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Dietary N-carbamylglutamate Supplementation in Reduced Protein Diet Affects Carcass Traits and Profile of Muscle Amino Acids and Fatty Acids in Finishing Pigs Changchuan Ye, Xiangzhou Zeng, JInlong Zhu, Ying Liu, Qianhong Ye, Shiyan Qiao, and Xiangfang Zeng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02301 • Publication Date (Web): 22 Jun 2017 Downloaded from http://pubs.acs.org on June 28, 2017

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Journal of Agricultural and Food Chemistry

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Dietary N-carbamylglutamate Supplementation in Reduced Protein Diet Affects

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Carcass Traits and Profile of Muscle Amino Acids and Fatty Acids in Finishing

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Pigs

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Changchuan Ye†, §, Xiangzhou Zeng†, §, Jinlong Zhu†, Ying Liu†, Qianhong Ye†, and

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Shiyan Qiao†, Xiangfang Zeng†,*

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†

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100193, PR China

State Key Laboratory of Animal Nutrition, China Agricultural University, Beijing

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§

contributed equally to this work.

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ACS Paragon Plus Environment


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ABSTRACT: The aim of this study was to investigate whether dietary N-

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Carbamylglutamate (NCG) supplementation in reduced protein diet affected carcass

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traits and meat quality in finishing pigs. One hundred and twenty gilts were randomly

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assigned to one of four treatments for 40 d, including standard protein diet (SP), reduced

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protein diet supplemented 1.7% L-alanine (RP + Ala), reduced protein diet

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supplemented with 1.0 % L-arginine (RP + Arg) and reduced protein diet supplemented

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with 0.1% NCG and 1.7% L-alanine (RP + NCG). NCG supplementation increased the

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endogenous synthesis of L-arginine. The RP + NCG diet significantly increased the loin

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eye area (P < 0.05) and tended to decrease the 10th rib fat depth (P = 0.08). NCG

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supplementation in reduced protein diet was effective to produce functional pork with

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high content of leucine (P < 0.05). The composition of several n-6 and n-3

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polyunsaturated fatty acids (PUFA) but not the ratio of n-6/n-3 PUFA in muscles was

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altered in finishing pigs with dietary NCG supplementation. In conclusion, the RP +

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NCG diet is effective to increase longissimus dorsi muscle area, decrease back fat

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accretion, and produce functional pork with high content of leucine but without

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negative impact on muscle fatty acid profile in finishing pigs.

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KEYWORDS: endogenous arginine synthesis; finishing pigs; pork quality; fatty acids;

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skeletal muscle; protein reduced diet

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INTRODUCTION

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Recently, the shortage of feedstuff and the increasing price of protein resources have

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affected the development of animal husbandry strongly, especially the intensive pig

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production in China. The whole society has paid close attention to the environmental

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contaminants released by pig production, including ammonia (NH3), nitrous oxide

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(N2O), methane (CH4) and carbon dioxide (CO2)1, 2. Reduced protein diet supplemented

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with crystalline amino acids has been reported to decrease the feed cost and NH3

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emission with no negative effects on growth performance2-5. Although reduced protein

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diet has positive impact on reduction in NH3 emission, it tends to increase back fat

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thickness and reduce muscle depth6. This is probably because reduced protein diet

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decreases the urinary energy and heat increment of metabolism, and this part of energy

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may be utilized for lipogenesis7. This is contrary to the market's demand for high lean

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meat yield, which also clashes with the goal of improving the proportion of lean meat

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in pork production.

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As a kind of functional amino acid, L-arginine is the precursor of some important

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active substances which could adjust metabolism of body tissue8. Dietary arginine

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supplementation reduces body fat mass in fatty rats and growing-finishing pigs, while

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enhances the growth performance in milk-fed young pigs and growing-finishing pigs9-

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to prevent acid-base imbalance13. With concerns about the negative effects of chronic

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provision of chloride on animal and human health and the short biological half-life of

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arginine, alternatives of arginine are necessary to be explored14, 15.

