All animal experimentation followed the regulations approved by the Institutional Animal Care and Use Committee of the University of Hawaii (Approval No. 17-2605-6). This research utilized animal experimentation from our previous studies (Al Amaz et al., 2024a). Briefly, six hundred fertilized Cobb 500 broiler eggs were procured from Asagi Hatchery Inc. (Honolulu, HI). The eggs were randomly allocated among three incubators (GQF Incubator, Savannah, GA), each containing 200 eggs, despite the total capacity of the incubators being 270 eggs. Following candling at ED 10, a total of 474 eggs with viable embryos were chosen for the experiment. From there, 236 eggs were selected for this experiment and transferred to one incubator. The remaining embryos were used for other experiments (Al Amaz et al., 2024a; Al Amaz et al., 2025a; Amaz et al., 2024b; Al Amaz et al., 2024c). All the eggs were incubated according to the standard protocol, which involved automated temperature control at 37.5°C and 55% relative humidity (RH), as well as egg rotation every 2 h.

The CAM tissues were collected on ED 12, ED 15, and ED 18 (n = 6/group). The eggs were initially broken into petri dishes, followed by the euthanization of the embryos through carbon dioxide asphyxiation for sampling purposes. The CAM tissue was collected, rapidly snap-frozen, and stored at −80°C until RNA extraction.

Total RNA was extracted from CAM tissue using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The RNA concentration was quantified using a NanoDrop™ spectrophotometer (ThermoFisher Scientific, Madison, WI) by measuring purity (A260/280∼2.0). The quality of the RNA was determined by running samples on 2% agarose gel. On the gel, high-quality RNA showed two distinct rRNA bands (28S and 18S), with the 28S band approximately twice as intense as the 18S band. The absence of smearing indicated minimal degradation, confirming the RNA was intact and suitable for downstream applications. Then, the RNAs were reverse transcribed into complementary DNA (cDNA), and the target genes were analyzed using qPCR following the established protocol as previously described (Chaudhary et al., 2023; Amaz et al., 2025b). The primer sequences utilized for gene expression analysis are enumerated in Table 1. The NCBI Primer-Blast tool created particular primer pairs for each gene’s detection. A High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) was used to reverse-transcribe 1 μg of total RNA (20 μL of reverse transcription reaction mixture) into cDNA, which was subsequently diluted with nuclease-free water (1:25). qPCR was carried out using the PowerUp SYBR Green Master Mix (Applied Biosystems, Foster City, CA) and real-time PCR equipment (Applied Biosystems). To achieve a final reaction volume of 10 μL, the qPCR reaction mixture included 3 μL of cDNA, 5 μL of PowerUp SYBR Green Master Mix, and 1 μL of each forward and reverse primer at a concentration of 5 μmol. The qPCR reaction was conducted using the standard cycling mode. The qPCR thermal profile consists of an initial denaturation at 95°C for 2 min, followed by 40 cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C for 30 s. A melting curve analysis was conducted to validate the SYBR Green-based amplicon. Furthermore, the specificity of each primer pair was evaluated through 1% gel electrophoresis of the qPCR products. The analysis was performed in triplicate for three housekeeping genes: GAPDH, β-actin, and TBP. The TBP expression was consistently stable across the CAM tissues. Post-amplification, the cycle threshold (Ct) values were documented, and gene expression levels were determined utilizing TBP as the reference gene, following the 2^−ΔΔCT method (Livak and Schmittgen, 2001).

The gene expression was evaluated utilizing GraphPad (GraphPad Software, San Diego, CA). After conducting a one-way analysis of variance (ANOVA), the Tukey-HSD test was employed to compare the means of the different treatment groups. All data are expressed as mean ± Standard error of the mean (SEM). SEM represents the variability of the sample mean and is calculated as the standard deviation (SD) divided by the square root of the sample size (n). The threshold for statistical significance was established at P < 0.05.

