Creatine Monohydrate Enhances Energy Status and Reduces

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Creatine Monohydrate Enhances Energy Status and Reduces Glycolysis via Inhibition of AMPK Pathway in Pectoralis Major Muscle of Transport-stressed Broilers Lin Zhang, Xiaofei Wang, Jiaolong Li, Xudong Zhu, Feng Gao, and Guanghong Zhou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02740 • Publication Date (Web): 02 Aug 2017 Downloaded from http://pubs.acs.org on August 3, 2017

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Journal of Agricultural and Food Chemistry

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Creatine Monohydrate Enhances Energy Status and Reduces Glycolysis

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via Inhibition of AMPK Pathway in Pectoralis Major Muscle of

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Transport-stressed Broilers

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Lin Zhang,† Xiaofei Wang,†,‡ Jiaolong Li,† Xudong Zhu,†,‡ Feng Gao,*,† Guanghong

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Zhou†

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†

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Origin Food Production and Safety Guarantee, Jiangsu Key Laboratory of

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Gastrointestinal Nutrition and Animal Health, Jiangsu Collaborative Innovation

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Center of Meat Production and Processing, Quality and Safety Control, Nanjing

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Agricultural University, Nanjing, Jiangsu 210095, China

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‡

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

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

ACS Paragon Plus Environment


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ABSTRACT: Creatine monohydrate (CMH) contributes to reduce transport-induced

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muscle rapid glycolysis and improve meat quality of broilers, but the underlying

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mechanism is still unknown. Therefore, this study aimed to investigate the molecular

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mechanisms underlying the ameliorative effects of CMH on muscle glycolysis

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metabolism of transported broilers during summer. The results showed that 3-h

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transport during summer elevated chicken live weight loss and plasma corticosterone

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concentration, decreased muscle concentrations of ATP, creatine and energy charge

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value, increased muscle AMP concentration and AMP/ATP ratio, upregulated muscle

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mRNA expression of LKB1 and AMPKα2, as well as protein expression of

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p-LKB1Thr189 and p-AMPKαThr172, which subsequently resulted in rapid glycolysis in

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the pectoralis major muscle and consequent reduction of meat quality. Dietary

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addition of CMH at 1200 mg/kg ameliorated transport-induced rapid muscle

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glycolysis and reduction of meat quality via enhancement of the energy-buffering

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capacity of intramuscular phosphocreatine/creatine system and inhibition of AMPK

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pathway.

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KEYWORDS: broiler, transport, creatine monohydrate, energy status, glycolysis


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INTRODUCTION

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In poultry industry, market-age broilers are inevitably transported from farms to

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slaughterhouses, which has been reported to result in poor welfare, physiological and

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metabolic changes.1-3 More importantly, pre-slaughter transport during hot summer

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have also brought huge economic losses owing to the increases in injuries, mortality,

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live weight loss, and poor meat quality.4-7 Therefore, there has been a growing interest

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in exploitation of the effective ways to reduce stress responses and improve meat

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quality of transported broilers. Some exogenous additives, such as creatine

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monohydrate (CMH), oregano, ascorbic acid, or chromium, supplemented in chicken

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diets have been proposed as an effective means of mitigating transport stress

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responses and reducing stress-induced deterioration of meat quality.6-9

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α-methylguanidine acetic acid, popularly known as creatine (Cr), is a naturally

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occurring nitrogen compound found primarily in skeletal muscle. The Cr is primarily

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synthesized endogenously in the liver, kidneys and pancreas from the amino acids

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arginine, glycine and methionine.10,11 Cr sources from both dietary and endogenous

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biosynthesis can be transported to skeletal muscle where it combines with inorganic

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phosphate to form phosphocreatine (PCr), which subsequently plays a pivotal role in

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energy metabolism by donating its phosphate groups to ADP to regenerate ATP

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catalyzed by creatine kinase, particularly when skeletal muscle are experiencing

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intense exhaustive exercise.12-14 Thus, the muscle Cr/PCr pool serves as an important

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cellular energy source for rapid resynthesis of ATP to meet the increased energy

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demands of intense activities. CMH is a primary additive form of Cr. We previously

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reported that dietary addition of CMH at 1200 mg/kg for 2 weeks before slaughter

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alleviated stress-induced deterioration of breast meat quality by reducing rapid muscle

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glycolysis.6 However, the detailed mechanism underlying this phenomenon remains



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unclear.

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AMP-activated protein kinase (AMPK) is an energy-sensing enzyme and metabolic

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transducer, which regulates both cellular and whole-body energy balance in response

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to metabolic stresses that inhibit ATP production or accelerate ATP consumption.15

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Several studies have reported that AMPK plays an important regulatory role in

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postmortem muscle glycolysis.7,16 In addition, as an AMPK upstream kinase, tumor

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suppressor liver kinase B1 (LKB1; also known as serine/threonine kinase 11, STK11)

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plays a pivotal role in activation of AMPK in response to increases in the intracellular

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AMP:ATP ratio.17,18 Thus, we hypothesize that the action of dietary CMH on muscle

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energy metabolism and postmortem glycolysis is highly mediated by the AMPK

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pathway. Therefore, the objective of this study was to further investigate the effects of

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dietary CMH on meat quality, muscle energy status, glycolysis, activities of key

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enzymes of glycolysis, and gene and protein expression of AMPK pathway in

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pre-slaughter transported broilers during summer.

