REPRODUCTIVE BIOLOGY OF CARPS

I.J. Singh, Professor Department of Fishery Biology

College of Fisheries, G.B.P.U.A.&T., Pantnagar

Onset of 1Onset of 1stst maturity and gonadal maturity and gonadal recrudescence recrudescence

Onset of 1Onset of 1stst maturity: maturity: Stage of 1Stage of 1st st gonadal development in gonadal development in

the life history of any fish.the life history of any fish.

Gonadal recrudescence:Gonadal recrudescence: Successive gonadal development after Successive gonadal development after

11stst maturity in subsequent breeding maturity in subsequent breeding seasonsseasons

Important factors for onset on 1st maturity

2 + years major carps

1 + year minor carps

6 months common carp

1- Age

Natural food level

Artificial feeding2-Nutritional status

Stimulants

Water quality

Pollution

3- Environmental Condition

Pattern of ovarian development in carps 1- Synchronous type (Synchronise total): All oocytes at the same stage of

development

2- Group Synchronous type (Synchronise par groups):

Atleast two populations of oocytes at different developmental stages.

Generally spawn once a year and heave relatively a short breeding season.

Carps except common carp

3- Asynchronous type (metachrone): Oocytes at all stages of development Common carp

Two patterns or types according to the distribution of spermatogonia.

1- Tubular type or restricted spermatogonial testis:

Spermatogonia are totally restricted to the distal terminus of the tubule immediately beneath the tunica albuginea.

Example - Atheriniformes

2- Lobular type or unrestricted spermatogonial testis:

Common type

Any portion of testis shows close relationship between germ cells at various stages of development and sertoli cells.

Example - Most of teleost groups including Cypriniformes (carps).

Pattern of spermatogenesis in teleosts

Activation of Brain – Pituitary – Gonad (B-P-G) or Hypothalamo - hypophyseal gonad axis

Most probably point of initiation in B-P-G axis is gonad. Pituitary gland from steroid injected immature fish released

gonadotropin when incubated with hypothalamus or GnRH/LHRH

Pituitary gland from immature fish without injection of steroid did not evoke such response.

First steroid produced by gonad activates both hypothalamus and pituitary.

Subsequently GnRH from hypothalamus stimulates pituitary for GtH release which in turn regulates steroidogenesis in fish.

Ultimately processes related with gonadal development move forward under the concerted regulation by B-P-G axis.

Neruohormonal regulation of onset of 1st maturity

Brain Brain HormonesHormones

1- Growth Hormone- Releasing hormone (GHRH)1- Growth Hormone- Releasing hormone (GHRH)

2- Gonadotropin Releasing Hormone (GnRH) 2- Gonadotropin Releasing Hormone (GnRH)

3- Corticotropin – Releasing Factor (CRF) 3- Corticotropin – Releasing Factor (CRF)

4- Thyrotropin Releasing Factor (TRF) 4- Thyrotropin Releasing Factor (TRF)

5- Gonadotropin Releasing Inhibitory Factor (GRIF) 5- Gonadotropin Releasing Inhibitory Factor (GRIF)

6- Thyrotropin Release Inhibitory Factor (TRIF) 6- Thyrotropin Release Inhibitory Factor (TRIF)

7- Somatotropin Release Inhibitory Factor (SRIF) 7- Somatotropin Release Inhibitory Factor (SRIF)

8- Neurohypophyseal Hormones8- Neurohypophyseal Hormones

Gonadotropin (GtH)Gonadotropin (GtH)

A dual GtH system has been proposed for several teleosts. A dual GtH system has been proposed for several teleosts.

1- 1- One preparation is designated as ConAI or GtH I or One preparation is designated as ConAI or GtH I or vitellogenic GtH or carbohydrate poor (CP) GtH.vitellogenic GtH or carbohydrate poor (CP) GtH.

2-2- Second preparation is designated as ConAII or GtH II or Second preparation is designated as ConAII or GtH II or maturational GtH / steroidogenic GtH or carbohydrate rich maturational GtH / steroidogenic GtH or carbohydrate rich (CR) GtH, adsorbed to Con-A.(CR) GtH, adsorbed to Con-A.

Normally two types of gonadotrops are attributed for above Normally two types of gonadotrops are attributed for above two GtHs. two GtHs.

