Orange essential oil in the diet of broilers: performance, organ biometrics, bone characteristics, and intestinal morphometry

Christiane Silva Souza Flávio Medeiros Vieites Lucas Rodrigo Justino Marcos Fabio de Lima Amália Saturnino Chaves Cibele Silva Minafra Cristina Amorim Ribeiro de Lima About the authors

ABSTRACT

Growth performance, organ biometrics, bone characteristics, and intestinal morphometry were evaluated in broilers fed a diet containing orange (Citrus sinensis L.) essential oil. A completely randomized design was used, with five treatments with orange essential oil (0, 100, 200, 300, and 400 mg kg−1 diet) and six replications with 20 birds per experimental unit. In the pre-starter phase, feed intake and weight gain of all birds linearly increased, while feed conversion decreased with the addition of orange essential oil in the feed. At day 21, bone density (Seedor Index) and body weight were higher in the birds that received the maximum level of essential oil (400 mg kg−1) compared with those not treated with essential oil. The observed effects resulted from the better functioning of the physiological mechanisms of digestion and absorption of nutrients, characterized by the increase in villus height. Glycemia and weights of gastrointestinal tract organs of broilers at 21 days of age were not influenced by the evaluated essential oil. The results show that the addition of phytogenic additives to the diet does not cause any physiological impairment in birds.

Keywords:
Citrus sinensis; jejunum; limonene; phytogenic additive; Seedor index

1. Introduction

The animal production industry has undergone countless and significant changes in recent years. There is an increasing concern with issues of food security for consumption, food impacts for human health, and bacterial resistance to the antimicrobials used as feed additives. Restrictions in the use of antibiotics to improve performance exacerbated the need of using alternative additives, such as phytogenics or phytobiotics (Seidavi et al., 2020Seidavi, A.; Zaker-Esteghamati, H. and Salem, A. Z. M. 2020. A review on practical applications of Citrus sinensis by-products and waste in poultry feeding. Agroforestry Systems 94:1581-1589. https://doi.org/10.1007/s10457-018-0319-2
https://doi.org/10.1007/s10457-018-0319-...
).

Phytogenic additives are substances derived from medicinal plants or spices, such as essential oils, which have positive effects on production and health of animals. Beneficial results of phytogenic additives are attributed to the presence of diverse classes of active substances conferring antimicrobial actions and stimulus to digestion and promoting production of endogenous enzymes in animals (Erhan and Bölükbaşi Aktaş, 2017Erhan, M. K. and Bölükbaşi Aktaş, Ş. C. 2017. Narenciye kabuk yağlarının etlik piliçlerde doku yağ asidi kompozisyonu ve raf ömrü üzerine etkileri. Atatürk Üniversitesi Veteriner Bilimleri Dergisi 12:157-166.; Aydin et al., 2018Aydin, A.; Korkunç, M.; Demirel, D. Ş. and Gül, S. 2018. Portakal kabuğu (Citrus sinensis L.) uçucu yağının kanatlı beslemedeki önemi. Uluslararası Doğu Akdeniz Tarımsal Araştırma Enstitüsü Dergisi 1:13-19.; Seidavi et al., 2020Seidavi, A.; Zaker-Esteghamati, H. and Salem, A. Z. M. 2020. A review on practical applications of Citrus sinensis by-products and waste in poultry feeding. Agroforestry Systems 94:1581-1589. https://doi.org/10.1007/s10457-018-0319-2
https://doi.org/10.1007/s10457-018-0319-...
; Sevim et al., 2020Sevim, B.; Olgun, O.; Şentürk, E. T. and Yıldız, A. Ö. 2020. The effect of orange peel oil addition to laying quail diets on performance, eggshell quality and some serum parameters. Turkish Journal of Agriculture – Food Science and Technology 8:1773-1777. https://doi.org/10.24925/turjaf.v8i8.1773-1777.3508
https://doi.org/10.24925/turjaf.v8i8.177...
). There is also evidence that essential oils can impact pathogen concentrations in the intestine and improve feed digestibility (Micciche et al., 2019Micciche, A.; Rothrock Jr., M. J.; Yang, Y. and Ricke, S. C. 2019. Essential oils as an intervention strategy to reduce Campylobacter in poultry production: a review 10:1058. Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2019.01058
https://doi.org/10.3389/fmicb.2019.01058...
).

In recent years, interest in using essential oils in feed has been increasing. Citrus oils contain high quantities of limonene (El Sawi et al., 2019El Sawi, S. A.; Ibrahim, M. E.; El-Rokiek, K. G. and El-Din, S. A. S. 2019. Allelopathic potential of essential oils isolated from peels of three citrus species. Annals of Agricultural Sciences 64:89-94. https://doi.org/10.1016/j.aoas.2019.04.003
https://doi.org/10.1016/j.aoas.2019.04.0...
; Sahu et al., 2019Sahu, A. C.; Manwar, S. J.; Gole, M. A.; Khose, K. K.; Wade, M. R. and Kuralkar, S. V. 2019. Effect of lemon and orange peel essential oils on performance of broiler chickens during summer. Indian Journal of Poultry Science 54:139-145. https://doi.org/10.5958/0974-8180.2019.00029.1
https://doi.org/10.5958/0974-8180.2019.0...
; Erhan, 2020Erhan, M. K. 2020. The potential of orange peel oil as a suppressor of cell proliferation in animal feed and human nutrition: an experimental study. Alinteri Journal of Agriculture Sciences 35:44-49. https://doi.org/10.28955/alinterizbd.740928
https://doi.org/10.28955/alinterizbd.740...
), to which most of their biological activities have been attributed, including antimicrobial (Bozkurt et al., 2017Bozkurt, T.; Gülnaz, O. and Kaçar, Y. A. 2017. Chemical composition of the essential oils from some citrus species and evaluation of the antimicrobial activity. Journal of Environmental Science, Toxicology and Food Technology 11:29-33.; Ambrosio et al., 2019Ambrosio, C. M. S.; Ikeda, N. Y.; Miano, A. C.; Saldaña, E.; Moreno, A. M.; Stashenko, E.; Contreras-Castillo, C. J. and Gloria, E. M. 2019. Unraveling the selective antibacterial activity and chemical composition of citrus essential oils. Scientific Reports 9:17719. https://doi.org/10.1038/s41598-019-54084-3
https://doi.org/10.1038/s41598-019-54084...
), antioxidant, anti-inflammatory, analgesic or anti-nociceptive, antidiabetic, induction of osteogenesis, and effects on the gastrointestinal tract (Soulimani et al., 2019Soulimani, R.; Bouayed, J. and Joshi, R. K. 2019. Limonene: natural monoterpene volatile compounds of potential therapeutic interest. American Journal of Essential Oils and Natural Products 7:1-10.).

