Preslaughter Transport Effect on Broiler Meat Quality and Post-mortem

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Preslaughter Transport Effect on Broiler Meat Quality and Postmortem Glycolysis Metabolism of Muscles with Different Fiber Types Xiaofei Wang, Jiaolong Li, Jiahui Cong, Xiangxing Chen, Xudong Zhu, Lin Zhang, Feng Gao, and Guanghong Zhou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04193 • Publication Date (Web): 07 Nov 2017 Downloaded from http://pubs.acs.org on November 10, 2017

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Journal of Agricultural and Food Chemistry

Preslaughter Transport Effect on Broiler Meat Quality and Postmortem Glycolysis Metabolism of Muscles with Different Fiber Types

Xiaofei Wang,†,‡ Jiaolong Li,† Jiahui Cong,† Xiangxing Chen,† Xudong Zhu,†,‡ Lin Zhang,*,† Feng Gao, † Guanghong Zhou†

†

College of Animal Science and Technology, Jiangsu Key Laboratory of Animal

Origin Food Production and Safety Guarantee, Jiangsu Key Laboratory of Gastrointestinal Nutrition and Animal Health, Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China ‡

College of Science, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China

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ACS Paragon Plus Environment


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ABSTRACT: Preslaughter transport has been reported to decrease quality of breast

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meat but not thigh meat of broilers. However, tissue-specific differences in glycogen

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metabolism between breast and thigh muscles of transported broilers have not been

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well studied. We thus investigated the meat quality, adenosine phosphates, glycolysis,

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and bound key enzymes associated with glycolysis metabolism in skeletal muscles

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with different fiber types of preslaughter transported broilers during summer.

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Compared to 0.5 h transport, 3 h transport during summer decreased ATP content,

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increased AMP content and AMP/ATP ratio, accelerated glycolysis metabolism via the

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upregulation of glycogen phosphorylase expression accompanied by increased

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activities of bound glycolytic enzymes (hexokinase, pyruvate kinase and lactate

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dehydrogenase) in pectoralis major muscle, which subsequently increased the

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likelihood of pale, soft, and exudative like breast meat. On the contrary, 3 h transport

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only induced a moderate glycolysis metabolism in tibialis anterior muscle, which did

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not cause any noticeable changes in quality traits of the thigh meat.

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KEYWORDS: Broiler; Transport; Meat quality; Fiber type, Glycolysis metabolism

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INTRODUCTION

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Over the past decades, the production of poultry meat has been growing rapidly

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worldwide. However, today's consumers have become increasingly concerned about

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the quality and safety of meat. As the pale, soft, exudative (PSE) pork, PSE-like

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poultry meat is characterized by its lighter appearance, softer texture, lower water

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holding capacity, excessive yield losses, and formation of soft gels in comparison to

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normal poultry meat.1,2 Nowadays, the PSE-like meat is a growing problem for

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modern poultry industry. It has been estimated that PSE-like meat represents 5 to 47%

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of chicken meat in Europe, with a greater occurrence during hot climate.3 The results

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of a survey study also showed that the average incidence of PSE-like chicken meat in

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China was more that 20%, and this number was greater in some regions during hot

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summer, resulting in huge economic losses to the Chinese poultry industry. 4

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The preslaughter transport, especially in summer, is considered to be one of the

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most important factors leading to PSE-like chicken meat.5,6 Previous studies showed

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that transport (more than 3 hours) during summer accelerated postmortem anaerobic

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glycolysis of muscle glycogen, especially in pectoralis major (PM) muscle richer in

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fast glycolytic fibers (IIb type fiber), resulting in the accumulation of lactate and

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further a rapid postmortem pH decline while carcass temperatures are still high.6,7 In

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general, the breakdown of muscle glycogen is the first step of glycolysis metabolism.

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Glycogen phosphorylase is a key enzyme involved in glycogen breakdown, which

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catalyzes the rate-limiting step of glycogenolysis, cleaves 1-4 linkages to remove

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glucose molecules from the glycogen chain.8,9 More importantly, the muscle

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glycolysis metabolic pathway is also highly regulated by some rate-limiting enzymes,

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including hexokinase, pyruvate kinase and lactate dehydrogenase.10,11 These enzymes

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catalyze diverse reactions of the glycolytic pathway, including the conversion of 3



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glucose to glucose-6-phosphate, phosphoenol-pyruvate to pyruvic acid, pyruvic acid

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to lactate, respectively.10,12

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In addition, it is known that the breast and thigh muscles of broiler chickens

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possess different fiber types with different energy metabolic characteristics.7,8,13 To

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market-age Arbor Acres broilers, their PM (main part of breast) muscle consists of

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100% type IIb fibers (fast twitch-glycolytic fiber), but the tibialis anterior (TA, main

