Physico-chemical properties of breast muscle in broiler chickens fed probiotics, antibiotics or antibiotic–probiotic mix

ABSTRACT

This study investigated the efficacy of antibiotics, probiotics and their combination on meat quality of breast muscle in broilers. A total of 480 male one-day-old Cobb chicks were randomly assigned to control (without additive), antibiotic growth promoter (AGP), Bioplus^® (probiotics), or AGP + Bioplus^® and raised for 42 d. Each treatment was replicated six times with 20 birds per replicate. At the end of rearing period, 10 birds were randomly selected from each treatment, slaughtered and the breast muscles were excised for meat quality analyses. The results showed that all additives influenced drip and cooking loses, pH, fat content and colour attributes of breast muscle but did not affect tenderness, muscle glycogen, moisture, crude protein and ash content of breast muscle. Both drip and cooking losses were lower in treatment groups than those in the control group. Birds fed sole probiotics had the least pH, drip loss and cooking loss compared with the other treatments. Sole probiotics-fed birds had higher lightness, redness and yellowness values and lower fat value than the other treatments on day 1 post-mortem.  The results indicate that probiotics are good substitutes for antibiotics in the diet of broiler chickens for the enhancement of meat quality.

KEYWORDS:

• Antibiotic
• antibiotic–probiotic mix
• broiler
• meat quality
• probiotic

1. Introduction

The use of antibiotics as growth promoter in livestock has been in practice for a long period (Choe et al. 2013). Due to beneficial effects of antibiotics on gut health and overall productivity of livestock, their use has become a necessary and regular practice in animal production. One of the major modes of action of antibiotics is their ability to alter gut microbial balance thereby reducing the population of pathogenic microbes (Butaye et al. 2003), and improving feed efficiency and growth. In addition, antibiotics play a vital role in improving the quality of livestock products such as enhancing and decreasing the protein and fat content of meat (Khaksefidi & Rahimi 2005). Nonetheless, there are many concerns about the safety of antibiotics on the health of human consumers. Antibiotics have residual effects on consumers as they lead to development of resistance to antibiotics (Rosyidah et al. 2011; Shazali et al. 2014).This instigated the ban on the use of antibiotics in livestock by the European Union in 2006 (Kabir 2009). The development of resistance to antibiotics created the spur to explore alternatives for improving animal health and performance without jeopardizing consumers’ health (Kabir 2009; Seo et al. 2010). Examples of alternatives to antibiotics are probiotics or directly fed microbials, prebiotics, phytobiotics, chemobiotics, symbiotics and so on (Seo et al. 2010; Pochop et al. 2011). In recent years, probiotics has been increasingly used thus expediting decline in the use of antibiotics (Seo et al. 2010). According to Fuller (1989), ‘Probiotics can be defined as a live microbial feed supplement that may beneficially affect the host animal upon ingestion by improving its intestinal microbial balance.’ In addition, probiotics organisms should be non-toxic, non-pathogenic and capable of resisting bile salts and low pH in the gastrointestinal tract in order to enhance its likelihood of survival in such environment (Fuller 1989; Fuller 1991; Stringfellow et al. 2011). Although several studies have demonstrated the beneficial effects of probiotics in maintaining gut health and promoting animal performance, there are few studies on how probiotics influence physico-chemical properties or quality of meat. Thus, the effect of probiotics on meat quality enhancement remains obscured and highly controversial. Some researchers reported beneficial effects of probiotics administration on meat quality (Ali 2010; Saad et al. 2013), while others found no improvement in meat quality when probiotics were administered (Owings et al. 1990). Given the importance of meat quality to all players involved in meat production chain, it is necessary to strike a balance between the conflicting results thus it needs further investigation to clarify the role of probiotics in meat quality in broiler chickens. Thus, the objective of this study was to investigate the effects of administering probiotics, antibiotics and their combination on physico-chemical properties of chicken breast meat.

