Effect of Leuconostoc mesenteroides KCCM35046 fermented aged garlic extract on egg production, egg quality, odour gas emissions, targeted E. coli colony, haematological characteristics and fatty acids composition of egg yolk in laying hens

ABSTRACT

The design of this study was to investigate the impacts of dietary supplementation with aged garlic extract (AGE) by Leuconostoc mesenteroides KCCM35046 on egg production, egg quality, odour gas emissions from excreta, excreta microbiota, haematological characteristics and fatty acids composition of egg yolk in laying hens. A total of 384, 54-week (ISA-brown) laying hens were randomly assigned to one of four treatments with eight replications per treatment and 12 adjacent cages as a replication. The experimental treatments were: (1) CON; basal diet, (2) AGE.05; CON + .05% aged garlic extract, (3) AGE.1; CON + .1% aged garlic extract, (4) AGE.2; CON + .2% aged garlic extract. No significant differences were observed in feed intake (FI) and egg production among treatments (P > .05). Laying hens fed with the AGE linearly improved yolk height at Week 3 (P = .02). The inclusion of .1% AGE had increased Haughunit (HU) at Week 4 (quadratic, P = .01). At the end of the experiment, AGE diet linearly increased yolk height and egg shell thickness (P = .01 and .03, respectively). No significant differences were observed in excreta gas emission (P > .05). The supplementation of AGE linearly reduced excreta E. coli concentrations (P = .02). Inclusion of AGE led to a greater lymphocyte and IgG concentrations (linear, P = .04 and P = .03, respectively), whereas blood total cholesterol (TC) was reduced by the AGE supplementation (linear, P < .01). PUFA: SFA was increased in AGE treatment at the end of the experiment (P < .05). In conclusion, the administration of AGE at a level of .1 and/or .2% can improve egg quality, blood lymphocyte, IgG concentrations and reduced excreta E. coli and blood TC in the laying hens.

KEYWORDS:

• Aged garlic extract
• blood cholesterol
• E. coli
• Leuconostoc mesenteroides KCCM35046
• PUFA: SFA

1. Introduction

Garlic (Allium sativum) has been used in many countries as spice and folk medicine for thousands of years (Rivlin 2001). Investigations have shown that garlic plays an important pharmacological role as an antibacterial, antiviral, antifungal agent as well as a remedy for intestinal disorders, flatulence, worms, respiratory infections, skin diseases, wounds, symptoms of ageing and many other ailments (Amagase et al. 2001). Moreover, garlic has several beneficial effects for either human or animals having antioxidant, antihypertensive properties (Sivam 2001) as well as prevents heart disease and cancer and diabetes (Konjufca et al. 1997; Amagase et al. 2001). It has also been reported that fresh dietary garlic can help to improve the immune stimulatory effect during Newcastle disease, avian influenza and bursal disease challenge in birds (Jafari et al. 2010). Sharma et al. (1979) observed that egg yolk cholesterol was decreased by 1 or 3% garlic powder supplementation to diets of laying hens for three weeks. Chowdhury et al. (2002) observed that garlic had no effect on egg production but significantly reduced serum cholesterol. However, those positive effects were mainly attributed to the bioactive components of garlic, including sulphur-containing compounds such as alliin and diallyl sulfides (Amagase et al. 2001), which may be altered during processing.

A fermentation could result out several advantages like as improves flavour, with desirable metabolites produced by the micro-organisms and enhance safety (Buckenhüskes et al. 1990). Results of previous studies have shown that supplementation of fermented garlic powder could benefit poultry and swine. Fermented garlic powder (2 g/kg) supplementation to the basal diet can increase average daily gain and average daily feed intake in finishing pigs up to six weeks of feeding (Yan et al. 2011). Moreover, Wang et al. (2011) observed that E. coli counts were significantly reduced by increasing amounts of fermented garlic by Weissella koreensis fed to growing pigs. Our previous studies (Wang & Kim 2011; Yan & Kim 2012) on herbs and fermented garlic powder indicated that intestinal microbiota and nutrient utilization influenced the faecal noxious gas emissions in animals. Thus, we have been focused on the impact of AGE on excreta microbiota and noxious gas emissions in laying hens in the current study. Moreover, fermented garlic powder increased white blood cells and lymphocyte counts but had no significant effect on body weight gain and feed conversion ratio in broilers (Ao et al. 2010). Therefore, based on the previous studies we hypothesize that aged garlic extract by Leuconostoc mesenteroides KCCM35046 supplementation can improve the performance and immune status in laying hens.

Since their modes of action are not fully understood and due to different processing methods, further scientific investigation is needed. To the best of our knowledge, effects of aged garlic extract by Leuconostoc mesenteroides KCCM35046 on laying hens have not been investigated yet now. Thereupon, the study was put to work to assess if supplementation of Leuconostoc mesenteroides KCCM35046 fermented aged garlic extract may exert conclusive effects on production performance, egg quality, odour gas emissions from excreta, targeted E. coli colony, haematological characteristics and fatty acids composition of egg yolk in laying hens.

