Effect of Essential Oil and Organic Acid on Performance, Gut Health, Bacterial Count and Serological Parameters in Broiler

H Iqbal A Rahman S Khanum M Arshad IH Badar AR Asif Z Hayat MA Iqbal About the authors

ABSTRACT

A total of 1500 day old broiler chicks were grouped into five treatments; each treatment group further consisted of six replicates. Group A was given a control diet having antibiotics. Group B and C were offered essential oil blend at the proportion of 0.1 g/kg and organic acid at the dose rate of 1g/kg. Group D was given both essential oil 0.1 g/kg and organic acid 1g/kg in combination. Group E was offered only a basal diet without antibiotics and considered as a negative control. The trial lasted for 35 days. Results indicated that the body weight gain and feed conversion ratio (FCR) exhibited significant improvement but insignificant in the case of feed consumption. Bodyweight and FCR were better in group B, followed by C. Carcass characteristics like eviscerated weight and giblet weight were also improved in group B while dressed weight showed insignificant results. Total cholesterol and high-density lipoproteins (HDL) were unaffected by the treatments; however, increased blood glucose levels and decreased low-density lipoproteins (LDL) concentration were significantly noticed. Treatment groups have no effect on antibody titer against ND at the end of the trial. In gut morphology, significantly higher villus height was observed in group C, but villus width and crypt depth remained unaffected. In conclusion, essential oil and organic acids have improved performance, carcass parameters, serum biochemistry, gut health, and decreased bacterial count.

Keywords:
Alternative additives; Intestinal morphology; Organic acids; Serum biochemistry; Total viable count

INTRODUCTION

The poultry sector is a progressing and well-flourishing business in the world. The poultry industry’s present status is the result of improvement in genetics through selection and advances in poultry nutrition, especially through feed formulation. Feed additives are an essential part of feed formulation to increase performance, growth, and production. Proper use of feed additives in feed can improve feed conversion and production (Khan & Iqbal 2016Khan SH, Iqbal J. Recent advances in the role of organic acids in poultry nutrition. Journal of applied animal research 2016;44(1):359-369.).

For decades, the poultry industry has been using antibiotic feed additives to boost the birds’ growth and production. A non-therapeutic dose of antibiotics being used as growth promoters has side effects of developing anti-microbial resistance in the birds (Robinson et al., 2019Robinson K, Becker S, Xiao Y, Lyu W, Yang Q, Zhu H, et al., Differential impact of subtherapeutic antibiotics and ionophores on intestinal microbiota of broilers. Microorganisms 2019;7(9):282.). This resistance is restricted to microbe and its progeny and transmitted to other irrelevant microbial species through the transfer of special genes. These resistant bacteria are then transferred to human beings and birds via the environment. Additionally, antibiotics residues in animal products seem to be a potential factor of drug resistance in humans. Hence anti-microbial resistance has become a global issue (Agyare et al., 2018). Owing to this anti-microbial resistance, the European Union officially prohibited the utilization of antibiotics for improving birds’ growth (Abudabos et al., 2017Abudabos AM, Alyemni AH, Dafalla YM, Khan RU. Effect of organic acid blend and Bacillus subtilis alone or in combination on growth traits, blood biochemical and antioxidant status in broilers exposed to Salmonella typhimurium challenge during the starter phase. Journal of Applied Animal Research 2017;45(1):538-542.). So, nutritionists’ quest becomes more intensified to find efficient alternates of antibiotics to prevent human beings from such harmful effects of antibiotics (Chand et al., 2014Chand N, Naz S, Shah Z, Khan S, Shah A, Khan RU. Growth performance and immune status of broilers fed graded levels of Albizia lebbeck seeds. Pakistan Journal of Zoology 2014;46:574-577.).

Various natural substitutes of antibiotics growth promoters have been found efficacious with different mechanisms of action, including enzymes (exogenous or recombinant), prebiotics, clay minerals, probiotics, phytobiotic, nucleotides, symbiotic, antibodies of egg yolk, polyunsaturated fatty acids, and organic acids. All these additives help improve the production and gastrointestinal health of the birds (Sethiya 2016Sethiya NK. Review on natural growth promoters available for improving gut health of poultry: an alternative to antibiotic growth promoters. Asian Journal of Poultry Science 2016;10:1-29.). Herbs and their additives are safe for animals, humans using animal products, and the environment. Herbs or herbal extract positively affect the broiler’s performance, immune system, and hematological characteristics. Due to these positive effects and zero safety issues, herbs and their derivatives are good replacers of antibiotics (Hassan et al., 2015Hassan H, Youssef AW, Ali H, Mohamed M. Adding phytogenic material and/or organic acids to broiler diets: Effect on performance, nutrient digestibility and net profit. Asian Journal of Poultry Science 2015;9(2):97-105.). Essential oils can be defined as volatile oils derived from different plants and have anti-microbial, antiviral, antifungal properties, immunomodulatory action, hypolipidemic effect, digestive stimulation effect and have a property to alleviate the heat stress (Gopi et al., 2014Gopi M, Karthik K, Manjunathachar HV, Tamilmahan P, Kesavan M, Dashprakash M, et al., Essential oils as a feed additive in poultry nutrition. Advances in Animal and Veterinary Sciences 2014;2(1):1-7.). These essential oils are recently being used in poultry feed because the digestion process is also improved and the increase in performance. This may occur due to the positive modulation of gastrointestinal microbiota of the birds (Wade et al., 2018Wade M, Manwar S, Kuralkar S, Waghmare S, Ingle V, Hajare S. Effect of thyme essential oil on performance of broiler chicken. Journal of Entomology and Zoology Studies 2018;6(3):25-28.).

