1. Husbandry of Aquatic Laboratory Animals Dr. Neil Ruane Marine Environment & Food Safety Services www.marine.ie

2. Responsible use of animals Knowledge of the biological characteristics and husbandry requirements of the species Animal must be suited to the research demands e.g. particular strain, physiology, behaviour Housing, feeding and care must be appropriate to the requirements of the species

3. Presentations topics Housing/rearing units Requirements Feeding Water quality Stocking density Biosecurity Fish welfare and stress

4. Major groups of modern fish GNATHOSTOMATA CHONDRICHTHYSOSTEICHTHYES ELASMOBRANCHISARCOPTERYGIIACTINOPTERYGII CHONDROSTEINEOPTERYGII TELEOSTEI

5. Diversity of fish species  Reflects their ancient history  Ability to speciate rapidly –In response to isolation (landlocked species) –Wide range of habitats (deep ocean, FW, SW) –Pelagic, benthic etc. Most of the diversity is found in FW even though 97% of all water is in the oceans. Physical and chemical characteristics of water impose a number of constraints on the functional design of fish – hence similar adaptations in body form and physical appearance

6. Fish Production in Ireland – ca 13,000t Atlantic salmon (90%) Rainbow trout (8%) Others (2%) COD PERCH TURBOT CHAR

7. Husbandry – rearing units  Rearing of fish based on life cycle – FW or marine  FW species have a less complex larval rearing cycle –Long egg incubation periods –Hatching of well developed larvae –Directly on to commercial diets  Marine species are more complex –Short incubation periods –Larvae less well developed –Often require live food prior to weaning on to commercial diets –Live food: Rotifer and Artemia (which also need to be fed)

8. Husbandry – FW rearing (e.g. trout, salmon)  Egg incubation units  Daily removal of dead eggs to prevent spread of infections (fungal/bacterial)

9. Husbandry – FW rearing (e.g. trout, salmon) 22 days 41 days 76 days 102 days

10. Rainbow trout are often on-grown in pond farms Husbandry – FW rearing (e.g. trout, salmon) Salmon and char are on-grown in tanks or cages in a lake

11.  Atlantic salmon, rainbow trout and Atlantic cod are reared at sea in cages – open system.  Juvenile cod and turbot are reared in pump ashore facilities – located on land, seawater is pumped into the facility Husbandry – SW rearing (e.g. trout, salmon, cod)

14. Recirculation Systems  Closed systems where the water is reused.  Allows better control of water quality and rearing environment.  Higher costs associated with set-up and maintenance.  Basic elements: –Physical filters (removal of large particles) –Biological filter (conversion of harmful waste) –Foam fractionation / Protein skimmer (seawater) –UV sterilisation

15. Physical filters Removing solid particulate matter from the systems. The bulk of these solids are in the form of faeces or uneaten feed. These wastes generate the greatest amount of pollution in a closed system. Settleable solids are those particles that will generally settle out of the water and can be removed by well placed drains, a sedimentation tank, mechanical filtration, or a swirl separator. Suspended solids are those small particles that will not settle to the bottom of the tank. These suspended solids can be removed by running the water through a fine screen or sand filter.

16. Biological filters The process of removing dissolved metabolic waste products (ammonia and nitrite) from the water. This process requires the water to pass through filters that have live bacteria living in them. The effectiveness of this process depends on the amount of bacteria that can be grown in the filter and maintaining optimum conditions for their survival.

17. Dissolved solids Removed by a process called foam fractionation or protein skimming. The fine and dissolved organic compounds cling to the bubbles used in the process. This results in foam, which can then be removed, from the system. This fine and dissolved matter is also removed during water changes. For most systems changing some water regularly will help avoid problems with dissolved solids build up. Zero discharge systems or integrated systems use animals at different trophic levels to clean the water e.g. plants or shellfish.

18. Requirements - feeding The majority of fish can be fed with commercially available diets Marine fish larvae have a more complex feeding strategy involving live feeds such as Artemia (brine shrimp) and rotifers

19. Requirements - feeding Marine larvae are too small to feed on commercial diets. Often fed with rotifers initially before switching to the brine shrimp. Rotifers need to feed on phytoplankton species. Rotifers can be enriched with essential fatty acids to aid growth and development.