The expression pattern of the Ca transporter-related genes (ATP2A2, ATP2A3, ATP2B1, ATP2B2, ATP2B4, ITPR1, RYR1, TRPV6, SLC8A1, and SLC8A3) in the CAM were summarized in Figure 1. The expressions of ATP2A2, ATP2A3, and ITPR1 were significantly increased at ED 15 compared to the other days (P < 0.05). ATP2B4, SLC8A1, and SLC8A3 were significantly lower at ED 12 than at ED 15 (P < 0.05), while TRPV6 was significantly lower at ED 12 compared to the other days (P < 0.05). ATP2B2 was significantly lower at ED 18 compared to the other days (P < 0.05), while RYR1 was significantly lower at ED 18 compared to ED 15 (P < 0.05). ATP2B1 expression remained unchanged across the time points.

The Na transporter-related genes (ATP1A1, ATP1B1, SCNN1A, SGK1, and SCNN1B) are shown in Figure 2. ATP1A1 was significantly higher at ED 15 than in ED 12 (P < 0.05), while SCNN1B was significantly higher at ED 15 compared to the other groups (P < 0.05). ATP1B1 and SGK1 were significantly higher at ED 18 than at ED 12 (P < 0.05). SCNN1A was significantly higher at ED 18 compared to the other groups (P < 0.05).

The HCO₃ ⁻ -transporter-related genes (SLCA4, SLC4A5, SLC4A7, SLC4A8, and SLC4A10) are shown in Figure 3. SLCA4 and SLC4A10 were significantly higher at ED 15 compared to ED 12 and ED 18 (P < 0.05). SLC4A5, SLC4A7, and SLC4A8 expression were not changed at any time points.

The K transporter-related genes (KCNJ15, KCNJ16, and KCNMA1) and H⁺ transporter-related genes (CA2, CA4, CA7, and CA9) are presented in Figures 4A,B. KCNMA1 was significantly higher at ED 15 compared to ED 12 (P < 0.05). CA2 was significantly higher at ED 18 than in the other groups (P < 0.05). CA7 and CA9 were significantly higher at ED 15 compared to ED 12 (P < 0.05). KCNJ15, KCNJ16, and CA4 were not changed at ED12,15 nad 18.

The Cl transporter genes (CLCN2, CLCN5, SLC4A1, SLC26A9, and CFTR) are presented in Figure 5. CLCN2, SLC4A1, and SLC26A9 were significantly higher at ED 12 than at ED 18 (P < 0.05). CLCN5 and CFTR remained unchanged across the time points.

Calcium is reabsorbed from the eggshell and transported to the embryo for bone development throughout the embryonic period (Johnston and Comar, 1955). Transporting calcium from the eggshell into the embryonic circulation requires Ca²⁺-activated ATPase. This membrane-bound enzyme, which necessitates ATP and Mg²⁺ and is activated by Ca²⁺, is specifically situated within the CAM ectoderm, the layer adjacent to the shell. Its activity markedly escalates during phases of swift calcium accumulation, underscoring its significance in skeletogenesis (Tuan and Knowles, 1984). In this study, ATP2A2 and ATP2A3 were significantly higher at ED 15 than at the other time points, while ATP2B4 was significantly higher at ED 15 than at ED 12. However, ATP2B2 was significantly higher at ED 15 and ED 12 than at ED 18. Taken together, it is evident that the peak initiation of calcium transportation happens at ED 12 and then peaks at ED 15 during bone mineralization. This coincides with the known period of active calcium mobilization and mineralization of the embryonic skeleton, which has been documented to intensify between ED12 and ED16 in chickens (Halgrain et al., 2022). IP3R1 encodes ITPR1, a member of the IP3 receptor family (IP3R1–3), and is essential for intracellular calcium signaling because it releases calcium ions into the cytosol from the endoplasmic reticulum (Sun et al., 2025). ITPR1 was significantly higher at ED 15 than in the other groups. The RYR1 codes ryanodine receptor 1, a protein essential for the release of calcium and contraction of skeletal muscle (Alvarellos et al., 2016). Significantly higher ITPR1 and RYR1 expressions at ED 15 indicate heightened intracellular calcium signaling. TRPV6 is crucial in initiating calcium transport in CAM (Huang et al., 2022). The SLC8A1 and SLC8A3, belonging to the solute carrier family 8, encode sodium/calcium exchangers in the plasma membrane. These exchangers function bidirectionally, crucially contributing to calcium homeostasis by facilitating the exchange of intracellular Ca²⁺ and extracellular Na⁺ ions (Gabellini et al., 2002). TRPV6, SLC8A1, and SLC8A3 were significantly higher at ED 15 than ED 12, allowing increased transcellular calcium influx and efflux.