2. Materials and methods

The Animal Care and Use Committee of Dankook University approved all of the experimental protocols used in the current study.

2.1. Animals and diets

A total of 384, 52-week-old (ISA-brown) laying hens were used in this five-week trial. Birds were randomly assigned to one of four treatments with eight replications (12 hens per replication). The hens were individually caged and provided with 16 h of light daily. The replications were allotted equally into the upper and lower cages to minimize the effects of cage level. The hens were housed in a windowless laying house at approximately 21°C. They were allowed to adjust to the environment for four days prior to the start of the experiment, during which the hens were fed on a control (CON) diet. All diets used in the present study were formulated to meet or exceed the nutrient recommendations of the NRC (1994). The diets were supplemented with 0 (control), .05, .10 or .2% aged garlic extract in powder form. All cages were equipped with nipple drinkers and common trough feed, experimental feed and water were provided ad libitum throughout the experimental period (Table 1). All diets were grounded through 1 mm screen in a Wiley mill before analysing for crude protein (CP), lysine, P and Ca (AOAC 2000).

Table 1. Basal diet composition (as-fed basis).

Ingredients	Percentage
Corn	50.40
Soybean meal (CP 46%)	18.70
Wheat grain	10.00
Corn gluten meal	2.00
Wheat bran	5.00
Animal fat	4.40
Limestone	7.50
Dicalcium phosphate (P 18%)	1.40
Salt	.30
DL-Met (50%)	.10
Vitamin premix^a	.10
Trace mineral premix^b	.10
Total	100
Calculated values (%)
ME (kcal/kg)	2904
CP	15.02
Ca	3.25
Available P	.38
Digestible lysine	.66
Digestible Met + Cys	.56
Digestible methionine	.38
Digestible threonine	.44
Digestible tryptophan	.16
Analysed values (%)
CP	15.00
Lysine	.74
Ca	3.23
P	.60

^aProvided per kilogram of premix: 125,000 IU retinylpalmitate; 2500 IU cholecalciferol; 10 mg α-tocopherol acetate; 2 mg menadfione sodium bisulfite; 1 mg thiamine; 5 mg riboflavin; 1 mg vitamin pyridoxine; 15 mg cobalamine; 500 mg folic acid; 35,000 mg niacin; 10,000 mg Ca-pantothenate and 50 mg biotin.

^bProvided per kilogram of diet: 8 mg Mn (as MnO₂); 60 mg Zn (as ZnSO4); 5 mg Cu (as CuSO₄·5H₂O); 40 mg Fe (as FeSO₄·7H₂O); .3 mg Co (as CoSO₄·5H₂O); 1.5 mg I (as KI) and .15 mg Se (as Na₂SeO₃·5H₂O).

2.2. Preparation of aged garlic extract

The AGE (40% garlic, 40% dextrin and 20% wheat powder) used in our study was provided by Eaglevet., Seongdong-gu, Seoul, Republic of Korea and the primary compositions of AGE had been analysed by the supplier, including 749.59 mg/kg alliin, 105.27 mg/kg allylmethyltrisulphide (AMTS), 33.23 mg/kg diallyltrisulphide (DATS), 4.49 mg/kgdiallyldisulphide (DADS) and .80 mg/kg 1, 2-VDT, 3-vinyl-[4H]-1,2-dithiin (1,2-VDT). In brief, after cracking the bulbs of garlic to separate the cloves, the defective and smallest cloves were discarded. The remaining cloves were peeled, washed with tap water and divided into two portions. The mixture of garlic and water (1:1; vol/vol) was filtered through a 45-μm sieve and the filtrate was then centrifuged (1000g for 40 min at 26°C) to get the suspension. After that, the suspension was inoculated with a starter culture of .10% Leuconostoc mesenteroides KCCM35046 (1.0 × 10⁷cfu/mL) in a room maintained at 25°C for 24 h.

2.3. Sampling and measurements

Daily records of egg production and feed intake were kept throughout the experimental period. The average daily feed intake and egg production were calculated during whole period of trial weekly. Egg production was expressed as an average production of hen per day, which was calculated from the total number of eggs divided by the number of experimental time (day as a unit) and summarized on an average basis. A total of 32 saleable eggs (no shell defects, cracks or double yolks) were randomly collected at 1700 h from each treatment (four per replicate, n = 32) on a weekly basis to determine the egg quality at 2000 h the same day. Eggshell colour was determined using a colour fan. Eggshell breaking strength was evaluated using a model II egg shell force gauge (Robotmation Co. Ltd, Tokyo, Japan). A dial pipe gauge (Ozaki MFG Co. Ltd, Tokyo, Japan) was used to measure egg shell thickness, which was determined based on the average thickness of the rounded end, pointed end and the middle of the egg, excluding the inner membrane. Finally, egg weight, egg yolk height, egg yolk colour and Haugh Unit (HU) were measured using an egg multi-tester (Touhoku Rhythm Co. Ltd, Tokyo, Japan).

