The Israeli Journal of Aquaculture – Bamidgeh 58(4), 2006.266

A Review of Ploidy Manipulations in Aquaculture: the Israeli Experience

Shmuel Rothbard*

YAFIT (R&D) Laboratory, Fish Breeding Center, Gan Shmuel 38810. Israel

Key words: androgenesis, aromatase inhibitor, chromosome set, dimensionless factor τ0,embryonic age, gynogenesis, neomales, ploidy manipulation, polyploidy

AbstractPloidy manipulation was introduced to Israel at the beginning of the 1980s. To improve existingmethods, Israeli fish centers conducted intensive research in this field for more than twodecades. Chromosome-set technology was adapted for ten fish species and varieties, includingtwo marine fish. Presently, though methodology achievements are remarkable, practical imple-mentation is applicable only for the common carp. The aim was to integrate gynogenesis andandrogenic sex-reversal to generate neomales with special genetic traits in this economicallyimportant species that is extensively cultured in Israel. Carp neomales can serve as parents forproduction of fast-growing all-female populations. This paper describes ploidy manipulationprogress and activities carried out in four Israeli fish centers. Future objectives include (a) imple-mentation of the existing know-how to produce tilapia YY-males as broodfish for producing male-monosex populations, (b) dissemination and promotion of export to foreign markets of theadvantageous female triploid grass carp and black carp, (c) continuation of genetic work withmarine fish to develop breeds that could be advantageous for mariculture, and (d) reconsidera-tion of the ploidy-manipulated koi as a genetic tool to be utilized to facilitate gene-mapping incarps.

IntroductionEarly investigations of ploidy manipulation infish have been reported by Stanley (1976) forgrass carp, Ctenopharyngodon idella V., byNagy et al. (1978) for common carp, Cyprinuscarpio L. and by Chourrout (1980) for rainbowtrout, Salmo gairdneri R. Techniques of chro-mosome-set manipulations were introducedto Israel in the early 80’s of the last century.

The ploidy manipulation technology wasbrought to attention of the Israeli aquacultur-ists when a group of three Hungarian scien-tists, headed by Dr. J. Bakos from FishCulture Research Institute in Szarvas, visited

Israel to meet Israeli aquaculture scientistsand fish farmers at Dor Aquaculture ResearchStation, and to participate in an round-tablediscussion on recent advancements inHungarian aquaculture. One of the major top-ics that stimulated deep interest of the Israeliparticipants was a lecture on ploidy manipula-tions held in Hungarian and simultaneouslytranslated into Hebrew.

In Israel, first attempts to introduce thetechnology of induction diploid gynogenesisand polyploidy, was carried out with tilapias inthe Gan Shmuel Fish Breeding Center

* Tel.: +972-6-6320008, fax: +972-4-6320404, e-mail: rot_bard@gan.org.il

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(Chourrout and Itskovich, 1983). Some yearslater, chromosome-set manipulation wasintensively researched at Dor Station (Cherfaset al., 1990) in order to adopt the technologyto Japanese ornamental (koi) carp.

In the last two decades four fish culturecenters were involved in investigation of chro-mosome-set manipulations, the AquacultureResearch Station – Dor (Ministry ofAgriculture), the YAFIT (R&D) Laboratory inkibbutz Gan Shmuel, the National Center forMariculture (IOLR - Eilat) and the Laboratoryof Fish Immunology and Genetics – Bar IlanUniversity.

Israeli scientists involved in this technolo-gy have investigated various aspects of ploidymanipulations during 25 years of research.The following subjects were selected forresearch: methods of shock application (type,level, duration, etc.), UV-irradiation (intensity,duration, conditions, etc.), embryonic agerequired for external intervention (τ0 factor),and the use of genetic markers (colors, scala-tion, fin shapes, chromosome structures,etc.). All these factors are essential to identifyand to achieve success at the process ofploidy manipulation. The possibility of storagecarp eggs prior to fertilization for short dura-tions was also investigated (Rothbard et al.,1996).

