Obtaining Embryos: hCG Injection

 

Ovulation is induced in X. tropicalis by injection of human chorionic gonadotropin (hCG). X. tropicalis requires a much smaller dose than does X. laevis. We use basically the same protocol as that described on the Grainger site .

We prime frogs 12-72 hours prior to when the eggs are desired. The priming dose is 10 units of hCG. Most often, we obtain frogs the night before we want to obtain eggs. For natural matings, we boost the male and female immediately without priming (100 u hCG for males; 200 u hCG for females). Overnight they will lay eggs which can be collected first thing in the morning.

However, for IVF, we prime females with 10 u hCG the night before we would like to obtain eggs. At the same time, we boost the males with 100 u hCG. Boosting the males early seems to improve the IVF the next day. We do keep the males separate from the females overnight to avoid amplexus and possible fertilizations.

Previously we used to inject 20 u hCG for priming and 100 u for boosting both males and females. However, once a colony of females is in a regular cycle of priming and boosting, we found that many of the females would lay many eggs after the priming dose. Therefore we reduced the priming dose to 10 u with less incidents of egg laying after priming. We increased the boosting dose because of a delay in egg laying. We found that in the larger, older females 100 u led to ovulation almost 6-8 hours after boosting which we found to be inconvenient. With 200 u, they again began to lay eggs at about 4 hours.

The frogs are kept at room temperature throughout the priming and boosting periods. Attempts to shorten the period between boost and egg laying by increasing the temperature (as high as 30ºC) have been unsuccessful. In a single attempt, frogs kept at warmer temperatures failed to mate naturally (Hayes lab).

In a single experiment, frogs boosted in the evening and then kept at 16ºC began laying eggs 2-3 hours after a morning boost (T. Grammer). However, the fertilized embryos had aberrant cleavages similar to those that we have seen when we raise embryos at 16ºC and were not viable .

We prime and boost both the males and females since it appears to improve fertilization rates (see in vitro fertilization for comments about fertilization rates). The hCG (Chorulon) comes as a powder of 10,000 units that we resuspend in 10 ml of sterile water (final concentration 1 unit/microliter). For the priming dose, we dilute the stock of hCG ten fold and inject 100 µl of this dilution per frog (10 units). For boosting, we inject 100-200 µl of the stock hCG (100-200 units). We find it difficult to accurately inject less than 100 µls with our syringes (we use 1 ml syringes).

We recommend using 30 gauge needles for injections, since we have found that a larger needle (25 gauge) is more difficult to use and can sometimes injure the frog. Also with the larger needle the hCG seems to leak out more often upon removal of the needle. We inject into the subcutaneous space of the back (avoiding the lateral line sutures). Keep the bevel of the needle up and the needle parallel to the surface of the skin to avoid hitting the underlying muscle. Sometimes when the skin is tough you may need to point the needle perpendicular to the skin to penetrate and then bring the needle parallel for injection. The needle should penetrate pretty easily. If it doesn't, then the needle has probably become blunt, and it is worth switching the needle or the frog will get an abrasion.

Be patient and careful with trops! X. laevis tend to be fairly docile but tropicalis are very active. Due to their hyperactivity and small size, it can be difficult to get a good grip on them. We have a number of strategies for immobilizing them for injection that work well.

NOTE: Whenever handling X. tropicalis, we recommend wearing gloves (powder-free). X. tropicalis have been shown to harbor mycobacterial infections. While we have had no instances of laboratory transmission of mycobacterial disease from frogs to humans, we recommend wearing gloves until the mycobacterial infection is fully characterized. Currently, we believe the risk of human infection of mycobacteria from frogs is quite low and the symptoms would be mild (cutaneous ulcer that can be treated with excision). Nevertheless, avoiding infection remains the goal so we recommend gloves as protection. For the protection of the frogs, it is important that the gloves be powder-free.

Frog On A Stick

"Frog on a stick" is likely the safest way to inject frogs with hCG, because the needle is always pointed away from the person injecting, In the other methods the user has to injec t so that the needle is pointing towards the restraining hand. To use this method, first wet a paper towel to provide a better grip. Use wet paper towels because dry paper towels can cause an abrasion to the frog (J. Parker unpublished observation). Then grasp the frog by the legs with the legs fully extended.

In this position the frog is completely immobilized and can be injected with the needle facing away from the person doing the injecting.

Inject the hCG into the dorsal lymph sac.

The most difficult part of this holding technique is getting the intial hold on the legs. Using the burrito method (see below) can help to get the legs extended and then allow the user to get a good grip on them.

Grainger Site )

For the burrito method, the frog is placed on a lab bench and covered with a wet paper towel. Again wetting the paper towel is important to avoid abrasions to the skin of the frog. Once the frog is covered with the wet paper towel, gently use your non-dominant hand to put pressure over the eyes and head of the frog. Covering the eyes of the frog appears to calm the animal so it stays still. Once the frog has settled down, using your non-dominant hand, continue to maintain gentle pressure over the head with the butt of your hand and reach over with your index finger and thumb and pinch the legs of the frog together. This will extend the legs of the frog and completely immobilize it.

Using your dominant hand, inject into the dorsum of the back. Often the skin will wrinkle making it easy to target the subcutaneous space and avoid the back muscles.

In our experience, this method works very well for beginners but can be a bit slow if there are a lot frogs that need to get hCG.

Iron Claw

We find the iron claw method to be the most convenient and efficient for hCGing large numbers of frogs but can be more technically difficult. There are two variants of the iron claw. In the first version, grasp the frog so that your index finger is between the legs, and your thumb and middle finger are flanking the legs.

The head of the animal will be buried in your palm. Once you have a good grasp let the frog settle down. At that point use your thumb to draw the leg back toward the body so the frog cannot use it to kick. The frog should now be completely immobilized and is ready to be injected.

Inject the frog into the dorsal lymph sac just below your index finger.

Grammer Variant of Iron Claw

In the Grammer variant of the iron claw, grasp the frog so that the belly of the frog is against the palm of the hand. The legs should be grasped between the index finger and thumb and the head held down with the ring finger and fifth digit. This should immobilize the frog and make it calm.

Lifting up the middle finger exposes the back of the frog for injection. Then inject the frog into the dorsal lymph sac carefully without injuring yourself.

 

contributed by Mustafa Khokha and Tim Grammer