Evaluation of Ruta graveolen , coriander and basil seed extracts as natural additives on productive performance, blood profiles and egg quality of White Leghorn layers

The study was conducted to investigate the effect of mixtures of Ruta graveolen (Rg), coriander (C), and basil (B) seed extracts on productive performance, blood profiles, egg quality and egg chemical composition of White Leghorn (WL) layers. One hundred eighty WL layers at twenty-six weeks of age were randomly distributed to four treatments, each replicated three times with fifteen layers per replications in a completely randomized design and kept on a deep litter system for eight weeks. The treatments (T) were water containing mixtures of Ruta graveolen , coriander


INTRODUCTION
Antibiotic growth promoters have undoubtedly improved animal performance and health status However, the inclusion of antibiotics as a principal growth promoter pose a serious health problem for consumer often resulted in the incidence of antibiotic resistance among pathogens and a source of residues in animal body tissues (Abd El-Hack et al., 2022). Hence, the European Union banned the use of antibiotics as a growth promoter in animal feeds. As a result, scientists searched for locally available phytogenic feed additives intended to improve gut health and functions by fighting pathogenic bacterial infection (Sapsuha et al., 2021;Abd El-Hack et al., 2022). Various types of phytogenic productssuch as, garlic, anise, cinnamon, coriander, oregano, chili, basil, pepper, rosemary, rosehip and thyme -may be used as additives to enhance performance and modulate gut health in poultry (Criste et al., 2017). Medicinal plant extracts that were used as natural antimicrobials have a positive effect on performance parameters, including egg production, egg quality (Khan et al., 2012), the immune system and antioxidant status (Abd El-Hack and Alagawany, 2015). In addition, the extract improved performance by increasing the digestibility and retention of nutrients, increasing the secretion of digestive enzymes and mucous production, and improving gut health status and microbial population to maintain production performance during heat stress (Criste et al., 2017).
The seed of Ruta graveolen is a rich source of secondary metabolites mainly: coumarins, alkaloids, volatile oils, flavonoids, and phenolic acids. It has been used to reduce inflammation abundantly worldwide due to its diverse medicinal properties or presence of essential oil obtained from this plant species have been shown to possess various pharmacological activities, such as antioxidant, anti-inflammatory, spasmolytic, sedative, antibacterial, antifungal, and antidiabetic effects (Szewczyk et al., 2023). Furthermore, it has anti-rheumatic, anti-diarrheic, anti-febrile, antiulcer, and antimicrobial properties reported in the recent pharmacological trials (Jianu et al., 2021).
The seeds of coriander (Coriandrum sativum) contain 0.5% -1.0% essential oil rich in beneficial phytonutrients including flavonoids, carvone, geraniol, limonene, borneol, camphor, lemon, and linalool. Flavonoids compound in coriander includes phenolic acid; it has been used to treat infections with worms due to its presence of anti-parasitic and antifungal (Matasyoh et al., 2009). Similarly, the seeds of basil (Ocimum basilicum) have biologically active compounds including ursolic acid, apigenin, and luteolin that activate the cell-mediated immune response and stimulate appetite responses and counterattack the deterioration of feed due to the presence of antioxidant (Shahrajabian et al., 2020). General-ly, as different researcher explained the use of medicinal plant extracts in poultry feeding have a positive effect on animal health, productivity, appetite stimulation, enhancement of enzyme activity and secretion related to diet digestion and absorption such as trypsin, amylase and jejunal chime (Windisch et al., 2008;Ghazaghi et al., 2014). In layer chickens, several studies indicated the possible beneficial effects of plant oils on egg production performance, improving egg quality and also supporting health status as a single compound or as mixed preparations (Al-Shaheen et al., 2023). According to Hadi and Jassim (2013) study, adding 1.5 g/kg of basil leaves powder to the diet of growing quail resulted in better body weights, less serum cholesterol and increase levels of serum total proteins and globulins from 1 st week up to 6th week of age compared to the control group. Basil seeds at 0.3% and 0.6% level also led to enhance the blood biochemical parameters and health status of broilers (Kadhim, 2016). Similarly, Supplementation of coriander seeds 1%-3% significantly increased yellowness in yolk color without affecting other quality parameters (Habiyah et al., 2016). On the other hand, synergistic effects of phytogenic compounds have been reported in studies with essential oils and a combination of herbal powders might tend to be more effective than a single herb administration (Khaligh et al., 2011). However, there is limited information on the utilization of mixtures of Ruta graveolen, coriander, and basil seed extracts in layers feeding. Therefore, this study was designed to evaluate the effect of mixtures of Ruta graveolen, coriander, and basil seed extracts on productive performance, blood profiles, egg quality, egg chemical composition, and economic benefit of WL layers.

