A study on induced breeding, embryonic and larval development of in semi-arid agro-climate Pangasianodon hypophthalmus

Aim : Breeding performance of Pangasianodon hypophthalmus in semi-arid agro-climatic conditions of northern India was evaluated in view of establishing hatchery to supply its seed. Methodology : P. hypophthalmuswere induced to breed using carp
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  DOI: JournalofEnvironmentalBiology  671-676Vol.392018September  Abstract Aim:Methodology:Results:Interpretation: Breeding performance of in semi-arid agro-climatic conditions of northernIndiawasevaluatedinviewofestablishinghatcherytosupplyitsseed.were induced to breed using carp pituitary gland (PG) extract. Femaleswere administered with priming dose of 2.5 - 3.0 mg kg ; after 6 hrs, a second resolving dose of 10-12 mgkg wasgiven,whilemaleswereadministeredwithsingledoseof2.5–3.0mgkg bodyweight.Eggsweredry fertilized after 14 hrs of second injection. The fertilized eggs were allowed to incubate in a circular hatchery maintaining a water flow of 30 l min . Embryonic and larval developments were assessed byobserving eggs at regular interval under a microscope. Optimum water quality parameters weremaintainedduringentireexperimentperiod.Theaveragefecunditywas57.5lakhkg ofbodywhilefertilizationandhatchingpercentagewas76.50 and 58.20, respectively.After fertilization, eggs became water hardened within 20 min. “Blastodiscstage” appeared after 00:25 hr and “Morula stage” after 03:45 hr of fertilization. “Blastula stage” wasobservedafter05:30hrpostfertilization.Twitchingmovementstarted24:00-26:00hrpostfertilizationandhatching started at 34:00-36:00 hr post fertilization. A newly hatched larva measured about 3.80 mm inlengthwithheadslightlybentandcompactyellowishyolksac.Duringtheageof5-10daysoflife,pectoral,caudal,ventralanddorsalfinsappeared.Stripped catfish can be successfully and commerciallybred in semi-arid agro-climatic conditions of northern India under controlled environmental conditionsusing PG as inducing agent. However, females were not observed to ovulate naturally but dry fertilizationtechnique. Pangasianodon hypophthalmusP. hypophthalmusPangasianodon hypophthalmus -1-1 -1-1-1 *Corresponding Author Email : PublicationInfo Paperreceived:13.10.2017Revisedreceived:10.12.2017Re-revisedreceived:20.02.2018 Accepted:04.04.2018© ,Lucknow(India) TriveniEnterprises Authors Info S.N. Datta*,A. Singh,G. Jassal andA. Pandey College of Fisheries, Guru Angad Dev Veterinary and Animal Sciences University,Ludhiana–141 004, India Key words Embryonic developmentInduced breedingLarval devlopmentPituitary extract Pangasianodon hypophthalmus p-ISSN:0254-8704e-ISSN:2394-0379CODEN:JEBIDP P Dlagiarism etector  White Smoke Just write. JournalHomepage:  E-mail: Journal of Environmental Biology A study on induced breeding,embryonic and larval development of insemi-arid agro-climate Pangasianodon hypophthalmus Original Research  JEB TM TM Breeding, embryonic and larval development of Asian striped catfish,  Pangasianodon hypophthalmus Induced breeding using pituitary gland extract and study of embryonic and larval developmentInduced breeding Stripping HatchingFirst time successful breeding of a new candidate species for aquaculture in Northern IndiaFertilized egg Blastula Gastrula Larva after 24 hrs of hatching  JournalofEnvironmentalBiology, September2018 Sciences University (GADVASU), Ludhiana. The juveniles of were procured from Kolkata, West Bengal andraised in the earthen pond of 600 m size during summer (April toNovember)andinthe poly-houses(4innumberwithsizeof80meach and equipped with electrical