Effect of fringed rue (Ruta chalepensis) leaf as feed additives on growth performance and carcass characteristics of broiler chickens

Abstract Ruta Chalepensis (fringed rue) has been used for therapeutic and aromatic purposes. However, using this plant as a natural additive in animals is scarce. A seven-week study was conducted to determine and elucidate the effects of fringed rue as a feed additive on broiler chickens growth performance and carcass characteristics. The average weight (40.08 ± 0.42 g) of 144 unsexed, day-old broiler chickens was arranged in a completely randomized design into four treatment groups. Each treatment group was divided into three replicates of twelve birds. Four treatments feed containing ground fringed rue leaves at different levels of T1 (basal diet+ the antibiotics Flavomycin at 1 g kg−1); T2 (basal diet + 60 g kg−1, T3 (basal diet + 120 g kg−1), and T4 (basal diet + 180 g kg−1. DM Intake was considerably high (P < 0.05) in the T3 group than in the antibiotics control group broilers. As a result, T3 groups have better improvement (P < 0.05) in final body weight, BW gain, and ADG compared to the antibiotics control in all phases. There were high improvements (P < 0.05) in dressing percentage, breast, thigh muscle, and drumsticks for chickens fed T3 and T2 than in the control group. Generally, supplementation of fringed rue up to 120 g kg−1 has no adverse effects on broiler chickens.

Abstract: Ruta Chalepensis (fringed rue) has been used for therapeutic and aromatic purposes.However, using this plant as a natural additive in animals is scarce.A seven-week study was conducted to determine and elucidate the effects of fringed rue as a feed additive on broiler chickens growth performance and carcass characteristics.The average weight (40.08 ± 0.42 g) of 144 unsexed, day-old broiler chickens was arranged in a completely randomized design into four treatment groups.Each treatment group was divided into three replicates of twelve birds.Four treatments feed containing ground fringed rue leaves at different levels of T1 (basal diet+ the antibiotics Flavomycin at 1 g kg −1 ); T2 (basal diet + 60 g kg −1 , T3 (basal diet + 120 g kg −1 ), and T4 (basal diet + 180 g kg −1 .DM Intake was considerably high (P < 0.05) in the T3 group than in the antibiotics control group broilers.As a result, T3 groups have better improvement (P < 0.05) in final body weight, BW gain, and ADG compared to the antibiotics control in all phases.There were high improvements (P < 0.05) in dressing percentage, breast, thigh muscle, and drumsticks for chickens fed T3 and T2 than in the control group.Generally, supplementation of fringed rue up to 120 g kg −1 has no adverse effects on broiler chickens.
Yonatan Kassu Yesuf ABOUT THE AUTHORS Yonatan Kassu Yesuf is an Assistant Professor in the Department of Animal Science, passionate about teaching and learning, research, and community service.His expertise lies in animal nutrition and health, and he is committed to working closely with stakeholders to improve livestock production.His research centers on sustainable solutions to enhance livestock production, focusing on animal welfare, nutrition, and food safety.He has also made significant contributions to the field of animal science, publishing extensively in reputable journals.Abera Anja is an assistant professor at Wolaita Sodo University's Department of Animal Science.His research and work have demonstrated a commitment to advancing knowledge and understanding in livestock production and nutrition.Melese Mengistu is a lecturer in the Department of Animal Science at Wolaita Sodo ATVET College.

PUBLIC INTEREST STATEMENTS
The study investigated the potential benefits of adding fringed rue leaves to the diet of broiler chickens.The experiment aimed to determine if this herb could improve the growth rate and meat yield in broiler chickens.The study's findings revealed that supplementing fringed rue leaves in broiler chicken feed significantly improved the chickens' growth performance and carcass characteristics.The study found that the broiler chickens fed with fringed rue leaves had similar feed conversion ratio, better body weight gain and carcass weight compared to those fed with synthetic additives (antibiotics growth promoter) diet broilers.The study suggests that fringed rue leaves could be a promising natural feed additive for the poultry industry, providing a potential alternative to synthetic additives.