At the end of the experiment, excreta samples were collected from six layers randomly selected from each replicate, then pooled and placed on ice for transportation to the laboratory, where analysis was immediately performed by the method of Wang and Kim (2011). One gram sample pooled was diluted 1:9 (wt/vol) in sterile saline. All samples were subjected to 10 sequential dilutions (mL) of 1% peptone broth 1:9 (vol/vol). Then serial dilution (10⁻⁴ to 10⁻⁷) of digesta samples was made using anaerobic diluents and placed on MacConkey agar plates (Difco Laboratories, Detroit, MI, USA) and lactobacilli medium III agar plates (Medium 638, DSMZ, Braunschweig, Germany) to isolate the E. coli and Lactobacillus, respectively. The lactobacilli medium III agar plates were then incubated for 48 h at 39°C under anaerobic conditions. The MacConkey agar plates were incubated for 24 h at 37°C. The E. coli and Lactobacillus colonies were counted immediately after removal from the incubator. The colonies appeared as large dome-shaped colonies with a double zone of haemolysis.

At the end of the experiment, fresh excreta samples were collected from each replication (12 hens per replication) for the analysis of noxious gas emission according to the method described by Cho et al. (2008) and Zhang and Kim (2013). A total of 300 g fresh excreta samples were stored in 2.6 L plastic boxes for replicates. The samples were fermented for five days at room temperature (28°C). After the fermentation period, the gases that formed were analysed using a Gastec (model GV-100) gas sampling pump (Gastec Corp., Gastec detector tube No. 3L and 3La for NH₃; No. 4LL and 4LK for H₂S; No. 70 and 70L for R.SH, Gastec Corp., detector tube, Japan). Prior to measurement, the excreta samples were manually shaken for approximately 30 s to disrupt any crust formation on the surface of the sample and to homogenize them. The adhesive plaster was then punctured and 100 ml of headspace air was sampled from approximately 2.0 cm above the sample surface.

At the end of the experiment, six layers randomly selected from each replicate and blood samples were then collected from the wing vein using a sterilized syringe with needle. Then the samples were transferred to either K3EDTA vacuum tubes or clot activator vaccum tubes (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ, USA). For serum analysis, the blood samples were centrifuged at 2000g at 4°C for 20 min to separate the serum. The IgG and total cholesterol (TC) in the serum were then analysed using an automatic biochemistry blood analyser (HITACHI747, Tokyo, Japan). Whole blood samples from the K₃EDTA vacuum tube were analysed to determine the WBC, RBC and lymphocyte concentrations using an automatic blood analyser (ADVIA 120, Bayer, Tarrytown, NY, USA) according to the method described by Yan et al. (2011) and Yan and Kim (2012).

In terms of fatty acids, 16 eggs of each treatment were collected randomly and cracked. The yolks were separated and divided into five samples. Samples were homogenized using an IKA Ultra Turrax T25, freeze-dried and stored at −20°C until analysis for fatty acid (FA) composition. Yolks were processed according to the method described by Folch et al. (1957) for the extraction of FA. Quantification of FA was conducted after the preparation of FA methyl esters (FAME) with 10% boron trifluoride methanol complex in methanolic solution. The fatty acid contents were determined using a HP6890 gas chromatograph (Agilent, Waldbronn, Germany) equipped with a flame ionization detector and an HP 19091 to 136 capillary column with a film thickness (.25 μm) of stationary phase. Helium was employed as the carrier gas. The oven temperature was programmed as follows: from140 to 160°C at 1.5°C/min; from 160 to 180°C at .50°C/min; and from 180 to 230°C at 2.50°C/min. The other chromatographic conditions were: injector and detector temperatures, 280°C; sample volume injected, 1 μL. Fatty acids were identified by matching their retention times to those of their relative standards, as well as by mass spectrometry (HP5973; Agilent, Waldbronn, Germany) of each peak.

2.4. Statistical analysis

Data were statistically analysed via ANOVA using the GLM procedure in the SAS (SAS Institute 2013) software package, version 9.4 for a completely randomized design. Differences among all treatments were separated by Tukey's range test. Before conducting statistical analysis of the microbial counts, the value was transformed logarithmically. The linear and quadratic effects of aged garlic among treatments were analysed using a contrast statement. The mean values and standard error (SE) were reported. Probability values of less than .05 were considered significant.