heating system) during winter (December to March) for three years 2014-17. Floating feed(28.0%crudeproteinand4.5%crudefatondryweightbasis)wasformulated with the help of extruder using agro-industrial by-productsi.e.,ricebran(30%),de-oiledgroundnut(30%),de-oiledsoybean (25%), fish meal (13%) and commercially availablemultivitaminandmulti-mineralmixture(2%).Fishwerefed@2%of body weight (two meals equally distributed at 9.00 am, 5.00pm). Total six numbers of breeding trials were conducted duringthe month of June to August, 2017. Three-year-old female(1500–2500 g) and male (1200-2000 g) reared in fish farm,College of Fisheries, GADVASU were selected for breeding.Before breeding, mature females were distinguished by their big,soft, distended belly with reddish, swollen ventral genital pore for the present study, while males were identified by their genitalpapilla which oozes milt with slight pressure on the abdomen.Fish were kept for one day before induced breeding in twodifferent circular cement tanks (3.0 media and 0.75 m depth) for maleandfemalefishwithcontinuousshoweringforconditioning.Pituitary glands of Indian major carpspreserved in absolute alcohol (100%) and stored in therefrigerator(at4 C)wereusedforextractpreparation.Atthetimeofbreeding,femaleswereadministeredanintramuscularprimingdose of 2.5 - 3.0 mg kg b.wt. of the pituitary extract (prepared inlaboratory).After 6 hrs, a second resolving dose of 10-12 mg kgofb.wt.wasgiven.Atthesametime,singledoses(2.5-3.0mgkgof b.wt.) of pituitary extract were given to males. Pituitary extractwas administered intramuscularly in the region of caudalpeduncleabovethelaterallineoffish.A2mlsyringegraduatedto0.1 ml division with 22 number needles were used for injection.Immediately after injection male and female fish were kept inseparate tanks under continuous showering. Females werefoundreadyforstrippingabout12-17hrsofsecondinjection.The ready-to-spawn females were taken out and their eggs were stripped on a dry plastic tray by gently pressing their bellies. The males were then stripped and their milt was mixedwitheggswiththehelpofacleanfeatherandgentleshaking.Miltfrom one male was sufficient to fertilize the eggs of two to threefemales.Thedrymethodofeggfertilizationwasfollowed.Asmallamount about 15 ml of distilled water was mixed and stirred for 1–2min.Afterthoroughmixing,thetraywasrinsedwith100–150mlofwatertoremoveexcessmiltandmucus.Eggsofthiscatfishis sticky in nature, they were washed with clay mud water for 3-4times before transferring it to Chinese circular hatchery. Eggincubationperiodvariedfrom36-40hrat27.30±0.28 C.Important parameters of water quality such as water temperature, pH, dissolved oxygen,total alkalinity, hardness, ammonia-nitrogen (NH -N), nitratenitrogen (NO -N), nitrite nitrogen (NO -N) and phosphate P.hypophthalmus 22o-10 Inducedbreeding:Studyofembryonicdevelopment: -1-133 2 IntroductionMaterials and MethodsExperimentalsetup:  Asian striped catfish isnative of Mekong basin of Indochina (Da 2011). It is nowrecognizedasasuperioraquaculturespeciesfortropicalregions,as well as a major food fish in world market because of it's whitefine grained sweet flesh (Pal, 2010). It has been introduced for cultivation in different countries of the world (Singapore,Philippines, Taiwan, Malaysia, China, Myanmar, Bangladesh,NepalandIndia)becauseofitshighgrowth,easyculturepractice,high disease resistance and high resistance to crowding and lowoxygen (Bui 2010;Ahmed 2013; Griffith 2010)hence there is a need to standardize the culture and breedingpractices as per the new environment conditions (Begum2012a). In India it was first introduced in the