Influence of Artemisia Annua on Broiler Performance and Intestinal Microflora

ABSTRACT

The present study aimed to investigate the effect of Artemisia (Artemisia annua) supplementation as essential oil and powder, in broiler diet on performance and intestinal microflora. One hundred and eighty Cobb 500 broiler chicks assigned to three experimental groups (six replicates with 10 broilers per replicate) were housed in an environment-controlled house. Compared to the control diet, the experimental diets included 0.05 g kg^-1 Artemisia essential oil (E1), 0.05 g kg^-1 essential oil plus 0.1 g kg^-1 powder of Artemisia (E2), respectively. Growth performance was monitored throughout days 14-42. Artemisia supplementation (E1, E2) did not influence growth performance of the chicks. Compared to the C and E1, the chicks from E2 group had a lower count of Enterobacteriaceae in the intestinal and caecal content, both at 35 and at 42 days. The Artemisia supplements did not influence the staphylococci populations from the intestinal content of the chicks (42 days), but in the caecal content samples, this count was lower in E2 (8.836 log10 cfu g^-1) than in C (8.876 log10 cfu g^-1) and E1 (8.870 log10 cfu g^-1). The count of lactobacilli increased in the intestinal and caecal contents of chickens fed the diet supplemented with Artemisia at the 35^th and 42^nd day. Diet supplementation with A. annua essential oil and powder could be an effective solution in maintaining the proper microflora balance in the chicks’ intestine.

Keywords:
Artemisia annua; broilers; performance; microflora

INTRODUCTION

Poultry production is currently the most efficient animal productive system (Clavijo & Florez, 2017). However, bacterial diseases still pose a serious problem in the intensive poultry production (Adaszyńska-Skwirzyńska & Szczerbińska, 2017). The focus of alternative strategies has been to prevent proliferation of pathogenic bacteria so that the health, immune status and performance are improved (Ravindran, 2006). Given the restrictions imposed on poultry production in terms of food safety and ethical aspects of husbandry, it seems appropriate to look for the use of natural substances in animal nutrition (Christaki et al., 2012). In recent years, there has been an increased interest in biologically active plant substances added to broilers diets, especially in the European countries, Japan, and the USA (Jafari et al., 2011). The phytogenic bioactive compounds had the potential to stimulate the proliferation and growth of absorptive cells in the gastrointestinal tract and to influence the production and activity of the digestive enzymes, to improve the growth performance of birds (Vidanarachchi et al., 2005; Jang et al., 2007).

One of these alternatives is Artemisia annua L. (A. annua), an annual herb, with several medicinal uses in human diseases as powder or essential oils (Pandey & Singh, 2017). The Artemisia annua L. essential oil has antimicrobial components that could substitute the use of antibiotics in poultry production (Fretté et al., 2011). The efficacy of Artemisia essential oils may be due to the presence of chemical components with effective antimicrobial and antioxidant properties (Dadasoglu et al., 2015). In this regard, Lopez-Lutz et al. (2008) reported that the essential oil of Artemisia has inhibitory effects on the growth of some microorganisms, such as Escherichia coli, Staphylococcus epidermidis, and Staphylococcus aureus.

Previous studies showed that the supplementation of A. annua powder to broiler diets could also enhance growth performance (Dragan et al., 2010; Gholamrezaie et al., 2013). Moreover, Khalaji et al. (2011) pointed out that the inclusion of Artemisia sieberi leaves had a positive effect on broiler gut health. Based on this background, and because the literature has no studies on the effects of using a combination of Artemisia essential oil and powder in broiler chicks, we designed a study that uses a combination of A. annua essential oil and powder, to assess whether it could enhance the growth performance and balance of the intestinal microflora.

