In Ovo Feeding of Creatine Pyruvate Increases the Glycolysis Pathway

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Bioactive Constituents, Metabolites, and Functions

In ovo Feeding of Creatine Pyruvate Increases Glycolysis Pathway, Glucose Transporter Genes Expression and AMPK Phosphorylation in Breast Muscle of Neonatal Broilers Minmeng Zhao, Daoqing Gong, Tian Gao, Lin Zhang, Jiaolong Li, Pengan Lv, Lanlin Yu, Guanghong Zhou, and Feng Gao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02557 • Publication Date (Web): 05 Jul 2018 Downloaded from http://pubs.acs.org on July 6, 2018

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Journal of Agricultural and Food Chemistry

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In ovo Feeding of Creatine Pyruvate Increases Glycolysis Pathway, Glucose

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Transporter Genes Expression and AMPK Phosphorylation in Breast Muscle of

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Neonatal Broilers

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Minmeng Zhao†, §, Daoqing Gong§, Tian Gao†, Lin Zhang†, Jiaolong Li†, Peng’an Lv†,

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Lanlin Yu†, Guanghong Zhou,† Feng Gao*, †

7 8

†

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Origin Food Production and Safety Guarantee, 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 210095, P. R. China

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§

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P. R. China

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

College of Animal Science and Technology, Yangzhou University, Yangzhou 225009,

1

ACS Paragon Plus Environment


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ABSTRACT: This study aims to investigate in ovo feeding (IOF) of creatine

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pyruvate

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5′-AMP-activated protein kinase (AMPK) pathway in breast muscle of embryos and

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neonatal broilers. Three treatments were: non-injected control, 0.75% NaCl treatment,

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and 12 mg CrPyr/egg treatment. The solution was injected on 17.5 day of incubation.

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At hatch, 120 male broilers from each treatment were chosen for a 7 days feeding trial.

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Compared with other treatments, CrPyr treated broilers enhanced insulin and

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thyroxine levels in plasma, adenosine triphosphate (ATP) concentration, hexokinase

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and pyruvate kinase activities, glucose transporter protein mRNA expressions, as well

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as protein abundances of phosphor-liver kinase B1 and phosphor-AMPK in breast

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muscle at hatch. In conclusion, IOF of CrPyr improved the energy status, increased

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gene expression of glucose transporter proteins, and facilitated glycolysis in breast

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muscle, which may be associated with the activated AMPK pathway.

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KEYWORDS: In ovo feeding, creatine pyruvate, glycolysis, glucose transporter

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gene, AMPK pathway

(CrPyr)

on

glucose

metabolism,

hormone

2


concentration,

and

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INTRODUCTION

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In contrast to mammals, embryonic growth and development of broiler chickens is an

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external process. The nutrient requirements for embryo are derived from the egg

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compartments. During late-hatching stage, the oxygen availability is limited, therefore,

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carbohydrates are preferentially metabolized by the chicken embryo rather than the

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lipid.1 The insufficiency of glycogen forces muscle proteins mobilization and

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produces amino acid for gluconeogenesis in embryos.2,3 Moreover, the broilers

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frequently have delayed access to feed after hatching, thereby aggravating the poor

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energy status and decreasing the growth performance of the broiler chickens on

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marketing age.4-6 Hence, increased the energy status during the few pre- and

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post-hatch days is critical for increasing the growth performance of broilers.

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In ovo feeding (IOF) was developed as an early nutritional technology to solve the

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problem. In this technique, the exogenous nutrients were injected into the amnion of

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eggs at the late stage of incubation.7,8 Towards hatch, the embryos can swallow and

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absorb the amniotic fluid, subsequently consume the injection nutrients.9 Several

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researches have indicated that injection of nutrients enhanced energy reserves, muscle

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weight, and growth performance of avian.10-13

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Pyruvate, an intermediate metabolite of glycolytic and gluconeogenesis pathway,

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plays an important role in energy metabolism.14 Creatine (Cr), which can be

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phosphorylated as phosphocreatine (PCr), has been used as a safe nutritional

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supplement for improving health and sports performance in athletic populations.15,16 3



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The Cr-PCr system maintains energy homeostasis by regulating adenosine

