Revised Spawning Plan
Featuring reduced crosses and pools
After showing Steven and Brent my preliminary spawning plan, I used their feedback and Joth’s comments from the July hatchery update meeting to make a revised plan. I was also able to think through the actual steps of spawning and materials needed for this plan because I shadowed a C. gigas spawn at Taylor Shellfish on Monday.
Crosses and pools
Here are the current oyster counts in my conditioning tanks.
Table 1. Oyster counts in each tank.
Assuming there is a 50/50 sex ratio, I calculated the number of males and females I’d have from each tank. I assumed I would have more males than females, just for the numbers.
Table 2. Sex ratios for each tank.
To make my crosses, I will pool all of the eggs from my six tanks and from the heat shock treatment. Keeping males separate, I will cross all of the males from Tanks 1-6 with the females from Tanks 1-6. Separately, I will cross the heat shock oysters with eachother. Therefore, I will have (45 males)(6 egg pools) + (6 males)(1 egg pool) = 276 crosses.
After measuring hatch rate for each cross, 24 hours post-fertilization, I will pool hatched D-hinge larvae from each cross according to Table 3.
Table 3. Treatment pools for crosses.
|Pool||Parent 1||Parent 2||Parent 1 Tank||Parent 2 Tank|
|1||Low pH Female||Ambient pH Male||Tanks 1-3||Tanks 4-6|
|2||Ambient pH Female||Low pH Male||Tanks 4-6||Tanks 1-3|
|3||Low pH Female||Low pH Male||Tanks 1-3||Tanks 1-3|
|4||Ambient pH Female||Ambient pH Male||Tanks 4-6||Tanks 4-6|
|5||Heat Shock Female||Heat Shock Male||Heat Shock Tank||Heat Shock Tank|
Once pooled, I will count the number of live and dead larvae in each pool. Based on these counts, I will calculate how many larvae I need to add to each bucket to get a density of 1/2 larvae per mL. I will then redistribute larvae into the static system.
Static system description
For the static system, I want to keep larvae in five gallon buckets. Each pool listed in Table 3 will have five replicates, meaning I need 25 buckets total. Buckets will be placed in larger totes with water and immersion heaters to keep water temperature at 25 ºC for the larval period. I think five buckets will fit nicely into a tote, but I need to check. I’m planning on keeping the totes on the middle table where Laura had her buckets.
- Shuck and sex all oysters
- Dry strip viable oysters
- IF NO COULTER COUNTER: Pool eggs together
- Use equal weights of gonads from multiple females
- Screen eggs using 80 and 20 micron screens
- Hydrate eggs
- IF COULTER COUNTER: Count number of eggs needed for fertilization
- Collect amount of sperm necessary for fertilization
- Hydrate sperm
- Examine under a microscope
- Fertilize eggs
- One tripour per cross
- Calculate hatch rate
- Pool alike treatments based on Table 3
- Calculate pool densities
- Redistribute larvae to static system
- Five gallon buckets in totes
- Five replicates/pool
- 1/2 larvae per mL
- Shucking knives: 4
- Shucking gloves: 4
- Oyster separators: Enough to hold 200 oysters
- Glass capilary tubes: 150
- Tripours: ~300
- Scoring knives: 4
- Spatulas: 4
- Microscope: 1
- 80 micron screens: 2
- 20 micron screens: 2
- 5 gallon buckets: 50
- Totes: 8
- Immersion heaters: 6
- Should I try and separate crosses between conditioning tanks (Tank A and Tank B)? I’ve had more mortalities in Tank A.
- Should I include Ambient x Heat shock crosses?
- Are we rearing larvae in a static system for the full duration, or are we going to switch to a flow-through at 100 microns?
Ask Molly and/or Rhonda
- How to calculate the amount of sperm and egg to use when fertilizing
- An easy way to calculate hatch rate