Tarragon (Artemisia Dracunculus L.) Could Alleviate Negative Effects of Stocking Density in Laying Hens

30/April/2020 Approved: 12/November/2020 ABSTRACT The study was conducted to determine the effects of stocking density (SD, 5 or 7 layers/cage) and tarragon ( Artemisia dracunculus L.) diet supplementation at four levels (0, 1.2, 6 and 12 g/kg feed) on performance, certain egg characteristics, serum, liver, egg yolk and small intestine bacteria parameters in laying hens. The experiment was carried out over a period of 8 weeks, with 192 Lohman Brown commercial hybrids at 50-w-age. The results showed that an increased SD reduced feed intake (FI; p< 0.01) and egg production ( p< 0.05), but had no effect on the weight gain, feed conversion ratio, damaged egg ratio, egg weight and egg quality ( p> 0.05) parameters. The supplementation of tarragon to the diet reduced the FI and damaged egg ratio ( p< 0.01), and improved egg production and FCR ( p< 0.01). While an increased SD reduced serum total antioxidants ( p< 0.05), it elevated corticosterone (CORT) and total oxidant serum (TOS) ( p< 0.05). Tarragon was found to enhance total immunoglobulin ( p< 0.05), but to decreased the CORT and TOS of the serum ( p< 0.05). An increased SD raised the malondialdehyde (MDA) in the serum ( p< 0.001), liver ( p< 0.05) and yolk ( p< 0.001). Tarragon supplementation reduced MDA of the serum ( p< 0.05), liver ( p< 0.001) and yolk ( p< 0.001). E. coli and total Mesophilic Aerobic Bacteria counts in the small intestine were raised ( p< 0.001) with increased SD. Tarragon decreased ( p< 0.05) mesophilic aerobic bacteria. It was thus found that, tarragon supplementation can be considered generally effective in improving performance parameters, alleviating stress-induced negativities, reducing lipid peroxidation, regulating the immune system and controlling


INTRODUCTION
A high (H) stocking density (SD) is one approach to increasing profitability in conventional poultry production, although HSD decreases animal welfare and increases stress. In the event of stress, the adrenocorticotropic hormone secreted from the anterior pituitary affects the adrenal cortex for the synthesis of glucocorticoids (corticosterone [CORT] and cortisol). The primary glucocorticoid released from the hypothalamic-hypophysis-adrenal (HPA) axis with the effects of stress factors is corticosterone in poultry (Ralph & Tilbrook, 2016). An increase in the CORT hormone in poultry has been found to decrease performance (Kutlu & Forbes, 1993;Mirfendereski & Jahanian, 2015;Sahin et al., 2002; and immunity parameters (Mirfendereski & Jahanian, 2015;von Eugen et al., 2019). Stress factors effect total serum antioxidant (TAS) and oxidant (TOS) concentrations (Sohail et al., 2011). While light and moderate stressors can be easily handled, severe stressors can cause pathological conditions (Moberg & Mench, 2000). That said, contradictory data has been presented with regards to the reaction of the HPA axis in connection with the period and intensity of the stressors in chickens (Ericsson, 2016).