. However, arginine is usually supplied with L-arginine-HCl to animals and humans



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N-Carbamylglutamate (NCG), the metabolically stable analogue of N-

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acetylglutamate (NAG), activates the key enzymes of endogenous arginine synthesis in

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enterocytes including carbamylphosphate synthase-1 (CPS-1) and pyrroline-5-

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carboxylate synthase (P5CS)16, ultimately resulting in the increase in endogenous

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synthesis of arginine and arginine family of amino acids. Compared with dietary

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arginine supplementation, dietary NCG supplementation offers unique and important

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advantages: (1) have no impact on intestinal absorption of dietary tryptophan, histidine,

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or lysine (2) a low dose to highly effectively stimulate endogenous arginine synthesis

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(3) a relatively long half-life in vivo (perhaps 8–10 h) (4) the substantially reduced

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cost16. Like dietary arginine supplementation, oral administration of 50 mg NCG/kg

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(body weight) twice daily for 7 days increases muscle protein synthesis in nursing pigs17.

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However, the effect of dietary supplementation with NCG on growth performance,

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carcass traits and meat quality in finishing pigs is still unknown. Our hypothesis was

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that reduced protein diet supplementation with NCG might increase muscle protein

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synthesis and reduce fat mass and improve carcass traits and meat quality. The objective

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of this study was to investigate whether dietary NCG supplementation in protein

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reduced diet could modulate the growth performance and carcass traits and meat quality

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in finishing pigs.

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MATERIALS AND METHODS

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All experimental procedures and animal care were approved by the China Agricultural University Animal Care and Use Committee (Beijing, China). Animals and Experimental Design. A total of 120 Duroc × Yorkshire × Landrace


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gilts, with an average initial body weight 75.00 ± 5.18 kg, were used in a 40-day

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performance trial. The experiments were conducted at the Pig Research Facility at the

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Swine Nutrition Research Centre of the National Feed Engineering Technology

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Research Centre (Chengde, Hebei Province, China). The pigs were allotted to one of

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four treatments based on initial body weight in a randomized complete block design

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with six replicates per treatment and five pigs per pen. The four treatments contained

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(1) standard protein diet (SP, total crude protein level: 13.6%), (2) reduced protein diet

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supplemented with 1.7% (wt:wt) L-alanine (RP + Ala, total crude protein level: 11.27%),

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(3) reduced protein diet supplemented with 1.0% (wt:wt) L-arginine (RP + Arg, total

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crude protein level: 11.40%), and (4) reduced protein diet supplemented with 0.1%

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(wt:wt) NCG + 1.7% (wt:wt) L-alanine (RP + NCG, total crude protein level: 11.26%).

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L-alanine was used to make the isonitrogenous diet among the three reduced protein

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diets. L-arginine was used as the positive control. The experimental diets were

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formulated based on corn, soybean meal and wheat bran (Table 1). Pigs were free access

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to feed and water. At the beginning and the end of the experiment, pigs were weighed

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after an overnight fasting to calculate the average daily gain. The feed was prepared

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before the start of the experiment, and packed with the amount of 25 kg in a plastic bag.

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Throughout the experiment, the number of feed bags were recorded. At the end of the

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experiment, feed remaining just before the start of the overnight fasting were weighed.

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The average daily feed intake was calculated as follows: (the number of feed bags × 25

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kg - feed remaining) / the number of days for the experiment. Based on the average

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daily gain and average daily feed intake, feed efficiency was calculated.



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Sample Collection. On the morning of d 40, blood samples from each pig were

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obtained by anterior vena cava puncture into a 5-mL uncoated vacutainer tube. After

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collection, the blood samples were placed on ice for 1 h and then centrifuged at 3000 ×

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g for 10 min and the serum was obtained and stored at -80℃ for the following

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biochemical analysis.

