
==== Front
Poult Sci
Poult Sci
Poultry Science
0032-5791
1525-3171
Elsevier

S0032-5791(24)00753-3
10.1016/j.psj.2024.104174
104174
GENETICS AND MOLECULAR BIOLOGY
Dynamic changes in insulin-like growth factor binding protein expression occur between embryonic and early post-hatch development in broiler chickens
Vaccaro Lauren A. 1
Herring Kyle
Wilson Abigail
England Emma
Smith Addison L.
Ellestad Laura E. lellestad@uga.edu
2
Department of Poultry Science, University of Georgia, Athens, GA 30602, USA
2 Corresponding author: lellestad@uga.edu
1 Present affiliation: Animal Science Department, University of Findlay, Findlay OH 45840, USA

05 8 2024
11 2024
05 8 2024
103 11 10417426 2 2024
31 7 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Somatotropic gene expression has been altered by genetic selection, and developmental changes in insulin-like growth factor (IGF) and IGF binding protein (IGFBP) expression may contribute to rapid growth and muscle accretion in commercial broilers. The objective of this study was to evaluate changes in somatotropic axis activity between embryonic day (e) 12 and post-hatch day (d) 21. Liver and breast muscle (pectoralis major) were collected to measure gene expression, and blood was collected post-hatch to measure circulating IGFs. Liver IGF1 rose rapidly post-hatch and, in muscle, IGF1 exhibited a dynamic expression pattern. Levels decreased from e14 to e20, returned to e14 levels at d3, decreased again at d10, and stayed low thereafter. In both tissues, mRNA levels of several IGFBPs changed between embryogenesis and post-hatch. Liver IGFBP2 increased between e12 and e20, returned to e12 levels on d1, and remained low. Conversely, liver IGFBP4 expression was greater post-hatch than during embryogenesis. Expression of select IGFBPs was depressed in liver during the peri-hatch period. Liver IGFBP1, IGFBP3, IGFBP5, and IGFBP7 mRNA levels all decreased around this time and returned to embryonic levels by d3. In breast muscle, expression of both IGFBP2 and IGFBP4 was reduced after hatch. Circulating insulin-like growth factor IGF1 and IGF2 levels did not change between hatch and d21. These data suggest that post-hatch IGF effects are likely modulated by target tissue IGFR1 and IGFBP expression rather than changes in circulating hormone levels, with promotion or restriction of IGF-receptor binding regulating growth. Downregulation of several IGFBPs synthesized in the liver may facilitate the metabolic transition from utilizing yolk lipids to dietary carbohydrates. Several IGFBPs produced in breast muscle appear to have growth-promotive effects during embryogenesis but restrict growth of this tissue after hatch, as their post-hatch downregulation could facilitate local IGF signaling. These developmental gene expression patterns suggest that somatotropic hormonal signaling regulating growth and muscle accretion might be controlled through differential actions of IGFBPs and provide a basis for future functional studies.

Key words

broiler
liver
breast muscle
insulin-like growth factor
insulin-like growth factor binding protein
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pmcINTRODUCTION

Modern commercial broiler chickens are capable of rapid growth and muscle accretion during post-hatch development (Bartov, 1982; Goddard et al., 1988; Havenstein et al., 1994; Berrong and Washburn, 1998; Havenstein et al., 2003; Collins et al., 2014). Though the molecular mechanisms behind these traits have yet to be fully elucidated, they are associated with highly conserved endocrine systems known to regulate vertebrate growth and metabolism. One of these systems is the somatotropic axis, which has been shown to be influenced by genetic selection of commercial broilers (Vaccaro et al., 2022a) and is known to induce growth via cellular proliferation and protein accretion in muscle and bone (Clark and Robinson, 1996; Levine, 2012; Gahete et al., 2016). Many of these processes are indirectly induced by growth hormone (GH) binding to the GH receptor (GHR) and increasing insulin-like growth factor (IGF) 1 and IGF2 production and signaling. Circulating IGFs are synthesized in the liver (Kajimoto and Rotwein, 1989; Stewart and Rotwein, 1996; Dewil et al., 1999; Woelfle et al., 2005) and influence growth by downregulating apoptosis and increasing cellular proliferation after binding the type I IGF receptor (IGFR1) (Girbau et al., 1989; Duclos and Goddard, 1990; D'Costa et al., 1998).