Study Area
The study was conducted at Haramaya University poultry farm, which is located 505 km from Addis Ababa, capital city of Ethiopia. The site is situated at an altitude of 1980 meters above sea level, 9 0 26 ' N latitude, and 42 0 3' E longitude. The area has an average annual rainfall of 741.6 millimeters the mean annual minimum and maximum temperatures are 8.25 0 C and 23.4 0 C, respectively (Mishra et al., 2004).

Collection and Preparation of Seed Extracts
Ruta graveolen (Rg), coriander (C), and basil (B) seeds were purchased from Harar market and the dried seeds were pulverized at Haramaya University's feed processing plant. The powder was preserved in an airtight plastic container until it was directly used for the preparation of water extract. Then 20 g Rg + 20 g C+ 20g B (a total of 60 g of the three seeds powder were added to 1L of distilled water and was shaken and infused overnight at room temperature, then filtered for all experimental days according to the protocol described by Mollah et al. (2012).\

Management of Experimental Chickens
A total of 180 White Leghorn layers were randomly selected for the study from Haramaya University Poultry farm. The birds were weighed and randomly distributed into four treatments, each treatment being replicated three times. Each replicate was kept in 2x2 m pens wire-mesh partition on deep litter housing covered with a wheat straw. Before the announcement of the actual experiment, watering, feeding troughs, and laying nests were thoroughly cleaned and disinfected. The experimental pen was sprayed against ecto-parasites. The birds were acclimated to rations for 7 days and then fed for 60 days for evaluation of egg parameters. During the experiment, feed was offered ad libitum and water was available all the time throughout the experimental period after they had finished Ruta graveolen, coriander and basil infusion offered in drinking water daily at 6:30 hours.

Experimental Design and Treatments
A completely randomized design with three replications was used for each treatment. Each treatment was replicated three times with fifteen layers (Table 1). Using commercial layer ration of the nutrient requirements of 2800-2900 kcal ME/kg DM and 16-17% CP (NRC, 1996).

