Modulation of Immunity, Antioxidant Status, Performance, Blood Hematology, and Intestinal Histomorphometry in Response to Dietary Inclusion of Origanum majorana in Domestic Pigeons’ Diet

This experiment was conducted to evaluate the effect of adding Origanum majorana (OM) powder to domestic pigeon diets on growth performance, feeding and drinking behaviour, blood hematology, blood biochemical parameters, blood inflammatory and oxidative markers, carcass characteristics, the weights of lymphoid organs, and and intestinal cecal, and bursa of Fabricius histology. A random distribution of fifty-four unsexed pigeon squabs (30 days old, average body weight; 321 g ± 7.5) into three groups was done. The first group was fed the grower basal diet without adding OM powder, while OM powder was added at levels of 0.5 and 1% to the basal diets of the second and third groups, respectively. The changes in growth performance parameters and feeding and drinking behavior under OM powder’s effect were insignificant. However, the lymphoid organs (spleen and thymus) significantly increased in weight (p < 0.05) in the OM-fed groups. Moreover, blood examination showed positive responses to OM powder in terms of blood cell counts (RBCs andWBCs), and the values of hemoglobin, hematocrit, mean corpuscular volume, lymphocyte numbers, levels of globulin, and glutathione peroxidase enzyme were significantly increased. The numbers of heterophils, the ratio of heterophil to lymphocyte, malondialdehyde levels were reduced (p < 0.05). Histomorphometry examination revealed increases in intestinal villi height, cecal thickness, and bursal follicle area and number. These results indicated that adding OM powder to the pigeon diet may improve their immunity, increase their antioxidant status, and correct some hematological disorders.


Introduction
Recently, the animal industry has expanded worldwide, including the production of livestock, companion animals, and poultry. Among these sectors, the most significant

Birds, Diets, and Design
Fifty-four unsexed pigeon squabs (age: 30 days; average body weight; 321 g ± 7.5) from a local source (El-Matieuh rural villages-Assiut, Egypt) were randomly distributed into three groups (n = 18, 3 replicates, n = 6/group). The grower basal diet without supplementing OM powder was offered for the 1st group, while OM was added to the basal diets of the 2nd and 3rd groups at levels of 5 and 10 g/kg diet, respectively. The mashed form of the diet was used. The ingredients of the grower basal diet, which was formulated based on the recommendations of [21,22], are shown in Table 1. The temperature was adjusted according to the bird's needs (18-23 • C). Natural and mechanical ventilations were supplied. Free access to both water and feed was provided. The schematic cartoon (Figure 1) of the experimental study was designed by BioRender.com.

Growth Performance
The body weight was recorded for each bird at the study beginning. After that, body weight (individual and cumulative), along with the feed intake of pigeon squabs, was recorded weekly. The feed conversion ratio (FCR), relative growth rate (RGR), and Euro-

Growth Performance
The body weight was recorded for each bird at the study beginning. After that, body weight (individual and cumulative), along with the feed intake of pigeon squabs, was recorded weekly. The feed conversion ratio (FCR), relative growth rate (RGR), and European production efficiency index (EPI) were calculated [23,24].

Feeding and Drinking Behavior Assessment
During the experiment, feeding and drinking behaviors were observed [25]. Pigeons involved in eating behavior (act\30 min) were recorded by observing their contact with feed and water, following the recommendation of Spudeit et al. [26].

Carcass Parameters and Lymphoid Organs
The experimental period was 45 days. At the experimental end, 3 birds per group were euthanized by slaughter after their random selection and weighing. The lymphoid organs (spleen, bursa of Fabricius, and thymus), liver, heart, and gizzard were weighed and expressed as a percentage of the live body weight [27].

Blood Examination
During slaughtering, blood was collected from the cervical vein and preserved in heparinized and non-heparinized tubes (Vacutainer, Becton Dickinson, Stuart, FL, USA).

