In-Ovo Technology in Poultry: Advancements, Benefits, and Challenges

Introduction

In-ovo technology involves the direct injection of substances into a developing avian embryo, a technique that has shown great promise in modern poultry production (El-Sabrout et al., 2019; Das et al., 2021; Akosile, Kehinde, et al., 2023). This method allows for the delivery of various beneficial compounds, such as vaccines, nutrients, and other bioactive substances, directly into the egg (Olanrewaju et al., 2019; Pandey et al., 2021; Akosile, Kehinde, et al., 2023). This is usually performed during the later stages of incubation, typically between days 17 and 19, when eggs are transferred from the setter to the hatcher (Siwek et al., 2018; El-Ghany & A, 2025). The in-ovo techniques aims to improve chick health and performance by manipulating the embryo's environment before hatching (El-Sabrout et al., 2019; Das et al., 2021). In-ovo feeding, for example, is a nutritional approach that can provide embryos with essential nutrients to support their development (Luqman, 2019; Givisiez et al., 2020; Akosile, Kehinde, et al., 2023). This is particularly useful as the embryo's carbohydrate reserves are limited at hatching (Shehata et al., 2021). Nutrients such as glucose, amino acids, and vitamins can be delivered to the embryo to improve its health and development (Das et al., 2021). In addition to nutrients, in-ovo technology is also used to administer vaccines. This method of vaccination has been shown to be effective in providing early protection against diseases such as Marek's disease, infectious bursal disease, and Newcastle disease (Peebles, 2018; El-Ghany & A, 2025). The in-ovo administration of vaccines provides earlier immune responses in young chicks compared to post-hatch vaccination (Oladokun & Adewole, 2020). Furthermore, in-ovo techniques can be used to deliver prebiotics and probiotics, which can help establish a healthy gut microbiome in the developing chick (Roto et al., 2016; Angwech, 2018; Oladokun & Adewole, 2020; Das et al., 2021; Akosile, Kehinde, et al., 2023). This is critical for proper immune system development and overall health, and can reduce the risk of disease (Roto et al., 2016; Rubio, 2019; Akosile, Kehinde, et al., 2023). The ability to deliver these various substances through in-ovo techniques represents a significant advancement in poultry farming by addressing nutritional deficiencies, improving disease resistance, and promoting overall bird health, making it a vital tool for both farmers and animal researchers (Roto et al., 2016; Oladokun & Adewole, 2020; Shehata et al., 2021; Akosile, Kehinde, et al., 2023; Oliveira et al., 2023).

Effect on Hatchability

In ovo-techniques, while promising, can have varied effects on hatchability, making it crucial to understand which substances and methods are most effective. Hatchability refers to the percentage of eggs that successfully hatch, a key metric for poultry production. Some substances, when injected in-ovo, have shown to enhance hatchability, while others can be detrimental (Oliveira et al., 2023). For instance, the in-ovo injection of vitamin E at a dose of 60.4 IU has been shown to provide the highest hatching rate and shortest hatch window (Araújo et al., 2019), while lower doses of clove and cinnamon extracts have shown improved hatchability (Akosile et al., 2023). In-ovo feeding with L-carnitine has also been shown to improve hatchability percentage and creatine pyruvate (Luqman, 2019). Additionally, the administration of vitamin C during incubation has been shown to combat heat stress, thereby increasing hatchability (Akosile et al., 2023).

However, it is not always the case that in-ovo feeding increases hatchability. There are instances where it has been reported to have no effect, or even a negative effect. Some studies have shown that injecting canthaxanthin in-ovo can result in lower hatching rates and longer hatch windows (Araújo et al., 2019). Similarly, high concentrations of Bacillus subtilis can significantly reduce hatchability, while Lactobacillus acidophilus and Bifidobacterium animalis do not affect hatchability (Peebles, 2018). It's also been noted that the method of injection itself can impact hatchability, with a 45° needle angle through the air cell hindering hatchability compared to a 90° angle that does not pass through the air cell (Groff-Urayama et al., 2019). The timing of in-ovo injection is crucial, with injections on day 17 of incubation potentially decreasing hatchability by 1-2% compared to day 18 (TAINIKA & BAYRAKTAR, 2021).

Furthermore, substances like nano zinc oxide and creatine monohydrate have been reported to negatively affect hatchability, while others like betaine hydrochloride and silver nanoparticles do not affect hatchability (Oliveira et al., 2023). Amino acids administered on day zero of incubation were also found to reduce hatchability, while the same amino acids injected into the yolk sac on day 7 did not (TAINIKA & BAYRAKTAR, 2021).

Substances That Improve Hatchability

For hatcheries looking to adopt in-ovo techniques, the following have been found to be successful and safe options:

• Vitamin E supplementation (Araújo et al., 2019).
• Lower doses of clove and cinnamon extracts (Akosile, Majekodunmi, et al., 2023).
• L-Carnitine and creatine pyruvate (Luqman, 2019).
• Vitamin C administration (Akosile et al., 2023).
• A 90° needle angle for injection, avoiding the air cell (Groff-Urayama et al., 2019).