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part of thigh) muscle consists of 19% type I (slow twitch-oxidative), 21% type IIa

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(fast twitch-oxidative-glycolytic) and 60% type IIb fibers.7,13 Type I fibers are

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responsible for slow speed contractions and utilize aerobic glycolysis for ATP

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production; IIa fibers make use of both both aerobic and anaerobic glycolytic

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pathways for energy production, whereas IIb fibers rely primarily on anaerobic

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glycolysis for energy production.8,14 To date, tissue-specific differences in meat

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quality, muscle glycolysis, and bound key enzymes associated with glycolysis

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metabolism in transported broilers have not been well studied. We thus hypothesize

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that the skeletal muscles with different fiber types differ in the amount of glycogen

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and glycogen metabolism in transported broilers. Therefore, the present study was set

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up to investigate the preslaughter transport effect on broiler meat quality, adenosine

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phosphates, glycolysis, and bound key enzymes associated with glycolysis

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metabolism in skeletal muscles with different fiber types.

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MATERIALS AND METHODS Add “studies and” after “animal”

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Birds and Transportation. All experimental procedures followed the ethical

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guidelines for animal studies that approved by the Institutional Animal Care and Use

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Committee of Nanjing Agricultural University. A total of 192 Arbor Acres male

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broilers (42-day-old) with the BW range from 2.4 to 2.6 kg were used for this

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transport trial. All these broilers were fed the same starter (days 1 to 21) and grower 4


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(days 22 to 42) diets from the first day to market age in a commercial farm (Nanjing,

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China). At time of transport, after an 8 h overnight feed withdrawal, the broilers were

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randomly categorized into 2 treatment groups: (1) a 0.5 h transport group (as a lower

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stress control group), and (2) a 3 h transport group. In the modern poultry industry,

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both chicken slaughter and meat processing are forbidden within the farms and

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therefore require transport to slaughterhouse, and we thus chose a 0.5 h of transport

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group as a lower-stress control, as previously reported.7,15 Each group consisted of 8

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replicates with 10 birds placed in one crate (0.73 m × 0.54 m × 0.26 m), and were

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randomly distributed in two identical commercial trucks. The total load was around

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1200 birds per truck. The transport durations were 0600 to 0630 h for the control

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group, and 0600 to 0900 h for the 3 h transport group, respectively. During the

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transport period, the temperatures and relative humidity (RH) inside trucks were

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27.5-29.3 °C and 78.5-82.6% in the control group, and 27.2-35.1 °C and 78.8-90.3%

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in the 3 h transport group. The transport distance was ∼240 km with an average speed

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of 80 km/h. There was no pause and break throughout entire transport process. No

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feed and water was supplied during the transport duration.

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Slaughter and Sampling Procedures. After transported to the slaughterhouse, all

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broilers were allowed to rest 1 h in a shady corner without feed and water supply.

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Immediately after rest, one male bird from each crate (replicate) of each treatment (n

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= 8) was randomly selected, stunned electrically (50 V: alternating current, 400 Hz for

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5 s each one) and then immediately slaughtered via exsanguination. Within 15 min,

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∼0.5 g muscle from left PM and TA muscles were put into 0.5-mL RNAase-free tubes,

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and then quickly frozen in liquid nitrogen and stored at -80 °C for RNA extraction.

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Two rectangular samples (0.3 cm × 0.3 cm × 0.5 cm in size) from left PM and TA

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muscles were also collected. Some were quickly frozen in liquid nitrogen and stored 5



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at −80 °C for nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR)

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staining, and the others were fixed overnight in 4% paraformaldehyde in 0.1 M PBS

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(pH 7.4) for periodic acid-Schiff (PAS) staining. Meanwhile, another ~5.0 g of sample

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from left PM and TA were frozen in liquid nitrogen for further analysis of muscle

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adenosine phosphates, glycogen and lactate contents as well as the activities of the

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key enzymes associated with muscle glycolysis metabolism. The entire right side of

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breast and thigh muscles were stored at 4 °C for determination of meat quality traits

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and low-field nuclear magnetic resonance (NMR) transverse relaxation (T2) times at

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24 h postmortem.

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Meat Quality Measurements. Muscle pH was measured at 45 min (pH45min) and

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24 h postmortem (pH24h) directly on the carcasses using a HI9125 portable waterproof

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pH/ORP meter (Hanna Instruments, Cluj-Napoca, Romania). The pH probe was

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inserted at an angle of 45 degrees into the samples, and washed with ultrapure water

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between samples. Each sample was measured 3 times at various points and their

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average value was taken as the final result.

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Meat color (L* = lightness, a* = redness, b* = yellowness) was measured at 24 h

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postmortem using a Minolta CR-400 chromameter (Konica Minolta Sensing Inc.,

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Osaka, Japan). Each sample was determined at two different locations on the freshly

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cut surface of each sample, and the average value was used.

