Advancing Embryonic Growth through Genetic Stimulation to Enhance Development in Chicken Embryos

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Abdulateef This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7087632/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Embryonic dormancy, a common issue during critical stages of development, leads to halted or slowed growth, negatively impacting the viability and weight of chicks. This study aims to reduce or eliminate this dormancy by using targeted genetic stimulation to enhance embryonic development. The study was conducted at the hatchery affiliated with the Department of Animal Production, Faculty of Agriculture, University of Anbar, with the objective of investigating embryonic dormancy at different developmental stages and improving overall efficacy. A total of 720 eggs from the Rose 308 breed were used the experiment was divided into six treatments, each replicated three times, across two incubators. The following treatments were applied: T1 (negative control, no injection), T2 (positive control, injection with distilled water), T3 (injection of siRNA targeting the SERPINA6 gene on day 3 of incubation), T4 (injection of siRNA targeting the SERPINA6 gene on day 7 of incubation), T5 (injection of siRNA targeting the SERPINA6 gene on day 14 of incubation), and T6 (injection of siRNA targeting the SERPINA6 gene on day 17 of incubation). The findings showed that the sixth treatment (T6) had a notably positive impact on embryo development characteristics when compared to the other treatments, including increased embryo weight and length, improved yolk and albumin consumption, and a significant increase in shell thickness (P ≤ 0.05). The findings suggest that using natural stimuli, such as siRNA, can effectively improve embryo development and increase chick weight at hatching. Embryonic dormancy siRNA SERPINA6 gene embryo development 1. Introduction The success and growth in the poultry industry begin with proper attention to the embryo within the egg. This initial phase is crucial for achieving optimal weight gain, accelerating growth, and ultimately increasing production at the marketing stage. chick embryos spend approximately 33–38% of their development time in the incubator, where the egg’s contents are transformed into a chick. The advent of In-Ovo technology in the 1980s significantly enhanced this process by allowing direct delivery of vaccines to the embryos [1]. However, a common issue arises as chicks enter a state of dormancy, particularly during the first critical stage of incubation at around five days, coinciding with the formation of the nervous system. This dormancy leads to a cessation or slowing down of vital processes, resulting in impaired growth of the embryos [2]. A similar halt in development can occur during the final stages of hatching due to the stress associated with the hatching process. Such interruptions negatively impact production outcomes, leading to weaker birds with reduced weights at the marketing stage, ultimately causing financial losses for breeders [3]. Given these challenges, current research has shifted towards understanding and enhancing incubation and embryonic development in chickens, recognizing that any errors during the incubation stage can adversely affect overall productivity [4]. The dormancy observed in embryos during these critical periods, particularly before hatching, can potentially be managed through the use of specific biostimulants or genetic interventions [5]. These methods may stimulate or suppress the expression of particular genes, such as the suppression of the SERPINA6 gene, which restricts the activity of the hormone corticosterone and reduces gene expression [6]. Given these challenges, current research has shifted towards understanding and enhancing incubation and embryonic development in chickens, recognizing that any errors during the incubation stage can adversely affect overall productivity [7]. The dormancy observed in embryos during these critical periods, particularly before hatching, can potentially be managed through the use of specific biostimulants or genetic interventions [8]. These methods may stimulate or suppress the expression of particular genes, such as the suppression of the SERPINA6 gene, which restricts the activity of the hormone corticosterone and reduces gene expression [9]. Small interfering RNA (siRNA) serves as an efficient method for gene silencing, effectively preventing the overexpression of targeted genes [10]. The SERPINA6 gene encodes the corticosteroid-binding globulin (CBG) protein, which is primarily synthesized in the liver [11]. Corticosteroid-binding globulin (CBG) binds to cortisol, a hormone that plays a crucial role in regulating blood glucose levels, protecting the body from stress, and reducing inflammation. When cortisol is bound to CBG, its activity is inhibited [12]. Under normal conditions, 80–90% of the body's cortisol is bound to CBG, with 5–10% remaining unbound and biologically active, while the rest is associated with albumin. CBG transports cortisol to target tissues, where it releases the hormone to become active and ready for physiological use [13]. In this case, CBG regulates the availability of cortisol in the body. Altering this mechanism through gene silencing may enhance embryonic activity, breaking the dormancy observed in embryos [14]. This study aims to identify the critical stages of embryo development, enhance their activity, and overcome the dormancy experienced during these stages. By optimizing the utilization of nutrients from the egg's contents, the goal is to achieve robust and healthy growth, resulting in chicks with higher weights at hatching. This improvement in early development is expected to positively influence marketing outcomes, yielding birds with desirable weights and enhancing profitability for breeders. 2. Materials and Methods This experiment was conducted at the hatchery of the faculty of Agriculture, University of Anbar. A total of 720 eggs from the ROS 308 strain were used. The average weight of the eggs was 54 g. Before placing the eggs in the incubator, the gas incubator was preheated to the appropriate temperature, and the humidity was adjusted to prevent shocking the eggs. In this study, siRNA primers designed for the SERPINA6 gene were sourced from OLIGO in South Korea. The primers were provided as a highly concentrated freeze-dried powder, with the sequence as follows: 5’-AACATATATAAAATACAGTCTTCAAT-3’ (25MER), consisting of 24 to 25 base pairs. The preparation involved adding 300 microliters of distilled water to the siRNA, followed by mixing using a vortex device for 30 seconds [15]. From the mixed solution, 10 microliters were taken and diluted to 240 microliters with distilled water in a sterile tube. Each egg was injected with 5 microliters of the prepared solution. This preparation process was repeated for each injection. The prepared injection solutions were kept at a temperature of at least 30°C to avoid embryo shock, and the injection chamber was heated to 37°C with a humidity level of 65% [5]. The first injection was performed on the second positive treatment group using distilled water, while the first treatment group served as the negative control with no injection. The injection area on the egg was sterilized with alcohol and cotton, and the egg was punctured using a drill that was also sterilized with alcohol. Care was taken to avoid fracturing the egg or damaging the blood vessels. A 1 ml syringe was used for the injections, administering 5 microliters per egg. After injection, the puncture site was sealed tightly with an insulating material, and the eggs were placed in the incubator [16]. The treatment is as follows: The positive control treatment group was injected with distilled water into the air chamber. The third treatment involved injecting siRNA into the air chamber on the third day of incubation, with 5 microliters