This post details overview of spawning activities and a general protocol for spawning at large scale at Point Whitney from 13 June 2024.

Overview

Today we went to Point Whitney learn about spawning and finish up organization from yesterday. We worked with Carrie, who taught us their large scale spawning protocol.

This is a write up of the protocol that they use.

1. Sexing oysters

• Prepare the group of oysters and bring them into the lab/hatchery
• Open a set of oysters using a shucking knife. Shuck by having the cup side of the oyster down, crack and insert the knife, swiping against the cup side towards you. This will preserve the most gametes and avoid tissue damage. The gonad will be on the flat side of the shell.
• Sex the oysters by taking a pairing knife, and adding a small smear of gonad to a microscope slide. Look in the microscope. You will either see round, large eggs or concentrated, tiny, vibrating sperm. They are very distinct to recognize.
• Decide on “good” vs “bad” males and females.
  • Good eggs are generally more round, concentrated, and low debris or tissue mixed in the eggs. Eggs will not always be round - it is normal for some to have square shapes. We will oxygenate the eggs which will round them out.
  • Good sperm will be concentrated and have some motility (vibrating, fuzzy appearance)

Here is a “good” female:

Here is a “bad” female:

Here is a sperm sample - as long as its concentrated they are generally all pretty good.

• Once you have your group of males and females, we can proceed to the next step.
• Today, we used 19 females and 4 males. Males and females were from different families.
• If you find hermaphrodites (both egg and sperm), don’t use these individuals.

2. Preparing eggs

• Remove the whole tissue of the selected females from the shell into a Pyrex/glass dish. Today, we had a dish with half of the females in each. This allows you to homogenize them evenly later on. Discard other females that you wont use.
• No water is added to the dish at this point.
• If low on sample size, you can use some of the less ideal females, but split evenly between dishes.
• Once all females are added to dishes, move one dish at a time into a plastic bag (quart sized) in groups of <10.
• Squish the tissue with your hands to break up all the gonad tissue of each animal.
• Once fully squished, repeat with the other dishes.
• Add all the squished tissue from all dishes to the seives. Use a 80um screen on top of a 20um screen on top of a stand to keep off the floor. The 80um screen will catch tissue and debris while the 20um screen will catch the eggs.
• Squish the tissue with your hands onto the screen to homogenize fully.

• Rinse the screen with warm seawater (25-27°C). Add water to the screen and rinse by rocking and rotating. Repeat this 2-3 times to rinse all eggs through the screen.

• Once all eggs are on the 20um screen, use warm seawater to rinse all eggs into smaller buckets (we used ~10L red buckets today). We split eggs into two buckets, because we had a lot of eggs.
• Fill the bucket about 3/4 full with warm seawater.
• Allow to sit in buckets for about 15 min, gently stirring every few minutes. This allows eggs to oxygenate and hydrate.

• After a few minutes in the bucket, remove a small sample with a clean glass pipette (rinse with bleach water and then fresh water) to view on the scope.
• You should see round, clean eggs.

• Prepare sperm while eggs are in the buckets.
• Keep eggs at 25-27°C, use a water bath if needed.

3. Preparing sperm

• Clean and rinse all dishes and supplies with bleach water and then fresh water.
• Fill two Pyrex dishes with about 1/2-1 inch of warm seawater.
• Use your hands to pinch and transfer sperm from the gonad to one dish. You do not need to use all the sperm from every male. We want to avoid having high sperm concentration.
• Once added to the dish, prepare a cleaned 20um screen (smaller in diameter than the previous step) - clean with bleach water and rinsed.
• Pour the sperm from the dish through the 20um screen into another cleaned dish. The sperm will pass through the screen into the second dish. Rub the bottom of the screen with your hand to help the sperm go through. Rinse with a little seawater.

• Check for motility with a small sample under the scope.

4. Fertilization

• Use a pasteur pipette to transfer 10 mL of sperm solution to each bucket. This volume is based on the approximate concentrations in the pictures above. If the sperm is in lower concentration, use a larger volume, for example.
• Carrie does this visually, but we can use a colorimeter for more exact measurements in the future.
• We estimated 10 mL of our sperm to 100 million eggs in each bucket.
• Stir once added to eggs.
• Keep in the bucket for about 5-10 minutes if adding directly to larval tanks (this is what we did today). If you want, you can do fertilization in the buckets for longer. Keep at 25-27°C and check for polar bodies at about 45 min.

5. Adding to larval tanks

• We added one bucket to a 5,000 L tank. We filled two 5,000L tanks today. Tanks were kept at 25-30°C for the first 3 days.

• Add EDTA to tanks to control microbial growth. Add at concentration of 1 g / 1,000 L
• We boiled fresh water in a kettle and added about 800 mL to 10 g of EDTA. This solution was split between our two 5,000 L tanks to achive 5 g per tank (1 g / 1,000 L).
• If the tanks are static, this can just be done once when fertilized eggs are added. If systems are flow through, repeat this EDTA addition every day.
• Target pH is about 8.2 (NBS) for larval growth. This is what we have inside the hatchery facility.

Timeline after fertilization

• 1 day after fertilization: first round of feeding (keep fluorescence at 15 until Day 3)
• 2 days after fertilization: continued feeding
• 3 days after fertilization: drop tanks onto 60-70um screen and keep larvae retained on screen

We didn’t talk about what happens after 3 days.