RESEARCH ARTICLE

Elevated Water Temperature Induces Germ Cell Proliferationin Loach Lepidocephalus guntea (Hamilton): Implicationfor Brood Stock Development

Sullip Kumar Majhi • Binod Kumar Choudhary

Received: 28 March 2013 / Revised: 30 July 2013 / Accepted: 22 October 2013 / Published online: 12 March 2014

� The National Academy of Sciences, India 2014

Abstract The effectiveness of elevated water tempera-

ture on growth and augmentation of gonadal maturation in

loach Lepidocephalus guntea is described here. Fish

(Mean ± SD 1.06 ± 0.29 g) were stocked at a density of

2.0 kg of fish per m3 under a constant light cycle. The

temperature in tanks was gradually increased at 1 �C/day

from 20 �C (control) to reach test temperature 25 �C

(elevated), with replications of each. The effectiveness of

the treatments was assessed by gonadal index (GSI) and

histology. The analyzes revealed a significant increase in

GSI at the end of 180 days in the group reared at 25 �C

(males 3.3 ± 0.54 %; females 7.5 ± 0.55 %) than their

peers at 20 �C (males 1.32 ± 0.25 %; females 2.92 ±

0.26 %; P \ 0.05). Further, 25 �C caused marked germ

cell proliferation in both the sex to attain gonadal matu-

ration in 180 days from the beginning of heat-treatment.

The findings in this study has implications for brood stock

development in loach and additionally, might provide

important clues for augmenting gonadal maturation in

other fish species originating from higher altitudinal habitat

that are yet to breed in captivity.

Keywords Lepidocephalus guntea � Growth � Germ cell �Reproduction � Brood stock

Introduction

The loach Lepidocephalus guntea (Ham.) belongs to Actin-

opterygii Cypriniformes Cobitidae (http://www.fishbase.org)

is a freshwater fish widely spread in Eastern Asia. Loaches are

generally abundantly found in calm hill streams, paddy fields

and low laying water bodies. They have omnivorous food

habit, tolerance to high stocking density and grow rapidly.

Loaches are small in body size and mostly spend their time

buried in sandy bottom and occasionally come to the surface

with swift movement to gulp atmospheric air. Thus they can

be grown in oxygen-depleted water bodies. Besides having

ornamental values, loaches are also used as folk remedies for

treatment of hepatitis, osteomyelitis, carbuncles, inflamma-

tion and cancer, as well as for restoration to health in debilities

caused by various pathogens and ageing [1]. Some of the

active compounds obtained from loach were found to be of

high medicinal value [2].

The commercial loach production in Asia is largely

depending on by-catch from natural water bodies. How-

ever, the continuous exploitation from natural sources

coupled with rapid environmental degradation has resulted

in sharp decline in its population in wild [3]. Consequently,

wild seeds are only seasonally available for culture. The

year-round production of fry under captivity could ensure

commercial production and meeting the growing consumer

demand in Asia. Thus, for expanded and consistent pro-

duction of loach ultimately require development of viable

brood stock for mass-scale seed production.

One of the prerequisite for successful breeding pro-

gramme is captive-development of brood stocks that are

capable of producing healthy gametes. Often nutritionally

rich diets supplementation is preferred to develop a brood

fish. However, this strategy alone might take considerably

long time to achieve the goal of gonadal maturation and,

might incur high expenditure. In wild, photoperiod and

temperature are two environmental factors known to play

pivotal role in gonadal maturation of teleost fish [4]. In this

maiden study, attempt was made to manipulate one of such

S. K. Majhi (&) � B. K. Choudhary

Indian Council of Agricultural Research (ICAR) for Eastern

Region, P.O. B.V. College, Patna 800 014, Bihar, India

e-mail: sullipkm@gmail.com

123

Natl. Acad. Sci. Lett. (March–April 2014) 37(2):107–111

DOI 10.1007/s40009-013-0208-1

environmental parameter viz. water temperature, to accel-

erate the gonadal maturation in L. guntea.

Materials and Methods

Fish Collection and Acclimation

Lepidocephalus guntea were collected using drag net of

suitable mesh size from streams, paddy fields and canal of

Meghalaya (90�55015 to 91�160 latitude and 25�400 to

25�210 longitude) and brought into wet laboratory of

Fisheries Division (900 m [ MSL), ICAR Complex for

NEH Region, Meghalaya, India in a aerated plastic con-

tainers (500 L capacity). Fishes were treated with 10 ppm

of KMnO4 (Sigma, St. Louis, MO, USA) and released into

a cemented cistern pool (4 m 9 2 m 9 1.5 m = 12 m3)

for acclimation in captivity.

