Effect of In Ovo Injection of Butyric Acid on Hatching, Physiological, and Productive Performance of Broilers

Modern fast-growing broiler strains are characterized by a high growth rate, and improper feeding of broiler breeder flocks can result in deficiencies in the nutritional content of eggs. As a consequence of genetic selection for rapid growth, the broiler rearing period has been shortened to approximately 35 days. When compared with the incubation period, the embryonic growth phase constitutes nearly 65% of the total rearing period, highlighting the critical importance of embryonic development. Therefore, considerable attention has been directed toward embryo growth and care to improve hatchability, post-hatch growth performance, and immune competence of chicks (Alsudani et al., 2025; Arkan et al., 2025).

Any defect in hatchability components can reduce embryo survival, leading to early embryonic mortality. In some cases, embryos complete development but fail to hatch successfully due to insufficient nutrient availability, which weakens their ability to break the eggshell (Kadhim, 2025). Consequently, in ovo injection technology has been adopted as an effective method to supply essential nutrients directly to the developing embryo. This technique represents a sustainable approach to broiler production through the use of environmentally friendly compounds (Ibrahim and Al-Jebory, 2020; Ajafar et al., 2024).

Organic acids are carbon-containing compounds with acidic properties that do not fully dissociate in water (Theron and Lues, 2007). They belong to the group of short-chain volatile fatty acids and are also referred to as weak acids containing one or more carboxyl groups; hence, they are known as carboxylic acids (Ricke, 2003). Their general chemical formula is RCOOH, where R represents an alkyl group or hydrogen (Hans, 2006). Butyric acid (BA), also known as butanoic acid (CH₃CH₂CH₂COOH), derives its name from the Greek word for butter due to its natural presence in butter, and its salts are referred to as butyrates. BA is produced through anaerobic fermentation in the colon and is characterized by a strong, unpleasant odor and pungent taste, detectable by humans at concentrations as low as 10 ppm (Lake, 2012).

Despite its odor, BA has several industrial and biological applications. Low-molecular-weight butyrate esters, such as methyl butyrate, possess pleasant aromas and are used in food and perfume industries. Additionally, BA has been reported to inhibit the proliferation of cancer cells in colon epithelial tissues, although its precise mechanism of action remains unclear (Vanhoutvin et al., 2009). In animal nutrition, BA has gained attention as a feed additive due to its antimicrobial properties and ability to suppress harmful intestinal bacteria. Chaveerach et al. (2004) demonstrated that the inclusion of organic acids such as butyric, acetic, propionic, and formic acids in drinking water reduced intestinal Enterobacteriaceae and Salmonella enteritidis populations. Furthermore, BA serves as an important energy source for intestinal epithelial cells and plays a role in metabolic regulation and lymphoid tissue development in the digestive tract (Navd et al., 2011).

The research has emphasized that embryonic growth and development reflect the management and nutritional status of broiler breeder flocks and represent the initial stage influencing subsequent growth performance and immune function of broilers (Salman et al., 2024a). As this stage accounts for nearly two-thirds of the bird’s productive lifespan, optimizing embryonic nutrition is crucial. Therefore, the present study aimed to investigate the effects of in ovo injection of butyric acid on hatchability traits, physiological and morphological characteristics of newly hatched chicks, and subsequent growth performance under farm conditions.

The present study was conducted through two consecutive trials, one at the hatchery and the other at the broiler farm of Al-Anwar Poultry Company, during the period from October 1 to November 11, 2024. All laboratory analyses were performed at the Department of Animal Production, College of Agriculture, Al-Qasim Green University. All experimental procedures involving animals were carried out in accordance with the ethical standards and guidelines for the care and use of animals.

In the first trial, a total of 540 fertilized broiler eggs, at 17.5 days of incubation, were used to evaluate the effects of in ovo injection of butyric acid (BA). The BA used in this study was of English origin with a purity of 98–99%. Butyric acid solutions were prepared by dissolving the required amounts of BA in 100 mL of distilled water, and the concentrations were adjusted according to previously described protocols (Zaki and Al-Jebory, 2021; Khalil et al., 2022; Al-Saeedi et al., 2023).

