Chickens are economically significant animals. Skeletal muscle constitutes the largest proportion and most valuable component of meat mass; its development is closely associated with the amount of meat production and its quality. Together with muscular histological traits, such as the type, diameter, density and size of muscle fibers, the deposition of intramuscular fat (IMF) can dramatically promote tenderness of meat and plays an important role in flavor of meat [1–3].

The development of skeletal muscle depends on myogenesis and, to some extent, also on adipogenesis. The muscle mass and IMF content are both determined by cell numbers and unit cell size. Hyperplasia refers to the increasing in cell number or muscle fiber number which occurs mainly in embryonic periods as the numbers of adipocytes and muscle fibers are fixed by the day of hatching [4, 5]. Velleman et al. (2007) observed that breast muscle organizational differences among breeds and between sexes begin to occur between embryonic 20 days (ED 20) and ED 25 in turkeys. Thus, embryonic muscle development has a dramatic impact on the postnatal accretion of muscle mass and its content of IMF [6]. Chartrin et al. (2007) investigated the effects of age (from D 1 to D 98) on IMF of mule ducks and showed that total lipid content in the breast muscle was high at D 1 and indicated that embryonic IMF was necessary for growth and energy requirements at this early stage [7]. Our previous study as reported by Liu et al. (2016) also showed that chicken IMF content was highest at D 1 and the protein expression profile at D 1 differed greatly from other post-hatching ages [8]. The previous molecular studies of muscle development and IMF deposition in poultry have been mainly focused on post-hatchling stages, except for Ouyang’s recent report on the proteomic analysis of embryonic muscle in a local breed [8–10]. Although the importance of embryonic muscle and IMF development was established, underlying molecular mechanisms remain poorly understood.

The Beijing-you (BJY) chicken, called Beijing-fatty chicken, is an indigenous breed in China with good meat quality [11, 12]. When compared with fast-growing commercial broilers, the BJY is favored by local consumers because of the taste, rich fragrance, and tenderness of the meat. The Cobb is a famous genetically improved commercial line with excellent growth rate, and yield, especially of breast muscle. Breast muscle yield could reach more than 20% of live weight after D 28 (www.Cobb-Vantress.com), which is double that (around 10%) of BJY of similar live weight [13].

With the aim of exploring the embryonic development-related proteome and metabolome signatures in breast muscle and its content of IMF, we quantitatively analyzed the proteomes and metabolomes of breast muscle of the two distinct chicken breeds at embryonic day12 (ED 12), ED 17, upon hatching (D 1) and day 14 post-hatching (D 14) using an Isobaric tags for relative and absolute quantitation (iTRAQ) and liquid chromatography/mass spectrometry (LC-MS) based approaches, and verified results from the discovery proteomics by Western blotting of selected proteins. Changing patterns of breast muscle growth and IMF contents at these critical stages of development were also measured.

Chickens are among the most important food animals of worldwide economic significance and are widely used as a model species for the study of embryonic development. To our knowledge, no study has systematically examined the proteomes and metabolomes of embryonic skeletal muscle and IMF development.

In the current study, obvious phenotypic patterns exist both in slow-growing BJY and fast-growing Cobb chickens which are in line with the three critical stages in muscle development: ED 12 to ED 17 (embryonic rapid growth, hyperplasia), ED 17 to D 1 (hyperplasia and embryo preparing for hatch), D 1 to D 14 (shift from myoblast-mediated growth to satellite cell-mediated growth by hypertrophy) [5, 6, 14].

For muscle development, interest centered on the striking divergence of slow-growing and fast-growing birds and the underlying molecular basis for it. Breast muscle mass during middle-embryo and post-hatch stages was dramatically increased, which was in accordance with that observed in previous reports [15, 16]. In the BJY with no history of genetic selection for pectoral muscle size, the muscle weight increased 1.8 fold during the hatchling period accompanied by increasing fiber diameter and fiber cross-sectional area (could not be measured for ED 12 reliably). The fiber diameter and fiber cross-sectional area decreased from ED 17 – D 1 in Cobb. Correspondingly, the fiber density increased marginally from ED 17 – D 1 in Cobb but decreased significantly in BJY. That indicated the growth of the embryonic muscle weight in Cobb is mainly a result of stronger hyperplasia process instead of hypertrophy, which contribute to the post-hatchling excellent muscle mass achievements in fast-growing Cobb (35.9 fold change from D 1 to D 14).