These have shown positive or neutral effects on hatchability, while research is ongoing for other substances. Techniques involving high doses of some substances and earlier injection times need further investigation to ensure they do not negatively impact hatchability (Araújo et al., 2020; Oliveira et al., 2023; TAINIKA & BAYRAKTAR, 2021).

Effect on Day-Old Weight and Growth Performance

In-ovo techniques can significantly influence both the day-old weight of chicks and their overall growth, particularly in broilers. Several studies have shown that specific in-ovo treatments can lead to increased chick weight at hatch (Hassan, 2018). For example, the in-ovo injection of nano-selenium has resulted in higher chick weight at hatch and a better chick weight to egg weight ratio (Hassan, 2018). Similarly, the in-ovo administration of L-glutamine can lead to heavier chicks at hatch, with a ratio of 0.59 chick to egg weight (Rahardja et al., 2018; Akosile, Majekodunmi, et al., 2023). In-ovo feeding with amino acids like lysine can increase the body weight of day-old chicks (Coskun et al., 2018). Furthermore, in-ovo injection with a combination of creatine monohydrate and glucose can positively affect hatchlings' somatic characteristics and energy status (Retes et al., 2018).

Beyond the immediate post-hatch period, in-ovo techniques can also positively impact the overall growth of broilers. Supplementation with nano-selenium increases the weight at hatch and leads to higher live body weight and weight gain throughout the rearing period (Hassan, 2018). In addition, in-ovo feeding with amino acids such as lysine and methionine has been reported to enhance chick growth (Coskun et al., 2018). This is because in-ovo feeding can improve the development of the digestive tract, which in turn improves the digestion and absorption of nutrients, and leads to better feed conversion ratios (Coskun et al., 2018; El-Sabrout et al., 2019; Luqman, 2019). Moreover, in-ovo supplementation of arginine and threonine has been shown to improve growth performance and increase the weight of the small intestine, further contributing to enhanced nutrient absorption (Luqman, 2019). In-ovo injection of L-Carnitine has also improved broiler performance and slaughter yield (Triplett et al., 2018). In-ovo feeding also has the potential to improve the utilisation of nutrients during the critical transition from egg nutrition to feed nutrition (Akosile et al., 2023). 

However, it is worth noting that while several studies report positive effects, some studies have found no significant impact of certain in-ovo treatments on growth performance. For example, in-ovo administration of trace minerals such as zinc and copper did not affect live weight gain or feed conversion ratios (Awachat et al., 2020). In contrast, studies focusing on in-ovo supplementation with amino acids showed significantly better results (Luqman, 2019). In summary, in-ovo techniques hold substantial potential for improving both day-old chick weight and overall broiler growth, with certain substances showing particularly promising results (Hassan, 2018; Triplett et al., 2018; Luqman, 2019). However, the efficacy of these techniques can depend on the specific substance used, highlighting the need for further research to optimize their use (Pandey et al., 2021).

Effect on Gut Development and Immunity

In-ovo techniques significantly influence gut development and immunity in chickens, which in turn affects their overall weight gain, growth, and production. The early establishment of a healthy gut is crucial for optimal performance, and in-ovo interventions can play a vital role in this process. Specifically, in-ovo administration of prebiotics and probiotics can promote the growth of beneficial bacteria, leading to improved gut health and immune function, primarily in broilers (Alves et al., 2020; Akosile et al., 2023). This is because the embryonic gut is sterile before hatching, and establishing a healthy microbiome is essential for proper immune system development and overall health (Akosile et al., 2023). In-ovo delivery of these substances can help reduce the risk of diseases by promoting competitive exclusion of harmful pathogens (Das et al., 2021).

In-ovo feeding can enhance the development of the gastrointestinal tract (GIT) and gut-associated lymphoid tissue (GALT). The GIT is responsible for nutrient digestion and absorption, while GALT plays a crucial role in immune function (Akosile et al., 2023). These systems can mature more effectively by delivering nutrients and other compounds in-ovo, improving the bird’s overall health and disease resistance (Akosile et al., 2023). In-ovo injection of prebiotics and synbiotics has also been shown to affect the digestive potency of the pancreas, which improves nutrient absorption (Retes et al., 2018; El-Sabrout et al., 2019). This improved digestive capacity directly contributes to better growth performance and feed efficiency.

Research indicates that the in-ovo delivery of prebiotics with or without antibiotics can reduce the severity of intestinal lesions and oocyst excretion induced by coccidiosis, especially in indigenous chickens like Kuroiler chickens (Angwech, 2018). Such interventions not only protect against diseases but also improve overall performance and meat quality (Angwech, 2018). The early establishment of the gut microbiome via in-ovo injection with prebiotics and probiotic strains has a positive impact on the development of the intestinal epithelium, enhancing the well-being of the chicken host (Alves et al., 2020) .

In broilers, in-ovo feeding with L-glutamine can also improve the intestinal villus height and crypt depth, enhancing nutrient absorption capacity (Luqman, 2019). This improvement in gut morphology has a direct impact on weight gain, growth, and overall productivity. The positive effects of in-ovo feeding on the gut also lead to a more efficient use of nutrients, which is crucial for rapid growth and production (Luqman, 2019; Givisiez et al., 2020).