Essential oils affect bacterial proliferation in four ways: affecting the cell wall by removing phospholipids and obstructing ions of passive passage through the passage obstruction of active ions and inhibition of ATP synthesis, destruction of bacterial cytoplasm, and inhibition of energy synthesis in mitochondria (Chouhan et al., 2017Chouhan, S.; Sharma, K. and Guleria, S. 2017. Antimicrobial activity of some essential oils - present status and future perspectives. Medicines 4:58. https://doi.org/10.3390/medicines4030058
https://doi.org/10.3390/medicines4030058...
; Namdeo et al., 2020Namdeo, S.; Baghel, R. P. S.; Nayak, S.; Khare, A.; Pal, R. P.; Chaurasiya, A.; Thakur, S. and Reddy, B. V. V. 2020. Essential oils: an potential substitute to antibiotics growth promoter in broiler diet. Journal of Entomology and Zoology Studies 8:1643-1649.).

Competitiveness in poultry farming is based on the obtention of better animal-performance indexes associated with the welfare and health of animals, as well as cost reduction and environmental impacts. Hence, it is relevant to study and evaluate the effect of different nutritional plans and breeding conditions on the physiology, biochemistry, and production of birds and combine these treatments to maximize benefits.

This study aimed to evaluate productive performance, organ biometrics, bone characteristics, and morphometry of the intestine of broilers fed a diet containing orange essential oil (Citrus sinensis L.).

2. Material and Methods

The procedures used in this study were approved by the Institutional Committee on Animal Use (case number 001/2018). The experiment was conducted in Pinheiral, Rio de Janeiro, Brazil (22°30'46" S, 44°00'02" W).

The statistical design was completely randomized, with five treatments, six repetitions, and 20 birds per experimental unit. The experiment utilized 600 commercial broilers from one to seven days of age. The treatments were 0, 100, 200, 300, and 400 mg of orange essential oil kg−1 diet.

The oil from the Pêra orange bagasse was obtained by hydrodistillation by using Clevenger equipment. The extraction yield was 4% and was determined through the ratio of the obtained oil mass by the plant mass used in the extraction (with an average of 100 g).

A gas chromatograph coupled with mass spectrometer (Shimadzu QP-2010 Plus) was used for separation, detection, and quantification of substances in the orange essential oil (Table 1). Essential oil (1.0 μL of 10 μL mL−1) was injected into the chromatograph in splitless mode. The components were separated in a fused-silica capillary column (5% diphenyl and 95% dimethylsiloxane), length, internal diameter, and film thickness of 30 m × 0.25 mm × 0.25 μm, respectively. Helium was used as carrier gas with the flux of 1 mL min−1. Temperatures of the injector and detector were 220 and 250 °C, respectively. The oven temperature was programmed at 60 °C for 2 min, with an increase of 5 °C min−1 until 110 °C, followed by an increase of 3 °C min−1 until 150 °C, and, finally, 15 °C min−1 until 290 °C, maintained constant for 15 min.

Table 1
Chemical composition of Pêra orange (Citrus sinensis L.) essential oil

Mass spectra were obtained using a quadrupole detector, operating at 70 eV with mass interval between 40-400 m z−1 and tax of 0.5 scan s−1. Identification of substances in the essential oil was based on the comparison of retention indexes and mass spectra of samples with data from the library of National Institute of Standards and Technology (NIST 2008) and from literature (Adams, 2007Adams, R. P. 2007. Identification of essential oil components by gas chromatography/quadrupole mass spectroscopy. 4th ed. Allured Publishing Corporation, Carol Stream.). The Retention Index was calculated based on the co-injection of alkane samples of C8 to C40 as described in the literature (van Den Dool and Kratz, 1963van Den Dool, H. and Kratz, P. D. 1963. A generalization of the retention index system including linear temperature programed gas-liquid partition chromatography. Journal of Chromatography A 11:463-471. https://doi.org/10.1016/S0021-9673(01)80947-X
https://doi.org/10.1016/S0021-9673(01)80...
).

During the initial experimental period, birds were heated using an automatic gas heater. Water was supplied by nipple drinkers and feed supplied by an infant tubular feeder, which was gradually replaced by a definitive tubular feeder. Wood shavings litter (10 cm deep) was used for floor covering of the aviary.

Lighting was maintained for 24 continuous hours (natural and artificial). The raising period was divided into two phases: pre-starter (1-7 days) and starter (8-21 days). Feeds were elaborated following the recommendations of Rostagno et al. (2017)Rostagno, H. S.; Albino, L. F. T.; Hannas, M. I.; Donzele, J. L.; Sakomura, N. K.; Perazzo, F. G.; Saraiva, A.; Teixeira, M. V.; Rodrigues, P. B.; Oliveira, R. F.; Barreto, S. L. T. and Brito, C. O. 2017. Tabelas brasileiras para aves e suínos: composição de alimentos e exigências nutricionais. 4.ed. Departamento de Zootecnia/UFV, Viçosa, MG. (Table 2). Daily, we registered the mortalities that occurred and other management procedures necessary for the welfare of birds.

Table 2
Composition of the experimental diets, in natural matter

Performance was evaluated in the phases described and the following characteristics were verified: feed intake (FI), weight gain (WG), feed conversion (FC), and viability. At the end of each period, birds from each experimental unit were weighed in groups, and the result was divided by the number of live birds, yielding the average WG and FI. Feed conversion ratio (FCR) was calculated by dividing FI by WG of birds. Viability was calculated using the total number of dead birds subtracted by total live birds.

At 21 days of age, a single fasting bird from each repetition (30 units in total) was taken for total blood glucose quantification (Rezende et al., 2019Rezende, M. S.; Silva, P. L.; Guimarães, E. C.; Lellis, C. G. and Mundim, A. V. 2019. Variações fisiológicas, influência da idade e sexo no perfil bioquímico sanguíneo de aves da linhagem pesada de frango de corte na fase de recria. Arquivo Brasileiro de Medicina Veterinária e Zootecnia 71:1649-1658. https://doi.org/10.1590/1678-4162-10661
https://doi.org/10.1590/1678-4162-10661...
), with the use of a portable digital glucometer (G-Tech Lite®, South Korea). Then, the birds were slaughtered and their organs (liver, pancreas, gizzard, and intestines) removed and weighed immediately. The length of small intestine was measured with a tape.

