Inferring the Skeletal Muscle Developmental Changes of Grazing and

Aug 26, 2016 - Key Laboratory for Agro-Ecological Processes in Subtropical Region, Hunan Research Center of Livestock & Poultry Sciences, South-Centra...
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Inferring the skeletal muscle developmental changes of grazing and barn-fed goats from gene expression data Jinyu Huang, Jinzhen Jiao, Zhi-Liang Tan, Zhixiong He, Karen. A. Beauchemin, Robert Forster, Xue-Feng Han, Shao-Xun Tang, Jinghe Kang, and Chuanshe Zhou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02708 • Publication Date (Web): 26 Aug 2016 Downloaded from http://pubs.acs.org on August 27, 2016

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Running head: Muscle development and feeding regime

Inferring the skeletal muscle developmental changes of grazing and barn-fed goats from gene expression data

J. Y. Huang†,‡, J. Z. Jiao†, Z. L. Tan†, Z. X. He†, K. A. Beauchemin§, R. Forster§, X. F. Han†, S. X. Tang†, J. H. Kang†, C. S. Zhou†,* †

Key Laboratory for Agro-Ecological Processes in Subtropical Region, Hunan

Research Center of Livestock & Poultry Sciences, South-Central Experimental Station of Animal Nutrition and Feed Science in Ministry of Agriculture, Institute of Subtropical Agriculture, The Chinese Academy of Sciences, Changsha, Hunan 410125, P.R. China ‡

University of the Chinese Academy of Sciences, Beijing 100049, China

§

Lethbridge Research Centre, Agriculture and Agri-Food Canada, Lethbridge, Alberta,

Canada T1J 4B1 *

Corresponding author: Chuanshe Zhou. Address: Institute of Subtropical Agriculture,

the Chinese Academy of Sciences, Changsha, Hunan 410125, P. R. China. E-mail: [email protected]; Tel: +86 731 4615236; Fax: +86 731 4612685.

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ABSTRACT: Thirty-six Xiangdong black goats were used to investigate age-related mRNA and

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protein expression levels of some genes related to skeletal muscle structural proteins, MRFs and

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MEF2 family, and skeletal muscle fiber type and composition during skeletal muscle growth

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under grazing (G) and barn-fed (BF) feeding systems. Goats were slaughtered at 6 time points

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selected to reflect developmental changes of skeletal muscle during non-rumination (d 0, 7 and

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14), transition (d 42) and rumination phases (d 56 and 70). It was observed that the number of type

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IIx in the longissimus dorsi was increased quickly while numbers of type IIa and IIb decreased

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slightly, indicating that these genes were coordinated during the rapid growth and development

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stages of skeletal muscle. No gene expression was affected (P > 0.05) by feeding system except

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Myf5 and Myf6. Protein expression of MYOZ3 and MEF2C were affected (P < 0.05) by age,

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while PGC1α was linearly decreased in the G group, and only MYOZ3 protein was affected (P


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0.05) the mRNA expression of skeletal muscle structural protein genes including myosin heavy

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chain isoforms (MYH1, MYH2 and MYH4), myosin regulatory light chain (MYLPF) and the

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myozenin family of skeletal muscle Z lines (MYOZ1, MYOZ2 and MYOZ3). Furthermore, there

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were no age × feeding system interactions for these variables except MYLPF. An interaction

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between age and feeding system (P = 0.045) for MYLPF was observed, with a greater level for G

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group than for BF group on d 42 and 56, but a lower level for G group than for BF group on d 70.

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Irrespective of feeding system, the mRNA expression of MYH1 was linearly increased (P = 0.017)

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with increasing age, whereas with a sharp decline occurring on d 56. However, MYH4 expression

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was decreased (linear and quadratic, P < 0.001) with increasing age. The expression of MYLPF

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tended to increase linearly (P = 0.077) with age for G group, while a trend for a quadratic increase

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(P = 0.092) was observed for BF group. The expression of both MYOZ1 (linear, P < 0.001) and

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MYOZ3 (linear, P < 0.001; quadratic, P = 0.011, least on d 14) were increased with age, with a

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sharp increment from d 14 to 42 and an immediately rapid decline from d 42 to 56 for both groups.

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Myogenic regulators for skeletal muscle growth. The feeding system affected the mRNA

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expression of Myf5 (P = 0.039) and Myf6 (P < 0.001) genes (Table 3), with greater er expression

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in the BF group than the G group. There were age × feeding system interactions for both MyoD (P

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= 0.047) and Myf6 (P = 0.002). Furthermore, from d 0 to 70, MyoD mRNA expression decreased

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linearly (P = 0.007) with increasing age in BF goats, and Myf6 expression increased (P < 0.05)

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with age for both groups.

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Feeding system did not markedly affect myocyte enhancer factors (MEF2s), but there was an

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age × feeding system interaction (P = 0.029) for MEF2C mRNA expression, with greater

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expression in the G group than the BF group on d 42 and 56. Regardless of feeding system, the

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expression of MEF2A was linearly increased (P = 0.001) with increasing age, and MEF2C

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expression displayed a quadratic effect (P = 0.045) for BF group from d 0 to 70, with the lowest

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value noted on d 56.

