The understanding of the gene functions, rapid and reliable quantification of gene expression, currently quantitative real-time PCR (qPCR) is one of the most sensitive techniques widely used. In quantitative gene expression, normalization is a key factor for accuracy and reliability, for that endogenous reference gene were used. According to the MIQE guidelines, the selection of suitable reference genes may vary for different species, different varieties, experimental conditions, and tissues and have to be validated before gene expression study [38]. Previous and recent studies also described different expression patterns of reference genes and focused on the validation of reference genes applied on different avian tissues [39,40,41]. However, so far validation of genes for their stable expression pattern in different embryo tissues such as 7th and 18th-day embryos of control broiler and improved Aseel was not performed.
Source, selection, primer design, verification of candidate reference genes
In the present study, to identify the suitable reference genes for 7th and 18th-day embryos of control broiler and improved Aseel, 24 candidate reference genes with a wide range of biological functions were selected based on previous studies of various avian and non-avian species. They are 18SrRNA, ALB, B2MG, β-ACT, EEF1A1, GAPDH, GUSB, HMBS, HSP10, HSP70, L-LDBC, MRPS27, MRPS30, PGK2, PPP2CB, RPL5, RPL13, RPL14, RPL19, RPL23, SDHA, TBP, TFRC, and DNAJC24. The chicken orthologous genes were obtained from NCBI, CDS region was found and amplified with gene-specific primers (Table S1). For all the primer pairs, melting curve analysis was performed to confirm the specific amplification for each reference gene, which showed presence of single peak indicating specificity of the amplification of the genes.
Expression stability and ranking of candidate reference genes
Expression levels of all candidate reference genes were measured in the samples collected from 7th and 18th-day embryos of the control broiler and improved Aseel. We observed that not all selected reference genes were expressing uniformly across different embryos of control broiler and improved Aseel (Fig. 3). Differential transcription abundance levels were observed among all 18 genes. The mean Ct values of the selected reference gene ranged from 12.34 to 28.27 irrespective of different embryos. 18S rRNA and DNAJC24 genes showed the most (Ct=12.34) and the least (Ct=33.88) abundant transcripts respectively. Each reference gene had different expression ranges across all sample sets. The results show that the GUSB reference gene had the least variation of expression with mean Ct values ranging from 17.78 to 22.52 whereas the RPL5 gene showed a much higher expression variation with mean Ct values ranging from 13.07 to 32.89 across all sample sets (Fig. 3). It is important to note that there was a wide range of variation among selected reference genes, and it shows that not a single reference gene expresses constantly across the 7th and 18th day of embryos of control broiler and improved Aseel in the present study (Fig. 3). Therefore, it is of pivotal importance to choose the most reliable reference gene for expression profiling of gene/s in different embryos of control broiler and improved Aseel.
For accurate gene expression, to select the best and most reliable reference gene and rank all the candidate reference genes according to their stability value, the most commonly used statistical programs were used i.e. geNorm, NormFinder, BestKeeper, Delta CT, and RefFinder [39,40,41,42,43,44,45,46,47,48]. These algorithms showed some differences in the stability ranking of stable reference genes, which may be due to the differences in each statistical program (Table 1). It is important to rank the stabilities of 18 genes and to confirm the number of reference genes necessary for accurate gene expression profiling under embryo initiation and developmental stages. The variation among the reference genes determined by geNorm is stability measure (M value) and pairwise comparison expression ratio and provides optima number of genes in a given experiment [49, 50]. According to geNorm stability criteria, the most stable genes in the 7th and 18th embryos of the control broiler and improved Aseel were PGK2 (0.38) and TFRC (0.38). NormFinder measures the reference gene stability by overall expression variation and across samples variation to reduce sensitivity towards co-regulation [49, 51]. According to NormFinder stability criteria, the most stable genes in the 7th and 18th embryos of the control broiler and improved Aseel were GAPDH (0.371) and TFRC (0.4). BestKeeper calculates the gene expression variation based on Ct values and also calculates the Pearson correlation coefficient by pairwise correlation analysis for all reference genes and found the stable genes. According to BestKeeper stability criteria, the most stable genes in the 7th and 18th embryos of the control broiler and improved Aseel were HSP70 (1.08) and PPP2CB (1.11). The Delta CT results supported geNorm and NormFinder results and it showed the best stable genes in 7th and 18th embryos of control broiler and improved Aseel i.e. TFRC (2.1) and PGK2 (2.13). RefFinder conclusive the calculations using the all above-mentioned algorithms and suggested the stable genes in 7th and 18th embryos of control broiler and improved Aseel i.e. TFRC (2.21) and PGK2 (2.85).
To overcome different software program limitations, the stability of candidate reference genes was determined based on the consensus ranking for gene expression normalization in 7th and 18th-day embryos of control broiler and improved Aseel. Our study identified the most stable genes and indicated that, TFRC and PGK2 for 7th and 18th-day embryos of control broiler and improved Aseel. Our observations further strengthen the necessity to analyze the stability of candidate reference genes as suitable references.
Differentially expressed transcripts during embryo development stages
To study the effect of muscle development, genome-wide expression analysis was carried out at muscle initiation (7th-day embryo) and muscle development (18th-day thigh muscle) stages (Fig. 1a, b). Labeled RNA was hybridized to Affymetrix GeneChip™ Chicken Genome Array. After statistical data analysis, transcripts with FDR adjusted P-value ≤ 0.01 and fold change ≥ 1 were considered as significantly DETs (Fig. 1c). The complete list of the DETs in improved Aseel during embryo development stages as compared to their respective control broiler samples is presented in Supporting Document 1 & 2. In total 19,022 transcripts (16,252 unigenes) which accounted for approximately 58% of the total transcripts present on the GeneChip™ Chicken Genome Array were showed differential expression in improved Aseel at various stages analyzed. The maximum number of transcripts (15,380, 46.9% of total DETs) showed differential expression on the 7th day of the embryo and the least number of transcripts (3642, 11.11% of total DETs) showed differential expression on the 18th day of the embryo. Commonly up-and down-regulated muscle responsive transcripts were identified among the embryo development stages to find out the degree of overlap (Fig. 1d, e). The maximum number of commonly up-regulated (9582) and down-regulated transcripts (4874) was observed in a 7th-day embryo. Less number of up-regulated (1557) and down-regulated (1157) transcripts were commonly differentially expressed on the 18th-day of the thigh muscle. Five commonly differentially expressed (545 up-regulated and 381 down-regulated) transcripts were identified among the embryo development stages, respectively (Fig. 1d, e; Supporting Document1&2).