In mammals, IGF1 contributes to growth and body weight (Stratikopoulos et al., 2008), and a lack of IGF1 is typically associated with dwarfism (Yakar et al., 2002). However, direct or correlative relationships between IGF signaling and growth in birds is less clear. Though exogenous administration of human recombinant IGF1, but not IGF2, has been shown to moderately increase average daily gain, feed efficiency, and lean tissue growth in young female chickens (Tomas et al., 1998), these effects are not consistently observed. Daily injection (McGuinness and Cogburn, 1991) or continuous infusion (Huybrechts et al., 1992) of human recombinant IGF1 over a 2-wk period did not influence body weight or feed efficiency in juvenile male or female broilers, respectively, but did decrease abdominal fat in females at the highest dose administered. Similarly, chronic infusion of chicken IGF1 did not influence growth rate but did reduce skeletal muscle weight in 3-wk-old male broilers (Czerwinski et al., 1998). While elevated levels of IGF1 mRNA have been demonstrated in chickens selected for high body weight when compared those selected for low body weight (Beccavin et al., 2001) and higher plasma IGF1 and IGF2 levels were also observed in broilers selected for high juvenile body weight (Scanes et al., 1989), another study found no differences in hepatic IGF1 mRNA or circulating IGF1 between broilers of differing growth potential through 42 days of age (Giachetto et al., 2004). Circulating IGF1 and IGF2 concentrations and levels of mRNA for these proteins in liver also did not differ between modern commercial Ross 308 and legacy Athens Canadian Random Bred (ACRB) broilers, despite Ross 308 chickens having significantly greater body weights from late embryonic development onwards (Vaccaro et al., 2022a; Vaccaro et al., 2022b).

IGF-binding proteins (IGFBPs) modulate IGF activity by influencing its stability in circulation, receptor affinity, and local tissue-specific effects (Baxter, 1991; Kelley et al., 2002; Kim, 2010; Baxter, 2023). There are seven IGFBPs in mammals (IGFBP1 – 7), though IGFBP7 exhibits a lower affinity for IGFs than the others (Kim et al., 1997); avian species lack IGFBP6 but do have genes for the other family members (Daza et al., 2011). Several tissue-specific and context-dependent effects of IGFBPs on IGF action have been observed in mammalian and some avian models, and IGFBPs have also been shown to have effects independent of the IGFs in mammalian cells. They can prevent binding of IGFs to their receptor, as is the case with IGFBP1 that blocks IGF1-induced protein synthesis in human skeletal muscle (Frost and Lang, 1999) and IGFBP2 and IGFBP4 that prevent long bone growth stimulated by IGF in mice and chicken embryos (Mohan et al., 1995; Fisher et al., 2005). They can also have differential effects depending on which IGF they are bound to. For example, IGFBP5 enhances rat myoblast differentiation into myotubes when bound to IGF1 but inhibits this process when bound to IGF2 (Ewton et al., 1998). Demonstrated IGF-independent effects of IGFBPs include upregulation of apoptosis in sarcoma and breast cancer cells by IGFBP2 (Schutt et al., 2004; Klaus et al., 2006) and stimulation of mouse osteoblast cell proliferation by IGFBP5 (Mohan et al., 1995). Studies investigating functionality of IGFBPs in birds are lacking, and understanding their developmental expression profiles in targets of the somatotropic axis could shed light on their actions in key tissues like liver and skeletal muscle.

The somatotropic axis is highly conserved across vertebrates, and IGFBPs exhibit a multiplicity of functions. As a result, it is important to understand their impact on economically important traits in poultry, such as growth, muscle accretion, and feed efficiency. In a previous study, no differences in circulating IGFs were observed between between modern commercial (Ross 308) and legacy [Athens-Canadian Random Bred (ACRB)] broilers, though IGFBP mRNA expression in liver and muscle did differ between lines during embryogenesis and post-hatch (Vaccaro et al., 2022a). Due to observed differences between the lines in expression of select IGFBPs in both tissues and a lack of difference in circulating hormone levels, it is likely that that the increased growth rate, greater muscle accretion, and improved feed efficiency observed in modern broilers is facilitated, at least in part, by IGFBP action. Further, based on gene expression patterns that were apparent between embryonic day (e) 10 and e18 and from post-hatch day (d) 10 through d40, it is possible that the influence of IGFBPs on broiler growth and body composition is distinct across developmental periods. The previous study examined developmental patterns in somatotropic axis gene expression and circulating IGF levels in 2 separate experiments (Vaccaro et al., 2022a) and, as a result, was not able to investigate changes that occurred between e18 and d10. This period represents critical times of rapid growth, proliferation and development of muscle satellite cells, and the metabolic transition in energy source from lipid-rich yolk to carbohydrate-rich diets (Noble, 1986; Noble and Cocchi, 1990; Halevy et al., 2000; Halevy et al., 2004). As such, understanding how the somatotropic axis, particularly the IGF-IGFBP system, influences these developmental processes is important to further determine its contribution to the aforementioned economically valuable traits. Therefore, the objective of this study was to evaluate developmental mRNA expression of GHR, IGFs, IGFR1, and IGFBPs in commercial broiler chickens between mid-embryogenesis and 3 wk post-hatch, as well as evaluate circulating IGF levels post-hatch.

