1. Events in Fish Maturation

2. 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

3. Jim Powell, PhD Product Development and Tech Support Doctorate in Neuroendocrinology of Fishes Former salmon farmer 18 yrs in aquaculture S&E trials Internationally

4. Introduction Overview of maturation process Types of spawners Methods of spawning

5. 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

6. Genesis of Maturation External Conditions  environmental  social Internal Conditions  physiology Perception Integration Reaction

7. Environmental Influences Temperate climates –Photoperiod –Temperature –Water quality Equatorial climates –Temperature –Rainfall –Water quality

8. Social Conditions Loading density –+ve and –ve –Sex ratios Nesting behaviour –Proper materials –Colouration Pheromones –Attraction –Cue

9. Internal Conditions Physiology –Puberty Fat content –Type and amount Nutrition –Availability and quality Disease Size and shape

10. Perception Changes detected Impulse transferred to brain Signal pathways inform areas of the brain Outside is now an inside thing Integration

11. 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

12. 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

13. 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

14. Endocrine Cascade Brain releases messengers Pit releases gonadotropins Gonads release steroids Gonad forms gametes

15. GtHs There are two types: –GtH I causes vitellogenesis – egg building –GtH II causes oocyte maturation Released consecutively

16. Vitellogenesis Ovary produces estradiol E2 Liver produces yolk platelets or globules Gonad incorporates yolk into eggs

17. Steroids Ovary –Makes estradiol, later progesterone –Egg building, then maturation Testis –Makes testosterone –Makes the sperm, then capacitates the sperm

18. Ovarian Cells Two cell layers Outside is theca Inside is granulosa Change function as egg goes from building to maturing

19. Maturation As GtHs change, so do the steroids E2 turns to progesterone P helps to mature the egg Makes fertilization possible

20. Summary Conditions permit maturation Process begins GnRH – GtH – steroids Eggs are built Eggs mature Correct environment Spawning proceeds

21. Stress and Spawning External influences Perception Integration Reaction

22. Stress Response Two stages: 1.Immediate –Flight or fight 2.Sustained –Extended energy supply –Change in metabolism –Puts all cell division on hold

23. Stress and Spawning Stressor is encountered Animal becomes stressed Endocrine cascade begins

24. Stress Effects Creates an assured supply of energy Depletes liver glycogen Releases sugars form protein wasting Represses immunity Inhibits growth

25. Stress and Reproduction Cortisol inhibits reproduction Direct and indirect effects Stops cell division Limit or mitigate stressors in breeders

26. Concept II: Spawning Specifics Role of the environment is covered Physiology is covered Task is to get the things to breed

27. Classifying Spawners Many ways: By spawning type By Linnaean methods By guilds By breeding patterns

28. Groups of Spawners 1)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.

29. 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

30. Fish Evolution

32. Where Is He Going With This? An ecological model for breeding won’t work A Linnaean model won’t work Is there anything else?

33. Spawning Fish Checklist 1.Environment is perfect 2.Fish health is excellent 3.Fish are gravid 4.Spawning them is easy

34. 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

35. A GnRH Primer: 1 1 st called LHRH Central role in reproduction Different forms are found in different species Linked through evolution

36. 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

37. 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

38. GnRH in Fishes

39. GnRH in Spawning Groups Salmonids Percs Ostariophyseans  Catfish  Tetras  Cyprinids  Characids  Pacu Lots of Others Boney tongues Eels Sturgeons and Paddlefish Seabream Salmon Catfish Mammalian

40. Ostariophysea Catfish Tetras Characids Silurids Carps Pacu Knifefish

41. Dopamine Inhibition Dopamine: –Blocks release of GnRH –Inhibits GtH release Released by environmental cues Must be blocked GnRH from the Hypothalamus Dopamine from the Hypothalamu s Pituitary release of GtHs -ve +ve

42. The Point Is: Unless the fish is an Ostariophysean, GnRH alone should work Based on principle

44. Steroids Act at the level of the gonad Augment natural levels to advance gamete release Overdose to get effect

45. Steroids Least desirable Most dangerous method for you and fish No control over dose Wastewater nightmare Can cause sterility, gynomastia

46. Gonadotropins Simulate or augment GtHs Act at level of gonad Stimulate ripening and release Causes increased steroids and PGs

47. CPE/LSP Oldest method Usually works Not truly reliable No dose conformation Quality can vary Stability issues HCG is human equivalent

48. Purity Fish GtHs have not been synthesized No recombinants to date Vary with species Large molecules mean stereo- conformity

49. GnRH Small molecule, decapeptide 16 known variants Central to reproduction Top of the cascade Robust

50. Methods: GnRHa Many forms: powder, liquid, implant Ovaprim is sterile solution with dopamine inhibitor Most natural stimulation Uses full HPG axis

51. sGnRHa vs. LHRHa Differs at three amino acids Affects receptor binding More potent in fish – similar to native

52. Delivery Systems Different applications = different method Depends on repro status of the fish Cost and application Farm friendly

53. 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

54. Sustained Release Bolus injections work in season Sustained release is needed in advance of the season Initial load – sustained release

55. 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!

56. 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

57. Rules of Thumb: Chondrostei mGnRH is the form in the brain LHRH Most analogues will work Inject 20-50µg/kg CPE to prime?

58. Rules of Thumb: Boneytongues Can have mGnRH or sGnRH in their brains sGnRHa will work well Dopamine inhib can’t hurt

59. Rules of Thumb: Ostariophysea Catfish, carp, charachins, silurids all need dopamine inhibition Pacu and the like do not

60. Rules of Thumb: Percaforms Three form of GnRH in brain Usually sbGnRH sGnRHa is best bet Dopamine inhibition is not normally required

61. Induction Fish must be mature Will not cause vitellogenesis Most injections are overnight for results IM or IP, sometimes immersion

62. 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

63. 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

64. Summary Be creative and resourceful Ask questions Respect the fish