Cluster analysis of differentially expressed transcripts
To profile the gene expression patterns in response to muscle slow growth during embryo development, the 19022 DETs were classified using hierarchical clustering. The expression patterns were separated into eight major clusters (I–VIII) based on tree branching (Fig. 2). Transcripts present in each stage within each cluster are presented in Supplementary Table S1. Among the eight major clusters, upregulated transcripts were enriched in cluster V, VI and VII and down-regulated transcripts were enriched in cluster II, III and IV.
Expression of muscle related genes
The difference in fast and slow growth is generally dependent on the combination of environment and genes. The embryos collected in this experiment are under the same growth environment. In this experiment, the DEGs related to the main cause of growth and development differences mainly include muscle system process, muscle tissue morphogenesis and muscle organ morphogenesis, etc. (Fig. 4). The genes enriched by these entries are mainly muscle-related genes such as TNNC1, TNNT2, MYL3, MYH7, and FBXO32. The contraction of skeletal muscle-related genes is TNNC1, TNNT2, MYL3, and MYH7. In animals, skeletal muscle contraction affects the growth traits like muscle protein, muscle fibre diameter, and muscle fibre density [52, 53]. In the corpus callosum, muscle is the most important component and the sarcomere is composed of thick and thin muscles. Myosin molecules are mainly involved in thick filament formation and the thin filament is a complex containing troponin, actin, and tropomyosin [54]. The skeletal muscle contraction is mainly based on the relative sliding of thick and thin filaments [55]. For controlling the poultry muscle, according to previous reports, the troponin family gene is an important candidate gene [52,56,57]. In this experiment, the DEGs obtained related to the troponin family and important components of skeletal muscle are troponin T type 3 (TNNT3), slow muscle troponin T (TNNT1), and troponin I type 1 (TNNI1). The myosin superfamily is a highly conserved family of proteins and is composed of a heavy chain, an alkaline light chain, and a light chain and that is widely present in eukaryotic cells [58]. In striated muscle, smooth muscle, and non-muscle, the myosin is involved in myofilaments as a class II. The MYH gene family is a key subunit in the myosin class II molecule and encoded the myosin heavy chain (MYHC) [59]. The studies revealed that MYH7 gene mutations could cause skeletal muscle disease or skeletal muscle disease with cardiomyopathy [60, 61]. In the chicken breast muscle, the MYH7 gene expression is high and it is a hypothesis that it has a regulatory role in muscle tissue growth and development [62]. MYL3 gene is primarily expressed in slow muscle and it is a member of the myosin light chain (MYL) gene family [63]. Inhibition of myosin light chain gene expression in cell lines showed myoblast proliferation [64]. The chicken embryonic leg muscles proteome analysis found that MYL3 protein is closely related to the regulation of muscle contraction and the long non-coding RNAs Inc00037615 and Inc00037619 together regulate the muscle development [65]. In this experiment, the DEGs obtained related to the myosin family are MYH7 and MYL3. In this study, five genes related to muscle contraction were screened i.e. TNNT3, TNNT1, TNNI1, MYL3, and MYH7. Transcriptome study showed that the expression of these five genes is high in improved Aseel slow growth compared with control broiler fast growth. Also, these genes simultaneously present in two significantly enriched pathways i.e. adrenergic signaling in cardiomyocyte and cardiac muscle contraction. Maybe due to the upregulation of these genes and down-regulation of the Adrenergic signaling in cardiomyocyte and cardiac muscle contraction, improved Aseel will grow slow compare to the control broiler. Previous studies showed that in animals under fasting, FBXO32 gene expression was significantly increased and muscles were degraded due to lack of food, and maybe it is associated with muscle atrophy [66, 67]. The FBXO32 gene expression was found in chicken’s leg muscle, heart, and chest muscle and plays an important role in the 7th-week growth of chickens [68]. In this experiment, the results of transcriptome data showed that the expression of FBXO32 and FBXO7 genes were significantly lower in slow-growth improved Aseel than that in the fast-growth control broiler, which was consistent with previous studies. The DEGs KEGG pathway enrichment analysis revealed three significantly enriched adrenergic signaling in cardiomyocytes, Cardiac muscle contraction, and tight junction signaling pathways. In cell junctions, the tight junction is an important component and it acts as a barrier for cells to pass ions and molecules, plasma membrane apical movement regulation, and basal proteins and lipids [69, 70]. In all eukaryotic cells, tight junction recognized as the actin cytoskeleton and it is involved in cell division, adhesion, movement, and phagocytosis and also found in chicken leg muscle transcriptome [71, 72]. The muscle growth epigenetic transcriptional regulators are differentially regulated in 7th such as protein arginine N-methyl transferase family (PRMT1, and PRMT3), histone lysine N-methyl transferases (EHMT1, and SETDB1), SWI/SNF chromatin-remodeling enzymes (SmarcB1, and SmarcaA4).