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diphosphate (ADP)/adenosine triphosphate (ATP).17 Additionally, Cr supplementation

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can participate in energy metabolism through increasing the glucose transporter

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protein (GLUT) expressions and activating the 5′-AMP-activated protein kinase

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(AMPK) pathway in muscle, which modulates both cellular and whole body energy

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balance.18-20 Previous researches have shown that Creatine pyruvate (CrPyr) is

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decomposed into pyruvate and Cr to participate in energy metabolism.21,22 Results in

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our lab found that CrPyr injection could improve energy reserves through increasing

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the levels of glycogen and PCr in liver or muscle tissue.23,24 However, the information

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about the impacts of CrPyr on energy status, glucose metabolism, and the detailed

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mechanism in muscle is generally limited. Therefore, this study aims to clarify the

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influences of CrPyr injection on glucose metabolism, hormone concentration,

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glycolysis enzyme activities, and protein abundances of AMPK signal pathway in

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breast muscle of broiler chickens.

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MATERIALS AND METHODS

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Incubation. The current experiment protocol was approved by the Institutional

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Animal Care and Use Committee of Nanjing Agricultural University. At first, 1200

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fertilized Arbor Acres broiler eggs (69.85 ± 0.74 g) were chosen. Eggs were incubated

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under standard conditions as previous report.23 On embryonic 6 day and 16 day,

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unfertilized and non-viable eggs were taken out. Next, 960 embryonated eggs were

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randomly assigned to three treatments of 320 eggs (n=8). 4


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Treatment Solutions and IOF Procedures. The CrPyr was purchased from

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Hubei Jusheng Technology Co., Ltd. (Wuhan, China). It was dissolved in 0.75%

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physiological saline buffer and prepared as the published reports in our lab.23,24 Three

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treatments were: non-injected control (Control), 0.75% NaCl treatment (Saline), 12

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mg CrPyr/egg treatment (CrPyr). On embryonic 17.5 day，the injection procedures

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were performed according to the previous methods.9,25 Briefly, the solution was

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injected into the amnion with a separate injector and the volume was 0.6 mL for each

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egg. Immediately, the hole was sealed with paraffin, and the eggs were placed back

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into the incubator. Eggs in control treatment were also taken out of the incubator to

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finish the same handling procedures except injection. The operation time was limited

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to 2 h.

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Birds Housing. At the end of the incubation period, all male chicks from one

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treatment were pooled and weighed. A total of 120 male broilers per treatment were

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chosen and randomly assigned into 8 replicates. The feeding and management were

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performed as our previous report and the trial ended on 7 day post-hatch.23 All of the

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chickens were provided with the same die (Table 1). Broilers were weighed after feed

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deprivation for 12 h and feed intake was recorded by replicate to calculate feed intake

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(FI), body weight gain (BWG) and feed/gain ratio (F:G) on 7 day.

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Sample Collection. On 19 day of incubation (19 E), eight male embryos were

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selected from each treatment. The gender identification was performed by observing

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the gonads morphology based on the method of Burke (1994).26 Then the breast 5



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muscle tissue of each bird was excised and frozen in liquid nitrogen.

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One bird per replicate was chosen at hatch, 3, and 7 day, respectively. Blood

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samples were collected in 2 mL tubes and centrifuged at 3000 × g for 15 min at 4°C.

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The tubes containing separated plasma was then stored at -20°C. Moreover, breast

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muscle samples were collected in liquid nitrogen.

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Adenosine Phosphate Concentration. The concentrations of ATP, ADP, and

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adenosine monophosphate (AMP) in breast muscle were determined by reverse-phase

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high performance liquid chromatography (HPLC) using a published procedure.27,28

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Frozen breast muscle sample of broiler chickens (300 mg) was homogenized in 1.5

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mL of 7% HClO4 (Sigma-Aldrich, St. Louis, MO, USA), centrifuged at 15000 × g for

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10 min at 4°C. The supernatant was mixed with KOH (Sinopharm Chemical Reagent

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Co., Ltd, Shanghai, China). The supernatant was filtered for HPLC analysis. The UV

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detection wavelength used in this study was 254 nm. The mobile phase consisted of

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13.5% methanol (Honeywell Burdick & Jackson®, Morristown, NJ, USA) and 86.5%

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phosphate buffer solution (vol: vol). Phosphate buffer solution contained 2.5 mM

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tetrabutylammonium sulfate, 0.04 M potassium dihydrogen phosphate, and 0.06 M

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dipotassium hydrogen phosphate (Sinopharm Chemical Reagent Co., Ltd, Shanghai,

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China).

