events in fish maturation. syndel international canadian owned private specialist company fish...
TRANSCRIPT
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
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 Response
• Two stages:1. Immediate
– Flight or fight
2. Sustained – Extended energy
supply– Change in
metabolism– Puts all cell division
on hold
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
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
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
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