11-KT Injection Protocol

Objectives

Prepare an injectable 11-ketotestosterone (11-KT) / cocoa butter dose (~20 µg/µL) from an ethanol stock, driving off the ethanol so the dose sets as a depot once delivered.
Anesthetize a mormyrid fish to a stable anesthetic plane using buffered MS-222.
Deliver a single intraperitoneal (IP) dose of warm cocoa butter with a Hamilton syringe.
Record and log a pre-treatment EOD baseline that all post-treatment measurements will be normalized against.

Background

Cocoa butter melts at body temperature, which makes it a useful slow-release vehicle for lipophilic steroids delivered into the peritoneal cavity. Implant-based steroid delivery of this kind is well established across fish taxa — for example, cocoa-butter implants in brown trout (Hoogenboom et al., 2011) and Silastic-capsule steroid implants in three-spined stickleback (Roufidou et al., 2019). Androgen action on the mormyrid electric organ was established by the Bass lab in the 1980s (Bass & Hopkins, 1983, 1985), and intraperitoneal cocoa-butter pellets of 11-KT were used to drive EOD change in Freedman et al. (1989). A single 11-KT cocoa-butter dose 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 procedure as it is run in our lab. Rather than surgically implanting a solid pellet, we deliver the cocoa butter as a warm IP injection through a Hamilton syringe. The steroid is the same and the depot behaves the same once it sets, but the delivery is faster and far less invasive — no incision, no closure. It assumes that the broader experimental schedule (acclimation, weighing, tank assignment, post-treatment recording cadence) is already defined elsewhere in the course materials.

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 non-reactive in the IP cavity and avoids the need for a DMSO/oil carrier injection.

Why inject instead of implant?

The injectable dose is roughly 10× more dilute than the old surgical pellet (~20 µg/µL versus the ~200 µg/µL of a 20% w/w plug), and delivered in a larger volume (30–40 µL versus ~5 µL). The reason is mechanical: a near-solid 20% w/w plug can be deposited through an incision but will not pass through a needle. Diluting the steroid into a fluid cocoa-butter mix lets the same depot chemistry be delivered by injection. The trade-off is that the per-fish dose is set by the injection volume, so volume control matters more here than it did with a hand-cut pellet.

Reagents

Cocoa butter chips
11-KT (Steraloids A6720, 25 mg vial)
100% (200 proof) ethanol — for the stock solution
70% ethanol — for needle wiping and between-fish syringe cleaning
1× sterile PBS — for between-fish syringe cleaning
MS-222 (tricaine methanesulfonate)
Recovery water (system water with NaCl supplement)

Equipment

Hamilton syringe (50–100 µL, gas-tight) with 26G ½″ needle
Dry bath (used at both ~40 °C for melting/holding and ~70 °C for ethanol evaporation)
Milligram balance (for stock preparation)
P20 / P200 pipette + tips (for dose preparation)
Kimwipes
Damp bench surface or foam fish holder (for restraint during injection)
Recovery containers
Fish-transfer net

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

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

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 inject — investigate first.

Step 1: Prepare 11-KT injection doses (~20 µg/µL in cocoa butter)

Confirm or prepare the 11-KT stock: 20 µg/µL in 100% ethanol (e.g., 7 mg dissolved in 350 µL ethanol).
Melt cocoa butter at ~40 °C on the dry bath.
For each batch, pipette your chosen volume of ethanol stock into a tube, then add an approximately equal volume of melted cocoa butter. Equal volumes hold the final concentration at ~20 µg/µL because the stock is already at that concentration (see the dose calculation below).
Evaporate the ethanol at 70 °C until it is fully gone — no ethanol odor remains, and the volume stabilizes at roughly the cocoa-butter volume you added. 70 °C sits just below ethanol’s boil, so it drives the ethanol off briskly without spattering cocoa butter out of the tube, and is well above the cocoa-butter melt point so the mix stays liquid.
Cool to just above the cocoa-butter melt point (~37–40 °C) and hold on the dry bath until injection.

Drive the ethanol off completely

Residual ethanol does two bad things: it adds volume (diluting your dose below target) and it can irritate the fish on injection. Confirm by odor and by a stable volume before you cool down — the volume should settle at the cocoa-butter volume you added, not somewhere between that and the original stock-plus-butter volume.

