed on the quality of meat or carcass characters of birds fed diets containing either powder or plant extracts [27,30,77–80]. However, little or no research has been conducted on the effect of CBP on TPA. The authors in [81] found that springiness and cohesiveness decreased as more cinnamon extract was added to sourdough bread. However, hardness, chewiness, and gumminess were reversed. It was thought that adding the cinnamon extract to a bread recipe would be beneficial. In our experiment, the hardness, springiness, cohesiveness, and chewiness decreased in CBP treatments compared with the salinomycin treatment, but there was no difference between the control groups.

Despite the fact that the supplemented CBP resulted in conflicting patterns in terms of SF and TPA, the treatment without CBP (NC) produced the best SF and hardness values. This was also the case for cohesiveness and springiness. The addition of CBP to broiler diets resulted in an increase in meat toughness in general. As a result, adding natural antioxidant compounds to meat can improve its quality, and cinnamon has the highest antioxidant capacity due to its high phenolic content [20,82]. Natural antioxidants, on the other hand, have been found to have little or no effect on the sensory characteristics of meat by some researchers. Supplementation with cinnamon oil, for example, had no effect on the quality of chicken meat, according to [55].

#### **5. Conclusions**

In summary, *Eimeria tenella* infection has a negative impact on growth performance, slaughter weight, carcass yield, and most carcass characteristics of broiler chicken; on the other hand, the use of cinnamon as alternatives to anticoccidials and ionophore coccidiostats, can mitigate these effects. Moreover, the addition of cinnamon was able to improve some physicochemical properties without affecting the meat's quality. However, in the

breasts of birds given cinnamon, MFI decreased and toughness increased, when compared with the other experimental groups. Although this is a consistent experimental paradigm that is highly applicable to commercial conditions, more research into the use of cinnamon to improve the meat quality and productivity of broiler chickens in both healthy and sick conditions is needed.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ani12020166/s1, Table S1: Ingredients and calculated nutrients of broilers starter and finisher diets.

**Author Contributions:** Conceptualization, M.M.Q. and G.M.S.; data curation, M.M.Q. and E.O.H.; formal analysis, M.M.Q. and A.H.A.; funding acquisition, M.A.A.-G. and A.H.A.; investigation, M.M.Q. and M.A.A.-G.; methodology, M.M.Q., A.A.A.-a. and G.M.S.; project administration, S.I.A.-M., M.A.A.-G., and A.H.A.; supervision, S.I.A.-M. and M.M.A.; validation, M.A.A.-G.; visualization, M.M.Q.; writing—original draft preparation, M.M.Q.; writing—review and editing, M.M.Q. and S.I.A.-M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the Research Supporting Project (RSP-2022R439), King Saud University (Riyadh, Saudi Arabia).

**Institutional Review Board Statement:** The study complied with the King Saudi Arabia standards on animal use (KSU-SE-20-44) and was approved by the local animal care and welfare committee of King Saud University.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data sets collected and analyzed during the current study are available from the corresponding author on fair request.

**Acknowledgments:** This work was supported by the Research Supporting Project (RSP-2022R439), King Saud University (Riyadh, Saudi Arabia). The authors extend their thanks to RSSU at King Saud University for their technical support.

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

#### **References**


### *Article* **Chicken Immune Cell Assay to Model Adaptive Immune Responses In Vitro**

**Filip Larsberg 1,\*, Maximilian Sprechert <sup>1</sup> , Deike Hesse <sup>1</sup> , Gudrun A. Brockmann <sup>1</sup> and Susanne Kreuzer-Redmer 2,\***


**Simple Summary:** Knowledge about the modes of action of immunomodulating compounds such as pathogens, drugs, or feed additives, e.g., probiotics, will allow the development of targeted nutrition strategies, prevent infectious diseases and the usage of antimicrobials, and promote the health of animals. To investigate the mechanisms of action of immunomodulating compounds, controlled in vitro systems using freshly isolated immune cells from blood represent a promising alternative to animal experiments. Immune cell isolation from the blood of chickens is a complex and difficult process since the immune cell fractions are significantly contaminated with red blood cells and platelets. To our knowledge, a robust protocol for immune cell isolation from chicken blood and the subsequent cultivation of immune cells is not available. Here, we established a protocol for blood sampling and immune cell isolation and cultivation from chicken blood, which could be applied for the investigation of direct effects of immunomodulating compounds. This protocol, combining different techniques of immune cell isolation, cultivation, and differentiation of distinct immune cell populations, will serve as a potential alternative to animal testing in vivo. By gaining knowledge about the mechanisms of action of immunomodulating compounds, this in vitro model will contribute to promote health and welfare in chicken farming.

**Abstract:** Knowledge about the modes of action of immunomodulating compounds such as pathogens, drugs, or feed additives, e.g., probiotics, gained through controlled but animal-related in vitro systems using primary cultured peripheral blood mononuclear cells (PBMCs) will allow the development of targeted nutrition strategies. Moreover, it could contribute to the prevention of infectious diseases and the usage of antimicrobials, and further promote the health of the animals. However, to our knowledge, a protocol for the isolation of PBMCs with reduced thrombocyte count from chicken blood and subsequent cell culture over several days to assess the effects of immunomodulating compounds is not available. Therefore, we established an optimized protocol for blood sampling and immune cell isolation, culture, and phenotyping for chicken PBMCs. For blood sampling commercial Na–citrate tubes revealed the highest count of vital cells compared to commercial Li–heparin (*p* < 0.01) and K3EDTA (*p* < 0.05) tubes. Using combined dextran and ficoll density gradient separation, the thrombocyte count was significantly reduced (*p* < 0.01) compared to slow-speed centrifugation with subsequent ficoll. For cell culture, the supplementation of RPMI-1640 medium with 10% chicken serum resulted in the lowest relative cell count of thrombocytes compared to fetal calf serum (FCS) (*p* < 0.05). To validate the ability of the cell culture system to respond to stimuli, concanavalin A (conA) was used as a positive control. The optimized protocol allows the isolation and cultivation of vital PBMCs with reduced thrombocyte count from chicken blood for subsequent investigation of the modes of action of immunomodulating compounds.

**Keywords:** chicken; PBMCs; primary cell culture; immunomodulating compounds

**Citation:** Larsberg, F.; Sprechert, M.; Hesse, D.; Brockmann, G.A.; Kreuzer-Redmer, S. Chicken Immune Cell Assay to Model Adaptive Immune Responses In Vitro. *Animals* **2021**, *11*, 3600. https://doi.org/ 10.3390/ani11123600

Academic Editors: Natalie Morgan and Youssef A. Attia

Received: 17 October 2021 Accepted: 15 December 2021 Published: 19 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/).

