Events in Fish Maturation. Syndel International Canadian owned Private specialist company Fish species only 25 years of service Domestic –Vaccines, parasiticides,

Events in Fish Maturation

Syndel International• Canadian owned• Private specialist company• Fish species only• 25 years of service

• Domestic– Vaccines, parasiticides, transport

disinfectants, sedatives, anesthetics

• International– Spawning agents, sedatives,

transport chemicals

Jim Powell, PhD

• Product Development and Tech Support

• Doctorate in Neuroendocrinology of Fishes

• Former salmon farmer• 18 yrs in aquaculture• S&E trials Internationally

Introduction

• Overview of maturation process

• Types of spawners

• Methods of spawning

Key Points

1. Two stages to getting quality gametes

2. Environment is the first step

3. Healthy animals is the second

4. Manipulation of spawning is third

5. There are different types of spawners

6. Mess with Nature, she’ll mess with you

Genesis of Maturation

External Conditionsenvironmentalsocial

Internal Conditionsphysiology

PerceptionIntegrationReaction

Environmental Influences

• Temperate climates– Photoperiod– Temperature – Water quality

• Equatorial climates– Temperature– Rainfall– Water quality

Social Conditions

• Loading density– +ve and –ve– Sex ratios

• Nesting behaviour– Proper materials– Colouration

• Pheromones– Attraction– Cue

Internal Conditions

• Physiology– Puberty

• Fat content– Type and amount

• Nutrition – Availability and

quality

• Disease• Size and shape

Perception

• Changes detected• Impulse transferred

to brain• Signal pathways

inform areas of the brain

• Outside is now an inside thing

• Integration

Integration

• Environmental and social cues are perceived and register

• Physiological cues are detected and registered

• The external and internal environments are integrated into a physiological response

Reaction

• Signals are coordinated in the hypothalamus

• The hypo sends out messengers to the pituitary

• The pit releases hormones into the blood

• The hormones effect target organs

HPG Axis

• Common to all vertebrates• Hypo coordinates signals• Releases GnRH• Pit releases GtHs• GtHs bind to gonads• Gonads produce steroids• Steroids cause gonad

growth

Endocrine Cascade

• Brain releases messengers

• Pit releases gonadotropins

• Gonads release steroids

• Gonad forms gametes

GtHs

• There are two types:– GtH I causes

vitellogenesis – egg building

– GtH II causes oocyte maturation

• Released consecutively

Vitellogenesis

• Ovary produces estradiol E2

• Liver produces yolk platelets or globules

• Gonad incorporates yolk into eggs

Steroids

• Ovary– Makes estradiol,

later progesterone– Egg building, then

maturation

• Testis– Makes testosterone– Makes the sperm,

then capacitates the sperm

Ovarian Cells

Diagrammatic representation of the follicle and oocyte during early vitellogenesis

Nagahama, 1983

• Two cell layers• Outside is

theca• Inside is

granulosa• Change

function as egg goes from building to maturing

Maturation

• As GtHs change, so do the steroids

• E2 turns to progesterone

• P helps to mature the egg

• Makes fertilization possible

Estradiol 11-KT DOHP Apr May June July Aug Sept Oct Nov Date

GTH-I GTH-II

Vitellogenesis Ovulation

Summary

• Conditions permit maturation

• Process begins• GnRH – GtH –

steroids• Eggs are built• Eggs mature• Correct environment• Spawning proceeds

Stress and Spawning

• External influences• Perception• Integration• Reaction

Stress Response

• Two stages:1. Immediate

– Flight or fight

2. Sustained – Extended energy

supply– Change in

metabolism– Puts all cell division

on hold

Stress and Spawning

• Stressor is encountered

• Animal becomes stressed

• Endocrine cascade begins

Stress Effects

• Creates an assured supply of energy

• Depletes liver glycogen

• Releases sugars form protein wasting

• Represses immunity• Inhibits growth

Stress and Reproduction

• Cortisol inhibits reproduction

• Direct and indirect effects

• Stops cell division• Limit or mitigate

stressors in breeders

Concept II: Spawning Specifics

• Role of the environment is covered

• Physiology is covered

• Task is to get the things to breed

Classifying Spawners

Many ways:• By spawning type• By Linnaean

methods• By guilds• By breeding

patterns

Groups of Spawners1) Synchronous- all oocytes develop

synchronously and ovulate at the same time.

2) Group-synchronous- oocytes ÷ into groups, ovulate over one breeding season.

3) Asynchronous- oocytes at all developmental stages are present in the ovary.

Ex: tropical fish.

Reproductive Guilds

• Proposed by Balon, 1975

• Still in use today• An ecological

approach• Non-Linnaean• Overlay with

physiology

Nonguarder

Guarders

Bearers

Open substrate

Brood hiders

Nesters

Substrate choosers

External

Internal

Fish Evolution

Where Is He Going With This?

• An ecological model for breeding won’t work

• A Linnaean model won’t work

• Is there anything else?

Spawning Fish Checklist

1. Environment is perfect

2. Fish health is excellent

3. Fish are gravid

4. Spawning them is easy

Remember This?