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Regulators of skeletal muscle fibre type and composition. The expression of some relevant

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genes on catalytic and regulatory subunits of calcineurin (CaN) (such as PPP3CA, PPP3CB and

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PPP3R1) was not affected (P > 0.05) by the feeding system, moreover no age × feeding system

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interactions was observed (Table 4). Meanwhile, from day 0 to 70, the mRNA expression of

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PPP3CB (P = 0.040), PPP3R1 (P = 0.030) and PPP3CA (P = 0.051) linearly increased with

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increasing age. Similarly, expression levels of PPP3CA, PPP3CB and PPP3R1 gradually 11

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increased from d 14 to 42, and instantly decreased from d 42 to 56, and again increased after d 56.

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There was also no age × feeding system interaction for NFATC1 mRNA expression. In terms of

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age, the expression of NFATC1 linearly increased (P = 0.013) from d 0 to 70.

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For the mRNA expression of PGC-1α, there was an age × feeding system interaction (P =

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0.020), with greater expression in the G group than the BF group on d 42 and less expression than

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the BF group on d 56 and 70. Moreover, from d 0 to 70, the expression of PGC-1α in the G group

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increased quadraticly (P = 0.003) , being the greatest on d 42; while the expression of PGC-1α in

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the BF group linearly increased (P = 0.002).

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Neither feeding system (P = 0.310) nor age (P = 0.276) had effect on the mRNA expression

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of myostatin (MSTN), and no age × feeding system interaction (P = 0.213) was observed. From d

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0 to 70, the mRNA expression of MSTN of the G group sharply increased from d 14 to 42 and

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then declined rapidly from d 42 to 56, whereas it slowly rose from d 14 to 56 in the BF group.

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Temporal Functional Protein Expression

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As illustrated by Western blotting results in Figures 1, 2 and 3, using GAPDH as the reference

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protein, feeding system affected (P = 0.001) MYOZ3 protein expression, and tended to exert an

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influence on the expression of MEF2C (P = 0.081) and PGC-1α (P = 0.070) proteins. Moreover,

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there were interactions between age and feeding system for these three proteins (MYOZ3, P =

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0.054; MEF2C, P = 0.054; PGC-1α, P = 0.002). When compared with the BF group, relative

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greater expression of MYOZ3 protein was observed in the G group from d 42 to 70; as for

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MEF2C and PGC-1α, greater protein expression was noted in the G group on d 42, but less was

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observed on d 56 and 70.;

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Furthermore, regardless of feeding system, animal age did affect (P < 0.05) the expression of

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MYOZ3, MEF2C and PGC-1α at protein level. From d 0 to 70, in two groups, the MYOZ3

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protein expression in both two groups was influenced (linear, P = 0.037; quadratic, P < 0.001,

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respectively) by age, with the greatest expression on d 42, and reduced gradually thereafter. The

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MEF2C protein expression presented a quadratic increasing curve as a whole (P < 0.001).

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Differently, the lowest expression occurred on d 56 for G group and on d 42 for BF group. The

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protein expression of PGC-1α in grazing goats decreased linearly (P = 0.004) with increasing age,

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while age didn’t exert a significant influence on that of BF goats. Interestingly, MEF2C and

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PGC1α proteins displayed disparate increasing and decreasing trends from 42 to 70 d, especially

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from d 56 to 70, PGC1α protein expression decreased but MEF2C increased. Moreover, from d 14

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to 42, the MYOZ3 protein expression rose in both groups, whereas the expression of MEF2C and

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PGC-1α proteins were decreased.

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DISCUSSION

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From the initiation of myogenesis to the acquisition of mature and functional contractile

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myofibres, an extremely complex signal regulatory network is involved in these biological events.

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Many different kinds of molecules participate in skeletal muscle growth at different levels, via

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multiple signal pathways and in various manners, which are as follows

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regulators, such as myogenic regulatory factors (MRFs) and myocyte enhancer factors 2 (MEF2s);

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2) growth factors and receptors such as fibroblast growth factors (FGFs) and receptors,

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insulin-like growth factors (IGFs) and receptors, TGFβ family signaling molecules (especially

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: 1) myogenic

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myostatin, MSTN), WNT-pathway signaling molecules, Notch and Hedgehog signaling pathway;

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3) hormones and receptors, such as follistatin, testosterone, leptin and growth hormone (GH) axis,

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thyroid hormone axis, and their receptors; 4) muscle structural protein and mitochondrial

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biogenesis genes, such as myosin heavy chains MYH1/2/4, myosin light chain, phosphorylatable,

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fast skeletal muscle (MYLPF), myozenin (MYOZ1/2/3) and peroxisome proliferator activated

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receptor gamma, and coactivator 1 alpha (PGC1α); 5) regulators of myofibre type and

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composition, such as calcineurin (CaN), calmodulin (CaM), nuclear factor of activated T cells

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(NFAT), MEF2s, MSTN and PGC1α; 6) molecules involved in matrix remodeling and epigenetic

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control. After birth, the majority of these molecules still exist, and they interact on each other and

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work together to exert a regulatory impact on myonucleus and myofibril contents, protein

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turnover, and transition of myofiber types.