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Determination of Hexokinase and Pyruvate Kinase Activities. For each chicken,

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breast muscle sample was homogenized with 0.75% saline (weight: volume, 1: 9).

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The activities of hexokinase and pyruvate kinase were determined using commercial 6


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kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

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Measurements of Pyruvic Acid, Glucose and Glycogen. The levels of pyruvate

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and glycogen were analyzed by commercial kits (Nanjing Jiancheng Bioengineering

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Institute, Nanjing, China). The glucose level in breast muscle was measured according

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to the procedure of commercial kit (Shanghai RongSheng Biotech Co. Ltd., Shanghai,

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China).

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Hormone Concentrations of Plasma. The concentrations of insulin, glucagon,

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triiodothyronine (T3), and thyroxine (T4) in plasma were determined by

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radioimmunoassay based on the commercially available kits (Beijing North Institute

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of Biological Technology, Beijing, China)

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Real-time PCR Analysis. Total RNA from breast muscle samples was obtained

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using RNAiso Plus reagent (TaKaRa Biotechnology Co. Ltd., Dalian, China). Total

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RNA was reverse transcribed to cDNA using kits (TaKaRa Biotechnology Co. Ltd.,

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Dalian, China). Real-time PCR was carried out on a QuanStudio5 detection system

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(Applied Biosystems, USA) using kit (Takara Biotechnology Co. Ltd., Dalian, China).

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The program was according to previous study in our lab.23 The 2−∆∆CT method was

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used to calculate relative mRNA level.29 The primer sequences can be found in Table

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2.

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Western Blot Analysis. The breast muscle sample was crushed into powder and

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centrifuged. The protein level was determined by commercial kit of Beyotime

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Biotechnology (Shanghai, China). Specific primary antibodies against total liver 7



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kinase B1 (LKB1, 1:1000), phosphorylated LKB1 (Thr189, 1:1000), total AMPK

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(1:1000), and phosphorylated AMPK (Thr172, 1:1000) were obtained from Cell

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Signaling Technology (Beverly, MA, USA), and β-actin (1:1500) from Santa Cruz

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Biotechnology (Santa Cruz, CA, USA). The amounts of protein used for separation

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was 30 ug. The densities of bands were quantified by Quantity One software (Bio-Rad

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Laboratories Inc., Hercules, CA, USA). The β-actin was considered as a standard.

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Statistical Analysis. Data were analyzed by one-way ANOVA using SAS 9.2 (SAS

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Institute 2010) software. Differences among the treatments were detected using

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Tukey’s test. Results were represented as the mean value ± standard error of eight

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sample birds per treatment (n=8). Data were considered significant at P < 0.05, and P

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values between 0.05 and 0.1 were considered as a trend.

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RESULTS

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Growth performance. No difference in FI or F:G was observed among treatments

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(P > 0.05, Table 3). Nevertheless, CrPyr injection tended to enhance BWG of broilers

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during the first week post-hatch (P = 0.09).

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Adenosine Phosphate Concentration. On 19 E, broiler chickens subjected to

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CrPyr group had higher ADP concentration and lower ATP concentration than those of

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other groups (P < 0.05, Table 4). Additionally, CrPyr injection individuals enhanced

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ATP concentration and decreased ADP concentration at hatch (P < 0.05).

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Hexokinase and Pyruvate Kinase Activities. On the day of hatch, the activities of

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hexokinase and pyruvate kinase in breast muscle of broilers in CrPyr treatment were 8


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found to be higher than those in other groups (P < 0.05, Table 5).

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Pyruvic Acid, Glucose and Glycogen levels. There was no difference in the

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concentrations of pyruvate, glucose, and glycogen in breast muscle of embryo and

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chicken among the three treatments (Figure 1).

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Hormone Concentration. As shown in Figure 2, CrPyr injection enhanced (P < 0.05) the insulin level at hatch and 3 day of age and T4 level in plasma at hatch.

