At the onset of egg production, commercial laying hens adapt physiological mechanisms that facilitate the utilization of calcium and phosphorus, primarily for the purpose of eggshell mineralization and medullary bone formation (Nys et al., 1986b; Wu et al., 1994). These adaptive changes begin to occur at the onset of sexual maturity between 17 and 18 weeks (Lei et al., 2020; Bahry et al., 2023) and involve specific organs important for absorption, transport, and utilization of calcium and phosphorus that are necessary for egg production (Bar, 2008). Described changes that occur at the onset of lay include increased intestinal mineral absorption (Nys et al., 1992a), renal mineral reabsorption and excretion (Wideman, 1987), calcium uptake and utilization by the shell gland (Corradino et al., 1993; Samiullah and Roberts, 2014), and medullary bone formation (Dacke et al., 1993; Kerschnitzki et al., 2014). This study provides a holistic physiological approach to understanding changes in the molecular mechanisms associated with calcium and phosphorus utilization before and after the onset of egg production.

Hormonal regulation of calcium and phosphorus homeostasis is influenced by multiple factors throughout the 24-h egg formation cycle (Sinclair-Black et al., 2023). Eggshell formation causes fluctuations in plasma ionized calcium levels (Sinclair-Black et al., 2024) that are detected by the plasma membrane protein calcium-sensing receptor (CASR) (Diaz et al., 1997; Hui et al., 2021). A decrease in calcium triggers the release of parathyroid hormone (PTH) that binds to parathyroid hormone receptor 1 (PTH1R) in target tissues (Pinheiro et al., 2012), stimulating renal calcium reabsorption and phosphorus excretion (Elaroussi et al., 1993), renal production of biologically active 1,25(OH)₂D₃ via 1α-hydroxylase (Fraser and Kodicek, 1973), intestinal calcium absorption (Nemere and Norman, 1986), and bone breakdown (Miller, 1978) releasing both calcium and phosphorus into the blood. As circulating calcium and phosphorus levels subsequently rise, calcitonin (CALC) opposes effects of PTH by acting through calcitonin receptor (CALCR), primarily by inhibiting bone remodeling (Warshawsky et al., 1980) and promoting renal calcium excretion (Cochran et al., 1970). Circulating levels of phosphorus are regulated by fibroblast growth factor 23 (FGF23), a hormone produced by bone in response to hyperphosphatemia in chickens (Wang et al., 2018; Gloux et al., 2020a). Its action is mediated by fibroblast growth factor receptors 1–4 (FGFR1, FGFR2, FGFR3, and FGFR4) in conjunction with the co-receptor Klotho (KL). In mammals (Shimada et al., 2004b; Perwad et al., 2005) and chickens (Ren et al., 2017), FGF23 induces renal excretion of excess circulating phosphorus. It has also been shown to inhibit PTH secretion (Ben-Dov et al., 2007) and production of 1,25(OH)₂D₃ (Shimada et al., 2004a) in mammals. Many of the hormonal mechanisms regulating calcium and phosphorus homeostasis during egg formation are not fully understood, particularly the roles of CALC and FGF23 (Yasuoka et al., 1998; Ogawa et al., 2003; Sinclair-Black et al., 2023).

The hormonal form of vitamin D₃, 1,25(OH)₂D₃, plays a major role in regulating calcium and phosphorus homeostasis. It can induce transcription of membrane mineral transporters and intracellular chaperone proteins involved in calcium and phosphorus absorption and utilization in intestine and kidney (Bar et al., 1990). To become active, dietary vitamin D₃ is first constitutively hydroxylated by hepatic 25-hydroxylase to produce 25(OH)D₃ (Tucker et al., 1973). These enzymes are encoded by the CYP2R1 (Watanabe et al., 2013) or CYP27A1 (Shang et al., 2023) genes. A second, rate-limiting hydroxylation is catalyzed by renal 1α-hydroxylase to produce biologically active 1,25(OH)₂D₃. This enzyme is encoded by CYP27B1 in mammals and fish (Monkawa et al., 1997; Shinki et al., 1997; Chun et al., 2014). However, this gene has not been identified in chickens to date, despite prior publications reporting measurements in its expression as reviewed in Sinclair-Black et al. (2023). Commercially available 1α-hydroxycholecalciferol is a pre-hydroxylated vitamin D₃ supplement (AlphaD3™, Iluma Alliance), requiring only the constitutive 25-hydroxylation referenced above. This could potentially improve the efficiency with which 1,25(OH)₂D₃ is produced. The inactive vitamin D₃ metabolite, 24,25(OH)₂D₃, is generated through an alternative hydroxylation step carried out by 24-hydroxylase, encoded by CYP24A1 (Armbrecht et al., 1997; Nemere, 1999).