#### **1. Introduction**

Today, there is growing concern about the resistance of pathogenic bacteria against antibiotics, the residual effects of antibiotics in meat products [1], and the public health risk from zoonotic pathogens like *Salmonella* and *Campylobacter*. With the ban of the subtherapeutic usage of antibiotics in farming (Council Regulation EC 70/524/ EEC), immunomodulating alternatives have arisen to improve animal and human health. The immune response to a given stimulus varies between different species [2]. The knowledge of immunomodulatory properties is of high importance, particularly regarding those immunomodulatory compounds with a high potential to improve animal health via improved defence against infection [3]. Therefore, knowledge about the specific modes of action of immunomodulating compounds is needed in order to develop specific diets as alternatives to widely used antimicrobials [4] on farms and to improve the health and welfare of animals, and thereby also humans [5–7]. Peripheral blood mononuclear cells (PBMCs) are often used as a cell model to investigate the direct effects of immunomodulating compounds. Methods are well established to isolate PBMCs from the blood of humans [8,9] and animals [5,10,11]. However, in avian species, although it is often performed [5,10,11], it is a complex and difficult process to isolate a population of PBMCs for subsequent cultivation and in vitro assays using common isolation methods, without contamination of nucleated thrombocytes and erythrocytes in the immune cell fractions [11–14]. Furthermore, erythrocytes, which can be eliminated by red blood cell lysis in mammals, are nucleated in avian species and show a high degree of resistance to common lysis procedures [15]. The majority of erythrocytes are usually eliminated using density gradient centrifugation with ficoll. Thrombocytes can be excluded in further analysis, e.g., by flow cytometry. In contrast to lymphocytes, they express lower levels of the pan-leukocyte marker CD45 [16–18]. Moreover, they lack the T-cell marker CD3, the B-cell marker Bu-1a, and the monocyte/macrophage marker Kul-01 [16,19,20], and appear positive for the fibrinogen receptor CD41/CD61 [21] and the CD51/CD61 integrin [17]. Therefore, a dual-labeling approach was established to eliminate thrombocytes [19]. However, as thrombocytes interfere with the quantification of avian leukocytes and can result in shifting percentages of the latter, further markers are needed for the exclusion of thrombocytes [19]. In cell culture, thrombocytes were shown to suffer apoptotic cell death when cultured together with monocytes. The addition of lymphocytes or their soluble factors could delay apoptosis [22]. However, functional ex vivo analyses of, for example, T-cell responses, which are used to determine responses to infections and vaccination in chickens, require an efficient and pure isolation of PBMCs without contaminating thrombocytes [12,20,23]. Furthermore, thrombocytes have been shown to be a major cytokine producer in chickens [24], which would distort the responses of the lymphocytes. The aim of this work was the establishment of a cell culture system with chicken PBMCs to assess the direct effects of potentially immunomodulating compounds on chicken immune cells in vitro, which requires the isolation of a PBMC population with reduced thrombocyte count. Recently, a combined dextran and ficoll density gradient separation was reported to yield large populations of chicken PBMCs without contaminating thrombocytes [25]. Furthermore, a few slow-speed centrifugation approaches have been described previously [12,14,17,26].

However, the isolation of adaptive immune cells from peripheral blood with subsequent primary cell culture over a period of several days for the assessment of the effects of immunomodulating compounds has not yet been described. In this study, different optimization steps were performed to establish an innovative in vitro approach to assess the properties of immunomodulatory compounds. The established in vitro model will help to investigate the modes of action of immunomodulatory compounds such as feed additives and other immune cell stimuli used to improve health in chicken farming and prevent infectious diseases and the usage of antimicrobials.

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

#### *2.1. Animals*

Five- to 6-week-old broiler chickens of the commercial layer variety Cobb500 (Cobb Germany Avimex GmbH, Wiedemar, Germany) were used for the establishment of an in vitro cell culture model with chicken PBMCs. The birds were stunned and decapitated. The blood was sampled in tubes containing an anticoagulant. All chickens were fed a starter diet from day 1 to day 14 post hatch, and a grower diet afterwards (H. Wilhelm Schaumann GmbH, Pinneberg, Germany). The ration was fed on an ad libitum basis and water was always available. The light duration was 24 h on days 1 and 2, followed by 16 h/day until sampling. The chickens were kept in groups of approximately 20 chickens in suitable 4 m<sup>2</sup> pens.

The study was approved by the local State Office for Health and Social Affairs, Landesamt für Gesundheit und Soziales Berlin (LaGeSo, T 0151/19).

#### *2.2. Blood Sampling Methods*

Different anticoagulants were tested for blood sampling. Commercial 9 mL tripotassium ethylenediaminetetraacetic acid (K3EDTA), lithium heparin (Li–heparin), and sodium citrate (Na–citrate) pre-filled polystyrene tubes (VACUETTE®) were used (all from Greiner Bio-One, Kremsmünster, Austria). Additionally, different volumes of 0.5 M EDTA (Carl Roth, Karlsruhe, Germany) in dH2O (200 µL, 1 mL, and 8 mL) were tested in 50 mL tubes. Per biological replicate, 30 mL of blood was used for investigation of the best anticoagulant. To test anticoagulants, subsequent cell isolation was performed via combined slow-speed centrifugation and density gradient centrifugation. Isolated PBMCs were resuspended in 5 to 10 mL RPMI-1640 medium (Gibco™, ThermoFisher Scientific, Waltham, MA, USA).

#### *2.3. PBMC Isolation Methods*

2.3.1. Isolation of PBMCs Using Combined Slow-Speed Centrifugation and Density Gradient Centrifugation

Blood samples were diluted 1:2 with PBS (Gibco™, ThermoFisher Scientific, Waltham, MA, USA) containing 2 mM EDTA (Carl Roth, Karlsruhe, Germany). The samples were mixed and centrifuged for 15 min at 60× *g*. Upon centrifugation, three layers could be observed. The lymphocytes, laid on top of the erythrocyte layer, were swirled up using a dropper and transferred into a new 50 mL tube. The cells were washed once with PBS/EDTA (the tube was filled up with PBS/EDTA to 50 mL), after which the pellet was resuspended in 10 mL PBS/EDTA upon centrifugation, layered 1:2 onto ficoll (Histopaque-1077, Sigma-Aldrich, St. Louis, MO, USA), and centrifuged without a break for 30 min at 400× *g*. The buffer layer was collected at the interface of the plasma and ficoll, transferred to a new 50 mL tube, and washed once by centrifugation at 350× *g* for 10 min. After centrifugation, cells were resuspended in 10 mL RPMI-1640 medium.

#### 2.3.2. Isolation of PBMCs Using Combined Dextran–Ficoll Separation

Blood samples were diluted 1:2 with PBS/EDTA. The diluted blood samples were mixed with 3% dextran in a ratio of 1:0.4 and centrifuged for 20 min at 50× *g*. The upper layer containing the PBMCs was carefully collected and layered onto ficoll in a ratio of 1:2 in a 50 mL tube. After centrifugation without a break for 30 min at 900× *g*, the buffer layer containing the PBMCs was collected, washed twice, and centrifuged for 10 min at 400× *g*. After centrifugation, cells were resuspended in 10 mL RPMI-1640 medium.

#### *2.4. PBMC Culture*

#### 2.4.1. Cell Counting

The number of isolated vital cells was counted using a Tali® Image-Based Cytometer (Invitrogen™, ThermoFisher Scientific, Waltham, MA, USA). Therefore, 25 µL of cell suspension was mixed with 1 µL of 1 mg/mL propidium iodide (PI) as a viability marker and transferred onto a Tali® Cellular Analysis Slide for measurement.

#### 2.4.2. Cell Seeding

For immune cell culture, cells were seeded in Nunc™ Non-treated T25 EasYFlasks™ (ThermoFisher Scientific, Waltham, MA, USA) at a density of 5 <sup>×</sup> <sup>10</sup><sup>6</sup> cells/mL and cultured for several days. For co-culture experiments, cells were seeded in nontreated, flat-bottomed 24-well plates (Eppendorf, Hamburg, Germany) at a density of 1 <sup>×</sup> <sup>10</sup><sup>6</sup> cells/mL and cultured from 24 h up to 72 h. All cells were cultured at 41 ◦C with 5% CO<sup>2</sup> in RPMI-1640 medium with 2 g/L glucose, 100 U/mL penicillin, 100 µg/mL streptomycin.