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mRNA Expressions of GLUT3, GLUT8, GYS1, and GP. Compared with broiler

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chickens in other groups, CrPyr-treated broiler chickens exhibited up-regulation of

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GLUT3 and GLUT8 expressions at hatch (P < 0.05, Figure 3A and 3B). Neither

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glycogen synthase 1 (GYS1) nor glycogen phosphorylase (GP) mRNA expression

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level was altered by CrPyr injection (P > 0.05, Figure 3C and 3D).

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AMPK Pathway Protein Abundances. As shown in Figure 4, CrPyr injection

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improved protein abundances of phosphor-LKB1 and phosphor-AMPK in breast

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muscle of broilers compared with other groups (P < 0.05, Figure 4B and 4D).

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DISCUSSION

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Previous studies showed that exterior nutrients injection increased the hatching and

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growth performance of broilers via influencing gastrointestinal development and

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motivating appetite.9,30,31 Similarly, injection CrPyr into amnion of broiler eggs

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increased the FI and BWG during 1 to 21, 22 to 42, and 1 to 42 days post-hatch and

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the higher FI of broilers in CrPyr group could induce the greater BWG.32 Another

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research found that CrPyr injection enhanced energy reserves, which was beneficial to 9



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the improvement of hatching weight and body weight on 7 day post-hacth.33 However,

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the results in this study suggested that CrPyr injection had no obvious impact on FI

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and F:G, but tended to increase the BWG of broilers from 1 to 7 day post-hatch.

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Previous study showed that arginine injection enhanced the average daily FI of

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chickens and speculated the reason might be attributed to that arginine solution

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stimulated the secretion of gastrointestinal hormones, thereby promoting the growth

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of gastrointestinal tract, which in turn increased the FI and BWG.34,35 Similarly,

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another research revealed that threonine and arginine injection could enhance the FI

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and BW of broilers possibly through improving gastrointestinal development.

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Hence, the possible explanation might be that the injection solutions containing CrPyr

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could stimulate the growth of gastrointestinal tract firstly, then the digestive and

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absorptive capacity, but the effects on FI and BWG required more than one week to

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reflect. Further research to explore the influences of CrPyr on digestive organs is

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needed.

31

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The embryos are preferentially metabolize the glycogen in liver and breast muscle

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over the lipid and protein due to the limited oxygen during the pre-hatch period, while

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there are insufficient carbohydrates in the chicken eggs.1 Consequently, the depleted

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carbohydrate reserves urge embryos to mobilize many proteins to produce amino

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acids for gluconeogenesis in breast muscle, thereby inhibiting the broilers

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development in post-hatch stage.3 IOF of carbohydrates, amino acid, or intermediate

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product of amino acid, could increase the energy reserves in poultry through 10


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increasing the glycogen concentration in liver or breast muscle.10,30,36 A previous

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research in our lab revealed that CrPyr injection improved the concentrations of

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pyruvate, glucose and glycogen in liver.24 In contrast, there were no detective changes

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in the levels of pyruvate, glucose, or glycogen in breast muscle of embryos and

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broilers in this study. This discrepancy suggested that the exogenous pyruvate from

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the injection solutions was mainly used as the substrate for gluconeogenesis in liver

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rather than absorbed and utilized in breast muscle of broilers. The reason may be

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attributed to that the gluconeogenesis process occurs mainly in liver instead of

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muscles.

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The impacts of CrPyr injection on breast muscle energy storage in this study were

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similar to the outcomes of previous studies, which showed that protein or amino acid

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individual injection did not change the glycogen concentration in breast muscle.36,37

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Conversely, IOF of carbohydrates (simpler sugars) enhanced the glycogen

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concentration in breast muscle of domestic pigeons.11 As indicated in another study,

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IOF of carbohydrates might stimulate the insulin secretion, thereby accelerating the

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glucose uptake in breast muscle.38 In our previous study, we also found that IOF of

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CrPyr had no obvious effect on glycogen or glucose concentration in breast muscle of

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broilers.33 Therefore, we speculated in the beginning that IOF of non-carbohydrate

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nutrient substrates, may did not increase insulin level, so, the glucose come from

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gluconeogenesis is primary stored in liver instead of breast muscle.33,36 However, the

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findings of the current research showed that CrPyr injection increased the plasma 11



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insulin concentration at hatch and 3 day post-hatch. Similarly, diet Cr supplementation

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could induce insulin secretion in rats.39 However, Cr monohydrate supplementation

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did not have effects on the insulin secretion in vivo in humans.40 The distinctions of

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experimental methods, Cr dosage, and animal species may explain these discrepancies

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in the obtained results.

