zebrafish thesis

8/4/2019 Zebrafish Thesis

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EFFECTS OFLEAD NITRATE ON THE

MORPHOLOGY OF THE EYES AND BODY OFTHE ZEBRAFISH (Danio rerio)

Jonathan U. DoloquePeter Paul R. Pascual


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Background Information

Commonly occurs as a colorless andwhite powder crystal.

Used in the manufacture of gasoline,batteries, paint as well as ammunition.

Has been linked to several eye diseasesand cardiovascular problems.

Lead Nitrate (Pb(NO3)2)


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Enters the aquatic habitat by mining,

agricultural runoffs and oil spills.

Must be handled and stored with

appropriate safety precautions to preventinhalation, skin contact and ingestion.

Limited applications are in scrutiny bythe International agency for research oncancer.


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Characterized by its prominent stripesthat extend lengthwise from head to tail.

Grows for about 5cm (2in) in length andhas a silver or golden with several bluish-black stripes.

Main benefits of using zebrafish is withregard to their size, husbandry andmorphology (Hill et. al, 2005).

Zebrafish (Danio rerio)


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Morphometrics are a set of statisticalprocedures for analyzing variability in sizeand shapes of organisms (Reyment,

2009).

Image J is a public domain java-basedimage program that was designed with anopen architecture to allow its extensibilityand efficient use.


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Statement of the Problem

There are very few studies that areconcerned with the effects of heavymetals in the condition of the adultorganism.

Studies have only focused ondeveloping embryo and have slightlyneglected the presence of adult

organisms in the aquatic habitat.


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General objective of the Study

Aims to determine the effects of thedifferent concentrations of Lead Nitrate(Pb(NO3)2) on the biometric landmarks ofthe adult zebrafish as well as its effect on

the overall morphology of the eyes.


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specific objectives of the Study

Aims to determine the specificconcentration of the compound Leadnitrate(PbNO3) which has affected the:

a. Morphology of the eyeb. Biometric landmarks

Total length(LT)

Fork length(LF)

Length from head to tail(LA)

Head length(LH)

Pelvic fin(LP)

Eye diameter(ED)


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Significance of the Study

Gives a proper idea of the toxicologicaleffects of Lead Nitrate that can affect theaquatic life present.

Can help predict the morphologicalchanges (damage) that has occurred onthe morphology of the different aquaticlife living in places with high

concentrations.


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Scope and Limitations

The study will only focus on the effectsof the chemical on the biometriclandmarks as well as the morphology ofthe eyes of the Zebrafish.

Will not deal with the physiology andmechanism of action of the Lead Nitrate.

Will not deal with the effects of otherbiotic and abiotic factors present.


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Flow chart of the methodology

Overnight acclimatization of zebrafish

, Exposure of zebrafish to four

concentration (Control, LC25,LC50,LC75)of lead nitrate(PbNO3) for 30 days

Sampling, biometric analysis, and histologicalanalysis of four zebrafish for the 5th, 10th, 15th,

20th, 25th, and 30th day.


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General Animal Husbandry

A mixed set of population of 300 Zebrafish

obtained will be obtained from the pet store.

They will be placed in 4 aquaria with 10 liters ofwater and 1 aerator each.

The concentrations will be Control (pure water),LC25 (12.5mg), LC50 (25mg) and LC75(37.5mg). There will be three replicates per

concentration except for the control.

The fishes will be acclimatized for 24 hours andwill be fed once every day.


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General Animal Husbandry

Lead Nitrate will be homogenized for 30

minutes prior to the exposure of thefishes.

They will be exposed to Lead Nitrate for30 days with sampling of 4 fishes on the5th, 10th, 15th, 20th, 25th and 30th day.


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Biometrical Measurement

Fig. 1: Biometric landmarks that will beused in the study.


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Microtome Section

The 4 fish sample sampling dates will be fixed

(at each sampling point) for 24 h in Bouinssolution.

Fixed specimens will be dehydrated and

subsequently embedded in paraffin.

Sagittal serial sections will be cut at 5-7 m,stained with haematoxylin and eosin (gross

histological).

Stained sections will be studied using asimple microscope (Osman, 1970).


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Statistical and Microtome Analysis

The data obtained from the Biometrics will be

statistically computed using the SPSS 16.0 atthe level of significance of = 0.05. Anovaand HSD will be used to determine if there willbe a significant correlation with the landmarks

and the concentrations used.

The microtome sections will be analyzed bygiving a description of the changes that will

occur during the period of exposure.


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zebrafish thesis

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