J. Exp. Zool. India Vol. 14, Supplement 1, pp. 27-32, 2011 ISSN 0972-0030

HISTOLOGICAL STUDY OF GONADS DURING BREEDING SEASON OF A THREATENED FISH RAINBOW ( BLEHERI) IN ASSAM

Samarendra Behera*, Rinku Gogoi and Sanjeev Kumar Department of Fisheries Resource Management, Faculty of Fishery Sciences, West Bengal University of and Fishery Sciences, 5, Budherhat Road, Chakgaria, P.O. Panchasayar, Kolkata – 700 094, India. *e-mail: [email protected] (Accepted 18 January 2011) ABSTRACT : The study was confined to the laboratory investigation during September 2010 to August 2011 for the study of gonad histologically during breeding of threatened fish rainbow snakehead (Channa bleheri) collected from natural environment in Assam. It was found that the genital material (milt and ova) their structure, number are regulated according to the influence of seasonal cycle and hormonal secretion. A single breeding season of Channa bleheri was found that is during May and June. Key words : Channa bleheri, gonad, histology and breeding seasons.

INTRODUCTION bleheri is considered as on threatened species, which As far as the export of ornamental fishes from India needs conservation through propagation. Propagation of is concerned, 90% of the total exports are wild caught fish is made by two ways as (i) Natural propagation and fishes of fresh water origin and arising from North-Eastern (ii) Artificial propagation. In natural propagation: fish region. The remaining 10% are either tank reared or breed enhances its population in the nature, which is already and reared varieties of exotic species of fresh water or destroyed and under risk now because of high exploitation marine origin. The important fresh water ornamental fish of this species from the natural resources for aquarium collection centers in India are located in the North Eastern fish trade. In artificial propagation: the species is supplied states. About 80% of ornamental fishes from India to with a simulated environment and allowed for breeding International market are exported via Kolkata Airport, of and rearing of spring in the laboratory condition. which the lion’s share (more than 80%) is contributed MATERIALS AND METHODS from North Eastern Region. A study on histological study of gonads during North-eastern India is rich in swamps and lakes, and breeding season of a threatened fish rainbow snakehead in Channid fauna. Under the All India Co-ordinated Project (Channa bleheri) in Assam was conducted in the on air-breathing fishes Dehadrai (1975) made extensive Department of Fishery Resources Management, Faculty studies on the cage culture of some of the species of this of Fishery Sciences, West Bengal, for one year. region. However, and phylogeny of the Channid Histological study fishes of the region is poorly understood. For histological study, the microscopic slides were Channa bleheri shows heigh density of occurance prepared by the following procedure as followed by of but restricted only to the upper Assam zone of the Agarwal (1996). The development stages of germ cells Brahmaputra, Dibru river basin (mainly Tinsukia and in the testes and the change of the oocytes in ovary were Dibrugarh districts of Assam) and can be considered rare. studied by following methods. This species encountered in the open water during flood Collection and fixation of tissue : For histological when they come out of their holes, but during winter the study, the middle parts of the gonadal tissues (testes and fish can be caught by digging the hole in the specific areas ovary) of Channa bleheri were collected as stated earlier. (Goswami et al, 2006). The tissues were trimmed into 5 to 6 mm size for better Channa bleheri species is caught from the natural penetration of fixatives into it. The tissues were put into resources for the aquarium fish trade (Ralf Britz, personal Formaldehyde Saline (Baker, 1944) (Appendix-I) for 24 commun., 2002). By which the population of this species to 48 hours as per size of tissues. is decreasing in a great extent and reached to a state of endemic and rare (Musikasinthern, 2000). Channa 28 Samarendra Behera et al Post fixation treatment tap water. After cleaning, the slides were air-dried and a Washing : The tissues (testes and ovary) were thin layer of Glycerin Egg Albumin (Appendix-IV) was removed from the fixatives and subjected to overnight rinsed over it. Then the ribbons with materials (about 10 washing with flowing clear tap water until the to 15 sections depending on the size) were spread over formaldehyde odour was vanished. the clean glass slides. Thin tissues were made wrinkle free and allowed to fix on slides by keeping them on hot Dehydration : The tissues were dehydrated perfectly plates (30°C) for 2 to 5 minutes. with graded alcohols, starting from 30%, 50%, 70%, 90% and absolute alcohol (100%) to avoid the brittleness of De-waxing and staining : Tissues fixed on slides the tissues. were de-waxed with descending order of alcohols (100%, 90%, 70%, 50% and 30%) and stained by the double De-alcoholization : Two changes of xylene (1 hr staining method with Haematoxylin and Eosin by using each) were made to clean the tissues from alcohol. For standard techniques as described by Agarwal (1996). better impregnation of wax into the tissue, the xylene penetrates into the tissue to become transparent and the Mounting : One or two drops of DPX (mountant) material comes up to float on the top. were put on the dried slide which one was ready for mounting. Then, a cover slip or slide was slowly lowered Infiltration : Paraffin wax (melting point 58-60°C when the mountant would flow ahead of the descending of B.D.H) was used for infiltration of tissue. Three glass without trapping air bubble between the cover slip changes of wax (45 min each) were made to make tissue and slide. The excess of mountant on the slides was xylene free. removed with xylene soaked cotton. After mounting, the Embedding : For the preparation of blocks, pure slides were allowed for drying. The excess of mountant paraffin wax was melted in water bath in between 58- on the slides was removed with xylene soaked cotton. 60°C. Metal ‘L’ moulds were adjusted according to the Labeling and storing : Labeling was done on the size of blocking materials. The melted paraffin was taken slide by glass marking pen to avoid future confusion. The from water bath and the blocking disc was filled. After slides were stored in slide box to protect them from dust permitting a layer of wax to be solidifying on the bottom and dirt. disc, the completely infiltrated tissues were carefully taken from the paraffin wax and put inside the different blocking Microscopic observation : The histological sections disc according to their size. Care was taken so that the on the prepared slides were thoroughly observed under wax on the top of the disc did not solidify during keeping Advanced Trinocular Microscope (Olympus, MODEL 8 the material in the blocking disc. For this reason, a heated x 51, Japan) microscope at different magnifications. The needle or forceps was put only the upper portion or inside developmental stages of germ cells in the testes and the wax of the disc. After the proper positioning of the changes of the oocytes of ovary were noticed carefully. tissues, the wax inside the disc was allowed to solidify. Colour photomicrographs of selected histological sections After few minutes, the ‘L’ moulds were removed from were taken as and when required. the wax block. Thus prepared blocks were kept separately RESULTS AND DISCUSSION inside the labeled polythene packets. Histological Observation Trimming and sectioning : The paraffin blocks Testicular cyclicity : Histological micro slides are 2 were trimmed carefully to 6 to 7 mm by sharp blades. observed for six months and found different levels of The trimmed blocks were fixed to the wooden holder (peg) testicular matters in it. On the basis of the testicular with the material facing away from it. Molten wax was matters the maturation stages of testes are also determined poured on the holder and the block was kept on it. The (Table 1). block was padded with more wax at the base to make it Histological structure of testes is variable from strong. After being confirm, the blocks were firmly fixed species to species. According to Gaurya et al (1997), with holder, the sectioning was done by using microtome when reproductive activity (spermatogenesis) is (SPENCER 820 TYPE). On the microtome, each section undergoing in the lobules of testes of fish, about six was cut into 5µ thickness. The ribbons containing tissues spermatogenic elements are produced from sperm mother were collected on clear glass slide (already a smear of cells of germinal epithelium and passes through different egg-albumin was kept on that slide) with the help of fine maturation stages as primary spermatogonia, secondary brush. spermatogonia, primary spermatocytes, secondary Spreading and fixing : Glass slides were cleaned spermatocytes, spermatids and spermatozoa (sperms). properly by Chromic-acid solution, soap and finally with Histological study of gonads during breeding season 29