Calculating the dose

You want to inject 2 fish at ~0.7 mg 11-KT each, ~35 µL per injection, with a little extra to cover the syringe dead volume. Starting from a 20 µg/µL ethanol stock, how much stock and cocoa butter do you combine, and what is your final concentration?

Solution

Required final concentration: 0.7 mg ÷ 35 µL = 20 µg/µL — the same as the stock. So the goal is simply to hold the concentration where it is, not to dilute or concentrate.
Combine equal volumes of ethanol stock and melted cocoa butter, then evaporate the ethanol. The steroid mass stays put while the ethanol leaves, and the final volume ≈ the cocoa-butter volume → concentration is unchanged at ~20 µg/µL.
For 2 × 35 µL plus margin (~100 µL final), use ~100 µL stock (= 2 mg 11-KT) + ~100 µL (~90 mg) cocoa butter → ~100 µL final dose at 20 µg/µL.
Two 30–40 µL injections then deliver 0.6–0.8 mg/fish; the small remainder is dead volume you discard.

Keep it warm, work fast

Cocoa butter sets at ~34–38 °C, so it will solidify inside a cold needle. Pre-warm the syringe, draw and inject one fish at a time, and return the dose tube to the dry bath between animals.

Step 2: Anesthetize fish & prepare the needle

Anesthetize the fish in MS-222 (0.1–0.2 g/L) until it loses its righting response but is still actively gilling.
Wipe the Hamilton needle with 70% ethanol and let it dry before use.

Anesthetic 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: Inject 11-KT

Draw 30–40 µL of the warm dose into the pre-warmed Hamilton syringe. Work quickly so the cocoa butter does not set in the needle.
Lift the fish from the anesthetic and restrain it belly-up on a damp surface (or in a wet gloved hand / foam holder). Blot the injection site dry with a kimwipe.
Insert the needle at a shallow angle into the peritoneal cavity — anterior to the anal pore and slightly off the ventral midline (to avoid major ventral vessels) — just through the body wall.
Inject the bolus smoothly, then pause a moment before withdrawing.
Withdraw the needle slowly and hold gentle pressure with a kimwipe over the site for a few seconds to limit backflow through the needle track.
Place the fish into fresh recovery water and wait ~20 minutes for it to return upright before returning it to its home tank.
Between fish, clear the syringe (you can do this while the injected fish recovers). Cocoa butter sets in the barrel and needle between animals and will clog the syringe over a run.
- Pull and expel ~3 quick volumes of 70% ethanol (onto a kimwipe or into the sink) to dissolve residual cocoa butter.
- Follow with ~3 quick pulls/expels of 1× sterile PBS to flush out the ethanol.
- Re-warm and reload for the next fish.

Injection site matters

Anterior to the anal pore, slightly off the ventral midline, keeps the needle clear of the major ventral vessels and deposits the dose into the IP cavity proper rather than the cloacal region.

Troubleshooting backflow

A minute after injecting you notice a small bead of cocoa-butter / 11-KT mix sitting at the needle entry site on the fish’s belly. What do you do, and how do you note it in the experimental log?

Solution

Blot the surface material off with a fresh kimwipe; do not try to push it back in.
Note the partial backflow in the log: the delivered dose 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.
For subsequent fish, withdraw the needle more slowly and hold brief pressure on the site to reduce backflow.

Recovery

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

Key Points

Cocoa-butter doses give a several-day, slow-release androgen exposure from a single Day 0 procedure.
Pre-treatment EOD recordings on Day −1 and Day 0 are the per-fish baselines — every post-treatment measurement is interpreted relative to them.
Because the stock is already at the target concentration (~20 µg/µL), combining equal volumes of stock and cocoa butter — with the ethanol evaporated off — holds the dose where you want it; the injection volume then sets the per-fish dose.
Keep the dose warm and work quickly: cocoa butter sets in a cold needle.
Between fish, flush the syringe (70% ethanol, then 1× PBS) — cocoa butter will clog the works over a run.
Inject IP anterior to the anal pore, slightly off the ventral midline, with a warm Hamilton syringe.
A dose that backflows to the surface is not a delivered dose — flag the animal in the log.

