Reproductive Biology of Punti, Punius sophore in Rajdhala Beel, Netrakona, Bangladesh

REPRODUCTIVE BIOLOGY OF PUNTI, PUNTIUS SOPHORE IN RAJHDHALA BEEL, NETRAKONA MS Thesis MD. ASHFAQ SADAT Department of Fisheries Management Bangladesh Agricultural University Mymensingh December (2021) (Examination held in November 2022) REPRODUCTIVE BIOLOGY OF PUNTI, PUNTIUS SOPHORE IN RAJHDHALA BEEL, NETRAKONA A Thesis Submitted to Bangladesh Agricultural University, Mymensingh In Partial Fulfilment of the Requirements for the Degree of Master of Science in Fisheries Management by MD. ASHFAQ SADAT Roll No.: 19FMJD-01M Registration No.: 43747; Session: 2014-15 Department of Fisheries Management Bangladesh Agricultural University Mymensingh December (2021) (Examination held in November 2022) REPRODUCTIVE BIOLOGY OF PUNTI, PUNTIUS SOPHORE IN RAJHDHALA BEEL, NETRAKONA A Thesis Submitted to Bangladesh Agricultural University, Mymensingh In Partial Fulfilment of the Requirements for the Degree of Master of Science in Fisheries Management Approved as to style and contents by Prof. Dr. Md. Mahfuzul Haque Supervisor Prof. Dr. Md. Shahjahan Co-supervisor Prof. Dr. Zoarder Faruque Ahmed Chairman, Defence Committee and Head, Department of Fisheries Management Bangladesh Agricultural University Mymensingh December (2021) (Examination held in November 2022) iv ACKNOWLEDGEMENTS Alhamdulillah, all praises are due to the Almighty Allah Rabbul Al-Amin for His gracious kindness and infinite mercy in all the endeavours the author to let him successfully complete the research work and the thesis leading to Master of Science. The author would like to express his heartfelt gratitude to his research Supervisor, Dr. Md. Mahfuzul Haque, Professor, Department of Fisheries Management, Bangladesh Agricultural University for his constant supervision, valuable suggestions, scholastic guidance, continuous inspiration, constructive comments, extending generous help and encouragements during his research work and guidance in the preparation of the thesis. The author also expresses his heartfelt gratitude to his mentor Dr. Saleha Khan, Professor, Department of Fisheries Management, Bangladesh Agricultural University for her continuous inspiration, advice, and good words. The author sincerely expresses his heartiest respect, deepest sense of gratitude and profound appreciation to his co-supervisor Prof. Dr. Md. Shahjahan, Department of Fisheries Management, and PI, Fish Ecophysiology Laboratory, Bangladesh Agricultural University for his constant encouragement, affable suggestions, helpful criticisms and valuable advice during the research period and preparing the thesis. The author would like to express his deepest respect and boundless gratitude to the head of the department Prof. Dr. Zoarder Faruque Ahmed and all the respected teachers of the Department of Fisheries Management, Bangladesh Agricultural University for their valuable teaching, sympathetic cooperation, and inspiration throughout the course of this study and research work. It is a great pleasure for the author to express his profound gratitude and deepest appreciation to his beloved parents for their sacrifices, never-ending prayers and dedicated efforts to educate the author to this level. The author is also grateful to his wife Mosrafa Nasrin Suma for her continuous inspiration and support throughout this journey. The author is acknowledging the sacrifices, encouragement, inspiration and blessing of his sisters throughout the whole period of the study. Finally, the author appreciates the assistance rendered by the staff members of the Fish Ecophysiology Laboratory and the Department of Fisheries Management. The Author v REPRODUCTIVE BIOLOGY OF PUNTI, PUNTIUS SOPHORE IN RAJHDHALA BEEL, NETRAKONA 19FMJD-01M ABSTRACT To identify the breeding season of Pool barb, Puntius sophore through observation of the gonadosomatic index (GSI), fecundity, oocytes diameter, and gonadal histology of the species and to suggest management recommendations, the reproductive biology was studied with individuals of the population from Rajdhala beel, Netrakona from July 2020 to June 2021. The fish specimens were collected monthly using a seine net. Three methods were employed to determine the spawning season. It is worth mentioning that, the monthly mean gonadosomatic index was found highest in June. Since the mean gonadosomatic index was highest in June, therefore it was opined that the spawning took place in June apparently. Various developmental stages of ovaries were classified considering their external features and the macroscopic views of eggs. The stages were immature, developing, maturing, and spawning. External morphology and macroscopic observation of female gonads showed that the spawning season of pool barb was between March and June. The microscopic examinations of gonad histology detected the presence of five stages of oocyte maturity which were early perinucleolus, yolk vesicle stage, primary yolk stage, migratory nucleus stage, and premature stage. Monthly plotting of percent data of histological stages revealed that the most advanced stage detected in this study occurred in April, May, and June. Based on histological investigation it was eventually concluded that the spawning season of P. sophore existed from April to June. Postovulatory follicles and hydrated eggs were absent in the mature ovaries over the study period which gave the support that P. sophore was a synchronous single spawner. The study also demonstrated that the standard length of the youngest female that bore mature eggs was 3056 mm, and its gonadosomatic index was 7.48. The range of relative fecundity was from 540 to 4352 per gm of mature females in terms of standard length having a range from 56 mm to 92 mm. Standard length and fecundity yielded a cubic relationship, the equation was F= 0.0232SL2.6877 (r2 = 0.8683). The relationship between fecundity and body weight was in the form of a straight line, and the estimated equation was F = 213.44BW + 125.24 (r2 = 0.892). High values of coefficients of determination in both cases described strong relationships between predictor and response variables in fecundity estimation. vi CONTENTS CHAPTER TITLE PAGE NO ACKNOWLEDGEMENTS IV ABSTRACT V CONTENTS VI LIST OF TABLES VIII LIST OF FIGURES IX LIST OF APPENDICES X ABBREVIATIONS XI INTRODUCTION 1 REVIEW OF LITERATURE 8 Breeding biology of Puntius spp. 8 Breeding biology of some other fishes 13 MATERIALS AND METHODS 18 Sampling site 18 Rajdhala Beel, Purbadhala, Netrakona. 18 Laboratory works 19 Collection of gonad and sex determination 19 Estimation of GSI (Gonadosomatic Index) 20 Observation of ovarian external features 20 Histological examination 21 Length at sexual maturity 22 Estimation of fecundity (F) 22 Gonad histology 23 Data analysis 26 RESULTS 27 Fish size 27 Standard length of fishes 27 Body weight of fishes 28 Determination of spawning season 30 Gonadosomatic index (GSI) method 30 External feature of ovary 32 Histology of ovary 34 Frequency of spawning and measurement of egg diameter 37 Minimum length at maturity 38 Fecundity 39 vii CHAPTER TITLE PAGE NO Relationship between fecundity and standard length 39 Relation between fecundity and body weight 40 DISCUSSION 42 SUMMARY AND CONCLUSION 47 REFERENCES 49 APPENDICES 57 viii LIST OF TABLES SL NO TITLE PAGE NO Table 1 Time schedule in the automatic tissue processor 24 Table 2 Staining procedure for histological study of gonad 25 Table 3 Standard length (mm) of monthly collected specimens (July 2019-June 2021) 27 Table 4 Body weight (g) of monthly samples specimens (July 2019-June 2021) 29 Table 5 Month-wise gonadosomatic index of P. sophore (July 2019-June 2021) 31 Table 6 Monthly fecundity of P. sophore 39 ix LIST OF FIGURES SL. NO TITLE PAGE NO Figure 1 Puntius sophore 6 Figure 2 A view of Rajdhala beel 18 Figure 3 Map showing the study area 19 Figure 