Although the methodologies and technolo-gy of ploidy manipulation have been welldeveloped and well established for variousfish species, the commercial implementationof the methods have been rather unsatisfacto-ry due to several constrains. For instance, inspite of generating the advantageous femaletriploid (XXX) grass carp, C. idella or blackcarp, Mylopharyngodon piceus the mainobstacles of marketing abroad such uniquefish were caused by local legislations thatblocked importation of these species.Introduction of black carp in the U.S. has beendiscouraged due to ecological concerns andits use in African countries as a biological con-trol of bilharziasis lacked appropriate funding.

In this paper, chromosome-set manipula-tion activities carried out in Israel are sepa-rately presented for each research center.Among these centers only the YAFIT

Laboratory is associated with a commercialfish breeding facility and fish farm. The otherthree institutions are public or national organi-zations: the Israeli Ministry of Agriculture, theIsrael Oceanic and Limnological Research,and Bar-Ilan University (Table 1).

Aquaculture Research Station – DorGynogenesis and polyploidy in Japanese(koi) carp (Cyprinus carpio). Exposing thezygote to shock is necessary to increase theincidence of diploidy or to induce polyploidy inthe haploid embryonic genome. A series ofstudies were carried out to evaluate and opti-mize early and late application of heat- andcold-shocks in koi (Cherfas et al., 1990).

Dor scientists used the τ0-parameter(Dettlaff and Dettlaff, 1961) to determine theinitiation of shocking and obtained the highestyields of diploid meiotic or mitotic gyn-geonotes by heat shocking when the τ0-dura-tion corresponded to the end of meiosis (sec-ond polar body retention – 2PB) or the firstmitotic cleavage at metaphase and anaphase,e.g. 0.15-025 τ0 or 1.5τ0 after insemination,respectively (Cherfas et al., 1994a). In cold-shock experiments, the highest yields of mei-otic and mitotic gynogenotes were obtainedby applying the shock at 0.05-010τ0 and 0.39-0.49τ0, respectively (Cherfas et al., 1994b).

Spontaneous diploids were detectedamong hatched larvae in haploid controls.Such diploids may have occurred due tospontaneous diploidization of the maternalgenome (Stanley, 1961; Cherfas et al., 1991)by non-disjunction occurring during the first orsecond meiotic division (Komen et al., 1991).The same phenomenon was detected in koi(Rothbard, 1991).

Triploid common carp. Triploidy in com-mon carps was induced by heat-shocking nor-mally fertilized carp eggs of the Dor-70 line.The triploidy was evaluated in larval stages bythe number of nucleoli per nucleus or quantifi-cation of DNA in the nuclei of erythrocytes andin the majority of the fish, and by comparingsizes and shapes of erythrocytes and theirnuclei to their respective diploid controls(Cherfas et al., 1994).

To assess growth potential of the triploid

BARD Workshop: Aquaculture Genetics – Status and Prospects

and diploid common carp (C. carpio), experi-mental groups of fish were examined in com-munal ponds (fish groups, marked by differentbrandings, cultured in the same pond; seeWohlfarth and Moav, 1985). In general,triploids grew slower than diploids and triploidmales grew slower than triploid females(Cherfas et al., 1994a).

Integration of gynogenesis and hormonalsex-inversion in common carp. Gynogeneticoffspring (female monosex) of carp and koiwere sex-inverted with methyl-testosterone(MT; 100 mg/kg food) and yielded high ratesof neomales (40-95%). The success of MT-treatment was dependent on water manage-ment (recirculated versus running water) and

268 The Israeli Journal of Aquaculture - Bamidgeh 58(4), 2006

Research center

Fish Culture Research Station,Israel Ministry of Agriculture,Dor

YAFIT (R&D) Laboratory, FishBreeding Center, Gan Shmuel

National Center forMariculture, IsraelOceanographic andLimnological Research, Eilat

Fish Immunology andGenetics, Bar Ilan University

Chromosome-set manipulation activity

Gynogenesis in koi

Triploidy in common carp

Integration of gynogenesis and sex-reversal in common carp

Gynogenesis and androgenesis in koi

Gynogenesis and polyploidy in black carp

Gynogenesis and polyploidy in grass carp

Gynogenesis in fantail goldfish

Computation of embryonic age in 9species of fishShort term preservation of carp (koi) eggs