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Temporal mRNA Expression of Genes Related to Muscle Growth

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Skeletal muscle structural protein genes. Myosins in skeletal muscle are the most abundant

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proteins and are hexamers composed of two myosin heavy chains (MHCs) and four myosin light

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chains (MLCs). Each of the MHCs associates with two MLC isoforms, one belonging to the alkali

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light chain (also called the essential light chain, ELC), and the other belonging to the regulatory

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light chain (RLC).16 Myosin isoforms are generally considered as the molecular markers of

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muscle fiber type.17 However, because of the existence of pure and hybrid fiber types, and other

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protein isoforms, there are some discrepancies between that of muscle fiber type and MHC

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isoform. For example, type IIa is characterized by the expression of MHC 2a isoform encoded by

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the MYH2 gene, type IIb is characterized by MHC 2b isoform encoded by MYH4, and type IIx is

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characterized by MHC 2x isoform encoded by MYH1.18 Hybrid fibers contain more than two

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MHC isoforms, including type I/IIa, IIa/I, IIa/IIx, IIx/IIa, IIx/IIb and IIb/IIx. Type I/IIa fibers

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express MHC 1 and 2a isoforms with MHC 1 in excess, whereas type IIa/I fibers also contain the

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MHC 1 and 2a isoforms, with MHC 2a in excess.19 The content of hybrid fibers is much higher in

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goat skeletal muscles. For instance, there are five fiber types (I, I/IIa, IIa, IIa/IIx and IIx) in

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semitendinosus and the hybrid fast-twitch fibers (IIa/IIx) are about total 21%.20 Thus, we may

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acquire an understanding of developmental changes of muscle fiber-type composition by

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detecting the mRNA expression of MYH1, MYH2 and MYH4 genes in goat longissimus dorsi

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muscle.

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The MYH1 gene is the most abundant isoform in the longissimus dorsi muscle in

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non-ruminants. The expression of MYH1 were greater than that of either MYH2 or MYH4 (Table

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2), indicating that it also was the most abundant isoform of structural protein genes in goat muscle.

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The MYH1 mRNA levels presented a linear increase with increasing age, suggesting that the

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number of type IIx fiber was increasing. The decreased MYH1 expression from d 42 to 56 in both

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groups might be resulted from weaning of the kid goats on d 40, this was in accordance with

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previous reports that weaning had a momentary influence on the number of type IIx fiber from

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pre-ruminant stage to ruminant stage in ruminants.21 However, the MYH2 expression remained

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stable during the whole experimental period, showing that the number of type IIa fiber in the

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longissimus dorsi muscle of goat kids was unaffected either by age or by feeding system in the

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early growth stage. The MYH4 was expressed at extremely low levels and decreased quadraticly

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with increasing age, implying the number of type IIb fiber was quite little and remarkably

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decreased with increasing age postnatal, furtherly hinted that the MYH4 gene might not play 15

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important roles in skeletal muscle growth after birth.. This was in accordance with the report by

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Vuocolo et al. found that expression of MYH4 was decreased from d 5 to 11-12 wk of age both in

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callipyge and wild-type sheep. 18 Furhtermore, Arguello et al. found that there was no MHC IIb

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isoform in semitendinosus from adult goats, suggesting that MYH4 was not expressed at the adult

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stage.20 The MYLPF is one of the three genes encoding the RLCs of striated muscle and may be a

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candidate gene that regulates skeletal muscle development and affects meat quality in livestock.

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Xu et al. found that MYLPF was highly expressed in the longissimus dorsi muscle of Tianfu goats

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and the expression was decreased from month 6 to 24.22 Their finding was in contrast with our

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results that MYLPF expression was not affected by age, which would due to differences in species

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and growth stage.

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The MYOZ may be a substrate to combine the CaN (a calcium/calmodulin (CaM)-regulated

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serine/threonine phosphatase) with contractile elements of muscle and participate in the

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Ca2+-mediated signal pathways, thereby regulating the gene expression of slow-twitch myofibres.

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The MYOZ genes, including MYOZ1, MYOZ2 and MYOZ3, are closely related to meat quality.

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The expression of MYOZ1 and MYOZ3 were observed in a similar trend that increased with

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increasing age but unaffected by feeding system, suggesting that MYOZ1 and MYOZ3 served

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similar functions in controlling muscle fiber types, and the skeletal muscle development was

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turned into slow-twitch types in postnatal goats. The MYOZ2 was expressed differently from

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MYOZ1 and MYOZ3, and it was notably unaffected by age and feeding system. Previous

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research in Tianfu goats found that MYOZ2 and MYOZ3 were expressed differently with age,

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and suggesting that MYOZ2 and MYOZ3 exerted different effects in muscles growth;23 MYOZ2

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was more highly conserved than MYOZ3 in nucleotide sequence, inferring that MYOZ2 occupied 16

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a larger role than MYOZ3. Obviously, our results were not in agreement with that of Tianfu goats,

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and further study is needed to figure out the exact role of MYOZ genes.

