Creatine Monohydrate and Guanidinoacetic Acid Supplementation

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Bioactive Constituents, Metabolites, and Functions

Creatine Monohydrate and Guanidinoacetic Acid Supplementation Affect the Growth Performance, Meat Quality and Creatine Metabolism of Finishing Pigs Jiaolong Li, Lin Zhang, Yanan Fu, Yanjiao Li, Yun Jiang, Guanghong Zhou, and Feng Gao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02534 • Publication Date (Web): 02 Sep 2018 Downloaded from http://pubs.acs.org on September 3, 2018

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Journal of Agricultural and Food Chemistry

Creatine Monohydrate and Guanidinoacetic Acid Supplementation Affect the Growth Performance, Meat Quality and Creatine Metabolism of Finishing Pigs Jiaolong Li, † Lin Zhang, † Yanan Fu, † Yanjiao Li, † Yun Jiang, § Guanghong Zhou, † Feng Gao†* †

College of Animal Science and Technology; Key Laboratory of Animal Origin Food

Production and Safety Guarantee of Jiangsu Province; Key Laboratory of Gastrointestinal Nutrition and Animal Health of Jiangsu Province; Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China §

Ginling College, Nanjing Normal University, Nanjing 210024, People’s Republic of

China *

Corresponding author: Feng Gao (Tel.: +86-25-84399007. Fax: +86-25-84395314.

Email: [email protected]).

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ACS Paragon Plus Environment


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ABSTRACT

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This study aimed to investigate the effects of creatine monohydrate (CMH) and

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guanidinoacetic acid (GAA) supplementation on the growth performance, meat

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quality and creatine metabolism of finishing pigs. The pigs were randomly allocated

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to three treatment groups: the control group, CMH group and GAA group. Compared

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with the control group, CMH treatment increased average daily feed intake, and GAA

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treatment increased average daily feed intake and average daily gain of pigs. In

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addition, CMH and GAA treatment increased pH45min, myofibrillar protein solubility

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and calpain 1 mRNA expression level, decreased the drip loss and shear force value in

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longissimus dorsi or semitendinosus muscle. Moreover, CMH and GAA

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supplementation increased the concentrations of creatine and phosphocreatine, and the

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mRNA expressions of guanidinoacetate N-methyltransferase and creatine transporter

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in longissimus dorsi, semitendinosus muscle, liver or kidney, and decreased the

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mRNA expressions of arginine: glycine amidinotransferase in kidney. In conclusion,

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CMH and GAA supplementation could improve the growth performance, meat quality

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and alter creatine metabolism of finishing pigs.

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KEY WORDS

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creatine monohydrate, guanidinoacetic acid, growth performance, meat quality,

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creatine metabolism, finishing pigs

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INTRODUCTION

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The creatine/creatine kinase system is an energy metabolic pathway to provide ATP

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for cellular function. 1 About 95% of the creatine is mainly located in skeletal muscle,

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and the remaining 5% is stored in the brain, liver, kidneys, and testes. Meanwhile,

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most of the energy is consumed by these organs and tissues. 2 Creatine is synthesized

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endogenously by a two-step process from glycine, arginine, and methionine. 3-5 In the

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first step, the amidino group in arginine is transferred to glycine to form ornithine and

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guanidinoacetic

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amidinotransferase (AGAT). This process mainly takes place in kidney and pancreas.

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As to the second step, the methyl group from S-adenosylmethionine is transferred to

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GAA to form creatine and S-adenosylhomocysteine. This process is mainly catalyzed

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by guanidinoacetate N-methyltransferase (GAMT), and occurs in kidney and liver.

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Then creatine enters the bloodstream and is subsequently transported to cells and

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tissues for energy supply. Once entering the cell, creatine is phosphorylated to

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phosphocreatine by creatine kinase, and functions to buffer changes in ATP during the

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altered energy status.

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excreted in urine.

acid

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(GAA),

which

is

catalyzed

by

arginine:

glycine

Finally, creatine is spontaneously converted to creatinine and

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It has been reported that the meat quality is related to the energy status in muscle. 7

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After slaughter, with the stop of blood circulation, the metabolic pathway of skeletal

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muscle turns into glycolysis and lactic fermentation, resulting in the accumulation of

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lactic acid, and a concomitant decrease in pH value. Several researches reported that

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dietary creatine or creatine monohydrate (CMH) supplementation could increase the 3



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concentrations of creatine and phosphocreatine,

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glycolysis, and then improve meat quality of pigs.

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precursor for creatine synthesis in animals, and has the similar functions as CMH.

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Given that GAA is less expensive, more chemically stable and more bio-available

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than creatine,

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human studies, dietary GAA increased total muscle creatine and phosphocreatine

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levels. 15,16 In addition, supplementation with GAA has also been reported to increase

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the muscle pH, improve the color, decrease the drip loss and shear force value of meat

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in pigs. 15,17

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decrease the rate of postmortem 10-13

GAA is the immediate

we presume it might be an alternative for CMH supplementation. In

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There are extensive reports on the effects of CMH and GAA supplementation on

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meat quality; however, there is limited information regarding the effects of dietary

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supplementation with CMH and GAA on the creatine metabolism of finishing pigs.