3. Results

3.1. Feed intake, egg production and egg quality

No significant differences were observed in feed intake and egg production in laying (Table 2). However, diets with aged garlic extract numerically (P > .05) increased the egg production in the whole experiment compared to basal diet. Laying hens fed the AGE.2 diet linearly increased yolk height (P = .01, P = .02 and P = .01 for Weeks 3, 4 and 5, respectively (Table 3). Haugh unit was increased in AGE.1 diet at Week 4 (quadratic, P = .01). During Weeks 4 and 5, laying hens fed the AGE.2 diet showed linearly higher eggshell thickness (P = .04 and .03, respectively). Moreover, laying hens fed the AGE.2 diet linearly increased HU at Week 5 (P = .04).

Table 2. The effects of aged garlic extract (AGE) supplementation on feed intake and egg production in laying hens.^a

Items	Diet					P-value^c
	CON	AGE.05	AGE.1	AGE.2	SE^b	T	L	Q
Feed intake
1 week, g	787	791	797	788	11.63	.32	.21	.13
2 weeks, g	784	792	800	795	6.27	.27	1.00	.45
3 weeks, g	793	789	794	784	8.36	.85	.09	.64
4 weeks, g	794	795	799	794	6.27	.12	.30	.47
5 weeks, g	795	795	793	797	5.01	.46	.11	.31
Egg production
1 week, %	90.41	90.81	92.91	91.80	<.01	.77	.08	.91
2 weeks, %	89.84	90.03	91.09	92.54	1.22	.15	.71	.42
3 weeks, %	91.16	91.32	92.02	92.87	.01	.96	.54	.07
4 weeks, %	89.90	90.99	91.43	91.99	<.01	.75	.64	.34
5 weeks, %	89.97	91.50	91.08	92.51	3.42	.81	1.01	.75

^aAbbreviations: CON, basal diet; AGE.05, CON + .05% aged garlic extract; AGE.1, CON + .1% aged garlic extract; AGE.2, CON + .2% aged garlic extract.

^bStandard error.

^cT represents the P-values of Tukey's range test; L and Q represent the P-values of Linear and Quadratic (polynomial contrasts) effect, respectively.

Table 3. The effects of aged garlic extract (AGE) supplementation on egg quality in laying hens.^a

Items	Diet					P-value^c
	CON	AGE.05	AGE.1	AGE.2	SE^b	T	L	Q
1 week
Eggshell colour	11.50	11.55	11.65	11.65	.32	.44	.42	.71
Egg weight, g	62.45	62.16	62.33	63.02	3.91	.36	.34	.55
Yolk height, mm	9.28	9.34	9.19	9.30	.51	.18	.79	.32
Yolk colour	9.00	8.95	9.00	9.05	.11	.48	.09	.45
Haugh Unit	94.22	94.67	94.43	94.50	3.52	.96	.37	.24
Eggshell strength, kg/cm^2	4.37	4.47	4.42	4.41	.18	.49	.91	.95
Eggshell thickness, mm^−2	40.44	40.75	40.62	40.49	.10	.65	.10	.13
2 weeks
Eggshell colour	12.00	12.20	12.25	12.20	.31	.21	.54	.95
Egg weight, g	63.30	63.41	63.72	63.52	1.68	.37	.34	.16
Yolk height, mm	9.30	9.29	9.35	9.35	.28	.14	.61	.56
Yolk colour	9.00	8.85	9.01	8.95	.11	.58	.12	.34
Haugh Unit	93.82	94.31	94.61	94.76	2.64	.26	.74	.27
Eggshell strength, kg/cm^2	4.40	4.41	4.42	4.43	.22	.83	1.01	.24
Eggshell thickness, mm^−2	40.50	40.62	40.49	40.64	.12	.58	.39	.84
3 weeks
Eggshell colour	11.95	12.10	12.21	12.10	.33	.82	.14	.58
Egg weight, g	62.14	63.25	63.60	63.78	3.67	.18	.51	.24
Yolk height, mm	9.17	9.38	9.47	9.49	.06	.02	.01	.52
Yolk colour	9.00	8.87	9.00	9.03	1.12	.34	.61	.09
Haugh Unit	93.76	94.01	94.31	94.41	5.44	.45	.24	.30
Eggshell strength, kg/cm^2	4.37	4.39	4.41	4.40	.06	.52	.31	.68
Eggshell thickness, mm^−2	40.52	40.67	40.69	40.71	1.26	.36	.42	.64
4 weeks
Eggshell colour	12.05	12.20	12.20	12.10	.31	.75	.71	.65
Egg weight, g	63.50	63.96	64.11	64.20	2.40	.28	.25	.64
Yolk height, mm	9.18	9.29	9.40	9.47	.06	.04	.02	.69
Yolk colour	9.05	9.05	8.95	9.00	.11	.74	.94	.85
Haugh Unit	93.99	95.85	96.22	96.08	.68	.01	.24	.01
Eggshell strength, kg/cm^2	4.40	4.43	4.42	4.43	.06	.43	.41	.42
Eggshell thickness, mm^−2	40.26	40.60	41.12	41.75	.20	.03	.04	.54
5 weeks
Eggshell colour	11.75	12.20	12.10	12.00	.38	.48	.21	.67
Egg weight, g	63.57	64.06	63.99	63.91	1.81	.71	.24	.42
Yolk height, mm	9.25	9.39	9.52	9.53	.06	.02	.01	.37
Yolk colour	8.95	9.00	8.95	9.00	.10	.25	.27	.29
Haugh Unit	94.47	95.54	96.18	96.42	.58	.02	.04	.91
Eggshell strength, kg/cm^2	4.39	4.40	4.40	4.41	.11	.36	.25	.82
Eggshell thickness, mm^−2	40.26	40.99	41.89	41.93	.27	.01	.03	.79