4 A: An adult female of Puntius sophore with isolated matured gonad; B: Taking body weight and gonad weight of samples in laboratory 20 Figure 5 Flow chart showing histology processes of gonad 26 Figure 6 Frequency distributions of standard length of P. sophore 28 Figure 7 Frequency distributions of body weight of P. sophore 29 Figure 8 Frequency distributions of gonadosomatic index of P. sophore 30 Figure 9 Monthly changes of gonadosomatic index of P. sophore (July 2019-June 2021) 32 Figure 10 Matured ovary of Puntius sophore 33 Figure 11 Monthly changes in the frequency of the occurrence of ovarian maturity 34 Figure 12 Maturity stages from histology of P. shopore's gonad 35 Figure 13 Frequency occurrence of maturity stages of oocytes 37 Figure 14 Size frequency distribution of oocytes of P. sophore 38 Figure 15 Relationship between gonadosomatic index (GSI) and standard length of female having mature gonad. Yellow dot indicates the GSI of youngest adult female having 56 mm SL in this study 38 Figure 16 The relationship between fecundity and standard length of P. sophore during spawning season 40 Figure 17 The relationship between fecundity and body weight of P. sophore during spawning season 41 Figure 18 Size frequency distribution of oocytes of P. sophore. Fecundity was counted with the eggs right to the arrow 41 5 possible alternative states, but the life history of a given species consists of only one of these states for each life-history period. Since each fish species evolves under a unique set of ecological conditions, it has a unique reproductive strategy with special adaptations including anatomical adaptations, developmental adaptations, behavioural adaptations, physiological adaptations, and energetic adaptations. The reproductive process allows species to perpetuate themselves. Almost all fishes reproduce sexually, thus permitting mixing of the genes of the two sexes. The reproductive processes of fishes form the basis for early life history studies. The great variety of these processes among fishes make their study worthwhile, but also determine how early life-history studies of various fishes can be conducted. For example, fishes reproduce in fresh and marine waters, have external and internal fertilization, have short annual reproductive periods, or produce gametes at regular intervals throughout the year. This study summarizes the reproductive patterns of fishes, with an emphasis on how these patterns affect early life-history studies. In life history of a fish species two major events are very important. One is reproduction another is recruitment. In fisheries studies, the particular event of interest in the reproductive cycle of fish species is time of spawning, when fully developed gametes are released. Study about reproductive biology of any fish is essential for evaluating the condition of its stock, life history, culture practice and actual management of its fishery (Lagler, 1956; Doha and Hye, 1970). Potentiality of reproduction of a population is one of the basic exigencies to designate the individuals of that population in respect to their gonadal conditions (Jhingran and Verma, 1972). It is important to assess the yearly breeding cycle of a fish species for successful fish culture. Spawning of fish occurs during a particular phase of reproductive cycle. Some of them breed once annually while some at regular intervals throughout the year. Knowledge of gonadal development and the spawning season of a species allow subsequent studies on spawning frequency of its population, which is important for its management. Another identification of the breeding activity of an exploited fish species provides management option for protection of its spawning stocks (Laroche and Richardson, 1988). Reproductive 6 parameters do not provide direct evidence of stock structure, but do provide information to assist in understanding biological processes that may be responsible for maintaining the underlying stock structure of a species. The most important aspect of reproduction is fecundity estimation. Reproductive studies have been important component of the biological basis of management for fish and fisheries. This study is undertaken to determine the spawning season, to estimate fecundity, the size at sexual maturity, age and pattern of egg release of punti fish. The fish species, Puntius sophore, one of the Cyprinid fishes under the family Cyprinidae are mostly available in ponds, small streams, beels, inundated fields and weedy ditches (Talwar and Jhingran, 1991a; Rahman, 1989, 2005) and largely distributed in Asia: Pakistan, India, Nepal, Bangladesh, Myanmar and Yunnan, China (Talwar and Jhingran, 1991b), Bhutan and Afghanistan (Petr, 1999). Moreover, they also remain in domestic aquaria and become mature at 7 to 8 cm (Talwar and Jhingran, 1991a). The English name of this fish species is pool barb and is locally known as Jati punti in Bangladesh. This is one of the most common, small-sized fishes caught in large quantities from various freshwater habitats. Morphologically, the body of Punti is elongated and compressed laterally with pointed head. Lower jaw is longer. The number of scales on the lateral line series are 20 to 25. The number of scales above the lateral line is 8 to 10. Scales number below the lateral line is 3 to 4. Scales number on the lateral line series mentioned by other writers are: 24 - 26 scales (Rahman, 2005), 22-27 scales (Talwar and Jhingran, 1991a). Figure 1 Puntius sophore 7 This small fish is well regarded as a quality food containing high amount of VitA, protein, zinc, calcium and mineral etc. (Bogard et al., 2015). In spite of having enormous economic and nutritional importance of this species to the rural and urban people as well as the fishing community, adequate studies on their reproductive biology, namely spawning season, minimum length at maturity and fecundity of this species are needed for appropriate management. Reproductive biology is an important parameter to determine the quality and quantity of fish stock in a natural waterbody as well as to determine the reproductive potential (King, 2013). Successful reproduction and recruitment ensure the sustainability of a fish stock in a natural waterbody. As punti is self-recruiting species in Rajdhala beel, a biological study can help to make decisions for maintaining its sustainability under the conditions of climatic change and natural productivity (Rahman and Haque, 2008). It is very important to know the effects of environmental variabilities and natural productivity on reproductive biology of this fish. Knowing the biology, the findings of the current study will be helpful in developing good management strategies as well as getting mitigation measures to increase the productivity of punti in order to maintain optimum stock and sustainable higher production benefiting the poor fisheries stakeholders around the area to a greater extent. In view of the above facts, the present study was undertaken on the reproductive biology of Pool barb (P. sophore) with the following objectives: 1. To determine the monthly gonadosomatic indices of the species 2. To determine the fecundity of the species 3. To estimate the spawning season of the species 8 REVIEW OF LITERATURE Before conducting any experiment, it is important to analyse previous research work relevant to the intended subject. The reproductive biology of punti is not well documented, but there is a few research on certain elements of the biology of several small fishes. The mentioned literature has limitations because there are few connections between it and the current study's scope and objectives. However, the suggested work's associated literature is examined. Despite the abundance of research on spawning frequency, breeding season, and fecundity of several fishes, knowledge on the reproductive biology of Puntius sophore is scarce. The