MATERIALS AND METHODS

Animals and Tissue Collection

Tissues used in this study were collected from embryonic and post-hatch male Ross 308 broilers hatched from a breeder flock raised at the University of Georgia's Poultry Research Center farm. All procedures using animals were approved by the University of Georgia's Institutional Animal Care and Use Committee.

Fertile Ross 308 eggs were obtained and incubated under standard conditions (37.5°C and 60% humidity, rotation every 2–3 h), with the day eggs were set defined as e0. After hatching, birds were raised in floor pens (n = 6 pens) with free access to water and a 2 phase commercial-type broiler diet. Birds were fed starter (21.3% crude protein, 1.2% digestible lysine, 3,050 kcal/kg metabolizable energy, 0.95% calcium and 0.48% available phosphorus) from d0 to d14 and grower (19.6% crude protein, 1.09% digestible lysine, 3120 kcal/kg metabolizable energy, 0.85% calcium and 0.43% available phosphorus) from d14 to d21.

On e12, e14, e16, e18, and e20, twelve embryos were euthanized by decapitation prior to collection of skin, liver, and breast muscle. Genomic DNA was extracted from skin as previously described (Vaccaro et al., 2022a; Vaccaro et al., 2022b), and embryos were sexed through PCR amplification of the chromo-helicase-DNA binding protein gene (Fridolfsson and Ellegren, 1999). Liver and breast muscle from six male embryos at each age (n = 6) were used for gene expression analysis as described below.

One bird was selected from each floor pen on d0 (day of hatch), d1, d3, d5, d7, d10, d14, and d21 from which blood and tissues were harvested. Blood was collected from a cardiac puncture into heparinized tubes, stored on ice until centrifugation at 1,500 x g for 10 min at 4°C, and plasma was collected and stored at -20°C prior to analysis of circulating hormone levels. After blood collection, birds were euthanized by cervical dislocation, sexed by visual identification of the gonads, and only males were used for liver and muscle collection (n = 6). Tissues were flash frozen in liquid nitrogen and stored at -80°C prior to total RNA extraction for gene expression analysis.

Reverse Transcription-Quantitative PCR

Total RNA was isolated from liver and breast muscle using RNeasy Mini kits (Qiagen, Valencia, CA) with modifications for lipid-rich or fibrous tissues, respectively and analyzed by RT-qPCR using primers as previously described (Vaccaro et al., 2022a; Vaccaro et al., 2022b). Transcripts were normalized to 18s ribosomal rRNA (18s rRNA), which was not affected by age in either liver or muscle (P > 0.05; Figure S1). For qPCR reactions where 18S levels were analyzed, cDNA was diluted an additional 50-fold beyond that for target gene amplification to ensure that CTs were comparable. The equation (2ΔCt)target/(2ΔCt)18s, where ΔCt = Ctno RT – CTsample, was used to transform and normalize data as previously described (Ellestad et al., 2009; Ellestad and Porter, 2013; Ellestad et al., 2015; Payne et al., 2019; Vaccaro et al., 2022a; Vaccaro et al., 2022b). Data are expressed relative to the age with the highest mRNA level. As a result, the age with the highest expression level is 100% in all cases.

IGF Enzyme-Linked Immunosorbent Assays

Samples were analyzed with commercial competitive-binding ELISAs (Cusabio, Houston, TX) for IGF1 and IGF2 as described previously (Vaccaro et al., 2022a). These assays were validated for parallelism and sensitivity using a series of five 2-fold dilutions of 4 independent broiler chicken sample pools that were initially diluted 1:3 (IGF1) or 1:100 (IGF2) in sample diluent. Parallelism was exhibited through the manufacturer's reported sensitivities of 125 pg/mL for IGF1 and 62.5 pg/mL for IGF2. All samples were analyzed in a single ELISA plate for IGF1 and 2 ELISA plates for IGF2. The intra-assay coefficient of variation (CV) for the IGF1 ELISA was determined to be 8.1%, and the and intra-assay and inter-assay CVs for IGF2 were determined to be 7.9% and 10.1%, respectively.