#### 2.4.3. Serum Supplementation of Culture Medium

For cultivation of chicken PBMCs over several days, different sera were tested as cell culture supplements. Porcine serum (Sigma-Aldrich, St. Louis, MO, USA), chicken serum (Gibco™, ThermoFisher Scientific, Waltham, MA, USA), or fetal calf serum (FCS, Gibco™, ThermoFisher Scientific, Waltham, MA, USA) was added to the cells cultured in T25 flasks in a concentration of 10% to the RPMI-1640 medium. Immune cells were cultured for 3 days. After 24 h and 72 h, cells were subjected to flow cytometric measurement.

#### 2.4.4. Supplementation of Culture Medium with Additional L-Glutamine

The effect of additional supplementation of 2 mM L-glutamine (Gibco™, ThermoFisher Scientific, Waltham, MA, USA) to the cell culture medium was tested. Therefore, PBMCs with or without additional L-glutamine were cultured in RPMI-1640 medium supplemented with 10% chicken serum for 24 h and subjected to flow cytometry.

#### 2.4.5. Response Capacity of PBMCs towards Immune Cell Stimulants

To validate the cell culture system's responses to stimuli, concanavalin A (conA, Vector Laboratories, Burlingame, CA, USA) was used as a positive control. Therefore, 5 µg/mL or 10 µg/mL conA was added to the cells cultured in 24-well plates. PBMCs were cultured in RPMI-1640 medium supplemented with 10% chicken serum for 24 h and subjected to flow cytometry.

#### *2.5. Immunophenotyping*

For immunophenotyping, 1 <sup>×</sup> <sup>10</sup><sup>6</sup> cells per antibody staining set were harvested, centrifuged for 10 min at 400× *g*, washed once with cold PBS/EDTA containing 0.05% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA), and stained with different panels of monoclonal antibodies. Therefore, 25 µL of the antibody mix, containing the diluted antibodies in the cold staining buffer PBS/EDTA, was added to the samples. After labeling, the samples were stored on ice in the dark for 30 min. The samples were washed with 600 µL PBS/EDTA/BSA and centrifuged for 10 min at 400× *g*. Subsequently, the supernatant was discarded, the samples were resuspended in 200 µL PBS/EDTA, and analyzed on a flow cytometer. Initially, the most suitable antibody concentration was determined by titration of the respective antibody from 1:25 to 1:400. In this study, immune cells were stained with mouse anti-chicken CD3-Allophycocyanin (APC) (CT-3, SouthernBiotech, Birmingham, AL, USA), CD4-Spectral Red (SPRD) (CT-4, SouthernBiotech, Birmingham, AL, USA), CD28-Phycoerythrin (PE) (AV7, SouthernBiotech, Birmingham, AL, USA), CD8-APC (CT-8, ThermoFisher Scientific, Waltham, MA, USA), CD45-Fluorescein isothiocyanate (FITC) (LT40, ThermoFisher Scientific, Waltham, MA, USA), CD41/CD61-(R)PE (11C3, ThermoFisher Scientific, Waltham, MA, USA), and human anti-chicken CD25-FITC (AbD13504, Bio-Rad Laboratories, Hercules, CA, USA) antibodies. Lymphocytes were gated using forward and sideward scatter by exclusion of debris, erythrocytes, and granulocytes. Dead cells were excluded using 1 µL 40 ,6-diamidino-2 phenylindole (DAPI, Sigma-Aldrich, St. Louis, MO, USA) (1 mg/mL). Thereafter, doublets were excluded and at least 20,000 cells in the vital lymphocyte region were acquired on a Canto II (Becton Dickinson (BD), Franklin Lakes, NJ, USA) flow cytometer.

#### *2.6. Statistical Analysis* The relative cell count of antibody-positive cells in the flow cytometer was calculated

*2.6. Statistical Analysis*

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Dickinson (BD), Franklin Lakes, NJ, USA) flow cytometer.

The relative cell count of antibody-positive cells in the flow cytometer was calculated relative to the number of vital lymphocytes. Statistical analysis for blood sampling, PBMC isolation, and PBMC culture was performed using an unpaired Student's *t*-test. All tests were executed using GraphPad Prism 8.0.2 (GraphPad Software, San Diego, CA, USA). Differences between groups were considered statistically significant at *p* < 0.05. relative to the number of vital lymphocytes. Statistical analysis for blood sampling, PBMC isolation, and PBMC culture was performed using an unpaired Student's *t*-test. All tests were executed using GraphPad Prism 8.0.2 (GraphPad Software, San Diego, CA, USA). Differences between groups were considered statistically significant at *p* < 0.05.

granulocytes. Dead cells were excluded using 1 µL 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St. Louis, MO, USA) (1 mg/mL). Thereafter, doublets were excluded and at least 20,000 cells in the vital lymphocyte region were acquired on a Canto II (Becton

#### **3. Results 3. Results**

#### *3.1. Blood Sampling 3.1. Blood Sampling*

Blood sampling in commercial Na–citrate tubes revealed the highest count of vital cells (1.11 <sup>×</sup> <sup>10</sup><sup>8</sup> ) compared to commercial K3EDTA (5.37 <sup>×</sup> <sup>10</sup><sup>7</sup> ) (*p* < 0.05) and heparin (2.42 <sup>×</sup> <sup>10</sup><sup>7</sup> ) (*p* < 0.01) tubes (Figure 1). We found no significant difference between blood sampling in commercial Na–citrate tubes and 200 <sup>µ</sup>L 0.5 M EDTA (6.81 <sup>×</sup> <sup>10</sup><sup>7</sup> ). Blood sampling in commercial Na–citrate tubes revealed the highest count of vital cells (1.11 × 10<sup>8</sup> ) compared to commercial K3EDTA (5.37 × 10<sup>7</sup> ) (*p* < 0.05) and heparin (2.42 × 10<sup>7</sup> ) (*p* < 0.01) tubes (Figure 1). We found no significant difference between blood sampling in commercial Na–citrate tubes and 200 µL 0.5 M EDTA (6.81 × 10<sup>7</sup> ).

**Figure 1.** Influence of the anticoagulants K3EDTA, Na–citrate, and Li–heparin as well as 200 µL 0.5 M EDTA on cell survival. After PBMC isolation, chicken immune cells were counted with a Tali image-based cytometer and the viability was assessed using propidium iodide (PI). Five biological replicates are displayed. All bars represent one experiment. A box-and-whisker plot is displayed. Significance is shown as \*\*; *p* < 0.01; \*, *p* < 0.05. NS: not significant. Significance was analyzed using **Figure 1.** Influence of the anticoagulants K3EDTA, Na–citrate, and Li–heparin as well as 200 µL 0.5 M EDTA on cell survival. After PBMC isolation, chicken immune cells were counted with a Tali image-based cytometer and the viability was assessed using propidium iodide (PI). Five biological replicates are displayed. All bars represent one experiment. A box-and-whisker plot is displayed. Significance is shown as \*\*; *p* < 0.01; \*, *p* < 0.05. NS: not significant. Significance was analyzed using an unpaired Student's *t*-test.

an unpaired Student's *t*-test. However, flow cytometric analysis of the relative cell count of CD45-high leukocytes and CD45-low and integrin CD41/CD61+ thrombocytes within the vital lymphocyte population revealed the highest thrombocyte count in blood sampled in commercial Na–citrate tubes after the cell isolation (Figure S1a,b). Since Na–citrate is often used in immunological studies and we also detected the highest total number of live cells after measurement with a Tali image-based cytometer (Figure 1) and BD FACS Canto II (Data not However, flow cytometric analysis of the relative cell count of CD45-high leukocytes and CD45-low and integrin CD41/CD61+ thrombocytes within the vital lymphocyte population revealed the highest thrombocyte count in blood sampled in commercial Na–citrate tubes after the cell isolation (Figure S1a,b). Since Na–citrate is often used in immunological studies and we also detected the highest total number of live cells after measurement with a Tali image-based cytometer (Figure 1) and BD FACS Canto II (Data not shown), we decided to find a more suitable isolation method to reduce the high thrombocyte count.

