Embryonic nano lauric acid delivery modulates lipid metabolism, oxidative balance, and gut morphogenesis in broiler chicks

Abstract

The in ovo-injection technique was employed as an early-life nutritional strategy to improve the health and productivity of birds by delivering nutrients and bioactive compounds directly to the developing embryo. This study explored the innovative use of in ovo administration of nano-lauric acid (NLA) as a strategy for the metabolic programming of broiler chicks. The goal was to improve hatchability, stimulate hepatic antioxidant activity, regulate growth-related genes, and support intestinal development in newly hatched chicks. A total of 400 fertile eggs from a 40-week-old Arbor Acres breeder flock were randomly divided into four treatment groups: a non-injected control group (CN), a vehicle-injected control group (CP; 0.1 mL of sterile distilled water), and two NLA-treated groups receiving either 2.5 mg/egg (NLA 2.5) or 5 mg/egg (NLA 5) of NLA, each dissolved in 0.1 mL of sterile distilled water. Injections were administered into the yolk sac on day 12 of incubation. Post-hatching, the hatchability percentage was recorded. Serum lipid profiles, hepatic redox status, and the expression level of hepatic genes, nuclear factor erythroid 2–related factor 2 (NRF2), mitochondrial superoxide dismutase 2 (mt-SOD2), and insulin-like growth factors 1 and 2 (IGF-1 and IGF-2) were evaluated. Additionally, the intestinal morphology of the newly hatched chicks was examined. Hatchability % was significantly reduced in the NLA 5 group (80%) compared to the CN (98%), CP (97%), and NLA 2.5 (96%) groups. The findings showed that in ovo injection of NLA at 2.5 mg/egg was therefore identified as optimal, significantly improving lipid metabolism by reducing serum triglycerides, LDL, VLDL, and cholesterol, while increasing HDL cholesterol compared to controls (P < 0.05). Hepatic antioxidant defense was significantly improved through the decrease of malondialdehyde (MDA) and increase of reduced glutathione (GSH) concentrations (P < 0.05). This enhancement was associated with the upregulation of NRF-2 and mt-SOD2 by (4.04; 3.69-folds, respectively) and stimulation of anabolic signaling genes IGF-1 and IGF-2 by (4.08 and 2.3-folds; respectively) (P < 0.05). In addition, intestinal development has been significantly promoted via increased villus height and crypt depth (P < 0.05). Our findings demonstrate that in ovo NLA supplementation at 2.5 mg/egg effectively enhances lipid utilization, activates NRF2-mediated antioxidant pathways, and stimulates anabolic signaling. This targeted nutritional strategy proves to be a safe and effective method for pre-hatch metabolic programming, with significant potential to improve post-hatch health and performance in broilers.

Introduction

In recent decades, the poultry industry has increasingly adopted early-life nutritional strategies to improve bird health and productivity. One such strategy is in ovo injection, a technique that has been used for many years. It involves delivering nutrients and bioactive compounds directly into the egg during embryonic development. This method has shown long-term benefits for post-hatch growth, physiology, and overall performance in poultry^1,2. Lauric acid, a medium-chain fatty acid, is well-recognized as an efficacious antioxidant and anti-inflammatory agent³. The new nano formulation of lauric acid offers the best cellular absorption and the highest efficacy in the tissues due to low particle size and a high tissue penetration⁴. It is well established that reactive oxidant molecules pose a significant threat to cellular health. To counteract their harmful effects, the body relies on antioxidant defense systems, which are regulated through a series of complex biochemical reactions. Interestingly, nuclear factor erythroid 2–related factor 2 (NRF2) serves as a key transcription factor in this defense mechanism. NRF2 activates the expression of a wide range of cytoprotective genes, including those encoding superoxide dismutase and glutathione-related enzymes. These genes play a critical role in neutralizing oxidative stress and detoxifying harmful xenobiotics^5,6. Medium-chain triglycerides (MCTs) from coconut oil (rich in lauric acid ) can effectively modulate growth pathways, including the somatotropic axis (insulin/IGF axis), which involves insulin, IGF-1, and IGF-2 ligands binding to their receptors (InsR, IGF1R, IGF2R)⁷. It is reported that insulin-like growth factors (IGF) are essential for cell growth and differentiation through the manipulation of many biological actions of growth hormone in chickens⁸. The synthesis of specific lipoproteins (LDL, VLDL, HDL) in the liver, together with hormonal regulation of lipid metabolism, plays a crucial role in maintaining metabolic balance. Advances in molecular biology techniques that elucidate the involvement of key enzymes further contribute to improved physiology and performance in birds⁹. In quail, in ovo injection of conventional lauric acid significantly decreased the hatchability, which prompted the use of nanoparticle-formulated lauric acid to mitigate this adverse effect¹⁰. Early development of intestinal villi is a crucial process during embryogenesis and early post-hatch. A recent study reported that in ovo injection of glycerin monolaurate improved intestinal morphology and antioxidant system in lipopolysaccharide (LPS)-challenged chicken embryos^11,12. The in ovo technique (IOT) was first introduced for vaccination against Marek’s disease¹³. The unrivaled success of in ovo vaccination (IOV) has virtually transformed the poultry industry, providing compelling evidence that it is indeed possible to deliver biologicals directly into an embryo. IOT has progressed beyond vaccination and is now also used for a variety of purposes, such as in ovo feeding (IOF) or injection (IOI), where nutrients and bioactive compounds known to improve embryonic development, gut function, and post-hatch performance are delivered¹⁴. Additionally, the technique has been employed for in ovo sexing to identify embryo gender¹⁵ and for epigenetic stimulation bringing about sustained growth and health benefits¹⁶. While much information has been gained on post-hatch nutrition, pre-hatch nutritional manipulation by IOT, as the critical factor which influences embryonic development and hatchability, is now acknowledged to influence performance thereafter¹⁷. Therefore, the use of IOF has been receiving noteworthy attention to enhance early chick performance and vitality. Numerous studies confirmed that in ovo supplementation with probiotics, amino acids, and vitamins is an effective practice^18,19. There are not many works focusing on lipid bioactive compounds. Recent achievements in nanotechnology have also expanded its potential; nano-formulations improve bioavailability, cellular internalization, and tissue penetration of the drug and potentially offer more effective performance at low doses²⁰. Nevertheless, the impact of lauric acid (LA) innovative nano-formulation on the molecular physiology of broiler embryo remains entirely unexplored. In particular, there is no available information on the impact of in ovo nano-lauric acid (NLA) on the NRF-2 antioxidant pathway, expression of hepatic growth factors (IGF-1 and IGF-2), systemic lipid metabolism, and intestinal development at hatch.