following information on the spawning frequencies, breeding season, and fecundity of related fishes that has been gathered from previous studies up to the present: Breeding biology of Puntius spp. According to Bhuiyan (1964), during the monsoon season, Puntius sophore reproduces. Male fish grow a bright red band along the middle of both flanks during the breeding season. In female fish, this is less obvious. Shafi and Quddus (1974a) observed that punti, Puntius stigma, with a short life cycle, lived only for a year or two and attained maturity very early. The sex ratio indicated that 60% of the fish were males and males were more numerous at lower lengthgroups (4-7 cm) and females in the higher group (8 cm and above). In another study, Shafi and Quddus (1974b) studied the fecundity of common punti, Puntius stigma, from Bangladesh waters and noted that the fecundity varied from 1,242 to 6,831. The relationship between fecundity and gonad weight was more significant than that of fecundity with other factors. Mustafa (1991) observed the fecundity and spawning frequency of Puntius chola in pond condition and reported that the fecundity of Puntius chola was 1,404 to 1,872 with an average of 1,581 ± 142. He also observed that the species had at least two distinct group of eggs in the ovary two breeding seasons. 9 In another study, Sobhana and Nadir (1974) observed the breeding season of Punlius sarana in Veli lake, Trivandrum extended from late May to November. Peak spawning occurred in July and October. Gonadosomatic index of both sexes showed the highest value in June and September and the fish matured at 183 mm in total length. The ova diameter frequency inferred that these fishes could breed twice a year (May and September). The maturity and fecundity of Puntius sarana were also studied by Sinha (1975) at Loni Reservoir, India. He reported the presence of a single batch of mature eggs with a mean model size of 99 mm. The cycle of maturation and coefficient of maturation indicated the spawning season of this fish was July-August. Fecundity of the species has been estimated to range from 58.327 to 139.984 with a total length of 230 to 326 mm. The fecundity of Puntius sarana with a length of 155 to 250 mm was found to range from 18,925 to 78,929. The mathematical relationships between fecundity and total length, fecundity and body weight, and fecundity and ovary weight were linear, whereas a curvilinear relationship was noted between fecundity and the standard length of the fish. According to Srivastava (2003), maximum value of gonadosomatic index was 1.849 of male and 12.075 for female in the month of July. The length at maturity was measured as 58 mm for the male and 66 mm for the female for Puntius sophore, which was discovered to be a single spawner in a year. Fish length, fish weight, ovary length, and ovary weight were found to have linear relationships with fecundity. Fecundity was significantly correlated with ovarian weight (r2 = 0.95479), fish length (r2 = 0.91047), and fish length (r2 = 0.88959) but not with ovary weight (r2 = 0.73668) or fish length (r2 = 0.88959). This demonstrates that ovarian and fish length and weight both affect fertility. Mitra et al. (2005) investigated length weight relation, reproductive characters and condition of Puntius sophore (Hamilton) from a floodplain wetland in West Bengal. This study found that while males considerably deviated from cube law, females followed it. The females matured when they were 6l-65 mm in length. The gender ratio was slightly in women's favour (av. 1:1.2). The fish had a fecundity of between 759 (85 mm and 7.66g size) and 29.650 (113 mm and 25.25g size). The diameter of 10 the ova varied from 0.l6 to 0.86 mm. The fish's spawning season lasted from February to August. Marina (2006) studied the reproductive biology and Puntius sophore of a perennial water body in Bangladesh. She suggested that the spawning seasons of Puntius sophore were started from April and continued up to September. Puntius sophore was a single spawned and its spawning frequency or strategy was synchronous type. The minimum standard length (SL) at maturity was 51 mm, and the mean SL at first reproduction estimated from the Logistic model was 66.5 mm for females. Fecundity estimates were 3494 to 31723 eggs from females of 23 to 91 mm standards length. The relations between fecundity (F) and standard length (SL), and between fecundity and body weight (BW) were estimated as F = 0.00109SL. (r2 = 0.708) and F = 1105.645BW - 1709.041 (r2 = 0.948) respectively. Tareque et al. (2008), studied the aspects of biology of Puntius sophore sampled from Mouri river, Khulna, Bangladesh. The mean weight of the bilobed gonads (WG) was 1.36 0.438 g. At the anterior, central, and posterior portions of both lobes, the mean ova diameter was 6.50.51 mm, 6.50.68 mm, and 6.310.56 mm, respectively. Fecundity ranged from 743 to 4013. (LT: 7.0-9.9 cm, W: 5.3-14.3 g). Positive but shaky connections existed between LT-F and W-F. The WG-F link was extremely solid. In August, the GSI was 15.25 ± 3.80. Mannan et al. (2010) observed the gonad development and reproductive cycle of Puntius filamentosus and observed that the spawning month of November and April coincided with North-East Monsoon (November) and South-West Monsoon (April) in India. The gross morphology of P. filamentosus showed 6 ovarian stages. The histological appearance of the ovaries showed ten stages (oogonia to atretic oocytes). The testes of P. filamentosus showed five morphological stages each. The progression of gonadal cycle of P. filamentosus collectively revealed that the maturation of males coincided with the maturation of females during the spawning period. Bithy et al. (2013) estimated the fecundity of Jat punti (Puntius sophore) from the experimental ponds of the Field Laboratory Complex, Faculty of Fisheries, 11 Bangladesh Agricultural University (BAU), Mymensingh. The estimated fecundity for the three fish groups was 7951 to 17670; 8135 to 23053; and 12461 to 15105 eggs, with a mean gonadosomatic index (GSI) of 23.44 2.50; 30.47 4.52; and 28.43 2.99, respectively. The association between fecundity and total length was linear, with Ln F= 1634 ln TL-1972, Ln F= 119405 ln TL-273.53, and Ln F= 1831.6 ln TL+ 825.31, of three groups respectively. The linear regression equation between body weight and total length was Ln W= 2.2128 ln TL0.7048, Ln W= 2.0207 ln TL+ 0.3493, and Ln W= 1.4438 ln TL+ 5.5694, respectively, in 3 group. The linear regression equation between body weight and fecundity was F= 656.84 W+199.96, F= 438.16 W+2470, and F= 1116.1 W-3433.5, respectively. The total length and body weight were closely linked with fecundity, suggesting that P. sophore was a moderately fecund fish. Using direct observation of sexual features (e.g., gonadosomatic index) and the indirect method of relative growth, Ahamed et al. (2015) determined the size of the female Puntius sophore at sexual maturity in the Old Brahmaputra River, North Eastern Bangladesh. Based on the link between standard length and gonadosomatic index, female P. sophore was determined to be 4.2 cm standard length at sexual maturity. Relative growth of fork length to standard length, on the other hand, indicated a changeover point at 4 cm standard length, indicating female P. sophore size at sexual maturity. According to Hossain et al. (2012) in the Padma River, Puntius sophore female size at first maturity was calculated to be 5.00 cm TL. Fecundity was calculated using specimens greater than 5.00 cm TL. Between 1580 to 16590, the mean total fecundity ranged from 5300 to 2700. Total length and total fecundity were found to be positively exponentially correlated (r2 = 0.421). In the Padma River, relative fecundity varied from 466 to 4036 (mean 1100 580). Langer et al. (2013) identified the breeding period of Puntius sophore is from JulyAugust. Puntius sophore was found to contain oocytes in seven stages. Throughout the entire reproductive cycle, oogonia were present. 