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Drip loss was measured as previous described.16 Briefly, ~30 g of regular-shaped

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muscle was weighted and placed in an airtight container. All samples were stored at

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4 °C. After 24 h, the surface moisture of fillets was absorbed with filter paper and

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reweighed. Drip loss rate (%) = [(initial weight-final weight)/initial weight] ×100%.

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Histological Analysis. Frozen muscles were embedded in optimal cutting

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temperature (OCT) compound (Tissue Tek, Sakura), and cross sections (10 µm thick) 6


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were cut with a cryostat (CM1900, Leica, Wetzlar, Germany) at a temperature of

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−27 °C. Some cryosections were mounted on 3-aminopropyltriethoxysilane

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(APTS)-treated glass slides and processed for NADH-TR staining, as previously

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described.17 Briefly, the cryosections were incubated at 37 °C for 2 h in staining

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solution containing 0.05 M Tris–HCl (pH 7.4), 1 mg/mL nitroblue tetrazolium (NBT;

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Solarbio Inc., Beijing, China), and 0.8 mg/mL nicotinamide adenine dinucleotide

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(ߚ-NADH disodium salt hydrate; Sigma-Aldrich Inc., St. Louis, MO, U.S.A.).

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Paraformaldehyde-fixed muscle was embedded in paraffin, and then serially sectioned

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into 8 µm cross sections. The dewaxing sections were PAS stained for glycogen as

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previously described.18 Images were taken using a Nikon Eclipse 80i microscope

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(Nikon Corporation, Tokyo, Japan) equipped with NIS-Elements F 3.00 imaging

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software.

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NMR Transverse Relaxation (T2) Measurements. The low-field NMR spin-spin

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relaxation measurements were performed on a Niumag pulsed NMR analyzer (PQ001;

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Niumag Electric Corporation, Shanghai, China) according to a previous method with

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some modifications.19,20 The analyser was operated at a resonance frequency of 22.6

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MHz at 32 °C. The NMR instrument was equipped with a 15 mm

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variable-temperature probe. A 2.0 g sample (weighed exactly) free of fat and

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connective tissues was cut from each PM and TA muscles 24 h post mortem and

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placed in a cylindrical glass tube (14 mm in diameter and 5 cm high) and then inserted

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into

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Carr–Purcell–Meiboom–Gill sequence with a ߬-value (time between 90° pulse and

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180 ° pulses) of 150 µs. Data from 4096 echoes were acquired as 32 scan repetitions

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with a repetition for 1 s with a multiexponential model using the programme of

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MultiExp Inv Analysis (Niumag Electric Corporation, Shanghai, China). Three

the

probe.

Transverse

relaxation

(T2)

was

7


measured

using

the


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relaxation times (T2b, T21 and T22) and their corresponding water proportions (P2b, P21

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and P22) were recorded.

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Measurement of Muscle Adenosine Phosphates. The contents of muscle

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adenosine phosphates (ATP, adenosine triphosphate; ADP, adenosine diphosphate; and

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AMP, adenosine monophosphate) were evaluated by high-performance liquid

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chromatography (HPLC) method as described previously with minor modifications.21

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Briefly, 0.3 g of frozen muscle sample was homogenized in 1.5 mL of precooled 7%

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perchloric acid at 13500 rpm for 30 s and then centrifuged at 15000 × g at 4 °C for 10

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min. The supernatant was neutralized with 1.03 M KOH and centrifuged again (15000

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×g, 4 °C for 10 min) to remove KClO4. After filtered with a 0.45 µm membrane, 10

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µL sample solution was injected into a Waters-2695 Alliance HPLC system (Waters

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Corporation, Milford, MA, U.S.A.). The chromatography was performed on a Waters

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SunFire C18 column (250 mm × 4.6 mm, 5 µm) at 30 °C. The mobile phase was a

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mixture of methanol and phosphate buffer (13.5:86.5, volume ratio), and the flow rate

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was 1 mL/min. UV detection was carried out at 254 nm. The standard samples of

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5′-adenosine triphosphate disodium salt, 5′-ADP sodium salt, and 5′-AMP sodium salt

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were purchased from Sigma-Aldrich, Inc. (St. Louis,MO, U.S.A.).

