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 1/9xMR+0.2% BSA.
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 poorer fertilization rates. Choosing males with prominent nuptial pads appears to improve fertilization rates significantly. We do not prime or boost the male. We keep these males separate from females.
Anesthetize the male in 0.05% benzocaine (ph 7.0-7.4). Once the male is completely anesthetized, he should euthanized according your instiutions protocols. Remove the testis via an incision in the abdominal wall. 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. Carefully cut the testes out with scissors or forceps and place in 1xMR+0.2%BSA.
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, Maura Lane and Mustafa Khokha