Plectranthus amboinicus and rosemary (Rosmarinus officinalis L.) essential oils effects on performance, antioxidant activity, intestinal health, immune response, and plasma biochemistry in broiler chickens

Abstract This work aimed to assess the effects of Plectranthus amboinicus essential oil (PAE) and rosemary (Rosmarinus officinalis L.) essential oil (ROE) as feed additives on performance, antioxidant activity, intestinal microbiota, intestinal morphology, immune response, and plasma biochemistry using 320 unsexed 1‐day‐old Ross 308 broiler chickens. The chickens were assigned randomly into four treatments containing eight replicates with 10 chickens each. Treatment diets included a basal diet as a control group, 100 mg/kg PAE, 200 mg/kg PAE, and 100 mg/kg ROE. ROE affected the growth performance in the starter phase by improving (p = .01) the feed conversion ratio (FCR) compared with the control diet. Glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity in the plasma were elevated (p < .0001) by both feed additives. Supplementation of additives could increase (p < .006) total antioxidant capacity (TAC). Furthermore, malondialdehyde (MDA) values in the breast (p < .0001) and thigh (p < .001) for all supplemented diets were less than the control group. The essential oils (EOs) reduced (p < .005) coliform counts in the ileum and increased (p = .029) lactic acid bacteria counts. In addition, villus height (VH) and crypt depth (CD) increased, whereas the density of goblet cells decreased in the small intestine when feed additives were included. Also, the antibody titers against sheep red blood cells (SRBC) and Newcastle disease virus (NDV) were increased (p < .0001) by EOs. Plasma total protein (p = .04) and globulin (p = .02) were increased, and cholesterol was reduced (p = .002) by supplemented diets. Our study revealed that PAE could effectively improve the antioxidant activity, intestinal microbiota population, intestinal morphology, immune response, and plasma biochemistry parameters in broiler chickens.


| INTRODUC TI ON
Phytobiotics, also known as phytogenics or phytochemicals, are an extensive subset of plant-based bioactive compounds. Over 5000 individual dietary phytobiotics have been recognized to date in fruits, whole grains, vegetables, legumes, herbs, nuts, and EOs Liu, 2004). Herbs and EOs from the Lamiaceae family have revealed antioxidant, antibacterial, and immunostimulatory effects and increased enzymatic secretions in the gastrointestinal tract of animals (Brenes & Roura, 2010;Ghalamkari et al., 2011).
These can potentially be an alternative to antibiotics, or used alongside (Aguiar et al., 2015). The characteristics of Plectranthus amboinicus (Lour.) Spreng (PA), a member of the Lamiaceae family, has been studied for effects including antibacterial (Aguiar et al., 2015), antifungal (Murthy et al., 2009), antivirus (Kusumoto et al., 1995), and antioxidant (Praveena & Pradeep, 2012) activities in vitro. However, there are few studies in vivo, particularly using farm animals. The leaf meal of PA enhanced the milk production of dairy Holstein cows (Fati et al., 2014) and PA extract was shown to have antioxidant, nephroprotective, and diuretic properties (Palani et al., 2010) in rats. Similarly, PAE increased antioxidant activity (Manjamalai & Grace, 2012) in mice. In other species, PA extract improved the nonspecific immune response and growth performance in white-leg shrimp (Huang et al., 2022). This herbal essential oil has the main bioactive compounds of carvacrol (Murthy et al., 2009) and thymol (Sabra et al., 2018), which can be compared with thyme and oregano EOs. Rosemary (Rosmarinus officinalis L.) is another aromatic plant from the Lamiaceae family with the main components including camphor, 1,8-cineole, and α-pinene (Sienkiewicz et al., 2013). ROE is more well known than PAE for farm animal research, with numerous studies utilizing ROE as a feed additive in poultry (Gharejanloo et al., 2017;Loetscher et al., 2013;Mahgoub et al., 2019;Mathlouthi et al., 2012;Yesilbag et al., 2011). Therefore, in the current study with PAE, its essential oil potential was assessed at 100 mg/kg, which for ROE was an effective dosage in some studies (Gharejanloo et al., 2017;Mathlouthi et al., 2012;Yesilbag et al., 2011). The present work aimed to determine the effect of feeding PAE compared with rosemary on performance, antioxidant activity, intestinal microbiota, intestinal morphology, immune response, and plasma biochemistry in modern commercial broilers.