state of West Bengalthrough Bangladesh during 1997 (Mukai, 2011). Culture of thisspecies is growing day-by-day in India (Lakra and Singh, 2010;SinghandLakra,2012;Kumar 2013).Itisextensivelybeingcultured in Andhra Pradesh and West Bengal (Moses2016). The culture of this species was earlier prohibited in Indiabut now the Government of India has permitted it's culture since2010-2011 (Moses 2016). Fish is cultivated both under monoculture as well as polyculture with Indian major carp.Monocultureof fetchesbetterperformanceandprofit to farmers. In India, culture production of is15-20tonha yr whichishigherthancarpproduction(8-10tonha yr )inthesamearea(SinghandLakra,2012).is highly fecund, seasonal spawner and breeds once a year in flooded rivers (Moses 2016).Females fish attain maturity at the end of third year while malematureintwoyears(Griffith 2010;VidthayanonandHogan,2013).PeopleinNorthernIndiaincludingPunjab,generallyprefer fish with less intramuscular spines, hence Pangas catfish has agreat potential in diversification as well as vertical and horizontalexpansionofaquaculturesectorsinthisregion.Forthecultureof Pangas at present fish farmers are entirely dependent on other states (mainly West Bengal) for the availability of seed.Transportation of seed increase the risk in the form of highmortality percentage caused due to change in agro-climaticconditions. Moreover, severe cold in northern India restricts thecultureofthisspeciesinoutdoorpondsfromApriltoOctoberonly.Thus, brood stock management for seed production requiresmore attention and special care. Maintaining brood stock inpolyhouse during winter season is one of the major practices tosave the brood stock for seed production in coming breedingseason. Therefore, in view of the increasing aquacultureimportance of in India, attempts were made tobreedthefishunderagro-climaticconditionsofPunjabforthefirsttime under controlled conditions along with the study onembryonicandlarvaldevelopment.ThestudywasconductedintheFishFarmof College of Fisheries, Guru Angad Dev Veterinary and Animal Pangasianodon hypophthalmuset al.,et al., et al., et al.,et al.,etal.,et al.,et al.,P.hypophthalmusP. hypophthalmusP. hypophthalmuset al.,etal.,P. hypophthalmus -1 -1-1 -1 672  S.N. Datta et al.  JournalofEnvironmentalBiology, September2018 Induced breeding, embryonic and larval development of P. hypophthalmus 673 may be attributed to prolong winter and occurrence of latemonsooninNorthernIndia.Ingeneral,eggincubationtimevaried12-14 hrs (Ferosekhan 2015); however, in the presentstudy it ranged between 12-17 hrs for final ovulation and releaseof gametes.This could be due to variation in maturity stages andagro-climatic condition. Parameters related with breeding of areshowninTable1.Theaveragefecunditywas57.5±0.0004lakhkg whilefertilizationpercentagewas76.50±0.60 kg of b.wt., respectively. spawns once ayear.Fertilizedeggswerestickyinnature,transparent,roundandincreased in diameter from 1.25 ± 0.03 to 1.80 ± 0.05 after fertilization and water hardening. Stickiness was removed bytreatingtheeggswithclaymudwater.Gonadosomaticindex(GSI)ofmaleandfemale duringbreedingseason(June- August) was recorded 7.024 and 10.08, respectively. Condition et al.,P.hypophthalmusP. hypopthalmusP.hypopthalmus -1-1 phosphorous (PO -P) were analyzed following the standardmethods (APHA, 2012). Parameters such as fertilization andhatching rate were calculated by the formula given byGunasekara (1996). Fulton's condition factor (K) wascalculated according to equation given by .Gonadosomatic Index was calculated by the formula given byBarber and Blake (2008).Afew number of egg ware collected inpetri