Essential oils from Capsicum and cinnamaldehyde causes certain modifications in the expression of genetic material. Capsicum oleoresin caused the changes in the genes which are involved in the metabolism and immunity of birds. Moreover, Cinnamaldehyde also changed genes’ expression in inflammatory conditions, antigen presentation, and immune response of humoral origin (O’Bryan et al., 2015O’Bryan CA, Pendleton SJ, Crandall PG, Ricke SC. Potential of plant essential oils and their components in animal agriculture - in vitro studies on antibacterial mode of action. Frontiers in Veterinary Science 2015;2(35).). Furthermore, increased thigh muscle percentage and less abdominal fat are the meat characteristics that are also improved by feeding essential oils to broilers (Mehdi et al., 2018Mehdi Y, Létourneau-Montminy M-P, Gaucher M-L, Chorfi Y, Suresh G, Rouissi T, et al., Use of antibiotics in broiler production: Global impacts and alternatives. Animal Nutrition 2018;4(2):170-178.).

Organic acids are another alternative that scientists have attracted because of their antibacterial effect against different pathogenic microorganisms. Moreover, it affects decreasing pH in the gastrointestinal tract, which leads to the improvement of the utilization of nutrients in the broiler birds (Kim et al., 2015Kim JW, Kim JH, Kil DY. Dietary organic acids for broiler chickens: a review. Revista Colombiana de Ciencias Pecuaria 2015;28(2):109-123.). Organic acids are safer feed additive, and the European Union has permitted to use of them in poultry feed (Elnaggar & EL-Maaty 2017Elnaggar AS, EL-Maaty HMA. Impact of using organic acids on growth performance, blood biochemical and hematological traits and immune response of ducks (Cairina moschata). Egyptian Poultry Science Journal 2017;37(3):907-925.; Sultan et al., 2015Sultan A, Ullah T, Khan S, Khan RU. Effect of organic acid supplementation on the performance and ileal microflora of broiler during finishing period. Pakistan Journal of Zoology 2015;47(3):635-639.). Moreover, organic acids can increase the digestibility of protein, increase the pancreas’ secretion, improve gut morphology, and decrease gut microbes’ (anti-microbial property) activity (Liu et al., 2017Liu Y, Yang X, Xin H, Chen S, Yang C, Duan Y, et al., Effects of a protected inclusion of organic acids and essential oils as antibiotic growth promoter alternative on growth performance, intestinal morphology and gut microflora in broilers. Animal Science Journal 2017;88(9):1414-1424.). Phytogenic feed additives, as well as organic acids, are together given in the diet have the capability of enhancing nutrient digestion resulting in increased broiler performance. So, the utilization of both of these compounds can be a better substitute for antibiotic growth promoters (Yang et al., 2018Yang X, Xin H, Yang C, Yang X. Impact of essential oils and organic acids on the growth performance, digestive functions and immunity of broiler chickens. Animal Nutrition 2018;4(44):388-393.). The interlinked phenomena behind both of these compounds’ synergism may be the positive modulation of gastrointestinal microflora. The hydrophobic nature of essential oils makes the bacterial cell membrane more permeable, resulting in an increased influx of organic acids into the cellular cytoplasm. Hence, the organic acids in their un-dissociated form make cellular pH more acidic, resulting in hampered bacteria’s cellular metabolism (Stefanello et al., 2020Stefanello C, Rosa DP, Dalmoro YK, Segatto AL, Vieira MS, Moraes ML, et al., Protected blend of organic acids and essential oils improves growth performance, nutrient digestibility, and intestinal health of broiler chickens undergoing an intestinal challenge. Frontiers in Veterinary Science 2020;6:491.). The public concerns about the availability of antibiotic residues free meat has a significant role in the consumption of poultry products. Both essential oils and organic acids can provide this antibiotic residue-free meat and will positively contribute to the development of meat industry. Keeping in view all the above facts, this study was designed to evaluate the efficacy of essential oil or organic acids as effective substitutes of antibiotics by investigating their effects on performance, gut health, total bacterial count in intestine and biochemical effects.