Plate 1 : Photomicrograph of T.S. of Testis of Channa bleheri, Stage Plate 2 : Photomicrograph of T.S. of Testis of Channa bleheri, Stage III developing phase during March (10×0.25). IV pr-spawning phase during April (10×0.25).

Plate 3 : Photomicrograph of T.S. of Testis of Channa bleheri, Stage Plate 4 : Photomicrograph of T.S. of Testis of Channa bleheri, Stage V spawning phase during May-June (10×0.25). VI spent phase during July (10×0.25).

Plate 5 : Photomicrograph of T.S. of Testis of Channa bleheri, Stage I resting phase during August (10×0.25).

The spermatozoa contains nucleus, cytoplasmic only during June, which is considered as breeding season. components and acquire flagellum for mobility This result is corroborated with the observation of (Nagahama, 1986; Agarwal, 1996). Mookherjee and Majumdar (1946) in the Anabas species. In the present study, testicular cyclicity of Channa Whereas Channa punctatus breeds twice in a year i.e. bleheri is concerned with the Pre- spawning phase or in early April and August- September (Srivastava et al, maturing phase, spawning or matured and post spawning 1992) and Channa striata breeds during most months of phase (Table 1, Plates-1, 2, 3, 4, 5). In the present study, the year with peak spawning season during peak rainfall it was observed that Channa bleheri attained maturity (Ali, 1999). 30 Samarendra Behera et al

Plate 6 : Photomicrograph of T.S. of Ovary of Channa bleheri, Stage Plate 7 : Photomicrograph of T.S. Ovary of Channa bleheri, Stage III developing phase during March (10×0.25). IV pre-spawning phase during April (10×0.25)..

Plate 8 : Photomicrograph of T.S. of Ovary of Channa bleheri, Stage Plate 9 : Photomicrograph of T.S. of Ovary of Channa bleheri, Stage V spawning phase during May-June (10×0.25). V spawning phase during May-June.

Plate 10 : Photomicrograph of T.S. of Ovary of Channa bleheri, Plate 11 : Photomicrograph of T.S. of Ovary of Channa bleheri, filled with ripe eggs during May-June (10×0.25). Stage VI spent phase during July (10×0.25).

