1. Module 3 Biology & husbandry + practical Disease and signs of ill health Introduction to anaesthesia and analgesia Conduct of minor procedures

2. Biology of Zebra Fish Zebra danio: Brachydanio rerio. A member of the carp family Cyprinidae. A small fish about 4-5 cm long. Striped, spotted and long-finned fish. Fresh water species, native to Pakistan, India, and Burma. Found in shoals in slow moving coastal streams and clear standing water of paddy fields. Relatively easy to keep and breed in captivity.

3. Biology of Zebra fish Males – smaller Females swollen abdomens Maximum lifespan 5.5 yrs. Av. 3.5yrs in the lab. Lateral line detecting water vibrations Hearing, olfaction and vision (UV photo pigment) Pattern detection – shoaling behaviour

4. Development Embryos: 0-72 hrs post fertilisation Early larvae: 72hrs – 13days post fertilisation Free feeding: 5 days post fertilisation Mid larvae: 14-29 days post fertilisation Juveniles: 30 days to 4 months Adults – when sexually mature

5. Biology of Xenopus Xenopus laevis, X. tropicalis and X. borealis Mainly aquatic & nocturnal Lungs for gaseous exchange Flattish antero-dorsal shape Skin smooth and glandular. Mucous glands secrete slimy protective layer. Serous glands (around head/shoulders)

6. Xenopus laevis South African clawed toad Family Pipidae Can live up to 25 yrs Tetraploid – 26 chromosomes Females >150mm, 300g body weight Males >60-100mm, 100g body weight Change colour according to surroundings In the wild live in stagnant water – mud substrate

7. Xenopus laevis Underwater acoustic communication – clicks. Pheromone communication possible Detect food odours and eat non living food Lateral line present

8. Xenopus tropicalis Diploid – used for genetic studies Smaller size 40-60mm Shorter generation time – 5 months Found in Ghana and the Ivory Coast Warmer environment required 24-27C

9. Housing. Traditional holding for research fish & xenopus used individual ‘stand alone’ tanks. A ‘fill and dump’ systems OR independently filtered and serviced. Labour intensive Frequent water changes Water quality varied markedly over time

10. Small Scale

11. Large Scale Holding Purpose built system is recommended. For all ‘furry animal only people’ think of them as essentially IVC’s for Zebra Fish & Xenopus. Substituting the need for clean air with the need for clean water!

12. Large Scale Holding - Mice

13. Large Scale Holding – Aquatics!

14. System requirements Maintain healthy Aquatic species. Keep them free of environmental stress Keep them free of disease. Containment Reliable & easy to service. Labour saving & cost effective. Meet research requirements. Allow flexibility for breeding Meet European directive requirements!!

15. What is involved Battery system of tanks – polycarbonate construction with mesh filters and lids plus non- corrosive support racking. Flow through or Re-circulating system of water. Series of filters to maintain water purity: Biological, Mechanical, Chemical U/V sterilisation.

16. Polycarbonate tank

17. Tanks Transparent tanks – polycarbonate Dark base of racks to reduce stress Able to observe inhabitants from the side Animals to be able to turn around easily

18. Battery of Tanks

19. What is involved Source of pure water (RO). This is obtained from a Reverse Osmosis unit.(Purite) Header tank for conditioning of water. Automatic monitoring of water quality. Semi-automated water quality maintenance. Temperature control and monitoring. Flow control of water. Electrical pumps and valves.

20. RO System

21. Header tank

22. Pumps & Filters

23. What is involved A flow system that ensures each tank is maintained as a separate entity to prevent spread of disease. Alarm systems and back up systems in place. Duplicate pipework and pumps, to allow immediate back up and isolation of faulty pumps for ease of repair and replacement.

24. Systems available Marine Biotech / Aquatic Habitats http://www.aquatichabitats.com/ http://www.aquatichabitats.com/ Tecniplast http://www.tecniplast.it/products.php?lineid=44&categoryid=14&familyid=1 http://www.tecniplast.it/products.php?lineid=44&categoryid=14&familyid=1

25. Emergency Code of Practice Emergency alarms and stand-by systems. A technologically-dependent animal facility is a vulnerable entity. It is strongly recommended that such facilities are appropriately protected to detect the breakdown of essential equipment

26. Monitors

27. Emergency Emergency call out and weekend cover. Animal care staff should be on a call out list Arrangements should be in place to ensure that local engineers can provide additional help. It is recommended that a service agreement and call out arrangement is in place for critical equipment e.g. RO Water Provision.