#### shown), we decided to find a more suitable isolation method to reduce the high thrombocyte count. *3.2. PBMC Isolation Method*

*3.2. PBMC Isolation Method* After blood sampling, the optimal PBMC isolation method was evaluated. In this step, we wanted to decrease the number of thrombocytes and increase the number of leukocytes in blood sampled in commercial Na–citrate tubes. Therefore, the relative cell counts of CD45-high leukocytes and CD45-low and integrin CD41/CD61+ thrombocytes in the vital lymphocyte population were assessed after the isolation of PBMCs using either a combined slow-speed and ficoll, or a combined dextran andficoll separation protocol (Figure 2).

(Figure 2).

After blood sampling, the optimal PBMC isolation method was evaluated. In this step, we wanted to decrease the number of thrombocytes and increase the number of leukocytes in blood sampled in commercial Na–citrate tubes. Therefore, the relative cell counts of CD45-high leukocytes and CD45-low and integrin CD41/CD61+ thrombocytes in the vital lymphocyte population were assessed after the isolation of PBMCs using either a combined slow-speed and ficoll, or a combined dextran andficoll separation protocol

**Figure 2.** Yield of leukocytes and thrombocytes using two immune cell isolation methods, combined slow-speed-ficoll and dextran–ficoll separation. After PBMC isolation, immune cells were stained with the pan-leukocyte marker CD45 and the thrombocyte marker CD41/CD61 and subjected to flow cytometry. (**a**) Leukocytes and (**b**) thrombocytes relative to the total live cell count. A total of 20,000 vital lymphocytes were recorded on a BD Canto II flow cytometer. DAPI was used as a viability marker. Data represent seven biological replicates and two technical replicates, each in two independent experiments. A box-and-whisker plot is displayed. Significance is shown as \*, *p* < 0.05. NS: not significant. Significance was analyzed using an unpaired Student's *t*-test. **Figure 2.** Yield of leukocytes and thrombocytes using two immune cell isolation methods, combined slow-speed-ficoll and dextran–ficoll separation. After PBMC isolation, immune cells were stained with the pan-leukocyte marker CD45 and the thrombocyte marker CD41/CD61 and subjected to flow cytometry. (**a**) Leukocytes and (**b**) thrombocytes relative to the total live cell count. A total of 20,000 vital lymphocytes were recorded on a BD Canto II flow cytometer. DAPI was used as a viability marker. Data represent seven biological replicates and two technical replicates, each in two independent experiments. A box-and-whisker plot is displayed. Significance is shown as \*, *p* < 0.05. NS: not significant. Significance was analyzed using an unpaired Student's *t*-test.

The dextran–ficoll separation did not change the relative cell count of vital leukocytes compared to the slow-speed–ficoll separation method (Figure 2a). Moreover, the mean of the relative cell count of thrombocytes was significantly lower after dextran–ficoll separation (27.23%) compared to the combined slow-speed–ficoll separation (41.19%) (*p* < 0.05) (Figure 2b). The latter was in line with the decrease of cells in the lymphocyte gate (*p* < 0.05, data not shown). The dextran–ficoll separation did not change the relative cell count of vital leukocytes compared to the slow-speed–ficoll separation method (Figure 2a). Moreover, the mean of the relative cell count of thrombocytes was significantly lower after dextran– ficoll separation (27.23%) compared to the combined slow-speed–ficoll separation (41.19%) (*p* < 0.05) (Figure 2b). The latter was in line with the decrease of cells in the lymphocyte gate (*p* < 0.05, data not shown).

#### *3.3. PBMC Culture Conditions 3.3. PBMC Culture Conditions*

#### 3.3.1. Medium Supplementation with Serum of Different Species 3.3.1. Medium Supplementation with Serum of Different Species

Chicken PBMCs were cultivated in RPMI-1640 medium supplemented with 10% porcine, chicken, or the standard fetal calf serum. The addition of chicken serum was associated with the highest mean of the relative cell count of leukocytes (84.21%) compared to the cultivation with the often-used FCS (54.55%) (*p* < 0.05); interestingly, there was no clear difference to porcine serum (77.04%) (Figure 3a). The thrombocyte count was low in cells cultivated in RPMI-1640 supplemented with chicken serum (13.44%) compared to those supplemented with FCS (43.01%) (*p* < 0.05), but not different to supplementation with porcine serum (21.17%) after 1 day of cultivation (Figure 3b). Chicken PBMCs were cultivated in RPMI-1640 medium supplemented with 10% porcine, chicken, or the standard fetal calf serum. The addition of chicken serum was associated with the highest mean of the relative cell count of leukocytes (84.21%) compared to the cultivation with the often-used FCS (54.55%) (*p* < 0.05); interestingly, there was no clear difference to porcine serum (77.04%) (Figure 3a). The thrombocyte count was low in cells cultivated in RPMI-1640 supplemented with chicken serum (13.44%) compared to those supplemented with FCS (43.01%) (*p* < 0.05), but not different to supplementation with porcine serum (21.17%) after 1 day of cultivation (Figure 3b).

**Figure 3.** Influence of the addition of different sera to the cell culture on the survival of immune cells. Isolated PBMCs were cultured in RPMI-1640 medium with glucose, 100 U/mL penicillin, 100 µg/mL streptomycin, and either 10% chicken, 10% porcine, or 10% FCS. The relative cell counts of leukocytes and thrombocytes were assessed after 24 h of cultivation. (**a**) CD45-high leukocytes, relative to the vital lymphocyte population; (**b**) CD45-low thrombocytes relative to the vital lymphocyte population. A total of 20,000 cells were recorded on a BD Canto II flow cytometer. DAPI was used as a viability marker. Data represent seven biological replicates and two technical replicates each. A box-and-whisker plot is displayed. Significance is shown as +, *p* < 0.1; \*, *p* < 0.05. NS: not significant. Significance was analyzed using an unpaired Student's *t*-test. **Figure 3.** Influence of the addition of different sera to the cell culture on the survival of immune cells. Isolated PBMCs were cultured in RPMI-1640 medium with glucose, 100 U/mL penicillin, 100 µg/mL streptomycin, and either 10% chicken, 10% porcine, or 10% FCS. The relative cell counts of leukocytes and thrombocytes were assessed after 24 h of cultivation. (**a**) CD45-high leukocytes, relative to the vital lymphocyte population; (**b**) CD45-low thrombocytes relative to the vital lymphocyte population. A total of 20,000 cells were recorded on a BD Canto II flow cytometer. DAPI was used as a viability marker. Data represent seven biological replicates and two technical replicates each. A box-andwhisker plot is displayed. Significance is shown as +, *p* < 0.1; \*, *p* < 0.05. NS: not significant. Significance was analyzed using an unpaired Student's *t*-test.