MATERIALS AND METHODS

A total of 1500 broiler (Ross 308) chicks were obtained from a commercial hatchery (Jadeed Group), and they were divided into 5 groups randomly. Each treatment had 300 chicks. Each group was further consisted of 6 replicates having 50 birds in each. Group A was given a normal diet having antibiotics (Enramycine; Enradin 8% MSD; 0.3 grams/Kg). Group B was given a basal diet and a commercially available essential oils product named Activo™ (EW Nutrition Germany; Marketed by Ghazi brothers in Pakistan) at the dose rate of 0.1 grams/Kg of basal diet. Activo™ is a blend of essential oils containing oregano, rosemary, cinnamon, and chili pepper extract as active ingredients. Group C consisted of organic acid (Sodium diformate, Formi NDF; Addcon Germany; Marketed by Ghazi brothers in Pakistan) at the dose rate of 1g/Kg of the basal diet. Sodium diformate was composed of formic acid 40%, formate 40%, and sodium 20%. Group D was offered with the mixture of both essential oils and organic acid at the dose rate of 0.1 gram/Kg of “Activo™” essential oils and 1g/Kg of organic acid in the diet. Group E contained only a basal diet considering it as a negative control. The whole experimental layout is given in the table 1. The birds were kept in a controlled environment containing pan feeders for feed and nipple drinkers for water availability. Pellet feed and fresh drinking water were givenad libitum.The shed’s temperature was maintained at 32°C for the early five days then gradually declined according to standard management protocols up to 25°C. The humidity of the shed was kept (65±5%). The lighting period was almost 23 hours per day. The bedding material was rice husk (8cm thickness), and floor space was 5.5×3.5×2ft (L×W×H) according to the standard requirement of the chicks. Birds were already vaccinated for Marek’s disease by the hatchery. According to standard procedures, the vaccine for Newcastle disease, infectious bronchitis, and infectious bursal disease was given. The whole flock was kept under serious observation until 35 days to complete. All other management procedures were performed according to the recommended protocols. Feed was offered in two phases, starter (0-22 days) and finisher (22-35 days) only. The ingredient and nutrient composition of the formulated diet is given in Table 2. The growth performance of the birds was measured weekly. Feed intake was measured by subtracting refused feed from the total offered feed on weekly basis. Weekly weight gain was measured by subtracting initial body weight from the final body weight. Moreover, the weekly feed conversion ratio was calculated as feed consumed in grams divided by body weight in grams. Carcass parameters such as dressed weight, eviscerated weight, and giblet weight were studied at the trial period’s termination. Four birds from each replicate were picked randomly and slaughtered. After slaughtering and removing blood, feathers, and visceral organs, the carcass was weighed to check the dressed weight. Eviscerated weight and giblet weight were recorded. For gut morphology, tissue samples from jejunum were taken from three birds per replicate. The Jejunum was cut about 3cm at its central region. Then these samples were preserved in the neutral buffer formalin (SJQW03140 Sigma-Aldrich, Merck; 10%) for 48 hours. After that, tissue samples were washed by using tap water and then treated with an alcohol solution (L850107 BDH). After sectioning of the tissues, samples were embedded in paraffin with the help of cassettes. Then sample tissue was cut down into 4-micrometer sections via microtome, mounted on the slide, and appropriately stained with HE (hematoxylin and eosin) stain (Medilines modified H 0706; E 920-921); after that, a light microscope was used for the examination of tissue sections and to determine the villus height and crypts depth. The measurement from the apex (tip of the upper border) of the villus till lamina propria was taken and recorded as villus height. Crypts depth was examined as the length between the crypts and villi according to the recommended protocol (Panda et al., 2009Panda A, Rao S., Raju M., Sunder G.S.: Effect of butyric acid on performance, gastrointestinal tract health and carcass characteristics in broiler chickens. Asian-Australasian Journal of Animal Sciences 2009;22(7):1026-1031.). At the study trial’s termination, the day after 35th, blood samples were taken during the birds slaughtering in non-heparinized tubes. Then centrifugation was done at 3000 RPM for 15 minutes for serum separation and kept in the freezer at -20°C until use. Then serum was thawed at room temperature. Serum sample was analyzed to determine total cholesterol, HDL and LDL by the method as described by Kamal & Ragaa (2014Kamal AM, Ragaa NM. Effect of dietary supplementation of organic acids on performance and serum biochemistry of broiler chicken. Natural Science 2014;12(2):38-45.) and Vinus et al. (2017Vinus, Sheoran N, Shunthwal J, Akbar MA, Tewatia BS. Effect of Supplementation of Salts of Organic Acids on Serum and Haematological Parameters of Broilers. International Journal of Current Microbiology and Applied Sciences 2017;6(11):4211-4218.) and glucose concentration was determined by the method as described by Adil et al. (2010Adil S, Banday T, Bhat GA, Mir MS, Rehman M. Effect of dietary supplementation of organic acids on performance, intestinal histomorphology, and serum biochemistry of broiler chicken. Veterinary Medicine International 2010;2010:479485.). Humoral immunity was estimated by using antibody titer of IgG against the NDV vaccine by standard hemagglutination inhibition test. Briefly, birds were vaccinated for NDV on day 1st, followed by 7th and 17th day via drinking water. Samples of blood were taken through slaughtering on the day after the 35th to determine antibody titer. Three ml blood sample was taken in a non-heparinized vacutainer and placed on a cold chain. After thawing and centrifugation of the sample at 3000 RPM for 15 minutes at 23°C, serum was separated and kept at -20°C for storage and further procedure. The NDV antibody titer was determined through a commercial HI-based kit as described by (Aksu & Bozkurt, 2009Aksu T, Bozkurt AS. Effect of dietary essential oils and/or humic acids on broiler performance, microbial population of intestinal content and antibody titres in the summer season. Kafkas Üniversitesi Veteriner Fakültesi Dergisi 2009;15:185-190.). For bacterial count, three birds were chosen randomly from each replicate at the experimental period’s termination, and slaughtering was performed. After slaughtering, part of the intestine from the duodenal distal end to the ileocaecal junction was severed and taken out. Digesta was taken out from the intestine of about one gram amount and diluted with 0.9% sodium chloride solution. Dilution was made up to ten-fold, and a 1 ml diluted solution from each dilution was taken and inoculated on an agar plate with the help of the spread plate method and the count was done according to the standard procedure (Hartemink & Rombouts 1999Hartemink R, Rombouts FM. Comparison of media for the detection of bifidobacteria, lactobacilli and total anaerobes from faecal samples. Journal of Microbiology Methods 1999;36:181-192.; Hassan et al., 2010Hassan H, Mohamed M, Youssef AW, Hassan ER. Effect of using organic acids to substitute antibiotic growth promoters on performance and intestinal microflora of broilers. Asian-Australian Journal of Animal Science 2010;23:1348-1353.); after that, the bacterial population was taken as log10 CFU/gram. The data analysis was performed with the help of one-way ANOVA (Analysis of variance) using the SPSS version 23.0. The difference of means among the treatments was measured through Duncan’s comparison test. The significance level was set at p≤0.05.