References

Bass, A. H., & Hopkins, C. D. (1983). Hormonal control of sexual differentiation: changes in electric organ discharge waveform. Science 220:971–974.
Bass, A. H., & Hopkins, C. D. (1985). Hormonal control of sex differences in the electric organ discharge (EOD) of mormyrid fishes. Journal of Comparative Physiology A 156:587–604.
Bass, A. H., Segil, N., & Kelley, D. B. (1986). Androgen binding in the brain and electric organ of a mormyrid fish. Journal of Comparative Physiology A 159:535–544.
Bass, A. H., & Volman, S. F. (1987). From behavior to membranes: testosterone-induced changes in action potential duration in electric organs. PNAS 84:9295–9298.
Freedman, E. G., Olyarchuk, J., Marchaterre, M. A., & Bass, A. H. (1989). A temporal analysis of testosterone-induced changes in electric organs and electric organ discharges of mormyrid fishes. Journal of Neurobiology 20(7):619–634.
Hoogenboom, M. O., Armstrong, J. D., Miles, M. S., Burton, T., Groothuis, T. G. G., & Metcalfe, N. B. (2011). Implantation of cocoa butter reduces egg and hatchling size in Salmo trutta. Journal of Fish Biology 79(3):587–596.
Roufidou, C., et al. (2019). Ovarian fluid in the three-spined stickleback Gasterosteus aculeatus: effects of egg overripening and sex steroid treatment. Journal of Fish Biology 94:446–457.

