Ovarian maturation and histological observations of Barilius bendelisis (Hamilton) in captivity

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1 Indian J. Anim. Res., 52(5) 2018: Print ISSN: / Online ISSN: AGRICULTURAL RESEARCH COMMUNICATION CENTRE Ovarian maturation and histological observations of Barilius bendelisis (Hamilton) in captivity Neha Saxena, Rabindar Singh Patiyal 1, Kiran Dube* and Virendra K. Tiwari Central Institute of Fisheries Education, Versova, Mumbai , Maharashtra, India. Received: Accepted: DOI: /ijar.B-3281 ABSTRACT Fish samples of Indian hill trout Barilius bendelisis, were collected from the Gaula river ( N Latitude, E) and reared in captive condition at Directorate of Coldwater Fisheries Research, Bhimtal ( N, E, altitude 1370 m MSL).Gonadosomatic index was found maximum in May for females (13.24 ± 0.92) and males (1.13 ± 0.07) respectively indicating a single spawning period of this species under captivity. Absolute fecundity (total number of eggs) varied from 320 to The average absolute fecundity was 1661 ± at a total length ± 0.30 cm and body weight of ± 1.23 g. Histological study of the ovary revealed the presence of oocytes at different stages of maturity with a maximum number of the mature oocytes during April to May. Results of the present investigation suggest that B. bendelisis belongs to a category of low fecund fish and spawns during April and May under captive condition. Key words: Barilius bendelisis, Captivity, Fecundity, Gonadosomatic index, Maturity stages, Ovarian maturation. INTRODUCTION Indian hill trout, Barilius bendelisis belongs to the family Cyprinidae, subfamily Danioniae, characterized by the pointed head, compressed body, blue/black vertical bands on the lateral side of body and origin of dorsal fin inserted behind the mid of the body.it is distributed in Brahmaputra and Ganga drainages along the Himalayan foothills (Talwar and Jhingran, 1991).The fish plays a significant role in the capture fishery in several parts of the Himalayan region of Arunachal Pradesh, where Indian major carps and exotic carps cannot be raised successfully (Sahoo et al., 2009). B. bendelisis is a popular ornamental species among aquarists and also known for its food value in Dehradun, Uttar Pradesh (Grover, 1971). As per IUCN red list (2012) this fish has been categorized as least concern. However, the population of this species has suffered a drastic reduction in the recent past (Sah et al., 2011). Therefore, to overcome this problem, culture and propagation of B. bendelisis is needed to replace the stock. Sufficient knowledge about the reproductive parameters like gonadosomatic index, fecundity and gonadal histology is essential to understand the gonadal maturation and reproduction under controlled condition, so that the spawning induction can be performed (Mylonas et al., 2010). Although, assessment of reproductive biology in terms of sex ratio, GSI, fecundity, spawning period etc. in different water bodies of Garhwal Himalayas have been reported (Badola and Singh, 1984; Dobriyal and Singh, 1987), however,reproductive biology of B. bendelisis in captivity has not been attempted. Therefore, the present study is aimed to observe the reproductive biology of B. bendelisis in captivity. MATERIALS AND METHODS Fish collection and culture: Fish specimens examined during present study were collected from river Gaula, Kumaon region of central Indian Himalayas ( N, E) in November and December These fishes were transported in live condition to Directorate of Coldwater Fisheries Research (DCFR) Bhimtal ( N, E, altitude 1370 m MSL) and reared in a cement pond (10m x 3m x 1.00m) after proper disinfection.to simulate a semi-natural environment to the fishes, few medium sized boulders were kept at the pond bottom facilitating periphyton growth. The fishes were fed ablibitum with formulated pellets containing crude protein 33%, fat 7.1%, and carbohydrate 33%. Feeding was maintained as per temperature variation and feeding intensity of the fishes. The fish stock were fed minimum at lower temperature and gradually