administered per egg. The fourth, fifth, and sixth treatments involved injecting siRNA on the 7th, 14th, and 17th days of incubation, respectively, with the injection made in the right lateral area midway through the yolk sac [17]. The experimental conditions were as follows: the temperature was set to 37.8°C with 65% humidity until the 18th day of incubation. After day 18, the temperature was reduced to 37.6°C, and humidity was increased to 80% as the eggs were transferred to hatching baskets. Additional evaporators were used to raise the humidity during this period [18]. After 3 days of incubation, the eggs were placed horizontally for 20 minutes and then examined for shell integrity, Transparent area/mm embryo length/mm, Blood vessels diameter/mm, and the number of somite pairs. A second assessment took place seven days into incubation, The egg contents were extracted by carefully cracking the shell, allowing for the measurement of the new plasma weight, along with the weights of the allantoic membrane and its fluid. Additionally, measurements were taken for the amniotic fluid and sac, albumen, yolk, embryo, and shell. A subsequent evaluation was performed after 14 days of incubation, during which the shell was broken to extract the contents. This evaluation included the measurement of the weights of the shell, embryo, yolk, yolk sac, amniotic fluid, membranes, and allantoic fluid. A fourth assessment was carried out at 17 days of incubation, during which the weights of the egg, shell, embryo, yolk, yolk sac, amniotic fluid, and the allantois and chorion membranes, along with their respective fluids, were measured and recorded [17, 19]. The study utilized a completely randomized design (CRD) comprising five treatments, each with three replicates. Data were analyzed using the General Linear Model (GLM) in the Statistical Analysis System (SAS), following the methodology outlined by [20]. Treatment means were compared using Duncan's multiple range test at various significance levels to identify significant differences between averages [21]. 3. Results and Discussion Table 1 : shows the effect of genetic stimulation on embryonic growth and development and early embryonic development indicators on 3 days of Incubation. There were no significant differences observed in blood vessel diameter (mm) among the six treatments, transparent area (mm), Somite number, and relative shell weight. However, a significant difference in embryo length (mm) was noted, with the T5 treatment showing the most favorable results, which outperformed all other treatments at the (P ≤ 0.05) significance level. This treatment involves the application of siRNA injection, resulting in an embryo length of 12.6 mm. Table 1 Early Embryonic Development Indicators on Day 3 of Incubation Trait (T1) (T2) (T3) (T4) (T5) (T6) P-Value Transparent area (mm) 30.3 ± 1.45* 34.3 ± 1.45 35.0 ± 1.52 31.3 ± 0.33 31.6 ± 2.96 32.33 ± 1.20 NS** Blood Vessels (mm) 16.3 ± 1.76 19.6 ± 0.33 22.0 ± 1.15 19.0 ± 0.57 19.0 ± 1.73 20.33 ± 0.33 NS Embryo Length (mm) 9.33 ± 0.33 c 11.0 ± 0.57 abc 10.3 ± 0.33 bc 10.6 ± 0.33 abc 12.6 ± 0.88 a 11.66 ± 0.88 ab 0.0341 Number of Somite 32.66 ± 1.85 30.0 ± 0 33.6 ± 2.33 35.33 ± 1.45 35.0 ± 1.00 34.66 ± 1.66 NS Sell (g) 7.33 ± 0.33 7.33 ± 0.33 6.66 ± 0.33 7.33 ± 0.33 7.66 ± 0.33 7.66 ± 0.33 NS “*Values are expressed as mean ± standard error.” ** NS: Indicates no significant differences between treatments at a significance level of (P ≤ 0.05). Different letters (a, b, c) within the same row indicate significant differences between treatments at a significance level of (P ≤ 0.05). Table 2 : Effect of genetic stimulation on embryonic growth and development, along with mid-term embryonic growth parameters measured on the seventh day of incubation, the table indicates a significant advantage in relative albumin weight for the T1 and T2 treatments (control treatments) compared to the other treatments, with a significance level of (P ≤ 0.05), with relative albumin weights of 42.95 g and 42.14 g, respectively. Furthermore, the relative shell weights of T1 and T4 were significantly higher than those of the other treatments, with values of 16.64 g and 16.2 g, respectively, at a significance level of (P ≤ 0.05). In contrast, no significant differences were observed in relative yolk weight, relative embryo weight, or the weights of the allantois and amnion (including both membrane and fluid) among the various treatments. Table 2 Mid-Term Embryonic Growth Parameters on Day 7 of Incubation Trait (T1) (T2) (T3) (T4) (T5) (T6) P-value Albumin (g) 42.95 ± 3.76a 42.14 ± 0.78a 40.72 ± 2.77ab 40.2 ± 1.44abc 34.75 ± 1.07bc 33.38 ± 1.58c 0.0386 Yolk (g) 30.33 ± 1.60 24.56 ± 1.57 28.54 ± 1.02 30.63 ± 3.33 26.66 ± 0.97 27.73 ± 3.56 N.S. Embryo Weight (g) 1.81 ± 0.14 1.75 ± 0.05 2.04 ± 0.100 2.59 ± 0.43 2.48 ± 0.20 2.96 ± 0.50 N.S. Amnion (g) 3.96 ± 0.22 3.48 ± 0.55 3.89 ± 0.16 4.35 ± 0.19 4.11 ± 0.42 3.16 ± 0.41 N.S. Allantois (g) 25.0 ± 1.51 22.0 ± 1.19 23.9 ± 2.01 27.4 ± 2.82 29.82 ± 1.92 29.13 ± 0.40 N.S. Shell (g) 16.64 ± 0.32a 15.2 ± 0.57ab 15.20 ± 0.90ab 16.2 ± 0.62a 14.06 ± 0.49b 13.39 ± 0.22b 0.0126 “*Values are expressed as mean ± standard error.” ** NS: Indicates no significant differences between treatments at a significance level of (P ≤ 0.05). Different letters (a, b, c) within the same row indicate significant differences between treatments at a significance level of (P ≤ 0.05). Table 3 : The effect of genetic stimulus on embryonic growth and development and advanced embryonic growth metrics on the fourteenth day of incubation is evident from the table. The T1 treatment showed significant superiority over all other treatments at the (P ≤ 0.05) significance level in the relative albumin weight, reaching 16.96 g. The T6 treatment also significantly outperformed the other treatments at the (P ≤ 0.05) significance level in relative embryo weight, recording 35.23 g. Additionally, the T6 treatment showed significant superiority in the relative weight of the amnion, recording 14.10 g, over all other treatments at a significance level of (P ≤ 0.05). For the relative shell weight, the T1 treatment showed significant superiority over the other treatments, recording 14.59 g. No significant differences were observed in the relative weight of the allantois or the relative yolk weight among the different treatments at the (P ≤ 0.05) significance level. Table 3 Advanced Embryonic Growth Metrics on Day 14 of Incubation Trait (T1) (T2) (T3) (T4) (T5) (T6) P-Value Albumin (g) 16.96 ± 0.30a 14.58 ± 0.66ab 12.86 ± 0.81bc 12.44 ± 0.11bc 10.46 ± 1.34cd 9.38 ± 0.89d 0.0003 Yolk (g) 31.10 ± 1.56 28.17 ± 1.53 30.97 ± 1.04 29.76 ± 0.29 30.68 ± 1.71 29.00 ± 0.77 N.S. Embryo Weight (g) 25.47 ± 0.47d 28.09 ± 1.87cd 28.54 ± 0.82cd 31.0 ± 0.21bc 32.34 ± 1.18ab 35.23 ± 1.07a 0.0005 Amnion (g) 10.59 ± 0.83b 8.75 ± 0.62b 10.39 ± 0.71b 10.62 ± 0.39b 10.90 ± 0.92b 14.10 ± 1.49a 0.0268 Allantois (g) 12.35 ± 2.34 16.94 ± 2.10 18.41 ± 4.26 14.19 ± 0.77 13.65 ± 1.01 15.41 ± 0.72 N.S. Shell (g) 14.59 ± 0.75a 13.1 ± 1.29ab 11.38 ± 0.24bc 10.39 ± 0.42c 10.11 ± 0.94c 9.86 ± 0.13c 0.0041 “*Values are expressed as mean ± standard error.” ** NS: Indicates no significant differences between treatments at a significance level of (P ≤ 0.05). Different letters (a, b, c) within the same row indicate significant differences between treatments at a significance level of (P ≤ 0.05). Table 4 : The effect of genetic stimulation on embryonic growth and development and late-stage embryonic development characteristics on the seventeenth day of incubation is clear from the table. The T1 treatment showed significant superiority over all other treatments at a significance level of (P ≤ 0.05) in relative yolk weight, which amounted to 33.16 g. The T6 treatment also showed significant superiority over all other treatments at the (P ≤ 0.05) significance level in relative embryo weight, recording 59.8 g. Additionally, the T1 treatment recorded significant