Screening of Temperatures for Heat-Treatment

The healthy L. guntea (n = 15) were stocked into single

insulated tanks (1 9 0.75 9 1 m3) with constant aeration

and flow-through. The temperature in tanks was gradually

increased at 1 �C/day from 20 �C (control) to reach test

temperatures of 25, 30 and 35 �C. The animals in their

respective temperature were daily fed twice with supple-

mentary diet (rice polish: mustard oil cake; 1:1 w/w) and

reared until 30 days from the beginning of attaining test

temperature. Every day animals in each tank were closely

observed for food acceptance and survivability. The dead

fish, if any, were removed with a hand net and discarded

following the standard protocol [5]. On termination of this

preliminary trial, survivability (%) was calculated and test

temperature was decided for heat-treatment.

Experimental Setup

Based on the preliminary experiment, a test temperature of

25 �C was selected for further investigation. The insulated

experimental tanks (1 9 0.75 9 1 m3) were provided with

2 cm sand bed, pebbles and aquatic plant water hyacinth to

simulate the natural environment in situ. A single tank for

each temperature treatment, control (20 �C) and test

(25 �C), was maintained with 200 L freshwater with con-

stant aeration and flow-through (1 l water/h.). Fish

(n = 50; Mean ± SD 1.06 ± 0.29 g) were stocked at a

density of 2.0 kg of fish per m3 under a constant light cycle

(12L12D). The fishes were daily fed with supplementary

feed (rice polish: mustard oil cake; 1:1 w/w) to apparent

satiation. Fishes were sampled at one-month intervals for

assessment of growth and biomass from randomly drawn

samples (n = 10), tanks were cleaned (25 % water

exchange), water samples collected and parameters viz.

dissolved oxygen, pH, free carbon dioxide, total hardness,

total alkalinity, nitrate: N and phosphate: P were analyzed

following the protocol prescribed in APHA 2005 [5].

Gonado Somatic Index and Histological Analysis

of Gonads

Fish (n = 5, each sex) were sacrificed at the end of

180 days from both control and treatment by an overdose

of anesthesia MS-222 (Tricaine methanesulfanate, Sigma,

St. Louis, MO, USA) and their body weight was recorded.

The gonads were excised, macroscopically examined and

weighed to the nearest 0.01 g. The middle portion of the

right and left gonads were then immersed in Bouin’s fix-

ative for 24 h and preserved in 70 % ethanol. Gonads were

processed for light microscopical examination following

routine histological procedures up to sectioning at a

thickness of 5 lm, staining with hematoxylin-eosin and

observation under a microscope at magnifications between

10 and 1009.

Statistical Analysis

Measured parameters were compared by one-way analysis

of variance (ANOVA) with Tukey’s multiple comparison

tests by using Graphpad prism ver. 5.0 for windows

(Graphpad software, San Diego, CA, USA). Data are pre-

sented as mean (replicates) ± Standard deviations (SD)

and differences between groups were considered as statis-

tically significant at P \ 0.05.

Results and Discussion

The preliminary trial on selection of temperature ranges for

heat-treatment in L. guntea revealed that, (1) the animal

Fig. 1 Survival of L. guntea (n = 15) at different temperature

regimes at the end of 30 days preliminary trial. Note, there was

significant mortality at 30 �C and beyond. Based on these observa-

tions, two temperature ranges (highlighted in box) were selected as

control and heat-treatment. Symbol with different letters vary

significantly (Tukey’s multiple comparison test, P \ 0.05)

108 S. K. Majhi, B. K. Choudhary

123

shows better adaptability in terms of feed acceptance with

nearly 100 % survivability at 20 �C (lower range) and 25 �C

(upper range) but not beyond, (2) the survivability of animal

significantly decreased at 30 and 35 �C (P \ 0.05; Fig. 1)

with development of pathology conditions like depigmenta-

tion of body surface and ulceration. Based on these obser-

vations, the present study explores the possible beneficial

effects of elevated water temperature (25 �C) on growth and

augmentation of gonadal maturation in loach L. guntea.

The survivability of animals at 20 and 25 �C ranged

from 98 to 100 % and was not significantly different

between the temperatures (P [ 0.05; results not shown).

The results of the physico-chemical parameters of water

during the experiment period were well within the

acceptable range for aquaculture throughout the study

period [6]; except the value of dissolved oxygen was found

to be slightly low (4.7–5.5 mg/l) in the tank with elevated

water temperature. Water quality is one of the vital factors

in fish farming and directly related to the growth and

production of farmed animals [7]. In this investigation,

healthy biotic parameters maintained at both the tempera-

ture regime, yet confirmed the optimum condition

requirement for captive rearing of L. guntea, as reported by

previous workers [8].

There was significant increase in body weight in both

the temperature regime (Fig. 2). However, increase was

much more prominent in elevated water temperature

(25 �C), especially between 90 and 180 days. Fish gener-

ally attains better growth and effectively utilize the feed

when reared at comparatively higher temperature ranges

versus the ambient range [9]. Probably at slightly elevated

temperature the activities of digestive enzymes increases

and facilitate the digestion of the nutrients, resulting in

good growth [10]. This was confirmed in this study and

found that in L. guntea, water temperature of 25 �C help in

efficient utilization of supplementary diet and results in

better growth attribute. However, future studies should

explore development of a balance diet for this species

considering the nutritional requirements, so as to boost up

the somatic growth in comparatively short time.

The gonado-somatic index (GSI), a proxy of gonadal

maturation, increased significantly in both the sex between

0 and 180 days and was more prominent in elevated water

temperature (Fig. 3). For instance, at the end of the study,

the GSI in males and females had reached 3.3 ± 0.54 and

7.5 ± 0.55 %, respectively at 25 �C than their peers at

20 �C (males 1.32 ± 0.25 %; females 2.92 ± 0.26 %;

P \ 0.05). Surprisingly, similar GSI values were also

Fig. 2 Body weight gain of L. guntea at 20 and 25 �C. Columns with different letters vary significantly (Tukey’s multiple comparison test,

P \ 0.05)

Fig. 3 Changes in the GSI of L.

guntea males and females at 20

and 25 �C. Columns with

different letters vary

significantly (Tukey’s multiple

comparison test, P \ 0.05)

Germ Cell Proliferation in Loach L. guntea (Hamilton) 109

123

obtained from sexually matured (age 2?) wild-type L.

guntea in the peak breeding season of April–July in open

water of Bangladesh [11]. The significant improvement in

GSI to match that of wild-type in considerably short time in

this case (approximately age 1?), infers to the mechanism

of heat-induced germ cells proliferation that generally

occur in summer season in wild to increase the gonad

weight [4]. Possibly, the treatment condition applied in this

study simulate the onset of ‘summer’ condition, yet con-

tinuous rearing at elevated water temperature has lead to

rapid proliferation of germ cells and add weight to the

gonad. It is known that teleost gonads began to respond to

the warmer water temperature and stimulate secretions of

the Sertoli cell [12], which then multiplies to expand the

Fig. 4 Histological appearance

of the gonads in control (20 �C)

and elevated water temperature

(25 �C) groups at the end of

180 days. a, b Testis from

20 �C male showing germinal

epithelium with small cysts of

spermatogonia at the periphery

of basement membrane

(highlighted b is a high

magnification of the box shown

in a). c, d Testis from 25 �C

male showing a thick germinal

epithelium with large cyst of

spermatogonia and other

spermatogenic cells

(spermatocytes, and

spermatids). Note, the lumen of

the efferent ducts is

accumulated with spermatozoa,

which indicate active

spermatogenesis (highlighted

d is a high magnification of the

box shown in c). e, f Ovary from

20 �C female showing small

cyst of oogonia and few

premature oocytes at various

stages of development (arrow

f is a high magnification of the

box shown in e). g, h Ovary

from 25 �C female showing the

presence of large cyst of

oogonia and mature

perinucleolar oocytes. Note,

germinal vesicle is at center of

oocyte and is surrounded by

yolk globules and small lipid

droplets (arrow h is a high

magnification of the box shown

in g). Scale bar 100 lm (a, c, e,

g), 30 lm (b, d, f, h)

110 S. K. Majhi, B. K. Choudhary

123

gonads size and promote the development of the smooth

endoplasmic reticulum [13]. The GSI values obtained in

this study in a short time could have imperative implica-

tions for aquaculture and seed production in the mid and/or

high altitude geographic regions where temperature

remains low throughout the year. Nevertheless, the future

study should aim at further shortening the maturation time

in L. guntea by combining the benefits of synthetic hor-

mones and elevated water temperature.

The histological analysis of testes revealed that all males

sampled at 180 days from the beginning of heat treatment

at 25 �C had mature spermatozoa in addition to large cysts

of spermatogonia, spermatocytes and spermatids, and thus

was considered as sexually mature individual with active

spermatogenesis (Fig. 4c, d). On the other hand, at the

same time, males at lower temperature (20 �C) had only

cysts of spermatogonia and spermatocytes in the lumen of

the seminiferous tubule and efferent ducts but lacked cysts

of spermatozoa and spermatids (Fig. 4a, b). In contrast,

females at 20 �C had ovary with some degree of hyper-

trophy at the vigorous lamellae with comparatively fewer

oocytes, particularly the cortical alveoli oocytes and cyst of

primitive egg mother cell i.e. oogonia (Fig. 4e, f). How-

ever, at 25 �C, there was significantly large cyst of oogonia

population with cortical alveoli oocytes and wide spread of

mature ovum in all section examined of five individuals

(Fig. 4g, h). Generally in low water temperature repro-

duction usually concentrate in summer season. Thus, with

increasing latitude the gonadal maturation in fish get

delayed [14]. Probably, this being the reason that many of

the fishes from mid or high altitude region, including loach,

depicts delayed sexual maturation by 1 or 2 years [11].