Blood Hematology
The heparinized tubes were used for evaluating red blood cells (RBCs), white blood cells (WBCs), blood hemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCH), hematocrit value (HCT), and differential white blood cell count. The ratio of heterophils/lymphocytes was calculated. Using a hemocytometer and staining blood films with the Wright-Giemsa stain, numbers of RBCs and WBCs were counted.

Blood Biochemical Parameters
The blood samples in the other tubes were centrifuged for 15 min, at 3000 rpm at 4 • C, and kept at −20 • C till further analysis. Total proteins, albumin, globulin, total cholesterol, urea, and creatinine were determined by using commercial kits (Biotechnology Company, Assiut, Egypt).

Histomorphometry Analyses
Eight pigeons were randomly chosen to collect samples from the intestine (duodenum), cecum, and bursa of Fabricius. Immediately after slaughtering, samples were dissected, fixed in Bouin's fluid, alcohol-dehydrated, cleared in methyl benzoate, and paraffin wax-embedded. After that, cutting at 4-5 µm thickness and staining with Harris hematoxylin were done [28]. Measurements of duodenal villi height/um, wall thickness/um, cecal muscle thickness/um, and follicle numbers/500 um and follicle area/um 2 of the bursa of Fabricius were done using ImageJ software. Measurement data are described as means ± SDM.

Statistical Analyses
Origanum majorana's effects on performance, behavior, carcass characteristics, blood hematology, blood parameters, and inflammatory and oxidative markers in pigeons were analyzed using SPSS [26.0]. For treatment comparison, Duncan's multiple range test was used. The 5% level was used as an indication of significance [29]. The statistical model was Y ij = µ + T i + E ij , where Y ij = response variables; µ = the overall mean; T i = treatment effect; E ij = the experimental error.

Growth Performance Parameters
Origanum majorana powder's effect on the performance of pigeon squabs is shown in Table 2. A numerical increase in growth performance parameters under the OM powder effect was observed. However, no significant differences were detected among groups in terms of body weight, weight gain, feed intake, feed conversion, production index, or relative growth rate.

Feeding and Drinking Behavior
The assessment of pigeons' feeding and drinking behavior under the effect of Origanum majorana powder is indicated in Figure 2. Adding OM powder increased the birds' feeding and drinking acts, but this increase was insignificant.
Life 2023, 13, x FOR PEER REVIEW 6 of 16

Feeding and Drinking Behavior
The assessment of pigeons' feeding and drinking behavior under the effect of Origanum majorana powder is indicated in Figure 2. Adding OM powder increased the birds' feeding and drinking acts, but this increase was insignificant.

Carcass Parameters and Lymphoid Organs
The effect of Origanum majorana powder on the carcass parameters and lymphoid organs is presented in Table 3. The dressing percentages for liver, gizzard, bursa, and heart did not show any significant changes among the experimental groups. However, there was a significant increase in spleen and thymus relative weights (p ≤ 0.05) in the OM

Carcass Parameters and Lymphoid Organs
The effect of Origanum majorana powder on the carcass parameters and lymphoid organs is presented in Table 3. The dressing percentages for liver, gizzard, bursa, and heart did not show any significant changes among the experimental groups. However, there was a significant increase in spleen and thymus relative weights (p ≤ 0.05) in the OM powder groups.

Hematological Parameters
The Origanum majorana powder effect on blood hematology is shown in Table 4.

Blood Biochemical Parameters, Serum Inflammatory Markers, and Oxidative Markers
Blood parameters and markers affected by adding Origanum majorana powder to pigeon diets are presented in Table 5. Supplementation of OM powder had no effect on serum total protein, albumin, creatine, or urea levels. In contrast, globulin was higher [p < 0.05], and the albumin-to-globulin ratio was lower [p < 0.05] in the treated groups. Moreover, adding OM powder to pigeon diets at both levels reduced [p < 0.05] the cholesterol level.

Bursal Follicle Histomorphometry
The number of follicles was about 8.8 follicles per 500 µm in the control group. By adding OM powder, the number was increased to about 9 follicles per 500 µm with 0.5% OM and about 12 follicles per 500 µm with 1% OM. Moreover, the area of the follicle in the control group was about 249.500 ± 3.190 µm 2 and increased to about 251 ± 5.332 µm 2 in the 0.5% OM group. The largest follicle area was demonstrated with a 1% OM group [about 254.500 ± 5.816 µm 2 ] [ Figures 4 and 5].

