INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)  
ISSN No. 2321-2705 | DOI: 10.51244/IJRSI |Volume XII Issue X October 2025  
Assessment of Lipid Peroxidation in Liver and Heart Of D-  
Galagctose Induced Chick Embryo  
Rahul B. Patil1 and Shreya R. Patil2  
Department of Zoology, Veer Wajekar Arts, Science and Commerce College, Phunde, Navi Mumbai-  
400702 (M.S.) India  
DOI: https://dx.doi.org/10.51244/IJRSI.2025.1210000266  
Received: 08 November 2025; Accepted: 16 November 2025; Published: 18 November 2025  
ABSTRACT  
Oxidative stress is a critical mediator of cellular injury, leading to lipid peroxidation and organ dysfunction.  
The present study evaluates D-galactose-induced oxidative damage in the liver and heart of 8-day-old chick  
embryos by quantifying lipid peroxidation through thiobarbituric acid reactive substances (TBARS) assay.  
Fertilized hen eggs were incubated and divided into control and experimental groups, with the latter receiving  
50 µg D-galactose/egg via the air sac. After 48 h, liver and heart tissues were analyzed for malondialdehyde  
(MDA), an indicator of lipid peroxidation. D-galactose administration significantly elevated (p < 0.001) MDA  
levels in both tissues, suggesting enhanced oxidative stress. The heart showed slightly higher peroxidation,  
possibly due to its post-mitotic nature. Histopathological interpretations (literature-based) support that  
oxidative injury could manifest as hepatocellular vacuolization and cardiomyocyte disruption. This study  
reinforces the chick embryo as a promising model for developmental oxidative stress and aging research,  
highlighting the pathophysiological impact of glycation-induced oxidative injury in early organogenesis.  
Keywords: oxidative stress, lipid peroxidation, D-galactose, chick embryo, MDA, aging, advanced glycation  
INTRODUCTION  
Aging and oxidative stress are closely intertwined biological processes characterized by increased production  
of reactive oxygen species (ROS) and impaired antioxidant defense (Halliwell & Chirico, 1993; Harman,  
2009). Lipid peroxidation, resulting from ROS attack on polyunsaturated fatty acids, leads to formation of  
reactive aldehydes such as malondialdehyde (MDA) — a key biomarker of oxidative injury (Ayala et al.,  
2014).  
Although rodent models are conventionally used for oxidative stress studies, the chick embryo provides a  
highly suitable in-ovo developmental model due to its accessibility, cost-effectiveness, and similarity to  
mammalian embryogenesis (Rengarajan et al., 2018). D-galactose, a reducing sugar, is widely employed to  
induce accelerated aging through formation of advanced glycation end products (AGEs), mitochondrial  
dysfunction, and chronic oxidative stress (Song et al., 1999; Ho et al., 2020).  
The liver and heart are particularly susceptible to oxidative damage owing to their high metabolic activity.  
Hepatic lipid peroxidation can lead to hepatocyte degeneration, while oxidative injury in cardiac tissue can  
impair contractility and mitochondrial function (Matsuda & Shimomura, 2013). Thus, investigating D-  
galactoseinduced oxidative changes in embryonic tissues can elucidate the early mechanisms of biochemical  
and structural alterations that underlie age-related degeneration.  
MATERIAL & METHODS  
Experimental Design  
Freshly fertilized zero-day hen eggs were obtained from a local hatchery (Panvel, Navi Mumbai, India). Eggs  
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INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)  
ISSN No. 2321-2705 | DOI: 10.51244/IJRSI |Volume XII Issue X October 2025  
were cleaned with distilled water and 70% ethanol, then incubated at 38 ± 0.5 °C and 58–60% relative  
humidity. Eggs were rotated manually twice daily to ensure uniform temperature exposure.  
At day 8 (E8), viable embryos were divided into two groups (n = 6 per group):  
1. Control group: received 50 µL sterile distilled water.  
2. Experimental group: received 50 µg D-galactose (Sigma-Aldrich) dissolved in 50 µL distilled water.  
Injection Technique and Contamination Control  
The injection site was localized by candling, avoiding visible blood vessels. A small hole (~1 mm) was drilled  
2 cm below the air sac, and injections were made using a sterile insulin syringe (26 G). The injection site  
was immediately sealed with sterile paraffin wax to prevent contamination and dehydration. All procedures  
were performed under aseptic conditions inside a laminar airflow cabinet.  
Incubation Consistency  
Post-injection, eggs were incubated for 48 h under controlled humidity and temperature, with continuous  
rotation to maintain even exposure. Non-viable embryos were removed daily.  
