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.

Tag Label	A	B	Total
1	7	9	16
2	8	8	16
3	6	6	12
4	6	7	13
5	9	8	17
6	5	8	13
Heat Shock	5	6	11

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.

Tag Label	M	F	Total
1	8	8	16
2	8	8	16
3	6	6	12
4	7	6	13
5	9	8	17
6	7	6	13
Subtotal	45	43	88
Heat Shock	6	5	11
Total	51	48	99

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.

Spawning steps

Day 1:

• 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

Day 2:

• 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

Materials needed

• 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

Questions

• 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