Tarragon (Artemisia Dracunculus L.) Could Alleviate Negative Effects of Stocking Density in Laying Hens
Basic free radicals deriving from oxygen in living systems play a role in oxidative stress and oxidative rancidity (Conforti et al., 2006). Oxidative stress damages important biological molecules, and when the body's ability to rid itself of radicals is reduced, at least one of the pathological findings in animals (Fellenberg & Speisky, 2006) lipid peroxydation in animal products (Galobart et al., 2001) occur. Today, medicinal aromatic plants with high phenolic and flavonoid content, as well as their extracts, are used to alleviate the negative effects of stress factors and synthetic antioxidants. It has been stated that the dietary supplementation of antioxidant substances regulates corticosteroid synthesis in the adrenal glands (Mirfendereski & Jahanian, 2015;Von Eugen et al., 2019) enhances immunity (Mirfendereski & Jahanian, 2015) and decreases the lipid peroxidation of egg yolk (Botsoglou et al., 2005;Karaalp et al., 2018). In addition, dietary supplementations of anti-microbial substances suppress pathogenic microflora in the digestive system (Wenk, 2000).
The initial humoral and cellular immune response takes the form of cytokines released from activated T cells. These cytokines are transformed into plasma cells that are capable of producing antibodies (IgG, IgA and IgM) through the stimulation of B lymphocytes (Taheri et al., 2005). There are three main antibody classes containing IgM, IgA and IgG (IgY) that have been identified in chickens (Ayaz et al., 2008). IgG in chickens accounts for approximately 75% of the total serum antibodies (Carlander et al., 2000).
One of the more common bacteria found in animal intestines is Escherichia coli (E. coli) (Omidpanah et al., 2016). Some serotypes of E. coli are pathogens for poultry (Dho-Moulin & Fairbrother, 1999; and some serotypes are pathogens for humans (Kassaify and Mine 2004). Aerobic mesophilic bacteria and their metabolites are the main pathogenic microorganisms causing environmental pollution (Witkowska & Sowińska, 2013).
The present study evaluates the effects of the dietary supplementation of Artemisia dracunculus L. in commercial laying hens housed in different SD on performance, certain egg quality characteristics, peroxidation, immune response and some intestinal bacteria.

Animals, Experimental Desing and Feed
All procedures were approved by the Animal Experiments Local Ethics Committee of Gumushane University (Approval date: 10.05.2017; Decision No: 01-07). A total of 192 Lohman Brown layer hens (at 50-w old) were used in a 2×4 factorial (with 4 replications) arrangement of treatments that included two cage densities [5, normal (N) SD and 7 (HSD) hens per cage; 90x45x35 cm], and four ratios of ground tarragon (0, 1.2, 6 and 12 g/kg diet, T0, T1.2, T6 and T12, respectively). The SDs were 810 and 580 cm 2 area per hen in the NSD and HSD groups, respectively. The ingredients and chemical compositions of the commercial feed are presented in Table 1. The tarragon was obtained from the village of Yedigözeler in the Bayburt Province, and was kept stored in a cool and dry environment. The total antioxidant amount of tarragon was measured as 0.833 mmol/g according to Erel (2004). The dietary supplementation of 0, 1.2, 6 and 12 g/kg of ground tarragon corresponded to 0, 1, 5 and 10 mmol of additional antioxidant, respectively. The first 2-w (48 to 50-w old) was the adaptation period. The main trial lasted for a total of 8-w, from 50 to 58-w old. The hens were exposed to 16 h light and 8 h darkness, feed and water were provided ad libitum, and the accommodation temperature was maintained at 21 to 24 ºC throughout the trial period. The diet was analyzed according to standard AOAC (2007), procedures for chemical composition.

Performance and Egg Quality Parameters
Weight gain (WG), feed intake (FI) and feed conversion ratio, FCR were determined every two weeks; the damaged egg ratio (DER), average egg weight (AEW) and egg weight (EW) were determined daily. In order to determine such egg quality parameters such as yolk height (YH), yolk color index (YCI), Haugh unit (HU), shell strength (SS), shell thickness (ST), shape index (SI) and shell weight (SW), three random egg samples were taken from each replication (12 from each group; total 96) at 4 and 8 w. After the samples were kept at room temperature for 24 hours, they were subjected to analysis with an advanced laser light method branded NABEL DET6000.

