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INDUCTION OF SPAWNING

Hydrogen Peroxide

(Morse, D. E., et al., 1977)

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The hydroperoxy free radical, HOO., or peroxy diradical, .OO. [the postulated biochemical electron transfer] can be produced chemically by the addition of peroxide to water. Hydrogen peroxide [H2O2] is the cheapest and most commonly available peroxide; however, reagent grade (30%) must be used, since 3% pharmaceutical solutions are unstable and give unreliable results.

Adults should be fully gravid and fed ad libitum for 2 weeks before spawning. Gravid individuals are placed singly in containers with enough water (ambient temperature) to cover them and the water is gently aerated. The pH of the water is first increased to 9.1 by adding 6.6 ml of 2 M Tris-(hydroxy-methylamino)methane (Tris-base, M.W. = 121.1) for each liter of water in the container. [To make a 2 M solution, dissolve 24.2 g of Tris in about 75 ml of water. After it has dissolved, add water to a final volume of 100 ml.]

Reagent grade hydrogen peroxide (30%) is diluted to make a 6% working solution. [To make a 6% solution, add 20 ml of 30% hydrogen peroxide to 80 ml of distilled water. This gives a final volume of 100 ml of 6% hydrogen peroxide.] Care should be taken when working with reagent grade hydrogen peroxide because it is caustic, unstable and should be stored at 0o - 4o C. Fifteen minutes after adding the Tris, 3 ml of freshly prepared 6% hydrogen peroxide solution is added for each liter of water in the container. Water in the container is then thoroughly stirred to mix the solution.

The animal is exposed to the hydrogen peroxide/Tris solution for 2.5 hours. After this period, the water is decanted and the container is thoroughly rinsed with clean isothermal sea water. It is very important to remove the hydrogen peroxide and Tris, and rinse out the container before spawning begins because the presence of these chemicals will cause gametes to be non-viable. After rinsing, the container is filled with clean isothermal sea water so as not to disturb the animal. Spawning will usually begin within 2.5 to 3.5 hours following the addition of hydrogen peroxide (2.5 hours at 18o C, 3.5 hours at 12o C).

Tris is not mandatory for successful artificial induction of spawning. Tris only increases the pH and is not required for any specific chemical reaction. Tris increases the percentage of individuals that spawn at a specific hydrogen peroxide concentration. Sodium hydroxide (1 x 10-3 M) has the same effect as Tris when used to increase the pH to 9.1. A high pH enhances the decomposition of the added hydrogen peroxide into highly reactive and short-lived free radical oxidants, which cause the induction of spawning.

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Step by Step Protocol

    1. Set up spawning tanks on night before spawning induction

    2. Before 10:00 a.m. -- select gravid individuals from conditioning tank (equal number of each sex)

    3. Select individual with Gonad Index of 3 [gonad swelling past rim of shell] or if necessary Gonad Index of 2 [gonad reaches edge of shell]

    4. Each animal -- measure shell length with a caliper, weigh, and record tag number

    5. 10 a.m. -- place animals between layers of damp cotton gauze for 1 hour at room temperature (about 20o C)

    6. 11 a.m. -- place animals singly in spawning tank with aeration and enough water (ambient temperature) to cover them

    7. Add 6.6 ml of 2 Molar Tris for each liter of water in tank

    8. Wait 15 minutes

    9. Add 4 ml of freshly prepared 6% hydrogen peroxide solution for each liter of water in tank

    10. Thoroughly mix water in tank

    11. Wait 2.5 hours

    12. Pour water out of each tank, and thoroughly rinse tank surfaces and animal with clean sea water

    13. Fill tank with clean sea water

    14. Wait 0.5 hour

    15. Begin observation of animals to detect spawning

    16. Collect and process gametes using Fertilization protocol

    17. Morning of next day -- return completely spawned adults to brood stock culture tank, return partially spawned or unspawned individuals to conditioning tank

    18. Record data on number of gametes spawned and fertilization rate of gametes for each animal


Morse, D. E., H. Duncan, N. Hooker, and A. Morse, 1977, Hydrogen peroxide induces spawning in mollusks, with activation of prostaglandin endoperoxide synthetase, Science, 196, 298-300.

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