raised as per increase in ambient temperature. GonadoSomatic Index: A total of 200 samples were collected randomly during Janurary 2014 to November 2014 at monthly interval. All the fresh specimens were weighed to the nearest 0.1 g, they were dissected and gonads were taken out individually from male and female and weighed with digital weighing balance (CITIZEN CG2201) up to nearest GSI was 0.01g.calculated month wise for both the sexes *Corresponding author s & address: kirandr@cife.edu.in Division of Aquaculture, ICAR- Central Institute of Fisheries Education, Off Yari Road, Panch Marg, Versova, Mumbai , Maharashtra, India 1 Directorate of Coldwater Fisheries Research, Bhimtal , India

2 696 INDIAN JOURNAL OF ANIMAL RESEARCH using the formula of De Vlaming et al.(1982).gsi= (Gonads weight)/(total weight of fish ) X 100 Histology of gonads:the gonads of female were collected from the individual fish and fixed in Bouin s solution for the histological studies. Dehydration, clearing and paraffin embedding were done using standard protocol (Humeson, 1979). Wax embedded sections were cut at 5 µ thickness and stained with hematoxylin-eosin for histological observations. The specific areas in each slide of gonad were marked and subsequently photographed by light microscope (NIKON, ECLIPSE, E100) at 10X and 40X magnification. Fecundity: Altogether 25fishes were examined during the preferable breeding period (April 2014 to June 2014). A sample of 0.01g of the ovary was taken separately from anterior, middle and posterior portions. The total fecundity was calculated by multiplying the number of eggs present in the 0.01g ovary and the total weight of the ovary, which gave the total number of eggs. Data analysis: Statistical analysis were performed with SPSS 16.0 software. The difference between monthly GSI was analyzed through analysis of variance (ANOVA). To establish the relationship regression coefficients were calculated between fecundity and other body parameters such as total length, total body weight and gonadal weight. RESULTS AND DISCUSSION Various physico-chemical parameters of pond water were recorded round the year. The averages of different physico-chemical parameters are summarized in Table 1. No abrupt changes were observed in the water quality of fish pond during the study period. B. bendelisis is a heterosexual fish with external fertilization. It exhibits some degree of sexual dimorphism during the spawning period. The females were easier to identify, as belly of a mature female is larger, total length of pectoral fin is short, whereas, males remain streamlined with prominent tubercles on upper and lower jaw. Total length of pectoral fin is larger and goes beyond the origin of pelvic fin. Sexual dimorphic characters during the breeding period are shown in Figure 1. Similar results were obtained by Badolaand Singh (1984) and Dobriyaland Singh (1987) in B. bendelisis collected from wild condition of Garhwal Himalayas. Macroscopic description of ovaries: Female reproductive system of B. bendelisis consists of a pair of ovary and oviduct. The ovaries of B. bendelisis were long covering the whole length of the abdominal cavity and attached by mesovaria. Depending upon the maturity stages, the macroscopic stages of ovary were classified into four stages (Table 2). Microscopic description of ovaries: The ovary of B. bendelisis is surrounded by the tunica albuginea membrane comprising of oogonia and oocytes of various developmental stages in the follicles without any specific arrangement. The oocyte diameter increased both in terms of enlargement of nucleus as well as addition of cytoplasm. Based on the shape and structure of oocytes, five developmental stages of oocyte were observed in the ovary. Chromatin nucleolus oocyte stage: These are smallest, spherical oocyte found in the ovary of B. bendelisis. The oocytes remained strongly basophilic and were deeply stained purple with haematoxylin. Perinucleolus oocyte stage: These oocytes nucleus contained several small peripherial situated nucleoli. (Figure 2). This stage is further divided