Corticosterone in ovo Injection Effects on the Development of Iraqi Native Chicken Embryos

Corticosterone is a major glucocorticoid hormone in the plasma of birds (Pu et al., 2019). It is produced in the adrenal gland and transferred to the eggs after 1-2 days of circulation in females  by distributing within the yolk (Almasi, 2012). It has been found that avian eggs contain a different maternal level of corticosterone as reported in domestic fowl (Groothuis et al., 2005; Rettenbacher et al., 2005) and Japanese quail (Hayward et al., 2006). This concentration is modified by different factors such as hen physiological status (Reed and Clark, 2011) and environmental conditions (Hayward et al., 2004). It is found that corticosterone serum levels increased with high environmental temperature via increasing levels of adrenal steroidogenic enzymes that led to an increase in the yolk (Pu et al., 2019). In general, glucocorticoid concentrations in blood plasma are widely used to monitor stress response in different species (Von Holst, 1988).
 
The levels of parents’ blood plasma corticosterone concentration in the yolk are influenced via passive diffusion (Hayward and Wingfielde, 2004). Corticosterone concentration in egg yolk and albumin differ between eggs produced from slow or fast-growing broiler chicks (Ahmed, 2013). Yolk corticosterone level also differs between breeds. Navara and Pinsion (2010) reported that corticosterone concentration levels in yolk were twice as higher in white hens than in brown hen’s eggs. Also, Ahmed (2016) recorded significantly differing yolk corticosterone levels between white Leghorn and Hy-Line brown eggs. Corticosterone high concentration orally gives a significant increase in yolk corticosterone (Almasi et al., 2012).
 
In contrast, parents’ injection of ACTH failed to induce detectable corticosterone deposition in chicken eggs (Rettenbacher et al., 2005). Early life experience or poor environments form a risk factor for post-hatch stress life disturbance by elevation of egg corticosterone that influences offspring growth rate, behavior and gene expression (Ahmed, 2016). The elevation of yolk corticosterone concentration makes a growth decrease in males but not female chicks with the reduced HPA axis responsiveness of adult females compared to males (Hayward et al., 2006). Few articles have focused on the effects of egg corticosterone levels in Iraqi native chick embryonic development; therefore, we conducted this study to evaluate the role of corticosterone hormone in the embryonic development of Iraqi native chickens.
 

In this study, we used 300 Iraqi native chicken eggs from 30-week-old breeds reared in the research poultry station, Abu Ghraib, Baghdad. Eggs were divided into five treatments and three replicates (20 eggs each). The injection surface was sterilized with an antiseptic (Dettol). Eggs were injected from the wide side with a micropipette by puncture (13 mlm) in the air sac. Then every egg was injected with corticosterone hormone (Sigma-Aldrich, 98.5%) dissolved with sesame oil as follows: Control group eggs were placed in the incubator without injection. Sesame oil group: Injected with 5 µl of sterile sesame oil only. Group 1: Injected with 0.25 ng corticosterone dissolved with 5 µl of sterile sesame oil. Group 2: Injected with 0.5 ng corticosterone dissolved with 5 µl of sterile sesame oil. Group 3: Injected one ng corticosterone dissolved with 5 µl of sterile sesame oil.
       
Punctures were closed by using dye pedicures. Eggs were incubated in an egg incubator (Weiqian 1050 brand) by distributed the groups randomly. The embryonic test was checked per the method described by Abdulateef (2010) described. After 72 hours of egg incubation, the egg was horizontally placed for 20 minutes, then checked for shell cutoff and test embryo length, vascular region and pairs of somites. The second test was conducted after seven days of incubation by taking out the egg contents after the shell breakout to study new plasma weight, allantois membrane and fluid weight, amnion fluid and amniotic sac albumin, yolk weight, embryo weight and shell weight. The third embryonic test was conducted after 14 days of incubation by breaking the eggs shell, wherein the contents of the egg were taken out. The weights of the shell, embryo, yolk and yolk sac, amniotic fluid and amniotic membrane, allantois fluid and allantois membrane and albumin were recorded. The fourth embryonic test was conducted at 17 days of incubation and the weight of the egg, shell, embryo, yolk and yolk sac, amniotic fluid and amniotic sac and allantois and chorion membrane with fluids were recorded. Hatchability from fertile eggs was measured as the number of hatched eggs/numbers of fertile eggs set in the incubator.
       
After hatching, early embryonic mortality (before seven days embryo mortality during incubation), intermediate embryo mortality (7-14 days embryo mortality during incubation), late embryonic mortality (14-17 days embryo mortality during incubation), life and dead pipped eggs were determined as a percentage of fertile eggs.
 