Statistical Analysis

Data were analyzed with a one-way analysis of variance (ANOVA) using the Fit Model Procedure of JMP Pro 14 (SAS Institute, Cary, NC). Gene expression data were log2-transformed prior to statistical analysis to correct for non-normal distribution of relative data. When ANOVA indicated a significant effect of age, post hoc means comparisons were performed using the test of least significant difference. All differences were considered significant at P ≤ 0.05.

RESULTS

IGF and Hormone Receptor Expression

Distinct developmental expression patterns were detected for IGF1, IGF2, IGFR1, and GHR in the liver (Figure 1; P ≤ 0.05). Expression of IGF1 began to increase on d5 and continued steadily rising through d21 (Figure 1A). Levels of IGF2 decreased between e12 and e16, increased transiently on e20 before dropping again on d0, and then steadily increased after hatch (Figure 1B). Unlike IGF1 and IGF2, IGFR1 expression dropped between e12 and d0 and increased again to intermediate levels on d5, after which it remained constant (Figure 1C). Expression of GHR did not change beween e12 and 18, decreased 10-fold between e18 and d1, and then steadily increased again after hatch through d21 (Figure 1D).Figure 1 Relative mRNA expression of (A) IGF1, (B) IGF2, (C) IGFR1, and (D) GHR in liver on embryonic days (e) 12, 14, 16, 18, and 20, day of hatch (d0), and post-hatch days (d) 1, 3, 5, 7, 10, 14, and 21 in Ross 308 male broilers. Relative expression levels were measured using RT-qPCR and normalized to 18S rRNA. The data (mean + SEM) are expressed relative to the age with the highest expression level (equivalent to 100%). Data were analyzed by one-way ANOVA followed by Fisher's least significant difference test. All genes exhibited an effect of age, and values without a common letter are significantly different (P ≤ 0.05; n = 6 replicate birds at each age).

Figure 1

As in liver, expression of these genes was also dynamic in breast muscle between mid-embryonic development and 3 wk post-hatch (Figure 2; P ≤ 0.05). A cyclical expression pattern was observed for IGF1 mRNA, with a transient decrease observed in the peri-hatch period and a second decline between d7 and d21 (Figure 2A). Levels of IGF2 increased slightly between e12 and e16 and remained at that level with the exception of subtle and inconsistent decreases observed on d5 and d14 (Figure 2B). Expression of IGFR1 in breast muscle was highest on e12 to e16, decreased through d3, and remained at that level through d21 (Figure 2C). Expression of GHR exhibited a similar pattern, though there was a transient increase on d1 and d3 and the overall difference in expression across the ages was smaller (Figure 2D).Figure 2 Relative mRNA expression of (A) IGF1, (B) IGF2, (C) IGFR1, and (D) GHR in breast muscle on embryonic days (e) 12, 14, 16, 18, and 20, day of hatch (d0), and post-hatch days (d) 1, 3, 5, 7, 10, 14, and 21 in Ross 308 male broilers. Relative expression levels were measured using RT-qPCR and normalized to 18S rRNA. The data (mean + SEM) are expressed relative to the age with the highest expression level (equivalent to 100%). Data were analyzed by one-way ANOVA followed by Fisher's least significant difference test. All genes exhibited an effect of age, and values without a common letter are significantly different (P ≤ 0.05; n = 6 replicate birds at each age).

Figure 2

IGFBP Expression

Dynamic expression patterns were exhibited between the developmental stages for all IGFBPs produced in the liver (Figure 3; P ≤ 0.05). There was a transient decrease in IGFBP1 on d0 and d1, with its expression at other ages remaining relatively stable (Figure 3A). Expression of IGFBP2 increased from e12 to d0, dropped sharply between d0 and d1 to levels not different from e12, and remained low through d21 (Figure 3B). A decrease was observed for IGFBP3 from e18 to d0, and it remained at low-to-intermediate levels after hatch (Figure 3C). Only IGFBP4 expression increased consistently in the liver throughout embryogenesis and after hatch, with significant increases occuring from e16 to e20, d3 to d5, and d10 to d21 (Figure 3D). A decrease in IGFBP5 mRNA was observed at and just after hatch, but expression was restored to embryonic levels by d5 (Figure 3E). Much like expression of IGFBP1 and IGFBP5, IGFBP7 levels decreased transiently after e20 before increasing again between d3 and d5 (Figure 3F).Figure 3 Relative mRNA expression of (A) IGFBP1, (B) IGFBP2, (C) IGFBP3, (D) IGFBP4, (E) IGFBP5, and (F) IGFBP7 in liver on embryonic days (e) 12, 14, 16, 18, and 20, day of hatch (d0), and post-hatch days (d) 1, 3, 5, 7, 10, 14, and 21 in Ross 308 male broilers. Relative expression levels were measured using RT-qPCR and normalized to 18S rRNA. The data (mean + SEM) are expressed relative to the age with the highest expression level (equivalent to 100%). Data were analyzed by one-way ANOVA followed by Fisher's least significant difference test. All genes exhibited an effect of age, and values without a common letter are significantly different (P ≤ 0.05; n = 6 replicate birds at each age).

