You are on page 1of 70

FISIOLOGI PEMBIAKAN INVERTEBRATA

KULIAH 4

Macrobrachium rosenbergii

 The ability to control production of viable eggs and their successful rearing through the larval stages are necessary for the reliable supply of the seedstock on which intensive culture systems depend.Intro Development of the aquaculture industry requires a reliable and adequate supply of high quality eggs from broodstock.  .

is needed for intensive culture.  .  In recent years procedures for stimulating ovarian maturation in penaeid shrimps and other aquaculture species under controlled conditions have been developed.  Environmental control of photoperiod and temperature were found to result in successful reproduction in Penaeus stylirostris and P. japonicus. especially the regulatory mechanisms involved in gonadal maturation of cultivable species of crustaceans.The understanding of the reproductive biology.

oocytes grow during oogenesis through the process of vitellogenesis. is synthesized and is taken in by the oocytes.Female reproduction 1. Vitellogenesis In crustaceans.  In the oocytes. vitellogenin.  During vitellogenesis.  Vitellin is utilized as a nutritional source during embryogenesis. 280-700 kDa).  . vitellin.  Vitellin and vitellogenin have been purified in several shrimps and determined to be large lipoprotein molecules (molecular weight. vitellogenin is processed and accumulated as vitellin. the precursor of the major yolk protein.

Oogonia start meiotic division I and become oocytes. GVBD). they accumulate RNA at the previtellogenic stage. oil globules and PAS (periodic acid-Schiff)-positive vesicles at the endogenous vitellogenic stage.  .  After the completion of yolk accumulation.  At the exogenous vitellogenic stage. and the germinal vesicle at the cell center disintegrates (germinal vesicle breakdown. oocytes recommence meiosis. oocytes grow rapidly by yolk accumulation. and yolk globules at the exogenous vitellogenic stage. While the oocytes remain arrested at prophase of meiotic division I.

by illuminating the internal body organs of the female by means of a bright underwater torch beam being passed along her side. The determination of ovarian development by hatchery technicians. only reveals the shadow of the ovary in the tail region and is scored from 1 to 5. Line drawing outline of female with ovary in situ.OVARIAN DEVELOPMENT 1  The ovary lies dorsal to the gut and extends from the cephalothorax (head and thorax region) along the entire length of the tail. .

.Female broodstock are graded for ovarian development by torchlight. The wide saddle of ovarian tissue directly behind the carapace (Stage IV) is indicative of an immediate pre-spawning female. A female scored as a Stage IV during the day is most likely to spawn that night.

underdeveloped and/or spent stage .Stage I.

Stage II. developing stage .

nearly ripe stage .Stage III.

Stage IV. The majority of the ovarian mass is within the cephalothorax region which cannot be observed by torchlight. . ripe stage The complete ovary extends from the head to the tail.

OVARIAN DEVELOPMENT 4 Schematic diagram of the major endocrine organs in P. monodon. . Magnified 8500x. (b) Electron microscopy section of the sinus gland demonstrating hormone filled vesicles (dark circles) which fuse and release their contents into the blood. The sinus gland is composed of the terminals from neurons which have their cell bodies in the X-organ and brain.

SECTION II DEVELOPMENT OF THE EGG: Post-Fertilization to Hatching .

TIME AFTER SPAWNING 0-30 MINUTES .

TIME AFTER SPAWNING 1 HOUR .

TIME AFTER SPAWNING 1 HOUR 30 MINUTES

Cell division is occurring rapidly. Within a sample of eggs a range of developmental stages will be seen (Fig. 2.3a, b, c). The majority will be in the 4-cell stage but will range between the 8-cell and 16-cell stage. At this time it is still possible to distinguish between fertilised and nonfertilised eggs due to the cleavage pattern on the outside edge of the egg.

2.3a

2.3b

2.3d

•Fertilised eggs have an even regular pattern of cell division whereas unfertilised eggs are uneven and nonsymmetrical (Fig. 2.3d).

2.3c

TIME AFTER SPAWNING 2 HOURS

The egg is well into embryogenesis at this time. This figure shows a number of stages of embryo development. The latest stage of development at this time is approximately the 6th (64-cell stage) and 7th (128-cell stage) cleavage. By this time a layer of cells has formed at the periphery of the egg, called the blastoderm. Differentiation of cells to form various tissues required for development of the nauplius has begun.

