In the present study birds were divided into six groups viz. A, B, C, D, E and F. Group-A served as control (basal diet) where as all the other groups B,C,D,E and F were treated with lead acetate @ 200 mg/kg. In addition the groups C,D,E and F were also supplemented with Ascorbic acid @ 200 mg/kg, Vitamin-E @ 100/mg/kg and Se @ 0.1 mg/kg, DL-methionine @ 100 mg/ kg , methanolic extract of Cissus quadrangularis (CQE) @ 400 mg/kg respectively for 42 days. It was observed that lead administration enhanced the lipid peroxide (LPO) level in erythrocytes, result of simultaneous administration of lead with ascorbic acid, vitamin-E and Se, DL-methionine and CQE led to conclusion that vitamin-E and Se, DL-methionine and CQE were very effective in limiting the enhanced LPO level in erythrocyte. Maximum restoration in values of LPO level was observed in vitamin-E & Se treated group. Decreased erythrocytic glutathione (GSH) level in lead treated group was significantly (P<0.05) increased with the treatment of ascorbic acid. At the end of the study, there was significant (P<0.05) reduction in erythrocytic super oxide dismutase (SOD) and Catalase (CAT) activity in lead exposed birds. All the treatment given was found very efficacious as they enhanced SOD and CAT activities. However maximum restoration was recorded with the treatment of vitamin-E & Se.
Lead is one of the extensive environmental pollutants that induce a wide range of physiological and biochemical dysfunctions in animals (Erdogan et al., 2005). Among all heavy metals lead is being an ubiquitous environmental pollutants, particularly widespread in industrial areas due its significant role in modern industry (Shalan et al., 2005). However, both occupational and environmental exposures remains a serious problem in many developing and industrializing countries causes health problems in man and its contingent including animals (Gurer and Ercal, 2000). Mahesar et al. (2010) reported that most of the poultry feed samples which they analyzed contained greater amount of Pb and cadmium (Cd) than the maximum tolerable levels for poultry. According to studies (Mateo et al., 2003), lead has a probable to induce oxidative stress and acts as a catalyst in the oxidative reactions of biological macromolecules. Hence, the toxicities associated with lead might be due to oxidative tissue damage (Ercal et al., 2001). To prevent peroxidative tissue damage, there are protective mechanisms in vivo, such as an enzymatic defense system (antioxidant enzymes) and free radical scavengers (antioxidants). The aim of the present study is to see the effect of lead intoxication on antioxidant defense in erythrocytes of broiler chicken.
Materials and Methods
The experiment was in accordance with animal welfare, and conducted under the protocols of Veterinary faculty, Anjora, Durg (Chhattisgarh) with the approval of Institutional Animal Ethics Committee (IAEC). For present study a total of 126 day old broiler chicks a of (Ven-Cobb strain) either sex along with broiler feed were procured from a well organized private hatchery of Indian Broiler Group, Rajnandgaon, Chhattisgarh. The diets were formulated according to the broiler chicken requirements suggested by the National Research Council guidelines (NRC, 1994). All treatments were given to chicks on the basis of per kg basal diet daily for the period of 42 days. In this small amounts of basal diet was first mixed with the respective amounts of lead acetate @ 200 mg/kg to induce lead toxicity and with treatment drugs viz. ascorbic acid @ 200 mg/kg, Vit-E @ 100 mg/kg and Se @ 0.1 mg/kg, DL-methionine @ 100 mg/ kg and methanolic extract of Cissus quadrangularis powder @ 400 mg/kg as per treatment groups in a small batch. Then it was mixed with a larger amount of basal diet, until the total amount of respective diets were homogeneously mixed and was treated as per the experimental design (Table 1).
Table 1: Experimental design
|Groups||No. of birds||Treatment given on the basis of per kg basal diet|
|B||21||Lead acetate @ 200 mg/kg|
|C||21||Lead acetate @ 200 mg/kg + Ascorbic acid @ 200 mg/kg|
|D||21||Lead acetate @ 200mg/kg + Vit-E @ 100 mg/kg and Se @ 0.1 mg/kg|
|E||21||Lead acetate @ 200 mg/kg + DL-methionine @ 100 mg/ kg|
|F||21||Lead acetate @ 200 mg/kg + Methanolic extract of Cissus quadrangularis(CQE) @ 400 mg/kg|
Blood samples were collected on 21st and 42nd day of experiment in the morning hours from 6 broilers that were randomly chosen from each group. Plasma was separated by centrifugation at 4000 rpm for 10 min at 4 oC and stored at -20 oC in clean polypropylene vials until analyzed while RBC plug was used for estimation of antioxidant parameters like lipid peroxidation (LPO), reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT). To test the difference between different groups, analysis of variance with one way classification followed by Dancan’s Multiple Range Test (DMRT) was applied as per the standard procedure outlined by Snedecor and Cochran (1994).