Table 1
Experimental design of the study.
Table 2
Ingredient composition of starter and finisher phase diets.

RESULTS

According to this study, non-significant results were noticed in case of feed intake in broilers. However, in 2nd-week feed intake manifested a remarkable difference among different groups (Table 3). Essential oils and organic acids increased the weight of broilers significantly except at the 4th weeks of age. The maximum weight was measured in group B having essential oils followed by group C having organic acids. However, the organic acids treated group manifested the highest weight in the first week, followed by an essential oil group. In the 4th week, no significant improvement was noticed, irrespective of the treatment group (Table 4).

Table 3
Effect of essential oil and organic acid on mean daily feed intake (g) of broilers.
Table 4
Effect of essential oil and organic acid on weekly body weight gain (g) of broilers.

The feed conversion ratio showed significant improvement in essential oil and organic acid groups compared with the antibiotic group. The most significant improvement was found in essential oil group B while the 4th week had no statistically significant results among all groups. After essential oil, the best improved FCR was noticed in group C (organic acids) at 2nd and 3rd week. (Table 5).

Table 5
Effect of essential oil and organic acid on feed conversion ratio in broilers.

Our study results exhibited a marked (p<0.05) increase in carcass characteristics, especially in eviscerated weight and giblet weight. Group B containing essential oils, revealed the highest eviscerated weight and giblet weight than all other groups, followed by group C (organic acids). In dressing percentage or dressed weight, no significant difference was found among the groups (Table 6).

Table 6
Effect of essential oils and organic acid on carcass characteristics of broiler.

Our study revealed no remarkable change in total cholesterol and high-density lipoprotein levels when birds were offered essential oil and organic acids in the diet. Low-density lipoprotein was decreased by feeding essential oils and organic acids. Group B containing essential oil has the lowest LDL level as compared to other groups, followed by group C and D (Table 9). Essential oil and organic acids raised the blood glucose level significantly compared to the control group. Glucose level was highest in the organic acid group C followed by group B having essential oil (Table 9). There was no significant effect on antibody titer noticed on the 35th days of age (Table 7).

