1. Introduction
The use of antibiotics selectively stimulates the growth of healthy intestinal microbiota, thus contributing to animals’ growth; however, the inclusion of this type of additive in the diet can cause problems related to the health of such animals due to resistance that develops over time, thus harming the immune system. However, the European Union’s 2006 ban on the use of antibiotics as growth promoters in poultry has increased the amount of research that has been conducted on the search for alternative phytogenic additives. However, animal nutrition plays an important role in the production of broiler birds through the use of high-quality ingredients with nutritional levels that fully meet this animal’s needs both for life and produce functions without the use of antibiotics [
1,
2,
3,
4].
Given the need to remove antibiotics from feed, studies in the area of nutrition have investigated possible natural substitutes. Among those identified are essential oils, which are lipophilic molecules with low molecular weights, consisting of mixtures of volatile organic compounds produced as secondary metabolites by plants, hydrocarbons (terpenes and sesquiterpenes), or oxygenated compounds (alcohols, ethers, aldehydes, ketones, lactones, and phenols) [
5].
Because the diversity of metabolites is immense, with approximately 320,000 secondary metabolites existing, and a single plant has a biosynthesis capacity of up to 25,000 compounds [
6,
7], essential oils have been widely used as bactericides, antioxidants, antivirals, fungicides, insecticides, acaricides, in antiparasitic activities, expectorants, and for their anticancer and cytotoxic properties [
5,
8,
9,
10].
As natural products, they have also been studied as phytogenic additives, since they contribute to the better use of nutrients and, consequently, improve performance, as they stimulate the appetite and the secretion of endogenous digestive enzymes, as well as improving the integrity of the intestinal epithelium and exhibiting antimicrobial, coccidiostatic, and anthelmintic activities [
11,
12,
13,
14,
15,
16,
17].
The biological activities of the species stand out in the
Citrus (Rutaceae) genus, in which studies have highlighted the antioxidant activity with the free radical reduction 2,2-diphenyl-1-picrilhhydrazyl (DPPH) using essential oils of
Citrus sinensis (orange) and
Citrus latifolia. In addition to a reduction in reactive oxygen species, the antioxidant potential of the essential oils of fruit peels of
Citrus sinensis, Citrus reticulata, and
Citrus paradisi, as well as their antimicrobial activity against strains of
Staphylococcus aureus, Escherichia coli, and
Salmonella typhi, among other pathogenic microorganisms (EROS), were highlighted [
18,
19].
Costa et al. [
20] identified the microbiological, antioxidant, and antitumor potentials of the essential oil of
Xylopia laevigata; Konan et al. [
21] highlighted the antioxidant activity of the essential oil of
Xylopia aethiopica; Tegang et al. [
22] identified excellent antifungal and antioxidant potentials of the essential oil of
X. aethiopica, with free radical (DPH) scavenging activity of up to 72%, highlighting the potential of this species of the genus; Wouatsa et al. [
23] identified a moderate biological activity for the essential oil of
X. aethiopica on
Bacillus subtilis,
E. coli, Micrococcus luteus, S. aureus, Pseudomonas aeruginosa, Raoultella planticola, and
Salmonella typhimurium. In addition to the
Citrus genus, the
Xylopia genus also stands out for its antioxidant and bactericidal activities.
The potential of essential oils for their antioxidant, antimicrobial, antiparasitic, and immunomodulatory activities, among others, which points to the benefits of their use as phytogenic additives in poultry feed, has been reported in the literature [
24,
25]. Wouatsa et al. [
23] evaluated the benefits of using the essential oils of lemon, orange, and bergamot peels as phytogenic additives, obtaining promising results regarding performance, histomorphology of the jejunum, and reduction in intestinal microbiota in broilers. Krishan et al. [
26] also evaluated the effect of a natural mixture of the essential oils of basil, caraway, laurel, lemon, oregano, sage, tea, and thyme as phytogenic additives in poultry feed, obtaining an improvement in weight gain and carcass quality in broilers. Erhan et al. [
27] included a mixture of essential oils in chicken feed, including oleoresin from carvacrol, cinnamaldehyde, and capsicum, checking the antioxidant potential of these essential oils, improving liver function, and increasing the concentration of carotenoids and coenzyme Q10.
Antioxidant and antimicrobial are properties of essential oils of species of the genera Citrus and Xylopia lead to future research on the use of these natural products as alternatives as phytogenic additives to replace growth promoters while maintaining performance and quality of the animal product.
