On oxidation resistance and meat quality of broilers challenged with lipopolysaccharide

Abstract

Effects of preslaughter immunological stress induced by lipopolysaccharide (LPS) on antioxidant performance and meat quality of broilers were investigated. Twenty-four broiler chickens (39 days old) with near-mean body weight (BW) were randomly divided into three following treatments: sham injection of saline (control), daily subcutaneous (s.c.) injection of LPS (3 mg/kg or 6 mg/kg of BW, respectively.) for three days. The results showed that average daily feed intake and daily gain of chickens were significantly decreased in the LPS treatment (P < 0.05). The F/G was significantly enhanced (P < 0.01). The LPS treatment had no significant influence on carcass performance (P > 0.05). The LPS treatment significantly decreased the pH at 24 h postmortem in breast muscle (P < 0.05) and significantly increased the water-holding capacity (WHC) of breast muscle (P < 0.01). It was the same as the change of yellowness (b*) at day 3 postmortem and lightness (L*) at day 6 postmortem in breast muscle (P < 0.05), but had no significant influence on shear force of breast muscle (P > 0.05). The pH at 45 min or 24 h postmortem of thigh muscle was significantly dropped in the LPS treatment (P < 0.01), however, the WHC and shear force of thigh muscle were not significantly different (P > 0.05). The yellowness (b*) significantly increased at day 3 postmortem of thigh muscle (P < 0.05). The plasma, breast muscle and thigh muscle MDA were significantly enhanced in the LPS treatment (P < 0.05). The total antioxidant capacity (T-AOC) level was significantly suppressed (P < 0.05); however, the plasma activity of glutathione peroxidase (GSH-Px) was slightly decreased (P > 0.05). The results of present study suggested that LPS-induced preslaughter immunological stress could increase body oxidative damage and decrease oxidation resistance, resulting in a decrease in growth performance and meat quality of broilers; also these adverse effects could be rested with the stress intensity.

Keywords:

• lipopolysaccharide
• immune stress
• oxidative damage
• meat quality
• broilers

1. Introduction

The modern broiler industry has been greatly expanding to meet increasing demands of the growing human population. Genetic improvement in growth rate and feed efficiency has allowed modern broilers to reach market weight in a shorter period of time (Flock et al. 2005). However, selection for these economically important traits has been accompanied by an increase in the number of problems encountered during the intensive production system, especially birds are easily susceptive to a variety of stressors (Cheng et al. 2004). This situation may affect bird welfare as well as increase stress-related diseases, resulting in poor performance, high mortality and inferior meat quality (Ngoka et al. 1982; Sackett et al. 1986; Küchenmeister et al. 2002; Ferguson & Warner 2008; Young et al. 2009; Vieira et al. 2011). Lipopolysaccharide (LPS) isolated from Escherichia coli bacteria is recognized by immune cells as a pathogen-associated molecular pattern and activates innate immune responses. Thus, LPS is often used as a model antigen to investigate the susceptibility of animals to the pathogen stimulus, especially non-specific components of micro-organism. LPS is an acute inflammatory stimulus, and the challenge with LPS stimulates the synthesis and release of glucocorticoids, inducing a rapid, short-lived increase in plasma corticosterone levels (Sternberg 2006). Furthermore, LPS administration causes a temporary reduction in feed intake (Star et al. 2008), which might have an influence on many physiological and metabolic processes, reflected by changes in oxidative stress parameters. Although the effects of LPS administration have been well-established on physiological responses such as fever, body temperature and body weight (BW) gain, the influence on oxidative stress responses and meat quality changes in broiler chickens is hardly described. In the present research, the effects of LPS-induced preslaughter stress on growth performance, oxidative stress responses, carcass characteristics and meat quality were investigated in broiler chickens from 39 to 42 days.

