69

ISSN: 0974 - 0376

KEYWORDS

NSave Nature to Survive

: Special issue, Vol. IV: 69-73: 2013

www.theecoscan.inAN INTERNATIONAL QUARTERLY JOURNAL OF ENVIRONMENTAL SCIENCES

Proceedings of International Conference onHarmony with Nature in Context of

Ecotechnological Intervention and Climate Change(HARMONY - 2013)

November 11 - 13, 2013, Gorakhpur,organized by

Department of Zoology,Biotechnology and Environmental Sciences

DDU, Gorakhpur University, Gorakhpurin association with

National Environmentalists Association, Indiawww.neaindia.org

Sangeeta Kumari and Kanaklata

Sodium Fluoride

Ovary Histology

Clarias batrachus.

HISTOARCHITECTURAL CHANGES IN OVARY OF THE SODIUMFLUORIDE TREATED FEMALE CAT FISH DURING BREEDINGPERIOD

70

SANGEETA KUMARI* AND KANAKLATADepartment of Zoology, Ranchi University, Ranchi - 834 008

E-mail: sangeeta0780@gmail.com

*Corresponding author

NSave Nature to Survive QUARTERLY

The effect of sodium fluoride on thehistoarchitectural changes of ovary duringbreeding period was studied. The lethal toxicityof sodium fluoride to freshwater fish Clariasbatrachus was carried out in which one groupof 10 fish served as control without anytreatment and the second group of 10 fishexposed to 1 mg/L of sodium fluoride whichwas the sub-lethal dose. Fishes from both thegroup were sacrificed to study histologicalchanges from the ovaries after 30 days ofexposure in all the three phases (pre-spawning,spawning and post-spawning) during breedingperiod. In experimental group the severity ofdamage due to exposure of sodium fluoridefound to increase in every phase of breedingperiod. The fishes after 30 days of exposure of1 mg/L showed a disorganization of ooplasm,inhibition of ovarian development, increasein atretic cells, lifting of ovarian wall, damagedoocyte and empty spaces of follicle wereprominent.

ABSTRACT

INTRODUCTION

Fishes are considered as very sensitive inhabitant and are known to be bioindicatorof aquatic environment. A slight change in the surrounding media effect the behavior,physiology and metabolism of fishes. The solid waste generated in large quantityenter into the water bodies which is adding to water pollution (Shashi Kant et al.,1990). Sodium fluoride has many uses in commerce, industry and it is soluble inwater which is readily taken up by plants and absorbed by animal (Bhatnagar et al.,2007). Fluoride is largely found in insecticide, rodenticide floor polishes in thepetroleum and aluminum industries glass etching and timber preservationsemiconductor industry and manufacture of chemical, solvent and plastic and inthe laundries. In freshwater environment naturally fluoride concentration are usuallylower than those expected to cause toxicity in aquatic organism. However aquaticorganisms might be adversely affected in vicinity of anthropogenic discharges.Fluoride has shown adverse affect on protein synthesis (Hangslow et al., 1980).The fluoride ion act as protoplasmic poison and living cell can tolerate this in lowconcentration (Pack et al., 1971). In the present investigation histoarchitecturalchanges in ovary of the NaF treated cat fish during Breeding period was studied.

MATERIALS AND METHODS

To study the effect of sodium fluoride Healthy fish C. batrachus of approximatelyequal size (15-20cm) and weight (50-60 gm) were procured from local market,brought to the laboratory and disinfected by treating with 0.01% potassiumpermanganate solution for 1-2 minutes. The fish were acclimated for 30 daysunder normal laboratory conditions for present experimental work at watertemperature of 26 ± 2ºC, pH 7.2 ± 0.5, total hardness 168 mg/L as CaCO3,dissolved oxygen content 5.0-5.5 mg/L and chloride content 28-32 mg/L. Dryfoods are available as granules which was given as food ad libitum. The fish wereexposed to natural photoperiodism (12L: 12D). During acclimation, water waschanged every day to discard the metabolic waste products.

After acclimation, fishes were divided into two groups, of 10 fishes each. One ofthe groups represented the control, while the other as experimental group. Theywere transferred to respective aquaria for the exposure period of 30 days. Thecontrol group was kept in sodium fluoride free water. The experimental group waskept in 1mg/L sublethal dose of sodium fluoride, which was 1/5th dose of 96h LC50

value for C. batrachus.

Six fishes from both control and experimental groups were sacrificed by decapitationafter 30 days of exposure in each phase of breeding period. The sacrificed fisheswere dissected to collect the ovaries and immediately fixed in bouin’s fixative.Paraffin embedding was carried out after in ascending series of alcohol grades.Sections of 4-5µ thickness were stained by eosin and haemotoxylin and thenmounted with Canada balsam. Sections were examined by light microscope andmicrophotographs (10Xmagnification) were used for histoarchitectural study.