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After blood sampling, one pig per pen for each treatment were randomly selected for

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slaughter. Pigs were killed under commercial conditions by exsanguination following

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electrical stunning at the Beijing Yuhang Meat Processing Facility (Beijing, China) and

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hot carcass weight was immediately recorded following carcass dressing.

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Dressing percentage was determined from live weight and hot carcass weight. The

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carcass was split before cooling at 2°C for 24 h and subsequently the right side was cut

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between 10th and 11th ribs to measure the longissimus dorsi muscle area and backfat

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thickness. Carcass fat-free lean gain and carcass fat-free lean index were calculated

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using the equations of the National Pork Producers Council (NPPC 1994). The

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longissimus dorsi muscle samples were stored at -80℃ for biochemical analysis.

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Muscle Quality Measurements. Muscle pH was measured at three locations on the

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10th rib interface using a hand-held pH meter (Model 2000; VWR Scientific Products

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Co., South Plainfield, NJ, USA). Drip loss was calculated by hanging a loin section on

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a hot carcass (100 g, longissimus dorsi muscle sample) in an inflated and closed plastic

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bag for 24 h at 4℃ (King et al. 2000). Loin muscle marbling score (NPPC standards

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are 7 steps, with 1-6 representing 1-6% intramuscular fat, and the 7th representing 10%)

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at 10th rib was determined according to NPPC 1994 guidelines. After chilling at 2°C


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for 24 h, the CIELAB L* (lightness), a* (redness) and b* (yellowness) color of the 10th

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rib was determined from 3 orientations (middle, medial, and lateral) with a colorimeter

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with 8 mm aperture and 0° viewing angle (Chromameter, CR410; Minolta, Tokyo,

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Japan). The illuminant condition was D65. The colorimeter was calibrated according to

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the manufacturer’s guide.

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Biochemical Analyses. Serum urea nitrogen concentration was determined with a

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blood urea nitrogen color test kit according to the manufacturer’s instructions (Nanjing

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Jiancheng Bioengineering Institute, Nanjing, China). Briefly, in presence of urease,

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urea can be hydrolyzed to ammonium anion and carbon dioxide. Ammonium in alkaline

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solution can generate blue product with chromogenic agent. The optical density (OD)

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at 640 nm is directly related to the urea concentration and thus can be calculated. Serum

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and muscular AA concentrations were determined by amino acid analyzer (S-433D

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Amino Acid Analyzer, Sykam GmbH, Eresing, Germany). About 100 mg muscle

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samples were dissolved in water with methanol (1:1) at 4℃ for 30 min and centrifuged

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at 10, 000 g for 10 min. The supernatant was filtered through glass wool. The muscular

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supernatant or serum were deproteinized with 120 mg of salicylic acid/mL. The samples

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were placed in an ice bath for 20 min. Thereafter, the reaction system was adjusted for

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pH by adding lithium hydroxide solution (2 mol/L), followed by centrifugation at 12,

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000 × g (L-80 XP; Beckman, Fullerton, CA, USA) for 30 min at 4℃. The supernatant

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was collected and filtered through a 0.1 μm filter before loaded on the amino acid

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analyzer. The composition of total fatty acids (FA) in longissimus dorsi muscle was

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performed by Agilent 6890N gas chromatographer. The longissimus dorsi muscles (0.5



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g) were Grinded in liquid nitrogen, followed by adding 4 mL Chloroacetic methanol, 1

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mL n-hexane, and 1 mL internal standard fatty acid solution (1 mg/mL eleven carbon

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fatty acid methyl ester). The samples were then vortexed for 1 min and kept in water

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bath at 75℃ for 2 h. After cooling down, 5 mL potassium carbonate solution (70 g/L)

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was added to the samples, and vortexed for 1 min, followed by centrifugation at 1,200

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rpm for 10 min. The supernatant was then loaded on the gas chromatographer. The

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concentration of individual fatty acid was quantified according to the following

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equation: Ci = m0*Ai*Fi*Ri/A0*m. Ci was the concentration of individual fatty acids

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(mg/g). m0 was the weight of internal standard fatty acid (mg). m was the weight of

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samples. Ai was the peak area of individual fatty acid in samples. A0 was the peak area

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of internal standard fatty acid. Fi was correction coefficient of fatty acid methyl ester

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to fatty acid. Ri was correction coefficient of the peak area.