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Indeed, GLUT4 is important in insulin stimulating glucose uptake from blood in

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mammals.41 In spite of the chickens are short of GLUT4 gene, some studies reported

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that the insulin responsive glucose transport mechanism is also exists in broilers.42-43

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Several reports revealed that the mRNA expressions of GLUT1, 3, 8, and 12 are

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detected in muscles, and speculated that these isoforms may play a role in the insulin

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stimulating glucose transport in broiler chicken muscle.43-45 In the present study,

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CrPyr injection increased GLUT3 and GLUT8 mRNA expression levels in breast

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muscle of broiler chickens at hatch. Cr supplementation could increase the GLUT4

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expression in human and rat skeletal muscles.18-19 A recent study has demonstrated

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that CrPyr injection enhanced the muscle Cr concentration of broilers at hatch.23

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Therefore, the enhanced mRNA expressions of GLUT3 and GLUT8 might be

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associated with the high Cr concentration of broiler chickens in CrPyr treatment.

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Interestingly, no differences in glucose concentration, GYS1, and GP mRNA

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expression level were observed among the treatments during the study. Nevertheless,

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the activities of hexokinase and pyruvate kinase, the key enzymes in the glycolysis

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pathway, were increased in CrPyr treatment, suggesting that IOF of CrPyr promoted 12


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glycolysis in muscle of chickens at hatch. These findings implied that the high Cr

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concentration might increase the mRNA expressions of GLUT3 and GLUT8 in breast

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muscle of birds in CrPyr treatment, thereby enhancing the glucose uptake from blood

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in chicken muscle. Subsequently, the glucose from blood was used to produce energy

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through glycolysis process before the broilers had access to feed and water. However,

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further investigation is needed to verify the speculation.

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The results in previous research indicated that CrPyr injection enhanced the levels

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of Cr and PCr in muscle.33 Therefore, we speculated that the increased ATP

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concentration of breast muscle in this study might partially result from the PCr

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producing Cr reaction which could replenish ATP. On the other hand, the outcomes in

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the current study suggested that CrPyr injection might facilitate the uptake of glucose

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from blood and accelerate glycolysis in breast muscle. Consequently, the increased

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ATP level could also come from the enhancement of glycolysis process. ATP can be

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used as the direct energy source of skeletal muscle cells. Hence, the increased ATP

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level enhanced the energy status in breast muscle, which might be useful to the

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development of late embryos and neonatal broilers.33

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AMPK signal pathway is involved in the regulation of energy homeostasis in

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mammals. Indeed, a functional LKB1/AMPK pathway as a central regulator of energy

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metabolism also present in broilers, similarly to that observed in mammals.46 Under

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conditions of stress, the body requires to consume large amounts of ATP, which leads

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to an increasing AMP/ATP ratio. Therefore, AMPK is activated and the activation of 13



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AMPK needs the phosphorylation of a threonine residue (T172) by an upstream

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kinase named LKB1.47 The activated AMPK pathway is responsible for regulation of

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GLUT4 gene expression in skeletal muscle.48 In addition, previous studies have

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demonstrated that oral Cr supplementation did not increase PCr concentration in the

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same proportion as Cr, leading to decreased PCr to Cr ratio, which is known to

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activate the AMPK pathway.20,49,50 CrPyr injection increased the phosphor-LKB1 and

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phosphor-AMPK protein abundances, suggesting that the AMPK pathway was

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activated in muscle of broiler chickens at hatch in this study. Consistent with our

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study, Ceddia and Sweeney (2004) found an approximately twofold increase in

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AMPK phosphorylation after 48 h of Cr supplementation in myocytes.51 Another

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research also reported that Cr feeding increased AMPK phosphorylation in rat

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muscle.19 IOF of CrPyr increased the Cr level but had no effect on the PCr level,

