Effect of heat stress on the gene expression of ion transporters/channels in the uterus of laying hens during eggshell formation

Figures & data

Table 1. Functions of uterine genes potentially involved in eggshell formation.

Name	Gene symbol	function
Transient receptor potential cation channel subfamily V member 6	TRPV6	Selective Ca^2+ channel (plasma membrane)
Calbindin 28 K	CALB1	Ca^2+ transporter (intracellular)
IP3 receptor3	ITPR3	a membrane glycoprotein complex acting as a Ca^2+ channel activated by inositol trisphosphate
Amiloride-sensitive sodium channel subunit gamma	SCNN1G	Non-voltage-gated Na^+ channel
Solute carrier family 4 member 4	SLC4A4	Na^+/HCO3^− co-transporter (plasma membrane)
Inward rectifier potassium channel 5	KCNJ15	voltage-gated K^+ selective ion channel (plasma membrane)
Solute carrier family 4 member 9	SLC4A9	Cl^−/HCO3^− exchanger
Chloride channel protein 2	CLCN2	Cl^− channel (plasma membrane)

Figure 1. Diagram of the processes in shell formation, with roles of genes measured in the present study.

Figure 2. Specificity of real time PCR amplification. Melting curves (dissociation curves) of the 8 target genes and 1 reference gene (β-actin) amplicons after the real time PCR reactions, all showing one peak. X-axis (horizontal): temperature (°C); Y-axis (vertical): negative derivative of fluorescence over temperature (−dF/dT).

Table 2. Primers used for quantitative real time PCR analysis of mRNAs in laying hen uterus.

Target	Primers sequence	PCR product	Accession No.
TRPV6	5′-CGCCACCCTCCTTATGCTCA-3′	145 bp	XM_004938142.2
	3′-CCAGAGACACCGTGGCAGTT-5′
CALB1	5′-TCTGGCACCACTACGACTCC-3′	148 bp	NM_205513.1
	3′-GCCTTGCCATACTGGTCCAC-5′
ITPR3	5′-CCGAGGTGGAGACCTTTGTC-3′	75 bp	XM_015298987.1
	3′-ACAGGTCCGAGAGGTAGTCC-5′
SCNN1G	5′-GGTGACATCGACAGGAGCCA-3′	131 bp	XM_015294500.1
	3′-GCTTGCGGGACCGTGTAAAG-5′
SLC4A4	5′-CGGCACCAGACCAAGAAGTC-3′	65 bp	XM_015276405.1
	3′-GCACTGGAGACGGTCTTTCC-5′
KCNJ15	5′-CTCAAAGCGGGAGCCATGTG-3′	73 bp	XM_425554.5
	3′-CTGCGACTCCAGAGAGAAGAGG-5′
SLC4A9	5′-CGAGGACTACGGTCTGGACT-3′	109 bp	XM_015293554.1
	3′-GTACTGCACCAGGTGACTCG-5′
CLCN2	5′-CGAAGAGCGTGGTCTGGGAT-3′	140 bp	XM_015291544.1
	3′-GGGTGCCACTGTCTTCATGC-5′
β-Actin	5′-AGCGAACGCCCCCAAAGTTCT-3′	139 bp	NM_205518.1
	3′-AGCTGGGCTGTTGCCTTCACA-5′

Table 3. Parameters of egg quality in last week of experiment.

	Number of hens	Egg weight (g)	Egg surface area (cm^2)	Egg volume (cm^3)	Eggshell weight (g)	Eggshell thickness (μm)	Eggshell ash weight (g)	Eggshell calcium (%)
Control	20	53.5 ± 1.4	78.75 ± 1.9	65.6 ± 1.6	7.3 ± 0.4	357.6 ± 25.2	7.1 ± 0.4	34.1 ± 0.1
Heat stress	17	39.1 ± 0.9*	53.03 ± 1.1*	36.7 ± 1.2*	4.5 ± 0.1*	244.1 ± 3.7*	4.3 ± 0.1*	30.3 ± 0.8*
p Value	–	0.031	0.029	0.025	0.013	0.018	0.009	0.010

Values are as mean ± standard error of mean (SEM). *p < 0.05 from corresponding control (statistically analyzed by t-tests).

Table 4. Relative gene expression of ion transporters in the uterine lining (glands + epithelium) of laying hens.

	Relative gene expression		Ratio (heat stress/control)	Pooled SEM
	Control	Heat stress	p Value
TRPV6	0.0033	0.0031	0.94	0.001	0.826
CALB1	0.027	0.925*	34.2	0.025	0.044
ITPR3	0.927	0.358*	0.39	0.114	0.044
SCNN1G	0.139	0.196	1.41	0.099	0.767
SLC4A4	2.726	0.925*	0.34	0.421	0.038
KCNJ15	0.289	0.21	0.73	0.091	0.639
SLC4A9	8.737	2.696*	0.31	1.082	0.032
CLCN2	0.598	0.481	0.8	0.015	0.719

Values are means. *p < .05 from corresponding control (statistically analyzed by t-tests; *significant differences, p < .05, in bold). Pooled SEM, pooled standard error of mean. Number of laying hens in each group =10. Values are relative to β-actin mRNA.