The coordinated expression of Na⁺ ion transport genes precisely controls the regulation of sodium homeostasis in the embryonic CAM. The ATP1A1 is essential for cation transport and generates and maintains the Na+/K+ ion electrochemical gradients throughout the cell membrane (Lin et al., 2021). In this study, ATP1A1 was significantly higher at ED 15 than at ED 12, suggesting enhanced sodium transport activity during mid-embryonic development. The Epithelial Sodium Channel consists of α, β, and γ subunits, encoded by the SCNN1A, SCNN1B, and SCNN1G, corresponding to the sodium channel epithelial 1 subunits α, β, and γ, respectively (Pierandrei et al., 2021). SGK1 regulates sodium retention, potassium excretion, and mineral and nutrient transport across renal, gastric, and intestinal tissues. It also influences salt appetite, blood coagulation, blood pressure, and insulin-dependent glucose uptake (Lang et al., 2006). SGK1 and SCNN1A were significantly higher at ED 18 than ED 12, indicating their role in the terminal phase of ENaC activation and effective sodium absorption in anticipation of hatching, when the demand for sodium transport is at its highest. However, SCNN1B was significantly higher at ED 15 than at the other time points. SCNN1B may facilitate channel assembly at an earlier stage, whereas SCNN1A may serve as a rate-limiting factor for the complete functionality of ENaC, which becomes essential as hatching approaches when the embryo necessitates optimal ion and fluid homeostasis. This temporal separation facilitates the sodium transport pathway’s synchronized accumulation and activation.

The solute carrier family 4 is a crucial protein that mediates various physiological processes, including molecular transduction, protein-to-protein interactions, and the transport of different ions across the cell membrane (Zhong et al., 2023). In chickens, SLC4A4 encodes the electrogenic sodium bicarbonate cotransporter 1. This protein is essential for transporting sodium and bicarbonate ions across cellular membranes (Bahadoran et al., 2018). Acid extrusion is mediated by SLC4A10, which explicitly uses the transmembrane gradient of Na⁺ to promote cellular net uptake of HCO₃ ⁻ (Maroofian et al., 2024). SLC4A4 and SLC4A10 were significantly upregulated at ED 15 compared to the other time points, signifying increased bicarbonate transport and pH buffering during this phase. In neuronal and epithelial tissues, SLC4A8 encodes the Na⁺-driven Cl⁻/HCO₃ ⁻ exchanger, which couples sodium influx with chloride–bicarbonate exchange to regulate intracellular pH and secrete bicarbonate (Remigante et al., 2022). SLC4A5 is a transmembrane protein that operates as an electrogenic cotransporter, facilitating the concurrent transport of HCO₃ ⁻ and Na⁺ ions in the same direction (Stütz et al., 2009). The SLC4A5, SLC4A7, and SLC4A8 showed numerically higher expression at ED 15 compared to the other time points, bolstering the higher bicarbonate-transporting activity during the mid-embryonic phase. These findings underscore a stage-specific activation of bicarbonate transport mechanisms, presumably to accommodate higher metabolic demands and preserve intracellular pH equilibrium during accelerated embryonic development. This aligns with previous studies indicating that embryonic metabolic rate and CO₂ production significantly increase during this window, requiring tight regulation of intracellular pH (Tazawa, 1980).

