Fipronil is a second-generation broad-spectrum phenyl pyrazole insecticide that is commonly used in veterinary, households, topical pet care products, and agricultural practices. The indiscriminate and inappropriate use of these insecticides causes various adverse effects in both humans and animals. Fipronil induces oxidative damage and impairs the functions of hepatic, nervous, nephrotic, reproductive, gastrointestinal, renal, hematologic, cardiovascular, and immune systems. To combat these complications, use of an herbal ameliorating agent with high antioxidant properties, fewer side effects, and better compatibility such as Pomegranate peel extract (Punica granatum) is one of them with enriched polyphenolic compounds.

The present study was carried out by procuring 24 male Wistar albino rats that were randomly assigned to four groups with six rats in each group. Fipronil was orally gavaged @ 10 mg/ kg b. wt. using distilled water as a vehicle to group II animals. To study the ameliorative effects, Punica granatum @ 200 mg/kg b. wt. was fed along with fipronil to group IV for 6 weeks. Group I and III served as vehicle control and Punica granatum control respectively. In the present study, no mortality was observed in any of the treated groups. Significant (P<0.05) reduction in Hb, TEC, PCV, TLC, percent lymphocyte counts, and a significant increase in percent neutrophil count was recorded in fipronil treated group (II). These counts were significantly improved in Punica granatum ameliorated rats (group IV) when compared to fipronil treated group (group II).

Serum biochemical analysis revealed a significant (P<0.05) increase in AST, ALT, BUN, creatinine and a decrease in total protein, serum albumin, and globulin levels in group II animals than control animals. Significant (P<0.05) increase in lipid peroxidation values and decreased levels of SOD, catalase, GPx, and GSH levels in the liver, kidney, brain, and testis were observed in fipronil treated rats (group II) than Group I rats. All these biochemical and oxidative stress parameters were significantly improved in the Punica granatum ameliorated group rats (group IV). A Significant (P<0.05) decrease in serum T3, T4, testosterone, and elevated levels of serum TSH was observed in (group II) rats when compared to control rats. Fipronil treated rats showed a significant (P<0.05) decrease in epididymal sperm count and an increase in abnormal sperm count than control rats (Group I). Significant improvement in these values was observed in Punica granatum ameliorated rats.

Grossly, the lesions including paleness of the liver, atrophied spleen, cerebral blood vessel congestion, and congested kidneys were observed in fipronil treated rats (group II) when compared to control rats. There were no specific pathological changes in the ameliorated animals (Group IV).

Histopathologically, the liver of the rats of Group II revealed congested and dilated blood vessels, severe degenerative changes in hepatocytes, disruption of hepatic cords, dilated and congested sinusoids, periportal and portal infiltration of MNCs and fibroblasts, bile duct epithelial hyperplasia, focal loss of hepatocytes with infiltration of MNC's, fatty degenerative changes, necrotic changes with perivascular infiltration of MNC's. Whereas in the Punica granatum ameliorated rats (group IV), all these changes were very mild and the liver came to near normal in appearance.

Kidneys of fipronil treated rats showed severe degenerative and desquamated changes in renal tubules, perivascular edema, intertubular, and pockets of hemorrhages. Hypertrophied, degenerated, atrophied, and hyperplastic glomeruli, infiltration of MNC's in interstitial and periglomerular spaces. In Punica granatum ameliorated rats (group IV), these lesions were less severe. Brain sections of fipronil treated rats (Group II) included sub-meningeal hemorrhages, capillary proliferation, neuronal degenerative changes like shrinkage, central chromatolysis, satellitosis and neuronophagia, gliosis, spongiosis in the cerebral cortex, and rounding, shrinkage, and loss of Purkinje cells of the cerebellum. In Punica granatum, ameliorated rats (group IV) showed similar changes with reduced intensity. Testis of Group II rats showed thickened tunica albuginea, interstitial edema, variation in the size of seminiferous tubules, and widened lumen with separation of germinal cell layers, reduced number of Leydig cells in testis. Similar lesions were observed in Punica granatum ameliorated rats (group IV) with reduced intensity. Lungs of Group II revealed widened interstitial space with infiltration of MNC's and eosinophils, adipogenicity, degenerated and desquamated bronchial epithelium. In the Punica granatum ameliorated rats, these changes were very mild in intensity. Heart of fipronil treated group showed focal areas of sarcolytic changes, congested and thickened blood vessels, hemorrhages in between cardiac muscles, and mild infiltration of MNCs and fibroblasts. In Punica granatum ameliorated group, mild changes were noticed. Mild to moderate lymphocyte depletion was more cons-picuous in the spleen, lymph node, and thymus. In Punica granatum ameliorated group, very mild changes were noticed in these organs. Pancreas of fipronil treated rats revealed atrophied islets of Langerhans, hyperplasia of ductular epithelium, and replacement of acinar cells with fibrous tissue. The thyroid sections showed disrupted follicular structure with desquamation of lining epithelium and lack of colloids in the lumen of the follicles. The lesions in epididymis of Group II rats included reduced sperm density in the lumen, inter-tubular, and loss of stereocilia. The severity of these lesions was of lesser intensity in Punica granatum ameliorated rats (group IV) when compared to fipronil treated rats.

Immunohistochemically, fipronil treated rats showed increased expression of BAX marker in liver, testis and thyroid tissues whereas Punica granatum ameliorated rats showed less immunoreactivity with BAX antigen.