Thus, the present study aimed to address this knowledge gap and provide the first insight into the impacts of in ovo injection of nano lauric acid (NLA) on hepatic redox system, antioxidant defense (NRF-2, SOD2), and growth-related genes (IGF1, IGF2), serum lipid profile, as well as intestinal morphology in newly hatched chicks.

Results

Effect of in ovo injection of NLA on hatchability

The effects of in ovo injection of NLA on hatchability are presented in Table 1. The results revealed that hatchability in the NLA 5 group (80%) was significantly lower than that of the non-injected control (CN, 98%; P = 0.0003), the solvent-injected control (CP, 97%; P = 0.0013), and the NLA 2.5 group (96%; P = 0.0046). No significant differences were observed among the CN, CP, and NLA 2.5 groups.

Table 1.

Effect of in ovo injection of nano-lauric acid (NLA) on hatchability.

Group	Eggs injected (n)	Hatched chicks (n)	Hatchability (%)	P-value
CN (Non-injected)	100	98	98	N/A
CP (Solvent control)	100	97	97	1
NLA 2.5	100	96	96	1
NLA 5	100	80	80	< 0.001

Data are presented as the percentage of hatched chicks relative to fertile eggs set. Statistical analysis was performed using Fisher’s exact test, followed by the Bonferroni test for correction of the P-value. Bold values indicate significant differences (P < 0.05) compared with the control group. CN (non-injected control); CP (solvent injected); NLA 2.5: injected with 2.5 mg nanolauric; NLA 5: injected with 5 mg nanolauric.

Effect of in ovo injection of NLA on chick weight and yields

Table 2 illustrates the effects of in ovo injection of NLA on chick weight and yield (%). The results showed that the NLA 2.5 group exhibited a significant increase (P ≤ 0.001) in both chick weight and chick yield (%) compared with the other groups, with a strong positive correlation between the two parameters. In contrast, the NLA 5 group demonstrated a negative correlation, accompanied by reduced values for both chick weight and chick yield (%).

Table 2.

Effect of in ovo injection of nano-lauric acid (NLA) on chick weight and yield%.

Group	Means of egg weight	Means of hatched chick weight	Chick yields%
CN (Non-injected)	65.11 ± 1.10	45.06b ± 0.41	69.21b ± 0.91
CP (Solvent control)	64.42 ± 1.42	44.50b ± 0.42	69.08b ± 1.05
NLA 2.5	65.13 ± 0.95	47.10a ± 0.51	72.32a ± 1.25
NLA 5	66.01 ± 1.13	43.18c ± 0.47	66.17c ± 1.11

Data are means ± SEM. Different superscript letters (a–c) within a column indicate significant differences among treatment groups’ means according to Tukey’s post-hoc test (P ≤ 0.001). CN (non-injected control); CP (solvent injected); NLA 2.5: injected with 2.5 mg nanolauric; NLA 5: injected with 5 mg nanolauric.

Effect of in ovo injection of NLA on serum lipid profile of newly hatched chicks

In ovo injection of nano-lauric acid significantly altered serum lipid fractions. Triglyceride (TG) levels were markedly reduced in both NLA-treated groups compared to CN and CP (P < 0.05), as shown in Table 3. Total cholesterol (TC) of the NLA 2.5 group was significantly decreased (P < 0.001) compared to both control groups. The difference in the levels of TC between NLA5 and both the controls was insignificant (P > 0.05). HDL cholesterol (HDL-C) levels decreased significantly (P < 0.001) in the NLA 5 group compared to CN, CP, and NLA 2.5, whereas no significant difference was found between the CN, CP, and NLA 2.5 groups. LDL cholesterol (LDL-C) was lowest in NLA 2.5 (P < 0.001) and highest in NLA 5 (P < 0.01). VLDL cholesterol mirrored triglycerides (TG) trends, with significantly lower levels in NLA 2.5 (P < 0.01) and NLA 5 (P < 0.05) compared to control groups.

Table 3.

Effect of NLA in ovo injection on serum lipid profile of hatchling chicks.

Parameters	CN	CP	NLA 2.5	NLA 5
Triglycerides (mg/dl)	47.91 ± 2.4a	47.87 ± 2.9a	35.11 ± 1.04b**	37.88 ± 1.44b*
Total cholesterol (mg/dl)	246.15 ± 6.64a	257.3 ± 2.72a	212.8 ± 4.48b***	251.7 ± 1.89a
HDL cholesterol (HDL-C) (mg/dl)	64.83 ± 1.1a	66.86 ± 0.93a	69.35 ± 0.65a	48.22 ± 2.2b***
LDL cholesterol (LDL-C) (mg/dl)	171.74 ± 6.1b	180.84 ± 2.04ab	136.32 ± 5.11c***	195.32 ± 3.07a**
VLDL cholesterol (VLDL-C) (mg/dl)	9.58 ± 0.48a	9.57 ± 0.58a	7.01 ± 0.2b**	7.57 ± 0.28b*

Different superscript letters (a–c) within a row indicate significant differences among treatment groups’ means according to Tukey’s post-hoc test (P < 0.05), where ns indicates non-significant. Asterisks (*) indicate the degree of statistical significance relative to the control group as follows: (*): P < 0.05; (**): P < 0.01; (***): P < 0.001. CN (non-injected control); CP (solvent injected); NLA 2.5: injected with 2.5 mg nanolauric; NLA 5: injected with 5 mg nanolauric. HDL cholesterol (HDL-C) (mg/dl): High-density lipoprotein cholesterol; LDL cholesterol (LDL-C)(mg/dl): Low-density lipoprotein cholesterol; VLDL cholesterol (VLDL-C) (mg/dl): Very low-density lipoprotein cholesterol. Data are means ± SEM (standard error of the mean). Number of sampled birds (n) = 9 hatchlings/group (3/ replicate).