For deposition of IMF in chicken embryo, this study is the first to show that the major time for accumulation of embryonic IMF was from ED 17 to D 1, the period when muscle growth was the least; the striking muscle growth occurred after D 1 (D 1 – D 14). These observed phenotypic patterns were supported by the proteomic and metabolomic profiles of breast muscle evident at ED 12, ED 17, D 1 and D 14 in the two breeds.

For proteomic profiling, mass spectrometry (MS)-based proteomics were applied by the 8-plex iTRAQ strategy, where each plex using two protein pools (n = 5 individuals). Pooling strategy with two biological replicates was applied, as suggested by Gan et al., (2007), which has the potential advantage of decreasing overall variability due to differences between individuals [17]. The correlation between DA fold changes for proteins in the two replicates were all high (R² > 0.94, Additional file 4: Figures S1, and S2). The consistency between the quasi-quantitative Western blots and the changing pattern of DA proteins from the iTRAQ analysis were acceptable. As metabonomics reflects, in part, the downstream consequence of proteomics, it is also regarded as the complement of other “omics” for interpretation of functional genomics [18]. For metabolomic profiling in the current study, ten biological replicates for each age were from the same batch of chickens used for proteomic profiling. The advantage of combined proteomic and metabolomic profiling is that more profound results could be acquired than independent proteome analysis.

Through analysis of DA proteins and metabolites in the context of development, a remarkable finding is that, for embryonic Cobbs, many DA molecules (NDUFS1, ATP5B, UQCRC2, ACO2, VDAC1, VDAC2 and palmitic acid) were concentrated in Oxidative Phosphorylation/Mitochondrial Dysfunction pathway; those are vital for energy generation and metabolism (Tables 5, 7, 8) [19–22]. The network analyses (Additional file 4: Figure S3) were consistent with the energy requirement being greater for the faster muscle growth of the Cobbs. ATP5B is the catalytic subunit of the rate-limiting enzyme of oxidative phosphorylation and its abundance increased from ED 12 – ED 17 in Cobbs, as shown by Western blotting (Fig. 7).

Different from the Cobbs, in BJY, numerous proteins related to protein processing (HSPA8, HSPA4, HSPA9, HSP90AA1 and FKBP4) showed highest abundance at ED 12 and decreased thereafter (Additional file 4: Figure S4). As chaperones, heat shock proteins are known to aid in the folding of newly synthesized proteins and have roles in the disaggregation and degradation of persistently misfolded proteins [23]. The similar changing pattern of molecular abundance in Purine metabolism pathways (inosine and xanthine), indicated that genetic information processing might be more active in the embryonic development of slow-growing breeds. Findings from this study, especially on the metabolites in the faster growing Cobbs, might supply extra evidence for Oliveira’s hypothesis that high yield meat birds may direct more caloric resources towards muscle growth than towards fueling the hatching process [14].

Specific for BJY chickens, a series of DA proteins (ACADL, APOA1, HSP90B1, PDIA3 and GOT2) were enriched in PPAR signaling after ED 17 (Tables 4, 7, 8), among which the increase in ACADL, verified by Western blotting, changing in a manner that is consistent with those of IMF from ED 17 to D 14. Recent studies proved that PPARs functions as a key regulator for adipocyte development and IMF regulation [9, 24, 25]. Those proteins might contribute to the high capacity for accumulation of IMF in BJY chickens. The high abundances of ACADL at D 1 compared to post-hatchling days were consistent with previous reports [8, 26]. The most obvious fat accumulation at ED 17 – D 1 was also supported by the most significant IPA network constructed from DA proteins and metabolites between ED 17 and D 1, in which 29/34 proteins were detected and enriched (Additional file 4: Figure S5). It is worth noting the role of the methionine pathway in early muscle development. Key enzyme proteins for methionine degradation were decreased from ED17 to D 14, including MAT1A, BHMT or AHCY (Tables 4, 8). It is well established that MAT1A is the main gene responsible of S-adenosylmethionine (SAM) synthesis. Inhibition of AHCY can make S-adenosyl-homocysteine (SAH) accumulation, consequently suppressing the use of SAM for transmethylation reactions [27]. BHMT is a methyl transferase that can catalyze the transfer of the methyl group from betaine to homocysteine, which supplies the methyl group to methylate DNA [28]. A consistent finding in the present study was the decreased abundance of betaine from D 1 to D 14. Taken together, it indicated that the transmethylation reaction was attenuated from ED 17 to D 14 in BJY, which might contribute to the little overlap found in DA proteins between periods for pre- and post-hatching muscle growth (ED 12 - ED 17 vs D 1 - D 14). Considering that the abundance of AHCY and BHMT were also decreased in Cobb from D1 to D 14, further study is needed to clarify the epigenetic changes underlying altered protein abundance in early stages of chicken muscle and IMF development.