Table 7
Effect of essential oil and organic acid on serological parameters of broilers.

A significant increase in jejunal villus height was observed by feeding essential oil and organic acids to the broilers. The maximum height was noticed in group C having organic acids followed by group B. In the case of villus width and crypts depth, and there was no remarkable difference among the groups (Table 8).

Table 8
Effect of essential oil and organic acids on gut health of broilers.

This study revealed that essential oil and organic acids significantly lower the total viable count in broilers’ intestine. Our results are showing that the lowest number of aerobic bacteria was counted in group D having both essential oil and organic acid, followed by group C that contains organic acids in the diet (Table 9).

Table 9
Effect of essential oil and organic acids on total bacterial count in broilers.

DISCUSSION

According to this study, non-significant results were noticed in feed intake in broilers except for 2nd-week. Our study results follow earlier research, which revealed no noticeable influence on weekly feed consumption among organic acid, essential oils, and antibiotics supplemented group (Bozkurt et al., 2012Bozkurt M, Küçükyılmaz K, Çatlı A, Çınar M, Çabuk M, Alçiçek A. Effects of administering an essential oil mixture and an organic acid blend separately and combined to diets on broiler performance. Archiv fur Geflugelkunde 2012;76(2):81-87.; Cabuk et al., 2014Cabuk M, Eratak S, Alçiçek A, Tuglu I. Effect of herbal essential oil mixture on intestinal mucosal development, growth performance, and weights of internal organs of quails. Journal of Essential Oil Bearing Plants 2014;17(4):599-606.; Mohammadi Gheisar et al., 2015Mohammadi Gheisar M, Hosseindoust A, Kim I. Evaluating the effect of microencapsulated blends of organic acids and essential oils in broiler chickens diet. Journal of Applied Poultry Research 2015;24(4):511-519.; Basmacioğlu-Malayoğlu et al., 2016Basmacioğlu-Malayoğlu H, Ozdemir P, Bağriyanik H. Influence of an organic acid blend and essential oil blend, individually or in combination, on growth performance, carcass parameters, apparent digestibility, intestinal microflora and intestinal morphology of broilers. British Poultry Science 2016;57:227-234.). Essential oils and organic acids increased the weight of broilers significantly except for 4th week than that of the antibiotic group. Several previous studies favor our results, showing the increased body weight by feeding essential oils and organic acid groups rather than a control group containing antibiotics (Basmacioğlu-Malayoğlu et al., 2016; Fathi et al., 2016Fathi R, Samadi MS, Qotbi A, Seidavi A, Martínez-Marín AL. Effects of feed supplementation with increasing levels of organic acids on growth performance, carcass traits, gut microbiota and pH, plasma metabolites, and immune response of broilers. Animal Science Papers Reports 2016;34(2);195-206.; Peng et al., 2016Peng Q, Li J, Li Z, Duan Z, Wu Y. Effects of dietary supplementation with oregano essential oil on growth performance, carcass traits and jejunal morphology in broiler chickens. Animal Feed Science and Technology 2016;214:148-153.; Elnaggar & EL-Maaty 2017Elnaggar AS, EL-Maaty HMA. Impact of using organic acids on growth performance, blood biochemical and hematological traits and immune response of ducks (Cairina moschata). Egyptian Poultry Science Journal 2017;37(3):907-925.). The feed conversion ratio manifested the significant improvement in essential oil and organic acid groups compared with the antibiotics group. Our results also match with previous studies which show that essential oils, as well as organic acids, have improved FCR in comparison with the control group (Fascina et al., 2012Fascina VB, Sartori JR, Gonzales E, Carvalho FBd, Souza IMGPd, Polycarpo GdV, et al., Phytogenic additives and organic acids in broiler chicken diets. Revista Brasileira de Zootecnia 2012;41(10):2189-2197.; Basmacioğlu-Malayoğlu et al., 2016), while some studies are against us showing no improvement in growth performance with essential oils as well as organic acids (Belenli et al., 2015Belenli D, Udum D, Cengiz SŞ, Polat Ü. Influence of various volatile oils as a dietary supplement on biochemical and performance parameters in broilers. Journal of Environmental Sciences 2015;9:47-55.; Pathak et al., 2017Pathak M, Mandal G, Patra A, Samanta I, Pradhan S, Haldar S. Effects of dietary supplementation of cinnamaldehyde and formic acid on growth performance, intestinal microbiota and immune response in broiler chickens. Animal Production Science 2017;57(5):821-827.). Stimulation of digestion, modulation of gut flora, and increased release of different endogenous digestive enzymes due to essential oils are the possible reasons for improved performance (Popović et al., 2016Popović S, Puvača N, Kostadinović L, Džinić N, Bošnjak J, Vasiljević M, et al., Effects of dietary essential oils on productive performance, blood lipid profile, enzyme activity and immunological response of broiler chickens. European Poultry Science 2016;80:1-12.). Along with the antibacterial effect, herbal extract’s antioxidant property may also be the possible reason for enhanced broiler’s performance (El-Shenway & Ali, 2016El-Shenway AM, Ali GI. Effect of Some Organic Acids and Essential Oils as Feed Additives on Growth Performance, Immune Response and Carcass Quality of Japanese Quail. Alexandria Journal for Veterinary Sciences 2016;51(1): 68-77.). In the case of growth performance, essential oils manifested controversy in several previous studies on broilers. Some studies are in favor, while others are against us. It is difficult enough to resolve these controversies and to generate biologically consistent results due to a lot of variations in inclusion levels, active ingredient concentrations, types of essential oil used, surrounding environment conditions, basal feed composition, infectious diseases outbreak, and origin of that herbal extract (Bozkurt et al., 2012; Basmacioğlu-Malayoğlu et al., 2016). Additionally, enhanced broilers’ weight and improved FCR may be due to the beneficial impact of organic acids on gut microflora and the bactericidal effect of organic acids because organic acids cause the interference of bacterial cell membrane and macromolecules of cells leading to hindrance in energy metabolism and nutrients transport (Elnaggar & EL-Maaty, 2017).