^aAbbreviations: CON, basal diet; AGE.05, CON + .05% aged garlic extract; AGE.1, CON + .1% aged garlic extract; AGE.2, CON + .2% aged garlic extract.

^bStandard error.

^cT represents the P-values of Tukey's range test; L and Q represent the P-values of Linear and Quadratic (polynomial contrasts) effect, respectively.

3.2. Excreta microbiota and excreta noxious gas emissions

The impacts of AGE on excreta microbiota are shown in Table 4. Lactobacillus counts were not positively affected (P > .05) among treatments, though E. coli counts were linearly reduced in AGE.2 diet (P = .02). There were no significant differences among treatments in NH3, R.SH and H2S gas emissions in laying hens. Average values (ppm) were 24.14 (SE = 5.56), 1.82 (SE = 1.52) and 1.47 (SE = 1.32) for NH3, R.SH and H2S, respectively (data not shown).

Table 4. The effects of aged garlic extract (AGE) supplementation on excreta microbiota in laying hens.^a

Items, log10cfu/g	Diet					P-value^c
	CON	AGE.05	AGE.1	AGE.2	SE^b	T	L	Q
Lactobacillus	7.52	7.54	7.59	7.60	.59	.22	.33	.91
E. coli	6.77	6.51	6.45	6.41	.05	.01	.02	.52

^aAbbreviations: CON, basal diet; AGE.05, CON + .05% aged garlic extract; AGE.1, CON + .1% aged garlic extract; AGE.2, CON + .2% aged garlic extract.

^bStandard error.

^cT represents the P-values of Tukey's range test; L and Q represent the P-values of Linear and Quadratic (polynomial contrasts) effect, respectively.

3.3. Haematological characteristics

The inclusion of AGE increased (linear, P = .04) lymphocyte concentrations compared with CON diet (Table 5). Moreover, IgG concentrations were linearly increased (P = .03) in AGE.2 diet, however, blood TC was linearly reduced compared with other diets in the current study (P < .01).

Table 5. The effects of aged garlic extract (AGE) supplementation on haematological characteristics in laying hens.^a

Items, ppm	Diet					P­-value^c
	CON	AGE.05	AGE.1	AGE.2	SE^b	T	L	Q
WBC, 10^3/µl	220.20	227.40	228.00	237.60	12.60	.33	.42	.95
RBC, 10^6/µl	2.77	2.79	2.81	2.83	.09	.74	.14	.27
Lymphocyte, %	75.20	80.30	81.20	81.80	.79	.01	.04	.64
IgG, mg/dL	2.61	3.81	4.27	4.31	.47	.02	.03	.52
TC, mg/dl	208.40	207.01	181.36	180.92	5.23	.02	<.01	.42

^aAbbreviations: CON, basal diet; AGE.05, CON + .05% aged garlic extract; AGE.1, CON + .1% aged garlic extract; AGE.2, CON + .2% aged garlic extract.

^bStandard error.

^cT represents the P-values of Tukey's range test; L and Q represent the P-values of Linear and Quadratic (polynomial contrasts) effect, respectively.

3.4. Fatty acid of yolk

In this study, laying hens fed AGE.2 diet reduced (linear, P = .01) total SFA compared to other diets (Table 6). The inclusion of AGE.2 had reduced C20:0 compared with the other diets (linear and quadratic, P = .03 and < .01, respectively). However, C20:1, total PUFA and C18:2n-6 were significantly increased in AGE.2 diet (P < .05). AGE.2 diet had higher C18:1n-9 than other diets (P < .05). In this study, PUFA: SFA was increased in AGE.2 diet compared with others (P < .05).