Introduction
The extensive practice of adding broiler diets with antibiotic growth promoters was primarily motivated by the compounds' positive effect on production performance.However, the exacerbated utilization of antibiotics in subtherapeutic dosage as growth promoters has led to increased microbial resistance and accumulation of antibiotic residues in animal products that affect the end-user or consumer health environment.This situation increased interest in natural alternatives (Hafeez et al., 2016).This situation required most countries to ban or limit AGP in poultry feeds and launch "antibiotic-free" labeled feeds (Hafeez et al., 2016).Nonetheless, the major concern related to rearing broilers without antibiotic growth promoters in the feed is poor production performance and the incidences of certain diseases leading to higher production costs (Khan et al., 2013).To alleviate the situation, interest in non-antibiotic feed additives that could help to eliminate the continuous use of AGPs has grown in recent decades.Several products based on probiotics, prebiotics, medicinal plants or phytogenics sources, and organic acids claim to improve broiler chicken productivity and health.
Medicinal plants or phytogenic feed additives (PFA) are compounds of plant origin (spices, herbs, plants, and products derived thereof) has been incorporated into animal feeds to enhance livestock productivity by improving digestibility, nutrient absorption, and elimination of pathogens residents in the animal gut that enhance performance feed conversion ratio, carcass meat safety, and quality in animals (Alloui et al., 2014;Dhama et al., 2015;Stanacev et al., 2011).PFA's principal method of action is the suppression of potential infections and the positive modulation of the gut flora.Antimicrobial, antiviral, anticoccidial, fungicidal, and antioxidant activities are found in medicinal plants and preparations (Applegate et al., 2010).The antimicrobial effects of PFA are primarily attributed to their phenolic and flavonoid components and their action on pathogenic cells (Burt, 2004).The content of active substances in these products can vary greatly depending on what part of the plant is used (seeds, leaves, roots or rhizome, bark, flowers, or buds), the harvest season, and geographical origins (Alloui et al., 2014;Yonatan, 2018).
Fringed rue (Ruta chalepensis L.) is a widely known medicinal herb belonging to the Rutaceae family.Fringed rue is a native evergreen subshrub of the Mediterranean region, but it is widely distributed in many parts of the world in temperate and tropical countries (Gonzalez-Trujano et al., 2006).Fringed rue is used in the traditional medicine of many countries, including Saudi Arabia, China, India, and Ethiopia, for treating fever, rheumatism, convulsions, and other nervous disorders and for its analgesic and antipyretic properties (Fullas, 2003;Iauk et al., 2004).In Ethiopia, fringed rue is typically acknowledged as the herb of grace and is locally known as "Tena Adam" which literally means Adam's health, signifying the plant's medicinal value to humankind.It is used to cure inconsolable crying newborns, diarrhoea, intestinal diseases, earache, heart discomfort, hemorrhoids, and influenza in Ethiopian folk medicine.In addition, a culinary application of the leaves of fringed rue is accustomed to flavor sour milk and cheese, coffee, and tea.Due to its strong smell, the fresh plant is an excellent insect repellant (Fullas, 2003).Some studies reported that fringed rue's chemical and active compounds are flavonoids, quinoline alkaloids, acridones, coumarins, chalepensin, and umbelliferone (El Sayed et al., 2000;Loizzo et al., 2018;Park & Lee, 2015).Even though fringed rue has medicinal value in humans, no study has been conducted as a natural feed additive and supplementation for farm animals.Therefore, the present study aims to evaluate the effect of fringed rue leaf as feed additives on broiler chicken's growth performance and carcass characteristics.

Experimental site
The feeding trial was carried out at the Poultry Farm, Wolaita Sodo ATVET College, Ethiopia.The study was carried out for seven weeks.

Preparation of fringed rue
A height of 50-60 cm fringed rue was collected from the university forage sites.The leaf part was detached, rinsed with tap water, and air-dried in the shade.The dried plant was ground using mortar and pestle, sieved through a 5 mm mesh screen, and stored over a desiccant at 4°C until used for laboratory analysis and incorporation into the basal diets.

