#1Studies on certain aspects of the reproductive biology of mouth-brooding tilapia, Oreochromis mossambicus (Peters) from Assam, India#2G.Hatikakoty Department of Biology, Nazira H.S. & M.P. School, Nazira - 785685, Assam, India S.P.Biswas Department of Life Sciences, Dibrugarh University, Dibrugarh - 786004, Assam, India#3INDIA LOCALITY-I LOCALITY-1] THE STUDY AREA ASSAM BHUTAN MEGHALAYA BANGLADESH PRADESH ARUNACHAL NAGALAND MANIPUR SIVASAGAR#494°30' SIVASAGAR LAKHIMPUR DISTRICT O 94 45 95 10' 950 15 DIBRUGARH DISTRICT SIVASAGAR SARAIDEW Dikhow river Simlugri JORHAT DISTRICT NAZUR NAGALAND ARUNACHA270 PRADESH The study area (pond) Fig. 1. Location map of the study area 26° 45'#5FIG p/3a. O.mossambicus Mature male Caudal fin (light pink). FIG p/3b. O.mossambicus Mature Female Plate - III#6Fig. p/1. Photograph of the Pond in WINTER#7Fig. p/1. Photograph of the Pond in MONSOON#8The experimental pond located at Nazira in Sivasagar District of Upper Assam, India. Latitude 26°54'36" N and Longitude 94°43'54” E at an altitude of 94 m above msl. The experiment was carried out for three years (1996 - 1999).#9The specimen of Oreochromis mossambicus (Peters) for the present study had been collected on a monthly basis from a small perennial domestic pond of Nazira Town of Sivasagar district in Upper Assam. Water of the pond was analyzed on a monthly basis. The pond bottom soils samples and the pond biota including fauna, flora and plankton had been recorded seasonally. The four season had been recorded according to climatic condition of Assam as winter (December - February); pre-monsoon (March-May); Monsoon (June - September) and autumn (October-November).#10Morphometric features of the pond Geographic location Latitude Longitude Altitude Туре Shape Area (m²) Water Colour Pond 26°54'36"N 94°43'54"E 94 (approximate) meters above mean sea level. Old perennial Square 91.54 Slightly transparent, slightly greenish from pre-monsoon to monsoon and light brown from autumn to winter. Muddy bottom mostly covered with decayed organic matter. Average maximum depth (m) Average minimum depth (m) Utility 4.98 1.98 Used for drinking and washing purpose#11Methods used: Seasonal progression of gonads recorded by physical examination of testes and ovary Assessment of various maturity stages - modified classification of Kesteven (1960) and Crossland (1977). • The GSR or Co-efficient of maturity -Hopkins (1979). ● Maturity stage of entire population - length maturity key (Kesteven, 1960). The attainment of sexual maturity (M50) - (Hopkins and Mann, 1978)#12Spawning periodicity - progressive change in the intra-ovarian ova diameter for a period of three years. • The absolute and relative fecundity - Bagenal (1967) and Hardisty (1964). Histological preparation of gonads - Histological techniques (Patki et.al., 1989 and Lal, 2001).#13RESULTS AND DISCUSSION#14Table 1. Degree of maturation and the morphology of the gonad in different stages of maturity of O.mossambicus Stage Degree of maturation Immature or virgin and resting adult Months of availability Throughout the year II Early maturing March to September III Developing March to October IV Developed / pre spawning March to October Ova diameter (mm) 0.045 0.055 0.056 -0.090 0.091-0.85 0.86 0.99 Spawning April to October 1.0 1.5 VI Spent April to late October 0.052 0.17 Description of the gonads Ovaries very small, thin, thread like pale in colour, occupying a small part of the body cavity. Testes is thin, slender translucent and pale in colour. Both the gonad invisible to the naked eye. Ovaries become slightly larger and increase in weight and volume with minute opaque whitish eggs occupied about half of the body cavity. Testes become enlarge, flat, increase in weight and volume, and creamy white in colour. Both the gonad are readily