Properties of Extracted Nagpur Essential Oil
Besides the range of valuable compounds, orange peels also contained d-limonene (DL), or (+)-limonene as a more chemically correct notation for the dextrorotary isomer. It is a secondary plant metabolite commonly found in essential oils from the citrus peel [50]. The essential oil was extracted from Nagpur orange waste using indigenous and patented extraction method. The chromatographic studies revealed that 91.88% limonene was present in the essential oil. Indeed, limonene was the major constituent which imparted potential benefits to the essential oil to be used for in-vivo studies in poultry feed. A critical observation of the composition of the oils has revealed that D-Limonene and other oxygenated compounds that contributed to the orange flavor and aroma. However, the presence of D-Limonene affected the quality of the orange essential oil. Oxygen compounds are more valuable than hydrocarbons in terms of their contribution to the fragrance and therapeutic properties of the essential oil and could be used as essential oil quality measures [25]. Ozturk et al. [46] showed that cyclopentyl methyl ether and 2-methyl-tetrahydrofuran bio-based solvents led to limonene yields up to 80%. The maximum product yield of Limonene, a major favorable component, were 77% and 100% in hydro-distillation and steam explosion processes, respectively.
Percent Proximate Analysis of Experimental Diets
The estimated CP % and ME (kcal/kg) was 23.04 and 3001.04 in pre-starter, 22.02 and 3100.40 in starter and 19.98 and 3203.74 in finisher diet respectively; which was in agreement with BIS [14] feeding standards for broilers. The essential oil had limonene in highest concentration (91.88%) followed by γ terpinene (5.02%), β phellandrene (1.37%) and other components in minor concentration. In agreement with present finding Aydin and Alcicek [10] reported 21.0 and 2988.0 and 19.0 and 3176.0 percent CP and Kcal/Kg ME in starter and finisher diet respectively. Alzawqari et al. [7] recorded 18.52 to 18.55 % CP and 2941.80 to 2946.91 kcal/kg of ME in their diets which were used for feeding broilers at age of 22 days.
Growth performance
Cumulative feed intake
The cumulative feed consumption during starter and overall period was significantly lower (P<0.05) in T3 than control and other treatment groups. Whereas during finisher period, control group had significantly higher (P<0.05) cumulative feed intake than other treatment groups. In agreement with present finding Aydin and Alcicek [10] recorded significantly reduced (P<0.05) feed intake in 150mg/kg OPEO supplemented group than control. They noted that with increase in OPEO supplementation from 50-150 mg/kg the feed consumption got depressed; which was in contrast with assumption that appetite and feed consumption gets improved owing to aromatic properties of essential oil. Similarly, Alcicek et al. [6] noticed decrease in feed consumption when dose rate of essential oil combination derived from selected herbs was increased from 24 to 72 mg/kg of diet in broilers. In present study the reduction in feed consumption during overall period was evident in 100g/ton OPEO supplemented group (T3). However, the depressing effect was not consistent when OPEO supplementation was given at higher level (150g/ton: T4). Moreover, the depression in feed intake remained consistent throughout the experimental period in T3 (OPEO 100g/ton) group only in present study. Alzawqari et al. [7] recorded comparable feed intake in dried sweet orange peel and lemongrass leaves supplemented broilers during finisher period. They further explained that the inconsistency in the effect of essential oil supplementation might be due to the difference in basal diet, housing management, physiological state, concentration of active ingredients in product used and mainly dose rates at which the feed additives are added along with experimental diets.