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HISTOARCHITECTURAL CHANGES IN OVARY

RESULTS

In the ovaries of control C.batrachus no abnormal histologicalfeatures were observed. In T. S. of ovary of freshwater ofC.batrachus large ovum impregnated with yolk globule wasseen. Ovary is composed of group of small ogonial cell andmature ovum. Developing oocytes are distributed in betweenthe mature oocyte. Oogonium has homogenous cytoplasmand central large nuclei. Ovary of control fish showed welldeveloped egg with intact follicular epithelium and zonapellucida. Several histoarchitectural changes were observed

in the ovaries of the sodium fluoride exposed fish (Table 1, 2,3).

Pre-Spawning PhaseDuring pre spawning phase, with 30 days of sodium fluorideexposure, the ovarian growth was inhibited significantly. Theovaries of C.batrachus showed degenerative changes (atresia)in some oocytes (Fig.1. 2) and proliferative changes in thegranulosa of some oocytes, resulting sometimes in adhesionof the cellular coat of the oocytes. Besides, some oocytescollapsed and became abnormally irregular in shape. Inaddition, separation of the follicular layers from the oocyteswas observed in the ovaries of C. batrachus.Spawning PhaseDuring spawning phase, the ovaries of C. batrachus showedsome ovarian growth was inhibited and oocyte degenerated.Growing oocyte undergo degeneration. Ooplasm wasdisorganized. Inhibition of yolk synthesis took place. Follicularlining was not intact. In the ovaries degenerative changes insome oocytes, separation of the follicular layers from theoocytes (Fig. 2. 2).

Post-Spawning PhaseDuring post spawning phase, the histopathological alterationsin the ovaries of the studied fish appeared more severe. In theovaries of C. batrachus showed presence of more number ofatretic cells. Damaged stroma was also observed. Lifting ofovarian wall occurred, freely floating oocyte were prominent.Some oocyte were degenerated. Yolk and cytoplasm werevacuolated. The histopathological changes includeddegenerative and necrotic changes in the oocytes (Fig. 3. 2),proliferative changes in the granulosa of most oocytes, resultingsometimes in adhesion of the cellular coat of the oocytes,separation of the follicular layers from the oocytes. Besides,Lifting of ovarian wall prominent and freely floating oocyteoccur.

Explanation of Fig.

Pre Spawning Phase

Table 1: A comparative summary of ovarian histology of control andsodium fluoride exposed Clarias batrachus Linn. in pre spawningphase of breeding period

Observation Control Experimental

Vitellogenic Oocyte Present PresentZona Pellucida Thick ThinOvarian Wall Thick and Healthy ThinFollicular Wall Intact Not IntactOvum Normal Hypertrophied

OvumFollicular Granule Compact LooseAtretic Oocyte Very Few MoreTissue Scars Absent PresentProliferation of Not Evident EvidentConnective Tissue

Observation Control Experimental

Vitellogenic Oocyte Present PresentZona Pellucida Thick Very ThinOvarian Wall Thick and Thin and

Healthy DegeneratingFollicular Wall Intact DamagedOvum Normal Not normalFollicular Granule Compact More LooseAtretic Oocyte Very Few AbundantTissue Scars Absent PresentYolk granule scattered Absent Presentin ovarian cavityProliferation of Not Evident EvidentConnective Tissue

Table 2: A comparative summary of ovarian histology of control andsodium fluoride exposed Clarias batrachus Linn. in spawning phaseof breeding period

Table 3: A comparative summary of ovarian histology of control andsodium fluoride exposed Clarias batrachus Linn. in post spawningphase of breeding period

Observation Control Experimental

Vitellogenic Oocyte Present PresentZona Pellucida Thick Very Thin and

DegeneratingOvarian Wall Thick and Thin and degenerating

HealthyFollicular Wall Intact Not intact or DamagedOvum Normal Hypertrophied OvumFollicular Granule Compact More LooseAtretic Oocyte Very Few More AbundantTissue Scars Absent PresentYolk granule scattered Absent Presentin ovarian cavityProliferation of Not Evident EvidentConnective Tissue

Figure 1.1: T.S. of ovary of cat fish Clarias batrachus showing growingfollicle impregnated with yolk globules (YG), intact follicular wall(FW) thick zona pellucida (ZP) and with immature follicle (IF) fromcontrol groupTreated

FW

YG

ZP

IF

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SANGEETA KUMARI AND KANAKLATA

Figure 2.2: T.S. of ovary of cat fish Clarias batrachus showing largemature follicle impregnated with yolk globules (YG), thin zonapellucida (ZP), more atretic cells (AT) and not intact follicular wall(NFW) from treated group in spawning phase.Post Spawning phase,Control

Control

DISCUSSION

The present study revealed variation in ovarian structure whencompared to control and fluoride treated freshwater fish Clariasbatrachus due to fluoride toxicity treatment.

