Shelly’s Notebook: Tues. Jan. 21, 2020 Pt Whitney broodstock conditioning

Strip spawn prep

  1. ID individuals with ripe gonads
    • last year we used punch biopsy tools for this, but decided they were too harmful to the animal. To get the biopsy this way, we had to really scrape and scoop the slippery tissue to prevent it from slipping out of the tool
    • This time, we did the following:
      1. Gently insert cut off 15mL falcon tube (1”) into pedal gape to be able to view gonad
        • had to move foot aside using finger first, then insert tube. uc?export=view&id=1o_4bPBj2SJ8-qBSfApBmgJHwT-fBV0T9
        • We tried a cut off pipette tip first shown here: uc?export=view&id=1pix6VYj70Pb63yig_ZPc02FUJbwiuguM
        • once inserted the animal would typically release water, so holding the tube in place, we would hold the animal with pedal gape aimed towards the floor and let it drain. Sometimes give is a gentle squeeze. We also used a transfer pipette to remove excess water before going in for the biopsy uc?export=view&id=173u5TOk1-TqglXGFBg1-RH_m7mFRL9iv
      2. Use 18 G needle on a 3mL syringe to pierce the gonad and withdraw some tissue. Barely piercing the surface should suffice. Too deep would pierce into digestive tissue. We repeated this procedure 3 times on some individuals to confirm they were indeed not ripe.
        uc?export=view&id=1KyURdM7csloGoJoPJpJ4a3HJTxcSFJBH
      3. Squirt biopsy out onto a glass slide. Sometime a drop of water is necessary to get the tissue from the biopsy out from the needle and onto the slide
      4. Visualize in the microscope under 4-10x magnification
        • eggs or sperm should be visual as shown here: Tank 2 male 10x mag: uc?export=view&id=1Dc6Bfj-wTNlyY9oaqwEsKirFQgps2lhK Tank 2 female 10x mag: uc?export=view&id=1L6R81u7dQTYNtsOzQxmY_Xz2crc1-opF Tank 2 female 4x mag: uc?export=view&id=1bttomsB6GeTyWoMOIVBeuHix3LNx5RfT Tank 4 male 10x mag: uc?export=view&id=1Vu6gIrNytQwD9djHphSJ7yMGSVSFER0o

    RESULTS: We checked a total of 2 individuals in tanks 1 (pH 7.2), 2 (pH amb), 4, (pH amb) and 5 (pH 7.2). We also checked 2 individuals in tank 6 (pH amb) from the broodstock cohert harvested in October and 3 individuals in tank6 from the broodstock cohert harvested in December. We found one ripe male and one ripe female in tank 2 (pH amb), one ripe male from tank 4 (pH amb), and one ripe male from tank 6 (pH amb).

  2. Prepare 50mM KCl solution
    • I made 200mL of 50mM KCl by diluting 5mL 2M stock solution in 195mL 15C? sea water
    • Once we decided to do a priming bath, I made 4L of 50mM KCl by diluting 100mL 2M stock solution in 3.9L 15C? sea water

Strip spawn steps

  1. Strip males and females
    • using a razor blade, score the gonad. Be careful about cutting too deep to avoid digestive tissue
    • Use back of razor blade to gently scrape sperm or eggs from gonad tissue into a clean 1L tripour beaker
      • rinse scored gonads with filtered sea water from squirt bottle to transfer excess sperm and eggs into tripour beaker
      • repeat gentle scraping and rinsing until most sperm or eggs have been extracted while avoiding the transfer of chunks of tissue male gonads that have been scored, scraped and rinsed: uc?export=view&id=1Csz2-VcW27PNmsJ5rAlZ9DlyKYKZsI2i Sperm collected from stripped males: uc?export=view&id=1wLzjRtzUbH51T9XsjdWx62HCraM5giOz
  2. Rinse eggs with warm seawater (15C?)
    • Transfer eggs from tripour to clean 100uM screen stacked on top a clean 20uM screen. The 100uM screen removes the tissue debris transferred during stripping. The eggs are caught on the 20uM screen. uc?export=view&id=1ekFfqNVgG7-TsGDIcvG2UZdiDRXkuVKy
    • rinse tripour and screen with warm filtered seawater (15C?) uc?export=view&id=1_zs5w4cPJVq-nVNExv1yiO2GzkoyIGh5
    • allow screen to drain. Screen should drain easily. If it doesn’t, it is likely clogged with eggs (as pictured below). The jelly coat on the eggs can get stuck in the mesh and clog the screen. To alleviate this, transfer onto a clean 20uM screen and spray the original screen from the back side to release eggs (this helped tremendously). If 2 20uM screens were used during rinsing, transfer eggs back to one screen. Clogged screen: uc?export=view&id=187dpVaR62Zqdcc2FdGIY5bskJBJVc8BU Transferring eggs onto clean 20uM screen: uc?export=view&id=1qz2CTkp06RA78V2c3rIwfziQe4-jZ1C4 Unclogged screen: uc?export=view&id=1FrLFSCdWjEP9mQeBssuFn7Khx039KFGX Screen speckled with eggs: uc?export=view&id=1pM7h95sRTxV7YVol_e2q3-vmsEHXf4f3 Combing eggs after splitting rinse on 2 20uM screens: uc?export=view&id=1YWaW24D8_HmJA2qmf1NUCvP6JgWN6EMr
  3. Prime eggs in KCl
    • Submerge screen with eggs in 50mM KCl solution uc?export=view&id=1sTkdd89Rctx4cxm0EZqB1ABWFjtFfypr
    • allow eggs to incubate in KCl solution for 20 minutes.
  4. Rinse eggs to remove KCl
    • rinse eggs on 20uM screen with warm filtered seawater (15C?) similarly to previous rinse step. After rinsing, transfer eggs to 2L easy-pour skinny beaker with handle and top off with warm filtered seawater (15C?) to a final volume that can be easily divided into the desired number of fertilizations
      • Since we had stripped 3 males, we made a final volume of 1200mL to be able to add 400mL eggs to each fertilization.
  5. Determine number eggs in final egg solution
    • Mix 1200mL egg solution with mixer while sampling for counts (mixer was a PVC pipe glued onto plastic circle studded with 1/2” holes and use almost as if a plunging while rotating it)
    • Microscope counts:
      • on microscope slide added 3 x 20uL egg solution and counted with tally counter
    • Cellometer counts:
      • added 2x 57uL egg solution to PD300 slide and counted cells on Nexcelom cellometer RESULTS:
    • Microscope counts:
      • 8.88 * 10^6 eggs = 148 eggs/20uL = 7400 eggs/mL * 1200mL
    • Cellometer counts:
      • 4.2*10^6 eggs = 3500 eggs/mL * 1200mL
  6. Combine sperm and eggs
    • check quality of sperm under microscope Discard any collections that seem non-viable RESULTS: One male sperm collection was not very active so we did not use it for fertilization
    • Mix eggs and aliquot into tripours for fertilization
      • We ended up splitting the eggs into 2 x 600mL aliquots for 2 fertilizations because one male collection seemed not viable
    • Added 6mL of sperm to each 600mL aliquot (@2:54pm)
    • Inubate for 15-20 minutes and transfer to LRT RESULTS:
    • 20min post-fertilization: sperm is all around the eggs but no polar bodies visible in 20uL drop uc?export=view&id=1mafmUN75wsXTl9P0fKxy3y703tGvvzag
  • 4-5 hours post-fertilization: starting to show cleavage uc?export=view&id=1y73_ofsgCAE-6SJSdliW8pAfsAhvKT_K

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