Results and Discussion
The present experimental outcomes appendages to adverse effects of lead intoxication on antioxidative states and their possible reversal by acquiring different antioxidants are explained hereunder. The results of erythrocytic antioxidant profile of lead exposed and for different treatment groups are summarized in the Table 2 for day 21 and Table 3 for day 42 and comparison between day 21 and 42 are presented in Table 4.
Lipid Peroxidation (LPO)
In present study mean Lipid peroxide (LPO) level, as expressed in terms of mole of malondialdehyde (MDA) produced per gram of haemoglobin, was elevated non-significantly (P>0.05) (Table 2) in the birds of lead treated group-B (19.43±0.51 mole/g Hb) as compared to control group A (17.39±0.38 mole/g Hb).
Table 2: Effect of lead and different antioxidants on erythroctic antioxidant profile in broiler chicken on day 21 (n=6)
|Antioxidative||Various Treatment Groups Showing Mean ± SE Values|
|SOD||165.90±13.33b||65.35 ±5.79a||161.71±11.64b||135.26 ±8.72b||133.79 ±6.79b||147.81±15.83b|
|CAT||336.47±10.94b||150.29±12.01a||507.63±5.29c||367.21±2.94b||359.81 ±8.83b||376.56 ±3.90b|
Means in the same row within categories with different superscript differ significantly (p<0.05)
Co-treatment with different alleviative agents prevented the elevation of LPO. The mean LPO in group C (17.96±0.73 mole/g Hb), group D (18.09±0.67 mole/g Hb), group E (18.28±0.80 mole/g Hb) and group F (18.15±0.63 mole/g Hb) was nonsignificantly (P>0.05) lower than group B. Comparison between the different treatment groups on day 21 showed non-significant (P>0.05) changes in LPO level. Although on day 42 LPO level was non-significantly (P>0.05) increased (Table 3) from the day 21 in all groups.
On day 42 the estimated LPO level was 18.41±0.52, 19.88±0.5, 18.47±0.58, 16.88±0.44, 17.92±0.66 and 17.96±0.21 mole/g Hb in group A, B, C, D, E and F respectively (Table 3).
Table 3: Effect of lead and different antioxidants on erythroctic antioxidant profile in broiler chicken on day 42 (n=6)
|Antioxidative||Various treatment groups showing mean ± SE values|
Means in the same row within categories with different superscript differ significantly (p<0.05)
A significant (P<0.05) reduction in LPO level was observed in group D, E and F treated with Vit-E & Se, DL-methionine and CQE. The present findings are in agreement with earlier workers who reported addition of lead to the diet of broiler significantly (P<0.001) increased the lipid peroxidation in terms of increasing malondialdehyde level and elevated MDA level was significantly reduced by addition of ascorbic acid to the diet containing lead (Knowlers and Donaldson,1996; Erdogan et al.,2005). Lipid peroxidation has been identified as a basic deteriorating mechanism of ageing process and cell damage. In the present study significantly elevated LPO level was observed in erythrocyte of the birds in lead treated group (B). The present finding was also in agreements with earlier worker who reported elevated LPO level in erythrocytes of calf (Patra and Swarup, 2000), hepatic cells of birds (Blazovics et al., 2002) following lead administration. In present study, highest therapeutic efficacy to reduce the LPO level in chronic lead intoxication was shown by Vitamin-E & Se followed by DL-methionine and CQ extract. They significantly (P<0.05) reduced the LPO level as compared to lead treated group and showed comparable result to normal group on day 21 and 42 respectively. But efficacy of vitamin-C to reduce the LPO was nonsignificant (P>0.05) in present study. Various workers have reported the therapeutic efficacy of Vit-E & Se and DL-methionine in lead induced elevation of LPO in erythrocyte and tissues of various experimental animals (Patra et al., 2001). However no published report on use of CQE in reducing the LPO level in birds is available to compare the present findings.