12 Fecundity of Puntius sophore was estimated by Kant et al. (2016) to be between 1560 and 2314 eggs, with a mean total length of 5.5 cm and a mean body weight of 5.47 gm. The absolute fecundity ranged from 6106.73 to 6942.35 eggs, whereas the relative fecundity varied from 390.85 to 480.18 eggs per fish. Absolute fecundity has a strong (r2=0.809) correlation with ovarian weight but a weak (r2=0.047) and moderate (r2=0.216) correlation with total length and body weight. Therefore, it would seem that ovarian weight had a greater influence on fertility than did bodyrelated factors. A straight-line link between relative and absolute fecundity and body parameters has been found during the current investigations. Choudhury et al. (2015), studied the reproductive features of Puntius sophore from the rivers of Tripura, India. Estimated breeding season was July-August. Fecundity was estimated during the peak spawning season (July) and the relative fecundity observed was 4073±310 g-1 body weight. The average GSI recorded in male and female populations was highest during July month (14.22±3.9 and 20.88±4.55% respectively) Hasan et al. (2018) identified the breeding season of Pool barb through observation of the gonadosomatic index (GSI), fecundity, oocytes diameter, and gonadal histology of the species. This study compared two populations of Pool barbs (one from Gazipur and the other from Jessore). The highest GSI value was 15.43 2.20 in April at Gazipur, while it was 15.60 1.74 in June at Jessore. The maximum fertility (5053 878.27) was observed in Gazipur in April, while the highest fecundity (5433 968.26) was recorded in Jessore in June. During March, histology of Pool barb's ovary revealed the presence of early and late peri nucleolar stage oocytes, indicating immature oocytes. Pool barb spawning season runs from March to July, with a peak in April to May for the Gazipur region and May to July for the Jessore region, according to histological data. Finally, these findings suggest that breeding season varies from region to region due to environmental and other variables. 13 Breeding biology of some other fishes Mookherjee and Basu (1946) who studied the life history of Amblypharyngodon mola was reported that the breeding season of the fish lasted from May to October and a single female can lay 500 eggs at a time. Qayyum and Qasim (1964) studied the biology of freshwater fishes including Barbus stigma from Indian waters and found the ovaries of this fish contained only once during the breeding season. Karamchandani et al. (1967) studied the fecundity of Tor tor in India and estimated 43,410 numbers of eggs in a specimen of 750 mm length and also estimated 30,420 eggs from a specimen of 625 mm total length. He reported that the fecundity of T. tor would increase with the increase of body length. Rastogi and Saxena (1968) studied annual changes in the ovarian activity of the cat fish Mystus tengara and found nine stages of development showing peak maturation from April to August. Karim and Hossain (1972) studied the biology of Mastacembelus pancalus (Spiny Eel, Hamilton) in artificial ponds. They found the fecundity vary from 1,296 to 3,246 with the mean 2,013. The correlation coefficient of the total length of M. pancalus was r=0.93. A straightline regression (Ye) of fecundity (Y) on weight（X) of ovary of M. pancalus was calculated to be Ye = 439.19+1.786 X. Dewan (1973) observed the spawning season of mola to last from April to November in the lake of Bangladesh Agricultural University Campus. The peak period of spawning occurred in August and September. The fecundity of fish ranged from 1,021 to 13,815. The number of eggs increased with the increase in length of the fish. Malhotra et al. (1978) studied ovarian cycle and spawning season of Ophiocephalus punctatus, inhabiting Jammu waters, India and reported six well defined stages of maturation and the fish had a prolonged spawning season extending from May to August. 14 According to Prasad et al. (1979) the male and female Macrognathus aculeatus become sexually mature at length of 15.5 cm (about 15 gm) and nearly 18 cm (22 gm) respectively. The fecundity estimated by them was ranging from 210 to 1828. De-silva and Chandrasoma (1980) studied the reproductive biology of Sarotherodon mossambicus, an introduced species, in an ancient a man-made lake in Sri Lanka. The reproductive biology of Sarotherodon mossambicus (Peters), a species exotic to Sri Lanka, was established in Parakrama Amudra an ancient man-made lake. Males mature at a length of 27.5 cm and females at a length less than 15 cm. S. mossambicus breeds throughout the year with four possible peak periods, which coincide with the tail end of the monsoon and inter monsoon rains. The egg diameter distribution indicates the presence of reserve oocytes and yolked oocytes, the later falling into a single mode between 1.2 to 3.6 mm. Fecundity varied between 360 and 1775 for fish ranging in length from 22 to 31.9 cm and 145 to 538 g in weight. Mustafa et al. (1980) studied on food, feeding habit and fecundity of freshwater Perch Nandus nandus (Hamilton), meni fish (Bangladesh). The fecundity ranged between 7,381 eggs for a fish with body length of 9.7 cm and 46,222 eggs for a fish with body length of 13.5 cm. Breeding season ranged from June to July. Mathematical relationship between body length-fecundity and weight-fecundity were found to be linear. The fecundity Colisa fasciata was studied by Banu et al. (1984), who mentioned that this fish spawned only at a particular time of the year, i.e., from March to April. The gravid female of this fish contained mature eggs, which ranged from 5,123 to 13,450. Relationship between fecundity and gonad weight was more significant than that between fecundity with others factors. Mustafa (1991) observed the fecundity and spawning frequency of Kholisa in pond condition and observed that the fecundity of Kholisa was 1,578 to 2,684 with an average of 2,175±431. He stated that the species at least two distinct group of eggs in the ovary indicating two breeding seasons. Kabir et al. (1998) determined the fecundity and gonadosomatic index of chapila, Gudusia chapra and observed that the male fish attained sexual maturity at 7.7 cm 21 Histological examination Conventional histological processing began with preservation of ovaries. Preserved ovaries were trimmed and oriented, dehydrated, infiltrated with paraffin, and formed into blocks of tissue surrounded by supporting medium. These blocks were then sectioned and slices were mounted on glass slides and stained. Once a cover slip was added, the sections were viewed microscopically. Punti ovaries were used for histological examination after the following procedure: 1. A transverse segment of about 4 mm thick from the middle part of left ovary was taken. 2. Dehydration of ovarian segment was achieved by the passage of tissue piece through a series of ethyl alcohol solutions in the room temperature according to the following schedule in which a repeated step meant a change of medium. 3. Impregnation of tissue with benzene, a solvent of paraffin was done twice successively for 1 hour each to remove traces of alcohol in order to have consistent paraffin block. 4. Infiltration by melted paraffin at 600 °C was done twice consecutively for a period of 40 minutes each. 5. Following dehydration, cleaning and infiltration by melted paraffin, ovarian segment was placed in a clay mold with melted paraffin for embedment. Care was given to ensure orientation of tissue within the result block, which was allowed to cool within the mold. 6. The paraffin block was cut to serial sections with a thickness of 5 μm each using a microtome and the sections were mounted on clean and adhesive coated slide, the slide was placed in rack within an oven and heated overnight at 200 °C. 7. The slides mounted with gonadal sections were stained by haematoxylin and eosin. 