UNIVERSITI PUTRA MALAYSIA. MORPHOLOGY AND GENERAL REPRODUCTIVE STAGES OF Pangasius nasutus FROM SG. PAHANG IN MARAN DISTRICT, PAHANG, MALAYSIA

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1 UNIVERSITI PUTRA MALAYSIA MORPHOLOGY AND GENERAL REPRODUCTIVE STAGES OF Pangasius nasutus FROM SG. PAHANG IN MARAN DISTRICT, PAHANG, MALAYSIA MOHD ZAFRI BIN HASSAN FP

2 MORPHOLOGY AND GENERAL REPRODUCTIVE STAGES OF Pangasius nasutus FROM SG. PAHANG IN MARAN DISTRICT, PAHANG, MALAYSIA MOHD ZAFRI BIN HASSAN MASTER OF SCIENCE UNIVERSITI PUTRA MALAYSIA 2006

3 MORPHOLOGY AND GENERAL REPRODUCTIVE STAGES OF Pangasius nasutus FROM SG. PAHANG IN MARAN DISTRICT, PAHANG, MALAYSIA By MOHD ZAFRI BIN HASSAN Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia in Fulfilment of Requirement for the Degree of Master of Science November 2006

4 Specially dedicated to my beloved parents ii

5 Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the Degree of Master of Science MORPHOLOGY AND GENERAL REPRODUCTIVE STAGES OF Pangasius nasutus FROM SG. PAHANG IN MARAN DISTRICT, PAHANG, MALAYSIA Chairman : Annie Christianus, PhD By MOHD ZAFRI BIN HASSAN June 2006 Faculty : Agriculture The present study was conducted to describe the morphological differences between P. nasutus and P. conchophilus, to describe and classify the histology of oocyte and spermatogenic cells of mature P. nasutus and to observe the seasonal patterns of the gonadal stages in twenty specimens of P. nasutus and P. conchophilus from Maran. In order to signify the species differences, twenty specimens of Thai-origin P. conchophilus from a cage culture at Sungai Pahang near Pekan, Pahang, Malaysia were also examined. The morphometric measurements were presented in percentage of standard length (%SL), predorsal length (%PDL) and head length (%HL). Based on the classification results, all P. nasutus specimens were different from P. conchophilus in both populations, in terms of snout length, eye diameter, lower and upper jaw length. A total of 205 P. nasutus were caught during the study period, from October 2004 to September 2005 for the gonadal stages study of P. nasutus. Nine distinctive iii

6 oocytes stages observed in the study were from oogonia to late vitellogenesis. Six microscopic oocytes developmental stages were assigned for the studied ovaries of P. nasutus, namely resting, maturing, mature, spawning, running and spent stages. Further, microscopic observation on the whole-section of the testes revealed that four testicular development stages could be distinguished namely early spermatogenesis, spermatogenesis, maturation and spent stages. In this study, it was found that the female and male P. nasutus were reproductively active at the beginning of rainy season which commenced in the middle quarter of 2005, from March to September coincided with the South-West monsoon. The study also revealed that P. nasutus exhibits a single-modal spawning season in a year, with multiple release of eggs during a single period. Such condition showed that P. nasutus is a multiple-spawner as supported by the group-synchronous oocyte development of the ovary. However, spawning did not commence synchronously within the population with the obvious presence of various female and male reproductive stages within a single sampling month. iv