Measurements and Observations
Chemical analysis. Representative samples of 20g of Ruta graveolen, 20g of coriander, and 20g of basil seed powder were further analyzed before starting the experiment for chemical composition. According to the proximate analysis method AOAC (1996) chemical analysis of experimental feeds were carried out for dry matter (DM), ether extract (EE), crude fiber (CF), ash, and Nitrogen (N) content was determined by Kjeldahl procedure and crude protein (CP) was calculated as Nx6.25. The analysis was conducted in the animal nutrition laboratory of Haramaya University. Metabolizable energy (ME) of the experimental diets was determined by indirect method according to the formula given by Wiseman (1987) as follows. ME (Kcal/kg DM) = 3951 + 54.4 EE -88.7 CF -40.8 Ash.
Feed intake. Feed was weighed and offered twice daily at 8:00 and 17:00 hours. The orts were collected the next morning and weighed after removing external contaminants by visual inspection and handpicking. The feed offers and refusal were recorded for each replicate. Feed intake was determined as the difference between the feed offered and refused.
. Body weight. The initial body weight of laying hens was individually measured and the final body weight was recorded at the end of the experimental study using sensitive balance to determine the body weight change and the mean value of the pen was taken. Bodyweight change per pen per bird was determined as the difference between the final and initial body weight. Average daily gain or loss was calculated as BW change divided by the number of experimental days.
Egg production. Eggs were collected twice a day from each pen at 10:00 and 15:00 hours. The sum of the two collections was recorded as daily egg production. The rate of lay for each replicate was expressed as the average percentage of hen-day egg production following the method of (Hunton, 1995).
Egg weight and egg mass. Daily collected eggs immediately after collection were individually weighed for each replication and average egg weight was computed by dividing the total sum egg weight by the total number of eggs. After the mean weight has been determined, the following formula was employed to calculate the egg mass per pen on daily basis (North and Bell, 1984).
Feed conversion ratio. The feed conversion ratio was calculated as the ratio of grams of feed to grams of egg mass according to the following formula.
Egg quality parameters. Egg quality was assessed in terms of egg weight, albumen quality (albumen height and albumen weight), yolk quality (yolk height, yolk weight, yolk diameter, yolk color, and yolk index), external quality of shell weight and thickness, and Haugh Unit Score (HUS). For internal egg quality measurement, (15 eggs per treatment and 5 eggs per replication) were taken randomly and the average was computed for each quality parameter once every two week.
Albumen height and (Haugh Unit). The sample eggs were individually weighed, coded, and broken on a flat tray, the height of the thick albumen of each egg was measured with a tripod micrometer, and the average Haugh Unit value for each replicate was calculated by using the formula given by (Haugh, 1937).
Haugh Unit (HU) =100 x log (H-1.7W 0.37 +7.6) Where, H= albumen height (mm); W= weight of egg (g) Eggshell weight and thickness. The shells of the broken eggs were separated from their shell membrane, air-dried, and weighed. The measurement of shell thickness was carried out with a micrometer gauge having a sensitivity of 0.001 mm, at three points of the eggshell (air cell, equator, and sharp end).
Yolk quality evaluation. The yolk of each of the sample eggs was separated from the white and yolk height and the diameter was measured using a tripod micrometer and ruler respectively. Then, the yolk was thoroughly mixed and a sample droplet from each egg was placed on a piece of white paper. The Roche color fan consisting of a series of fifteen colored plastic strips were used as a reference to determine yolk color, with 1 rated as very pale yellow and 15 as deep reddish-orange. The mean for each replicate was calculated by taking the average reading from the three sample eggs. Yolk index was also computed using the following formula: Yolk index = (yolk height) / (yolk diameter) x 100 Hematological and serum biochemical parameters. At the end of the experiment, blood samples (5 ml each) were collected from the wing vein of 12 hens from each treatment. Hematological and serum biochemical analysis was conducted at Haramaya University veterinary physiology laboratory and Higher clinic laboratory, respectively. For, the blood analysis, 2.5 ml of blood was collected using EDTA (Ethylene Diamine tetra acetic acid) tube while the remaining 2.5 ml was collected in a plain tube and left to coagulate. Blood samples were analyzed for total red blood cells (RBC), hemoglobin (Hb), packed cell volume (PCV), white blood cell (WBC), total protein (TP), and serum cholesterol % Hen egg production = Egg mass (g/hen/day) = concentration. RBC and WBC were determined by using an improved Neubauer hemocytometer chamber (Dacie and Lewis, 1991). Hemoglobin concentration was determined by using acid hematin or Sahli's methods. The packed cell volume (PCV) by microhematocrit (capillary) tubes method and centrifuged at 3000 rpm for 5 minutes. Finally, Serum was harvested from blood collected in a plain tube which was transferred to an Endorphin tube and stored at -20c o and analyzed for serum chemistry parameters (serum total protein and albumin, total cholesterol count, HDL-C, and LDL-C) with an automated chemistry analyzer (Douglas et al., 2010). The globulin value was determined by the difference between serum total protein and albumin (Doumas et al., 1981).
Egg chemical composition. A total of 48, twelve eggs per treatment (four per replication) were taken for proximate analysis. The egg yolk and albumen were separated carefully and then each component was mixed thoroughly and poured into the pan and covered with aluminum foliate. The egg yolk and albumen collected were heated in an oven at 55 o C for about 72 hours for partial drying. The sample was ground, homogenized, packed, and stored for further proximate analysis. The partially dried samples were weighed and dried in an oven at 105 o C for about 12 hours for the determination of the dry matter. The ash, protein, and lipid content of the egg white and yolk were analyzed following the AOAC methods (AOAC, 2000). Egg nitrogen content was determined by the Kjeldahl method and crude protein was calculated using the formula; egg nitrogen × 6.25.