2. Materials and methods

2.1. Experimental design and bird management

This experiment was conducted according to protocols approved by the Northwest A& F University Animal Care and Use Committee. The design used was one-factor randomized block design. Twenty-four 39-day-old broiler chicks (Cobb 500) with near-mean BW (2108.6 g) were randomly divided into three following treatments: sham injection of saline (control), daily subcutaneous (s.c.) injection of E. coli LPS 3 mg/kg or 6 mg/kg of BW, respectively, for three days. LPS (serotype O55:B5) was provided by Sigma Chemical Ltd. Co., USA. The birds were fed corn-soybean meal basal diet (Table 1). Water and feed were provided ad libitum, and feed intake and BW were recorded. Birds were kept in two-layer cages. The photoperiod was set at 22L:2D throughout the whole experimental period.

Table 1. Ingredients and nutrient levels of the basal diets.

Ingredients (%)	0–21 days	21–42 days
Corn	60.6	64.43
Soybean meal	31	27
Fish meal	2.2	1.5
Limestone	1.38	1
Dicalcium phosphate	1.1	1.1
Salt	0.16	0.20
L-Lysine	0.16	0.14
Methionine	0.16	0.1
L-Threonine	0.04	0.03
Soybean oil	1.7	3
Premix^a	1.5	1.5
Nutrient level
ME kcal/kg)	2900	3030
CP (%)	21	19
Ca (%)	1.07	0.91
TP (%)	0.63	0.58
AP (%)	0.42	0.39
Lys (%)	1.17	1.03
Met (%)	0.50	0.42
Met+ Cys (%)	0.84	0.72
Thr (%)	0.83	0.74

^aProvided per kilogram of diet: retinyl acetate, 2970 μg; cholecalciferol, 75 μg; dl-α-Tocopherol Acetate, 10 mg; menadione, 2 mg; vitamin B12, 0.04 mg; folic acid, 1.96 mg; d-pantothenic acid, 15.64 mg; riboflavin 8, mg; niacin, 39.2 mg; thiamin, 2.76 mg; d-biotin, 0.3 mg; pyridoxine, 4.9 mg; Cu, 10 mg; Fe, 72 mg; Zn, 89.7 mg; Mn, 101.76 mg; Se, 0.15 mg; I, 0.31 mg.

2.2. Processing procedure and sampling

On day 42 of age, all birds were euthanized under anaesthesia and exsanguinated after a 12-h fast and access to water ad libitum to measure eviscerated carcass percentage, breast meat percentage, leg meat percentage, abdominal fat percentage, etc. Left pectoralis major muscle and left leg muscle were sampled for determining meat quality and blood was sampled for measuring body antioxidant capacity parameters.

2.3. Muscle pH measurement

At 45 min and 24 h postmortem, the breast and leg muscle pH were, respectively, determined at a depth of 2.5 cm below the surface by using a Model PH-211 metre equipped with a spear electrode.

2.4. Colour measurement

The surface colour of chicken rolls was measured in packages using a Hunter LabScan colorimeter and expressed as colour L*- (lightness), a*- (redness) and b*- (yellowness) values. A colour reading was taken from both sides of the rolls. The same packaging materials were used to cover a white standard plate in order to eliminate the influence of packaging material on meat colour.

2.5. Water-holding capacity measurement

Water-holding capacity (WHC) was estimated by determining expressible juice using a modification of the filter paper press method described by Wierbicki and Deatherage (1958). Briefly, a raw meat sample weighing about 1000 mg was placed between 18 pieces of 11-cm diameter filter paper and pressed at 35 kg for 5 min. Expressed juice was defined as the loss in weight after pressing and presented as a percentage of the initial weight of the original sample (Bouton et al. 1971).