7 Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk Ijazah Master Sains MORFOLOGI DAN PERINGKAT PERKEMBANGAN PEMBIAKAN Pangasius nasutus DARI SG. PAHANG DI DALAM DAERAH MARAN, PAHANG, MALAYSIA Pengerusi : Annie Christianus, PhD Oleh MOHD ZAFRI BIN HASSAN Jun 2006 Fakulti : Pertanian Kajian ini telah dijalankan untuk menerangkan perbezaan morfologi antara P. nasutus dan P. conchophilus, menghurai dan mengkelaskan histologi oosit dan sel-sel spermatogenik bagi P. nasutus matang dan seterusnya untuk melihat corak bermusim untuk peringkat-peringkat gonad bagi P. nasutus. Dua puluh spesimen P. nasutus dan P. conchophilus dari Maran, Pahang telah dikaji. Untuk melihat perbezaan spesies, dua puluh spesimen P. conchophius berasal dari Thailand tetapi dikultur dalam sangkar di Sungai Pahang, dekat Pekan, Pahang, Malaysia, turut dikaji. Ukuran-ukuran morfometrik diberikan dalam bentuk peratusan panjang piawai (%SL), panjang pra-dorsal (%PDL) dan panjang kepala (%HL). Berdasarkan keputusan pengkelasan, semua specimen P. nasutus adalah berbeza dari P. conchophilus pada kedua-dua populasi, terutamanya dari segi panjang muncung, diameter mata, panjang rahang bawah dan atas. v

8 Sejumlah 205 spesimen P. nasutus ditangkap sepanjang waktu kajian yang bermula dari Oktober 2004 sehingga September Sembilan peringkat kematangan oosit telah diperhatikan dalam kajian ini adalah dari peringkat oogonia sehingga peringkat akhir vitellogenesis. Enam peringkat perkembangan oosit yang diperhatikan secara mikroskopik untuk keseluruhan ovari P. nasutus dibahagikan kepada kepada peringkat rehat, sedang matang, matang, bersedia untuk membiak, sedang membiak dan selesai membiak. Berdasarkan pemerhatian mikroskopik bagi keseluruhan testis P. nasutus menunjukkan terdapat empat peringkat perkembangan testis yang dapat dikenalpasti iaitu, spermatogenesis awal, spermatogenesis, kematangan dan selepas pembiakan. Kajian ini mendapati, betina dan jantan P. nasutus adalah aktif membiak pada permulaan musim hujan yang bermula pada suku pertengahan tahun 2005, dari Mac sehingga September serentak dengan musim monsun tenggara. Kajian ini juga mendapati, P. nasutus mempunyai satu musim pembiakan khusus dalam satu tahun, yang dapat dilihat dengan pelepasan telur yang berperingkatperingkat pada sesuatu tempoh masa. Keadaan ini menunjukkan bahawa P. nasutus merupakan pembiak yang melepaskan telurnya secara berperingkatperingkat yang jelas dapat dilihat dari perkembangan oosit secara serentak dalam dua atau tiga kumpulan di dalam satu-satu ovari. Walaubagaimanapun, pembiakan tidak berlaku serentak di dalam populasi berdasarkan kehadiran pelbagai peringkat perkembangan reproduktif betina dan jantan semasa penyampelan di dalam satu-satu bulan. vi

9 ACKNOWLEDGEMENTS In the name of Allah, the Lord that I always put my belief in. Thanks to my parents and siblings for being my pillars and strength all my life. I would like to express my deepest gratitude to the chairman of my supervisory committee, Dr. Annie Christianus for her tremendous encouragement, financial assistance on the research expenses and camaraderie throughout the study period. Not forgetting, many thanks to my co-supervisors Assoc. Prof. Dr. Siti Shapor Siraj and Assoc. Prof. Dr. Siti Khalijah Daud for sharing their invaluable expertise, ideas and comments on the research conducted and write-up. Thanks to UPM for the scholarship and granting me study leave off my duty at UPM Bintulu Campus. The study could not have completed without the support and co-operations from many parties, namely Assoc. Prof. Dr. Khir Bejo and his lab assistant Saipu for allowing and helping me to use facilities in the Pathology Laboratory, Faculty of Veterinary UPM; Assoc. Prof. Dr. Mohd Salleh Kamaruddin for his permission to use Aquatic Biotechnology Laboratory, Faculty of Agriculture UPM, En. Jasni Yusof at the UPM hatchery, En. Mat Piah and his family for their friendly entertainment on the lodging and helping hands during sampling activities; and last but not least, million thanks to my all-time friends Ikram, Sazahar, Halim and Saufi to whom I owe their time and energy very much. vii