seen without any aid. Ovaries distended occupied, about 2/3 of abdominal cavity with large pale yellow eggs. Testes enlarge, increase in weight and volume, light pinkish and thicker in size and look more vascular. Blood capillaries become conspicuous. Ovary becomes more enlarged occupying almost entire body cavity, with large number of big, turgid, spherical, translucent, deep yellow riped ova. Testes become soft turgid pinkish red and increase in weight and volume. Blood capillaries prominent. Roe to milt run with slight pressure. Ovary walls become thin almost transparent. Riped eggs are visible through the ovarian wall and some riped eggs are present in the oviduct. Testes become flabby, thin and dull white in colour. Gonad shrunken having loose walls. Ovaries are flaccid, shrinked and sac like, reduced in volume. Ovary contains ripped unspawned darkened eggs and a large number of small ova. Testes become flabby, thin and dull white in colour.#15MORPHOLOGICAL PICTURES#16Fig p/4 a. Genital papilla of mature male (TL 17 cm). Light red coloured Ventral Fin Anus Genital Papilla Ureter Ventral Fin Anus Genital Papilla Fig p/4b. Genital Papilla of mature female (TL 21 cm).#17Fig.p/4c. Dorsal fin Male Female Red margin Male Female Fig.p/4d. Pectoral fin#18Fig. p/4e. Ventral fin Female Male Female Male Fig p/4f. Anal fin#19Fig. p/4g. Caudal fin Red margin in the dorsal fin Red corner in the caudal fin Male Female Red Corner Fig.p/4h. Caudal fin with red corner & dorsal fin with red margin in the mature male.#20Fig.p/10a. Testes Stage I Immature phase Testes Plate - XIX#21Pair of testes Fig.p/10b. Testes Stage II Early maturing phase Fig. p/10c. Testes Stage III Advanced / late maturing phase Testes#22Fig. p/10d. Testes Stage IV pre Spawning phase Blood capillary Plate - XX Testes in the abdominal cavity 03 2 Fig. p/10e. Testes Stage V Spawning phase#23Fig. p/10f. Testes Stage V Spawning phase Fig.p/12a. Ovary - Stage II Early maturing phase Common sperm duct Pair of Ovary#24Pair of ovary Fig. p/12b. Ovary - Stage III Advanced / Late Maturing Phase Fig. p/12c. Ovary - Stage IV Pre-spawning Phase Common oviduct Blood capillaries#25Blood capillary Ripe eggs visible through the ovarian wall 2 2 Fig.p/12d. Ovary - Stage V Spawning Stage, length 2.9 cm METER Fig.p/12e. Ovary - Stage - V Spawning, Length 3.5 cm. Mature ovary occupied most part of the abdominal cavity#26TESTES Non-zonal, lobules loosely organised anterior middle and posterior parts of testes showed morpho-histological variations in different maturity stages. All the parts showed similar stages of development in a particular maturity stage#27Seminiferous Lobules Spermatogonial cells Interstitial cells Fig. 1a. Photomicrograph of T.S.of Testes. Stage I immature phase (5x X 10) IMMATURE: Numerous SPERMATOGONIA observed inside the small SEMINIFEROUS LOBULES. Spermatogonia are large spherical cells with a large round central nucleus with distinct nucleolus.#28Spermatogonia Seminiferous lobules Fig. 1b. Photomicrograph of Testis Resting Stage (5x X 10) RESTING: Numerous SPERMATOGONIA observed inside the small SEMINIFEROUS LOBULES. Spermatogonia are large spherical cells with a large round central nucleus with distinct nucleolus.#29Sperm mother cells Fig. 1c. Photomicrograph of T.S.of Testes. Stage II early maturing phase (5x X 10) EARLY MATURING: SLOW MITOTIC ACTIVITY. Sparmatogonia start dividing and transformed into SPERM MOTHER CELLS.#30Primary spermatocytes Secondary spermatocytes Fig. 1d. Photomicrograph of T.S. of Testes Stage III developing phase (10x X 10 middle part) DEVELOPING :Intense SPERMATOGENESIS seen during later part of his phase. SPERMATOGONIA DECRESES IN NUMBER. Numerous PRIMARY AND SECONDARY SPERMATOCYTES visible.