Muscle Development and Myostatin Signaling
The muscle development and differentiation-related genes like MYOD1, MYF6, MYF5, Myoz2, MAP2K6, MAP3K7, CAV1, CAV2, CAV3, HSP70, and NCF2 were differentially regulated in slow-growing improved Aseel then compared to fast-growing control broiler (Fig. 4). In muscle differentiation, MYOD1 promotes the muscle-specific gene expression and function together with MYF5 and MYOG [73]. MYOD1 combined with transient placeholder protein, prevents the binding of other transcription factors to DNA, and retains the inactive conformation of the DNA [74]. One of the key functions of MYOD is to stop the differentiated myocytes proliferation by enhancing the transcription of p21 and myogenin to remove cells from the cell cycle [75]. Altogether, up-regulation of MYOD1 involved in skeletal muscle phenotype establishment by regulation of precursor cell proliferation and promoting irreversible cell cycle arrest, facilitate differentiation and sarcomere assembly by activation of sarcomeric, and muscle-specific genes [76]. In this study, transcriptome data shows down-regulation of MYOD1 in the 7th day improved Aseel embryo, maybe due to this reason the muscle-specific gene proliferation is slow in improved Aseel. The myozenin is a α-actinin- and γ-filamin-binding protein of Z line skeletal muscle binds to calcineurin, and involved in skeletal muscle myocyte differentiation [77, 78]. Therefore, the up-regulation of MYOZ2 and MYOZ3 genes in muscle tissues suggested that they are involved in muscle growth and development and directly influence meat quality. Besides, MYOZ1 and MYOZ2 genes expressed in mice showed a significant reduction of calcineurin gene expression [79,80,81]. Our transcriptome study shows less expression of the MYOZ2 gene and high expression of regulator of calcineurin 1 gene in 7th day improved Aseel embryo, maybe this is the region the muscle development is slow in improved Aseel. Mitogen-activated protein kinases are major components of pathways controlling embryogenesis, cell differentiation, cell proliferation, cell death, muscle development, and response to environmental stress (Fig. 8) [82,83,84,85]. The MAP2K6 and MAP3K7 activate MAP kinase and nuclear factor-kappa β (NFκB) and play an important role in its signal transduction pathway, respectively [86, 87]. In a proteomic study, predicted the expression of MAP2K1, MAP2K2, and MAP2K4, MAP4K4 gene may inhibit the low feed efficiency phenotype [88]. In our transcriptome data, the mitogen-activated protein kinase family genes are up-regulated in the 7th-day embryo of improved Aseel. Our results correlating with these results and maybe in this region the improved Aseel has less feed efficiency and slow muscle growth.
In this study, the expression of caveolin family genes like CAV1, CAV2, and CAV3 was downregulated in the 7th-day embryo of improved Aseel. In cell signaling, the caveolin genes act as sub-cellular structures by assisting the attribute of hormonal signals after binding hormone to the target receptor on the cell surface. The CAV3 is acting as a muscle-specific isoform for the caveolin protein and mutations or different expressions of CAV3 can result in muscle myopathies [89, 90]. In pigs, the CAV3 expression was up-regulated during muscle hyperplasia and it may use as a genetic marker for meat production in swine [91]. In mouse, the high or low expression of CAV3 made muscle cells more susceptible to oxidative stress and reduced survival through PI(3)K/Akt signaling [92]. In the low FE PedM broiler phenotype, the higher expression of CAV3 contributed to higher oxidative stress and enhance muscle development [93]. In high FE breast muscle, the CAV1 protein is involved in insulin signaling [88]. Based on previous reports, down-regulation of caveolin family genes in the 7th-day embryo of improved Aseel, maybe the improved Aseel muscle development will be reduced. In high FE breast muscle, the up-regulation of HSP70 maintains muscle fibre integrity and enhances muscle regeneration and recovery from damage [94]. HSP70 is also responsible for correct folding and assembly of nuclear-encoded proteins an important chaperone for mitochondrial DNA-encoded proteins as components of the mitochondrial electron transport chain targeted for import into the mitochondria [95, 96]. In low and high FE phenotype, higher expression of HSP90 and HSPB2 was in response to oxidative stress, respectively [3, 88]. In our transcriptome study correlated with previous studies and we observed up-regulation of HSP70 and HSPB1 on the 7th-day and down-regulation of HSP90 on the 18th-day of improved Aseel embryo. Maybe the improved Aseel muscle is growing this region slowly compared to the control broiler. In NADPH oxidase 2 (NOX2), the NADPH/NADH oxidase is a critical component and encoded by neutrophil cytosolic factor 2 (NCF2) [97]. In muscle, superoxide was generated by NOX2 in the sarcoplasmic reticulum, and it is a major source of oxidative stress [97, 98]. In neutrophils, NADPH generated superoxide during phagocytosis. The nuclear factor erythroid 2-like 2 (NFE2L2) is a downstream target for NOX2 and activates genes that contain an antioxidant response element in their promoter regions [99, 100]. In high FE animals, predicted the NFE2L2 expression should be upregulated [5, 88]. In a high FE commercial broiler, the up-regulation of NCF2 was associated with muscle remodeling and hypertrophy [5]. In our transcriptome data, the NCF1, NOX1, and NOX3 were up-regulated on the 7th-day and NCF2 was down-regulated on the 18th-day of improved Aseel embryo. Maybe due to the differential expression of these genes, the improved Aseel is more resistant to oxidative stress, low FE, and slow growth.
