Non steroidal anti-inflammatory drugs (NSAIDs) are commonly used to reduce pain in various diseases and postoperative conditions¹. Toxic side effects of diclofenac have also been demonstrated in various animals, even at the therapeutic doses. Recently, its use has declined in the veterinary medicine due to the reports of diclofenac toxicity in vultures. There have been very little published reports of systematic studies done on the patho-physiological effects of diclofenac in the avian species. Various workers had also reported nephrotoxicity in man, animals and vultures, induced by administration of diclofenac and some other NSAIDs. Diclofenac was found to be causing pathological changes in the kidneys of the vultures, which ultimately lead to the gout². There may be many patho-physiological aspects of the diclofenac metabolism in the avian species. The present study was carried out to determine the adverse effects of widely used diclofenac sodium in Japanese quails after oral administration on the hematological, biochemical and immune response.

A total of 120 apparently healthy Japanese quails, of either sex, aged 4-weeks, having 150 to 200 gm body weight, were procured from Central Avian Research Institute (CARI), Izatnagar, Bareilly. Prior to housing, the poultry shed was thoroughly cleaned, white washed and disinfected by 2.5% phenolic solution. Faecal and blood samples of all the birds were regularly examined for any parasitic infestations. The birds were given feed and fresh water ad libitum.Quails were divided into 2 groups comprising 80 in group-I (toxicity) and 40 in group-II (control). The quails of group-I were administered with 0.5 mg/kg body weight diclofenac sodium through oral route daily for 60 days. The quails of group-II were provided with plain drinking water for the period of 60 days. The haematological parameters were carried out at the intervals of day 15, 30, 45 and 60. Two ml blood was collected in a clean dry vial containing 2.0 mg ethylene diamine tetraacetic acid (EDTA) as an anticoagulant, during sacrifice of the bird. Total leukocyte count (TLC) and total erythrocyte count (TEC) were done as described by Natt and Herick³. The blood smears were stained with Wright stain and MayGrunwald-Giemsa stain⁴ with slight modification for differential leukocyte count (DLC). Hemoglobin (Hb) and packed cell volume (PCV) was analysed as described by Schalm et al⁵. Erythrocyte indices viz. mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) were also calculated.

For estimation of different biochemical parameters, blood samples were collected at 15, 30, 45 and 60 days intervals in a clean and dry test tube without using any anticoagulant. The serum samples were separated and centrifuged at 3000 rpm for 10 minutes at room temperature and stored at 0°C till further analysis. Serobiochemicals analysed includes serum creatinine, serum uric acid, blood urea nitrogen (BUN), alanine amino transferase (ALT), aspartate amino transferase (AST), total protein and albumin (TP & Alb) were estimated by using diagnostic kits.

Cell mediated immune response was evaluated on 25^thday post feeding as per the method described by Rajan et al.⁶.Immune response was measured by alteration in skin thickness following sensitization and challenge with DNCB by epicutaneous route. The data obtained were analyzed statistically to observe the test of significance.

The mean values of Hb, PCV, TEC, MCV, MCH, MCHC, TLC and DLC on day 15, 30, 45 and 60 are presented in Tables 1 and 2.

Hemoglobin was significantly decreased on 45th day and on 60^th day of trial in birds of groups-I, while the mean values of PCV and TEC revealed significant decreases on 30^th and 45^th day and highly significant decrease on 60^th day during trial period in the birds of group-I as compared to group-II. There was significant increase in the values of MCV on 45^th and 60^th day of experiment in the quails of group-I as compared to group- II but did not show significant variation in the mean values of MCH, MCHC and TLC in birds of group-I. Heterophil and lymphocyte counts were gradually increased from 45^th day onwards in the quails of group-I.

The mean values of creatinine, urea, uric acid, albumin, total protein, ALT and AST on day 15, 30, 45 and 60 are presented in Table 3.

The values of creatinine, uric acid and BUN showed significant increase in the quails of group-I at 45^th and 60^th day of intervals while there was significant decrease in the mean values of total protein and albumin on 30^th and 45^th day which was highly significant at 60^th day of experimentation. ALT and AST values were almost comparable to control at all the intervals.

There was less thickness of skin at all the intervals of post challenge of DNCB in toxicity group as compared to the mean skin thickness of control group.

The mean values of Hb, PCV and TEC indicated anemia on 45^th and 60^th day of intervals. Similar results in diclofenac toxicity have also been reported in cattle @ 2.5mg/kg body weight⁷ and @ 3.0mg/kg body weight⁸. It appears that the prolonged uses of diclofenac sodium are toxic to proliferating hemopoietic tissue leading to development of significant anemia in the quails. Increase in MCV in the present study suggested release of large immature size RBCs in circulation from bone marrow leading to macrocytic anemia as a compensatory mechanism.

In the present study there was no significant change in the total leucocyte count in the birds of toxicity group. However, differential leucocyte count showed significant lymphocytopenia on 45th and 60^th day of experiment with relative heterophilia. These findings indicate depletion of lymphoid reserve in the body as also supported by decreased density of lymphoid cells in the malpighian corpuscles of spleen in the present study. Similar observations in rats induced with diclofenac toxicity have also been reported⁹.

The observation of increased value of serum creatinine, uric acid and BUN in the present study amply suggested that the Diclofenac sodium induces nephrotoxic effects leading to renal dysfunction. Determination of creatinine has been described to be a reliable index of renal function¹⁰. The decrease in the level of serum proteins in this study may be attributed to increased catabolism of proteins, loss of serum proteins in urine, hepatic and renal dysfunctions. The observation of increased serum creatinine, uric acid, BUN, hypoproteinemia and hypoalbuminemia due to diclofenac sodium toxicity in this study conforms the finding of the diclofenac toxicity in rabbits¹¹.

No change in the levels of AST, and ALT in the blood may be due to the fact that these enzyme containing organs are not so severely damaged in diclofenac toxicity to cause significant increase in the level of these enzymes as also reported in Mongrel dog¹².

The increase in the thickness of skin was due to development of variable degree of indurations, redness and vesicles formation at the test site of DNCB deposition in birds of group-II as compared to group-I. The decrease in the skin thickness of diclofenac fed group indicated poor reactivities of host to the mitogen by cell mediated immune response. This may be due to toxic and immunosuppressive properties of diclofenac on lymphoid organs. The same can also be evidenced by lymphocytopenia in the present study.

The Authors are thankful to the Dean, Veterinary College, Mathura for providing necessary financial support and other technical and non technical staffs of the Department for providing necessary help

* Significant at P<0.05 in between the groups at different intervals, ** Significant at P<0.01 in between the groups at different intervals

* Significant at P<0.05 in between the groups at different intervals, ** Significant at P<0.01 in between the groups at different intervals

*Significant at P<0.05 in between the groups at different intervals, ** Significant at P<0.01 in between the groups at different intervals

REFERENCES