It’s time to think about crosses!

After my oysters are done conditioning, I will strip spawn them and rear larvae. Understanding which males and females to cross, and how many to use, are important to ensure genetic diversity in the F1 generation.

From each tank, I will pool eggs, but keep sperm separate. I will cross the pooled eggs from one tank with sperm from all males from all tanks. The next day, I will measure hatch success, then pool D-hinge larvae by treatment.

Table 1. Number of males available for crosses, assuming there is a 50/50 male-to-female ratio in each tank. Tanks 1-3 were exposed to low pH conditions, and tanks 4-6 had ambient water conditions. Number of oysters from each tank can be found here.

Tank	Number of Males
1	9
2	9
3	7
4	8
5	9
6	8
Heat Shock	6
Total	56

With 56 males to cross and 7 different “tanks”, there are 392 possible crosses. If I were to limit the number of males used, I would have less crosses.

Table 2. Number of crosses for varying numbers of males used.

Number of Males	Number of Crosses
1	49
2	98
3	147
4	196
5	245

After measuring hatch rate, I can pool my individual crosses into groups based on treatments.

Table 3. Treatment pool combinations. In total, there are nine possible pools.

Parent 1	Parent 2
Low Female	Ambient Male
Low Male	Ambient Female
Low Female	Heat Shock Male
Low Male	Heat Shock Female
Ambient Female	Heat Shock Male
Ambient Male	Heat Shock Female
Low Female	Low Male
Ambient Female	Ambient Male
Heat Shock Female	Heat Shock Male

Each pool will be transferred to 28 micron silos, so I will need replicates. Assuming I can fit four totes on each level of the table at Manchester, and each tote can fit 8 silos, I can have a maximum of 64 silos. The number of replicates I need will depend on how many larvae hatch and my stocking density. Molly recommends having 15-25 larvae per mL of silo volume.

Table 4. Number of silos needed based on replicates used for each pool.

Replicates	Total Silos Needed
2	18
3	27
4	36
5	45
6	54
7	63

Here’s what I need to do next:

• Consult with Steven and Brent on the number of crosses, and where I can trim crosses
• Obtain drippers (1 L/hr drippers for first few days, 1 gallon/hr drippers for remainder of larval period
• Determine flow rate and pipe diameter needed for manifolds
• Build manifolds
• Ensure I have enough tubing to feed silos
• Find a method to volumetrically count eggs and sperm