Improvement of a twice collection method of mouse oocytes by surgical operation

Abstract

Mouse oocytes are generally collected after euthanasia. However, if oocytes were collected without euthanasia, then mice could be used to collect oocytes again after recovery. This condition is especially useful for mice that are genotypically rare. In this study, we examined the reusability of mice after collecting oocytes via a surgical operation. When oocytes were collected using medetomidine/midazolam/butorphanol combination anesthesia and examined for the quality of oocytes after in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), they could develop to full term at the same rate as controls. When oocytes were collected from those mice a second time, the average number of oocytes was reduced by nearly 1/3. However, the blastocyst and offspring rates of those oocytes after IVF or ICSI were the same as those of the control regardless of the recovery day period. Although germinal vesicle (GV) oocytes can be collected from all reused mice, the final number of offspring did not increase. Interestingly, when oocytes were collected from the front position of the ampulla, 76% of the oviducts possessed oocytes after reuse, and the average number of oocytes significantly increased to a level comparable to that of the control. Finally, we examined whether reused mice can be used as recipient females, and then healthy offspring were obtained similarly as the control recipients. In conclusion, we provide a new method to collect a sufficient number of oocytes from reused mice without concern.

Mouse <i>in vivo</i>-derived late 2-cell embryos have higher developmental competence after high osmolality vitrification and −80°C preservation than IVF or ICSI embryos

“…The excised specimens were compressed to release a dense mass of spermatozoa from which drops (approximately 2 µl) were collected and placed in a dish containing 0.4 ml HTF medium and cultured at 37°C in a CO 2 incubator for 20–30 min. IVF was performed as previously described [ 15 , 16 ]. Five to ten microliters of sperm suspension were collected and introduced into each drop containing COCs.…”

“…The vitrified-warmed 2-cell embryos derived from in vivo or IVF were cultured for 1–2 h to recover from vitrification damage. Thereafter, some randomly selected embryos were transferred to day 0.5 pseudopregnant mice that had been mated with vasectomized males the night prior to the transfer [ 16 ]. Five to eight embryos were transferred to each oviduct.…”

Mailing viable mouse freeze-dried spermatozoa on postcards

“…Pronucleus formation was verified 6 h after ICSI. Embryos at the 2-cell stage were transferred to a day-0.5 pseudo-pregnant ICR female mice (body weight: 28-40 g) that had been mated with a vasectomized male the night before transfer ( Inoue et al., 2020 ). Eight to eighteen embryos were transferred into each oviduct.…”

Optimised CO2-containing medium for in vitro culture and transportation of mouse preimplantation embryos without CO2 incubator

“…In vitro fertilization was performed as previously described [ 31 ]. Briefly, spermatozoa were collected from the cauda epididymitis of ICR, C57BL/6N or B6D2F1 male mice (>10 weeks) into 200 μL of human tubal fluid (HTF) medium [ 32 ] that was then covered with sterile mineral oil and capacitated by incubation for 1 h at 37°C under 5% CO 2 in a CO 2 incubator.…”

“…Embryo transfer was performed as previously described [ 31 ]. Blastocyst-stage embryos derived from embryos cultured in sealed plastic tubes were transferred into the uteri of pseudo-pregnant ICR female mice at 2.5 dpc, which were previously mated with vasectomised ICR males.…”