--- title: "11-KT Injection Protocol" mode: protocol teaching: 10 labwork: 30 reading: 15 # optional pre-class reading, in minutes (blank = N/A) optional: true location: Loeb 160 # room shown in the schedule group heading, e.g. Loeb 160 --- ::: {.callout-tip title="Objectives"} - Prepare an injectable 11-ketotestosterone (11-KT) / cocoa butter dose (~20 µg/µL) from an ethanol stock, driving off the ethanol so the dose sets as a depot once delivered. - Anesthetize a mormyrid fish to a stable anesthetic plane using buffered MS-222. - Deliver a single intraperitoneal (IP) dose of warm cocoa butter with a Hamilton syringe. - Record and log a pre-treatment EOD baseline that all post-treatment measurements will be normalized against. ::: ## Background Cocoa butter melts at body temperature, which makes it a useful slow-release vehicle for lipophilic steroids delivered into the peritoneal cavity. Implant-based steroid delivery of this kind is well established across fish taxa — for example, cocoa-butter implants in brown trout (Hoogenboom et al., 2011) and Silastic-capsule steroid implants in three-spined stickleback (Roufidou et al., 2019). Androgen action on the mormyrid electric organ was established by the Bass lab in the 1980s (Bass & Hopkins, 1983, 1985), and intraperitoneal cocoa-butter pellets of 11-KT were used to drive EOD change in Freedman et al. (1989). A single 11-KT cocoa-butter dose 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 procedure as it is run in our lab. Rather than surgically implanting a solid pellet, we deliver the cocoa butter as a warm IP **injection** through a Hamilton syringe. The steroid is the same and the depot behaves the same once it sets, but the delivery is faster and far less invasive — no incision, no closure. It assumes that the broader experimental schedule (acclimation, weighing, tank assignment, post-treatment recording cadence) is already defined elsewhere in the course materials. ::: {.callout-note title="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 non-reactive in the IP cavity and avoids the need for a DMSO/oil carrier injection. ::: ::: {.callout-note title="Why inject instead of implant?"} The injectable dose is roughly **10× more dilute** than the old surgical pellet (~20 µg/µL versus the ~200 µg/µL of a 20% w/w plug), and delivered in a larger volume (30–40 µL versus ~5 µL). The reason is mechanical: a near-solid 20% w/w plug can be deposited through an incision but will not pass through a needle. Diluting the steroid into a fluid cocoa-butter mix lets the same depot chemistry be delivered by injection. The trade-off is that the per-fish dose is set by the injection *volume*, so volume control matters more here than it did with a hand-cut pellet. ::: ## Reagents - Cocoa butter chips - 11-KT (Steraloids A6720, 25 mg vial) - 100% (200 proof) ethanol — for the stock solution - 70% ethanol — for needle wiping and between-fish syringe cleaning - 1× sterile PBS — for between-fish syringe cleaning - MS-222 (tricaine methanesulfonate) - Recovery water (system water with NaCl supplement) ## Equipment - Hamilton syringe (50–100 µL, gas-tight) with 26G ½″ needle - Dry bath (used at both ~40 °C for melting/holding and ~70 °C for ethanol evaporation) - Milligram balance (for stock preparation) - P20 / P200 pipette + tips (for dose preparation) - Kimwipes - Damp bench surface or foam fish holder (for restraint during injection) - Recovery containers - Fish-transfer net ## Step 0: EOD baseline (Day −1 and Day 0, pre-injection) 1. Record an EOD waveform from each fish on Day −1 (24 h prior) and again on Day 0 immediately before injection. These pre-treatment recordings are the per-fish baselines that all post-treatment measurements will be normalized to. 2. Confirm fish ID, weight, and tank assignment are logged before proceeding to Step 1. ::: {.callout-note title="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 inject — investigate first. ::: ## Step 1: Prepare 11-KT injection doses (~20 µg/µL in cocoa butter) 1. Confirm or prepare the 11-KT stock: **20 µg/µL in 100% ethanol** (e.g., 7 mg dissolved in 350 µL ethanol). 2. Melt cocoa butter at ~40 °C on the dry bath. 3. For each batch, pipette your chosen volume of ethanol stock into a tube, then add an **approximately equal volume of melted cocoa butter**. Equal volumes hold the final concentration at ~20 µg/µL because the stock is already at that concentration (see the dose calculation below). 4. **Evaporate the ethanol at 70 °C** until it is fully gone — no ethanol odor remains, and the volume stabilizes at roughly the cocoa-butter volume you added. 70 °C sits just below ethanol's boil, so it drives the ethanol off briskly without spattering cocoa butter out of the tube, and is well above the cocoa-butter melt point so the mix stays liquid. 5. Cool to **just above the cocoa-butter melt point (~37–40 °C)** and hold on the dry bath until injection. ::: {.callout-important title="Drive the ethanol off completely"} Residual ethanol does two bad things: it adds volume (diluting your dose below target) and it can irritate the fish on injection. Confirm by odor and by a stable volume before you cool down — the volume should settle at the cocoa-butter volume you added, not somewhere between that and the original stock-plus-butter volume. ::: ::: {.callout-important title="Calculating the dose"} You want to inject **2 fish at ~0.7 mg 11-KT each**, ~35 µL per injection, with a little extra to cover the syringe dead volume. Starting from a **20 µg/µL** ethanol stock, how much stock and cocoa butter do you combine, and what is your final concentration? :::: {.callout-tip title="Solution" collapse="true"} - Required final concentration: 0.7 mg ÷ 35 µL = **20 