MATERIAL AND METHODS

The feeding trial was conducted in the experimental house of a research institute from Balotesti, Ilfov, Romania (44º36’46”N 26º4’43”E). The experiment was conducted according to the guidelines of the Commission of Ethics of the institute (case no. 3620/31.05.2017). A total of 180, day-old Cobb 500 broiler chicks, (average weight 39.31± 2.99 g) were obtained from a commercial hatchery and housed in an environment-controlled house (16 broilers/m² capacity). The broilers were reared on permanent wood shaves litter (10-12 cm thick). During the first 14 days, all broilers received a control diet (C) with corn, soybean meal, gluten and sunflower oil. After 14 days, the chicks were individually weighed and assigned to 3 groups (C, E1, E2) homogenous in terms of bodyweight: C (461.899 ± 8.15 g), E1 (469.265 ± 6.60 g), E2 (454.42 ± 7.46 g). Each group consisted of six replicates (10 chick/ replicate). The environmental conditions in the house were 27.02±2.79ºC air temperature, 61.05±13.55% humidity; 33.71±27.38% ventilation; 0.857±0.91 ppm NH₃ level and 0.641±0.20 ppm CO₂ level. The light regimen was adequate to broiler age, i.e. 23h light/1h darkness.

The broilers had free access to the feeds and water. Compared to the control diet C (Table 1), during the grower (14-35 days) and finisher (35-42 days) stages, the experimental diets (E1 and E2) included 0.05 g kg^-1 Artemisia annua (A. annua) essential oil (E1) or 0.05 g kg^-1essential oil plus 0.1 g kg^-1 A. annua (E2) powder. Diet formulations were calculated to meet or exceed the minimal requirements for broiler chicks, in agreement with The Management guide of Cobb 500 hybrid (2015). The following parameters were monitored throughout the experimental period (14-42 days, broiler age): bodyweight (g); average daily feed intake (g feed/broiler/day); average daily weight gain (g/broiler/day); feed conversion ratio (g feed/g gain). Mortality was recorded throughout the experimental period.

Table 1
Composition (g kg ^-1 as fed) and chemical composition of the experimental diets.

Ingredients	Grower stage (14-35 days)			Finisher stage (35-42 days)
	C	E1	E2	C	E1	E2
	g kg-1			g kg-1
Corn	620	620	610	604.5	604.5	594.6
Soybean meal	265.9	265.8	265.8	255.4	255.4	255.4
Gluten	40	40	40	60	60	60
Sunflower oil	25	25	25	32	37.2	37.2
Artemisia essential oil	-	0.05	0. 05	-	0.05	0.05
Artemisia powder	-	-	0.1	-	-	0.1
Calcium carbonate	14	14	14	13.3	13.2	13.2
Monocalcium phosphate	13.6	13.6	13.6	11.3	11.3	11.3
Salt	3.7	3.7	3.7	3.3	3.3	3.3
Methionine	2.6	2.6	2.6	2.5	2.5	2.5
Lysine	4.8	4.8	4.8	2.0	2.0	2.0
Choline	0.05	0.05	0.05	0.05	0.05	0.05
Vitamin-mineral premix*	10	10	10	10	10	10
Total	1000	1000	1000	1000	1000	1000
Calculated
Metabolisable energy, MJ**	13.598	13.598	13.598	13.003	13.003	13.003
Chemical composition
Dry matter, %	88.07	88.17	87.80	89.71	89.85	89.75
Crude protein, %	21.78	21.30	21.42	20.40	20.50	20.10
Ether extractives, %	4.80	5.04	5.10	5.60	5.64	5.72
Crude fibre, %	3.76	3.68	3.95	3.47	4.14	4.18
Ash, %	5.60	5.40	6.95	4.97	5.13	5.72
Calcium, %	0.93	0.93	0.93	0.90	0.91	0.90
Phosphorus, %	0.72	0.70	0.80	0.88	0.99	0.82
Available phosphorus, %	0.45	0.45	0.44	0.43	0.47	0.42

• *1kg premix contains: = 1100000 IU/kg vit. A; 200000 IU/kg vit. D3; 2700 IU/kg vit. E; 300 mg/kg vit. K; 200 mg/kg Vit. B1; 400 mg/kg vit. B2; 1485 mg/kg pantothenic acid; 2700 mg/kg nicotinic acid; 300 mg/kg vit. B6; 4 mg/kg Vit. B7; 100 mg/kg vit. B9; 1.8 mg/kg vit. B12; 2000 mg/kg vit. C; 8000 mg/kg manganese; 8000 mg/kg iron; 500 mg/kg copper; 6000 mg/kg zinc; 37 mg/kg cobalt; 152 mg/kg iodine; 18 mg/kg selenium.
• ** Metabolisable energy of the diet was calculated using optimization program HybriminFutter 5.