The period between medullary bone formation that begins at the onset of sexual maturity around 18 weeks of age (Hudson et al., 1993; Bahry et al., 2023) and peak egg production around 31 weeks of age assumes significance, as hens must develop and sustain the metabolic capacity to meet high mineral requirements throughout their productive life. Therefore, this study sought to elucidate physiological changes associated with calcium and phosphorus uptake and utilization that occur during the onset of lay between 18 and 31 weeks of age and determine if supplementation with AlphaD3 influenced these changes. The objectives were to measure 1) expression profiles of genes for hormone receptors mediating calcium and phosphorus homeostasis, enzymes involved in vitamin D₃ metabolism, and mineral transporters in kidney, shell gland, ileum, and liver; 2) circulating vitamin D₃ metabolites; and 3) parameters associated with skeletal integrity in hens fed control (18 and 31 weeks) or AlphaD3-supplemented (31 weeks) diets.

At the onset of egg production, laying hens undergo many physiological changes necessary to facilitate uptake, storage, and utilization of large amounts of calcium for eggshell calcification. These start to occur around 2 weeks prior to the onset of lay and involve specialized tissues such as intestine, kidney, and shell gland that play major roles in calcium and phosphorus absorption, retention, and utilization (Bar, 2008). This study identified potential physiological mechanisms related to hormonal signaling and mineral transport in these tissues by measuring gene expression, as well as evaluated bone parameters prior to and after the onset of lay. The effect of dietary supplementation with AlphaD3 on these changes was also evaluated. Changes in gene expression were evaluated by measuring mRNA levels without further examination into whether protein levels of the same transcripts changed in a similar manner. Since variations in mRNA expression do not necessarily reflect functional modifications at the cellular level, caution should be exercised when interpreting the results. However, these data are useful at identifying systems and networks potentially involved in mediating changes that allow for enhanced handling of minerals necessary to support daily egg production. Transcriptional findings suggest PTH could be a major hormonal driver regulating calcium homeostasis across all tissues following the onset of lay, and this is particularly apparent in ileum and shell gland. Similarly, hormonal sensitivity to FGF23 appears to be upregulated after the onset of lay in kidney and shell gland, with the latter being identified as a novel target of this hormone. In addition to its role in mineral retention and excretion, kidney could be involved in the 25-hydroxylation of vitamin D₃ and DBP synthesis. Calbindin was demonstrated to have a primary role in assisting with calcium flux in all tissues, especially the shell gland, while calcium transporter NCX1 does not seem to be influenced by these adaptative changes in any of the tissues examined. Phosphorus excretion and absorption appears to occur primarily through P _i T-1 in kidney and NaP _i llb in the ileum, respectively. Similarly, phosphorus utilization for hydroxyapatite and cuticle deposition within the eggshell seems to be mediated by P _i T-1 and NaP _i llb in the shell gland. The above findings related to hormonal signaling and mineral transport are depicted in Figure 9. It should be noted that hens were only sampled prior to lay at 18 weeks and during peak production at 31 weeks. Including ages between these would allow for a more comprehensive examination of the transition that occurs immediately around the onset of lay. Most mRNA expression profiles appeared unaffected by dietary supplementation with AlphaD3, a finding that could be result of the high efficiency with which hens at peak production are able handle the daily mineral turnover necessary for eggshell mineralization. Including intermediate ages during early lay may have revealed ways in which tissues other than bone were influenced by AlphaD3 in the diet. Despite few observable impacts on mRNA levels in the tissues examined, several bone mineralization parameters were improved by feeding hens AlphaD3 during the critical period of medullary bone formation between 18 and 31 weeks of age.