8. Finally, Canada balsam, a mounting medium was applied and cover slip was lowered into place, completely covering the sections. 22 9. The sections were examined under light microscope (x 200). At least three sections were examined for recording the occurrence of various histological stages of ovary. Length at sexual maturity Female classified into various spawning stages by ovarian histology were used to determine minimum length at sexual maturity. For the determination of minimum length at maturity, standard length and GSI values of all female specimens were plotted and female with the lowest standard length having final spawning stage was considered capable of spawning and its standard length was the minimum standard length at maturity of the population. Estimation of fecundity (F) Fecundity was estimated using the volumetric method (Diaz et al., 1983). Weight of ovaries were taken using the electronic balance. Portions of ovaries weighed and placed in Gilson’s fluid and shaken periodically to release the oocytes. After suspending the oocytes in a volume of (1-1.5 L) water subsamples were taken. In each subsample, oocytes were counted at 50x magnification. The reliability of subsamples tested through analysis of variance (ANOVA) test. The total number of oocytes in the ovaries will be estimated using the following formula: N= (V V1)n × (W W1) Where, V= Volume of sample, V1= Volume of subsample, W= Weight of ovary, W1= Weight of portion of ovary, n is the number of oocytes in the sub-sample. There are several methods for the estimation of fecundity of fish of which are actual counting method was found to be the most accurate one. But this method is very tedious and time consuming and to certain extent rather impossible in case of high fecund fishes. When the actual counting of eggs is impracticable, approximate fecundity may be obtained by one of the following methods as outlined by (Lagler, 1952). a) Volumetric method b) Gravimetric method c) Von Vayer method 23 Volumetric method and Von vayer method have been found to be suitable for relatively large eggs. But the eggs of P. sophore are comparatively smaller and which may lead to error in the estimation of fecundity by Von vayer method and volumetric method. Gravimetric method was found to be more efficient than those of other methods and gave fairly accurate results. The gravimetric or weight method has been successfully used by Doha and Hye (1970), Shafi and Quddus (1974) etc. Therefore, gravimetric method was thought, may offer the best possibility of minimizing error due to its simple and easy sampling technique. Fecundity is defined as the number of oocytes shed by a female in a spawning season was estimated gravimetrically. In this method, prior to estimation, oocytes from samples (0.0001 g) obtained from three portions (anterior, middle and posterior) of five ovarian lobes randomly were counted and measured to determine whether they had significance differences between locations. Results showed that oocytes were uniformly distributed, and in the succeeding analysis, sample was taken from the middle portion. A section of about 4 mm long was cut from middle part of the right ovary, weighted to nearest 0.0001 g (gw) after removal of formalin from the ovary surface with tissue paper, and put into a Petri dish with a small amount of water. All the eggs were separated from each other with needles and measured along their longest axis under stereomicroscope. A frequency distribution of egg size by 1 mμ interval was constructed, the number of eggs in the largest modal group, b, was summed. Fecundity, F was calculated using the following formula: F= B × GW gw Where, F = Fecundity of fish, GW = Gonad weight, B = Number of eggs in sample and gw = Sample weight Gonad histology To get solid evidence of the findings, histology of female gonads was done to determine the gonadal development. Histology process was done at Ecophysiology 24 Laboratory, Department of Fisheries Management, Bangladesh Agricultural University. 3.2.7.1 Process of gonad histology: Histological processes were undertaken at the Fish Ecophysiology Laboratory, Department of Fisheries Management, Bangladesh Agricultural University. The gonad of each fish was preserved in 10% buffered formalin with labeled vials for further study. The preserved samples after cutting into square size were taken out in a perforated plastic holder, which was covered by perforated steel plates. Cleaning, infiltration and dehydration process will be carried out in an automatic tissue processor using a series of alcohol of increasing concentrations, two changes of xylene and finally through molten wax (three series) as mentioned in Table 1 Time schedule in the automatic tissue processor Paraffin embedded blocks were cut by microtome knife at 4-5 µm size and left the sections into a water bath at a temperature of 40 ºC. The sections were placed on a glass slide and kept overnight on a slide drier hot plate at a temperature of 20 ºC. Then the sections were stained routinely with haematoxylin and eosin (Humason, 1962) as per the schedule given in Table 2. The consecutive steps of conducting histology of gonad of P. sophore are demonstrated in Figure 5. Table 1 Time schedule in the automatic tissue processor Sl. No. Steps of process Time (hour) Process 1 50% Methylated spirit 1 Dehydration 2 80% Methylated spirit 2 3 100% Methylated spirit 2 4 100% Methylated spirit 2 5 100% Methylated spirit 2 6 100% Alcohol 2 7 100% Alcohol 2 8 Xylene 2 Clearing 9 Xylene 1 10 Molten wax 1 Infiltration 11 Molten wax 2 12 Molten wax 2 Total 21 25 Table 2 Staining procedure for histological study of gonad Sl. No. Staining process Solution Times (min) 1 2 Clearing Xylene Xylene 3 3 3 4 5 6 Rehydration 100% Alcohol 100% Alcohol 95% Alcohol 870% Alcohol 2 2 2 2 7 Running tap water 5 8 Staining Haematoxylin 1 dip 9 Running tap water 25 10 Counter stain Eosin 1 11 12 13 14 Dehydration 70% Alcohol 95% Alcohol 100% Alcohol 100% Alcohol 1 2 2 2 15 16 Clearing Xylene Xylene 2 2 26 Figure 5 Flow chart showing histology processes of gonad Data analysis Microsoft Excel 2019 (Microsoft Corporation, 2020) was used to tabulate the raw data and analysed using the R programming language (R Core Team, 2021). The analyses were done using R-Studio (RStudio Team, 2022), and ggplot2 (Wickham, 2016) package was used to make graphs and charts. A one-way ANOVA (Analysis of Variances) was performed to determine whether there were significant differences in body indices between months. The Duncan's Multiple Range Test (DMRT) was utilized to determine the differences (Duncan, 1955). The models of regression regarding different relationships were compared and the best model were selected for measuring the dependencies of fecundity. The relationships between bodyweight and standard length were measured and related analysis were done in R-studio IDE. R-scrips of data analysis has been provided into the Appendix -2. Preserved gonads  Taking in perforated plastic holder  Automatic tissue processor (Dehydration, cleaning, infiltration process)  Paraffin embedded block (Embedding)  Cutting into 5 µm thick sections (Sectioning)  Water bath for 1-2 minuets @ 40 °C  Placing the sections on glass slides  Slide dryer (Drying the slides overnight @ 20 °C  Routine staining with stains (haematoxylin and eosin)  Mounting on glass slides, mounting the sections on glass slides using Canada balsam and covering with a cover slip  Examination under microscope and keeping photographic records. 