Table 6. The effects of aged garlic extract (AGE) supplementation on fatty acid of yolk in laying hens.^a

	Diet					P-value^c
Items, %	CON	AGE.05	AGE.1	AGE.2	SE^b	T	L	Q
Total SFA	36.16	36.55	35.95	33.46	1.11	.01	<.01	.74
C14:0	.41	.41	.40	.41	.03	.77	.85	.58
C16:0	26.07	27.12	25.47	25.12	1.12	.29	.25	.48
C18:0	9.59	9.96	8.73	8.28	4.21	.41	.41	.45
C20:0	.09	.08	.06	.05	.01	.01	.03	<.01
Total MUFA	49.46	49.57	49.66	49.97	.36	<.01	.87	<.01
C14:1n-5	.09	.09	.09	.10	.02	.96	.75	.81
C16:1n-7	2.98	2.88	3.01	3.14	.27	.55	.65	.44
C18:1n-9	45.99	44.81	46.12	46.37	.32	.01	.02	<.01
C20:1	.11	.13	.18	.20	.04	.02	.94	.02
Total PUFA	12.03	12.09	13.01	13.04	.45	<.01	<.01	<.01
C18:2n-6	11.05	11.11	11.95	12.02	.41	<.01	<.01	<.01
C18:3n-3	.03	.02	.03	.03	<.01	.73	.41	.54
C20:2n-6	.15	.15	.15	.16	.02	.27	.57	.98
C20:4n-6	.01	.01	.01	.02	<.01	.71	.54	.48
C20:5n-3	.19	.19	.20	.20	.02	.85	.82	.42
C22:6n-3	.60	.61	.61	.61	.02	.49	.54	.51
PUFA:SFA	.33	.33	.36	.39	.02	.01	<.01	<.01

^aAbbreviation: CON, basal diet; AGE.05, CON + .05% aged garlic extract; AGE.1, CON + .1% aged garlic extract; AGE.2, CON + .2% aged garlic extract.

^bStandard error.

^cT represents the P-values of Tukey's range test; L and Q represent the P-values of Linear and Quadratic (polynomial contrasts) effect, respectively.

4. Discussion

4.1. Feed intake and egg production

In the current study, no significant differences were observed among the treatments on feed intake and egg production, which is in agreement with Lim et al. (2006), who reported that there was no difference in the feed consumption of laying hens fed 0, 1, 3 and 5% garlic powder. Similarly, Chowdhury et al. (2002) observed that feed consumption, egg production and feed efficiency were not significantly affected by 0–100 g/kg sun-dried garlic paste over six weeks. According to Reddy et al. (1991), egg production and feed efficiency were not affected during or at the end of eight weeks of feeding garlic oil to the Babcock layer. Canogullari et al. (2009) fed layer diets containing 0, .5, 1 and 2% garlic powder for 12 weeks period and also reported that feed consumption and feed efficiency were not affected by the diets. Recently, Safaa (2007) indicated that laying hen production performance was not affected by 2.0% garlic addition. Ao et al. (2010) reported that 1, 2 and 3% fermented garlic powder supplementation had no significant effects on egg production in 44 weeks ISA brown. Moreover, Aporn and Adcharatt (2008), who reported that there were no significant differences in feed intake, weight gain and feed conversion ratio across treatments in broilers fed .7–1.3% garlic powder diets. In addition, Yalçin et al. (2006) found no effects of garlic addition (5–10 g/kg) on body weight, feed consumption or feed efficiency in laying hens. In contrast with the current study, Qureshi et al. (1983) reported that garlic supplementation in diets decreased the feed intake because of the specific odour of garlic. It is reported that the pungent smell of garlic is responsible for reduced feed intake in pigs (Ferket 1991). Also, garlic contains a precursor, such as alliin, which is easily converted to an odourous component, such as allicin or DADS, imparting an unfavourable taste (Langendijk et al. 2007; Hossain et al. 2014). Cullen et al. (2005) indicated that the inclusion of garlic at greater amounts (1 or 10 g/ kg) caused a reduction in feed intake. Allicin and other thiosulfinates readily decompose via several pathways, depending on the reaction conditions, such as medium polarity, pH and temperature and can yield various biologically active aliphatic and heterocylic sulfides (Velišek et al. 1997). In our study, AGE contained much less alliin when compared with fresh garlic. The content of AMTS, DATS, DADS and 1, 2-VDT in the AGE was greater than that in fresh garlic. Khan et al. (2007) showed that the inclusion of 0, 2, 6 and 8% garlic powder significantly increased feed intake with the increasing levels of dietary garlic in laying hens. Yan et al. (2011) also reported that 2 and 4 g/kg fermented garlic powder supplements increased average daily feed intake in finishing pigs. These different results may be due to the use of different garlic sources, active garlic nutrients, concentration and methods of preparation of aged garlic extract and strains or age of laying hens used in the experiments. Thus, more researches need to evaluate the effect of aged garlic extract on laying hen, especially fermentation with Leuconostoc mesenteroides KCCM35046 with specific age.

