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

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 some intestinal microorganisms.

Keywords:
Antioxidant; bacteria; IgG; stocking density; tarragon

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).

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).

Artemisia dracunculus L., which is one of the 22 types of the Artemisia species grown in Turkey, has both antioxidant and antibacterial features. The dominant ingredients in the oil of Artemisia dracunculus L. plant, which is a multi-year woody plant, are (Z)-anethole (81%), (Z)-β-ocimene (6.5%), (E)-β-ocimene (3.1%), limonene (3.1%) and methyleugenol (1.8%) (Kordali et al. 2005). In another study conducted in the same period and in the same region, the β-ocimene (1.237.21 arbitrary units, AU×10−6), α-pinene (114.4 AU×10⁻⁶ ), β-thujene (166.92 AU×10−6), D-limonene (366.2 AU×10⁻⁶), γ-terpinene (187.27 AU×10^-6), Terpinolene (129.92 AU × 10⁻⁶) and Estragole (11 242 AU × 10⁻⁶) levels were stated (Yilmaz et al. 2019). There have to date been several studies examining the effects of Artemisia annua (Brisibe et al., 2008) Artemisia sieberi (Khalaji et al., 2011; Kheirabadi et al., 2014) and Artemisia dracunculus (Gharetappe et al., 2015; Hosseinzadeh & Moghaddam, 2014), all of which are Artemisia spp, in broiler chickens; and while there have been a few studies examining effects of Artemisia annua in laying hens (Brisibe et al., 2008; Li et al., 2016), no study has been found with specific focus on Artemisia dracunculus.

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.

MATERIALS AND METHODS

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 cm² 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.

Table 1
Composition of experimental diet.

Ingredients	g/kg
Corn	352.40
Triticale	175.00
Wheat	75.00
Soybean meal, 340 g CP/kg	114.60
Sunflower meal, 330 g CP/kg	103.60
Hazelnut, 420 g CP/kg	35.00
Corn gluten meal, 600 g CP/kg	25.00
Vegetable oil	8.30
Limestone	95.50
Dicalcium phosphate	7.20
NaCl	3.20
Premix	2.50
L-Lysine HCl	1.50
Naturabind-S	1.20
Chemical analysis of feed
Crude matter	892.20
Crude protein	167.50
Ether extract	46.60
Crude fiber	52.90
Crude ash	131.70
Starch	339.90
Calculated contents of feed (…/kg)
Metabolizable energy, MJ2	11.45
Methionine, g	3.70
Methionine + Cystine, g	6.70
Lysine, g	7.50
Linoleic acid, g	22.10
Ca, g	39.00
Available P, g	3.50
Na, g	1.50

• ¹
  Premix provided per kilogram of diet: vitamin A (retinyl acetate), 6.7 mg; vitamin D₃ (cholecalciferol), 1.6 mg; vitamin E (α-tocopherol), 30 mg; vitamin K₃ (menadione), 2.5 mg; vitamin B₁ (thiamine), 3 mg; vitamin B₂ (riboflavin), 7 mg; vitamin B₃ (niacin), 40 mg; vitamin B₅ (Ca-D-pantothenate), 8 mg; vitamin B₆ (pyridoxine), 4mg; vitamin B₈ (D-biotin), 0.1 mg; vitamin B₉ (folic acid), 1 mg; vitamin B₁₂ (cyanocobalamin) , 0.02 mg; vitamin C (ascorbic acid), 50 mg; choline chloride, 125 mg; Mn, 80 mg; Fe, 60 mg; Zn, 60 mg; Cu, 5 mg; Co, 0.10 mg; Se, 0.15 mg.
• ²
  Metabolisable energy (ME) is calculated according to the method provided by TSE (1991).

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 -82⁰C 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.

RESULTS

Performance and Egg Quality Criteria

Effects on performance of SD and of different doses of tarragon in the diet are given in Table 2. SD and 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).

Table 2
The effects of tarragon supplementation to diet on performance parameters.

S	T, g/kg feed	BWBE, g	BWEE, g	WG, g	FI, g/hen/d	FCR, kg /kg	EP, %	AEW, g	DE, %
N	0	1617	2123	506	127.0a	2.59b	73.5bc	66.8	4.42bc
	1.2	1659	2088	429	123.5b	2.38ab	78.5ab	66.4	3.77ab
	6	1621	2050	429	124.5b	2.26a	84.0a	65.9	3.07a
	12	1662	2107	445	127.5a	2.55b	76.3bc	65.8	4.17bc
H	0	1659	2133	473	115.8cd	2.57b	71.3c	63.4	4.85c
	1.2	1661	2058	398	113.8d	2.19a	79.0ab	65.9	4.02abc
	6	1629	2004	374	114.3cd	2.39ab	75.0bc	63.9	3.66ab
	12	1646	2000	354	116.5c	2.43ab	73.3bc	65.6	4.51bc
	SEM	9	15	18	1.0	0.04	0.04	0.4	0.13
S	N	1640	2092	453	125.6a	2.44	78.1a	66.2	3.86
	H	1649	2049	400	115.1b	2.40	74.6b	64.7	4.26
T, g/kg feed	0	1638	2128	490	121.4a	2.58c	72.4b	65.1	4.64c
	1.2	1660	2073	413	118.6b	2.28a	78.8a	66.2	3.90ab
	6	1625	2027	402	119.4b	2.33ab	79.5a	64.9	3.36a
	12	1654	2053	400	122.0a	2.49bc	74.8ab	65.7	4.34bc
		p-value
S		NS	NS	NS	0.01	NS	0.05	NS	NS
T		NS	NS	NS	0.01	0.01	0.01	NS	0.01
S×T		NS	NS	NS	NS	NS	NS	NS	NS