The variation is mostly according to time and dose dependent.It was observed that fluoride treatment cause vacuolization ofoocyte, inhibiton of yolk synthesis, empyting of follicle anddamaged oocyte was significant. Similar observation was alsoreported in the sunfish Lepomis macrochirus due to theexposure of endosulfan (Dutta and Dalal, 2008). Thehistopathological effects of different pollutants on gill, blood,liver, brain, gonads and other organ have been studied by

many researchers (Girija et al., 1985; Inbamani and Seenivasa,1998). Kamel (1990) recorded reduced number of oocytewith large follicular spaces and pycnotic nuclei of perinucleolusstage were observed in ovaries from the fish, oreochromisniloticus. He attributed these alterations to change inenvironmental conditions. Jobling et al. (2002) stated that,atresia was recorded in Rutilus rutilus living in rivers thatreceived treated sewage effluents. Saxena and Agarwal (1986)recorded cadmium chloride blocked oogonial activity fromfish, Clarias batrachus and reported that due to metal treatmentthere was retardation in proliferation, growth of oocyte andincrease in number of atretic follicle prominent. In presentstudy after 30 days of exposure in spawning phase, sodiumfluoride treated fishes exhibited occurrence of large numberof atretic follicle, degeneration of growing oocyte, vacuolated

Figure 1.2: T.S. of ovary of catfish Clarias batrachus showing Ateticcell (AT), lifting of follicular wall (FW), thin zona pellucida (ZP) andloosely arranged follicule (LAF) from experimental (treated) group inpre-spawning phase, spawning phase control

ZP

LAF

FW AT

Figure 2.1: T.S. of ovary of cat fish Clarias batrachus showing largemature follicle (MF) impregnated with yolk globules (YG), intactfollicular wall (FW), less atretic cells (AT) and from control group,treated

FW MF

YG

AT

YG

ZP MF

NFW

AT

Figure 3.1: T.S. of ovary showing ovulated follicles (OF), intactfollicular wall (FW)and less atretic oocyte (AT) from control groupin post-spawning phase, treated

OFAO

FW

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REFERENCES

Bhatnagar, C., Bhatnagar, M., Regar Bhag, C. 2007. Fluoride inducedhistopathological

Changes in gill, liver, kidney and intestine of freshwater teleost Labeorohita. Research Report Fluoride. 40(1): 55-61.

Datta, H. M. and Dalal R. 2008. The effect of endosulfan on ovary ofblugill sunfish: A Histopathological study (Lepomis macrochirus). Int.J. Environ res. 2(3): 215-224.

Girija, Moses, R. and Jayantha, K. 1985. Histopathological changesinduced by heptachlor in gills of freshwater fish,Tilapia mosambicca.Environ. Ecol. 3: 74-75.

Hangslow, C. F., Hanslow, J. K. and Holland, R. I. 1980. Fluoridesensitivity of cells from different organs. Aceta. pharmacol. ET. Toxicol.2: 46-73.

Inbamani, N. and seenivasan, R.1998. Effect of phophamedon toxicityand pesticidal histopathology of fish.Serotherodon mossambica. J.Ecotoxicol. Eviron. Monit. 8: 85-95.

Jobling, S., Beresford, N., Nolan Grey, T. R., Brigthy, G. C. andSumpter, J. P. 2002. Altered sexual maturation and gamatic productionin wild Roach (Rutilus rutilus) living in rivers that receive treatedsewage effluents. Biological Reproduction. 66: 272-281.

Kamel S. A. 1990. Study of artesian in the ovary of Nile BoltyOreochromis niloticus during its annual reproductive cycle. Fifthscientific conference proceedings of zoological society, Faculty ofscience, Cairi University, Egypt.

Nuehold, J. M. and Singler, W. F. 1960. Effect of sodium fluoride oncarp and rainbow trout. Trans. Am. Fish. Soc. 89: 358-370.

Narayanaswamy, S. Y. and Ramchandran, M. 2009. Melathion inducedchanges in the ovary of Gobid fish Glossogobius giuris (HAM) Indian.J. Comp. Animal Physiol. 27(2): 44-47.

Pack, M. R. 1971. Effect of hydrogen fluoride on production abdorganic reserve of been seed. Environ. Sci. Technol. 5: 1128-1132.

Shasi, K. 1990. Pollution of aquatic ecosystem human contribution.Symp. Env. Poll and Res. Land and water. pp. 1-13.

Sexena, D. N. and Agarwal, A. 1986. Current Science. 15: 397-398.

ooplasm empty follicular spaces and inhibition of yolksynthesis. Fluoride might have induced violent movement ofeggs, cause rupture of vitelline membrane. As reported by(Neuhold and Singler, 1960) this phenomenon might befollowed by immediate coagulation of yolk protein.Naryanswamy and Ramchandran Mohan (2009) reportedexposure of fish reported after 72 and 96 hours of 0.5mmmelathion indicated disorganization of ooplasm andhypertrophy of oocyte with earlier stage of oocyte becomingatretic. In present study also due to fluoride treatment, due tofluoride toxicity from fish, Clarias batrachus showed similarcondition in ovarian organ. It can be concluded that sodiumfluoride acute toxicity induced histopathological changes thereby severely affecting the overall reproductive capacity of fish.Further insight is needed to know the impact of sodium atcellular and sub-cellular level.

HISTOARCHITECTURAL CHANGES IN OVARY

Figure 3.2: T.S. of ovary of cat fish Clarias batrachus showing ovulatedfollicle (OF), not intact follicular wall (FW), more atretic cells (AT)and loosely arranged follicle from treated group in post- spawningphase

LAF

AT

FW

OF

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