The C. sinensis essential oils can be obtained through the use of waste from the juice industry, being a great alternative for reusing raw materials. Furthermore, the use of X. aromatica essential oils added to animal feed has not yet been reported in the literature, highlighting the innovative nature of this research.
Thus, the objective of the present study was to determine the effects of adding essential oils from the fruits of C. sinensis and X. aromatica in broiler diets on performance, biometrics, biochemistry, and duodenal histomorphology in broilers from 1 to 14 days.
4. Discussion
The
C. sinensis essential oil has Limonene as the major compound and the essential oil of species belonging to
Xylopia genus has the major compounds as Sylvestrene, α-Pinene and β-Pinene. These major compounds were also identified by other authors for species of
Citrus [
19] and
Xylopia general [
22,
36], both from Rutaceae family. The species of this family are extensively studied, mainly the
Citrus genus, which includes lemons, oranges, and tangerines, whose major compound is Limonene [
37,
38]. As for
Xylopia genus, it is a little less studied, as it comprises less-known wild plants, and α-Pinene, β-Pinene, and Myrcene are among the major compounds of its fruits [
20,
39]. Despite presenting similar compounds, none of those studies mentioned the presence of Sylvestrene, the major compound identified here. Jamwa et al. [
40] identified Sylvestrene among the major compounds of the essential oil of
Zanthoxylum caribaeum (Rutaceae) leaves; the authors of [
41] also identified the compound Sylvestrene among the majority of compounds in the essential oil of
Z. Bungeanum (Rutaceae) from different regions of China.
In the present study, supplementation with
C. sinensis and
X. aromatica essential oils did not lead to significant results on animal performance. Similar results were reported by [
42], who found there was no significant effect on the performance of broilers treated with essential oil of Mexican oregano in 7, 14, and 21 days of the experiment.
However, several authors report the positive impact of essential oils on broiler performance [
43,
44]. Some authors observed that supplementation with essential oils of orange, lemon, and bergamot peel significantly improved the feed conversion rate in broilers [
26]. Al-Yasiry et al. [
45] found better weight gain in birds fed with
Boswellia serrataresina essential oil (Indian tree popularly known as frankincense) from 1 to 42 days. Chalghoumi et al. [
46] found no significant effect in the initial phase (8–14 days) in treatments with essential oil of garlic and
cinnamon but highlighted an improvement in performance after 35 days of experiment. Wade et al. [
47] reported an improvement in the performance of broilers fed with thyme essential oil, reporting greater gain in body weight, better feed conversion, viability, and profit in the production of broilers. El-Latif et al. [
48] observed improvement in the performance of broilers fed with essential oils of
Rosmarinus officinalis (rosemary) and
Allium sativum (garlic) for 42 days, in addition to noticing a stimulation of innate immunity by increasing the phagocytic capacity of heterophile.
Differentiation in feed consumption was expected, as essential oils stimulate appetite and release digestive enzymes, in addition to increasing nutrient absorption by increasing villi and decreasing crypts [
12]. However, in the present test, there was no evidence of the effects of the essential oils of
C. sinensis and
X. aromatica and, consequently, of their major compounds on the performance of birds.
The metabolization of nutrients showed a significant effect at 14 days, although the feed consumption was not affected, there was greater use of nutrients. Catalan et al. [
49] and Yang et al. [
50] reported that essential oils improve palatability, stimulating appetite, and increasing food intake, influencing the speed of passage of food through the gastrointestinal tract, increasing the secretion of saliva, bile and mucus, and increasing enzyme activity. The
X. aromatica essential oil has Sylvestrene, α-Pinene, and β-Pinene among its major chemical compounds, while the
C. sinensis essential oil had an abundance of limonene, compounds that may have influenced both palatability and hydrolysis of nutrients present in the feed, which did not influence the animal performance. The metabolism coefficient reflects the digestibility of nutrients; that is, an increase in that coefficient indicates greater absorption of nutrients from a diet [
51]. In this sense, it is possible to observe that a greater absorption of these nutrients was shown in diets with essential oils concerning the control group in this phase of animal development, at 14 days of age.