Morphometric analyses of the intestine followed the procedures described in Faveri et al. (2015)Faveri, J. C.; Murakami, A. E.; Potença, A.; Eyng, C.; Marques, A. F. Q. and Santos, T. C. 2015. Desempenho e morfologia intestinal de frangos de corte na fase de crescimento, com e sem adição de nucleotídeos na dieta, em diferentes níveis proteicos. Pesquisa Veterinária Brasileira 35:291-296. https://doi.org/10.1590/S0100-736X2015000300013
https://doi.org/10.1590/S0100-736X201500...
and Erhan and Bölükbaşi (2017)Erhan, M. K. and Bölükbaşi, Ş. C. 2017. Citrus peel oils supplementation in broiler diet: effects on performance, jejunum microflora and jejunum morphology. Brazilian Journal of Poultry Science 19:15-21. https://doi.org/10.1590/1806-9061-2016-0274
https://doi.org/10.1590/1806-9061-2016-0...
. Fragments of the birds’ jejunum were collected and fixed in a 10% buffered formaldehyde solution. Then, segments were transferred to 70% alcohol, dehydrated in a growing series of alcohols, diaphanized in xylol, and embedded in paraffin. The slides with the cuts were stained using the hematoxylin and eosin technique. In the captured images, the villus height (VH) and crypt depth (CD) were measured using ImageJ. Villus height was measured from the basal region coinciding with the superior portion of the crypt to the tip of the villus, while the CD was measured from the basal region of the villus to the bottom of the crypt.

As regards bone analysis, in the tibiotarsus in natura, all adherent tissue was removed with the aid of scissors and tweezers, weighed on an analytical balance (±0.0001 g), and the diameters (horizontal and vertical) and length were measured using a digital pachymeter (0-150 mm, to an accuracy of 0.001 mm). The Seedor Index (SI; Seedor et al., 1991Seedor, J. G.; Quarraccio, H. H. and Thompson, D. D. 1991. The biophosphonate alendronate (MK-217) inhibits bone loss due to ovariectomy in rats. Journal of Bone and Mineral Research 6:339-346. https://doi.org/10.1002/jbmr.5650060405
https://doi.org/10.1002/jbmr.5650060405...
) was calculated by the bone weight (mg) divided by its length (mm).

In the analysis of bone resistance, bones were subjected to a flexion test, with the use of a texturometer (Stable Micro System, TA.XT.plus® model). All bones were tested in the same position, with the extremities resting on supports, and the load was applied in the central area (bone diaphysis). The value corresponding to bone rupture was expressed as kilogram-force (kgf).

Data were subjected to variance and regression analysis in SAS (Statistical Analysis System, version 9.0) software, using PROC REG with α = 0.05. Additionally, a Dunnett's test (α = 0.05) was used to compare the treatment without orange essential oil (control) to the other treatments.

The following statistical model was adopted:

Y i j = μ + α i + ε i j ,

in which Yij = value observed for the variable response obtained for the i-th treatment (i = 0, 100, 200, 300, and 400 mg kg−1 of orange essential oil) in its j-th repetition, μ = general average, αi = effect of treatment i on the observed value Yij, and εij = random error related to each observation.

3. Results

The use of Pêra orange essential oil in rations influenced the pre-starter performance of broilers (Table 3). Broiler chicken FI, WG, and, consequently, the total weight at seven days of age increased in a linear trend. Feed conversion ratio was reduced with the addition of orange oil in rations, which indicates the beneficial effect of the additive.

Table 3
Performance of broilers from one to seven days of age fed diets containing orange (Citrus sinensis L.) essential oil

At 21 days of age, broilers that received 400 mg kg−1 of orange essential oil showed higher body weight (BW) compared with birds fed diets without essential oil (Table 4, P<0.05).

Table 4
Performance of broilers from one to 21 days of age fed diets containing orange (Citrus sinensis L.) essential oil

The addition of orange essential oil in diets had no influence on the weights of gastrointestinal tract organs and glucose of broilers at 21 days of age (Table 5). Bone characteristics of broilers (Table 6) were modified with the addition of Pêra orange essential oil in diets. The in natura weight and length were higher in the tibia of birds that received, respectively, 300 and 400 mg kg−1 of orange essential oil, when compared with the treatment without the essential oil (P<0.05).

Table 5
Organ biometrics and glucose of broilers at 21 days of age fed diets containing orange (Citrus sinensis L.) essential oil
Table 6
Characteristics of tibias of broilers at 21 days of age fed diets containing orange (Citrus sinensis L.) essential oil

The morphometry of the jejunum of broilers fed diets with orange essential oil was significantly changed (Table 7). Villus height was adjusted in increasing quadratic model (P<0.05), which indicated an increase in the absorption surface in the intestine, demonstrated by the higher CD in birds at 21 days of age.

Table 7
Morphometry of the jejunum of broilers at 21 days of age fed diets containing orange (Citrus sinensis L.) essential oil

4. Discussion

The use of Pêra orange (Citrus sinensis L.) essential oil in diets influenced the pre-starter performance of birds (Table 3). A lower FCR value was observed, similar to findings of Erhan and Bölükbaşi (2017)Erhan, M. K. and Bölükbaşi, Ş. C. 2017. Citrus peel oils supplementation in broiler diet: effects on performance, jejunum microflora and jejunum morphology. Brazilian Journal of Poultry Science 19:15-21. https://doi.org/10.1590/1806-9061-2016-0274
https://doi.org/10.1590/1806-9061-2016-0...
, who studied the inclusion of citrus oils (bergamot, orange, and lemon) in diets (1, 2, and 3 mL kg−1) for broilers. The authors obtained positive results in FCR with the use of orange essential oil. Similarly, Aydin and Alçiçek (2018)Aydin, A. and Alçiçek, A. 2018. Effects of the supplementation of essential oil isolated from orange peel (Citrus sinensis L.) to broiler diets on the performance. Türk Tarım ve Doğa Bilimleri Dergisi 5:127-135. evaluated orange essential oil (0, 50, 100, and 150 mg kg−1) in rations of broilers until six weeks of age. These authors recommended the quantity of 150 mg kg−1, since higher rates of productive efficiency were verified.