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MATERIALS AND METHODS

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Animal Care and Diets. All experimental and animal care procedures were

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approved by the Institutional Animal Care and Use Committee of Nanjing

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Agricultural University. Arbor Acres male broilers (n=288, mean initial weight:

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1303.17 ± 6.81 g) fed with same starter and grower diets for the first 27 days were

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randomly distributed into 2 dietary treatments: 1) a basal control diet (192 birds), and

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2) a basal diet supplemented with CMH at 1200 mg/kg (96 birds) from 28 to 42 d of

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age. There were 8 replicated cages per treatment with 12 broilers per cage (1.10 m ×

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0.70 m × 0.43 m), except the control had 24 broilers in each replicate with 2 cages.

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CMH was purchased from Tianjin Tiancheng Pharmaceutical Co. Ltd (Tianjin, China).

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Diets were fed in mash form. Chickens consumed feed and clean water ad libitum.


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The basal diet composition and nutritional values are summarized in Table 1. All

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chickens were weighed at 42 d of age. Feed intake was recorded and a feed

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conversion ratio (g feed intake/g gain) was calculated.

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Transportation and Sample Collection. At time of transport, after an 8-h

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overnight feed withdrawal, the birds in the basal diet group were divided into 2 equal

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groups, resulting in 3 groups of 80 birds (2 groups control, 1 group treated). All birds

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in these 3 groups were transported from the rearing house to the laboratory according

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to a designed protocol: 1) a 0.5-h transport of birds on basal diet (as a lower stress

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control group), 2) a 3-h transport of birds on basal diet (T3h group), and 3) a 3-h

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transport of birds on 1200 mg/kg CMH supplemental basal diet (CMH+T3h group).

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Ten birds from the same replicate were placed into one crate (0.73 m × 0.54 m × 0.26

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m), and all 24 crates were randomly distributed in the same truck. The transport

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durations were 0600 to 0630 h for the control group, and 0600 to 0900 h for the 3-h

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transport groups, respectively. In modern poultry industry, both chicken slaughter and

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meat processing are forbidden in farm and have to transport to slaughterhouse, and we

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thus chosen a 0.5-h of transport group as a lower stressful control as previously

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reported.3 The transport distance is about 240 km with an average speed of 80 km/h.

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During the transport period, the temperatures and humidity (RH) inside of the trucks

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were 26.8∼31.5°C and 77.6∼83.1% in the control group, and 26.8∼34.8°C and

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77.6∼87.3% in the 3-h transport groups, respectively. No feed or water was supplied

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during the transport.

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After arrival, the broilers were allowed to rest 1 hour in a shady corner without

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feed and water supply. Immediately after rest, one bird from each crate (replicate) of

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each treatment (n = 8) was randomly selected, stunned electrically (50 V: alternating

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current, 400 Hz for 5 s each one) and immediately slaughtered via exsanguination.



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Blood samples were collected to get plasma. Muscle samples (5.0 g) of the left

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pectoralis major (PM) were snap-frozen in liquid nitrogen, and stored at -80 ◦C until

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analysis. The entire right PM muscle was collected and stored at 4°C for

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determination of meat quality, and NMR relaxometry at 24 h postmortem.

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Plasma Corticosterone Analysis. Plasma corticosterone (CORT) concentration

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was measured using a commercial ELISA kit, validated for use in chickens (Cusabio

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Biotech. Co., Ltd., Wuhan, China).

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

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24 h (pH24h) postmortem using a calibrated portable waterproof pH/ORP meter

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(HI9125; Hanna Instruments, Cluj-Napoca, Romania). Meat color at 24 h postmortem

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was measured using a Minolta chromameter (CR-400; Konica Minolta Sensing Inc.,

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Osaka, Japan) with a CIE D65 illuminant, 8 mm aperture diameter and 0° viewing

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angle. The values of lightness (L*), redness (a*), and yellowness (b*) were collected

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from three different locations on the freshly cut surface of each sample. Drip loss,

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cooking loss and shear force value at 24 h postmortem were determined as previously

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described.3,6

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

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relaxation measurements were conducted using a previously published procedure with

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minor modifications.19,20 Briefly, 2.0 g sample was cut along the direction of the

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myofiber from the PM sample, placed in a cylindrical glass tube (14 mm in diameter

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and 5 cm high) and then inserted in the NMR probe of a PQ001 Niumag Pulsed NMR

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analyzer (Niumag Electric Corporation, Shanghai, China). The analyzer was operated

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at resonance frequency of 22.6 MHz at 32◦C. Transverse relaxation (T2) was

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measured using the Carr-Purcell-Meiboom-Gill sequence with a τ-value of 150 µs.

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Data from 4096 echoes were acquired as 32 scan repetitions for a 1-s with a


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multiexponential model using the program MultiExp Inv Analysis (Niumag Electric

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Corporation, Shanghai, China). Three relaxation times (T2b, T21 and T22) and their

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corresponding water proportions (P2b, P21 and P22) were recorded.

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Measurement of Muscle Lactic Acid, Glycogen and Glycolytic Potential.

