Practical # 1Serial sections of frog

 At 26 hours of development:

Fig. 1.1 at 26 hours of fertilization gastrulation starts in the frog embryo. The figure shows the
gastrulation stages of fro embryo. It also shows the clear crescent shaped dorsal lip.

 At 34 hours of development:

Fig.1.2 shows that the formation of middle semicircular dorsal blastoporal lip of frog embryo.

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 At 42 hours of development:

Fig.1.3 in the late gastrulation circular blastopore is form. From which the future anus is
develop.

Fig. 1.4 shows the ventral lip of blastopre. It formed at 42 hours of development.
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 At 50 hours of development:

Fig.1.5 shows the neurulation in frog embryo at 50 hours of development. Frog

embryo begin to form the nervous system.

 At 50 hours of development:

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 Fig. 1.6 shows the formation of early medullary plate. In frog embryo the nervous
system begin to form at 50 hours after fertilization. Medullar plate also form.

 At 62 hours of development:

Fig.1.7 shows the convergence of middle neural fold to form the neural tube.
Convergence of neural groove into neural tube begin at 62 hours of development.

 At 67 hours of development:

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 Fig.1.8 shows the formation of late neural tube. At 67 hours of development late neural
tube forms in frog embryo and ciliation starts.

 At 84 hours of development:

Fig.1.9 shows the formation of tail bud. At the 84 hours after fertilization frog embryo
begin o form tailbud.

 At 96 hours of development:
At this stage the frog embryo start muscular response to tactile stimulation.

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 Fig. 1.10 shows muscular response to tactile stimulation.

Development stages in frog:

Fig.1.11 show the complete life cycle of frog

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Practical # 2

Serial sections of chick embryo

At 24 hours of development:

Fig.2.1 demonstrate the chick embryo at 24 hours of development

At 25 hours of development:

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 Fig. 2.2 represents the beginning of heart formation

At 25 to 29 hours of development:

Fig. 2.3 chick embryo developmental stages from 25 to 29 hours

At 33 hours of development:

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 Fig. 2.3 shows chick embryo at 33 hours of development

At 35 hours of development:

Fig. 2.4 development of ear in chick embryo

At 36 hours of development:

Fig. 2.5 future development of heart is no mesodermal

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At 40 hours of development:

Fig.2.6 heart tube form at 40 hours of development

At 42 hours of development:

Fig.2.6 shows the beginning of heart beating

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At 48 hours of development:

Fig.2.7 shows the chick embryo at 48 hours of development

At 55 hours of development:

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 Fig.2.8 shows the development of chick embryo. It shows brain development

At 56 hours of development:

Fig.2.9 chick embryo develop the tail bud

At 60 hours of development:

Fig.2.10 chick development at 60 hours. Beginning of nose.

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At 72 hours of development:

Fig.2.11 chick embryo at 72 hours of incubation

At 80 hours of development:

Fig.2.12 At 80 hours of development chick embryo

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At 96 hours of development:

Fig.2.13 chick embryo at 96 hours of development. Wing buds appears anteriorly, heart
chambers form.

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 Practical # 3

Induced spawning in frog

Summary of spawning in frog:

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 Practical # 3

Induced spawning in frog

Summary of spawning in frog:

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Fig. 3.1 the figure shows the whole process of inducing eggs in frog. The hormones which are
used in the breeding also indicate there location in body.

Step 1: Anesthetize frog

Fig.3.2 Anesthetize the frog. At left is female frog and at right is male frog. female frog is
slightly larger than male and have pear shaped body and prominent cloaca. Males often have
dark nuptial pads on the inner surface of their forelimbs. Place both male and female in tank
containing water at a depth of approximately 2 inch. Leave the anesthetic frog for 25-30 minutes.
Do not leave the frog in anesthetic condition for a long time. Do not Keep the temperature below
than 23°C because it can lead to death. Ideally, water temperatures should be maintained
between 25°C (77.0°F) and 28°C.

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Step 2: Inject the both frog sexes

Fig. 3.3 Inject the frog to induce spawning. Inject each frog with 0.1 ml of a 100 U/ml solution
of HCG. Make the injection into a dorsal lymph sac using a 1-mL syringe and a 25-gauge needle.
Once each injection is complete, allow the frog to recover from anesthesia for 30–60 min. in
shallow water. Return active frogs to normal housing conditions for 20–24h.

Step 3: transfer of frog

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Fig.3.4 Transfer the individual female frogs to tanks at a depth of approximately 2 inch And
allow them to lay eggs. Tanks should be situated in a quiet place if possible, and covered with a
sheet to prevent any disturbance to the frogs during egg-laying. Females typically begin laying
eggs approximately 2 h after HCG injection and continue for several hours.

Step 3: frog eggs

Fig.3.4 figure shows the frog eggs.

Step 4: collect the frog eggs

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Fig.3.5 Collect the eggs from the tanks using a plastic transfer pipette that has been precoated
with Leibovitz L-15 medium containing 10% calf serum, and transfer them to a Petri dish.
Remove as much of the buffer as possible, allowing the eggs to form a monolayer in the dish.

Step 6: collect the testis

Fig.3.6 kill the frog and dissect to remove the testis from frog.