Two types of gonadotrops differing in their location within Two types of gonadotrops differing in their location within PPD and synthetic activity during the reproductive cycle are PPD and synthetic activity during the reproductive cycle are identified. identified.

The synthetic activity of these cell- types vary during the The synthetic activity of these cell- types vary during the reproductive development. reproductive development.

Immunocytochemical localisation of GtH I Immunocytochemical localisation of GtH I and GtH II producing two distinctly and GtH II producing two distinctly different gonadotrop cell- types in PPD of different gonadotrop cell- types in PPD of salmonids.salmonids.

Immunoreactive GtH II localised in cells Immunoreactive GtH II localised in cells located mainly in the central regions of the located mainly in the central regions of the glandular cords of the PPD. glandular cords of the PPD.

GtH I was found in cells located in the GtH I was found in cells located in the periphery of the glandular cords of the periphery of the glandular cords of the PPD.PPD.

The synthetic activity of these cell- types The synthetic activity of these cell- types vary during the reproductive development. vary during the reproductive development.

Two Cell Model for Steroidogenesis

Gonadotropin and Steroidogenesis

1. Testis- Spermatogenesis, spermiogenesis and spermiation in males

2- Ovary- Oogenesis including vitellogenesis, final oocyte maturation and ovulation in females.

Processes associated with the gonadal development

Spermatogenesis Spermatogenesis

In teleosts, resting single cells in testis lobule or tubule are In teleosts, resting single cells in testis lobule or tubule are referred as primary spermatogonia. referred as primary spermatogonia.

Primary spermatogonia in the tubule/lobule walls proliferate Primary spermatogonia in the tubule/lobule walls proliferate to form the clusters of secondary spermatogonia, each cluster to form the clusters of secondary spermatogonia, each cluster gets enclosed in a cyst.gets enclosed in a cyst.

The secondary spermatogonia in each cyst divide The secondary spermatogonia in each cyst divide synchronously mitotically (2synchronously mitotically (212 12 or 2or 21616) to form primary ) to form primary spermatocyte. spermatocyte.

First meiotic division of primary spermatocyte produces First meiotic division of primary spermatocyte produces secondary spermatocyte and 2secondary spermatocyte and 2nd nd meiotic division produces meiotic division produces haploid spermatids. haploid spermatids.

Spermiogenesis Spermiogenesis The metamorphosis of spermatids to spermatozoa is The metamorphosis of spermatids to spermatozoa is

referred as spermiogenesis.referred as spermiogenesis.

Spermiogenesis takes place in the lumen of testis tubule. Spermiogenesis takes place in the lumen of testis tubule.

It involves transformation of immotile spermatids into It involves transformation of immotile spermatids into motile spermatozoa.motile spermatozoa.

SpermiationSpermiation

Milt formation involving hydration, change in electrolytes Milt formation involving hydration, change in electrolytes and pH etc.and pH etc.

Decrease in density and osmolarity by dilution required Decrease in density and osmolarity by dilution required for facilitating release of milt to exterior.for facilitating release of milt to exterior.

Hormonal control of Hormonal control of steroidogenesis, Spermatogenesis, steroidogenesis, Spermatogenesis,

and Spermiation and Spermiation GtH II controls steroidogenesis in testis.GtH II controls steroidogenesis in testis.

It increases levels of of 11-Ketotestosterone and It increases levels of of 11-Ketotestosterone and testosterone in male fish.testosterone in male fish.

GtH I has no steroidogenic effect.GtH I has no steroidogenic effect.

11-ketotestosterone is found only in fishes and is 11-ketotestosterone is found only in fishes and is responsible for spermatogonial proliferation responsible for spermatogonial proliferation (spermatogenesis).(spermatogenesis).

Maturational steroid (MS, 17Maturational steroid (MS, 17αα, , 2020ββ-DP -DP ),), regulates the regulates the processes of spermiation and milt hydration.processes of spermiation and milt hydration.

OogenesisOogenesis Ovigerous lamellae are the seat for the development of oocyte, Ovigerous lamellae are the seat for the development of oocyte,

the germ cells or oogonia. the germ cells or oogonia.

Two phases of initial growth are recognized- Two phases of initial growth are recognized-

1- Increase in number by mitotic division 1- Increase in number by mitotic division

2- Increase in size2- Increase in size

Increase in number takes place by large number of mitotic Increase in number takes place by large number of mitotic division. division.

