Matt, Brent, Nate (from hatchery in Lilliwaup?), and Josh present

Biopsies of animals from treatment and control groups (tanks B1, B2, B4, and B5)

Grabbed 5 animals from each tank and labeled with pencil and marker:

  • 1A-1E
  • 2A-2E
  • 4A-4E
  • 5A-5E

Used 15mL falcon tube “speculum” to hold pedal gap open and sampled gonad with 23G 1.5” needle on a 5mL syringe.

Gave each animal shellfish tag

Tank Treatment Written Label Tag Sex
1 pH 7.2 1A 019 NA
1 pH 7.2 1B 018 NA
1 pH 7.2 1C 013 male
1 pH 7.2 1D 012 male
1 pH 7.2 1E 009 NA
2 ambient 2A 031 male
2 ambient 2B 028 NA
2 ambient 2C 027 NA
2 ambient 2D 022 NA
2 ambient 2E 025 female
4 ambient 4A 057 NA
4 ambient 4B 040 NA
4 ambient 4C 059 NA
4 ambient 4D 044 NA
4 ambient 4E 058 NA
5 pH 7.2 5A 097 male
5 pH 7.2 5B 085 female
5 pH 7.2 5C 093 female
5 pH 7.2 5D 088 female
5 pH 7.2 5E 090 male

pictures of gonad biopsies:

  • biopsies for tank 1 animals cross-contaminated on the slide before I could take pictures so no tank 1 animals are pictured below
Animal Label Image

Because all animals seemed mostly immature, we decided to biopsy animals from Tank 6

Biopsies of animals from tank B6

Biopsied all animals from October harvest and found 5 ripe females and 4 ripe males.

Images of ripe females:

Decided to strip spawn these and not the animals from the control and treatment tanks

Strip spawning animals from tank B6

  • Followed the protocol outlined here:

  • Pooled all eggs together in one 4L beaker
  • Rinsed in 4 batches so to not clog the screens
  • Did 2 KCl priming baths at a time to save time
    • made KCl as follows:
  • Rinsed KCl off and transferred back into beaker and Josh counted eggs
    • 15 M eggs
  • Divided eggs up in 4 equal volumes and added sperm from each male
  • Transferred fertilized eggs to LRT
  • SIDE EXPERIMENT: Did a fertilization with leftover eggs from gonads WITHOUT KCl. Let fertilization sit in container until d-hinge count a few days later.

End of the day notes

At the end of the day, tank 2 had a volitional spawn. Matt and Josh saved 4M eggs.

Noticed a couple ports in manifold in the silo set up in tank B3 were clogged. I took pictures of animals in these silos, they look alive and ok. I flushed the manifold out with fresh water, reconnected, and made sure each port had flow before I left.