Centaurian gonadotropin frog mating protocol Preparation of hormones: Gonadotropin, pregnant mare serum (PMSG) from Sigma: 2000IU in 2mL 0.9% saline Gonadotropin, human chorionic (HCG): Make up HCG solution of 500IU per 1 mL. (in 2500IU vial add 5ml 0.9% saline) Store hormones at 4C for up to three months. Day 1: Prepare tank for female or place the animal in one of the tanks in the rack (if available). Inject 50µl (50UI) PMSG into female frog (this is only 0.05ml which is less than 0.1ml). The PMSG can be injected intraperitoneally into the ventral side of the frog or into the lymph sac on the dorsal side (see pictures below). Squeeze the frogs firmly in the net to avoid their moving. Make sure to hold the syringe at an angle nearly parallel to the skin. If you see blood after removing the syringe you went too deep!!!! Always change the number written in whiteboard marker on the laminated tag on the tank (indicating the number of frogs) when you remove or add a frog. Tell Anne what you did for her to record. Day 2: Clean tank by exchanging 90% of the water with fresh water from the system. Day 4 morning: Inject 150 UI (0.3ml) of hCG into the dorsal lymph sac of the male. (This step is recommended but optional) Day 4 (around 4pm): Make sure that tank of female is clean if not exchange 90% of water. Inject 150 UI (0.3ml) hCG into the male and 400UI HCG (0.8ml) into the female and place animals together in tank. If the two animals are not in amplexus one day after injection, wait another day or repeat the Day 4 injections. Note: It is ok to push the Day 3/4 injections to Days 4/5. Frog Breeding Schedule
Friday Saturday Sunday Monday
Prime Clean
Nothing AM:
female tank of
Prime
female
male
PM: Inject
and put 1st
couple
together.
Prime 2nd
female.
Tuesday
Collect
eggs. If
there are
no eggs
you can
inject
again (as
on day 4)
Thursday
Collect
eggs. Put
female and
male from
injection 1
into male
and female
“last used”
tanks.
Friday
AM:
Prime
male
PM: Inject
and put 2nd
couple
together
Saturday
Collect
eggs of
2nd
couple.
Sunday
Come
into lab
to clean
eggs!!!!
How to move labels of the frog tanks: When all frogs of the current group (tank labeled “current males”/ “current females”) have been used change the labels as follows: The now empty tank that is labeled “Current” becomes “last used”. The tank “last used” becomes waiting 3. The tank “waiting 3 (next3 up)” becomes “waiting 2”. The tank “waiting 2 (next2 up)” becomes “waiting 1” also known as “next up”. “Waiting 1 (next up)” becomes “current”. There might not always be 4 groups of males or females, however the same logic applies. (e.g., that there is no “waiting 3” tank for males). Remember to keep count of frogs for each tank. That means change the count on each label to match the number of frogs that this tank contains. Let Anne know what you did so she can update the database. Don’t be scared. You can do it! The sites at which frogs may be injected to administer the priming dose of pregnant mare serum gonadotropin PMSG into either the peritoneum or the dorsal lymph sac are indicated (red arrows and circles): Ventral: Dorsal: