Toxic Effect of Environmental Acid-Stress on the Sperm of aHill-Stream Fish Devario aequipinnatus: A Scanning ElectronMicroscopic EvaluationSUDIP DEY,1* S.M. KHARBULI,2 RAHUL CHAKRABORTY,1 S.P. BHATTACHARYYA,3 AND U.C. GOSWAMI41Electron Microscope Division, Sophisticated Analytical Instrumentation Facility, North Eastern Hill University,Shillong-793003, India2Department of Pisciculture, St. Anthony’s College, Shillong-793003, India3Department of Zoology, G.C. College, Silchar-78804, Assam, India4Depatment of Zoology, Gauhati University, Guahati-14, Assam, India

KEY WORDS SEM; acid stress; sperm; Danionin; hill-stream fish

ABSTRACT Environmental stress due to acidic pH of water was found to be one of the majorfactors leading to toxic effects on the sperm of a hill-stream fish Devario aequipinnatus of Megha-laya, India. The Scanning Electron Microscopy of the transverse section of testes of the fish col-lected from its natural habitat with acidic pH (5.6–6.0) showed that the sperms were clumped to-gether and their tails were either absent or were of extremely small length. The acrosome and mid-piece were also not well differentiated. When the fingerlings from the natural habitat were rearedto maturity in aquarium with water from natural habitat after changing the pH to alkaline range(8.0–8.2), the clumping of the sperm was not observed. The sperm tail was found to be well-devel-oped along with well-differentiated acrosome and midpiece. Since the only change in the waterquality parameters of the experimental aquarium as compared to those of the natural habitatwas the pH, it is evident that the abnormal features of the sperm observed in fish from naturalhabitat is mainly because of environmental acid stress. Microsc. Res. Tech. 72:76–78, 2009. VVC 2008

Wiley-Liss, Inc.

INTRODUCTION

The Northeastern part of India is one of the hot-spotsof biodiversity. The region is well-known for fish diver-sity especially for ornamental fish (Biswas et al., 2000).One of the popular groups of ornamental fish availablein Meghalaya (a state in the Northeastern part of India)is the Danionin, which are lively, energetic, and unde-manding shoaling fish. A wide variety of Danionin spe-cies have been reported from different parts of the worldwhich include Zebra-Danio (Danio rerio), Pearl-Danio(Danio alboineatus), Giant-Danio (Devario aequipinna-tus) Spotted-Danio (Danio nigrofasciatus) etc.

The Giant-Danio (Devario aequipinnatus) is the larg-est of all the species of Danionin, in which the adultgrows to about 4–4.5 inches in length. The body is lat-erally compressed and the profile of the underside ismore strongly convex than the back. The head ispointed with one pair of barbels. There are three to fiveblue–black stripes from head toward the tail.

Zebra-fish, in general, is important not only as orna-mental fish but also as vertebrate models for toxicitytests and different biological studies (Alestrom et al.,1995; Braunbeck et al., 1992; Driever et al., 1994). Thetransgenic technique for studying gene function has alsobeen developed in the Zebra-fish (Kawakimi et al., 1996).

The Giant-Danio species (Devario aequipinnatus)were abundantly found in the state of Meghalaya untilrecent years. Unfortunately, the population of this im-portant bioresource of the state is gradually declining.

As a part of the detailed studies on the possible causesfor the gradual decline in population of Devario aequi-

pinnatus, the present investigation was aimed at under-standing the structural features of the gonads of the fishin its natural habitat. To begin with, studies were car-ried out on testes and sperm of Devario aequipinnatus.

MATERIALS ANDMETHODSFish

The fish used for the study was Devario aequipinna-tus, a hill-stream ornamental fish, which is consideredone of the most important bioresources and an impor-tant vertebrate model for toxicity test.

Collection of Samples

A set of 10 male fish was collected from their naturalhabitat at each monthly collection made during thecourse of the study (2001–2005). The collection wasmade from ‘‘Sumer’’ stream, ‘‘Mawkynroh’’ stream, and‘‘Umroi’’ stream, situated in and around Shillong, thecapital of Meghalaya, a state in the Northeastern partof India.

Measurement of pH

Water pH of the streams was measured with thehelp of Lovibond 2000 Comparator.

*Correspondence to: Dr. Sudip Dey, Electron Microscope Division, Sophisti-cated Analytical Instrumentation Facility, North Eastern Hill University, Shil-long-793003, India. E-mail: sudipdeyrsic@yahoo.com

Received 1 July 2008; accepted in revised form 8 August 2008

DOI 10.1002/jemt.20640

Published online 20 October 2008 in Wiley InterScience (www.interscience.wiley.com).

VVC 2008 WILEY-LISS, INC.

MICROSCOPY RESEARCH AND TECHNIQUE 72:76–78 (2009)

Experimental Design

A group of 10 advanced juvenile fish was reared ineight separate aquariums with water collected fromnatural habitat. The water pH of four aquariums wasacidic (5.5–6) as the water from natural habitat wasused in these.

The water in the remaining four aquariums wasmaintained at alkaline pH (8–8.2) by the addition oflime (Atland and Barlaup, 1991) to the water collectedfrom the natural habitat. The juveniles were reared inthe aquariums until they attained maturity.

Microscopic Preparation

The testes of mature fish collected from natural habi-tat and also raised in aquarium were processed forscanning electron microscopy.

