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INFORMATION FOR

Natural Matings

Natural mating is ideal for collecting a variety of developmental stages at one time, or where the stage of the embryos is not critical. Examples would be collecting embryos to fix at a series of stages, such as for in situ or immunohistochemistry, or when collecting embryos to raise. A natural mating is less labor intensive than in vitro fertilization, requires less handling of the animals, and does not require sacrificing a male.

Factors important for optimal fertilization in a natural mating are water temperature (23.5 – 24.5°C), pH (6.8–7.0), and conductivity (800-1200µS). The mating can be set up in system water, or in 1/9XMR if low conductivity (typically results in soft eggs) is an issue. Equally critical is choosing mature frogs, ideally from a proven line, as discussed in the Introduction. In general, plump females with prominent cloacas and males with obvious nuptial pads are the most successful.

For natural matings, single pairs of boosted males and females are placed together. We usually keep them on a darkened shelf away from disturbances, but where they can be easily monitored. Three to four hours after boosting, the pair should begin responding to the HCG and should assume the typical amplexus mating position (see below). Our best success with fertilization rates comes from carefully choosing mature frogs. Females with prominent cloacas and males with obvious nuptial pads are the most successful. We have developed proven lines of females that lay high quality eggs and males that fertilize well.

We do our natural matings at room temp (23.5-24.5°C). When we have attempted to do matings at warmer temperatures (25°C or warmer), we have had poor results. We have not done a careful comparison however. Nevertheless based on our experience, we recommend natural matings be done at 23.5-24.5°C.

The bucket can be left undisturbed until the following day when raising embryos or collecting later stages. This allows production and fertilization of the maximum number of eggs. The number of fertilized embryos produced can be well over 3000. Allow embryos destined for raising to develop undisturbed in the mating bucket, at 24 – 25°C, to stage 31-34.

The percentage of developing embryos is found to be improved when frogs are left in the bucket for up to 36 hours after boosting, instead of being removed the morning following boosting. The reason for this is unknown, but it is thought perhaps the movement of the frogs aids in water aeration. Embryos ready to be removed will be flat, may “flutter” when disturbed, but will return to resting; they are not actively swimming. They are often seen attached to the side of the mating bucket by the cement gland.

Another methodfor raising embryos is to collect them first thing in the morning following boosting. The sticky jelly coat surrounding the embryos is partially removed by swirling in 3% cysteine for 30 seconds - 1 minute in an Erlenmeyer flask. (see Cysteine Embryos). Partial de-jellying makes sorting easier, but leaves some protection for the embryo. These embryos are then raised in 1/9X MR in 150 mm petri dishes and transferred to main housing.

Amplexus

Male and female tropicalis in amplexus.

If contamination of embryos in the dishes becomes a problem, evidenced by fungal growth or disintegration of embryos, 1/9XMR media can be supplemented with gentamicin antibiotic at 100 µg/ml (Sigma, G3632).

Contributed by Tim Grammer, Maura Lane and Mustafa Khokha