Integration of gynogenesis and sex-reversal in common carp

Gynogenesis in gilthead seabream

Allotriploidy in gilthead bream, using redseabream as sperm donor

Gynogenesis and triploidy in tilapia

Androgenesis in tilapia

Current status

TechnologyavailableRetarded growthof 3N fishCommerciallyapplicable

TechnologyavailableTechnologyavailableTechnologyavailableTechnologyavailableτ0 ApplicationprovenApplicationprovenCommerciallyapplicable

TechnologyavailableTechnologyavailable

TechnologyavailableTechnologyavailable

Table 1. Chromosome-set manipulation activities in fish, as carried out in Israeli researchcenters (1980-2005).

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day of initiation of feeds containing hormones(27 versus 40 days post-hatching). Similarresults were obtained for koi as well as forcommon carps (Gomelsky at al., 1994).

Gynogenetic females were sex-invertedwith MT to produce neomales (XX-genotype;F1-generation). Sperm of these neomales wasused to fertilize eggs obtained from normalcarp to produce monosex females (the F2-generation) on large scale. The F2 femaleswere again sex-inverted using Fadrozole (aro-matase inhibitor - AI). The efficiency of sex-inversion using feeds containing MT (40 and100 mg/kg food) or AI (200 mg/kg food) wasevaluated by the yield of neomales. The rateof neomales resulting from AI-treated fish washigher (58.6%) than for fish treated by MT(4.7-9.4%).

Growth performance of carp female-monosex offspring derived from neomalescrossed with normal females was assessedfor commercial fish culture. The growth of thefemales prior to sexual maturation was about15% faster than of the males (Cherfas et al.,1996). These findings served as a significantstimulus to implement the technology andmake it available for commercial aquaculture.

YAFIT (R&D) LabPioneer work on ploidy manipulation in tilapia.The first attempts in Israel to induce chromo-some-set manipulations in tilapia were carriedout in the Gan Shmuel Fish Breeding Center,by Chourrout and Itskovich (1983). The deter-mination of ploidy levels in different groups offish was carried out by simple nuclear exami-nation. Eggs of Oreochromis niloticus werefertilized with homologous sperm and heat-shocked for 2-7 min at 30.5-41.0°C, four min-utes post-fertilization to retain the secondpolar body (2PB). High rates of triploid off-spring were obtained that reached the feedingstage.

An interesting hybridization experimentwas carried out involving a mouthbreedingfemale of O. niloticus and a male of the sub-strate spawner Tilapia rendalli. Although sur-vival of embryos was low in this intergenerichybridization, the yield of viable larvaeincreased dramatically when they were

triploidized. It is possible that O. niloticuseggs, activated with T. rendalli sperm andshocked, generated not only triploids but alsogynogenetic offspring of O. niloticus. Thisphenomenon was verified by karyotyping(Chourrout and Itskovich, 1983); the numberof chromosomes in tilapia of normal fish anddiploid gynogenotes was 44, but 66 intriploids.

The goal of these experiments wasachieved with successful induction of gyno-genesis in O. niloticus obtained from O. niloti-cus eggs inseminated with UV-irradiatedsperm of red Taiwanese tilapia (probably anO. mossambicus hybrid derivative), then heat-shocked at 41-41.5°C for 3.5 min, starting theshock 5 min after fertilization. Non-shockedzygotes yielded only haploids that did not sur-vive beyond the yolk-sac stage. The diploidgynogenotes were gray-pigmented and hadthe maternal phenotype, indicating successfulinduction of gynogenesis since the gray col-oration of O. niloticus dominates the recessivered coloration of the Taiwanese fish.However, at present, post factum, we wonderwhether these were truly gynogenotes or justnormal hybrids.

Meiotic and mitotic gynogenesis inJapanese (koi) carp. Experiments aimed toinduce mitotic gynogenesis (endomitosis) bylate shock were carried out in the YAFIT labo-ratory to generate broodstock that could serveas parents for the second generation ofgynogenotes, presumable clones (Rothbard,1991). Koi eggs were inseminated with UV-irradiated sperm of wild-type (dominant) col-ored carp that served as a color marker. Carpof the Dor-70 line with the recessive gene forgold coloration (Wohlfarth et al., 1975) wereexcluded from these experiments. The domi-nant wild-type coloration allowed evaluation ofsuccessful irradiation of sperm. Survival ofgynogenetic embryos from late-shocked eggsat the embryological age of 1.3τ0 (32.5%) washigher than those shocked at 1.6τ0 (14.7%).Spontaneous diploidization of the maternalgenome was detected among non-shockedcontrol embryos, as previously reported byCherfas et al. (1991) and Komen et al. (1991).