The dietary Artemisia essential oil (obtained by solid-liquid extraction) used in the experimental diets (E1, E2) was purchased from Jiangxi Xuesong Natural Medicinal Oil Co., Ltd. A. annua plant material used in the E2 diet was harvested when plants were in the late vegetative stage from Livezeni, Târgu-Mureş, Romania (46.55º N, 24.63º E). Whole plants were dried for three weeks under shade at ambient temperature (20ºC) and ground finely to obtain A. annua powder. Samples were taken from both A. annua essential oil and powder and assayed for volatile compounds. Gas chromatography (GC) coupled with a mass spectrometer was used to determinate the profile of volatile compounds of Artemisia oil and whole plant powder. Each sample was prepared and analysed as described previously by Crisan et al. (2015) using a Thermo Electron system - Focus GC chromatograph coupled with a Polaris Q ion trap mass detector, both controlled with Xcalibur^® software. DB-5MS capillary column (25 m length, 0.25 mm i.d., and 0.25 μm of film thickness) was used.

Samples were taken from each batch of compound feeds and assayed for the basic chemical composition (dry matter, crude protein, ether extractives, crude fibre, ash) and for calcium and phosphorus. These assays observed standardized methods according to Regulation (CE) no. 152/ 2009 (Methods of sampling and analysis for the official inspection of feeds): dry matter (DM) was determined by the gravimetric method, according to SR ISO 6496:2001; crude protein (CP) was determined by the Kjeldahl method, according to SR EN ISO 5983-2:2009; ether extract (EE) was determined by extraction in organic solvents, according to SR ISO 6492:2001; crude fibre (CF) was determined by successive hydrolysis in alkali and acid environment, according to SR EN ISO 6865:2002; ash (Ash) was determined by the gravimetric method, according to SR EN ISO 2171:2010; calcium was determined by the titrimetric method according to SR ISO 6490-1/1996, phosphorus was determined by the spectrophotometric method according to SR ISO 6491:1983.

According to the protocol approved by the Commission of Ethics of the institute, at 35 days (grower stage) and 42 days (finisher stage), six broilers (with live weight similar to the average bodyweight of the group) from each group were slaughtered by cervical dislocation and immediately bled. Carcasses were eviscerated manually and the gastrointestinal tract was excised. Small intestinal (duodenum, jejunum, ileum) and caecal contents (2 caeca per bird) were collected aseptically in sterilized plastic tubes and preserved at -20ºC until the bacteriological analyses (Enterobacteriaceae, E. coli, lactobacilli, staphylococci, Salmonella spp). The samples were prepared and analysed as described previously by Criste et al. (2017). The results were expressed as log base 10 colony-forming units (cfu) per gram of intestinal/caecal contents. The colony forming units from Enterobacteriaceae, E. coli, staphylococci and lactobacilli was determined by a colony counter (Scan 300, Interscience France).

The effects of treatments were tested by analysis of variance using the GLM procedure of the Minitab software (version 17, Minitab^® Statistical Software), with treatment as fixed effect. When overall F-test was significant, the differences between means were declared significant at p<0.05 using the test of Tukey.

RESULTS

Table 2 shows the composition of volatile compounds in the A. annua essential oil and powder. The main volatile compounds in Artemisia oil are the monoterpenes, dominated by eucalyptol (29.17%) and α-pinene (11.32%). Ketones were the second major class of compounds observed in A. annua essential oil, including camphor (9.38%) and Artemisia ketone (7.38%).

Table 2
Volatile compounds (%) determined in the Artemisia annua essential oil and powder used in broiler diets.