Myostatin is a member of tumor growth factor β (TGF-β) and is known as growth/differentiation factor 8 (GDF-8) [101, 102]. In the myostatin (MSTN) signaling pathway, MSTN binds to its receptors ActIIA/ActIIB and activates ALK4 and ALK5 that phosphorylate Smad2/3 leads to its binding with Smad4 and translocation of the complex to the nucleus and where it blocks the transcription of genes responsible for the myogenesis [103,104,105,106,107] (Fig. 8). The myostatin is solely expressed in skeletal muscle during embryogenesis to control the differentiation and proliferation of the myoblasts [101]. However, in the adult stage, it is not only expressed in skeletal muscle but also expressed in other tissues like the heart, adipose tissue, mammary gland [101,108,109,110,111,112]. In turkey satellite cells, MSTN is a strong negative regulator for skeletal muscle growth, differentiation, and proliferation [101, 113]. The relation between MSTN and growth performance studies in broilers shows that MSTN is a polymeric gene in which different alleles of the gene can affect performance [114,115,116]. In PedM broiler, the FE differences may be due to different haplotypes of the MSTN gene [107]. Myostatin knockdown by RNAi shows muscle growth enhancement in transgenic sheep and chicken [117,118,20]. In the present study, MSTN has differentially regulated in the 7th-day improved Aseel embryo. Maybe differential regulation of myostatin is need for myoblasts differentiation and proliferation in initial embryogenesis. Follistatin (FSTN) regulates the MSTN by inhibiting or limit the MSTN activity. Follistatin-like 1 (FSTL1) is a glycoprotein and rich in cysteine (SPARC) family and comprises a secretion signal, a Follistatin- and a Kazal-like domain, two EF-hand domains, and a von Willebrand factor type C domain [119] (http://www.uniprot.org/uniprot/Q12841). In mouse, the FSTL1 were broadly expressed throughout the entire embryo and restricted most of the tissues at the end of gestation but in the adult mouse, it is highly expressed in heart, lung, and subcutaneous white adipose tissue [120, 121]. In this present study, FSTL1 was up-regulated and follistatin/kazal down-regulated in the 7th and 18th-day of improved Aseel embryo, respectively. Initial up-regulation and later down-regulation of FSTL1 may initiate muscle proliferation in the 7th-day embryo and 18th-day embryo slowdown the muscle development in improved Aseel. In humans, activin receptor type-1B (ACVR1B) or ALK4 is a protein that acts as a transducer of activin or activin-like ligand signals [122]. ACVR1B forms a complex with ACVR2A/ACVR2B and goes on to recruit the SMAD2/SMAD3 [123]. Besides, ACVR1B transduces nodal, GDF-1, and Vg1 signals combined with other coreceptor molecules like protein cripto [124]. Transforming Growth Factor-β (TGFβ) is a key player in cell proliferation, differentiation, and apoptosis and TGFβ receptors are single-pass serine/threonine kinase receptors and can be eminent by their structural and functional properties [125]. Due to the similar ligand-binding affinities, the transforming growth factor beta receptor I (TGFβR1)/ALK5 and TGFβR2 can be distinguished from each other by peptide mapping only. In mouse, the TGFβ1 mRNA/protein has been present in cartilage, endochondral, membrane bone, and skin and play a role in the growth and differentiation of these tissues [126] (Fig. 8). In the present study, activin A receptor type IB (ALK4) and transforming growth factor beta receptor II (TGFBR2) were up-regulated, and transforming growth factor beta receptor I (TGFBR1/ALK5) was down-regulated in the 7th-day embryo of improved Aseel. The differential expression of ALK4 and ALK5 may control the myostatin signaling pathway. The SMADs are important for regulating cell development and growth and they have structurally similar proteins and main signal transducers for TGFβ receptors (Fig. 8). The eight SMAD genes are distributed into three sub-types of SMADs, they are R-SMADs, Co-SMADs, and I-SMADs [127, 128]. The R-SMADs consist of Smad1, Smad2, Smad3, Smad5, and Smad8/9 and are involved in direct signaling from the TGFβ receptor [129, 130]. The Co-SMADs consist of the only SMAD4 and working jointly with R-SMADs to recruit co-regulators to the complex [131]. R/Co-SMADs are primarily located in the cytoplasm, following TGFβ signaling later accumulate in the nucleus, where they can bind to DNA and regulate transcription. I-SMADs consist of SMAD6 and SMAD7 and are predominantly found in the nucleus, and they can act as direct transcriptional regulators. SMAD6 has specifically associated BMP signaling and SMAD7 is a TGFβ signal inhibitor and suppresses the activity of R-SMADs [132,133,134]. In the present transcriptome study, the SMAD family member 1 (SMAD1), SMAD specific E3 ubiquitin protein ligase 2, SMAD family member 3 (SMAD3), SMAD family member 5 (SMAD5), TGF-beta signal pathway antagonist Smad7 (SMAD7B) up-regulated in 7th day of improved Aseel embryo and they may control the myostatin signaling pathway in improved Aseel embryonic stage. Summarizes the initial steps in the MSTN signaling pathway in the present study that would potentially exert a negative effect on muscle differentiation and proliferation in the slow-growing improved Aseel.
Energy Sensing
In humans and animals, the adenosine monophosphate-activated protein kinase (AMPK) gene regulates diverse biological functions [135]. The mammalian 5′ AMPK gene has two α subunits i.e. AMPKα1 and AMPKα2 that are encoded by Prkaa1 and Prkaa2 gene, respectively. The knockout mouse clearly demonstrated that the AMPKα2 controls the homeostasis in skeletal muscle [136, 137]. Also observed reduction of fibre numbers (~25%) and sizes (~20%) in the soleus muscle of AMPKα1 knockout mice [138]. However, in AMPKα2 knockout mice, both fibre size and muscle mass were significantly increased, while the muscle fibre number has remained similar to WT animals. The muscle mass reduced and increased differentially expressed alternative polyadenylation sites (DE-APSs) were down-regulated in AMPKα1 knockout mice, but up-regulated in AMPKα2 knockout mice, respectively [46]. The five genes i.e. carbonic anhydrase 3 (Car3), myosin light chain kinase family, member 4 (Mylk4), nebulin (Neb), obscurin (Obscn), and phosphofructokinase, muscle (Pfm) are utilized different APSs showed potential effects on muscle function [46]. The high FE phenotype birds show up-regulation of both AMPKα1 and AMPKα2 [3]. In low energy level conditions the AMPK genes expression increases for ATP production by inhibiting the ATP consuming pathways like fatty acid synthesis, protein synthesis, and gluconeogenesis and stimulates the ATP producing pathways like mitochondrial biogenesis and oxidative phosphorylation, glycolysis, and lipolysis [139,140,141]. In the present study, the AMPK genes like 5'-AMP-activated protein kinase gamma-1 non-catalytic subunit variant 1 (PRKAG1), protein kinase cAMP-dependent regulatory type I alpha (tissue specific extinguisher 1) (PRKAR1A), protein kinase AMP-activated beta 2 non-catalytic subunit (PRKAB2), protein kinase AMP-activated gamma 2 non-catalytic subunit (PRKAG2), carbonic anhydrase XIII, myosin light chain kinase (MYLK), atrial/embryonic alkali myosin light chain, are down-regulated in the 7th-day of improved Aseel embryo, maybe due to the down-regulation of energy-producing pathways, the improved Aseel muscle will grow slowly compare to control broiler. Curiously, creatine kinase (muscle isoform, CK-M) and creatine kinase (brain isoform, CK-B) were up-regulated in high and low FE phenotype, respectively [88,142,143]. The reason for this discrepancy may be the high FE phenotype broiler breast muscle has enhanced capabilities for mitochondrial oxidative phosphorylation as well as creatine and phosphorylated creatine shuttle in and out of mitochondria [143]. In the present study, the creatine kinase muscle (CKM), creatine kinase mitochondrial 1A (CKMT1A), creatine kinase brain (CKB), creatine kinase, mitochondrial 2 (sarcomeric) (CKMT2) genes were up-regulated in the 7th-day embryo of improved Aseel. In skeletal muscle, nitric oxide was synthesized by nitric oxide synthases and it is regulated key homeostatic mechanisms like mitochondrial bioenergetics, network remodeling, mitochondrial unfolded protein response (UPRmt), and autophagy (Fig. 4; Fig. 5). In mice, nitric oxide synthase deficiency inhibits the Akt-mammalian target of the rapamycin pathway and dysregulated the Akt-FoxO3-mitochondrial E3 ubiquitin-protein ligase 1 (Mul-1) axis [144]. Thus, the mitochondrial biogenesis and body energy balance were controlled by the nitric oxide-cGMP-dependent pathway [145]. In detail, the inhibition of nNOS/NO/cGMP-dependent-protein kinases enhanced the FoxO3 transcriptional activity and increased the Mul-1 expression. The absence of the nitric oxide synthases significantly impaired muscle fibre growth with muscle force, decreased resistance to fatigue, and degeneration/damage post-exercise. In our study, nitric oxide synthase 2 was up-regulated and, cGMP-dependent protein kinase type I, and FOXO1 was down-regulated in the 7th-day of improved Aseel embryo, maybe this is the region, the improved Aseel muscle strength was high and they are more energetic compared to the control broiler.