Experimental diets
The trial used conventional maize and soybean meal-based broiler diets free of coccidiostat and antibiotics.The diets had metabolizable energy (ME) of 3050 kcal/kg DM and 21% CP for the starter phase (1-21 days) and ME of 3250 kcal/kg DM and 18% CP for the finisher phase (22-49 days).The treatment groups were fed a basal diet supplemented with fringed rue leaf at 0% T1 (basal diet+ the antibiotics Flavomycin at 1 g kg −1 ), T2 (basal diet +60 g kg −1 fringed rue, T3 (basal diet +120 g kg −1 fringed rue) and T4 (basal diet +180 g kg −1 fringed rue).

Ethical consideration
The experimental chicks were treated or managed according to an experimental animal protocol approved by the Ethics Commission of the Wolaita Sodo University (reference number WSU/41/14/ 1023).

Experimental design
A total of 144 unsexed day-old Cobb-500 broiler chicks with an average weight of 40.08 ± 0.42 g (Mean±SD) were randomly assigned into four dietary treatment groups (T1, T2, T3, and T4), with three replications per treatment arranged in a completely randomized design (CRD).

Experimental chickens and management
A seven weeks feeding trial was conducted to determine and elucidate the effects of fringed rue as a feed additive on broiler chickens.The chicks were reared in a deep litter housing system covered with sawdust as a litter material at a depth of 10 cm.The house had portioned into 12 pens with a dimension of 150 cm×150 cm.Before the commencement of the experiment, the room, waterer, feeder, and other utensils were thoroughly washed, cleaned, and sprayed with disinfectant chemicals, and the room was fumigated by mixing potassium permanganate (KMnO4) powder with 10% formalin solution.The bulbs were hanged just above the chicks' level at the center of each pen, and the temperature was maintained at 35°C during the first week and then incrementally lowered each week until it reached an ambient temperature of 24°C.The chicks were vaccinated against Newcastle disease (NCD) and infectious bursa disease and were administered according to the vaccine company National Veterinary Institute (NVI) protocol.

Growth performance measurement
Daily records of the amounts of feed offered and refused for each pen were kept.Feed intake was determined by subtracting the amount of refused feed from the amount of feed offered and was expressed on a feed basis.Body weights of the birds were taken as a group at the beginning of the experiment and weekly until the end of the trial using a bench balance with a sensitivity of 0.001 grams (Xianghai, model ACS-C).The average body weight gain (BW gain) was calculated by dividing the difference between two consecutive weights by the number of chickens.The average daily gain (ADG) was calculated by dividing the final BW change by the number of experimental days.The feed conversion ratio (FCR) was determined by dividing the average feed intake by the average BW gain.

Carcass characteristics measurement
At the termination of the study, two chickens were selected randomly from each replication to undergo a carcass examination.Before slaughter, the broiler chickens were weighed on a highly sensitive balance (Xianghai, model ACS-C, with a sensitivity of 0.001 grams) after fasting for 12 hours.Exsanguinations were carried out, the jugular vein was severed, and the broilers' feathers were plucked by hand.The procedure followed to identify carcass features was described by Kubena et al. (1974).The cut of the eviscerated carcass and the non-edible offal were determined.The breasts, wings, drumsticks, thighs, and back were used to evaluate the carcass yield/weight.The amount of carcass weight to slaughter weight, multiplied by 100, was used to compute the dressing percentage.The separated and weighed parts of the eviscerated carcass include the breast, wings, thighs with drumsticks, and back.The giblets (heart, gizzard and liver) were weighed in grams.The fat around the proventriculus, gizzard, abdominal wall and cloacae was collected and weighed.

Chemical analysis
Fringed rue leaf samples were ground using a Wiley mill (Thomas ® Wiley Cutting Mill) to pass through a 1 mm screen for proximate analysis at National Veterinary Institute in the animal nutrition laboratory.The fringed rue leaf and feed offered and refusal was sampled and analyzed for DM according to the procedure of AOAC.(Association of Official Analytical Chemist) (1998).