28. Where to put the System A room with a strong enough floor! 1000 Litres of water = 1000 kg A room with enough space. Strong floor. Waterproof non-slip floor. Drainage & sink. Hot & Cold water.

29. Requirements (cont) Electrical supply Waterproof sockets, switches and electrical connections Circuit breakers and earth wires Installed by professional electricians

30. Requirements (cont) Room temperature control. Helps control water temp without overloading system heaters. Provides a safeguard if system fails.

31. Requirements (cont) Humidity contol. Prevention of water loss by evaporation. Ventilation to remove smells and maintain environmental temp & humidity. >8-12 air changes OK (UFAW) Light Control Light cycle for breeding Light level for algal growth. Noise & vibration – habituation to vibration of system?

32. Module 3 Introducing Fish & Xenopus to the aquarium

33. WATER QUALITY Vital – essential to get right Aquatic species are ectothermic animals. Completely dependent upon the quality and stability of their immediate environment – the water.

34. Environmental Parameters Water Quality Temperature pH Ammonia (NH3 /NH4) Nitrite (NO2) Nitrate (NO3) Dissolved Oxygen (DO) Conductivity

35. Introducing fish New system – Test before use. Leave to run and stabilise for 2 weeks. Check for leaks. Check RO water supply. Check environmental parameters.

36. A Biological System Seed the biological filters Add culture of bacteria: Nitrosomonas & Nitrobacter spp. Introduce sentinel fish

37. Water Quality Achieved by filtration Aim: To remove the uneaten food, skin debris and solid waste products of digestion, plus dissolved faeces and ammonia. Fish are extremely susceptible to a build up of ammonia, toxic effects are reported at 0.2 – 0.5 mg/L of free ammonia (NH3) (Andrews et al, 1998).

38. Mechanical filters Pre-filters of mesh, or rough filter material e.g. white polyester pad. These are used for the removal of gross high density and suspended solid debris including food and faeces from the water before it enters the biological filters

39. Pre-filters The pads require regular cleaning to remove the rapid build up of soiled on their surface Can block the filter with consequent backflow of water. At Nottingham this is done 3 times per week to prevent contamination and maintain efficiency of the biological filters.

40. Particulate Filter 5-20micron Particulate cartridge filter removes suspended particles generated by or passing through the biological filter. This is an in line filter similar to a HEPA filter. Replaced around every 6 months or if flow rate drops.

41. Sand Scrubber Sand filter that remove macro-parasites from the water as it passes through the filter. This type of filter assists in water clarification. Fluidized bed biofilters – a highly efficient nitrification process takes place in such filters

42. Biological Filter Biological filters remove ammonia and nitrite using biological filters Seeded with beneficial nitrifying bacteria (Nitrosomonas and Nitrobacter species) Convert the ammonia into nitrite and then nitrate via the nitrogen cycle. Pure RO water protects the biological filters from chlorine present tap water.

43. Biological Filter

44. Biological Filters Newly installed or cleaned biological filters need time to mature. Build up of a nitrifying bacterial population required to become fully effective in removing ammonia and nitrite.

45. Biological Filters Mature in around 2 weeks. During this time monitoring of ammonia levels is crucial. Reduce toxic levels by removing quantities of contaminated water and adding more clean system water.

46. Biological Filter If flow rate or ammonia, nitrite and nitrate levels cannot be controlled. Gravel to be removed and rinsed before being replaced. Ensures that the bacteria receive sufficient oxygen and nutrients. Only one filter is cleaned at a time to allow maintenance of the beneficial bacteria within the system.

47. Chemical Filters Chemical filters - activated Charcoal or Carbon. Remove organic compounds (including ammonia) from the system water. Remove discoloration and odour by binding organic chemicals in suspension.

48. Ultraviolet (U/V) Filters Sterilising/disinfecting filter that will destroy bacteria and other micro-organisms Recommended dosage of 30,000-60,000 m wattsec/cm 2 or 240-280nm wavelength (UFAW). Protozoa may not be affected. Change at appropriate intervals

49. U/V Filters

50. Ultraviolet (U/V) Filters Most effective in clear water. Water must not pass through the sleeve to quickly or the treatment will be less effective. The sleeve will need cleaning every week and the bulb replacing every 8-10 months. Note – U/V will kill off the bacteria from the biological filters therefore ‘turn off’ the U/V while the colony establishes.