When an oogonium enters the prophase of the 1When an oogonium enters the prophase of the 1stst meiotic meiotic division it is called primary oocyte. division it is called primary oocyte.

After this stage enters 2After this stage enters 2ndnd growth phase and vitellogenesis growth phase and vitellogenesis takes place. takes place.

Primary oocyte passes through two discrete growth phases. Primary oocyte passes through two discrete growth phases.

1- Endogenous vitellogenesis:1- Endogenous vitellogenesis:

Also known as non-vitellogenic which includes deposition of yolk vesicle in Also known as non-vitellogenic which includes deposition of yolk vesicle in the peripheral region the peripheral region

2- Exogenous vitellogeneis: 2- Exogenous vitellogeneis:

Vitelogenin formation (vitellogenesis) takes place in the liver under the Vitelogenin formation (vitellogenesis) takes place in the liver under the stimulation of 17stimulation of 17ββ-estradiol produced by ovary. -estradiol produced by ovary.

1717ββ-estradiol in ovary is produced under the control of steroidogenic GtH -estradiol in ovary is produced under the control of steroidogenic GtH (GtH II).(GtH II).

Vitellogenin is transported to ovary through blood where it gets Vitellogenin is transported to ovary through blood where it gets incorporated into ovary through micropinocytosis under the control of incorporated into ovary through micropinocytosis under the control of vitellogenic gonadotropin (GtH I)vitellogenic gonadotropin (GtH I)

Uptaken vitellogenin is deposited in the centre of oocyte as yolk granule. Uptaken vitellogenin is deposited in the centre of oocyte as yolk granule.

VITELLOGENESIS VITELLOGENESIS

It involves two important developments. It involves two important developments.

1- Germinal Vesicle Migration (GVM)1- Germinal Vesicle Migration (GVM)

2- Germinal Vesicle Break-down (GVBD)2- Germinal Vesicle Break-down (GVBD)

When vitellogenesis followed by yolk deposition is almost When vitellogenesis followed by yolk deposition is almost complete the nucleus migrates (GVM) to the animal pole. complete the nucleus migrates (GVM) to the animal pole.

Nuclear break-down (GVBD) takes place there and 1Nuclear break-down (GVBD) takes place there and 1stst meiotic meiotic division is complete forming a secondary oocyte and a polar division is complete forming a secondary oocyte and a polar body. body.

22ndnd meiotic division which usually takes place after fertilization meiotic division which usually takes place after fertilization in teleosts, converts the secondary oocyte into the ovum and in teleosts, converts the secondary oocyte into the ovum and another polar body. another polar body.

Final Oocyte Maturation

Maturation Promoting Maturation Promoting Factor (MPF)Factor (MPF)

The presence of MS receptors at the oocyte surface is The presence of MS receptors at the oocyte surface is indicative of the existence of a cytoplasmic factor responsible indicative of the existence of a cytoplasmic factor responsible for mediation of oocyte maturation. for mediation of oocyte maturation.

This factor is designated as MPF and is possibly produced in This factor is designated as MPF and is possibly produced in fish oocytes under the control of 17fish oocytes under the control of 17αα, 20, 20ββ-DP. -DP.

MPF activity has been demonstrated in goldfish oocytes MPF activity has been demonstrated in goldfish oocytes matured by matured by in vivo in vivo treatment of HCG. treatment of HCG.

Similarly, MPF activity was also detected in oocytes matured Similarly, MPF activity was also detected in oocytes matured in vitroin vitro by by 1717αα, 20, 20ββ-DP. -DP.

Immature goldfish oocytes matured faster when injected with Immature goldfish oocytes matured faster when injected with MPF extracted from goldfish compared to oocyte induced to MPF extracted from goldfish compared to oocyte induced to mature mature in vitroin vitro by 17 by 17αα, 20, 20ββ-DP. -DP.

In MPF injected oocytes, though GVM did not take place but In MPF injected oocytes, though GVM did not take place but the GVBD occurred at the centre. the GVBD occurred at the centre.

MPF activity has been reported to increase before GVBD in MPF activity has been reported to increase before GVBD in goldfish during goldfish during in vitroin vitro induction of oocyte maturation by induction of oocyte maturation by 1717αα, 20, 20ββ-DP, peaking at 1-DP, peaking at 1stst meiotic metaphase, decreasing at meiotic metaphase, decreasing at 11stst polar body elimination and increased again and remained polar body elimination and increased again and remained high till insemination. high till insemination.