After one day of cultivation, the highest number of lymphocytes (Figure S2a) and

vital cells (Figure S2b) was found in PBMCs cultured in RPMI-1640 medium with FCS (60.96%). The relative lymphocyte count was higher compared to culture with porcine (43.16%) (*p* < 0.01) or chicken serum (44.58%) (*p* < 0.05). The live cell count of cells cultivated in FCS (91.16%) was higher compared to those with porcine serum (86.74%) (*p* < 0.05), but did not differ between FCS and chicken serum (89.64%). The relative cell count of leukocytes cultured in RPMI-1640 supplemented with porcine serum (77.04%) was higher compared to cells cultured in medium with FCS (54.55%) (*p* < 0.1). The thrombocyte count was lower in cells cultured in medium with porcine serum (21.17%) compared to FCS (43.01%) (*p* < 0.1). The supplementation of porcine and chicken serum did not differ significantly for leukocytes and thrombocytes. After one day of cultivation, the highest number of lymphocytes (Figure S2a) and vital cells (Figure S2b) was found in PBMCs cultured in RPMI-1640 medium with FCS (60.96%). The relative lymphocyte count was higher compared to culture with porcine (43.16%) (*p* < 0.01) or chicken serum (44.58%) (*p* < 0.05). The live cell count of cells cultivated in FCS (91.16%) was higher compared to those with porcine serum (86.74%) (*p* < 0.05), but did not differ between FCS and chicken serum (89.64%). The relative cell count of leukocytes cultured in RPMI-1640 supplemented with porcine serum (77.04%) was higher compared to cells cultured in medium with FCS (54.55%) (*p* < 0.1). The thrombocyte count was lower in cells cultured in medium with porcine serum (21.17%) compared to FCS (43.01%) (*p* < 0.1). The supplementation of porcine and chicken serum did not differ significantly for leukocytes and thrombocytes.

#### 3.3.2. Medium Supplementation with Additional L-Glutamine Supplementation with an additional 2 mM L-glutamine to the RPMI-1640 medium 3.3.2. Medium Supplementation with Additional L-Glutamine

with glucose did not affect the viability of cultured PBMCs (Figure 4a). Furthermore, the relative cell count of leukocytes did not change after the addition of L-glutamine, compared to the control without additional L-glutamine (Figure 4b). Supplementation with an additional 2 mM L-glutamine to the RPMI-1640 medium with glucose did not affect the viability of cultured PBMCs (Figure 4a). Furthermore, the relative cell count of leukocytes did not change after the addition of L-glutamine, compared to the control without additional L-glutamine (Figure 4b).

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**Figure 4.** The effect of additional L-glutamine on cell viability. Isolated chicken PBMCs were cultured in RPMI-1640 medium with glucose, 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% chicken serum with or without additional L-glutamine (2 mM) for 24 h. (**a**) Vital cells relative to the lymphocytes; (**b**) CD45-high leukocytes, relative to the vital lymphocyte population. A total of 20,000 cells were recorded on a BD Canto II flow cytometer. DAPI was used as a viability marker. Data represent seven biological replicates and two technical replicates each. A box-and-whisker plot is displayed. NS: not significant. Significance was analyzed using an unpaired Student's *t*-test. **Figure 4.** The effect of additional L-glutamine on cell viability. Isolated chicken PBMCs were cultured in RPMI-1640 medium with glucose, 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% chicken serum with or without additional L-glutamine (2 mM) for 24 h. (**a**) Vital cells relative to the lymphocytes; (**b**) CD45-high leukocytes, relative to the vital lymphocyte population. A total of 20,000 cells were recorded on a BD Canto II flow cytometer. DAPI was used as a viability marker. Data represent seven biological replicates and two technical replicates each. A box-and-whisker plot is displayed. NS: not significant. Significance was analyzed using an unpaired Student's *t*-test. **Figure 4.** The effect of additional L-glutamine on cell viability. Isolated chicken PBMCs were cultured in RPMI-1640 medium with glucose, 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% chicken serum with or without additional L-glutamine (2 mM) for 24 h. (**a**) Vital cells relative to the lymphocytes; (**b**) CD45-high leukocytes, relative to the vital lymphocyte population. A total of 20,000 cells were recorded on a BD Canto II flow cytometer. DAPI was used as a viability marker. Data represent seven biological replicates and two technical replicates each. A box-and-whisker plot is displayed. NS: not significant. Significance was analyzed using an unpaired Student's *t*-test.

3.3.3. ConA as a Positive Control for the Validation of the Response Capacity of the Cell Culture System 3.3.3. ConA as a Positive Control for the Validation of the Response Capacity of the Cell Culture System 3.3.3. ConA as a Positive Control for the Validation of the Response Capacity of the Cell Culture System

To test if the cell culture system was a valid system to examine the direct effects of potentially immunomodulating compounds, the effect of conA, a well-known lymphocyte mitogen which stimulates mainly T-cells, was examined via measurement of CD8+ cytotoxic T-cells (Figure 5a,b) and CD4+ T-helper cells (Figure 5c,d). To test if the cell culture system was a valid system to examine the direct effects of potentially immunomodulating compounds, the effect of conA, a well-known lymphocyte mitogen which stimulates mainly T-cells, was examined via measurement of CD8+ cytotoxic T-cells (Figure 5a,b) and CD4+ T-helper cells (Figure 5c,d). To test if the cell culture system was a valid system to examine the direct effects of potentially immunomodulating compounds, the effect of conA, a well-known lymphocyte mitogen which stimulates mainly T-cells, was examined via measurement of CD8+ cytotoxic T-cells (Figure 5a,b) and CD4+ T-helper cells (Figure 5c,d).

**Figure 5.** *Cont*.

**Figure 5.** Effects of conA on CD8+ cytotoxic T-cells and CD4+ T-helper cells. The effects of two concentrations (5 µg/mL and 10 µg/mL) of conA on the activation and proliferation of cytotoxic T-cells and T-helper cells relative to the vital lymphocyte population were assessed after 24 h of cultivation. (**a**) CD8+ cytotoxic T-cells; (**b**) CD8+CD25+ activated cytotoxic T-cells; (**c**) CD4+ T-helper cells; (**d**) CD4+CD25+ activated T-helper cells. A total of 20,000 cells were recorded on a BD Canto II flow cytometer. DAPI was used as a viability marker. Data represent four biological replicates and two technical replicates each. A box-and-whisker plot is displayed. Significance is shown as +, *p* < 0.1; \*, *p* < 0.05. NS: not significant. Significance was analyzed using an unpaired Student's *t*-test. **Figure 5.** Effects of conA on CD8+ cytotoxic T-cells and CD4+ T-helper cells. The effects of two concentrations (5 µg/mL and 10 µg/mL) of conA on the activation and proliferation of cytotoxic T-cells and T-helper cells relative to the vital lymphocyte population were assessed after 24 h of cultivation. (**a**) CD8+ cytotoxic T-cells; (**b**) CD8+CD25+ activated cytotoxic T-cells; (**c**) CD4+ T-helper cells; (**d**) CD4+CD25+ activated T-helper cells. A total of 20,000 cells were recorded on a BD Canto II flow cytometer. DAPI was used as a viability marker. Data represent four biological replicates and two technical replicates each. A box-and-whisker plot is displayed. Significance is shown as +, *p* < 0.1; \*, *p* < 0.05. NS: not significant. Significance was analyzed using an unpaired Student's *t*-test.