#31Primary spermatocytes Secondary spermatocytes Fig. p/11c.Photomicrograph of T.S.of Testes. (Stage III developing phase 5x X 10) DEVELOPING : Primary spermatocytes are smaller than spermatogonia and posses a darkly stained nucleus. They give to secondary spermatocytes, which are still smaller than primary spermatocytes with clump chromatin material.#32Testicular wall Blood vessel Interstitial cell Seminiferous lobules Spermatogonia Secondary Spermatocyte Primary Spermatocyte Sperm Spermatid Fig. 1e. Photomicrograph T.S. of Testes Pre spawning phase Stage IV (10x X 10) PRE-SPAWNING :Blood capillaries become conspecuous, seminiferous lobules are larger in size and full of sperm. Spermatogia are few. All stages of spermatogenesis seen in various lobules#33Fig.p/11e. Photomicrograph T.S. of Testes (Interior part Stage IV pre spawning phase) Spermatozoa Seminiferous lobules#34Sperm Fig.p/11h. Photomicrograph T.S.of Testes Stage V spawning phase 10x X 10 SPAWNING :Smaller deeply stained SPERMATIDS with eliptical nucleus and slightly reduced SPERMS seen. Seminiferous lobules became empty because of release of sperms.#35Fig. 1f. Photomicrograph T.S. of Testes Stage V spawning phase (100x X 10) Sperm#36Fig. p/11g. Photomicrograph T.S. of Testes Stage V spawning phase 10x X 10 (oil) Sperm#37Collapsing seminiferous tubules Residual sperm Fig.1g. Photomicrograph of T.S.of Testes (anterior part) Stage VI Spent phase (5x X 10) SPENT: Empty & collapsing seminiferous lobules are seen, some of which contain residual or unexpelled sperm.#38OVARIES * Ovary wall is fairly thick during non-breeding season. * Becomes thin and highly vascular during the spawning period. * Ovarian lumen is loosely organised and zonation is not apparent.#39Ovigerous lamellae Nest of oogonia Connective tissue Developing oocyte Peritoneum Fig.2a. Photomicrograph T.S. of Ovary Stage I Immature phase (5x X 10) IMMATURE: Germ cells, oogonium are found in bunch in the ovigerous lamellae.#40Peritoneum Nests of oogonia Ovigerous Lamella Immature oocyte Fig.2b. Photomicrograph T.S. of Ovary Resting phase (5x X 10) RESTING: Ovigerous lamellae having nest, of oogonia and immature oocytes Stage I and Stage II are visible#41Ooplasm Chromatin Threads Nucleus Fig. 2c. Photomicrograph T.S. Ovary showing Oogonium (5x X 10) In the early stage of oogenesis oogonium is a large cell with large nucleus and prominent nucleolus surrounded by narrow rim of ooplasm which ar chromophotic.#42-Oocyte II Developing Oocyte Nucleus Oocyte II Nucleus Oogonium Oocyte I Oocyte IV Yolk vesicle Membrane Oocyte V Vitelline Oocyte III Yolk vesicle Ovigerous Lamella Non fibrous connective tissue Fig.2d. Photomicrograph T.S. Ovary - Stage II Early Maturing Phase (5x X 10) EARLY MATURING: Numerous oocytes in different stages of development. Oocytes Stage I to Stage III are in large number. Stage IV and Stage V they are few in number.#43Oocyte VI Oocyte VII Oocyte V Theca Docyte V Fig.2e. Photomicrograph T.S. of Ovary Stage III developing phase (5x X 10) Ovigeous Lamellae Theca Zone granulosa Blood Capillary Mature Oocyte Yolk Vesicle Migrating Nucleus Tunica Albuginea Zone Radiata Yolk Globules Peritoneum DEVELOPING: Vascular supply increased blood capillaries become conspicuous. Immature oocytes reduced. Stage IV and Stage V oocytes large in number. Stage VII may also seen.#44Fig.2f. Photomicrograph T.S. Ovary - Stage IV pre-spawning (5x X 10) Ripe eggs Oocyte VII Small ball of Non-hypertrophic Atresia PRE-SPAWNING :Large number of OVA in Stage VII, Ripe eggs and Non-hypertrophic Atresia also observed.