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Frozen muscle sample (0.50 g) was homogenized in 4.5-mL ice-cold saline, and the

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supernatant fraction was used for measurement of lactic acid concentration using a

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commercial kit (Nanjing Jiancheng Biochemical Institute, Nanjing, China). Glycogen

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concentration was measured as previously described.3 The glycolytic potential (GP)

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was calculated according to the formula: GP = 2 × (glycogen) + (lactic acid), and

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expressed as µmol of lactic acid equivalent per g of fresh muscle.21

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Activity Analysis of Muscle Glycolytic Key Enzymes. Approximately 0.5 g of

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frozen muscle sample was homogenized in a centrifuge tube with 4.5 mL of 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 determined with their relative commercially available kits

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(Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The detection principle

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of HK activity is based on the coupling ribulose-5-phosphate formation from glucose

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6-phosphate to the reduction of NADP+.22 The detection principle of PK and LDH

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activities are based on the decrease rate of NADH during the conversion of

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phosphoenol-pyruvate into pyruvate, and the conversion of pyruvate into lactate.23,24

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Measurement of Muscle Cr and PCr. The concentrations of muscle Cr and PCr

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were determined by HPLC method as previously described with minor

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modifications.25 Briefly, 300 mg of frozen muscle sample was homogenized in 2.0

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mL of ice-cold 5% perchloric acid for 1 min. After being kept for 10 min in an ice

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bath, the homogenate was centrifuged at 10000 × g at 4°C for 10 min. The



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supernatant was adjusted to a pH of 7.0 with 0.8 M K2CO3. The mixture was kept in

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an ice bath for 10 min, and then centrifuged at 15000 × g at 4°C for another 10 min.

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Supernatant was filtered with a 0.45 µm membrane, and 10 µL of this sample solution

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was injected into the Alliance HPLC system (Model 2695, Waters Corporation,

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Milford, MA, USA) with ultraviolet detection at 210 nm. The chromatography was

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performed on a Waters SunFire C18 column (250 mm × 4.6 mm, 5 µm) at 25°C. The

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mobile phase was a mixture of methyl cyanides and 29.4 mM KH2PO4 buffer (2:98,

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volume ratio) and the flow rate was 1 mL/min. The creatine and phosphocreatine

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disodium salt (Sigma-Aldrich Inc., St. Louis, MO, USA) were used as standards.

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Measurement of Muscle Adenosine Nucleotides. The concentrations of muscle

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ATP, ADP and AMP were analyzed by HPLC method as previously described with

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minor modifications.26 Briefly, 300 mg of frozen muscle sample was homogenized in

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1.5 mL of ice-cold 7% perchloric acid for 1 min. After being kept for 15 min in an ice

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bath, the homogenate was centrifuged at 15000 × g at 4°C for 10 min. The

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supernatant was adjusted to a pH of 6.5 with 1.03 M KOH. The mixture was kept in

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an ice bath for 10 min, and then centrifuged at 15000 × g at 4°C for another 10 min.

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Supernatant was filtered with a 0.45 µm membrane, and 10 µL of this sample solution

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was injected into the Alliance HPLC system (Model 2695, Waters Corporation,

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Milford, MA, USA) with ultraviolet detection at 245 nm. The chromatography was

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performed on a Waters SunFire C18 column (250 mm × 4.6 mm, 5 µm) at 30°C. The

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mobile phase was a mixture of methanol and phosphate buffer (13.5:86.5, volume

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ratio) and the flow rate was 1 mL/min. The standard samples of 5’-Adenosine

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triphosphate disodium salt, 5’-ADP sodium salt, and 5’-AMP sodium salt were

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purchased from Sigma-Aldrich Inc (St. Louis, MO, USA).

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RNA Extraction and Real-time Quantitative PCR Analysis. Total RNA was


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extracted from frozen samples using RNAiso Plus reagent (TaKaRa Biotechnology

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Co. Ltd., Dalian, China). Reverse transcription and real time quantitative PCR were

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respectively conducted using the PrimeScriptTM RT Master Mix (TaKaRa

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Biotechnology Co. Ltd., Dalian, China) and SYBR Premix Ex Taq (TaKaRa

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Biotechnology Co. Ltd., Dalian, China) according to the manufacturer’s instruction.

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All of the specific primers used are listed in Table 2. Real-time quantitative PCR was

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performed using ABI 7500 Real-Time PCR System (Applied Biosystems,Foster City,

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CA) as follows: one cycle at 95°C for 30 s; 40 cycles at 95°C for 5 s, and 60°C for 30

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s. All of the samples were run in triplicate. The 2-△△Ct method was used for the

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quantification with β-actin as a reference gene, and the relative abundance was

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normalized to the control (as 1).27

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Western Blot Analysis. The specific primary antibodies of anti-p-LKB1Thr189,

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anti-p-AMPKαThr172 and anti-α-Tubulin were purchased from Cell Signaling

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Technology (Beverly, MA, USA) and were validated previously for use with chicken

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samples.28,29 A frozen muscle sample (40 mg) was homogenized in 0.5-mL ice-cold

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RIPA lysis and extraction buffer (20 mM Tris–HCl, pH 7.4, 1 mM PMSF, 0.8 µM

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Aprotinin, 20µM leupeptin, 0.015 µM pepstatin A, 5 mM NaF, 5 mM EDTA, 1 mM

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sodium pyrophosphate,1 mM β-glycerophosphate, 1 mM sodium orthovanadate).

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After centrifugation, the supernatant was collected and protein concentration was

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determined by using a BCA protein assay kit (Beyotime Biotechnology, Haimen,

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China). Then the proteins were boiled at 100 °C for 5 min with 6 × SDS sample buffer.

