Hormones II: Dosage

Dr. Craig Kasper

Introduction

• Last time we discussed tank/hatchery spawning methods when using hormones.

• Timing is always and issue, but so it dosagedosage.

• You don’t want to overdose your fish to death, but you don’t want to shoot ineffective dosage of hormones either! (Why not??)

• Today we’ll learn how to calculate dosage of common hormones used in fish spawning.

Hormone Preparation

• Three pieces of information must be known to prepare the correct injection.

1. The recommended dosage (treatment level).

2. Weight of the fish

3. Desired injection volume

Sterilization

• All equipment to contact fish should be sterilized including needles, syringes, saline solution to be mixed with hormones, etc.

• Hormones can be stored in bacteriostatic (sterile) saline for years with no loss of potency.

• Label everything!! You don’t want to make a mistake with expensive chemicals!

Dosage Units

• Hormone dosage units are often given as weight (kg, g), volume (ml, ul) and biological

activity (IU or International Units).

• Doses of commonly used hormones can be viewed in Table 1 of your handhout.

• When first evaluating dosage of a pituitary hormone, it’s better to use a little too much, but only on a few fish.

Hormone Preparation: Pituitary

• It’s really easy!

Hormone Concentration =

Recommended Dose (ml or µl) x Fish Weight (g) or HC= RD X FW Desired Injection Volume IV

• If pituitary material is dry, then it can be crushed with a mortar/pestle.

• After making a suspension with saline, then the top mixture is drawn off and stored in cold storage until used.

Injection Volumes

• Dried carp pituitary extract is usually injected at 4 mg /kg fish body weight for many fish. (If the fish is 6 kg, injection volume of 1 mg, then 24 mg/cc should be administered.)

• What if you have multiple fish to be injected all at once??

• Just use the entire fish weight to calculate the total hormone volume to be used, then adjust the injection volume for each individual fish by weight.

Total Weight of Hormone* = Recommended Dose x Total Weight of Hormone* = Recommended Dose x Total Weight Total Weight of Fishof Fish

* Contingency: It is often a good practice to overestimate by 10-15% due to loss of injection volume within the syringe or due to mixing/filling errors.

Problem: How much dried pituitary do you require?Given:

Females First injection:0.3 mg/kgSecond injection:3.5 mg/k

MalesOne injection:2.0 mg/kg

Example 1: You are planning to treat 34 females (average weight 4 kg) and 17 males

(average weight 2.5 kg).

You will require the following quantities of dried pituitary (also called hypophysis):

(a) for the first injection of the females: 34 fish x 4 kg x 0.3 mg/kg = 40.8 mg

(b) for the second injection of the females: 34 fish x 4 kg x 3.5 mg/kg = 476 mg

(c) for the injection of the males: 17 fish x 2.5 kg x 2 mg/kg = 85 mg

Mixing Hormone to Be StoredExample 2: • What if you intend to store the hormone in a freezer?

Again, hormone is mixed with sterile saline.

-By multiplying the hormone concentration calculated in example 1 (24 mg/cc) by volume of the vial (30 cc); 720 mg of hormone are then weighed and mixed in a 30 cc vial.

Formula• Quantity of Hormone=

Hormone Concentration x Volume

Quantity of Hormone=24 mg/cc x 30 cc = 720 mg

Human Chorionic Gonadotropin (HCG)

• Unlike pituitary, HCG is mixed according to biological activity (Usually 5-10K IUs/vial).

• Vials must be kept at 35-42°F.• Intramuscular injections of 300-1,800 IU HCG/kg

fish bw recommended.

What volume of liquid solvent should be added to a vial of HCG?Example 3: Step 1

-10,000 IU/vial of HCG -Recommended dose (RD): 400 IU/kg

-Fish weight: 1 kg-Desired Injection Volume (IV): 0.2cc (mL)

• First, determine needed hormone concentration!

• Hormone Concentration = RD x FW IV

• Hormone Concentration = 400 IU/kg / 1 kg = 2,000 lU/cc 0.2 cc

What volume of liquid solvent should be added to a vial of HCG?Example 3: Step 2

• Hormone Concentration = 400 IU/kg / 1 kg = 2,000 lU/cc 0.2 cc

• What volume of water must be mixed with the 10,000 IU vial to get the desired concentration?

• The required volume of liquid is equal to the amount of hormone in the vial (10,000 IU), divided by the desired concentration (2,000 IU/CC). This equals 5 cc.

Volume of Liquid = Amount of Hormone = 10,000 IU = 5 cc Hormone Concentration 2,000 lU/cc

Luteinizing Hormonereleasing Hormone Analog (LHRHa)• LHRHa is usually injected in the muscle at 5-10 μg/kg FW.

1 mg (1,000 μg) vial of LHRHa RD 10 μg/kgFW 50 g each IV 0.1 cc.

• Hormone Concentration =10 μg/kg X 0.05 kg = 5 μg/cc

0.1 cc• Volume of Liquid = Weight Of Hormone = 1,000 μg = 200 cc!!

Hormone Concentration 5 μg/cc

Whoa!! Dr. K, I thought you said this stuff is expensive!!!

You must dilute it…

1. Draw 10 cc of sterile water in a syringe, mix the liquid with the vial of hormone (1000 μg).

2. Nine cc of this mixture are then placed in sterile 1 cc vials and labeled (100 μg/cc).

3. The remaining 1 cc of hormone is mixed with 9 cc of sterile water.

4. Nine cc of this mixture are then placed in sterile 1 cc vials and labeled (10 μg/cc).

5. This time, the remaining 1 cc of hormone is mixed with 1 cc of sterile water to obtain the desired concentration of 5 μg/cc.

10 cc H20 +1 mg LHRHa

Result:9 cc H20 +1cc LHRHa solution

1 ccH20

Result:1 cc H20 +1cc LHRHa solution

9 cc H20

5μg/cc LHRHa

Injections are given either intramuscularlyor intraperitoneally.

For larger fish a 19-23 gauge needle is used, while smaller fish demand a steady hand and a 25-30 gauge needle.

What Are We Manipulating

• Whew!!! That was a lot of info…

• Where do the hormones we utilize act?

• How do we actually influence the spawning cycle?

Normal Endocrine Cascade

• Brain releases messengers

• Pit. releases gonadotropins

• Gonads release steroids

• Gonad forms gametes

Another ViewAnother View• Conditions permit maturation Conditions permit maturation • Process begins Process begins • GnRH – GtH – steroids GnRH – GtH – steroids • Eggs are built Eggs are built • Eggs mature Eggs mature • Correct environment Correct environment • Spawning proceedsSpawning proceeds

• All the hormones you administer take action at specific points in the hormonal cascade.