Bursal Follicle Histomorphometry
The number of follicles was about 8.8 follicles per 500 μm in the control group. By adding OM powder, the number was increased to about 9 follicles per 500 μm with 0.5% OM and about 12 follicles per 500 μm with 1% OM. Moreover, the area of the follicle in the control group was about 249.500 ± 3.190 μm 2 and increased to about 251 ± 5.332 μm 2 in the 0.5% OM group. The largest follicle area was demonstrated with a 1% OM group [about 254.500 ± 5.816 μm 2 ] [ Figures 4 and 5].

Effect of Origanum majorana Powder on Growth Performance Characteristics
The numerical values showed that pigeons fed OM powder had higher feed intake, body weight, weight gain, and relative growth rate than the control group; no significant differences were detected among the three groups. Our results agreed with those obtained by Khattab et al. [30]; they investigated feeding different levels of Origanum majorana, Pimpinella anisum, and Mentha piperita in relation to growth performance improvements in broiler chicks. They indicated that neither feed conversion nor body weight were affected by adding Origanum majorana to the broiler diet. Moreover, Ali [19] reported a reduction in the daily feed intake of broilers by supplementing Origanum majorana at levels of 0.5, 1.0, and 1.5%. Contrary to our results, Shawky et al. [20] and Abdel-Wahab [31] indicated that adding dietary Origanum majorana to broiler diets improved their weight and weight gain.
No clear explanation was found for the variant effect of Origanum majorana on growth performance among the different research works. Still, it may be related to the variation in the level used in each experiment or other factors, such as stress. Vase-Khavari et al. [32] indicated that the efficacy of herbal plants and probiotics is correlated to different factors, such as their level and concentration used, the composition of the diet, environmental factors, and the hygiene of the poultry houses.

Effect of Origanum majorana Powder on Feeding and Drinking Behavior
Bird physiological conditions, diet composition, feeder space, and heat stress can affect birds' feeding and drinking behavior [33][34][35]. The assessment results for feeding and drinking behavior were consistent with the performance results, as OM powder increased feeding and drinking behavior, but this increase was insignificant. Ramadan [36] and Harrington et al. [37] indicated that aromatic herbs and their extracts could increase feeding behavior in poultry. Ramadan [36] reported the presence of a negative correlation between fear and feeding behaviors in poultry, as a decrease in the fear response will increase feeding behavior. Scientists suggested that aromatic plants have a depressing effect on neural activity through the activation of GABA receptors, resulting in reduced fear behavior and increased feeding times [36][37][38]. The fear response was not assessed in our experiment.

Effect of Origanum majorana Powder on Growth Performance Characteristics
The numerical values showed that pigeons fed OM powder had higher feed intake, body weight, weight gain, and relative growth rate than the control group; no significant differences were detected among the three groups. Our results agreed with those obtained by Khattab et al. [30]; they investigated feeding different levels of Origanum majorana, Pimpinella anisum, and Mentha piperita in relation to growth performance improvements in broiler chicks. They indicated that neither feed conversion nor body weight were affected by adding Origanum majorana to the broiler diet. Moreover, Ali [19] reported a reduction in the daily feed intake of broilers by supplementing Origanum majorana at levels of 0.5, 1.0, and 1.5%. Contrary to our results, Shawky et al. [20] and Abdel-Wahab [31] indicated that adding dietary Origanum majorana to broiler diets improved their weight and weight gain.
No clear explanation was found for the variant effect of Origanum majorana on growth performance among the different research works. Still, it may be related to the variation in the level used in each experiment or other factors, such as stress. Vase-Khavari et al. [32] indicated that the efficacy of herbal plants and probiotics is correlated to different factors, such as their level and concentration used, the composition of the diet, environmental factors, and the hygiene of the poultry houses.

