Valine Supplementation in a Reduced Protein Diet Regulates Growth

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Bioactive Constituents, Metabolites, and Functions

Valine supplementation in a reduced protein diet regulates growth performance partially through modulation of plasma amino acids profile, metabolic responses, endocrine and neural factors in piglets xiao ya zhang, Xiangfang Zeng, xutong liu, hongmin jia, pingli he, Shiyan Qiao, and Xiangbing Mao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01113 • Publication Date (Web): 10 Mar 2018 Downloaded from http://pubs.acs.org on March 13, 2018

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Journal of Agricultural and Food Chemistry

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Valine supplementation in a reduced protein diet regulates growth performance

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partially through modulation of plasma amino acids profile, metabolic responses,

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endocrine and neural factors in piglets

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Xiaoya Zhang,†, § Xutong Liu,† Hongmin Jia,† Pingli He,† Xiangbing Mao,‡ Shiyan Qiao† and

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Xiangfang Zeng†,*

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†

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China

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‡

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State Key Laboratory of Animal Nutrition, China Agricultural University, Beijing 100193,

Animal Nutrition Institute, Sichuan Agricultural University, Sichuan, Chengdu 611130,

China

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ACS Paragon Plus Environment


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ABSTRACT: The objective of this study was to investigate whether valine (Val)

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supplementation in a reduced protein (RP) diet regulates growth performance associated with

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the changes in plasma amino acids (AAs) profile, metabolism, endocrine and neural system in

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piglets. Piglets or piglets with a catheter in the precaval vein were randomly assigned to two

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treatments, including two RP diets with standardized ileal digestible (SID) Val: Lysine (Lys)

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ratio of 0.45 and 0.65, respectively. The results indicated that piglets in the higher Val:Lys

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ratio treatment had higher average daily feed intake (ADFI) (P < 0.001), average daily gain

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(ADG) (P = 0.001), and feed conversion ratio (FCR) (P = 0.004) and lower plasma urea

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nitrogen (P = 0.032) and expression of gastric cholecystokinin (CCK) and hypothalamic

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pro-opiomelanocortin (POMC). Plasma AAs profiles including postprandial plasma essential

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AAs (EAAs) profile, and in serum, muscle and liver involved in metabolism of AAs and fatty

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acids were significantly different between two treatments. In conclusion, Val influenced

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growth performance associated with metabolism of AAs and fatty acids and endocrine and

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neural system in piglets.

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KEYWORDS: amino acids profile, cholecystokinin, growth performance, metabolites,

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pro-opiomelanocortin, piglets, feed intake, valine

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INTRODUCTION

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Branched chain amino acids (BCAAs) valine (Val) is an important nutrient for

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animals and humans and plays crucial roles in growth,1 metabolism homoeostasis

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including lipid and protein metabolism modulation,2 energy homoeostasis,3 cell

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function and immunity.4 High amount of dietary Val supplementation has no

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detrimental effect on laying productivity or immune function in laying hens.5

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Optimum dietary Val supplementation increases body weight gain (BWG) and feed

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conversion ratio (FCR), which is due to the specific response of Val and not related

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with the increase in total nitrogen.6 Val deprivation markedly decreases blood glucose

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level, partially because of downregulation of glucose-6-phosphatase expression.7

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Besides, dietary Val deficiency impairs intestinal physical barrier function and

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intestinal immune function through decreasing tight junction protein expression and

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anti-inflammatory cytokines in fishes.8 Under Val deprivation status, monocytes were

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not able to differentiate into mature dendritic cells as well as a decrease in

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phospho-S6 kinase and CD83 expression.9 Val has the capacity to increase

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macrophage phagocytosis in mice, ultimately reducing the load of pathogens through

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stimulation of PI3K/Akt1 pathway and the increase in NO production.10

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Val, the fifth limiting AA for growing pigs fed corn-soybean meal-based diets,11

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is commonly added to the low protein (LP) diets. Our previous data showed that Val

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supplementation to the LP diet (16.9% crude protein (CP)) had a comparable effect in

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improving growth performance of weaned pigs, compared with the standard protein

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diet (20.9% CP). 12 While dietary Val deficiency appeared to reduce the average daily

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feed intake (ADFI), resulting in a subsequent reduction in growth of weaned pigs.13

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The feed intake in animals and humans is regulated by the hypothalamic signaling

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pathways14 and endocrine system.15,16 Nevertheless, there is lack of evidence that

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whether Val modulates feed intake through endocrine and neural system in RP diet.