Serum CORT, IgG, TAS and TOS
At the end of the experiment, a total of 80 samples, 10 from each group, were taken for blood, yolk and tissue analysis. CORT, TAS, TOS and IgG in the blood; malondialdehyde (MDA) in the blood, yolk and liver; and E. coli and total mesophilic aerobic bacteria in the small intestine were determined. At the end of the experimental period, serum was separated by centrifuging approximately 7 ml blood samples taken from the vena jugularis after 12-hours of fasting, and kept at -82 0 C until the analysis. CORT concentrations were determined using commercially available radio immune assay sets (Byk-Sangtec Diagnostica, Dietzenbach-Germany; Immulite 2000, DPC, LA) (Sahin et al., 2003). Serum IgG concentrations were determined using the ELISA method, as mentioned by Li et al. (2000). linearity of the standard IgG was calculated using the Excel program. Serum TOS was measured spectrophotometrically and calibrated with hydrogen peroxide (Erel, 2005). Serum TAS level was measured using the colorimetric method developed by Erel (2004). The eggs that were selected for yolk MDA analysis were stored for 21 days at +6 ºC, after which, 10 samples were taken from the liver and yolk, and stored until the time of analysis at -82 ºC after being homogenized. The MDA analyses were carried out based on the method put forward by .

Small Intestine E. coli and Total Mesophilic Aerobic Bacteria
A total of 10 small intestine content samples were collected from each experiment group for total mesophilic aerobic bacteria and E.coli counts. The total E.coli and mesophilic aerobic bacteria counts reproduced at the end of the incubation period were determined in line with.

Statistical analysis
The data was subjected to an analysis of variance (General Linear Model procedure) with a completely randomized design using SPSS software (2002). Data on the egg quality parameters was analyzed according to the 2x2x4 factorial design. A Duncan multiple range test was applied to compare the effects of the different doses of tarragon.
2 Metabolisable energy (ME) is calculated according to the method provided by TSE (1991).

Tarragon (Artemisia Dracunculus L.) Could Alleviate Negative Effects of Stocking Density in Laying Hens
tarragon doses had no effect on body weight between the beginning and end of the test, WG and AEW (p>0.05). An increase in SD had no effect on FCR and DER (p>0.05), but decreased the daily FI (p<0.01) and EP (p<0.05). T1.2 and T6 diets decreased FI and DER (p<0.01) and improved FCR (p<0.01). The effects on egg quality characteristics of SD and different tarragon doses in the diet are given in Table 3. It was determined that age, SD and tarragon dietary supplementation had no significant effect on the YH, HU, SS, SI or SW egg quality parameters. Age and SD had no effect on YCI (p>0.05), while YCI was decreased by tarragon supplementation (p<0.01). The interactions of D×S and D×S×T on YCI, on the other hand, were significant (p<0.01), and SD and changes in the tarragon doses in the diet had no effect on ST (p>0.05), but decreased as the age increased (p<0.05).

Serum CORT, IgG, TAS and TOS
The effect of SD and tarragon doses on serum CORT, IgG, TAS and TOS quantities are presented in Table  4. Serum CORT, which was high (p<0.001) in HSD, decreased (p<0.001) in a parallel with tarragon dietary supplementation. Furthermore the S×T interaction on serum CORT was significant (p<0.001). SD had no effect on the serum IgG (p>0.05). T6 and T12 increased the serum IgG more than in the control, and the T12 effect was higher (p<0.001). HSD decreased serum TAS (p<0.01), and tarragon supplementation increased the TAS amount only numerically (p>0.05). While SD increased serum TOS (p<0.001), the tarragon doses decreased this parameter (p<0.001). T12 was more effective (p<0.05) in decreasing serum TOS when compared to T1.2 and T6.

Serum, Liver and Egg MDA
The effects of SD and tarragon doses on serum, liver and egg MDA quantities are presented in Table  5. HSD raised the MDA in the serum (p<0.001), liver (p<0.05) and egg yolk (p<0.001). T6 and T12 diets decreased serum MDA. All doses of tarragon decreased the liver MDA (p<0.001), and T6 and T12 were more effective than T1.2 on this parameter. Moreover S×T interaction was significant in the quantity of liver MDA (p<0.05). The T6 and T12 diets decreased the yolk MDA (p<0.001), and T12 was more effective on the yolk MDA when compared to T6 (p<0.05).