Hormonal regulation of calcium homeostasis is primarily mediated by the synchronized activity of CASR, PTH, and CALC, though specific involvement of each of these during the onset of lay is not fully understood. Observations from this study found expression of CASR to change between 18- and 31-week in kidney and liver, with liver exhibiting a pronounced 60-fold increase. Although renal involvement in calcium homeostasis has been widely described (Sommerville and Fox, 1987; Wideman, 1987), liver is not canonically associated in this process beyond 25-hydroxylation of vitamin D₃ (Shang et al., 2023). CASR is not specific to calcium and has been demonstrated to contain binding sites for other divalent ions including magnesium (Quinn et al., 2013), zinc, and iron (Chanpaisaeng et al., 2021), as well as phosphate (Centeno et al., 2019) and aromatic amino acids (Conigrave et al., 2000). Hence, it is possible that hepatic expression of this gene may be related to the vitellogenesis and influenced by estrogen levels (Gloux et al., 2019; Cui et al., 2020). There are very few studies in chickens related to hepatic expression of CASR and, in contrast to this study, Deng et al. (2010) reported that this gene was not detected in liver or other tissues such as heart, spleen, lungs, pancreas, or stomach of laying hens. However, hepatic expression of this gene has been shown in mammals, and studies with mouse hepatocytes demonstrated CASR’s capability to sense and mobilize calcium associated with bile secretion (Canaff et al., 2001) and induce intracellular calcium elevation (Xing et al., 2010).

The influence of PTH on regulation of calcium and phosphorus uptake and utilization was demonstrated in this study, where all tissues evaluated had heightened PTH1R mRNA expression after the onset of lay, an event that was only observed for this gene. Although function of PTH have been well described in kidney (Wideman, 1987; Elaroussi et al., 1993), the most apparent upregulation in this study was observed in ileum. For decades, the influence of PTH on intestinal calcium and phosphorus absorption has been described as indirect, as it occurs primarily via activation of 1,25(OH)₂D₃ (Tanaka et al., 1971; Bar et al., 1990). However, other authors have suggested that PTH may directly stimulate calcium absorption in mice (Picotto et al., 1997), fish (Rotllant et al., 2006), and chickens (Nemere and Norman, 1986). Thus, ileal upregulation of PTH1R in this study could indicate a direct effect of PTH on the intestine to meet increasing calcium demands after the onset of lay.

In the case of shell gland, fewer studies into the role of PTH signaling have been conducted. In the research presented here, PTH1R was found to be upregulated in the shell gland of hens at 31 weeks. Consistent with this, Thiede et al. (1991) measured PTH1R mRNA levels in the laying hen oviduct and found greatest expression in the shell gland. A possible role for PTH in oviduct differentiation and development as well as regulation of smooth muscle activity in this tissue was hypothesized in the aforementioned study. Furthermore, receptor binding affinity for PTH in the shell gland of laying hens (Ieda et al., 2000) and guinea fowl (Ogawa et al., 2000) was found to increase during periods of eggshell calcification, while no variations were observed in non-laying birds. This indicates a strong association of PTH with the oviposition cycle that was also evidenced by Sinclair-Black et al. (2024), where upregulation of PTH1R in the shell gland was observed between 21 and 24 HPOP. This correlates with findings from the present study, where tissue collection occurred at 21 HPOP and upregulation of PTH1R was observed. Together, this suggests that PTH signaling through PTH1R may play an important role in providing shell gland with the capacity to transfer minerals for eggshell formation.

