Effect of Treating Hatching Eggs with Aromatase Inhibition in Regulating the Balance of Estrogens- Androgens and Some Physiological Traits of Broiler

The crucial stage at the transformation of estrogen and testosterone (C 19 steroids) into ESTs (C 18 amphetamines) (Al-Jebory and Ibrahim, 2021; Al-Jebory et al., 2024), also known as aromatization, is catalyzed by the enzyme or group of enzymes known as cytochrome P450 as aromatase, a gene governs it, and sexual differentiation depends on the hormone EST and its receptors, identifying a female’s sex, early analysis of a bipotential chick’s embryonic gonads (Shimada, 2002; Ajafar et al., 2024a). Anti-Mullerian hormone (AMH), which testosterone is produced by the testicles, causes survival. The male ducts are alive, although both sexes’ embryonic gonads (testes and ovaries) are present to determine sex in birds in general and domestic birds in particular, Embryonic Anti-Müllerian Hormone (AMH), produced by the testes of developing male embryos, results in the regression of Müllerian (female) ducts which leads to the disappearance of Müllerian (female) ducts, which is known as anti-M; only male fetuses experience Müllerian duct regression due to high levels of AMH. The estradiol (E2) is what gives female chickens their ovarian structure and secondary sexual traits, including short, thin legs and a smaller comb; unlike mammals, male birds have homologous “ZZ” Chromosomes, while female birds have heterozygous “ZW” chromosomes, with additional feathering along the mid-back (Al–Daraji et al., 2010; Matsushita et al., 2006; Salman et al., 2024a). The female genotype’s W chromosome regulates the production of the early aromatase enzyme, which causes the production of ESTs (aromatase regulation is complex and involves gene expression, not just presence of the W chromosome) (Shimada, 1998, 2002). It is well known that male broiler chickens weigh more than females; the male broiler weighs more than the analogs due to this differential in body weight at marketing age, which has a live weight difference between them of approximately 400-500 g because the female reaches the marketable body weight a few days earlier, feed consumption is reduced, leading to a higher economic return (Verapeen and Driver, 2000; Khalil et al., 2022). Therefore, this study aims to investigate the impact of treating hatching eggs (spraying and dipping) with (AIS) on the level of androgen and EST hormones and some physiological traits for the production of commercial broilers.

This study was carried out in the hatchery of Al-Anwar Poultry Company in the Al-Mouradia area, Babylon Governorate for the period 2-17-2022 until 3-8-2022; 300 eggs were incubated and divided into five experimental groups, each containing 60 eggs The eggs used in the experiment were treated before inserting them into the incubators by the following two methods it is the spray and dip method, the hatching eggs groups were divided into five groups which were: G1: Control group without any treatment, G2: Spray eggs with 1 mg/100 ml from (AIS), G3: Dipping eggs with 1 mg/100 ml from (AIS), G4: Spray eggs with 2 mg/100 ml from (AIS), G5: Dipping eggs with 2 mg/100 ml from (AIS).

The second experiment was carried out at the poultry farm in the Department of Animal Production, College of Agriculture, Al-Qasim Green University during the period from 8-3-2022 to 12-4-2022 for 35 days, 180 chicks were selected from the total number of hatched chicks (Chicks were randomly selected from the replicates of hatched eggs mentioned in the first experiment), 36 chicks were placed for each group, the chicks were distributed into 5 groups, with 3 replicates for each group, and each replicate contained 12 chicks. The groups for the hatched chicks were divided according to the first experiment’s groups into five groups and according to what was mentioned in the first experiment.

The chicks were fed three diets: starter, grower and finisher, containing protein (21.29, 19.34, 19.22 %) and energy (3027.37, 3054.67, 3093.47 kacl), respectively. followed the temperature, humidity, and ventilation guide according to the Ross 2023 Breeding Guide. As for the vaccination schedule, follow it according to the epidemiology of the region and as shown in the Table 1.

Table 1: The study vaccination schedule.

Anastrozole a medical preparation was obtained in the form of tablets (pills) manufactured by AstraZeneca, and it was ground and converted into powder using a sensitive balance. The concentrations used in this study were weighed, and after grinding, the concentrations were dissolved in distilled water at the concentrations mentioned in the groups to use.

