Zebrafish Course, Gupta and Mullins, 2009

Protocol for Adult Fish Dissection 1) Anesthetize a male fish in 0.2% Tricaine, then transfer with a spoon to ice water to euthanize. Rinse the fish in fish water to remove the Tricaine. Lightly pat the fish dry on a paper towel so that it is just damp. 2) Pin the fish onto the dissecting mat with the left side of the fish facing up. 3) Cut the skin from the anal fin along the belly of the fish to the operculum (Figure 1, step 1). Remove the operculum and the pectoral fin, including the pectoral girdle (Figure 1, step 2). Cut the skin beginning from above the now exposed gills posteriorly along the side of the fish and then down to the anal fin (Figure 1, step 3). Figure 1: Illustration of how to cut the skin to reveal the organs.

4) Remove the skin from the side of the fish. Identify the testis, the anterior and posterior chambers of the swim bladder, the liver and the intestine (Figure 2). Remove the testis and place it in the PBS.

Zebrafish Course, Gupta and Mullins, 2009

Figure 2: Photo and illustration of internal organs. Illustrations are from the American Association for Laboratory Animal Science, Zebrafish Anatomy Poster.

5) Carefully remove the liver and intestine. You should be able to identify the spleen and gall bladder within the viscera. Straighten the intestine out and identify the anterior, mid and posterior regions. Place a piece of the anterior intestine in PBS and observe the epithelial folds.

Zebrafish Course, Gupta and Mullins, 2009

6) Remove and discard the swim bladder. Unpin the fish and re-pin it ventral side up. Identify the head, body, and tail regions of the kidney (Figures 3 and 4). Dissect out a piece of the kidney and place it in PBS. Look for the renal tubules. Figure 3: Illustration of internal organs with digestive tract removed.

Figure 4: Photograph of kidney showing head, body and tail regions.

Zebrafish Course, Gupta and Mullins, 2009

7) Unpin the fish and cut the head off with a razor blade. Pin the head onto the dissecting mat ventral side up. Remove all of the soft tissue surrounding the skull. Carefully break the skull open with your forceps and remove as much of the bone as possible. Identify the optic nerves and optic chiasm. 8) Remove the tissue surrounding the forebrain being careful not to damage the olfactory bulbs. Gently place the brain into PBS and carefully remove the skin and bone on the dorsal side of the brain. Identify the olfactory bulbs, telencephalon, habenula, optic tectum, cerebellum and medulla (see Figure 5). Figure 5: Lateral and Dorsal views of the zebrafish brain. Illustrations are from Neuroanatomy of the zebrafish brain : a topological atlas by Wullimann, Rupp, and Reichert. Birkhäuser Verlag, 1996.

Zebrafish Course, Gupta and Mullins, 2009

9) Anesthetize and then euthanize a female fish, as above. Remove skin and muscle as above, then dissect out the ovary (Figure 6) and place it in PBS. Identify stage I, II, III and IV oocytes. Examine the testis that you dissected in step 4. Figure 6: Illustration of internal organs of a female fish.

Figure 7: Images of ovary (A-C) and testis (D-E) tissue.

Zebrafish Course, Gupta and Mullins, 2009

10) Find the heart (Figure 6), which is located posterior and ventral to the gill. The heart is delicate, so it is helpful to remove most of the surrounding tissue before trying to remove it from the fish. Put the heart into a dish of PBS. Identify the atrium, ventricle, and bulbous arteriosis (Figure 8). Figure 8: Schematic of the adult zebrafish heart. Illustration from Hu et al., 2001. Cardiac morphology and blood pressure in the adult zebrafish. The Anatomical Record 264: 1-12.

11) If you wish, you may try dissecting the brain of the female fish from the dorsal side in order to better preserve the ventral structures.