Figure 3

Expression of all IGFBPs except IGFBP7 changed between developmental stages in breast muscle (Figure 4; P ≤ 0.05). Transcripts of IGFBP1 could not be detected in this tissue, which is consistent with our previous findings (Vaccaro et al., 2022a). Levels of IGFBP2 mRNA increased between e12 and e16, decreased between e20 and d3, and remained low through d21 (Figure 4A). Expression of IGFBP3 diminished between e14 and e16 and returned to e14 levels on d1. A second decrease occurred between d1 and d5 and expression remained relatively lower through d10 before increasing again on d14 and d21 (Figure 4B). Levels of IGFBP4 mRNA were highest between e12 and e18, decreased to intermediate levels on e20 through d1, and further decreased to its lowest levels on d3 through d14 (Figure 4C). For IGFBP5, expression steadily declined from e20 to d3 and tended to be lower between d3 and d21 than at earlier ages (Figure 4D). No significant differences were detected for IGFBP7 in bresast muscle (Figure 4E; P > 0.05).Figure 4 Relative mRNA expression of (A) IGFBP2, (B) IGFBP3, (C) IGFBP4, (D) IGFBP5, and (E) IGFBP7 in breast muscle on embryonic days (e) 12, 14, 16, 18, and 20, day of hatch (d0), and post-hatch days (d) 1, 3, 5, 7, 10, 14, and 21 in Ross 308 male broilers. Relative expression levels were measured using RT-qPCR and normalized to 18S rRNA. The data (mean + SEM) are expressed relative to the age with the highest expression level (equivalent to 100%). Data were analyzed by one-way ANOVA followed by Fisher's least significant difference test. For genes demonstrating an effect of age, values without a common letter are significantly different (P ≤ 0.05; n = 6 replicate birds at each age). Transcripts of IGFBP1 were not detected in muscle.

Figure 4

Circulating IGFs in Post-Hatch Plasma

Circulating IGF concentrations did not change during the first 3 wk of post-hatch development (Figure 5; P > 0.05). However, changes in levels of IGF2 approached significance (Figure 5B; P = 0.0661), with concentrations appearing to rise between d1 and d3 before dropping after d7.Figure 5 Circulating (A) IGF1 and (B) IGF2 in Ross 308 male broilers on post-hatch days (d) 1, 3, 5, 7, 14, and 21 were determined using an IGF1 and IGF2 ELISA, respectively. Data were analyzed by one-way ANOVA followed by Fisher's least significant difference test. The data (mean + SEM) are presented as the average hormone level at each age (pg/mL). No significant age effects were observed for (A) IGF1 or (B) IGF2 (P > 0.05; n = 6 replicate birds at each age).

Figure 5

DISCUSSION

A comprehensive evaluation of somatotropic gene expression from mid-embryogenesis through the first 3 wk post-hatch has provided insight into how this axis regulates growth and metabolism during distinct developmental periods. The ages examined were chosen based on prior work demonstrating that the somatotropic axis becomes active during the last week of embryogenesis, although IGF1 production does not appear GH-dependent until the axis is fully mature during the early post-hatch period (Porter et al., 1995; Ellestad et al., 2011; Vaccaro et al., 2022a). Additionally, expression of genes within the somatotropic axis was observed to differ between Ross 308 modern commercial broilers and ACRB legacy broilers during both embryonic and post-hatch development (Vaccaro et al., 2022a), indicating that this axis is involved in regulating growth and development of modern broilers to such a degree its activity has been altered by commercial genetic selection. By examining additional ages that encompass times of rapid growth, development of muscle, and the metabolic transition between yolk and grain-based diets (Noble, 1986; Noble and Cocchi, 1990; Halevy et al., 2000; Halevy et al., 2004), potential roles for the IGF system in these processes can be inferred.

