ASIAN J. EXP. BIOL. SCI. VOl 1 (3) 2010 :677-680 © Society of Applied Sciences ORIGINAL ARTICLE Impact of Neem Oil on Ovarian Changes in the Fresh Water Fish Glossogobius Giuris S.Y.Narayanaswamy and Ramachandra Mohan M. Dept. of Zoology, Jnanabharathi Campus, Bangalore University, Bangalore – 560 056. India. ABSTRACT The fish G. giuris were exposed to various concentrations of neem oil (0.05 ppm, 0.25 ppm, 0.50 ppm) for 24, 48, 72 and 96 hrs. The results revealed that the lower dosage of neem oil for 24 hrs, treatment for 0.05 ppm showed yolk vesicles increases and few stage I and II of oocytes got degenerated and brings about a reduction in the ovarian weight. A higher dosage of neem oil for 78 hrs and 96 hrs, in for 0.50 ppm the nucleus of stage of I and II oocytes were more and also more atretic follicles, liquification, swelling of follicular wall, ruptured follicles and necrosis. Neem oil appears to inhibit ovarian development. KEY WORDS: G. giuris, Neem oil, Atretic follicle, Necrosis INTRODUCTION The biologically active compound is azadirachtin (extract of neem seed), repellent for a broad spectrum of agricultural and household insects. The neem extracts can affect aquatic life including fish and tadpoles. Patnaik et al., (1987) [1] observed that higher larval mortality of Crocidolomia binotalis with neem oil for laboratory condition. Azam (1991) [2] reported that neem oil causes more than 80 percent larval mortality of Liriomyza trifolii in cucumbers. Shanmugapriyan and Kingsly (2001) [3] reported that neem oil at 0.5, 1.0 and 2.0 % of concentration was effective on third instar larvare of earias vitteila than in the fourth and fifth instars. Neem has been used world wide in traditional medicine for various therapeutic purposes, antibacterial, antifungal and antifertility properties (Jegede and Fagbenro, 2007) [4] Sinna and Riai (1985) [5] reported that in Rhesus monkey and human, spermatozoa became totally immobile in 30 seconds of contact with undiluted neem oil. Termitope Jegede and Oyedapo fagbenro (2008) [6] reported histological observation of testes and ovaries in T. zillii fed diet containing NLM, revealed that neem leaves may be effective as determination of the contraceptive efficacies of dietary NLM. In combating problems of Tilapia overproduction in ponds. Fish is one of the major sources of animal proteins in many countries and for many millions, particularly poorer sections. Since, fish contain less connective tissue than other animals. Therefore an attempt has been made to investigate the histopathological changes in the ovary of fish G. giuris in response to various concentration of neem oil. MATERIALS AND METHODS The fresh water gobiid fish Glossogobius giuris (HAM) undergoes four phases of reproduced cycle i.e., preparatory, pre spawning, spawning and post spawning. In the present study fish were collected during pre spawning phase for experimentations. They were kept in 50 l. aquaria and acclimated to laboratory conditions for 15 days prior to using them in experiments. The large and sexually mature females were identified. The body ASIAN J. EXP. BIOL. SCI. VOl 1 (3) 2010 677 Impact of Neem Oil on Ovarian Changes in the Fresh Water Fish Glossogobius Giuris S.Y.Narayanaswamy and Ramachandra Mohan M. length and weight of each fish were recorded. The female fish weighing about 20-50 gms and length 110-220 mm were selected. The neem oil was dissolved in acetone and then added to the test water to obtain the desired concentration. The stock solution of 1 mg/l was prepared and the desired concentration was obtained by adopting the dilution technique out lined by APHA 197 [7]. The fishes were placed in 0.05, 0.25 and 0.5 ppm of neem oil. The fourth group was kept in fresh water to serve as control. For all experiment the acclimated fishes were starved for 24 hrs prior to their exposure