Statistical Analysis
The experimental design used in this study was a completely randomized design. The data collected during the period of the study were subjected to analysis of variance using (SAS 2009 version 9.4) computer software. When the analysis of variance indicates the existence of a significant difference between treatment means, then the least significant difference method was used to locate the treatment means that are significantly different (p < 0.05).

Feed Intake, Egg Production and Egg Mass
Feed intake (FI) was significantly differ (P<0.05) among the different levels of mixtures of Ruta graveolen, coriander, and basil seed extracts in drinking water. Higher feed intake was recorded in T3 followed by T2, T1, and, T4 ( Table 2). The improvement in feed intake with the addition of polyherbal could be due to essential oils and their main component, which stimulated the appetizing and digestive process in animals. Mudalal et al. (2021) reported that medicinal plant extracts have appetite and digestionstimulating properties and antimicrobial and antioxidant effects. The possible reason for lower feed intake in T4 as compared to T2 and T3 might be due to the high concentration of essential oils which is toxic when used at higher doses (Lee et al., 2004). Feed intake and water intake have positive relationship (Leeson and Summers, 2005). Plant extract that was given in drinking water can affect the feed intake of the animal. As Sigolo et al., (2021) indicated, different plant extracts were delivered to broiler in drinking water and they had a significant impact on the feed intake of the animal. Body weight gain and egg production in T4 were not significantly different compared to control that could be the feed intake similarity between these treatments (Yaman et al., 2020).
Egg production of White Leghorn layers was significantly higher (P<0.05) in T3 than in T2 and T1. T4 was numerically higher than in the T1 (control) but, it did not indicate any statistical difference (P>0.05) ( Table 2 and Figure 1). The result in this study is in line with the study of Ooi et al. (2018) who reported dietary local medicinal herbs as feed additives on production performance and fecal parameters in laying hens level of 1% was effective to increase egg production. Similarly better egg production was obtained by using supplementation of medicinal polyherbal extracts supplements at a level of 1%-2% in the production and egg quality of laying Japanese quail hens (Zeweil et al., 2006). The exact mechanism through which egg laying performance enhanced is not known. However, according to Zhao et al. (2011) the higher egg production performance of the laying hens may be due to antioxidant, antimicrobial and other activities such as increased blood circulation and secretion of digestive enzymes and reduction in the oxidation of feed due to the bioactive components in the plant. In addition the bioactive components play a vital role in the digestion and absorption of nutrients that might have improved the performance parameters of laying hens Hen-day Egg Production Figure 1. Weekly average hen-day egg production of White Leghorn chick administerd on mixtures of Rg, B and C seed extract via drinking water (Windisch et al., 2008).
The egg mass of layers in T3 was higher (P<0.05) than that of layers in T2, T4, and control group. Similarly, Guler et al. (2006) reported that significant (P<0.05) increase in the highest egg mass were in 1% and 2% coriander seed extracts on egg production performance and nutrient retention in laying Japanese Quails. These results are in contrast with the findings of Ooi et al. (2018) who reported dietary local medicinal herbs as feed additives on production performance in laying hens level of 1% was effective to increase egg weight but no significant change in egg mass. A possible reason for higher egg mass might be due to positive influence of the extract on the conversion of digested feed into eggs.

Yolk Color
A high yolk color score was recorded in T3, followed by T2, T4, and T1. But yolj color score of T3 was statistically similar to T2 (Table 3). Likewise, Kazem (2013) stated that dietary supplementation of 1%-3% medicinal herbs on diets including 2% medicinal herbs increased egg yolk color. In addition, Kopsell et al. (2005) explained that, basil was shown to rank highest among spices and herbal crops for carotenoids. Similarly, supplementation of coriander seeds into the feed was able to increase the absorption of betacarotene contained in the feed (Habiyah et al., 2016). So, the changes in color values, i.e., the increase in yolk color fun (YCF) score from the experimental treatments in the present study are consistent with the changes in the content of carotenoid fractions in the diets (Kljak et al., 2021).  As Hernandez et al. (2014) reported the carotenoid in such medicinal herbs supported the high yolk color score since it had the same function as xanthophyll.