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Measurements of Muscle Lactate, Glycogen and Glycolytic Potential. About

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0.50 g frozen muscle (weighed exactly) was homogenized for 1 min in 4.5-mL ice

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cold saline, and then centrifuged for 10 min at 2700 × g at 4 °C. The supernatant was

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used for measurement of lactate content using a commercial lactate kit (Nanjing

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Jiancheng Bioengineering Institute, Nanjing, China). Measurement method of muscle

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glycogen content was conducted as previously described.15 The GP was calculated

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according to the formula: GP = 2 × [glycogen] + [lactate], and expressed as µmol of

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lactate equivalent per g of fresh muscle.22 8


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RNA Extraction and Real-Time PCR Analysis. Total RNA was extracted from

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frozen muscle samples using RNAiso Plus reagent (TaKaRa Biotechnology Co. Ltd.,

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Dalian, China). The purity of the total RNA was measured by a Nanodrop ND-1000

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spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, U.S.A.). The integrity

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of the total RNA was checked by evaluating the 28S and 18S bands in 1.5% agarose

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gels stained with ethidium bromide. Reverse transcription was conducted with the

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PrimeScriptTM RT Master Mix (TaKaRa Biotechnology Co. Ltd., Dalian, China).

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The pairs of oligonucleotide primers specific to glycogen phosphorylase (forward 5’-

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GTTTGACTCCTTCCCTGACC -3’, reverse 5’- AATGTCTGCTTGGTGATGTCC

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-3’; amplicon size, 141 bp), and the housekeeping gene β-actin (forward 5’-

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ATCCGGACCCTCCATTGTC -3’, reverse 5’- AGCCATGCCAATCTCGTCTT -3’;

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amplicon size, 120 bp) were used. Real-time PCR was performed using ABI 7500

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Real-Time PCR System (Applied Biosystems, Foster City, CA, U.S.A.) with SYBR®

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Premix Ex TaqTM Kit (Takara Biotechnology Co. Ltd., Dalian, China) as follows:

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one cycle at 95 °C for 30 s; 40 cycles at 95 °C for 5 s, and 60 °C for 30 s. All of the

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samples were run in triplicate, and the expression of target genes relative to β-actin

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were analyzed using 2-△△Ct method.23

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Activity

Analysis of Muscle Glycolytic Key Enzymes. Approximately 0.5 g

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frozen muscle sample was homogenized in a centrifuge tube with 4.5 mL 0.85%

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ice-cold saline and then centrifuged at 3500 × g for 10 min at 4 °C. The enzyme

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activities of hexokinase (HK), pyruvate kinase (PK), and lactate dehydrogenase (LDH)

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in the supernatant were evaluated using commercial kits (Nanjing Jiancheng

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Bioengineering Institute, Nanjing, China). The detection principle of HK activity is

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based on the coupling ribulose-5-phosphate formation from glucose 6- phosphate to

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the reduction of nicotinamide adenine dinucleotide phosphate (NADP+).24 The 9



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detection principles of PK and LDH activities are based on the decreased rate of

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nicotinamide adenine dinucleotide hydrate (NADH) during the conversion of

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phosphoenol pyruvate into pyruvate, and the conversion of pyruvate into lactate.25,26

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Statistical analysis. The statistical analysis of the data was performed using SAS

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statistical software (SAS, 2002; SAS Institute, Inc., Cary, NC). Data from eight

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replications were used for calculating means and standard errors (SE) and were

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presented as mean ± SE. The normal distribution of data was initially tested by the

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Shapiro–Wilk test. All data were normally distributed. The statistical significance of

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comparisons between the 2 experimental treatments (control vs. 3 h transport group)

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or 2 muscles (PM vs. TA) was performed by Student's t-test using the JMP version 5

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software at the significant levels of P < 0.05.

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RESULTS AND DISCUSSION

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Meat Quality. During the preslaughter transport, the broilers may be exposed to a

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variety of potential stressors, including the thermal changes of transport

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microenvironment, fasting, withdrawal of water, acceleration, vibration, motion,

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impacts, social disruption and noise, which influence their degree of comfort, energy

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metabolism, meat quality, and even mortality.27 Previous studies have showed that the

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breast muscles of 3 h transported Arbor Acres broilers showed a lower pH, higher

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cooking loss and shear force value at 24 h postmortem.6,28 On the contrary, another

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study reported that 4 h transport induced higher pH at 0, 2 and 24 h postmortem, in

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breast meat of both Ross 308 and Naked Neck strains.)29 Inconsistent results from

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these previous studies may be ascribed to the differences in transport seasons (hot

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summer, vs. comfortable spring).

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In the current study, 3 h transport decreased (P < 0.05) pH at 45min postmortem,

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and increased (P < 0.05) the L* value and drip loss of breast muscle at 24 h 10


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postmortem in comparison to those of the control group (Table 1), indicating that the

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breast muscle are prone to likelihood of PSE-like meat owing to an acceleration of

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anaerobic glycolysis at slaughtering which persists in muscle after animal death while

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the carcass is still hot, leading to accumulation of protons from ATP-hydrolyzing

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along with a rapid pH drop early postmortem.30,31 After slaughtered, accumulation of

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lactate and protons, sourced from transport-induced rapid anaerobic glycolysis, cannot

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be timely removed from the blood by liver or other tissues, which may explain why

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pH at 24 h postmortem in the PM muscle of 3 h transport broilers was still lower.