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Moreover, BCAA deficiency is demonstrated to cause disturbances in nutrients

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metabolism.17 However, the metabolic changes in different organs and the

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corresponding contributions to feed intake and growth is still unclear in response to

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BCAA supplementation in RP diet.

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Therefore, the objective of this study was to investigate whether Val

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supplementation in a RP diet influenced growth performance through changes in

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plasma AAs concentrations, expression of endocrine factors and metabolic profile of

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serum, muscle and liver in piglets.

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MATERIALS AND METHODS

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The animal handling procedures used in this study were in accordance with the

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Chinese Guidelines for Animal Welfare and were approved by the China Agricultural

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University Animal Care and Use Committee (Beijing, China).

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Animals, Experimental Design and Sampling. In order to investigate the

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different changes of metabolites in serum, liver and muscle, plasma amino acids

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concentrations, as well as neural and endocrine system factors in two levels of Val

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treatments, twelve healthy piglets (Duroc x Landrace x Large White) with similar

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initial body weight (18.9 ± 2.3 kg) were randomly assigned to one of two dietary

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standardized ileal digestible (SID) Val: Lys ratios (0.45 and 0.65) for 14 days, based

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on body weight and sex. The diets were formulated based on corn and soybean meal

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(16.9 % CP, Table 1). The other essential amino acids (EAAs) were balanced to meet

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or exceed the recommendations of the National Research Council (NRC, 2012).

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Piglets had free access to feed and water throughout the experiment. Piglets were

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weighed on the morning of day 1 and 14 in the fasted state (overnight fasting) to

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calculate average daily gain (ADG). Feed intake was recorded daily during the

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experiment to calculate ADFI and FCR. On the morning of days 14 (overnight fasting

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state), blood samples from all piglets were collected into 10 mL heparinized

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vacutainer tubes and vacuum blood collection tubes (Becton Dickinson Vacutainer

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Systems, Franklin Lakes, NJ) via the jugular vein, respectively. Blood was

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centrifuged at 1,000 × g for 15 min at 4℃ within 2 h after sampling. An aliquot of

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plasma was stored at -20℃ until AA analysis. Serum was obtained after

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centrifugation and stored at -80℃ until use. Thereafter, all the piglets were killed by

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electrocution. Mucosa of the gastric fundus were stripped at the middle of the bottom

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position of the stomach and duodenal mucosa at a point 2 cm from the pylorus was

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also collected. Samples from hypothalamus, liver and longissimus dorsi muscle were

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also collected. All samples were stored at -80℃.

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To investigate the differences of postprandial amino acid profiles in two levels of

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Val treatments, six healthy piglets (Duroc x Landrace x Large White) with similar

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initial body weight (15.7 ± 0.8 kg) were surgically fitted with a catheter in the

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precaval vein and assigned to one of the same two dietary treatments as in Exp. 1

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based on body weight and sex. After a 7-d recovery period from the surgery, blood

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samples were collected from the catheters every 2 days in a 6-d experiment. At 07:50

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h collected blood sampling, piglets were fed their respective diets (12 g/kg BW0.75)

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and for each piglet, all feed was ingested within 10 min.18 After that, blood samples

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were taken every 20 min during the first hour and every 1 hour during the following 3

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h (total of 6 time points). Blood was stored in heparinized tubes (Becton Dickinson

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Vacutainer Systems, Franklin Lakes, NJ) kept on ice and centrifuged at 1,000 × g for

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15 min at 4℃ within 2 h after sampling. An aliquot of plasma was stored at -20℃

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until AA analysis.