Meiotic gynogenotes of the second gener-

BARD Workshop: Aquaculture Genetics – Status and Prospects

ation of koi were produced by early heat(40°C/2 min) or pressure (ca. 7500 psi/2 min)shock from gametes of three 2-year-oldfemales previously generated by late-shockinduction. Survival of diploid gynogenetic lar-vae was significantly lower (2.1-7.6%) than inthe respective fertilization controls (39.8-64.8%). The yield of gynogenotes from thethree females was about 10,000 larvae.Although these should have been expected tobe clones, with colors similar to their mothers’(tri-color), when they reached fry size (2-4cm), the colors and color patterns were vari-able and not uniform. The color variationamong these putative cloned fish suggeststhat other factors affect coloration.Presumably, a combined effect of genetic(polygenic inheritance), environmental, andphysiological (hormonal) factors may changecolors even in mature and large size fish.

Evaluation of the mitotic duration (Dettlaffand Dettlaff, 1961) in fish species by using theτ0 parameter for timing ploidy manipulationsis an important tool for ploidy manipulation,since it provides a dimensionless measurethat incorporates the temperature effect onthe developmental rate, relative to the initia-tion of shocking the developing zygotes.Since τ0 is species-specific, this important cri-terion was defined for common carp, grasscarp, and black carp and computerized fornine different fish species (Shelton andRothbard, 1993; Rubinshtein and Rothbard,1997).

Androgenesis in koi. There are two objec-tives for inducing androgenesis in koi: (a) gen-erating YY-males capable of yielding exclu-sively Y-sperm and (b) preserving sperm ofunique koi in a sperm bank (Lubzens et al.,1993).

Carp females of the Yugoslavian (Nasice)line were selected as egg donors for koiandrogenesis. This strain, contrary to Dor-70carp, does not carry a recessive gene for goldcoloration (Wohlfarth et al., 1975) that inter-acts with the coloration of koi and thereforecan serve as a color marker for the identifica-tion of androgenetic offspring.

The highest survival of androgenetic koilarvae resulted from common carp eggs irra-

diated with a UV-dose of 2500 J/m2 andimmersed in natural ovarian fluid to preventpost-activation stickiness. The inseminatedeggs were heat-shocked (40°C for 2 min) dur-ing metaphase of the first mitotic division atthe embryonic age of 1.5-1.7τ0 (survival 5.4-7.8%). Most of the hatched larvae were mal-formed, probably due to increased homozy-gosity. Although the fry survived for severalmonths, all eventually died. The last fish fromthe group died at 5-7 cm (Rothbard et al.,1999).

Gynogenesis in fantail goldfish. Attemptswere made to induce gynogenesis in the red-cap fantail, a variety of goldfish (Carassiusauratus). Methods used for koi and commoncarp (heat-shock duration and level, τ0, etc.),were successfully applied (unpubl. data).Since the goldfish does not become fully col-ored until the age of 2-3 months, we neededto find an effective genetic (phenotypic) mark-er to assess the success of gynogenesis atthe earliest possible stage of development.

During the investigation it was observedthat the single tail of the comet goldfish domi-nates over the fantail of the redcap and there-fore it can serve as a reliable phenotypicmarker. As a result of this finding, UV-irradiat-ed sperm of comet was used to activate red-cap eggs. The fantail characteristic could beclearly detected among the gynogenetic prog-eny after only a few days and well before thecolor normally develops in the larvae, indicat-ing successful gynogenesis.