2.6. Shear force measurement

The muscles were refrigerated overnight at 0–4°C and then brought to room temperature before cooking. The breast muscle from each bird was cooked to an internal temperature of 70°C on a digital thermostat water bath (HH-4, Jiangbo instrument, Jiangsu, China). Endpoint internal temperature was monitored with a thermometer. Cooked muscle was cooled to room temperature. Slices of 1 cm × 1 cm were cut perpendicular to the fibre orientation of the muscle. Ten 1 cm × 1 cm cores about 3 cm thick were removed parallel to the fibre orientation through the thickest portion of the cooked muscle. Warner-Bratzler shear force was determined by using an Instron Universal Mechanical Machine (Instron model 4411, Instron Corp., Canton, MA). A Warner-Bratzler apparatus was attached to a 50 kg load cell, and tests were performed at a crosshead speed of 127 mm/min. Signals were processed with the Instron Series ninth software package.

2.7. Oxidation products

The levels of the lipid peroxidation product TBAR, mainly malondialdehyde, were determined according to the modified method of Li and Chow (1994) spectroflourometrically at 515 nm excitation and 550 nm emission following isobutyl alcohol extraction. 1,1,3,3-Tetraethoxypropane was used as the standard. The levels of conjugated dienes, another indicator of lipid oxidation, were measured spectrophotometrically. The content of protein-bound carbonyls, which is used to assess protein oxidation, was determined spectrophotometrically at 375 nm by the 2, 4-dinitrophenylhydrazine method of Levine et al. (1990).

2.8. Antioxidant status

Activity of glutathione peroxidase (GSH-Px) and level of total antioxidant capacity (T-AOC) in plasma were determined by using the kits offered by Nanking Jiancheng Biological engineering research institute.

2.9. Statistical analysis

All the data were analysed statistically using the general linear model procedure (SAS 1996) and the treatment means were separated by Duncan's multiple range test (p = 0.05).

3. Results

3.1. Growth performance

Effects of LPS-induced preslaughter immunological stress on growth performance of broilers from 39 to 42 days are shown in Table 2. The results indicated that average daily feed intake (ADI) of birds challenged with LPS significantly decreased (P < 0.05) compared to control treatment, and average daily gain (ADG) was significantly decreased (P < 0.01), while feed/gain ratio (F/G) was significantly enhanced (P < 0.01).

Table 2. Effects of preslaughter immunological stress on growth performance of broilers from 39 to 42 days.

LPS (mg /kg BW)	Average daily feed intake (g)	Average daily gain (g)	Feed:gain
0	151.67 ± 5.76^a	58.33 ± 2.64^A	2.59 ± 0.02^C
3	115.21 ± 5.14^b	32.92 ± 1.64^B	3.50 ± 0.02^B
6	110.21 ± 7.01^b	29.17 ± 2.36^B	3.78 ± 0.07^A

^a–bMeans with difference superscript letters in same row differ significantly (P < 0.050);

^A–CMeans with difference superscript letters in same row differ significantly (P < 0.001).

3.2. Carcass characteristics

Effect of preslaughter immunological stress on the slaughter parameters of broilers is showed in Table 3. There were no significant differences in the slaughter parameters of broilers between the LPSs – challenged treatment and the control treatment (P > 0.05).

Table 3. Effect of preslaughter immunological stress on the slaughter parameters of broilers.

	LPS (mg/kg BW)
Items (%)	0	3	6
Dressing percentage	85.36 ± 2.35	85.22 ± 0.69	85.07 ± 0.55
Eviscerated percentage	77.13 ± 0.58	76.92 ± 1.61	76.86 ± 0.70
Semi-eviscerated percentage	89.61 ± 1.04	89.39 ± 0.97	89.21 ± 0.76
Breast muscle percentage	22.48 ± 1.93	22.37 ± 1.74	22.31 ± 1.09
Leg muscle percentage	18.83 ± 1.08	18.71 ± 0.84	18.57 ± 0.87
Abdominal fat percentage	2.67 ± 0.80	2.46 ± 0.54	2.16 ± 0.65