Small Intestine E. coli and Total Mesophilic Aerobic Bacteria
The effects of SD and tarragon doses on E. coli and total mesophilic aerobic bacteria counts in the small intestine are presented in Table 5. HSD raised the counts of E. coli and the total Mesophilic Aerobic Bacteria in the small intestine (p<0.001). Tarragon doses decreased E. coli only numerically (p>0.05); and T6 and T12 decreased significantly (p<0.05) the total mesophilic aerobic bacteria count when compared to the control.

Performance
In the present study, SD and tarragon doses had no effect on body weight between the beginning and end of the test, WG and AEW (p>0.05). There are also studies indicating that the HSD has a negative effect on the WG (Onbaşılar & Aksoy, 2005;Nahashon et al., 2006;Sahin et al., 2007;Sarica et al., 2008) and has no effect as in the present study (Jalal et al., 2006;Onbaşılar et al., 2009;Fidan, 2010). The differences between the results of the present study and those in literature could be attributed to the lack of standardization of area allocated per hen. In the present study, the addition of 40% more space per animal to the HSD group than in the NSD group was not sufficient to affect weight parameters (p>0.05). It is stated that the dietary supplementation of 1% Artemisia sieberi (Khalaji et al., 2011), 2 and 4% Artemisia annua (Cherian et al., 2013) and 0.4% Artemisia dracunculus to broilers (Gharetappe et al., 2015) had no effect on WG. (Hosseinzadeh & Moghaddam, 2014) reported that the dietary supplementation of 0.125 and 0.25% of Artemisia dracunculus did not affect WG, while a 0.5% dose decreased it. Kheirabadi et al. (2014) reported that the incorporation of 5 mg/kg of Artemisia sieberi granulated extract (GEAS) into the diet reduced live weight at 42 days when compared to control group. It was reported that the incorporation of 10 and 20% of Artemisia annua (Brisibe et al., 2008); 0.5, 1 and 1.5% of Artemisia annua leaf powder with 2000 and 4000 ppm methanolic extract (Gholamrezaie et al., 2013); 1.5% of Artemisia annua (Drǎgan et al., 2014); and 100, 150 and 200 g/kg of Artemisia

Tarragon (Artemisia Dracunculus L.) Could Alleviate Negative Effects of Stocking Density in Laying Hens
absinthium (Kostadinović et al., 2015) into broiler diets increased WG. In the present study, the incorporation of different levels of tarragon into diets of laying hens had no significant effect on live weight or live weight gain (p>0.05).
In addition to the findings that SD had no effect on the FI of laying hens (Anderson et al., 2004;Simsek & Kilic, 2006), similar findings to those of our study that it decreased FI have been reported (p<0.01) (Sohail et al., 2004;Jalal et al., 2006;Sahin et al., 2007;Onbaşılar et al., 2009;Fidan, 2010;Mirfendereski & Jahanian, 2015). It is considered that the reduction in feeder space per bird in HSD is the main factor reducing FI. T1.2 and T6 diets reduced FI when compared to the control