as early perinucleolus oocyte stage and late perinucleolus oocyte stage. In late perinucleolus stage, oocytes were more spherical in shape and cytoplasm was found to be less reactive to hematoxylin stain. At this stage, nucleus was observed in the center with two or more nucleolus initially at the center, moving towards the periphery as the development progressed. Although, this oocyte has been found in all the ovarian development stages, with higher percentage in developing phase. Table 1: Physico-chemical characteristics of pond water during January to December Months Temperature ph Dissolved Alkalinity Ammonia N Nitrite- N Nitrate- N ( o C) oxygen ( mg L -1 ) ( mg L -1 ) ( mg L -1 ) ( mg L -1 ) (mg L -1 ) January 9.9± ± ± ± ± ± ±0.01 February 11.2± ± ± ± ± ± ±0.01 March 17.4± ± ± ± ± ± ±0.02 April 21.2± ± ± ± ± ± ±0.01 May 25.3± ± ± ± ± ± ±0.02 June 25.2± ± ± ± ± ± ±0.02 July 24.9± ± ± ± ± ± ±0.01 August 21.4± ± ± ± ± ± ±0.01 September 20.6± ± ± ± ± ± ±0.02 October 20.8± ± ± ± ± ± ±0.01 November 17.5± ± ± ± ± ± ±0.01 December 10.3± ± ± ± ± ± ±0.02 Data presented as Mean±Standard Error

3 Volume 52 Issue 5 (May 2018) 697 (A) (B) Fig 1: Male and Female Identification characteristics of Barilius bendelisis during breeding period. A- Male having prominent tubercle; B- left Female, right male Table 2: Macroscopic and histological observations of ovaries of Barilius bendelisis ovaries at various stages during maturation. Stages Macroscopic appearance Histological Developing stage - Ovaries were pale in color Chromatin nucleolus oocytes and perinucleolus - Oocytes were small, visible by the naked eyes. oocytes predominate and a few yolk vesicle - Thick ovarian wall was observed. oocytes present Fully Developed (Vitellogenic ) - Ovary was yellowish in colour. Late perinucleolus oocytes and yolk vesicle stage - Bulky lobes with well-developed oocytes present in the ovaries. All stages of vascularisation were noticed. vitellogenesis were present. Spawning stage - Ovary turgid with oocytes and fills the Ovaries mostly composed of mature oocytes. entire abdominal cavity. Oocytes dark yellow Few vitellogenic oocytes were present. in colour, loosely attached to ovigerous lamellae. - Oocytes easily shed on application of slight pressure on the ovary. - Ovarian wall was very thin. Resting/ Spent Stage - Ovary flaccid with few vitellogenic oocytes Vitellogenic oocytes in advanced stages of visible and several small oocytes were noticed. atresia; primary growth and cortical alveoli oocytes were noticed.

4 698 INDIAN JOURNAL OF ANIMAL RESEARCH Fig 2: Transverse section of ovary of Barilius bendelisisshowing Perinucleolus oocyte stage (PVO). N-Nucleus, Nc- Nucleolus Fig 3: Micrographs of oocytes in the cortical alveolus stage from Barilius bendelisis. CA- Cortical alveolus; N-Nucleus; VE- Zona pellucida is present between the oocyte and its overlying follicular cells. Cortical alveoli stage: Cells in this stage are characterized by three components viz., cortical alveoli, zona pellucid and lipids (Selman and Wallace, 1989). The sizes of oocytes were enlarged due to deposition of fat and yolk. The ooplasm was less basophilic in nature and nucleus still occupied the central position. Initially cortical alveolus was found in two layers,one near to oocyte membrane and another near to nucleus. As the oocyte grows the cortical alveoli, increase in number as well as in size and fill the whole cytoplasm. The content of these yolk vesicles got dissolved during dehydration with different solvents used and appeared empty with staining (Figure 3). Vitellogenic oocytes stage: Cells in this stage are characterized by increased number of yolk vesicles, which were in ooplasm periphery at the beginning of the vitellogenic phaseand further occupied the entire central area of ooplasm. The vitelline membrane (zona pellucid) was thicker. The nucleus initially was located at the center of the oocyte and was irregular in shape.as the oocyte grew further, the nucleus moved toward the animal pole of the egg, and a few larger oil droplets were also seen (Figure 4). Mature