3.3. Meat quality

Effect of preslaughter immunological stress on breast muscle colour of broilers is displayed in Table 4. LPS-induced preslaughter immunological stress did not significantly influence fresh breast muscle colour L*, a*, b* value (P > 0.05); Injection of LPS at 6 mg/kg BW significantly affected breast muscle colour b* value (P < 0.05) at day 3 postmortem compared to control treatment, while injection of LPS at 3 mg/kg BW did not significantly affect breast muscle colour b* value (P > 0.05); LPS-induced preslaughter immunological stress did not significantly influence breast muscle colour L*, a* values at day 3 postmortem (P > 0.05). At day 6 postmortem, injection of LPS at 6 mg/kg BW significantly increased breast muscle colour L* value (P < 0.05), while injection of LPS at 3 mg/kg BW did not significantly affect breast muscle colour L* value (P > 0.05); LPS-induced preslaughter immunological stress did not significantly influence breast muscle colour a*, b* values at day 6 postmortem (P > 0.05).

Table 4. Effect of preslaughter immunological stress on breast muscle colour of broilers.

		LPS (mg/kg BW)
Items		0	3	6
1st day	L*	46.11 ± 5.65	49.17 ± 4.31	45.27 ± 8.57
	a*	14.90 ± 1.18	10.78 ± 2.58	16.45 ± 6.13
	b*	23.15 ± 6.25	20.29 ± 1.67	22.69 ± 3.87
3rd day	L*	53.05 ± 2.08	55.01 ± 3.99	54.48 ± 4.83
	a*	14.91 ± 1.90	14.25 ± 1.51	13.58 ± 2.27
	b*	23.64 ± 2.07^b	24.56 ± 2.76^ab	26.50 ± 1.68^a
6th day	L*	50.30 ± 3.03^b	52.61 ± 3.35^ab	54.78 ±4.38^a
	a*	8.76 ± 5.34	7.98 ± 2.06	6.94 ± 2.91
	b*	20.33 ± 3.06	21.31 ± 1.05	22.00 ± 2.14

^a–bMeans with difference superscript letters in same row differ significantly (P < 0.05).

Effects of preslaughter immunological stress on breast muscle pH, shear force and WHC of broilers are shown in Table 5. Injection of LPS did not significantly affect breast muscle pH value (P > 0.05) at 45 min postmortem compared to control treatment, but significantly decreased pH value at 24 h postmortem (P < 0.01). Injection of LPS slightly increased shear force of breast muscle (P > 0.05). LPS-induced preslaughter immunological stress significantly decreased WHC of breast muscle (P < 0.01).

Table 5. Effect of preslaughter immunological stress on breast muscle pH, shear force and WHC of broilers.

LPS (mg/kg BW)	pH		Shear force (N)	Water-holding capacity(%)
	45 min	24 h
0	6.80 ± 0.26	6.06 ± 0.10^A	6.99 ± 1.75	94.45 ± 0.83^B
3	6.74 ± 0.14	5.91 ± 0.13^B	7.67 ± 1.91	92.16 ± 1.42^A
6	6.67 ± 0.21	5.85 ± 0.11^B	7.79 ± 1.31	91.87 ± 1.00^A

^A–BMeans with difference superscript letters in same row differ very significantly (P<0.001).

Effect of preslaughter immunological stress on thigh muscle colour of broilers is shown in Table 6. LPS-induced preslaughter immunological stress did not significantly influence thigh muscle colour at days 1, 3 and 6 postmortem (P > 0.05), only thigh muscle colour b* value at day 3 postmortem was significantly increased (P < 0.05) in birds injected with LPS compared to control treatment.

Table 6. Effect of preslaughter immunological stress on thigh muscle colour of broilers.