Tarragon (Artemisia Dracunculus L.) Could Alleviate Negative Effects of Stocking Density in Laying Hens
group (p<0.01), while the T12 diet had no effect on FI (p>0.05) in the present study, which concurs with the findings of Gholamrezaie et al. (2013), who reported that the incorporation of Artemisia annua extract and leaf powder mixture into broiler diets reduced FI. The incorporation of Artemisia sieberi (Khalaji et al., 2011), Artemisia dracunculus (Gharetappe et al., 2015;Hosseinzadeh & Moghaddam, 2014), Artemisia annua (Cherian et al., 2013) and GEAS (Kheirabadi et al., 2014) into broiler diets did not affect FI, although Brisibe et al. (2008) reported that the incorporation of Artemisia annua leaves increased FI.
In our study, it was found that HSD reduced EP as in previous studies (Onbaşılar & Aksoy, 2005;Jalal et al. 2006;Sarica et al., 2008;Onbaşılar et al., 2009;Fidan, 2010;Mirfendereski & Jahanian, 2015) (p<0.05), and reduced feeder space per bird (Onbaşılar & Aksoy, 2005;Jalal et al., 2006) and stress conditions caused by high stocking densities (Sarica et al., 2008;Mirfendereski & Jahanian, 2015; ) were found to be responsible for this result. Mirfendereski & Jahanian (2015) found a reduction of EP in HSD to be associated with blood CORT levels. In some studies, it was reported that SD had no effect on EP (Simsek & Kilic, 2006;Sahin et al., 2007). Brisibe et al. (2008) reported that the incorporation of 20% Artemisia annua leaves into laying hen diets increased EP. It has further been reported that the incorporation of an extract composed of 70% pine needle and 30% Artemisia annua into laying hen diets (Li et al., 2016) improved EP. In the present study, the incorporation of tarragon into the diet increased EP in the T1.2 and T6 diets when compared to the control diet (p<0.01). This improvement can be explained by the fact that tarragon, which has antioxidant properties, reduces serum CORT and TOS levels, thus reducing stress. It has further been reported that the incorporation of salvia officinalis, thymbra spicata, menthae piperitae extracts, vitamin E (Kaya & Turgut, 2012) and Coriander Oil extract (Çiftci & Macit, 2018) into laying hen diets increased EP, while Rosmarinus officinalis L. (Çimrin & Demirel, 2016) incorporation did not affect EP.
It has also been reported that HSD reduced EP and increased AEW (Onbaşılar et al., 2009), while HSD reduced feeder space per bird, and thus AEW (Sohail et al., 2004;Sahin et al., 2007;Fidan, 2010). In addition, there have been studies reporting that increasing SD has no effect on AEW (Guesdon et al., 2006;Sarica et al., 2008;Mirfendereski & Jahanian, 2015 ), as in the present study, or affected AEW (Sahin et al., 2007;Onbaşılar et al., 2009;Fidan, 2010). In the present study, the tarragon levels incorporated into the diet had no effect on AEW (p>0.05). It has been reported that the incorporation of Artemisia annua leaves into laying hen diets does not affect egg weight (Brisibe et al., 2008;Li et al., 2016), and that the incorporation of different plant extracts (Kaya & Turgut, 2012;Çimrin & Demirel, 2016) into laying hen diets did not affect or reduced (Çiftci & Macit, 2018) egg weights. It has been stated that effects of phytogenic products on poultry performance data may differ depending on the origin, content and processing of the plant, as well as the species and age of the animal and environmental hygiene (Windisch et al., 2008).
In the present study, laying hens at 50 weeks of age were kept for 8 weeks. It is stated that it can be advantageous to provide antioxidant sources to old hens (Karaalp et al., 2018) whose egg shell quality may be more problematic due to many factors (Światkiewicz et al., 2010). Contrary to the finding that HSD causes a significant increases in DER (Guesdon et al., 2006), there have also been reports of no such effects on DER (p>0.05) (Sarica et al., 2008;Onbaşılar et al., 2009), similar to the present study. It has been found that the incorporation of an extract composed of pine needle and Artemisia annua into laying hen diets reduced DER, similar to the present study (Li et al., 2016). It has been further reported that the incorporation of different plant extracts into laying hen diets reduced (Kaya & Turgut, 2012) or had no affect (Çimrin & Demirel, 2016;Çiftçi & Macit, 2018) on DER. In the present study, T1.2 and T6 reduced DER when compared to the control diet (p<0.01).