The eggs were treated according to the concentrations mentioned in the groups above. The dipping process was carried out as follows: The eggs were placed in a container after being removed from the cold storage and left to reach room temperature. A solution containing an AIS was then added to them according to the concentrations mentioned in the groups. They were left in the solution for one minute, then removed to dry completely before being placed in the incubator (Baylan et al., 2018; Alsudani et al., 2025). As for the spraying process, the eggs were placed in an egg tray, and a water sprayer was used to spray the solution on the eggs. They were then left to dry completely before being placed in the incubator (Al-Shammari, 2017; Batkowska et al., 2018). The method of spraying and dipping used for the hatching eggs with an aromatase inhibitor before introducing the eggs into the incubators, assuming that the treated substance would enter through the pores of the egg shell into the egg, as is the case when the embryo breathes, and that the time during the spraying or dipping treatment was sufficient for the aromatase inhibitor to pass through the pores of the shell and for the reproductive system in the embryo to be formed at the age of 6 days. Therefore, it is assumed that the sex of the embryos will change to males and not just a hormonal change. In general, this matter requires a comprehensive histological study to follow the changes in the reproductive organs in the treated embryos.

TES and EST hormone level serum were measured using ELISA technology by a kit) from the Chinese company (Bioassay Technology Laboratory) (Salman et al., 2025).

Blood samples were collected from chicks at the ages of 14 and 21 days from the age of chicks (Days 14 and 21 of the chicks’ ages were used to take blood samples and measure blood parameters to further clarify the possibility of the physiological parameters of the chicks being affected by treatment with AIS and whether any other hormonal or physiological changes occur with age). Reed blood cells are calculated according to Pierson (2000); Arkan et al., (2025).

The level of glucose (GLU), CHO, ALT and AST enzymes, TRI-G, and GPX were calculated by using (Kit) from the German company Roche according to (Coles, 1986; Franney and Elias, 1968; Ritman and Frankel, 1957; Fossati and Prencipe, 1982; Hafemann et al., 1974) respectively, while the (HDL), (LDL), and (VLDL) was calculated according to the LDL concentration according to the equation indicated by Grundy et al. (2004).

Completely random design (CRD) was used to analyze data according (SAS, 2012; Duncan, 1955).

Table 2 and Figures 2, 3 shows the effect of treating hatching eggs with (AIS) on the level of androgen and EST hormones for broiler chickens at the age of 14 and 21 days. There was a significant superiority (P≤0.05) of group G4 compared to groups G1, G2, and G5 at the age of 14 days. At the age of 21 days, group G3 was significantly height (P≤0.05) than the group. G1, G2, and G5 group: The G5 group was significantly height (P≤0.05) than group G1 and G2 in EST concentration at the age of 14, and no significant differences appeared between groups G3 and G4 compared to the rest of the groups, and no significant differences appeared between groups in EST concentration at the age of 21.

Table 2: Effect of (AIS) in TES and EST of broiler chickens (Ross 308).

Means including different litter defer at level *(P≤0.05); NS, Not Significant.

Table 3 indicates that there were no differences between the study groups in the numbers of red and white blood cells at the ages of 14 and 21 days.

Table 4 shows a highly significant decrease (P≤0.0١) in CHO concentration in the G2, G3, and G4 groups compared to the G1 group, and also a highly significant height (P≤0.0١) was obtained in the G2 group compared to the G3 and G4 groups, as well as the G3 group outperformed the G4 group at the age of 14 days, while no significant differences between the groups at the age of 21 days for the same trait.

Table 3: Effect of (AIS) in red and white blood cells of broiler chickens (Ross 308).

NS, Not Significant.

Table 4: Effect of (AIS) in serum CHO of broiler chickens (Ross 308).

Means including different litter defer at level **(P≤0.01): NS, Not Significant.

Table 5 indicates the effect of the studied groups on the level of TRI-G. A highly significant increase (P≤0.0١) in this characteristic is observed in the group G1 compared to the experimental groups. The concentration of TRI-G increased in the group G2 compared to the groups G3 and G5 at the age of 14 days, and at the age of 21 days, it increased significantly. The concentration of TRI-G in group G1 compared to the rest of the groups, and this characteristic increased significantly in group G2 compared to groups G3, G4, and G5, and this trait also increased significantly in group G3 compared to G4 and G5 at the age of 21 days.

Table 5: Effect of (AIS) on the level of TRI-G of broiler chickens (Ross 308).