to neem oil and use in experiment and were not fed during the course of experiment the neem oil treated fishes at the end of experiment i.e. 24, 48, 72 and 96 hrs were sacrificed by decapitation. After studying the ovary in situ, they were carefully extirpated, weighed and processed for histopathological studies. The GSI was also calculated. RESULTS Control Histological sections of control ovary showed that the cytoplasm of the cells laden with yolk vesicles with fewer atretic follicles. The large number of mature oocytes along with a few vitellogenesis was also noticed. The stage I oocyte had large rounded nucleus and vitellogenesis almost completed (Fig. 1). Treated In 24 hrs of 0.05 ppm neem oil, ovarian weight was lost when compared to control and it was reflected in GSI (control : 5.97 + 0.97, treated 5.75 + 0.62 the number and size of yolk vesicles are increases along with the development of the oocytes. Few stage I and II oocytes showed degeneration (Fig. 2). The C.S of ovaries of 0.25 ppm of neem oil treated fish exhibits increasing trend of yolk vesicles when compare to that of controls. Further ovaries shows signs of degeneration with an abundance of stage IV and V oocytes and increases number of atretic follicles (Fig. 3). The ovaries of 24 hrs 0.50 ppm of neem oil exhibits growing oocytes of stage IV and V oocytes undergone degeneration (Fig. 4). Table 1 Effect of various concentrations of neem oil on Gonado somatic index of G giuris Time in hrs Concentration(ppm) Control 24hrs 48hrs 72hrs 96hrs GSI 5.97 ± 0.97 0.05 5.75 ± 0.62 0.25 5.70 ± 0.23 0.50 3.98 ±.0. 06 0.05 4.17 ± 0.97 0.25 5.05 ± 0.99 0.50 4.42 ± 0.82 0.05 4.35 ± 0.34 0.25 4.34 ± 0.11 0.50 3.96 ± 0.06 0.05 3.23 ± 0.14 0.25 2.59 ± 0.08 0.50 2.18 ± 0.28 After treatment with 0.05 ppm neem oil, the ovaries exhibits number of yolk vesicles increases, atretic follicles are more in number, yolk granules were found, extensive vacuolization, presence of necrotic tissues in some of ovarian follicles (Fig. 5). The C.S ovary of 0.25 ppm neem oil treated fish exhibits degeneration of oocytes, presence large number of immature oocytes stage I and II, its indirectly indicated impairment of vitellogenic 678 ASIAN J. EXP. BIOL. SCI. VOl 1 (3) 2010 Impact of Neem Oil on Ovarian Changes in the Fresh Water Fish Glossogobius Giuris S.Y.Narayanaswamy and Ramachandra Mohan M. synthesis (Fig. 6). The ovaries of 0.50 ppm of neem oil treatment exhibited deleterious changes marked initially by perinuclear ooplasm become less dense due to lysis and breakdown of nuclear membrane (Fig. 7). The exposure of fishes to 0.05 ppm for 72 hrs of neem oil, change are observed in the nucleus and stage I and II oocytes are more susceptibility of the particular stage of oocyte development, significant reduction in GSI (4.35 + 0.34) (Fig. 8). Further 0.25 ppm of neem oil exhibited granulated yolk, phagocytic oocytes. Oocytes loosely arranged, the nucleoli were detached from nuclear membrane and had their free distribution within nuclear space (Fig. 9). Treatment with 72 hrs. 0.50 ppm neem oil exhibited complete vacuolization in the matured follicle along with bursting in the follicular wall (Fig. 10). Expose to 0.05 ppm for 96 hrs. neem oil showing increased atretic follicles, decreases number and size of stage I and II oocytes and necrosis(Fig. 11). The C.S of 0.25 ppm of neem oil yolk globules are fused to form irregular masses, atretic follicles number slightly increases, disorganized zonapellucida (Fig. 12). The C.S of ovaries of 0.50 ppm neem oil showed cytoplasmic liquification, swelling of follicular wall and its separation from the cytoplasm (Fig. 13). DISCUSSION The ovarian recrudences due to exposure of neem oil was reflected in the decreases the weight of ovary similar observations has been made in few other teleosts (Shukla et al., 1984) [8]. Present observation