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MRFs and MEF2s for skeletal muscle growth. It has been previously reported that MyoG is

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significantly correlated with Myf5 (r = 0.869).24 Furthermore, Myf5 and Myf6 are reversely

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co-expressed, meaning that when either Myf5 or Myf6 was activated, the other will be inactivated.

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In contrast, MyoG and MyoD are positively co-expressed in long term studies.25 In the current

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study, we found that the mRNA expression of both Myf5 and Myf6 were increased from d 0 to 70;

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and from d 0 to 56, one of them was expressed to a greater extent than the other, namely when one

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of them increased the other decreased, which was seemingly coincident with the above-mentioned

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results. However, the relationships aforementioned between Myf5 and MyoG, and between

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MyoG and MyoD were not demonstrated in this study, whereas our observed changing trends

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were almost in according with the MRFs expression of Nanjiang Yellow goats at 3-90 d.10 Among

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MRFs, the Myf5 and Myf6 mRNA expression were remarkably affected by feeding system.

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Compared with grazing goats, both Myf5 and Myf6 were expressed to a greater extent in BF

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group, indicating that their mRNA expression was possibly correlated with nutrition and activity

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levels.

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The MRFs initiate the expression of MEF2s, and MEF2s enhances the transcriptional

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regulation of MRFs, thus MEF2s are indispensable for the MRFs. Moreover, MEF2s are essential

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for the specific gene expressions of slow-twitch fibers by linking the activated NFAT and specific

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transcriptional factors in muscles. In the current study, the expression of MEF2A, MEF2C and

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MEF2D tended to increase from d 0 to 70, implying they are involved in transcriptional regulation

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in skeletal muscle development after birth. The MEF2B tended to increase in G group, but 17

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decrease in BF group, which may suggest that MEF2B serves different functions from the other

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three.

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Regulations of skeletal muscle fiber type and composition. The CaN , is a heterodimer made

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up of CaM-binding catalytic A subunit (CnA, encoded by PPP3CA and PPP3CB) and

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calcium-binding regulatory B subunit (CnB, encoded by PPP3R1). The NFATs are one kind of the

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most crucial substrates of CaN, and inhibition of CaN leads to NFATC1 cytoplasm translocation

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from nuclear, which suggests NFATC1is a CaN-dependent sensor,26 and CaN/NFATC1 signaling

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pathway is considered to be vital in controlling skeletal muscle hypertrophy.27 Parsons et al.

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reported that CnAα (encoded by PPP3CA) and CnAβ (encoded by PPP3CB) existed in skeletal

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muscles and CnAα was much richer than CnAβ.28 We found that the expression of PPP3CA was

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also higher than PPP3CB from d 0 to 70. The expressions of PPP3CA, PPP3CB and PPP3R1

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tended to increase with increasing age, in detail, rose from d 14 to 42, then sharply decreased from

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d 42 to 56, and afterwards increased again. This suggested that they were affected by weaning but

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not by feeding system. That NFATC1 successively decreased from d 14 to 56 but increased

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afterwards. We inferred that the starter, weaning and physiological transition probably affected its

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expression, but feeding system did not exert a notable influence on it.

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The MSTN is believed to function both pre- and postnatally, controlling postnatal muscle

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growth by regulating satellite cell activation and number, and regulating muscle fiber-type

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composition postnatally. We noticed that MSTN transcript level had an increasing trends with age

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in both groups, this was in accordance with the findings of Tripathi et al. that MSTN mRNA was

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increased in contractile myotubes compared to myoblasts. 24

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The PGC1α is considered as a crucial factor of regulating mitochondrial gene expression for it 18

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activates mitochondrial biogenesis and oxidative metabolism.29 The PGC1α cooperates with CaN

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pathway and interacts directly with several MEF2 proteins, consequently promoting the formation

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of slow-twitch or oxidative fibers.30 Our data showed that the PGC1α expression for G group

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presented a quadratic changing trend from d 0 to 70 and started to increase after d 56, and it was

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linearly increased for BF group. Similarly, MEF2C showed a similar changing trend at d 42 to 70

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for G group and likewise increased after d 56 for BF group, indicating that PGC1α and MEF2C

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may interplay during these stages.

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Temporal Protein Expression Profiles

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We found that MYOZ3 protein expression was significantly affected by age and feeding system.

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It quickly increased from d 14 to 42 and sharply decreased from d 42 to 70 in both G and BF

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groups, with lower levels in BF goats. The results implied that MYOZ3 protein expression was

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affected more seriously by weaning than by starter, and these factors seemed to exert more

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influences on BF group than G group. Our results were a little different from the finding that

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MYOZ3 protein expression gradually increased from d 1 to 70 reported by Wan et al..23 The

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difference between studies was possibly associated with goat breed, growth stage and artificial

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management.