Amad et al. [
52] observed that the metabolizability of crude protein, ether extract, calcium, and phosphorus was significantly higher in birds fed with diets containing essential oils of thyme and anise than in the control group. According to [
53,
54], one of the reasons why essential oils improve nutrient absorption may be the fact essential oils reduce the bacterial load by acidifying the intestinal lumen and, thereby, reduce the competition of intestinal bacteria with the host for energy supplying nutrients. For this reason, in the present work, better metabolism coefficients were observed for crude protein, dry matter, and ether extract when
C. sinensis and
X. aromatica essential oils were added.
The addition of
C. sinensis and
X. aromatica essential oils in the diet of broilers did not influence the biometrics of the gastrointestinal tract. The data in the literature on the effect of essential oils on the biometrics of the gastrointestinal tract of birds is still very controversial. Contrary to the present study, [
55] found that quails that received thyme essential oil as a phytogenic additive had a larger intestine length and weight than the control treatment. Çabuk et al. [
56] reported that the blend of essential oils from
Origanum sp.,
Laurus nobilis L.,
Salvia triloba L.,
Myrtus communis, Foeniculum vulgare, and
Citrus sp. did not result in a significant difference in organ biometrics, whereas [
52] observed an increase in the relative weight of the liver in broilers from the inclusion of essential oils. The authors of [
57] found a reduction in proventriculus and gizzard and an increase in the length of the duodenum in broilers fed with a diet supplemented with 3% of
Boswellia serrata, resin, which reflected in better use of nutrients. Contradictorily, [
58] found no effects between treatments with
pepper oil on the relative weight of the organs, which is also in agreement with the results of the present study.
The size of the bursa of birds fed with
X. aromatica essential oil was smaller than that of birds fed with the control diet at seven days of age. At 14 days, birds fed with diets containing
C. sinensis and
X. aromatica essential oils had bursa with small size, showing that the essential oil can have a positive effect on animal immunity, acting on the reduction in lymphocyte production. Knowledge of the typical morphology of primary and secondary lymphoid organs and tissues is an essential morphometric analytical tool for determining the intensity of the immune response in these sites [
59]. The Bursa of Fabricius is a lymphoepithelial organ found only in birds. It is a rounded pouch located just above the cloaca and reaches its largest size about one or two weeks after hatching and then decreases as the bird ages; it is hardly identified in older birds [
60,
61,
62,
63]. Tizard et al. [
64] state that Bursa of Fabricius is a primary lymphoid organ that functions as a place for maturation and differentiation of the cells that make up the antibody-producing system such as B and T lymphocytes, despite the involution with age.
Histomorphometric studies of Bursa of Fabricius in birds subjected to stress conditions concluded that stress affects the development of bursa with increasing size; thus, the weight of lymphoid organs such as Bursa of Fabricius, for example, reflects the body’s ability to produce lymphoid cells during the immune response [
65].
The literature presents different data regarding the effects of essential oils on lymphoid organs. Mohammad et al. [
66] evaluated the addition of
Satureja khuzistanica essential oil in the diet of broilers and noted no significant difference in the development of lymphoid organs such as Bursa, thymus, and spleen at 21 and 42 days, in the different doses tested and in the control treatment. El-Latif et al. [
48] also found no significant difference in the weight of the Bursa between control and treatments containing different doses of essential oils of
Rosmarinus officinalis (rosemary) and
Allium sativum (garlic). Yang et al. [
50] found that the mixture of organic acids and essential oils had no significant effect on the performance and size of Bursa of the 21- and 42-day-old broilers. Contradictorily, when analyzing the immune-stimulating effect of essential oils of
peppermint and
eucalyptus added to water at a dose of 0.25 mL/L [
67] found an increase in the weight of Bursa of chickens treated with those essential oils and concluded they are capable of implementing an immune response in broilers.
Bone mineral density can be measured by the Seedor index that represents the weight/length ratio of the organ [
68]. The Seedor index of tibia and its diameter was not influenced by the diet containing
C. sinensis and
X. aromatica essential oils. Murakami et al. [
69] studied the effect of adding flaxseed oil to the feed of broilers and observed a positive influence of these on the bone development of the animal, verifying greater mineralization of the femur and tibia bones. The addition of lipids in broiler diets is known to promote a significant reduction in the rate of bone calcification [
70]. However, there are no records in the literature on the influence of essential oils and their major compounds on bone metabolism.
Calcium (Ca) and phosphorus (P) ions are essential to maintain animal homeostasis in the 2:1 ratio and are related to bone formation [
71]. However, the 2:1 ratio was not observed in the present experiment, not even for the control treatment. In this experiment, at 14 days, a significant differentiation in the levels of calcium and phosphorus was shown for the essential oil of
C. sinensis, a result that is not reflected in the differentiation of bone biometrics. Cardoso-Teixeira et al. [
72] observed that monoterpenes such as limonene have vasorelaxant activity due to electromechanical coupling, which promotes the influx of Ca
2+ ions into the intracellular medium, which may have reflected in the imbalance of these ions.