dish to calculate the rate of fertilization and embryonicdevelopmentbyusingflorescentmicroscopeLeicaDM3000LEDmodel. Egg diameter, functional and morphological changeswereassessedaspertheproceduredescribedbyFerosekhan(2015). All the statistical analysis was performed by usingsoftwareSPSSVer.16.Water quality parameters of hatching pool such astemperature, pH and dissolved oxygen were ranged from 26.90-27.90 C,7.01-7.14,6.48-7.86mgl ,respectively.Similarrangeof temperature, pH and dissolved oxygen was also recorded byChand (2011) for successful induced breeding of where temperature, pH and dissolved oxygenranged from 28.7–29.3 C, 7.2-7.4, 6.0-6.20 mg l , respectively.Whereas, alkalinity and hardness of water ranged higher (286.0-360.0and296.0-324.0mgl ,respectively)inthepresentstudyascompared with the result of Chand (2011) (alkalinity : 71-78mgl andhardness72-76mgl ,respectively)Moses (2016) reported May to July as inducedbreeding season of Pangas in different parts of world such asVietnam,BangladeshandThailandetc.InPunjab,midofJunetomid ofAugust was found suitable for breeding. Delayed maturity 4 et al. P.hypophthalmuset al. Htun-Han (1978) Results and Discussion o -1o -1-1-1 -1 Table 2 :StagesTime (hr : min)Description Embryonic developmental stages of Fertilization 0.0 hr Single cell at animal pole, periviteline space smallBlastodisc formation 0:25 ± 0.01 hr Polar body developedTwo cell 0:35 ± 0.02 hr Blastodisc divided to form two cell i.e., first cleavageFour cell 0:50 ± 0.04 hr Second cleavage perpendicular to the 1 cleavage plane, Four cell formedEight cell 1: 00 ± 0.05 hr Third cleavage parallel to that of the 1 cleavage plane, Eight cell formedSixteen cell 2:00 ± 0.08 hr Fourth cleavage stage parallel to 2 cleavage plane, 16 cell formedThirty two cell 2:15 ± 0.10 hr Fifth cleavage. Blatoderm divided into thirty two cellSixty four cell 2:30 ± 0.12 hr Sixth cleavage. Sixty four cells in 2-3 layers formedMorula 3:45 ± 0.15 hr Blastodisc consisted of many blastomeres. Numerouscells with flower like appearance of animal poleBlastula 5:30 ± 0.24 hr Blastoderm spreading gradually lowered and flattened. Appearance of epiboly with formation of blastocoelGastrula 7:45 ± 0.30 hr Blastoderm becomes flattened down onto the yolk sphere.Blastoderm covered almost 3/4th of yolk sac‘C ’  shape embryo 10.15 ± 0.50 hr Embryo looked like letter   ‘ C ’  over the yolk sphere. Head and tail become clear Twitching movement 25.00 ± 1.00 hr Tail become free from yolk sphere and beating movementto rupture the egg membraneHatching 34.00 ± 2.00 hr Transparent larvae with straight body and becomes freeswimming after a few moments of motionless stateValues are mean ±SE P. hypopthalmus ststnd Table 1 :Hatching time after pituitary injection12-17 hrs Parameters related with breeding of  Average fecundity 57.5 ± 0.0004 lakh kgColour of normal egg Creamish yellowColour of fertilized egg TransparentColour of unfertilized egg Opaque whiteFertilization rate 76.50 ± 0.60%Hatching rate 58.20 ± 1.35%Hatching time 34.00 ± 2.00 hr (at 27.33 ± 0.18 C)Diameter of egg before fertilization 1.25 ± 0.03 mmDiameter of egg after fertilization 1.80 ± 0.05 mmhardeningValues are mean ±SE P. hypophthalmus -1o andwater   JournalofEnvironmentalBiology, September2018 674  S.N. Datta et al. fertilization and 87-92% hatching rate, respectively. Relativelylesshatchingrateinthepresentstudymaybeduetohigherlevelof alkalinity and hardness in water (286.0-360.0 and 296.0-324.0mg l ) which might have deposited over the surface of the eggsrestricting supply to the developing embryo. Thedevelopmental stage of eggs was achieved up to C-embryonicstage. The