2. Materials and methods

2.1. Ethical note

This study was conducted following the animal ethics guidelines of the Research Policy of Universiti Putra Malaysia.

2.2. Experimental design and management of birds

Four hundred and eighty 1-day-old male Cobb broiler chicks were raised on deep litter for 42 d. The experiment consisted of three treatment groups and one control group as outlined below:

(A) Basal diet (Control); (B) basal diet supplemented with 0.1 mg/kg antibiotic growth promoter (AGP); (C) basal diet supplemented with commercial probiotic 5 g/kg; (D) basal diet supplemented with AGP 0.1 mg/kg + commercial probiotic 5 g/kg. Antibiotic (AGP) used was a combination of oxytetracycline and neomycin at the concentration of 100 ppm (w/w) (Sunzen Berhad, Malaysia). The commercial probiotic (Gallipro^®) was obtained from Evonik (SEA) Pte. Ltd., Singapore; containing Bacillus subtilis (DSM 17299) at a minimum 1.6 × 10⁹ viable spores/g.

All treatments were replicated six times. Each replicate (pen) consisted of 20 chicks and they were randomly assigned to the open house with wood shavings litter. Chicks were vaccinated at the age of 7 days for Newcastle Disease Infectious Bronchitis vaccine was followed by Infectious Bursal Disease vaccine (Gumboro) at the age of 14 days by means of eye drop. The birds were provided with ad libitum feed and water. The birds were fed starter diet during 0–21 d of age while finisher diet was fed during 22–42 d of age. Diets were formulated to meet the nutrient requirements of broiler chickens in accordance to NRC (1994), as shown in Table 1. At the end of the trial, 12 birds from each treatment were slaughtered for meat quality analyses. The live weights of broiler chicken were 2.09 ± 0.01, 2.13 ± 0.01, 2.15 ± 0.01 and 2.18 ± 0.01 kg for A, B, C and D treatments, respectively.

Table 1. Composition and nutrient content of starter (d 1–21) and finisher (d 22–42) basal diets for broiler chicks (%, as fed-basis).

Ingredients	Starter	Finisher
Corn	50.6	54.9
Soybean	29.4	26.9
Wheat pollard	6.1	6.4
Crude palm oil	3.6	3.2
Fish meal	7.6	5
L-lysine	0.25	0.25
DL-methionine	0.2	0.2
Monodicalciumphosphate 21	1	1.4
Calcium carbonate	0.68	0.99
Choline chloride	0.06	0.058
Salt	0.25	0.25
Mineral mix^a	0.1	0.1
Vitamin mix^b	0.06	0.06
Antioxidant^c	0.01	0.08
Toxin binder^d	0.135	0.15
Total	100	100
Calculated analysis
Crude protein (%)	22.50	20.34
Metabolizable energy (MJ/kg)	12.22	12.18

^aMineral mix contains Fe 100 mg, Mn 110 mg, Cu 20 mg, Zn 100 mg, I 2 mg, Se 0.2 mg, Co 0.6 mg.

^bVitamin mix contains retinol 2 mg, cholicalciferol 0.03 mg, α-tocopherol 0.02 mg, menadione 1.33 mg, cobalamin 0.03 mg, thiamine 0.83 mg, riboflavin 2 mg, folic acid 0.33 mg, biotin 0.03 mg, panthothenic acid 3.75 mg, niacin 23.3 mg, pyridoxine 1.33 mg.

^cAntioxidant contains butylated hydroxyanisole (BHA).

^dToxin binder contains natural hydrated sodium calcium aluminium silicates (HSCAS).

2.3. Slaughter procedure

Upon completion of the feeding experiment, 12 birds per treatment were randomly selected and weighed prior to slaughter. The slaughter process was conducted at the Department of Animal Science abattoir, Faculty of Agriculture, Universiti Putra Malaysia. The birds were slaughtered according to a Halal slaughter procedure as outlined in the Standards Malaysia 1500: (MS1500 2009).