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Equal amounts of proteins (30 µg) from each sample were then separated on 10%

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SDS-PAGE gels and then transferred to polyvinylidene difluoride membranes

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(Millipore, Billerica, MA, USA). To avoid non-specific binding, membranes were

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blocked with Tris-buffered saline (TBS) that contained 0.1% (w/w) Tween 20 (TBST)



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and 5% (w/v) bovine serum albumin (BSA) at room temperature for 1 h. After a brief

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wash in TBST, the membrane was incubated with the specific primary antibodies,

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including a rabbit polyclonal anti-p-LKB1Thr189 antibody (1:1000), a rabbit polyclonal

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anti-p-AMPKαThr172 antibody (1:1000) and a rabbit monoclonal anti-α-Tubulin

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antibody (1:2000) at 4 °C overnight. After washing three times in TBST, membranes

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were incubated for 1 h with secondary anti-rabbit HRP-conjugated antibodies (1:1000)

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in TBST containing 5% BSA. The blots were detected with the Tanon-3900

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Chemiluminescent Imaging System (Tanon Science & Technology Co.,Ltd. Shanghai,

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China) after reactions with ECL Plus detection reagents (Tanon Science &

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Technology Co., Ltd. Shanghai, China). The band density was quantified with the use

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of Quantity One software (Bio-Rad Laboratories Inc., Hercules, CA, USA). All results

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were normalized to α-Tubulin and expressed as the relative values to those for the

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control group.

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Statistical Analysis. Data were analyzed with the use of SAS version 9.1 (SAS

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Institute Inc. Cary, NC, USA; 2004). The growth performance data were analysed

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with the cage as the experimental unit, and other data were were analysed with the

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individual chicken from each replicate as the experimental unit (n = 8). One-factor

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ANOVA followed by Tukey’s post hoc test were used to assess differences between

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transport groups for the different measured variables, except the data on growth

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performance between basal and CMH supplementation diet groups was subjected to

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Student's t-test. Results are expressed as means ± standard error of the mean (SEM).

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Differences were considered significant at P < 0.05.

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RESULTS

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Growth Performance. Compared with the basal control diet group, dietary

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addition of CMH at 1200 mg/kg in finishing diets did not affect the average BW gain,


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average feed intake and FCR of broilers from 28 to 42 days of age (Table 3).

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Live Weight Loss and Plasma CORT Concentration. The bird live weight loss

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and plasma CORT concentrations were higher in the T3h group than those in the

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control group (P < 0.05; Figure 1A, B). The CMH+T3h group had a lowered

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concentration of plasma CORT compared with the T3h group (P < 0.05; Figure 1B).

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There was no significant difference in live weight loss between the T3h and CMH+T3h

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groups (Figure 1A).

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Meat Quality. The pectoralis major muscle of T3h group had a lower pH24h, higher

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lightness, drip loss and cooking loss than were seen in the control group (P < 0.05;

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Table 4). CMH+T3h treatment increased muscle pH24h and reduced drip loss in

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comparison to the T3h group (P < 0.05). No significant differences among all 3

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treatment groups were observed in redness, yellowness and shear force values of

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pectoralis major muscle.

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NMR Relaxometry. The distributed water proton NMR relaxation times (T2) curve

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is presented in Figure 2. Three peaks (T2b, T21 and T22) were detected, which

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respectively represent 3 types of water components in meat: T2b proportions located in

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the region of 1-2 ms is considered to be bound water tightly associated with

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macromolecules such as proteins, T21 proportions located in the region of 40-150 ms

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represented immobile water, and T22 proportions located in the region of 200-400 ms

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is known as free water.19

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Table 5 shows the data of NMR spin-spin relaxation times (T2) and proportions (P2)

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in pectoralis major muscle at 24 h postmortem. The pectoralis major muscle of T3h

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group had a lower P21 proportion (P < 0.01) and a higher P22 proportion (P < 0.05)

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than those were seen in the control group. In comparison to the T3h group, CMH+T3h

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treatment increased muscle P21 proportion although not reaching the level of the



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control group (P < 0.05). There were no significant treatment effects on T2b, T21 and

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T22 times, nor on P2b proportion of pectoralis major muscle.

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Concentrations of Muscle Lactic Acid, Glycogen, GP, and Activities of

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Glycolytic Enzymes. Compared with the control group, T3h treatment decreased the

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concentration of glycogen (P < 0.05; Figure 3A), increased the concentrations of

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lactic acid (P < 0.05; Figure 3B) and GP (P < 0.05; Figure 3C) in pectoralis major

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muscle, which were accompanied by the increased activities of HK (P < 0.001; Figure

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3D), PK (P < 0.01; Figure 3E) and LDH (P < 0.05; Figure 3F), respectively. Whereas

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CMH+T3h treatment decreased the concentrations of lactic acid and GP in pectoralis

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major muscle compared with the T3h group (P < 0.05; Figure 3B, C). There were no

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significant differences in activities of HK, PK and LDH between the T3h and

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CMH+T3h groups (Figure 3D, E, F).

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Concentrations of Muscle ATP, ADP, AMP, Cr and PCr. Compared with the

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control group, T3h treatment decreased muscle concentrations of ATP (P < 0.05;

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Figure 4A), EC value (P < 0.05; Figure 4E) and Cr (P < 0.001; Figure 4F), and

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increased concentration of AMP (P < 0.05; Figure 4C) and AMP/ATP ratio (P < 0.05;

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Figure 4D), respectively. CMH+T3h treatment increased muscle concentrations of Cr

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(P < 0.001; Figure 4F) and PCr (P < 0.001; Figure 4G) compared to both control and

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T3h groups, and decreased muscle ADP concentration (P < 0.05; Figure 4B) compared

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only to the T3h group. No significant differences in concentrations of muscle ATP and

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AMP, AMP/ATP ratio, EC and PCr/Cr ratio were observed between the T3h and

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CMH+T3h groups (Figure 4).