Integration of gynogenesis and sex inver-sion, female triploidy, and androgenesis ingrass carp. Attempts were made in a series ofexperiments to produce meiotic (2PB) andmitotic gynogenotes, triploid females (XXX),and androgenetic (YY) grass carp (Fig. 1).Tetraploidy in grass carp was induced to gen-erate broodfish capable of producing diploidgametes that, when coupled with haploidgametes, yield triploid offspring. Neverthe-less, the preliminary experiment to inducetetraploidy yielded weak tetraploid larvae thatdid not survive beyond the larvae stage (7-10days; Rothbard et al., 2000).

The τ0-curve for grass carp (Shelton andRothbard, 1993) served to define the precise

270 The Israeli Journal of Aquaculture - Bamidgeh 58(4), 2006

271BARD Workshop: Aquaculture Genetics – Status and Prospects

Fig. 1. Ploidy manipulations carried out in the YAFIT laboratory on grass carp (Rothbard et al., 2000).

Late-shock

¢ Albino grasscarp (XY)

Sperm (X & Y) UV-irradiated eggs UV-irradiated sperm

Fertilization

Shock

Fertilization

Late-shock

(Paternal origin offspring)

¢¢ (YY) ™™ (XX)Homozygousprogeny

™™ XXFemale monosex

(2nd task)

MT sex-inversion

¢¢ XXGynogenetic

males

Fertilization

Early-shock

™™ XXXFemale triploids

(3rd task)

™™ XXXX4N-females(4th task)

2N gametes

Fertilization

™™ XXXFemale triploids

(5th task)

¢¢ XYMale monosex

(1st task)

™ Albino grasscarp (XX)

™ Wild-typegrass carp (XX)

¢ Common carp(XY)

Spermbank

Spermbank

¢™

timing for application of early and late shocks.Female albino grass carp were used asfemale broodstock to take advantage of therecessive color marker in ploidy manipula-tions. Cold (10°C/10-30 min), heat(40±0.5°C/2 min) and pressure (7000-9000psi/35-120 sec) shocks were tested to inducethe expected ploidy levels (Rothbard et al.,2000)

Ploidy levels of various groups were ana-lyzed by fluorocytometric methods and areshown in Fig. 2. Tetraploid fish were success-fully generated and confirmed by fluorocytom-etry, however, the fish did not survive beyondthe larvae stage and therefore tetraploidyrequires further investigation.

There is an advantage to producingtriploid females (XXX). Unlike triploid males(YXX) that possess testicular fragments thatoccasionally produce a very low number ofviable spermatozoa (Allen et al., 1986) andfertilization ability (Van Eenennaam et al.,1990), XXX-females are totally sterile, lackinggonads and exhibiting almost all-somaticdevelopment. Gynogenetic grass carp finger-lings (ca. 10 cm body length) were sex-invert-ed with MT-implants (Jensen et al., 1983;Shelton, 1986) and grown to maturity as neo-males that produce exclusively X-bearingspermatozoa. Viable sperm from 2-year-oldneomales was easily stripped and used to fer-tilize eggs of XX females to produce sterile

272 The Israeli Journal of Aquaculture - Bamidgeh 58(4), 2006

Fig. 2. Fluorocytometric analysis of diploid and triploid grass carp erythrocytes, carried out in YAFIT lab-oratory: (a) diploid larva, (b) triploid fingerling, (c) diploid larva, and (d) triploid fingerling (Rothbard et al.,2000).

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XXX-triploid females; some sperm was cryop-reserved in a sperm bank.

Gynogenesis and female triploidy in blackcarp. The first attempt to induce meiotic gyno-genesis in black carp ova (2N = 48) was per-formed using UV-irradiated sperm of heterolo-gous species, koi and goldfish, which possessa different number of chromosomes (2N =100). Fertilization of black carp eggs with suchsperm results in non-viable offspring(Rothbard and Shelton, 1993). However,exposure of activated eggs to heat or pres-sure shocks restored diploidy. Another set ofchromosome-set manipulations followed thefirst successful gynogenetic induction(Rothbard et al., 1997). In this follow-on study,the highest survival of gynogenetic larvae wasobtained when eggs were early heat-shocked(41±1°C/1 min) or pressure-shocked (7500-7600 psi/1.5 min) at the embryonic age of0.08-0.16τ0.

Triploid black carp were obtained fromnormally fertilized eggs that were early (0.15-0.16τ0) heat or pressure shocked to retain thesecond polar body. The ploidy level of diploidsand triploids was evaluated by fluorocytome-try (Fig. 3).