The comparative muscle transcriptome analysis between high and less pH chicken showed that most of the glycolysis pathway genes are up-regulated in the less pH chicken [146]. The previous study shows Aseel and broiler chicken’s meat do not have any significant pH variation but the heavier bird’s meat had a significantly higher pH [147]. In this study, glycolysis metabolism-related genes are differentially regulated on the 7th-day of improved Aseel embryo. The glucose-6-phosphate isomerase, fructose bisphosphate aldolase, phosphoglycerate kinase, and enolase were down-regulated and GAPDH, phosphoglycerate mutase, and pyruvate kinase were up-regulated in the 7th-day embryo of improved Aseel. Fructose bisphosphate aldolase is a key enzyme in glycolysis as well as gluconeogenesis and involved in the stress-response pathway during hypoxia [148]. The high pH chickens have increased oxidative stress, maybe the higher expression of fructose bisphosphate aldolase is linked to its function in the stress-response pathway rather than to its role in ATP biosynthesis [149]. In our study, down-regulation of fructose bisphosphate aldolase enhanced the ATP synthesis, maybe this is the region improved Aseel birds have more energy than control broiler. Noteworthy, the up-regulation of glycolysis pathway genes are increased the pyruvate levels and entering the citric acid cycle, and thus, higher levels of ATP are produced in improved Aseel. The protein phosphatase-1 regulatory subunit 3A (PPP1R3A) binds glycogen with high affinity, and activates glycogen synthase (GYS), and inhibits glycogen phosphorylase kinase (PHK) by dephosphorylation through the protein phosphatase-1 catalytic (PPP1C) subunit. In this study, the glycogen metabolism genes i.e. protein phosphatase-1 regulatory subunit 2 (PPP1R2), glycogenin 1, glycogen phosphorylase, and protein phosphatase-1 catalytic subunit beta (PPP1CB) were down-regulated in the 7th-day embryo of improved Aseel, maybe this is the region that the improved Aseel muscle have more glycogen. The AMP-activated protein kinase (AMPK) complex is another key regulator of glycogen turnover and it consists of one α catalytic and two non-catalytic subunits, β, and γ. The β subunit binds to glycogen along with α and γ subunits and forms a heterotrimeric AMPK complex. In the muscle cell, the γ subunits of the AMPK complex act as energy sensors and binding to AMP and ATP [150]. In our study, AMP-activated protein kinase beta 2 non-catalytic subunit (PRKAB2), cAMP-dependent protein kinase regulatory type I alpha (tissue-specific extinguisher 1) (PRKAR1A), AMP-activated protein kinase gamma 2 non-catalytic subunit (PRKAG2) were down-regulated, and 5'-AMP-activated protein kinase gamma-1 non-catalytic subunit variant 1 (PRKAG1), AMP-activated protein kinase gamma 3 non-catalytic subunit (PRKAG3), AMP-activated protein kinase alpha 2 catalytic subunit (PRKAA2) were up-regulated. The down-regulation of β subunits and up-regulation of α and γ subunits may balance the glycogen accumulation and increase the ATP molecules for energy production in improved Aseel muscle, maybe this is the region the improved Aseel has more strong than the control broiler. Apart from these, several other genes indirectly influence glycogen storage in muscle. The phosphodiesterase 3B (PDE3B) gene activated by insulin and induce antiglycogenolytic effects and the mitochondrial creatine kinase (CKMT2) transfers the high-energy phosphate from mitochondria to creatine. In our study, phosphodiesterase 3A (PDE3A), phosphodiesterase 4D, phosphodiesterase 8A, mitochondrial creatine kinase 2 (CKMT2), mitochondrial creatine kinase 1A (CKMT1A), mitochondrial creatine muscle (CKM) were up-regulated in the 7th-day of embryo improved Aseel, maybe this is the region, the higher expression of these genes in muscle, improved Aseel birds are more energetic compared to control broiler. To produce energy and compensate for the lack of energy due to carbohydrates and glycolysis, the high pH chickens muscle asks for more intense oxidative pathways, such as lipid β-oxidation and ketogenic amino acid degradation [149]. In the high pH muscle line, the 3-hydroxymethyl-3-methylglutaryl-CoA lyase (HMGCL) catalysis the final step of leucine metabolism and ketone metabolism, acetyl-CoA acetyltransferase-2 (ACAT2) involved in β-oxidation or degradation of ketogenic amino acids, and nudix hydrolases (NUDT7, NUDT12, NUDT19) hydrolyse a nucleoside di and triphosphates, dinucleoside and diphosphoinositol polyphosphates, nucleotide sugars and RNA caps, were up-regulated. In the present study, 3-hydroxymethyl-3-methylglutaryl-CoA lyase like-1 (HMGCLL1), acetyl-CoA acetyltransferase 2 (ACAT2), nudix type motif 7 (NUDT7), and nudix type motif 21 (NUDT21) were down-regulated and carnitine/palmitoyl-transferase 1 (CPT1) were up-regulated in the 7th-day embryo of improved Aseel (Fig. 6). They may regulate the β-oxidation in peroxisomes as well as mitochondria, this is the region may be fats required for initial embryo development and excess fats involved in β-oxidation and finally provide the energy for embryo development.