The pre-starter phase constitutes a critical stage in poultry farming, since the animals have more accelerated physiological, metabolic, and body development. It is equivalent to μ of the production cycle of broilers, being decisive in the profitability of the enterprise. The performance results in the referred phase denote the differentiation of enterocytes, as well as the capacity of absorption and transport of nutrients and production and secretion of digestive enzymes.

Further, a Dunnett's test was run to identify possible differences between the control treatment (without orange oil) and those containing different amounts of the phytogenic additive (orange essential oil). It was found that broilers at 21 days of age receiving 400 mg kg−1 of orange essential oil in their diet had higher BW (P<0.05). The highest WG and BW resulted from the improvement in the physiological processes in the digestive tract, as well as the possible inhibition of intestinal pathogenic organisms in the evaluated raising period (Table 4). Thus, the better feed efficiency achieved with the use of orange essential oil may be due to positive effects on the promotion of endogenous enzyme activity, as well as the antimicrobial activity of orange essential oil (Aydin and Alçiçek, 2018Aydin, A. and Alçiçek, A. 2018. Effects of the supplementation of essential oil isolated from orange peel (Citrus sinensis L.) to broiler diets on the performance. Türk Tarım ve Doğa Bilimleri Dergisi 5:127-135.) and nutrient digestibility and intestinal health (Sahu et al., 2019Sahu, A. C.; Manwar, S. J.; Gole, M. A.; Khose, K. K.; Wade, M. R. and Kuralkar, S. V. 2019. Effect of lemon and orange peel essential oils on performance of broiler chickens during summer. Indian Journal of Poultry Science 54:139-145. https://doi.org/10.5958/0974-8180.2019.00029.1
https://doi.org/10.5958/0974-8180.2019.0...
).

The mechanism of action of essential oils on bacterial cells comprises a series of events that can destabilize the cellular architecture, leading to the breakdown of membrane integrity and, thus, increased permeability of the cellular constituents (Ambrosio et al., 2019Ambrosio, C. M. S.; Ikeda, N. Y.; Miano, A. C.; Saldaña, E.; Moreno, A. M.; Stashenko, E.; Contreras-Castillo, C. J. and Gloria, E. M. 2019. Unraveling the selective antibacterial activity and chemical composition of citrus essential oils. Scientific Reports 9:17719. https://doi.org/10.1038/s41598-019-54084-3
https://doi.org/10.1038/s41598-019-54084...
). This disrupts many cellular activities, including energy production, membrane transport, and other metabolic regulatory functions. Furthermore, this action can alter the membrane fatty acid composition and membrane proton motive force and affect proteins in the cytoplasmatic membrane (Ambrosio et al., 2019Ambrosio, C. M. S.; Ikeda, N. Y.; Miano, A. C.; Saldaña, E.; Moreno, A. M.; Stashenko, E.; Contreras-Castillo, C. J. and Gloria, E. M. 2019. Unraveling the selective antibacterial activity and chemical composition of citrus essential oils. Scientific Reports 9:17719. https://doi.org/10.1038/s41598-019-54084-3
https://doi.org/10.1038/s41598-019-54084...
).

The main constituent of the orange essential oil is limonene (95.64%), a terpene with the ability to release short-chain fatty acids (fermentation products), which lower the pH and prevent the growth of harmful microorganisms. Bozkurt et al. (2017)Bozkurt, T.; Gülnaz, O. and Kaçar, Y. A. 2017. Chemical composition of the essential oils from some citrus species and evaluation of the antimicrobial activity. Journal of Environmental Science, Toxicology and Food Technology 11:29-33. characterized different citrus essential oils obtained by the hydrodistillation method. The authors showed that the amount of limonene in each essential oil was directly proportional to the antimicrobial effect (E. coli, B. cereus, S. aureus, S. Thyphimurium, E. faecalis, and L. monocytogenes).

Further, orange essential oil contains myrcene, sabinene, α-sabinene, linalool, and other compounds in minor amounts (Table 1). The antimicrobial properties of orange essential oil can be assigned to each constituent feature of isolation (Guimarães et al., 2019Guimarães, A. C.; Meireles, L. M.; Lemos, M. F.; Guimarães, M. C. C.; Endringer, D. C.; Fronza, M. and Scherer, R. 2019. Antibacterial activity of terpenes and terpenoids present in essential oils. Molecules 24:2471. https://doi.org/10.3390/molecules24132471
https://doi.org/10.3390/molecules2413247...
) and also the synergy that occurs between them (Ambrosio et al., 2019Ambrosio, C. M. S.; Ikeda, N. Y.; Miano, A. C.; Saldaña, E.; Moreno, A. M.; Stashenko, E.; Contreras-Castillo, C. J. and Gloria, E. M. 2019. Unraveling the selective antibacterial activity and chemical composition of citrus essential oils. Scientific Reports 9:17719. https://doi.org/10.1038/s41598-019-54084-3
https://doi.org/10.1038/s41598-019-54084...
; Berdejo et al., 2020Berdejo, D.; Pagán, E.; Merino, N.; Pagán, R. and García-Gonzalo, D. 2020. Incubation with a complex orange essential oil leads to evolved mutants with increased resistance and tolerance. Pharmaceuticals 13:239. https://doi.org/10.3390/ph13090239
https://doi.org/10.3390/ph13090239...
). Ambrosio et al. (2019)Ambrosio, C. M. S.; Ikeda, N. Y.; Miano, A. C.; Saldaña, E.; Moreno, A. M.; Stashenko, E.; Contreras-Castillo, C. J. and Gloria, E. M. 2019. Unraveling the selective antibacterial activity and chemical composition of citrus essential oils. Scientific Reports 9:17719. https://doi.org/10.1038/s41598-019-54084-3
https://doi.org/10.1038/s41598-019-54084...
demonstrated that limonene could collaborate with the selective activity of citrus oils when present in the gut, promoting the beneficial bacteria, while other minor compounds could act to inhibit pathogenic bacteria.