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Similarly, Simões et al. also reported that preslaughter transport during summer

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increased the occurrence of PSE-like breast meat in broilers.32 However, 3 h transport

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did not affect the overall quality traits of thigh meat (P > 0.05; Table 1). Moreover,

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breast meat had a higher drip loss, and lower pH24h and a* value than those in thigh

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meat (P < 0.05). The most probable reason for the difference in meat quality between

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breast and thigh meat under stressful conditions was that they possess different

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muscle fiber types and different energy metabolic characteristics.7,8 Figure 1 shows

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the characteristics of fiber types between PM and TA muscles of market-age Arbor

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Acres broilers. The PM muscle is composed of only type IIb fibers (Figure 1A), but

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the TA muscle is composed of type I, type IIa and type IIb fibers (Figure 1B), which

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is consistent with findings reported previously.7,13 Type I fibers are small fibers

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containing many mitochondria and myoglobin, which rely primarily on aerobic

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glycolysis; IIa fibers, with a mid-size, make use of both aerobic and anaerobic

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glycolytic pathways for energy production, whereas type II fibres are large fibers

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containing few mitochondria and myoglobin, which produce energy primarily by

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anaerobic glycolysis.8,14 In this study, we chose these two different muscles because

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they possess different muscle fiber distributions and energy metabolism patterns. 11



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NMR Transverse Relaxation (T2) Times. The proton NMR relaxation is regarded

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as a very powerful tool for determination of water-holding capacity (WHC) of fresh

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meat because it gives a direct measure of the proportion of water in the meat that is

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susceptible to be lost as drip.33 Figure 2 presents a typical NMR spin-spin relaxation

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times (T2) curve. As shown in Figure 2, we detected three relaxation proportions T2b,

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T21 and T22 in both breast and thigh meat, reflecting the bound water, immobilized

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water and free water in chicken meat, respectively, which also represents the mobility

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of water fractions from most tightly bound to most loosely bound.19,34 Approximately

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94% water in fresh meat was immobilized water, which mainly exists in spaces

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between the thick filaments of myosin and the thin filaments of actin/tropomyosin,

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and is highly correlated with the WHC of meat.20,35,36

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The data of NMR spin-spin relaxation times (T2) and proportions (P2) in the PM

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and TA muscles are presented in Table 2. There were no significant differences in T2b,

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T21 times, and T2b corresponding proportion (P2b) both in PM and TA muscles

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between the control and 3 h transported broilers (P > 0.05), suggesting that the bound

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water is so stable that it cannot be influenced by preslaughter transport. However, the

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PM muscle of 3 h transport group presented lower (P < 0.05) P21 proportion and lower

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T22 time, as well as a higher (P < 0.05) P22 proportion than those in PM muscle of

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control group (P < 0.05), suggesting that 3 h transport reduced the WHC of breast

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meat by decreasing the proportion of immobilized water and increasing the proportion

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of free water. In this study, the result of NMR is in accordance with the higher drip

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loss of breast meat in the 3 h transported broilers (Table 1). Moreover, PM muscle

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had lower (P < 0.05) P21 proportion, and greater (P < 0.05) P22 proportion than those

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of TA muscle (Table 2). These results suggested that breast muscle is more vulnerable

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to induce PSE-like meat than thigh muscle when birds are subjected to transport stress 12


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during summer.

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Contents of Muscle Adenosine Phosphates, Glycogen, Lactate and Glycolytic

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Potential. The ATP contents and AMP/ATP ratio are typically used to indicate the

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energy status of cells, tissues, organs, and the entire body.37 Various studies have

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shown that a lower ATP content, and/or a higher AMP/ATP ratio in PM muscle were

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observed in turkeys or broilers subjected to heat stress, or preslaughter transport.6,38,39

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In accordance with these previous reports, the results of the present study showed that

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3 h transport decreased (P < 0.05) ATP content, increased (P < 0.05) AMP content and

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AMP/ATP ratio in PM muscle compared with the control group (Table 3), indicating

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that transport accelerates ATP consumption in PM muscle. There were no differences

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on the contents of ATP, ADP and AMP, and AMP/ATP ratio in TA muscle between

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two treatments (P > 0.05; Table 3). More interestingly, contradictory to our

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hypothesis, ATP content was greater (P < 0.05) for PM muscle than TA muscle (Table

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3). This is probably because PM muscle (type IIb fibers) has much higher glycogen

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content than that of TA muscle before slaughtering, which thus generates more ATP

281

by anaerobic glycolysis during early postmortem.

