Expression of chemerin and its receptors in extra-embryonic annexes and role of chemerin and its GPR1 receptor in embryo development in layer and broiler hens

Abstract

Intensive genetic selection of broiler breeders and layer hens resulted in differences in the mechanisms of growth and also in cell metabolism during embryogenesis. Previous research has shown that an adipokine named chemerin and one of these receptors, CMKLR1 were potentially involved in broiler embryo development. Here, our objectives were 1) to compare the expression of chemerin and its receptors CMKLR1, GPR1, and CCRL2 and chemerin concentration in extra-embryonic annexes (allantoic and amniotic membranes and fluids and plasma) in broiler and layer fertile eggs during the development (embryonic day (ED) 7, 14, and 18) by RT-qPCR and specific chicken ELISA and 2) to investigate the role of chemerin and one of its receptors GPR1 in embryo development after in ovo injections of neutralizing antibodies against chicken chemerin and GPR1. We found that chemerin expression in amniotic membranes was higher in layer than broiler eggs at ED7 and ED14 whereas the expression of the 3 receptors was higher in layer than broiler in the allantoic membranes at ED14 and ED18. Chemerin concentration was more important in layer than broiler at ED14 and ED18 in amniotic liquid and at all the studied stages in blood plasma. We also showed positive correlation between amniotic chemerin concentration and chemerin amniotic membrane expression, chemerin plasma concentration and embryo body weight in both breeds. Finally, in ovo injection of chicken chemerin and GPR1 neutralizing antibodies increased embryo mortality in both layer and broiler eggs. Taken together, even if chemerin concentration and chemerin system expression in embryonic membranes are mainly higher expressed in layer than in broiler, chemerin potentially through GPR1 could promote embryo development in both breeds.

Keywords: broiler; chemerin; embryo annexes; embryo development; layer.

Figure 1

Weight of broiler and layer eggs and embryos during the embryo development. Determination of the egg (A) and embryo (B) weights from 7 (ED7) until 18 (ED18) d of incubation of broiler (n = 9–10 for each ED) and layer (n = 7–10 for each ED) eggs/embryos. Data are shown as the mean ± SEM. Two-ways ANOVA were used to determine an effect of incubation time (T, ED7 until ED18), an effect of genotype (G, layer vs. broiler) and an effect of the interaction between time and genotype (G × T). Different asterisks indicate significant differences between layer and broiler breeds at: *P ≤ 0.05 and ****P < 0.0001.

Figure 2

Weight of broiler and layer organs during the embryo development. Determination of the subcutaneous white adipose tissue (WAT) (A), heart (B), liver (C) and digestive system (D) weights from 14 (ED14), 16 (ED16), and 18 (ED18) embryonic days of broiler (n = 10 for each ED) and layer (n = 7–10 for each ED) embryos. Data are shown as the mean ± SEM. Two-ways ANOVA were used to determine an effect of incubation time (T, ED7 until ED18), an effect of genotype (G, layer vs. broiler) and an effect of the interaction between time and genotype (G × T). Asterisks indicate significant differences between layer and broiler breeds at *P ≤ 0.05.

Figure 3

Chemerin system expression profile in broiler and layer embryonic membranes during the incubation. Expression of chemerin in allantoic (all) and amniotic (amn) membranes of broiler and layer eggs at 3 embryonic days (ED7, ED14, and ED18) determined by RT-qPCR (n = 10 for each breed at each ED). Data are shown as the mean ± SEM. For each embryonic day (ED7, ED14, and ED18), a genotype (G, layer vs. broiler) effect, a membrane (M, all vs. amn) effect, and an interaction between genotype and time (G × M) were calculated. For each embryonic day (ED7, ED14, and ED18), different asterisks indicate significant differences between layer and broiler breeds for each embryonic membrane (all and amn) at *P ≤ 0.05 and ***P < 0.001. For each embryonic day (ED7, ED14 and ED18), different $ indicate significant differences between embryonic membranes (all and amn) for each breed (layer and broiler) at ^$$P < 0.01 and ^$$$$P < 0.0001.

Figure 4

Chemerin receptors expression profile in broiler and layer embryonic membranes during the incubation. Expression of CMKLR1 (A), GPR1 (B), and CCRL2 (C) in allantoic (all) and amniotic (amn) membranes of broiler and layer eggs at 3 embryonic days (ED7, ED14, and ED18) determined by RT-qPCR (n = 10 for each breed at each ED). Data are shown as the mean ± SEM. A genotype (G, broiler vs. layer) effect, a membrane (M, all vs. amn) effect and an interaction effect between genotype and membrane (G × M) were calculated separately at ED7, ED14, and ED18. Different asterisks indicate significant differences between layer and broiler breeds for each embryonic membrane at *P ≤ 0.05, **P < 0.001 and ****P < 0.001 separately at ED7, ED14 and ED18. Different $ indicate significant differences between embryonic membranes (all vs. amn) for each breed (layer and broiler) at ^$$P < 0.01, ^$$$P < 0.001, and ^$$$$P < 0.0001.

Figure 5

Chemerin concentration in extraembryonic fluids (allantoic and amniotic) and in blood plasma during the incubation. Chemerin concentrations in allantoic (all) and amniotic (amn) fluids (A) of broiler and layer eggs at 3 embryonic days (ED7, ED14, and ED18) and in blood plasma (B) at 4 embryonic days (ED11, ED14, ED16, and ED18) determined by ELISA (n = 10 for each breed at each ED). Data are shown as the mean ± SEM. (A) Chemerin concentration in extraembryonic fluids: A genotype (G, broiler vs. layer) effect, a fluid (F, all vs. amn) effect and an interaction effect between genotype and fluid (G × F) were calculated separately at ED7, ED14, and ED18. Different asterisks indicate significant differences between layer and broiler breeds for each embryonic fluid (all and amn) separately at each ED (ED7, ED14, and ED18) at ****P < 0.001. Different $ indicate significant differences between embryonic fluid (all vs. amn) for each breed (layer and broiler) at ^$$$$P < 0.0001. (B) Chemerin concentration in blood plasma: 2-ways ANOVA were used to determine an effect of incubation time (T, ED11 until ED18), an effect of genotype (G, layer vs. broiler) and an effect of the interaction between time and genotype (G × T). Different asterisks indicate significant differences between layer and broiler breeds at ****P < 0.0001.

Figure 6

Effect of in ovo injections of neutralizing chicken chemerin and GPR1 antibodies on embryo mortality rates. Percentage of embryo mortality of fertilized layer and broiler eggs injected at ED7 with about 100 μL of Immunoglobulin G (IgG, n = 100 eggs for each breed), phosphate buffer saline (PBS, n = 100 eggs for each breed) and different amounts of antichemerin (A, B, n = 100 eggs for each breed and each amount of antibodies) and anti-GPR1 (C, D, n = 100 eggs for each breed and each amount of antibodies) antibodies (0.01, 0.1, and 1 μg). Embryo mortality was measured by candling and breaking eggs at E14. Data are shown as the mean ± SEM and different letters indicate significant differences treatments at P ≤ 0.05.