Overall, it can be surmise here that elevated water tem-

perature significantly augments gonadal maturation in

loach and many other teleost fishes [13].

In conclusion, the fishes survived well in both 20 �C

(control) and 25 �C (elevated) water temperatures. How-

ever, animals reared at 25 �C exhibited significant gonadal

growth in comparatively short period over their peers

reared at 20 �C. Further, the strategy of elevated water

temperature treatment appears to be a viable option for

advancing gonadal development in L. guntea brood stock

for captive breeding and aquaculture, although it should be

noted that viable gametes were not produced in this study

from fish in any of the treatments.

Acknowledgments Authors are indebted to the field staff of Fishery

Division, ICAR Complex for NEH Region, Barapani, Meghalaya for

kind support during the study period.

Conflict of interest The authors declare that they have no conflict

of interest.

References

1. Qin CG, Huang KX, Xu HB (2002) Protective effect of poly-

saccharides from the loach on the in vitro and in vivo peroxi-

dative damage of hepatocyte. J Nutr Biochem 13:592–597

2. Kimura M, Hama Y, Sumi T, Asakawa M, Rao BNN, Horne AP,

Li SC, Li YT, Nakagawa H (1994) Characterization of a deam-

inated neuraminic acid-containing glycoprotein from the skin

mucus of the loach, Misgurnus anguillicaudatus. J Biol Chem

269:32138–32143

3. Beveridge MCM, Phillips MJ, Macintosh DJ (1997) Aquaculture

and the environment: the supply of and demand for environ-

mental goods and services by Asian aquaculture and the impli-

cations for sustainability. Aquac Res 28:797–807

4. Okuzawa K, Furukawa K, Aida K, Hanyu I (1989) Effects of

photoperiod and temperature on gonadal maturation, and plasma

steroid and gonadotropin levels in a cyprinid fish, the honmoroko

Gnathopogon caerulescens. Gen Comp Endocrinol 1:139–147

5. APHA (2005) Standard methods for examination of water and

wastewater, 21st edn. APHA, AWWA, WPCF, Washington, DC

6. Boyd CE (ed) (1982) Water quality. Water quality management

for pond fish culture. Elsevier, Oxford, pp 6–50

7. Majhi SK, Das A, Mandal BK (2006) Growth performance of

organically cultured grass carp C. idella (Val.) under mid-hill

conditions of Meghalaya: North Eastern India. Turk J Fish Aquat

Sci 6:105–108

8. Fujimoto T, Toshiya N, Rie GK, Yutaka K, Etsuro Y, Katsutoshi

A (2010) Sexual dimorphism of gonadal structure and gene

expression in germ cell-deficient loach, a teleost fish. Proc Natl

Acad Sci USA 107:17211–17216

9. Gadowaski DM, Caddell SM (1991) Effects of temperature on

early-life-history stages of California halibut Paralichthys cali-

fornicus. Fish Bull 89:567–576

10. Ray AK, Roy T, Mondal S, Ringø E (2010) Identification of gut

associated amylase, cellulase and protease producing bacteria in

three species of Indian major carps. Aquac Res 41:1462–1469

11. Rahman MH, Hossain MA, Kawai K, Hossain MA (1997)

Morphometric characteristic and reproductive periodicity of

freshwater fish, Lepidocephalus guntea (Hamilton). Bull Mar Sci

Fish Kochi Univ 17:141–147

12. Franca LR, Bartke A, Borg KE, Cocim M, Fadden CT, Yagi A,

Russell LD (1994) Sertoli cells in testes containing or lacking

germ cells: a comparative study of paracrine effects using the W

(c-kit) gene mutant mouse model. Anat Rec 240:225–232

13. Schulz RW, Franca RL, Lareyre JJ, LeGac F, Garcia HC, Nob-

rega RH, Miura T (2010) Spermatogenesis in fish. Gen Comp

Endocrinol 165:390–411

14. Baker JR (1938) The relation of breeding seasons. In: De Beer

GR (ed) Evolution, essays or aspects of evolutionary biology.

Oxford University Press, Oxford, pp 161–177

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