Chemical composition of fringed rue
Proximate analysis was conducted at National Veterinary Institute in the animal nutrition laboratory, Bishoftu.The results of the chemical composition of fringed rue leaf are given in Table 1.

Statistical analysis
The collected data were subjected to the analysis of variance test for each parameter using the general linear models (GLM) procedures of SAS Version 9.3.Duncan's Multiple Range Test was used to separate between group means at a 5% level of significance.The statistical model used was:

Yi= µ+α+ℇ ij
Where: Y ij = the dependents variables observed of the fringed rue supplementation i and the replication, j within the level of the treatment µ = overall mean effect αi= the effect of i th fringed rue supplementation at (0, 60, 120, and 180 g kg −1 ) e ij = the random error variation of the ij th (mean 0 and variance σ)

Feed intake and feed conversion ratio
The feed intake of chickens fed with different levels of fringed rue leaf as feed additives is presented in Table 2.A highly significant difference (P < 0.001) was observed in the broiler's daily and total feed intake among treatments during the starter, finisher, and entire experimental period.The total and daily feed intake increased (P < 0.05) in the T3 group than in the antibiotics control (T1), T2, and T4 group during the starter, finisher, and entire period (P < 0.05).This improvement in feed intake might be due to fringed rue compounds like, alkaloids, flavonoids, phenols, furocoumarins, coumarins, saponins, and amino acids.Phenolic compounds originated from aromatic plants may improve the flavor and palatability of the feed by stimulate saliva secretions and bile and enhance enzyme activities thus increase the feed intake.
Even though no farm animal experiment is conducted on fringed rue to contrast the current findings, other herbs and plants are considered for comparison.For instance, Daramola (2019) reported improved daily and total feed intake of broilers fed with 2% bitter leaf meal Moringa leaf meal and blending of both bitter leaf meal and Moringa leaf, respectively.Similarly, this finding also agrees with Khaligh et al. (2011), who reported differences among treatments in average daily and total feed intake of broilers fed with 10 g kg −1 thyme, rosemary leaf, anise, cinnamon, and garlic.However, the findings of this study differ from those of Alhajj et al. (2015), who claimed that adding 1 and 2 g kg-1 of anise to a broiler's diet had no impact on feed intake.The inconsistent reports may be explained by variances in the amount of primary and secondary metabolites in various medicinal plants and distinct parts of the plants used, as well as environmental factors, including soil type, climate, and harvesting time.
The FCR was not affected (P > 0.05) when using fringed rue as feed additives on broiler chicken.Similar experiments showed that adding thymol, cinnamaldehyde, and commercially prepared essential oil to broiler chicken did not affect feed intake and FCR (Lee et al., 2004;Ren et al., 2019).When used as a feed additive, a rue helps broiler chickens perform similarly to antibiotic growth promoters (AGP) and may improve broiler digestibility by increasing enzyme activity and mucus production in the gut.
An increase in feed intake and body weight gain among different treatment diet groups without a significant difference in feed conversion ratio (FCR) or not similar to the antibiotics control group in the current study could be due to the treatment diets may contain more digestible nutrients,  ); T2 (basal diet +60 g kg −1 fringed rue, T3 (basal diet +120 g kg −1 fringed rue) and T4 (basal diet +180 g kg −1 fringed rue); FCR= Feed conversion ratio; SEM = standard error mean; p=probability value.
such as protein or energy, compared to the control diet.This can increase feed intake and body weight gain, as the animals can consume more nutrients without experiencing digestive or metabolic limitations (Owusu-Asiedu et al., 2006).However, if the additional nutrients are not efficiently converted into body weight gain, there may not be a significant difference in FCR compared to the control group.In addition, the treatment diet groups containing fringed rue were more palatable than the control diet due to the fringed rue aroma, which affects the stimulation of saliva and digestive enzymes and can lead to an increase in feed intake and body weight gain (as seen in Table 3).However, suppose the broilers are consuming more feed solely because of its palatability and not because it contains more nutrients or energy.In that case, there may not be a significant difference in FCR compared to the control group (Rojo et al., 2007).