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This study was carried out to investigate the effects of CMH and GAA

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supplementation on the growth performance, meat quality and creatine metabolism of

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finishing pigs.

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MATERIALS AND METHODS

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Animal Managements and Experimental Diets. All pigs were provided and

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managed by Changzhou Meinong Farming Technology Co., Ltd. (Changzhou, China).

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A total of 180 healthy cross castrated male pigs (Duroc × Landrace × Yorkshire, 90.88

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± 2.91 kg) were individually weighed and randomly allocated to three treatment

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groups, and each treatment consisted of three replicates (pens) of twenty pigs each. 4


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The pigs in three groups were fed with basal diets (control group), basal diets

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supplemented with 0.8% CMH (CMH group), or basal diets supplemented with 0.1%

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GAA (GAA group), individually. CMH and GAA were obtained from Tianjin

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Tiancheng Pharmaceutical Co., Ltd. (Tianjin, China). Feed and water were available

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ad libitum during the experiment for 15 days. The compositions and nutrient levels of

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the basal diet are presented in Table 1. At the beginning and the end of the experiment,

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the body weight and feed intake of each replicate pen were recorded to measure the

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average daily feed intake (ADFI), average daily gain (ADG) and feed to gain ratio

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(F/G). The experimental design and procedures were approved by the Animal Care

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and Use Committee of Nanjing Agricultural University.

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Sample Collection. After the 15 days of dietary treatment, three pigs per pen with the

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similar average body weight of the pen (twenty-seven in total) were selected and

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transferred to the slaughterhouse for slaughter with bleeding after electrical stunning

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after the 12 h fasting. After eviscerating, the samples from the longissimus dorsi

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muscle at the last rib and semitendinosus muscle were placed in vacuum bags at 4°C

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in order to measure meat quality traits. Meanwhile, samples from the longissimus

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dorsi muscle, semitendinosus muscle, liver and kidney were immediately frozen in

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liquid nitrogen, and stored at −80 °C for further analysis. After 24h chilling, samples

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from the muscles were collected and stored at −80 °C for protein solubility

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measurement.

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Meat Quality Measurement. The pH values of the longissimus dorsi muscle and

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semitendinosus muscle were measured at 45min (pH45 min) and 24h (pHu) postmortem 5



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using a pH Meter (HI9125, HANNA Instrument, Italy). Each sample was tested three

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times at different locations, and the average values were used.

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The meat color was measured with a CR410 Chroma Meter (Konica Minolta

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Sensing Inc., Japan) at 24 h postmortem, and the values were described as CIE L*

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(lightness), a* (redness) and b* (yellowness). Measurements were performed in

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triplicate for each sample, and the values were averaged.

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Drip loss was measured by the method of Li et al. (2016)

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with minor

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modifications. Approximately 20 g samples of the longissimus dorsi muscle and

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semitendinosus muscle were cut like a strip parallel to the longitudinal orientation of

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the myofibers. After removing the surface water, the samples were initially

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weighed ,and hanged in the plastic bag in a refrigerator at 4 °C for 24 h. Then the

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surface water was removed again, and the samples were reweighed to calculate the

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drip loss percentage.

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The 48 h postmortem samples were used to measure the cooking loss and shear

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force value in accordance with the method described by Li et al. (2017). 18 About 20 g

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of samples were weighed and placed in vacuum bags, and cooked in a water bath at

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75 °C until the internal temperature of samples reached 70 °C. After cooling in the

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running water, the surface water was removed, and the samples were reweighed to

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calculate the cooking loss percentage. Then the cooked samples were shaped to a size

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of 3 × 1 × 1 cm, and sheared perpendicular to the muscle fiber direction in triplicate to

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measure the shear force value using a Digital Meat Tenderness Meter (C-LM3B,

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Northeast Agricultural University, Harbin, China). 6


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Protein Solubility Measurement. Total protein solubility (TPS) and sarcoplasmic

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protein solubility (SPS) in longissimus dorsi muscle and semitendinosus muscle were

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analyzed as described by Niu et al. (2015).

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muscle samples were homogenized in 10 mL of cooled 1.1 M potassium iodide in 0.1

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M phosphate buffer (pH 7.2) for 30 s, and about 0.5 g of muscle samples were

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homogenized in 5 mL of cooled 0.025 M phosphate buffer (pH 7.2) for 30 s to

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measure the SPS. The homogenates were extracted at 4 °C for 12h, and then

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centrifuged for 20 min at 1500 × g at 4 °C to obtain the supernatants, and the protein

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concentrations in the supernatants were determined using commercial kits by

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Bradford method (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

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Myofibrillar protein solubility (MPS) was calculated by the difference between total

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and sarcoplasmic protein solubility.

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Creatine and Phosphocreatine Determination. The concentrations of creatine and

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phosphocreatine in the muscle, liver and kidney were evaluated by reverse phase high

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performance liquid chromatography (HPLC) according to the method of Liu et al.