• GnRH has a primary role in reproduction

• It is common to all things with a spine

• Must be some connection to spawning groups

A GnRH Primer: 1

• 1st called LHRH• Central role in

reproduction• Different forms are

found in different species

• Linked through evolution

A GnRH Primer: 2

• All vertebrates have at least two forms in their brains:– One for nervous

function– One for reproduction

• Some fish have three forms

• The one in the pit is the repro one

Locations of GnRH Forms in the Brain

• Two populations of neurons– Hindbrain– Midbrain

• C-II always in the hindbrain

• Pit form controls the GtH release

GnRH in Fishes

1 2 3 4 5 6 7 8 9 10

Mammal pGLU –HIS – TRP – SER – TYR – GLY – LEU – ARG – PRO – GLY – NH2

Chicken – 1 pGLU –HIS – TRP – SER – TYR – GLY – LEU – GLN – PRO – GLY – NH2

Herring pGLU –HIS – TRP – SER – HIS – GLY – LEU – SER – PRO – GLY – NH2

Catfish pGLU –HIS – TRP – SER – HIS – GLY – LEU – ASN – PRO – GLY – NH2

Seabream pGLU –HIS – TRP – SER – HIS – GLY – TRP – TYR – PRO – GLY – NH2

Salmon pGLU –HIS – TRP – SER – TYR – GLY – TRP – LEU – PRO – GLY – NH2

Chicken – II pGLU –HIS – TRP – SER – HIS – GLY – TRP – TYR – PRO – GLY – NH2

Dogfish pGLU –HIS – TRP – SER – HIS – GLY – TRP – LEU – PRO – GLY – NH2

Lamprey - III pGLU –HIS – TRP – SER – HIS – ASP – TRP – LYS – PRO – GLY – NH2

Lamprey – I pGLU –HIS – TYR – SER – LEU – GLU – TRP – LYS – PRO – GLY – NH2

GnRH in Spawning

Groups

SalmonidsSalmonids

PercsPercs

OstariophyseansCatfishTetrasCyprinidsCharacidsPacu

OstariophyseansCatfishTetrasCyprinidsCharacidsPacu

Lots of OthersLots of Others

Boney tonguesEelsBoney tonguesEels

Sturgeons and PaddlefishSturgeons and Paddlefish

SeabreamSalmonCatfishMammalian

SeabreamSalmonCatfishMammalian

Ostariophysea

• Catfish• Tetras• Characids• Silurids• Carps

• Pacu• Knifefish

Dopamine Inhibition

• Dopamine:– Blocks release

of GnRH– Inhibits GtH

release

• Released by environmental cues

• Must be blocked

GnRH from the Hypothalamus

Dopamine from the Hypothalamus

Pituitary release of GtHs

-ve+ve

The Point Is:

• Unless the fish is an Ostariophysean, GnRH alone should work

• Based on principle

Steroids

• Act at the level of the gonad

• Augment natural levels to advance gamete release

• Overdose to get effect

Steroids

• Least desirable• Most dangerous

method for you and fish• No control over dose• Wastewater nightmare• Can cause sterility,

gynomastia

Gonadotropins

• Simulate or augment GtHs

• Act at level of gonad• Stimulate ripening

and release• Causes increased

steroids and PGs

CPE/LSP

• Oldest method• Usually works• Not truly reliable• No dose conformation• Quality can vary• Stability issues• HCG is human

equivalent

Purity

• Fish GtHs have not been synthesized

• No recombinants to date

• Vary with species• Large molecules

mean stereo-conformity

GnRH

• Small molecule, decapeptide

• 16 known variants• Central to

reproduction• Top of the cascade• Robust

Methods: GnRHa

• Many forms: powder, liquid, implant

• Ovaprim is sterile solution with dopamine inhibitor

• Most natural stimulation

• Uses full HPG axis

sGnRHa vs. LHRHa 1 2 3 4 5 6 7 8 9 10 Salmon LHRH

NEt

NEt

• Differs at three amino acids• Affects receptor binding• More potent in fish – similar to native

Delivery Systems

• Different applications = different method

• Depends on repro status of the fish

• Cost and application• Farm friendly

Bolus Injection

• IM or IP• Causes pit to

release stored GtH• Works only at the

time injected• Used to get

stubborn or reluctant fish going

Sustained Release• Bolus injections work

in season• Sustained release is

needed in advance of the season

• Initial load – sustained release

Recap

• GnRH is a good method to induce final maturation

• Inhibit dopamine in some species

• Bolus injection to push them over the edge

• Sustained release to bring them along

• Fish must be ready!

Induced Maturation

• Only final maturation can be induced

• Eggs can’t be induced, manipulate the conditions

• Many methods have been developed

• Some are effective

• Not all fish need inducing

• Some fish spawn willingly

• Some like Ostariophysids need dopamine blockers

• Work with the HPG

Rules of Thumb: Chondrostei

• mGnRH is the form in the brain

• LHRH• Most analogues will

work• Inject 20-50µg/kg• CPE to prime?

Rules of Thumb: Boneytongues

• Can have mGnRH or sGnRH in their brains

• sGnRHa will work well

• Dopamine inhib can’t hurt

Rules of Thumb: Ostariophysea

• Catfish, carp, charachins, silurids all need dopamine inhibition

• Pacu and the like do not

Rules of Thumb: Percaforms

• Three form of GnRH in brain

• Usually sbGnRH• sGnRHa is best bet• Dopamine inhibition

is not normally required

Induction

• Fish must be mature• Will not cause

vitellogenesis• Most injections are

overnight for results• IM or IP, sometimes

immersion

Summary

• Outside and inside cues must be correct

• HPG axis works in an endocrine cascade

• Two parts, growing and maturing gametes

• Induction will only mature gametes

Summary

• There are different forms of GnRH in fish

• Some fish need dopamine inhibition

• The type of fish will indicate the best method of induction

Summary

• Be creative and resourceful

• Ask questions• Respect the fish