Regarding health challenge, birds were raised on reused litter. The viability in the pre-starter phase was 100%, and in the total period of the experiment (one to 21 days of age), the viability was not significantly affected with the dietary supplement of orange essential oil. In addition to the superior results on nutrient digestibility and the consequent performance of poultry (Aydin et al., 2018Aydin, A.; Korkunç, M.; Demirel, D. Ş. and Gül, S. 2018. Portakal kabuğu (Citrus sinensis L.) uçucu yağının kanatlı beslemedeki önemi. Uluslararası Doğu Akdeniz Tarımsal Araştırma Enstitüsü Dergisi 1:13-19.; Sahu et al., 2019Sahu, A. C.; Manwar, S. J.; Gole, M. A.; Khose, K. K.; Wade, M. R. and Kuralkar, S. V. 2019. Effect of lemon and orange peel essential oils on performance of broiler chickens during summer. Indian Journal of Poultry Science 54:139-145. https://doi.org/10.5958/0974-8180.2019.00029.1
https://doi.org/10.5958/0974-8180.2019.0...
), the use of phytogenics could improve viability. However, in this study, there was no change in this productive parameter, similar to findings reported by Aydin and Alçiçek (2018)Aydin, A. and Alçiçek, A. 2018. Effects of the supplementation of essential oil isolated from orange peel (Citrus sinensis L.) to broiler diets on the performance. Türk Tarım ve Doğa Bilimleri Dergisi 5:127-135..

The orange essential oil evaluated did not influence the weights of the gastrointestinal tract organs and glucose of broilers at 21 days of age (Table 5). Similarly, Erhan and Bölükbaşi (2017)Erhan, M. K. and Bölükbaşi, Ş. C. 2017. Citrus peel oils supplementation in broiler diet: effects on performance, jejunum microflora and jejunum morphology. Brazilian Journal of Poultry Science 19:15-21. https://doi.org/10.1590/1806-9061-2016-0274
https://doi.org/10.1590/1806-9061-2016-0...
and Aydin and Alçiçek (2018)Aydin, A. and Alçiçek, A. 2018. Effects of the supplementation of essential oil isolated from orange peel (Citrus sinensis L.) to broiler diets on the performance. Türk Tarım ve Doğa Bilimleri Dergisi 5:127-135. did not observe any changes in the liver weight of broilers fed citrus oils at 42 days of age. Dhanapal et al. (2014)Dhanapal, S. K.; Rao, S.; Govindaraju, P. K. P.; Hukkeri, R. and Mathesh, K. 2014. Ameliorative efficacy of citrus fruit oil in aflatoxicosis in broilers: a growth and biochemical study. Turkish Journal of Veterinary and Animal Sciences 38:207-211. https://doi.org/10.3906/vet-1303-9
https://doi.org/10.3906/vet-1303-9...
studied essential oil of citrus fruits (2.5 g kg−1) in diets, with or without aflatoxin for broilers, and did not find adverse effects of the essential oil on relative weight of lymphoid organs, kidneys, heart, and serum biochemistry of healthy birds.

Biometrics of digestive system organs is an instrument to be used in physiological and economic evaluation of the use of additives in diets. The viscera are commercially important and can be used as a signal for metabolic effects of dietary manipulation. Weights and percentages of the intestines (small and large), as well as length of the small intestine, were not changed by the treatments, which demonstrate no physiological changes of birds.

Glycemic levels were within the normal range (200-500 mg dL−1) for healthy birds (Schmidt et al., 2007Schmidt, E. M. S.; Locatelli-Dittrich, R.; Santin, E. and Paulillo, A. C. 2007. Patologia clínica em aves de produção – uma ferramenta para monitorar a sanidade avícola – revisão. Archives of Veterinary Science 12:9-20. https://doi.org/10.5380/avs.v12i3.10906
https://doi.org/10.5380/avs.v12i3.10906...
) or birds that received the orange essential oil (Sevim et al., 2020Sevim, B.; Olgun, O.; Şentürk, E. T. and Yıldız, A. Ö. 2020. The effect of orange peel oil addition to laying quail diets on performance, eggshell quality and some serum parameters. Turkish Journal of Agriculture – Food Science and Technology 8:1773-1777. https://doi.org/10.24925/turjaf.v8i8.1773-1777.3508
https://doi.org/10.24925/turjaf.v8i8.177...
). In an experiment by Karabayir et al. (2018)Karabayir, A.; Öğütcü, M.; Acar, U. and Arifoğlu, N. 2018. Effects of orange peel oil on quail (Coturnix coturnix japonica) growth-performance, egg quality and blood parameters. New Knowledge Journal of Science 7:127-136., broilers fed diets containing 600 ppm of orange essential oil (Citrus sinensis, 94.74% of limonene) exhibited high serum glucose values. The authors reasoned that there was a breakdown of glucose mobilization of the tissues and, thus, the molecules migrated from the tissues directly into the blood. The maximum quantity of orange essential oil evaluated (400 mg kg−1) was safe and possibly insufficient to cause a change in the glycemia of the animals.

The in natura weight and length (P<0.05) were higher in the tibia of chickens that received 300 and 400 mg kg−1 of orange essential oil in their diet, demonstrating the effects of phytogenics on mineral metabolism. Sevim et al. (2020)Sevim, B.; Olgun, O.; Şentürk, E. T. and Yıldız, A. Ö. 2020. The effect of orange peel oil addition to laying quail diets on performance, eggshell quality and some serum parameters. Turkish Journal of Agriculture – Food Science and Technology 8:1773-1777. https://doi.org/10.24925/turjaf.v8i8.1773-1777.3508
https://doi.org/10.24925/turjaf.v8i8.177...
observed a serum modification of calcium and phosphorus (increase) in broilers given orange essential oil, likely because of limonene, which lowers the pH of the digestive system, increasing endogenous digestive enzymes and the intestinal surface area, thereby, enabling improvement in mineral absorption that was observed in this assay.

Seedor Index indirectly indicates bone density, and a higher index shows a higher density of the bone and vice versa. Broilers fed 400 mg kg−1 of orange essential oil had a higher dry weight and density in the tibia (SI), when compared with birds that did not receive the essential oil (Table 6). Sabbieti et al. (2011)Sabbieti, M. G.; Agas, D.; Maggi, F.; Vittori, S. and Marchetti, L. 2011. Molecular mediators involved in Ferulago campestris essential oil effects on osteoblast metabolism. Journal of Cellular Biochemistry 112:3742-3754. https://doi.org/10.1002/jcb.23306
https://doi.org/10.1002/jcb.23306...
described that essential oils have lipophilic properties, cross cell membranes easily, and affect the function of bone cells by stimulating or inhibiting specific metabolic pathways. Furthermore, they can modulate the proliferation of osteoblasts, probably by alternative signaling, which depends on the maturation stage of the cells, and increases mineral density and bone strength (Olgun, 2016Olgun, O. 2016. The effect of dietary essential oil mixture supplementation on performance, egg quality and bone characteristics in laying hens. Annals of Animal Science 16:1115-1125. https://doi.org/10.1515/aoas-2016-0038
https://doi.org/10.1515/aoas-2016-0038...
). Limonene can be considered a promising compound for bone healing through the induction of osteogenesis (Soulimani et al., 2019Soulimani, R.; Bouayed, J. and Joshi, R. K. 2019. Limonene: natural monoterpene volatile compounds of potential therapeutic interest. American Journal of Essential Oils and Natural Products 7:1-10.).