Levels of IGF1 mRNA in liver increased almost 50-fold after hatch, while in breast muscle, expression diminished about 5- to 10-fold during the peri-hatch period but increased again during the first week post-hatch. Of the 2 IGFs, circulating IGF1 is thought to be the more important regulator of post-natal growth in mammals (Stratikopoulos et al., 2008). Our results revealed that, in broilers, the pattern of hepatic IGF1 mRNA expression is consistent with previous reports that the highest levels occur during the 2.5 to 3.5-wk post-hatch period in which weight gain is most rapid in broilers (Giachetto et al., 2004; Liu et al., 2016; Vaccaro et al., 2022a). Additionally, low embryonic expression of IGF1 in the liver coincided with greater GHR mRNA in the same tissue. This indicates that GH signaling via GHR likely does not control IGF1 production until after hatch, when the somatotropic axis is thought to become fully mature (Ellestad et al., 2011). Additional evidence for the development of negative feedback comes from decreases in muscle IGFR1 mRNA post-hatch, which is consistent with observations that binding of radiolabeled IGF1 to partially purified IGFR1 on breast and leg muscle plasma membranes decreased between 1 and 7 weeks of age (Oudin et al., 1998). Levels of IGF2 mRNA in the liver were expressed at comparable levels both pre- and post-hatch, while hepatic IGF1 mRNA was much lower during embryonic development than after hatch. Based on relative levels of hepatic IGF1 and IGF2 mRNA during each developmental phase, IGF2 could have a reduced role in post-hatch growth and development as IGF1 production increases, similar to what is seen in mammals (DeChiara et al., 1991). In chickens, the cation-independent mannose 6-phosphate receptor does not bind IGF2 as it does in mammals (Canfield and Kornfeld, 1989), and IFGR1 exhibits similar affinities for both IGF1 and IGF2 (Oudin et al., 1998). Therefore, relatively higher levels of IGF1 than IGF2 after hatch that were reflected in hepatic mRNA expression and circulating hormone concentrations measured here and previously observed (Radecki et al., 1997) would indicate that it is the primary IGF regulating growth after hatch.

The production of IGF1 in breast muscle also suggests that local synthesis of IGF1 is critical for growth and development of this tissue, as previous data have implicated in chickens (Guernec et al., 2003; Guernec et al., 2004; Vaccaro et al., 2022a). This is consistent with studies in mice, where it has been demonstrated via tissue-specific knockout of hepatic IGF1 that liver is the main source of circulating IGF1 but does not appear to control overall body growth (Sjögren et al., 1999; Yakar et al., 1999). Circulating IGF levels have sometimes (Scanes et al., 1989; Beccavin et al., 2001) but not always (Giachetto et al., 2004; Vaccaro et al., 2022a) correlated with growth rate in chickens, suggesting that autocrine or paracrine action might also be important in controlling growth or body weight in this species. A drop in IGF1 levels in muscle was observed between e12 and e20 and levels increased greatly again between e20 and d3. This was followed by a decrease after d7. As the first week post-hatch represents a period of rapid satellite cell expansion (Halevy et al., 2000; Halevy et al., 2004), this increase in IGF1 suggests its importance in local regulation of this process. Comparatively, IGF2 may also facilitate muscle growth and satellite cell mitotic activity, as expression of IGF2 mRNA in breast muscle rose between e12 and e16 and remained constant thereafter. Thus, rapid muscle accretion induced by satellite muscle cell proliferation observed shortly post-hatch in broilers (Halevy et al., 2000; Halevy et al., 2004) could be facilitated by locally produced IGF1, whereas IGF2 might perform a role in maintaining muscle tissue throughout both embryogenesis and post-hatch. Additionally, IGFR1 expression may change in the wake of increased paracrine IGF1 signaling. By d3, IGFR1 levels decreased in breast muscle, concomitant with the increase in IGF1 observed during the same time. This suggests that IGFR1 mRNA production diminishes post-hatch in breast muscle following increased IGF1 production, potentially as part of a negative feedback loop.

Expression of IGFBPs demonstrated dynamic changes in both tissues between developmental stages. In the liver, IGFBP1, IGFBP3, IGFBP5, and IGFBP7 dropped transiently during the peri-hatch period. On the other hand, hepatic IGFPB2 levels dropped substantially just after hatch, while IGFBP4 increased steadily from mid-embryogenesis until 3 wk post-hatch in this tissue. A similar decrease in expression shortly after hatch was observed for IGFBP2 in breast muscle, though the change was more gradual. In contrast to liver, IGFBP4 expression in muscle also decreased shortly after hatch on d3 and remained relatively low. Despite these dynamic changes in expression in liver and breast muscle, circulating IGF1 and IGF2 concentrations in plasma did not change throughout the first 3 wk post-hatch. Collectively, these results provide further evidence that IGFBPs, rather than circulating IGF levels, play a substantial role in mediating signaling (Vaccaro et al., 2022a). Dynamic fluctuations in IGFBP expression indicate that the activity of these proteins varies based on developmental stage, with transient or consistent downregulation of several in the liver and muscle necessary for the transition between embryogenesis and post-hatch growth and development.