Protein Synthesis
To promote cell growth, the mTORC1 complex increases protein synthesis and lipid metabolism, and autophagy inhibition, and regulates the transcription of several genes [151]. In high FE phenotype, the cDNA microarray data shows higher expression of mTORC1 [152, 153]. The mTORC1 complex has two major components i.e. mTOR and RAPTOR [154]. The RAPTOR and mTOR were up and down-regulated in high and low FE birds, respectively, and the up-regulation of RAPTOR could be a positive effect on protein synthesis [154]. In low FE phenotype, the PRKAR1A and GLUT-8 were up-regulated. The p70S6k and eukaryotic translation initiation factor 4E (EIF4E) are the key downstream targets for mTORC1 and involved in enhancing the protein synthesis. In low FE birds, the expression o p70S6k was higher [155]. The muscle tissue of RNAseq transcriptomic data showed higher expression of eukaryotic initiation, elongation, and translation genes in high FE compared to low FE PedM phenotype [142, 156]. In the present study, the late endosomal/lysosomal adaptor MAPK and MTOR activator 3 (LAMTOR3), protein kinase, cAMP-dependent, regulatory, type I, alpha (tissue specific extinguisher 1) (PRKAR1A), solute carrier family 2 (facilitated glucose transporter) member 8 (SLC2A8)/ GLUT8/ GLUTX1, solute carrier family 2 (facilitated glucose transporter), member 3 (SLC2A3), ribosomal protein S6 kinase, 90kDa, polypeptide 1 (RPS6KA1), ribosomal protein S6 kinase, 90kDa, polypeptide 3, ribosomal protein S6 kinase, 52kDa, polypeptide 1, ribosomal protein S6 kinase, 90kDa, polypeptide 6, ribosomal protein S6 kinase-like 1, KIAA1328, KIAA1324, and eukaryotic translation initiation factor 4E binding protein 1 gene were down-regulated in the 7th-day embryo of improved Aseel compared to control broiler (Fig. 4). May be due to the down-regulation of mTORC1 complex and ribosomal machinery genes, the protein synthesis for muscle growth is less in improved Aseel compared to control broiler.
Insulin Signaling
In chicken, the SHC1 is only activated by nutritional changes and suggesting that insulin signaling in chickens have a tissue-specific manner [157, 158]. When insulin binds to the insulin receptor, both IRS-1 and SHC1 are activated by a phosphoinositide-3 kinase (PI3K) mediated tyrosine phosphorylation activity [3]. The skeletal myoblast is mainly differentiated by two key modulators i.e. insulin-like growth factor 1 (IGF1) and fibroblast growth factor 2 (FGF2) [159, 160]. In L6 and C2C12 myoblasts, a high concentration of IGFs reduces their differentiation, whereas a low concentration enhances their differentiation [161, 162]. The IGF binding proteins (IGFBP-1 to IGFBP-6) have highly conserved regions and binds with high affinity to IGF-1 and IGF-2. In the extracellular matrix, IGFBP3 may regulate the interaction of IGFs and it is present in rat soleus muscle (type I muscle fibre) [163, 164]. In humans, IGFBP3 was also playing a role in myoblasts differentiation [165]. The fibroblast growth factor 2 (FGF2), transforming growth factor beta (TGFb), and oncogenic Ras also inhibit the skeletal myoblast differentiation [160]. In 23A2 myoblast cell lines, showing the inhibition of 23A2 myoblasts differentiation by IGF1 and FGF2 by stimulating the signaling through mitogen-activated protein kinase (MAPK) kinase (MEK) to MAPK [166]. In our study IGF2, FGF2, IGFBP2, IGF2BP1, IRS-1, IRS-2, INSIG1, PIK3CA, PIK3CD, TGFb, and Ras oncogene family genes are up-regulated in the 7th-day embryo of improved Aseel (Fig. 7). Maybe this is also one of the regions for improved Aseel muscle is differentiated slowly when compared to the control broiler.
Expression of plumage development genes
In vertebrate coloration, melanin pigmentation is an important component and regulated by strong genetic control [167, 168]. In chicken, the plumage coloration development is extremely complex and can be categorised as structural or pigment-based [169]. For animal colouration, melanin is a common component, synthesized in melanocytes, and deposited in various organs as granules [170]. Different pigment patterns are formed based on the presence of melanocytes modulating, arrangement or differentiation, and associated with a series of functional genes [171, 172]. The melanogenesis genes such as HOX, CHAC1, GPX3, BMP5, PITX2, RGN, MITF, TYR, KIT, OCA2, ASIP, MCIR, KITLG, IRF4, SLC24A4, SLC45A2, EDN, TYRP1, and TYRP2 are involved in melanin pigmentation [173,174,175,176,177]. The homeobox (HOX) genes are transcription factors and involved in skin appendages development including hair follicles and feathers [178,179,180,181,182,183] (Fig. 8). In black-bone chickens, four HOX genes i.e. HOXB9, HOXC8, HOXA9, and HOXC9 were identified for melanin pigmentation [172]. The Wnt signaling is essential for skin organogenesis and its appendages like hairs, feathers, and scales, melanocyte development, and differentiation [184,185,186,187]. The HOXB9 is identified as a target gene for Wnt signaling and HOXC8 is expressed in the first stage of feather morphogenesis like dorsal dermal and epidermal cells [188, 189]. In this study, HOXA2, HOXA9, HOXB3, HOXB5, HOXB7, HOXB8, HOXB9, HOXC11, HOXD1, and HOXD3 are up-regulated in the 7th-day improved Aseel embryo, maybe this is the region the improved Aseel plumage has multiple colors.