yolk material migrated towards vegetative pole withtheprogressofembryonicdevelopmenttoprovideextraspacefor division of blastomere at the animal pole. Similar observationswithrespecttoovariandevelopmentwasalsoreportedbyMoses -1 oxygenFactor(K) 1indicatesgeneralwell-beingoffishundertheexistingwater quality. It also indicates health status and physiologicalactivity. In the present study, K value calculated 1.051 ± 0.06 and1.048 ± 0.15 in male and female fish, respectively indicating thatcultureenvironmentwassuitableforfish. Average 58.20 ± 1.35% hatching was achieved under agro-climatic conditions of Punjab with pituitary extract.Successful breeding of using same hormone was alsoreported by Chattopadhyay and Mazumdar (2002) with 77- 90% ≥ P. sutchi  FertilizationBlastodiscb.c.Two celld.Four celle.Eight cellf.Sixteen cellg.Thirty two cellh.Sixty four cellMorula j. Blastulak. Gastrulal.  ‘ C ’ shaped embryom. Immediate after hatchingn. 24 hrs. after hatchingo. 48 hrs. after hatchingp. 72 hrs. after hatching a.i. Fig. 1 :  Embryonic and larval development in  P. hypophthalmus  JournalofEnvironmentalBiology, September2018 Induced breeding, embryonic and larval development of P. hypophthalmus 675 around5.50-6.00hrofhatchingwithincreasingthesizeofembryoabout 4.75 mm. Alimentary canal was formed about 12 hr of hatching.After13-15hrsofpost-hatching,pigmentationineyewasrecognized. Yolk sac gradually reduced in size and yolk wascompletely absorbed about 60 hrs after hatching. Islam (2005)observed gradual reduction of yolk sac till 4-7 days after thoselarvaewerereadyforacceptingexternalfeeding.In ,firstfeeding started (diluted and filtered boiled egg yolk enriched withmultivitamins)after40hrsofhatching.Duringthistime,ifenoughfood particles of suited size were not provided, the larvae did notaccustom themselves to external food, became extremelycannibalistic within the next 24 hrs and died after sometime.Cannibalistic nature of young larvae was also reported inby Chattopadhyay and Mazumdar (2002) andIslam (2005) whereas in by Ferosekhan(2015).Boiled egg-yolk mixed with multivitamins was foundsuitable as starter feed at around 40 hr after hatching at 3-4 hr interval. Gradual administration of freshly filtered zooplanktonalongwithdilutedboiledegg-yolkat3-4hrintervalsfrom60-96hr after hatching revealed better growth of larvae. Diluted boiledchickenliverenrichedwithmultivitaminswereprovidedfourdaysonward till 12-14 days. Chattopadhyay and Mazumdar (2002)reported lactogen as starter feed (48 hrs after hatching) followedbyearthwormdustupto5-8daysandsoybeandustafter10daysonwards as larval feed of Hossain and Rahman (2014)reported boiled fish flesh paste enriched with multivitamins asless preferred starter feed, but diluted boiled egg-yolk wasacceptable and helped the larvae to accustom with externalfeeding habit at around 30 hr after hatching in Administration of a mixture of live but blended and Artificial plankton (APR) rather than blendedalone at 3-4 hr intervals during 36-40 hr after hatching wasfound better with negligible amount of larval mortality.Therefore, it can be concluded that variability in larval feed maybe adjusted as per the feed acceptability of larvae andavailability of larval feed in particular region and at the sametime, time of first acceptance of feed by newly hatched larvaeshould be observed critically to reduce the mortality due tocannibalism. Successful culture and brood stock developmentof through overwintering under polyhousecondition and successful induced breeding trials in Punjabclearly indicate potentiality of this species for productivityenhancement, as well as vertical