Impact of in ovo NLA injection on hepatic oxidative stress in chicks

Hepatic malondialdehyde (MDA) levels-a key indicator of lipid peroxidation-differed significantly among treatment groups, as illustrated in Fig. 1A. Treatment with 2.5 NLA significantly decreased MDA levels compared to both control groups (CN and CP) (P < 0.001), suggesting decreased lipid peroxidation and oxidative stress. Conversely, a significant increase ( P < 0.001) in levels of MDA concentration was observed in the NLA5 group, indicating enhanced oxidative damage at the higher dose. No significant differences were observed between the CN and CP groups.

Fig. 1.

Effect of in ovo injection of NLA with different concentrations on hepatic oxidative stress markers, (A) Malondialdehyde (MDA) (nmol/gT), (B) Reduced glutathione (GSH) (μM/gT). CN (non-injected control); CP (solvent injected); NLA 2.5: injected with 2.5 mg nanolauric; NLA 5: injected with 5 mg nanolauric. Data are presented as mean ± standard error (n = 10 hatchlings/group). Significant differences were set by Tukey’s Post hoc test, where ns: indicates non-significant; (*): P < 0.05; (**): P < 0.01; (***): P < 0.001. Number of sampled birds (n) = 9 hatchlings/group (3/ replicate).

The level of reduced glutathione (GSH) of liver tissue significantly increased in the NLA 2.5 treatment group when compared to CN, CP, and NLA5 groups, respectively (P < 0.01), as shown in Fig. 1B, indicating enhanced antioxidant capacity. In contrast, the NLA 5 group did not exhibit any significant differences in GSH levels relative to the controls. The differences in the mean values were not significant when comparing the CN and CP groups.

In ovo NLA injection and hepatic growth gene expression in chicks

As illustrated in Fig. 2, Quantitative RT-PCR analysis revealed treatment-specific modulation of hepatic gene expression. In ovo NLA injection significantly upregulated hepatic expression of growth-promoting genes; insulin-like growth factor (IGF-1 and IGF-2). The fold change expression of both IGF-1 and IGF-2 genes of NLA 2.5 group was greater (P < 0.001) by (4.08 ± 0.06, and 2.3 ± 0.052-folds, respectively) when compared to CP and CN groups. Treatment with a higher dose also led to a significant increase in the expression of the IGF genes, but the fold change in expression was less in the NLA5 group when compared to the NLA2.5 group; IGF-1 (1.8 ± 0.09-fold) and IGF-2 (1.5 ± 0.032 fold) (P < 0.001).

Fig. 2.

Effect of in ovo injection with NLA with different concentrations on hepatic relative expression of growth-related genes; IGF-1: insulin growth factor-1, and IGF-2: insulin growth factor-2. CN (non-injected control); CP (solvent injected); NLA 2.5: injected with 2.5 mg nanolauric; NLA 5: injected with 5 mg nanolauric. Data are presented as mean ± standard error (n = 10 hatchlings/group). Significant differences were set by Tukey’s Post hoc test, where ns: indicates non-significant; (*): P < 0.05; (**): P < 0.01; (***): P < 0.001. Number of sampled birds (n) = 9 hatchlings/group (3/ replicate).

The difference in the expression levels of the two genes between the CN and CP groups was not significant (P > 0.05). These findings indicate that moderate-dose (NLA 2.5) more effectively stimulates hepatic expression of key anabolic growth factors in newly hatched chicks than the higher dose (NLA 5).

In ovo NLA injection and hepatic antioxidant gene expression in chicks

The treatment with lower or higher doses of NLA upregulated the expression of antioxidant enzyme genes (NRF-2 and mt-SOD2) (P < 0.001) when compared to the expression level of both the genes of CN and CP groups (Fig. 3), respectively. Whereas the change in the expression level of antioxidant genes when compared between the control groups was observed to be not significant (P > 0.05, Fig. 3). The lower dose brought out greater change in the expression level of genes (4.04 ± 0.08; 3.69 ± 0.05-folds) when compared to the higher dose of NLA (2.11 ± 0.08, 2.88 ± 0.22-folds) for NRF-2 and mt-SOD2, respectively.

Fig. 3.

Effect of in ovo injection of NLA with different concentrations on hepatic relative expression of growth-related genes; NRF-2: Nuclear factor erythroid 2-related factor 2, and mt-SOD2: Mitochondrial superoxide dismutase-2. CN (non-injected control); CP (solvent injected); NLA 2.5: injected with 2.5 mg nanolauric; NLA 5: injected with 5 mg nanolauric. Data are presented as mean ± standard error (n = 10 hatchlings/group). Significant differences were set by Tukey’s Post hoc test, where ns: indicates non-significant; (*): P < 0.05; (**): P < 0.01; (***): P < 0.001. Number of sampled birds (n) = 9 hatchlings/group (3/ replicate).

Effects of in-ovo injection of NLA on intestinal development

The in ovo-injection of NLA at 2.5 mg/egg significantly improved villi length and crypt depth compared to CP and CN (P < 0.05), while the higher dose (5 mg/egg) did not produce significant changes. No significance was detected among treatments in the villi length: crypt depth ratio (P > 0.05) (Table 4 and Fig. 4).

Table 4.

Effect of in ovo injection of NLA with different concentrations on jejunal villi length, crypt depth, and villi length: crypt depth ratio of the hatching chicks.