Integration of gynogenesis and hormonalsex-reversal in common carp. Meiotic (2PB)gynogenesis was induced in the Yugoslavianstrain of common carp eggs. UV-irradiatedsperm of comet goldfish provided a geneticmarker, i.e., the scale coverage of the gold-fish, and was used to inseminate commoncarp eggs. The activated eggs were cold-shocked (ca. 5°C) for 45-50 min to diploidizethe haploid zygote. The amphimictic hybrids(intact carp eggs fertilized with intact goldfishsperm) served as a scaled control (Fig. 4).

Gynogenetic carp fry (1.5-2 g avg body wt)were fed for two months on feed that con-tained Fadrozole, an aromatase inhibitor (AI;CGS 16949A, Novartis Pharma AG,Switzerland), at 200 mg AI/kg feed (Rothbardet al., unpubl. data). The AI-treated fish (neo-males) continued to grow to about 20-50 gwhen they were examined for sperm by gentlepressure on the abdomen and the sperm wasmicroscopically examined for motility. About50% of the fish (200-250 fish) released drops

BARD Workshop: Aquaculture Genetics – Status and Prospects

Fig. 3. Fluorocytometric analysis of black carpcells from (a) hatchlings, (b) young fry, and (c) ery-throcytes. MN = mean channel position calibratedcells taken from homologous controls, 2n = diploids,3n = triploids (Rothbard et al., 1997).

Rel

ativ

e no

. cel

s pe

r ch

anne

l

Channel numbe

of viable and motile sperm. Sperm sampleswere cryopreserved and stored in a spermbank; some was used for commercial produc-tion of all-female carp populations. The otherfish were discarded due to body malformationscaused by the haploid syndrome occurring atgynogenesis (Fig. 5). At present, about 1.5million putative female offspring of three Dor-70 females are being nursed in earthen ponds.When the fish attained the size appropriate forsexing (20-50 g), a sample of 30 fish wasexamined to verify the monosex population. Allfish in the sample were female.

National Center for Mariculture (IOLR) –Eilat

Gynogenesis and allotriploidy in giltheadseabream (Sparus aurata). The aim of ploidyinvestigation in gilthead seabream was togenerate inbred lines that, when crossed withfish from different and distant populations,would have improved growth due to theadvantage of hybrid vigor.

274 The Israeli Journal of Aquaculture - Bamidgeh 58(4), 2006

Fig. 4. Amphimictic hybrids of common carp xgoldfish. The fish served as a control in experimentsin which gynogenesis was induced by inseminationof common carp eggs with goldfish UV-irradiatedsperm.

Fig. 5. Malformed carp fry resulting from haploid syndrome or insufficient application of physical para-meters at ploidy manipulation (Rothbard, unpubl.)

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UV-treated sperm of red seabream,Pagrus major, served as genetic marker. Thesperm was used to activate giltheadseabream eggs and produce gynogeneticdiploids. Ploidy levels of offspring derivedfrom various chromosome-set manipulations(haploids, gynogenetic or amphimicticdiploids, allotriploids; 24, 48, 72 chromo-somes, respectively) were identified by thedifferences in the structures of metacentricand sub-metacentric chromosomes of thesetwo heterologous species and by karyotyping(Gorshkov et al., 1998).

Ploidy manipulations in the Europeanseabass, Dicentrarchus labrax. Europeanseabass is a commercially important marinefish, endemic to the Mediterranean Sea basin.Extended research on chromosome-setmanipulations has been conducted with thisspecies. The long-term final target of investi-gation was to generate all-female seabasspopulations, characterized with advantageousgrowth performance.

Gynogenetic seabass were obtained byexposing eggs to early heat-shock (35°Capplied 3-5 min post fertilization). UV-irradiat-ed homologous sperm, karyologically exam-ined for success of irradiation, was used toactivate eggs. Non-shocked eggs developedas haploid embryos that did not survive while shocked eggs yielded gynogenetic fish. Since there are structural differencesbetween male and female chromosomes, thesuccess of manipulation was confirmed bykaryological examination (Gorshkova et al.,1995, 1996).