27 RESULTS Fish size Standard length of fishes Standard length of collected fishes ranged from 49 to 96 mm over the study period Table 3 and Figure 6). Fish specimens collected on June 2021 were largest (P < 0.01) in size in terms of standard length ranging from 58-87 mm and 61.00-96.00 mm respectively. Fish samples caught on March 2021, April 2021, May 2021 and July 2020 were medium in terms of standard length varied from 56.00–72.10 mm, 56-82 mm, 58 - 87 mm and 55 – 82 mm respectively. On other months, standard length ranged from 52.50–59.00 mm in January-2021, 49.30–61.50 mm in August, 50.80– 60.60 mm in September 2020, 50.60–78.00 mm in October 2020, and 49–70 mm in December 2021 which were statistically smallest (P <0.01) throughout the study period. Table 3 Standard length (mm) of monthly collected specimens (July 2020June 2021) Months Min. SL Max. SL Mean SL SD No of female fish F value LSD (5%) Jan 52.50 59.00 55.64d 0.16 20 42.454 4.95 Feb 50.80 66.00 57.94cd 0.46 20 Mar 56.00 72.10 66.59b 0.47 22 Apr 56.00 82.00 69.38b 0.77 13 May 58.00 87.00 66.33b 0.69 24 Jun 61.00 96.00 83.18a 0.79 28 Jul 55.00 82.00 65.10b 0.76 21 Aug 49.30 61.50 55.24d 0.34 22 Sep 50.80 60.60 55.12d 0.27 20 Oct 50.60 78.00 55.62d 0.66 15 Nov 51.07 77.00 60.59c 0.91 20 Dec 49.00 70.00 57.45cd 0.56 20 SL: Standard Length; SD: Standard Deviation; LSD: Least significant difference; In a column figure with same letter do not differ significantly whereas figures with dissimilar letter differ significantly (as per DMRT). 28 Figure 6 Frequency distributions of standard length of P. sophore Body weight of fishes During the sampling period, the body weight of fish specimens captured ranged from 3.8-19.5 g (Table 4 and Figure 7). Specimens caught in August 2020, September 2020, October 2020 and December 2020 had the lowest average body weight with one percent level of significance (P < 0.01) (Table 4). Fish samples collected in November 2020, February 2021 and March 2021 comprised medium weight ranging from 4.59-10 g, 5.37-8.66 g and 5.35-9.19 g respectively, though there were some large and small fish but these were few in number. Fish sample collected on July 2020, April 2021, May 2021, June 2021 and were large in weight which range varied from 4.6-15.75 g, 5.84-15 g, 6.34-17 g and 7.65-19.50 g respectively. Among the specimens with high body weight specimens from June 2020 were statistically higher than all other months (p < 0.01). 29 Table 4 Body weight (g) of monthly samples specimens (July 2020-June 2021) Months Min. BW Max. BW Mean BW SD No of samples F value LSD (5%) Jan 5.07 6.43 5.89de 0.34 20 48.268 1.73 Feb 5.37 8.66 6.54cde 0.94 20 Mar 5.35 9.19 7.51c 1.18 22 Apr 5.84 15.00 9.74b 3.15 13 May 6.34 17.00 10.06b 2.92 24 Jun 7.65 19.50 15.77a 3.14 28 Jul 4.66 15.75 9.00b 3.10 21 Aug 4.48 6.32 5.74de 0.62 22 Sep 4.00 7.30 6.03de 0.72 20 Oct 4.65 9.95 5.83de 1.20 15 Nov 4.59 10.00 7.17cd 2.27 20 Dec 3.84 9.00 5.50e 1.37 20 BW: Body Weight; SD: Standard Deviation; LSD: Least significant difference; In a column figure with same letter do not differ significantly whereas figures with dissimilar letter differ significantly (as per DMRT). Figure 7 Frequency distributions of body weight of P. sophore 30 Determination of breeding season The month-wise gonadosomatic index, external features, fecundity of P. sophore were determined and established the relationship between standard lengthfecundity, body weight-fecundity to study the reproductive biology of this species. Ovarian developments were observed to determine the breeding season. Gonadosomatic index (GSI) method The gonadosomatic index, the indicator of the status of gonadal development and maturity of individuals of experimental species, was calculated for fish P. sophore during July 2020 to June 2021. The frequency distributions and the values of gonadosomatic index of fish are shown in Figure 8, and Table 5. Figure 8 Frequency distributions of gonadosomatic index of P. sophore The GSI values in the month of March, April, May, June and July were ranged from 4.63-14.45, 8.12-13.57, 8.78-18.10, 12.50-17.17, and 8.8-12.64 respectively. From August 2020 to February 2021, there was very small GSI values varied from 1.784.03, 0.23-4.27, 1.88-4.42, 0.34-0.82, 0.08-0.74, 0.37-0.82, and 0.37-0.82 respectively. Only in March the GSI value was medium. 37 Figure 13 Frequency occurrence of maturity stages of oocytes Frequency of spawning and measurement of egg diameter For the measurement of egg diameter about 100 eggs were taken randomly from the mixed sample of eggs for mature fish species. These were arranged in several rows on a glass slide and diameter of individual ova was taken with the help of stereomicroscope. The diameter of ova along whatever axis they lay parallel to the graduation of the micrometre, were measured to ensure random nature of the readings and unbiased values as suggested by Clark (1934). The measurements of egg diameter were taken in millimetre along the longest axis of the egg. Frequency distributions of egg diameter of ovaries were ranged from 0.4 mm to 0.8 mm in size and fecundity were counted as from 0.6 mm to 0.8 mm. The size range of 0.4 mm were not included as fecundity estimation because of their very smaller in size (Figure 18). The above facts concluded that Puntius sophore was a single spawner, i.e., synchronous species and it spawned once in a spawning season. 38 Figure 14 Size frequency distribution of oocytes of P. sophore Minimum length at maturity Female ovaries classified to premature stages were used to determine the minimum length at maturity. Female having premature oocyte was in standard length of 56 mm and its gonadosomatic index was 7.48. The relationship between gonadosomatic index (GSI) and standard length (SL) of female having mature gonad has been demonstrated in Figure 15. Figure 15 Relationship between gonadosomatic index (GSI) and standard length of female having mature gonad. Yellow dot indicates the GSI of youngest adult female having 56 mm SL in this study 39 Fecundity After estimating the breeding season, depending on the egg diameter 133 female specimens were taken for fecundity estimation. The range of relative fecundity was from 450 to 4352 per gm of mature female in terms of standard length from 56 mm to 92 mm respectively (Table 6). Table 6 Monthly fecundity of P. sophore Months Mean fecundity Min. of fecundity Max. of fecundity No of Fish examined Fvalue LSD (5%) Mar21 1713.129c 520 2315 22 65.904 386.76 Apr21 1827.909c 720 2781 11 May-21 2284.833b 1440 4005 12 Jun-21 3358.654a 1494 4352 26 Jul-20 1546.35c 520 2621 20 Aug-20 1262d 998 1478 22 Sep-20 1092.85d 816 1500 20 LSD: Least significant difference In a column figure with same letter do not differ significantly whereas figures with dissimilar letter differ significantly (as per DMRT). Relationship between fecundity and standard length The scatter diagram obtained from the fecundity and standard length showed a non-linear cubic relationship (Figure 16). In the determination of this relation, standard length was taken as independent variable, while fecundity as dependent variable. Standard length and fecundity yielded cubic relationship, the equation was F= 0.0232SL2.6877 (r2=0.8683) and the yielded equation indicated that fecundity was highly correlated with the standard length of fish. 40 Figure 16 The relationship between fecundity and standard length of P. sophore during spawning season Relation between fecundity and body weight The scatter diagrams obtained from the fecundity and body weight showed a linear relationship (Figure 17). In the determination of this relation, body weight was taken as independent variant, while fecundity as dependent variant. The relationship between fecundity and body weight was in the form of straight line, and the estimated equation was F = 213.44BW + 125.24 (r2 = 0.892) and it was concluded that fecundity was highly correlated with the body weight of fish. Figure 18 shows the frequency distribution of egg diameters of the experimental units and the diameter of egg from where fecundity counting was started. y = 0.0232x2.6877 