1. Sucul Hayvan deneyleri Deney hayvanlar kullanım şekilleri, sucul hayvan test sistemleri http://hdek.trakya.edu.tr

2. Fish and invertebrates Fish and invertebrates from marine and freshwater environments have long provided valuable models for the study of basic biological processes. Some of the earliest studies of non-self recognition and phagocytosis were conducted using sea urchins (leading to the 1908 Nobel prize for Mechnikov) while the principles of signal propagation in axons were elucidated from studies of the giant axon of squid (leading a Nobel prize in 1963 for Hodgkin & Huxley). Aquatic invertebrates and fishes have proved to be valuable model organisms based on specific physical features, such as giant axons, as well as by providing an evolutionary perspective on structure and function in higher vertebrate systems.

3. Introduction Laboratory animal medicine is the specialty field within veterinary medicine that is concerned with the diagnosis, treatment, and prevention of diseases in animals used in research, testing, and teaching. Laboratory animal science is the body of scientific and technical information, skills and techniques that apply to laboratory animal care and use This includes husbandry, nutrition, behavior, health, production and management of laboratory animals

4. Laboratory animals Human beings use animals for a wide variety of purposes, including research. The approximately 260 million people in the United States keep about 60 million cats and about 52 million dogs as pets. Including birds and horses, just these types of American pets total nearly 130 million. More than five billion animals are consumed each year as food. It’s estimated that about 17 million animals are used for biomedical research annually.

5. WHY USE ANIMALS? In research, animals are used to learn more about biological systems and the illnesses that afflict human beings and other animals. They serve as surrogates for humans in obtaining information that cannot be gained in any other way. Some animals have biological similarities to humans that make them particularly good models for specific diseases such as rats for cancer, rabbits for atherosclerosis and nonhuman primates for polio.

6. Laboratory Animal Testing What’s animal testing? Animals used in Lab. Facilities* Test’s performed on some of the animals* Places where experiments take place* Standards and Restrictions

7. What is Animal Testing ??? Animal Testing is the use of non- human animals in scientific experimentation

8. What kind of animal are used? Small Animal (dogs, cats) Large Animal (pigs, sheep, goats, etc) Exotic Animal (ferrets, nonhuman primates, guinea pig, rabbits, mice, snakes, geckos, etc) Avian (chickens, pigeons, songbirds, etc) Wildlife (armadillos, owls, bats, etc) Aquatic (zebrafish, frogs, goldfish, etc)

9. Choose one

10. WHAT ARE THE MOST COMMONLY USED ANIMAL MODELS? Animal: Cat System/Condition: Auditory Why Is It Studied?: Like humans, cats have very well- developed hearing systems and brain mechanisms for hearing. They can be trained to respond to many behavioral cues given through auditory stimuli. Cats also experience naturally occurring hearing defects and are susceptible to environmentally induced defects, as are humans.

11. WHAT ARE THE MOST COMMONLY USED ANIMAL MODELS? Animal: Primate System/Condition: Immune Why Is It Studied?: Primates possess striking immunological similarities to humans. They are susceptible to similar diseases and often react to the same infectious agents as humans.

12. WHAT ARE THE MOST COMMONLY USED ANIMAL MODELS? Animal: System/Condition: Cardiovascular Why Is It Studied?: A dog’s cardiovascular system is structured quite similarly to humans. They suffer from many inherited cardiovascular defects that affect humans. Since they possess inherited defects nearly identical to those seen in humans, hematology, the study of the blood, is also practiced using dogs. System/Condition: Endocrine Why Is It Studied?: Dogs naturally experience diabetes like humans. Diabetes can also be easily induced in dogs to aid research. In addition, dogs share other diabetes-induced deficits such as glaucoma, that occur in humans.

13. WHAT ARE THE MOST COMMONLY USED ANIMAL MODELS? Animal: Mouse System/Condition: Aging Why Is It Studied?: Mice age 30 times more rapidly than humans, with several body systems declining with age in the same manner as those systems do in humans. Genetic composition and environmental conditions can be precisely and easily duplicated and controlled — a vital consideration in interpreting data.

