11-Ketotestosterone Implantation in Mormyrid Electric Fish

Last updated on 2026-05-08 | Edit this page

Estimated time: 95 minutes

Background

Cocoa butter melts at body temperature, which makes it a useful slow-release vehicle for lipophilic steroids implanted into the peritoneal cavity. Bass and colleagues established this approach in the 1980s for studies of androgen action on the electric organ in mormyrids (Bass et al., 1986; Bass et al., 1987). A single 20% w/w cocoa-butter implant of 11-KT yields elevated circulating androgen for several days and produces measurable shifts in EOD waveform within ~24–72 h, depending on species and dose.

This episode walks through the Day 0 implant procedure as it is run in our lab. It assumes that the broader experimental schedule (acclimation, weighing, tank assignment, post-implant recording cadence) is already defined elsewhere in the course materials.

Callout

Why cocoa butter?

Cocoa butter is solid at room temperature, liquefies at 35–40 °C, and re-solidifies inside the fish — trapping the steroid in a slow-release depot. It is also non-reactive in the IP cavity and avoids the need for a DMSO/oil carrier injection.

Reagents

  • Cocoa butter chips
  • 11-KT (Steraloids A6720, 25 mg vial)
  • MS-222 (tricaine methanesulfonate)
  • Vetbond (tissue adhesive)
  • Recovery water (system water with NaCl supplement)
  • Maintenance-dose MS-222 (for gill drip during surgery, if used)

Equipment

  • Scalpel (sharp, fresh blade)
  • Kimwipes
  • Dry bath
  • Milligram balance
  • P200 pipette + tips
  • Dissecting tray + dissecting pins
  • Recovery containers
  • Fish-transfer net

Step 0: EOD baseline (Day −1 and Day 0, pre-implant)

  1. Record an EOD waveform from each fish on Day −1 (24 h prior) and again on Day 0 immediately before implantation. These pre-treatment recordings are the per-fish baselines that all post-implant measurements will be normalized to (Bass et al., 1986).
  2. Confirm fish ID, weight, and tank assignment are logged before proceeding to Step 1.
Callout

Why two baselines?

Day −1 captures the fish’s “settled” waveform; Day 0 confirms it has not drifted overnight. If the two recordings disagree by more than your normal day-to-day variability, do not implant — investigate first.

Step 1: Prepare 11-KT doses (20% w/w in cocoa butter)

  1. Melt 1 cocoa-butter chip (≈ 1.75 mL) at 40 °C.
  2. Weigh out 6.5 mg 11-KT.
  3. Combine the 11-KT with 35 µL of melted cocoa butter and pipette up and down thoroughly while maintaining heat at 35–40 °C, until 11-KT crystals are fully dissolved and the solution appears clear/translucent. If particles persist after 2 min at temperature, hold longer before pipetting.
  4. Keep the dose mix on the dry bath until ready for implantation.
Callout

Don’t rush dissolution

11-KT does dissolve fully in cocoa butter at this loading and temperature, but it isn’t instantaneous. Pipetting before the crystals are gone gives non-uniform doses across your fish — the first fish gets a heavy bolus, the last gets diluted vehicle.

Challenge

Calculating the dose

You want to implant 5 fish at 1 mg 11-KT per dose, with ~30% extra prep volume to absorb pipetting losses. Working at 20% w/w, how much 11-KT do you weigh out, and how much cocoa butter do you combine it with?

  • Per-dose 11-KT mass: 1 mg.
  • For 5 fish + 30%: ≈ 6.5 mg of 11-KT.
  • At 20% w/w, the matching cocoa-butter mass is ≈ 26 mg, which corresponds to roughly 35 µL of melted cocoa butter (density ≈ 0.9 g/mL).

This is the recipe used in Step 1 above.

Step 2: Anesthetize fish & sterilize equipment

  1. Anesthetize the fish in MS-222 (0.1–0.2 g/L) until it loses its righting response but is still actively gilling.
  2. Spray tools with 70% ethanol and let sit for 5 minutes, then dry with a kimwipe. Alternatively, use a heat sterilizer — just make sure the tools are properly cooled before they touch the fish.
Callout

Surgical plane, not deep anesthesia

You want gilling to continue throughout the procedure. If gill movement stops, you have gone too deep — move the fish into fresh water immediately.

Step 3: Implantation of 11-KT

  1. Remove the fish from the anesthetic and place it in the dissecting tray, belly-up, leaning against two dissecting pins.
  2. Blot the surgical area dry with a kimwipe.
  3. With a sharp, fresh scalpel blade, make a small incision anterior to the anal pore and slightly off the ventral midline (to avoid major vessels), passing through skin and muscle wall into the peritoneal cavity. The cut should be just wide enough to fit a P200 pipette tip, and should feel “deep” enough that you are clearly through the muscle wall.
  4. Draw 5 µL of the 11-KT / cocoa-butter mix from the heated solution and immediately deposit it through the incision. A successful implantation leaves all material deep within the cut, not on the surface.
  5. If any blood or fluid is on the outside of the fish, blot once more with a kimwipe.
  6. Apply a small amount of Vetbond over the incision and wait for it to skin, checking that the seal is complete. Apply more Vetbond if necessary, but take care that it is not “wicked” onto the pectoral fins or gills nearby.
  7. Place the fish into fresh recovery water and wait ~20 minutes for it to return upright before returning it to its home tank.
Callout

Incision location matters

Bass’s original phrasing was “small incision was made anterior to the anal pore.” Going off-midline keeps you out of the major ventral vessels, and staying anterior to the anal pore puts the dose into the IP cavity proper rather than the cloacal region.

Challenge

Troubleshooting a failed seal

Five minutes after implantation you notice a small bead of cocoa-butter / 11-KT solution sitting on the fish’s belly, outside the Vetbond. What do you do, and how do you note it in the experimental log?

  • Blot the surface dose off with a fresh kimwipe.
  • Apply a second layer of Vetbond and let it skin.
  • Note the partial extrusion in the log: dose delivered is uncertain. This animal should be flagged for exclusion or, at minimum, tracked separately when EOD changes are analyzed — the slow-release pharmacology assumes the depot is fully internal.

After surgery

Once each fish is upright and swimming, return it to its labeled home tank and resume the post-implant EOD recording schedule defined in the broader course protocol. Do not feed for 24 h.

Key Points
  • Cocoa-butter implants give a several-day, slow-release androgen exposure with a single Day 0 procedure.
  • Pre-treatment EOD recordings on Day −1 and Day 0 are the per-fish baselines — every post-implant measurement is interpreted relative to them.
  • Full dissolution of 11-KT in warm cocoa butter is what makes the dose uniform across animals; rushing this step is the most common avoidable source of variance.
  • Incise anterior to the anal pore, slightly off the ventral midline, and seal cleanly with Vetbond.
  • A surface-extruded dose is not a delivered dose — flag the animal in the log.

References

  • Bass, A. H., et al. (1986). Androgen binding in the brain and electric organ of a mormyrid fish.
  • Bass, A. H., et al. (1987). From behavior to membranes: testosterone-induced changes in action potential duration in electric organs.