Compound	CAS	Essential oil, (%)	Powder, (%)
α-Pinene	80-56-8	11.32	11.95
Camphene	79-92-5	2.83	22.52
Sabinene	3387-41-5	3.04	0.88
β-Pinene	127-91-3	4.52	3.55
Yomogi alcohol	30458-12-9	1.45	ND
α-Terpinene	99-86-5	1.18	ND
p-Cymene	99-87-6	1.28	3.07
Eucalyptol	470-82-6	29.17	9.28
Artemisia ketone	546-49-6	7.38	1.33
Trans- sabinene hydrate	17699-16-0	ND	0.53
Artemisia alcohol	27644-04-8	1.19	ND
α-Terpinolene	586-62-9	0.57	ND
trans-Pinocarveol	547-61-5	3.16	ND
Camphor	76-22-2	9.75	46.21
Borneol	507-70-7	ND	0.68
Pinocarvone	34-41-3	2.98	ND
Terpinen-4-ol	562-74-3	3.63	ND
α-Terpineol	98-55-5	1.13	ND
Myrtenal	564-94-3	1.11	ND
Pulegone	89-82-7	0.45	ND
Thymol	89-83-8	0.56	ND
α-Terpineol acetate	80-26-2	0.28	ND
Eugenol	97-53-0	0.35	ND
Butyl benzoate	136-60-7	0.20	ND
α-Copaene	3856-25-5	1.15	ND
Caryophyllene<Z>	118-65-0	4.37	ND
Caryophyllene<E>	87-44-5	0.80	ND
Humulene	6753-98-6	0.26	ND
γ-Muurolene	30021-74-0	3.38	ND
α-Selinene	473-13-2	1.23	ND
α-Muurolene	31983-22-9	0.72	ND
δ-Cadinene	483-76-1	0.31	ND
Caryophyllene oxide	1139-30-6	0.24	ND
Total		100	100

• Where: CAS: Chemical Abstract Service; ND: Not detected.

Table 3 shows the effects of Artemisia oil and combination of Artemisia essential oil and powder on broiler’s performance. There were no significant differences (p>0.05) throughout the experimental period among the three groups in terms of broiler body weight.

Table 3
Effect of Artemisia annua (essential oil/ essential oil and powder) on broiler performance (14-42 days).

Item	Period (days)	C	E1	E2	SEM	p-value
Body weight (g/broiler)	14	461.9	469.26	454.42	4.288	0.371
	35	2165.51	2200.2	2124.87	17.922	0.231
	42	2634.77	2727.89	2667.5	23.553	0.256
Average daily feed intake (g/broiler/day)	14-35	131.42	138.47	128.86	4.213	0.617
	35-42	165.58	173.38	158.14	3.253	0.230
	14-42	139.68	146.81	135.95	0.360	0.465
Average daily weight gain (g/broiler/day)	14-35	81.12	82.42	79.54	0.793	0.264
	35-42	67.04	75.38	77.52	3.615	0.505
	14-42	77.60	80.66	79.04	0.859	0.309
Feed conversion ratio (kg CF/kg gain)	14-35	1.62	1.68	1.63	0.140	0.109
	35-42	2.46	2.30	2.19	0.127	0.525
	14-42	1.80	1.82	1.73	0.022	0.223

• Where: SEM: standard error of the mean.

Nevertheless, it can be noticed that both at 35 days, and at 42 days, the broilers treated with A. annua essential oil (E1) had a higher body weight (p>0.05) than the other two groups (C and E2). Also, according to Table 3 data, during the period 14-42 days, the average of daily feed intake and the average of daily weight gain of E1 broilers were higher than in groups C and E2 (p>0.05), but the difference was not statistically significant. The broilers treated with the mixture of A. annua essential oil and powder (E2) had a better feed conversion ratio (Table 3) than those which received the conventional diet and E1 diet, but not statistically significant (p>0.05).

The lowest number of E. coli colony-forming units was detected in the intestinal content of E2 broilers, lower (p<0.05) than in C and E1 (Table 4). The Enterobacteriaceae count in the intestinal content, both at 35 and 42 days (Table 5) was lower (p<0.05) in E1 broilers than in C broilers. Similarly, to the data concerning the intestinal content, the Enterobacteriaceae count was lower (p< 0.05), at 35 days, in groupE2 compared to C.

Table 4
Effect of Artemisia annua supplements (essential oil/ essential oil and powder) to broiler diets on the intestinal and caecal microflora at 35 days (log₁₀cfug^-1intestinal or caecal content).