superiority in relative shell weight, with a value of 12.9 g. There were no significant differences in the relative weight of the allantois and amnion among all treatments at a significance level of (P ≤ 0.05). Table 4 Late-Stage Embryonic Development Characteristics on Day 17 of Incubation Trait (T1) (T2) (T3) (T4) (T5) (T6) P-Value Yolk (g) 33.16 ± 1.04a 28.40 ± 0.68bc 30.4 ± 1.26b 26.74 ± 0.52cd 25.5 ± 0.14d 25.1 ± 0.52d 0.0001 Embryo (g) 41.95 ± 1.90d 48.33 ± 1.02c 49.1 ± 1.51c 54.80 ± 1.90b 54.4 ± 0.82b 59.8 ± 0.64a 0.0001 Amnion (g) 5.11 ± 0.69 10.09 ± 2.76 6.95 ± 1.23 7.65 ± 1.92 8.05 ± 0.19 8.60 ± 0.33 N.S. Allantois (g) 6.73 ± 0.42 9.33 ± 1.08 10.16 ± 1.73 8.21 ± 1.31 10.6 ± 0.89 10.2 ± 0.43 N.S. Shell (g) 12.9 ± 1.15a 11.13 ± 1.18ab 11.42 ± 0.06ab 10.0 ± 0.80bc 9.66 ± 0.31bc 8.36 ± 0.60c 0.0231 “*Values are expressed as mean ± standard error.” ** NS: Indicates no significant differences between treatments at a significance level of ((P ≤ 0.05)). Different letters (a, b, c) within the same row indicate significant differences between treatments at a significance level of ((P ≤ 0.05)). Injecting siRNA into chicken embryos has proven highly effective in stimulating embryonic growth by silencing target genes, thereby enhancing fetal activity and reducing dormancy that may hinder development [22]. This technique elevates free cortisol levels in the body by silencing the SERPINA6 gene, which encodes the corticosteroid-binding globulin (CBG) [12]. Silencing this gene reduces CBG levels, thereby increasing free cortisol, a hormone essential for metabolism and energy supply to developing tissues [11]. This increase in free cortisol stimulates metabolic processes within the embryo, promoting the consumption of nutrients, particularly albumen, which is converted into energy and new tissues, contributing to increased embryonic weight and reduced albumen and shell mass [17]. The upregulation of proteins such as C-fos and C-JUN is a direct consequence of siRNA-induced genetic stimulation. C-fos is essential for cell differentiation and development and also plays a role in promoting angiogenesis, enhancing blood flow and oxygen delivery to growing tissues within the embryo [23]. C-JUN is critical during the early phases of embryonic development, aiding in the growth of neurons and glial cells, which are crucial for the proper formation of the central nervous system. This genetic stimulation accelerates embryo growth and improves cellular differentiation, resulting in enhanced morphological characteristics [24]. An increase in amniotic fluid volume, particularly on the fourteenth day of incubation, is indicative of improved embryonic development. Amniotic fluid plays a vital role in protecting the embryo from adhering to the eggshell, thereby providing an ideal environment for growth and reducing the risk of developmental abnormalities or mechanical damage. This fluid acts as a barrier between the embryo and the shell, maintaining the integrity of the embryo and enhancing its ability to utilize nutrients effectively [25]. Genetic stimulation through siRNA injection also regulates neural metabolism within the embryo. Acetylcholine, a critical neurotransmitter, is secreted in greater quantities as a result of genetic stimulation, which promotes neurodevelopment and the growth of neural structures [26]. Acetylcholine is essential for regulating neurological and cellular functions, ensuring the proper development of the nervous system. Increased acetylcholine secretion improves neuronal and morphological functions, promoting neuron proliferation, differentiation, and development in embryos [27]. Genetic stimulation not only affects physical development but also promotes neurodevelopment by increasing the secretion of neurotransmitters such as acetylcholine [26]. Acetylcholine is crucial for the development of the central nervous system, serving as a primary neurotransmitter at synapses. It binds to receptors on neurons, triggering calcium and sodium ion influxes, which initiate electrochemical signals that drive the formation of axons and dendrites, essential components for nerve signal transmission between cells [28]. Furthermore, Acetylcholine facilitates neurodevelopment by stimulating the activation of neurotrophic factors such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Improved acetylcholine secretion also promotes synaptogenesis [29], the process by which connections between neurons are formed, enhancing neural communication efficiency and ensuring proper nervous system development [30]. The complete consumption of nutrients, including albumen and yolk, by the embryo indicates the success of genetic stimulation in enhancing metabolic processes [14]. This ensures the embryo receives all necessary elements for growth, contributing to increased weight and improved bone formation and muscle tissue development [31]. The utilization of mineral elements from the shell, such as calcium and phosphorus, is crucial for skeletal formation, indicating that genetic stimulation has enhanced the absorption of these minerals, promoted bone formation and hardened [32]. Furthermore, genetic stimulation increases blood flow to the pituitary gland, which is crucial for promoting the secretion of growth hormones such as growth hormone (GH). GH is vital for regulating overall embryonic development by binding to GH receptors on specific cells and activating multiple signaling pathways [33], This includes the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway, which triggers the activation of genes involved in producing proteins that promote tissue growth and enhance body mass, such as insulin-like growth factor-1 (IGF-1) [34]. IGF-1, primarily secreted by the liver in response to GH stimulation, promotes cell division and proliferation, particularly in bones and muscles. IGF-1 enhances bone matrix formation and muscle protein synthesis, leading to significant increases in bone and muscle mass [35]. Additionally, GH and IGF-1 together stimulate lipolysis in adipose tissue, providing an additional energy source for tissue growth [36]. Thus, genetic stimulation through siRNA technology not only enhances the physical development of embryos but also promotes neurodevelopment, leading to the birth of chicks with robust physiological and neurological characteristics [37]. This improves their chances of survival and adaptation post-hatching. The effects of genetic stimulation using siRNA clearly indicate that this technique significantly contributes to enhancing embryonic growth and development by increasing metabolic activity and the efficient consumption of nutrients within the egg. The results suggest that siRNA technology can be effectively utilized to improve hatching rates and produce stronger, healthier chicks, thereby enhancing poultry production efficiency and breeder profitability [5]. 4. Conclusions The study demonstrated that injecting siRNA into chicken embryos significantly improved embryonic development by silencing the SERPINA6 gene and increasing free cortisol levels, thereby boosting metabolic processes and nutrient consumption. Genetic stimulation also enhanced the expression of proteins critical for angiogenesis and neuronal development, improving the central nervous system's development. Additionally, the increase in growth hormone and neurotransmitter release contributed to the growth of bone and muscle tissue. Based on these findings, siRNA technology can be considered an effective tool for improving hatching rates and increasing poultry productivity. Declarations Author Contribution Oady Lafthali:Conceptualization; Methodology; Data Collection; Laboratory Work; Formal Analysis; Visualization; Drafting the Original Manuscript.Prof. Dr. Salwan M. 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Available from: https://www.sciencedirect.com/science/article/pii/S0032579124005248 Vaccaro LA, Porter TE, Ellestad LE. The Effect of Commercial Genetic Selection on Somatotropic Gene Expression in Broilers: A Potential Role for Insulin-Like Growth Factor Binding Proteins in Regulating Broiler Growth and Body Composition. Front Physiol. 