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In both experiments, all the piglets were housed in the Metabolism Laboratory of

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the Ministry of Agriculture Feed Industry Centre (China Agricultural University,

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Beijing, China). All the piglets were housed individually in stainless steel metabolic

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cage (1.4 m x 0.7 m x 0.6 m) equipped with a water nipple and a feed trough in an

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environmentally controlled room (22 ± 2℃).

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Chemical Analysis. Analysis of dietary CP content (Table 1) was conducted

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according to the method of the Association of Official Analytical Chemists (AOAC)

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(2003). For the analysis of most AAs, diets were hydrolyzed in 6 N HCl at 110℃ for

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24 h (AOAC, 2003). The content of sulfur AAs was determined after performic acid

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oxidation followed by acid hydrolysis and tryptophan content was determined after

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alkaline hydrolysis (AOAC, 2003). AA analysis was conducted by High Performance

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Liquid Chromatography (Hitachi L-8800 Amino Acid Analyzer, Tokyo, Japan).

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Plasma AA content was using an Ion-Exchange Chromatography (S-433D

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Amino Acid Analyzer, Sykam, Germany).46 Plasma urea nitrogen was determined

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using a blood urea nitrogen color test kit (Nanjing Jiancheng Bioengineering Institute,

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Nanjing, China).

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RNA Extraction and RT-PCR Analysis. Total RNA was extracted from the

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gastric, duodenal and hypothalamic tissues using Trizol Reagent (Invitrogen Life

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Technologies, Carlsbad, CA) according to the manufacturer’s instruction. RNA

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concentrations were measured using a Nanodrop. First-strand cDNA was synthesized

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using a PrimeScript 1st Strand cDNA Synthesis Kit (Takata, Ostu, Japan, Cat: 6110A).

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Primers were designed using Oligo 7.0 Software (Table 2). Real-time PCR was

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

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CA, USA) with SYBR Green PCR Master Mix (Takara, Japan) in 10 µL reaction with

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1/20 (for β-actin) or 1/10 (for target gene) of the first-strand cDNA. The PCR

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program was set for the denaturation at 95℃ for 10 min, followed by 40 cycles of 94℃

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for 15 s, 55℃ for 20 s, and 72℃ for 30 s. Each sample was measured in triplicate.

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Relative gene expression was calculated according to the △△Ct method using

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β-actin as the reference gene.

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Non-Targeted Metabolic Fingerprinting Analysis. Serum samples (200 µL)

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were deproteinized and metabolites were extracted with ice-cold acetonitrile and

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methanol mixture (1:1, v: v) at 1:4 sample: extract solution ratio. After vortexing, the

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samples were centrifuged at 13000 rpm for 10 min at 4℃. Then 500 µL of the

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supernatants were removed and evaporated to dryness in a vacuum concentrator. The

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residues were resuspended in 100 µL of methanol: water (1:1), vortexed and

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centrifuged again at 13000 rpm for 10 min at 4℃. The supernatants were transferred

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to sample vials for HPLC-QTOF-MS analysis.

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Muscle and liver samples were initially crushed and extracted using ice-cold

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extraction mix (methanol : water, 8: 2, v/v). The samples were vortexed and then

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stand on dry ice for 4 h. The aqueous fractions were centrifuged at 13000 rpm for 10

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min at 4℃ and the supernatant were removed. The mixture of the precipitate and 400

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L methanol were vortexed for 1 min. After standing on dry ice for 30 min, the

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samples were centrifuged again at 13000 rpm for 10 min at 4℃ and the supernatants

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were combined with the supernatant from the first extraction. The supernatants were

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evaporated to dryness in a vacuum concentrator. The residues were resuspended in

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100 mL of methanol: water (1:1) solution, vortexed, and centrifuged again at 13000

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rpm for 10 min at 4℃. The supernatants were transferred to sampler vials for LC-MS

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analysis.