The biochemical constituents of blood, liver, and pancreas reflect the physiological responses resulting from internal (age and sex) and external (food and environment) factors, providing information on the metabolism and health of animals. Thus, the feed consumption and diet composition affect blood, enzymatic, and metabolic parameters [
73]. The present study analyzed the biochemical parameters of blood, liver, and pancreas, which revealed the animals’ metabolic response to the diet supplemented with essential oils of
C. sinensis and
X. aromatica.
The protein concentration in birds consists of 40 to 50% of albumin, in addition to transport and coagulation proteins, enzymes and hormones produced in the liver, and immunoglobulins synthesized by B lymphocytes and plasma cells [
74]. This parameter was observed in the blood of broilers fed with
X. aromatica essential oil at 14 days of age, which had levels of total proteins higher than those of the control diet. As essential oils can have immunomodulatory effect, a stimulus to the immune system can increase the bird’s ability to synthesize antibodies, represented by immunoglobulins, which would lead to an increase in serum levels of proteins such as albumin, alpha globulins (1 and 2), beta globulins, and gamma globulins, among others [
75,
76].
The primary lipids present in the blood are triglycerides and cholesterol. Triglycerides are an essential source of energy for the body and the main lipids in adipose tissue and circulate through the blood of the intestine to adipose tissues, where they are stored to supply energy to the muscles during fasting periods [
77].
Both cholesterol and triglycerides, as they are insoluble in the blood, need an adjuvant to be transported within the bloodstream, such as HDL (high-density lipoprotein), LDL (low-density lipoprotein), IDL (intermediate-density lipoprotein), VLDL (very-low-density lipoprotein), and chylomicrons. VLDLs are synthesized in the liver and transport triglycerides from the liver to peripheral tissues. LDL and IDL are synthesized in plasma by the action of lipoprotein lipase. As the VLDL deposit their triglycerides, there is an increase in density due to the release of triacylglycerol fatty acid. These fatty acids enter the cell where they are re-esterified for storage or are metabolized for energy. The glycerol of triglyceride returns to the liver, where it is metabolized [
78,
79].
Therefore, the concentration of circulating triglycerides reflects the balance between its intestinal absorption, its synthesis, and secretion in hepatocytes, and its absorption in adipose tissue, influenced by the fat content in the diet and the production of hormones [
80,
81]. In this work, triglycerides in blood serum were reduced in treatments with
C. sinensis and
X. aromatica essential oils, at 7 and 14 days, as well as in the liver at 7 days, being a good indicator of animal health. Another indication of the animal’s health was verified with the reduction in cholesterol in the liver of chickens fed with essential oils of
C. sinensis and
X. aromatica at 14 days.
The use of natural compounds as essential oils in the feed can lead to hypocholesterolemia, by inhibiting the regulatory enzyme of cholesterol synthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, produced in the liver [
78,
82]. The inhibition of cholesterol synthesis requires two regulators that modulate HMG-CoA reductase activity, LDL-derived cholesterol, and non-steroidal products derived from mevalonic acid [
78,
83]. The active principles of some essential oils like Timol, Carvacrol, and Borneol can cause hypocholesterolemia in broilers inducing non-steroidal regulatory products like mevalonate that inhibit HMG-CoA reductase [
84,
85].
The mechanism of action of limonene has been studied by [
86], who report its potential to inhibit 70% of HMG-CoA reductase at a post-transcriptional level, thereby interfering with cholesterol synthesis. Limonene is metabolized in the liver and transformed into perillyl alcohol, perillyl aldehyde, and perillyl acid, all of which can prevent protein isoprenylation by interfering with the isoprenoid pathway [
87]. These data justify the benefits of adding the essential oils of
C. sinensis, rich in Limonene, and of
X. aromatica in the feed of broilers. However, no reports were found in the literature on the mechanism of action of the major compounds of