Growth performance
There were highly significant differences (P < 0.001) in final body weight, BW gain, and ADG of broiler among treatments during starter, finisher, and the entire periods as shown in Table 3.The initial BW of the day-old chick had no significant differences (P > 0.05) among the treatment.The final body weight and growth of chickens fed fringed rue as a feed additive improved considerably (P < 0.05) over the antibiotics control group.During the overall growing period, there was a (3.3% −10.5%) and (3.3-11.29%)increase in BW and ADG in the fringed rue additive group compared to the control group.In addition, the DM intake was higher in broilers fed with 120 g kg −1 fringed rue.
According to Khan et al. (2012), the improved growth results seen in broilers fed a diet supplemented with fringed rue may be due to an increase in the production of digestive enzymes and an improvement in the way the liver processes nutrients.The antibacterial action of the fringed rue component may inhibit the growth of pathogenic bacteria in the gut while promoting the development of beneficial bacteria (Barreto et al., 2008).However, higher inclusion of fringed rue with 180 g kg −1 did not produce linear BW and BW gain results with the feed intake.In the current study, fringed rue leaf increased BW gain without affecting FCR, similar to Azzam et al. (2020) on broiler chickens fed 1,000-3,000 mg kg kg −1 Rumex nervosus leaves improve BW and BW gain in female broiler chickens.Moringa oleifera leaves, on the other hand, have been shown to boost BW and BW gain without influencing FCR (Khan et al., 2017).Furthermore, El Tazi et al. (2014) indicated that 0, 0.5, 0.75, and 1.0% of black pepper (Piper nigrum L.) to broiler feed resulted in better body weight and gain.Al-Kassie (2008) found that supplementing the feed with 0.5 and 1% rosemary herb enhanced broiler growth performance at 42 days.However, Amad et al. (2011) reported that supplementing a phytogenic compound including thyme and star anise at 150, 750, and 1,500 mg kg −1 in the diet did not alter body weight and gained favourably compared to the control diet and had no beneficial production outcomes as supplementation increased.Medicinal plants or active compounds in the chicken feed may increase appetite and feed intake, increase endogenous digestive enzyme synthesis, promote immunity, and have antiviral, antibacterial, anthelminthic, and antioxidant properties.Furthermore, isoprene derivatives, flavonoids, glucosinolates, and other herbal metabolites may impact the poultry gut's physiological and chemical function (Reda et al., 2021).Moroverr, the fringed rue in the broiler chicken diet might have improved the production and activities of digestive enzymes.