Item	CN	CP	NLA 2.5	NLA 5
Villi length (µm)	320.71 ± 6.9b	321.48 ± 7.1b	369.19 ± 3.2a	348.28 ± 8.7ab
Crypt depth (µm)	67.92 ± 3.7b	68.98 ± 3.6b	87.69 ± 2.5a	73.75 ± 3.02ab
V length: C. depth ratio	4.85 ± 0.3a	4.78 ± 0.32a	4.24 ± 0.08a	4.76 ± 0.16a

Different superscript letters (a–c) within a row indicate significant differences among treatment groups’ means according to Tukey’s post-hoc test (P < 0.05). CN (non-injected control); CP (solvent injected); NLA 2.5: injected with 2.5 mg nanolauric; NLA 5: injected with 5 mg nanolauric. V length: Villus length, C.. depth: Crypt depth. Data are presented as mean ± standard error. Number of sampled birds (n) = 9 hatchlings/group (3/ replicate). A minimum of 10 villi and 20 crypts were measured from 3 to 5 sections from each chick. CD was measured from the base of the crypt to the end of Olfm4 staining along the villi. VH was measured from the end of the crypt to the tip of the villi. The VH/CD ratio was calculated based on these measurements.

Fig. 4.

Microscopic examination with H&E of jejunal villi (length, crypt depth, and villi length: crypt depth ratio) of one-day-old chicks. CN (non-injected control); CP (solvent injected); NLA 2.5: injected with 2.5 mg nanolauric; NLA 5: injected with 5 mg nanolauric. Number of sampled birds (n) = 9 hatchlings/group (3/ replicate). Images were taken using light microscopy at 10X magnification. Scale bar indicates 200 μm.

Discussion

Lauric acid, a major medium-chain fatty acid, is increasingly recognized for its bioactive benefits in animal nutrition²¹ and enhancing the metabolic performance²². Moreover, LA is a promising alternative to antibiotics in poultry and aquaculture feeds, as it improves gut integrity and hepatic antioxidant properties²³. The application of nanotechnology can further enhance lauric acid’s effectiveness by improving its bioavailability, facilitating rapid cellular uptake, and enabling broader tissue distribution²⁴. The nanolauric acid formulation used for in ovo delivery is designed to improve the solubility, stability, and tissue availability of lipophilic compounds²⁵. In addition, it aims to reduce the required effective dose by enhancing cellular uptake and distribution compared with conventional free-lipid forms²⁶, especially across extra-embryonic membranes during critical developmental timings²⁷.

The most relevant finding in this study concerning hatchability is that NLA in ovo at 2.5 mg/egg caused no adverse effects and was well-tolerated by the developing embryo; in contrast, hatchability was significantly reduced when eggs were injected with 5 mg/egg. The hatchability values between the CN (98%) and CP (97%), NLA 2.5 (96%) are high and similar, confirming the soundness of our methodology, hereby dismissing major stress or contamination induced by the injection process¹⁴. The marked decrease in hatchability to 80% in the NLA 5-group indicates that this dose was above the toxicological threshold that mediates energy metabolism and immunity²⁸. Additionally, the NLA 2.5 group showed a significant increase in both chick weight and chick yield (%), with a strong positive correlation between the two parameters. Conversely, the NLA 5 group exhibited a negative correlation, along with decreased chick weight and yield (%). The reduction in hatchability and yield percentages may result from metabolic disturbances caused by lipotoxicity, either due to excessive β-oxidation of fatty acids or disruption of the delicate lipid-dependent structure of the embryonic membrane²⁹.

In this study, in ovo administration of NLA at a dose of 2.5 mg (NLA 2.5) significantly lowered serum TC, TG, LDL, and VLDL, while maintaining HDL levels. In contrast, the 5 mg dose (NLA 5) resulted in reduced TG and VLDL levels but was also associated with decreased HDL and elevated LDL, suggesting a possible threshold beyond which metabolic regulation is disrupted. These findings align with previous reports demonstrating lauric acid can reduce body fat and serum triglycerides in animal models³⁰. In this respect, the in ovo injection of polyunsaturated fatty acid proportion as linoleic acid has been shown to alleviate lipid accumulation in newly hatched chicks by suppressing fatty acid synthesis and stimulating lipolysis in the liver³¹. Our NLA injections appear to have mimicked these lipid‐modulating effects, perhaps by upregulation of hepatic lipid catabolism or altering lipoprotein secretion³². Mechanistically, MCFAs bypass the carnitine shuttle (CPT I/II), allowing direct mitochondrial β-oxidation³³, and enhancing the excretion of cholesterol-rich bile, thereby reducing serum cholesterol levels³⁴.

At the molecular level, displayed reduced activities of lipoprotein lipase and HMG-CoA reductase, along with an increased HMG-CoA/mevalonate ratio, comparable to that of the statin drug atorvastatin in rats³⁵. In humans, lauric acid reduces blood cholesterol levels³⁶ and mitigates both high-fat diet-induced liver injury and metabolic dysfunction³⁷. In broilers, 1 ml of coconut oil supplementation significantly reduced TG and VLDL³⁸, and similar improvements in lipid profile were observed by Attia et al.³⁹. Moreover, Saeidi et al.⁴⁰ reported similar effects in quail chicks, with reduced TC, TG, LDL-C, and elevated HDL-C. Common carp fed high-fat diets with added GML showed lower plasma triglyceride levels, although total cholesterol remained unchanged³². Interestingly, the hypolipidemic effect of lauric acid is dose-dependent. In crabs, low-dose LA (0.8 mg/g) downregulated lipid synthesis and upregulated lipid catabolism genes, whereas high doses (2.91 mg/g) induced lipid accumulation and metabolic stress³³. Some studies reported that a portion of lauric acid may be stored in body fat rather than being rapidly metabolized, allowing it to contribute to physiological regulation³³. In black sea bream, 0.8% lauric acid supplementation elevated TG levels significantly, further highlighting species-specific responses⁴¹. While some studies reported that LA raises both HDL and LDL, the net cardiovascular risk depends on the TC: HDL ratio. Ramya et al.⁴² and Hewlings⁴³ found that although lauric acid elevates LDL-C, it also significantly increases HDL-C, improving lipid ratios. However, uncontrolled elevations in free fatty acids have been linked to insulin resistance, cardiovascular risk, and hepatic stress⁴⁴. The enhanced efficacy of NLA at 2.5 mg could also stem from its nanoparticulate form, through increasing cellular uptake and bioavailability of lipophilic molecules⁴⁵.