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The MEF2C protein expression was increased quadratically from d 0 to 70 in both groups, but

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feeding system did not exert prominent effects. Moreover, we found that the MEF2C transcript

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level and protein level gradually increased with age, demonstrating that MEF2C did play critical

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roles in promoting the development of slow-twitch fibers in skeletal muscle of postnatal goats.

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The expressions of PGC1α as well as MYOZ3 exhibited different increasing and decreasing

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trends at transcript and protein levels, and the difference was much larger for PGC1α at d 14 to 70

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and for MYOZ3 at d 56 to 70. The reason for the differences may be due to their respective

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biological functions as they might be regulated by many other molecules at different levels.

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Besides, PGC1α protein and MEF2C protein displayed disparate increasing and decreasing trends

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from d 42 to 70, especially between d 56 to 70 d, with PGC1α protein expression reduced but

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MEF2C increased, from which we inferred PGC1α and MEF2C protein may interact and

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coordinate between each other to promote skeletal muscle growth. The mechanisms for this

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response warrant further examination.

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To sum up, we conclude that the growth of skeletal muscle of goats from d 0 to 70 after birth

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was mainly due to myofiber hypertrophy and differentiation; the number of type IIx in the

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longissimus dorsi muscle quickly increased, whereas the number of type IIa and IIb decreased

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slightly. These regulating genes related to 1) skeletal muscle structural proteins, 2) MRFs and

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MEF2 family, and 3) skeletal muscle fiber type and composition, which demonstrated a degree of

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coordination being expressed during fast growing and developing stages. Only the expression of

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Myf5 and Myf6 was remarkably affected by feeding system. The MYOZ3 protein expression was

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affected by age and feeding system, but MEF2C protein as well as PGC1α protein in grazing goats

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were affected only by age. The MEF2C was critical in manipulating skeletal muscle growth after

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weaning. Moreover, MEF2C and PGC1α protein may interact and coordinate with each other to

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promote the skeletal muscle growth.

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AUTHOR’S CONTRIBUTION

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J. Y. Huang carried out mRNA expression and functional protein expression analysis and

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drafted the manuscript. J. Z. Jiao participated in functional protein expression analysis. Z. L. Tan

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participated in the design of the study, Z. X. He participated the data analysis, K. A. Beauchemin

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and R. Forster helped to revise the manuscript, X. F. Han and J. H. Kang participated slaughtered

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experiment, and S. X. Tang performed the statistical analysis. C. S. Zhou conceived of the study,

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and participated in its design and coordination and helped to revise the manuscript. All authors

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read and approved the final manuscript.

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ACKNOWLEDGEMENTS

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The authors acknowledge Key Laboratory of Subtropical Agro-ecological Engineering,

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Institute of Subtropical Agriculture, Chinese Academy of Sciences (CAS) for providing all the

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experimental materials and apparatus.

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FUNDING SOURCES

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This study was financially supported by the project National Natural Science Foundation of

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China (Grant No. 31320103917, 31201826, 31372342), "Strategic Priority Research Program -

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Climate Change: Carbon Budget and Relevant Issues" (Grant No. XDA05020700), “CAS

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Visiting Professorship for Senior International Scientists (Grant No. 2010T2S13), and Hunan

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Provincial Creation Development Project (2013TF3006).

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CONFLICT OF INTEREST STATEMENTS

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The authors declared no competing interests.

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(Capra pyrenaica) in meat mixtures. J AOAC Inte 2008, 91, 103-11. (8) Ran T.; Li H.Z.; Liu Y.; Tang S.X.; Han X.F.; Wang M.; He Z.X.; Kang J.H.; Yan Q.X.; Tan Z.L., Zhou C.S., Expression of genes related to sweet taste receptors and monosaccharides transporters along the gastrointestinal tracts at different development stages in goats. Livest Sci 2016, 188, 8. (9) Livak, K. J.; Schmittgen, T. D., Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001, 25, 402-8. (10) Zhong, T.; Jin, P. F.; Dong, E. N.; Li, L.; Wang, L. J.; Zhang, H. P., Caprine sex affects skeletal muscle profile and MRFs expression during postnatal development. Anim Sci J 2013, 84, 442-8. (11) Edmondson, D. G.; Lyons, G. E.; Martin, J. F.; Olson, E. N., Mef2 gene expression marks the cardiac and skeletal muscle lineages during mouse embryogenesis. Development 1994, 120, 1251-63. (12) Cheng, B. Study on cloning and tissue expression of MEF2 gene family in goat. Sichuan Agricultural University, Ya’an, 2012. (13) Shen, L. Y.; Zhang, S. H.; Wu, Z. H.; Zheng, M. Y.; Li, X. W.; Zhu, L., The influence of satellite cells on meat quality and its differential regulation. Hereditas (Beijing) 2013, 35, 10811086. (14) Braun, T.; Gautel, M., Transcriptional mechanisms regulating skeletal muscle differentiation, growth and homeostasis. Nat Rev Mol Cell Biol 2011, 12, 349-61. (15) Palacios, D.; Puri, P. L., The epigenetic network regulating muscle development and regeneration. J Cell Physiol 2006, 207, 1-11. 24