14. WHAT ARE THE MOST COMMONLY USED ANIMAL MODELS? Animal: Rat System/Condition: Aging Why Is It Studied?: Rats are available in a number of purpose-bred strains and have been the focus of intensive physiological and biochemical research. Rats show major, spontaneously developing and age- related damage in most major systems of the body that commonly are seen in humans. However, in rats these deficits occur faster and are easily studied during a rat’s lifetime.

15. WHICH ANIMALS ARE STUDIED FOR THEIR UNIQUE CHARACTERISTICS? Organism: Armadillo System Studied: Reproduction, Developmental biology What is Studied & Why: Armadillos can give birth up to three years after fertilization. They experience delayed implantation of the fertilized ovum and give birth to identical quadruplets. Their low body temperature allows leprosy to grow. Human Counterpart: Twinning, menopause, infertility, leprosy

16. WHICH ANIMALS ARE STUDIED FOR THEIR UNIQUE CHARACTERISTICS? Organism: Ferret System Studied: Immune system What is Studied & Why: Both ferrets and humans are affected by closely related bacteria. Human Counterpart: Gastritis and stomach ulcers

17. WHICH ANIMALS ARE STUDIED FOR THEIR UNIQUE CHARACTERISTICS? Organism: Seal System Studied: Respiratory and Nervous systems What is Studied & Why: Young seals exhibit similar characteristics to humans when they hold their breath for long dives or when sleeping under water. Human Counterpart: Sudden infant death syndrome (SIDS)

18. What kind of tests are performed ? Toxicology Tests Experiments Cosmetic Tests And More !!! Genetics

19. Aquatic animal assay systems Research on the subject has different aquatic test systems, depending on the studied species. Generally, there are four basic assay system used in aquatic animal experiments. 1. Static test system: test system during the experiment is that all the water and changing the applied chemical. 2- The recirculation system experiments: experiments with water and chemicals used in a pump or a similar system moves continuously from the filter returns to the door then try again. This filter system (or air conditioning, sterilizers) arrested and investigated the effects of chemicals, to come back and try to connect the aquarium has a problem.

20. Aquatic animal assay systems

25. 3-Renewed test system: This aquatic system in static test system prepared in accordance with the test conditions in a particular period of time (usually 24 hours) is replaced with water prepared again. In this way all the aquatic animal is exposed to chemicals in the same quantities, as well as the removal of metabolites of aquatic animals had left the medium is provided. 4- Flow test system: a certain chemical in the test system enters a certain amount of test solution containing a constant speed as quickly flask and interests. During the experiment with the constant washing of the test solution is placed in the door of aquatic animal, the same amount of material affect aquatic animals and aquatic animal has all the advantages that can be removed from the environment of the metabolite flow. In contrast, large amounts of chemicals are used, which limits the use requires special equipment and experience.

26. Aquatic animal assay systems

28. Test systems test system costs water consumption Experiences accumulation of metabolites Stres constant environment al conditions Long term work special equipment Static test system ++++++++++ Recirculat ion test system ++++ ++ Renewed test system ++++ +++++ + Flow test system +++ ++ ?: No or very difficult, +: very hard and / or too little, ++: Available and / or a sufficient amount, +++: easy and / or high amounts of

29. The Aquatic Environment in aquatic animal experiments Critical Parameters dissolved oxygen temperature pH un-ionized ammonia nitrite nitrate carbon dioxide alkalinity solids

30. Parameter Interactions Parameters ar not static. parameters affect each other, CO 2 and dissolved oxygen concentrations pH versus ammonia-nitrogen concentration Temperature and growth rate and health

31. Quantity Amount of water needed will depend on: species density management practices production technology degree of risk one is willing to accept Rule of Thumb 20% water exchange of total system volume per day(Semistatik or static systems)

32. Recirculating Aquaculture Systems Short Course Quantity – Reuse Systems Three Categories of Reuse Systems Serial-reuse Systems – Serial flow through Partial-reuse systems – 80-90% water reuse Fully recirculating systems – >95% water reuse Low High