Testes were excised from male fish and were cuttransversely into small pieces, washed thoroughly indouble distilled water, fixed in 2.5–3% glutaraldehydeprepared in 0.1 M Na-cacodylate buffer (pH 7.2) for4 h. at 48C. Following the primary fixation, the tissueswere washed in Na-cacodylate buffer for 15–30 min,dehydrated in ascending grades of acetone and dried inTetramethylsilane following the method of Dey et al.(1989). The dried samples were secured horizontally tobrass stubs (10 mm 310 mm) with double coated adhe-sive tape connected via a patch of silver paint to ensurecharge conduction. A conductive coating of gold wasapplied to the sample using JFC-1100 (Jeol) Ion-Sput-ter Coater, by establishing a low vacuum (1023 Torr) inthe sputtering chamber.

Microscopic Analysis

The coated samples were examined in JSM-35CF(Jeol) Scanning Electron Microscope using the second-ary electron emission mode at an accelerating voltageof 15–20 kV. Tilt control was fixed at 08 for settingspecimen stage in a horizontal position, and workingdistance (WD) was set at 15 mm by turning the WD-selector.

A total of 125 testes samples comprising 25 fromeach collection during the year 2001–2005 havebeen examined by SEM. The same number of sam-ples from each experimental group was used for SEMassessment.

RESULTS AND DISCUSSION

The scanning electron microscopy has been proved tobe versatile in assessing the effect of environmentalstress, pollution, and toxic chemicals on surface mor-phology of various tissues in a number of animals (Deyet al., 2001, 2005). The present study on SEM of thetransverse section of the testes of the fishes collectedfrom their natural habitat with acidic pH and alsofrom adults raised in aquarium with acidic pH revealedsome remarkable abnormal features. The sperm headexhibited an average diameter of 0.6 lm in acidic pHgroup (Fig. 1a) in contrast to 1.70 lm in alkaline pHgroup (Figs. 1b and 1c). The sperm tail in most of thecases in acidic pH group of fish was found to beextremely small in length (about 1.66 lm) (Figs. 1a and1d) in contrast to those in alkaline pH group (about6.36 lm) (Figs. 1b and 1c). The acrosomes and midpie-ces were also found to be poorly differentiated in fish

Fig. 1. (a) Distortion of sperm-head and short sperm-length insome Devario aequipinnatus fromits natural habitat with acidic pH(Bar5 1 lm). (b) Sperm of Devarioaequipinnatus grown at alkalinepH of water showing absence ofclumping and long sperm-tail(Bar 5 1 lm). (c) A single sperm ofDevario aequipinnatus grown inalkaline pH of water (Bar 5 1 lm).(d) Clumping of sperms andvery short sperm tails in Devarioaequipinnatus collected from itsnatural habitat with acidic pH.(Bar5 1 lm).

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77TOXIC EFFECT OF ENVIRONMENTAL ACID-STRESS

collected from the natural habitat with acidic pH ofwater.

The sperm-head in acidic pH group from naturalhabitat and also from those raised in the aquarium atacidic pH were found to be clumped (Fig. 1d). Further,some fish collected from the natural habitat with acidicpH revealed remarkable distortion of the sperm-head(Fig. 1a).

The observations on the clumping of spermatozoa(Fig. 1d), poorly developed sperm-tails and distortion ofsperm-head in some cases in Devario aequipinnatuscollected from its natural habitat suggests that spermmotility is adversely affected in the natural habitat ofthe fish due to the acidic pH of water. This was con-firmed through our observations that fish grown inalkaline pH of water in the experimental aquariumexhibited well-differentiated acrosome and sperm-tailof normal length. In this context, it is to be noted thatnormal sperm-length is extremely important for spermmotility, because sperm-length parameter is correlatedpositively with ATP, energy charge and fertilizationsuccess (Vladic et al., 2002).

The movement of spermatozoa in most of the fish iscontrolled by energetic and cytoplasmic ionic condi-tions which are responsible for marked changes in cellmorphology (Dreanno et al., 1999). This supports ourobservations on the absence of well-differentiated acro-somes, mid-piece and the existence of very shortsperm-tails in Devario aequipinnatus collected fromtheir natural habitat with acidic pH. It is worthwhileto mention here that sperm motility in some fish wasreported to be very low in acidic pH of water (Kime andTvieten, 2002). However, attempts to correlate micro-structural features of sperm with low water pH are notavailable in the existing literature. Our study thusseems to be the first attempt in understanding theadverse affect of acidic pH on sperm morphologythrough an ultrastructural approach.

As high motility of sperm is a prerequisite for fertil-ization and since it correlates strongly with fertiliza-tion successes (Rurangwa et al., 2004), our present ob-servation on poorly developed sperm tail in Devarioaequipinnatus grown in the natural habitat with acidicpH of water is certainly one of the major causes forimpairment of reproductive function in the fish leadingto its gradual population decline.

Further, our observation on distortion of sperm-headin some of the Devario aequipinnatus collected from

the natural habitat suggests that the fish species in thepresent study area is under severe stress as far as itsreproductive efficiency is concerned.

ACKNOWLEDGMENTS

We are thankful to Prof. B. Myrboh, Head, Sophisti-cated Analytical Instrument Facility, North EasternHill University, Shillong, for his encouragement.S.M.K. is thankful to the Principal, St. Anthony’s Col-lege, Shillong, for his kind permission to carry out thework.

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