11) ISO-PERCOLL:

1) Solution A: 0.21 g NaHCO3 + 10 ml EBSS [10x]. (Filter)

2) Use 3.75 ml of Solution A + 33.75 ml Percoll.

3) Adjust pH to be 7,4 in small tubes. (The Percoll-solution must not leave the Flowbench).

4) Adjust osmolarity to 300 mOsm by adding Percoll. Can be stored for 2 weeks at 4°C (Fridge 1).

12) 55% + 90% PERCOLL:

55%    1100 µl  IsoPercoll (see no_11) + 900 µl SPERM-TALP .

90%    1800 µl. IsoPercoll (see no_11) + 200 µl SPERM-TALP.

Note: Make this in a 14 ml Falcontube.

Note:

- Place these two and the Sperm-TALP at 39°C and 5% CO2 (Heraeus-incubator) overnight.

You can also buy 90% Percoll and then adjust part of it to a lower density (55% mentioned above or less, going down to 40% or so). Having said that, a number of studies on sexing by density gradient failed to show any real success. The difference between the X and Y sperm is too minor for an insensitive technique such as density gradient to separate between these two populations.

Influence of Discontinuous PureSperm® and

OptiPrep™ Gradient Centrifugations on Bovine

Sperm Quality and the Sex Ratio of in vitro Produced

Embryos

Practical use of percoll density gradient centrifugation on sperm sex

determination in commercial dairy farm in Thailand

thanks for suggestion and help for both of you