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leading to a decline of PCr to Cr ratio.23 CrPyr-treated broilers had increased ATP

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concentration and similar AMP concentration on the day of hatch, inducing a decrease

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of AMP/ATP ratio in this study. Hence, the activation of AMPK pathway was caused

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possibly by the decreased PCr to Cr ratio, not the AMP/ATP ratio in the breast muscle

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of broilers in CrPyr treatment at hatch. Nevertheless, dietary addition of Cr

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monohydrate ameliorated 3 hours transport-induced rapid glycolysis through

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inhibition of AMPK pathway in breast muscle of chickens. The discrepancy might be

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explained through the different Cr supplement products and different physiological

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status of the broilers.52 14


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In summary, IOF of CrPyr improved the energy status of neonatal broiler chickens

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by enhancing the ATP concentration in muscle. In addition, IOF of CrPyr could

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increase the gene expression of glucose transporter proteins (GLUT3 and GLUT8)

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and facilitate the muscle glycolysis possibly through up-regulating the activities of

285

hexokinase and pyruvate kinase, which might be associated with the activated AMPK

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signal pathway.

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AUTHOR INFORMATION

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Corresponding Author

289

*

290

Funding

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This research was supported by the National Natural Science Foundation of China (no.

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31572425), the National Key Research and Development Program of China (no.

293

2016YFD0500501),

294

SXGC(2017)281), and China Postdoctoral Science Foundation (no.2017M621840).

295

Notes

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The authors declare that there are no competing financial interests in the work

297

described.

298

ABBREVIATIONS USED

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IOF, in ovo feeding; CrPyr, creatine pyruvate; Cr, creatine; PCr, phosphocreatine;

300

ADP, adenosine diphosphate; ATP, adenosine triphosphate; AMP, adenosine

301

monophosphate; AMPK, 5′-AMP-activated protein kinase; FI, feed intake; BWG,

Tel.: +86-25-84399007. Fax: +86-25-84395314. Email: [email protected].

Three

Agricultural

Projects

of

15


Jiangsu

Province

(no.


302

body weight gain; F:G, feed/gain ratio; 19 E, 19 d of incubation; HPLC, reverse-phase

303

high performance liquid chromatography; GAPDH, glyceraldehyde-3-phosphate

304

dehydrogenase; T3, triiodothyronine; T4, thyroxine; LKB1, liver kinase B1; GLUT3,

305

glucose transporter protein 3; GLUT8, glucose transporter protein 8; GYS1, glycogen

306

synthase 1; GP, glycogen phosphorylase.

16


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Table 1. Composition and Nutrient Content of the Basal Diet Items

Value

Ingredients (%) Corn

57.61

Soybean meal

31.00

Corn gluten meala

3.29

Soybean oil

3.11

Limestone

1.20

Dicalcium phosphate

2.00

L-Lysine

0.34

DL-Methionine

0.15

Sodium chloride

0.30

Premixb

1.00

Calculated nutrient levels Apparent metabolizable energy (MJ/kg)

12.56

Crude protein (%)

21.10

Calcium (%)

1.00

Available phosphorus (%)

0.46

Lysine (%)

1.20

Methionine (%)

0.50

Methionine + cysteine (%)

0.85 25



a

Crude protein content was 60%.

b

The premix contained (per kilogram of diet):

retinyl acetate for vitamin A, 12000 IU; cholecalciferol for vitamin D3, 2500 IU; DL-α-tocopheryl acetate for vitamin E, 20 IU; menadione sodium bisulphate, 1.3 mg; thiamin, 2.2 mg; riboflavin, 8.0 mg; nicotinamide, 40 mg; choline chloride, 400 mg; calcium pantothenate, 10 mg; pyridoxine HCl, 4 mg; biotin, 0.04 mg; folic acid, 1 mg; vitamin B12 (cobalamin), 0.013 mg; Fe (from ferrous sulfate), 80 mg; Cu (from copper sulphate), 8.0 mg; Mn (from manganese sulphate), 110 mg; Zn (from zinc sulfate), 60 mg; I (from calcium iodate), 1.1 mg; Se (from sodium selenite), 0.3 mg.

