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Alternative IVF Protocol

Alternative IVF Protocol

Day 1 – PMS Injection

  1. Between 10:00 am to 12:00 pm, administer PMS via intraperitoneal (i.p.) injection to female mice of appropriate strain and age.

Day 3 – hCG Injection and Dish Preparation

  1. 48 hour after PMS, administer hCG via i.p. injection.

Prepare IVF Dishes

  1. For fresh sperm IVF, prepare one culture dish per male containing 1 mL IVF medium.
  2. Prepare one culture dish per three females containing 3 mL IVF medium for oocyte collection.
  3. Prepare one culture dish per three females containing one 400 μl IVF medium drop (for fertilization) and four 150 μl IVF medium wash drops (post-fertilization).  Cover these dishes with mineral oil and do not use lids on these dishes.
  4. Keep all dishes at 37ºC under mixed gas (5% CO2, 5% O2, 90% N2).

Day 4 – IVF Procedure

Sperm Preparation

  1. Collect sperm (see sperm thawing protocol).
  2. Add sperm to the fertilization drop:
  • Frozen sperm: 10 µL
  • Fresh sperm: 5–10 µL depending on motility

  1. Incubate under mixed gas for 1 hour.  Fresh sperm may be used immediately.

  2. Record the time sperm was added.

Oocyte Collection (13–14.5 hours post‑hCG)

  1. Collect oviducts from superovulated females. Work quickly—≤5 minutes per group of three females.
  2. Dissect egg clutches and transfer to the IVF+sperm drop using a 10 µL pipette, minimizing extra medium.
  3. Record the time eggs were added. Repeat until all eggs are transferred.
  4. Incubate 4–6 hours at 37°C under mixed gas.

Post‑Fertilization Washing

  1. Move eggs/zygotes into the first 150 µL wash drop.
  2. Select the healthiest embryos and transfer to the next drop, leaving behind dead or fragmented cells.
  3. Distribute healthy embryos between the final two drops.
  4. Record counts of:
  • Live
  • Fragmented
  • Dead
  • Zona‑free cells
  1. Complete this step in ≤5 minutes and return dishes promptly to the incubator.
  2. Incubate overnight under mixed gas.

Day 5 – Scoring IVF

For each IVF dish, count and record:

  • Two‑cell embryos
  • One‑cell embryos
  • Fragmented oocytes
  • Dead oocytes

Next Steps

  1. Embryos may be cryopreserved, transferred to 0.5 dpc pseudopregnant females, or cultured to blastocysts stage with the use of KSOM or another appropriate medium.