Our study results exhibited a marked (p<0.05) increase in carcass characteristics, especially in eviscerated weight and giblet weight. This is also favored by Rehman et al. (2016Rehman ZU, Haq AU, Akram N, Abd El-Hack ME, Saeed M, Rehman SU, et al., Growth performance, intestinal histomorphology, blood hematology and serum metabolites of broilers chickens fed diet supplemented with graded levels of acetic acid. International Journal of Pharmacology 2016;12(8):874-883.), who found insignificant results in dressing percentage. Previous studies are also in favor of us, showing that essential oils and organic acids exert better overall carcass traits (Khattak et al., 2013Khattak F, Ronchi A, Castelli P, Sparks N. Effects of natural blend of essential oil on growth performance, blood biochemistry, cecal morphology, and carcass quality of broiler chickens. Poultry Science 2013;93(1):132-137.; Peng et al., 2016Peng Q, Li J, Li Z, Duan Z, Wu Y. Effects of dietary supplementation with oregano essential oil on growth performance, carcass traits and jejunal morphology in broiler chickens. Animal Feed Science and Technology 2016;214:148-153.; Ragaa et al., 2016Ragaa NM, Korany RM, Mohamed F. Effect of thyme and/or formic acid dietary supplementation on broiler performance and immunity. Agriculture and Agricultural Science Procedia 2016;10:270-279.). While some researches are against our findings, no significant effect on carcass parameters was noticed (El-Shenway & Ali, 2016El-Shenway AM, Ali GI. Effect of Some Organic Acids and Essential Oils as Feed Additives on Growth Performance, Immune Response and Carcass Quality of Japanese Quail. Alexandria Journal for Veterinary Sciences 2016;51(1): 68-77.; Özsoy et al., 2017Özsoy B, Ölmez M, Karadağoğlu Ö, Şahin T. Determination of the effect of mixture of essential oil and organic acid containing at different levels on broiler rations on fattening performance, carcass parameters and some internal organ weights. Lalahan Hayvancılık Araștırma Enstitüsü Dergisi 2017;57(2):77-82.; Gomathi et al., 2018Gomathi G, Senthilkumar S, Natarajan A, Amutha R, Purushothaman MR. Effect of dietary supplementation of cinnamon oil and sodium butyrate on carcass characteristics and meat quality of broiler chicken. Veterinary World 2018;11(7):959.). This conflict of results may be due to variation in diet composition and breeds of broilers (Peng et al., 2016). The improvement in carcass characteristics is due to the existence of antioxidants and phenolic compounds in essential oil, decreasing the harmful microbial population in the digestive tract of birds and increasing the absorbed amino acid level. Moreover, an active ingredient of essential oil like carvacrol has triggering action on secretions of the pancreas. This high digestive secretion can improve digestion and absorption of certain nutrients (amino acids), leading to improved carcass characteristics (Ragaa et al., 2016). Better digestion and slower transit rate through the intestine due to organic acid feeding make certain improvements in the absorption of specific nutrients leading to better carcass parameters. Moreover, better proteolysis and digestion of proteins and amino acids cause better musculature. This may be the other reason for improved carcass parameters by organic acids (Hossain & Nargis, 2016Hossain M, Nargis F. Supplementation of organic acid blends in water improves growth, meat yield, dressing parameters and bone development of broilers. Bangladesh Journal of Animal Science 2016;45(1):7-18.).