Potassium voltage-gated channel subfamily J member 16 (KCNJ16) protein encodes the inward rectifier potassium channel Kir5.1, dimerizes with Kir4.2 (encoded by KCNJ15) and Kir4.1 (encoded by KCNJ10) to form functional heteromeric channels that help control membrane potential and potassium ion transport (Zhang et al., 2021). The “Big K+” (BK) large-conductance calcium and voltage-activated K+ channel’s pore-forming α subunit is encoded by KCNMA1. Both excitable and non-excitable cells are found in tissues with a large distribution of BK channels (Bailey et al., 2019). In this study, KCNJ15 expression remained stable across developmental stages, suggesting a constitutive role in baseline K⁺ conductance that may not require transcriptional regulation during this period. In contrast, KCNJ16 showed a trend toward upregulation at ED15, which may reflect a supportive role in enhancing Na⁺/K⁺-ATPase function or accommodating increased ion exchange demands as CAM ion transport activity peaks. This subtle change may indicate post-transcriptional regulation or gradual recruitment of K⁺ channels to meet the increased ionic flux during mid-embryogenesis. However, KCNMA1 was significantly upregulated at ED 15 compared to ED 12. This upregulation indicates a developmentally regulated activation of BK channels, presumably facilitating rapid cellular activity, volume regulation, or signal transduction during the mid-embryonic phase. Carbonic anhydrases (CAs) are widespread metalloenzymes that facilitate the crucial reaction of converting carbon dioxide (CO₂) and water (H₂O) into bicarbonate (HCO₃ ⁻) and protons (H⁺) (Occhipinti and Boron, 2019). The calcium carbonate (CaCO₃) is broken down into Ca²⁺ and HCO₃ ⁻ by the protons generated by CA2, which acidify the region surrounding the eggshell. Specialized cells in the CAM subsequently absorb the released calcium ions and transfer them to the growing chick embryo (Huang et al., 2022). The CA7 may enhance acid-base equilibrium and CO₂ transport in the CAM, which is essential for embryonic gas exchange and pH regulation (Supuran, 2008). The transmembrane enzyme CA9 catalyzes the reversible hydration of CO₂ to bicarbonate and protons, preserving pH homeostasis in hypoxic environments (Swietach et al., 2010). In this study, CA2 was significantly upregulated at ED 18, whereas CA7 and CA9 were significantly upregulated at ED 15 compared to ED 12. The initial increase in CA7 and CA9 suggests an upregulation of respiratory and pH homeostasis facilitation. In contrast, the subsequent induction of CA2 is associated with calcium extraction for osseous development, indicating a sequential functional transition in CA isozyme activity as embryogenesis progresses.

A voltage-gated chloride channel (CLC-2), widely expressed and distributed throughout the body, is encoded by chloride voltage-gated channel 2 (CLCN2). This encoded transmembrane protein is essential for electrogenesis, homeostatic cell volume regulation, and ion gradient maintenance. It also stabilizes chloride ion homeostasis in various cells (Thiemann et al., 1992). The voltage-gated chloride channel (ClC-5) encoded by CLCN5 mainly involves ion transport and endosomal acidification, controlling vesicular trafficking and electrolyte balance in the epithelium (Jentsch and Pusch, 2018). In this study, CLCN2 was significantly upregulated at ED 12 compared to ED 18, suggesting an aid in the electrogenesis and initial stabilization of chloride ions during the early stages of vascular and epithelial growth and development in CAM. In erythrocytes and epithelial tissues, anion exchanger 1, a transmembrane protein that facilitates chloride–bicarbonate exchange and is necessary for CO₂ transportation and pH regulation, is encoded by SLC4A1 (Alper, 2006). SLC26A9 either physically or functionally interacts with the CFTR chloride channel to control CFTR activity or is controlled by CFTR (Remigante et al., 2022). SLC4A1 and SLC26A9 were significantly decreased from ED 12 to ED 18. The decrease in ED 18 expression indicates a developmental shift from chloride-driven acid-base regulation as mineral demands and calcium mobilization become more important in later stages.