The cultivation of many economically important species has been helped greatly by the growing use of the

technique of artificial fertilization and incubation. The discovery of artificial fertilization is supposed to be very old and

this technique is said to have been practiced in the middle ages by the monk Don Pinchon in trout. The rediscovery of

wet artificial fertilization in salmonids in 1842 by two anglers Gehin and Remy and the discovery of dry fertilization by

the Russian Vrassky and its application between 1856 and 1870 led to great technical progress. Artificial spawning

was first achieved in striped mullet in 1930 in Italy. In general the technique of hypophysation has triggered rapid

progress in the induced breeding of cultured species, in the controlled rearing of fish larvae and in selective breeding

Krishnan, L

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Course ManualCentral Marine  Fisheries Research Institute(Indian Council of Agricultural Research)P.B.No.1603, Marine Drive North Extension,Ernakulam  North ,P.O.Cochin, KERALA – INDIA - 682018Winter School  onRecent Advances in Breeding  and Larvicultureof Marine Finfish and Shellfish30.12.2008 -19.1.2009CMFRICompiled and  Edited byDr. K. Madhu, Senior Scientist  and  Director,Winter school&Dr. Rema Madhu, Senior  Scientist and Co-ordinatorCentral Marine Fisheries Research InstituteCMFRI - Winter School Course Manual on “Recent Advances in Breeding and Larviculture of Marine Finfish  and Shellfish”.30.12.2008 - 19.1.200947ASSESSMENT OF MATURATION AND INDUCTION OFSPAWNING IN MARINE FINFISHL. Krishnan,Principal Scientist (Retd. ), Central Marine Fisheries Research Institute, Kochi - 682 018.E -mail : lordkrishnan2001@yahoo.co.inIntroduction     The cultivation of many economically important species has been helped greatly by the growing use of thetechnique of artificial fertilization and incubation. The discovery of artificial fertilization is supposed to be very old andthis technique is said to have been practiced in the middle ages by the monk Don Pinchon in trout. The rediscovery ofwet artificial fertilization in salmonids in 1842 by two anglers Gehin and Remy and the discovery of dry fertilization bythe Russian Vrassky and its application between 1856 and 1870 led to great technical progress. Artificial spawningwas first achieved in striped mullet in 1930 in Italy. In general the technique of hypophysation has triggered rapidprogress in the induced breeding of cultured species, in the controlled rearing of fish larvae and in selective breeding.Maturity determinationThe effectiveness of hypophysation technique for artificial spawning depends mainly on the selection of suitablerecipient fish at the proper stage of ovarian development. For finfish species which undergo normal gonad developmentbut fail to spawn in captivity, identification of the correct stage of development is important and critical. There arevarious methods for maturity determination. External anatomical characteristics have been described and used ex.,depth and fullness of belly, colour and state of swelling of the cloaca, softness and resilience of the belly, roughness ofpectoral fins or presence of head tubercles.. Other methods include microscopic appearance of the oocytes, histologicalstructures of eggs, etc.Physiological parameters associated with sexual maturity such as elevated plasma proteins and calciumconcentrations are of little practical use. Shehadeh, Kuo & Milison,1973) described a method for assessment ofovarian biopsy in vivo which is accurate and reliable. In this method intra-ovarian oocytes are removed in vivo from anunanaethestised female through a polyethylene canula of known orifice diameter. The canula is inserted into theoviduct for a distance of 6-7 cm from the cloaca, and the oocytes sucked orally into the tube by the operator as thecanula is withdrawn. Oocytes from the mid portion of the ovary are the most representative and sampling error isminimized by avoiding extremities.The oocytes are removed form the canula, washed and preserved in a solution of 1% formalin in 0.6% Sodiumchloride solution. They are then placed in a plexiglass plate and measured with an ocular micrometer. Fine grooves cutin the plate align the oocytes and facilitate measurement. Egg diameters are measured along the horizontal axis andthe measurements grouped into 50 mm. class intervals. The sexual maturity of the fish is expressed in terms of meanegg diameter, calculated from the egg diameter frequency distribution.  