4.2. Egg quality

In this study, the yolk height, HU and eggshell thickness were increased without any change in the egg weight, eggshell colour, yolk colour and eggshell strength by supplementation of aged garlic extract in the diet. In agreement with our results, Ao et al. (2010) reported that addition of fermented garlic powder increased yolk colour, yolk height and HU but had no significant effects on egg shell thickness in the laying hens during the fifth week of trail. Moreover, Lim et al. (2006) reported that, with an increasing level of dietary garlic powder, the HU linearly increased after two weeks of storage. A study conducted by Safaa (2007) reported that 2.0% addition of dietary garlic increased yolk weight, yolk colour and HU. Olobatoke and Mulugeta (2010) stated that .81 mm in albumen height and 2.71 HU of fresh eggs were increased at 3% garlic powder addition in the diet. Yalcin et al. (2007) also reported a significant increase in egg weight when the diet of laying quails was supplemented with garlic powder. However, these results are different from the findings reported by Sakine and Onbasilar (2006) who observed that the supplementation of garlic powder had no significant effect on egg breaking strength, shell thickness, albumen index, yolk index and HU. Mahmoud et al. (2010) observed that garlic juice supplementation improved albumen height and HU in the 40-week-old Hi-sex laying hen. Kaya and Macit (2012) found that supplementation of dried garlic powder had no positive effect on egg shell thickness and HU in the laying hen. Ashfaq (2001) and Khan et al. (2007) also found no significant effect of garlic powder on egg weight. The differences in garlic preparation and administration methods may have been responsible for some of the variations seen in trial outcomes. Although the reasons for the improvement in yolk height, HU and eggshell thickness noted in this trial cannot be fully explained, it is thought that the absorption of aged garlic extract components with their subsequent incorporation into tissues and egg shell thickness is possible. Generally, garlic, a plant rich in amino acids and selenium, possess increased ability to readily take up selenium from the soil (Arnaulta & Augerb 2006). Selenium in garlic occurs in the form of selenium compounds such as selenomethionine and selenocysteine. Selenomethionine, a stored form of organic selenium that is actively absorbed from the intestinal tract, is known to be significantly retained in animal tissues (Shan & Davis 1994). Thus, in this study, egg shell thickness, yolk height and HU may be attributed due to the AGE supplementation in the diet of laying hens.

4.3. Excreta microbiota

Previously, Michael de and Marteau (2007) had presented that gastrointestinal and lymphoid system is the largest immunologically competent organ in the body as well as the development and composition of the indigenous microflora are the principal factors influencing maturation and optimal development of gastrointestinal and lymphoid system. Therefore, excreta Lactobacillus and E. coli in laying hens were investigated. Historically, garlic has been used worldwide to fight bacterial infections. It is well documented that garlic preparation exhibit a wide spectrum of antibacterial activity against gram-negative and gram-positive bacteria such as Escherichia coli, Salmonella, Staphylococcus, Streptococcus, Bacillus and Clostridium (Ankri & Mirelman 1999; Sivam 2001). El-Astal (2004) also reported the high antibacterial efficacy of garlic extract on certain pathogenic gram-positive bacteria (Staph. aureus, Staph. saprophyticus) and gram-negative bacteria (E. coli, E. cloacae and Pseudomonas aeruginosa) and suggested that those functions are mainly attributed to the sulphur-containing compound of garlic (Amagase et al. 2001). The analysis data of our study suggested that AGE has a higher content of sulphur-containing compound (1, 2-VDT, DADS, AMTS and DATS) than the intact garlic, which may confirm that the AGE has a higher antimicrobial activity than that of intact garlic (Kakimoto et al. 2000). Indeed in our study, laying hens fed AGE supplemented diets reduced excreta E. coli compared to basal diet, indicating the intestine microbial status is affected by the AGE administration. But interestingly, no difference was observed in the excreta Lactobacillus numbers. The reason is likely to be the different sensitivity of bacteria towards garlic extract (Ankri & Mirelman 1999), as the E. coli are the highly sensitive micro-organisms, while lactic acid bacteria (beneficial) are the least sensitive micro-organisms to the inhibitory effects of garlic (Rees et al. 1993). Moreover, it is well known that the presence of most E. coli is negatively related to the gut health (Kaper et al. 2004). Our previous studies also confirmed this observation (Wang et al. 2011; Yan & Kim 2012; Hossain et al. 2014). Therefore, we hypothesized the AGE supplementation might increase the gut health.

4.4. Excreta noxious gas emissions

Poultry manure is one of the major sources of nitrogen pollution (Song et al. 2012), in which NH₃ and H₂S are major aerial pollutant (Kristensen & Wathes 2000) with adverse effects on the production of birds (Miles et al. 2004). Therefore, our emphasis has been placed to observe the impact of AGE on excreta noxious gas emission in laying hens. There were many researches carried out on different aspects and goal of garlic but in our best knowledge we did not find any related observation of AGE on excreta gas emission in laying hens. However, Cho et al. (2006) and Huang et al. (2010) have suggested that some herbs extract could reduce the faecal noxious gas content in pigs manure. Wenk (2003) also observed that the inclusion of herbs or herbs extract could reduce the faecal noxious gas content in growing pigs by manipulating the microflora in the gastrointestinal tract of pigs. Ferket et al. (2002) have previously suggested that the ultimately faecal noxious gas emission of animals is related to intestinal microflora and nutrient utilization. In this study, we found that AGE supplementation had reduced excreta E. coli count but no effect on excreta gas production compared to basal diet. The reason for the different result is likely to be the dose, preparation method and different supplementation employed in the current study. Therefore, further study is still necessary to investigate the effect of AGE on excreta gas emission on laying hens.