In animals, melanogenesis is regulated by GSH and it is closely associated with melanin deposition in the skin of humans and other mammals [190,191,192,193,194]. The low and high level of GSH indicates eumelanin-type pigmentation and phaeomelanin-producing melanocytes found in skin, respectively [195]. Two feather melanin pigmentation genes were identified in black-bone chickens such as ChaC glutathione-specific gamma-glutamylcyclotransferase 1 (CHAC1) and glutathione peroxidase 3 (GPX3) [172]. The CHAC1 cleavage GSH into 5-oxoproline and Cys-Gly dipeptide and GSH over-expressed mammalian cells caused GSH depletion [196, 197]. Hence, CHAC1 expression is associated with GSH metabolism and plays an important role in the melanogenesis process. In eumelanin and phaeomelanin synthesis, the hydrogen peroxide reduced by GSH-dependent peroxidase and GPX3 belongs to the GSH peroxidase family and catalyzes the GSH to glutathione disulphide (GSSG) [194, 198]. The melanoma cells pigmentation regulated by GSH levels, glutathione peroxidase, and glutathione reductase suggesting that GSH mediated redox process plays an important role in melanogenesis regulation [199]. Hence, the expression of GPX3 plays an active role in chicken feather melanogenesis. In this study, CHAC1, CHAC2, gamma-glutamylcyclotransferase (GGCT), and GPX8 were down-regulated on the 7th-day of improved Aseel embryo, maybe this is the region the improved Aseel has multiple colors on their feathers. In black-bone chicken, two pathways i.e. TGF-β signaling pathway, and ascorbate and aldarate metabolism were identified for plumage melanogenesis [172]. The TGF-β involved in the regulation of chicken retinal pigment epithelial cell proliferation and melanin synthesis [200]. The BMP5 and PITX2 genes are involved in the TGF-β signaling pathway and play a role in chicken melanin synthesis, and the BMP5 and PITX2 were highly expressed in white feather and black feather bulbs, respectively [200]. The BMP3 gene was highly expressed in embryonic and post-embryonic stages of control layer when compare to broiler chicken and BMP4 gene was differentially expressed in juvenile stages of broiler and layer chicken, respectively [201, 202]. The regucalcin (RGN) is a calcium-binding protein involved in the ascorbate and aldarate metabolism pathway and plays a crucial role in intracellular calcium homeostasis maintains [203]. In this study, transforming growth factor beta receptor II (TGFBR2), BMP1, BMP1A, BMP4, BMP7, BMPR1A, BMPR2, PITX3 up-regulated and RGN was down-regulated in the 7th-day embryo of improved Aseel, maybe this is the region the improved Aseel plumage has multiple colors.
In melanin synthesis, TYR is a rate-limiting enzyme and involved in different oxidative steps [204, 205]. In black vs. white skin chicken, the TYR is highly expressed and it is consistent with sheep coat color studies [173,206,207]. In black-coated vs. white-coated sheep, the TYRP1 gene was highly expressed [207]. KIT is a receptor tyrosine kinase, the mutation in KIT showed piebaldism and auburn hair color in humans and it plays an important role in UVB-induced melanogenesis in the epidermis, and inhibition of KIT expression may result in the lighten human skin color [208, 209]. In black skin chicken, KIT is highly expressed and black skin color is due to increased melanin compared to the white skin color [173]. In melanocytes development, microphthalmia-associated transcription factor (MITF) is playing a role and mutations in the MITF gene is responsible for Japanese quail and chicken plumage color and it is supported to alternative splicing of MITF gene in the skin of sheep [210, 211]. In ducks, the TYR and MITF expression may involve in the formation of black and white plumage [212]. Melanocortin-1 receptor (MC1R) is binding to melanocyte stimulating hormone (MSH) and initiates the melanogenesis cascade and regulates the mammalian skin pigmentation and hair color [213,214,215,216]. The agouti signaling protein (ASIP) is responsible for the skin color of both white and black-coated sheep and mutations in ASIP cause black and tan pigmentation phenotype in pigs [206, 217]. The ASIP binds to MC1R and reduced the MITF and TYR gene expression and finally, the phaeomelanin would be reduced in epidermal tissues. In black-skinned chickens, the expression of ASIP is higher than compared to the white-skinned chicken and it can suppress the MC1R gene expression in black-skinned chickens [173]. The Oculocutaneous albinism type 2 (OCA2) is a common skin pigmentation disorder caused by a mutation in the OCA gene. In black chickens, the OCA2 was up-regulated and it may be related to black skin color [173]. In chickens, the endothelins (EDN1, EDN2, and EDN3) and their receptors (EDNRA, EDNRB, and EDNRB2) are involved in the regulation of pigmentation and plumage [218]. The expression of EDNRB2 was significantly different between adult black and non-black chicken [219]. In this study, TYRP1, KITLG, MITF, MC1R, AGRP, YRK, and P56LCK were up-regulated and EDNRA and EDN1 were down-regulated in the 7th-day of improved Aseel embryo and this is the region the improved Aseel plumage has multiple colors.