TIME AFTER SPAWNING 3-4 HOURS The embryos continue to undergo rapid cell division. An invagination can be seen on the surface of the egg that is due a process called gastrulation. can be seen. At this time a secondary envelope. accompanied by tissue differentiation. Time 3 hours Time 4 hours There appears to be very little difference in external appearance of the embryo even though cell division continues at a rapid rate. the number of cleavages is difficult to estimate by this stage. The edges of the eggs have a corrugated appearance and it is difficult to visualise individual cells. within the external hatching envelope. It is increasingly difficult to distinguish between a fertilised and unfertilised egg. . It involves a dynamic morphological change from a monolayered to a multilayered embryo.

Depending on the orientation of the individual egg the limb buds may or may not be visible. Time 5 hours Time 6 hours . Limb buds may be distinguishable which will form the major appendages of the nauplii. Some embryos will appear similar to those at 5 hours after spawning. Further embryonic development has occurred.TIME AFTER SPAWNING 5-6 HOURS A wide range of embryonic development may be observed.

including setae.TIME AFTER SPAWNING 7-8 HOURS The appendages are more defined as the naupliar body develops into a more distinguishable form. The naupliar body has thickened and further development. it may be possible to distinguish the formation of setae on the tips of the developing appendages. may be observed. Time 7 hours Major developmental changes are distinguishable. Depending on orientation. Time 8 hours .

Time 9 hours : Embryos progressively develop with little change in observable form . Depending on orientation. the pairs of major naupliar appendages are clearly distinguishable.TIME AFTER SPAWNING 9-10 HOURS Time 10 hours : Further differentiation of appendages are observable on the naupliar body.

TIME AFTER SPAWNING 11-12 HOURS Time 11 hours : The naupliar form is recognisable. Time 12 hours : The nauplii begin to hatch. A single reddish pigmented spot can be seen which is the naupliar eye. typically around 12. . Appendages are well developed. Commercial hatcheries have reported that occasionally vigorous active nauplii can be seen within the hatching envelope but are unable to ‘hatch’ and die within the shell. In this figure the bottom of the hatching envelop has been broken and the nauplii tears their way out using their appendages. The reason for this is unknown but may be due to poor quality and weak nauplii that are unable to tear open the hatching envelope.5 hours after spawning.

This figure (2. In addition. if hatching has not occurred by 15 hours post-spawning the nauplii may be of inferior quality.15b Time 13 hours : Not all eggs will have hatched by this time. However.15b) shows the 1st naupliar stage with appendages fully extended.15a 2. .TIME AFTER SPAWNING 13 HOURS 2. Hatching does not occur synchronously and its timing is temperature dependent. if the eggs do not hatch out within a few hours of each other then the nauplii will be of questionable quality.

.

 VIH has been purified as a peptide in the American lobster.2. Eyestalk hormones It is well-known that eyestalk ablation induces ovarian development and oviposition.  . Homarus americanus and in the isopod. This is because the source (X-organ-sinus gland complex) of the vitellogenesis. Armadillidium vulgare.inhibiting hormone (VIH) is removed by the ablation.

inhibited proteinsynthesis activity of ovaries. including VIH. VIH may act on the vitellogenin synthesis sites directly.  Previous study that purified eyestalk peptides.  .The regulatory mechanism of VIH is partially understood.

.

and they are converted to 20hydroxyecdysone. . the Yorgan produces and secretes ecdysone and 3dehydroecdysone. the biologically active ecdysteroid. Ecdysteroids   Ecdysteroids are known as a molting hormone in crustaceans and insects. In crustaceans.3.

. Vertebrate-type steroid hormones    Several vertebrate-type steroid hormones such as estradiol17β and progesterone have been identified in crustaceans. Previous studies suggesting that vertebrate-type steroid hormones are not involved in ovarian development. but the results are varied and sometimes inconsistent. The administration of the steroids has been attempted.4.

.

Spermatogenesis In crustaceans.  . and spermatocytes differentiate into spermatozoa through spermatids. as in vertebrates. spermatozoa are produced through spermatogenesis.  Spermatogonia start meiotic division after proliferation and become spermatocytes.Male reproduction 1.

.

.