R² = 0.8683 0 1000 2000 3000 4000 5000 6000 020 40 60 80 100 120 No of Eggs SL (mm) 41 Figure 17 The relationship between fecundity and body weight of P. sophore during spawning season Figure 18 Size frequency distribution of oocytes of P. sophore. Fecundity was counted with the eggs right to the arrow y = 212.76x - 110.14 R² = 0.8891 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 0 5 10 15 20 25 No of Eggs BW (g) 42 DISCUSSION The knowledge on reproductive biology of fish is essential for evaluating the commercial potentialities of its stock, life history, culture practice and management of its fishery. In order to achieve success in fish culture it is important to assess the yearly breeding cycle of culturable fishes. Some of them breed once a year while some at regular intervals throughout the year. Information of gonadal development and the spawning season of a species is also important for its management. The aim of the study of reproductive biology of Pool barb, P. sophore was to identify the spawning seasons, determine the minimum length at maturity, frequency of spawning, maturation stages of oocytes, frequency distribution of eggs and fecundity of the species. Therefore, the investigation was carried out from July 2020 to June 2021 in a natural reservoir of the Rajdhala beel, Purbadhala, Netrokona, Bangladesh. The gonadosomatic index (GSI), which is indicative of the breeding season of the fish, had been calculated from March to June. There was a sudden rise in the values in March and gradually increased till June. The highest gonadosomatic index of P. sophore was in June and the lowest was in December. This indicates that P. sophore may breed in March to June. These data suggest that one spawning season might occur from March to July. These findings agree with the findings of Mitra et al. (2005) and Hasan et al. (2018). Alam et al. (2014) found the breeding season of pool barb from May to August while Bithy et al. (2013) reported the months from June to August. Bithy et al. (2013) also determined July as a breeding month of P. sophore. Moreover, Mitra et al. (2005) observed that the breeding season of this fish extended February to August and May-June as spawning months. Hasan et al. (2018) found that the breeding season runs from March to July, peaking in April and May in Gazipur Bangladesh. The same study also identified May to July as the breeding season in Jessore, Bangladesh. Due to the fact that Jessore is a 43 baor area with numerous baors and beels, the current study location Rajdhala beel has similar hydrographic characteristics, which could be one explanation for the parallels between the two studies. Peaks of the GSI index value in June might be due to completion of maturity and subsequent steep fall in the index value clearly indicated alternatively the spawning and spent condition of fish. The gonadosomatic index increased with the maturation of fish, being maximum during the period of peak maturity and declined abruptly thereafter, when fish became spent (LeCren, 1951). Treasurer and Holiday (1981) in studies with Perca fluvintilis, Piska et al.（1991) with Salmostona phulo stated that GSI was useful for establishing spawning period. The monthly changes in GSI reflect the ovarian activity of fish and it must be noticed that GSI is a reliable tool to determine peak spawning season (Vladimir, 2000). However, the present results were fully agreed with that of Mitra et al. (2005). Additionally, the occurrence of spawning ovaries examined by external characteristics began to appear in March and continued up to July. Percent occurrence of ovaries started to increase in March and peaked in June. It appeared in March to some extent and gradually peaked in June and steadily declined in August. After that it was not found in September to February. In the present study, monthly changes of gonadosomatic index for P. sophore revealed that mean GSI increased once in a year in June suggested one peaks of GSI. Various developmental stages of ovaries were classified considering their external features and the macroscopic views of eggs. The stages were immature, developing, maturing and spawning. External morphology and macroscopic observation of female gonads showed that the spawning season of pool barb was March to July. This finding is similar to findings of Mitra et al. (2005); Bithy et al. (2013) and Alam et al. (2014) who found one peak breeding season for P. sophore in different regions. Alam et al. (2014) found the season to be May to August in Bangladesh while Bithy et al. (2013) reported the season to be June to August with July as peak month in Bangladesh. These results are similar to the finding of this study, where March to July is the breeding period of Pool barb in Rajdhala beel in Netrakona, Bangladesh. 44 Histology is a powerful tool for biological assessment. In this study, oocytes maturity stages were classified into five stages as early perinucleolus stage, yolk vesicle stage, primary yolk stage, migratory nucleus stage and premature stage. The premature stage was observed as the most advanced stage in the study. Most advanced mature stages indicated that probability of spawning might be occurred within short time. Other advanced maturity stages were not found surely because the sampling time was during the morning, when ovaries might not supposed to be ready for ovulation and spawn yet. It is believed that if samples are taken in late afternoon and evening during the spawning season, more advanced maturity stages may appear (Vladimir, 2000). However, females classified into premature stage were supposed to ready for capable of spawning or near to spawn. The most advanced premature stage oocyte i.e., premature oocyte was present from March to June. There was no premature oocyte observed in the month of September. These results indicated one spawning season of P. sophore precisely lasted from March to June. Ovary of P. sophore was a synchronous type that contained almost similar oocyte maturation stages. This type of oocyte maturity was by far the most common strategy among teleost (Wallace and Selmanm, 1981; Lee et al., 2005) studied with Leiognathus equulus and found eight maturity stages of oocytes as chromatin nucleolus, perinucleolus, yolk vesicle, primary yolk, secondary yolk, tertiary yolk, migratory nucleus and ripe stage by histological examination of ovaries. Spawning frequency may be determined on the basis of the occurrence or presence of postovulatory follicle, hydrated eggs (De Martini and Fountain, 1981; Hunter and Macewicz, 1985) and mode of frequency distribution of egg diameter (Sherry et al., 1996). In fact, hydrated eggs and postovulatory follicles were not observed in the ovaries throughout the study period. The presence of almost same oocyte maturity stages in the histological section of matured female ovaries were observed during the study period which indicated that female P. sophore were single spawner (i.e., may spawn only once during a single spawning season). While in case of multiple or serial spawner, fish may 45 spawn more than once during a single spawning season. Post ovulatory follicles or hydrated eggs are the indications of a serial spawner (George, 1996). But no postovulatory follicles or hydrated eggs were found in the current study. Hence, this information revealed that the experimental fish spawned once in a spawning season. The study found that the standard length of the youngest female that bore mature eggs was 56 mm, and its gonadosomatic index was 7.48. Minimum size of maturity is not an independent criterion, but is one that closely associated with the other features of growth, food supply, etc. of a particular stock or population. In general, minimum size of maturity is also related to the longevity and the rate of growth. When the sexes differ in longevity that with the lower longevity tends to have