Effect on Disease Control

In-ovo techniques offer a promising approach to disease control in poultry, particularly through early intervention strategies. One of the most established applications is in-ovo vaccination, which provides early immune protection to chicks, especially against diseases like Marek's disease (Peebles, 2018; Oladokun & Adewole, 2020; El-Ghany & A, 2025). This method is more effective than post-hatch vaccination because it stimulates an earlier immune response (Oladokun & Adewole, 2020). In-ovo vaccination is now standard commercial practice for several economically important viral diseases, such as Newcastle disease, infectious bursal disease, infectious laryngotracheitis, infectious bronchitis, avian influenza and avian metapneumovirus vaccines for mycoplasmosis and coccidiosis can also be administered in-ovo (El-Ghany & A, 2025). The accuracy of in-ovo injection is vital for effective vaccination, with the amniotic sac being a primary target (Triplett et al., 2018; El-Sabrout et al., 2019; El-Ghany & A, 2025).

Beyond vaccines, in-ovo delivery of other bioactive compounds also controls disease. For instance, in-ovo administration of probiotics and prebiotics can promote the growth of beneficial gut bacteria, reducing the risk of disease through competitive exclusion of pathogens, particularly in broilers (Roto et al., 2016; Alves et al., 2020; Oladokun & Adewole, 2020; Akosile et al., 2023). This approach also helps in the development of the gut-associated lymphoid tissue (GALT), which is crucial for immune function (Siwek et al., 2018; Akosile, Kehinde, et al., 2023). Furthermore, in-ovo delivery of phytobiotics has been shown to possess antibacterial and anti-inflammatory properties, offering an alternative to traditional antibiotic use, especially in broilers (Akosile, Kehinde, et al., 2023). Research has shown that in-ovo delivered prebiotics can reduce the severity of intestinal lesions and oocyst excretion caused by coccidiosis, particularly in indigenous breeds like Kuroiler chickens (Angwech, 2018).

Moreover, in-ovo stimulation with probiotics, such as Leuconostoc mesenteroides, has demonstrated potential in reducing Campylobacter jejuni colonisation in the ceca of broiler chickens (Wishna et al., 2024). The combination of probiotics with prophybiotics, such as garlic aqueous extract, can further enhance this effect. These findings indicate that in-ovo techniques can be a valuable tool in reducing the reliance on antibiotics and improving the overall health and disease resistance of poultry (Oladokun & Adewole, 2020; Das et al., 2021). The automation of in-ovo delivery systems reduces the risk of pathogenic contamination and ensures uniform dosing, which is vital for disease control (Das et al., 2021).

Research Gaps and Future Prospects

In-ovo technology, which involves injecting substances into eggs, is gaining traction in poultry production, yet several research gaps need addressing to harness its potential fully. While in-ovo techniques offer a promising avenue for delivering vaccines, nutrients and other bioactive compounds, there's a need for standardized methods for its application in commercial settings. Current research shows inconsistent outcomes from in-ovo feeding, highlighting the need to determine the most effective nutrient composition, dosages, and timing of injections for optimal post-hatch results. The impact of different injection sites on hatchability and chick quality needs further investigation, as some techniques, such as a 45° needle axis passing through the air chamber, can hinder hatchability. While the amniotic site is considered most effective, the practicality of combining in-ovo vaccination with other in-ovo applications at the same time point (day 18 of incubation) needs further research.

Looking ahead, future research should focus on the long-term benefits of in-ovo techniques, exploring how they influence gene expression and if they can be combined with vaccine diluents to make the process more efficient. New compounds with multiple functions such as organic acids and nutraceuticals, need to be evaluated for in-ovo use. There is also a need to improve injection systems, with automated systems being vital for commercial implementation, as these can ensure better precision and reduce labor costs. Moreover, research should investigate the potential of in-ovo techniques to address animal welfare concerns, such as in-ovo sexing, to avoid culling male chicks. Furthermore, understanding the impact of in-ovo techniques on the gut microbiome and overall immune system development of poultry is crucial. Studies focusing on in-ovo delivery of prebiotics and probiotics to establish a healthy gut microbiome early in life, will also provide more avenues to address the reduction in use of antibiotics in poultry production. Finally, collaborative research networks involving different countries, researchers, and the poultry sector are essential to overcome the current limitations and to develop standardised protocols for the industry.

Conclusion

In conclusion, in-ovo technology holds significant promise for poultry production, with the potential to enhance hatchability, growth, and overall health. However, research gaps exist regarding optimal application methods, dosages, and the long-term effects of in-ovo interventions. Future prospects lie in developing standardised protocols for various substances, exploring novel compounds, and improving injection systems. Focusing on the gut microbiome, immune system, and animal welfare through in-ovo techniques will be crucial for optimising poultry production. Collaborative efforts between researchers and the poultry industry are essential to translate research into practical applications, ensuring a sustainable and ethical poultry production system.

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