Ovarian cyclicity : The histology of ovaries are period, it remained thicker. The germinal epithelium consist studied for six months. During the study, different stages of a single layer of cuboidal cells which posses very little of over are identified in the ovary. On the basis of that amount of cytoplasm. the maturity stages of ovary are differentiated which is The maturation cycle of Channa bleheri based on represented in Table 2. the histology of ovary and Gonado Somatic Index, was Histological observation of ovaries showed that the divided into viz: Mature or Pre-spawning Phase (March- different stages of ova in different quantity, which in turn April), Spawning Phase (May-June), Spent Phase (July), regulate the ovarian cycle. It is covered by an ovarian Resting phase (August) (Table 2, Plates- 6,7,8,9,10,11,12). wall and encloses a cavity ovocoel. During the spawning In the pre-spawning season, the ovary of Channa period, the tunica albuginea became thin but during other Histological study of gonads during breeding season 31

Plate 12 : Photomicrograph of T.S. of Ovary of Channa bleheri, Stage I resting phase during August (10×0.25).

Table 1 : Histological changes and testicular cycle of Channa bleheri. Month Histological changes observed Testicular cycle March Primary and secondary spermatocytes were seen. Pre-spawning April More number of primary and secondary spermatocytes were seen. May Testicular wall become thinner and thin lobular wall and full of spermatozoa were present. Spawning June Testicular wall become thinner and thin lobular wall and full of spermatozoa were present. July Empty and collapsing seminiferous tubules were seen some of which contain residual Spent or unexpelled sperm.

August Seminiferous tubules were small and full of spermatogonia. Resting Table 2 : Histological changes and ovarian cycle of Channa bleheri. } Month Histological changes observed } Ovarian cycle March Large number of ova in stage VII were seen Pre-spawning

April Large number of ova in stage VII and ripe eggs were seen. May Ovary was filled with ripe eggs along with ova in stage VII. Spawning June Ovary was filled with ripe eggs.

July Atretic eggs and discharged follicles along with stage I and II oocytes were seen. Spent August Immature oocytes in stage I and II were seen. Resting bleheri is categorized into two process as slow oogenesis Hatikakoti et al (2002) in Tilapia mossambica. From the and rapid oogenesis. During slow oogenesis, a large above study, it can be concluded that Channa bleheri number of stage-III and stage-IV oocytes were found. attained maturity only during June, which is considered The time required from stage-III to stage-IV was as breeding season with a single annual breeding season. comparatively more. However, in the case of rapid REFERENCES oogenesis, the stage-IV, stage-V and stage-VI were seen. Agarwal N K (1996) Fish Reproduction, APH Publishing Corporation, During, the oocytes of stage-VI and stage-VII were seen. New Delhi, 264. However, stage-VII was found comparatively more than Ali A B (1999) Aspects of reproductive biology of female snakehead stage-VI. During the peak breeding phase, a large number (Channa strita Bloch) obtained from irrigated rice agro ecosystem, of matured oocytes was seen in the ovary. Malaysia. Hydrobiologia 411, 71-77. Less numbers of unspawned ova were noticed in the Baker J R (1944) Quart. J. Micr. Sci. 85, i. spent phase and the ovary became flaccid; ovary. In the Dehadrai P V (1975) Derelict waters for air breathing fish culture. resting phase, the ovary became thin and the ovarian wall Indian Farming 2, 19- 20. became thick. This result can be corroborated with Gauraya S S, Toor H S and Kumar S (1997) Morphology of ovarian 32 Samarendra Behera et al changes during the reproductive cycle of the Cyprinus carpio and rearing of Anabas testudineus (Blotch). J. Dep. Sci. Calcutta communis (Linn). Zool. Beitr. 23, 405-437. Univ. 2, 101-140. Goswami M M, Borthakur A and Pathak J (2006) Comparative Musikasinthorn P (2000) Channa aurantimaculata, a new Channid biometry, habitat structure and distribution of four endemic fish from Assam (Brahmaputra River Basin), India, with snakehead (Teleostei : Channidae) species of Assam, India. J. designation of a neotype for C. amphilebus (McClelland, 1845). Inland Fish. Soc. India 38(1), 1-8. Icthyol. Res. 47, 27-37. Hatikakoti G and Biswas S P (2002) Studies on certain aspects of the Nagahama Y (1986) Testes. Vertebrate Endocrinology Fundaments reproductive biology of mouth-breeding tilapia, Oreochromics and biomedical implications (eds.Pang K T and Schreibmen M mossambicus (Peters) from Assam, India.Dibrugarh University, P), Academic Press, New York, I, pp. 399-426. Dibrugarh, Ph. D. Thesis, p. 130-133. Srivastava S J and Singh R (1992) Effects of constant photoperiod- Mukherjee H K and Mazumdar S R (1946) On the life history, breeding teperature regims on the testicular activity of Channa punctatus. Asian Fisheries Science 5, 231-240.