Expression of genes related to egg production
In chicken, the reproductive system is regulated by hypothalamic-pituitary-ovarian (HPO) axis hormones, while ovulation, the GnRH-I triggers the pituitary gland to release FSH and LH, and stimulates the secretion of estradiol and progesterone in the ovary [220]. Several reproductive hormone regulation genes were identified between high and low egg production chickens, such as HADH, HMGCR, RAB11FIP1, and FAM3D [221]. In the pituitary gland, lipid metabolic process, prolactin and, MAPK signaling pathway genes i.e. HMGCR, HMGCS1, NFKB1, VAV3, SOS1, IL1R1, MEF2C, STK3 were highly expressed. In chicken, the anterior pituitary gland synthesized and released prolactin and involved in reproduction, laying eggs, metabolism, development, and hypothalamic-pituitary-gonadal axis regulation [222, 223]. In chicken, the HMGCR gene variants (G-789-A, C-937-G, and A-2316-C) and high and low concentrations of VLDL showed higher and lower egg production, respectively [224]. In laying chickens, the APOB is a primary organizing protein for chylomicrons and VLDL and responsible for the transport of lipoprotein and circulating in the plasma, and stored into the oocytes to form an egg yolk [225, 226]. In our study, GNRHR, HADHB, HMGCS1, HMGCS2, RAB11FIP2, RAB11FIP3, RAB11FIP4, NFKB2, VAV2, SOS2, MEF2D, STK3, PRL, PRLR, PRLH, and PRLHR2 genes were up-regulated, and FSHR, VAV3, IL1RL1, and IL1RAPL2 genes are down-regulated, and family with sequence similarity genes and apolipoprotein B were differentially regulated in the 7th-day embryo of improved Aseel. Maybe this is the region the improved Aseel has less egg production than the commercial chicken. In avian species, the genes SPP1, BPIFB3, and EDIL3 are mainly involved in egg and oviposition, development of reproduction system, and vesicle-mediated eggshell calcication, respectively [227,228,229,230]. In this study, secreted phosphoprotein 1 (SPP1) and secreted phosphoprotein 2 (SPP2) genes are down-regulated, and EDIL3 is up-regulated in the 7th-day of improved Aseel embryo, due to this, egg and oviposition are less and eggshell calcication is more in improved Aseel. In nandan-yao chicken, FN1, FGF7, SOX2, ALDOB, HSPA2 genes are expressed in the ovary, and UQCRH, COX5A, FN1, TGFB, ACTN1 genes are expressed in the uterus and involved in egg production [231]. In this study, FN1, FNDC3A, FGFR1, FGFR3, FRS3, FRS2, FGFR2, FGFRL1, FGF8, FGF18, FGF3, FGF12, SOX2, SOX3, SOX4, SOX5, SOX7, SOX8, SOX9, SOX11, SOX17, HSPA2, HSPA4, COX1, COX2, COX3, TGFB4, and ACTN1 were up-regulated and ALDOB, HSPA5, HSPA8, HSPA9, HSP12A, UQCRFS1, UQCRB, were down-regulated in the 7th-day improved Aseel embryo. The differential expression of the ovary and uterus related genes are differentially expressed in the 7th-day of improved Aseel embryo, due to this region the egg production is less in improved Aseel. The DEGs related to the pituitary gland between high and low egg production chickens are mainly involved in mTOR and Jak-STAT signaling pathways [221]. In mouse, the mTOR signaling pathway will regulate the granulosa cell proliferation and differentiation [232]. In this study, the mTOR and Jak-STAT signaling pathways were up-regulated in the 7th-day improved Aseel embryo.
In high egg production chickens, several embryonic development genes are up-regulated such as GDNF, HOXD9, MEF2C, STK3, CLRN1, IRX5, LBX1, CSNK1A1, LGR5, PRDM15, and DAB2IP [221]. In this study, the GDNF, HOXA2, HOXB3, HOXB5, HOXB7, HOXB8, MEF2D, STK3, STK16, STK25, STK32B, IRX1, IRX2, IRX5, IRX6, LBX1, LBX3, PACSIN2, RGR, PRDM4, PRDM8, and DAB2IP genes are up-regulated in the 7th-day of the embryo, these genes are involved in embryo development. In the ovary, the tryptophan metabolism and PI3K-Akt signaling pathways were enriched and they are important for egg production [221] (Fig. 8). In stressful conditions, peripheral and brain tryptophan levels can alter by stimulating the immune system and activating the hypothalamic-pituitary-adrenal axis [233, 234]. In this study, tryptophan metabolism was down-regulated in the 7th-day of the embryo and up-regulated in the 18th-day thigh muscle, this is the region the improved Aseel has less egg production. In high egg productions, the hypothalamus genes are highly expressed such as EXFABP, SNRNP25, FAM114A1, and SIX1 [221]. In the hypothalamus, nerve growth factor response, lipid metabolism, canonical Wnt signaling pathway genes were highly expressed i.e. SIX1, RPS15, IGFBP7 thus play a role in chicken egg production. In laying hens, the dietary corticosterone treatment shows low levels of extracellular fatty acid-binding protein (EXFABP) and suggested that the egg white proteins synthesis and secretion may effect by environmental stress [235]. Many studies reported that ovarian follicular development is stimulated by IGFBPs and plays a role in the ovary to FSH action [236, 237]. In chicken adipose tissue, the lipid metabolism gene like insulin-like growth factor binding protein 7 (IGFBP7) was highly expressed and it is correlated with egg production [238, 239]. In this study, FAM114A, FAM116A, FAM116B, FAM117A, FAM117B, SIX1, SIX2, RPS13, RPS24, IGFBP2, IGFBP3, and IGFBP5 genes are up-regulated and FABP1, FABP2, FABP3, FABP5, SNRPB, SNRPB2, IGFBP1, and IGFBP7 genes are down-regulated in the 7th-day embryo of improved Aseel, these differentially expressed genes may cause less egg production in improved Aseel (Fig. 7). The cuticle or organic matrix of the eggshell-related genes i.e. MEPE, BPIFB3, RARRES1, and WAP are highly expressed in oviposition [240,241,242]. In this study, RARA, RARB, POSTN, CDH4, CDH13, CDH8, CDH11, and CDH20 were up-regulated, and RARRES1, and CDH1, were down-regulated in the 7th-day improved Aseel embryo. Due to the differential expression of these genes may be the improved Aseel eggshell thickness is more than commercial laying eggs.
The mitochondrial oxidative phosphorylation, active transport, and energy-metabolism related genes such as NADH dehydrogenase, ND4, ND1, ND2, ND5, ACTB, GAPDH, ATP6, and ATP1A are required for a large amount of energy, and active secretion of proteins and minerals [243]. A recent report shows differential expression of these genes like MEPE, COX1, COX3, COX2, BPIFB3, Cytochrome b, ATP6, ND5, ATP1A1, ND4, ND2, EIF4A2, UBB, Novel mitochondrial gene, IGLL1, HSPA8, RASD1 in GNRH1 vs. AVT study [244]. In this study, ND1, ND2, ND3, ND4, ND4L, ND5, ND6, GAPDH, ATP6, ATP1A2, COX1, COX2, COX3, and CYTB were up-regulated, and NDUFAF1, NDUFA4, NDUFA5, NDUFA9, NDUFA10, NDUFAF4, ATP1A1, EIF4A2, COX15, COX19, COX20, CYB5A, CYB5B, and CYB5R2 were down-regulated in the 7th-day of the improved Aseel embryo. Most of the energy metabolism genes are up-regulated and may be the region improved Aseel is stronger than commercial birds.