HAWAU-G-99-004 C2 I.OAlilCOPV OSLO' Aquafarmer Information Sheet Spawning the tinfoil barb, Barbodes schINanenfeldi in Hawaii by Richard Bailey and Brian Cole Aquaculture Extension Agents, Sea Grant Extension Service School of Ocean and Earth Science and Technology, University of Hawaii
Introduction or aviulability of niitural feeds phytoplankton and zooplank- ton! Ior the developingfry. The tinfoil barb,like other related The tinfoil barb, Brir/rodes sclivva»arly Mi, is a common species, is an cgg scattcrcr, laying eggs over a substrate such fish found in thc international aquarium trade and food mar- as plants or gravel to help hide and protect the eg s and larvae kets in Southeast Asia. Tinfoil barbs have a dorsal, ventrally from predators. f!attened body covered with moderately large, silver, chrome- Unfortunately, the tinfoil barb will not reproduce natu- like scales and rcd fins with a red leading fui ray Figure I!. rail> in a captive cnvironmcnt. Certain cnvironmcnta! cucs, Tinfoil barbsare a peacefulfish thatcan reacha lengthgreater suchas lunar phase, photopcriod, temperature, feeds, substrate, than 40 centimctcrs or 16 inches, and like other, larger barbs, cic,, stimulate a complex of'hormonal interactions that con- can often live morc than 10 years in captivity. In nature, tinfoil trol reproduction in fish. These environmental cucs inust be barbsare primarily macrophages;that is, they feed on vascu- presentfor most fish or the fish specieswill not matureand lar aquaticplants. However, they will cat mostconunercialfy spawn in captivity. Fortunately, the endocrine system of many prepared diets consisting of vegetable and fish meal proteins. fish is relatively well understood. Thc complex hormonal mechanismscontrolling matunition of'oocytes oocytes are Figure 1. maturingeggs inside thc femaleovaries; once spawned, they A Female arc called eggs! and ovulation in fish can be bypassedthrough induction with hormones. Ifhormoncs are administered to the tinfoil barb. fish by injection at the right time and dose, thc fish can be deceived into responding as though thc right environmental stiinuli are present.This will initiate a sequenceof natural hormonal changes that will lead to final oocyte maturation. ovulation and spawning. Some fish will naturally spawn eggs Thc genus Barbodesis closely related to the genera in captivity after hormone induction, while others must be Calioetaand Punriu» found in the subfamily Cyprininac.Thc "hand stripped" of eggs and milt. taxonomy of'these three gcncra are often treated as being syn- onymous.These closely relatedspecies inhabit the freshwater lakes, rivers and tributaries of Southeast Asia. Water quality Facilities parameters of the tinfoil barb's natural habitat ranges from soft to moderatelyhard water Cicrmanscale dH 5- 12', or 100to Successin hormonally inducedspawning in thc tinfoil 200 milligrams per liter [equivalent to parts pcr tnillion] cal- barb and other related species results from a good working cium carbonate!. pH of 6.5 to 7.0, and a temperature of 22 to knowledge ofhormone induction as well as adcquatc support 25 C [72' to 78 F]! Tinfoil barbsare a hardyfish that cando facilitics. Broodfish,holding hatchingand larval rearing tanks well in conditions exceeding the water quality parameters of are important to thc successof producing larvae. the i r natural habitat. Broodstock tanks or ponds should be large enough In I lawaii, tinfoil barbs become sexually mature from >1,000 gallons! to hold two to three times the number of May tlirough July during the onset of rising water tempera- broodstock required to produce the desired number of eggs. ture, 25' to 27'C 7' to III'F!. Many fish naturally spawn Two scparatc.static 50- to 150-gallonholding tanks during seasonalchanges, such as an increasein tcrnperature
CTSA Publication Number 136 January 1999 Spawning the tinfoil barb Page 2