10 I certify that an Examination Committee has met on 28 November 2006 to conduct the final examination of Mohd Zafri Bin Hassan on his Master of Science thesis entitled Morphology and General Reproductive Stages of Pangasius nasutus from Sg. Pahang in Maran District, Pahang, Malaysia in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations The committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows: Che Roos Saad, PhD Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Chairman) Sharr Azni Harmin, PhD Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Internal Examiner) Mustafa Kamal Abdul Satar, PhD Lecturer Faculty of Agriculture Universiti Putra Malaysia (Internal Examiner) Mazlan Abdul Ghaffar, PhD Associate Professor Faculty of Science and Technology Universiti Kebangsaan Malaysia (External Examiner) HASANAH MOHD GHAZALI, PhD Professor/Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date: viii

11 This thesis submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfillment of the requirement for the degree of Master of Science. The members of the Supervisory Committee are as follows: Annie Christianus, PhD Lecturer Faculty of Agriculture Universiti Putra Malaysia (Chairman) Siti Shapor Siraj, PhD Associate Professor Faculty of Science Universiti Putra Malaysia (Member) Siti Khalijah Daud, PhD Associate Professor Faculty of Science Universiti Putra Malaysia (Member) AINI IDERIS, PhD Professor/Dean School of Graduate Studies Universiti Putra Malaysia Date: 8 March 2007 ix

12 DECLARATION I hereby declare that the thesis is based on my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions MOHD ZAFRI BIN HASSAN Date: 15 February 2007 x

13 TABLE OF CONTENTS DEDICATION ABSTRACT ABSTRAK ACKNOWLEDGEMENTS APPROVAL DECLARATION LIST OF TABLES LIST OF FIGURES LIST OF ABBREVEATIONS Page ii iii v vii viii x xiii xv xix 1. INTRODUCTION 1.1 Background of Patin buah in Malaysia 1.2 Statement of problems 1.3 Significance of the study 1.4 Objectives LITERATURE REVIEWS 2.1 Taxonomy 2.2 Distribution of the family Pangasiidae 2.3 Catfishes as an Economic commodity 2.4 Morphological characters of the genus Pangasius nasutus 2.5 The biology of catfishes 2.6 Reproductive dynamics Spawning pattern and cycle of ovarian development in catfishes Testicular development of catfishes 2.7 Histology of the gonadal development MORPHOLOGICAL VARIATIONS BETWEEN Pangasius nasutus AND Pangasius conchophilus 3.1 Introduction 3.2 Materials and methods Study area Samples collection Sampling methods Morphometric and meristic characters Statistical analysis xi

14 3.3 Results Morphological features for characterizing P. nasutus and P. conchophilus Meristic characters Morphometric characters 3.4 Discussion GAMETOGENESIS IN Pangasius nasutus 4.1 Introduction 4.2 Materials and methods Field sampling Gonads collection and macroscopic observations Histological preparation for gonad studies Oocyte and spermatogenic stages Fecundity estimation Statistical analysis 4.3 Results Samples landed and standard length-weight relationship Water parameters, rainfall and water level of Pahang River near Maran Biological indices Macroscopic observation of the ovaries of P. nasutus Macroscopic observation of the testes of P. nasutus Microscopic observations on the individual stage of oocytes in P. nasutus Microscopic observations of the whole-section of ovarian development of P. nasutus Microscopic observations on the spermatogenic cells of P. nasutus Microscopic observations of the whole-section of testicular development of P. nasutus Seasonal development in the gonadal stages of P. nasutus Homogeneity of the ovary and fecundity of P. nasutus 4.4 Discussion GENERAL DISCUSSION AND CONCLUSION 5.1 General discussion 5.2 Conclusion REFERENCES APPENDICES BIODATA OF AUTHOR xii