Effect of Origanum majorana Powder on Feeding and Drinking Behavior
Bird physiological conditions, diet composition, feeder space, and heat stress can affect birds' feeding and drinking behavior [33][34][35]. The assessment results for feeding and drinking behavior were consistent with the performance results, as OM powder increased feeding and drinking behavior, but this increase was insignificant. Ramadan [36] and Harrington et al. [37] indicated that aromatic herbs and their extracts could increase feeding behavior in poultry. Ramadan [36] reported the presence of a negative correlation between fear and feeding behaviors in poultry, as a decrease in the fear response will increase feeding behavior. Scientists suggested that aromatic plants have a depressing effect on neural activity through the activation of GABA receptors, resulting in reduced fear behavior and increased feeding times [36][37][38]. The fear response was not assessed in our experiment.

Effect of Origanum majorana Powder on Carcass Characteristics and Lymphoid Organs
The dressing percentage and liver, heart, gizzard, and the bursa of Fabricius did not differ among pigeons supplemented with OM powder and the control group. The absence of the OM effect on carcass traits was expected, because of the similar growth performances among the three groups. Several studies reported that adding herbal plants either in powder or oil extract did not affect broilers' internal organ weights [39]. Moreover, Shawky et al. [20] reported that the weights of the liver, heart, and gizzard did not show any significant difference when dietary supplementation of OM was used in broiler diets.
The avian immune response can be affected by several extrinsic or intrinsic factors; one of the significant extrinsic factors affecting bird immunity is the diet and its composition [40]. The lymphoid organs responsible for avian immunity include primary and secondary organs. The primary organs are the thymus and bursa of Fabricius [41]. These organs are the sites for maturation, differentiation, and immunocompetence of T and B types of lymphocytes [42]. Functional T and B cells depart from the primary to the secondary lymphoid organs, including the bone marrow and spleen [43]. Ahsan et al. [44] indicated that the relative weight of lymphoid organs reflects the bird's immunity status. In our experiment, the thymus and spleen showed significant increases in their weights with the OM powder supply, which means that the pigeon immune responses were improved under the effect of Origanum majorana. The impact of OM may be related to the presence of flavonoids and phenolics, with their antibacterial, antioxidant, and immune-modulating effects. However, Ali [19] indicated that the spleen weight in broilers was not affected by adding variant Origanum majorana powder levels.

Effect of Origanum majorana Powder on Blood Hematology
Nutrition's effect on bird physiology and metabolism can be indicated by examining blood hematology and biochemical blood parameters [45]. As a result, adding Origanum majorana to birds' diet affected the pigeons' hematology. Origanum majorana powder significantly increased both RBC (with 1% OM) and WBC counts, which are responsible for oxygen transfer and protection against infection, respectively. The elevation in the numbers of RBCs with OM may refer to its antioxidant activity, preventing lipid peroxidation in blood cell membranes. Moreover, the increased WBC number may be related to thymol (active component in OM), which is responsible for immune response enhancement [46]. The favorable effect of Origanum majorana on blood hematology also included raising the values of Hb, HCT, MCV, and lymphocyte percentage. The positive impact of OM on Hb may be related to its higher content of iron, which is considered an essential nutrient for hemoglobin production [47]. At the same time, the increased MCV suggests that OM has a hematopoietic impact, as RBCs (both new and young) are more prominent and contain a higher Hb amount [48]. According to Altan et al. [49], the H/L ratio is a valuable tool for explaining the different stress factors to which birds are exposed. In our experiment, adding OM powder to the pigeon diet decreased heterophils, increased lymphocytes, and, consequently, decreased the H/L ratio, which means that it may play a vital role in alleviating bird stress. Stef et al. [50] indicated that lymphocytosis could enhance interferon production. Furthermore, it was reported that hematological disorders caused by toxins, metals, or bacterial infections in different animals could be corrected by adding herbs to their diets [51,52].