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(16) Choi, Y. M.; Kim, B. C., Muscle fiber characteristics, myofibrillar protein isoforms, and meat quality. Livest Sci 2009, 122, 105-118. (17) Bottinelli, R.; Reggiani, C., Human skeletal muscle fibres: molecular and functional diversity. Prog Biophys Mol Bio 2000, 73, 195-262. (18) Vuocolo, T.; Byrne, K.; White, J.; McWilliam, S.; Reverter, A.; Cockett, N. E.; Tellam, R. L., Identification of a gene network contributing to hypertrophy in callipyge skeletal muscle. Physiol Genomics 2007, 28, 253-272. (19) Pette, D.; Staron, R. S., Myosin isoforms, muscle fiber types, and transitions. Microsc Res Techniq 2000, 50, 500-509. (20) Arguello, A.; Lopez-Fernandez, J. L.; Rivero, J. L. L., Limb myosin heavy chain isoproteins and muscle fiber types in the adult goat (Capra hircus). Anat Rec 2001, 264, 284-293. (21) Wardrop, I. D.; Coombe, J. B., Development of Rumen Function in Lamb. Aust J Agr Res 1961, 12, 661-&. (22) Xu, H.; Xu, G.; Wang, D.; Ma, J.; Wan, L., Molecular cloning, sequence identification and expression analysis of novel caprine MYLPF gene. Mol Biol Rep 2013, 40, 2565-72. (23) Wan, L.; Ma, J.; Wang, N.; Wang, D.; Xu, G., Molecular cloning and characterization of different expression of MYOZ2 and MYOZ3 in Tianfu goat. PloS one 2013, 8, e82550. (24) Tripathi, A. K.; Ramani, U. V.; Rank, D. N.; Joshi, C. G., In vitro expression profiling of myostatin, follistatin, decorin and muscle-specific transcription factors in adult caprine contractile myotubes. J Muscle Res Cell Motil 2011, 32, 23-30. (25) Hudson, N. J.; Reverter, A.; Wang, Y.; Greenwood, P. L.; Dalrymple, B. P., Inferring the 25

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transcriptional landscape of bovine skeletal muscle by integrating co-expression networks. PloS one 2009, 4, e7249. (26) Jiao, Q. Z. Gene expression studies of pig postnatal skeletal muscle hypertrophy and myofiber transformation pathway. Huazhong Agricultural University, Wuhan, 2009. (27) Schulz, R. A.; Yutzey, K. E., Calcineurin signaling and NFAT activation in cardiovascular and skeletal muscle development. Dev Biol 2004, 266, 1-16. (28) Parsons, S. A.; Millay, D. P.; Wilkins, B. J.; Bueno, O. F.; Tsika, G. L.; Neilson, J. R.; Liberatore, C. M.; Yutzey, K. E.; Crabtree, G. R.; Tsika, R. W.; Molkentin, J. D., Genetic loss of calcineurin blocks mechanical overload-induced skeletal muscle fiber type switching but not hypertrophy. J Biol Chem 2004, 279, 26192-26200. (29) Wu, Z. D.; Puigserver, P.; Andersson, U.; Zhang, C. Y.; Adelmant, G.; Mootha, V.; Troy, A.; Cinti, S.; Lowell, B.; Scarpulla, R. C.; Spiegelman, B. M., Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1. Cell 1999, 98, 115-124. (30) Lin, J.; Wu, H.; Tarr, P. T.; Zhang, C. Y.; Wu, Z. D.; Boss, O.; Michael, L. F.; Puigserver, P.; Isotani, E.; Olson, E. N.; Lowell, B. B.; Bassel-Duby, R.; Spiegelman, B. M., Transcriptional co-activator PGC-1 alpha drives the formation of slow-twitch muscle fibres. Nature 2002, 418, 797-801. (31) Zhou, Z.; Wan, Y.; Zhang, Y.; Wang, Z.; Jia, R.; Fan, Y.; Nie, H.; Ying, S.; Huang, P.; Wang, F., Follicular development and expression of nuclear respiratory factor-1 and peroxisome proliferator-activated receptor gamma coactivator-1 alpha in ovaries of fetal and neonatal doelings. J Anim Sci 2012, 90, 3752-3761. 26

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Figure captions Fig 1. (A) The relative postnatal expression of MYOZ3 protein in the longissimus dorsi muscle under grazing (G) and barn-fed (BF) condition; (B) representative lanes of Western blot analysis of MYOZ3 protein, lane from the left to the right represents the exression level on d 0, 7, 14, 42 grazing, 42 barn-fed, 56 grazing, 56 barn-fed, 70 grazing and 70 barn-fed, respectively. Fig 2. (A) The relative postnatal expression of MEF2C protein in the longissimus dorsi muscle under grazing (G) and barn-fed (BF) condition; (B) representative lanes of Western blot analysis of MEF2C protein, lane from the left to the right represents the exression level on d 0, 7, 14, 42 grazing, 42 barn-fed, 56 grazing, 56 barn-fed, 70 grazing and 70 barn-fed, respectively. Fig 3. (A) The relative postnatal expression of PGC-1α protein in the longissimus dorsi muscle under grazing (G) and barn-fed (BF) condition; (B) representative lanes of Western blot analysis of PGC-1α protein, lane from the left to the right represents the exression level on d 0, 7, 14, 42 grazing, 42 barn-fed, 56 grazing, 56 barn-fed, 70 grazing and 70 barn-fed, respectively.