26


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Table 2. Primers Sequences for Real-Time quantitative PCR Analysis Gene namea Genbank number

Primer sequence(5'to3')

Product Size (bp)

F:ATGCTCTTCCCCTATGCTGA GLUT3

NM_205511

123 R:AAAAGTCCTGCCCTTGGTCT F:GCAAGGGGTGTATCAAGCAG

GLUT8

NM_204375

126 R:GGCAGAGAAGAGCCAGAATG F:CACGCACCAACAACTTCAAC

GYS1

XM_015275065

122 R:CACCAGCAGCGACTCATAGA

GP

XM_015274646

F:CCAACGACTTCAACCTCAAAG

126

R:GCTCCTTCCCCTCAAAGAA F:GAGGGTAGTGAAGGCTGCTG GAPDH

NM_204305.1

113 R:CATCAAAGGTGGAGGAATGG

a

GLUT3, glucose transporter protein 3; GLUT8, glucose transporter protein 8; GP,

glycogen phosphorylase; GYS1, glycogen synthase 1; GAPDH, glyceraldehyde 3 phosphate dehydrogenase.

27



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Table 3. Effects of IOF of CrPyr on the Growth Performance of Broilers from 1 to 7 day of Agea Treatmentsb Itemsc Control

Saline

CrPyr

FI (g/bird)

119.55 ± 2.02

125.04 ± 2.30

124.33 ± 2.34

BWG（g/bird）

102.37 ± 1.90

104.17 ± 1.01

106.86 ± 1.14

1.17 ± 0.01

1.20 ± 0.02

1.16 ± 0.03

F:G (g:g) a

IOF, in ovo feeding; CrPyr, creatine pyruvate. bControl is the non-injected

treatment. Saline is the treatment injected with 0.6 mL of physiological saline (0.75%) per egg. CrPyr is the IOF treatment injected with 0.6 mL of physiological saline (0.75%) containing 12 mg CrPyr per egg. Results are represented as the mean value ± standard error of eight sample birds per treatment (n=8). cFI, Feed intake; BWG, body weight gain; F:G, feed:gain.

28


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Table 4. Effects of IOF of CrPyr on the Concentrations of Adenosine Phosphate in Breast Muscle of Embryos and Broilers (µmol/g)a Treatmentsb Itemsc Control

Saline

CrPyr

ATP

0.52 ± 0.03a

0.50 ± 0.02a

0.41 ± 0.02b

ADP

0.69 ± 0.04b

0.68 ± 0.03b

0.82 ± 0.02a

AMP

1.01 ± 0.04

1.07 ± 0.05

1.03 ± 0.03

ATP

0.31 ± 0.01b

0.31 ± 0.01b

0.40 ± 0.02a

ADP

0.55 ± 0.03a

0.57 ± 0.02a

0.46 ± 0.01b

AMP

0.93 ± 0.04

0.93 ± 0.05

1.02 ± 0.05

ATP

0.88 ± 0.03

0.86 ± 0.04

0.84 ± 0.04

ADP

1.04 ± 0.08

0.97 ± 0.02

1.01 ± 0.06

AMP

1.16 ± 0.04

1.24 ± 0.06

1.23 ± 0.04

ATP

3.15 ± 0.20

3.17 ± 0.18

3.45 ± 0.13

ADP

1.29 ± 0.05

1.21 ± 0.09

1.23 ± 0.06

AMP

0.50 ± 0.02

0.51 ± 0.03

0.48 ± 0.03

19 E

Hatch

3 day

7 day

a

IOF, in ovo feeding; CrPyr, creatine pyruvate. bControl is the non-injected 29



treatment. Saline is the treatment injected with 0.6 mL of physiological saline (0.75%) per egg. CrPyr is the IOF treatment injected with 0.6 mL of physiological saline (0.75%) containing 12 mg CrPyr per egg. 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). c19 E, 19 day of incubation; ATP, adenosine triphosphate; ADP, adenosine diphosphate; AMP, adenosine monophosphate.