2.4. Evaluation of meat physico-chemical properties

All physico-chemical parameters were assessed in the breast muscles, taken at 45 min post-mortem from the dressed carcasses. Immediately after removal, approximately 30 g of the breast muscle was collected, properly labelled, vacuum packed and stored in a 4°C chiller and assigned for the determination of drip loss. The remaining muscle was cut into three equal portions. Each portion was assigned to one of three different ageing periods at 0, 1 and 7 d post-mortem. Samples from day 0 post-mortem were snap frozen in liquid nitrogen and stored at −80°C for subsequent analyses. All samples for 1 and 7 d post-mortem ageing were temporarily kept on ice, vacuum-packaged, labelled and stored in a chiller (4°C). At each ageing period, appropriate packs were removed from the chiller, and the muscle samples for each different ageing period were further cut to subsamples. All subsamples were stored at −80°C frozen storage until further analysis.

2.5. Drip loss

Fresh samples from the breast muscle at day 0 were individually weighed (approximately 30 g) and recorded as initial weight (W₁). The samples were then placed in sealed polyethylene plastic bags, vacuum-packaged, placed within a container and were stored in a chiller at 4°C. After 1 and 7 d of storage, the samples were immediately removed from the bags, gently blotted dry, weighed and recorded as W₂ (final weight). The percentage of drip loss was calculated and expressed as the percentage of differences of sample initial weight. The sample weight after 1 and 7 d of storage was divided by sample initial weight (Honikel 1998) using the following equation:

2.6. Cooking loss

The frozen subsamples of breast muscles at days 1 and 7 were transferred from the −80°C freezer into a 4°C chiller overnight to thaw. The thawed samples were individually weighed and recorded as initial weight (W₁). The samples were then placed in plastic bags and cooked in a pre-heated water bath at 80°C for 20 min in a water bath. The cooked samples were removed from the plastic bags, cooled in ice slurry for 20 min. The samples were then re-weighed and recorded as W₂ (cooked weight). The cooking loss was calculated based on the difference between the weight of raw meat and cooked meat by using the following equation:

2.7. Shear force values

Subsamples for shear force measurement were taken from the breast muscle samples that were previously cooked at 80°C for 20 min for cooking loss determination then kept in a chiller at 4°C overnight for shear force evaluation. Subsamples of 1 cm high × 1 cm width × 2 cm length dimension were sheared by the Volodkevitch bite jaw attached to a texture analyzer (TA.HD plus^®, Stable Micro System, Surrey, UK) in the centre and perpendicular to the longitudinal direction of the fibres (Sazili et al. 2005). Following overnight storage at 4°C, the cooked samples were cut into subsamples for textural analysis. Shear force values were recorded as the average of all subsamples value for each individual sample.

2.8. Colour measurement

Samples were removed from the −80°C freezer and subjected to overnight thawing at 4°C. They were removed from the packaging and allowed to bloom in air for 20 min prior to colour measurement. Meat colour measurement was conducted using a Colour Flex spectrophotometer (Hunter Lab, Reston, VA, USA). The device was initially calibrated against black and white reference tiles prior to use. L* (lightness), a* (redness) and b* (yellowness) were measured in triplicate on each sample at 1 and 7 d post-mortem. The ratios of hue angle [tan⁻¹(b*/a*)], and saturation index or chroma √(a² + b²) were also determined (Onenc & Kaya 2004).

2.9. pH muscle determination

The pH of breast muscles at three post-mortem condition times (day 0, 1 and 7) were indirectly determined using a portable pH meter (Mettler Toledo, AG 8603, Switzerland). The pH meter was calibrated at pH 4.0 and pH 7.0 prior to each use. Approximately 0.5 g of each crushed muscle sample was homogenized (Wiggen Hauser^® D-500, Germany) for 20 s in 10 ml ice cold double distilled water. The electrode attached to the pH meter was used to measure the pH of homogenates. Each sample was measured in triplicate and the average pH value calculated for each treatment.