2022;13. Vaccaro LA, Herring K, Wilson A, England E, Smith AL, Ellestad LE. Dynamic changes in insulin-like growth factor binding protein expression occur between embryonic and early post-hatch development in broiler chickens. Poult Sci [Internet]. 2024;104174. Available from: https://www.sciencedirect.com/science/article/pii/S0032579124007533 Chang HS, Lin CH, Chen YC, Yu WCY. Using siRNA technique to generate transgenic animals with spatiotemporal and conditional gene knockdown. American Journal of Pathology. 2004;165(5):1535–41. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7087632","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":483530196,"identity":"778ad8e6-d5fb-400c-9ab1-c629e4e0faa3","order_by":0,"name":"Oady Lafthali","email":"","orcid":"","institution":"University of Anbar","correspondingAuthor":false,"prefix":"","firstName":"Oady","middleName":"","lastName":"Lafthali","suffix":""},{"id":483530197,"identity":"df41de11-528e-4955-a23c-c514a3ba23d6","order_by":1,"name":"Salwan M. Abdulateef","email":"data:image/png;base64,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","orcid":"","institution":"University of Anbar","correspondingAuthor":true,"prefix":"","firstName":"Salwan","middleName":"M.","lastName":"Abdulateef","suffix":""}],"badges":[],"createdAt":"2025-07-09 23:38:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7087632/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7087632/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86747421,"identity":"d35fc070-cb93-4ec9-b8bf-bd591f5999ff","added_by":"auto","created_at":"2025-07-15 07:54:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":462605,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7087632/v1/cc5e9816-3720-415f-a074-b6d77a67d532.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Advancing Embryonic Growth through Genetic Stimulation to Enhance Development in Chicken Embryos","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe success and growth in the poultry industry begin with proper attention to the embryo within the egg. This initial phase is crucial for achieving optimal weight gain, accelerating growth, and ultimately increasing production at the marketing stage. chick embryos spend approximately 33\u0026ndash;38% of their development time in the incubator, where the egg\u0026rsquo;s contents are transformed into a chick. The advent of In-Ovo technology in the 1980s significantly enhanced this process by allowing direct delivery of vaccines to the embryos [1].\u003c/p\u003e\u003cp\u003eHowever, a common issue arises as chicks enter a state of dormancy, particularly during the first critical stage of incubation at around five days, coinciding with the formation of the nervous system. This dormancy leads to a cessation or slowing down of vital processes, resulting in impaired growth of the embryos [2]. A similar halt in development can occur during the final stages of hatching due to the stress associated with the hatching process. Such interruptions negatively impact production outcomes, leading to weaker birds with reduced weights at the marketing stage, ultimately causing financial losses for breeders [3].\u003c/p\u003e\u003cp\u003eGiven these challenges, current research has shifted towards understanding and enhancing incubation and embryonic development in chickens, recognizing that any errors during the incubation stage can adversely affect overall productivity [4]. The dormancy observed in embryos during these critical periods, particularly before hatching, can potentially be managed through the use of specific biostimulants or genetic interventions [5]. These methods may stimulate or suppress the expression of particular genes, such as the suppression of the SERPINA6 gene, which restricts the activity of the hormone corticosterone and reduces gene expression [6]. Given these challenges, current research has shifted towards understanding and enhancing incubation and embryonic development in chickens, recognizing that any errors during the incubation stage can adversely affect overall productivity [7]. The dormancy observed in embryos during these critical periods, particularly before hatching, can potentially be managed through the use of specific biostimulants or genetic interventions [8]. These methods may stimulate or suppress the expression of particular genes, such as the suppression of the SERPINA6 gene, which restricts the activity of the hormone corticosterone and reduces gene expression [9]. Small interfering RNA (siRNA) serves as an efficient method for gene silencing, effectively preventing the overexpression of targeted genes [10]. The SERPINA6 gene encodes the corticosteroid-binding globulin (CBG) protein, which is primarily synthesized in the liver [11]. Corticosteroid-binding globulin (CBG) binds to cortisol, a hormone that plays a crucial role in regulating blood glucose levels, protecting the body from stress, and reducing inflammation. When cortisol is bound to CBG, its activity is inhibited [12]. Under normal conditions, 80\u0026ndash;90% of the body's cortisol is bound to CBG, with 5\u0026ndash;10% remaining unbound and biologically active, while the rest is associated with albumin. CBG transports cortisol to target tissues, where it releases the hormone to become active and ready for physiological use [13]. In this case, CBG regulates the availability of cortisol in the body. Altering this mechanism through gene silencing may enhance embryonic activity, breaking the dormancy observed in embryos [14].\u003c/p\u003e\u003cp\u003eThis study aims to identify the critical stages of embryo development, enhance their activity, and overcome the dormancy experienced during these stages. By optimizing the utilization of nutrients from the egg's contents, the goal is to achieve robust and healthy growth, resulting in chicks with higher weights at hatching. This improvement in early development is expected to positively influence marketing outcomes, yielding birds with desirable weights and enhancing profitability for breeders.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003eThis experiment was conducted at the hatchery of the faculty of Agriculture, University of Anbar. A total of 720 eggs from the ROS 308 strain were used. The average weight of the eggs was 54 g. Before placing the eggs in the incubator, the gas incubator was preheated to the appropriate temperature, and the humidity was adjusted to prevent shocking the eggs. In this study, siRNA primers designed for the SERPINA6 gene were sourced from OLIGO in South Korea. The primers were provided as a highly concentrated freeze-dried powder, with the sequence as follows: 5\u0026rsquo;-AACATATATAAAATACAGTCTTCAAT-3\u0026rsquo; (25MER), consisting of 24 to 25 base pairs. The preparation involved adding 300 microliters of distilled water to the siRNA, followed by mixing using a vortex device for 30 seconds [15]. From the mixed solution, 10 microliters were taken and diluted to 240 microliters with distilled water in a sterile tube. Each egg was injected with 5 microliters of the prepared solution. This preparation process was repeated for each injection. The prepared injection solutions were kept at a temperature of at least 30\u0026deg;C to avoid embryo shock, and the injection chamber was heated to 37\u0026deg;C with a humidity level of 65% [5]. The first injection was performed on the second positive treatment group using distilled water, while the first treatment group served as the negative control with no injection. The injection area on the egg was sterilized with alcohol and cotton, and the egg was punctured using a drill that was also sterilized with alcohol. Care was taken to avoid fracturing the egg or damaging the blood vessels. A 1 ml syringe was used for the injections, administering 5 microliters per egg. After injection, the puncture site was sealed tightly with an insulating material, and the eggs were placed in the incubator [16].