| Preparation and extract of essential oils
The PA was raised in the greenhouse of Tarbiat Modares Agriculture Faculty. We purchased ROE from a local company.
The fresh aerial parts of PA were collected and exposed to hydrodistillation for 3 h via a Clevenger-type apparatus to extract the essential oil. Dehydrating the essential oil used sodium sulfate, and it was stored after filtration at 4°C and in darkness to safeguard its components.

| GC/MS analysis
Plectranthus amboinicus essential oil (PAE) and rosemary (Rosmarinus officinalis L.) essential oil (ROE) analysis was performed using a Thermoquest-Finnigan Trace GC-MS tool equipped with a DB-5 fused silica capillary column (film thickness 0.25 mm, 60 m × 0.25 mm i.d.). The temperature of the oven was programmed to increase from 60 to 250°C at a rate of 4°C per minute. It was ultimately held for 10 min; at a transfer line temperature of 250°C. The carrier gas was helium with a split ratio of 1/50 at a 1.1 mL/min flow rate. Over 35-465 amu (atomic mass units) the mass spectrometer was scanned via the quadrupole, with an ionizing voltage of 70 eV and an ionization current of 150 mA. The constituents were identified based on their retention indices by comparison of the mass spectra with those reported in the studies and Willey (Chem Station data system) libraries (Table 1).

| Animals, diets, and experimental design
This study was conducted at the poultry farm of Tarbiat Modares Agriculture Faculty (latitude 35°44′N; longitude 51°9′E and altitude 1284 m above sea level) using 320 unsexed 1-day-old broiler chickens (Ross308, Aviagen Inc.). The chickens were randomly allocated into TA B L E 1 Identification of essential oil components by GC-MS. four treatments with eight replicates of 10 chickens each. The treatments were the basal diet as the control group, basal diet +100 mg/ kg PAE, basal diet +200 mg/kg PAE, and basal diet +100 mg/kg ROE.

| Growth performance assay
The weight gain and feed intake were assayed per pen at the end of each week for the broiler chickens. Then, the average daily feed intake (ADFI), average daily gain (ADG), and the ratio of those as FCR were calculated for the different phases of starter, grower, and finisher. Chick mortality was reported and considered in calculations for all treatments.

| Plasma antioxidant assay
At day 42, the blood of two chickens per pen (16 chickens per treatment) was drawn using heparinized syringes via the jugular vein.
The blood plasma was separated to assay the activity of antioxidant enzymes using SOD and GPx commercial kits (Navand Salamat Company) according to the manufacturer's instructions. Inhibition of pyrogallol autoxidation and decrease in the absorbance at 405 nm were used to detect SOD activity. GPx activity was determined by monitoring the level of the consumption of NADPH with decreasing absorbance at 340 nm. The measurement of TAC was conducted by the ferric reducing ability of plasma (FRAP) assay. This assay is based on the reduction of Fe 3+ to Fe 2+ by antioxidants present in the sample, with the absorbance monitored at 593 nm (Benzie & Strain, 1996).

| Lipid oxidation assay
At day 42, after slaughter, the breast and thigh meat of eight chickens per treatment were removed and used for the lipid oxidation assay. Measuring MDA was performed as a secondary oxidation product based on the thiobarbituric acid method as explained by Botsoglou et al. (1994).

| Intestinal microbiota assay
At day 42, eight chickens were slaughtered from each treatment.
The small intestine (from the jejunum distal end to the ileocecal junction) was dissected to assess the numeral of total aerobic, coliform, and lactic acid bacteria. Immediately postmortem, the small intestine was longitudinally opened. A sterile knife was used to scrape the digestive contents and mucosal surface, and the ileal contents were then transferred using aseptic technique into sterile tubes.
One gram of the homogenized ileal content was diluted from 10 1 to 10 8 in sterile phosphate-buffered saline (PBS). Agar media, plate count agar, MacConkey agar, and MRS agar were used to enumerate total aerobic, coliform, and lactic acid bacteria, respectively. Each duplicate sample dilution was inoculated on agar plates and incubated at 37°C for 24 h before counting. The counts were stated as log 10 CFU/g.