The efficiency of triploid seabass produc-tion was rather high; the rate of triploid fishexceeded 80%. Exposure of seabass eggsfertilized with homologous sperm to heat-shock, similar to the shock used to inducegynogenesis, induced the triploidy. The GSI (gonado-somatic index) of 2-3 year-oldtriploid females was much lower than of thediploid fish. Moreover, preliminary data indi-cated that the triploids were characterized byretarded growth when compared to theirrespective diploid control (Gorshkova et al.,1996).

Laboratory of Fish Immunology andGenetics – Bar Ilan University

Gynogenesis in tilapia, a model of tilapia sexdetermination. The Hertwig effect was exam-ined to define the optimal UV dose effectivefor inactivation of tilapia sperm, which canserve for inducing gynogenesis in tilapia (Donand Avtalion, 1988a). UV-irradiated sperm ofNile tilapia, O. niloticus, characterized bydominant genetic markers (serum esteraseand tail stripes), was used to inseminate O.aureus eggs. The gynogenetic offspring suf-fered from body malformations and very lowsurvival. Higher embryo survival was obtainedfrom the F2 rather than the F1 generation(0.36% and 3.6%, respectively). This phe-nomenon was explained as a result ofreduced levels of recombination in the secondgynogenetic generation and lower expressionof lethal and defective genes. The experimen-tal conditions required to induce ploidy andgynogenesis in O. niloticus and O. aureus arereported by Don and Avtalion (1988b) and byShirak et al. (1998).

Triploidy and tetraploidy in tilapia.Triploidy was induced in two tilapia species,O. niloticus and O. aureus, by exposing fertil-ized eggs to early heat (about 40°C/3.5-4 min)or cold (11°C/60 min) shock. Higher survivalof O. aureus triploids (60% vs. 13%) wasattributed to the genetic contribution of thematernal inheritance (Don and Avtalion,1988c). Better results were obtained when theeggs were cold-shocked (50%-60%), proba-bly due to the possibility that cold shock inter-feres with two distinct meiotic cell divisions.Nevertheless, the authors noticed that coldand heat shock are equally effective in induc-ing triploidy in O. aureus.

In another study, diploid (normal) embryosof O. aureus, incubated in 25-26°C, werecold-shocked at the zygotic age of 92 min andduring zygotic age intervals lasting between80 to 104 min. Shocking at the zygotic age of92 min resulted in higher yields of tetraploids.Ploidy of the derived embryos was assessedby karyotyping and by orange stained FACS(fluorescent activated cell sorter) analysis ofthe adult fish (Don and Avtalion, 1988d).

BARD Workshop: Aquaculture Genetics – Status and Prospects

Degree of inbreeding in gynogenetictilapia. The rate of inbreeding in tilapia pro-duced by gynogenesis was compared toamphimictic offspring. Fish of the first genera-tion of gynogenetic O. aureus were comparedto their female parents and to O. niloticus byexamining immune rejection of transplantedscale grafts (Avtalion et al., 1988). No rejec-tion of transplanted scales from O. niloticusparents was observed among the gynogenet-ic O. niloticus offspring. A similar response toskin allografts was reported in common carpby Komen et al. (1990). However, acute rejec-tion of transplanted scales was observedamong allogeneic O. aureus, probably due tousing fish from strongly selected inbred lines.Nevertheless, the results were not unequivo-cal for other examined groups and furtherinvestigation of this promising tool for measur-ing inbreeding in tilapia was suggested.

Summary and ConclusionsThe progression of chromosome-set manipu-lation practice in Israeli aquaculture centers isremarkably impressive. Successful technolo-gies have been developed for ten fish speciesand varieties, including two commerciallyimportant marine species. Nevertheless, thepractical and commercial impact of ploidymanipulation research, carried out in fourIsraeli laboratories, is quite limited. Onenotable exception is the production of com-mon carp broodstock (YAFIT Laboratory andDor Station) that yield all-female populationson a commercial scale. In this technology, fishobtained by gynogenesis are used to gener-ate neomales with the female genotype (XX-sex determination mechanism).