Items		LPS (mg/kg BW)
		0	3	6
1 st day	L*	41.09 ± 9.07	44.65 ± 2.41	46.33 ± 3.85
	a*	17.38 ± 2.67	19.56 ± 5.09	19.99 ± 3.90
	b*	28.69 ± 4.35	21.19 ± 3.12	23.27 ± 4.86
3rd day	L*	48.27 ± 2.88	49.79 ± 1.92	48.36 ± 3.89
	a*	15.29 ± 1.18	17.01 ± 2.42	17.05 ± 1.86
	b*	19.68 ± 2.20^b	26.01 ± 5.84^a	26.36 ± 4.00^a
6th day	L*	47.49 ± 2.95	48.49 ± 3.18	49.32 ± 3.26
	a*	13.79 ± 4.24	16.42 ± 2.98	14.68 ± 2.40
	b*	18.15 ± 2.69	19.17 ± 1.75	18.39 ± 2.89

^a–bMeans with difference superscript letters in same row differ significantly (P < 0.05).

Effects of preslaughter immunological stress on thigh muscle pH, shear force and WHC of broilers are shown in Table 7. Injection of LPS significantly decreased pH value of thigh muscle at 45 min or 24 h postmortem (P < 0.01). LPS-induced preslaughter immunological stress did not significantly influence high muscle shear force and WHC (P > 0.05).

Table 7. Effect of preslaughter immunological stress on thigh muscle pH, shear force and WHC of broilers.

LPS (mg/kg BW)	pH		Shear force (N)	Water-holding capacity (%)
	45 min	24 h
0	6.72 ± 0.14^A	6.46 ± 0.11^A	14.13 ± 2.51	93.25 ± 2.66
3	6.38 ± 0.27^B	6.29 ± 0.15^B	15.38 ± 2.27	91.75 ± 2.67
6	6.35 ± 0.11^B	6.21 ± 0.09^B	16.21 ± 2.65	90.87 ± 2.75

^A–BMeans with difference superscript letters in same row differ very significantly (P < 0.001).

3.4. Antioxidant capacity

Effects of preslaughter immunological stress on plasma antioxidation and muscle lipid peroxidation level of broilers are shown in Table 8. Injection of LPS significantly increased plasma MDA (P < 0.05), slightly decreased plasma GSH-Px activity (P > 0.05), and significantly decreased plasma T-AOC (P < 0.01). Injection of LPS significantly increased thigh muscle MDA at day 1 postmortem, while it did not affect thigh muscle MDA at day 3 or 6 postmortem (P > 0.05). Injection of LPS very significantly increased breast muscle MDA at day 1, 3 or 6 postmortem (P < 0.01).

Table 8. Effect of preslaughter immunological stress on plasma antioxidation and muscle lipid peroxidation level of broilers.

Items		LPS (mg/kg BW)
		0	3	6
Plasma	MDA (nmol/ml)	1.71 ± 0.59^b	2.15 ± 0.35^a	2.63 ± 0.56^a
	GSH-Px (U/ml)	1449.64 ± 65.54	1352.14 ± 74.13	1340.89 ± 68.36
	T-AOC (U/ml)	16.56 ± 1.50^A	12.04 ± 2.84^B	12.49 ± 1.84^B
Thigh muscle MDA (nmol/mg prot.)	1st day	2.34 ± 0.64^b	2.49 ± 0.23^a	2.96 ± 0.29^a
	3rd day	3.62 ± 1.52	3.74 ± 1.93	3.65 ± 2.16
	6th day	7.21 ± 0.76	7.61 ± 1.42	8.14 ± 1.79
Breast muscle MDA (nmol/mg prot.)	1st day	1.62 ± 0.23^B	2.21 ± 0.38^A	2.62 ± 0.62^A
	3rd day	3.16 ± 1.01^B	5.77 ± 0.93^A	5.93 ± 1.56^A
	6th day	6.43 ± 0.51^B	7.83 ± 0.78^A	8.39 ± 0.90^A

^a–bMeans with difference superscript letters in same row differ significantly (P < 0.05);

^A–BMeans with difference superscript letters in same row differ significantly (P < 0.001).