15 LIST OF TABLES Table Page 1 Subgeneric classification of genus Pangasius 8 2 The presence of genus Pangasius recorded in Malaysia 9 3 Morphometric data taken were all according to Pouyaud et. al. (1999). With exception for measurements with * were after Gustiano et. al. (2004), after Lowe-McConnell (1975) and *** additional measurements in the present study 4 Number of specimens with their respective meristic counts Morphometric data of P. nasutus (n = 20) 41 6 Morphometric data of P. conchophilus caught from Maran (n = 20) 7 Morphometric data of P. conchophilus caught from Pekan (n = 20) 8 Variables that were significantly different (P < 0.05) in percentage of standard length (in % SL) (n = 20). 9 Variables that were significantly different (P < 0.05) in percentage of predorsal length (in % PDL) (n = 20). 10 Variables that were significantly different (P < 0.05) in percentage of head length (in % HL) (n = 20). 11 Standardized canonical discriminant function coefficients for ratio percentages 12 Structure matrix of canonical discriminant function coefficients for ratio percentages Classification results based on ratio percentages Stepwise dehydration with a series of alcohol and subsequent paraffin wax infiltration 59 xiii

16 15 Step-by-step staining procedure Mean±SD biological indices of P. nasutus females caught from the Pahang River near Maran from October 2004 to September Mean±SD biological indices of P. nasutus males caught from Pahang River off Maran from October 2004 to September Size range of oocyte stages of P. nasutus Estimated fecundity and frequency of spawning of P. nasutus collected from the Pahang River near Maran during the sampling period from October 2004 to September xiv

17 LIST OF FIGURES Figure 1 Taxonomic hierarchy leading to the Family Pangasiidae (Source: Systema Naturae, 2000) Page 7 2 Map of study area 25 3 Lateral view of the body; a) P. nasutus, b) P. conchophilus caught in Maran 4 Methods used for samples collection; a) drift-net; b) longlines; c) fishing rod; and d) cage containment in the vicinity of study area 5 Diagram of fishing gears used, a) drifting gillnet; b) longlines 6 Diagrammatic representative of measurements taken on specimens (adapted from Pouyaud et. al. (1999) with modifications) Counts of gill raker of the first gill arch 35 8 Lateral view of the head; a) P. nasutus, b) P. conchophilus 9 Side by side comparison of dorsal view of the head; a) P. nasutus, b) P. conchophilus 10 Ventral view of the head; a) P. nasutus, b) P. conchophilus 11 Gutted belly showing swim bladder position located at the abdominal cavity of the body in P. nasutus; a) male, b) female 12 Canonical discriminant functions plots using proportional values Diagrammatic procedures of tissue histology process Length-size class of total specimens caught throughout the study period 66 xv

18 15 Log 10 -transformed length-weight relationship of P. nasutus caught from Pahang River near Maran 16 Water surface temperature, dissolved oxygen (D.O) and specific conductivity (SpC) taken from the Pahang River near Maran during the sampling period from October 2004 to September Mean±SD are subject to Tukey HSD in which dissimilar letters are significantly different (P<0.05) 17 Turbidity, ph and total dissolved solids (TDS) taken from the Pahang River near Maran during the sampling period from October 2004 to September Mean±SD shown subject to Tukey HSD in which dissimilar letters are significantly different (P<0.05) 18 Rainfall (mm) and water level (m) data collected at Maran Meteorological Station from October 2004 to September Biological indices of the females of P. nasutus caught from the Pahang River near Maran during the sampling period from October 2004 to September Biological indices of the males of P. nasutus caught from the Pahang River near Maran during the sampling period from October 2004 to September The macroscopic morphology the ovaries; a) 2.02 GSI from 1050 g BW and 41 cm SL; b) 8.24 GSI from 800 g BW and 46 cm SL; c) 2.37 GSI from 700 g BW and 36 cm SL ; d) 0.30 GSI from 780 g BW and 45 cm SL; e) 0.25 GSI from 1600 g BW and 49.2 cm SL 22 The macroscopic morphology of the testes; a) 1.31 GSI from a sample of 37 cm in SL and 660 g in BW; b) 6.11 GSI from a sample of 46 cm in SL and 900 g in BW 23 Stages of oogonia stage; a) the presence of oogonia within the ovarian wall (10x); b) closer view of oogonia (100x). Primary follicles stage; a) overall view of the ovarian cavity (10x), primary follicles are in dark colour; b) primary follicles encapsulated in the nest and nucleus is formed with the presence of a single nucleoli (100x) xvi

