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
2A
2B
2C
2D
2E
4A
4B
4C
4D
4E
5A
5B
5C
5D
5E

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: https://shellytrigg.github.io/225th-post/

  • 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.