Of the tissues examined, CALCR was found to only increase in kidney and shell gland after the onset of egg production, the two tissues found to have the most capacity for mineral transport in this study. Tissue collection from this study occurred when a hard-shelled egg was present in the shell gland; thus, increased CALCR expression in kidney and shell gland observed here are likely to ensure sensitivity to the regulatory response by calcitonin at the end of the oviposition cycle, when eggshell formation is almost complete and calcium demands for this process decrease. Yasuoka et al. (1998) demonstrated increased binding affinity of calcitonin to its receptor in the laying hen kidney between 3 h before oviposition and 2 h post-oviposition, consistent with times of lower calcium demands and when tissues were collected in the present study. Similar findings were observed in the shell gland of laying hens by Ieda et al. (2001) and guineafowl by Ogawa et al. (2003), where calcitonin binding affinity increased at later stages of eggshell formation. These results correlate with observations from Sinclair-Black et al. (2024), where mRNA levels of shell gland CALCR in laying hens had greater expression between 2 h before oviposition and 1 h post-oviposition. During this time, mineral requirements in the shell gland switch from high amounts of calcium to phosphorus for hydroxyapatite and cuticle deposition within the eggshell, and CALC might play an important role in this process.

Hormonal regulation of phosphorus homeostasis exerted by FGF23 signaling also appears to be upregulated in kidney and shell gland after the onset of lay, specifically observed through increased expression of FGF receptors and KL, their co-receptor. In mammals (Shimada et al., 2004b; Perwad et al., 2005) and chickens (Ren et al., 2017), FGF23 has been associated with renal excretion of excess circulating phosphorus. In the current study, renal expression of FGFR1, FGFR4, and KL was upregulated at 31 weeks. When measured throughout the 24-h oviposition cycle in Sinclair-Black et al. (2024), renal expression of FGFR1 and KL had no variation, while FGFR2,FGFR3, FGFR4 were influenced by time. In that study, a decrease in FGFR3 was observed after 21 HPOP, so it is likely that FGFR3 did not change here due to the time of tissue collection; however, these and other findings support the role of FGF23 signaling in this tissue to promote renal excretion of phosphorus (Kerschnitzki et al., 2014; Gloux et al., 2020a).

Findings presented here suggest that shell gland is a novel target for FGF23 signaling, as indicated by increased expression of FGFR2 - 4 and KL after the onset of egg production. Signaling of FGF23 in this tissue may be associated with phosphorus uptake and utilization at the later stages of eggshell formation, as it was observed in Sinclair-Black et al. (2024) that upregulated expression of these genes occurred between 18 and 24 HPOP. Signaling by FGF23 could regulate expression of specific phosphorus transporters. Its pattern in the shell gland was followed by increased expression of P _i T-1 (Sinclair-Black et al., 2024), and our findings also indicated upregulation of P _i T-1, P _i T-2, and NaP _i llb in the shell gland concurrently with mediators of FGF23 signaling. These changes support that FGF23 could be involved in mediating phosphorus deposition within the hydroxyapatite layer of the eggshell immediately below the cuticle that has been suggested to be required for halting calcification of the shell (Cusack et al., 2003) or formation of the cuticle itself. Taken together, these findings suggest that after the onset of lay, hens acquire greater sensitivity to hormones regulating calcium and phosphorus homeostasis in target tissues involved in mediating increased demands of these minerals for bone remodeling and eggshell formation.