As expected, conA treatment increased the relative cell count of CD8+ cytotoxic Tcells (Figure 5a), although the result was not significant. However, at a concentration of 10 µg/mL, conA increased the mean of the relative cell count of CD8+CD25+ activated cytotoxic T-cells from 0.29% to 1.32% (*p* < 0.05) (Figure 5b). For T-helper cells, conA stimulation increased the relative cell count of CD4+ T-helper cells numerically (Figure 5a), but this also did not reach significance. However, looking at CD4+CD25+ activated Thelper cells, conA treatment in a concentration of 10 µg/mL increased the mean relative cell count from 1.40% to 3.54% (*p* < 0.05) (Figure 5d). These effects were visible for two different organs, blood (Figure 5) and spleen (data not shown). For validation, the effect of 10 µg/mL conA was tested on a higher number of biolog-As expected, conA treatment increased the relative cell count of CD8+ cytotoxic Tcells (Figure 5a), although the result was not significant. However, at a concentration of 10 µg/mL, conA increased the mean of the relative cell count of CD8+CD25+ activated cytotoxic T-cells from 0.29% to 1.32% (*p* < 0.05) (Figure 5b). For T-helper cells, conA stimulation increased the relative cell count of CD4+ T-helper cells numerically (Figure 5a), but this also did not reach significance. However, looking at CD4+CD25+ activated T-helper cells, conA treatment in a concentration of 10 µg/mL increased the mean relative cell count from 1.40% to 3.54% (*p* < 0.05) (Figure 5d). These effects were visible for two different organs, blood (Figure 5) and spleen (data not shown).

ical replicates (Figure 6). Therefore, the relative cell count of conA-treated PBMCs was assessed by measurement of T-helper cells (Figure 6a), activated T-helper cells (Figure 6b), and all T-cells, except γδ T-cells, via an additional marker, CD28-PE (Figure 6c). For validation, the effect of 10 µg/mL conA was tested on a higher number of biological replicates (Figure 6). Therefore, the relative cell count of conA-treated PBMCs was assessed by measurement of T-helper cells (Figure 6a), activated T-helper cells (Figure 6b), and all T-cells, except γδ T-cells, via an additional marker, CD28-PE (Figure 6c).

(**a**) (**b**) (**c**)

different organs, blood (Figure 5) and spleen (data not shown).

(**c**) (**d**) **Figure 5.** Effects of conA on CD8+ cytotoxic T-cells and CD4+ T-helper cells. The effects of two concentrations (5 µg/mL and 10 µg/mL) of conA on the activation and proliferation of cytotoxic T-cells and T-helper cells relative to the vital lymphocyte population were assessed after 24 h of cultivation. (**a**) CD8+ cytotoxic T-cells; (**b**) CD8+CD25+ activated cytotoxic T-cells; (**c**) CD4+ T-helper cells; (**d**) CD4+CD25+ activated T-helper cells. A total of 20,000 cells were recorded on a BD Canto II flow cytometer. DAPI was used as a viability marker. Data represent four biological replicates and two technical replicates each. A box-and-whisker plot is displayed. Significance is shown as +, *p* < 0.1; \*, *p* < 0.05. NS: not significant. Significance was analyzed using an unpaired Student's *t*-test.

As expected, conA treatment increased the relative cell count of CD8+ cytotoxic Tcells (Figure 5a), although the result was not significant. However, at a concentration of 10 µg/mL, conA increased the mean of the relative cell count of CD8+CD25+ activated cytotoxic T-cells from 0.29% to 1.32% (*p* < 0.05) (Figure 5b). For T-helper cells, conA stimulation increased the relative cell count of CD4+ T-helper cells numerically (Figure 5a), but this also did not reach significance. However, looking at CD4+CD25+ activated Thelper cells, conA treatment in a concentration of 10 µg/mL increased the mean relative cell count from 1.40% to 3.54% (*p* < 0.05) (Figure 5d). These effects were visible for two

For validation, the effect of 10 µg/mL conA was tested on a higher number of biological replicates (Figure 6). Therefore, the relative cell count of conA-treated PBMCs was assessed by measurement of T-helper cells (Figure 6a), activated T-helper cells (Figure 6b), and all T-cells, except γδ T-cells, via an additional marker, CD28-PE (Figure 6c).

**Figure 6.** Effects of conA on activation and proliferation of CD4+ T-helper cells and CD28+ T-cells. The effect of 10 µg/mL conA on the activation and proliferation of T-helper cells relative to the vital lymphocyte population was assessed after 24 h of cultivation. (**a**) CD4+ T-helper cells; (**b**) CD4+CD25+ activated T-helper cells; (**c**) CD28+ T-cells. A total of 20,000 cells were recorded on a BD Canto II flow cytometer. DAPI was used as a viability marker. Data represent eight biological replicates and two technical replicates each. A box-and-whisker plot is displayed. Significance is shown as \*\*, *p* < 0.01; \*, *p* < 0.05. NS: not significant. Significance was analyzed using an unpaired Student's *t*-test.

As shown by testing two different conA treatment concentrations on PBMCs (Figure 5), the validation of the effect of 10 µg/mL conA on PBMCs by testing more biological replicates produced similar results (Figures 6 and S3). ConA treatment increased the mean relative cell count of CD4+ T-helper cells significantly from 46.93% to 54.81% (*p* < 0.05) (Figures 6a and S3a,d). Furthermore, conA increased the mean relative cell count of CD4+ CD25+ activated T-helper cells from 5.33% to 8.44% (*p* < 0.01) (Figures 6b and S3b,d). Moreover, the mean relative cell count of CD28+ T-cells increased significantly after conA treatment from 76.79% to 88.54% (Figures 6d and S3c,e), indicating T-cell proliferation.

In summary, conA induced T-cell activation and proliferation and can be used as a positive control.

#### *3.4. Immune Cell Phenotyping Using Flow Cytometry*

To assess the percentage of immune cell subsets, the following gating strategy was used (Figure 7a–d). First, lymphocytes were gated with a SSC/FSC plot (Figure 7a). From the lymphocyte population, only live cells were considered for further analysis (Figure 7b). Doublets were gated out (Figure 7c). Immune cell subsets were analyzed with antibodies labeling leukocytes (CD45-FITC), thrombocytes (CD41/CD61-(R)PE), T-helper cells (CD4- SPRD), cytotoxic T-cells (CD8-APC), T-cells (CD28-PE and CD3-APC), and activated T-cells (CD25-FITC) (example CD45-FITC; Figure 7d and Table 1).

**Figure 6.** Effects of conA on activation and proliferation of CD4+ T-helper cells and CD28+ T-cells. The effect of 10 µg/mL conA on the activation and proliferation of T-helper cells relative to the vital lymphocyte population was assessed after 24 h of cultivation. (**a**) CD4+ T-helper cells; (**b**) CD4+CD25+ activated T-helper cells; (**c**) CD28+ T-cells. A total of 20,000 cells were recorded on a BD Canto II flow cytometer. DAPI was used as a viability marker. Data represent eight biological replicates and two technical replicates each. A box-and-whisker plot is displayed. Significance is shown as \*\*, *p* < 0.01; \*, *p*

*3.4. Immune Cell Phenotyping Using Flow Cytometry*

As shown by testing two different conA treatment concentrations on PBMCs (Figure 5), the validation of the effect of 10 µg/mL conA on PBMCs by testing more biological replicates produced similar results (Figure 6 and Figure S3). ConA treatment increased the mean relative cell count of CD4+ T-helper cells significantly from 46.93% to 54.81% (*p* < 0.05) (Figure 6a and S3a,d). Furthermore, conA increased the mean relative cell count of CD4+CD25+ activated T-helper cells from 5.33% to 8.44% (*p* < 0.01) (Figure 6b and S3b,d). Moreover, the mean relative cell count of CD28+ T-cells increased significantly after conA treatment from 76.79% to 88.54% (Figure 6d and S3c,e), indicating T-cell proliferation.