Production of common carp neomales hasbeen dramatically facilitated by introduction ofFadrozole, an aromatase inhibitor, that verysuccessfully and efficiently replaced the for-merly used MT in androgenic sex-inversion.

Although ploidy manipulation of the twomarine species, gilthead seabream andEuropean seabass, has promising futureapplication, it must still be proven and extend-ed to fill missing gaps, e.g. field experimenta-tion as well as progeny testing.

Much work has been invested in develop-

ment of technology for chromosome-setmanipulations in tilapia. Two of the most impor-tant tilapia species worldwide, O. aureus andO. niloticus, were selected for investigation.The basic research aimed at understanding theintriguing puzzle of sex-determination intilapias. Ploidy manipulation technology inte-grated with hormone treatment may permitgeneration of YY-males (in O. niloticus) or ZZ-males and ZZ-neofemales (O. aureus neofe-males were reported by Rosenstein andHulata, 1993) that, in appropriate mating com-binations, may yield the desired all-male popu-lations. Nevertheless, results have not yetbeen commercially implemented. Most com-mercially applied all-male production of tilapiasexclusively uses androgen sex-inversed tech-nology, however, concerns about human con-sumption of hormone-treated fish has causedincreasing constraints. Using genetically modi-fied fish could provide a solution if the basis forsex determination in fishes can be resolved.The technology was extended and hormonalsex-inversion was implemented on one Israelifish farms (Nir David Fish Breeding Farm) toproduce O. aureus neofemales with the male(ZZ) genotype. Such females can yield male-monosex populations when mated withamphimictic males (Lahav, 1993).

Since 1987, intensive research has beencarried out on chromosome-set manipulationswith the Japanese ornamental (koi) carp. Theaim of these investigations was to produce koiclones through two-step gynogenesis. Duringthe first stage, gynogenetic and totally homozy-gous koi were obtained by induction of mitotic(late shock) gynogenesis. The resultinggynogenotes served as ancestors for a secondgeneration of meiotic gynogenotes. The off-spring obtained in the second gynogenetic gen-eration are expected to be clones due tohomozygosity and the absence of recombina-tion and crossing-over at meiosis. Nevertheless,in spite of success in gynogenesis, the colors ofoffspring indicated that there are other factorsthat are difficult to control, e.g. possible geno-type-environment interaction or polygenic inher-itance which probably affects uniformity of col-oration and color patterns of the derived proge-nies (Rothbard and Wohlfarth, 1995). At pre-

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sent, it seems that further progress to improvecoloration of koi can be achieved only with theassistance of DNA markers. The onset of suchlong-term research was recently reported byDavid et al. (2001, 2004).

Generation of unique fish during theprocess of ploidy manipulation such as koi,common carp, and grass carp neomales orthe need to preserve unique genetic traits wasa major stimulus for establishing a fish spermbank. Such a bank allows long-term preserva-tion of gametes with unique characteristicsthat have been consolidated or developedthrough genetic manipulation (Lubzens et al.,1993). Sperm of various lines of koi, carp, andgrass carp have already been stored in thebank for possible prospective use.

Possession of albino grass carp and theoption to use sperm of heterologous specieshave facilitated ploidy manipulations with bothgrass and black carp. Male triploids (YXX-triploids) can develop gonads with testicularfragments (Allen et al., 1986) and viable sper-matozoa and are therefore considered only par-tially sterile. Therefore, a method to producetotally sterile female triploids (XXX-triploids)was developed by integrating gynogenesis andMT sex-inversion to produce grass or blackcarp neomales as broodfish. Male triploids ofgrass carp have been widely used in the USAfor many years as a biocontrol of water vegeta-tion (Allen and Wattendorf, 1987). In spite ofbeing extensively documented as a commer-cially important species and component of bio-logical control, export of these advantageousfemale triploids was halted by strict govern-mental legislation or lack of funding.

Although considerable research has beencompleted, only a small amount of the devel-oped technology has been commerciallyimplemented. It usually takes 10-20 yearsfrom being experimentally established untilapplications are adopted and practiced byindustry (Hulata, 2001).

AcknowledgementI wish to thank Prof. W.L. Shelton (OklahomaState University, Norman, OK, USA) for hisvaluable comments and suggestions in prepa-ration of this manuscript.

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