Tissue Preparation  
Liver and heart tissues were excised, rinsed in ice-cold phosphate buffer (75 mM, pH 7.0), and homogenized  
(2 mg tissue/mL). A 10 ppm chlorotetracycline solution was used as an antibiotic control to prevent microbial  
contamination during incubation.  
Estimation of Lipid Peroxidation (TBARS Assay)  
Lipid peroxidation was estimated following Wills (1966). Homogenates were incubated with 1 mM ascorbic  
acid, 1 mM FeCl₃, and 75 mM phosphate buffer. After heating in a boiling water bath for 10 min, samples were  
cooled, and absorbance was recorded at 532 nm. MDA concentration was expressed as nmol MDA/mg tissue.  
Statistical Analysis  
Data were expressed as mean ± SD (n = 6) and analyzed using Student’s t-test, with significance set at p <  
0.001.  
RESULT  
Table no. 1: Effect of induced oxidative stress by D-galactose on Lipid Peroxidation of Heart in chick  
embryo (n mols MDA/mg tissue)  
Type of LPO  
Total LPO  
Control Group 1  
51.924±1.665  
D-galactose injected Group 2 Statistical Significance 1:2  
92.3072±2.3552  
4.570±0.220  
t=7.000 p<0.001  
t=3.9596 t<0.001  
Mitochondrial LPO 4.1526±0.084  
Effect of induced oxidative stress by D-galactose on Lipid Peroxidation of Heart in chick embryo The MDA  
were found elevated significantly (p<0.001) in D-galactose stressed heart of chick embryo compared to  
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INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)  
ISSN No. 2321-2705 | DOI: 10.51244/IJRSI |Volume XII Issue X October 2025  
control. Mitochondrial LPO in the form of MDA increased but not found significant compared to total LPO.  
Table no. 2: Effect of induced oxidative stress by D-galactose on Lipid Peroxidation of Liver in chick  
embryo (n mols MDA/mg tissue)  
Type of LPO  
Total LPO  
Control Group  
1
D-galactose injected  
Group 2  
Statistical Significance 1:2  
37.4998±1.4422 95.1918±2.7617  
t=9.2590 p  
<0.001  
Mitochondrial Peroxidation 3.2720±0.166  
5.703±0.115  
t=2.804 p<0.001  
Effect of induced oxidative stress by D-galactose on Lipid Peroxidation of liver in chick embryo Total lipid  
peroxidation in the form of MDA were found elevated significantly (p<0.001) in D-galactose stressed heart of  
chick embryo compared to control. Mitochondrial LPO in the form of MDA increased but not found significant  
compared to total LPO  
Graph No. 1: Effect of D-galactose on level of MDA heart and liver of Chick embryo  
160  
140  
120  
100  
80  
D-galactose induced  
Control  
60  
40  
20  
0
Total LPO  
Heart  
Total LPO  
Liver  
Mito. LPO  
Heart  
Mito. LPO  
Liver  
DISCUSSION  
D-galactose administration induces oxidative stress by generating ROS and AGEs, leading to mitochondrial  
dysfunction and lipid peroxidation (Song et al., 1999; Ho et al., 2020). The observed elevation of MDA  
indicates increased peroxidative damage to membrane lipids, particularly in high-energy tissues like heart and  
liver.  
Histopathological analyses in related studies revealed cytoplasmic vacuolization, hepatocellular swelling, and  
cardiomyocyte disorganization in D-galactose-treated embryos and rodents (Zhao et al., 2018; Chen et al.,  
2021). These morphological outcomes align with the biochemical findings of elevated lipid peroxidation in the  
present study, suggesting structural disruption secondary to oxidative injury.  
The slight difference between total and mitochondrial LPO levels indicates compartment-specific vulnerability.  
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INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)  
ISSN No. 2321-2705 | DOI: 10.51244/IJRSI |Volume XII Issue X October 2025  
Post-mitotic cells in cardiac tissue accumulate oxidative damage more readily than hepatocytes, which possess  
stronger regenerative and antioxidant capacities (Matsuda & Shimomura, 2013). Thus, the chick embryo  
provides a suitable model for assessing oxidative stress during early development.  
CONCLUSION  
D-galactose exposure significantly enhances lipid peroxidation in chick embryonic liver and heart tissues,  
validating its oxidative and aging potential even during early developmental stages. Incorporating  
histopathological and ultrastructural evaluation in future studies would provide direct visual correlation with  
biochemical alterations. The chick embryo model demonstrates promising utility for exploring mechanisms of  
embryonic oxidative stress, offering biomedical insights relevant to developmental toxicity, aging, and  
metabolic disorders.  
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