Status of mean Glutathion (GSH) level expressed in terms of mole/g Hb is given in the Table 2. The GSH level decreased nonsignificantly (P>0.05) as compared to control group A (0.027±0.003 mole/g Hb), group B (0.015±0.003 mole/g Hb), group D (0.032±0.038 mole/g Hb), group E (0.028±0.009 mole/g Hb) and group F (0.03±0.004 mole/g Hb). However, significant (P<0.05) increase in the GSH level was recorded in group C (0.038±0.005 mole/g Hb) on day 21. Comparison between the different treatment groups on day 21 showed non-significant (P>0.05) changes in GSH level. After 42 days (Table 3) GSH level decreased significantly (P<0.05) in group C. On day 42 lead treated group B (0.018±0.004 mole/g Hb) showed non-significant (P>0.05) change (Table 3) in GSH level as compared to group A (0.022±0.005 mole/g Hb), C (0.023±0.005 mole/g Hb), D (0.033±0.003 mole/g Hb), E (0.025±0.005 mole/g Hb) and F (0.023±0.005 mole/g Hb). No significant (P>0.05) comparative changes were observed in between the group A, C, D, E and F. In the present study protection against the toxic effects of lead was observed in all the groups, however simultaneous administration of ascorbic acid administration resulted in significant increase in GSH level in erythrocytes. Earlier studies have concluded that GSH was involved in detoxification of heavy metal by binding them and other intermediates of organic compounds (Kaplowitz et al., 1985). Latta and Donaldson (1986) also found increased glutathione (GSH) concentrations by simultaneous supplementation of methionine and lead, which is also in confirmative with our finding. In the present study, highest therapeutic efficacy to increase the GSH level was shown by vitamin-E & Se followed by DL-methionine > CQ extract ≥ vitamin C on day 42 but on day 21 highest therapeutic efficacy was shown by vitamin C followed by vitamin E & Se > CQ extract > DL methionine. They increases the GSH level as compare to lead treated group and showed comparable result to normal group on day 21 and 42 respectively. Glutathion is considered to be master antioxidant of the body and is found in all living cells. Body utilizes GSH chiefly for reducing oxidized Vi-E and Vit-C to their reduced state. Further, they have the property to detoxify many toxins, to maintain cellular redox potential and to maintain erythrocyte cellular membrane integrity (Kidd, 1997), supplementation of Vit-C and Vit-E & Se have a significant effect on raising glutathione level in liver tissues. Thus the result of present study suggestive of beneficial effect of these antioxidants used in the present study along with lead, there by undesirable effect of lead in term of formation of free radicals that were generated could be compromised successfully.
Superoxide Dismutase (SOD)
Findings of present study showed that mean SOD level expressed in terms of Unit/mg Hb was significantly (P<0.05) reduced (Table 2) in the lead treated group B (65.35 ±5.79 U/mg Hb) as compared to control group A (165.90 ±13.33 U/mg Hb). Co-treatment with different antioxidants significantly (P<0.05) enhanced the SOD level on day 21. Mean SOD activity estimated in group C, D, E and F was 161.71 ±11.64 U/mg Hb, 135.26 ±8.72 U/mg Hb, 133.79 ±6.79 U/mg Hb and 147.81 ±15.83 U/mg Hb respectively on day 21 and maximum restoration of SOD activity was achieved in group C (161.71 ±11.64 U/mg Hb). Comparison between 21 and 42 days SOD level showed significant (P<0.05) decrease (Table 4) from the day 21 in treatment group-C and F except group-B, D and E which showed non-significant (P>0.05) decreased.
Table 4: Comparative effect of lead and different antioxidants on erythroctic antioxidant profile in broiler chicken between day 21 and 42
|Day||Various Treatment Groups Showing Mean ± SE Values|
At the end of the study (42 days) erythrocytic activity of SOD decreased significantly (P<0.05) (Table 3) in lead treated group B (47.61±5.74 U/mg Hb) as compare to control group A (157.90±3.11 U/mg Hb), group C (96.26±16.33 U/mg Hb), group D (146.77±7.47 U/mg Hb), group E (97.63±6.60 U/mg Hb) and group F (92.27±7.12 U/mg Hb). The SOD is one of the primary antioxidant defense enzymes in cells that help to detoxify a particular ROS. The SOD enzyme removes O2 by accelerating its conversion to H2O2. Because SOD enzyme generates H2O2, so it works in collaboration with H2O2 removing enzyme. In the present study lead decreased the SOD activity significantly (P<0.05) in birds. Earlier reports suggested that lead diminished antioxidant enzymes (Flora et al., 2008) and supplementation of various vitamins and minerals enhance the antioxidant enzyme level against lead induced depression (Patra et al.,2001). Also in the present study activity of SOD was significantly (P<0.05) increased with various therapeutic agents used along with lead in birds. Maximum restoration of erythrocytic SOD activity was observed in Vit-E & Se treated group on day 42.