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Metabolomics Profiling. Isolating the substances in 5 µL of the extracted

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sample were performed by the Agilent 1290 Infinity HPLC system, coupled to an

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Agilent 6520 quadrupole-time of flight mass spectrometer (QTOF-MS) with a heated

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electrospray ionization (ESI) source. An Agilent Eclipse Plus C18 column (2.1×100

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mm, 1.8 µm) was also used. The column temperature was set at 35℃ and the flow

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rate was 0.3 mL/min with solvent A (0.1% formic acid water solution) and solvent B

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(0.1% formic acid acetonitrile solution). Gradients were used from 5-30% solvent B

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from 0 to 6 min; 95% solvent B from 6 to 9 min; 95% solvent B from 9 to 14 min; 5%

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solvent B from 14 to 18 min before the loading of the next sample. The temperature

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of the drying gas process was set at 350℃. The flow rate of desolvation was set at 12

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L/min. And the nebulizer pressure was at 60 psig. The capillary voltages were set at

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3500V, with fragmentor voltages of 150 V. Data acquisition were up to 2 spectra/s in

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a full scan mode. The metabolites were collided straight into MS at a rate of 4

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spectra/s. The precursor ions and their isotopes were selected by quadrupole of 4.0

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a.m.u..

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Data Processing. Molecular feature extraction from the raw data were

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performed by Masshunter Profinder software (version B.06.00, Agilent Technologies,

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Inc., Santa Clara, USA). Data were processed using algorithm followed by data

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reduction. The data were corrected by chromatographic dimension and time

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normalization. The window-wise summing of 0.15 min and 2 mDa produced m/z

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values. Serum and tissues’ metabolic differences in two treatments were analyzed

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using ANOVA. Significant changed metabolites were selected through P < 0.05 and a

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fold change > 1.5 and then used the METLIN software to generate formulas.

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Statistical Analysis. Statistical analysis of Exp.1 was performed using MIXED

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procedure in SAS (SAS 9.3 software, SAS Inst. Inc., Cary, NC). The model included

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diet as fixed effects and piglets as a random effect. Polynomial contrasts were

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conducted to determine linear and quadratic effects of increasing SID Val:Lys ratios.

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The data in Exp. 2 were analyzed using repeated measures of MIXED Procedure, and

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the model included diet, time and their interaction. An alpha of P < 0.05 was the

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criterion for statistical significance.

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RESULTS

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Growth Performance. Dietary Val levels affected growth performance of piglets

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(Table 3). Piglets fed SID Val:Lys ratio of 0.45 had lower ADG (P = 0.001) and

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ADFI (P < 0.001), and poorer FCR (P = 0.004), compared with piglets fed SID

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Val:Lys ratio of 0.65 treatment.

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Plasma Concentrations of Urea Nitrogen and Amino Acids. In Exp. 1, plasma

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urea nitrogen decreased (P = 0.032) with increased dietary SID Val: Lys ratio (Table

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3). Plasma Val increased (P = 0.004) with the addition of dietary Val. For other

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plasma EAAs, methionine (P = 0.017) and threonine (P = 0.001) decreased with an

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increase in SID Val:Lys ratio (Table 4). For plasma nonessential AA (NEAAs),

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asparagine (P = 0.007) and glutamine (P = 0.012) were significantly lower with the

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addition of Val (Table 4).

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The postprandial plasma profiles for the piglets fed the diets with SID Val:Lys

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ratio of 0.45 and 0.65 are shown in the Fig.3. The plasma profiles of these five

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essential amino acids isoleucine, leucine, methionine and threonine displayed

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variations over two groups at 40 min time point (P < 0.05). 2, 3 or 4 h after feeding,

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Val concentration was higher (P < 0.05) in piglets fed the diet with SID Val:Lys ratio

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of 0.65. Other NEAAs were not different among the two treatments.

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Relative Gene Expression of Endocrine Factors in Stomach, Duodenum, and

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Hypothalamus. Compared with dietary SID Val:Lys ratio of 0.45, the expression of

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gastric CCK was lower (P = 0.032) in pigs fed the diet with dietary SID Val:Lys ratio

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of 0.65 (Fig. 2). Dietary Val level had no impact on duodenal CCK level as well as

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gastric and duodenal ghrelin level (Fig. 2). The expression of hypothalamic POMC

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was lower (P = 0.045) in pigs fed the diet with a SID Val:Lys ratio of 0.65 compared

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with that in dietary Val:Lys ratio of 0.45 group (Fig.1). Hypothalamic neuropeptide Y

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level was not affected by dietary Val content (Fig.1).