oocyte stage:this stage is the final stage of oocyte maturation. The main characteristic feature of this stage is that nucleus totally migrated to the cell periphery and the ooplasm is characterized by the full of yolk globules (Figure 5). Histological examination showed the presence of oocytes of different maturity stages in ovary. Transformation of immature oocytes into developing and mature stage oocytes is associated with the accumulation of lipid globules. In the present study, resting phase of ovary is observed short with prolonged developing and vitellogenic stageunder captive condition. It may be ascribed to the failure of completion of vitellogenesis and oocyte maturation of few developing oocytes. Similar results of reproductive dysfunction were observed in several captive-reared females (Mylonas and Zohar, 2001; Lee and Yang, 2002; Yang and Chen, 2004; Agullerio et al., 2006). According to Mylonas et al.(2010) the failure of completion of vitellogenesis and oocyte maturation is due to the dysfunctional endocrine release of LH, which may be related to various environmental as well as social factors. In the present investigation, maturing and spawning stages were highly abundant during March to May, indicating preferable spawning season of fish. Gonado Somatic Index (GSI): The development and maturation of primordial germ cells of fish gonads is directly associated with the increase in weight of gonads, which is expressed as gonadosomatic index. GSI is a reliable indicative of fish spawning in temperate and tropical region (Bouain and Sian, 1983). In the present study, minimum GSI for female was observed in November (4.51 ± 0.60) whereas for male it was in July (0.01 ± 0.02).Whereas,maximum value for female and malewas observed in May (13.24 ± 0.92) and April (1.13 ± 0.07 ) respectively (Table 3), therefore,indicating a single spawning period of fishes under captivity. However, Dobriyal and Singh (1987) reported two peaks in the GSI value of B. bendelisis, one in March and another in September from natural conditions of Garhwal Himalayas. Variation in breeding season in different fish species have been reported by different researchers and even for the same species at different locations (Thomas et al.2003). Fecundity: The number of total eggs (absolute fecundity) varied from 320 to 4000 with an average number of 1661 ± for mean total length ± 0.30 cm and mean body weight ± 1.23 gm. Relative fecundity per gram of body weight varied from to eggs with an

5 Volume 52 Issue 5 (May 2018) 699 (A) Fig 4: Micrographs of Oocytes showing vitellogenic oocytes of Barilius bendelisis. (A Vitellogenic oocytes (VO) of Barilus bendelisis showing migratory Nuclueus (N) (B) The initiation of vitellogenesis in the appearance of yok globules (gl) lying in between the cortical alveoli (CA). (B) (A) Fig 5: Micrographs of oocytes showing the mature stage of oocyte (MO) of Barilius bendelisis. Follicular cell (F), Zona pellucida (Z) and Yolk globules (gl) Table 3: Gonado Somatic index (GSI) of Barilius bendelisis under captivity. Months Female Male January 5.96 cd ± bc ± 0.01 February 6.42 cd ± bc ± March 9.12 bc ± ab ± 0.01 April a ± a ± 0.07 May a ± a ± 0.01 June ab ± bc ± 0.04 July cd ± c ± 0.02 August 5.74 cd ± c ±0.01 September 6.71 cd ± c ± 0.05 October 5.92 cd ± c ± 0.07 November 4.51 cd ± c ± 0.8 Data presented as Mean ± Standard Error (n=10). Figures with same superscript letters in rows are not significantly different from each other at p>0.05. (B) average of ± ( ) eggs (Table 4). However, Dobriyal and Singh (1987) reported comparatively higherfecundity ( eggs) in B. bendelisis from Garhwal Himalayan region. Similarly, Gaur and Pathani (1996) reported the fecundity of B. vagra ranging from 800 in fish measuring from 65.0 mm in total length to 3400 in fish measuring from mm in total length. The breeding potential of Bariliusvagra was estimated by Bahuguna et al. (2009) from Garhwal region ranging from 510 to 7214 eggs in the fish measuring 55 mm to 89 mm in total length and 407 mg to 4,260 mgovarian weight. In the present study low fecundity was observed in fishes reared in pond condition as compared to fishes under wild condition. This can be ascribed due to environmental factors like temperature and food