Vitamin D₃ metabolism by 25-hydroxylase, encoded by CYP2R1 or CYP27A1, was found to be either downregulated or not influenced by the onset of lay in all tissues examined except kidney, which exhibited a fold increase of approximately 1.5 or higher after the onset of lay for both genes. It was only recently that CYP27A1 was confirmed to encode for an alternative 25-hydroxylase (Shang et al., 2023), and CYP2R1 has been catalogued as highly conserved between avian and mammalian species (Watanabe et al., 2013). Liver has been identified to be the major tissue responsible for conversion of circulating vitamin D₃ into 25(OH)D₃ in mammals (Ponchon et al., 1969). In avian species, early studies demonstrated in vitro 25-hyroxylase activity in liver homogenates (Tucker et al., 1973), as well as identified liver as an important tissue in this process through in vivo studies (Bhattacharyya and Deluca, 1974). However, it appears that liver is not the exclusive tissue with 25-hydroxylase activity in chickens. Tucker et al. (1973) first demonstrated that renal 25-hydroxylase from white Leghorn hens can play this role in vitro, which would give kidney the ability to perform the full two-step transformation of vitamin D₃ into its active form. Similar to results presented here, Sinclair-Black et al. (2024) observed minor changes in hepatic CYP2R1 mRNA expression over the oviposition cycle, while renal expression of this gene was noted to increase towards periods of peak eggshell calcification. Furthermore, Bhattacharyya and Deluca (1974) evaluated regulation of liver 25-hydroxylase in chickens and discussed the possibility that, although kidney may have the capacity to convert dietary vitamin D₃ into 25(OH)D₃, it may not necessarily contribute to circulating levels of this metabolite. This is likely associated with the fact that when synthesized in the kidney, 25(OH)D₃ can be immediately hydroxylated into 1,25(OH)₂D₃ by renal 1α-hydroxylase and utilized for renal calcium reabsorption. Investigation into tissue distribution of CYP27A1 in both male and female 8-week-old broiler chickens demonstrated an approximate 20-fold difference between liver and kidney, with liver levels being the highest amongst all tissues examined (Shang et al., 2023). This would suggest that liver remains the primary site of CYP27A1 25-hydroxylase activity, while the kidney may exhibit 25-hydroxylase activity by both enzymes for local production of 25(OH)D₃ and, ultimately, 1,25(OH)₂D₃. Thus, results presented in the current study provide support for the notion that the conversion of dietary vitamin D₃ into 25(OH)D₃ can take place at the renal level, particularly during periods characterized by elevated calcium demands such as eggshell formation and medullary bone replenishment. Furthermore, it was observed that, although expression of genes encoding 25-hydroxylase in kidney were upregulated, circulating levels of 25(OH)D₃ decreased at 31 weeks. This is presumably a result of greater conversion of this metabolite to 1,25(OH)₂D₃ in the kidney, as is evidenced by an increase in circulating 1,25(OH)₂D₃ and an accompanying decrease in the inactive 24,25(OH)₂D₃ metabolite. Collectively, activation of dietary vitamin D₃ is upregulated by the onset of lay as a responsive mechanism to escalating calcium and phosphorus demands. Additionally, the kidney may undertake a local role in both activating hydroxylation steps that ultimately facilitate calcium reabsorption and phosphorus excretion in this tissue.

Both 25(OH)D₃ and 1,25(OH)₂D₃ bind to circulating DBP to be transported to target tissues (White and Cooke, 2000). In mammals, the major synthesis site of this binding protein is the liver (Cooke et al., 1991), although expression of the gene encoding for this protein is present in several other tissues, including kidney, testis, placenta, and adipose, but it has not been detected in either intestine or uterus (McLeod and Cooke, 1989). However, to the authors’ best knowledge, no literature has been published elucidating DBP mRNA tissue distribution in chickens, though the protein has been detected in chicken serum (Bouillon et al., 1980). Findings from the current study confirm expression of DBP in laying hen kidney and liver, and unpublished data revealed levels in liver to be over 600-fold higher than those in other tissues (R.A. Garcia-Mejia, M. Sinclair-Black, and L.E. Ellestad). Similar to lack of detection in mammalian uterus, DBP was not detected in the shell gland in this study; however, it was found to be expressed in the ileum. As with 25-hydroxylase expression, kidney was found to be the only tissue where DBP was influenced by sexual maturation, with a 2-fold increase after the onset of lay. Research from Nys et al. (1986a) first demonstrated a marked increase in circulating DBP and 1,25(OH)₂D₃ in hens between 8 and 24 weeks as they approached the onset of egg production, specifically starting 2 weeks prior to the first oviposition. Further research by Nys et al. (1989) evaluated the effect of the onset of lay on plasma DBP levels by inducing sexual maturity in 12 weeks old pullets via administration of estrogen alone or in combination with testosterone, which induced an increase in DBP levels after treatment. In addition, circulating DPB levels were analyzed in mature laying hens and these were found to be even higher than those of induced immature pullets. These findings suggest a close relationship between the onset of lay and both DBP and 1,25(OH)₂D₃ in circulation. From the present study, the upregulation of renal DBP could indicate that the kidney plays a role in production of this protein following the onset of lay, which might allow this tissue to respond to 1,25(OH)₂D₃ more efficiently.