Cumulative body weight gain
It was noticed that during starter, finisher and overall period of experiment the cumulative body weight remained significantly lower (P<0.05) in control than OPEO supplemented group. In contrast with present findings of significantly improved (P<0.05) body weight; Khattak et al. [34] reported no influence (P>0.05) during starter phase followed by improved performance in grower, finisher period due to supplementation of natural blend of EO in broilers. They attributed this improvement in performance to the increased endogenous digestive enzyme secretion and thereby improved nutrient utilization and digesta flow in chicken. Akbarian et al. [4] noted no effect (P>0.05) of dietary orange peel and lemon peel extract supplementation on weight gain, intake and FCR in broilers exposed to high ambient temperature during finisher period. However, the present findings reveal no such adverse effect on performance due to OPEO supplementation. On the contrary, the significant improvement (P<0.05) in cumulative body weight of OPEO supplemented broilers over control in present study; ratifies its utilization as an alternative growth promoter to antibiotic in broiler diet. In support of present finding, Sahu et al. [48] reported significant improvement (P<0.05) in weekly gain as well cumulative body weight in heat stressed broilers supplemented lemon and orange peel essential oil separately or in combination during summer.
Cumulative feed conversion ratio (FCR)
In consonance with present findings, Sahu et al. [48] found significantly improved (P<0.05) FCR in broilers supplemented lemon peel, orange peel essential oil or their combination than control of 6th week of age. They attributed this improvement in performance to efficient nutrient utilization via action of essential oil. The significantly improved (P<0.05) FCR in T3 group in present study corresponded well with significantly reduced feed consumption and significantly improved (P<0.05) body weight in said group. Erhan and Bolukbasi [22] in congruence with present finding recorded significant improvement (P<0.05) in FCR of citrus peel oil supplemented broilers. They further noted that out of oils used in their study; the improvement in FCR was best in broilers supplemented 3ml/kg of OPEO in diet. Also, improvement in intake and FCR due to supplementation herbal essential oil mixture in breeder flocks was reported [18]. In contrast with present findings, Akbarian et al. [4] recorded comparable (P>0.05) FCR, intake and weight gain in dietary orange peel and lemon peel extract supplemented broilers. However, Aydin and Alcicek [10] recorded significantly improved (P<0.05) FCR in broilers supplemented 150 mg/kg OPEO than control. They suggested that utilization of volatile oil mixture positively affected feed conversion efficiency [6]. Similarly, Hong et al. [30] recorded significantly improved (P<0.05) survival rate and FCR in essential oil supplemented broilers over the control. Hence, they concluded that in view of economic consideration; the 100 mg/kg essential oil supplementation would provide adequate growth promoting effect in broilers from 1-36 days of age. In present study the best FCR with corresponding lowest feed consumption and highest cumulative body weights were recorded in T3 group with OPEO supplemented at 100g/ton in broiler diet.
Nutrient Metabolisability (%)
The percent nutrient metabolisabilty was not affected (P>0.05) due to dietary OPEO supplementation except for nitrogen free extractives. Attia et al. [8] proposed that use of essential oil enhanced nutrient utilization through stimulation of digestive enzymes and thereby increasing nutrient availability for achieving growth in broilers and observed improved crude fibre, crude protein and ether extract digestibility due to essential oil supplementation. Cao et al. [19] suggested that the ileal energy digestibility as well as use of nitrogen gets augmented through cinnamaldehyde and thymol supplementation in broiler diet. Sahu et al. [48] elaborated that the improved feed efficiency (P<0.05) due to supplementation of lemon and orange peel essential oil in their study was attributed to positive influence of essential oil mixture on nutrient digestibility and gut health. They further explained that a balance in microbiota gets restored in the gut; thereby enhancing nutrient absorption and boosting animal performance due to essential oil supplementation [40]. The use of herbal mix [35] and essential oils [27] is considered to promote nutrient utilization through producing more digestive enzymes and also increase protein digestion through enhancement in pepsin and HCL secretion. Similarly, the improved performance (P<0.05) in OPEO supplemented broilers might be due to probable positive effect of limonene contained in essential oil used in present study.