The intestinal microbiota of birds has probably influenced bone variables (in natura weight, length, and SI) in the present study. According to Lunedo and Pedroso (2017)Lunedo, R. and Pedroso, A. A. 2017. Microbiota intestinal: a microbiota intestinal e seus efeitos sobre a fisiologia da ave. In: Fisiologia das aves comerciais. Macari, M. and Maiorka, A., eds. Funep/Fapesp/Facta, Jaboticabal., the effects of the microbiota on bone mass occur via the immune system, particularly from the role of T cells, which regulate the formation of osteoclasts. In addition, the presence of microorganisms can impact mineralization, density, and bone strength. No changes were observed in mineral composition (ash) and in the breaking strength of the tibia; however, the density (SI) was higher with the use of orange essential oil, which showed the integrity in the filling of the bone matrix.

The morphometric characteristics of the broiler jejunum were significantly altered (Table 7). It was found that the villus height showed a quadratic response, indicating an increase in the absorption surface in the intestine, also confirmed by the greater depth of crypts. Erhan and Bölükbaşi (2017)Erhan, M. K. and Bölükbaşi, Ş. C. 2017. Citrus peel oils supplementation in broiler diet: effects on performance, jejunum microflora and jejunum morphology. Brazilian Journal of Poultry Science 19:15-21. https://doi.org/10.1590/1806-9061-2016-0274
https://doi.org/10.1590/1806-9061-2016-0...
also found differences in the histological variables of the jejunum (increase in villus length) of chickens, when using orange essential oil in diets (3 mL kg−1).

The increase in height of the villi observed can also be correlated with the antioxidant properties of the essential oil studied. According to Namdeo et al. (2020)Namdeo, S.; Baghel, R. P. S.; Nayak, S.; Khare, A.; Pal, R. P.; Chaurasiya, A.; Thakur, S. and Reddy, B. V. V. 2020. Essential oils: an potential substitute to antibiotics growth promoter in broiler diet. Journal of Entomology and Zoology Studies 8:1643-1649. oxygen radicals released during digestion attack the superficial mucosa of the intestine and can shorten the intestinal villi. Antioxidant enzymes such as catalase, glutathione peroxidase, and superoxide dismutase can attract oxygen radicals and extinguish free radicals (Namdeo et al., 2020Namdeo, S.; Baghel, R. P. S.; Nayak, S.; Khare, A.; Pal, R. P.; Chaurasiya, A.; Thakur, S. and Reddy, B. V. V. 2020. Essential oils: an potential substitute to antibiotics growth promoter in broiler diet. Journal of Entomology and Zoology Studies 8:1643-1649.). Limonene, a constituent of orange essential oil, prevents oxidative damage and can protect lymphocytes against oxidative stress and stimulate cell proliferation (Soulimani et al., 2019Soulimani, R.; Bouayed, J. and Joshi, R. K. 2019. Limonene: natural monoterpene volatile compounds of potential therapeutic interest. American Journal of Essential Oils and Natural Products 7:1-10.).

The long villi correlate with the improvement of the intestinal health of birds, which provides better uniformity and integrity of the mucosa, besides the greater the capacity to absorb nutrients (Borsatti et al., 2020Borsatti, L.; Broch, J.; Avila, A. S.; Schneiders, J. L.; Rocha, C. S.; Oxford, J. H. and Nunes, R. V. 2020. Essential oils and prebiotic on broiler diets as feed additives. Semina: Ciências Agrárias 41:1307-1316. https://doi.org/10.5433/1679-0359.2020v41n4p1327
https://doi.org/10.5433/1679-0359.2020v4...
). The intestinal mucosa must have adequate morphofunctional characteristics since the absorption processes depend on the integrity of the epithelium. Thus, stimulation in the development of the intestinal mucosa, with trophic agents in the diet such as essential oils, has become an important nutritional strategy.

The use of orange essential oil in rations also influenced the CD, the value of which was greater in treatments of 300 and 400 ppm. The verified results may be due to the possible modulation of the immune system resulting from limonene (Wang et al., 2019Wang, L.; Zhang, Y.; Fan, G.; Ren, J. N.; Zhang, L. L. and Pan, S. Y. 2019. Effects of orange essential oil on intestinal microflora in mice. Journal of the Science of Food and Agriculture 99:4019-4028. https://doi.org/10.1002/jsfa.9629
https://doi.org/10.1002/jsfa.9629...
), as well as the greater need for renewal of the intestinal epithelium. Bayrakdar et al. (2017)Bayrakdar, A.; Dalkiliç, B.; Yaman, M.; Şimşek, Ü. G. and Çiftçi, M. 2017. Effect of dietary orange peel essential oil and thermotolerance on histo-morphometry and serotonin-immunoreactive endocrine cell numbers in the small intestines of heat stressed japanese quails. Kafkas Universitesi Veteriner Fakultesi Dergisi 23:177-184. investigated histomorphometric characteristics and endocrine cells immunoreactive to serotonin (CEIR) in crypts of the jejunum of Japanese quails under heat stress and fed diet containing the orange essential oil (300 ppm). Their results showed an increase in CEIR and in CD of the jejunum with the orange essential oil supplement.

The increase in CD can denote an accelerated villus renewal rate, which leads to a greater energy expenditure by the cells. According to Faveri et al. (2015)Faveri, J. C.; Murakami, A. E.; Potença, A.; Eyng, C.; Marques, A. F. Q. and Santos, T. C. 2015. Desempenho e morfologia intestinal de frangos de corte na fase de crescimento, com e sem adição de nucleotídeos na dieta, em diferentes níveis proteicos. Pesquisa Veterinária Brasileira 35:291-296. https://doi.org/10.1590/S0100-736X2015000300013
https://doi.org/10.1590/S0100-736X201500...
, the mitotic divisions in the crypts account for about 60% of cell proliferation, the middle region of villus is responsible by 32% of cell proliferation, and the apical region by 8%.