Means including different litter defer at level **(P≤0.01)

Note from Table 6 that there were no differences in the level of GLU at the age of 14 and 21 days due to the group of hatching eggs with AIS.

Table 6: Effect of (AIS) in GLU of broiler chickens (Ross 308).

NS, Not Significant.

It is noted from Table 7 that there were no significant differences in the level of GPX and the value of peroxide at the age of 14 and 21 days when the hatching eggs were treated with aromatase inhibitors.

Table 7: Effect of (AIS) in GPX level of broiler chickens (Ross 308).

NS, Not Significant.

Controversial data Table 8 indicate that there are no significant differences in the level of liver enzymes in broiler chickens at the age of 14 and 21 days, resulting from the group of hatching eggs with aromatase inhibitors.

Table 8: Effect of (AIS) in AST and ALT enzyme of broiler chickens (Ross 308).

NS, Not Significant.

Most aromatase inhibitors (such as letrozole, anastrozole, and fadrozole) are relatively small, lipophilic compounds (molecular weight 200–300 Da), which means they can penetrate the microscopic pores of the shell. Passive diffusion occurs across the membrane layers toward the lower concentration, provided there is sufficient contact between the shell and the solution for an appropriate period of time (Abdulateef et al., 2021; Han et al., 2023), short immersion (1–3 minutes) in a suitable solute of the aromatase inhibitor provides better penetration and ensures the compound reaches the egg interior, provided the solvent is safe and non-toxic to the embryo (Samson et al., 2020), spraying may also be suitable for rapid practical applications, but requires a higher concentration of the substance to achieve the same effect (Deepika et al., 2017). The increase in testosterone in the aromatase inhibitor groups and the EST hormone in the control group may be due to the role of anastrozole (the aromatase inhibitor) in inhibiting the action of aromatase, and thus the increase in the testosterone and the decrease in the EST, as explained by Ali (2017) when using (AIS) led to inhibition of the aromatase enzyme Thus, an increase in the level of testosterone and a decrease in the level of EST, and (AIS) prevent the conversion of testosterone into EST (Joseph, 2019), as the testosterone hormone turns into EST in a process called aromatization, which is carried out by the aromatase enzyme (Simpson et al., 2002; Bardal et a., 2011; Al-Daraji et al, 2002a, b; Joseph, 2019) and therefore the group of hatching eggs with (AIS)limits this process, as indicated by Vaillant et al. (2001), exposure of embryos to (AIS) before the age of sexual differentiation (4-6 days of embryonic age) leads to sex change in chickens and an increase in testosterone levels. It also reduced the enzyme activity on the fourteenth and eighteenth day after hatching. Many studies have shown that the sex difference between females and males is based on the lack of aromatase and EST. While EST receptors are available in embryos before sexual differentiation, in males, both endocrine pathways and performance factors modulate the steroid hormone receptor, which in turn modulates insulin-like growth factors and estimated glomerular filtration rate (Dabrosin et al., 2002). It reduces cell proliferation in both EST receptor-positive and EST receptor-negative cell lines for the inhibition of aromatase and 17-hydroxysteroid dehydrogenase and collaborates with (AIS) to lessen EST receptor outgrowth (Wang et al., 1994); this explains the process of changing sex from females to males in the hatched chicks Mokarrami (2020), Abdulateef et al. (2021) indicated that injecting hatching eggs with a concentration of 0.1 mg/egg Anastrozole significantly improved the sexual ratio, embryonic growth rate, and hatched chicks characteristics. Anastrozole also reduces blood CHO and triglyceride levels, which improves the physiological status of birds.

Anti-aromatase compounds (anastrozole play roles in sex change, as well as their effect in improving the production traits of broiler chickens through improving physiological indicators, such as reducing the concentration of CHO and TRI-G in groups that were treated with aromatase inhibitors; this could also improve the characteristics of the carcass. The future implications of the study include treating chicken broilers with aromatase inhibitors or early feeding in the egg to increase the percentage of hatched male broilers and improve their subsequent performance, the best groups were G3, G4, and G5.

The authors express their gratitude to Al-Anwar Poultry Company and the Physiology Laboratory at the College of Agriculture, Al-Qasim Green University.

The study received no external funding.

The Animal Production Department Council of Al-Qasim Green University, Iraq approved the study (8-2022)

The authors confirm that they used artificial intelligence in preparing the manuscript, however the mansucript is carefully reviewed by the authors.

The authors have declared no conflict of interest.