2.10. Glycogen assay

Total glycogen content in the meat samples was determined using a Glycogen Assay Kit K646–100 (Bio Vision, USA). In the assay, glycogen was hydrolysed to glucose by glucoamylase enzyme, and was then oxidized to produce a product, which reacted with OxiRed probe. The colour generated from this reaction was measured at 570 nm wavelength using an auto microplate reader (infinite M200, Tecan, Austria). The concentrations of glycogen in the samples were calculated according to the formula below:

C = Ay/Sv (µg/µl, or mg/ml),

where C is the glycogen concentration, Ay the glycogen amount in the sample from the standard curve, and Sv the sample volume (µl) added to the sample well

2.11. Proximate composition

Proximate composition of the breast meat was determined following the procedures of AOAC (2000). Moisture was determined by drying 1 g of meat in an oven at 100–105°C until a constant weight was obtained. Crude protein was determined by the Kjeldahl method. The crude protein was obtained as 6.25 × N%. Fat content of the meat was determined by Soxhlet extraction method using petroleum ether. Ash content of the meat was determined by igniting the sample in a muffle furnace at 550°C for 3 h.

2.12. Statistical analysis

Statistical analysis was performed by SAS package Version 9.2 software (SAS 2007) using a completely randomized design procedure. A two-way design was chosen to verify the influence of probiotics, antibiotic and combination between both supplementation and storage on the data obtained for pH, muscle glycogen, shear force, drip loss, cooking loss and colour. Data obtained for proximate composition was subjected to ANOVA model of completely randomized design. Means were separated by Duncan's multiple range tests.

3. Results and discussion

3.1. Drip and cooking loss

The effect of probiotic and antibiotic supplementation on drip and cooking losses in chicken breast meat are shown in Table 2. Irrespective of the treatment, cooking loss increased from day 1 to 7 d post-mortem. No difference (p > .05) in drip loss was observed in breast muscles at 24 h post-mortem among birds fed the control and those fed diet with additives. At 7 d post-mortem, drip loss was lower (p < .05) in the birds fed sole probiotics (diet C) than other treatments.

Table 2. Drip loss and cooking loss of breast meat in broiler chickens fed probiotics, antibiotics and antibiotic–probiotic mix (mean ± SE, n = 12).

Diet	Drip loss (%)		Cooking loss (%)
	1 day	7 day	1 day	7 day
A	3.13 ± 0.23^ay	4.99 ± 0.26^ax	20.16 ± 0.89^ay	22.11 ± 0.67^ax
B	2.56 ± 0.16^ay	4.61 ± 0.16^abx	20.14 ± 0.95^ax	20.43 ± 1.04^abx
C	2.98 ± 0.23^ay	4.38 ± 0.10^bx	18.91 ± 0.44^ax	18.35 ± 0.70^bx
D	2.87 ± 0.21^ay	4.72 ± 0.13^abx	18.50 ± 0.39^ay	20.85 ± 0.46^aby

Notes: A = basal diet (control); B = basal diet supplemented with 0.1 mg/kg AGP; C = basal diet supplemented with commercial probiotic 5 g/kg; D = basal diet supplemented with AGP 0.1 mg/kg + commercial probiotic 5 g/kg.

^a,bMeans within the same column for each parameter with different superscripts are significantly different (p < .05).

^x,yMeans within the same row for each parameter with different superscripts are significantly different (p < .05).