In present study mean catalase (CAT) level expressed in terms of unit milligram hemoglobin, was significantly (P<0.05) low (Table 2) in the birds of lead treated group B (150.29±12.01 U/mg Hb), as compare to group C (507.63±5.29 U/mg Hb), group D (367.21±2.94 U/mg Hb), group E (359.81 ±8.83 U/mg Hb), group F (376.56 ±3.90 U/mg Hb) and control group A (336.47±10.94U/mg Hb) on day 21. Comparison among the different treatment groups showed non-significant (P>0.05) changes in CAT activity in group D, E and F but group C showed significant (P<0.05) high CAT activity than the other treatment groups on day 21. Although on day 42 CAT activity decreased significantly (P<0.05) (Table 3) from the day 21 in group A, B, D and E but significantly (P<0.05) in group C and F. On day 42 lead treated group B (107.60±12.84 U/mg Hb) showed significant (P<0.05) decrease in CAT activity from (Table 3) group C (393±7.92 U/mg Hb), group D (707.99±15.51 U/mg Hb), group E (438.03±3.39 U/mg Hb), group F (290.21±9.00U/mg Hb) and non significantly (P>0.05) from control group A (173.41±10.63 U/mg Hb). Significant (P<0.05) comparative changes were observed in the values of group C, D and F but not in between the group C and F. It is reported by several workers that simultaneous use of antioxidants provides a better therapeutic efficacy in the management of Plumbism (Patra et al.,2000b). It has been observed that lead caused oxidative stress by enhancing free radical formation, reducing the antioxidative defense enzymes like catalase, SOD and/or increasing the susceptibility of cells to oxidative attack (Gurer and Ercal, 2000). In the present study (on day 42) erythrocytic catalase activity was declined though nonsignificantly (P>0.05) in group B as compared to healthy birds (group A). This could be correlated with the adoptive response towards the detrimental effect of lead on erythrocyte. Several studies have shown that lead alters the antioxidant enzymes like SOD, catalase etc. (Flora et al., 2008). In erythrocytes from workers exposed to lead. Since there is no report pertaining to effect of lead on erythrocytic catalase activity of birds to compare the present findings. Highest therapeutic efficacy to enhanced the antioxidative defense enzymes CAT against lead intoxication was shown by vitamin-E & Se followed by DL-methionine > vitamin-C > CQ extract on day 42 but on the 21 days highest therapeutic efficacy was showed by vitamin-C and followed by Q extract > vitamin-E & Se > DL-methionine. They enhanced the CAT level as compared to lead treated group and brought the activity comparable to control group on day 21 and 42 respectively. Vit-E known to be one of the most potent antioxidants that checks lipid peroxidation through limiting the propagation of free radicals reaction and ascorbic acid an antioxidant acts as scavenger of free radicals and play an important role in regeneration of α-tocopherol and DL-methionine serves as a precursor for low molecular mass antioxidant glutathione (Young, 2001). The antioxidant effect of CQE is believed to originate from ability to stimulate glutathione synthesis and scavenging reactive oxygen species (Jainu and Devi, 2005).
Lead exposure enhanced the lipid peroxide (LPO) level in erythrocytes, simultaneous administration of lead with ascorbic acid, vitamin-E and Se, DL-methionine and CQE led to conclusion that vitamin-E and Se, DL-methionine and CQE were very effective in limiting the enhanced LPO level in erythrocyte. Maximum restoration in values of LPO level was observed in vitamin-E & Se treated group followed by ascorbic acid treated group.
IB Group, Rajnandgaon, Chhattisgarh for providing free of cost Ven-Cobb strain broiler chicks; Dean, Veterinary College Anjora, Durg (Chhattisgarh) for providing necessary facilities and Head, Department of Animal Nutrition for feed formulation.