19 24 Nucleolar stage. Chromatin nucleolar stage; a) nucleoli in the nucleus present as chromatin form and more numbers of nucleoli are visible (100x); b) nucleoli are moving toward the nucleus periphery. Follicular layer appear as purple stained layer surrounding the oocyte (40x). Perinucleolar stage; a) Nucleoli allocated peripherally to the nucleus. Cytoplasm is stained light blue (20x); b) Dark spot granules can be observed alongside the follicular layer. A sign of the formation of granulosa layer (40x) 25 Cortical alveoli stage, a) Early cortical alveoli stage with translucent dots at the peripheral oocyte indicating the presence of yolk vesicles (40x); b) Follicular envelope showing the developing vitelline membrane (100x); c) More round yolk vesicles are surrounding the ooplasm periphery (20x); d) The prominent yolk vesicles are a result of the coalescence of the prior yolk vesicles (100x) 26 Early vitellogenic stage; a) Formation of yolk globules forming an eosin-stained ring around the nucleus (20x); b) Yolk vesicles still remained at the peripheral ooplasm (100x) 27 Vitellogenesis stage; a) Yolk globules filling up the ooplasm (5x); b) Peripheral ooplasm is filled up with smaller yolk globules, striated vitelline envelope is evident (100x) 28 Late vitellogenesis stage; a) oil droplets present in the ooplasm and the start of germinal vesicle breakdown (10x); b) germinal vesicle leaves the nucleus and moves towards the animal pole at the peripheral ooplasm and yolk begins to coalesce (10x) 29 Atretic oocytes; a-b) atresia during cortical alveoli (20x); c-d) atresia during vitellogenesis (40x); e-f) final stage of atresia (100x and 20x, respectively) 30 Resting ovarian stage (from a sample of 1600g BW, 43cm SL and 0.25 GSI) 31 Ovarian development at GSI 1.0; a) Progressing - maturing (from a sample of 910g BW, 45cm SL and 0.14 GSI); b) Regressing - spent (from a sample of 710g BW, 43cm SL and 0.44 GSI) xvii

20 32 Ovarian development at GSI ; a) Progressing - mature (from a sample of 1050g BW, 41cm SL and 2.02 GSI); b) Regressing - running (from a sample of 700g BW, 36cm SL and 2.38 GSI). 33 Ovarian development at GSI 5.1. Spawning (from a sample of 1300g BW, 45cm SL and 7.95 GSI). 34 Developmental stages of spermatogenic cells; a) spermatogonia; b) spermatocytes; c) spermatids; appearance is blotchy and in clutch form; spermatozoa; mature spermatozoa have distinct basophilic spherical heads 35 Testicular development of P. nasutus; a) Early spermatogenic stage; (from a sample of 45cm SL, 930g BW and 0.14 GSI); b) Spermatogenic stage; (from a sample of 54cm SL, 1450g BW and 0.85 GSI); c) Maturation stage; (from a sample of 41cm SL, 780g BW and 3.35 GSI); d) Spent stage; (from a sample of 44cm SL, 770g BW and 1.06 GSI). 36 Frequency of occurrences of ovarian development of the females and males of P. nasutus caught from the Pahang River near Maran from October 2004 to September Number in parenthesis ( ) represents the number of specimens caught 37 Frequency of occurrences of ovarian development of the females and males of P. nasutus caught from the Pahang River near Maran from October 2004 to September Number in parenthesis ( ) represents the number of specimens caught xviii

21 LIST OF ABBREVIATIONS Analysis of Variance Body weight ANOVA BW Celsius degree C Centimeter Department Fatsomatic index Gonadosomatic index Gut repletion index Gram Head length Hepatosomatic index Kilogram Meter Milimeter cm Dept. FSI GSI GRI g HL HSI kg m mm Micrometer µm Percent % Predorsal length Standard deviation Standard length Universiti Putra Malaysia PDL SD SL UPM xix