Egg Quality Criteria
The advancement of the trial period (50-58 weeks of age) had no effect on any egg quality criteria other

Tarragon (Artemisia Dracunculus L.) Could Alleviate Negative Effects of Stocking Density in Laying Hens
than ST (p>0.05). The increase in age caused a decrease in ST in the present study, concurring with the findings of and Onbaşılar et al. (2018). In the present study, the fact that SD did not affect egg quality parameters was consistent with earlier studies (Sahin et al., 2007;Sarica et al. 2008;Fidan, 2010). In poultry, it was reported that SD did not affect egg YH (Karabayir et al., 2010), SS (Sahin et al., 2007;Sarica et al., 2008;Karabayir et al., 2010), ST (Onbaşılar & Aksoy, 2005;Nahashon et al., 2006;Sahin et al., 2007;Sarica et al., 2008;Fidan, 2010), SI (Sarica et al., 2008;Fidan, 2010) or SW (Sahin et al., 2007;Karabayir et al., 2010). That said, it has been reported that SD has different effects on parameters such as YCI (Sarica et al., 2008;Fidan, 2010) and HU (Nahashon et al., 2006;Sahin et al., 2007;Sarica et al., 2008;Fidan, 2010). It has been described that some antioxidant components of plants can improve calcium accumulation in the uterus, and thus increases shell weight and thickness (Ramírez-Tortosa et al., 1999). Our study has shown that the incorporation of tarragon into the diet has no effect on egg quality parameters, aside from reducing YCI (p<0.01). Li et al. (2016) reported that the incorporation of an extract composed of pine needle and Artemisia annua into laying hen diets did not affect EW, ST, HU, SS or SI. The increase in tarragon levels darkened the color of the diet, which had an unexpected effect on the egg yolk, but the cause of this could not be understood.

Serum CORT, IgG, TAS and TOS
Results of the present study showed that HSD, which is a social stressor affected some stress parameters (CORT, TAS, TOS) negatively (p<0.05). In laying hens, it was detected that HSD lead to an increase in the blood corticosteroid level (Mirfendereski & Jahanian, 2015). The glucocorticoids (cortisol and corticosterone) released by HPA with the effect of stressors when the animal is under stress are known to be immunosuppressive, and inhibit the production and effect of antibodies, deteriorate lymphocyte function and cause leukocyte proliferation (MUnck et al., 1984). Sohail et al. (2011) described that stress increases total oxidants and antioxidants in broiler serum. However, increases in serum antioxidants depending on the severity and duration of stressors is a controversial issue. Accordingly, in the present study, HSD increased serum CORT and TOS (p<0.05), but reduced TAS (p<0.05). The reduction in serum TAS concentration is considered to be related to the severity and duration of stressors (24 hours/day in this study) as well as the use of serum TAS in sweeping away the free radicals that are formed due to the oxidative stress that occurs.
The increases in the levels of tarragon, which contains antioxidant substances (Kordali et al., 2005), as well as the continuous overall reduction of serum CORT and TOS (first reduction in the T1.2 diet) (p<0.05), the increase in serum IgG (first increase in T6 diet) (p<0.05) and the fact that it does not affect serum TAS levels (p>0.05) confirm our finding.
It is being suggested that antioxidant substances regulate corticosteroid synthesis in the adrenal glands and improve certain immune responses significantly (Gholamrezaie et al., 2013;Pardue & Thaxton 1984). High egg production (physiological stress) often causes a degeneration of lymphoid organs (Gray et al., 1989) and the suppression of humoral and cell-mediated immune response (Murray et al., 1987). Furthermore, it has been noted that cell-mediated immunity drops after 45 weeks of age (Fahey & Cheng, 2008). In the present study, the fact that SD did not affect serum IgG (p>0.05) suggests that reducing the space per bird is not sufficient to reduce this parameter. In addition, the T6 diet increased serum IgG (p<0.05). The overall increase in serum IgG with the increase of tarragon levels in the diet (p<0.001) can be associated with the immune-enhancing effect of tarragon. It is suggested that Artemisia annua leaf powder and methanolic extract affect performance by increasing the cellular and humoral immunity of broilers (Gholamrezaie et al., 2013). Furthermore, it has been reported that a 1% incorporation of Artemisia sieberi into broiler diets has no effect on plasma lymphocytes, eosinophils, basophils, primary or secondary antibody response against sheep red blood cells, but increases monocyte percentage (Khalaji et al., 2011). Gholamrezaie et al. (2013) reported that the effects of different natural products on the immune system were complicated, and that the stimulation of the lymphatic tissue of the digestive system was a direct effect, while the alteration of microbial population of the gastrointestinal system lumen was an indirect effect.