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Metabolic Profiles in Plasma, Liver, and Muscle. Serum metabolome in pigs

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fed diets with SID Val: Lys ratio of 0.45 and 0.65 were differentially expressed (Table

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5). To identify which variables accounted for such a distinct separation, we calculated

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the P value and fold change (FC), defined as the SID Val: Lys ratio of 0.45 to 0.65

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group for a given expression profile. Taken these two variables into account, we

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identified 14 metabolites shown in Table 5. Intriguingly, 10 of the metabolites

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detected were found to be upregulated in the SID Val: Lys ratio of 0.45, while 3 were

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downregulated. In muscle, 7 metabolites were shown in Table 6. 5 of the metabolites

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detected were found to be upregulated in the SID Val: Lys ratio 0.45 group, while 2

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were downregulated. In liver, 7 metabolites were shown in Table 7. 5 of the

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metabolites detected were found to be upregulated in the SID Val: Lys ratio 0.45

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group, while 2 were downregulated.

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DISCUSSION

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Our results indicated that with Val supplementation to the LP diets, feed intake

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and FCR improved compared to the Val-deficient diets. As a result, piglets growth

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performance was different between the two treatments. These results are consistent to

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a previous experiment in which piglets fed a LP diet showed positive response to Val

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supplementstion.19 This raises the question how piglets react to the change of Val

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levels in LP diet.

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Plasma urea nitrogen can be considered as a marker of protein utilization

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efficiency.20 Because of an imbalance of EAAs available for tissue protein synthesis,

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excess AAs can serve as metabolic molecules in different metabolic pathways.21 Also,

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excess AAs cannot be stored, therefore, undergo the urea production22. The present

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results showed that plasma urea nitrogen was higher in the Val deficient group than

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the Val supplemented group, which means that Val deficiency in LP diets breaks

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down the balance among AAs causing body’s metabolic changes.

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Therefore, we measured the content of metabolites in serum, liver and muscle

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between the dietary SID Val: Lys ratio of 0.45 and 0.65. Results of present study

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demonstrated that dietary Val supplementation decreased serum concentrations of

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N-stearoyl valine and N-(Ammonioacetyl) glycylglycine and increased serum

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concentrations of ketoisovaleric acid which are related to BCAA metabolism.

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Research has shown that when rats were fed a Val-deficient diets, plasma

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concentrations of Val, Leu and Ile decreased significantly.18 These results suggested

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that a Val-deficient diet activated Leu and Ile catabolism. BCAAs are circulated in the

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whole body acting as nutrient signals that involve in protein metabolism.23 We can

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also see that the concentration of Nalpha-Methylhistidine decreased significantly in

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the dietary SID Val: Lys ratio of 0.65 revealing that the ratio of protein proteolysis

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decreased.

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Muscle can acutely sense and identify deficiencies in AAs because it storages

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the largest protein and turns over protein to AAs.24 Val deficiency can result in the

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elevation of threonine in muscle.17,25 Study has shown that the excess dietary

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threonine can lower protein synthesis in tissues causing the growth and development

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failure.25 Our results indicated that L-Homoserine, intermediate product of the

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biosynthesis of threonine, increased in SID Val: Lys ratio of 0.45 treatment, which

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means that Val deficiency actives threonine metabolism process and may result in a

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significant reduction in tissue protein synthesis.

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Data on liver metabolites suggested coordinate regulation of nutrient

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metabolism that favors fatty acids metabolism and protein synthesis in piglets. Our

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results indicated that oleoylethylamide (OEA) was much higher in SID Val: Lys ratio

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of 0.45 treatment. OEA belongs to the monounsaturated fatty that is released by

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enterocytes26,27 and further combines with peroxisome proliferator-activated

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receptor-alpha (PPAR-α) to enable fat oxidation in the liver.28 OEA might be

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responsible for reduced satiety and hyperphagia.26 Therefore, the differences in

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metaboliscs between two Val levels in LP diets remain us that some metabolics may

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be essential elements of the physiology and metabolic system regulating food intake

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and then affecting growth performance.