.

sex ratio. thoracic ganglion and ovary) and their functions which are closely related to the release of vitellogenesis-stimulating hormones and ovarian hormone. and nutrition. photoperiod.Eyestalk ablation has been used to mature female shrimp in captivity in conjunction with management of water temperature.  . light intensity.  The recent research has focused mostly on organs (brain. density.

and – gonad stimulating hormone (GSH).  There are two types of crustacean reproductive neurohormones.  the neurosecretory cells. and  neurohemal organs. The crustacean endocrine system consists of – classical ephitelial-type endocrine glands. .  – gonad inhibiting hormone (GIH). This neuroendocrine component is of major significance with respect to both the number of hormones (neurohormones) and their broad array of roles. and – endocrine structures of neural origin.

this inhibitory hormone is also found in male prawn.  GSH is present in the brain and thoracic ganglia of females with maturing ovaries. but absent (or  removal results in precocious gonadal development. . found in the brain and thoracic ganglia. The decapod sinus gland is the source of a gonad-inhibiting hormone (GIH). is the gonad-stimulating hormone (GSH). in low quantity) in the brain and thoracic ganglia of immature females. eyestalk A second decapod reproductive neurohormone.

 GIH has a molecular weight of 9135 Da.  .This suggest that thoracic ganglia from maturing female shrimp could be used to induce ovarian maturation. in addition to its hyperglycemic activity. GSH has a molecular weight of 1000-2000 Da.  The three forming a family of neuropeptides unique to crustaceans. and its structurally related to the crustacean hyperglycemic hormone (CHH) and the moltinhibiting hormone (MIH). GSH activity is present in the same fraction as CHH and raised the possibility that CHH may have. a stimulatory action on the reproductive system.

these hormone appear to exert their effect on the testes only undirectly by directly affecting the androgenic glands. But in males. – functioning and the development of the male secondary sexual characteristics.   In females GSH and GIH act directly on the ovaries. and one gland is attached to each sperm duct.  Male crustaceans have a pair of androgenic glands. . The androgenic gland hormone controls: – differentiation of the male reproductive system.

– Vitellogenesis. and – final maturation  germinal vesicle breakdown (GVBD)  ovulation in oocytes. Vitellogenesis occurs in hardshelled shrimp at the intermolt stage C4 . reproductive maturation involves two main processes. In penaeid shrimp. shortly after molting and continues until immediately before final maturation.  .  Oil globule stage is an initial stage of primary vitellogenesis.

the surface of the shrunken follicle cells was relatively smooth compared to the irregular surface of the oocyte. which had numerous well-developed microvilli. Furthermore. . This suggests that follicle cells on yolk granule stage oocytes are not related to the route of egg yolk protein uptake in this prawn.     Follicle cells on the oil globule stage oocytes expand rapidly and reach maximum size during oogenesis. The follicle cells are possibly responsible for ovarian vitellogenin (Vg) synthesis in the kuruma prawn Penaeus japonicus. Vg appears to be secreted only from enlarged follicle cells surrounding oil globule stage oocytes and not from shrunken follicle cells related to yolk granule stage.

 . have migrated to the peripheral cytoplasm of the oocytes. shrunken during the late prematuration phase. which in P.  Ovulation occurs when nuclei. involved: the appearance of ripe ova.Final maturation of ovarian oocytes immediately precedes spawning. and germinal vesicle breakdown (GVBD) in preparation for fertilization after spawning.  Two phases are. japonicus occurs after dark.

over a period of several hours. still in metaphase. Immediately after release from the female gonopore. japonicus. implying that GVBD is initiated in the evening and completed during the night. meiotic metaphase is arrested and remains visible just beneath the cytoplasmic membrane of the ovarian oocyte. the mature eggs. are fertilized by sperm released into the seawater from the spermatophore held in the thelycum. . indicating that GVBD is completed after ovulation.00 in P.00 and 03.   In the late phase of the maturation cycle. Rapid shrinkage of the nucleus can be observed in the ovary after sunset and meiotic metaphase follows between 21.

