THE EFFECTIVENESS OF SOME MEDICINAL PLANTS ON BODY PERFORMANCE, HEMATOLOGICAL, ILEUM MORPHOLOGY AND IMMUNE STATUE OF JAPANESE QUAIL

This study was aimed to investigates and compares the effect of medicinal plants extract: Hero flowers ( Alcea kurdica ) in the north of Iraq, Thyme ( Thymus bovie ), and Bay leaf ( Laurus nobilis ) extract with two antibiotic drugs Rifaximine and Ranitidine in quail’s drinking water by emphasizing the productive performance, and physiological status of the quails at grower and developer stages till 10 th weeks of age, a total of 90 quails were distributed into six treatments, each treatment with three replicates of 15 chicks in each. Data analysis showed significantly increased body weight gain (237.5, 224.1, and 234.9) g for Hero, thyme, and Bay leaf, respectively. In comparison with the control (188.3) g. quail’s drunk plant extract mixed water had better hematological and the ileum profiling (goblet cells numbers, Villus height, and Crypt depth) and significantly higher serum concentrations of total protein, globulin, and antibodies titers against some bird virus diseases in comparison with positive control. Also, significantly lower level of albumin, ileum content of E. Coli, and Enterococcus count in plant treated quails in comparison with the control T0.


INTRODUCTION
Poultry meat is a very important source of protein; therefore, in many countries increased capital has been invested in the poultry industry and especially in quails. As a member of the pheasant family, the quail known as the smallest bird. The amount of protein in the meat is 5-10 percent more than other birds and ruminants (15). Quail meat is also a good source of niacin, phosphorus, copper, thiamine, riboflavin, vitamin B6, zinc, iron, and selenium (32). Nowadays, natural diet additives gained more popularity because of increased health issues regarding synthetic nutrients. Therefore, researcher's persuasion for alternatives to replace synthetic sources (antibiotics and drugs) as diet enhancement in poultry farming becomes an important subject (1). Rifaximin and ranitidine were known as gastrointestinal-selective antibiotics with antiulcer and antimicrobial activities (22,23). But after the ban of nutritive antibiotics in the European Union, interests in plant additives as poultry feed ingredients become risen continuously (3,16). Medicinal plants have been known as a source of natural antioxidants, such as phenolic acids, flavonoids, and tannins (12,36). Numerous research works have shown the efficacy of these natural compounds in stopping the onset and/or progression of many human diseases (17). The curative enhancement efficacy of antioxidants from plants may be related to their role in counteracting reactive oxygen species (ROS) (34). Previous research also suggested the need of adding antioxidants to animal's diets to minimize lipid peroxidation and maximize productivity (8). Alcea kurdica (Gule Hero) and Thymus bovie (Thyme) are medicinal plants with antioxidant and antimicrobial activities, at which both of them have been used traditionally as antiinflammatory, anthelmintic, expectorant, antispasmodic, antiseptic agents because of flavonoids, polyphenols, and oxygenated Monoterpenoid contents (2,31). Laurus Nobilis (bay leaf) also known as a traditional medicinal plant with antioxidant activity because of phenolic and flavonoid contents at which its essential oil served as antifungal, the antibacterial activity against various gram positives, negative bacteria, and fungus species in the various extracts (10). Another study also reported that bay leaf essential oils have been used to increase appetite and enhance immunity (20). In that regard, on that regard, the present study was set to investigate and compare the effects of Hero flower, Thyme and bay leaf extracts in quail drinking water by emphasizing growth performance, intestinal morphology as well as immune, bacterial and physiological status.

MATERIAIS AND METHODS Sample collection and powder preparation
The plant samples were collected in a wide geographic range near Shaqlawa-Erbil/Iraq. Identification and authentication of the plants done by Biology Deptarment/ College of Education/ Salahaddin University-Erbil. The plant part: Hero flowers (A. kurdica), Bay leaf (L. Nobilis), and Thyme (T. Bovie) extracts were prepared as mentioned by Mombeini (25).

Body performance and histological traits
The birds from each replicate were randomly selected to measuring body weight, and gain, feed intake and Feed Conversion Ratio, water consumption, mortality, and feasibility or economic profit were calculated (30).

