• Plan to strip oysters on day of arrival.

There is some experience that results are not as good after 48hours. If necessary store cool (10oC) and damp and allow 'recuperation' in 20oC seawater (Caution: may spawn!)

• Make visual assessment of gonad maturity .

Good oysters have large area of gonad and this is mature when it is deeply veined. The gametes should ooze out easily when incised. Report assessment to your supplier !

• Strip oysters individually and examine a small sample of gametes from each.

Good eggs have uniform pear shapes which round up on contact with seawater. Good sperm is uniformly active on contact with sea water.

• Pool good sperm
• Fertilise individual beakers of eggs.

Aim for 5 to 10 per microlitre. Fertilise within two hours of stripping.

• Amount of sperm is very important.

Aim to see 3-10 sperm per egg. Sperm can vary greatly in concentration and activity. Essential to examine eggs within 5 minutes to check then easily add more. However, avoid too much - can cause abnormal development ('polyspermy'). Some labs. check egg and sperm densities with Coulter counter

• Keep eggs in suspension by stirring or decanting every 10 minutes

Check eggs again after 30 minutes (at 25oC) for >80% with first polar bodies

If less, start again with fresh gametes. Do not add more sperm. No need to wait 2 or 4 hours. Try taking eggs from different oysters and/or different parts of the gonad.

• Pool best quality eggs with uniform development.

Adjust density as required and add to test containers according to protocol procedure.

REFERENCES

THAIN J.E. (1991) Biological effects of contaminants: the oyster (C. gigas) embryo bioassay In: Techniques in Mar.Env. Sci., ICES

JOHNSON I. (1994) WRc /EA R&D Note 322