1- Department of Pathobiology, Faculty of Veterinary Science, Bu-Ali Sina University, Hamedan, Iran.
2- Department of Animal Science, University of Guilan, Rasht, Iran
3- Department of Animal Science, University of Mohaghegh Ardabili, Ardabil, Iran
4- Department of Range and Watershed Management, University of Mohaghegh Ardabili, Ardabil, Iran
5- North Region Branch, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research, Education and Extension Organization (AREEO), Rasht, IRAN
2- Department of Animal Science, University of Guilan, Rasht, Iran
3- Department of Animal Science, University of Mohaghegh Ardabili, Ardabil, Iran
4- Department of Range and Watershed Management, University of Mohaghegh Ardabili, Ardabil, Iran
5- North Region Branch, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research, Education and Extension Organization (AREEO), Rasht, IRAN
Abstract: (51 Views)
Introduction and Objective: The global poultry industry is actively seeking natural, safe, and effective alternatives to growth-promoting antibiotics, which have faced severe restrictions due to concerns about bacterial resistance and drug residues. In this context, phytogenic compounds and their derivatives have garnered significant attention due to their multiple biological properties, low toxicity, and biodegradability. D-limonene, a natural cyclic monoterpene abundantly found in citrus essential oils, is known for its antioxidant, anti-inflammatory, antimicrobial properties, and particularly its unique ability to inhibit the aromatase enzyme (the key enzyme in the conversion of androgens to estrogens). Furthermore, in ovo injection technology is a novel method for intervening during critical embryonic developmental stages and directly delivering bioactive compounds. This study was designed to investigate the effect of in ovo injection of two different levels of D-limonene on the morphology (histomorphometry) of the digestive and immune organs in broiler chicken embryos. The primary hypothesis was that D-limonene could dose-dependently influence the growth and differentiation of these tissues through various mechanisms, including hormonal modulation and antioxidant protection.
Materials and Methods: This research was conducted on 160 fertile, homogeneous broiler breeder eggs (Ross 308) with an average weight of 65-68 grams. The eggs were randomly assigned in a completely randomized design into four treatment groups of 40 eggs each: Control group (C) receiving no injection, Sham group (CW) injected with 0.1 mL of sterile distilled water, DL1 group injected with 0.1 mL of pure D-limonene extract, and DL2 group injected with 0.2 mL of the same D-limonene extract. Injection was performed on the fifth day of incubation via the air cell under fully sterile conditions. On the eighteenth day of incubation, tissue samples were collected from the digestive organs, including the esophagus, proventriculus, gizzard, small intestine (jejunum), and liver, as well as the primary immune organs, including the bursa of Fabricius, spleen, and thymus. After fixation in 10% buffered formalin, the samples underwent standard processing steps of dehydration, clearing, and paraffin embedding. Tissue sections of 5-7 micrometers thickness were prepared and stained with Hematoxylin and Eosin (H&E). Histomorphometric examination was performed using a light microscope equipped with a digital camera and image analysis software. Numerous parameters, including epithelial thickness, muscular layer thickness, intestinal villus height, gland diameter, hepatocyte dimensions, and the dimensions of lymphoid structures in immune organs, were accurately measured. Data were analyzed using one-way analysis of variance (ANOVA) after ensuring normal distribution with the Shapiro-Wilk test; if significant, means were compared using Duncan's multiple range test at a significance level of P < 0.05.
Results: The findings of this research revealed significant and dose-dependent changes in response to D-limonene injection. In the digestive system, the high dose of D-limonene (DL2) caused a significant decrease in the thickness of the muscular layer in the esophagus and proventriculus, while simultaneously significantly increasing the diameter of the secretory glands in these organs. In the gizzard, the low dose (DL1) significantly increased the thickness of the muscular layer, whereas the high dose (DL2) led to an increase in the depth of the mucosal glands. In the small intestine (jejunum), D-limonene injection at both doses caused a significant increase in epithelial thickness, villus height, and muscular layer thickness; the increased villus height indicates a strong potential for improving the surface area for nutrient absorption. In the liver, the diameter of hepatocytes and their nuclei significantly increased in the D-limonene treated groups, suggesting hypertrophy of liver cells. In the immune system, the results were even more notable. The high dose of D-limonene (DL2) caused a significant increase in the diameter of the lymphoid nodules in the bursa of Fabricius, the diameter of the spleen's white pulp (lymphocyte-producing areas), and the thickness of the thymic lobules' medulla. These results clearly demonstrate that D-limonene affects not only the digestive tissues but also the development and early maturation of the immune system.