equipped with thermostatic controlled heirter s! are neededto the males by gently pinching thc fish between one's fingers and maintain a constant temperature to hold sexed broodstock fish sliding down the abdominalregion towardsthe anterior genital duringthe induction process. Fish are uoi fcdv hilc theyare in opening or papillae. whitish clear fluid or milt will come out of thc hoMingtanks. Static tank systemsv ork well if the density the rnafc'sgcnit;il papillaewhen it i» sexuallymature and ripe. is kept low, for example,I kilogram pcr 189 liter» or 4 to 5 Tinfoil barb»can achicvc»cxualmaturity by their first year. fish pcr 5 ! gallons. High w«ter quality shouldbc maintained I lowever, two-year-old fish are most commonly used for induced during spawning induction, spawningbecause they producea largernumber of eggsper spawn An cgg hatching systemthat consistsol a hatchingjar and often a grc«icr fertilization rate. Onc-year-oldtinfoi! barbs anda Iarv«1rearing tank is nccdcd.Usc of a flow-throughsys- canbe usedfor»pawning if the femaleshove f'ully developedoo- tem i» recominendcdto eliminatewater quality problems.IKc- cytcs and if the males show milt. Female tinfoil harb» 200 io 300 circulatingsystems are not rccom»iendedbecause they arenot gram»in sizecan produce5,000-plus eggs pcr»paivn. often biologically c»tablishcdand rcquirc additionalmanage- ment skills. Hatchingjar»ysteiiis require,on the avcragc,I to 2 liters per minute of water flow, basednn the egg density in Determining Female Maturity the jar. In Hawaii, I'emaletinfoil barbsbegin to developoocyte» in The larval rearingtank shouldbc largeenough to handle March or April and maturein IVlaythrough July. Farmersshould thc potentia!amount of !arvaefrom a spawn,For example,a harvest brood»tock from thc broodstock ponds and select a num- 30-gallon glass aquarium can hold two spawns of tinfoil barb ber of large femalesthat havevisibly well roundedabdominal re- larvae 0,00 ! or 88 pcr liter! for a periodof'onc weekwith a gion or ovariesand weigh within approximately10 percent of each waterexchange rate nf 21itcrspcr minute 60 gallonspcr day other. The broodfish shouM bc handled gently, and their time out pcr tank! bctbre requiring a reductionin density. of water should be limited by pl ~cingthem into bucket »! with Conical fiberglassor acrylic tanks are now becoming quinaldinc for quinaldine mixture seeTable I !. Sedatingbrood morecommonly used duc to their improvedwater circulation fish helps limit »tres»«nd rn!ury during handling at this critical andefficient removalol wastes,which improveswater quiility stageprior to»pawning, and larval management. Determiningoocyte maturity rcquircs removing «»ample of oocytcs -30! from the ovariesot the fish using 1.14millimeter I.D. cannulatubing also called "microborc tubing" in chemical Broodstot:k supplycatalogs!. The tubing is madefrom flexib!c tygon. sihcon, One-year-oldflsli should bc isolatedf'rom growout fa- or polycthylcncmaterial that can be gently in»citedinto the gc»i- cilitiesand placed into separatebrood»tock rearing ponds or tal ventand movedup the oviductto the ovariesof'thc fish without I;irgetanks at a stockingdensity of 2 ki1ogramsper cubic meter causing injury. I fish per 25 gallons!. The fi»h arc kept at this density for a After the fish arecompletely sedated, which they exhibit by year io achievefully maturesize, to reduce»tressand discase being upsidedoivn and not reactiveto touch. they can bc staged prob!cm»,and to ensureproper nutrition and water quality, lor maturity.Thc cannulatubing shouldbc inscrtcd into the geni- Two brood»tock ponds or tank~ should bc maintained for each tal vent while gently tv i»tingand working thc tubedown ihc oi i- sex to ensurequality stock for spawningat a scx ratio of ai duct 2 to 3 inches to the ovary Figure 2!. Once in, the cannula least two malesto onc female. Broodfish