Oxidative stress arises when the production of reactive oxygen species (ROS) surpasses the capacity of endogenous antioxidant defenses, leading to damage of lipids, proteins, and nucleic acids, and ultimately triggering cell dysfunction or death⁴⁶. Central to this defense system are enzymatic antioxidants, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), which mitigate ROS and preserve cellular homeostasis^32,47. In this study, NLA enhanced antioxidant defenses in hatchlings, as evidenced by increased hepatic NRF2 mRNA expression, particularly at the 2.5 mg dose, indicating activation of the key antioxidant regulator. Consistently, in ovo administration of NLA significantly upregulated hepatic SOD expression, reflecting a strengthened antioxidant defense system. SOD, a critical NRF2-regulated enzyme, catalyzes the dismutation of superoxide radicals, thereby mitigating oxidative stress in hepatocytes⁴⁸. This upregulation was accompanied by an increase in the embryo’s glutathione (GSH) pool and a reduction in malondialdehyde (MDA), a marker of lipid peroxidation—particularly at the 2.5 mg NLA dose. The resulting improvement in the GSH: MDA ratio indicates enhanced redox balance and reduced oxidative damage, which is especially critical for developing chicks that depend on yolk-derived lipids for growth and energy⁴⁹. NLA may enhance its antioxidant efficacy by more effectively modulating intracellular signaling pathways such as NRF2, thereby boosting the expression of antioxidant genes and increasing the activity of associated enzymes.

Mechanistically, the antioxidant effect of lauric acid and its nanoform may be attributed to activation of the NRF2 pathway. NRF2 is a redox-sensitive transcription factor that regulates genes involved in antioxidant defense, including those for GSH biosynthesis, SOD1, CAT, GPx, thioredoxins, and glutathione S-transferases, which together orchestrate cellular detoxification and defense against oxidative insults⁵⁰. The activation of the NRF2–SOD axis may be attributed to the increased availability of medium-chain fatty acids from NLA, which are known to stimulate mitochondrial β-oxidation and redox signaling⁵¹. More specifically, Kong et al.⁵² reported that glycerol monolaurate (GML) rescued the LPS-induced suppression of SOD expression, and GML not only restored antioxidant enzyme expression but also reduced MDA and enhanced SOD activity in LPS-challenged broilers, demonstrating its ability to restore redox equilibrium under inflammatory oxidative stress. Similarly, glyceryl monolaurate (GML), a lauric acid derivative, elevated serum SOD and GSH-Px activities and total antioxidant capacity (T-AOC), while reducing MDA in laying hens⁵³ and broilers⁵⁴. Complementary evidence from aquatic and terrestrial species confirms the dose-dependent and species-specific effects of lauric acid. In broiler chickens, dietary alpha-monolaurin decreased lipid peroxidation (LPO), improved feed efficiency, and enhanced immune response in birds, suggesting its potential as a natural immune enhancer in the broiler industry⁵⁴. Similarly, dietary lauric acid in black sea bream reduced serum LPO levels^41,55. However, in gilthead sea bream, plasma antioxidant capacity remained unchanged with coconut fatty acid distillate supplementation, suggesting species-specific responses⁵⁶. Contrarily, and in alignment with our findings regarding the NLA 5 mg, higher dietary lauric acid levels (0.5% and 1%) in some European Seabass Juveniles increased hepatic LPO and decreased GPx activity, possibly due to pro-oxidant effects at excessive doses³⁰. In swimming crab, dietary lauric acid (up to 1.0 mg/g) enhanced glutathione peroxidase activity in the hepatopancreas, and antioxidant gene expression (CAT, GPx) peaked at 1.0 mg/g lauric acid³³. Moreover, in rodent models of oxidative stress, lauric acid administration upregulated NRF2 and its downstream extracellular SOD, and reduced markers of lipid peroxidation, particularly in cerebral microvasculature^57,58. Overall, the results highlight NLA’s dual antioxidant role: it strengthens endogenous enzyme defenses through NRF2-driven gene activation and decreases oxidative damage by lowering MDA levels. These outcomes support its promise as a safe, natural antioxidant—assuming proper dose control. Additionally, key metabolic and redox-related genes in the cecum were identified as responsive targets. Malic acid also contributes by helping restore redox balance, reducing gut inflammation, and improving feed efficiency in broilers with inflammation-related intestinal challenges⁵⁹.

The insulin-like growth factors IGF-1 and IGF-2 play central roles in regulating post-embryonic growth, development, and protein synthesis in poultry through their interaction with growth hormone⁶⁰. Our study shows that in ovo delivery of NLA significantly influenced hepatic IGF expression, with the 2.5 mg group displaying notable upregulation of both IGF-1 and IGF-2 alongside improved metabolic indicators. This suggests that NLA enhances hepatic anabolic signaling and supports growth-related nutrient regulation. Additionally, in ovo sodium butyrate given on day 12 of incubation has potential as a broiler supplement, with its effects depending on dosage and influencing gut microbiota, immune-related genes, and intestinal barrier function⁶¹. Medium-chain triglycerides (MCTs) rich in lauric acid can regulate growth by influencing the somatotropic (insulin/IGF) axis. Through interactions among insulin, IGF-1, IGF-2, their receptors, and IGF-binding proteins, these pathways stimulate cell growth and suppress apoptosis, mainly via the PI3K–Akt–mTOR and MAPK/ERK signaling cascades⁷. Watanabe and Tsujino⁶² stated that dietary MCT (from coconut oil) increases ghrelin and GH, raising IGF-1, which in turn activates Akt/mTOR signaling to boost protein synthesis.