33. Recirculating Aquaculture Systems Short Course Quantity – Serial-reuse Systems Serial-reuse Systems Trout and Salmonid raceways Limiting Factor – Dissolved Oxygen Systems limited by ammonia concentrations

34. Recirculating Aquaculture Systems Short Course Quantity – Partial-Reuse Systems Partial-reuse Systems Circulation Production Tanks – Swirl Separators Solids removed from center drain (15-20 % flow) Ammonia controlled by dilution and system pH pH controlled by controlling CO 2 level in tanks

35. Recirculating Aquaculture Systems Short Course intermittent cleaning flow primarydischarge (180-390 L/min) air O2O2 H2OH2O backwashslurry Partial-Reuse Fingerling System (Courtesy of PRAqua Technologies) 1000-1900 L/min

36. Quantity – Fully Recirculated Systems Fully Recirculating Systems Circulation Production Tanks – Dual Drain Solids controlled with microscreen filters Ammonia controlled by biofiltration Aeration or oxygenation required for high densities Sophisticated backup and alarm systems required.

37. Recirculating Growout System Fully-recirculating system 4 - 8% make-up rate on a flow basis (0.5-1.0 day HRT) 4,800 lpm recir. water flow 150 m 3 culture volume 7% through bottom drain 93% through side drain 200 kg/day feed (Courtesy of Marine Biotech Inc.)

38. Water Sources Groundwater Surface Water Municipal Water Supplies chlorine removal activated carbon Sterilization

39. Water Sources – Ground Water Advantages: Constant Temperature Disadvantages: Dissolved H 2 S and CO 2 Low Dissolved Oxygen Supersaturation High Iron Concentration

40. Water Sources – Municipal Water Designed and treated to safeguard the health of humans, not fish! Disadvantage Chlorine Fluorine Cost Advantages Availability Reliability

41. Water Quality Standards ParameterConcentration (mg/L) Alkalinity (as CaCO 3 ) 50-300 Ammonia (NH 3 -N unionized) <0.0125 (Salmonids) Ammonia (TAN) Cool-water fish <1.0 Ammonia (TAN) Warm-water fish <3.0 Carbon Dioxide (CO 2 ) Tolerant Species (tilapia) <60 Sensitive Species (salmonids) <20

42. Water Quality Standards ParameterConcentration (mg/L) Hardness, Total (as CaCO 3 ) >100 Iron (Fe) <0.15 Nitrogen (N 2 ) <110% total gas pressure <103 % as nitrogen gas Nitrite (NO 2 ) <1, 0.1 in soft water Nitrate (NO 3 ) 0-400 or higher

43. Water Quality Standards ParameterConcentration (mg/L) Oxygen Dissolved (DO)>5 > 90 mm Hg partial pressure Ozone (O 3 )<0.005 pH 6.5-8.5 Salinity <0.5 to 1 Total dissolved solids (TDS) <400 Total suspended solids (TSS) <80

44. Water Quality Parameters Dissolved Oxygen Temperature Ammonia/Nitrite/Nitrate pH Alkalinity/Hardness Salinity Carbon Dioxide Solids Critical Parameters Important Parameters

45. Dissolved Oxygen Saturation concentration of dissolved oxygen: highest at low temperature lowest at high temperatures But demand for basic metabolism and food conversion: highest at high temperatures lowest at low temperatures

46. Temperature Three Classifications: cold-water species below 15 ° C cool-water species between 15 °- 20° C warm-water species above 20° C

47. Ammonia/Nitrite/Nitrate Nitrosomones Bacteria Nitrobacter Bacteria 2 NH 4 + + OH - + 3 O 2  2 H + + 2 NO 2 - + 4 H 2 O 2 NO 2 + 1 O 2  2 NO 3 - NH 4 + + 2 HCO 3 + 1.9 O 2  NO 3 + 2.9 H 2 O + 1.9 CO 2 +0.1 CH 2 O Nitrifying Bacteria – Overall Reaction

48. Ammonia - Nitrogen Equilibrium Reaction - Ammonia NH 4 + + OH -  NH 3 + H 2 O Note: NH 4 + -N + NH 3 -N  TAN NH 4 - -N  Ammonia - nitrogen Increase in pH Increase in temperature