The VH:CD ratio was not influenced by the essential oil levels (P<0.05) added to the diets. This ratio is a good indicator of the proliferation and development of enterocytes in the villi. In other words, the digestive efficiency of the animal and a decrease in this ratio is not acceptable in terms of digestion and absorption, and vice versa (Namdeo et al., 2020Namdeo, S.; Baghel, R. P. S.; Nayak, S.; Khare, A.; Pal, R. P.; Chaurasiya, A.; Thakur, S. and Reddy, B. V. V. 2020. Essential oils: an potential substitute to antibiotics growth promoter in broiler diet. Journal of Entomology and Zoology Studies 8:1643-1649.). The desirable relationship occurs with high villi and shallow crypts, since there would be less energy losses with cell renewal and better nutrient absorption.

5. Conclusions

The addition of Pêra orange (Citrus sinensis L.) essential oil in diets promotes the improvement of productive variables (weight gain, feed intake, and feed conversion) of broilers in the pre-starter phase. At 21 days of age, birds that received 400 mg kg−1 of orange essential oil showed higher weight gain and, consequently, higher body weight. The verified results are a consequence of the improvement in physiological processes in the digestive tract, which is characterized by the increase in villus height. Bone characteristics are influenced by the phytogenic additive, with an increase in natura weight, length, and bone density. The biometrics of gastrointestinal tract organs and glycemia of birds are not altered by the treatments, indicating that there is no physiological impairment to the birds.

Acknowledgments

The authors would like to thank the Programa Nacional de Pós-Doutorado/Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (PNPD/CAPES) – Brazil, for supporting the first author with a fellowship.