20. Requirements - feeding General rules: maintenance levels for feeding are 1% body weight per day. Can be increased to 3 – 5% for growth. Growth trials require more complicated calculations e.g. allometric growth. Fish can be fed by continuous belt feeders or divided into meals – never feed more than the fish can eat. Regular observation of feeding is necessary (sick fish do not eat) Feeding level can be adjusted by weighing fish and readjusting the feeding level.

21. Requirements - feeding Terms: Food conversion ratio: ratio of weight gain to feed (optimal is an FCR of 1 or less i.e. 1 g food equals 1 g increased weight) Feed intake: actual amount of feed digested by the fish Growth rate: weight gain by fish e.g. Xg per day

22. Requirements – water quality As fish are in intimate contact with their environment a reduction or sudden change in the quality of the water can have serious implications for the health and welfare of the animal Important parameters to monitor include: pH, temperature, salinity, oxygen, ammonia, nitrite, water flow carbon dioxide, turbidity, aluminium, water depth

23. Water quality: pH Water pH is essential as it affects the acid/base balance within the fish. Sudden changes in pH can lead to fish kills from hydromineral imbalance (fish lose minerals e.g. Na, Cl, maintaining acid/base balance). High dissolved CO 2 levels can lead to blood acidosis. Seawater has a higher buffering capacity than freshwater. Generally pH levels should be slightly basic, but can range from 6 – 8.5

24. Water quality: temperature Fish are poikilothermic, therefore temperature regulates all aspects of life – development, metabolism, oxygen levels. Sudden changes in water temperature are stressful – need to keep temperatures stable. Each species will have a different optimal temperature range: Atlantic salmon: 4 – 15 o C Rainbow trout: 4 – 20 o C Zebrafish: 18 – 24 o C Carp 4 – 25 o C

25. Water quality: salinity Stenohaline – tolerate a narrow range of salinities (carp, cod) Euryhaline – tolerate a wide range of salinities (salmon, tilapia) Marine fish operate hypo-osmotically, maintaining blood osmolarity at 33% of seawater – drink seawater and excrete ions. FW fish operate hyper-osmotically, excrete water (dilute urine) and actively take up ions.

26. Water quality: oxygen Dissolved oxygen (DO) is probably the most critical factor in maintaining good water quality. Expressed as mg/L or % saturation. DO levels decrease during active periods (feeding) and oxygen dissolves better in cold water. Cyprinids are more tolerant than salmonids. DO must be > 5 mg/L, levels below 2 mg/L can lead to death

27. Water quality: ammonia Ammonia is a natural product of fish metabolism, majority is excreted through the gills. High levels cause stress, damage gills, increase susceptibility to pathogens and cause fish kills. Ammonia occurs in two forms: un-ionized (NH 3 ) and ionized (NH 4 + ). NH 3 is highly toxic.

28. Nitrogen Cycle

29. Calculating Un-ionized Ammonia

31. Requirements – stocking density Stocking density refers to the number of fish per tank i.e. g fish / L water. Loading density: g fish / L water per hour (i.e. includes the flow rates. High densities affect water quality and fish behaviour. Some species like high densities (catfish), some are solitary (grouper, tilapia when reproducing). Salmonids – high and low densities lead to stress.

32. Requirements – biosecurity Defined as a set of management practices to prevent the introduction of a disease causing pathogen into the rearing system. Prevent pathogen entry – External barriers Prevent pathogen spread – Internal barriers + Ensure the health and welfare of animals at all times

33. Biosecurity – external barriers All equipment and vehicles should be cleaned and disinfected before entering the site. Fish coming in to the site should be certified as disease free, it is not allowed to move sick fish. Other aspects – visitor logbook, clear signs indicating procedures

34. Biosecurity – internal barriers Clothing and equipment should be cleaned and disinfected, before and after use. Wash hands after handling fish. Regular cleaning schedules for rearing system. Logs of disinfectants and cleaning agents.

35. Cleaning Protocol 1. GROSS RINSE/REMOVE SOILING 2. APPLY DETERGENT * 5. RINSE WITH CLEAN WATER & DRY 4. APPLY DISINFECTANT * 3. RINSE WITH CLEAN WATER & DRY * Used according to the manufacturers instructions

36. Examples Detergents - Chlorine based – Halamid Iodine based – FAM30, Buffodine Peroxy compounds – Virkon Aquatic Peracetic acid/acetic acid/hydrogen peroxide – Vanodox, Proxitane