Effect of Origanum majorana Powder on Biochemical Parameters, Inflammatory and Oxidative Markers
Total protein, albumin, creatinine, and urea were not changed by adding OM powder. Similarly, Shawky et al. [20] reported no significant difference in total protein and urea between the OM-supplemented group and the control group. Globulin was increased, while the ratio of albumin/globulin was decreased with OM powder. The same author suggested that the significant elevation in globulin indicates the Origanum's ability to enhance the immunity of broiler chicks. Origanum majorana has been reported to induce hypocholesterolemia [15,31]. Our cholesterol result agreed with these reports. It was indicated that carvacrol and thymol present in OM could reduce cholesterol levels by inhibiting hepatic 3-hydroxy-3-methyl-glutaril198 CoA reductase [46,53].
In our experiment, interleukin 6 was not affected, while tumor necrosis factor-α levels tended to be increased with OM powder. Substances that promote leukocytosis may stimulate cytokine secretion from these cells, such as interleukin 6 and TNF-α [54]. Therefore, the tendency of TNFα to increase may be a compensatory reaction due to leukocytosis. Contrary to our results, Arranz et al. [55] indicated that the essential oil extracted from Origanum majorana has anti-inflammatory activity, as it contains terpineol and sabinene hydrate, which adversely affect cytokine production.
Malondialdehyde is considered a lipid peroxide. When its level increases, it can impair nucleic acid metabolism and function, destroy membrane proteins, and lead to autoimmune diseases [56]. To overcome lipid peroxidation and toxic free radicals, the secretion of some enzymes such as superoxide dismutase and glutathione peroxidase is enhanced, which play essential roles in the body's defense mechanism against peroxidation. In the current study, OM powder significantly decreased the MDA level and increased the glutathione peroxidase level. The association between OM's richness in phenolics and flavonoids (such as carnosol, carnosic acid, and hydroxycinnamic acid) and its antioxidant effect was investigated [9]. Therefore, Origanum majorana can play an essential role in maintaining the normal physiology, production, health, and welfare of animals.

Effect of Origanum majorana Powder on Duodenal, Cecal, and Bursal Follicle Histomorphometry
Feed utilization efficiency depends on feed digestion and absorption, which are affected by the intestinal surface [57]. Our histological results indicated that the intestinal villi length was slightly increased with OM powder. Abdelatty et al. [58] reported that improvements in growth performance are associated with increases in intestinal villi length and intestinal absorption. It was observed that the slight increase in intestinal villi length in the Origanum groups was associated with a numerical increase in body weight.
In the muscular layer, the cecal wall was thickened with OM powder supplementation. Scarce studies take morphometrical measurements for the cecum, despite its role in immune response, water absorption, digestion, and fermentation [59][60][61].
As a primary lymphoid organ, the bursa has a crucial role in B cell maintenance and establishment [41]. The area and number of follicles in the bursa of Fabricius were increased with OM powder. Attia et al. [62] reported that induction of humoral immunity and B lymphocyte production is associated with increases in the area of the bursal follicle.

Limitations of the Study
There was no possibility to confirm the results through q-PCR or to perform LC-MS analysis of serum, in order to see whether feeding OM increased the metabolites related to brain modulators, antioxidants, and immune modulators. In addition, the fear response was not assessed in our experiment in order to confirm as to whether it is correlated with increased feed intake. However, this research is essential and acts as the first step to realizing the influence of OM on improving the health status and welfare of pigeons; therefore, further experiments are required to emphasize the neural activity of birds.

Conclusions
Adding OM powder to the pigeon diet increased the relative weights of the lymphoid organs (spleen, thymus, and the number and area of bursal follicles), the WBC count, the lymphocyte count, and the serum globulin level. These effects suggest that OM powder may enhance bird immunity. The increased Hb, HCT, and MCV may have a hematopoietic effect. A decreased H/L ratio and MDA, as well as increased GPx, indicated that OM powder might have an antioxidant effect. Histological examination of the intestine suggested that nutrient absorption may be affected by adding OM powder, but this point needs further investigation. In conclusion, adding OM powder to the pigeon diet may play an essential role in alleviating stress, correcting some hematological disorders, and maintaining the physiology, metabolism, health, and welfare of birds, but more future work is still required.