Effect of Lead Toxicity on Japanese Quail and its Alleviation with Dietary Selenium

Lead is a heavy metal that can be found in a variety of forms in soil, water, and plants. It is a natural component of the Earth’s crust. Lead is well as -known heavy metal contaminant with significant toxicity and a long-term environmental persistence due to its non-biodegradable nature (Flora et al., 2012). Lead poisoning in birds is a serious problem (Yazdanparast et al., 2022), when it comes to birds, As a result of the exposure to lead in the liver, histopathology liver and kidney damage, as well as a reduction in bone mineralization (Binkowski et al., 2013). Lead poisoning can influence laying hens’ performance in reproduction by reducing fertility (Flora et al., 2012). Lead Poisoning has a variety of negative consequences in the body including problems with the brain; kidneys, liver, circulation, and immune system are all affected (Offor and Orisakwe, 2017). Furthermore, low-dose lead exposure can cause considerable apoptosis in the liver and kidneys, eventually impairing their function (Francisco et al., 2003).

Lead poisoning has resulted in a large number of bird deaths, and it continues to be a serious hazard to both terrestrial and aquatic species (Francisco et al., 2003). It is a major cause of death of many endangered species like the California condor (Gymnogyps californianus); while many other species are at high risk (Bakker and Demerout, 2017). Lethargy, anorexia, crop paralysis, vomiting, diarrhea, ataxia, anemia, osteoporosis, emaciation, were all symptoms of lead poisoning. Moreover it may cause deterioration of sperm quantity and quality in male birds. Additionally, lead poisoning can cause memory loss in young birds (Vallverdu et al., 2016;Kou et al., 2020).

Selenium is a mineral that is required by microorganisms, animals, and humans to some extent. Due to its’ potent antioxidant activity it has been observed beneficial for growth performance, reproduction, health, and immunity of farm animals (Abdel-Hamid et al., 2020;Ahn et al., 2021). Selenium is found in a variety of selenoproteins, including glutathione peroxidases (GPx), which are antioxidant enzymes found in both animals and humans (Savitha et al., 2014). Protective effects of selenium against acute and chronic toxicity has been well established in the literature (Qiao et al., 2022; Yu et al., 2023). It has been reported that like other poultry species, quails are extremely vulnerable to lead poisoning, altering the weight of the body and its organs as well as the blood, serum biochemistry and immunity (Wang et al., 2022). The goal of this research was to investigate the harmful effects of long-term exposure of lead acetate and its protection with selenium in quails.

This animal experiment was conducted in line with Animal Ethical Standards and was approved by Sindh Agriculture University Tandojam. The current research was conducted during March and April 2021 to evaluate the effects of lead acetate and ameliorative efficacy of selenium on liver and kidney functions of Japanese quail. In this experiment, 3- week old female Japanese quails (n=36) were obtained from a commercial hatchery and brought to the Faculty of Animal Husbandry and Veterinary Sciences. All the Birds were weighed immediately upon arrival. Japanese quails were randomly assigned to each five treatments, with six birds per cage. Treatment groups includes viz., A (control group, without any treatment), B (selenium 1.0 mg/L), C (lead acetate 200 mg/L), D (lead acetate 400 mg/L ), E (lead acetate 200 + selenium 1.0 mg/L) and F (lead acetate 400 + selenium 1.0 mg/L). Both selenium (sodium selenite) and lead acetate were given by adding in the drinking water. The birds were allowed for water and feed ad libitum. Commercial feed as well as all environmental conditions were provided according to standard (NRC, 1994). The experiment lasted for 20 days.

All the birds were observed daily for behavioral changes, body weight and development of clinical signs like dullness, morbidity and mortality etc. At the end of trial, 4 birds were randomly selected and blood was collected from the wing vein aseptically using a 1 ml sterile needle and was placed into 1.5 ml tube (without anticoagulant) for the biochemical analysis. After the completion of blood collection all the birds were slaughtered by halal method. Liver and kidney samples were collected, washed with normal saline twice and preserved in formalin for histopathology.

Biochemical tests

Commercially available kits (HUMAN Diagnostic Co., Wiesbaden, Germany) were used to perform liver and kidney function tests in accordance with given protocol methods of manufacturer. The kits includes gama- glutamyl transferase (GGT), bilirubin, albumin, alkaline phosphate, uric acid and creatinine. Prescribed protocols of IFCC (International Federation of Clinical Chemistry) were adopted according to methods of Talpur et al. (2022).