6 700 INDIAN JOURNAL OF ANIMAL RESEARCH Table 4 : Fecundity of Barilius bendelis is under captivity. Month Average Average Average Absolute Fecundity Fecundity Fecundity length of weight of ovary Fecundity per gram of per cm of per gram of fish (cm) fish (gm) weight (gm) bodyweight body length ovary weight April ± ± ± ± ± ± ± 143 ( ) ( ) ( ) ( ) ( ) ( ) ( ) May 10.1 ± ± ± ± ± ± ± 219 ( ) ( ) ( ) ( ) ( ) ( ) ( ) June 9.81 ± ± ± ± ± ± ± ( ) ( ) ( ) ( ) ( ) ( ) ( ) Total ± ± ± ± ± ± ±99.03 ( ) ( ) ( ) ( ) ( ) ( ) ( ) Data presented as Mean ± Standard Error (n=4). Figures with same superscript letters in rows are not significantly different from each other at p>0.05 availability. Doha and Hye (1970) reported that the change in fecundity among two populations is common and also concluded that the fecundity of fish by an individual female is dependent upon various factors like age, size, food availability, space, climatic condition, season, environmental factors, habitat, nutritional status and genetic potential. Relationship between fecundity and total length :The linear relationship between fecundity and total length of fish showed a positive correlation (r= 0.817) (figure 6). The regression equation of fecundity and total length of fish is expressed as Log 10 F = Log 10 TL (R 2 = ) Relationship between fecundity and body weight: The linear relationship between fecundity and total length of fish showed a positive correlation (r= 0.880) (figure 7). The regression equation of fecundity and total length of fish is expressed as Log 10 F = Log 10 TL (R 2 = ). Relationship between fecundity and ovary weight: The linear relationship between fecundity and total length of fish showed a positive correlation (r= 0.904) (figure 8). The regression equation of fecundity and total length of fish is expressed as Log 10 F = Log 10 TL (R 2 = ). Fig 7: Relationship between fecundity and body weight of Barilius bendelisisunder captivity Fig 6: Relationship between fecundity and total length of Barilius bendelisisunder captivity Fig 8: Relationship between fecundity and ovary weight of Barilius bendelisisunder captivity

7 In the present investigation it has been observed that the number of eggs increased linearly with the increase in body length, body weight and ovary weight of fish. There appeared to be greater relationship between fecundity and ovary weight of fish than the fish total length and body weight because the correlation coefficient is higher in ovary weight and fecundity (R2= ) than fish length and body weight. Therefore ovary weight may be better index of fecundity in comparison to other body parameters. Similar relationships between body measurements and fecundity have been reported in T. putitora (Pathani, 1981), B. bendelisis (Dobriyal and Singh, 1987), B. vagra (Gaur and Pathani, 1996; Gandotra et al., 2008), Botiaalmorhae (Joshi, 2007) and Botiadayi (Kumar et al., 2006). Volume 52 Issue 5 (May 2018) 701 Results showed that B. bendelisis has responded to one breeding season for a short period during April- May in captive condition, as indicated by of large number of ripe fish and ripening individuals with advanced stages of oocytes in the ovary, appearance of spent individuals, presence of mature ova and the high GSI. Further present study also provides the baseline information on the biological aspect of this fish for commercial production and development of artificial propagation techniques under controlled conditions. ACKNOWLEDGEMENT We sincerely, thank Dr. W.S. Lakra, Director, CIFE, Mumbai and Dr. A. K. Singh, Director DCFR Bhimtal, for providing necessary facilities. Department of Science and Technology is greatly acknowledged for providing INSPIRE fellowship to Neha Saxena to carry out the research work. REFERENCES Agullerio, M.J., Anguis, V., Cañavate, J.P., Martínez-Rodríguez, G., Mylonas, C.C. andcerdá, J. (2006). 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