49. Unionized Ammonia-Nitrogen Percent unionized Ammonia-nitrogen pH Temp.6.0 6.5 7.0 7.5 8.0 9.0 10-0.10.20.61.815.7 15-0.10.30.92.721.5 20-0.10.41.23.828.4 250.10.20.61.85.436.3 300.10.30.82.57.544.6

50. Nitrite-Nitrogen Equilibrium Reaction – Nitrite NO 2 - + H 2 O  HNO 2 + OH - Note: NO 2 - -N  Nitrite - nitrogen (mitigated by adding salt (chlorides) Decrease in pH

51. Recirculating Aquaculture Systems Short Course Nitrate - Nitrogen Equilibrium Reaction – Nitrate NO 3 -N Note: NO 3 - -N  Nitrate - nitrogen Non-toxic (freshwater systems)

52. Recirculating Aquaculture Systems Short Course pH pH value expresses the intensity of the acidic or basic characteristic of water. Seawater: 8.0- 8.5 Freshwater: 6.5 – 9.0

53. Alkalinity Alkalinity (50 -150 mg/l as Ca CO 3 ) FormulaCommon NameEquivalent Weight NaOHsodium hydroxide40 Na 2 CO 3 sodium carbonate53 NaHCO 3 sodium bicarbonate83 CaCO 3 Calcium Carbonate50 CaOslaked lime28 Ca(OH) 2 hydrated lime37

54. pH, alkalinity and CO 2 The relationship between pH, alkalinity, and CO 2 concentrations. Alkalinity 100 mg/L

55. Hardness soft (0-75 mg/L moderately hard (75 – 150 mg/L) hard (150-300 mg/L) very hard (> 300 mg/L) Classified as: Recommended range: 20 to 300 mg/L CaCO 3

56. RecirculatingAquaculture Systems Short Course Carbon Dioxide Exposure to high carbon dioxide concentrations reduces respiration efficiency and decreases the tolerance to low dissolved oxygen concentrations. Carbon dioxide is a highly soluble in water. Concentration in pure water: 0.54 mg/L at 20° C. Groundwater concentrations range from 0-100 mg/L.

57. Solids – settleable, suspended, dissolved Three categories: settleable suspended fine or dissolved solids upper limit: 25 mg TSS/L normal operation (species dependent) 10 mg/L for cold water species 20 – 30 mg/L for warm water species Rule of Thumb Solids produced by fish : 0.3 to 0.4 kg TSS for every 1 kg of feed fed

58. Recirculating Aquaculture Systems Short Course Salinity Osmoregulation Rule of Thumb To reduce stress and reduce energy required for osmoregulation, freshwater aquaculture systems are maintained at 2-3 ppt salinity. Usually reported as parts per thousand, ppt.

59. Recirculating Aquaculture Systems Short Course Measurements – Dissolved Oxygen Winkler Titration DO Meters – polarographic -galvanic

60. Recirculating Aquaculture Systems Short Course Measurements - Temperature Off-the-self-components and hardware. Included with most DO, pH, conductivity meters. NOT RECOMMENDED! Mercury thermometers

61. Measurements - pH Both laboratory and field instruments readily available.

62. Recirculating Aquaculture Systems Short Course Measurement – CO 2 Measurement of pH and Alkalinity yields CO 2 Alkalinity 100 mg/L

63. Measurement – Salinity Measurement of a physical property: Conductivity Density - hydrometer Refractive index

64. Recirculating Aquaculture Systems Short Course Chemical Analysis Test Kits and Colorometers

65. Recirculating Aquaculture Systems Short Course Chemical Analysis – Dissolved Oxygen Winkler Method: manganous sulfate, potassium iodide, sodium hydroxide manganous ion + oxygen  manganous dioxide (proportional to dissolved oxygen concentration) sulfuric acid causes the oxidation of iodide to iodine by the manganous dioxide. Titration with sodium thiosulfate with starch indicator (iodine concentration proportional to DO concentration

66. Chemical Analysis – CO 2 CO 2 Carbon Dioxide Free CO 2 reacts with sodium hydroxide (0.0227 N) to form sodium bicarbonate; completion indicated using a pH meter (8.3) or phenolphthalein indicator. 1 ml of NaOH equals 1 mg/LCO 2.