• S, stocking; N, normal; H, high, T, tarragon; BWBE, body weight at the beginning of the experiment; BWEE, Body weight at the end of the experiment; WG, weight gain; FI, feed intake; EP, egg production; AEW, average egg weight; FCR, feed conversion ratio; DE, damaged egg; NS, not significant.
• ^a-d, Values within a column with different superscripts differ significantly (Duncan’s test).

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).

Table 3
The effect of tarragon supplementation to diet on the egg shell quality parameters.

D	S	T, g/kg feed	EW, g	YH, mm	YCI	HB	SS, Kgf	ST, mm	SI	SW, g
28	N	0	65.8ab	6.3	9.7a	75.7	2.19ab	0.37ab	73.6	8.0 ab
		1.2	64.4ab	6.1	6.8bc	74.1	2.43a	0.38ab	73.4	8.0 ab
		6	65.3ab	6.1	6.6bc	74.3	2.46a	0.38ab	74.8	8.1a
		12	64.5ab	5.9	6.6bc	73.3	2.34ab	0.34ab	73.9	7.8 ab
	H	0	61.9ab	6.2	7.5bc	76.9	2.38a	0.36ab	74.4	7.7 ab
		1.2	65.4ab	6.6	7.1bc	77.5	2.42a	0.39a	73.5	7.9 ab
		6	61.3b	5.7	6.6bc	71.5	2.35ab	0.38ab	74.1	7.7 ab
		12	63.3ab	6.0	7.6bc	74.9	2.39a	0.35ab	74.3	7.7 ab
56	N	0	68.2a	5.6	7.5bc	71.7	2.49a	0.33b	73.9	7.6 ab
		1.2	65.3ab	6.4	6.8bc	76.9	2.40a	0.34ab	73.3	7.7 ab
		6	61.8ab	6.1	5.8c	73.8	2.33ab	0.35ab	73.7	7.6 ab
		12	65.2ab	5.7	6.7bc	71.7	2.37a	0.36ab	73.8	7.2b
	H	0	64.8ab	5.8	10.4a	73.4	1.97b	0.34ab	74.0	7.4 ab
		1.2	65.4ab	6.4	6.4bc	77.1	2.38a	0.36ab	73.1	8.0 ab
		6	62.1ab	5.6	5.8bc	73.1	2.43a	0.36ab	74.1	8.0 ab
		12	65.8ab	5.6	6.6bc	70.4	2.04a	0.35ab	72.9	7.7 ab
		SEM	0.5	0.1	0.2	0.8	0.03	0.01	0.2	0.1
D		28	64.0	6.1	7.3	74.8	2.37	0.37a	74.0	7.9
		56	64.8	5.9	7.1	73.5	2.35	0.35b	73.6	7.6
S		N	65.0	6.0	7.0	73.9	2.38	0.36	73.8	7.7
		H	63.7	6.0	7.4	74.3	2.34	0.36	73.8	7.7
T, g/kg feed		0	65.1	6.0	8.8a	74.4	2.26	0.35	74.0	7.7
		1.2	65.1	6.3	6.8b	76.4	2.41	0.37	73.3	7.9
		6	62.6	5.8	6.4b	73.2	2.39	0.37	73.2	7.8
		12	64.7	5.8	6.9b	72.6	2.37	0.35	73.7	7.6
		p-value
		D	NS	NS	NS	NS	NS	0.05	NS	NS
		S	NS	NS	NS	NS	NS	NS	NS	NS
		T	NS	NS	0.01	NS	NS	NS	NS	NS
		DxS	NS	NS	NS	NS	NS	NS	NS	NS
		DxT	NS	NS	NS	NS	NS	NS	NS	NS
		SxT	NS	NS	NS	NS	NS	NS	NS	NS
		DxSxT	NS	NS	0.01	NS	NS	NS	NS	NS

• D, day; S, stocking; N, normal; H, high, T, tarragon; EW, egg weight; YH, yellow height; YCI; yellow color index NS, not significant; HB, haugh birimi; SS, shell strength; ST, shell thickness; SI, shape index; SW, shell weight.^a-c,
• Values within a column with different superscripts differ significantly (Duncan’s test).