282

In chickens, the major glycogen reserves are in both liver and skeletal muscle. In

283

situ periodic acid-Schiff staining for glycogen in PM and TA muscles are shown in

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Figure 3. Nuclei were counterstained using hematoxylin (blue-violet). The PM

285

muscle of control birds showed higher glycogen contents (dark red-purple cytoplasm,

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Figure 3A), whereas PM muscle of 3 h transported group exhibited lower glycogen

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contents (reddish purple cytoplasm, Figure 3B). Compared with the PM muscle, the

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TA muscle of both control (Figure 3C) and 3 h transport (Figure 3D) groups showed

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lower glycogen contents (few purple cytoplasm). Table 3 shows the contents of

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muscle glycogen, lactate and GP. Compared with 0.5 h transport group, 3 h transport 13



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deceased (P < 0.05) glycogen content, increased (P < 0.05) lactate content and GP in

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the PM muscle compared to birds in the control group (Table 3), which was

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consistent with our previous periodic acid-Schiff staining result. Previous studies have

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reported that transport firstly accelerated the breakdown of liver glycogen with a

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transient increase of blood glucose level to regulate the stability of blood glucose to

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provide energy for multiple organs.7,40 However, the muscle glycogen is primary used

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to directly generate ATP for muscular contractions by glycolytic pathway under

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stressful condition. The muscle anaerobic glycolysis generates a small amount of ATP

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(2 mol of ATP/1 mol of glucose) and 2 molecules of lactate.12 It is generally

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recognized that anaerobic glycolysis generates lactate along with H+ ions released

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from ATP-hydrolyzing proton pum, and the accumulation of protons causes a

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subsequent rapid decline of muscle pH postmortem, which is the direct cause of

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PSE-like meat.30,31 Therefore, both lactate content and glycogen reserves in the

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glycolytic fibers at slaughter time, as well as its postmortem anaerobic glycolysis rate

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are more strongly associated with meat quality traits. In the current study, 3 h

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transport significantly decreased (P < 0.05) glycogen content, increased (P < 0.05)

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muscle lactate content and GP in PM muscle (Table 3) with concomitant lower pH24h

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in breast meat (Table 1), indicating that 3 h transport during summer caused a rapid

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anaerobic glycolysis rate in PM muscle during the early postmortem. These findings

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are in agreement with previous reports.6,7 Interestingly, 3 h transport did not affect

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(P > 0.05) the lactate content and GP although glycogen content was significantly

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lower in the TA muscle, which may be primarily because the proportion of fast-twitch

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glycolytic fibers (type IIb fiber) in TA muscle is much lower (approximately 60% in

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TA vs. 100% in PM) than that in PM muscle.7,13 In addition, PM muscle had greater

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(P < 0.05) contents of glycogen, lactate and GP than those in TA muscle (Table 3), 14


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suggesting that the difference in the fiber composition between the TA and PM

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muscles led to different response to glycolysis metabolism when broilers experience

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preslaughter transport.

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Expression of Glycogen Phosphorylase, and Activities of Glycolytic Key

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Enzymes. The glycogen synthase and glycogen phosphorylase are two main enzymes

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of glycogen metabolism, which are responsible for synthesis and mobilization of

322

glycogen, respectively.12 Glucose released from muscle glycogen stores is catalyzed

323

by glycogen phosphorylase, which can be activated by Ca2+, epinephrine and AMP.41

324

The results of our present study showed that 3-h transport upregulated (P < 0.05) the

325

mRNA expression of glycogen phosphorylase (the key enzyme involved in glycogen

326

breakdown) in both the PM and TA muscles (Table 4), which was consistent with the

327

lower glycogen content and higher lactate content in PM muscle (Table 3). These

328

results indicated that 3 h transport accelerated the breakdown of muscle glycogen and

329

subsequent glycolysis metabolism. In addition, PM muscle had higher (P < 0.05)

330

mRNA expression of glycogen phosphorylase compared to TA muscle (Table 4),

331

indicating anaerobic glycolysis in PM muscle is faster than that in TA muscle.

332

The glycolysis metabolic pathway is mediated by some key enzymes of anaerobic

333

metabolism. The HK is the first enzyme in glycolytic pathway, which converts

334

glucose to glucose-6-phosphate. As the key terminal enzymes, both PK and LDH are

335

involved

336

phosphoenol-pyruvate to pyruvate, and pyruvate to lactate under anaerobic conditions,

337

respectively.10,12

338

net molecules of ATP and 2 molecules of lactate per glucose molecule. Some recent

339

studies reported that the decrease of muscle HK activity contributed to the

340

improvement of pork quality by reducing the glycolysis rate.42,43 In our present study,

in

the

last

The

steps

whole

of

the

glycolytic

glycolytic

pathway,

reaction

15


pathway

which

convert

generates

2


341

the activities of HK in the PM muscle of 3 h transported broilers were increased (P
0.05; Table 4). This

349

result suggests that tissue-specific differences in glycogen metabolism do exist in the

350

form of activities of glycolytic key enzymes.