Effect of fringed rue on carcass characteristics
The results showed that dietary treatments significantly affected (P < 0.001) slaughter weight, dressing percentage, commercial carcass component, and giblet parts except for heart weight (P > 0.05), as shown in Table 4.The dressing percentage was highly affected (P < 0.001) by treatment and higher in both T3 and T2, followed by T4 and T1 (antibiotics control) (P < 0.05).
The improvement could be attributed to the active compound in fringed rue, such as flavonoids, which can alter feed intake, metabolism, and uptake by enhancing the synthesis of digestive enzymes, leading to higher body weight growth and dressing percentage.These findings agree ); T2 (basal diet +60 g kg −1 fringed rue, T3 (basal diet +120 g kg −1 fringed rue) and T4 (basal diet +180 g kg −1 fringed rue);SEM = standard error mean; p=probability value.with those of Kadhim (2018) and Saleh et al. (2020), who studied the effects of dietary Marjoram (Origanum vulgare) and rosemary (Rosmarinus officinalis L.) in Japanese quail and broiler chicken.Dietary treatments significantly affected the breast, drumsticks, thighs, and back (P < 0.001).The birds fed T3 had particularly (P < 0.05) higher breast weight, drumsticks, thighs, and back, followed by T2, T4, and T1 in all phases.This result agrees with Natasa et al. (2016), indicated broilers with phytobiotics in feed produced more meaty carcasses than control broilers.The presence of essential compounds flavonoids in the medicinal plants may be related to the increased breast weight, which prevents lipid oxidation, allowing high amounts of lean cut to accumulate in the organs and proportions of the organs, reducing the risk of hyperlipidemia in consumers and increasing acceptability.The current result differs with Alabi et al. (2017), who reported no influence of aqueous Moringa oleifera leaf extract on the weight of the breast meat, thighs, wings, and drumsticks.
The treatments had a significant effect on the weight of the liver and gizzard (P < 0.001).Their weight was substantially higher (P<0.05) in the T4 group than in the T3 group.T2 and T1 (control group) are, however, comparable (P>0.05).This indicates that their weight grows with the amount of fringed rue in their diet increases, which may be related to the liver's detoxification (protection against toxins and free radicals) process as the amount of fringed rue in their diet increases.Medicinal plants or herbs contain large amounts of flavonoids, phenolic acids, tannins, and other hepatoprotective effects (Albert, 2018;Salam et al., 2014).As a result, fringed rue should be fed at an acceptable dosage (60-120 g kg −1 ) to avoid affecting broiler metabolic activity and production.Yet, in this study, the liver weight ranged from 2.01% to 2.57% of the overall carcass weight, which is still within the acceptable liver weight range.Ologhobo et al. (1993) estimate that the average liver weight in broilers ranges from 2.64% to 4.40% of total carcass weight.Nevertheless, the liver weight in this study ranged from 2.01% to 2.57% of the total carcass weight, which is still within the normal liver weight range.
The enlargement of the gizzard could be due to the response of broiler chickens to metabolic activity.According to Svihus (2011), the increasing size of the gizzard was an effort by broilers to improve nutritional digestibility through prolonged retention time and grinding and mixing the diets with digestive enzymes.This finding contradicts the findings of Al-Sagan et al. (2020), who found no change in the weight of broiler gizzards fed diets containing 1.6% and 3.2% fennel seed powder.The dietary treatment groups showed that the abdominal fat weight was highly affected (P < 0.001).The fringed rue as a feed additive in T3 and T4 had significantly lower abdominal fat (P < 0.05) than the control and T2 broiler chickens.The abdominal fat content of broilers decreases when the dietary supplementation of fringed rue level increases (P < 0.05).The reduction in abdominal fat could be attributed to fringed rue active compounds such as acridones, coumarins, chalepensin, and umbelliferone, which have the potential to inhibit hepatic fatty acid synthase activity and increase oxidation of fatty acids (stimulated glycogenesis), resulting in an effective reduction in fat storage (El Sayed et al., 2000;Loizzo et al., 2018;Park & Lee, 2015;Sugiharto et al., 2011).This result agrees with El Tazi et al. ( 2014), broilers supplemented with 1% black pepper had significantly lowered abdominal fat percentage than the control group.

Conclusion
This study found that supplementing broiler chicken meals with up to 120 g kg −1 fringed rue leaf boosts DM intake, live body weight, and weight gain.Consequently, the dressing percentage and significant carcass cuts have improved due to their digestive stimulating and antibacterial properties or activities.Therefore, it should be considered a possible natural growth promoter for broiler chicken production.

Table 3 .**
Effect of fringed rue additives on growth performance of broiler Mean values within the same row having different superscript letters are significantly different (P < 0.05); T1 (basal diet+ the antibiotics Flavomycin at 1 g kg −1 ); T2 (basal diet +60 g kg −1 fringed rue, T3 (basal diet +120 g kg −1 fringed rue) and T4 (basal diet +180 g kg −1 fringed rue); IBW=Initial body weight; FBW= Final body weight; BWG= Body weight gain; ADG=Average daily gain; SEM= standard error mean; p=probability value.Table 4. Effect of fringed rue additives on carcass and visceral organs of broiler Mean values within the same row having different superscript letters are significantly different (P < 0.05); T1 (basal diet+ the antibiotics Flavomycin at 1 g kg −1

Table 2 . Effect of fringed rue additives on feed consumed and FCR of broiler chicken
*Mean values within the same row having different superscript letters are significantly different (P < 0.05); T1 (basal diet+ the antibiotics Flavomycin at 1 g kg −1