It is well known that in penaeid shrimp many small clusters of immature eggs often become attached to the walls of the spawning tank.  The coating of immature eggs is not yet formed in the early phase of prematuration in the ovary. immediately after spawning.  These immature oocytes. are interconnected. which are at early and late perinucleolus stages.  . together with separated fertilized eggs.  Histological studies of the maturation cycle revealed eggs surrounded by many immature oocytes. forming a coating around the egg.

females releasing batches of eggs from the ripe ovary and sperm from the spermatophore into the seawater. where fertilization takes place.  Once begun.  Probably.  Therefore. female shrimp have to repeat the process of molting.This suggests that immature oocytes may increase rapidly after the late phase of prematuration and then quickly surround the mature oocytes immediatley prior to spawning. and sexual maturation in order to achieve several spawnings during their life. spawning is continuous. the coating of immature eggs lubricates the mature oocyte during release from the ovary.  . mating.

vannamei did not initiate vitellogenesis even after eyestalk ablation. vitellogenesis is controlled by two factors.Thoracic ganglion hormone effects on vitellogenesis Smaller size P.  Thus.  . one which inhibits and the other which stimulates. the vitellogenesis-stimulating principle is absent or not yet functioning.  In previtellogenic (immature) females.

Homarus americanus. .  Vitellogenesis in shrimp can be stimulated by implantation of pieces of thoracic ganglion tissue prepared from the female lobster. with vitellogenic ovaries. The accumulation of yolk granules in oocytes was stimulated by repeated implantation of pieces of thoracic ganglion in the immature female crab Potamon dehaani.

 Injection of thoracic ganglion extract prepared from vitellogenic females is effective in increasing serum Vg in the kuruma prawn. japonicus. also known as the gonad-stimulating hormone.  . P.This result indicated that vitellogenesis could be stimulated by a vitellogenesisstimulating hormone (VSH).

 . also known as the gonad-inhibiting hormone (GIH).  This treatment reduces the production of a vitellogenesis-inhibiting hormone (VIH). and thus permits maturation of the ovaries in female penaeid shrimp.Eyestalk hormone directly inhibits vitellogenin synthesis Eyestalk ablation stimulates ovarian maturation in penaeid shrimp.

which is located in the medulla terminalis of penaeid shrimp and the other crustaceans. the acidophilic sinus gland is connected with axons from the neurosecretory cell of the X-organ.  In this complex.  In general.Reproductive maturation in penaeid shrimp is regulated by a VIH from the X-organ-sinus gland complex in the eyestalks. surrounding the optic ganglia. the sinus gland is easily recognizable on the neurilemma.  .

 These findings suggest that VIH. vannamei. . inhibits Vg synthesis and secretion into the blood in female penaeid shrimp. Unilateral eyestalk ablation stimulates Vg synthesis and its secretion into the blood in previllogenic immature P. secreted by the X-organ-sinus gland complex. japonicus. Eyestalk ablation also induces a rapid increase in yolk protein synthesis in P.

In the kuruma prawn.  These results suggest that Vg synthesis in the ovary may be inhibited directly by VIH secreted by the X-organ-sinus gland complex of eyestalks. japonicus. Vg synthesis in previtellogenic ovary can be initiated when the ovary is not affected directly by VIH from eyestalks.  . P.

 This suggests that the VIH level decreases quickly immediately before the initiation of vitellogenesis and stays at a low level until after vitellogenesis is completed.  Therefore.  .Unilateral eyestalk ablation is not effective in increasing serum Vg in vitellogenic female kuruma prawn. eyestalk ablation may no longer be effective in regulating the production of VIH after vitellogenesis has been initiated.

 Evidence has been presented to show that 17-hydroxy-progesterone is effective in increasing serum Vg in the kuruma prawn.  In addition.Induction of vitellogenin synthesis in ovary by estradiol-17 Vg synthesis in incubated ovarian pieces can be stimulated by estradiol-17 in the kuruma prawn. oil globule stage oocytes were found in incubated previtellogenic ovarian pieces after treatment with estradiol-17  in vitro. japonicus. P. .

that estradiol-17 secreted from ovarian follicle cells. 17-hydroxy-progesterone can be converted intro the estradiol-17.  17-hydroxy-progesterone may be worked into the Vg synthesis mechanism in kuruma prawn as a precusor of estradiol-17.The hormone estradiol-17 or 17-hydroxyprogesterone is generally distributed in the ovary of crustaceans. induces Vg synthesis in the ovary as a Vg-stimulating ovarian hormone in penaeid shrimp.  In crustaceans.  .  Probably.