\u003c/p\u003e\u003cp\u003eThe treatment is as follows: The positive control treatment group was injected with distilled water into the air chamber. The third treatment involved injecting siRNA into the air chamber on the third day of incubation, with 5 microliters administered per egg. The fourth, fifth, and sixth treatments involved injecting siRNA on the 7th, 14th, and 17th days of incubation, respectively, with the injection made in the right lateral area midway through the yolk sac [17].\u003c/p\u003e\u003cp\u003eThe experimental conditions were as follows: the temperature was set to 37.8\u0026deg;C with 65% humidity until the 18th day of incubation. After day 18, the temperature was reduced to 37.6\u0026deg;C, and humidity was increased to 80% as the eggs were transferred to hatching baskets. Additional evaporators were used to raise the humidity during this period [18].\u003c/p\u003e\u003cp\u003eAfter 3 days of incubation, the eggs were placed horizontally for 20 minutes and then examined for shell integrity, Transparent area/mm embryo length/mm, Blood vessels diameter/mm, and the number of somite pairs. A second assessment took place seven days into incubation, The egg contents were extracted by carefully cracking the shell, allowing for the measurement of the new plasma weight, along with the weights of the allantoic membrane and its fluid. Additionally, measurements were taken for the amniotic fluid and sac, albumen, yolk, embryo, and shell. A subsequent evaluation was performed after 14 days of incubation, during which the shell was broken to extract the contents. This evaluation included the measurement of the weights of the shell, embryo, yolk, yolk sac, amniotic fluid, membranes, and allantoic fluid. A fourth assessment was carried out at 17 days of incubation, during which the weights of the egg, shell, embryo, yolk, yolk sac, amniotic fluid, and the allantois and chorion membranes, along with their respective fluids, were measured and recorded [17, 19].\u003c/p\u003e\u003cp\u003eThe study utilized a completely randomized design (CRD) comprising five treatments, each with three replicates. Data were analyzed using the General Linear Model (GLM) in the Statistical Analysis System (SAS), following the methodology outlined by [20]. Treatment means were compared using Duncan's multiple range test at various significance levels to identify significant differences between averages [21].\u003c/p\u003e"},{"header":"3. Results and Discussion","content":"\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e: shows the effect of genetic stimulation on embryonic growth and development and early embryonic development indicators on 3 days of Incubation. There were no significant differences observed in blood vessel diameter (mm) among the six treatments, transparent area (mm), Somite number, and relative shell weight. However, a significant difference in embryo length (mm) was noted, with the T5 treatment showing the most favorable results, which outperformed all other treatments at the (P\u0026thinsp;\u0026le;\u0026thinsp;0.05) significance level. This treatment involves the application of siRNA injection, resulting in an embryo length of 12.6 mm.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEarly Embryonic Development Indicators on Day 3 of Incubation\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTrait\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e(T1)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(T2)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(T3)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(T4)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e(T5)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e(T6)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eP-Value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTransparent area (mm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e30.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e34.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e35.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e31.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e31.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e32.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNS**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBlood Vessels (mm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e16.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e19.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e22.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e19.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e19.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e20.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEmbryo Length (mm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33 c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57 abc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33 bc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33 abc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e12.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e11.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.0341\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of Somite\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e32.66\u0026thinsp;\u0026plusmn;\u0026thinsp;1.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e33.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e35.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e35.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e34.66\u0026thinsp;\u0026plusmn;\u0026thinsp;1.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSell (g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e7.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e7.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"8\"\u003e\u0026ldquo;*Values are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error.\u0026rdquo;\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e** NS: Indicates no significant differences between treatments at a significance level of (P\u0026thinsp;\u0026le;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003eDifferent letters (a, b, c) within the same row indicate significant differences between treatments at a significance level of (P\u0026thinsp;\u0026le;\u0026thinsp;0.05).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e: Effect of genetic stimulation on embryonic growth and development, along with mid-term embryonic growth parameters measured on the seventh day of incubation, the table indicates a significant advantage in relative albumin weight for the T1 and T2 treatments (control treatments) compared to the other treatments, with a significance level of (P\u0026thinsp;\u0026le;\u0026thinsp;0.05), with relative albumin weights of 42.95 g and 42.14 g, respectively. Furthermore, the relative shell weights of T1 and T4 were significantly higher than those of the other treatments, with values of 16.64 g and 16.2 g, respectively, at a significance level of (P\u0026thinsp;\u0026le;\u0026thinsp;0.05). In contrast, no significant differences were observed in relative yolk weight, relative embryo weight, or the weights of the allantois and amnion (including both membrane and fluid) among the various treatments.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMid-Term Embryonic Growth Parameters on Day 7 of Incubation\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTrait\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e(T1)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(T2)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(T3)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(T4)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e(T5)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e(T6)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eP-value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAlbumin (g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e42.95\u0026thinsp;\u0026plusmn;\u0026thinsp;3.76a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e42.