There was no significant difference (p > .05) in the cooking loss among the treatments after 24 h chilling storage. However, birds fed diet C (18.35%) had significantly lower cooking loss than group A (22.11%) at 7 d post-mortem (p < .05). On the other hand, cooking loss was not significantly different (p > .05) among birds fed diet B, D in comparison with birds fed basal diet at 7 d post-mortem. Drip and cooking losses serve as useful indicators for the water holding capacity (WHC) of meat. WHC influences meat appearance prior cooking, meat cooking characteristics and as well as juiciness. Among the additives, the higher WHC observed in birds fed sole probiotics (Diet C) supported the findings of Zhou et al. (2010) who reported reduction in drip loss in breast muscle of Guangxi yellow chicken birds fed Bacillus coagulan. Regardless of the treatment, drip loss increased across the ageing period. This could be due to the disruption of the collagen and other myofibrillar protein matrix during the process of ageing which makes the myofibrillar proteins lose their ability to hold water. In addition, Lawson (2004) reported that water could be forced out of myofibrils due to contraction during rigor mortis and enters into channels formed between the muscle fibre and the cell membrane because of the loss of intracellular matrix by action of calpain; such water could flow to the exterior as weep or drip. Cooking loss in breast meat was not influenced by dietary treatments at 24 h post-mortem. However, differences in cooking loss were observed on 7 d post-mortem. Ali (2010) reported similar findings where birds fed probiotics had lower drip loss than the control birds from 0 to 8 d post-mortem. In contrast, Pelicano et al. (2003) reported that probiotic supplementation had no effect on cooking loss in broiler breast meat. Among the additives, birds fed sole probiotics (Diet C) had lower cooking loss than other treatments. The profound influence of various additive on cooking and drip losses could be an advantage in enhancing the juiciness of the breast meat since higher juiciness is mostly derived from meat that exhibits lower drip and cooking losses. In addition, lower drip loss observed in birds fed additives could guard against loss of water soluble nutrients associated with drip loss.

3.2. Shear force values

In relation to the texture of the breast meat (Table 3), no significant differences (p > .05) were found among treatments at 1 and 7 d post-mortem. There was a general decrease in shear force as ageing progressed though it was not significant. Tenderness is one of the most important meat quality traits influenced by various factors. The decrease in shear force (increase in tenderness) observed on 7 d post-mortem for all treatments was due to rigor resolution caused by enzymatic breakdown of collagen holding muscle fibre together in the course of ageing. The results obtained in the present study corroborate earlier findings of Pelicano et al. (2003), and Ali (2010) who observed that probiotic supplementation does not influence shear force values in chicken breast meat. However, the shear force values observed in this study were higher than values reported in the findings of Lyon and Lyon (1990) and Simpson and Goodwin (1974) but were in range of values reported by Ali (2010).

Table 3. Shear force values of breast meat in broiler chickens fed probiotics, antibiotics and antibiotic–probiotic mix (mean ± SE, n = 12).

Diet	Shear force (N)
	1 day	7 day
A	10.08 ± 0.01	9.18 ± 0.01
B	9.55 ± 0.02	8.48 ± 0.01
C	9.41 ± 0.01	8.29 ± 0.01
D	9.38 ± 0.01	8.32 ± 0.02

Notes: A = basal diet (control); B = basal diet supplemented with 0.1 mg/kg AGP; C = basal diet supplemented with commercial probiotic 5 g/kg; D = basal diet supplemented with AGP 0.1 mg/kg + commercial probiotic 5 g/kg.