#45Fig. Photomicrograph T.S Ovary Stage IV PRE-SPAWNING 5x X 10 Oocyte VII Ripe eggs Non-hypertrophic Atresia#46Ripe eggs Fig 2g. Photomicrograph T.S. of Ovary Stage V Spawning phase (5x X 10) SPAWNING: RIPES OVA come out by rupturing of follicular epithelium#47Ripe eggs SPAWNING: RIPES OVA come out by rupturing of follicular epithelium#48Atretic eggs Oocyte II Discharge follicles Oocyte I Fig.2h. Photomicrograph T.S. of Ovary, Stage VI Spent phase (5x X 10) SPENT : ATRETIC AND DISCHARGED FOLLICLES along with STAGE I AND STAGE II OOCYTE#49Table 1. Degree of maturation and the morphology of the gonad in different stages of maturity of O.mossambicus Stage Degree of maturation Immature or virgin and resting adult Months of availability Throughout the year II Early maturing March to September III Developing March to October IV Developed / pre spawning March to October Ova diameter (mm) 0.045 0.055 0.056 -0.090 0.091-0.85 0.86 0.99 Spawning April to October 1.0 1.5 VI Spent April to late October 0.052 0.17 Description of the gonads Ovaries very small, thin, thread like pale in colour, occupying a small part of the body cavity. Testes is thin, slender translucent and pale in colour. Both the gonad invisible to the naked eye. Ovaries become slightly larger and increase in weight and volume with minute opaque whitish eggs occupied about half of the body cavity. Testes become enlarge, flat, increase in weight and volume, and creamy white in colour. Both the gonad are readily seen without any aid. Ovaries distended occupied, about 2/3 of abdominal cavity with large pale yellow eggs. Testes enlarge, increase in weight and volume, light pinkish and thicker in size and look more vascular. Blood capillaries become conspicuous. Ovary becomes more enlarged occupying almost entire body cavity, with large number of big, turgid, spherical, translucent, deep yellow riped ova. Testes become soft turgid pinkish red and increase in weight and volume. Blood capillaries prominent. Roe to milt run with slight pressure. Ovary walls become thin almost transparent. Riped eggs are visible through the ovarian wall and some riped eggs are present in the oviduct. Testes become flabby, thin and dull white in colour. Gonad shrunken having loose walls. Ovaries are flaccid, shrinked and sac like, reduced in volume. Ovary contains ripped unspawned darkened eggs and a large number of small ova. Testes become flabby, thin and dull white in colour.#50G.S.R and seasonal cycle of maturation The cycle of maturation and monthly variation of gonadosomatic ratio provide good indication of the extent of development of gonad with respect to the time of year. Gonad staging on a descriptive scale allows a rapid qualitative assessment of the breeding state and gonad weight give a quantitative record of changes in the gonad condition (Crossland, 1977).#51Table 2. Average monthly fluctuations in the K, GSR and GSI Months K G.S.R. G.S.I. January 1.77 0.28 7.1 February 1.85 0.37 6.5 March 2.05 0.54 5.9 April 1.88 0.48 5.5 May 2.36 0.55 5.7 Jun 2.27 0.51 4.9 July 2.60 0.60 5.2 August 2.55 0.58 4.5 September 2.37 0.59 4.7 October 2.25 0.56 3.9 November 2.09 0.55 4.2 December 1.67 0.18 7.5 G.S.R VARIED FROM 0.18 (DECEMBER) TO 0.60 (JULY). High correlation between GSR and condition factor ( r = 0.88). Negative correlation between GSR and GSI ( r = -0.84).#52High correlation between GSR and condition factor, K (r = 0.88) and occurrence ripe specimen stage IV and stage VI during March to October is the indication of maturity in these months. Negative correlation between GSR and GSI (r = -0.84) is the indication that during maturation of the gonad the fish takes less amount of food suggesting that the developing of the gonads are highly effected the feeding habits of the fish during breeding season.