In poultry, information on IGF regulation remains limited. For example, supplementing broiler diets with 1–1.5 mL/kg coconut oil has been shown to significantly improve body weight gain and enhance hepatic antioxidant enzyme activity, although growth hormone and IGF levels were not assessed in that study. Similarly, glycerol monolaurate, a lauric acid derivative administered at 0.9–1.2 g/kg, increased feed intake but did not produce significant improvements in early (0–14 day) weight gain⁵⁴. In summary, although MCFAs such as monolaurate commonly enhance broiler performance, no studies have evaluated IGF-1 or IGF-2 expression in poultry to verify an endocrine-mediated effect. Mechanistic evidence suggests that MCFAs could influence IGF signaling through multiple pathways; for instance, MCTs are known to increase ghrelin and growth hormone, which in turn stimulate IGF-1 production⁶². Although it is well known that IGF-1 is a postnatal growth factor in the poultry⁶³, IGF-2 also plays an important, yet likely underestimated, role during embryonic development. Recent findings show that IGF-2 plays a role in myoblast differentiation and mitochondrial dynamics in chicken embryos, connecting IGF-2 function with early tissue metabolic programming⁶⁴. Comparative analyses show that IGF-2 is expressed across more developmental stages in birds than IGF-1 and may play a larger role in certain embryonic tissues. The simultaneous upregulation of IGF-1 and IGF-2 in this study suggests coordinated activation of anabolic pathways and mitochondrial maturation, supporting improved protein deposition, lipid metabolism, and gut development at hatch. Mechanistically, enhanced antioxidant activity (via NRF-2 and mt-SOD2) may lower oxidative stress and strengthen IGF signaling and mitochondrial β-oxidation. Increased IGF activity may further stimulate mitochondrial biogenesis, creating a positive feedback loop that supports early metabolic programming. In addition, the greater efficiency exerted by NLA is probably due to its higher bioavailability as well as cellular uptake in comparison with native lauric acid. Nano-sizing enhances both surface area and stability, enabling effective absorption and hepatic delivery, which is the site of growth and metabolic signaling^66–68. Enhanced dispersion facilitates interaction with, for example, GPR84 or TLR4 sensors that then activate PI3K/Akt/mTOR and ERK signalling pathways to down-regulate IGF transcription⁶⁹. These mechanisms may explain the marked increases in IGF-1 and IGF-2 observed in the NLA 2.5 group. The nano-formulation’s sustained-release properties could prolong activation of anabolic and antioxidant pathways, enhancing lauric acid’s influence on IGF signaling and early metabolic programming in embryonic tissues. Future studies should examine IGF-2 localization, receptor activation, and downstream mitochondrial markers to clarify these interactions. In summary, lauric acid and its derivative monolaurate have been shown in mammals to enhance IGF-1 signaling—often through growth hormone (GH) stimulation—thereby improving growth performance. Although MCFA supplementation benefits poultry productivity, its influence on IGF expression remains poorly characterized. At the cellular level, MCFAs can activate IGF-related pathways, including Akt/mTOR and ERK, through receptors such as TLR4 and GPR84⁶².

During the perinatal period, rapid gut development occurs, with the early formation of intestinal villi being a critical process for embryonic and early post-hatch growth in broilers¹¹. The current study revealed that in ovo injection of both NLA 2.5 and NLA 5 doses improves villi length and crypt depth significantly if compared with controls. Our study is in agreement with a recent study that illustrated that glycerin monolaurate in-ovo injection had improved intestinal morphology and antioxidant system in LPS-challenged chicken embryos¹². Similarly, in an aquatic study, a diet supplemented with Lauric acid increased the height of villi and the number of goblet cells/villus significantly⁵⁵. Generally, in the same manner, it was reported that a diet supplemented with MCFAs significantly increased villus length and crypt depth in the duodenum and caecum of broilers⁷⁰. From the previous studies and our study, it can be concluded that lauric acid can efficiently improve gut histomorphometry, and this improvement may lead to improved gut health and performance.

While some mechanistic insights are drawn from studies in aquatic species, they are presented for comparative purposes only, recognizing potential species-specific metabolic differences between avian and aquatic organisms. Despite the promising findings, several limitations should be acknowledged. This study primarily focused on molecular and biochemical responses during the embryonic and early post-hatch periods, without evaluating subsequent effects on growth performance, feed efficiency, carcass traits, or immune function. Only two in ovo nanolauric acid doses (2.5 and 5 mg/egg) were tested, which may be insufficient to establish a comprehensive dose–response relationship or determine the optimal and maximum safe formulation for practical use. Additionally, potential nanotoxicological concerns such as nanoparticle accumulation, metabolic clearance, and effects on organ histoarchitecture were not assessed.

Conclusion

It is concluded that in ovo injection of 2.5 mg/egg of NLA is a safe and feasible approach for early post-hatch performance and improves serum lipid profiles in broiler chicks. Moreover, NLA strengthened hepatic antioxidant defenses via upregulation of NRF-2 and mt-SOD2, increased expression of growth-related genes (IGF-1 and IGF-2), and improved intestinal histomorphology. Conversely, a higher dose (5 mg/egg) negatively affected hatchability and induced metabolic stress. Overall, 2.5 mg/egg was identified as the optimal and safe dose, highlighting the potential of in ovo interventions to support embryonic-to-post-hatch transition in poultry production. A limitation of the study is that it was performed on day-old chicks, and long-term carry-over of these effects in profitable terms of overall productivity awaits investigation. Further research is desired using lower dosages (< 2.5 mg/egg) to evaluate dose-dependent responses and confirm whether 5 mg/egg is truly toxic.

Future recommendations and perspectives

In ovo injection of 2.5 mg/egg nano-lauric acid (NLA) shows promise for early-life metabolic programming in broilers by upregulating hepatic IGF-1 and IGF-2, enhancing anabolic signaling, and improving antioxidant gene expression and plasma lipid profiles. Further research is needed to elucidate underlying mechanisms, including downstream pathways like PI3K/Akt/mTOR, mitochondrial β-oxidation, and redox interactions. The 2.5 mg dose was effective and well-tolerated, whereas 5 mg/egg reduced hatchability, highlighting the need for dose–response and safety studies. Long-term trials should assess post-hatch growth, feed efficiency, carcass traits, intestinal morphology, and gut microbiome interactions. Exploring synergistic effects with other bioactives, such as probiotics or prebiotics, could support more advanced in ovo strategies to enhance gut health, oxidative capacity, and overall productivity.

Material and methods

Nano lauric acid preparation

The nano-lauric acid (NLA) suspension was procured from Arcos Organics (Fair Lawn, NJ, USA). According to the supplier, the suspension contained 82% (w/w) lauric acid in its nano-particulate form. The preparation method is proprietary to the manufacturer.