3.3. Colour values

The colour coordinates of the breast meat of broiler chickens fed different dietary treatments are presented in Table 4. At day 1 post-mortem, the control samples indicated lower lightness (L*) and this was significantly different (p < .05) from other treatments. There were no significant differences (p > .05) among birds fed probiotics, antibiotics and their mix. In the same vein, at 7 d post-mortem, there was no significant difference among the treatments. There were significant differences in redness (a*) of breast meat among the treatments at 1 d post-mortem. However, the redness (a*) of breast meat did not differ among the treatments at 7 d post-mortem. Yellowness (b*) of breast meat was not significantly different (p > .05) among the treatments at 7 d post-mortem but differed (p < .05) at 1 d post-mortem. For the Chroma (C*) and Hue (H*), there were significant differences (p < .05) among the treatments at 1 d post-mortem with no difference (p > .05) observed at 7 d post-mortem. Colour or visual appearance is certainly one of the most important sensory attributes that influence consumers’ acceptance of meat and meat products (Adeyemi & Sazili 2014). Supplementation of antibiotics, probiotics and their mix had positive impact on colour attributes of breast meat in broiler chickens. However, this positive effect was limited to 1 d post-mortem as the control samples exhibited the same colour attributes at 7 d post-mortem. This observation corroborates the report of Ali (2010) who observed that dietary supplementation of probiotics positively influenced colour attributes in chicken breast meat. However, the present results contradict the findings of Pelicano et al. (2003), who reported no difference in the colour of breast meat among the control and various probiotic treatments. The profound influence of antibiotics, probiotics and their mix could be due to their effects on reduction of muscle glycogen content through anaerobic glycolysis which resulted in lactic acid accumulation and subsequent decline in muscle pH at 24 h post-mortem. The lack of significant difference in colour attributes among the treatments at 7 d post-mortem could be explained by the ultimate pH (pHu) which is normally attained within the first 24 h in birds. Thus, no subsequent pH decrease was possible.

Table 4. Colour coordinates of breast meat in broiler chickens fed probiotics, antibiotics and antibiotic–probiotic mix (mean ± SE, n = 12).

Colour coordinate	Ageing (day)	Dietary treatments
		A	B	C	D
L* (Lightness)	1	52.14 ± 0.50^by	53.93 ± 0.96^ax	54.42 ± 0.34^ax	54.56 ± 0.40^ax
	7	55.73 ± 0.97^ax	55.48 ± 0.55^ax	55.50 ± 0.73^ax	54.30 ± 0.35^ax
a* (Redness)	1	6.89 ± 0.16^ay	6.02 ± 0.16^bx	5.83 ± 0.16^bx	6.18 ± 0.18^by
	7	5.26 ± 0.28^ax	5.00 ± 0.17^ax	4.79 ± 0.15^ax	5.08 ± 0.18^ax
b* (Yellowness)	1	15.35 ± 0.49^bx	16.59 ± 0.57^abx	17. 55 ± 0.26^ax	17.91 ± 0.60^ax
	7	16.97 ± 0.35^ax	17.24 ± 0.21^ax	17.14 ± 0.49^ax	17.97 ± 0.65 ^ax
C* (Chroma)	1	16.43 ± 0.46^bx	16.43 ± 0.46 ^bx	19.49 ± 0.25 ^ax	18.97 ± 0.57^ax
	7	17.79 ± 0.33^ax	17.79 ± 0.33^ax	17.80 ± 0.50^ay	18.68 ± 0.66^ax
H* (Hue)	1	1.04 ± 0.01^bx	1.041 ± 0.01^bx	1.09 ± 0.01^ax	1.08 ± 0.01^ax
	7	1.01 ± 0.01^ax	1.01 ± 0.005^ax	1.02 ±  0.01^ay	1.01 ± 0.01^ay

Notes: A = Basal diet (Control); B = Basal diet supplemented with 0.1 mg/kg AGP; C = Basal diet supplemented with commercial probiotic 5 g/kg; D = Basal diet supplemented with AGP 0.1 mg/kg + commercial probiotic 5 g/kg.

^a,bMeans within the same column for each parameter with different superscripts are significantly different (p < .05).

^x,yMeans within the same row for each parameter with different superscripts are significantly different (p < .05).