#53Certain physical parameters of water such as turbidity, water temperature, dissolved oxygen, free carbon-di-oxide and total alkalinity also studied to examine the effect of these abiotic parameters on gonadal maturation. It was observed that in both aquarium and pond condition the species spawns four times in a year, between March and October, when temperature was favourable spawning activity of the species has not been observed during winter (November to February) when water temperature drops down below 18°C. It has further been noticed that seasonal peaks in the G.S.R. values coincided with the peaks in the percentage of occurrence of matured individuals. The result on GSR indicates that both the males and females mature at the same time of the year, the peak breeding period being June and July.#54Parameters Water Temperature Seasonal variation of water quality parameters (Physical) Seasons Winter Pre-monsoon Monsoon Autumn 17.63 23.13 28.98 21.4 (°C) pH 7.75 7.78 8.13 7.83 Turbidity 28.48 7.89 4.92 5.29 (ppm) Conductivity 36.07 33.9 21.35 23.13 TDS 385 334 152 164 Suspended 108 73 55 63 Solids (SS)#55Seasonal variation of water quality parameters (Chemical) Parameters Seasons Winter Pre-monsoon Monsoon Autumn TH 127.1 116.6 60.0 63.0 DO 1.5 2.6 4.5 3.0 FCO2 16.01 8.13 7.13 11.31 HCO2 113.3 122.8 151.8 140.6 ΤΑ 114.6 149.6 182.6 179.6 CL 27.07 29.53 54.22 39.88 K 7.4 13.3 37.0 21.8 TP 8.69 6.43 2.95 3.4 ΝΑ 26.3 28.0 44.8 30.0 SILICATE 10.83 7.59 6.6 7.1 NO3N 0.547 0.413 0.320 0.410 NO2N 0.041 0.065 0.092 0.070 AMMONIA 0.111 0.124 0.180 0.145 AMMONICAL 0.43 0.32 0.25 0.30 NITROGEN SO4 22.64 22.75 119.36 54.10 MA 24.41 19.13 6.93 17.18 CA 18.8 18.2 8.8 15.1 IRON 3.16 3.03 1.72 2.82 COD 71.67 44.50 34.67 38.70 BOD 14.0 8.63 4.77 8.15#56G.S.R 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 January February March April May Months June July August September October Female -Male Fig. 3. Monthly Variations of Gonadosomatic ratio (GSR) of Male & Female fish G.S.R. value showed 4 peaks in March, May, July & September coinciding with the spawning of the species in North-Eastern India. November December#57Ovadiameter and seasonal cycle of maturation * In the present study it was observed that the ova-diameter increased (0.045 1.5 mm) alongwith the progression of the maturity stage (Table.1). - * Measurement of ova-diameter and their frequency polygon distribution at different time of the months of the year was a common method for determination of maturity cycle of the fish (Macer, 1974). * The progressive change observe in the intra-ovarian diameter for a period not less than a year can give an idea of the spawning periodicity of the fish studies (Biswas, 1993). * From the percentage occurrence of mature ova in different months (Fig.4) it is inferred that in O.mossambicus the mature ova showed 4 peaks suggesting 4 times of spawning in a year. In other words, the fish was a batch spawner and only gravid ova are released at one time during spawning season.#58percentage 100 90 80 70 60 50 40 30 20 10 0 January February March April May June July August months Fig.4. Frequency polygon of mature ova-diameter in different months September October November December#59Length at first maturity and determination of M Close relationship between maturity and the length of the fish O.mossambicus attains maturity length - 6.5 to 180 mm and age 2 and 5 months (Chimitz 1955, Pongsuwana, 1956 and Mironova, 1969) -50- • In the present study sexual maturity observed in the length group: female length 5-10 cm, male length 10 - 15 cm. - 50% male mature -> average length 12.5 cm • 50% female mature -> average length 7.5 cm Female matures at the shorter length than their male counterparts (Table.3)#60Size groups (cm) Sex Immature Stage I Table 3. Percentage of maturity of various length groups Mature Maturing Stage II & III Stage IV & V Female 100 0-5 Male 100 - - Female 25 25 50 5-10 Male 75 Female 25 10-15 Male 228 20 5 75 50 50 Female 100 >15 Male 100#61Fecundity of Tilapia (O.mossambicus) showed wide variation: - 1. Hora & Pillay (1962): The female tilapia lays 75 - 250 eggs at a time. • 2. Mironova (1969): Fecundity tilapia ranged between 80 and 1000 eggs. 3.Chimitz (1955) & Chang kong Tam (1962): 8 - 9 cm long females incubate 80 eggs. 15 cm long female nursed 800 eggs. A six months old female incubate 180-300 eggs whereas a eighth month old incubate 350-500 eggs.