Our study revealed no remarkable change in total cholesterol and high-density lipoprotein levels due to feeding essential oil and organic acids in the diet. Low-density lipoprotein was decreased by feeding essential oils and organic acids. Our results are according to previous research in which essential oil and organic acids decreased the serum LDL concentration. At the same time, HDL remained unchanged (Elnaggar & El-Tahawy, 2018Elnaggar AS, El-Tahawy W. Productive performance, physiological and immunological response of broiler chicks as affected by dietary aromatic plants and their essential oils. Egyptian Poultry Science Journal 2018;38(3).; Yildirim et al., 2018Yildirim BA, Tunc MA, Gül M, Yildirim F, Yıldız A. The effect of Rosemary (Rosmarinus officinalis L.) extract supplemented into broiler diets, on performance and blood parameters. GSC Biological and Pharmaceutical Sciences 2018;2(3):1-9.), and the total cholesterol level also remained unchanged (Popović et al., 2016Popović S, Puvača N, Kostadinović L, Džinić N, Bošnjak J, Vasiljević M, et al., Effects of dietary essential oils on productive performance, blood lipid profile, enzyme activity and immunological response of broiler chickens. European Poultry Science 2016;80:1-12.; Vinus et al., 2017Vinus, Sheoran N, Shunthwal J, Akbar MA, Tewatia BS. Effect of Supplementation of Salts of Organic Acids on Serum and Haematological Parameters of Broilers. International Journal of Current Microbiology and Applied Sciences 2017;6(11):4211-4218.). The reduction in low-density cholesterol is the inhibition of specific enzymes like peroxidase and dehydrogenase by herbal extracts (essential oils). Moreover, active ingredients of these essential oils like carvacrol and thymol have a limiting effect on lethal metabolites, resulting in increased blood cholesterol levels (Hedayati & Manafi, 2018Hedayati M, Manafi M. Evaluation of anherbal compound, a commercial probiotic, and an antibiotic growth promoter on the performance, intestinal bacterial population, antibody titers, and morphology of the jejunum and ileum of broilers. Brazilian Journal of Poultry Science 2018;20(2):305-316.).

Essential oil and organic acids raised the blood glucose level significantly compared to the control group. Our findings match several past studies showing that essential oil and organic acid increased glucose levels compared with the control group (Rahman et al., 2015; Elnaggar & EL-Maaty, 2017Elnaggar AS, EL-Maaty HMA. Impact of using organic acids on growth performance, blood biochemical and hematological traits and immune response of ducks (Cairina moschata). Egyptian Poultry Science Journal 2017;37(3):907-925.; Elnaggar & El-Tahawy, 2018). However, some studies are against our findings showing no noticeable influence on broilers’ glucose level (Belenli et al., 2015Belenli D, Udum D, Cengiz SŞ, Polat Ü. Influence of various volatile oils as a dietary supplement on biochemical and performance parameters in broilers. Journal of Environmental Sciences 2015;9:47-55.). There was no significant effect on antibody titer noticed at 35th days of age (Table 9). Our results match previous studies with no significant influence of dietary essential oils and organic acids separately or in combination with antibody titer at the end of the trial (Fascina et al., 2017Fascina VB, Pasquali G, Carvalho F, Muro E, Vercese F, Aoyagi M, et al., Effects of phytogenic additives and organic acids, alone or in combination, on the performance, intestinal quality and immune responses of broiler chickens. Brazilian Journal of Poultry Science 2017;19(3):497-508.).