51. Water testing & recording. Electronic meters allowing continuous monitoring of system conditions. Aquanodes that can be linked to alarms Ensure that the system is correctly calibrated and that probes are maintained in optimum condition. The’aquanode’ does not give levels of ammonia, nitrite or nitrate. Use the appropriate test kit. Monitor twice per week and record details. System service – increase monitoring for 2 wks

52. Aquanode monitor

53. Temperature Aquatic species keep as close to natural environmental temperature as practicable (CoP). ( Zebra fish Range 6C- >38C! ) Zebra fish require a water temperature of 28.5C Above 31C and below 25C, decreased breeding Room temperature at 25C as safeguard. X. laevis require a water temperature of 18-20C Room temperature of 20C X. tropicalis water temp of 24-27C

54. Temperature (cont) It is important to maintain a constant temperature as changes greater than 2C in 24 hours are not recommended (UFAW). Water temperature affects the oxygen carrying capacity of the water.

55. Affect of temperature change. Fish May lead to osmo-regulatory dysfunction. Suppression of the immune system. Loss of equilibrium High temp. - Increased activity. Low temp - Sudden violent spasms and coma Xenopus Low temp – stop eating. Depressed metabolism and immune system

56. pH Levels recommended for the Zebra fish are pH 6.8-7.5 and 6.5-8.5 for X.laevis pH should be monitored daily, kept constant and buffering salts added as required to maintain the desired levels. Sodium bicarbonate may be added to raise the pH as the nitrification cycle gradually reduces pH. High pH and increased toxicity of NH3

57. pH (cont) Levels maintained at Nottingham: around pH 7 Note: pH is measured on a logarithmic scale Each unit change in pH value is equivalent to a 10 fold change in hydrogen ion concentration.

58. Ammonia levels (NH 3 /NH 4 + ): Either dissolved as ammonium ions or more dangerously to the fish as free ammonia. Levels of free ammonia increase with increased pH or temperature. Level of free ammonia that can be tolerated without toxic effects is 0.01-0.03mg/L In a well established system, levels should be at zero or at least below 0.01ppm

59. Nitrite levels (NO 2 - ) Nitrite is less toxic than ammonia. Toxicity occurring from 0.5-1.0mg/L with death at levels in excess of 10mg/L. In a well established system, levels should be at zero. Nottingham: <0.1mg/l

60. Nitrate levels (NO 3 - ): Affected by the level of bacterial nitrification occurring, as it is the final product in the nitrogen cycle. Recommended maximum nitrate level is 50mg/L (Schlotfeldt and Alderman). Nottingham <12.5mg/l

61. Test kits

62. Dissolved Oxygen (DO) Levels. Automatic systems help in this process by creating turbulence as the water circulates in the system. Zebra fish - recommended DO level of 6.0 ppm, levels below 3.0ppm will induce stress. Levels maintained at Nottingham: 3.5ppm. If fish are at the surface and gulping air then DO levels should be checked and increased.

63. Dissolved Oxygen (DO) Levels. Too much turbulence, increased amounts of dissolved oxygen. Gas bubbles in capillaries of the gills. Red gills and nose. Death in older fish. Xenopus – bubbles under the skin

64. Conductivity This determines the levels of dissolved solids in the system – measures ion content. Indication of the general water quality. RO water is free of all contaminants. Synthetic Sea Salts (Instant Ocean) are added to the purified water to maintain conductivity. Recommended level of 0.49 – 0.51 milliSiemens. Levels maintained at Nottingham: 544 microSiemens (Moderately hard water) Adjusted by addition of salts.

65. Other Toxins If not using RO – obtain analysis of water. Chlorine & chloramines in the supply. Requires additional filtering and aeration before use.

66. Water changes Required to maintain water quality. Recommended 3 changes per hour in tanks. Re-circulating system = 10% change per day. Xenopus – if ‘fill & dump’ system. Change water after feeding at least 3 times per week.

67. Light Levels Strong circadian pattern – daytime activity The photoperiod will influence the physiological regulation of the fish, affecting the activity, breeding behaviour and feeding pattern of the fish. A light cycle of 14L:10D is used at Nottingham. Special light controlled cabinets surrounding the tanks can also be created. Spawning triggered by light.