For histopathological examination, the liver and kidney samples were dissected into small pieces using scalpel blade and were preserved in formalin. Tissue samples were dehydrated in ethanol, cleared in xylene then infiltrated / embedded in hard paraffin wax. It was cut into small sections (5 μm), and stained using HandE (Hematoxylin and Eosin) stains (Khan et al., 2022).

The data was statistically analyzed using Statistix 8.1 software. Groups were compared by analysis of variance (ANOVA) and LSD test to know level of significance (p<0.05).

The weight gain results are depicted in Figure 1. At the end of experiment, the weight was highest (132 g) in group A, while it was lowest in the lead induced group C (66.66g). Selenium treatments significantly increased weight when compared with positive control groups (group C, and D), but group E showed a better amelioration. The statistical analysis indicated significant (p<0.05) differences between all groups except group B and D.

The findings of different dosages of lead and selenium are presented in Figure 2 for GGT level in quails. The results indicated that maximum GGT (3.36 U/L) was recorded in group D followed by group F (2.98 U/L), group C (2.81 U/L), and group E (2.68 U/L); while minimum GGT level was observed in group A (1.78 U/L) and B (1.82 U/L). Selenium treated group F showed a significant reduction (p<0.05) in GGT level as compared to its’ corresponding positive control (group D).

The effects of different dosages of lead and selenium on albumin level in quails was presented in Figure 3. The results indicated that maximum albumin (13.84 g/L) was recorded in group A followed by group B (12.94 g/L), group E (11.3 g/L), group C (9.55 g/L), group F (8.58 g/L) and group D (6.57 g/L). Albumin values differences between various groups were significant (p<0.05) except between group A and B. Albumin level was significantly improved (p < 0.05) in selenium treated groups ( group E and F) as compared to their corresponding positive controls (group C and D respectively).

Mean serum bilirubin values obtained in quails at the end of trial were depicted in Figure 4. Significant difference (p < 0.05) in serum bilirubin level was recorded between various groups. Selenium treated groups (group E and F) exhibited significant reduction in the bilirubin level as compared to their corresponding positive control groups (group C and D respectively).

The impact of dietary lead and selenium on the blood uric acid level was illustrated in Figure 5. The uric acid concentrations were increased significantly (p < 0.05) in all the lead acetate supplemented groups. Selenium treated groups (group E and F) reduced the uric acid concentration (p < 0.05) as compared to both negative and their corresponding positive controls (group C and D respectively).

The effects of selenium against dietary lead on the blood creatinine concentrations were illustrated in Figure 6. Dietary lead significantly increased (p < 0.05) creatinine level as compared to the control group. However, creatinine level was significantly reduced (p < 0.05) by the dietary selenium in both groups E and F when compared with their corresponding positive control groups.

Gross pathology of liver of quail treated with lead acetate and selenium was shown in Figure 7. Livers of lead-treated groups exhibited high intensity of discoloration and inflammation, while selenium-treated groups showed recovery towards normal color and texture. Histopathology of slides shows collapsed tubules with pinkish proteinaceous and sloughed tubular cells from basement membrane were suggestive of chronic degenerative changes. Liver tissues in lead acetate groups showed that hepatocytes were severely swollen, and have granule denaturation and macrovesicular steatosis, however such deteriorative changes in liver were milder in selenium treated groups (Figure 8). Tubular necrosis, aggregates of mononuclear cells in parenchyma along with increased cellular infiltration around proximal convoluted tubules in kidneys of lead treated birds. 

Lead considered as the harmful or deleterious heavy metal which produce toxicity in humans, animals and birds (Rice and Silbergeld 2023). This present research study was undertaken to investigate the probable effect of lead acetate toxicity on biochemical and histopathological changes in quail and its protection via selenium.

Our results exhibited that body weight was decreased in quail received lead acetate @ 200 400 mg/L in contrast with control group. The selenium supplementation ameliorated the deleterious effects of lead which is evidenced by raise in body weight in E group. Our present findings corroborates with findings reported by Kou et al. (2020). Decrease in body weight in lead acetate supplemented groups could be due to decrease in the feed consumption or owing to metabolic disorders associated with lead toxicity production, such as inhibition of enzyme involved in the catabolism process or the oxidase reduction system resulting in loss of cellular functions and tissue damage (Kou et al., 2020).