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In the current study, increasing Val content decreased the expression of CCK in

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the gastric fundus. CCK, endocrine hormone secreted from gastrointestinal tract, is

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primarily released from the upper small intestine29 and may be transported to the

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blood as important signals to disturb regulation of neurons in the hypothalamic.30-33

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Neuropeptide Y (NPY) and pro-opiomelanocortin (POMC) neurons are located in the

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arcuate nucleus of the hypothalamus which are related to the feeding behaviors. In our

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study, the expression of POMC was decreased in the SID Val: Lys ratio of 0.65

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treatment. This supports the fact that the expression of PMOC neurons can be

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activated by CCK, which is important in the regulation of satiety31.

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On the other hand, the AAs imbalance that results from a Val deficient diet

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might be a signaling transferred to the hypothalamus to induce the suppression of feed

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intake.35Our data indicated that postprandial plasma leucine, and isoleucine

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concentration were higher at 40 min time point in the SID Val: Lys ratio of 0.45 RP

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diet then decreased rapidly. These results indicated that the body can rapidly detected

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the Val deficiency. BCAAs share the same enzymes that be utilized to compete for

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transamination and oxidative decarboxylation catalyzed. With higher Val diets, the

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activity of enzyme complex can be simulated causing catabolism of Leu and Ile37,

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therefore, the leucine, and isoleucine concentration was lower than the SID Val: Lys

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ratio of 0.45 RP diet. And that AAs imbalance would be a critical factor in the

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regulation of some metabolic pathway, which can result in a reduction in feed intake.

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Our data indicated that in Val deficiency treatment, plasma Val content was lower,

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and methionine, threonine, asparagine and glutamine were significantly higher. Study

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has shown that Val deficiency elevated plasma threonine levels, which is thought to

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be a body protection mechanism.36,37 Threonine can be as amino radicals in the body

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which is less toxic than ammonia, whose concentration would be increased

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significantly when dietary Val is deficient as protein proteolysis is increased.24

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In conclusion, LP diets supplemented with crystalline Val increased feed intake

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and growth performance, partly through the modulation of metabolic profiles in serum,

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liver and muscle, endocrine factors and the AAs profiles during the postprandial

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period which may act as signals for neural system.

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AUTHOR INFORMATION

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Corresponding Authors

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*(X.Z.)

Phone:

+86

10-62733588.

Fax:

+86

10-62733688.

E-mail:

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[email protected].

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Funding

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This study was supported by the Beijing Advanced Innovation Center for Food

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Nutrition and Human Health, China Agricultural University and the Modern

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Agro-Industry Technology Research System of Beijing. The authors thank the

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Dacheng Group (Changchun, China) and Health and Nutrition of Evonik Industries

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(AG Germany) for providing the crystalline amino acids.

312

Notes

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The authors declare no competing financial interest.

314 315

ABBREVIATIONS USED

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RP: reduced protein; Val, valine; SID, standardized ileal digestible; Lys, Lysine;

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ADFI, average daily feed intake; ADG, average day gain; G:F, gain: feed; CCK,

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cholecystokinin; POMC, pro-opiomelanocortin; MS, mass spectrometer; BCAA,

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branched-chain amino acids; AA, amino acids; BWG, body weight gain; FCR, feed

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conversion ratio; NEAA, nonessential amino acids; NPY, Neuropeptide Y; AGRP,

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agouti-related protein; CART, cocaine- and amphetamine-regulated transcript; FC,

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fold change; AOAC, Association of Official Analytical Chemists; QTOF-MS,

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quadrupole-time of flight mass spectrometer; ESI, electrospray ionization; mTOR,

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mammalian target of rapamycin; BCAT, branched chain aminotransferase; BCKDH,

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branched chain α-keto acid dehydrogenase; CP, crude protein.

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436

Table 1.

