When we first started using X. tropicalis, in vitro fertilization had an extremely poor efficiency. However, with the careful selection of a mature male and the use of L15/CS (L15 Leibovitch's Mediasupplemented to 10% calf serum), the in vitro fertilization efficiency has been improved drastically. Direct comparison of different medias (1xMR, L15 alone, L15/CS), clearly showed that L15/CS gives the best rates of fertilization with L15 alone being better than 1xMR. Others have reported that an albumin solution works well although we have not tested this directly against L15/CS. Once we used goat serum instead of calf serum which seemed to work well although not tested in a side by side comparison. Other types of serum (fetal calf serum for example) have not been tried. (many thanks to J. Gerhart for suggesting the serum and L. Zimmerman for the L15).
Another improvement for IVF efficiency is to boost the males with 100 u hCG the night before you do the IVF. We usually prime females with 10 u hCG as well at the same time (we used to prime with 20 u hCG but noticed that the females sometimes laid a fair number of eggs at that dose). We haven't scientifically tested whether boosting the male the night before actually improves fertilization, but that is our general feeling.
Once the eggs are squeezed and ready for fertilization, we macerate the testis in 500 µl cold (on ice) L15/CS in a 1.5 ml eppendorf tube using a 1.5 ml microtube pestle. Unless haploids are being generated (see "making haploids" section), we immediately pipet the sperm suspension onto the eggs and gently mix well to ensure exposure of all the eggs to the sperm. Allow the sperm to attach to the eggs for about two-three minutes, then "activate" the fertilization by flooding the dish of embryos with 1/9xMR. The low salt solution activates the sperm. Formicroinjection, flood with 3% Ficoll + 1/9xMR.
Unlike X. laevis, we find that "turning", or the upright rotation of the animal hemisphere in the vitelline envelope, is not always a good indication of successful X. tropicalis egg fertilization. Many times we have had very good fertilization rates with little obvious turning. We often do see cortical contraction of the animal hemisphere in fertilized eggs, but the absence of either contraction or turning is not truly predictive of a failed fertilization. Usually the first cell division occurs about an hour and ten minutes after flooding the eggs. If two hours have elapsed and no cell division is seen, then the fertilization was unsuccessful.
If one needs to obtain one cell stage embryos for manipulation and therefore cannot wait until first cleavage to determine fertilization, we recommend dejellying the eggs about 30-40 minutes after flooding. Once dejellied, the fertilized eggs can usually be identified based on their rigidity, presence of sperm entry point (see microinjection protocol), and (sometimes) cortical contraction of the animal hemisphere.
Contributed by Tim Grammer and Mustafa Khokha
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