In summary, conA induced T-cell activation and proliferation and can be used as a

To assess the percentage of immune cell subsets, the following gating strategy was used (Figure 7a–d). First, lymphocytes were gated with a SSC/FSC plot (Figure 7a). From the lymphocyte population, only live cells were considered for further analysis (Figure 7b). Doublets were gated out (Figure 7c). Immune cell subsets were analyzed with antibodies labeling leukocytes (CD45-FITC), thrombocytes (CD41/CD61-(R)PE), T-helper cells (CD4-SPRD), cytotoxic T-cells (CD8-APC), T-cells (CD28-PE and CD3-APC), and acti-

vated T-cells (CD25-FITC) (example CD45-FITC; Figure 7d and Table 1).

< 0.05. NS: not significant. Significance was analyzed using an unpaired Student's *t*-test.

positive control.

**Figure 7.** Example of the gating strategy for chicken PBMCs (**a**) *y* axes: SSC-A, *x* axes: FSC-A, the egg-shaped gate represents the lymphocyte gate; (**b**) *y* axes: DAPI, *x* axes: FSC-A, DAPI was used as a live/dead marker, the rectangle gate represents the vital lymphocyte population; (**c**) *y* axes: FSC-H, *x* axes: FSC-A, the rectangle gate represents the single cell population out of the vital lymphocyte population in B; (**d**) *y* axes: CD45-FITC, *x* axes: FSC-A, the two rectangle gates represent the CD45 high leukocyte and CD45-low thrombocyte populations out of the single cell and vital lymphocyte populations in B and C. Data represent one biological replicate. A total of 20,000 cells were recorded on a BD Canto II flow cytometer.


**Table 1.** Antibodies with the conjugated fluorophore, its isotype, and the concentration used after antibody titration.

All antibodies used in this study were titrated and evaluated by mean fluorescence intensity (MFI) to obtain optimal concentrations for the staining assays (Table 1 and Figure S4). The following antibody sets were used: CD45-FITC, CD41/CD61-(R)PE, CD3-APC; CD4- SPRD, CD28-PE, CD25-FITC; CD8-APC, CD28-PE, CD25-FITC.

#### **4. Discussion**

To investigate the mode of action of immunomodulating compounds such as pathogens, drugs, or feed additives, e.g., probiotics, primary cell culture systems are inevitable and represent a good alternative to in vivo models. Our aim was the establishment of an in vitro cell culture model with chicken PBMCs, without contaminating thrombocytes, to assess the properties of immunomodulating compounds, especially feed additives. The study will help to increase knowledge about precise mechanisms of action, which will allow the development of targeted nutrition strategies, prevent infectious diseases and the usage of antimicrobials, and further improve the health in poultry production. Chicken PBMCs are often isolated using common methods such as density gradient centrifugation using ficoll, which result in the isolation of immune cells contaminated by nucleated thrombocytes and erythrocytes [11–14]. Therefore, the isolation of PBMCs without either nucleated thrombocytes or erythrocytes is complex and difficult in avian species. Concerning this matter, a dextran–ficoll separation method was recently published [25]. However, PBMCs were not cultivated for a longer period after the isolation. Here, we present a robust protocol for longer cultivation of chicken PBMCs, which will enable functional in vitro studies in chicken PBMCs.

#### *4.1. Blood Sampling*

Our data suggest that, despite a higher relative thrombocyte and a lower relative leukocyte count, the cell number and viability in Na–citrate tubes was highest compared to commercial K3EDTA and Li–heparin tubes, as well as to 200 µL 0.5 M EDTA. In other species, it has been shown that a slightly purer population of PBMCs is obtained by using EDTA as an anticoagulant compared to heparin [27]. Furthermore, for the purification of mononuclear cells from other sources than peripheral blood, heparin was shown to promote clumping and pre-activation of unstimulated control cells [28]. Citrate as an anticoagulant may result in better quality of RNA and DNA compared with other anticoagulants and furthermore produce a higher yield of mononuclear cells, which is consistent with the results of the current study. Li–heparin, which revealed the highest leukocyte and the lowest thrombocyte count in this study, is reported to affect T-cell proliferation and to bind to many proteins. RNA yields from EDTA-treated blood have been shown to be higher compared to heparin-sampled blood [29]. Furthermore, EDTA was shown to affect PBMCs by a progressive and irreversible loss of antigen-specific lymphoprolerative responses when PBMCs were exposed to EDTA for a longer time period [30]. Therefore, ethyleneglycol-bis- (beta-aminoethylether)tetraacetate (EGTA) was suggested [30]. However, in the current study, K3EDTA revealed lower relative lymphocyte and leukocyte counts and a lower count of total isolated vital cells. Taking these findings from the literature and our results into account, we choose Na–citrate as the best choice for anticoagulation.

#### *4.2. PBMC Isolation Method*

The reduction or elimination of thrombocytes, which are a large part of chicken PBMCs, is of high importance in immunomodulatory studies, since chicken thrombocytes have been shown to play roles in inflammation and antimicrobial defence [24,31,32]. Initially, we used a combined slow-speed centrifugation with subsequent density gradient centrifugation using ficoll, which was modified according to Viertlboeck and Göbel (2007) [17], Lavoie et al. (2005) [14], Sundaresan et al. (2005) [26], and Gogal et al. (1997) [12]. In comparison to those studies, we were not able to reduce the thrombocyte and erythrocyte counts in our samples (Figure 2). Other studies used only density gradient centrifugation to isolate PBMCs from chickens, without considering the thrombocyte count [10,27,33,34]. However, in this study, a method by Jergovi´c et al. (2017) [25] was additionally tested. The method included the use of dextran, a slow-speed centrifugation step and a density gradient centrifugation step afterwards. Dextran, a high-molecular polysaccharide, is often used for the purification of neutrophils [35]. Other studies used 1% methylcellulose instead of dextran [36,37]. Our results showed a significant decrease of the relative cell count of thrombocytes by about

14% when using the dextran–ficoll separation method (Figure 2b). However, compared to the study of Jergovi´c et al. (2017), where the thrombocyte count was about 2.85% compared to 96.9% of PBMCs, we found a thrombocyte count of 27.23%. However, the thrombocyte count was lower compared to the slow-speed centrifugation and ficoll separation method, in which we obtained 41.19% thrombocytes. However, the leukocyte count remained unaffected in our study (Figure 2a). One possible explanation is the dextran used in the different studies, which was provided by different companies. Furthermore, we centrifuged for 20 min after the addition of dextran, because we could not discriminate the phases after 10 min of centrifugation. In the study of Jergovi´c et al. (2017), the centrifugation step was 10 min. All in all, we could reduce the thrombocyte count in our samples and start the cell culture with a low number of thrombocytes when we used the combined separation method based on the addition of dextran, a slow-speed centrifugation step, and a density gradient centrifugation.