MPF is considered a protein consisting of two components, MPF is considered a protein consisting of two components, one catalytic subunit, a homologue of the serine / threonine one catalytic subunit, a homologue of the serine / threonine protein kinase (cdc2 kinase) and another a regulatory subunit, protein kinase (cdc2 kinase) and another a regulatory subunit, cyclin.cyclin.

The mature oocytes induced by 17The mature oocytes induced by 17αα, 20, 20ββ-DP were observed to -DP were observed to have 35- KDa inactive and 34-KDa active cdc2 kinase and have 35- KDa inactive and 34-KDa active cdc2 kinase and cyclincyclinββ. .

The appearance of the 34- KDa active cdc2 kinase coincided The appearance of the 34- KDa active cdc2 kinase coincided with that of cyclin with that of cyclin ββ just before GVBD. just before GVBD.

It is suggested that 17It is suggested that 17αα, 20, 20ββ-DP induces synthesis of cyclin -DP induces synthesis of cyclin ββ by oocytes which activates 35- KDa cdc2 kinase to by oocytes which activates 35- KDa cdc2 kinase to produce 34- KDa active cdc2 kinase and thus induces produce 34- KDa active cdc2 kinase and thus induces GVBD. GVBD.

The available information also indicates that MPF is The available information also indicates that MPF is similar among vertebrates and invertebrates and it is not similar among vertebrates and invertebrates and it is not specific. specific.

It may also be a more general factor responsible for It may also be a more general factor responsible for initiation of breakdown of nuclear membrane and initiation of breakdown of nuclear membrane and subsequent cell division. subsequent cell division.

Actual expulsion of oocyte from the follicle.Actual expulsion of oocyte from the follicle.

Follicular separationFollicular separation

Following final maturation detachment of microvillar Following final maturation detachment of microvillar connections between oocyte and follicular layer.connections between oocyte and follicular layer.

Follicular ruptureFollicular rupture

After separation formation of a distinct hole in the follicular After separation formation of a distinct hole in the follicular layer through which the oocyte leaves.layer through which the oocyte leaves.

A very specific area of the follicle is weakened and involved in A very specific area of the follicle is weakened and involved in hole formation at ovulation. hole formation at ovulation.

Oocyte expulsion Oocyte expulsion

Expulsion of oocyte occurs by active contraction of the follicle Expulsion of oocyte occurs by active contraction of the follicle with involvement of smooth muscles, pushing the oocyte out. with involvement of smooth muscles, pushing the oocyte out.

OVULATIONOVULATION

Mechanism- Mechanism-

Enzymic control-Enzymic control- Protease plasmin Protease plasmin

Hormonal control-Hormonal control-

Plasminogen activator Plasmin

Stimulated by GtH

Plasminogen system

Prostaglandins (PG) are involved in ovulation by stimulating follicular contraction.

Indomethacin treatment.

Increase in PG at ovulation.

Migrated Germinal Vesicle and Follicular Separation Migrated Germinal Vesicle and Follicular Separation

Pituitary-ovarian relay for the stimulation of Pituitary-ovarian relay for the stimulation of final maturation (MS, maturational steroid) final maturation (MS, maturational steroid)

Neurohormonal regulation of final Neurohormonal regulation of final oocyte maturation and ovulationoocyte maturation and ovulation

GnRH II

GnRH

Hypothalamus

GnRH GRIF

Pituitary

GtH II

Ovary

MS (17αα, 20β-DP)Final maturation and ovulation

Annual changes in Photoperiod, Temperature and Rainfall

Gonado-somatic index (GSI) in female of Cirrhinus mrigala (Singh, I.J. and T.P. Singh, 1984)

0

2

4

6

8

10

12

14

16

18

20

22

24

March April May June July August Sep. Oct. Nov. Dec. Jan. Feb.

Months

GSI

Gonado-somatic index (GSI) in female of Cirrhinus mrigala (Singh, I.J. and T.P. Singh, 1984)

0

2

4

6

8

10

12

14

16

18

20

22

24

March April May June July August Sep. Oct. Nov. Dec. Jan. Feb.

Gonado-somatic index (GSI) in male Cirrhinus mrigala (Singh, I.J. and T.P. Singh, 1984)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

March April May June July August Sep. Oct. Nov. Dec. Jan. Feb.