The expression of genes associated with calcium uptake and utilization exhibited a discernible increase following the onset of egg production, particularly in kidney, shell gland, and ileum that are all actively engaged in mineral transport. These included upregulated expression of PMCA1 and CALB1 in kidney, shell gland, and ileum, as well as increased TRPV6 in the kidney. However, the calcium transporter NCX1 was found to be downregulated in all tissues examined. Renal NCX1 was found to be unaffected by time within the oviposition cycle in laying hens (Sinclair-Black et al., 2024) or throughout different production stages (San et al., 2021). In addition, expression of TRPV6 in this study was only detected in the kidney, which is consistent with findings from other authors where TRPV6 was not detected in shell gland (Sinclair-Black et al., 2024) or ileum (Proszkowiec-Weglarz and Angel, 2013). There are no reports to date for liver that the authors are aware of, though unpublished data in our lab indicated it is not detected in this tissue (R.A. Garcia-Mejia, M. Sinclair-Black, and L.E. Ellestad). In the intestine, the calcium chaperone CALB1 has been previously reported to increase 5-fold after sexual maturity in the intestine of laying hens (Nys et al., 1992a) and 12-fold in the intestine after an intramuscular dose of 1,25(OH)₂D₃ (Theofan et al., 1986). The increase observed in the present study is consistent with those findings.

In the shell gland, Jonchère et al. (2012) measured ion transporter expression during the presence or absence of a calcifying egg and observed downregulation of NCX1 and upregulation of PMCA1 and CALB1 during eggshell calcification, which correlates with findings from the current study. Ieda et al. (1995) reported an increase in both VDR and CALB1 in the shell gland with the presence of a calcifying egg, and increased mRNA expression of CALB1 after the onset of lay has been demonstrated in prior studies (Nys et al., 1989; Bar et al., 1990), as observed here. Furthermore, findings presented in the current study exhibited upregulation of VDR in shell gland following the onset of egg production, which is supported by previous research (Ieda et al., 1995; Yoshimura et al., 1997). Similarly, findings from Sinclair-Black et al. (2024) described expression of shell gland VDR to increase towards times of peak eggshell calcification and decline around oviposition and ovulation. However, whether shell gland CALB1 expression is vitamin D₃-dependent is not clear (Corradino, 1993; Yoshimura et al., 1997; Ohira et al., 1998), and it appears its expression could be driven by estrogen (Navickis et al., 1979; Corradino et al., 1993) or calcium flux (Bar et al., 1992; Nys et al., 1992b; Bar et al., 1996). Required for CaCO₃ synthesis, expression of CA in the shell gland was upregulated after the onset of egg production; however, the bicarbonate transporter SLC26A9 was found to be downregulated. This is contradictory to findings from other authors that suggest eggshell formation induces upregulation of this gene (Jonchère et al., 2012). However, the difference between results presented here and findings from the aforementioned study are likely related to the fact that tissues were collected in this study at 21 HPOP, when calcification is complete. This is supported by observations in Sinclair-Black et al. (2024), where expression of SLC26A9 increased between ovulation and late calcification but was downregulated between 21 and 24 HPOP.