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Relative Expression of Gene mRNA and Protein in AMPK Pathway. Compared

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with the control group, T3h treatment upregulated muscle relative mRNA expression

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of LKB1 (P < 0.001; Figure 5A) and AMPKα2 (P < 0.001; Figure 5C), and protein


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expression of p-LKB1Thr189 (P < 0.05; Figure 5D) and p-AMPKαThr172 (P < 0.01;

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Figure 5E). CMH+T3h treatment downregulated the relative mRNA expression of

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muscle LKB1 and AMPKα2 (P < 0.001; Figure 5A, C), and protein expression of

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p-AMPKαThr172 (including α-1 & -2) (P < 0.01; Figure 5E) in comparison to the T3h

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group. There was no significant difference in relative mRNA expression of AMPKα1

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in pectoralis major muscle among all 3 treatment groups (Figure 5B).

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DISCUSSION

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In the present study, dietary supplementation with CMH at 1200 mg/kg for 2 weeks

288

prior to slaughter had no effect on the growth performance of broilers, which is in

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accordance with previous findings that dietary addition of CMH at different levels for

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14 days (600 or 1200 mg/kg) or 21 days (250, 500 or 1000 mg/kg) before slaughter

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did not influence the growth performance of broilers.6,30 In contrast, another study

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reported that dietary addition of creatine at 3.0% decreased average daily feed intake

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and average BW gain from 22 to 42 days.31 The reason for this inconsistent result may

294

be ascribed to the differences in creatine supplement types, bioavailability,

295

supplementation dose and experimental duration.

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The plasma CORT concentration has been considered as a sensitive indicator in

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response to many types of stress.32,33 As expected, we observed significant elevated

298

plasma CORT concentration accompanied by an increase live weight loss in chicken

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of 3-h transported group, which is consistent with findings reported previously.6,32

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These observations indicated that broilers suffered from strong psychological or

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physiological stress during 3-h transport duration. In addition, we also found that

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broilers in CMH+T3h group showed a lower plasma CORT concentration compared to

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the T3h group, indicating that dietary supplementation with CMH at 1200 mg/kg for 2

304

weeks prior to slaughter is a potential effective means of relieving stress response of



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transported broilers during summer.

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Previous studies have demonstrated that the muscle containing a higher proportion

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of fast glycolytic fibers (type IIb fibers) are more prone to be pale, soft, and exudative

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(PSE) pork or PSE-like chicken meat due to its anaerobic glycolysis nature, higher

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glycogen concentration and lower ultimate pH.34,35 In chickens, the pectoralis major

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muscle only consists of IIb type fibers, which rely primarily on glycolytic pathways

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for energy production.3,36,37 Broilers experienced a 3- to 4-h transport showed a lower

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ultimate pH and higher cooking loss in pectoralis major muscle at 24 h

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postmortem.6,38 Similarly, our present results showed that 3-h transport lowered pH24h

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and increased lightness, drip loss and cooking loss at 24 h postmortem, indicating a

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typical PSE-like syndrome in breast muscle according to the criteria values for

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PSE-like meat (pH24h < 5.7, L*24h > 53) as previously recommended.39 Also, our

317

results demonstrated that dietary supplementation with CMH prior to slaughter for 2

318

weeks is helpful for alleviating the deterioration of meat quality by increasing water

319

holding capacity and reducing postmortem rapid pH decline, and thereby reducing the

320

occurrence of transport-induced PSE-like breast meat.

321

Low-field proton NMR relaxation measurements is regarded as a useful approach

322

to estimate the water-holding capacity (WHC) of fresh meat since it gives a direct

323

measure of the proportion of water in the meat that is susceptible to be lost as drip.19

324

As shown in Figure 2, three relaxation proportions T2b, T21 and T22 were detected in

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all samples, which represent three states of water in fresh meat: bound water,

326

immobilized water and free water, respectively. A higher P21 proportion

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(approximately 95%, Table 5) suggested that the most of the water in the pectoralis

328

major muscle was the immobile water. In the present study, the 3 h transport

329

significantly reduced the WHC of breast meat by deceasing P21 proportion and


Page 14 of 36

Page 15 of 36


330

increasing P22 proportion, which is accordance with the higher drip loss in the T3h

331

group as shown in Table 5. Nevertheless, broilers in CMH+T3h group had a significant

332

higher P21 proportion compared to the T3h group, indicating that dietary CMH may

333

have beneficial effects in the increase of muscle WHC. There is evidence that creatine

334

can increase the intracellular volume by elevating water uptake in the muscle, thereby

335

resulting in an increase of water content and WHC.40

336

In chickens, the glycogen is mainly stored in glycolytic fibers and liver. The data of

337

the present study demonstrated that transport stress accelerated the breakdown of

338

muscle glycogen and subsequent anaerobic glycolysis metabolism when oxygen is

339

limited, resulting in lactate accumulation, rapid decline of muscle pH and subsequent

340

poor meat quality as compared with the birds in the control group. The glycolysis

341

metabolic pathway is mediated by some key enzymes of anaerobic metabolism. The

342

first enzyme in glycolytic pathway is HK, which converts glucose to

343

glucose-6-phosphate; the other two key glycolytic enzymes, PK and LDH, are

344

involved

345

phosphoenol-pyruvate to pyruvate, and pyruvate to lactic acid under anaerobic

346

conditions, respectively.41,42 Our present results showed that 3-h transport increased

347

the activities HK, PK and LDH in pectoralis major muscle compared to the control

348

group, suggesting that exhaustion of muscle ATP may trigger glycolysis mechanism

349

by activating HK activity, following higher activities of PK and LDH with the

350

accumulation of the metabolites phosphoenol-pyruvate and pyruvate. In spite of this,

351

CMH supplementation significantly decreased the activity of HK, and the

352

concentration of lactic acid and GP compared to the T3h group, suggesting that the rate

353

of glycolysis reaction was reduced.