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e40.72\u0026thinsp;\u0026plusmn;\u0026thinsp;2.77ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e40.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.44abc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e34.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07bc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e33.38\u0026thinsp;\u0026plusmn;\u0026thinsp;1.58c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.0386\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eYolk\u003c/p\u003e\u003cp\u003e(g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e30.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e24.56\u0026thinsp;\u0026plusmn;\u0026thinsp;1.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e28.54\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e30.63\u0026thinsp;\u0026plusmn;\u0026thinsp;3.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e26.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e27.73\u0026thinsp;\u0026plusmn;\u0026thinsp;3.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eN.S.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEmbryo Weight (g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eN.S.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAmnion (g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eN.S.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAllantois (g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e25.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e22.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e23.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e27.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e29.82\u0026thinsp;\u0026plusmn;\u0026thinsp;1.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e29.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eN.S.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eShell\u003c/p\u003e\u003cp\u003e(g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e16.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e15.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.90ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e16.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e14.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e13.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.0126\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"8\"\u003e\u0026ldquo;*Values are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error.\u0026rdquo;\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e** NS: Indicates no significant differences between treatments at a significance level of (P\u0026thinsp;\u0026le;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003eDifferent letters (a, b, c) within the same row indicate significant differences between treatments at a significance level of (P\u0026thinsp;\u0026le;\u0026thinsp;0.05).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e: The effect of genetic stimulus on embryonic growth and development and advanced embryonic growth metrics on the fourteenth day of incubation is evident from the table. The T1 treatment showed significant superiority over all other treatments at the (P\u0026thinsp;\u0026le;\u0026thinsp;0.05) significance level in the relative albumin weight, reaching 16.96 g. The T6 treatment also significantly outperformed the other treatments at the (P\u0026thinsp;\u0026le;\u0026thinsp;0.05) significance level in relative embryo weight, recording 35.23 g. Additionally, the T6 treatment showed significant superiority in the relative weight of the amnion, recording 14.10 g, over all other treatments at a significance level of (P\u0026thinsp;\u0026le;\u0026thinsp;0.05). For the relative shell weight, the T1 treatment showed significant superiority over the other treatments, recording 14.59 g. No significant differences were observed in the relative weight of the allantois or the relative yolk weight among the different treatments at the (P\u0026thinsp;\u0026le;\u0026thinsp;0.05) significance level.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAdvanced Embryonic Growth Metrics on Day 14 of Incubation\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTrait\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e(T1)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(T2)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(T3)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(T4)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e(T5)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e(T6)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eP-Value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAlbumin\u003c/p\u003e\u003cp\u003e(g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e16.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e14.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e12.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81bc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e12.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11bc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10.46\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34cd\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e9.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89d\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.0003\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eYolk\u003c/p\u003e\u003cp\u003e(g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e31.10\u0026thinsp;\u0026plusmn;\u0026thinsp;1.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e28.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e30.97\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e29.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e30.68\u0026thinsp;\u0026plusmn;\u0026thinsp;1.71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e29.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eN.S.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEmbryo Weight (g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e25.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47d\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e28.09\u0026thinsp;\u0026plusmn;\u0026thinsp;1.87cd\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e28.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82cd\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e31.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21bc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e32.34\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e35.23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.0005\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAmnion\u003c/p\u003e\u003cp\u003e(g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e14.10\u0026thinsp;\u0026plusmn;\u0026thinsp;1.49a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.0268\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAllantois\u003c/p\u003e\u003cp\u003e(g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e12.35\u0026thinsp;\u0026plusmn;\u0026thinsp;2.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e16.94\u0026thinsp;\u0026plusmn;\u0026thinsp;2.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e18.41\u0026thinsp;\u0026plusmn;\u0026thinsp;4.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e14.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e13.65\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e15.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eN.S.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eShell\u003c/p\u003e\u003cp\u003e(g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e14.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e13.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e11.