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.

Table 4
The effect of the tarragon supplementation on the blood serum CORT, IgG, TAS and TOS.

S	T, g/kg feed	CORT nmol/L	IgG mg/dL	TAS µmol/L	TOS µmol/L
N	0	43e	1.09bc	761ab	21.9d
	1.2	39e	1.13bc	776ab	19.1e
	6	39e	1.20ab	787ab	19.8de
	12	37e	1.30a	798a	18.4e
H	0	102a	1.08c	710b	30.8a
	1.2	93b	1.13bc	728ab	28.9ab
	6	71c	1.16bc	735ab	27.7bc
	12	61d	1.20ab	749ab	26.1c
SEM	SEM	3	0.01	9	0.6
S	N	40b	1.18	781a	19.8b
	H	82a	1.14	730b	28.4a
T	0	73a	1.08c	735	26.4a
	1.2	66b	1.13bc	752	24.0b
	6	55c	1.18b	761	23.7bc
	12	49d	1.25a	773	22.3c
		p-values
S		0.001	0.109	0.004	0.001
T		0.001	0.001	0.467	0.001
S×T		0.001	0.541	1.000	0.550

• S, stocking; N, normal; H, high; T, tarragon; CORT, corticosterone; TIgG, total immunoglobulin G; TAS, total antioksidant of serum; TOS, total oxidant of serum; SEM, standart error of the mean.
• ^a-e, Values within a column with different superscripts differ significantly (Duncan’s test).

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).

Table 5
The effect of the tarragon supplementation on the MDA and intestine microorganisms.

S	T, g/kg feed	SMDA mmol/L	LMDA mmol/L	EMDA mmol/L	E. coli Log, TBX	TMAB Log, PCA
N	0	3.60c	95bc	221cd	3.65bc	6.2bcd
	1.2	3.49c	94bc	218cd	3.61c	6.0cd
	6	3.50c	91bcd	216d	3.52c	5.9d
	12	3.46c	85d	211d	3.42c	5.8d
H	0	4.25a	107a	254a	4.05a	6.8a
	1.2	3.99ab	96b	248a	4.03a	6.7ab
	6	3.73bc	90bcd	237b	3.94ab	6.4abc
	12	3.79bc	88cd	227bc	3.92ab	6.4abc
	SEM	0.05	1	2	0.04	0.1
S	N	3.51b	91b	217b	3.55b	6.0b
	H	3.94a	95a	242a	3.98a	6.6a
T	0	3.92a	101a	237a	3.85	6.5a
	1.2	3.74ab	95b	233ab	3.82	6.3ab
	6	3.61b	90c	227b	3.73	6.1b
	12	3.62b	87c	219c	3.67	6.1b
		p-values
S		0.001	0.011	0.001	0.001	0.001
T		0.019	0.001	0.001	0.280	0.040
S×T		0.234	0.022	0.056	0.972	0.976

• S, stocking; N, normal; H, high; T, tarragon; STP, serum total protein; LTP, liver total protein; SMDA, serum malondialdehyde; LMDA, liver malondialdehyde; EMDA, egg malondialdehyde; TMAB, total mesophilic aerobic bacteria; SEM, standart error of the mean.
• ^a-d, Values within a column with different superscripts differ significantly (Duncan’s test).

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.

DISCUSSION

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 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 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.

There have been several studies indicating that SD affected (Onbaşılar et al., 2009), had no effect (Anderson et al., 2004; Simsek & Kilic, 2006; Fidan, 2010; Mirfendereski & Jahanian, 2015)) or improved (Sahin et al., 2007) FCR in laying hens. In the present study, HSD had no effect on FCR (p>0.05), while T1.2 and T6 diets improved FCR when compared to control diet (p<0.05). There have been studies that the incorporation of different Artemisia species into the diets of broilers improved FCR (Gholamrezaie et al., 2013; Drǎgan et al., 2014; Kheirabadi et al., 2014; Kostadinović et al., 2015) or not affected (Khalaji et al., 2011; Cherian et al., 2013; Gharetappe et al., 2015). Hosseinzadeh & Moghaddam (2014) reported that the incorporation of 0.5% tarragon into the diet affected FCR negatively, while lower levels (0.125% and 0.25%) had no effect.

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 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., 2012 Drǎgan et al., 2014; Kheirabadi 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.

ACKNOWLEDGMENT

This study was supported by Scientific Research Projects Coordinatorships of Bayburt University (Project No: 2017/01-69001-09) and Gumushane University (Project No: 17.B0121.02.01). This study was presented oraly at the 1st International Erciyes Agriculture, Animal and Food Sciences Conference, AGANFOS-2019, 24-27 April 2019, Kayseri/TURKEY. No potential conflict of interest was reported by the author(s). Thanks to the Aydın Doğan Foundation for their contribution

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Publication Dates

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

History

• Received
  30 Apr 2020
• Accepted
  12 Nov 2020