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Table 1 Primer sequences for the mRNA expression analysis of genes in this study Gene MYH1 MYH2 MYH4 MYLPF MYOZ1 MYOZ2 MYOZ3 Myf5 MyoD Myf6 MyoG MEF2A MEF2B MEF2C MEF2D MSTN PGC1α NFATC1 PPP3CA PPP3CB PPP3R1 β-ACTIN

2 3

Primer sequences (5’ - 3’) F: CAAGGCAGGGTCTTTGATTG R: CTTTCGGAGGTAAGGAGCAG F: GCAGAGAAGGTGGGAAAGTG R: TTGTCGAATTTGGGAGGATT F: GGAAACTGGAGGATGAATGC R: CTTGTTCTCTGTGGCGTGTT F: GAAGACCTGCGGGACACT R: GATCACATCCTCAGGGTCAG F: GCTTTCGCTGCTCACTAACC R: CCGACACTGCTCTTGCTGTA F: TAAGATGCGACAAAGAAGAT R: TAGGAGGAGTAAATGGTGCT F: CTGGGCAAGAAACTGAGCG R: GAGGCGGGATAGATGTGGAG F: CACGACCAACCCTAACCAGAG R: TCTCCACCTGTTCCCTTAGCA F: GCCTGAGCAAAGTCAACGAG R: GAGTCGCCGCTGTAGTGTTC F: CGGAGCGCCATTAACTACAT R: AAATCCGCACCCTCAAGATT F: GCAGCGCCATCCAGTACATAG R: GAAGGCCGCAGTGACATCC F: TGAAAGGAATCGACAGGTCAC R: CAGTGCTGGCATACTGAAACA F: CCTTCCCTTACCCTTTGCTC R: TAACTGCTGCGTCTTCTCCA F: CAGTCATTGGCTACCCCAGT R: GCGGTGTTAAAGCCAGAGAG F: GGTCTCCCAGTCTACCCACTC R: TGAACTGAAGGCTGGTAAGGA F: GTGTTGCAAAACTGGCTCAA R: TCATCACAATCAAGCCCAAA F: CACCCACAACTCCTCCTCAT R: GCCTTCCTTTCCTCGTGTC F: GGCAAAGACTGAAGGAGACC R: GCTGGTACTGACTCCGCTTC F: GAAGATGGATTTGATGGAGCA R: TGAGAACCGAGAACACTCTGG F: CTCCACATCCTTATTGGTTGC R: TGGTCTTCGCCTTCAGTCAT F: CGATGGGAATGGAGAAGTAGA R: GATACGGAAAGCAAACCTCAA F: ATGGCTACTGCTGCGTCGT R: TTGAAGGTGGTCTCGTGGAT

Product size

GenBank

101 bp

XM_005693577

100 bp

XM_005693576

101 bp

XM_005693575

159 bp

NM_001145183 [22]*

195 bp

XM_005699214

155 bp

JX573191 [23]

254 bp

KC537058 [23]

101 bp

JF829004 [10]

229 bp

JF829005

134 bp

NM_001285602

284 bp

JF829006 [10]

143 bp

JN967621 [12]

204 bp

JN967622

152 bp

JN967623 [12]

160 bp

JN967624 [12]

191 bp

GU377303 [24]

232 bp

AY321517 [31]

140 bp

XM_005696948

100 bp

XM_005681369

142 bp

XM_005699205

103 bp

XM_005686814

161 bp

XM_005694067

* numbers shown in brackets are corresponding references

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Table 2 The mRNA expression of skeletal muscle structural proteins Item MYH1 MYH2 MYH4 MYLPF MYOZ1 MYOZ2 MYOZ3

System Grazing Barn-fed Grazing Barn-fed Grazing Barn-fed Grazing Barn-fed Grazing Barn-fed Grazing Barn-fed Grazing Barn-fed

Age, d 0

7

14

1.00

1.69

4.72

1.00

0.47

1.03

1.00

0.89

0.07

1.00

2.51

1.02

1.00

1.82

0.82

1.00

2.24

2.17

1.00

1.41

0.61

42

56

70

5.61

1.96

5.39

8.14

2.01

7.85

1.11

0.83

0.55

1.10

0.50

0.50

0.04

0.02

0.07

0.04

0.03

0.04

1.95

2.49

1.85

1.47

0.95

2.69

4.45

1.52

3.89

2.85

2.41

6.51

1.95

2.43

2.11

2.51

0.62

2.53

3.70

0.63

5.30

2.26

1.39

5.62

SEM 1

P value 2 Age

System

Age × System

1.682

0.015

0.237

0.706

0.267

0.061

0.502

0.010

0.023

0.436

SEM 3

P value for age 4 L

Q

1.318

0.017

0.258

0.748

0.253

0.439

0.230

0.465

0.264

0.081

< 0.001

< 0.001

0.361

0.285

0.045

0.349

0.077

0.375

0.423

0.260

0.092

1.032

0.020

0.459

0.150

0.702

< 0.001

0.207

0.692

0.470

0.629

0.188

0.678

0.572

0.618

0.850

< 0.001

0.846

0.316

0.539

< 0.001

0.011

1

Standard error of least squares means for Age (d 42 to 70) × System.