This study convincingly demonstrated that in ovo injection of D-limonene on the fifth day of incubation is an effective strategy for modulating the growth and development of digestive and immune tissues in broiler chicken embryos. The observed effects, which were predominantly dose-dependent, are exerted through multiple interconnected mechanisms, including aromatase inhibition, antioxidant activity, and stimulation of secretion. In summary, the low dose of D-limonene is more beneficial for strengthening the muscular structures of the digestive tract and improving the intestinal absorptive potential, while the higher dose has greater potential for enhancing secretory capabilities and, particularly, strengthening and developing the primary immune system.
Conclusion: These findings provide a solid scientific basis for considering D-limonene as a natural growth promoter and immune modulator in poultry breeding programs, especially through the novel technology of in ovo injection. However, to translate these laboratory findings into practical applications at the industrial level, further studies are essential to evaluate the impact of this intervention on growth performance, feed conversion ratio, general health, and response to immune challenges during the post-hatch period, as well as to gain a deeper understanding of the underlying molecular mechanisms of these effects.
Materials and Methods: This research was conducted on 160 fertile, homogeneous broiler breeder eggs (Ross 308) with an average weight of 65-68 grams. The eggs were randomly assigned in a completely randomized design into four treatment groups of 40 eggs each: Control group (C) receiving no injection, Sham group (CW) injected with 0.1 mL of sterile distilled water, DL1 group injected with 0.1 mL of pure D-limonene extract, and DL2 group injected with 0.2 mL of the same D-limonene extract. Injection was performed on the fifth day of incubation via the air cell under fully sterile conditions. On the eighteenth day of incubation, tissue samples were collected from the digestive organs, including the esophagus, proventriculus, gizzard, small intestine (jejunum), and liver, as well as the primary immune organs, including the bursa of Fabricius, spleen, and thymus. After fixation in 10% buffered formalin, the samples underwent standard processing steps of dehydration, clearing, and paraffin embedding. Tissue sections of 5-7 micrometers thickness were prepared and stained with Hematoxylin and Eosin (H&E). Histomorphometric examination was performed using a light microscope equipped with a digital camera and image analysis software. Numerous parameters, including epithelial thickness, muscular layer thickness, intestinal villus height, gland diameter, hepatocyte dimensions, and the dimensions of lymphoid structures in immune organs, were accurately measured. Data were analyzed using one-way analysis of variance (ANOVA) after ensuring normal distribution with the Shapiro-Wilk test; if significant, means were compared using Duncan's multiple range test at a significance level of P < 0.05.
Results: The findings of this research revealed significant and dose-dependent changes in response to D-limonene injection. In the digestive system, the high dose of D-limonene (DL2) caused a significant decrease in the thickness of the muscular layer in the esophagus and proventriculus, while simultaneously significantly increasing the diameter of the secretory glands in these organs. In the gizzard, the low dose (DL1) significantly increased the thickness of the muscular layer, whereas the high dose (DL2) led to an increase in the depth of the mucosal glands. In the small intestine (jejunum), D-limonene injection at both doses caused a significant increase in epithelial thickness, villus height, and muscular layer thickness; the increased villus height indicates a strong potential for improving the surface area for nutrient absorption. In the liver, the diameter of hepatocytes and their nuclei significantly increased in the D-limonene treated groups, suggesting hypertrophy of liver cells. In the immune system, the results were even more notable. The high dose of D-limonene (DL2) caused a significant increase in the diameter of the lymphoid nodules in the bursa of Fabricius, the diameter of the spleen's white pulp (lymphocyte-producing areas), and the thickness of the thymic lobules' medulla. These results clearly demonstrate that D-limonene affects not only the digestive tissues but also the development and early maturation of the immune system.
This study convincingly demonstrated that in ovo injection of D-limonene on the fifth day of incubation is an effective strategy for modulating the growth and development of digestive and immune tissues in broiler chicken embryos. The observed effects, which were predominantly dose-dependent, are exerted through multiple interconnected mechanisms, including aromatase inhibition, antioxidant activity, and stimulation of secretion. In summary, the low dose of D-limonene is more beneficial for strengthening the muscular structures of the digestive tract and improving the intestinal absorptive potential, while the higher dose has greater potential for enhancing secretory capabilities and, particularly, strengthening and developing the primary immune system.
Conclusion: These findings provide a solid scientific basis for considering D-limonene as a natural growth promoter and immune modulator in poultry breeding programs, especially through the novel technology of in ovo injection. However, to translate these laboratory findings into practical applications at the industrial level, further studies are essential to evaluate the impact of this intervention on growth performance, feed conversion ratio, general health, and response to immune challenges during the post-hatch period, as well as to gain a deeper understanding of the underlying molecular mechanisms of these effects.
Keywords: Broiler chicken, D-limonene, Digestive system, Embryo, Histomorphometry, Immune syste, In ovo injection
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