#62• 4. De Silva and Chandrasoma (1980): Fecundity of O.mossambicus varied between 360-1775 for fish ranging from 20 to 31.9 cm in length and 145 to 538 gm in weight. In the present study absolute fecundity 100 - 850 for a size range of 7.6 to 19.9 cm (6.45 gm to 155.73 gm in weight)#63• FECUNDITY The low fecund could well be attributed to the parential care (Anon 2000). Low fecundity of O.mossambicus in these region might be due to prolonged breeding season. • The logarithmic relationship between fecundity and different body parameters were found to be linear. Fecundity and body length was found to the most closely related (r = 0.99).#64The relative fecundity ranged between 6-16 (Table.4): - • Anon (2000): The fecundity of O.mossambicus varied from 431 - 1012 eggs / 100 gm body weight. • Riedel (1965): The fecundity of O.mossambicus varied from 660 - 1750 eggs/100 gm body weight. In the present study the fecundity varied from 546 - 1550 eggs / 100 gm body weight similar to those recorded elsewhere.#65Table 4. Absolute and relative fecundity Total Length Body Weight Ovary Weight Absolute Relative (cm) (gm) (gm) Fecundity Fecundity 7.6 6.45 0.028 100 16 9.8 12.92 0.042 150 12 10.2 26.12 0.113 179 7 11.7 28.32 0.327 200 7 12.6 35.57 0.330 250 7 13.5 45.2 0.380 414 9 14.2 50.71 0.486 511 10 15.0 53.82 0.575 569 11 17.3 108.65 0.725 620 6 19.9 155.73 0.835 850 6#66• Abstract: Certain aspects of reproductive biology Morpho-histological studies of gonads revealed the existence of six maturity stages. Gonadosomatic index (GSR) indicated that the breeding season extended from March to October. Frequency polygon of ova-diameter showed 4 peaks suggesting 4 times of spawning of the fish in this part of the country. • First sexual maturity observed - 5 to 10 cm length groups in female and 10 to 15 cm in male.#67• . 50% of male mature at an average length of 12.5 cm and 50% of female mature at an 7.5 cm length groups. Absolute fecundity 100 to 850, Relative fecundity 6 to 16. Positive correlation between GSR and condition factor (r = 0.88). Negative correlation between GSR and GSI (r=0.84). High correlation was observed between Fecundity and total length (r=0.99), Fecundity and body weight (r=0.85), Fecundity and body weight (r = 0.62).#68ACKNOWLEDGEMENTS Authors are thankful to the Head, Dept. of Life Sciences, Dibrugarh University for providing necessary facilities. Authors are also greatly indebted to the Dr. V.V.Sugunan, Director, CIFRI, Barrackpore, Kolkata and Dr.Manash Kr. Das, Dr.U.Bhaumik and Mr.S.Bhaumik, the Scientist for providing necessary laboratory facilities for _ microphotography. Sincere thanks are due to Director and Laboratory Staffs Dr. Arun Salmon, Dr.(Mrs.) Arumina Hazarika Challang, Mr. Mahesh Borpujari, Mr.Juesh Sona, Mr.Rupam Rajkhowa & Mr.Bisheswar Murmu, Tata Referral Hospital & Research Centre, Chubwa, Assam, Mr. Prafulla Bhattacharjyya and Mr. Someswar Dihingia - Pathology Department, Assam Medical College, Dibrugarh and Dr.Jagadish Mahanta Director, Dr. P.K.Mahapatra, Dr.A.Prakash, Dr.D.R.Bhattacharjyya and Mr.Basanta Kr.Goswami, Scientists, ICMR, Lahowal for microscopic photographs. Sincere thanks to Deepak, Rias and Biplav Sinha. Also special thanks to Dr.Kevin Fitzsimmons, University of Arizona for publication of the paper.#69THANKS TO : - * THE GOVERNMENT OF INDIA. * THE CHIEF MINISTER AND THE STATE GOVT. OF ASSAM, INDIA. * ALSO SPECIAL THANKS TO : Dr. Remedios Boliever, Mr.Kevin Fitzsimmons, the Organisers, ISTA 6, Manila, Philippines and the Philippose.