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Hormones II: Dosage

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 dosage. You don’t want to overdose your fish to death, but you don’t want to shoot ineffective dosage of hormones either!

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Hormones II: Dosage

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  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

  10. 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.

  11. 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

  12. 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

  13. 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!!!

  14. 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.

  15. 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

  16. 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

  17. 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?

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

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

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

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