Fig 1.
Experiment 1) Oocytes will be collected from stimulated (experimental) and unstimulated (control) animals prior to IVF. Experiment 2) Oocytes will be fertilised with fresh spiny mouse spermatozoa and cultured to the two cell stage prior to vitrification. Experiment 3) Embryos will be vitrified, stored and then thawed prior to ET. Experiment 4) Embryos will be collected from stimulated (experimental) and unstimulated (control) animals prior to vitrification. Experiment 5) Embryos will be thawed and transferred to surrogate female spiny mice. Dams will be monitored for pregnancy (~30 days) and for live births (~40days). A group of sham-transfer controls have affigel beads transferred in place of embryos.
Table 1.
Research timeline for ART development in Acomys cahirinus.
Fig 2.
Control unstimulated oocyte collections.
Mice are lavaged daily until the late follicular phase (~day 6 of the menstrual cycle) when they will be killed and oocytes collected for use in IVF (Experiment 2).
Fig 3.
Superovulation protocol and oocyte collections in Deslorelin implanted mice.
Mice are vaginally lavaged until the menstrual phase (~ day 1–3 of the cycle) and a Deslorelin implant then subcutaneously implanted into each mouse. All mice will be given 3 cycle lengths (27 days total) to recover from surgery and for the agonist implant to have taken effect. Mice will then be injected IP once daily for 4 days with Gonal-F followed by a single injection of Ovidrel on day 5. Females are culled 36 hours later and oocytes collected for use in IVF.
Fig 4.
Rotation of injection sites for hormone IP injections.
Injection sites are rotated across the lower abdomen of spiny mice to prevent injury from repeated injection sites.
Fig 5.
Embryo collections from unstimulated spiny mice.
Females are lavaged daily until the late follicular phase (~day 6 of the cycle) when they will be paired with a fertile male and left undisturbed to mate overnight. Female mice are then killed 48hrs later and embryos collected for immediate vitrification (Experiment 3).
Fig 6.
Superovulation protocol and embryo collections in Deslorelin implanted spiny mice.
Females are vaginally lavaged until the menstrual phase (~ day 1–3 of the menstrual cycle) and a Deslorelin implant then subcutaneously implanted into each mouse. Females will be allowed 3 cycle lengths (27 days total) to recover and for the agonist implant to have taken effect. They will then be injected IP once daily for 4 days with Gonal-F followed by a single injection of Ovidrel on day 5. Females are then paired with a fertile male and left undisturbed to mate overnight. Forty-eight hours after the Ovidrel injection, the females are killed, embryos collected and immediately vitrified (Experiment 3).
Fig 7.
NSET transfer device and handling procedure.
A) Both speculum (*) and the NSET cannula, B) Insertion of the smaller speculum, C) Speculum fully inserted in the vaginal canal, D) Insertion of NSET needle through the speculum and cervical opening.
Fig 8.
Embryo transfer procedure for surrogate spiny mice.
Mice are vaginally lavaged until the luteal phase (~ day 7 of the menstrual cycle) and a maximum of 4 embryos will be transferred per female using the NSET device. Surrogate mice are presumed pregnant and will be monitored for external signs of pregnancy (~30 days). If mice are considered non-pregnant, they are removed from experimentation and returned to colony. If mice are pregnant, they are left to litter-down (~8–10 days later).