should be given a shouldbe placedin th» farmer'smouth and gent!esuction should propercommercial dict consistingof at least30-plus percent be applied.The endof the tubing shou!iIbc held with one'sthumb protein in two or morerations ai a rateof I to 2 percentol'ih» while retractingthc tubecontaining the oocyte sample, which shoukl tot«l biomass weight TI3W! per day, Most commercial cat- be 30-plusoocytcs, from the fish, 'I' he fish shouldbe placedinto a lish and trout productiondiets are suitable,Floating feeds are numbered, aerated bucker that is marked "r! I. ' The fish will re- often used; these allow a farmer to oh»eree thc animals' feed- vive tully from thc quinaldine sedation within 2 to 5 minutes. ing response and gauge the population health. Placethe dry oocyte sampleon a graduatedglass micro- scope»lidc to determinethc oocyte'sdiameter. Ripe tinfoil barb oocytes are mature at«mean size of 1.12 millimctcrs. If the oo- Sexing Broodstock cytcsarc 1.12mill imetersin size,they shouldfir»t bc checkedfor Maturetwo-yc«r-old fish, 200 to 300 gramsin size,can overripeoocytcs, which can be distingui»hcdby the presenceof be visually sexedusing externalch iracteri»ties,Ripe Ii:male space betv een the oocytes' internal content and thc mcmbranc tuzforf barbs can bc identified by their»wollen abdominal re- Figurc 3!. indicatedby a withdrawa!of the oocyte'sinternal con- gion and smooth»cafes,M«lc fi»h are morc slenderin body tentsfrom ihc outcr wall. If the oocytcsarc not overripeand have shapeand havedi»tinctive rough tubcrcles pimple or sand- a good density,a couple of drops of "oocyte cgg clearing solu- p«pcr-like bumps! on their scales. One can extrude milt from tion" Table 2! shouId he added to the oocytes, ivhich will iillow the farmer to betterjudge migration of the nucleus.Vithi» 10
Center for Tropical and Subtropical Aquaculture Publication Number 136 January 1999 Spawning the tinfoil barb Page 3
n
b c
Figure 3. Oocyte staging a. Centered nuclei immature!; b. Off-center nucleus ripe!; c. Yolk materiai pulled away from oocyte wall overripe!.
the middle or peaknl the seasona»d then taperingofT.
Figure 2. Extracting oocytes from a tinfoil barb. Male Brood Fish Selection A sex ratio of one f'emalc to two male» v ill bc needed for artificial sp nvning.Thc I'armershould select large i»ales exhibiting roughtiiberclcs or scalesfrom the broodstocktank. minutes.the oocyteswill clear, and their maturity can be staged. The i»ales should be checked for milt by the procedure stat»d Thc nucleusin thc oocyte.which is»»cn;isa densespherical mass previously.'I hc averageweight for the male fisli should hc within rhc oocyte.»ho«id be located.Oocytcs with centcrcdnuclei dctermirred. tire» the fish should bc placed into a holding tank arenot yet ripe, Ripe nocytcscan bc idcntificd by nuclei that have at a density not greaterthan l kilogram per 189 liters 0 migratedoft'-center toward the outercell waII of'thc oocyte Figurc gallo»s!at ambient temperature. Tlie Iish should»nt be fed at 3!, I'ish that have cc»iered nuclei should be returned to thc this time. brondstnck pond and rechecked in two wccks. The hestf'ish with highestdcgrcc of nuclei migration should bc sclcclcdI'rom thc sampledfish, I.rom that »ubsct,frsfi thatweigh within !0 percentof'each other shouldbe selectedso that one hor- Hormone Mixture Preparation mone mixtiirc can he made f'or thc entire sct of fish, thus simplify- Tinfoil barbscan bc hormonally inducedto ovulateby ing thc induction steps. usins>a mixt«re of Human Chorionic ionadntrnpin HCC>! Severalpubliccitions describe oocyte staging;iii is avail- Selectedripe fish are sedated,towel dried and vverghedto ablc in vials of 10,000 I.L!., and CPI. is often sold by thc gram the nearestgram, An avcragcweight is calculatedby totali»g the in whole or ground form. weight anddihiding by the numberof fish. Fish that weitrh 10 per- The tnial volume injected I'or male and fbmalc tiiiloil cent moreor lessthan the averagebody weight shouldbc returned barbs is 0,5 cubic centimeters or 0.5 milliliiers The total in- to thc brood»tocktank and u»edI'or the next »p;iwningcvcirt. jection h olumeis dele»»i»edhy body v eight.The total injec- The idcntificd ripe female lish are then placed into ii »tatic tion volume should not exceed 0.5 cubic»entimctcrs I.'or fish hokling lani