4.5. Blood profile

Laying hens fed with the AGE supplemental diets had higher blood lymphocyte counts and IgG and lower TC concentrations, which is in agreement with Ao et al. (2011) who found that fermented garlic powder increased the lymphocyte counts and IgG and reduced TC concentrations in the broilers. Ao et al. (2010) found a significant reduction in serum cholesterol concentration when the dietary level of fermented garlic powder was increased from .0 to 3.0% in the 41 weeks ISA brown laying hens. Previous studies have found that garlic and its contents could activate the immune function such as proliferation of lymphocyte, cytokine release, natural killer cell activity and phagocytosis (Amagase et al. 2001; Ross et al. 2001; Wang et al. 2011). Furthermore, it was suggested that garlic or allicin supplementation exerted positive influence on early age animals mainly due to the improvement in immunity (Wang et al. 2011). These results were consistent with many studies which demonstrated that garlic addition could induce a positive effect on cholesterol metabolism (Chowdhury et al. 2002; Kim et al. 2009; Yan et al. 2011). This affects the health because high levels of triglyceride, LDL-cholesterol and TC may induce the development and progression of atherosclerosis and cardiovascular diseases. A study conducted by Singh et al. (2006) indicated that the compounds containing an allyldisulfide or allyl-sulfhydryl group were most likely responsible for the inhibition of cholesterol synthesis by garlic. Also, our results are consistent with the beneficial effects of garlic on cholesterol metabolism and agreed well with previous study (Ao et al. 2010). Chowdhury et al. (2002) reported that serum cholesterol concentrations were decreased on average by 15, 28, 33 and 43% with increasing levels of dietary garlic paste of 2, 4, 6 or 8%, respectively. Garlic powder supplementation reduced significantly the serum cholesterol concentration when laying hens were fed .5 and 1.0% garlic powder (Sakine & Onbasilar 2006). Reduction of serum cholesterol concentration by the addition of garlic powder was also observed in rats and broilers (Qureshi et al. 1983). The decrease of serum cholesterol concentration by garlic powder supplementation might be due to the reduction of synthetic enzymes. Significant decreases in hepatic 3-hydroxy-3- methylglutaryl-CoA reductase, cholesterol 7α-hydroxylase, fatty acid synthetase and in representative pentose phosphate pathway activities accompanied the feeding of petroleum ether, methanol and water-soluble fractions of garlic (Qureshiet al. 1983). In contrast, Reddy et al. (1991) reported that feeding .02% garlic oil did not have any effect on serum and yolk cholesterol in the Babcock B-300 strain. This result might be due to the strain used in the experiments because serum cholesterol concentrations differ significantly among strains (Chowdhury et al. 2002). Besides, different analytical methods, bacteria culture and garlic products might also result in this difference.

4.6. Fatty acid of yolk

In this study, the total SFA concentration of fatty acids in egg yolk decreased in response to AGE.2 diet compared to other diets. Monounsaturated fatty acids (MUFA) were higher in AGE.2 treatment than in CON, AGE.05 and AGE.1 treatments. Furthermore, compared with other treatments, PUFA concentration was higher in AGE.2 diet. This was mainly due to an increase in linoleic acid (C18:2n-6). Further, .2% fermented garlic powder supplementation resulted in a higher PUFA: SFA ratio. In our best knowledge only one experiment was carried out on relation to garlic powder on laying hen, where Ao et al. (2010) stated that addition of 3.0% garlic powder decreased SFA but increased PUFA and PUFA: SFA ratio in egg yolk. This is beneficial for human health in that clinical data strongly support a relationship between SFA and the risk of coronary heart disease and, therefore, there is a need to decrease the consumption of SFA and increase that of PUFA. However, no comparisons with other studies could be made because investigations of the use of AGE by Leuconostoc mesenteroides KCCM35046 in relation to fatty acids of yolk have not yet been reported. Therefore, further research is needed to study the effect of garlic powder on fatty acid composition in egg yolk.

5. Conclusion

In conclusion, this study showed that dietary AGE exerted no significant egg production performance enhancing effect when incorporated into layer diet. However, yolk height, HU and eggshell thickness of fresh eggs were significantly improved by AGE supplementation in the diet. Inclusion of AGE increased the blood lymphocyte counts and IgG and reduced TC and excreta E. coli without any change of excreta noxious gas emission in the laying hen. Moreover, dietary .2% of AGE addition decreased SFA and increased PUFA and PUFA: SFA ratio in egg yolk. However, further studies are needed to evaluate the mechanism by which dietary AGE effects on egg quality and egg yolk composition in laying hens.

Disclosure statement

No potential conflict of interest was reported by the authors.