354

in

the

last

steps

of

the

glycolytic

pathway,

which

converts

ATP is the direct energy source of skeletal muscle cells. Generally, oxidative



Page 16 of 36

355

phosphorylation generates 38 mol of ATP/1 mol of glucose, whereas anaerobic

356

glycolysis only produces 2 mol of ATP/1 mol of glucose.42 In addition, as an

357

important energy-buffering

358

phosphocreatine circuit) plays an essential role in regulating the cellular energy

359

homeostasis in periods of high-energy demand or energy supply fluctuations.12,43

360

Once intracellular ATP level is below a threshold value, as an immediate energy

361

reserve, a proportion of muscle Cr is phosphorylated to PCr, which subsequently

362

phosphorylates ADP to regenerate ATP with the catalysis of creatine kinase as follow:

363

PCr + ADP + H+ ↔ ATP + Cr.44 Thus, the muscle Cr/PCr pool serves as an important

364

cellular energy source for rapid resynthesis of ATP to meet the increased energy

365

demands of intense activities.

system,

muscle

Cr/PCr

pool (also

known

as

366

Some previous studies reported that muscle ATP concentration, ATP:ADP ratio and

367

EC were significantly decreased in broilers experiencing pre-slaughter transport or

368

heat stress.3,7,45 Similarly, our present study showed that 3-h transport decreased the

369

concentrations of muscle ATP and Cr, and EC, increased AMP concentration and

370

AMP/ATP ratio, suggesting that transport stress during summer accelerated muscle

371

ATP exhaust accompanied by the activation of Cr/PCr shuttle system. The lower ATP

372

and higher AMP/ATP ratio in muscle of 3-h transported broilers also indicated that

373

mitochondrial oxidative respiration and endogenous phosphocreatine circuit were not

374

able to generate enough ATP to meet muscle contraction demand, and then anaerobic

375

glycolysis becomes a critical pathway for ATP supply during intense muscle

376

contraction. Dietary supplementation with CMH could increase the concentrations of

377

muscle Cr or/and PCr in broilers or finishing pigs.46,47 In accordance with these

378

previous findings, we found that CMH supplementation increased concentrations of

379

muscle Cr and PCr by 20.31% and 27.45%, respectively, compared with that of birds


Page 17 of 36


380

in the T3h group. This suggested that dietary CMH supplementation increased

381

intramuscular anaerobic (Cr and PCr) energy-buffering capacity when muscle ATP is

382

depleted in broilers subjected to transport during hot summer.

383

AMPK is activated classically within the cell in response to decreased ATP levels

384

and increased intracellular AMP:ATP ratio.48 In eukaryotic cells, heterotrimeric

385

protein AMPK is formed by α catalytic subunit and two regulatory subunits, β and γ.49

386

The catalytic α subunit of AMPK has two isoforms, α1 and α2, which display

387

differential expression patterns.28 AMPK in chicken tissues is activated in response to

388

environmental or nutritional stress factors, which deplete intracellular ATP levels.7,29

389

Similar to mammals, the AMPK activity in chicken tissues is also activated mainly via

390

LKB1.28,29 In chicken, both the mRNA and protein levels of LKB1 and AMPKα in the

391

pectoralis major muscle (fast glycolytic fibers) were decreased in response to fasting

392

stress.29 In addition, higher AMPK activity and its phosphorylated protein were

393

previous found in transport-induced PSE-like chicken meat and PSE pork.7,16 In our

394

present study, compared with birds in the control group, 3-h transport upregulated

395

muscle relative mRNA expression of LKB1 and AMPKα2, and protein expression of

396

pLKB1Thr189 and pAMPKαThr172 (including α-1 & -2), which was accompanied by the

397

decrease of ATP concentration, the increase of AMP/ATP ratio and the rapid

398

postmortem glycolysis in pectoralis major muscle. These results further confirmed

399

that AMPK pathway is an important molecular target for the control of muscle

400

glycolysis and incidence of transport-induced chicken PSE-like meat during hot

401

summer. However, we did not find significant change in the mRNA expression of

402

AMPKα1 among 3 treatment groups. A probable reason for this unexpected result is

403

that AMPKα1 isoform is predominantly expressed in the adipose tissue but the

404

AMPKα2 isoform is highly expressed in skeletal muscle.28 Nevertheless, dietary



Page 18 of 36

405

CMH downregulated the mRNA expression of muscle LKB1 and AMPKα2, and

406

protein expression of p-AMPKαThr172, suggesting that dietary addition of exogenous

407

Cr (as CMH form) inhibits the transport stress-induced activation of AMPK pathway.

408

In conclusion, dietary addition of CMH at 1200 mg/kg improved muscle energy

409

status by enhancing energy-buffering capacity of intramuscular PCr/Cr system,

410

inhibited the transport-induced activation of AMPKα pathway, which was beneficial

411

for improving meat quality by reducing rapid muscle glycolysis of transport-stressed

412

broilers during summer.

413

AUTHOR INFORMATION

414

Corresponding Author

415

*Tel: +86-25-84399007. Fax: +86-25-84395314. E-mail: [email protected].