Center for Tropical a»d Subtropical Aquaculture Publication Number 136 January 1999 Spawning the t nfoil barb Page 4
Preparing a Stock Mixture of HCG Example: UsnLempty HCG bottleswork well for storingand hold- Eight mature female broodstock that v'eigh within 10 per- mg mixtures of hormones. Thc bottle is opened, washed,dried centof eachother aud leave fully migratednuclei haveb««n placed and seaIedwith thc rubber stoppers«cured with elastictape in a holding tank fitted with aeration and thermostatically con- electricaltape works well'!. Ivlicrolitcr syringes I cubic c«n- troll«d heaters sct at 27.2' .'. 82'F!. Sixteen mature males have timctcr = 1milliliter = 1,000microliters! work well for taking beenselected and placed into a holdingtank at ambienttempera- »mallamount» of hormone from stock solution bottles. When ture. spawning a few tish, the amount ol hormone needed from thc Calculating Female Hormone stock solutions of hormone i» often quite small. The stock so- Average weight of each tcmal« I'ish lution canbe further diluted to allow Ibr great«rvolumes to bc used to gain more accuracy in mixing hormone amounts when 1,680grams total weight di vid«dby 8 fish =-210 grams spawning just a few tish. However, be aware of thc total vol- average weight pcr fish umeof llorlilolie to inject, ~ One "ghost fish" is added to make up for hormone loss in Farmers should moke a stock mixture of I ICG so that syringe and bottle small amounts ofhormone can be removed iit ii time, while not risking contaminationor spoilageof the remainingbulk mate- Thus, 8+ I = 9 fish 1,680 210 1.890 gramsor I.89 rial. The hormone comes in dry form in a scaled vial and is kilogram» total weight used in calculating hotmonc often accompanied with bacteriostatic water. Ten cubic centi- volit l1lc meter» of »tcril«, bactcrio»tatic water should be added to the Amount of HCG Needed dry 10,000 I.U. vial of HCG. This will result in a conc«ntra- tion in thc vial of I cubic centiinctcrequaling 1,0001.U.HC'G 1.89kilograms mrrliiplierl by 60 InternationalUnits I,U,! or 0.1 cubic ccntimct«r«qualing 100 I,U. Thc vial shouldbe per kilogram 113,4 I.U. HCG stock I FCC!vial concen- marked"HCG I cc/1,0001U" with a greasepencil. tration = 1cc = 1,000 1. !.! Preparing a Stock Mixture of CPE 113.4I.U. IICG diiidcd by 1,000I.U..'I cubic centimeters = 0,1134 cubic centimeters A stock mixture of carp pituitary extract CPE! can be madeby placing 250 milligrams of finely groundCPF. into a Take 0,1134 cubic centimeters, or 0,! I cubic centimeters clean vial. A tissue grinder can bc used to grind CPE into a of stock11CG solution with a»yringe andplace it into a fine powder.Care must be taken to prevent thc ti»sucfrom ncw clean vial marked "female' getting warm while grinding with a tissue grinder. Overheated Amount CPE Needed tissuewill result in a lossinpotcncy. Fivecubic ccntimctersof bacteriostatic water should then be added to the vial and mixed ~ 4 milligram» per kilogram CPE mrrltil>lied by 1,89 thoroughly. This will make a stock ~olution with a concentra- kilogram 7 56 milligram» CPE stock C'.PEvial concen- tion of 1«ubiccentimctcr equaling 50 milligrams CPE. Thc tration = 1 cubic centimeter= 50 milligrams CPF! vialshould be marke Center for Tropical and Subtropical Aquaculture Publication Number ! 