revealed ovarian growth was more (Mohan 1991 and 2000) [9]. The ovaries of treated fish at 0.05 ppm neem oil showed degeneration of stage I and II oocytes and considerable increase of atretic follicles. Such abnormalities in the ovary have been recorded of other fishes Dutt (1996) [10], Ternpitope Jegede and Oyedapo Fagbenro, (2008) [11]. The ovaries of treated with 0.05 ppm neem oil for 48 hrs, large number of atretic follicles and degeneration of stage II and III, were noticed. Similar observations were made by Ram and Sathyanesan (1983) [12] after exposing fish to mercury, Ram, Singh and Singh (2001) [13] after exposing them to carbofuron in Channa punctatus. Saxena and Garg (1978) [14] pointed out that arrest of ovarian growth in C. punctatus occurred due to its exposure to fenthion and carboxyl. Our results are also in agreement with above authors. G. giuris after exposed to neem oil for 72 and 96 hrs with 0.5 ppm. Further, the neem oil also brought out significant other changes like disorganization of ooplasm, rupture follicles, necrosis atretic follicles of the early stage of oocytes like stage I and II. REFERENCES [1]. [2]. [3]. [4]. [5]. [6]. [7]. [8]. [9]. [10]. [11]. [12]. Patanaik NC, Panda N, Patro ER and Misra BK (1987). Effect of Neam Oil (Azadirachta Indica A. Juss) on mustrd web-worm, Crocidolomia binotalis Zell. (Lepidoptera: Pyralidae). Neem New Letter, 4(2): 15-17. Azam KM (1991). Toxicity of Neem Oil against leafminer, Liriomyza trifolii (Burgess) on cucumber. Pl. Port. Qrtly., 6: 196-197. Shanmugapriyan R and Kingsly (2001). Bioefficacy of Neem Oil on Larvae of Bitter Gourd Beetle Epilachna Vigintioctopunctata (Coccinellidae: Volropyrts). J. Ecotoxicol. Environ. Monit., 11(3): 215-219. Jegede T and 0 A Fagbenro (2007). Tilapia husbandry vs. midwifery: use of antifertility plants. pp.118-122, in G.E. Onibi, S.O. Agele, V.AJ. Adekunle and M.O. Olufayo (eds.) Proceedings of the Third Annual Conference of School of Agriculture and Agricultural Technology, Federal University of Technology Akure, Nigeria. Sinna KC and S S Riai (1985). Neem oil: an ideal contraceptive. Biological. Termitope J and Oyedapo F (2008). Histology of Gonads in Tilapia Zillii (Gervais) fed neem (Azadirachta Indica) Leaf Mead Diets. 8th International Symposium on Tilapia in Aquaculture. APHA, AWWAH WPCF (1971). Standard methods for the examination of water and wastewater, 13th end. (TARUS, ed.) (A.P.H.A., Washington, D.C.). Shukla L, Srivastava A, Merwani D and Pandey AK (1984). Effect of sublethal malathion on ovarian histopathology in Sarotherodon mossambicus. Comp. Physio.. Physiol. Ecol., 9: 13-17. Mohan (1991). Experimental studies on the hypothalmo hypophyseal – ovarian system of fresh water Gobiid fish, G. giuris, Ph.D thesis Bangalore University, Bangalore. Dutt HM (1996). A composite approach for evaluation of the effects of pesticides on fish. Fish morphology. pp. 249-277. Ram RN and AG Sathyanesan (1983). Effect of merucuric chloride on the reproductive cycle of the teleostean fish, Channa punctatuys, Bull. Environ. Contam. Toxicol., 30:24-27. Ram RN, IJ Singh and DV Singh (2001). Carbofuran induced impairment in the hypothalamo-neurohypophyseal-gonadal comples in the teleost, Channa punctatus (Bloch). J, Environ. Biol., 22 (3): 193-200. ASIAN J. EXP. BIOL. SCI. VOl 1 (3) 2010 679 Impact of Neem Oil on Ovarian Changes in the Fresh Water Fish Glossogobius Giuris [13]. S.Y.Narayanaswamy and Ramachandra Mohan M. Sexena PK and Garg M (1978). Effect of inspecticidal pollution on ovarian recrudence in the freshwater teleost. Channa punctatus. Indian J. Exp. Biol., 16: 689-691. CORRESPONDENCE TO AUTHOR: S.Y.Narayanaswamy, Dept. of Zoology, Jnanabharathi Campus, Bangalore University, Bangalore – 560 056, India. E-mail: adityasy06@rediffmail.com 680 View publication stats ASIAN J. EXP. BIOL. SCI. VOl 1 (3) 2010