Nano particle characterization

Dynamic light scattering (DLS)

The size distribution of the Lauric acid nanoparticles was determined using a particle size analyzer (Nano-ZS, Malvern Instruments Ltd., UK). Approximately 90% of the particles were found to have a size of about 76 d.nm.

High-resolution transmission electron microscopy (HR-TEM)

A suspension of the Lauric acid nanoparticles was sonicated for 10 min using an ultrasonicator (Crest Ultrasonics Corp., New Jersey, USA). Then, a few drops were loaded on a carbon-coated copper grid, stained with phosphotungstic acid (1%), and left to dry. The grid was then examined by HR-TEM (JEOL, JEM-2100, Tokyo, Japan). The characteristics of the NLA, as shown in Fig. 5, revealed a uniform spherical morphology with particle sizes ranging from 122 to 156 nm.

Fig. 5.

Identification of lauric acid nanoparticle by High-Resolution Transmission Electron Microscopy (HR-TEM).

Zeta potential

The zeta potential for the sample was − 13.70 mV, determined using a dynamic light scattering instrument (NICOMP 380 ZLS), suggesting moderate stability of the nanoparticles in colloidal solution. The study confirmed that the NLA exhibited a hydrodynamic diameter of 76 nm and a zeta potential of − 13.70 mV.

Hatchery management and egg incubation

All experiments were approved by the Ethics Committee of the Faculty of Veterinary Medicine, Cairo University, Egypt (VET.CU.IACUC.EG; Approval no. VET CU110520251104), following the National Research Council’s Guide for the Care and Use of Laboratory Animals, the EU Directive 2010/63/EU for the protection of animals used for scientific purposes, and the UK Animals (Scientific Procedures) Act of 1986 and related guidelines. Furthermore, all experimental techniques were documented following the ARRIVE guidelines⁷¹, ensuring adherence to the highest standards of animal welfare and scientific integrity.

A total of 400 fertilized eggs laid by a 40-week-old Arbor Acres breeder flock were obtained from Cairo Poultry Company (CPC). Immediately after egg collection, eggs were weighed, and the average egg weight was 65 ± 1.50 g. Eggs were then sanitized by ammonium tetrachloride and distributed in trays within the incubator (PTO-Italy). The incubator was thoroughly disinfected by formalin fumigation, and the eggs were incubated under optimal incubation conditions of (37.5 °C and 60% RH) with automatic trays turning every hour.

Rationale for nanolauric acid (NLA) dosage selection

The doses of 2.5 mg and 5 mg nanolauric acid per egg were selected based on biological relevance, embryonic safety, and practical feasibility, as no prior studies have examined the in ovo application of nanolauric acid. Given that the nanolauric formulation contained 82% lauric acid, these doses correspond to approximately 2.05 and 4.10 mg lauric acid per egg, respectively. Compared with typical post-hatch dietary exposure (approximately 50 mg/day), these represent 4–8% of a normal daily intake, thus constituting a conservative, physiologically safe range consistent with accepted in ovo injection limits¹⁴.

In ovo injection protocol

On the eleventh day of incubation, all the eggs were carefully removed and candled; unfertilized eggs and dead embryos were discarded. On the 12th day of incubation, fertilized eggs were sanitized using 70% ethyl alcohol, then allocated into 4 experimental groups, each containing 3 replicates (30 eggs per replicate). Sterile water for injection was used to dissolve the Lauric acid nanoparticles (NLA). The experimental groups were a non-injected control group (CN); diluent-injected control (CP, 0.1 ml sterile water for injection; NLA-injected group at a dose of 2.5 mg /egg, dissolved in 0.1 ml sterile water (NLA 2.5); NLA-injected group at a dose of 5 mg/egg, dissolved in 0.1 ml sterile water (NLA 5). The eggs were sanitized with 70% ethanol, a hole was drilled in the eggshell at the narrow end using the tip of a sterile 22-gauge needle⁷² at 45 angle according to the technique of Abdel-Moneim et al.⁷³, and 0.1 ml of the treatment was injected into the yolk by using an insulin syringe and a 16 mm long preterm infant needle. Melted paraffin was then used to seal the holes, and eggs were returned to the incubator. On the 18th day of incubation, the eggs were placed into the hatching boxes under optimal hatching conditions of 37 °C and 65% RH.

Hatchability assessment

After the injection, all eggs were returned to the incubator. On day 18 of incubation, eggs were transferred to hatching trays. The hatch was monitored from day 20 to day 22. A chick was regarded as hatched when it had completely left the shell and displayed no gross abnormality. Percentage hatchability was determined for each group using the formula:

Chick yield

After hatching, chicks were labeled and weighed individually to the nearest 0.1 g. Chick yield was calculated for each group according to the following equation:

Blood sampling (from hatchlings)

Blood samples were randomly collected from one-day-old chicks of both sexes with an average body weight of 45 ± 0.50 g. For each treatment, 3 chicks per replicate were used. Samples were collected in gel separator tubes and centrifuged at 5000 rpm for 20 min to obtain serum for lipid profile analysis. The blood collection was performed by the cardiac puncture method according to Cook et al.⁷⁴.

Serum lipid profile assessment

The lipid profile—including total cholesterol, triglycerides, and HDL cholesterol—was assessed according to Fayed et al.⁷⁵ and following the methods described by Lopes-Virella et al.⁷⁶, Richmond⁷⁷, and Tietz⁷⁸, respectively. Commercial reagent kits (Spectrum Company, Cairo, Egypt) were used according to the manufacturer’s protocol, with absorbance measured at 546 nm. LDL and VLDL cholesterol levels were calculated using the Friedewald formula⁷⁹, where VLDL = triglycerides ÷ 5, and LDL = total cholesterol − (HDL + VLDL).

Tissue sampling

Three hatchlings/replicates were picked, injected with thiopental sodium [Thiopentone, 2.5%, Egyptian International Pharmaceutical Industries Co., Egypt], which was diluted with normal saline solution (0.9% NaCl) at a dose of 35 mg/kg. The anaesthetic was administered intraperitoneally (IP), following the protocol described by Mousa et al.⁸⁰ in chicks. The chicks were then euthanized by cervical dislocation, followed by a surgical incision. A 100 mg of liver specimens were cut, gathered on liquid nitrogen for redox status assessment and on RNAlater™ Stabilization Solution (Invitrogen, USA, Cat. #AM7020) for RNA extraction, then kept at − 80 °C until usage.