Estimation of hematological parameters
At the end of the study10 th weeks blood samples were collected from nine birds by cutting the jugular vein of each group using EDTA tubes for counting total Red Blood Cells and White Blood Cells by using Nutt & Herrick solution, blood collected in capillary tubes to estimate Packed Cell Volume, mean corpuscular volume, mean corpuscular hemoglobin concentration Mean corpuscular hemoglobin (MCH), and Drabkin's Reagent is used for the quantitative, colorimetric determination of hemoglobin (Hb) concentration in whole blood at 540 nm, heterophil and lymphocyte with Wright-Giemsa stains to determine the heterophils and lymphocytes (H/L) ratio (28). Serum protein and immunology profile Serum separated from collected blood and determined total protein, globulin, and albumin, according to the instructions of the kits included in the Buyer's Guide for Life Science Bio-compare. Thus, antibody titers against Gamboro, Newcastle, Infectious bronchitis viral diseases were measured by hemagglutination inhibition assay (HI) test (6). Ileum bacterial count and histological profile: At age 10th week, ileum contents were cautiously kept in sterile petri dishes at -20°C until analyses in the laboratory. One gram of each homogenized sample was collected and transferred into 10 ml sterile saline solution for dilution, then each sample was spread on selective agar plates as follows. De Man, Rogosa and Sharpe (MRS) agar medium was used for lactobacillus bacteria and the Nutrient and McConkey agars were utilized for E. Coli (19) and Enterococcus were cultured in semisolid Agar and incubated at 37°C for 24 h for the determining colonies. Intestinal ileum samples were dehydrated, cleared, and embedded in paraffin. Serial sections were cut at 5 µm and placed on glass slides. For all assays, sections were deparaffinized in xylene and rehydrated in a graded alcohol series. Sections were examined by light microscopy (35) for determining number of goblet cells, villus height, crypt depth and villus to crypt (V/C) ratio (30).

Statistical analysis
All data were analyzed using CRD (Completely Randomized Design) by the SAS institute program (33). Duncan's multiple range tests were used to compare differences among the treatments.