Item	C	E1	E2	SEM	p-value
Small intestine content
Enterobacteriaceae, log10	7.26a	7.25b	7.22c	0.01	<0.001
Escherichia coli, log 10	5.89a	5.89a	5.82b	0.01	<0.001
Staphylococci, log10	5.61a	5.86b	5.76c	0.03	<0.001
Lactobacilli, log 10	6.42a	6.93b	7.06c	0.07	<0.001
Salmonella spp.	ND	ND	ND	-	-
Caecal content
Enterobacteriaceae, log10	11.35a	11.16b	11.06c	0.03	<0.001
Escherichia coli, log 10	9.93a	9.96b	9.92a	0.01	0.002
Staphylococci, log10	8.64a	8.78a	8.54a	0.07	0.367
Lactobacilli, log 10	11.41a	11.40b	11.43c	0.04	<0.001
Salmonella spp.	ND	ND	ND	-	-

• Where: cfu=colony-forming units; SEM: standard error of the mean; ND: Not detected; n=6;
• ^a,b,c Mean values within a row having different superscripts are significantly different by least significant difference test (p<0.05).

Table 5
Effect of Artemisia annua supplements (essential oil/ essential oil and powder) to broiler diets on the intestinal and caecal microflora at 42 days (log₁₀cfug^-1 intestinal or caecal content).

Item	C	E1	E2	SEM	P-value
Small intestine content
Enterobacteriaceae, log10	7.35a	7.33b	7.32c	0.01	<0.001
Escherichia coli, log 10	6.02a	5.99b	5.95c	0.04	<0.001
Staphylococci, log10	5.94a	5.95a	5.95a	0.04	0.121
Lactobacilli, log 10	7.03a	7.01b	7.37c	0.02	<0.001
Salmonella spp.	ND	ND	ND	-	-
Caecal content
Enterobacteriaceae, log10	11.32a	11.27b	11.21c	0.01	<0.001
Escherichia coli, log 10	10.02a	10.00b	9.96c	0.01	<0.001
Staphylococci, log10	8.88a	8.87a	8.84b	0.04	<0.001
Lactobacilli, log 10	11.30a	11.47b	12.41c	0.09	<0.001
Salmonella spp.	ND	ND	ND	-	-

• Where: cfu=colony-forming units; SEM: standard error of the mean; ND: Not detected; n=6;
• ^a,b,c Mean values within a row having different superscripts are significantly different by least significant difference test (p< 0.05).

At 35 days, the caecal count of E. coli decreased (p<0.05) in E2 compared to E1, but it was not significantly (p>0.05) different from C.

The number of staphylococci colony-forming units was higher (p<0.05) in the intestinal content of the experimental groups (E1 and E2) compared to the control group (Table 4). On the contrary, a lower (p>0.05) staphylococci count was determined in the caecal content of E2 broilers (Table 4). Both in the samples of intestinal and caecal content collected at 35 days, the lactobacilli count was higher (p<0.05) in E2 broilers than in E1 and C broilers. Moreover, the lactobacilli populations from the intestinal content of E2 broilers were 10% higher than in C and 1.9% higher than in E1.

At 42 days, the Enterobacteriaceae and E. coli counts were lower (p<0.05) in the intestinal and caecal contents of E1 and E2 broilers compared to C broilers (Table 5).

While no significant differences (p>0.05) were recorded in terms of the staphylococci count from the small intestine among groups, the caecal staphylococci populations were lower (p<0.05) in E2 broilers compared to C and E1 broilers.

The A. annua supplements to broiler diets had a positive influence on the lactobacilli count from the intestinal content. Thus, the lactobacilli count was higher (p<0.05) in E1 (7.098 log10 cfug^-1 intestinal content) and E2 (7.372 log10 cfug^-1 intestinal content) than in C (7.028 log10 cfug^-1 intestinal content). Similar results were reported for the samples of caecal content. Salmonella spp. were absent in all samples of intestinal and caecal content.