The IGFBPs can inhibit growth by preventing IGF access to IGFR1, ultimately interfering with IGF-IGFR1 mediated siganling (Baxter, 1991). Therefore, diminished expression of multiple IGFBPs in liver or muscle post-hatch could allow for greater access of circulating or local IGFs to IGFR1, ultimately promoting growth in a holistic or tissue-specific manner. Expression of IGFBP2 decreased at or shortly after hatch in both liver and muscle; IGFBP3 and IGFBP4 expression was reduced in liver or breast muscle, respectively. These developmental patterns suggest that hepatic IGFBP2 and IGFBP3 could have an inhibitory effect on post-hatch growth in chickens. Similar results were found in a prior study, where post-hatch expression of IGFBP2 mRNA was higher in the liver and IGFBP3 mRNA was higher in the breast muscle of slower growing ACRB broilers when compared to faster growing modern Ross 308 broilers (Vaccaro et al., 2022a). Overexpression of IGFBP2 in mice slows the development of myofibers, causing total lower body protein and reduced muscle mass (Rehfeldt, 2008; Rehfeldt et al., 2010). In this study, IGFBP2 mRNA was observed to increase between e12 and e16 in breast muscle but began to decrease afterward to very low levels throughout the post-hatch period. Levels of IGFBP4, which also decreased in this study throughout late embryonic development and after hatch, is also involved in myofiber development through blocking differentiation in mouse myoblasts (James et al., 1993; Rotwein et al., 1995; Mukherjee et al., 2008). If IGFBP2 and IGFBP4 act similarly in avian muscle as they do in mammalian muscle, a reduction in expression would be required to facilitate rapid development and accretion of this tissue that is observed after hatch in broilers. Taken together, IGFBP2 and IGFBP4 appear to act in a paracrine, inhibitory fashion in breast muscle prior to hatch and are subsequently downregulated to allow for rapid muscle growth after hatch.

In addition to acting in an inhibitory fashion, select IGFBPs can also promote IGF action and have effects independent of IGF (Schutt et al., 2004; Klaus et al., 2006). The results observed in this study suggest that IGFBP4 functions as an promoter or inhibitor of growth in a tissue-specific manner. Throughout embryonic and post-hatch development, IGFBP4 mRNA levels increased in liver and decreased in breast muscle. When released into circulation from the liver, IGFBPs bind to IGFs in plasma to extend their half-life. Therefore, higher IGFBP4 expression in broiler liver post-hatch suggests it may promote growth via stabilizing IGF in circulation and, therefore, promoting its signaling, similar to mammals (Awede et al., 1999). However, IGFBP4 mRNA decreased at d3 in breast muscle. A reduction in local IGFBP4 activity in broiler muscle could facilitate growth of this tissue as described above, because less IGFBP4 would allow for increased IGF access to IGFR1. This inhibitory effect of IGFBP4 in mammals has been established to occur via paracrine signaling, including acting as an IGF antagonist in mouse smooth muscle cells (Jones and Clemmons, 1995; Florini et al., 1996; Wang et al., 1998) and rat skeletal muscle cells (Silverman et al., 1995). These data and our results suggest that IGFBP4 has multiple roles in the context of growth, including both positive and negative IGF interactions that may be tissue-specific (Ning et al., 2008), and these effects appear conserved between mammals and birds.

The effects of IGFBPs on IGF signaling can affect biological processes outside of cellular proliferation and differentiation. Hepatic IGFBP1, IGFBP5, and IGFBP7 transiently decreased between e20 and d1, only to return to embryonic levels within the first week of hatch. These changes during the peri-hatch period may be the result of the known metabolic switch from pre- to post-hatch. The majority of energy utilized by the chick embryo is sourced from lipoproteins in the yolk. This occurs until around d3 (Noble, 1986; Noble and Cocchi, 1990), after which energy is obtained from a carbohydrate-based diet (Sklan, 2003). The peri-hatch downregulation of these IGFBPs coincides with this metabolic transition, as the IGFs begin to function in glucose homeostasis via GH suppression (Clemmons, 2006). There is precedence for IGFBP involvement in mediating glucose utilization in mammals, and this may ultimately impact hepatic metabolism and growth. Overexpression of human IGFBP3 in fasted mice induced hyperglycemia and impaired glucose tolerance (Silha et al., 2002), and in a mouse model where IGFBP3 was deleted, knockouts had larger livers and higher body weights than controls (Yakar et al., 2009; Yamada et al., 2010).