Phosphorus metabolism in laying hens is also heavily influenced by egg production, as the mechanisms required for eggshell formation involve medullary bone breakdown. The kidney has been demonstrated to play major role in regulation of calcium and phosphorus homeostasis (Wideman, 1987), and excretion of phosphorus has been shown to increase during periods of peak eggshell calcification associated with medullary bone remodeling for eggshell formation (Nys et al., 1986b; Kerschnitzki et al., 2014). In the kidney, P _i T-I appears to be the primary transporter mediating phosphorus flux during egg production, as demonstrated by a 15-fold increase in expression between 18 and 31 weeks. This finding correlates with increased renal P _i T-1 and P _i T-2 expression during times of peak (Sinclair-Black et al., 2024) or late (Gloux et al., 2020b) eggshell formation, probably to support renal phosphorus excretion. A similar increase was observed for shell gland P _i T-I levels; however, this tissue seemed to have an even greater increase in NaP _i llb expression, with a near 40-fold increase in at 31 weeks compared to 18 weeks. Phosphorus transport in the shell gland could increase in order to synthesize the eggshell cuticle (Sinclair-Black et al., 2024), composed of a large concentration of phosphorus (Cusack et al., 2003), as well as a thin layer of hydroxyapatite crystals beneath the cuticle, known as vertical crystal layer (Dennis et al., 1996; Kulshreshtha et al., 2022). Furthermore, as indicated previously, upregulation of phosphorus transporters in the shell gland could be mediated by FGF23 signaling. In general, hens at 31 weeks exhibited greater capacity for mineral transport, utilization, and uptake in kidney, ileum, and shell gland as calcium and phosphorus demands escalate to fulfill eggshell formation requirements.

Dietary supplementation with AlphaD3 maintained bone mineralization and breaking strength after the onset of egg production. Hens fed AlphaD3 exhibited conservation of humerus BMD and tibia breaking strength at 31 weeks, as well as higher tibia BMD and BMC at this age. Changes in bone mineralization in this study may be correlated with improved medullary bone formation in AlphaD3-fed hens compared to control-fed hens. Although medullary bone was not measured in this study and further investigation would be needed to confirm this, DEXA is considered a reliable tool to measure bone density, which is indirectly associated with medullary bone (Hester et al., 2004). The increase in BMD and BMC that was observed between 18 and 31 weeks could be attributed to changes in medullary bone, considering that cortical bone formation is limited following the onset of sexual maturity (Hudson et al., 1993; Whitehead, 2004). Given that medullary bone formation induced by the onset of sexual maturity acts as one of the major calcium reservoirs in laying hens, enhancement in this process would hold significant importance. Frost et al. (1990) reported increased tibia weight in 53-week-old laying hens after supplementation with 1α-hydroxycholecalciferol. Similar findings have been observed in broiler chickens, where increased tibia ash (Biehl et al., 1995) and strength (Han et al., 2009) were reported with 1α-hydroxycholecalciferol in the diet. Thus, our findings suggest that supplementation with AlphaD3 may facilitate medullary bone formation during early stages of the production cycle. As such, it has the potential to reduce fracture incidences as hens age, thereby countering skeletal health issues associated with high mineral demands during extended production and improving animal welfare. As AlphaD3 did not have any other major effects in this study, mechanisms by which it improved skeletal parameters are unknown but could involve direct actions on bone itself. Elucidation of AlphaD3 effects on bone could be uncovered by investigation into gene expression at the mRNA or protein level, biochemical markers for remodeling, and high-resolution structural analyses.

In conclusion, findings from this study revealed tissue-specific involvement of select transporters and hormonal signaling mechanisms potentially influencing functional changes associated with eggshell formation and bone mineralization in laying hens following the onset of egg production. The tissues evaluated exhibited a remarkable capacity to alternate between highly demanding states of increased mineral absorption, retention, or excretion in order to maintain homeostasis between eggshell formation and bone mineralization. Key regulators of mineral homeostasis in kidney and shell gland that were identified represent potential targets for strategies aimed at enhancing mineral utilization by laying hens. In addition, targeted nutritional approaches that improve efficiency of utilization and activation of dietary vitamin D₃ could be used to optimize development of medullary bone, ultimately improving skeletal integrity and hen welfare at all stages of production.

Five Future studies that expand on the current findings should incorporate additional methods such as transcriptomics and metabolomics that allow for a more robust identification of the broader physiological pathways and processes involved. Functional validation of pathways and genes identified by analysis of protein expression and activity, as well as the use of in vitro methods that allow a detailed investigation into molecular and cellular processes, will ultimately provide fundamental information necessary to develop successful strategies allowing hens to efficiently utilize minerals for skeletal remodeling and eggshell deposition throughout all stages of egg production. CONFLICT OF INTEREST

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.