Carcass Characteristics
In agreement with present findings Alzwaqari et al. [7] reported comparable (P>0.05) carcass characteristics such as dressing percentage and relative weight of spleen, bursa, gizzard and liver in dried sweet orange peel and lemongrass leaves supplemented broilers during finisher phase. Similarly comparable carcass traits in response to lemongrass oil supplementation to the diet were also reported [42]. However, in present study the relative weight of gizzard was significantly increased (P<0.05) and relative weight of heart was significantly decreased (P<0.05) in OPEO supplemented groups than control. Erhan and Bolukbasi [22] recorded comparable relative weights of liver and heart in citrus peel oil supplemented broilers. In contrast, Aydin and Alcicek [10] reported significantly higher (P<0.05) yield and weight of carcass, abdominal fat and relative weights of wing, back, breast, heart and thighs respectively due to OPEO supplementation in broilers. They observed that as the dose of OPEO increased from 50 to 150 mg/kg; the carcass weight also increased significantly (P<0.05) indicating dose dependent improvement in carcass weight. Similarly present findings also revealed that the live weight was significantly higher (P<0.05) in birds fed 100g/ton OPEO. In present study though the relative weights of liver were comparable (P>0.05), significant improvement (P<0.05) in gizzard weight was visible in OPEO supplemented groups. In congruence with present findings, Denli et al. [21] reported significantly higher (P<0.05) length and weight of intestine in quails supplemented thyme and black seed essential oil. Hosseini et al. [31] recorded comparable (P>0.05) relative weights of internal organs such as small intestine, gizzard and liver.
Hematological Parameters, Erythrocytic Antioxidant Enzyme Status and Serum Metabolite Concentration
Analogous to present findings no significant influence (P>0.05) on glucose, cholesterol, total protein, albumin and globulin was recorded of up to 40% inclusion of sundried sweet orange peel in broilers [45]. In broilers treated with EO and ZnB, Attia et al. [8] found equivalent A:G ratio, albumin, total cholesterol, and triglycerides. The addition of ZnB, cinnamaldehyde, and thymol had no effect on the haematological parameters of RBCs, Hb, PCV, MCH, MCHC, lymphocytes, monocytes, basophils, eosinophils, heterophils, and WBCs. Whereas, Alzawqari et al. [7] and Akbarian et al. [4] noted significantly higher (P<0.05) globulin and total protein due to dietary dried sweet orange peel and lemongrass leaves supplementation in broilers. In present study also significant increase (P<0.05) in globulin concentration was recorded in T3 (100g/ton OPEO) than control. In agreement with present findings, Alzawqari et al. [7] noted significantly higher (P<0.05) total antioxidant capacity in dried sweet orange peel supplemented broilers. They justified that the orange peel being rich in phenols, Vit C and β carotene, is supposed to improve antioxidant potential and relieve oxidative stress in broilers during finisher phase. Jeon et al. [33] suggested that the OPEO is considered donor of H atoms that helps in stabilizing and decreasing number of free radicals, hence three OH-groups possessed by OPEO are highly associated to its significant antioxidant potential [7]. Akbarian et al. [4] recorded no significant influence (P>0.05) of dietary orange peel or lemon peel extract supplementation on glucose, bilirubin and albumin concentration, however increased total protein was recorded in 200mg/kg orange peel extract to supplemented birds.
Immune Response
In contrast with present findings of improved immune response; Attia et al. [8] recorded no significant influence (P>0.05) of cinnamaldehyde and thymol EO supplementation on relative weights of lymphoid organs. In support of present findings of improved immune response against RD virus in 100g/ton OPEO supplemented group (T3); Popovic et al. [47] recorded significant improvement (P<0.05) in IgA and IgG concentration in thyme, rosemary and oregano EO supplemented broilers. On the other hand, Hosseini et al. [31] recorded no significant effect (P>0.05) of lemon EO on SRBC antibody titre, IgM and IgG titres in broilers. The present findings are supported by Gole et al. [26]; who recorded comparable antibody titres at 21 days of age but significantly improved (P<0.05) titres against RD virus at day 42 in clove EO or organic acid supplemented broilers. Gole et al. [26] further explained that the efficacy of vaccination gets improved due to EO supplementation through increased antibody production [9].