437

as-fed)

Ingredient composition and calculated nutrient content of the basal diet (%

treatment (SID Val to Lys ratio) item 0.45

0.65

69.60 4.00 9.00 3.00 8.00 1.00 0.60 1.20 0.30 0.50 0.84 0.30 0.37 0.10 0.00 0.22 0.30 0.23 0.12 0.32

69.53 4.00 9.00 3.00 8.00 1.00 0.60 1.20 0.30 0.50 0.84 0.30 0.37 0.10 0.23 0.22 0.30 0.23 0.12 0.16

16.89 1.22 0.73 0.80 0.22 0.58 2490 1.15 0.70 0.75 0.20 0.52

16.90 1.24 0.74 0.80 0.23 0.80 2490 1.15 0.70 0.75 0.20 0.75

ingredient composition (%) corn soybean meal peanut meal fish meal whey powder soybean oil limestone dicalcium phosphate salt vitamin-mineral premix1 L-Lysine HCl DL-Methionine L-Threonine L-Tryptophan L-Valine L-Isoleucine L-Leucine L-Phenylalanine L-Histidine L-Alanine calculated nutrition content (%) crude protein Lys Met + Cys Thr Trp Val NE (MJ/kg)2 SID3 Lys SID Met + Cys SID Thr SID Trp SID Val

21



438

1

439

vitamin A acetate; vitamin D, 3,000.00 IU as vitamin D3; vitamin E, 64.00 IU as DL-α-tocopheryl

440

acetate; vitamin K3, 3.00 mg; vitamin B1, 1.50 mg; vitamin B2, 5.50 mg; vitamin B6, 3.00 mg as

441

pyridoxine hydrochloride; vitamin B12, 12.00 µg; nicotinic acid, 40.00 mg; pantothenic acid, 15.00

442

mg as calcium pantothenate; folic acid, 0.80 mg; biotin, 100.00 µg; choline chloride, 0.55 g; Fe,

443

100.00 mg as as Ferrous sulfate monohydrate; Cu (CuSO4·5H2O), 150.00 mg; Zn, 100.00 mg as

444

zinc sulfate; Mn, 40.00 mg as zinc sulfate; I, 0.30 mg as calcium iodate; Se, 0.30 mg as sodium

445

selenite. 2NE: net energy (MJ/kg). NE content was calculated using energy values for the

446

ingredients obtained from the NRC (2012). 3SID: standardized ileal digestible. SID values for the

447

diets were estimated by multiplying the analyzed AA levels by the SID of the corresponding AA

448

in those feedstuffs obtained from the NRC (2012).

Vitamin-mineral premix provided the following per kilogram of diet: vitamin A, 9,000.00 IU as

22


Page 22 of 35

Page 23 of 35

449


Table 2. The primer sequences for quantitative Real Time-PCR gene

primer sequence 5'-3'

product length (bp)

Tm (ºC)

accession No.

238

60.00

KU_672525

268

61.80

AH_013312

118

60.80

NM_214237

269

60.80

XM_008686403

267

58.90

XM_021085834

TGCGGGACATCAAGGAGAAG β-actin

AGTTGAAGGTGGTCTCGTGG GGAGTCCAAGAAGCCAGCAG

ghrelin

ACAGAGGTGGCTGGTCTCAG GGCCAGATACATCCAGCAGG

cholecystokinin

CATCCAGCCCATGTAGTCCC ACCCTCGCCCTGTCCCTGCT

neuropeptide Y

ATGTGGTGATGGGAAATGAG CGGTGAAGGTGTATCCCAAC

Pro-opiomelanocortin

AGGTCATGAAGCCGCCGTAG

23



Page 24 of 35

450

Table 3. Effect of dietary standardized ileal digestible (SID) Val to Lys ratios on the performance and plasma urea nitrogen concentrations of

451

piglets1 treatment (SID Val to Lys ratio) item

452

1

453

2

SEM2

P

0.45

0.65

ADG (g/d)

341a

713b

47.00

Valine Supplementation in a Reduced Protein Diet Regulates Growth

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