Chemerin abundance in egg white and its expression with receptors in extra-embryonic annexes of Pekin ducks: implications for embryo development

Abstract

Embryonic mortality is a significant problem in the commercial duck industry worldwide. Therefore, identification of new biomarkers for duck embryo development is necessary. In the chicken (order Galliformes), we previously showed that chemerin is a hormone locally produced by the reproductive tract in hens, particularly in the magnum area, leading to its accumulation in the egg white and within the embryo annexes during embryonic development. We therefore hypothesized that the chemerin concentration in egg white could be a biomarker of egg performance and reproductive parameters in Pekin ducks (order Anseriformes). Thus, we collected eggs from Pekin ducks over a 5-d period at three stages of the laying period (before the laying peak, after the laying peak, and at the end of the laying period) to measure the chemerin concentrations in egg white by enzyme-linked immunosorbent assay. The chemerin concentration in egg white decreased during the laying period and was not associated with reproductive parameters. We found negative correlations between the chemerin level in egg white and the albumen weight. Reverse-transcriptase quantitative polymerase chain reaction showed that chemerin and its three receptors CMKLR1, GPR1, and CCRL2 were expressed in the reproductive tract and within allantoic and amniotic annexes during embryo development. Chemerin concentrations strongly increased in amniotic fluid on embryonic day 16 (ED16) when the egg white was transferred into the amniotic sac. Finally, chemerin inhibition in egg white by in ovo injections of anti-chemerin antibodies (0.01, 0.1, and 1 µg) increased the embryo mortality rate. These data demonstrate the important role of the chemerin system during egg formation and embryo development in Pekin ducks, suggesting their potential use as biomarkers for determining the quality of poultry eggs and embryo development.

Keywords: Pekin; chemerin; duck; egg white; female reproduction.

Figure 1

Experimental design. Fifty ducks were followed at three time points of the laying period: before the laying peak, after the laying peak, and at the end of laying. For each time point and for 5 successive laying days, eggs were collected to weigh the whole egg, egg white (albumen), and yolk individually. For each egg, an albumen sample was stored at −20°C until use.

Figure 2

Egg performances during the laying cycle. (A) Egg, (B) yolk, and (C) albumen weights are shown during the laying cycle (n = 42 eggs). Data are shown as mean ± SEM. Different letters indicate significant laying period effects.

Figure 3

Chemerin concentration in duck egg white during laying cycle. The concentration of chemerin in egg white during different periods of laying was determined by ELISA. Data are shown as mean ± SEM. Different letters indicate a significant laying period effect.

Figure 4

Expression of chemerin and its receptors in magnum of oviduct of ducks. Relative mRNA expression of (A) RARRES2 (chemerin gene) and its three receptors (B) CMKLR1, (C) GPR1, and (D) CCRL2 in the magnum of the duck oviduct quantified by RT-qPCR (n = 5). Values are expressed as mean ± SEM. Different letters indicate significant differences between parts of the reproductive tract.

Figure 5

Weight of duck embryos during development. Determination of embryo weights from ED9 to ED26 in duck embryos (n = 10 for each ED). Data are shown as mean ± SEM. Different letters indicate significant differences between incubation times.

Figure 6

Chemerin concentrations in extraembryonic fluids (allantoic and amniotic) and blood plasma during incubation. Chemerin concentrations (A) in allantoic and amniotic fluids of duck eggs on ED9, ED16, and ED24 and (B) in blood plasma on ED16, ED20, ED24, and ED26 as determined by ELISA (n = 10 on each ED. Data are shown as mean ± SEM. (A) Chemerin concentrations in extraembryonic fluids: a fluid effect F (allantoic vs. amniotic), a time effect T of incubation, and an interaction effect between fluid and time (F × T) were calculated. Different asterisks indicate significant differences between extraembryonic fluids at each ED at ****p < 0.0001. (B) Chemerin concentrations in blood plasma: different letters indicate significant differences during embryonic days.

Figure 7

Expression of chemerin and its receptors in duck extraembryonic membrane and extraembryonic fluids (allantoic and amniotic) during incubation. Expression of (A) chemerin, (B) CMKLR1, (C) GPR1, and (D) CCRL2 in allantoic and amniotic fluids of duck eggs on ED9, ED16, and ED24 determined by RT-qPCR (n = 10 on each ED). Data are shown as mean ± SEM. A membrane effect M (allantoic vs. amniotic), a time effect T of incubation, and an interaction effect between membrane and time (M × T) were calculated. Different asterisks indicate significant differences between extraembryonic fluids at each ED at ****p < 0.0001.

Figure 8

Effect of in ovo injections of neutralizing duck chemerin and receptor antibodies on embryo mortality rates. Embryo mortality rates of fertilized duck eggs injected on ED9 with 100 µL of mouse IgG (1μg), PBS, and different amounts of anti-chemerin antibodies (0.01, 0.1, and 1 µg) (63–85 eggs for each treatment). Embryo mortality was measured by candling and breaking eggs on ED26. Data are shown as mean ± SEM. Different letters indicate significant differences between treatments at p ≤ 0.05.