416

ORCID*

417

Lin Zhang:

418

Funding

419

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

420

(31402094), the Fundamental Research Funds for the Central Universities of China

421

(KYZ201641), the Science and Technology Innovation Fund for the Youth of Nanjing

422

Agricultural University (KJ2013017), the National Key Research and Development

423

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

424

Province (SXGC2017281).

425

Notes

426

The authors declare no competing financial interest.

427

ABBREVIATIONS USED

428

ADP,

429

AMP-activated protein kinase; ATP, adenosine triphosphate; BW, body weight; CMH,

0000-0003-1555-1086.

adenosine

diphosphate;

AMP,

adenosine

monophosphate;


AMPK,

Page 19 of 36


430

creatine monohydrate; CORT, corticosterone; Cr, creatine; EC, energy charge; FCR,

431

feed conversion ratio; GP, glycolytic potential; HK, hexokinase; LDH, lactate

432

dehydrogenase; LKB1, liver kinase B1; NMR, nuclear magnetic resonance; PCr,

433

phosphocreatine; PK, pyruvate kinase.

434

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435

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Table 1. Basal Diet Formulation and Nutritional Values 1 to 21 days

22 to 42 days

corn

57.61

61.87

soybean meal

31.00

23.00

corn gluten meal

3.29

6.00

soybean oil

3.11

4.00

dicalcium phosphate

2.00

2.00

Ingredient (%)

limestone

1.20

1.40

L-Lysine HCl

0.34

0.35

DL-Methionine

0.15

0.08

salt

0.30

0.30

1.00

1.00

metabolisable energy (MJ/kg)

12.56

13.19

crude protein

21.10

19.60

a

premix

Nutrient level (calculated, %)

calcium

1.00

0.95

available phosphorus

0.46

0.39

lysine

1.20

1.05

methionine

0.50

0.42

methionine + cystine

0.85

0.76

a

Premix provided per kilogram of diet: vitamin A, 12,000 IU; vitamin D3, 2,500 IU; vitamin E, 20 mg;

menadione, 1.3 mg; thiamine, 2.2 mg; riboflavin, 8 mg; nicotinamide, 40 mg; calcium pantothenate, 10 mg; pyidoxine·HCl, 4 mg; biotin, 0.04 mg; folic acid, 1 mg; vitamin B12, 13 µg; 50% choline chloride, 400 mg; iron, 80 mg; copper, 8 mg; manganese, 110 mg; zinc, 60 mg; iodine, 1.1 mg; selenium, 0.3 mg.


Page 24 of 36

Page 25 of 36


Table 2 Nucleotide Sequences of Specific Primers for Real-Time Quantitative PCR Analysisa genes

Primer sequences (5’ to 3’ direction)

Amplicon

size

GenBank accession no.

(bp) LKB1

F: TGAGAGGGATGCTTGAATACGA

138

NM_001045833.1

125

NM_001039603.1

215

NM_001039605.1

120

NM_205518.1

R: ACTTGTCCTTTGTTTCTGGGC AMPKα1

F: ATCTGTCTCGCCCTCATCCT R: CCACTTCGCTCTTCTTACACCTT

AMPKα2

F: GGGACCTGAAACCAGAGAACG R: ACAGAGGAGGGCATAGAGGATG

β-actin a

F: ATCCGGACCCTCCATTGTC

R: AGCCATGCCAATCTCGTCTT LKB1, liver kinase B1; AMPKα1, adenosine 5’-monophosphate-activated protein kinase α1; AMPKα2,

adenosine 5’-monophosphate-activated protein kinase α2.



Table 3. Effects of Dietary CMH Supplementation on the Growth Performance of Broilers from 28 to 42 Days of Agea,b diet treatments control

1200 mg/kg CMH

1300.90 ± 11.39

1306.25 ± 9.12

BW gain (g/bird)

1143.57 ± 15.95

1129.71 ± 20.58

feed intake (g/bird)

2268.13 ± 14.25

2259.02 ± 18.51

FCR (feed:gain, g:g)

1.98 ± 0.03

2.00 ± 0.04

initial BW at 28 d (g/bird) 28-42 days

a

Results are represented as the mean value ± standard error of eight replicates per treatment.

b

CMH, creatine monohydrate; BW, body weight; FCR, feed conversion ratio.


Page 26 of 36

Page 27 of 36


Table 4. Effects of Dietary CMH Supplementation on Meat Quality of Pectoralis Major Muscle of Transport-stressed Broilersa,b

a

control

T3h

CMH+T3h

pH45min

6.47 ± 0.03

6.39 ± 0.03

6.44 ± 0.04

pH24h

5.82 ± 0.03a

5.67 ± 0.02c

5.74 ± 0.02b

L*

48.86 ± 1.03b

53.94 ± 1.36a

51.40 ± 0.97ab

a*

4.42 ± 0.22

3.81 ± 0.20

4.20 ± 0.16

b*

14.95 ± 0.61

15.43 ± 0.72

16.50 ± 0.79

drip loss (%)

2.04 ± 0.09b

2.85 ± 0.14a

2.35 ± 0.11b

cooking loss (%)

13.31 ± 0.29b

15.13 ± 0.45a

14.35 ± 40ab

shear force (N)

14.47 ± 0.56

16.06 ± 0.47

15.42 ± 0.65

Results are represented as the mean value ± standard error of eight sample birds per treatment

(n=8). Means in a row without a common superscript letter significantly differ (P < 0.05). bControl, broilers fed the basal diet and experienced a 0.5-h transport; T3h or CMH+T3h, broilers fed the basal diet or the basal diet supplemented with CMH at 1200 mg/kg from 28 to 42 days of age and experienced a 3-h transport. CMH, creatine monohydrate; pH45min, pH at 45 min postmortem; pH24h, pH at 24 h postmortem; L*, lightness; a*, redness; b*, yellowness.



