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 (HCO 3 − ), 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 ( CLCN2, SLC4A1, and SLC26A9 ) were significantly upregulated compared to ED 18 ( P < 0.05). At ED 15, calcium transporters ( ATP2A2, ATP2A3, ITPR1, ATP2B4, SLC8A1, SLC8A3, TRPV6, and RYR1 ) were significantly upregulated compared to ED 12 and/or ED 18 ( P < 0.05). Sodium transporters ( ATP1A1 and SCNN1B ), bicarbonate transporters ( SLC4A4 and SLC4A10 ), potassium transporters ( KCNMA1) , and proton transporters ( CA7 and CA9 ) were also significantly upregulated at ED 15 compared to ED 12 ( P < 0.05). At ED 18, sodium transporter ( ATP1B1, SCNN1A, and SGK1 ) and proton transporter ( CA2 ) were significantly upregulated compared to ED 12 and/or ED 15 ( P < 0.05). The results indicate a synchronized, stage-dependent transcriptional activation of ion transporter genes in the CAM, especially at the mid-embryonic stage.