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24bc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e9.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.0041\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"8\"\u003e\u0026ldquo;*Values are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error.\u0026rdquo;\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e** NS: Indicates no significant differences between treatments at a significance level of (P\u0026thinsp;\u0026le;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003eDifferent letters (a, b, c) within the same row indicate significant differences between treatments at a significance level of (P\u0026thinsp;\u0026le;\u0026thinsp;0.05).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e: The effect of genetic stimulation on embryonic growth and development and late-stage embryonic development characteristics on the seventeenth day of incubation is clear from the table. The T1 treatment showed significant superiority over all other treatments at a significance level of (P\u0026thinsp;\u0026le;\u0026thinsp;0.05) in relative yolk weight, which amounted to 33.16 g. The T6 treatment also showed significant superiority over all other treatments at the (P\u0026thinsp;\u0026le;\u0026thinsp;0.05) significance level in relative embryo weight, recording 59.8 g. Additionally, the T1 treatment recorded significant superiority in relative shell weight, with a value of 12.9 g. There were no significant differences in the relative weight of the allantois and amnion among all treatments at a significance level of (P\u0026thinsp;\u0026le;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eLate-Stage Embryonic Development Characteristics on Day 17 of Incubation\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTrait\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e(T1)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(T2)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(T3)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(T4)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e(T5)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e(T6)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eP-Value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eYolk\u003c/p\u003e\u003cp\u003e(g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e33.16\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e28.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68bc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e30.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52cd\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e25.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14d\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e25.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52d\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.0001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEmbryo\u003c/p\u003e\u003cp\u003e(g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e41.95\u0026thinsp;\u0026plusmn;\u0026thinsp;1.90d\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e48.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e49.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.51c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e54.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.90b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e54.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e59.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.0001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAmnion\u003c/p\u003e\u003cp\u003e(g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10.09\u0026thinsp;\u0026plusmn;\u0026thinsp;2.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.95\u0026thinsp;\u0026plusmn;\u0026thinsp;1.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e7.65\u0026thinsp;\u0026plusmn;\u0026thinsp;1.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e8.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eN.S.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAllantois\u003c/p\u003e\u003cp\u003e(g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10.16\u0026thinsp;\u0026plusmn;\u0026thinsp;1.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e8.21\u0026thinsp;\u0026plusmn;\u0026thinsp;1.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eN.S.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eShell\u003c/p\u003e\u003cp\u003e(g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e12.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e11.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80bc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e9.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31bc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e8.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.0231\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"8\"\u003e\u0026ldquo;*Values are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error.\u0026rdquo;\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e** NS: Indicates no significant differences between treatments at a significance level of ((P\u0026thinsp;\u0026le;\u0026thinsp;0.05)).\u003c/p\u003e\u003cp\u003eDifferent letters (a, b, c) within the same row indicate significant differences between treatments at a significance level of ((P\u0026thinsp;\u0026le;\u0026thinsp;0.05)).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eInjecting siRNA into chicken embryos has proven highly effective in stimulating embryonic growth by silencing target genes, thereby enhancing fetal activity and reducing dormancy that may hinder development [22]. This technique elevates free cortisol levels in the body by silencing the SERPINA6 gene, which encodes the corticosteroid-binding globulin (CBG) [12]. Silencing this gene reduces CBG levels, thereby increasing free cortisol, a hormone essential for metabolism and energy supply to developing tissues [11]. This increase in free cortisol stimulates metabolic processes within the embryo, promoting the consumption of nutrients, particularly albumen, which is converted into energy and new tissues, contributing to increased embryonic weight and reduced albumen and shell mass [17].\u003c/p\u003e\u003cp\u003eThe upregulation of proteins such as C-fos and C-JUN is a direct consequence of siRNA-induced genetic stimulation. C-fos is essential for cell differentiation and development and also plays a role in promoting angiogenesis, enhancing blood flow and oxygen delivery to growing tissues within the embryo [23]. C-JUN is critical during the early phases of embryonic development, aiding in the growth of neurons and glial cells, which are crucial for the proper formation of the central nervous system. This genetic stimulation accelerates embryo growth and improves cellular differentiation, resulting in enhanced morphological characteristics [24].\u003c/p\u003e\u003cp\u003eAn increase in amniotic fluid volume, particularly on the fourteenth day of incubation, is indicative of improved embryonic development. Amniotic fluid plays a vital role in protecting the embryo from adhering to the eggshell, thereby providing an ideal environment for growth and reducing the risk of developmental abnormalities or mechanical damage. This fluid acts as a barrier between the embryo and the shell, maintaining the integrity of the embryo and enhancing its ability to utilize nutrients effectively [25].\u003c/p\u003e\u003cp\u003eGenetic stimulation through siRNA injection also regulates neural metabolism within the embryo. Acetylcholine, a critical neurotransmitter, is secreted in greater quantities as a result of genetic stimulation, which promotes neurodevelopment and the growth of neural structures [26]. Acetylcholine is essential for regulating neurological and cellular functions, ensuring the proper development of the nervous system. Increased acetylcholine secretion improves neuronal and morphological functions, promoting neuron proliferation, differentiation, and development in embryos [27].