351

In summary, 3 h transport during summer decreased ATP content, increased AMP

352

content and AMP/ATP ratio, accelerated glycolysis metabolism via upregulation of

353

glycogen phosphorylase expression, and the increased activities of bound glycolytic

354

enzymes in PM muscle, which subsequently increased the likelihood of PSE-like

355

breast meat. On the contrary, however, 3 h transport only induced a moderate

356

glycolysis metabolism in TA muscle, which did not cause any noticeable changes in

357

these key enzymes associated with glycolysis metabolism and subsequent changes in

358

quality traits of the thigh meat. These results imply that breast muscle is prone to

359

PSE-like meat owing to its sole fiber composition and rapid glycolysis metabolism

360

when broilers subject to long-term preslaughter transport during hot summer.

361

AUTHOR INFORMATION

362

Corresponding Author

363

*Tel: +86-25-84399690. Fax: +86-25-84395314. E-mail: [email protected].

364

ORCID*

365

Lin Zhang:

0000-0003-1555-1086.

16


Page 16 of 31

Page 17 of 31


366

Funding

367

This work was supported by the National Natural Science Foundation of China

368

(31402094, 31601957), the Fundamental Research Funds for the Central Universities

369

of China (KYZ201641), the National Key Research and Development Program of

370

China (2016YFD0500501), and the Three Agricultural Projects of Jiangsu Province

371

(SXGC2017281).

372

Notes

373

The authors declare no competing financial interest.

374

ABBREVIATIONS USED

375

ADP, adenosine diphosphate; AMP, adenosine monophosphate; ATP, adenosine

376

triphosphate; GP, glycolytic potential; HK, hexokinase; LDH, lactate dehydrogenase;

377

NADH-TR, nicotinamide adenine dinucleotide tetrazolium reductase; NMR, nuclear

378

magnetic resonance; PAS, periodic acid-Schiff; PK, pyruvate kinase; PM, pectoralis

379

major; PSE, pale, soft, exudative; TA, tibialis anterior.

380

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381

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FIGURE CAPTIONS Figure 1. Characteristics of fiber types between PM (A) and TA (B) muscles by nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR) staining. PM = pectoralis major; TA = tibialis anterior; I = slow twitch-oxidative fiber; IIa = fast twitch-oxidative-glycolytic fiber; IIb = fast twitch-glycolytic fiber. Magnification, 200×. Figure 2. Distribution of low-field NMR transverse relaxation (T2) times in the PM and TA muscles of the control and 3 h transported broilers measured at 24 h postmortem. PM = pectoralis major; TA = tibialis anterior. Figure 3. Periodic acid-Schiff (PAS) staining of glycogen in situ. Cross section of the PM muscles in control (A) and 3 h transport group (B), and the TA muscles in control (C) and 3 h transport group (D). Nuclei were counterstained with hematoxylin (blue-violet). Glycogen storage is indicated by pink or dark red-purple cytoplasms. PM = pectoralis major; TA = tibialis anterior. Bar: 100 µm.

23



Table 1. Meat Quality Traits of the Control and 3 h Transported Broilersa,b

pH45min

pH24h

L* (lightness)

a* (redness)

b* (yellowness)

drip loss (%) a

control group

3 h transport group

breast

6.50 ± 0.04 a

6.39 ± 0.02 b

thigh

6.44 ± 0.04

6.35 ± 0.04

breast

5.89 ± 0.03 a,y

5.66± 0.04 b,y

thigh

6.17 ± 0.05 x

6.04 ± 0.06 x

breast

49.45 ± 0.68 b

53.57 ± 0.72 a,x

thigh

47.70 ± 0.69

49.68 ± 0.81 y

breast

3.53 ± 0.42 y

2.76 ± 0.29 y

thigh

5.18 ± 0.89 x

6.77 ± 1.09 x

breast

11.64 ± 1.01

12.66 ± 0.51

thigh

11.30 ± 0.89

12.86 ± 0.53

breast

2.45 ± 0.20 b

3.59 ± 0.27 a,x

thigh

2.18 ±0.14

2.46 ±0.10 y

Values are presented as mean ± standard error (n = 8). Means of a parameter in each row with

different letters (a, b) differ significantly (P < 0.05), and means of a parameter in each column with different letters (x, y) differ significantly (P < 0.05). bpH45min = pH at 45 min postmortem; pH24h = pH at 24 h postmortem; meat color (L*, a*, and b* values) and drip loss was measured at 24 h postmortem.