A significant increase in jejunal villus height was observed by feeding essential oil and organic acids to the broilers. Several previous studies’ results are similar to our results showing improvement in villus height, i.e. (Liu et al., 2017Liu Y, Yang X, Xin H, Chen S, Yang C, Duan Y, et al., Effects of a protected inclusion of organic acids and essential oils as antibiotic growth promoter alternative on growth performance, intestinal morphology and gut microflora in broilers. Animal Science Journal 2017;88(9):1414-1424.; Sukandhiya et al., 2017Sukandhiya K, Mani K, Rajendran K, Sangli Vikram Kumar K, Prabhakaran U, Sasidhar T. Influence of dietary supplementation of sodium diformate on the intestinal histo: morphology of broilers in environmentally controlled housing system. International Journal of Chemical Studies 2017;5(6):842-843.). If villus length and crypt depth are shorter, the bird has a lower nutrient absorption rate due to less surface area available for absorption, and enterocytes are less mature. Thus less nutrient absorption leads to the poor performance of the bird (Paiva et al., 2014Paiva D, Walk C, McElroy A. Dietary calcium, phosphorus, and phytase effects on bird performance, intestinal morphology, mineral digestibility, and bone ash during a natural necrotic enteritis episode. Poultry Science 2014; 93(11):2752-2762.). Moreover, essential oils and organic acids cause regulation and balancing effect on gastrointestinal microflora. This effect may be beneficial in improving gut morphology (Zeng et al., 2015Zeng Z, Zhang S, Wang H, Piao X. Essential oil and aromatic plants as feed additives in non-ruminant nutrition: a review. Journal of Animal Science and Biotechnology 2015;6(1):7.). Furthermore, improved intestinal morphology may be due to the anti-inflammatory and anti-oxidation mechanism of essential oils and organic acids (Du et al., 2016Du E, Wang W, Gan L, Li Z, Guo S, Guo Y. Effects of thymol and carvacrol supplementation on intestinal integrity and immune responses of broiler chickens challenged with Clostridium perfringens. Journal of Animal Science and Biotechnology 2016;7:19.; Gao et al., 2019Gao Y-Y, Zhang X-L, Xu L-H, Peng H, Wang C-K, Bi Y-Z. Encapsulated blends of essential oils and organic acids improved performance, intestinal morphology, cecal microflora, and jejunal enzyme activity of broilers. Czech Journal of Animal Science 2019;64(5):189-198.).

This study revealed that essential oil and organic acids have a significant lowering effect on the total viable count in broilers’ intestine. Our results are showing that the lowest number of aerobic bacteria was counted in the group “D,” which contains both essential oil and organic acid, followed by group “C” that contains organic acids in the basal diet. Our results are matching with previous researches, which exhibited that essential oil and organic acids have a significant decreasing effect on bacterial gut count (Basmacioğlu-Malayoğlu et al., 2016Basmacioğlu-Malayoğlu H, Ozdemir P, Bağriyanik H. Influence of an organic acid blend and essential oil blend, individually or in combination, on growth performance, carcass parameters, apparent digestibility, intestinal microflora and intestinal morphology of broilers. British Poultry Science 2016;57:227-234.; Ndelekwute et al., 2018Ndelekwute EK, Enyenihi GE, Essien EB. Effect of organic acid treated diets fed during finisher phase on gut microbiota and blood profile of broiler chickens. Journal of Dairy & Veterinary Sciences 2018;5(4).) and Chowdhury et al. (2018Chowdhury S, Mandal GP, Patra AK, Kumar P, Samanta I, Pradhan S, Samanta AK. Different essential oils in diets of broiler chickens: 2. Gut microbes and morphology, immune response, and some blood profile and antioxidant enzymes. Animal Feed Science and Technology 2018;236:39-47.) also observed that pathogenic bacterial count likeEscherichia coliwas reduced by feeding cinnamon essential oil, but beneficial bacterial species likeLactobacillus spp. was unaffected. However, Pathak et al. (2016Pathak M, Mandal G, Patra A, Samanta I, Pradhan S, Haldar S. Effects of dietary supplementation of cinnamaldehyde and formic acid on growth performance, intestinal microbiota and immune response in broiler chickens. Animal Production Science 2016;57(5):821-827.) observed no noticeable effect on a total viable bacterial count by feeding cinnamaldehyde and formic acid to the broilers. The lipophilic nature of organic acids in their un-dissociated form causes them to enter the bacterial cell through passive diffusion. This intracellular acid affects cells’ normal physiology because the bacterial cell cannot bear acidic pH, causing the stoppage of microbial enzymes and transport systems, leading to inhibition of bacterial growth (Ragaa & Korany, 2016Ragaa NM, Korany RM, Mohamed F. Effect of thyme and/or formic acid dietary supplementation on broiler performance and immunity. Agriculture and Agricultural Science Procedia 2016;10:270-279.). This may be the reason behind the antibacterial activity of organic acids.

CONCLUSION

Hence, we concluded that essential oil and organic acids have improved performance, carcass parameters, serum biochemistry, gut health, and decreased bacterial count. The consequences of group B (Essential oil supplemented) were the best among all treatment groups. According to our research, almost no synergism between essential oil and organic acids was noticed except for the gut microbial count parameter. So essential oil or organic acids can be a better substitute for antibiotics separately rather than in combination.

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  • FINANCIAL DISCLOSURE STATEMENT

    There was financial support for this research.
  • ANIMAL RIGHTS STATEMENT

    The experiments on animals were conducted following the local Ethical Committee laws and regulations regarding the care and use of experimental animals.

Publication Dates

  • Publication in this collection
    23 July 2021
  • Date of issue
    2021

History

  • Received
    03 Jan 2021
  • Accepted
    19 Mar 2021
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