Statistical analysis
 
Complete random design (CRD) inside five medicines and three repeats were utilized in this analysis. Information was examined by utilizing the GLM model strategy of SAS (Statistical analysis system) (Fernandez, 2010). Means for treatments are thought about by utilizing Duncan’s polynomial utilizing different significance levels to decide massive contrasts between the averages (Duncan, 1955).
 

Table 1 shows a significant increase (P<0.05) in embryo length for (0.5 ng/5 µL sesame oil) bunch contrasted and control gathering and there are no massive contrasts in other embryonic development parameters at 72 hours of incubation.


 
Table 2 shows a significant decrease (P<0.05) in amniotic fluid and amniotic weight percentage for (0.5 ng/5 µL sesame oil) in the gathering contrasted and the benchmark group and there are no huge contrasts in other embryonic development parameters at seven days of incubation.


 
Table 3 shows no significant differences in all embryonic development parameters between all treatments and the control group at 14 days of incubation.


 
Table 4 shows a significant decrease (P<0.05) in amniotic fluid and amniotic sac membrane percentage for (1 ng/5 µL sesame oil) in the gathering contrasted and the benchmark group. The results showed no significant differences in all embryonic development parameters between all treatments and the control group in 17 days of incubation.


 
Fig 1 shows a significant decrease (P<0.05) in hatchability percentage for corticosterone (1 ng/5 µL sesame oil) compared with the control group.


 
In Table 5, the results indicate that significant decrease (P<0.05) in the early embryonic mortality percentage of the (1 ng/5 µL sesame oil) group compared with the control group. The results showed no significant differences between all treatments and control groups in intermediate and last embryo mortality stage and dead and life pipped eggs.


 
Vassallo et al., (2014) showed that only 0.4% of corticosterone injected in quail Coturnix japonica eggs reached embryos, with the first being metabolized. Offspring can be exposed to maternal glucocorticoid deposition into the yolk or through placental transfer, then make interaction with embryonic tissues (Hayward et al., 2006; Love and Williams, 2008). In addition, maternal stress effects can arise without direct exposure to glucocorticoids in embryos (Carter et al., 2018). In this study, a decrease in the weight of allantoic membrane fluid, hatchability percentage and increase in the percentage of embryonic mortality for the high doses of corticosterone groups is a result of corticosterone affection in the chicken embryo. The embryos are capable of metabolizing corticosterone with less than 1% of the original dose accumulated in embryos (Carter et al., 2018).
 
In addition, the results indicate that corticosterone injection increases with embryos’ height compared with the control group. This increase may be due to the role of increasing growth hormone concentration in embryos. These results agree with Yu et al., (2018), who showed that low doses of corticosterone in eggs significantly induce goose embryos’ somatotroph differentiation. In addition, corticosterone makes for stimulates and differentiates somatotroph differentiation of chicken embryonic development (Sato and Watanabe, 1998). In vitro corticosterone increases the number of cells that secrete growth hormones in chicken embryo pituitary cell cultures (Bossis et al., 2004). In addition, the high concentration of corticosterone makes to induce growth hormone secretion by e14 of chicken embryos, as it turns out that a single inovo injection of corticosterone increased the level of GHmrna and plasma growth hormone significantly in pituitary somatotrophs and blood (Yu et al., 2018).
 
But it seems that a high dose of corticosterone concentration in an egg may cause embryonic abnormality and death in the early and late stages of embryonic development (Al-Bayar, 2016). Because the high concentration of corticosterone in eggs has poisoning effects on avian embryos and may negatively affect embryonic biological systems (Pavlik et al., 1986; Mashaly, 1991; Kaltner et al., 1993), among breeds may there is a different ability to bear difficult and stressful situations and may be differences in the embryonic ability to corticosterone metabolism because low concentrations of maternal corticosterone in unmanipulated yolks can metabolize maternal corticosterone in natural systems to avoid fitness consequences (Carter et al., 2018). So, we need to do more research to study the ability of Iraqi native chicken embryos to arrive at safe levels of egg corticosterone concentration.
 

In conclusion, a high concentration of corticosterone hormone in Iraqi native chicken hatching eggs causes an increase in embryo length, amniotic fluid and amniotic sac weight percentage and decreasing in amniotic fluid, amniotic sac membrane percentage, hatchability and early embryonic mortality percentage.
 

The authors wish to thank the College of Agriculture, University of Anbar and Environment Research Center, University of Technology, Iraq, for their valuable support and scientific assistance.