30


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Table 5. Effects of IOF of CrPyr on the Activities of Hexokinase and Pyruvate Kinase in Breast Muscle of Embryos and Broilersa Treatmentsb Itemsc Control

Saline

CrPyr

Activity of hexokinase (U/g of protein) 19 E

30.19 ± 1.13

29.08 ± 0.94

29.31 ± 0.97

Hatch

24.40 ± 1.21b

25.60 ± 1.18b

32.04 ± 1.53a

3 day

33.63 ± 1.07

34.20 ± 1.28

35.16 ± 0.92

7 day

28.70 ± 0.57

27.82 ± 1.05

27.07 ± 0.78

Activity of pyruvate kinase (U/g of protein)

a

19 E

437.05 ± 16.28

419.97 ± 11.37

432.09 ± 14.81

Hatch

526.97 ± 14.17b

534.41 ± 10.11b

581.65 ± 16.18a

3 day

514.88 ± 9.47

508.87 ± 9.93

505.03 ± 13.04

7 day

486.09 ± 11.74

472.67 ± 14.32

480.00 ± 15.69

IOF, in ovo feeding; CrPyr, creatine pyruvate. bControl is the non-injected

treatment. Saline is the treatment injected with 0.6 mL of physiological saline (0.75%) per egg. CrPyr is the IOF treatment injected with 0.6 mL of physiological saline (0.75%) containing 12 mg CrPyr per egg. 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). c19 E, 19 day of incubation. 31



Figure captions Figure 1. The effects of in ovo feeding (IOF) of creatine pyruvate (CrPyr) on the concentrations of pyruvate (A), glucose (B), and glycogen (C) in breast muscle of embryos and broilers on 19 d of incubation (19 E), the day of hatch, 3, and 7 d post-hatch. Control is the non-injected treatment. Saline is the treatment injected with 0.6 mL of physiological saline (0.75%) per egg. CrPyr is the IOF treatment injected with 0.6 mL of physiological saline (0.75%) containing 12 mg CrPyr per egg. Results are represented as the mean value ± standard error of eight sample birds per treatment (n=8). Different letters within the same time points indicate significant differences among the three treatments (P < 0.05). Figure 2. The effects of in ovo feeding (IOF) of creatine pyruvate (CrPyr) on the level of plasma hormone in broilers on the day of hatch, 3 and 7 d post-hatch. Control is the non-injected treatment. Saline is the treatment injected with 0.6 mL of physiological saline (0.75%) per egg. CrPyr is the IOF treatment injected with 0.6 mL of physiological saline (0.75%) containing 12 mg CrPyr per egg. Results are represented as the mean value ± standard error of eight sample birds per treatment (n=8). Different letters within the same time points indicate significant differences among the three treatments (P < 0.05). Figure 3. The effects of in ovo feeding (IOF) of creatine pyruvate (CrPyr) on the relative mRNA expressions of GLUT3 (A), GLUT8 (B), glycogen synthase 1 (GYS1, C), and glycogen phosphorylase (GP, D) in the breast muscle of embryos 32


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and broilers on 19 d of incubation (19 E), hatch, 3 and 7 d post-hatch. Control is the non-injected treatment. Saline is the treatment injected with 0.6 mL of physiological saline (0.75%) per egg. CrPyr is the in ovo feeding (IOF) treatment injected with 0.6 mL of physiological saline (0.75%) containing 12 mg CrPyr per egg. Results are represented as the mean value ± standard error of eight sample birds per treatment (n=8). Different letters within the same time points indicate significant differences among the three treatments (P < 0.05). Figure 4. The effects of in ovo feeding (IOF) of creatine pyruvate (CrPyr) on the abundances of total Liver kinase B1 (LKB1, A) and phosphor-LKB1, total 5′-AMP-activated protein kinase (AMPK) and phosphor-AMPK. The representative Western blot images are shown. β-actin was used as a standard to normalize the protein abundances. Control is the non-injected treatment. Saline is the treatment injected with 0.6 mL of physiological saline (0.75%) per egg. CrPyr is the in ovo feeding (IOF) treatment injected with 0.6 mL of physiological saline (0.75%) containing 12 mg CrPyr per egg. Results are represented as the mean value ± standard error of eight sample birds per treatment (n=4). Different letters within the same time points indicate significant differences among the three treatments (P < 0.05).

33



Figure 1

34


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Figure 2

35



Figure 3

36


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Figure 4

37



TOC Graphic

38


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In Ovo Feeding of Creatine Pyruvate Increases the Glycolysis Pathway

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