Pathway of steroid hormone in decapod crustacean animals Cholesterol Pregnenolone Progesterone 17-hydroxy-progesterone Androst-4-ene-3. 17-dione Testosterone Estradiol-17 .

and red swamp crayfish.Serotonin stimulates the release of vitellogenesis-stimulating hormone (VSH) It is well known that biogenic amines release peptide neurohormones from neuroendocrine structures in several crustaceans.  Serotonin is found widely distributed throughout the nervous system in decapod crustaceans.  .  Serotonin (5-hydroxytryptamine) stimulates gonadal maturation in male and female sand fiddler crabs. serotonin apparently stimulating release of VSH that is present in the brain or thoracic ganglia.  This action of serotonin is indirect.

 .  Therefore.Facts suggest that the release of VSH from thoracic ganglion may be induced by serotonin present in the central nervous system in crustaceans. neurohormonal serotonin is nominated as a vitellogenesis-stimulating hormone-releasing hormone (VSH-RH) responsible for ovarian Vg systhesis in penaeid shrimp.

induced and accelerated final maturation in P.  A formulated.Prostaglandine stimulates final maturation Final maturation may be induced by certain nutrients obtainable only from a combination of foods. pelleted diet rich in vitamin E and containing fish oil.  . monodon.

from oxidation during digestion or peroxidation in the shrimp body.  A high level of vitamin E or C is thought to protect the eicosapentaenoic acid (EPA). pelleted diet containing fish oil also induced and accelerated final maturation.  . a precusor of prostaglandin which is essential for reproduction in crustacean or other invertebrates.Vitamin C-rich.

 . which is essential for the induction of final maturation in crustaceans. under high levels of vitamin E or C. may therefore be stimulated by mating behaviour and subsequent spermatophore transfer in open-thelycum species.EPA is one of the essential fatty acids in crustaceans and so spawners must take this up as a precusor of the prostaglandin required for final maturation.  Probably. active EPA could be converted to prostaglandin. Secretion of prostaglandin.

The release of serotonin (VSH-RH) from central nervous system. respectively. japonicus. japonicus. Even in eyestalk ablated P.Reproductive maturation is affected by water temperature and photoperiod    It is well known that ovarian maturation is affected by water temperature and photoperiod in female P. VIH from the Xorgan-sinus gland complex and estradiol-17B from the ovary may be be induced directly or indirectly by the water temperature and light stimuli via the antennule and eye. in female penaeid shrimp. VSH from the thoracic ganglia. . a photoperiod of 14 to 16 h (light) and temperature of 24 to 26 oC stimulate ovarian maturation. Hormonal control of reproductive maturation is affected by the water temperature and photoperiod in penaeid shrimp. full sexual maturation did not occur below 17 oC.

some methods for blocking VIH activity. only eyestalk ablation for the induction of female maturation is practically used in shrimp farming. such as anti-VIH antibody and antagonists.Perspectives on hormonal manipulation      At present. the factors may be synthesized by molecular biological techniques as recombinant molt-inhibiting hormone and recombinant androgenic gland hormone. This method is not repeatable and sometimes causes high mortality. however. Recently. Methyl farnesoate may be used to stimulate female maturation in the near future. If the mechanism of VIH function is fully understood. In addition. After characterization. vitellogenin was purified and its cDNA was cloned. can be anticipated. Subsequently. additional stimulating factors may be newly characterized in the future. . further studies are necessary. These assay methods are very sensitive. To characterize additional hormones. enzyme-immunoassay of vitellogenin and quantitative reverse-transcription (RT)-polymerase chain reaction (PCR) of vitellogenin have been developed. and will be used for bioassay of the characterization process. a suitable bioassay is required.

 .. In some shrimps.  New techniques for controlling sexual differentiation will improve economic gains in shrimp farming.  The androgenic gland hormone may be used to control sexual differentiation. growth rate is different between sexes (e. administration of the androgenic gland will become available. although purification of the hormone in shrimp is required.For male reproduction. high growth rate in males in Macrobrachium species and in females in Penaeid shrimps).g.

Conclusion To develop hormonal manipulation techniques. Characterizing new hormones should be given priority. Only a few hormones have been discovered so far. much needs to be done.  Many hormones are still unidentified.  .

and be generally useful for obtaining disease-free spawners. serve in developing selective breeding programs.Control of reproductive maturation is a major problem in the development of commercial aquaculture programs for penaeid shrimp.  .  Controlling reproductive maturation in captivity could help to provide a reliable year-round supply of juveniles.

Thanks .