67. Chemical Analysis - Alkalinity Titration Method Titration with 0.02 N Sulfuric Acid with methyl orange indicator end point (4.5 pH) 1 ml titrant equals 10 mg/L CaCO 3.

68. Chemical Analysis – Ammonia, Nitrite and Nitrate Ammonia: colorimetric Nesslerization ion specific electrodes Nitrite:colorimetric Nitrate:reducing to nitrite with cadmium catalyst, measure nitrite. ion specific electrode

69. Chemical Analysis - Solids Solids A well-mixed sample is filtered through a weighed standard glass-fiber filter and the residue retained on the filter is dried to a constant weight at 103 to 105 °C. The increase in the weight of the filter represents the total suspended solids.

70. Chemical Analysis - Orthophosphorus P Phosphorus Ammonium molybdate and potassium antimonyl tartrate react to form a heteropoly acid, which is reduced with to intensely colored molybdenum blue by ascorbic acid..

71. 3R "Reduction" concept, the necessary scientific practice to get away from the current results include a method for reducing the number of animals required. "Recovery" includes the use of the process that could lead to less pain and stress in animals. "Replacement", the lower species as phylogenetic methods instead of animals or animal non defines the use of the system.

72. What are the criteria for selecting the appropriate test organisms in aquatic animals? If possible, native species should be represented ecosystem. Should be provided easy species to be selected should be sufficient in number during the test Should be Similar characteristics (age, gender, etc.) Species should have ecological and economic importance. Test for aquatic has a wide sensitivity range as possible is intra-species differences in sensitivity between species and appropriate experimental conditions show animal should be selected.

73. Should have high, rapid, easy adaptability to aquatic animal experiments, İt should be made of economic production and culture. Aquatic animal experiments should be able to live under test conditions for 1 week -a month or more aquatic life needs of the biology of experimental animals, salinity, knowledge of the physico-chemical requirements, such as pH and temperature and should be provided. Organism level in the food chain, the importance of the economic aspect and the most delicate phase must be known. Will be used in the experiment should be of a suitable size and weight of aquatic animal experiments. should be used at least 100 times the rate of the aquarium containing water.. What are the criteria for selecting the appropriate test organisms in aquatic animals?

74. To be answered by researchers in all aquatic animals studies 1. Identify which issues will be answered by the study, 2 of which will be used in the study tissue (cell types, tissues, organs, organ interpersonal communication etc.) And determining how the need for tissue amounts of this tissue, 3. Which animal species / descendant in to investigate, 4. This feature is suitable animal species or which is technically more advantageous and descendant "from experimental procedures" which will determine the minimum level of discomfort, 5. supply of animals, shelter and care, adoption, follow-up, to make the appropriate infrastructure, the literature, the review identified the practical factors, such as experience with animals, 6. Selection of an appropriate animal model of ethical and scientific applications

75. Choice of Suitable aquatic animals Sensitivity Adaptation, Native species B and tissue to ob lood tain, long-term suitability study adequate number costs special Papers Vertebrate Salmo sp. ++++++ + (Türe vd. 2014) Oncorhynchus mykiss ++++++ + (Atamanalp vd. 2003) Cyprinus carpio +++++ +++ (De Boeck vd. 2004) Danio rerio +++++- +++++ (Griffitt vd. 2007) Oreochromis niloticus +++-+++ ++ + (Alim ve Matter 2015) Poecilia sp. ++ - +++ (Gallo vd. 1995) Gambusia sp. ++++-++ +++ + (Guner 2012a,b) İnvertebrate Artemia sp. ++++-+?+ (Dağlıoğlu vd. 2016) Daphnia sp ++++++?+ +++ (Lavorgna vd. 2016) Gammarus sp. +++++++?+++++++ (Türevd. 2014) Ostracoda ++++++?+ + (Sevilla vd. 2013) Astacus sp. +++++++++++ +++ (Güner 2010)