Early and mid-embryonic upregulation of chloride, calcium, and sodium transporter genes mark functional maturation of the chorioallantoic membrane in broiler embryos

The chorioallantoic membrane (CAM) is essential for ion transport, acid-base homeostasis, and respiratory gas exchange during chicken embryonic development. The temporal expression of ion transporter-related genes in CAM is still inadequately investigated. This study examined the developmental regulation of genes associated with the transport of calcium (Ca), sodium (Na), bicarbonate (HCO₃ ^-), potassium (K), protons (H⁺), and chloride (Cl^-) in the CAM at embryonic days (ED) 12, 15, and 18. Six hundred fertilized Cobb 500 broiler eggs were obtained from a local hatchery. After candling, on ED 10, a total of 236 eggs were selected for this experiment. All the eggs were incubated in an automated temperature regulation of 37.5°C, 55% relative humidity (RH), and egg rotation every 2 h. On days 12, 15, and 18 of the embryonic period, CAM tissues were collected (n = 6 per group), total RNA isolated, and target genes were analyzed using qPCR. At ED 12, chloride transporter genes (<i>CLCN2, SLC4A1,</i> and <i>SLC26A9</i>) were significantly upregulated compared to ED 18 (<i>P</i> < 0.05). At ED 15, calcium transporters (<i>ATP2A2, ATP2A3, ITPR1, ATP2B4, SLC8A1, SLC8A3, TRPV6,</i> and <i>RYR1</i>) were significantly upregulated compared to ED 12 and/or ED 18 (<i>P</i> < 0.05). Sodium transporters (<i>ATP1A1</i> and <i>SCNN1B</i>), bicarbonate transporters (<i>SLC4A4</i> and <i>SLC4A10</i>), potassium transporters (<i>KCNMA1)</i>, and proton transporters (<i>CA7</i> and <i>CA9</i>) were also significantly upregulated at ED 15 compared to ED 12 (<i>P</i> < 0.05). At ED 18, sodium transporter (<i>ATP1B1, SCNN1A,</i> and <i>SGK1</i>) and proton transporter (<i>CA2</i>) were significantly upregulated compared to ED 12 and/or ED 15 (<i>P</i> < 0.05). The results indicate a synchronized, stage-dependent transcriptional activation of ion transporter genes in the CAM, especially at the mid-embryonic stage.