Step 7: mixing of eggs with testis

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Fig.3.7 Transfer both testes to the Petri dish, place them alongside the eggs, and macerate
them using forceps to release the spermatozoa. Holding the macerated testis tissue with
forceps, pass it over the eggs systematically. Ensure that all eggs come into direct contact
with the testis tissue. Allow the eggs to sit for exactly 4 min before flooding the dish with
0.1X MBS.

Step 8: de-jellying eggs

Fig.3.8 After fertilization, remove the jelly coat surrounding the embryos by discarding
the buffer and replacing it with 25 ml of 2% (w/v) cystein hydrochloride de-jellying
solution. Gently agitate the embryos to ensure even exposure to the de-jellying solution.
Dejellying should take 3–5 min. The clear separation between abutting embryos, which
results from the presence of the outer jelly coat, will disappear when the dejellying
process is complete. Embryos in dejellying solution must be monitored continuously by
eye or by dissection microscope to avoid lethal overexposure.

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Step 9: production of eggs

Fig. 3.9 frog are produced. Figure shows the larvae of frog

Step 10: larval to adult stages

Fig.3.10 figure shows the developmental stages of frog

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Practical # 4

Induced spawning in African catfish fish

Step 1: collect catfish

Fig 4.1.farmer collect the catfish at the shallow parts of river

Step 2: African catfish

Fig 4.2 photograph of Catfish. Catfish has biting nature. so when putting in holding tank

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Stitch their mouths closed to prevent them from biting one another.

Step 3: identify and separate the sexes

Fig.4. 3 this diagram shows that female and male catfish. After collecting the fish, separate

Both sexes in separate container.

Step 3:Determining the separate sexes

Fig.4.4 both sexes can be easily recognized. Female catfish has swollen genital opening and
sexually ready female has the swollen and usually reddish genital opening. Male catfish has

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distinct sexual papilla elongated and just behind the anus. This sexual papilla is usually red at the
tip for sexually ready males. It is absent in females.

Step 4: select and check for gravid female

Fig 4.5 this photograph shows the method of pressing eggs out of gravid female. Gently press the
abdomen from front to rear to strip the eggs.

Step 5: weigh the female fish

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Fig.4.6 eight the female fish. The fish should be healthy. Fish should have the appropriate
weight to lay egg and sexually ready for spawn.

Step 6:preparation of pituitary extract

Fig4.7 Dried African catfish pituitary gland

Step 7: putting in mortar

Fig.4.8 put the dried pituitary gland in laboratory mortar

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Step 8:grinding

Fig.4. 9 grind the pituitary with the help of a pestle until it becomes powder.

Step 9: Addition of saline solution

Fig.4.10 Add 1ml saline solution in the pituitary powder to make the pituitary extract injection.
This is a synthetic hormone used to induce the fish. Collect the solution and inject the female fish
using an hypodermic syringe.

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Step 11: ova prim

Fig. 4.11 it is a synthetic hormone which is use in the induced spawning of fish

Step 12: inject the fish

Fig. 4.13 Photograph showing how to inject the African Catfish. The fish should also be injected
above the lateral line with the needle at 45 degrees to body of the fish. Inject intraperitoneally.

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Step 13:isolation of injected fish

Fig4.14. Isolate the injected fish in a comfortable bowl and wait for 10 to 12 hours. inject again
if necessary and wait.

Step 14: set up of incubator

Fig. 4.15 incubator setup

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Step 15: escape out the fish

Fig.4.16 Bring the fish out and cover with soft and moist towel.

Step 16: wipe the body of fish

Fig 4.17 this shows that how to dry the fish gently. clean fish with wet towel to remove the
anesthesia before stripping. It prevents the egg from killing.
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Step 17: stripping

Fig. 4.18 it shows the method of pressing the fish gently to strip the eggs. It also show the
stripped eggs. Repeat the process until the blood come with the eggs. Stop the process.

Step 18: bring the male out to extract the milt

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 Fig 4.19. Bring the male out and kill it. Then turn the belly up, cut it and extract the milt sac.

Fig.4.20 This photograph shows the milt sac of male African fish

Step 20: remove the milt sac

Fig. 4.21 extract the milt sac to remove the sperm.

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 Fig.4. 22 cut the testicles into bits to remove the sperm

Fig.4. 23 Add the saline solution into milt and mix it. It forms a mixture and used to fertilize the

egg of fish.

Step 21: pouring of milt

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Fig4. 24. Pour the saline solution mixed with milt into the eggs. Then mix it with stirrer.

Prevent the eggs from mixing with water. If water mix then it prevents from fertilization.

After mixing the eggs with milt wait for 2-3 minutes.

Step 22: Addition of water

Fig.4.25 Add clean water and mix it. Continue stirring to prevent the eggs from sticking togather.

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Step 23: Add into incubator

Fig.4. 26 set up the incubator. The spawning sponge is completely immersed into water. It sits on
the spawning net which keeps it suspended in water.

Step 24: remove after 36 hours

Fig. 4.27 after 36 hours remove the sponge an spawning net.

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Step 25: observe the newly hatched eggs

Fig.4.28 observe the newly hatched eggs with yolk still attached with fry.

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