\u003c/p\u003e\u003cp\u003eGenetic stimulation not only affects physical development but also promotes neurodevelopment by increasing the secretion of neurotransmitters such as acetylcholine [26]. Acetylcholine is crucial for the development of the central nervous system, serving as a primary neurotransmitter at synapses. It binds to receptors on neurons, triggering calcium and sodium ion influxes, which initiate electrochemical signals that drive the formation of axons and dendrites, essential components for nerve signal transmission between cells [28].\u003c/p\u003e\u003cp\u003eFurthermore, Acetylcholine facilitates neurodevelopment by stimulating the activation of neurotrophic factors such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Improved acetylcholine secretion also promotes synaptogenesis [29], the process by which connections between neurons are formed, enhancing neural communication efficiency and ensuring proper nervous system development [30].\u003c/p\u003e\u003cp\u003eThe complete consumption of nutrients, including albumen and yolk, by the embryo indicates the success of genetic stimulation in enhancing metabolic processes [14]. This ensures the embryo receives all necessary elements for growth, contributing to increased weight and improved bone formation and muscle tissue development [31]. The utilization of mineral elements from the shell, such as calcium and phosphorus, is crucial for skeletal formation, indicating that genetic stimulation has enhanced the absorption of these minerals, promoted bone formation and hardened [32].\u003c/p\u003e\u003cp\u003eFurthermore, genetic stimulation increases blood flow to the pituitary gland, which is crucial for promoting the secretion of growth hormones such as growth hormone (GH). GH is vital for regulating overall embryonic development by binding to GH receptors on specific cells and activating multiple signaling pathways [33], This includes the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway, which triggers the activation of genes involved in producing proteins that promote tissue growth and enhance body mass, such as insulin-like growth factor-1 (IGF-1) [34].\u003c/p\u003e\u003cp\u003eIGF-1, primarily secreted by the liver in response to GH stimulation, promotes cell division and proliferation, particularly in bones and muscles. IGF-1 enhances bone matrix formation and muscle protein synthesis, leading to significant increases in bone and muscle mass [35]. Additionally, GH and IGF-1 together stimulate lipolysis in adipose tissue, providing an additional energy source for tissue growth [36].\u003c/p\u003e\u003cp\u003eThus, genetic stimulation through siRNA technology not only enhances the physical development of embryos but also promotes neurodevelopment, leading to the birth of chicks with robust physiological and neurological characteristics [37]. This improves their chances of survival and adaptation post-hatching. The effects of genetic stimulation using siRNA clearly indicate that this technique significantly contributes to enhancing embryonic growth and development by increasing metabolic activity and the efficient consumption of nutrients within the egg. The results suggest that siRNA technology can be effectively utilized to improve hatching rates and produce stronger, healthier chicks, thereby enhancing poultry production efficiency and breeder profitability [5].\u003c/p\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eThe study demonstrated that injecting siRNA into chicken embryos significantly improved embryonic development by silencing the SERPINA6 gene and increasing free cortisol levels, thereby boosting metabolic processes and nutrient consumption. Genetic stimulation also enhanced the expression of proteins critical for angiogenesis and neuronal development, improving the central nervous system's development. Additionally, the increase in growth hormone and neurotransmitter release contributed to the growth of bone and muscle tissue. Based on these findings, siRNA technology can be considered an effective tool for improving hatching rates and increasing poultry productivity.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eOady Lafthali:Conceptualization; Methodology; Data Collection; Laboratory Work; Formal Analysis; Visualization; Drafting the Original Manuscript.Prof. Dr. Salwan M. Abdulateef:Supervision; Project Administration; Validation; Writing \u0026ndash; Review \u0026amp; Editing; Resources; Scientific Guidance and Final Approval of the Manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eLizio M, Deviatiiarov R, Nagai H, Galan L, Arner E, Itoh M, et al. Systematic analysis of transcription start sites in avian development. PLoS Biol. 2017;15(9).\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eAbdulateef SM, Atalla OK, Q A L-Ani M, Mohammed THT, Abdulateef FM, Abdulmajeed OM. Impact of the electric shock on the embryonic development and physiological traits in chicks embryo. Vol. 90, Indian Journal of Animal Sciences. 2020.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eAbdulateef SM, Atalla OK, Al-Ani MQ, Mohammed THT, Abdulateef FM, Abdualmajeed OM, et al. The effect of the electric shock on embryonic development and neurophysiological traits in the chick\u0026rsquo;s embryo. 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American Journal of Pathology. 2004;165(5):1535\u0026ndash;41. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Embryonic dormancy, siRNA, SERPINA6 gene, embryo development","lastPublishedDoi":"10.21203/rs.3.rs-7087632/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7087632/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEmbryonic dormancy, a common issue during critical stages of development, leads to halted or slowed growth, negatively impacting the viability and weight of chicks. This study aims to reduce or eliminate this dormancy by using targeted genetic stimulation to enhance embryonic development. The study was conducted at the hatchery affiliated with the Department of Animal Production, Faculty of Agriculture, University of Anbar, with the objective of investigating embryonic dormancy at different developmental stages and improving overall efficacy. A total of 720 eggs from the Rose 308 breed were used the experiment was divided into six treatments, each replicated three times, across two incubators. The following treatments were applied: T1 (negative control, no injection), T2 (positive control, injection with distilled water), T3 (injection of siRNA targeting the SERPINA6 gene on day 3 of incubation), T4 (injection of siRNA targeting the SERPINA6 gene on day 7 of incubation), T5 (injection of siRNA targeting the SERPINA6 gene on day 14 of incubation), and T6 (injection of siRNA targeting the SERPINA6 gene on day 17 of incubation). The findings showed that the sixth treatment (T6) had a notably positive impact on embryo development characteristics when compared to the other treatments, including increased embryo weight and length, improved yolk and albumin consumption, and a significant increase in shell thickness (P ≤ 0.05). The findings suggest that using natural stimuli, such as siRNA, can effectively improve embryo development and increase chick weight at hatching.\u003c/p\u003e","manuscriptTitle":"Advancing Embryonic Growth through Genetic Stimulation to Enhance Development in Chicken Embryos","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-11 06:42:42","doi":"10.21203/rs.3.rs-7087632/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fdf7fa69-a886-443c-8da0-fb8dc3a5df89","owner":[],"postedDate":"July 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-15T07:54:29+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-11 06:42:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7087632","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7087632","identity":"rs-7087632","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}