DISCUSSION

The characterisation of A. annua essential oil (Table 2) shows a high content in volatile compounds. As table 2 data shows, a higher number of compounds (31 compounds) were detected in the A. annua essential oil than in the powder (10 compounds). A higher number of monoterpenes were detected in A. annua powder, especially: camphor (46.21%), camphene (22.52%), α-pinene (11.95%), eucalyptol (9.28%). These monoterpenes are well-known chemicals for their pronounced antimicrobial properties. This profile of volatile compounds detected in the plant powder supports its use as phytoadditive with effect on broiler growth performance and gut health assurance. Perazzo et al. (2003) reported that A. annua plant included camphor, β-cubebene and trans-caryophyllene. On the other hand, Nezhadali & Parsa (2010) detected 72 compounds in Artemisia absinthium leaves, including camphor (14.83%), p-cymene (10.35%), isoledene (8.52%), caryophyllene (6.92%). The concentration of volatile compounds in Artemisia depends on the plant material (cultivar, variety), age, growing conditions, as well as on the seasonal and geographical variations (Olsson et al., 2009). Factors such as temperature changes and stress treatments can be applied to affect the content of secondary metabolites of plants (Malik et al., 2009). However, as Marinas et al. (2015) shows, the essential oil from A. annua plant grows in Romania, had contained camphor (17.74%), α-pinene (9.66%), germacrene D (7.55%), 1,8-cineole (7.24%), α-caryophyllene (7.02%), artemisia ketone (6.26%) as main compounds. In Bosnia, Artemisia ketone (30.7%) and camphor (15.8%) are the major constituents in the essential oil of A. annua (Cavar et al., 2012). Radulovic et al. (2013) in a characterization study of A. annua essential oil from Serbia, have shown that it contains Artemisia ketone (35.7%), alpha-pinene (16.5%), 1,8-cineole (5.5%). Different from it, Kazemi (2015) identified in an oil from Iran, α-Pinene (7.33%), camphene (5.68%), sabinene (4.78%), α-myrcene (22.41%), 1,8-cineole (17.17%), camphor (20.41%). The composition of essential oils from the same species varied depending on the different geographical origin (Tzenkova et al., 2010; Verdian-Rizi Mohammadreza, 2008; Pandey & Singh, 2017). Variation in the volatile components of these plants may occur during plant ontogeny or growth at different altitudes (Pandey & Singh, 2017). The strong antimicrobial activity of A. annua (Atta-Ur-Rahman, 2008) is due to the high content in monoterpene hydrocarbons.

The supplement of A. annua did not influence broiler performance, as seen from Table 3. Neither the plant, nor the essential oil of A. annua did not improve broiler appetite, although feed intake was higher in the group treated with A. annua essential oil (E1) than in the other two groups, but the difference was not statistically significant. It is noteworthy that the performance of E2 broilers (mixture of A. annua essential oil and powder) was comparable with that of E1 broilers, which could be due to the rather low dietary essential oil level (0.05 g kg^-1) knowing that the action of this phytoadditive depends on the level of inclusion in the diet. However, the combination of Artemisia essential oil and powder (group E2), generally produced lower values of the average daily feed intake both in groups C and E1, but the differences are not statistically significant (p>0.05). In the present work, the higher average of daily feed intake and the higher average of daily weight gain of E1 broilers substantiate the statements of Windisch et al. (2008) and Grashorn (2010), that phytobiotics, especially those from the group of essential oils, have been reported to improve the flavour and palatability of the feed. The data from table 3 were consistent with those reported by Khalaji et al. (2011). They showed that the supplementation of 0.1 g kg^-1 Artemisia sieberi leaves in Ross 308 chicken diets did not affect the body weight and feed conversion ratio of broilers during the entire experimental period (1-42 days). Broiler weight (42 days) reported by Habibi et al. (2016) who used Ross 308 broilers treated with diets which included 100, 200, 300 mg/kg A. absinthium oil were 14.06%, 12.88%, and 14.70% lower, respectively, compared to the results of our study, while the expected difference among the two hybrids is about 1.71% (according to Management Guide of Cobb 500 Hybrid and Management Guide of Ross 308 Hybrid). Pop et al. (2017) noticed that the use of lower concentrations of artemisinin (5 ppm) in Ross 308 broilers (1-28 days) increased the average daily feed intake, the average daily weight gain and improved the feed conversion ratio, while the higher concentration of artemisinin (500 ppm) caused reduced weight gain, inefficient feed conversion, and a lower feed intake.