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(2015).

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0.5% perchloric acid for 1 min, and extracted at 4 °C for 15 min. Then the

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supernatants of homogenates were obtained by centrifugation for 10 min at 8500 × g

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at 4 °C. After that, the supernatants were transferred into a new centrifuge tube, and

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neutralized with 900 µL of 0.8 M K2CO3 for 10 min, then recentrifuged for 10 min at

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8500 × g at 4 °C. The supernatant was filtered through 0.45µm filtration membrane

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before injection into the Waters 2695 Alliance HPLC system (Waters Corporation,

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To measure the TPS, about 0.5 g of

Briefly, about 300 mg of samples were homogenized in 2 mL of ice-cold

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Milford, MA, USA). The chromatographic parameters were set as follow: The

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injection volume was 10 µL; the column temperature was 25 °C; the flow rate was

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kept at 1.0 mL/min; the ultraviolet detection was 210 nm; the mobile phases were

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methyl cyanides and 29.4 mM KH2PO4 buffer, and the volume radio was 2 : 98.

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Real-Time Quantitative PCR Analysis. Total RNA were extracted from the frozen

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muscle, liver or kidney samples using Trizol reagent (TaKaRa Biotechnology Co. Ltd.,

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Dalian, China) in accordance with the manufacturer’s instructions. The purity and

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concentration of total RNA were measured using Nanodrop 1000 Spectrophotometer

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(Thermo Scientific, Wilmington, USA). After that, the RNA was reversed to cDNA

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using commercial kits (TaKaRa Biotechnology Co. Ltd., Dalian, China). Then,

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Real-Time PCR was performed in optical 96-well plates using the 7500 Quantitative

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PCR Step One Plus

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Premix Ex Taq™ kits (TaKaRa Biotechnology Co. Ltd., Dalian, China) according to

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the manufacturer’s instructions. The primers used for mRNA expression were

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synthesized by Sangon Biotech Co. Ltd. (Shanghai, China), and the sequences of

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target genes and housekeeping gene are exhibited in Table 2. The reaction system

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volume of Real-Time PCR was 20 µL, and each sample was repeated in triplicate. The

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program was as follows: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and

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60 °C for 34 s, and 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s. The relative

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gene expression was calculated using the 2−∆∆Ct method according to Livak and

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Schmittgen (2001). 20

TM

system (Applied Biosystems, Foster City, USA) with SYBR®

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Statistical Analyses. Data analysis was carried out using one-way analysis of

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variance (ANOVA) with the SPSS statistical software (version 20.0 for Windows;

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SPSS Institute Inc., Chicago, USA), and Tukey’s test was performed. All data were

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analyzed using pen as the experimental unit (n = 3). Data were shown as mean values

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and standard error of the mean (SEM), and the result with P 0.05).

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As indicated in Table 4, the pigs subjected to CMH and GAA treatments had higher

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pH45min and lower drip loss in longissimus dorsi muscle (P < 0.05). Meanwhile, CMH

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supplementation decreased the shear force value in longissimus dorsi muscle

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compared with the control group (P < 0.05). There were no differences in pHu, L*, a*,

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b* and cooking loss among three groups (P > 0.05). Similarly with longissimus dorsi

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muscle, the pigs fed with CMH had higher pH45min and lower drip loss in

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semitendinosus muscle (P < 0.05), and dietary GAA reduced the drip loss compared

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with the control group (P < 0.05). There were no significant treatment effects on pHu,

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L*, a*, b*, cooking loss and shear force in semitendinosus muscle (P > 0.05). In

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addition, dietary CMH supplementation increased the MPS of semitendinosus muscle 9



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compared with the control group (P < 0.05, Table 5). Moreover, compared with the

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control group, CMH and GAA treatment showed significant up-regulation of CAPN1

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mRNA expression levels (P < 0.05, Fig. 1A), while exerted no effect on CAST

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mRNA expression levels in the longissimus dorsi muscle (P > 0.05, Fig. 1A). Similar

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tendency was found with the mRNA expressions of CAPN1 and CAST in the

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semitendinosus muscle (Fig. 1B).

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As shown in Table 6, the concentrations of creatine were elevated in longissimus

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dorsi muscle and liver by CMH and GAA supplementation compared with the control

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group (P < 0.05), as well as the concentrations of phosphocreatine in longissimus

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dorsi muscle and semitendinosus muscle (P < 0.05). Neither the concentrations of

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creatine nor phosphocreatine in kidney were altered by CMH or GAA

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supplementation (P > 0.05). Meanwhile, compared with the control group, CMH and

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GAA treatment decreased the AGAT mRNA expression levels in kidney (P < 0.05,

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Fig. 2D). Dietary CMH supplementation showed significant up-regulation of GAMT

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mRNA expression levels in semitendinosus muscle, liver and kidney (P < 0.05, Fig.

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2B, 2C, 2D), and the similar effect was also found in liver of the GAA group (P

Creatine Monohydrate and Guanidinoacetic Acid Supplementation

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