and horizontal expansion of aquaculturesectorinnorthernstatesofIndia.The authors are thankful to the University GrantsCommission, New Delhi, India for providing financial assistanceduring the course of this research work and to the Dean, Collegeof Fisheries, Guru Angad Dev Veterinary and Animal SciencesUniversity,Ludhianaforprovidingfacilitiestocarryoutthiswork. P.sutchi Pangasius sutchi P. hypothalamus et al.P. sutchi.P. sutchi.Tubifex Rotifer Tubifex P. hypophthalmus Acknowledgments et al. P. hypophthalmusP. hypopthalmuset al. Pangasius pangasiuset al. Hemibagrus nemurus (2016) who stated that fertilized eggs of were sticky in nature and with the advancement of embryonicdevelopment; egg membrane got separate giving birth to theuniform perivitelline space. The yolk sphere migrated towardsvegetativepolewithfurtherembryonicdevelopment.Embryonic developmental stages of ispresented in Table 2 and Fig. 1. Cleavage in fertilized eggsoccurred only in the blastodisc region. The cleavage wasmeroblastic type. Cytoplasm of the blastodisc became theembryo, thus meroblastic cleavage was discoidal (Harrison,2011). About 20 minutes after fertilization, eggs became water hardened.Blastodiscformationstartedat0:25±0.01hr(Fig.1b).The 1 division took place in 0:35 ± 0.02 hr. minutes after fertilization (2-celled stage) and the 2 division within 15 minthereafter(4-celledstage).The3rddivisiontakesplacewithin1hr of fertilization (8 celled stages). Sixteen (Fig. 1f), thirty two (Fig.1g)andsixtyfour(figure1hr.)cellstageappearedat2:00±0.08,2:15±0.10and2:30±0.12hr,respectively.Morula stage appeared at 3:45 ± 0.15hr after fertilizationwithaballofcells,clearpolarprojectionandtheconvexbase.Theblastomere was divided into many cells at this stage andappearedasacompactmassofcells(Fig.1i).Blastulastagewasobservedat5:30±0.24hrwhichischaracterizedbyformationof  ‘ blastoderm ’  and  ‘ periblast ’ . The embryo was developed fromblastoderm,andperiblasthelpedinchannelizationofyolkmaterials(Fig. 1j). Gastrulation was characterized by formation of three-layered structure (ectoderm, mesoderm and endoderm) known asgastrula(Harrison,2011).Gastrulastageappearedat7:45±0.30h(Fig. 1k) after fertilization. The  “ C ”  shaped stage was observed at10.15 ± 0.50 hr. (Fig. 1l).At this stage, head and tail of the embryowas recognized and further progression of time of another 6-7 hr differentiationoffins,headandtailregionwereclear.TimerequiredfordevelopmentofeggsfromfertilizationtoC-embryonic stage varied. Ferosekhan (2015) reported  ‘ C ’ shape embryonic stage at 09:29 hr. Moses (2016) reportedthesamestageabout12hrafterfertilizationfor In the present study, time required to reach  ‘ C ’  shape embryonicstage was about 10.15 hr these differences may be due tovariation in age, maturity stages of ova and agro-climaticconditions. Twitching and hatching stage was observed at 21.30hrofpostfertilization.Thestageischaracterizedbyyolkattachedwithhead,beatingoftailofembryovigorouslyforapanof5-8secwith a pause of 2-5 sec. Hatching started at 25.27 hr of postfertilization. Immediate by after hatching, the larvae remained atthe bottom and remained motionless. After few moments,activenessoflarvaewasobservedwithincreasingtailmovement(Fig. 1m). Immediate after hatching, larvae measured about 3.80mm characterized by bent head attached with yolk sac. Mouthwas not visible though mouth cleft was visible after 15-17 hr.TheambiguityofmouthformationwasalsoreportedbyFerosekhan(2015) in (varied 12-13 hr) and by Adebiyi (2013) in (immediate by after hatching). Eye rudiments, optic and auditory vesicles appeared stnd
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