2

P value for Age (d 42-70), System, and Age (d 42 to 70) × System. Age (d 42 to 70) × System is the interaction between Age (d 42 to 70) and System.

3

Standard error of least squares means for Age (d 0 to 70).

4

P value for Age (d 0 to 70); L = Linear effects, Q = Quadratic effects.

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Table 3 The mRNA expression of genes of MRFs family and MEF2 family Item Myf5 MyoD Myf6 MyoG MEF2A MEF2B MEF2C MEF2D

System Grazing Barn-fed Grazing Barn-fed Grazing Barn-fed Grazing Barn-fed Grazing Barn-fed Grazing Barn-fed Grazing Barn-fed Grazing Barn-fed

Age, d 0

7

14

1.00

1.16

1.04

1.00

4.82

1.24

1.00

0.26

0.53

1.00

0.85

0.48

1.00

2.17

2.16

1.00

2.05

2.12

1.00

1.55

1.35

1.00

2.25

4.20

42

56

70

0.82

0.59

1.22

1.09

1.19

2.50

0.64

0.24

1.97

0.24

0.74

0.44

0.73

1.61

1.81

2.13

1.81

5.05

1.24

0.42

0.73

0.73

0.67

1.14

3.32

2.14

2.90

2.90

2.92

4.02

0.65

1.47

1.59

1.19

0.65

0.35

2.03

1.08

1.45

1.49

0.48

2.76

3.22

1.94

2.14

2.34

3.93

3.37

SEM 1

P value 2 Age

System

Age × System

0.396

0.043

0.039

0.449

0.376

0.104

0.137

0.047

0.365

< 0.001

< 0.001

0.002

0.260

0.204

0.818

0.552

0.262

0.399

SEM 3

P value for age 4 L

Q

0.261

0.211

0.052

0.766

0.108

0.198

0.686

0.007

0.673

0.228

< 0.001

0.018

0.296

< 0.001

< 0.001

0.193

0.400

0.524

0.431

0.288

0.357

0.415

0.001

0.464

0.943

0.136

0.095

0.596

0.385

0.503

0.367

0.006

0.858

0.029

0.280

0.563

0.128

0.344

0.083

0.045

0.594

0.948

0.124

0.068

0.531

0.467

0.093

1

Standard error of least squares means for Age (d 42 to 70) × System.

2

P value for Age (d 42-70), System, and Age (d 42 to 70) × System. Age (d 42 to 70) × System is the interaction between Age (d 42 to 70) and System.

3

Standard error of least squares means for Age (d 0 to 70).

4

P value for Age (d 0 to 70); L = Linear effects, Q = Quadratic effects.

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Table 4 The mRNA expression of skeletal muscle fiber type and composition genes Item PPP3CA PPP3CB PPP3R1 NFATC1 PGC-1α MSTN

System Grazing Barn-fed Grazing Barn-fed Grazing Barn-fed Grazing Barn-fed Grazing Barn-fed Grazing Barn-fed

Age, d 0

7

14

1.00

1.89

1.41

1.00

0.91

1.67

1.00

1.22

1.73

1.00

0.38

2.73

1.00

1.08

0.45

1.00

1.79

0.93

42

56

70

2.79

1.75

2.35

3.14

1.58

2.72

2.03

1.44

1.73

2.06

1.21

3.03

2.37

1.64

2.19

3.23

1.59

4.74

1.83

1.11

2.60

1.61

1.27

2.49

4.30

0.89

1.33

2.07

2.19

3.08

2.68

1.13

2.18

1.11

1.39

2.11

SEM 1

P value 2 Age

System

Age × System

0.418

0.007

0.548

0.706

0.489

0.116

0.371

0.720

0.056

0.287

SEM 3

P value for age 4 L

Q

0.511

0.051

0.315

0.278

0.394

0.040

0.352

0.074

0.215

0.547

0.030

0.393

0.001

0.814

0.801

0.316

0.013

0.798

0.695

0.086

0.633

0.020

0.475

0.103

0.003

0.543

0.002

0.412

0.600

0.276

0.310

0.213

0.428

0.133

0.322

1

Standard error of least squares means for Age (d 42 to 70) × System.

2

P value for Age (d 42-70), System, and Age (d 42 to 70) × System. Age (d 42 to 70) × System is the interaction between Age (d 42 to 70) and System.

3

Standard error of least squares means for Age (d 0 to 70).

4

P value for Age (d 0 to 70); L = Linear effects, Q = Quadratic effects.

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