µg/µL** — the same as the stock. So the goal is simply to *hold* the concentration where it is, not to dilute or concentrate. - Combine **equal volumes** of ethanol stock and melted cocoa butter, then evaporate the ethanol. The steroid mass stays put while the ethanol leaves, and the final volume ≈ the cocoa-butter volume → concentration is unchanged at ~20 µg/µL. - For 2 × 35 µL plus margin (~100 µL final), use ~100 µL stock (= 2 mg 11-KT) + ~100 µL (~90 mg) cocoa butter → ~100 µL final dose at 20 µg/µL. - Two 30–40 µL injections then deliver **0.6–0.8 mg/fish**; the small remainder is dead volume you discard. :::: ::: ::: {.callout-note title="Keep it warm, work fast"} Cocoa butter sets at ~34–38 °C, so it will solidify inside a cold needle. Pre-warm the syringe, draw and inject one fish at a time, and return the dose tube to the dry bath between animals. ::: ## Step 2: Anesthetize fish & prepare the needle 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. Wipe the Hamilton needle with 70% ethanol and let it dry before use. ::: {.callout-note title="Anesthetic 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: Inject 11-KT 1. Draw **30–40 µL** of the warm dose into the pre-warmed Hamilton syringe. Work quickly so the cocoa butter does not set in the needle. 2. Lift the fish from the anesthetic and restrain it belly-up on a damp surface (or in a wet gloved hand / foam holder). Blot the injection site dry with a kimwipe. 3. Insert the needle at a shallow angle into the peritoneal cavity — **anterior to the anal pore and slightly off the ventral midline** (to avoid major ventral vessels) — just through the body wall. 4. Inject the bolus smoothly, then pause a moment before withdrawing. 5. Withdraw the needle slowly and hold gentle pressure with a kimwipe over the site for a few seconds to limit backflow through the needle track. 6. Place the fish into fresh recovery water and **wait ~20 minutes** for it to return upright before returning it to its home tank. 7. **Between fish, clear the syringe** (you can do this while the injected fish recovers). Cocoa butter sets in the barrel and needle between animals and will clog the syringe over a run. - Pull and expel ~3 quick volumes of 70% ethanol (onto a kimwipe or into the sink) to dissolve residual cocoa butter. - Follow with ~3 quick pulls/expels of 1× sterile PBS to flush out the ethanol. - Re-warm and reload for the next fish. ::: {.callout-note title="Injection site matters"} Anterior to the anal pore, slightly off the ventral midline, keeps the needle clear of the major ventral vessels and deposits the dose into the IP cavity proper rather than the cloacal region. ::: ::: {.callout-important title="Troubleshooting backflow"} A minute after injecting you notice a small bead of cocoa-butter / 11-KT mix sitting at the needle entry site on the fish's belly. What do you do, and how do you note it in the experimental log? :::: {.callout-tip title="Solution" collapse="true"} - Blot the surface material off with a fresh kimwipe; do not try to push it back in. - Note the partial backflow in the log: the delivered dose 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. - For subsequent fish, withdraw the needle more slowly and hold brief pressure on the site to reduce backflow. :::: ::: ## Recovery Once each fish is upright and swimming, return it to its labeled home tank and resume the post-treatment EOD recording schedule defined in the broader course protocol. Do not feed for 24 h. ::: {.callout-tip title="Key Points"} - Cocoa-butter doses give a several-day, slow-release androgen exposure from a single Day 0 procedure. - Pre-treatment EOD recordings on Day −1 and Day 0 are the per-fish baselines — every post-treatment measurement is interpreted relative to them. - Because the stock is already at the target concentration (~20 µg/µL), combining equal volumes of stock and cocoa butter — with the ethanol evaporated off — holds the dose where you want it; the **injection volume** then sets the per-fish dose. - Keep the dose warm and work quickly: cocoa butter sets in a cold needle. - Between fish, flush the syringe (70% ethanol, then 1× PBS) — cocoa butter will clog the works over a run. - Inject IP anterior to the anal pore, slightly off the ventral midline, with a warm Hamilton syringe. - A dose that backflows to the surface is not a delivered dose — flag the animal in the log. ::: ## References - Bass, A. H., & Hopkins, C. D. (1983). Hormonal control of sexual differentiation: changes in electric organ discharge waveform. *Science* 220:971–974. - Bass, A. H., & Hopkins, C. D. (1985). Hormonal control of sex differences in the electric organ discharge (EOD) of mormyrid fishes. *Journal of Comparative Physiology A* 156:587–604. - Bass, A. H., Segil, N., & Kelley, D. B. (1986). Androgen binding in the brain and electric organ of a mormyrid fish. *Journal of Comparative Physiology A* 159:535–544. - Bass, A. H., & Volman, S. F. (1987). From behavior to membranes: testosterone-induced changes in action potential duration in electric organs. *PNAS* 84:9295–9298. - Freedman, E. G., Olyarchuk, J., Marchaterre, M. A., & Bass, A. H. (1989). A temporal analysis of testosterone-induced changes in electric organs and electric organ discharges of mormyrid fishes. *Journal of Neurobiology* 20(7):619–634. - Hoogenboom, M. O., Armstrong, J. D., Miles, M. S., Burton, T., Groothuis, T. G. G., & Metcalfe, N. B. (2011). Implantation of cocoa butter reduces egg and hatchling size in *Salmo trutta*. *Journal of Fish Biology* 79(3):587–596. - Roufidou, C., et al. (2019). Ovarian fluid in the three-spined stickleback *Gasterosteus aculeatus*: effects of egg overripening and sex steroid treatment. *Journal of Fish Biology* 94:446–457.