22 CHAPTER ONE INTRODUCTION 1.1 Background of Patin buah in Malaysia Patin buah, a vernacular river catfish name for Pangasius nasutus in Malaysia, is one of the favourite food fish and commands a good price in the local market. Information on this native fish is lacking probably attributed to the dominant interest in the other patin, Pangasius hypopthalmus, a catfish of Thai origin introduced to Malaysia for aquaculture purpose. Popularity of the native patin buah then had been brought up by the recent awareness of its economic value and the declining of its population in the Pahang River. The occurrence and distribution of P. nasutus in Malaysia are literally not conclusive. Catfishes are known to occupy habitats ranging from upland streams to large river channels and to seasonal floodplain lagoons (Winemiller and Kelso- Winemiller, 1996). Therefore it is not a surprise that P. nasutus was known to occur in the Pahang River where the richness of the biodiversity is at its best among the rivers in Malaysia (Haslawati et al., 2004). Roberts and Vidhthayanon (1991) also reported the occurrence of this species in Chenderoh Dam, Perak.

23 A number of species of the family Pangasiidae are of great economical importance in Southeast Asia (Hung et al., 2004) and have high potential for aquaculture in Malaysia. At present, the wild catch of patin buah (Pangasius nasutus) and patin muncung (Helicophagus wandersii) demands as much as RM 50kg -1 and RM 100kg -1 in the market, respectively. Due to its white and tender flesh, Pangasiid catfish was ranked among the top five preferred species for five consecutive years in terms of consumer preferences in Malaysia (Fisheries Annual Report, 2002). 1.2 Statement of problems Recently, the decline in the abundance of patin buah in the local rivers has been reported. In fact, it is supported by the declining numbers in landing data of the species in Maran, a district along the Pahang River, which is known for patin buah (Department of Fisheries, 2004). The first successful artificial propagation however, was reported by the local Department of Fisheries in March 2004 after several years of attempt at propagating it. Despite the success, however continuous fry supply however cannot be guaranteed due to low survival rate of repeated attempt. P. conchophilus that is found in the vicinity of Pahang River was recently brought in from Thailand by the Cambodian immigrants residing in Pekan, Pahang (Haslawati et al., 2004). Due to its resemblance to P. nasutus, it was named by 2

24 the local people as patin buah Kemboja (Cambodian patin buah). These two species are commonly mistakenly misidentified for each other due to morphological features of both species (Roberts and Vidthayanon, 1991). Although induced breeding has been successful, studies on the reproductive biology of the species sampled from its natural environment are crucial so as to gain insight and more understanding on the species. Even for fish that has been established in a farming system, the information on the reproductive aspects including the absolute and relative fecundities would be helpful to ensure a continuing success in its culture (Ali, 1993). These guidelines also constitute major approach towards establishing biodiversity management as well as breeding programme (Corriero et al., 2003; Utoh et al., 2003; Micale et al., 1996; Legendre, 1986). 1.3 Significance of the study The population of P. nasutus is currently declining. In the present situation, the marketable supply patin buah is entirely dependant on the wild catch. This increases the chances for the fish to be over-exploited. Effort at culturing this species has never been attempted and the population silently decreasing. Therefore, a study on the gonadal cycle of the species is necessary as an initial step towards husbandry management. 3

25 Reproductive biology has served numerous advantages in the study of a particular species or population. Manosroi et al. (2003) stated that in order to conserve a threatened Mekong giant catfish P. gigas, maturation induction by hormone injection and seasonal endocrine regulation expressed as gonadosomatic index (GSI) are crucial. Knowledge in the reproductive biology can also lead to a better understanding and advantage in breeding a particular species before being introduced for aquaculture purposes (Hassin et al., 1997). Temporal difference in reproductive maturity at individual level allows an efficient broodstock management (Dahle et al., 2003). The study on annual cycles in the gonadal stages of wild animals will provide baseline information on body composition, which is useful in the evaluation of quality and physiological condition of animals before being cultured (Craig et al., 2000). Conservation, catch size restrictions, controlled fishing seasons or improvements of breeding technologies in hatcheries can be inferred from the study. 1.4 Objectives The main objective of this study is to describe the distinctive morphological characteristics of the native P. nasutus and to differentiate these characteristics from those of the introduced P. conchophilus. Apart from that, monthly observation and assessment on the gonadal activity of P. nasutus caught from its 4