Table 5. Effects of Dietary CMH Supplementation on Low-field NMR Spin-spin Relaxation Times (T2) and Proportions (P2) in Pectoralis Major of Transport-stressed Broilersa,b

a

control

T3h

CMH+T3h

T2b (ms)

1.16 ± 0.05

1.24 ± 0.06

1.25 ± 0.07

T21 (ms)

43.62 ± 2.14

41.99 ± 1.49

42.08 ± 1.41

T22 (ms)

209.42 ± 9.87

195.36 ± 9.05

202.95 ± 10.27

P2b (%)

3.02 ± 0.15

3.17 ± 0.17

3.10 ± 0.20

P21 (%)

95.11 ± 0.19a

94.20 ± 0.25c

94.76 ± 0.25b

P22 (%)

1.87 ± 0.13a

2.63 ± 0.15b

2.14 ± 0.15ab

Results are represented as the mean value ± standard error of eight sample birds per treatment

(n=8). Means in a row without a common superscript letter significantly differ (P < 0.05). bControl, broilers fed the basal diet and experienced a 0.5-h transport; T3h or CMH+T3h, broilers fed the basal diet or the basal diet supplemented with CMH at 1200 mg/kg from 28 to 42 days of age and experienced a 3-h transport. CMH, creatine monohydrate.


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Figure captions Figure 1. Effects of dietary CMH supplementation on live weight loss (A) and plasma CORT (B) concentration of transported broilers. Results are represented as the mean value ± standard error of eight sample birds per treatment (n=8). Means without a common letter significantly differ (P < 0.05.) Control, broilers fed the basal diet and experienced a 0.5-h transport; T3h or CMH+T3h, broilers fed the basal diet or the basal diet supplemented with CMH at 1200 mg/kg from 28 to 42 days of age and experienced a 3-h transport. CMH, creatine monohydrate; CORT, corticosterone.

Figure 2. Distribution of low-field NMR transverse relaxation (T2) times in the pectoralis major muscles of the control and 3-h transported broilers measured at 24 h postmortem.

Figure 3. Effects of dietary CMH supplementation on concentrations of glycogen (A), lactic acid (B), glycolytic potential (C), and activities of HK (D), PK (E) and LDH (F) in pectoralis major muscle of transport-stressed broilers. Results are represented as the mean value ± standard error of eight sample birds per treatment (n=8). Means without a common letter significantly differ (P < 0.05). Control, broilers fed the basal diet and experienced a 0.5-h transport; T3h or CMH+T3h, broilers fed the basal diet or the basal diet supplemented with CMH at 1200 mg/kg from 28 to 42 days of age and experienced a 3-h transport. CMH, creatine monohydrate; GP, glycolytic potential (GP = 2 × [glycogen] + [lactic acid]); HK, hexokinase; PK, pyruvate kinase; LDH, lactate dehydrogenase.

Figure 4. Effects of dietary CMH supplementation on concentrations of ATP (A), ADP (B), AMP (C), AMP/ATP ratio (D), EC (E), and concentrations of Cr (F), PCr (G) and PCr/Cr ratio (H) in pectoralis major muscle of transport-stressed broilers. Results are represented as the mean value ± standard error of eight sample birds per treatment (n=8). Means without a common letter significantly differ (P < 0.05). Control, broilers fed the basal diet and experienced a 0.5-h transport; T3h or CMH+T3h, broilers fed the basal diet or the basal diet supplemented with CMH at 1200 mg/kg from 28 to 42 days of age and experienced a 3-h transport. CMH, creatine monohydrate; ATP, adenosine triphosphate; ADP, adenosine diphosphate; AMP, adenosine monophosphate; EC, energy charge (EC = ([ATP] + 0.5[ADP])/([ATP] + [ADP] + [AMP]); Cr, creatine; PCr, phosphocreatine.

Figure 5. Effects of dietary CMH supplementation on relative mRNA expression for LKB1 (A), AMPKα1 (B), AMPKα2 (C), and protein abundances for p-LKB1 (D) and p-AMPK (E) in pectoralis major muscle of transport-stressed broilers. Results are represented as the mean value ± standard error of eight sample birds per treatment for mRNA expression analysis (n=8), and four sample birds per treatment for protein abundance analysis (n=4). Means without a common letter significantly differ (P < 0.05). Control, broilers fed the basal diet and experienced a 0.5-h transport;



T3h or CMH+T3h, broilers fed the basal diet or the basal diet supplemented with CMH at 1200 mg/kg from 28 to 42 days of age and experienced a 3-h transport. CMH, creatine monohydrate; LKB1, liver kinase B1; AMPKα1, adenosine 5’-monophosphate-activated protein kinase α1; AMPKα2, adenosine 5’-monophosphate-activated protein kinase α2; p-LKB1, phospho-liver kinase B1; p-AMPKα, phospho-adenosine 5’-monophosphate-activated protein kinase α (including α-1 & -2).


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Figure 1



Figure 2


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Figure 3



Figure 4


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Figure 5



Table of contents (TOC) Graphic


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Creatine Monohydrate Enhances Energy Status and Reduces

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