IsolatingTestesForIVF

In order to get the best in vitro fertilization, two criteria appear to be critical: first, selection of a mature male and second, use of L15/CS (L-15 Medium [Leibovitz] supplemented to 10% calf serum).

Select a male that has prominent (deep purple) nuptial pads. In our experience, males that have subtle nuptial pads (even if they are large animals) will give poor fertilization rates. We tend to raise our own males so it may be that these animals are just immature. Regardless, choosing males with prominent nuptial pads appears to improve fertilization rates significantly. We've also noticed that we get a better fertilization rate if we boost the males with 100 u hCG the night before fertilization (at the same time that we prime the females). We keep these males separate from females.

Anesthetize the male in 0.05% benzocaine. If this is done at room temperature it can sometimes take quite a long time before the male is adequately anesthetized. We find that placing the male in benzocaine at 12°C significantly hastens adequate anesthesia (about 5-6 minutes). Once the male is completely anesthetized, we remove the testis via an incision in the abdominal wall. However be aware that the testis lie deep in the back of the abdomen flanking the aorta (see photo). Sometimes we dissect out the testes under the dissecting scope which does make it a bit easier. Carefully cut the testes out with scissors or forceps and place in ice cold L15 (Leibovitch) Media supplemented with calf serum to 10% (L15/CS). Be sure to keep the testes on ice at all times.

Compared to X. laevis, tropicalis sperm have a much shorter window in which they remain viable. We tend to use an entire testis or pair of testes per ovulation to ensure good fertilization rates and don't store testes for more than a few hours before use. We have not tried storing testes for prolonged periods of time.

Abdomen of male frog (ventral view with the head at the top of photo). The skin and intestines have been lifted anteriorly to reveal testes (white arrows).

Contributed by Tim Grammer and Mustafa Khokha