In many fishes where the oocytes develop insynchrony, this method helps in determining the ovarian development accurately without sacrificing the female fish.This method also provides a means to observe and record oocyte development in individual fish and thus precludesvariations between females in the brood stock. Apart from the ova diameter this method also helps in assessing/observing the shape, quality and stage of maturity of the ova. In mullets ova in the tertiary oil globule stage areselected for experimentation on induced breeding. Ova which are dark, opaque and perfectly round are found to beideal to initiate experimentation. Fishes wherein ova are in the process of disintegration are to be avoided.Apart from the knowledge of ova diameter, the knowledge of the exact stage of maturity of ova thus sampled isa pre-requisite. Most of the researchers have accepted the ova maturity classification described by Yamamoto (1956)and Yamamoto et.al, (1965) Kuo et. al ( 1974 ) have classified the oogenesis in oocytes of Mugil cephalus along theCMFRI - Winter School Course Manual on “Recent Advances in Breeding and Larviculture of Marine Finfish  and Shellfish”.30.12.2008 - 19.1.200948pattern of Yamamoto and compared them microscopically and histologically. As per the classification there are fivestages which are primary oocyte stage (12-170m  in dia.), yolk vesicle stage (170-210 m ) yolk globule stage ( 200-700m), ripe stage and atresia. . Kuo et.al. (1974) advocated initiation of hypophysation in M.cephalus when the ovaare in the tertiary yolk globule stage in which the intraovarian eggs are fully yolk laden. . In milk fish an ova dia. of 850mis ideal to start with. In mullets,  fishes with ova dia. of above 600m  alone are taken for inducement. The actualrelationship between initial mean ova diameter of recipient females and the amount of gonadotropin required to inducespawning in M.cephalus has been worked out.Source of breeders usedA review of earlier works on induced spawning of cultivable fishes will show that the experiments on breedingwere performed in wild ripe fish caught just before natural spawning or enroute their spawning migration. Later workersrelied on brood stock reared in captivity. This has the advantage of easier availability and the breeders are less activecompared to wild caught breeders. Pond reared fish are easier to spawn since they are docile and free from injuries.Wild caught brood stock is restless and easily prone to handling stress. This may sometimes lead to degeneration ofova.Hormones and their useThe influence of environmental factors on gametogenesis of fishes is mediated through the hypothalamo-pituitary-gonad axis. The hormones involved in the process of maturation are gonadotropin releasing hormones, gonadotropinsand steroids. It is this basic knowledge which enables us to manipulate gametogenesis by exogenous hormones. Therole of pituitary glands in reproduction of vertebrates was understood quite early. Many workers give elaborate accountsof the techniques of collection, processing and storage of pituitary and their methods of application with reference tocultured fishes. Usage of pituitary glands in hypophysation suffers in lacking standardisation especially on the doses.So also are the inherent problems connected with the variations in fish pituitary gonadotropin activity which in turn isrelated to the phylogeny, sex and season. It is generally believed that pituitary from either sexes are equally potent andthe difference if any can be minimized by the preparation of mixed pituitary extracts from both the sexes. . Thecommon practice is to collect pituitary from mature fishes during spawning season. This leads to sacrifice of valuablebreeders. The use of salmon pituitaries revolutionized the further use of pituitary gonadotropins. A partially purifiedform of salmon gonadotropin has also been described. (PPSG). This led to the preparation of purified salmongonadotropin. Different preparations of pituitary such as  in the form of powders  LPE ( lyophilized salmon pituitaryextract ) , ADP ( acetone dried salmon pituitary powder ) , acetone dried pituitary powder of carp ( CPH ) are available.Information on spawning of fishes using pituitary glands alone or in combination with other hormones is in plenty.Whatever hormones are used in spawning a fish, a priming dose with pituitary extract (30 –50mg. CPH/Kg.) helps inachieving good spawning. The dosages used by workers on different species vary.The use of mammalian hormones for inducement of cultured fishes has many advantages such as reliablesources, readily available always, easily stored, no sacrifice of valuable breeders etc.  . Their use revolutionized thebreeding of many cultivable fishes. Beginning with Human pregnancy urine and pregnant mare’s serum, the availablemammalian hormones are in plenty such as H.C.G., F.S.H., L.H, puberogen and Synahorin (a mixture of chorionichormone and mammalian pituitary extracts). HCG is widely used in many food fishes in concentrations beginning from20 –30 IU/ gm body wt. Many workers use HCG in combination with CPH to get spawning. HCG can also be used toinduce spermiation in males as well as seminal thinning. Mammalian hormones have been used in getting successfulspawning of mullets, rabbit fishes, milk fish, sea bass , many species of catfishes, striped bass, groupers, etc.The use of synthetic hormones came into existence in recent years. The most important of these is the syntheticform Leutinizing hormone Releasing Hormone analogue ( LHRH-a). The effective doses begin from 200 to 300 mgm/Kg. wt. of fish .Another advancement in hormone therapy is the use of steroids such as D.O.C. (Deoxycorticosterone).The steroids are cheap and are available as pure preparations. The use of hormones such as Methyl testosterone inprolonging spermatogenesis in males is quite common. Similarly the use of MT in sex reversal of protogynous speciessuch as groupers to obtain functional males is well established.ASSESSMENT OF MATURATION AND INDUCTION OF SPAWNING IN MARINE FINFISHCMFRI - Winter School Course Manual on “Recent Advances in Breeding and Larviculture of Marine Finfish  and Shellfish”.30.12.2008 - 19.1.200949Administration of hormonesAdministration of hormones is mainly intramuscular and sometimes intraperitoneal. But other methods of hormoneadministration such as fixation of silastic tubing, coelomic administration via a catheter, intraperitoneal implantation ofosmotic pumps or intramuscular implantation of cholesterol hormone pellets are all in use in modern laboratories.These new avenues of hormone administration reduce the stress due to repetitive injections. . The use of plastichoods in covering the head portion of the breeder while injecting is helpful. The number and frequency of hormoneinjections vary and are related to the response of the breeder and in other words the maturity stage of the breeder, theenvironmental conditions and the type of hormone used. Live ovarian biopsy gives a clear understanding of theresponse of the ova to the hormone used. Visible reactions such as the enlargement of the belly or production calciumsalt depositions as seen in mullets and milkfish are the direct results of the hydration of ova. The release of a fewhydrated eggs by the female shows the onset of spawning. The males are generally released along with the femalesat the time of giving the resolving dose. Male female ratio is also important and varies from species to species. Naturalspawning is always preferred compared to stripping since it gives better quality eggs which in turn result in bettersurvival of larvae. Stripping can be done either through the dry or wet method. Stripping has to be performed at theright time mainly through observation of the breeder through visible symptoms of courtship or release of hydrated eggsby the female.Egg collection and incubationThe collection of spawned eggs in hatchery bred fishes is done with the help of appropriate scoop nets. Butcollection is difficult in caged brood stock in open enclosures or in ponds. The quality of the incubating systems, thecontrol of environmental conditions especially water flow, aeration etc help in better hatching.Preservation of sex productsMethods for storage of fish sperm and ova have several advantages mainly in selective breeding and hybridization.Short term storage of sperm by chilling at 5 °c up to 23 days helps in extending their fertile period in tropical andsubtropical species. Many descriptions of cryogenic preservation of sex products in liquid nitrogen are available.Cvryoprotective agents such as glycerine, DMSO are used. Shiro Hara et al. ( 1982 )  studied various extenders forpreservation of milk fish sperm. The work by Withler and Crim (1982) throw much light on the storage of groupersperm.ASSESSMENT OF MATURATION AND INDUCTION OF SPAWNING IN MARINE FINFISH

Assessment of maturation and induction of spawning in marine finfish- Winter School on Recent Advances in Breeding and Larviculture of Marine Finfish and Shellfish

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Assessment of maturation and induction of spawning in marine finfish- Winter School on Recent Advances in Breeding and Larviculture of Marine Finfish and Shellfish

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