| Histological morphometric assay
At the end of the experimental period, eight chickens per treatment were randomly selected and slaughtered to prepare tissue samples from the small intestine. The small intestine was removed, and 2 cm sections of the duodenum, jejunum (between the entry of the bile duct and Meckel's diverticulum), and ileum were excised, rinsed, and stabilized using physiological serum in 10% saline-formalin solution for 24 h. Tissue was dehydrated, clarified, and embedded in paraffin in order before cutting into 5μm-thick sections using a rotary microtome. After mounting on glass slides, the sections were stained with both hematoxylin-eosin and alcian blue. The determination of villus height, crypt depth, and the density of goblet cells (per 100 μm villus height) was conducted with an Olympus light microscope using imaging software (Dino Capture 2.0). At last, the ratio of villus length to crypt depth (VH/ CD) was calculated.

| Immune response assay
At day 9, two chickens per pen were subcutaneously vaccinated (0.2 mL per chicken) with inactivated bivalent NDV and H9N2 avian influenza virus (AIV) vaccines. At day 19, the blood of these chickens was drawn using heparinized syringes to assess the antibody titers for AIV and NDV. A hemagglutination inhibition assay to assess antibody titers was conducted on the collected blood plasma.
In addition, at day 28, 0.1 mL of 5% SRBC in sterile PBS was injected into the breast muscle of two chickens per pen. A booster injection was also given per chicken at day 35 to measure the secondary anti-SRBC antibody response. After 7 days, the blood of each chicken receiving the SRBC was drawn with heparinized syringes, and the plasma was assayed for anti-SRBC antibody titers via a microhemagglutination test using 96-well microplates. The assessment methods were carried out according to Sedaghat and Karimi Torshizi (2017).

| Lymphatic organs assay
At day 42, eight chickens were slaughtered from each treatment, and the spleen and bursa were removed and quickly weighed after slaughtering. Then, their relative weight percent was determined.

| Plasma biochemistry assay
Blood samples were randomly collected from two chickens per pen using heparinized syringes via the jugular vein. Then, the samples were centrifuged within 20 min at 1000 × g at room temperature to separate the plasma. The plasma samples were stored at −20°C in Eppendorf tubes until further analysis. Plasma biochemistry measures, such as total protein, uric acid, albumin, globulin, cholesterol, triglycerides, and glucose, were spectrophotometrically analyzed with analytical kits (Pars Azmun Company) and a microplate reader (Stat Fax 3200; Awareness Technology Inc). Ultimately, the ratio of albumin to globulin was determined.

| Statistical analysis
This work was performed in a completely randomized design. The obtained data were exposed to ANOVA via the SAS software (SAS 9.4, 2014) GLM Proc. The statistical significance was compared among the means at a 95% significance level through Duncan's multiple-range test. Results are presented as the mean value.

| Growth performance
There was no difference among trial treatments in ADFI, ADG, and FCR for the different phases with the exception that in the starter phase there was an improvement (p = .01) in FCR for the ROE treatment compared to the control group (Table 3).

| Antioxidant activity
In the present study, GPx and SOD activities were elevated in the plasma (p < .0001) by both feed additives compared to the control, except 200 mg/kg PAE which showed no significant effect on GPx. In addition, supplemented diets had increased (p = .006) TAC.
Moreover, all the supplemented diets reduced MDA in both breast (p < .0001) and thigh (p < .001) when compared to the control group (Table 4).

| Intestinal microbiota
In this study, dietary supplementation with both levels of PAE decreased (p < .005) coliform counts. Moreover, both EOs increased (p = .029) lactic acid bacteria counts in the ileum ( Table 5).

| Intestinal morphology
This study showed that PAE increased (p < .036) VH in the duodenum and that all the supplemented EOs increased (p < .041) VH in the jejunum. Also, VH (p = .023) was increased by 200 mg/kg PAE in the ileum. The ratio of VH to CD was not significant in the different parts of the intestine. Furthermore, the CD was increased (p = .022), and the density of goblet cells was decreased (p = .046) by ROE and 200 mg/kg PAE in the ileum (Table 6). The number of colonies was counted, and all the data are expressed as log 10 CFU/g.