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Statistical Analysis. Data for growth performance, meat quality, fatty acid

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composition and blood variables were subjected to ANOVA suited for a randomized

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complete block design using the General Linear Model (GLM) procedure (version 9.2;

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SAS Institute, Inc., NC, USA). For the growth performance, pen served as the

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experiment unit. Results are expressed as mean + SEM. Statistical differences among

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groups were separated by Bonferroni Multiple Comparisons Test. P values < 0.05 were

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considered to be significant for all data in this manuscript.

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RESULTS


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Growth

Performance.

Similar

to

arginine

supplementation,

dietary

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supplementation with NCG did not affect (Table 2) average daily gain, average daily

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feed intake, or feed to gain ratio, compared with the SP or RP + Ala diets.

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Carcass Traits and Meat Quality. There were no significant effects for slaughter

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weight, carcass weight and dressing percentage among these groups (Table 3). The fat

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depth tended to be lower (P = 0.08) in pigs fed the RP + Arg or RP + NCG diets

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compared with pigs fed the SP or RP + Ala diets. The RP + Arg or RP + NCG diets

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significantly increased the longissimus dorsi muscle area of pigs, compared with the SP

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or RP + Ala diets (P < 0.05). The fat-free lean index and the fat-free lean gain were

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markedly enhanced in pigs fed the RP + Arg or RP + NCG diets, compared with pigs

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fed the SP or RP + Ala diets (P < 0.05). The pH45min, pH24h, drip loss, meat color traits

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and marbling of meat did not differ significantly among the treatments (Table 4).

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Concentrations of Serum Amino Acids and Urea Nitrogen. Serum concentrations

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of amino acids and urea nitrogen were presented in Table 5. Like dietary arginine

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supplementation, serum concentration of urea nitrogen was lower (P < 0.01) in pigs fed

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the RP + NCG diet compared with the pigs fed the SP or RP + Ala diets. Serum

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concentration of tryptophan in pigs fed the RP + Arg or RP + NCG diets was

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significantly higher (P < 0.05) than pigs fed the RP + Ala diet, but was not different

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from the pigs fed the SP diet. Compared with the SP diet, serum concentration of

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isoleucine was markedly decreased in pigs fed the RP + Ala diet, which was recovered

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in pigs fed the RP + Arg diet, but not the RP + NCG diet. Serum concentration of

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arginine in pigs fed the RP + Arg or RP + NCG diets is dramatically higher (P < 0.01)



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than pigs fed the SP or RP + Ala diets (P < 0.05). Serum concentration of lysine in pigs

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fed the RP + Arg or RP + NCG diets was significantly increased in comparison with

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that in pigs fed the SP or RP + Ala diets (P < 0.01). The highest concentration of

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glutamine was obtained in pigs fed the RP + Arg diet, which has highly significant

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difference from pigs fed the SP diet (P < 0.01). Serum concentrations of other amino

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acids did not show significant difference among the treatments (P > 0.05).

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Muscular Amino Acid Concentration. Amino Acid concentration in muscle are

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shown in Table 6. Except for Leucine, other amino acids do not show significant

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difference (P > 0.05) in longissimus dorsi muscle. The concentration of leucine of

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longissimus dorsi muscle in pigs fed the RP + NCG diet but not RP + Arg diet was

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significantly increased, compared with that in pigs fed the SP or RP + Ala diets (P

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