3.4. pH values

The pH values of breast meat in broiler chickens fed diet supplemented with different probiotics and antibiotics are shown in Table 5. pH values significantly decreased (p < .05) from 0 to 1 d post-mortem and increased afterwards. The rise of the pH value during post-mortem storage (7 days) of meat could be due to the progressing alkalization caused by spoilage organisms and the release of basic products of protein breakdown throughout the post slaughter endogenous changes (Stanisic et al. 2012). At days 0 and 1 post-mortem, birds fed probiotic, antibiotics and their mix had lower pH (p < .05) than those from the control group. At 7 d post-mortem, the birds fed probiotic (diet C) presented lower pH value that was not significantly different (p > .05) in comparison with birds fed antibiotic (diet B). The decrease in pH is due to conversion of muscle glycogen to lactic acid post-mortem. Normally, when a bird is exsanguinated and consequently suffer hypoxia, the muscle cells will eventually have to resort to anaerobic glycolysis for their remaining metabolic activities which serves as the only source of energy for the post-mortem muscles (Sabow et al. 2015). Because of this, glycogen stores are depleted as they are converted to lactic acid causing the pH to drop. The muscle pH is an important indicator of various quality traits in meat. In this study, the pH values observed for all treatments were within the range reported earlier in chickens (Castellini et al. 2002; Qiao et al. 2002; Karaoğlu et al. 2006). The significantly lower pH values observed in probiotic, antibiotic and their mix groups at 24 h post-mortem were in line with the report of Karaoğlu et al. (2006) who found that probiotic treatment influenced pH decline in broiler breast muscles particularly during the first 24 h post-mortem. The low pH values observed for probiotic, antibiotic and probiotic–antibiotic mix contradict the findings of Aksu et al. (2005) where high muscle pH values were observed following probiotic treatments.

Table 5. pH values of breast meat in broiler chickens fed probiotics, antibiotics and antibiotic–probiotic mix (mean ± SE, n = 12).

Diet	pH values (unit)
	0 day	1 day	7 day
A	6.47 ± 0.05^ax	5.99 ± 0.05^ay	6.00 ± 0.04^ay
B	6.41 ± 0.04^abx	5.83 ± 0.04^by	5.87 ± 0.03^by
C	6.34 ± 0.02^bx	5.77 ± 0.05^cz	5.86 ± 0.04^by
D	6.38 ± 0.04^abx	5.76 ± 0.03^cz	5.90 ± 0.03^aby

Notes: A = Basal diet (Control); B = Basal diet supplemented with 0.1 mg/kg AGP; C = Basal diet supplemented with commercial probiotic 5 g/kg; D = Basal diet supplemented with AGP 0.1 mg/kg + commercial probiotic 5 g/kg.

^a,b,cMeans within the same column for each parameter with different superscripts are significantly different (p < .05).

^x,y,zMeans within the same row for each parameter with different superscripts are significantly different (p < .05).

3.5. Glycogen content

The effect of probiotics and antibiotics supplementation on glycogen concentration was significant at 1 d post-mortem but insignificant at 0 and 7 d post-mortem (Table 6). At 1 d post-mortem, control birds had lower glycogen concentration (p < .05) than those subjected to the other dietary treatments. The conversion of muscle glycogen to lactic acid post-mortem is necessary for the attainment of pHu (Adeyemi & Sazili 2014). The pHu in turn affects myriad biochemical and physiological processes in the post-mortem quality, which influence various meat quality traits (Adeyemi & Sazili 2014). Regardless of the treatment, there was a general decline in muscle glycogen from 0 to 7 d post-mortem. This is expected as the only source of energy to muscle cells following exsanguination of a bird is through the anaerobic glycolysis, which demands the conversion of glycogen to lactic acid in the post-mortem muscle (Adeyemi & Sazili 2014).

Table 6. Glycogen contents of breast meat in broiler chickens fed probiotics, antibiotics and antibiotic–probiotic mix (Mean ± SE, n = 12).