Prior to injection, eggs were candled to identify fertilized eggs. The wide end of each egg was disinfected using an appropriate antiseptic, and a small hole was made using a sterile egg piercer. A sterile needle was then inserted vertically into the egg, and the prepared solution was injected following standard in ovo injection procedures. After injection, the puncture site was immediately sealed with paraffin wax to prevent contamination and moisture loss.

The fertilized eggs were randomly allocated into six experimental groups, each comprising 90 eggs, with three replicates per group (30 eggs per replicate), as follows:

• C1: Non-injected control
• C2: NaCl-injected control
• C3: 0.3% BA injection
• C4: 0.4% BA injection
• C5: 0.5% BA injection
• C6: 0.6% BA injection

The inclusion of two control groups was scientifically justified to allow comparison between natural incubation conditions (C1) and the effect of the injection process and diluent solution (C2), as NaCl may influence embryonic development due to its nutrient properties.

Following hatching, hatchability parameters and physical characteristics of chicks were evaluated according to the methodology described by Ajafar et al. (2024). Data related to hatchability, chick weight, chick length, and other physical traits were recorded immediately post-hatch.

After hatching, chicks from each treatment group were transferred to the farm and reared under standard commercial conditions. Physiological traits were assessed at one week of age, following the procedures outlined by Al-Saeedi et al. (2024), Salman et al. (2024b), and Alsudani et al. (2025). Productive performance parameters, including body weight, weight gain, feed intake, and feed conversion ratio, were recorded throughout the rearing period up to 35 days of age, in accordance with Al-Jebory and Ibrahim (2021) and Al-Jebory et al. (2024).

Birds were fed starter, grower, and finisher diets formulated to meet the nutritional requirements of broilers as recommended in the Ross 308 Broiler Management Guide. The diets contained crude protein levels of 23.00%, 21.50%, and 20.08%, and metabolizable energy values of 2975, 3050, and 3100 kcal/kg, respectively.

All collected data were statistically analyzed using SAS Software (2012). Differences among treatment means were determined using Duncan’s Multiple Range Test (1955), and statistical significance was declared at p ≤ 0.05.

The effects of the experimental treatments on hatchability traits are presented in Tables 1 and 2. A significant increase (p ≤ 0.05) in hatchability percentage was observed in the C6 group compared with the C1 and C2 groups. In contrast, the hatch mortality rate was significantly higher (p ≤ 0.05) in the C2 group than in the C4 and C6 groups. Hatch chick weight was significantly increased (p ≤ 0.01) in the C3, C4, C5, and C6 groups compared with the C1 and C2 groups (Table 1).

Regarding the pipped egg ratio, a significantly higher value (p ≤ 0.05) was recorded in the C2 group compared with the C4 and C6 groups. The live pecked chick percentage was significantly higher (p ≤ 0.05) in the C3 and C5 groups than in the C1 and C2 groups. Meanwhile, the pecked chick mortality rate was significantly increased (p ≤ 0.05) in the C3, C4, C5, and C6 groups compared with the C1 and C2 groups. Furthermore, the incidence of abnormal chicks was significantly higher (p ≤ 0.05) in the C2 group than in the C3, C4, and C6 groups (Table 2).

Table 1: Effect of in ovo butyric acid (BA) injection on hatchability traits of broilers.

* p ≤ 0.05; ** p ≤ 0.01. Values are expressed as mean ± standard error. Different superscript letters within the same column indicate significant differences.

Table 2: Effect of in ovo butyric acid (BA) injection on hatching quality traits of broilers.

* p ≤ 0.05; ** p ≤ 0.01. Values are expressed as mean ± standard error. Different superscript letters within the same column indicate significant differences.