4. Discussion

Chickens treated with LPS reduced feed consumption, daily gain and feed efficiency during the period 1–4 days after treatment. These data are in agreement with other investigations that found a reduction in growth and BW (Koh et al. 1996; Xie et al. 2000; Mireles et al. 2005) in birds treated with LPS. In contrast, Parmentier et al. (1998) and Cheng et al. (2004) demonstrated that the effect of LPS injection on BW gain is strain- and time-dependent. Similarly, our findings suggest that the reason for a different regulation of growth performance in birds could be related to the strain's unique characteristics and age. However, no significant reductions in daily gain were observed by Shini et al. (2008).

Colour is essential for the appearance of meat and hence consumer preference. In the present study, effect of the preslaughter stress on colour was investigated in tow muscles. In general, preslaughter stress affected the colour, measured as L*, a* and b* value. LPS-induced preslaughter immunological stress did not significantly influence fresh breast muscle colour or fresh thigh muscle colour L*, a*, b* value. Injection of LPS at 6 mg/kg BW significantly affected breast or thigh muscle colour b* value at day 3 postmortem. Injection of LPS at 6 mg/kg BW significantly increased breast muscle colour L* value, whereas it did not significantly influence thigh muscle colour L* value. These results are similar to other studies (Juncher et al. 2001; Rosenvold & Andersen 2003).

The rate of decline of postmortem pH has been considered the most important predictor of meat quality. Muscle pH does not only intuitively indicate muscle acidity but also directly affects meat tenderness, drip loss and meat colour. Muscle WHC, i.e., muscle water-retaining capacity, which is assessed in terms of drip loss or water loss, directly exert influence on meat taste, succulence, colour, nutrients and flavour and more greatly affect processed meat yield, structure and colour. Muscle tenderness, a meat quality indicator that can be expressed in terms of meat shear force, can indicate internal structure of meat as well as muscle myofibril content, and fat content, distribution and chemical structure of connective tissue to some extent. Normally, there exists a negative correlation between meat shear force and tenderness. The results of present research indicated that injection of LPS significantly decreased pH value of thigh muscle at 45 min or 24 h postmortem and breast muscle at 24 h postmortem. A strong correlation between pH_24h and meat quality traits was observed in the current study. Deduction of pH resulted in decreasing WHC, similar to other investigations (Ngoka, et al. 1982; Juncher et al. 2001; Küchenmeister et al. 2002).

In their normal life activities, poultry can generate reactive oxygen species inside their bodies, which attack biological membranes inside the bodies, thus causing lipid hydroperoxide formation and tissues damages. The method for assessing oxidative damages to living beings is to determine TBARS or MDA content. It is statistically shown that TBARS content of meat products are more capable of indicating their rancidity than their peroxide content (Barbut 1997) and as a result TBARS contents of meat products are indirect quantifying indicators of their lipid peroxidation (Li 1999). Living beings have their own anti-oxidation mechanism, employing their blood or muscle SOD and GSH-Px, and T-AOC to enhance their antioxidative capacities, reduce their reactive oxygen species-caused damages and improve their immune capacities. The results of present study showed that LPS-induced preslaughter immunological stress reduced the T-AOC of broiler blood and muscles and tended to decrease their blood GSH-Px activities of the broilers. Breast or thigh muscle or plasma MDA content of the broiler tended to be higher when injection of LPS. Furthermore, muscle content MDA of the broilers gradually increased with the time after slaughtering. By now, there are no research reports on effects of injection of LPS on antioxidative capacities of broilers.

5. Conclusion

The results of present study suggested that preslaughter LPS-induced immunological stress could increase body oxidative damage and decrease oxidation resistance, resulting in decreasing in growth performance and meat quality of broilers; also these adverse effects could be rested with the stress intensity.

Acknowledgement

To the Chinese Government.

Disclosure statement

No potential conflict of interest was reported by the authors.