Diet	Glycogen concentration (mg/kg meat)
	0 day	1 day	7 day
A	0.66 ± 0.03^az	0.31 ± 0.02^by	0.17 ± 0.01^ax
B	0.67 ± 0.03^az	0.38 ± 0.013^ay	0.16 ± 0.01^ax
C	0.68 ± 0.02^az	0.38 ± 0.02^ay	0.15 ± 0.01^ax
D	0.66 ± 0.02^az	0.37 ± 0.01^ay	0.16 ± 0.01^ax

Notes: A = Basal diet (Control); B = Basal diet supplemented with 0.1 mg/kg AGP; C = Basal diet supplemented with commercial probiotic 5 g/kg; D = Basal diet supplemented with AGP 0.1 mg/kg + commercial probiotic 5 g/kg.

^a,bMeans within the same column for each parameter with different superscripts are significantly different (p < .05).

^x,y,zMeans within the same row for each parameter with different superscripts are significantly different (p < .05).

3.6. Proximate composition

Except for fat content, proximate components were not significantly different (p > .05) across dietary treatments (Table 7). The breast meat from the control birds had the highest fat content which was significantly different (p < .05) from other treatments. Nevertheless, there was no significant difference in fat contents (p > .05) among birds fed diets B, C and D. The lower (p < .050 fat content observed in the probiotic, antibiotic and antibiotic–probiotic mix birds as compared to the control group are in line with the reports of Pietras (2001), Khaksefidi and Rahimi (2005), Endo and Nakano (1999). Contrary to earlier reports on increase in protein and ash contents in probiotic fed birds (Nahashon et al. 1992, 1994; Mohan et al. 1996; Thomke & Elwinger 1998; Khaksefidi & Rahimi 2005), the present results suggest that probiotics, antibiotics and probiotic–antibiotic mix did not influence the protein, moisture and ash content of breast meat in broiler chickens. The implication of this result is that mineral retention and protein efficiency ratio were unaffected by the probiotic, antibiotic or antibiotic–probiotic mix treatments. The disposition of the present findings supports the report of (Joy & Samuel 1997) that carcass protein was not influenced by supplementation of Lactobacillus sporogenes in broiler diets.

Table 7. Proximate composition of breast meat in broiler chickens fed probiotics, antibiotics and antibiotic–probiotic mix (Mean ± SE, n = 12).

Diet	Proximate composition (%)
	Moisture	Crude protein	Fat	Ash
A	69.99 ± 0.61^a	23.38 ± 0.25^a	5.27 ± 0.12^a	1.35 ± 0.02^a
B	70.45 ± 0.40^a	23.85 ± 0.19^a	4.26 ± 0.14^b	1.39 ± 0.05^a
C	70.68 ± 0.38^a	23.94 ± 0.39^a	3.95 ± 0.14^b	1.34 ± 0.05^a
D	70.50 ± 0.38^a	24.20 ± 0.23^a	3.92 ± 0.05^b	1.31 ± 0.05^a

Notes: A = Basal diet (Control); B = Basal diet supplemented with 0.1 mg/kg AGP; C = Basal diet supplemented with commercial probiotic 5 g/kg; D = Basal diet supplemented with AGP 0.1 mg/kg + commercial prob

^a,b,cMeans within a column with different superscripts are significantly different (p < .05)

4. Conclusion

This study showed that among the additives, birds fed diet supplemented with commercial probiotic 5 g/kg beneficially influenced on meat quality traits in terms of pH, WHC and colour coordinates in comparison with those fed basal diet. However, all additive influenced positively on muscle glycogen concentration at d 1 post-mortem as well as carcass fat content. Thus, it is suggested that probiotics alone or mix with antibiotic could be used in the diet of broiler chickens without deleterious effect on meat quality traits.

Acknowledgements

Thanks to Dr Girish from Evonik (SEA) Pte. Ltd., Singapore for allowing us to publish this work. Authors’ contribution: LTC contributed to the idea, design, and execution of the study. ABS, NRA and KYK performed the meat quality analysis, while, ABS, NRA and AQS did the glycogen assay. ANMZ and SN assisted in all broiler chicken procedures for the experiment. ABS and NRA were responsible for the statistical analysis. All authors contributed equally to the write-up of the final manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.