Table 3 shows the effects of the experimental treatments on chick physical traits. Chick length was significantly increased (p ≤ 0.01) in the C3, C4, C5, and C6 groups compared with the C1 and C2 groups. No significant differences were observed among the experimental groups in wing length. Leg length was significantly greater (p ≤ 0.01) in the C4 group compared with all other groups. In addition, tonic immobility was significantly improved (p ≤ 0.01) in the C3, C4, C5, and C6 groups compared with the C1 and C2 groups.

Tables 4 and 5 illustrates the effects of in ovo injection of butyric acid on selected physiological traits of broiler chicks. No significant differences were observed among the experimental groups in serum triglycerides, total cholesterol, high-density lipoprotein (HDL), or total protein concentrations (Table 4). Regarding antioxidant status and immune response (Table 5), glutathione peroxidase (GSH-Px) activity was significantly increased (p ≤ 0.01) in the C3, C4, C5, and C6 groups compared with the C1 and C2 groups. Superoxide dismutase (SOD) activity showed a significant increase (p ≤ 0.01) in the C5 group compared with the C1, C2, and C3 groups.

Table 3: Effect of in ovo butyric acid (BA) injection on physical traits of broiler chicks.

N.S: not significant; ** p ≤ 0.01. Values are expressed as mean ± standard error. Different superscript letters within the same column indicate significant differences.

Table 4: Effect of in ovo butyric acid (BA) injection on physiological traits of broiler chicks.

N.S: not significant (p > 0.05). TG = Triglycerides; TC = Total cholesterol; HDL= High-density lipoprotein; TP= Total protein.

Table 6 shows the impact of in ovo injection of BA on the live body weight of broilers. During the first week, C3, C4, C5, and C6 groups had significantly higher body weight (p ≤ 0.05) than C1. In the second week, C3 and C4 were significantly higher (p ≤ 0.05) compared to C1 and C2. In the third week, C3, C4, and C6 groups showed a significant increase (p ≤ 0.05) over C2. In the fourth and fifth weeks, C3, C4, and C5 groups exhibited significantly higher body weights (p ≤ 0.05) than the control groups.

The changes in weight gain (WG) are presented in Table 7. There was no significant difference between groups in the 1st and 5th weeks. In the 2nd week, the C5 group showed a significant increase (p ≤ 0.05) compared to C1, C2, C3, and C6 groups. In the 3rd week, WG was significantly higher (p ≤ 0.05) in the C3 group compared to C2 and C5. During the 4th week and for total weight gain, C3, C4, C5, and C6 groups exhibited a significant increase (p ≤ 0.05) compared to C1 and C2 groups.

In feed intake, there was no significant difference between groups during the 1st, 2nd, and 3rd weeks. In the 4th week, C1 showed significantly higher feed intake (p ≤ 0.05) compared to the other groups. In the 5th week, C5 had significantly higher feed intake (p ≤ 0.05) than C1, C2, C3, and C4. The total feed consumption over the 5-week period was significantly higher (p ≤ 0.05) in C6 compared to C1, C2, C3, and C4 (Table 8).

The impact of in ovo BA injection on feed conversion ratio (FCR) is presented in Table 9. In the 1st week, significant improvement (p ≤ 0.05) was observed in C3 and C5 compared to the other groups. In the 2nd week, C5 showed a significant improvement (p ≤ 0.05) over all other groups. No significant differences were observed among groups in the 3rd week. During the 4th week, C2, C3, C4, and C5 exhibited significant improvement (p ≤ 0.05) compared to C1. In the 5th week and for total FCR, all BA-injected groups showed significant improvement (p ≤ 0.05) compared to the non-injected control (C1).

Table 5: Effect of in ovo butyric acid (BA) injection on antioxidant and immune status of broiler chicks.

N.S: not significant; ** p ≤ 0.01. Values are expressed as mean ± standard error. Different superscript letters within the same column indicate significant differences. GSH-Px = Glutathione peroxidase; SOD = Superoxide dismutase; MDA = Malondialdehyde; IgM = Immunoglobulin M; IgG = Immunoglobulin G.

Table 6: Impact of in ovo injection of butyric acid (BA) on live body weight of broiler (g/bird).