TA B L E 5
Effect of the supplemented diet with essential oils on intestinal microbiota of broiler chickens (42 days of age).

| Immune response
In the present study, anti-NDV antibody titers, with the exception of the 200 mg/kg PAE treatment, increased (p < .0001) with feed additives. Total anti-SRBC antibody titers in supplemented diets were higher (p < .0001) when compared to the control group (Table 7).
However, IgY and anti-SRBC antibody titers, anti-AIV antibody titers, and lymphatic relative weight percent were not significantly affected by the feed additive treatments.

| Plasma biochemistry
In the current study (Table 8) This study showed that EOs reduced MDA values in the breast and thigh meat. Hashemipour et al. (2013) reported that thymol and carvacrol supplementation made no difference in breast muscle's MDA value, but did in thigh muscle. Also, Placha et al. (2019) expressed that the available thymol in the diet did not affect lipid oxidation and fatty TA B L E 6 Effect of the supplemented diet with essential oils on the performance of broiler chickens (42 days of age Note: a,b Means with different letters within the same row differ significantly (p < .05).
acid composition due to a low residue of it in breast muscle. However, Loetscher et al. (2013) and Abbasi et al. (2020) both revealed that the MDA value of the breast meat was decreased significantly by ROE and thyme oil supplementation when compared to a control group.
The phytobiotic antimicrobial activity is accepted to be due to the lipophilic property of their components permeating the cell membranes and the mitochondria of microorganisms, which inhibits energy metabolism and membrane-bound electron flow.
Thus, a collapse occurs in the proton pump and then a draining  (Kırkpınar et al., 2011). It has been inferred that EOs are more effective on Gram-positive than Gram-negative bacteria (Trombetta et al., 2005); however, in contrast, Aguiar et al. (2015) observed that PAE was more effective on E. coli than on S. aureus.
The chemical composition of EOs is known to be affected by the geographical origin and harvesting period, which may explain the various effects of EOs against Gram-positive and Gram-negative bacteria (Brenes & Roura, 2010). Goblet cells are secretory epithelial cells that secrete mucus.
Mucin glycoproteins, as the principal part of mucus, create a The ratio of Albumin to Globulin.

TA B L E 8
Effect of the supplemented diet with essential oils on plasma biochemistry parameters of broiler chickens (42 days of age).
preservative barrier to pathogenic bacteria. They also lubricate the intestinal surface and form a layer to prevent damage to cells by endogenous digestive enzymes, gastric acid, and ingested feed (Alemao et al., 2021). Previous experiments showed an increase in the density of goblet cells in chickens with EOs (Liu et al., 2018) or no effect with mixed EOs and organic acids (Abdelli et al., 2020).
However, as in our work, Amer et al. (2021)

| CON CLUS ION
In this study, we tried to investigate PAE as a newer supplement compared to the more commonly used ROE. We observed that PAE and ROE improved antioxidant activity, intestinal microbiota, intestinal morphology, immune response, and plasma biochemistry parameters. PA is widely used as a medicinal and culinary herb. Therefore, it may be used in the poultry industry, in the same way as thyme and oregano, to improve the growth and health of the animal. In the future, more studies need to be carried out to elucidate the mechanisms of action of this herb in poultry.

ACK N OWLED G M ENTS
The authors are thankful to Tarbiat Modares University (Tehran, Iran) for providing facilities and financial support for this study and to Ahmad Reza Kamaliun for technical assistance.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare that they do not have any conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding authors (above). The data are not publicly available due to privacy or ethical restrictions.

E TH I C S S TATEM ENT
The study protocol was evaluated and accepted by the Tarbiat Modares University's Ethics Committee for animal use.