Hi Hana,

Difficult to have a comprehensive answer without a video of some sort. I work in human IVF so things may be a bit different. The most obvious answer is: can you watch your supervisor inject? What is different between your technique and his?

One thing that you didn't mention in the protocol is sperm immobilisation. This is extremely important as 1) it activates the sperm, 2) it stops it from moving within the oocyte.

Make sure that the sperm you are using is:

1) alive (i.e., fully progressing or at least twitching), this will maximise the chances of oocyte fertilisation.

2) fully immobilised by swiping it across the tail with the injection needle (however, this is the method for human ICSI and there may be alternative effective methodologies for animals). When properly swiped, the sperm tail usually makes a kink that disappears within a few seconds. This procedure also ensures that the sperm won't be moving inside of the oocyte after injection (which obviously would wreck internal organelles and likely result in degeneration.

Hope this helps.

Hereditics of Interspecific Fertilization

Dear Hana,

Interspecific fertilization usually is failed. The mice cell contains 40 chromosomes while goat cell contains 60 chromosomes. The fertilization needs chromosome number pairs with the said microtubules inside the cell. If they are not paired, it will fail. Heredity is not only decided by genome, but also decided by cytoplasm. That is: Hereditics included both Genetics and Cytohetics. We did man-cow tissue cloning test in 2000, and it failed even the embryos grew up to blastocyst stage. Please read Preface part in hereditics.net.

Best wishes,

Ke-Hui Cui

Maurizio Poli Thank you for your answer :)

Yes, I can watch my supervisor and I have already done it many times. He also watched me during the micromanipulation. He had some good points, for example injecting some dose of handling medium with sperm into the oocyte (potential toxicity after some time), not enough washing of injection pipette from PVP. He also mentioned that "my" oocytes look stiff or stony (sorry for my English). They are not as flexible and elastic as they should be. So I focused on culture and handling medium. But we both use the same product and concentrations.

To sperms:

We use frozen sperms which are thawed in a handling medium and put into a centrifuge to get to rid of cryoprotectants.

I do this whole process for evaluating sperm DNA damage and fertilization ability after cryopreservation. And If their ability to move corresponds with their ability to fertilize. My supervisor uses separated sperm heads from tested ID (some from centrifuge, some from piezo pulses or pairing of sperm head from its tail by pressing microinjection pipette).

He does it routinely and he regularly acquires zygotes.

I don´t think that potential fertilization disability would cause instant breaking of the oocyte. But maybe I´m wrong.

If the oocyte didn´t break, it often dies in the incubator and I tried to use MEM and KSOM culture medium too.

We use micropipettes that we made in a micropipette puller and Microforge. I´m trying my best to make it just a bit wider in inner diameter than the sperm head is - to make injection trauma as small as possible.

I truly believe that the critical step could be somewhere between the isolation of oocytes and catching each of them on the holding pipette.

I focus on oocyte position for injection too, If it is possible and I can see it. Especially on the polar body and "metaphase area"(?).

I know that my problem is most likely just skills. But "a drowning man will clutch at a straw".

Thank you again :)

Hana

Ke-Hui Cui Hi :) thank You for your answer.

As I mentioned in the previous comment I do this whole process for evaluating sperm DNA damage and fertilization ability after cryopreservation. And If their ability to move corresponds with their ability to fertilize.

The goal is to acquire zygotes (and It has been done many times, but not by me ;).

To be honest, I still don´t know lots of things about the fertilization process and its genetic and biological aspects. Especially the timing of each development phase.

Thank you again and have a nice day :)

Hana

For your hyaluronidase step, do you use a supplemented PBS or just plain PBS? In human IVF we use mHTF (with HEPES) for that step on a warm (37degree surface) Osmolarity very important.