68. Light Levels Light levels are kept low during normal activity, in order to reduce algal growth in the tanks. Recommended levels 54-324 lux at the surface of the water for fish: –200 Lux at bench height. –<20 Lux on top of the tanks Xenopus – poss. require UV light for Vit D: –100 lux at bench height –40 lux at the top of the tanks – avoid retinal damage

69. Module 3 Feeding Only use approved food that will not introduce pathogens to the system. SDS aquatic diets Do not overfeed OR underfeed. Will affect the health of the fish and the quality of the water. Uneaten food will start to decay and also block up the filters. Fish should be eager to feed and consume the food quickly. Too little food and egg production decreases

70. Feeding fish The aim to have a slight deposit of food at the bottom of the tank when initial feeding has finished – with ideally this extra food being cleared by the next day.

71. Feeding – early larvae SDS 100: Fed twice daily. For very young fry from free feeding stage Day 5 post fertilisation until around day 14. Note; this is dependent upon actual growth of fry, leave on the diet if any doubt regarding the ability of the fry to eat the SDS 200 diet.

72. Feeding Mid-larvae: SDS 200 Fed twice daily. For fry from around day 14 until around day 42. Note; dependent upon actual size of fish. Juveniles: SDS 300: Fed twice daily. From around day 42 until the fish mature at around 4 months of age. Adults: SDS 400 Fed daily. For mature adult fish in the main system.

73. Supplementary feeding Ground Krill: Feeding restricted to adult fish on Tuesday and Thursday afternoon. This is fed as a nutritional and environmental enrichment supplement. Decapsulated brine shrimp: Fed twice daily and once per day at weekends. Is a high protein supplementary feed fed to juvenile (SDS 300 stage) and adult fish.

74. Supplementary feeding (cont.) Paramecium: Paramecium is fed once a day in the afternoon (am at weekends) to fry fish being fed SDS 100. Paramecium are dispensed by pipettes and a tank of 35 fry should be given around 15 ml (3 pipettes full)

75. Brine Shrimp These are produced in the purposely designed brine shrimp hatcheries. Brine Shrimp Hatchery Option to keep at 37C Increase lighting

76. Brine Shrimp (cont) Brine Shrimp preparation. Obtained as cysts from Mackay Marine, 30mg into each hatchery and use after 3 days. Water in hatchery has increased salt concentration – 20 parts per 1000 - Use ‘Instant Ocean’ Recommended pH 8.0-8.5

77. Xenopus feeding Blades biological Xenopus diet- Size number 3 The Xenopus are fed twice or three times weekly Adult Xenopus should be given enough food so that there is some remaining in the tank after two hours. Any remaining food should be removed after the three - five hours. Beef liver – as part of the diet up to twice per week

78. Cleaning and disinfection: The hard way by hand! Any debris or uneaten food should be removed from the holding tanks daily. Achieved by means of a siphon – adapted squeezee bottle or by netting. Small particles are flushed into the pre filters by the circulating water. Pre filters are washed 3 times per week.

79. Cleaning (cont) Full clean of adult tanks takes place about every 6 wks. Gross debris removed when emptying the tank of it’s contents. Wipe away most of the algal staining Rinse with tap water and finally add system water before replacing fish.

80. Cleaning (cont) Embryo’s and developing fish are attended to daily. Any surface film is removed by soaking into absorbent tissue. The tank contents are then decanted into a clean tank. Any dead embryos are then removed and the old tank cleaned and rinsed out before re-use. Now – embryo’s left until fully viable free swimming fish ‘Graze’off the food on the bottom of the tank

81. Disinfection Equipment is immersed in chlorine solution. Sodium hypochlorite diluted to 100ppm is recommended. At Nottingham ‘MILTON’ is used according to instructions on the bottle.

82. Disinfection (cont) Iodophors diluted according to the manufacturers directions to give 100ppm of available iodine may also be used. E.g. Buffodyne® and Wescodyne Not used at Nottingham.

83. Important All traces of the chemicals used must be removed by rinsing and/or soaking in system water before use with the fish. This also applies to any soaps and detergents used in the facility.

84. Sterilisation Autoclaving Equipment must be robust enough to withstand the autoclaving process. Tanks are not normally autoclaved, as this may result in reduced visibility and cracking of the tanks. ‘Dirty steam’

85. Room clean The facility should also be cleaned with a chlorine based disinfectant on a regular basis. However as most disinfectants are toxic to fish, it is imperative that the aquarium system is not contaminated.