Serum, Liver and Egg MDA
In the present study, HSD, which is a social stressor, affected MDA, which is a lipid peroxidation indicator (in serum, liver, egg yolk), and some bacteria in the small intestine (E. coli and total mesophilic aerobic bacteria counts) desirably (p<0.05). Furthermore, HSD in the present study caused an increase in serum, liver and egg MDA (p<0.05) by increasing serum CORT and reducing serum TAS, resulting from the increased stress in hens. There are studies reporting that the incorporation of plants and plant products containing antioxidant substances into laying hen diets reduced  (Botsoglou et al., 1997;Botsoglou et al., 2005;Karaalp et al., 2018;Krause & Ternes, 2000;Radwan et al., 2008) or did not affect (Galobart et al., 2001) egg yolk lipid peroxidation. Kostadinović et al. (2015) found that the incorporation of 20% Artemisia absinthium into the diet reduced blood MDA in broilers, while Cherian et al. (2013) reported that the incorporation of 2% and 4% Artemisia annua into broiler diets reduced MDA in the thigh and breast muscles of broilers. In the present study, serum, liver and egg MDA levels reduced (p<0.05) in parallel to the reduction in serum CORT and TOS under the effect of the antioxidant substances found in the tarragon incorporated into the diets (Kordali et al., 2005). Serum and egg MDA started to reduce with the T6 diet, while liver MDA started to reduce with the T1.2 diet, when compared to the control diet (p<0.05).

Small Intestine E. coli and Total Mesophilic Aerobic Bacteria
The findings of the present study suggest that the increase in E. coli and total mesophilic aerobic bacteria counts in the small intestine due to HSD are related to contamination (p<0.001). In literature, the antimicrobial effects of artemisia species were mostly researched on coccidiosis, and these species were reported to be used for coccidiostatic purposes (Brisibe et al., 2008;De Almeida et al., 2012;Kostadinovic et al., 2012Drǎgan et al., 2014Kheirabadi et al., 2014). Lopes-Lutz et al. (2008) reported that the oils of different Artemisia species are effective at different levels against microorganisms such as Escherichia coli, Staphylococcus epidermidis and Staphylococcus aureus. The fact that the incorporation of 1% Artemisia sieberi into broiler diets reduces cecal coliform and Escherichia coli populations (Khalaji et al., 2011), in addition to the findings in our study that the incorporation of different levels of tarragon into the diet has no effect on E. coli levels in the gut (p>0.05), can be associated with the difference in artemisia species used in the studies. Furthermore, the presence of 6 g or more tarragon in the diet was found to affect the reduction of total mesophilic aerobic bacteria counts when compared to the control diet (p<0.05).

CONCLUSIONS
With the incorporation of different levels of Artemisia dracunculus L. into the diets of laying hens in the second production cycle and housed under different SDs, it was found that: HSD reduced FI and EP, the T1.2 and T6 kg diets reduced FI and DER, increased EP and improved FCR. All levels of tarragon were found to reduce YCI levels. HSD increased serum CORT and TOS, as well as MDA levels in serum, liver and egg yolk, and reduced serum TAS, which was associated with the oxidative stress occurring resulting from the increasing social stress. The reduction of serum CORT and TOS (with all diets), serum and egg yolk MDA (T6 and T12 diets) and liver MDA (T1.2 diet) with the incorporation of tarragon into the diet was associated with the antioxidant substances found in tarragon. Serum IgG (with T6 and T12 g diets) increased with the incorporation of tarragon into the diet, indicating that tarragon has immune-enhancing substances. Total mesophilic aerobic bacteria counts reduced with the T6 and T12 diets, when compared to control diets, suggesting that tarragon contains a certain level of antibacterial substances.