a higher rate of growth and minimum size of maturity and vice versa. In the west coast spart the growth rate and longevity of the sexes have been shown to be almost identical (De-Silva, 1973) and hence, as expected, both sexes have the same minimum size and age at maturation. This finding is similar to the findings of Hossain et al. (2012) and Mitra et al. (2005). The range of relative fecundity was from 540 to 4352 per gm of mature female which maintained standard length from 56 mm to 92 mm. It was observed that female specimens belonging to the same size group had different number of eggs in their ovaries. Lagler et al. (1967) reported that the number of eggs produced by an individual female was dependent on various factors like size, age, condition and type of species of the fish. It was also observed in some cases that the fecundity of some larger fishes was much less than that of some smaller fishes. This type of variation was also reported by different workers (Doha and Hye, 1970; Karim and Hossain, 1972). The relationship between the fecundity and standard length of P. sophore found to be nonlinear cubic relation and was expressed as F= 0.0232SL2.6877 (r2=0.8683) during spawning season (i.e., from March to July). The equation yielded r2=0.8683 indicated that fecundity was highly correlated with the standard length of fish. Similar relationship for P. sophore was found by Tareque et al. (2008). Some other studies found linear relationship between fecundity and length of P. sophore (Bithy et al., 2013; Srivastava, 2003; Kant et al., 2016). 46 The relationship between the fecundity and body weight of P. sophore was linear and it was expressed as F = 213.44BW+125.24 (r2 = 0.892) during spawning season. From the linear value r2=0.892, it was concluded that fecundity was highly correlated with the body weight of fish which is related to the findings of Srivastava (2003); Tareque et al. (2008); Bithy et al. (2013) and Kant et al. (2016). The findings of this study can be said reliable as all the findings were more or less matches with the related literatures and findings. Most of the dissimilarities occurred due to variation of regional and environmental factors. 53 Marina A 2006: Reproductive biology of Pool barb Puntius sophore (Hamilton, 1822) of a perennial water body in Bangladesh. M. S. 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Wallace RA and Selman K 1981: Cellular and dynamic aspects of oocyte growth in teleosts. American Zoologist 21 (2) 325–43. WHO 2011: World Health Organization. Vitamin and mineral nutrition information system (VMIS). Micronutrient database. Wickham H 2016: Ggplot2: Elegant graphics for data analysis. Springer-Verlag New York. https://ggplot2.tidyverse.org. Ziki TB 2006: Reproductive biology of Mystuas vittatus of a perennial water body in Bangladesh, M.S. Thesis, Department of Fisheries Management, Bangladesh Agricultural University, Mymensingh. 57 APPENDICES Appendix1 Analysis of Variance (One-way ANOVA) of monthly standard length (SL), body weight (BW), gonadosomatic index and fecundity Sum of Squares df Mean Square F Sig. SL(cm) Between Groups 184.153 11 16.741 42.454 <0.001 Within Groups 91.882 233 .394 Total 276.035 244 BW (g) Between Groups 2377.303 11 216.118 48.268 <0.001 Within Groups 1043.257 233 4.477 Total 3420.560 244 GSI Sum of Squares df Mean Square F Sig. Between Groups 5635.013 11 512.274 236.455 <0.001 Within Groups 418.130 193 2.166 Total 6053.143 204 Fecundity Sum of Squares df Mean Square F Sig. Between Groups 90681264.315 6 15113544.052 65.904 <0.001 Within Groups 28895369.363 126 229328.328 Total 119576633.678 132 58 Appendix2 Source code (R) of data analysis DATA ANALYSIS FOR REPRODUCTIVE BIOLOGY OF PUNTI (P. SOPHORE) IN RAJHDHALA RESERVOIR, NETROKONA Md. Ashfaq Sadat Loading Data from Raw data library(readr) data_punti <- read_csv("data_punti.csv", col_types = cols(Month_NO = col_factor(levels = c("1", "2", "3", "4", "5", "6", "7", "8", "9", "10", "11", "12")), Sex = col_skip(), Month = col_factor(le vels = c("January", "February", "March", "April", "May", "June","July", "August","September","October","November", "December")))) # Loading monthly Gonadal Stage Frequency Data gonadFreq <- read_csv("Frequncy of Gonad Stages.csv", col_types = cols(Month = col_factor(levels = c("January","February", "March", "April", "May","June", "July", "August", "September","October" , "November", "December")),Stages = col_factor(levels = c("Resting", "Developing", "Ripe", "Spawning", "Spent")), Percent = col_integer())) ## Warning: One or more parsing issues, see `problems()` for details # Loading monthly histology Frequency Data histFreq <- read_csv("histFreq.csv", col_types = cols(month = col_factor(levels = c("January","February", "March", "April", "May","June", "July", "August", "September","October" , "November", "December")),stages = col_factor(levels = c("EP", "YV", "PY", "MN", "PM")), percent = col_integer())) Distribution of Total length along months library(ggplot2) ggplot(data=data_punti, aes(SL_mm)) + geom_histogram(bins=5)+facet_wrap( data_punti$Month)+ylab("Frequency")+xlab("Standard Length (mm)") 59 Distribution of Body Weight along months library(ggplot2) ggplot(data=data_punti, aes(BW)) + geom_histogram(bins = 5)+facet_wrap(d ata_punti$Month)+ylab("Frequency")+xlab("Body Weight (g)") Distribution of GSI along months library(ggplot2) ggplot(data=data_punti, aes(GSI)) + geom_histogram(bins=6)+facet_wrap(da ta_punti$Month)+ylab("Frequency")+xlab("Gonadosomatic Index") ## Warning: Removed 40 rows containing non-finite values (stat_bin). 60 library(ggplot2) ggplot(data=data_punti, aes(Diameter)) + geom_histogram(bins = 7, bounda ry = 0)+ylab("Frequency")+xlab("Egg Diameter") ## Warning: Removed 118 rows containing non-finite values (stat_bin). Monthly Changes in Gonadosomatic index Frequency of Gonadal Development ggplot(gonadFreq,aes(x=Month, y = Percent, fill= Stages))+geom_col()+gui des(fill=guide_legend(reverse=FALSE))+ 61 theme(axis.text.x = element_text(angle = 45, hjust=1))+labs(x ="Months ", y = "Frequency Percent (%)") ## Warning: Removed 16 rows containing missing values (position_stack). Frequency of Histology Development ggplot(histFreq,aes(x=month, y = percent, fill= stages))+geom_col()+guid es(fill=guide_legend(reverse=TRUE))+ theme(axis.text.x = element_text(angle = 45, hjust=1))+labs(x ="Months ", y = "Frequency Percent (%)") ## Warning: Removed 27 rows containing missing values (position_stack). 62 Models data_punti%>% filter(!is.na(Fecundity)) -> df # sl_model <- lm(log(df$Fecundity) ~ log(df$SL_mm)) summary(sl_model) ## ## Call: ## lm(formula = log(df$Fecundity) ~ log(df$SL_mm)) ## ## Residuals: ## Min 1Q Median 3Q Max ## -0.78290 -0.13309 0.00608 0.14184 0.51336 ## ## Coefficients: ## Estimate Std. Error t value Pr(>|t|) ## (Intercept) -3.8297 0.4827 -7.934 8.29e-13 *** ## log(df$SL_mm) 2.6877 0.1153 23.318 < 2e-16 *** ## --- ## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1 ## ## Residual standard error: 0.2228 on 131 degrees of freedom ## Multiple R-squared: 0.8059, Adjusted R-squared: 0.8044 ## F-statistic: 543.8 on 1 and 131 DF, p-value: < 2.2e-16 coef(sl_model) ## (Intercept) log(df$SL_mm) ## -3.829672 2.687672 # The overall F-value of the model is 511.2 and the corresponding p-valu e is extremely small (< 2.2e-16), which indicates that the model as a wh ole is useful. # # Using the coefficients from the output table, we can see that the fitt ed power regression equation is: # # ln(F) = -3.7614 + 2.6718*ln(SL) # # Applying exp to both sides, we can rewrite the equation as: # # F = exp (-3.7614 + 2.6718*ln(SL)) # calculating exp(2.6718) exp (-3.7614) ## [1] 0.02325117 # if we plot the value on the equation # We get the final equation is F = 0.02325 SL^2.6877 # We can use this equation to predict the response variable, y, based on the value of the predictor variable, x. # plotting the data