X. aromatica essential oil.
Literature data on the effects of natural products such as essential oils on biochemical parameters are very different. The data obtained here corroborate those found by [
88], who found a reduction in cholesterol and triglycerides in quail eggs fed with 0.5% saffron (
Curcuma longa). Gerzilov et al. [
89] also indicated a significant reduction in the concentrations of triglycerides and total cholesterol in the blood of ducks fed with a mixture of herbs, a reduction in cholesterol and triglyceride levels with the addition of turmeric in quail diets at doses starting from 0.05 g/100 g of feed, highlighting the ability to inhibit the synthesis of triglycerides in liver cells. According to the authors, the expression of genes involved in energy metabolism may have been influenced by turmeric, resulting in reduced expression, decreasing the accumulation of fats in the blood and tissues, with decreased intracellular levels of lipids [
90,
91]. Domingues et al. [
58] also found a reduction in triglyceride levels in broilers fed with Piper cubeba. Manan et al. [
92] observed a decrease in triglyceride levels when supplementing the diet of broilers with medicinal plants. Contradictorily, some authors have not verified the effect of rosemary and garlic essential oils on broiler performance, besides causing an increase in serum triglyceride levels, total cholesterol, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) [
48].
In the present experiment, blood lipase levels were lower at seven days in broilers fed with essential oil of
C. sinensis, which was not observed at 14 days. In this initial phase, the levels of lipase and amylase were slightly lower in broilers fed with essential oil of
X. aromatica than broilers fed with other treatments. However, such variations were not observed in the biochemical analysis of the pancreas. In broilers, essential oils can also stimulate the activity of certain digestive and pancreatic enzymes [
93]. Another explanation for the increase in enzyme activity and pancreatic secretion is the increase in the relative weight of pancreas that those oils could induce [
93,
94]. However, such a change was not observed in the present study.
The levels of glutamate-oxaloacetate transaminase (TGO) and glutamate-pyruvate transaminase (TGP) enzymes in the blood were lower in broilers fed with essential oils of
C. sinensis and
X. aromatica than broilers fed with control diet at 7 days, a result that was not observed at 14 days. In the liver, only the levels of glutamate-pyruvate transaminase (TGP) were lower in broilers fed with essential oils of
C. sinensis and
X. aromatica than broilers fed with control diet at seven days. The glutamate-oxaloacetate transaminase (TGO) and glutamate-pyruvate transaminase (TGP) enzymes are essential in the diagnosis of liver damage caused by drugs or infections, since after these events several enzymes, including aminotransferases, leak from the injured cells and pass into the blood [
95]. However, such lesions were not observed in the present study.
Borsa et al. [
96] defined the following values for glutamate-pyruvate transaminase (TGP) in serum: 2–13 and 9–22 (U/dL) for birds at 7 and 14 days, respectively. Minafra et al. [
97] found values of 203–267 (U/dL) at 7 days, with values of 251–375 (U/dL) at 14 days for glutamate-oxaloacetate transaminase (TGO) levels; they also found values around 73.3–79.5 (U/dL) at 7 days, with 72.2–75.6 (U/dL) at 14 days for glutamate-pyruvate transaminase levels in the liver tissue of chickens fed with diets supplemented with glutamic acid and vitamin K. These results are divergent in relation to the biochemical parameters of normality for this enzyme in liver tissue.
In another study, the addition of a mixture of essential oils containing Timol and Carvacrol in the diet of ducks also did not change the biochemical parameters regarding the levels of total proteins, glycemia, calcium, triglycerides, cholesterol, and the enzymes glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase, but they significantly improved phagocytic activity when compared to the control [
97].
In the present study, the addition of
C. sinensis and
X. aromatica essential oils to the broiler feed positively influenced the intestinal health of animals, promoting an increase in the villus height and depth of crypts, mainly in the initial phase of animal development, which evidences the maintenance of the intestinal integrity of the animal. According to El-Katcha et al. [
98], the villus height and the depth of crypts are considered indicators of proper intestinal development.
The results found in the present study corroborate the literature data regarding the effect of essential oils on the intestine histomorphometry of broilers. Kuzmuk et al. [
99] also found greater villus height and less depth in the ileum crypts of 21- and 42-day-old broilers fed with essential oils of oregano (
Origanum onites), cloves (
Syzygium aromaticum), and cumin (
Cuminum cyminum). Basmacioğlu-Malayoğlu et al. [
100,
101], found greater villus height and depth of crypts in the jejunum of chickens fed with a mixture of organic acids and essential oil at 42 days of age. Yang et al. [
50] also found greater villus height and depth of duodenal crypts of birds fed with a mixture of sorbic acid, fumaric acid, and thymol at 42 days of age, highlighting the potential of using essential oils and their active principles in improving the health of birds.