Albumen Weight, Eggshell, Yolk Weight, Yolk Height, Yolk Index and Yolk Diameter
The result of this study revealed that eggshell, albumen, yolk weight, yolk height, yolk index, and yolk diameter did not show significant difference (p>0.05) among the different treatments (Table 3). Likewise, Habiyah et al. (2001), reported no significant (P>0.05) difference in the shell, albumen, and yolk weight by supplementation of the deferent ratio of coriander seed extracts in Lohmann Brown. The current study disagrees with the study of Guler et al. (2006) who reported that significantly (P < 0.05) increase in the highest yolk and albumen weights were in 1% and 2% coriander seed extracts on egg production performance and nutrient retention in Laying Japanese Quails. .

Egg Yolk and Albumen Chemical Composition
There were no significant (P>0.05) differences in egg yolk and albumen chemical compositions across all the treatments combination except for albumen protein content (Table 4). Higher (P<0.05) albumen protein content was recorded in T4 than those in T3, T2, and T1. But, T3, T2, and T1 were statistically similar. The increase in albumen protein might be the synergetic effect of the active compounds in polyherbal seed (furanocoumarins, flavonoids, and furoquinolines) which have strong, antioxidant, antiinflammatory, and anti-helminthic properties maybe reduce protein oxidation (Brenes and Roura, 2010). Besides, Rg, C, and B mixtures might have abundant photolytic enzymes, which promote protein digestion, deposition, and improve the transportation of metabolic protein in birds (Wasiyati et al., 2018).

Blood Analysis
The total serum cholesterol concentration was significantly lower (P < 0.01) in T4 compared with control, T2, and T3. As supplementation of Rg, C, and B seed extracts increased, serum HDL-cholesterol significantly (P < 0.01) increased from 47.57 mg/dl in T1 to 55.95 mg/dl in T 4. In the present study, the serum LDLcholesterol concentration decreased significantly (P <0.05) from 101.65 mg/dl in T1 and 96.75 mg/dl in T4 (Table 5). The finding in the present study was in line with Khubeiz and Shirif (2020) who observed that coriander as a feed additive to broiler increased HDL-cholesterol level and decreased their LDL-cholesterol level. The decrease in cholesterol levels might be due to the polyherbal oil is an inhibitor of the hepatic 3hydroxy-3-methylglutaryl coenzyme A (HMG- CoA) reductase activity, which is a key regulatory enzyme in cholesterol synthesis by hepatocytes or the fractional reabsorption from the small intestine (Lee et al., 2004). Statistically significant difference was not seen on PCV and total RBC count of the experimental birds among the different treatments. However, significant differences (P<0.05) were noted on total WBC count, Hb concentration, serum total protein and globulin. Likewise, Zeweil et al. (2006) noted significantly higher serum total protein and globulin with 1% supplementation of medicinal polyherbal extracts to laying Japanese quail hens as compared to a control diet. A higher value serum total protein and globulin might indicate there is enzyme hydrolysis of dietary proteins explained that the blood pool serves as a major source of amino acids needed for the synthesis of proteins as indicated by Scott (1970). Significant difference in blood constituents of hens may be due to associated with the effects of herbs bioactive compounds on improving the antioxidant status of the bird (Rababah et al., 2004) CONCLUSION From this study, it was concluded that inclusion of 4ml of Ruta graveolen, coriander, and basil seed extracts (mixed medicinal herbs powder) in one liter of drinking water significantly increased hen-day egg production, yolk color, feed intake, egg weight, egg mass and HDLcholesterol level while, it can significantly decrease serum LDL-cholesterol level. Further research was recommended to assess the identification of active chemical compounds in the seeds of Ruta graveolen, coriander, and basil as well as their effect at higher proportions on the performance of layers.

ACKNOWLEDGMENTS
Authors would like to thank Haramaya University Research Office for the financial funding to the research project. A special gratitude is going to Haramaya University Animal nutrition and food science laboratory technicians and poultry farm workers for their kind support with critical research inputs and the help in the farm and laboratory.