1. Hormones II: Dosage Dr. Craig Kasper

2. Introduction • Last time we discussed tank/hatchery spawning methods when using hormones. • Timing is always and issue, but so it dosage. • You don’t want to overdose your fish to death, but you don’t want to shoot ineffective dosage of hormones either! • Today we’ll learn how to calculate dosage of common hormones used in fish spawning.

3. Hormone Preparation • Three pieces of information must be known to prepare the correct injection. 1. The recommended dosage. 2. Weight of the fish 3. Desired injection volume

4. 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!

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

6. 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.

7. Injection Volumes • Dried pituitary extract is usually injected at 4 mg /kg fish body weight. (If the fish is 6 kg, injection volume of 1, 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 Fish * 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.

8. How much dried pituitary do you require? Given: Females First injection:0.3 mg/kg Second injection:3.5 mg/kg Males One 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 hypophysis (pituitary): (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

9. Mixing Hormone to Be Stored Example 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 the example above (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

11. 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.

12. 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 400 IU/kg -Fish weigh 1 kg -Desired Injection Volume 0.2cc • Hormone Concentration = RD x FW IV • Hormone Concentration = 400 IU/kg / 1 kg = 2,000 lU/cc 0.2 cc

13. 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

14. 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/kg FW 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!!!

15. You must dilute it… • Draw 10 cc of sterile water in a syringe, mix the liquid with the vial of hormone (1000 μg). • Nine cc of this mixture are then placed in sterile 1 cc vials and labeled (100 μg/cc). • The remaining 1 cc of hormone is mixed with 9 cc of sterile water. • 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.

16. 9 cc H20 1 cc H20 10 cc H20 + 1 mg LHRHa 5μg/cc LHRHa Result: 9 cc H20 + 1cc LHRHa solution Result: 1 cc H20 + 1cc LHRHa solution

17. Note 1: How much dried hypophysis do you require? Injections are given either intramuscularly or intraperitoneally. For larger fish a 19-23 gauge needle is used, while smaller fish demand a steady hand and a 25-30 gauge needle. Example: 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 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

18. 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?

19. Normal Endocrine Cascade • Brain releases messengers • Pit. releases gonadotropins • Gonads release steroids • Gonad forms gametes

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

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