24


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Table 2. Low-Field NMR Spin-Spin Relaxation Times (T2) and Proportions (P2) in PM and TA Muscles of the Control and 3 h Transported Broilers Measured at 24 h Postmortema,b

T2b (ms)

T21 (ms)

T22 (ms)

P2b (%)

P21 (%)

P22 (%) a

control group

3 h transport group

PM

1.12 ± 0.06

1.21 ± 0.05

TA

1.31 ± 0.08

1.34 ± 0.09

PM

46.07 ± 1.25

43.18 ± 0.90

TA

46.96 ± 1.44

44.64 ± 1.68

PM

227.81 ± 7.23 a,y

198.63 ± 8.26 b,y

TA

327.64 ± 32.53 x

307.62 ± 14.00 x

PM

3.88 ± 0.23

3.91 ± 0.15

TA

3.54 ± 0.18

3.75 ± 0.13

PM

94.28 ± 0.19 a,y

93.62 ± 0.21 b,y

TA

95.62 ± 0.11 x

95.26 ± 0.25 x

PM

1.84 ± 0.13 b,x

2.47 ± 0.17 a, x

TA

0.83 ± 0.15 y

0.98 ± 0.16 y

Values are presented as mean ± standard error (n = 8). Means within each row with different

letters (a, b) differ significantly (P < 0.05), and means within each column with different letters (x, y) differ significantly (P < 0.05). b PM = pectoralis major; TA = tibialis anterior.

25



Table 3. Contents of Muscle Adenosine Phosphates, Glycogen, Lactate and Glycolytic Potential in PM and TA Muscles of Control and 3 h Transported Broilers (on a Fresh-Tissue Basis) a,b

ATP (µmol/g)

ADP (µmol/g)

AMP (µmol/g)

AMP/ATP ratio

glycogen (µmol/g)

lactate (µmol/g)

GP (µmol/g) a

control group

3 h transport group

PM

3.30 ± 0.09 a,x

2.83 ± 0.14 b,x

TA

1.29 ± 0.10 y

1.18 ± 0.08 y

PM

0.92 ± 0.03

0.85 ± 0.05

TA

0.83 ± 0.04

0.87 ± 0.05

PM

0.42 ± 0.03 a, y

0.55 ± 0.04 b

TA

0.55± 0.03 x

0.57 ± 0.03

PM

0.13 ± 0.01 b,y

0.19 ± 0.02 a,y

TA

0.43 ± 0.04 x

0.48 ± 0.05 x

PM

19.76 ± 1.41 a,x

11.68 ± 1.12 b

TA

16.29 ± 1.06 a,y

13.57 ± 1.02 b

PM

95.79 ± 4.22 b,x

126.67 ± 4.60 a,x

TA

51.24 ± 3.52 y

55.94 ± 3.05 y

PM

134.10 ± 4.57 b,x

150.03 ± 5.11 a,x

TA

84.22 ± 2.43 y

83.08 ± 3.04 y


letters (a, b) differ significantly (P < 0.05), and means within each column with different letters (x, y) differ significantly (P < 0.05). bPM = pectoralis major; TA = tibialis anterior; ATP = Adenosine triphosphate; ADP = adenosine diphosphate; AMP = adenosine monophosphate; GP (glycolytic potential) = 2 × glycogen + lactate (Monin and Sellier, 1985).

26


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Table 4. Expression of Glycogen Phosphorylase (the Key Enzyme Involved in Glycogen Breakdown) and Activities of Bound Glycolytic Enzymes in PM and TA Muscles of Control and 3 h Transported Broilersa,b

glycogen phosphorylase mRNA

control group

3 h transport group

PM

1.00 ± 0.07 a,x

1.72 ± 0.13 b,x

TA

0.51 ± 0.09 a,y

0.73 ± 0.08 a,y

PM

12.70 ± 0.79 b,y

15.58 ± 0.63 a,y

TA

17.91 ± 0.66 x

19.32 ± 0.87 x

PM

10.60 ± 0.59 a,x

12.56 ± 0.35 b,x

TA

7.51 ± 0.39 y

8.23 ± 0.23 y

PM

3.10 ± 0.21 b,x

4.06 ± 0.15 a,x

TA

2.56 ± 0.14 y

2.80 ± 0.15 y

abundance (arbitrary unit)

HK activity (U/g of protein)

PK activity (U/g of protein)

LDH (U/mg of protein) a


letters (a, b) differ significantly (P < 0.05), and means within each column with different letters (x, y) differ significantly (P < 0.05). bPM = pectoralis major; TA = tibialis anterior; HK = hexokinase; PK = pyruvate kinase; LDH = lactate dehydrogenase.

27



Figure 1

28


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Figure 2

29



Figure 3

30


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Graphic for table of contents (TOC)

31


Preslaughter Transport Effect on Broiler Meat Quality and Post-mortem

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