RESULTS AND DISCUSSION
Medicinal plants as diet additives in poultry farming have been used widely due to multiple biological activities that the herbs that play in stimulate the digestive system and enhancing growth rate (18). The results in Table 1 show that adding medicinal plant extract (Hero flower, bay leaf, and thyme) extracts in the drinking water of quail caused significantly (P≤0.05) higher body weight and body weight gain in T3, T5, and T4 groups. Data analysis also showed significantly (P≤0.01) higher economic profit of the marketing age of quails' drunk water mixed plant extracts compared with the other treated groups (T0, T1, and T2). As well as feed conversion ratio (FCR) improved in T5, T3, and T4 in comparison with T2, T1, and T0. While nonsignificant differences among all groups in feed intake. Previous researches reported that Hero flower (9), Thyme (21), and Bay leaf (26) extracts possess great antioxidant activity because of their phenolic, flavonoid, and monoterpenoid contents. A study by Abd El-Galil (1) concluding that medicinal plants with characteristics as an anti-inflammatory, restoratives, antibacterial and anti-parasitic will facilitate the performance productivity in poultry. Studies have shown that expansion of the animal's metabolism by herbal plant powder, generally carbohydrates and proteins in the major nutrient would increase growth rates (31). Another cause of weight gaining of quail treated with plant additives may be because of the plant's aromatic content that stimulating the appetite as known that aroma capable of activating the olfactory nerves and gustatory papillae (5). The results of this study accompanied by previous work on the antioxidant efficacy of plants toward increasing egg and body weight as well as improved Feed Conversion Ratio (FCR) in quails (4). No significant changes have been seen in feed intake of all experimented treatments with no mortality during the experimented period Table 1.  The results in Table 2. show that erythrocytes profile (total counts of RBCs, Hb concentration, and PCV%) and leukocytes profile (total counts of WBCs and lymphocyte%) were significantly (P≤0.01) increased in the T3 group followed by T4 and T5 experimented treatments. While the lowest erythrocyte and leukocyte profiles were seen in T0, T1, and T2 groups, respectively. The level of MCV, MCH, heterophil%, and H/L ratio were significantly (P≤0.05) lower in the T3, T5, T4, T2, and T1 treated groups, respectively in comparison with the normal control T0. The level of MCHC was significantly (P≤0.05) decreased in the T5, T3, T4 groups in comparison with T1 (Rifaximine 2g/L).
Positive feedback of Three experimented plants on the erythrocyte and leukocyte profiles may be linked with their anti-inflammatory and antioxidant activities because of their natural phenolic, flavonoid, and terpenoid contents (9,21,26). Scientists previously have revealed that adding medicinal plants to poultry drinking water will significantly improve their hematological profile due to the plant's capability in facilitating the erythropoietin process that stimulated kidneys to promote the production of red blood cells and hemoglobin (28). Researchers also have shown the rise of leukocyte production in animals by using medicinal plants and suggest that it could be serve as an immune booster (24). The same results regarding improvement of erythrocyte and leukocyte profiles were found when quails were treated with natural diet additives (27).  Protein profile (total protein and globulin) concentrations in Table 3 (18). Regarding our data, studies have shown the antioxidants activity of medicinal herbs can significantly delay or prevent lipid peroxidation as well as decrease protein and carbohydrate denaturation (24).The antioxidant and antimicrobial activities of Hero flower by Bouayed (9), Thyme by Jaradat (21), and Bay leaf by Muñiz-Márquez (26) have been explained previously, at which their phytochemical contents (flavonoids and polyphenols) aid them in scavenging free radicals. Studies also concluded that herbs containing flavonoids, vitamin C, and phenolic compounds will aid in stimulating the immune system by improving lymphocytes, macrophages, and NK cells activities leading to an increase in phagocytosis or interpterion synthesis process (17). The results of this study were similar to the previous study done on the medicinal plant's efficacy on the quail's immunity (27,37). Similarly, a previous report also showed plant additives caused Physiological and reproductive improvements in Japanese quail (7).  The results of the ileum bacterial profile in a  (9), Thyme (Thymol) (21) and Bay leaf (kaempferol and quercetin glycosides) (26) could be the reasons behind their inhibitory effect against the growth of E. coli and Enterococcus bacteria in the ileum of quail's drunk water mixed medicinal plants. Several studies have reported that phenolic compounds mediate the antimicrobial activities of various plant extracts (6,14,29). The ileum histological study was present in Table 4. The villi height and crypt depth were increased in the plant extract, and antibiotics treated groups. Of which, quails drunk Hero flower extract in the water had significantly (P≤0.01) higher villus height (865) μm than that (843, 815, 774, 752 and 495) μm for T5, T4, T1, T2, and T0, respectively. The T3 groups also had significantly (P≤0.01) higher crypt depth (91.92) μm and goblet cells (13.17) numbers than that of T5, T4, T1, T2, and T0, respectively. Quails with no water treatments (T0) showed poor histological traits regarding villus height (495) μm, crypt depth (80.49) μm, V/C (6.15) μm, and goblet cells number (7.03) μm compared with other treated quails that drunk mixed water additives. Different parts of the small intestine are the areas for absorption of various nutrients, and the structure is related to their function. So, changes in feed and feed additives could be affect the morphology of the intestine at different parts. It is well known that improvement of nutrient absorption occurs by increasing the villi height and decreasing crypt depth. Also, increased intestinal villi height and height to crypt depth ratio and decreased crypt depth by plant extracts could be attributed to the decreased renewal rate of intestinal cells as a result of antioxidant properties and antimicrobial actions of plant extracts (29). Therefore, Hero flower, Thyme, and Bay leaf extracts could be increasing the absorption of nutrients by affecting the height and crypt depth of the intestine villi and increasing goblet cell numbers. In accordance with this study, supplementation of different levels of thyme oils in quail's diet due to their thymol content (35%) improved intestinal morphology (11). In contrast, the results of a study on rats showed that the villi width significantly increased in the duodenum and jejunum of the rats fed thyme volatile oil (23). Accordingly, previous reports showed a significantly increased villi height to crypt depth ratio by thymol compared to the basal diet in a state where birds are exposed to pathogens (13).

CONCLUSION
The current research draws attention to compare three traditional medicinal plant additives in quail's drinking water with two antibiotic drugs and a control group. Their effect caused an increase in the body weight and gain, economic profit (feasibility) of birds by maximizing the absorbed nutrients based on their positive effect on the hematology, immune status, intestinal morphology, and Lactobacillus population in the ileum of quails. A.kurdica , L.nobilis, and T.bovie extracts were more influential due to their higher contents of gallic acid, quercetin, and kaempferol glycosides compared with the antibiotic drugs and control groups. Moreover, A.kurdica extract could compete with the antibiotics to fight intestinal microbes such E.Coli and Enterococcus. As result, adding plant extracts to quails drinking water could be a better choice for increasing poultry production than antibiotics with fewer adverse effects, which positively reflects on the profitability of birds.