References

  • Adams, R. P. 2007. Identification of essential oil components by gas chromatography/quadrupole mass spectroscopy. 4th ed. Allured Publishing Corporation, Carol Stream.
  • Ambrosio, C. M. S.; Ikeda, N. Y.; Miano, A. C.; Saldaña, E.; Moreno, A. M.; Stashenko, E.; Contreras-Castillo, C. J. and Gloria, E. M. 2019. Unraveling the selective antibacterial activity and chemical composition of citrus essential oils. Scientific Reports 9:17719. https://doi.org/10.1038/s41598-019-54084-3
    » https://doi.org/10.1038/s41598-019-54084-3
  • Aydin, A. and Alçiçek, A. 2018. Effects of the supplementation of essential oil isolated from orange peel (Citrus sinensis L.) to broiler diets on the performance. Türk Tarım ve Doğa Bilimleri Dergisi 5:127-135.
  • Aydin, A.; Korkunç, M.; Demirel, D. Ş. and Gül, S. 2018. Portakal kabuğu (Citrus sinensis L.) uçucu yağının kanatlı beslemedeki önemi. Uluslararası Doğu Akdeniz Tarımsal Araştırma Enstitüsü Dergisi 1:13-19.
  • Bayrakdar, A.; Dalkiliç, B.; Yaman, M.; Şimşek, Ü. G. and Çiftçi, M. 2017. Effect of dietary orange peel essential oil and thermotolerance on histo-morphometry and serotonin-immunoreactive endocrine cell numbers in the small intestines of heat stressed japanese quails. Kafkas Universitesi Veteriner Fakultesi Dergisi 23:177-184.
  • Berdejo, D.; Pagán, E.; Merino, N.; Pagán, R. and García-Gonzalo, D. 2020. Incubation with a complex orange essential oil leads to evolved mutants with increased resistance and tolerance. Pharmaceuticals 13:239. https://doi.org/10.3390/ph13090239
    » https://doi.org/10.3390/ph13090239
  • Borsatti, L.; Broch, J.; Avila, A. S.; Schneiders, J. L.; Rocha, C. S.; Oxford, J. H. and Nunes, R. V. 2020. Essential oils and prebiotic on broiler diets as feed additives. Semina: Ciências Agrárias 41:1307-1316. https://doi.org/10.5433/1679-0359.2020v41n4p1327
    » https://doi.org/10.5433/1679-0359.2020v41n4p1327
  • Bozkurt, T.; Gülnaz, O. and Kaçar, Y. A. 2017. Chemical composition of the essential oils from some citrus species and evaluation of the antimicrobial activity. Journal of Environmental Science, Toxicology and Food Technology 11:29-33.
  • Chouhan, S.; Sharma, K. and Guleria, S. 2017. Antimicrobial activity of some essential oils - present status and future perspectives. Medicines 4:58. https://doi.org/10.3390/medicines4030058
    » https://doi.org/10.3390/medicines4030058
  • Dhanapal, S. K.; Rao, S.; Govindaraju, P. K. P.; Hukkeri, R. and Mathesh, K. 2014. Ameliorative efficacy of citrus fruit oil in aflatoxicosis in broilers: a growth and biochemical study. Turkish Journal of Veterinary and Animal Sciences 38:207-211. https://doi.org/10.3906/vet-1303-9
    » https://doi.org/10.3906/vet-1303-9
  • El Sawi, S. A.; Ibrahim, M. E.; El-Rokiek, K. G. and El-Din, S. A. S. 2019. Allelopathic potential of essential oils isolated from peels of three citrus species. Annals of Agricultural Sciences 64:89-94. https://doi.org/10.1016/j.aoas.2019.04.003
    » https://doi.org/10.1016/j.aoas.2019.04.003
  • Erhan, M. K. and Bölükbaşi Aktaş, Ş. C. 2017. Narenciye kabuk yağlarının etlik piliçlerde doku yağ asidi kompozisyonu ve raf ömrü üzerine etkileri. Atatürk Üniversitesi Veteriner Bilimleri Dergisi 12:157-166.
  • Erhan, M. K. and Bölükbaşi, Ş. C. 2017. Citrus peel oils supplementation in broiler diet: effects on performance, jejunum microflora and jejunum morphology. Brazilian Journal of Poultry Science 19:15-21. https://doi.org/10.1590/1806-9061-2016-0274
    » https://doi.org/10.1590/1806-9061-2016-0274
  • Erhan, M. K. 2020. The potential of orange peel oil as a suppressor of cell proliferation in animal feed and human nutrition: an experimental study. Alinteri Journal of Agriculture Sciences 35:44-49. https://doi.org/10.28955/alinterizbd.740928
    » https://doi.org/10.28955/alinterizbd.740928
  • Faveri, J. C.; Murakami, A. E.; Potença, A.; Eyng, C.; Marques, A. F. Q. and Santos, T. C. 2015. Desempenho e morfologia intestinal de frangos de corte na fase de crescimento, com e sem adição de nucleotídeos na dieta, em diferentes níveis proteicos. Pesquisa Veterinária Brasileira 35:291-296. https://doi.org/10.1590/S0100-736X2015000300013
    » https://doi.org/10.1590/S0100-736X2015000300013
  • Guimarães, A. C.; Meireles, L. M.; Lemos, M. F.; Guimarães, M. C. C.; Endringer, D. C.; Fronza, M. and Scherer, R. 2019. Antibacterial activity of terpenes and terpenoids present in essential oils. Molecules 24:2471. https://doi.org/10.3390/molecules24132471
    » https://doi.org/10.3390/molecules24132471
  • Karabayir, A.; Öğütcü, M.; Acar, U. and Arifoğlu, N. 2018. Effects of orange peel oil on quail (Coturnix coturnix japonica) growth-performance, egg quality and blood parameters. New Knowledge Journal of Science 7:127-136.
  • Lunedo, R. and Pedroso, A. A. 2017. Microbiota intestinal: a microbiota intestinal e seus efeitos sobre a fisiologia da ave. In: Fisiologia das aves comerciais. Macari, M. and Maiorka, A., eds. Funep/Fapesp/Facta, Jaboticabal.
  • Micciche, A.; Rothrock Jr., M. J.; Yang, Y. and Ricke, S. C. 2019. Essential oils as an intervention strategy to reduce Campylobacter in poultry production: a review 10:1058. Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2019.01058
    » https://doi.org/10.3389/fmicb.2019.01058
  • Namdeo, S.; Baghel, R. P. S.; Nayak, S.; Khare, A.; Pal, R. P.; Chaurasiya, A.; Thakur, S. and Reddy, B. V. V. 2020. Essential oils: an potential substitute to antibiotics growth promoter in broiler diet. Journal of Entomology and Zoology Studies 8:1643-1649.
  • Olgun, O. 2016. The effect of dietary essential oil mixture supplementation on performance, egg quality and bone characteristics in laying hens. Annals of Animal Science 16:1115-1125. https://doi.org/10.1515/aoas-2016-0038
    » https://doi.org/10.1515/aoas-2016-0038
  • Rezende, M. S.; Silva, P. L.; Guimarães, E. C.; Lellis, C. G. and Mundim, A. V. 2019. Variações fisiológicas, influência da idade e sexo no perfil bioquímico sanguíneo de aves da linhagem pesada de frango de corte na fase de recria. Arquivo Brasileiro de Medicina Veterinária e Zootecnia 71:1649-1658. https://doi.org/10.1590/1678-4162-10661
    » https://doi.org/10.1590/1678-4162-10661
  • Rostagno, H. S.; Albino, L. F. T.; Hannas, M. I.; Donzele, J. L.; Sakomura, N. K.; Perazzo, F. G.; Saraiva, A.; Teixeira, M. V.; Rodrigues, P. B.; Oliveira, R. F.; Barreto, S. L. T. and Brito, C. O. 2017. Tabelas brasileiras para aves e suínos: composição de alimentos e exigências nutricionais. 4.ed. Departamento de Zootecnia/UFV, Viçosa, MG.
  • Sabbieti, M. G.; Agas, D.; Maggi, F.; Vittori, S. and Marchetti, L. 2011. Molecular mediators involved in Ferulago campestris essential oil effects on osteoblast metabolism. Journal of Cellular Biochemistry 112:3742-3754. https://doi.org/10.1002/jcb.23306
    » https://doi.org/10.1002/jcb.23306
  • Sahu, A. C.; Manwar, S. J.; Gole, M. A.; Khose, K. K.; Wade, M. R. and Kuralkar, S. V. 2019. Effect of lemon and orange peel essential oils on performance of broiler chickens during summer. Indian Journal of Poultry Science 54:139-145. https://doi.org/10.5958/0974-8180.2019.00029.1
    » https://doi.org/10.5958/0974-8180.2019.00029.1
  • Schmidt, E. M. S.; Locatelli-Dittrich, R.; Santin, E. and Paulillo, A. C. 2007. Patologia clínica em aves de produção – uma ferramenta para monitorar a sanidade avícola – revisão. Archives of Veterinary Science 12:9-20. https://doi.org/10.5380/avs.v12i3.10906
    » https://doi.org/10.5380/avs.v12i3.10906
  • Seedor, J. G.; Quarraccio, H. H. and Thompson, D. D. 1991. The biophosphonate alendronate (MK-217) inhibits bone loss due to ovariectomy in rats. Journal of Bone and Mineral Research 6:339-346. https://doi.org/10.1002/jbmr.5650060405
    » https://doi.org/10.1002/jbmr.5650060405
  • Seidavi, A.; Zaker-Esteghamati, H. and Salem, A. Z. M. 2020. A review on practical applications of Citrus sinensis by-products and waste in poultry feeding. Agroforestry Systems 94:1581-1589. https://doi.org/10.1007/s10457-018-0319-2
    » https://doi.org/10.1007/s10457-018-0319-2
  • Sevim, B.; Olgun, O.; Şentürk, E. T. and Yıldız, A. Ö. 2020. The effect of orange peel oil addition to laying quail diets on performance, eggshell quality and some serum parameters. Turkish Journal of Agriculture – Food Science and Technology 8:1773-1777. https://doi.org/10.24925/turjaf.v8i8.1773-1777.3508
    » https://doi.org/10.24925/turjaf.v8i8.1773-1777.3508
  • Soulimani, R.; Bouayed, J. and Joshi, R. K. 2019. Limonene: natural monoterpene volatile compounds of potential therapeutic interest. American Journal of Essential Oils and Natural Products 7:1-10.
  • van Den Dool, H. and Kratz, P. D. 1963. A generalization of the retention index system including linear temperature programed gas-liquid partition chromatography. Journal of Chromatography A 11:463-471. https://doi.org/10.1016/S0021-9673(01)80947-X
    » https://doi.org/10.1016/S0021-9673(01)80947-X
  • Wang, L.; Zhang, Y.; Fan, G.; Ren, J. N.; Zhang, L. L. and Pan, S. Y. 2019. Effects of orange essential oil on intestinal microflora in mice. Journal of the Science of Food and Agriculture 99:4019-4028. https://doi.org/10.1002/jsfa.9629
    » https://doi.org/10.1002/jsfa.9629

Publication Dates

  • Publication in this collection
    05 Feb 2021
  • Date of issue
    2021

History

  • Received
    18 May 2020
  • Accepted
    02 Dec 2020
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