#### *4.3. PBMC Culture Conditions*

We tested sera from different species as cell culture supplementation. Fetal calf serum is very often used even for nonbovine species such as mice, humans, and chickens [38–41]. Since we detected an increased number of thrombocytes in the cell culture with RPMI-1640 medium supplemented with FCS compared to supplementation with chicken serum, we do not recommend the addition of FCS. Thrombocyte cytokine expression was reported up to 18 h in culture [42], which could interfere with assays that are carried out to investigate immunomodulatory functions. DaMatta et al. [22] and Lam [43] showed that thrombocytes cultured in DMEM with 10% fetal bovine serum displayed cytoplasm and chromatin condensation and were suggested to suffer an apoptotic cell death in culture of about 70% after 24 h and 85% after 48 h of cultivation. Apoptosis of thrombocytes was additionally reported by Kaspers and Kaiser (2014), who showed that cells die by apoptosis after 48–72 h [44]. Despite the fact that thrombocytes are active for a short period, our aim was to reduce the thrombocyte count as much as possible to ensure the least interference with subsequent immunomodulatory assays. However, in our study, thrombocytes were not cultured under agitation to prevent adherence. Interestingly, there seemed to be a difference between the adherence in FCS-supplemented medium to the adherence in porcineand chicken-serum supplemented media. Whether the serum influences the adhesion of thrombocytes still needs to be elucidated. In addition to the reduced thrombocyte count in cultures supplemented with chicken serum, we found that supplementation with chicken serum resulted in higher counts of vital leukocytes. Therefore, we showed that chicken serum is the most suitable supplement for long-term chicken PBMC cultures with reduced thrombocyte counts.

As L-glutamine is very unstable in medium, the supplementation of additional Lglutamine has been reported in different studies for PBMC culture in RPMI-1640 medium [10,45–47] in different concentrations. In our study, we added L-glutamine in a concentration of 2 mM to the cell cultures. We could not detect differences for cell viability or cell counts between media with and without additional supplementation of L-glutamine. Hence, we did not include the supplementation of additional L-glutamine in our protocol.

It is of high importance to validate the response capacity of cultured chicken immune cells to a stimulus. We chose conA, since it is a well-reported T-cell mitogen [12,13,48–50]. We observed a clear effect on the proliferation and activation of CD8+ cytotoxic T-cells (Figure 5a,b) and CD4+ T-helper cells (Figure 5c,d) in the presence of conA. Furthermore, we validated the effect on T-cell activation and proliferation (Figure 6a–c and Figure S3a–f). In a study by Alvarez et al. (2020), it was shown that chicken splenocytes have a low viability after conA treatment in a concentration of 1 µg/mL for 3 days [51]. In fact, the cell viability decreased also in our study after treatment with 10 µg/mL conA, but not with 5 µg/mL. However, we aimed initially to validate the responsiveness of the chicken PBMCs in culture as a positive control. Therefore, we were able to validate the presented protocol for a system to culture chicken immune cells.

#### **Summarized brief protocol:**


#### **5. Conclusions**

After several optimization steps, we established a valid in vitro cell culture system to assess the direct effects of potentially immunomodulating compounds.

Here, we tested and optimized blood sampling, PBMC isolation, PBMC culture, and immune cell phenotyping of chicken PBMCs using monoclonal antibodies. This cell culture system will help to evaluate and understand the underlying mechanisms of the immunomodulatory properties of potentially immunomodulating compounds, e.g., feed additives, which could serve as potential alternatives to antibiotics, and may further serve as an alternative to animal testing in vivo. Besides testing feed additives, immunomodulation by challenges with pathogenic bacteria like *Salmonella* or *Campylobacter*, viruses, or particular drugs could be tested in our established chicken immune cell assay to model adaptive immune responses in vitro.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/ani11123600/s1, Figure S1: Influence of the anticoagulants K3EDTA, Na-citrate, and Li-heparin as well as 200 µL 0.5 M EDTA on leukocyte and thrombocyte count in isolated PBMCs. After cell isolation, cells were resuspended in PBS/EDTA and counted with Tali image-based cytometer and the viability was assessed using propidium iodide (PI). 1 <sup>×</sup> <sup>10</sup><sup>6</sup> cells/mL in duplicates per anticoagulant were labelled with the thrombocyte marker CD41/CD61-(R)PE and the leukocyte marker CD45-FITC. 20,000 cells were recorded on a BD Canto II flow cytometer. DAPI was used as a live/dead marker. Data represent 5 biological replicates. A box-and-whisker plot is displayed. Significance is shown as + *p* < 0.1; \*\* *p* < 0.01. NS: not significant. Significance was analyzed using an unpaired Student's *t*-test, Figure S2: Comparison of sera in cell culture of chicken PBMCs. Isolated PBMCs were cultured in RPMI-1640 medium with glucose and 100 U/mL penicillin and 100 µg/mL streptomycin and either 10% chicken, 10% porcine, or 10% fetal calf serum. The relative cell count of lymphocytes and vital lymphocytes was assessed after 24 h of cultivation (a) Gated lymphocytes relative to the measured cells; (b) Vital cells relative to the lymphocyte population. 20,000 cells were recorded on a BD Canto II flow cytometer. Data represent 7 biological replicates and two technical replicates each in two independent experiments. A box-and-whisker plot is displayed. Significance is shown as \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001. NS: not significant. Significance was analyzed using an unpaired Student's *t*-test, Figure S3: Effect of conA on PBMCs. (a–c) the upper row shows the relative cell count of CD4+ (a), CD4+CD25+ (b), and CD28+ (c) cells in untreated PBMCs as negative controls; (d–f) the low-er row displays the relative cell count of CD4+ (d), CD4+CD25+ (e), and CD28+ cells in conA-treated PBMCs as positive controls for activation and proliferation. After 24 h of cultivation, the relative cell count was investigated on a BD Canto II flow cytometer. The populations of interest out of the vital lymphocyte population, are represented in rectangle gates. Data represent 1 bio-logical replicate. 20,000 cells were recorded, Figure S4: Example of a serial antibody dilution. 1 <sup>×</sup> <sup>10</sup><sup>6</sup> PBMCs were labelled with CD4-SPRD in different concentrations. The y-axes of each plot represent the CD4-SPRD fluorescence signal and the x-axes show FSC-A. (a) unlabelled negative control; (b–f) cells labelled

with CD4-SPRD in a concentration 1:25 (b), 1:50 (c), 1:100 (d), 1:200 (e), and 1:400 (f). CD4-SPRD in a concentration of 1:25 will be used in further experiments since this concentration enabled clear discrimination between the positive and negative population. The relative cell count of CD4-SPRD labelled cells was investigated on a BD Canto II flow cytometer. The populations of interest out of the vital lymphocyte population, are represented in rectangle gates. Data represent 1 biological replicate. 20,000 cells were recorded.

**Author Contributions:** Conceptualization, G.A.B. and S.K.-R.; Data curation, F.L.; Formal analysis, F.L.; Investigation, F.L. and M.S.; Supervision, D.H., G.A.B. and S.K.-R.; Writing—original draft, F.L.; Writing—review and editing, D.H., G.A.B. and S.K.-R. All authors will be informed about each step of manuscript processing including submission, revision, revision reminder, etc. via emails from our system or assigned Assistant Editor. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Federal Ministry of Education and Research, Bundesministerium für Bildung und Forschung (BMBF, FKZ 031B0743).

**Institutional Review Board Statement:** The study was approved by the local State Office for Health and Social Affairs, Landesamt für Gesundheit und Soziales Berlin (LaGeSo, T 0151/19).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data supporting reported results will be provided upon request.

**Acknowledgments:** We thank K. Männer (Freie Universität Berlin, Berlin, Germany) for providing Cobb500 broiler chicken. We acknowledge support by the German Research Foundation (DFG) and the Open Access Publication Fund of Humboldt-Universität zu Berlin.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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