36 January !999 Spawning the tinfoil barb Page 5 marked "fern,tie' !gin Place the vial in thc refrigerator until first injection at 2 AM Calculating Male Hormone Male broodstockfish requireonly 20 percentof the fern;il« hormonedosage and onc injection. Thc proceduresto obtain the niale dosage are the same for calculating thc dosage 1'or thc fe- males and multiplied by 0.2 0 percent!. Horinones are used io siiinulaic an increase in the total volume of'milt to incrcasc fertiliz- ationn success and sychronize final maturation in both male and fcmalc fish. 1 sing the example above. 16 male fish were selectedby gcn- lly applying pressureanterior toward thc vent which produced milt. The fish werc weighedto the nearesttenth of;i gr;im, 16 males = 3,680 grams = 230 grams average weight each ~ Two "ghost fish' ;iverage weight of'males used! arc added to tlie make up for hormoneloss during volumetransfers I 8 males = 4,140 gram» 36b0 + 230 + 230! ~ 4,140 grams = 4,14 kilograms Figure 4. nj ecting the tinfoil barb with hormones. Amount of HCG Needed 60 I.U. Hali pci kilogram multiplied by 4.14 kilogiams = 248.4 ,U, Add 8,42 cubic centimeters bacteriostatic water to thc stock HCC! vial concentration = lee = 1,0001.U.! male vial to bring the total volume to 9.0 cubic centimeters,Pla«c thc vial in thc refrigeratoruntil 248,4 1.U. 1 fC'Cidivided by 1,000 EU. per 1 cubic needed at 8 AM. centimeters 0.24 cubic centimeters When using very small volumes in setting up mixtures Take 0.24 cubic centimeters of stock HC'Ci soluiion with a and for administeringsmall dosages,use of a micro-syringe syringe and place it into a nevv,clean vial m irked "male" of 100 microlitcr , lee! to 500 microlitcrs is recommended to morc accurately withdruvv the proper amount of. hormone Amount CPE Needed from stock bottles.A standard,1-eubic-centimeter insulin sy- 4 milligrams C'PE p«r kilogram multiplied by 4.14 ringe with a one-half-inch ¹22-gauge ncedlc works well for kiiograms = 16,56 milligrams CPE stock CPE vial inj eel ing tin!oil barbs. eonecnlration 1 cubic centimeters 50 milligrams C 'PE! 16.56 milligrams CPE divided by 50 milligrams / cubic ccnliiiieier = 0.33 cubic eeniiineicrs CPE Hormone Injection At 2 AM, the feniale brood fish should be netted from ~ Take 0.33 cubic «entimeters of stock O'Pl-.'solution with a the tcmpcraturc-controlled holding tank and placed into a syriiige and place it into the vial marked "m«le" bu«ket«ontuining water from thc holding tank with quinal- Volume Needed for Males dine 00 to 150 mi]liliters of'stock quinaldine solul.ion per 2 Total injection volunie per mule 0.50 cubic centimeters! to 3 gallons water! to sedate thc lish. Once sedated, the first iiijcciion ol O.l cubic centimeters 0 percent uf 0.5 cubic 0.25 cubic centimeters HCG 0.33 cubic centimeters C'PE centimeters! should bc administered by inserting the needle = 0.58 cubic ceiitii11eters under a scale near thc muscular areajust belovv the dorsal fin 18 fish multiplied by 0.5 injection volume per fish! 9.0 ray about0,5 inchesin Figure 4!. The syringeplunger shoufd cubic centimeters tota1 hormone solution needed bc slowly depressedto inject thc homione. The needle should bc vvithdrawn and a finger should bc placed over thc injection 9.0 cubic centimeters - 0.58 cubic centiineters Center for Tropical and Subtropical Aquaculture Publication Number 136 January 1999 Spawning the tinfoil barb Page 7 ucd until the eggs arc clean of blood or rhc water is clear, A portion of thc water from thc cgg hatching jar sometimes calleda "McDonald'sjar," Figure6! shouldbc removedby placing a handinto the jar and displacingsome water. Then thc eggsshould be poured into the jar, Thc water tiow into thc jar should be adjustedso thc eggsare gently tumbling and not forced near the top of thejar. Thc eggs will expand up to ten times their volume after fertili- x;ition. As the eggsswell, they becomeless dense, so the wa- ter flow rateinto thejar will requirefine adjustmentto keep the eggsfrom overflowing into the larval