OPU-IVF PROTOCOLS

Oocytes Aspiration Plus

SAR IVF

SAR IVC

 

IVF for Large Scale Embryo Production PROTOCOLS

SAR IVF

SAR IVC

 

IVF For Large Scale Embryo Production PROTOCOL

 

SAR IVF

 

A. Making Sperm Washing Medium

1. Add one tube containing 800 L of SAR IVF Stock II to 40 mL of SAR IVF Stock I to make 40 mL of SAR sperm washing medium. Filter the medium using a 0.22 m syringe filter. Aliquot 10 mL of this medium into each of two 15 mL conical tubes (Cat# FALCON 352099, Fisher) and two 14 mL round-bottom tubes (Cat# FALCON 352057, Fisher), and label as SAR-Sperm. A volume of 20 mL of SAR-sperm (two tubes, 10 mL/tube) is used for washing one straw of conventional semen. Prior to washing sperm, loosen the caps on the tubes, set the tubes in a humidified, 5% CO2 incubator set at 39C and pre-warm the medium for 23 h.

2. Place 3537C tap water in a glass tray. Take one straw of semen out of the LN2 tank, gently shake it 10 s in air before immersing it into the tap water for 1 min.

3. Take the conical tube containing SAR-Sperm out of incubator. Wipe the scissors and the straw, cut one end of the straw (the non-stuffed end) and put the end into the conical tube, touching the SAR-Sperm solution. Cut the other end of the straw (the cotton end), drain the semen into sperm washing medium and wash the straw with 0.5 mL of SAR-Sperm medium.

4. Invert the tube several times to mix the semen. Centrifuge the sperm at 1200 rpm (about 700 g) for 8 min. Carefully remove top supernatant of SAR-Sperm using a 1mL pipet, leave about 1.52 mL of the bottom sperm layer without disturbing the sperm pellet.

    5. Take the round-bottomed tube with the SAR-Sperm out of the incubator, pour SAR-sperm into the conical tube. Invert the tube several times. Centrifuge the sperm at 1200 rpm for 8 min. Carefully remove the top layer of SAR-sperm washing solution.

    6. For conventional semen, leave about 0.5 mL of the bottom layer in the tube, add 0.5 mL of SAR-Sperm into the tube, and mix by pipetting up and down, total volume is 1.0 mL for conventional semen. For sexed semen, leave about 0.35 mL of the bottom layer in the tube; suspend the sperm pellet by pipetting up and down in the remaining solution, total volume is 0.35 mL for sexed semen.

 

B. Making fertilization medium and droplets

1. Add one tube of 770 L of Stock III to 10 mL of Stock I to make the solution for fertilization. Filter the medium using a 0.22 m syringe filter. Aliquot 5 mL of this medium into each of two 5 mL round-bottom tubes (Cat# FALCON 352058, Fisher) and label as SAR-Fert. A volume of 5 mL/tube SAR-Fert is used for each fertilization. Loosen the cap, set the tubes in a humidified, 5% CO2 incubator set at 39C and pre-warm the medium for 23 h before washing the sperm.

2. Before fertilization, prepare the appropriate number of 35 mm petri dishes (Cat# FALCON 351008, Fisher). Place 7 droplets of SAR-Fert in each petri dish, using a 200 L pipetman to yield 50 L/droplet (350 L/dish, 700 L total). Slowly layer 33.5 mL of mineral oil (Cat# M8410, Sigma) over the droplets. Set the dishes in a humidified, 5% CO2 incubator set at 39C. The rest of the fertilization medium (4.3 mL) can be used for pre-washing maturated oocytes.

 

C. Fertilization procedures

1. At 22 h (2224 h) in vitro maturation (IVM), during the sperm centrifugation, prepare the appropriate number of 60 mm petri dishes (Cat# FALCON 351007, Fisher) and add 4 drops of SAR-Fert (the remaining SAR-Fert after making enough droplets) in each petri dish.

2. Wash 25 matured oocytes in each drop for a total of four times and then transfer 2025 oocytes/drop into the 50 L/droplet of SAR-Fert medium.

    3. Add 50 L of purified sperm into each 50 L/droplet of SAR-Fert medium containing washed matured oocytes. Check the activity of the sperm at the edge of each droplet under the microscope. The sperm should be very active, swimming fast. The final concentration of sperm for IVF should be 1 106/mL when prepared from conventional semen and 1 105/mL when prepared from sexed semen.

    4. Incubate the dishes at 39C in 5% CO2 for 18 h.

 

IVF For Large Scale Embryo Production Protocol (pdf format)