Gut Health
The gut health in experimental birds showed no significant (P>0.05) effect of dietary OPEO supplementation on total microbial, coliform and lactobacilli count among the treatment groups at both 21 and 42 days. In agreement with present findings Gole et al. [26] reported significantly decreased (P<0.05) total viable count and E. coli count in clove EO and fumaric acid supplemented groups over control. They further stated that antimicrobial and antioxidant properties of EO are mainly due to their specific structure; wherein 831g/kg eugenol is contained in clove EO. Jang et al. [32] also recorded significantly reduced E. coli count and significantly increased (P<0.05) lactobacilli count in broilers supplemented 50 ppm EOs. Similarly, Aksu and Bozkurt [5] recorded significant reduction in E. coli along with significant increase (P<0.05) in lactobacilli in broilers supplemented with EO blend with or without humic acid. Sahu et al. [48] also reported the significant decrease (P<0.05) in E. coli population and total viable count in lemon and OPEO supplemented broilers. In present study also a numerical (P<0.05) increase in lactobacillus population was recorded in OPEO supplemented broilers. The variation in response to EO regarding its effect on population of pathogenic as well as beneficial microflora in intestine may be attributed to the type and dose of EO used, the type of basal diet, management of poultry house and ambience as justified earlier [17, 48].
pH and morphometry of intestine and lungs and histology of liver
In agreement with present findings Hong et al. [30] recorded comparable (P>0.05) pH in different segments of intestine. They further noted that the villus height was significantly higher (P<0.05) in duodenum due to EO supplementation; however, the villus height and crypt depth remained comparable (P>0.05) in ileum and jejunum samples. Michiels et al. [38] noted increased villus height of small intestine in broilers supplemented with carvacrol in the diet. Erhan and Bolukbasi [22] recorded best feed conversion ratio as well as feed consumption in 3ml/kg orange peel oil supplemented group which corresponded well with increased length, villi density and blood and lymphatic capillary density in the said group. The significant improvement in parabronchial dimension in OPEO supplemented groups hint towards positive impact of limonene in OPEO in terms of improved lung capacity; which to our knowledge has been recorded for the first time and might help birds to relieve probable stress of respiratory acidosis or alkalosis. The EOs are understood to promote digestive secretions, blood circulation, antioxidant activity [27]; anti-inflammatory, antibacterial and growth promoting properties [30]. Apart from this, the effect of OPEO or any other EOs on capacity or function of lungs or respiratory system hitherto is not reported. However, the significantly improved parabronchial morphometry corroborate well with earlier findings of betterment in growth performance, feed conversion efficiency, erythrocytic LPO activity, immune response and gut health respectively due to OPEO supplementation. Nonetheless further studies are warranted to assess probable positive impact of OPEO supplementation on parabronchial morphometry in particular and lung capacity in general.
The present findings seem to confirm earlier results of improved immune response and erythrocytic LPO enzyme status due to dietary OPEO supplementation. Though findings regarding effect of dietary EO supplementation on liver histology are hardly studied; an effort is made to discuss current observations in comparison with related studies earlier. The histological changes of liver are supposed to be indicative of diet quality, nutritional status of broilers and their nutrient metabolism. Mousa et al. [41] reported moderate alterations in histomorphology of kidney, duodenum and liver in broilers fed 1-2% crude protein reduction diet than recommended level. They observed that augmenting lysine supplementation by 10% above the recommended level alleviated the moderate alterations like disorganization of hepatocytes, vacuolated hepatic cells, excessive connective tissue deposition and moderate degenerative changes. However, the results of histological analysis of hepatic tissue in present study reveal no such degenerative changes in hepatic parenchyma. The minimal lipid droplets present in OPEO supplemented groups indicate efficient utilization of lipid/dietary fat arriving at liver when compared to antibiotic supplemented and control group with higher accumulation of fatty changes. The normal histology of intestine, lungs and liver in present study indicates confirmative positive influence of dietary OPEO supplementation on health of broilers.