In vitro fertilization is ideal for obtaining a large number of embryos developing at the same rate, as when using embryos for injection, for example. In addition to selection of a mature male with dark nuptial pads, mature proven females and optimal water quality conditions, other critical factors for IVF success include proper pH of IVF solutions, and use of proper storage solution for harvested testes, as will be explained in this section.
Four hours prior to the in vitro fertilization procedure, a number of primed females (2 – 4) are boosted with 250 – 300u hCG. The are immediatelyplaced in a holding bucket (Rubbermaid, #6304 and # 6509) with approximately 2 - 3 liters of system water or 1/9X MR. Using multiple females will ensure eggs are obtained even if one or more of the females do not produce eggs. The females are left at room temp (23.5 – 24.5°C) in a quiet place. Keeping frogs at temperatures above 25°C or below 22°C usually results in delayed egg production and poor quality eggs.
Approximately three to four hours after boosting, the female’s cloacae should become swollen and red. The frogs should begin producing eggs within an hour, visible as clumps or individual eggs in the bucket. At this point, the females are ready for use in egg collection. Note: Before collecting the eggs, euthanize the male and harvest the testes as described in Isolating Testes. If the male is important however, express a small number of eggs from the females and check egg quality as described below before euthanizing the male.
Occasionally the female’s cloacae may be red but no eggs are visible at four hours. In this case, egg production can sometimes be gently encouraged as described in Manual Expression of Eggs below. If no eggs are obtained, wait 15 – 20 minutes and check for eggs again. Trying to force egg production by excessive squeezing will only stress and possibly injure the frog. Occasionally a frog will appear ready to lay, but never produce eggs.
Use one 60 mm Petri dish (#351007) per female, to allow separate evaluation of egg quality. Coat the dishes with just enough 1xMBS + 0.2%BSA to cover the bottom. This prevents eggs from sticking to the dish. Coat the inside of the transfer pipets (Globe Scientific, # 89209-802) with the 1xMBS + 0.2%BSA also.
Squeeze the female so that her cloaca is closest to the dish. This prevents the eggs from running down your fingers and her legs.
After at least an hour of rest after the first squeeze, the females can be re-squeezed for a second fertilization if desired. A good female can expel over 1000 eggs in a squeeze, and with several squeezes, we can get 2000-3000 eggs. In a natural mating, the number of fertilized embryos can be well over 3000.
Contributed by Tim Grammer and Mustafa Khokha
Manual Expression of Eggs
Using a gloved hand, hold the female as described in the Handling section, with legs pulled back and the cloaca at the vertex (see photo). Keep the cloaca at the lowest point to allow the eggs fall out and land in the dish. Gentle pressure on the lower back and abdomen will expel the eggs from a ready female. Applying a gentle downward pressure while moving fingers toward the cloaca may induce production of eggs if they are not immediately expelled. Allowing the female to move slightly in the hand, thus contracting her abdominal muscles, often aids in egg production. The eggs are sticky and may run onto the frog and the handler’s fingers. These eggs can be recovered for use by scraping them from the fingers onto the lip of the dish.
Place used females in a new bucket with clean water or 1/9X MR after expressing eggs. This allows tracking of which animals have been used, and avoids over handling. If the females have not been overly stressed, they can be tried again after a few minutes rest. If eggs are not readily expressed, do not apply excessive pressure. The female may not be ready to lay, and too much pressure can result in stress and injury.
Determining Egg Quality
Examine the eggs under a stereomicroscope . They should beuniform in appearance, with a smooth, round shape. There should be a nicely pigmented animal pole and a pale vegetal pole. The pigmentation should not be mottled or splotchy. Production of “stringy” eggs, or the presence of many white eggs in the dish indicates poor quality. This dish of eggs should not be used. While they may fertilize and appear normal initially, poor quality eggs often fail to divide or develop normally.