Several researchers have shown that the essential oils act against Gram-negative bacteria, such as Campylobacter jejuni, Escherichia coli, Mycoplasma gallisepticum, Mycoplasma synoviae, Pseudomonas aeruginosa, Salmonella enteridis, or Klebsiella sp. (Solorzano-Santos & Miranda-Novales, 2012; Kurekci et al., 2013; Nimbarte et al., 2013; Alali et al., 2013; Cerisuelo et al., 2014; Zengin & Baysal, 2014). The results of the present work (Tables 4 and 5) were in agreement with those. The dietary A. annua essential oil decreased (p<0.05) the Enterobacteriaceae and E. coli populations in the intestinal content, both at 35 days and at 42 days. This beneficial action could be due to the higher concentration of terpenoids, including monoterpenes in the A. annua essential oil (Table 2). These compounds are well-known to cause membrane disruption in microorganisms by the action of lipophilic compounds in the oils, thereby impairing the antibacterial activity (Sahoo et al., 2012). Khalaji et al. (2011) and Ghazanfari et al. (2015) reported that Artemisia sieberi leaves (1%), and oil (300 mgkg^-1) given to Ross 308 broilers (1-42 days) reduced (p<0.05) the caecal E. coli populations. On the contrary, Saracila et al. (2018) showed that broilers (14-35 days) treated with Artemisia oil (0.05 g kg^-1) had a significantly higher number of Enterobacteriaceae and E. coli in the caecal content than those treated with mix of Artemisia oil and powder. In addition, there are studies that highlight the action of essential oils against Gram-positive bacteria: Bacillus cereus, Bacillus subtilis, Clostridium colinum, Clostridium septicum, Listeria monocytogenes, Staphylococcus aureus, or Streptococcus gallolyticus (Jerzsele et al., 2012; Muthayian et al., 2012; Solorzano-Santos & Miranda-Novales, 2012; Nimbarte & Kulkarni, 2013; Zengin & Baysal, 2014).

The balance between the count of intestinal beneficial and ”bad’’ bacteria (at least 85% of the total bacterial count should be beneficial) is vital for the host, and the impact on gut health often comes from bacterial imbalance in broiler gut (Choct, 2009). Lactobacilli produce a wide range of short-chain fatty acids (SCFAs), which have bacteriostatic activity on some bacterial strains, either directly, or by reducing the intestinal pH; they produce bacteriocins which have microbiocid or microbiostatic properties and thereby contribute to the resistance to pathogens by modifying the receptors they use (Adil & Magray, 2012; Rinttila & Apajalahti, 2013). This microbial imbalance is maximized when antibiotics are withdrawn from the feed (Choct, 2009). It is possible to manipulate nutritionally the intestinal microbial population, concomitantly with the increase of the number of beneficial bacteria in broiler gut (Adil & Magray, 2012).

The addition of A. annua (essential oil and powder) to broiler diets determined the multiplication of lactobacilli populations in the intestinal and caecal contents at 35 and 42 days. Similar results were reported by Ghazanfari et al. (2015), using A. sieberi oil (300 mgkg^-1) given to Ross 308 broilers (1-42 days). On the contrary, Khalaji et al. (2011) showed that Artemisia sieberi leaves (1%) given to Ross 308 broilers had no effect on the caecal lactobacilli populations. The increase of the lactobacilli populations in the intestine of broilers treated with A. annua (essential oil and powder) diet maintained the balance between the bacteria colonizing the gastrointestinal tract of the broilers.

CONCLUSIONS

The dietary supplementation of A. annua (essential oil or combination of essential oil and powder) did not influence broiler performance (14-42 days).

Supplementing A. annua as a combination of essential oil and powder to broiler diets can improve the balance of gut microflora (as measured by changes in populations of Escherichia coli and Lactobacillus).

Both at 35 and at 42 days, the broilers treated with the combination of A. annua essential oil and powder had the lowest count of Enterobacteriaceae in the intestinal and caecal content from all groups. The number of lactobacilli colony-forming units was higher, in the intestinal and caecal contents of chickens (on days 35 and 42), fed the diet supplemented with A. annua (essential oil and powder).

ACKNOWLEDGEMENTS

This work was supported by a grant of the Romanian Ministry of Education and Research (Project PN 18 20 0102).

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Publication Dates

• Publication in this collection
  20 Dec 2019
• Date of issue
  2019

History

• Received
  05 July 2019
• Accepted
  24 Sept 2019