Sucul Hayvan deneyleri

Sucul Hayvan deneyleri

Deney hayvanlar kullanım şekilleri, sucul hayvan test sistemleri

http://hdek.trakya.edu.tr

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.

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

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.

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.

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

What is Animal Testing ???

• Animal Testing is the use of non- human animals in scientific experimentation

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)

Choose one

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.

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.

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.

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.

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.

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

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

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)

What kind of tests are performed ?

Toxicology Tests Experiments

Cosmetic Tests

And More !!!

Genetics

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.

Aquatic animal assay systems

Aquatic animal assay systems

Aquatic animal assay systems

Aquatic animal assay systems

Aquatic animal assay systems

Aquatic animal assay systems

• 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 .

Aquatic animal assay systems

Aquatic animal assay systems

Aquatic animal assay systems

Test

systems

test syste m costs

wate r cons ump tion

Expe rienc es

accu mula tion of meta bolit es

Stres

const ant envir onm ental cond ition s

Long term work

speci al equi pme nt

Static test

system + + + +++ + + + +

Recircula tion 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

The Aquatic Environment in aquatic animal experiments

Critical Parameters

• dissolved oxygen

• temperature

• pH

• un-ionized ammonia

• nitrite

• nitrate

• carbon dioxide

• alkalinity

• solids

Parameter Interactions

• Parameters ar not static. parameters affect each other,

•CO

and dissolved oxygen concentrations

• pH versus ammonia-nitrogen concentration

• Temperature and growth rate and health

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)

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

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

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 CO₂ level in tanks

Recirculating Aquaculture Systems Short Course

intermittent cleaning flow

primary discharge (180-390 L/min)

air

O₂

H₂O

backwash slurry

Partial-Reuse Fingerling System

(Courtesy of PRAqua Technologies)

1000-1900 L/min

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.

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 m³ culture volume

• 7% through bottom drain

• 93% through side drain

• 200 kg/day feed

(Courtesy of Marine Biotech Inc.)

Water Sources

• Groundwater

• Surface Water

• Municipal Water Supplies

• chlorine removal

• activated carbon

• Sterilization

Water Sources – Ground Water Advantages:

• Constant Temperature

Disadvantages:

• Dissolved H₂S and CO₂

• Low Dissolved Oxygen

• Supersaturation

• High Iron Concentration

Water Sources – Municipal Water

Designed and treated to safeguard the health of humans, not fish!

Disadvantage

• Chlorine

• Fluorine

• Cost Advantages

• Availability

• Reliability

Water Quality Standards

Parameter Concentration (mg/L)

Alkalinity (as CaCO₃) 50-300

Ammonia (NH₃-N unionized) <0.0125 (Salmonids) Ammonia (TAN) Cool-water fish <1.0

Ammonia (TAN) Warm-water fish <3.0 Carbon Dioxide (CO₂)

Tolerant Species (tilapia) <60 Sensitive Species (salmonids) <20

Water Quality Standards

Parameter Concentration (mg/L)

Hardness, Total (as CaCO₃) >100

Iron (Fe) <0.15

Nitrogen (N₂) <110% total gas pressure <103 % as nitrogen gas Nitrite (NO₂) <1, 0.1 in soft water Nitrate (NO₃) 0-400 or higher

Water Quality Standards

Parameter Concentration (mg/L)

Oxygen Dissolved (DO) >5

> 90 mm Hg partial pressure

Ozone (O₃) <0.005

pH 6.5-8.5

Salinity <0.5 to 1

Total dissolved solids (TDS) <400 Total suspended solids (TSS) <80

Water Quality Parameters

•Dissolved Oxygen

•Temperature

•Ammonia/Nitrite/Nitrate

•pH •Alkalinity/Hardness

•Salinity

•Carbon Dioxide

•Solids

Critical Parameters

Important Parameters

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

Temperature

Three Classifications:

•

• cool-water species between 15 °- 20° C

• warm-water species above 20° C

Ammonia/Nitrite/Nitrate

Nitrosomones Bacteria

Nitrobacter Bacteria

2 NH₄⁺ + OH ^- + 3 O₂ ^ 2 H ⁺ + 2 NO₂^- + 4 H₂O

2 NO₂ + 1 O₂  2 NO₃^-

NH₄⁺ + 2 HCO₃ + 1.9 O₂ ^

NO₃ + 2.9 H₂O ⁺ 1.9 CO₂ +0.1 CH₂O Nitrifying Bacteria – Overall Reaction

Ammonia - Nitrogen

Equilibrium Reaction - Ammonia

NH

+ OH

 NH

+ H

O

Note: NH₄⁺-N + NH₃-N  TAN NH₄^--N  Ammonia - nitrogen

Increase in pH

Increase in temperature

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 25 0.10.20.61.85.436.3 30 0.10.30.82.57.544.6

Nitrite-Nitrogen

Equilibrium Reaction – Nitrite

NO

+ H

O

 HNO

+ OH

Note: NO₂^--N  Nitrite - nitrogen (mitigated by adding salt (chlorides)

Decrease in pH

Recirculating Aquaculture Systems Short Course

Nitrate - Nitrogen

Equilibrium Reaction – Nitrate

NO

-N

Note: NO₃^--N  Nitrate - nitrogen

Non-toxic (freshwater systems)

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

Alkalinity

Alkalinity

Formula Common Name Equivalent Weight

NaOH sodium hydroxide 40

Na₂CO₃ sodium carbonate 53 NaHCO₃ sodium bicarbonate 83 CaCO₃ Calcium Carbonate 50

CaO slaked lime 28

Ca(OH) ₂ hydrated lime 37

pH, alkalinity and CO

0 10 20 30 40 50 60 70 80 90 100

6.50 7.00 7.50 8.00 8.50

pH CO2, mg/L

The relationship between pH, alkalinity, and CO₂ concentrations.

Alkalinity 100 mg/L

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 CaCO₃

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.

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

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.

Recirculating Aquaculture Systems Short Course

Measurements – Dissolved Oxygen

Winkler Titration

DO Meters – polarographic -galvanic

Recirculating Aquaculture Systems Short Course

Measurements - Temperature

Off-the-self-components and hardware.

Included with most DO, pH, conductivity meters.

NOT RECOMMENDED!

Mercury thermometers

Measurements - pH

Both laboratory and field

instruments readily available

.

Recirculating Aquaculture Systems Short Course

Measurement – CO

0 10 20 30 40 50 60 70 80 90 100

6.50 7.00 7.50 8.00 8.50

pH CO2, mg/L

Measurement of pH and Alkalinity yields CO₂

Alkalinity 100 mg/L

Measurement – Salinity

Measurement of a physical property:

• Conductivity

• Density - hydrometer

• Refractive index

Recirculating Aquaculture Systems Short Course

Chemical Analysis

Test Kits and Colorometers

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

Chemical Analysis – CO

CO₂ Carbon Dioxide

Free CO₂ 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.

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 CaCO₃

.

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

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.

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. .

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

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.

• 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?

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

Choice of Suitable aquatic animals

Sensitivit y

Adaptatio n,

Native species

Native species

Band tissue to oblood tain,

long- term suitabili ty study

adequa te 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)