* p ≤ 0.05. Values are expressed as mean ± standard error. Different superscript letters within the same column indicate significant differences.

Table 7: Impact of in ovo injection of butyric acid on weekly weight Gain (g) of broilers.

N.S: not significant; * p ≤ 0.05. Values are expressed as mean ± standard error. Different superscript letters within the same column indicate significant differences.

Table 8: Effect of in ovo injection of butyric acid on feed intake (g) of broilers.

N.S: not significant; * p ≤ 0.05. Values are expressed as mean ± standard error. Different superscript letters within the same column indicate significant differences.

Table 9: Effect of in ovo injection of BA on feed conversion ratio (FCR) of broilers.

N.S: not significant; * p ≤ 0.05. Values are expressed as mean ± standard error. Different superscript letters within the same column indicate significant differences.

Improvement in hatching characteristics and the physical traits of hatched chicks was observed in groups injected with BA, which supports the energy and growth of embryos. Injecting BA into the amniotic sac allows it to be absorbed through the digestive tract via the yolk diverticulum, leading to a decrease in the pH of the digestive tract. This increased acidity favors the growth of beneficial bacteria in the early intestinal tract of hatched chicks, while reducing harmful bacteria that thrive in more basic environments, thereby decreasing microbial competition. The enhanced microbial balance stimulates the secretion of digestive enzymes, improving the breakdown of proteins and energy sources, including fats and carbohydrates. Lactobacillus species, for example, promote the secretion of amylase, which aids in starch digestion (Jang et al., 2004).

The increase in the surface area of intestinal villi may also contribute, as this enhances yolk absorption and results in higher chick weight (Jahanian, 2011). Furthermore, the acidic environment created by BA stimulates the release of digestive hormones, including gastrin and cholecystokinin (CCK), which regulate protein digestion and absorption. These hormones influence gallbladder contractions, exocrine pancreatic secretions, and gastric acid production, thereby improving feeding behavior, nutrient utilization, and post-hatch growth performance (SAlAhi et al., 2015).

The development of intestinal epithelial tissue may also explain these effects (Born, 2002). Supplementation with organic BA increases villi length (Jahanian, 2011), expanding the surface area available for nutrient absorption (Ricke, 2003). Organic acids enhance nutrient digestibility, improving blood serum quality and increasing antioxidant enzyme activity, including SOD and GSH-Px. These effects result from increased acidity, stimulation of pancreatic enzyme secretion, and improved absorption of minerals such as calcium, phosphorus, magnesium, and zinc, all of which enhance the function of the antioxidant system.

Moreover, the presence of BA promotes the utilization of digested nutrients and supports the growth of beneficial gut bacteria, which in turn stimulate pancreatic and digestive enzyme activity for more efficient protein digestion (Ghazalah et al., 2011). Lowering intestinal pH increases the availability of nutrients for beneficial bacteria, reduces harmful bacteria, and minimizes protein degradation into ammonia by pathogenic microbes during fermentation (Adil et al., 2010; Al-Hussany and Kadhim, 2025).

The current study demonstrated that in ovo injection of BA enhanced embryo development, resulting in higher hatchability, increased chick weight, and improved physical traits. These improvements translated into better growth performance on the farm, highlighting the role of BA in supporting broiler growth. Among the treatments, the C6 group (0.6% BA) performed best in hatching, physiological, and productive traits.

The authors thank the College of Agriculture, Al-Qasim Green University and Al-Anwar Poultry Hatchery for their continuous support of scientific research and studies.

This study is unique in that it is the first to use butyric acid to support the growth and development of chicken embryos in the egg.

Noor Ali Hussein and Hashim Hadi Al-Jebory performed the field and laboratory work and prepared the initial project, while Mohammed Khalil Ibrahim Al-Saiddi and Asha Fajer Al-Jebory contributed to the laboratory work, manuscript writing, and statistical analysis.

Artificial intelligence tools were used to improve the language quality of the manuscript. The authors confirm that all scientific content, data analysis, interpretations, etc. are solely their own.

The authors have declared no conflict of interest.