Hepatic redox status assessment

Before measurement of hepatic redox status, 100 mg of liver samples were homogenized in 50 mM phosphate buffer, pH 7.5. Homogenates were centrifuged at 4000 rpm for 15 min at 4 °C; supernatants were collected and stored at − 80 °C until analysis⁸¹.

Malondialdehyde (MDA) concentration as an index of lipid peroxidation was determined as described by Ohkawa et al.⁸². MDA was evaluated by measuring the thiobarbituric acid reactive species (TBARS). The absorbance of the resultant pink product was measured spectrophotometrically at 534 nm using commercial kits (Bio Diagnostic, Giza, Egypt). GSH concentration was determined based on its reaction with 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), forming a yellow-colored chromophore measurable at 405 nm. The GSH levels were expressed as mg/g of tissue⁸³.

Quantitative real-time RT-PCR analysis

Total RNA was extracted using a total RNA purification kit (Jena Bioscience, Germany, Cat. #PP-210S) following the manufacturer’s instructions. Using a Thermo Scientific Nanodrop ND-1000 Spectrophotometer, the concentration and purity of RNA were determined. Next, the whole RNA was reverse-transcribed using the Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, USA, Cat. #K1622). The fluorescence-based real-time detection technique utilizing iQ SYBR® Green Supermix (Bio-Rad 1708880, USA) was used to quantify the amount of mRNA expression of each gene concerning β-actin (ACTB) as an endogenous reference gene, as per the manufacturer’s instructions⁸⁴.

Real-time RT-PCR was performed for each gene using the primers specified in Table 5. The sample was denatured for 3 min at 95 °C as part of the cycling technique. This was followed by 40 cycles of denaturation (15 s at 95 °C), annealing (30 s at 60 °C), and extension (30 s at 72 °C). The specificity of the PCR products was assessed by inspecting the melting curves after each experiment. Each experiment had a no-template negative control (NTC) and was conducted in three replicates. Equation 2-ΔΔCT was used to calculate the expression relative to the control⁸⁵.

Table 5.

Primer sequences used for qRT-PCR analysis.

Gene symbol	Amplicon size (bp)	Gene description	Accession number/REF	Primer sequence
1 Housekeeping gene
β-actin	177 bp	Beta-actin	Abdelfatah et al.84	F:-5′-CCCACACCCCTGTGATGAAA-3′ R:-5′-TAGAACTTTGGGGGCGTTCG-3′
2 Antioxidant genes
NRF-2	148 bp	Nuclear factor erythroid 2-related factor 2	NM_001396902.1	F:5′-GAGCAATGGGCTCTGTGGAA-3′ R:5′-AGATCCGCCATGATCGAATACAA-3′
Mt-SOD2	175 bp	mitochondrial superoxide dismutase 2	NM_204211.2	F: 5′-GTGACTTTGGTTCCTTCGCA-3′ R: 5′- CCCAGCAATGGAATGAGACC-3′
5 Nutrient-sensing pathway
IGF-1	194 bp	Insulin-like growth factor-1	NM_001004384.3	F: 5′- TTGGCCTGTGTTTGCTTACC-3′ R: 5′-ACTCTGGAAGCAGCACTCAT-3′
IGF-2	150 bp	Insulin-like growth factor-2	NM_001030342.5	F: 5′-TGGCTGAGATGGGACCTTTT-3′ R: 5′-GGGTTTCCAGATCCACTCCA-3′

Intestinal morphology

Jejunal specimens (n = 10) were immediately fixed by immersion in 10% neutral buffered formalin. Following fixation, the tissues underwent graded dehydration using ascending concentrations of alcohol, and then were cleared in xylene. Subsequently, the samples were embedded in paraffin (melting point: 56 °C) and blocked. Tissue sections of 4 µm thickness were then prepared using a microtome (Leica, Germany) and processed for hematoxylin and eosin (H&E) staining. The stained slides were examined under a light microscope (LEICA DM500), and images were captured using a Leica ICC50 HD camera mounted on the microscope. The captured images were analyzed using Leica Microsystems image analysis software (LAS version 3.8.0 [build:878], Leica Ltd). Computer-assisted morphometric measurements were performed, including: Villus height (µm) was measured from the tip to the base of each villus. Villus width (µm) was measured at three levels: the apex, middle, and base. Additionally, the villus height-to-width ratio was calculated to assess intestinal morphology⁸⁶.

Statistical analysis

All statistical analysis and graphs were conducted using RStudio-2023.06.1-524⁸⁷ and the R programming language v4.3.1 (R Core Team, 2023). A one-way analysis of variance (ANOVA) was conducted to assess statistical differences between groups. Before the analysis, ANOVA assumptions were verified using the Shapiro and Wilk⁸⁸ for normality and the Levene⁸⁹ test for homogeneity of variance. Subsequently, ANOVA was performed. When significant effects were detected, Tukey’s Honestly Significant Difference (HSD) test was applied as a post-hoc multiple comparison procedure, with P-value adjustment using the Benjamini and Hochberg⁹⁰. with P-value adjustment. Finally, data were plotted as bar graphs and tables representing the mean ± standard error. Significant differences between groups were indicated on bar graphs and tables using letter annotations above each group. Groups not sharing the same letters were considered significantly different at a P-value < 0.05. Asterisks (*) indicate the degree of statistical significance relative to the control group as follows: P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***). Differences in hatchability among treatment groups were analyzed using Fisher’s test with Bonferroni correction to control the family-wise error rate. A P-value of less than 0.05 was considered statistically significant.

Author contributions

M.M.S, S.N.E, K.A.E, G.A.S : Conceptualization, investigation, methodology, funding acquisition, project administration, writing the original draft, data analysis, and interpretation. A.M.Y. : conducted the antioxidant and RT-PCR analysis. M.I. : Project administration and egg supplementation. All authors read and approved the manuscript.

Funding

Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.