tank. Determining Fertilization Rate 1'irstcell division, or cleavage,can be seenone hour afterfertilization. At thattime, bctwcen 20 and 30 eggs should bc takenfrom the hatchingjar andplaced on a microscope slide. A couple of drops ot'cgg clearing solution should bc added,AAer waiting a fewminutes for theeggs to clear,they canb» checked for thepercentage of'fertilization. Dead eggs will appearopaque white in coloras compared to viableeggs, whichwill betranslucent. Divide the number of viableeggs by thcnumber of deadeggs in thesample to estimatepercent- ageof ferailrzation.Eggs will hatchin 24hours at 28' 82'F!. A largenumber of deadeggs will greatlyaffect the viability ol' th» remainingfev. Conclusions Techniquesusing hormones to inducespawning of fish havebccn around for morethan 40 years,Common fish spe- ciessuch as koi, goldfish, kuli loaches,redtail blackand rain- bowsharks arc routinelyinduced to spawnusing hormones. 1 owever,no one standard protocol works for all species.Fish reproduction is regulated by both external environrncnlal cucs and internal endocrine mechanisms. Hormones are used to intervenein a chainof internalcvcnts that control reproduc- tion in fish, Althoughthe preparationol hormones.screening cgg quality iu broodlish, establishingholding tanksand a hatch- ery requireextra effort andresources, the basictechniques arenot overly involved. Using hormones to inducespawning in f'isht'acilita esrhc production of a largenumber of eggs simultaneously,which can incrcasc hatchery ct7icierrcy and pfit. CenterI'0forTropical andSubtropicalJanuary Aquaculture 1999 PublicationNumber136 Spawning the tinfoil barb Page 8 Recommended Reading Acknowledgments Rottmann, R,. Shircman, J., 8r.Chapman, F., 1991. Introduc- All photos were taken by Rich Bailey and Clyde Tamaru, tion to Hormone-Induced Spawning of Fish. USDA University of Hawaii Sea Grant Extension Service. SRAC pub. ¹421 This work was part of a project titled "Expansion and Diversificationof FreshwaterTropical Fish Culture in Hawaii," Rottmann, R., Shireman, J,, k Chapman, F., 1991. Captur- which was funded by thc Ccntcr for Tropica1 and Subtropical ing, Handlmg, Transporting, Injecting and HoMing Brood Fish tor InducedSpawning. USDA SRAC pub. Aquaculture through a grant from thc U.S. Department of Ag- riculture Cooperative State Research, Education and Exten- ¹422 sion Service USDA grant ¹96-38500-2743!. Rottmann, R., Shireman, J., k. Chapman, F., 1991. Deter- This publication was funded in part by a grant/coopera- mining Sexual Maturity of Broodstock for induced tive agrecmcntfrom the National Oceanicand Atmospheric Spawning of Fish. USDA SRAC pub. ¹423 Administration, project ¹A/AS-I, which is sponsored by the Rottmann, R., Shireman, J., k Chapiuan, F., 1991. Hor- University of Hawaii Sea Grant College Program, SOEST, monal Control of Reproduction in Fish for Induced under Institutional Grant No. NA36RG0507 from NOAA Of- Spawning, USDA SRAC pub,¹ 424 fice of Sea Grant, Department of Commerce, This publication was funded in part by thc Aquacullurc kottmann, k,. Shireman, J,, &. Chapman, 1',, 1991, Hormone Development Program, Hawaii Department of Agriculture, as Preparatioii, Dosage Calculation, and injection Techniques f'or induced Spawning in Fish. USDA part of the Aquaculture Extension ProJcct with the University SARC pub. ¹ 425 of Hawaii Sea Grant Extension Service Thc views expressed in this publication arc those of the Rottmann, R,, Shireman. J., & Chapman, F,, 1991.Tech- authors and do not necessarily reflect the views of CTSA, niquesfor Taking and Fertilizing the Spav,n of 1'ish. USDA, NOAA, ADP or any of their sub-agencies UNIHI- USDA SRAC pub.¹ 426. S EA CrR A NT- TR-98-04!. Sea Grant Extension Service University of Hawaii School of Ocean aod Earth Science and Technology 2525 Oorrea Rd., HIG 237 Honolulu, Hl 96822 CTSA Publication Number 136 January '1999