There are many clinical examples where exposure to lead might result in diminished aminotransferase action when the enzyme pyridoxal-5-phosphate become lower under stress (Ashrafizadeh and Rafiei 2018). It is reported that when the lead acetate contaminated feed was fed to the chickens, significant increase in apoptotic cell number in liver was recorded (Jahromi et al., 2017). It is explained that infiltration of provocative cells within the hepatic structure due to lead toxicity produce cellular disruptions that leads to disruption of the liver morphology (Boey et al., 2020). Similar results have seen in mice (Ali et al., 2021). In our current research, various doses of lead acetate (200 and 400 mg/L) raised the values of gamma-glutamyltransferase (GGT) in Japanese quails. Al-Wabel et al. (2007), reported that when 50 mg/kg lead acetate was available to rats, the serum concentration of GGT was gradually elevated from 24 IU/L to 38.3 IU/L. Gani et al. (2016) revealed the familiar research conclusion when exposed the feed of rats with lead acetate. The authors reported elevated serum values of glutamate pyruvate transaminase and recorded detrimental effects on the hepatic system.

Uric acid is a crystalline pure white organic compound which is barely soluble in water and insoluble in ether and alcohol. It is a waste product of body that results from the breakdown of purine within the liver and expelled through the kidney (Suleiman et al., 2011). While creatinine is a chemical compound produced during the energy-producing processes as a breakdown of creatinine phosphate in muscles and metabolism of protein. Healthy kidneys filter creatinine out of the blood and excrete it through urine (Canaud et al., 2020). The creatinine evaluation assessment is usually carried out to monitor the kidney functions. If the kidney function performance is found irregular, creatinine concentration level will be increased within blood, owing to decline in excretion of waste creatinine of the urine (Nissl and Terra, 2004). Creatinine concentration in plasma or serum is considered to be a high sensitive indicator point of kidney function test against the blood urea nitrogen (Taha et al., 2013). The presence of high serum urea and creatinine concentration within blood, recommends the incapability of the urinary system to expel these waste products (Kalantar et al., 2004). In our present experiment, different dosages of lead acetate increased creatinine and uric acid values, which were again reduced by selenium. Similar results have been reported by (Ashour et al., 2007), who cited that lead acetate boost the intensity of creatinine in lead acetate toxicitiy in rats. The findings of this study align with previous research emphasizing the protective effects of selenium against heavy metal toxicity.  A rat study reported beneficial effects of selenium when supplemented with N-Acetylcysteine on the toxicological effects of lead acetate. The study reported a significant increase in uric acid and creatinine levels in lead exposed groups that was markedly reduced when selenium was given (Sharma et al., 2014). Several other workers have also reported protective role of lead-induced toxicity in chickens (Jiayong et al., 2020) and murine models (Apaydin et al., 2014).

Heavy lead can causes a variety of functional and structural changes in liver cells, which is indicated by hepatocytes damage (Valko et al., 2005). Gross pathological results showed pale, congested and enlarged liver. Histopathological deterioration induced within kidneys of quails by lead acetate was congestion, mild to moderate tubular dilatation and degeneration. Other changes involves were degeneration of glomeruli and proliferative variation in epithelial cells of Bowman capsule. Glomerulonephritis, proliferative cellular changes in intertubular spaces of tubules also seen in chickens (Tahir et al., 2022; Abdel Moneim et al., 2007) previously demonstrated kidneys cellular swelling. Heavy metals are known to cause severe damage to vital organs like, kidney, liver, pancreas etc. Cd produced renal toxicity evidenced by dilated peri-tubular capillaries, endothelial vacuolar degeneration and disruption in tubules (Olsvik et al., 2016). Outcome of our research study are very indicative that lead acetate has severe nephrotoxic and hepatotoxic effects, while the selenium has the capability to reduce these toxic effects. Selenium role in protecting the lead induced toxicity was evidenced by biochemical, and gross and histological changes. Selenium’s ability to scavenge free radicals and enhance antioxidant defense mechanisms may explain its efficacy in counteracting the toxic effects of lead.

Based on the present findings, it can be concluded that, lead acetate had acute toxicity in liver and kidney of Japanese quail. Our study suggests that the addition of 0.1 mg/L selenium can ameliorate the toxicological effects induced by lead up to 200 mg/L. These results have implications for the development of strategies to mitigate lead toxicity in avian species, such as Japanese quail, through dietary selenium supplementation.

We acknowledge all lab staff and coworkers to help during the experimentation.

We declare no conflict of interest.

This study have explored the toxicological effects of long term exposure (20 day) of lead acetate in Japanese quails and its’ amelioration using dietary selenium. Our results demonstrated that selenium supplementation could be a practical option against toxicological effects of heavy metals.

NH carried out all experimental work under the supervision of her supervisor (SNK) who conceived this study. FPS and SB act as co-supervisors and helped in analytical protocols. AAK and BRT helped in manuscript writing, revision and publishing.