Circulating IGF levels did not change significantly during post-hatch development, although IGF2 tended to decrease over time. This suggests that, as IGF1 concentrations stay relatively stable in plasma, its effects are modulated largely by IGFBP activity and IGFR1 sensitivity. It should be noted that the lack of developmental changes in circulating IGF1 is in contrast to prior studies that have shown an increase after the early post-hatch period, with levels plateauing sometime between 3 and 6 weeks of age (Radecki et al., 1997; Beccavin et al., 2001; Giachetto et al., 2004; Guernec et al., 2004; Vaccaro et al., 2022a). The absolute values measured in the current study and Vaccaro et al. (2022a) were also about an order of magnitude lower than those in prior reports, which ranged from approximately 5 to 60 ng/mL (Radecki et al., 1997; Beccavin et al., 2001; Giachetto et al., 2004; Guernec et al., 2004). It is possible that genetic differences in the chickens used contribute to the inconsistencies, as the previous studies used non-commercial broilers divergently selected for body weight (Beccavin et al., 2001), commercial and non-commercial broilers from the early 2000s (Giachetto et al., 2004; Guernec et al., 2004), or layers (Radecki et al., 1997). The older studies also used a heterologous radioimmunoassays validated for chickens (Huybrechts et al., 1985) and not an ELISA as used here, which could have contributed to differences. The slight decrease observed in circulating IGF2 suggests that IGF-induced post-hatch growth is primarily carried out by signaling and modulation of IGF1. Relatively stable or reducing circulating IGF2 levels post-hatch that are somewhat lower than IGF1 have been noted previously (Radecki et al., 1997; Vaccaro et al., 2022a) and support this. The IGFBPs may alter endocrine IGF signaling by directly binding to IGFs and either preventing them from binding IGFR1 or facilitating their transport to IGF-sensitive tissues expressing the receptor (Baxter, 1991; Kelley et al., 2002; Kim, 2010; Baxter, 2023), though these functions have not been established in chickens. Therefore, the IGFBPs are critical for controlling endocrine IGF action. However, as both IGF1 and IGF2 were detected in breast muscle tissue, locally-produced IGFs that signal in a paracrine fashion are likely important for breast muscle growth, as discussed earlier.

Results presented here indicate the dynamic and tissue-specific nature of broiler somatotropic gene expression between mid-embryogenesis and the first 3-wk post-hatch. This suggests these genes play important roles in regulating growth, development, and metabolic transitions during this period. Post-hatch, IGF1 appears to become the primary regulator of growth, particularly at the paracrine level, as its expression patterns are much more dynamic in both liver and muscle at this time. Effects of IGFBPs likely differ depending on developmental stage, tissue of origin, and mode of action. As the results are limited to changes in mRNA levels, inferences surrounding potentional functionality should be interpreted with caution but do provide a basis for hypothesis generation to guide future investigations, especially since functional data pertaining to the IGF-IGFBP system are lacking in chickens. For example, hepatic IGFBP4 expression was unique in increasing in a manner similar to hepatic IGF1, suggesting it could promote IGF signaling and ultimately growth, perhaps through extending the half-life of IGF1 in circulation. Similarly, the transient decrease in hepatic expression of several IGFBPs around hatch indicates that this might be necessary to facilitate the metabolic switch between lipid and carbohydrate utilization that must occur at this time. In breast muscle, IGFBPs with downregulated expression after hatch would be more likely function in an inhibitory, paracrine fashion since a decrease in their levels would allow for enhanced sensitivity to circulating and locally-produced IGFs necessary for the rapid accretion of this tissue in modern commercial broilers. In conclusion, differential expression of most IGFBPs across developmental stages reinforces the idea that they are critical regulators of IGF signaling that contribute to broiler growth, metabolism, and muscle accretion and provide a basis for future studies investigating their functionality.

DISCLOSURES

The authors declare no conflicts of interest.

Appendix Supplementary materials

Image, application 1

ACKNOWLEDGMENTS

This work was supported by the USDA National Institute of Food and Agriculture competitive grant #2021-67034-35185 . The funding source did not participate in the study design, collection, analysis and interpretation of data, the writing of the report, or the decision to submit the article for publication. Special thanks to Jason Payne and Brett Marshall for their assistance in sample collection, and to Brett Marshall, Shailes Bhattrai, and Ky Meeks for constructive feedback during preparation of the manuscript.

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.psj.2024.104174.
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