For the common features between the two breeds, several pathways, key proteins and metabolites (Tables 6, 7, 8) are known to be involved in muscle development. Some pathways, including FXR/RXR and LXR/RXR Activation, Calcium Signaling, Glycolysis I/Gluconeogenesis I, TCA Cycle II and Actin Cytoskeleton Signaling, were enriched in more than two stages. It was interesting that the abundance of androsterone, a weak androgen, decreased from D 1 to D 14 in both breeds. As an activator of the nuclear hormone receptor farnesoid X receptor (FXR) [29], the changing pattern is consistent with that of the FXR/RXR activation pathway, which was inactive from D 1 – D 14. Additionally, consistent with the increased protein abundance of TCA cycle enzymes, including ACO2 and/or MDH2, there were increases in L-malic acid (in BJY from ED 17 to D 14) and succinic acid (in both breeds from D 1 to D 14).

It is also worth noting the roles of the DA proteins enriched in Protein Kinase A Signaling in muscle development. Six to eight DA proteins and metabolites were enriched in that pathway in more than two stages in both breeds indicating the likelihood of their contributing to muscle glycogen metabolism. The breast muscle of the avian embryo is metabolically important mainly because of its large storage capacity for glycogen [6]; an important source of energy for skeletal muscle. The enriched proteins included YWHAE and YWHAZ, which belong to the 14–3-3 family of proteins, and were found to decrease from fast growing stages (D 1 - D 14) in both breeds. That is perhaps consistent with the report that YWHAE inhibited hypertrophy of cardiomyocytes [30]. Additional study of the function and mechanism of action of 14–3-3 proteins in development of skeletal muscle is needed. Acute Phase Response Signaling was also enriched in both breeds from ED 17 to D 14. The abundance of most of the enriched proteins, including APOA1 and APOH in Cobbs and PLG, C3 and APOA1 in BJY showed reciprocal changes between ED 17 – D 1 and D 1 – D 14 similar to that of IMF content. The function of complement C3 precursor has been reported to relate to insulin secretion and glucose homeostasis [31], but its possible relationship with IMF has not been described.

As for IMF accumulation, according to the findings in cattle, the level of IMF at the start of the growth period is likely a key determinant of the final level of IMF after finishing [32]. In chickens, the early period of change in IMF was observed between ED 17 and D1, when changes in muscle growth were least obvious. This suggested that myocytes and adipocytes influencing each other during development. The accretion rate of IMF depends on the number of adipocytes and their net accumulation of stored lipid. Aspects of muscle growth and its metabolic activity are also important. For example, animals having a high muscularity with a high glycolytic activity display a reduced development of IMF [2]. In the current study, several proteins altered in abundance between ED 17 and D 1 and reciprocal changes were demonstrated from D 1 to D 14 and this changing pattern was exactly consistent with the changes in IMF. Of special interest, four were rate-limiting enzymes for mitochondrial beta-oxidation of fatty acid (ACADL, ACAD9, HADHA, HADHB), which highlight the important role of this pathway playing in IMF accumulation at early stage. The ACADL catalyze the initial step of mitochondrial beta-oxidation of straight-chain fatty acid and its changes from iTRAQ analyses were verified by Western blotting. The results were consistent with previous report that lipid oxidation was very active from mid incubation until 2 or 3 days before internal pipping because adequate oxygen and yolk fatty acids could be supplied. Additionally, the abundant of apolipoprotein A1 was increased from D12 to D1, which might contribute to lipoproteins taking up cholesterol from the yolk sac membrane [14]. Consistent with Cui’s report [9], genes and pathways related to muscle development and ECM were also involved in IMF deposition, including the muscle related protein LAMA2 [33], an extracellular protein TPM2 [34] and the calcium-binding protein SRL [35]. The changes in TPM2 from iTRAQ analyses were verified by Western blotting. As it is difficult to quantify IMF content during embryonic development in chickens, it emphasizes the utility of suitable bio-markers.

In summary, this study found that ED 17 - D 1 was the earliest period for IMF accumulation. Through integrated analysis of the protein and metabolite profiles of breast muscle, pathways related to protein processing and PPAR signaling were found to be enriched to support high capacity of embryonic IMF accumulation in Beijing-You. Pathways related to ATP production were enriched to support the fast muscle growth in Cobb. Mitochondrial beta-oxidation of fatty acid is identified as the key pathway related to chicken IMF deposition at early stages.