GSI

Months

Gonado-somatic index (GSI) in male Cirrhinus mrigala (Singh, I.J. and T.P. Singh, 1984)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

March April May June July August Sep. Oct. Nov. Dec. Jan. Feb.

Gonado-somatic index (GSI) in female Labeo rohita (Kumar, A., I. J. Singh and R.N. Ram, 2001)

0

2

4

6

8

10

12

14

Ocober Dec. Jan. Feb. March April May June August

Months

GSI

0

2

4

6

8

10

12

14

Ocober Dec. Jan. Feb. March April May June August

Gonado-somatic index (GSI) in male Labeo rohita(Singh, A.K., A.Kumar, I.J.Singh and R.N. Ram,2004)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

October Nov. Jan. Feb. March April May June August

Months

GSI

Gonado-somatic index (GSI) in male Labeo rohita(Singh, A.K., A.Kumar, I.J.Singh and R.N. Ram,2004)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

October Nov. Jan. Feb. March April May June August

Gonado-somatic index (GSI) in male Labeo gonius

0

0.5

1

1.5

2

2.5

Nov. Dec Jan. Feb. March April May June

Months

GSI

Gonado-somatic index (GSI) in female Labeo gonius

0

1

2

3

4

5

6

7

8

Nov. Dec. Jan. Feb. March April May June

Months

GSI

Principles of Induced breeding Principles of Induced breeding

1.1. Gonadal development in major carps is completed Gonadal development in major carps is completed in captivity but spontaneous spawning does not in captivity but spontaneous spawning does not occur due to lack of stimulatory factors and occur due to lack of stimulatory factors and presence of some inhibitory factors such as excess of presence of some inhibitory factors such as excess of excretory metabolites. excretory metabolites.

2.2. Sudden upsurge of GtH is must for spawning in Sudden upsurge of GtH is must for spawning in carps.carps.

3.3. This is induced to happen with the help of inducing This is induced to happen with the help of inducing agents i.e. pituitary extract (PE) or ovaprim and its agents i.e. pituitary extract (PE) or ovaprim and its alikes. alikes.

Hypothalamus

Gonadotropin

Releasing

Hormone (GnRH)

Pituitary Gland

Gonadotropin (GtH)

Ovary

Oocyte Final Maturation

Ovulation

ova

Antiestrogens Dopamine Antagonists

GnRH and GnRH Analogues

Pituitary Extracts GtH Preparations

ProgestinsConticosteroids

ProstaglandinsCatecholamines

Levels of external intervention in the hypothalamic-pituitary-ovarian axis for inducing maturation and ovulation

Principles of Induced Breeding Principles of Induced Breeding

HYPOPHYSATIONHYPOPHYSATION

Use of pituitary gland for induced breeding. Use of pituitary gland for induced breeding.

First priming low dose of PE to females triggers GVM and / First priming low dose of PE to females triggers GVM and / or initiation of GVBD. or initiation of GVBD.

GtH present in PG targets ovary / oocyte directly. GtH present in PG targets ovary / oocyte directly.

Second resolving high dose of PE induces GVBD and Second resolving high dose of PE induces GVBD and ovulation in female fish.ovulation in female fish.

Low dose of PE to male with resolving dose to female Low dose of PE to male with resolving dose to female induces spermiation. induces spermiation.

OVAPRIMOVAPRIM

Constituents- SGnRHa (20 μg) and domperidone (10 mg) in Constituents- SGnRHa (20 μg) and domperidone (10 mg) in each ml. each ml.

SGnRHa directly acts upon pituitary to induce synthesis SGnRHa directly acts upon pituitary to induce synthesis and release of GtH II.and release of GtH II.

Domperidone (Dopamine-antagonist) counters negative Domperidone (Dopamine-antagonist) counters negative impact of dopamine thus facilitating release of more GnRH impact of dopamine thus facilitating release of more GnRH and increasing sensitivity of pituitary to GnRH and and increasing sensitivity of pituitary to GnRH and SGnRHa. SGnRHa.

As a result release of GtH II from pitutary is increased and As a result release of GtH II from pitutary is increased and level of MS is elevated causing GVM, GVBD and ovulation level of MS is elevated causing GVM, GVBD and ovulation in female and spermiation in male leading to spawning. in female and spermiation in male leading to spawning.