IVF recovery of mutant mouse lines using sperm cryopreserved with mtg in cryovials.

BACKGROUND: Modification of cryoprotective medium (CPM) R18S3 (18% raffinose and 3% skim milk) by addition of monothioglycerol (MTG) or L-glutamine (Glu) has been shown to improve in vitro fertilization (IVF) using mouse sperm cryopreserved in cryostraws. However, whether these CPMs can be applied effectively to sperm cryopreserved in cryovials is unknown. OBJECTIVE: The study was to determine the comparative effectiveness of using R18S3, R18S3+Glu (100mM and 87 mM), or R18S3+MTG (477 µM) to cryopreserve various sample volumes of mouse sperm in cryovials and cryostraws. METHODS: This study compared the effects of different CPMs on motility of fresh and frozen-thawed C57BL/6J sperm and on IVF rate of C57BL/6J sperm cryopreserved in different CPMs and containers with different volumes, and then used technologies developed to cryopreserve and recover sperm of knockout mouse lines on inbred C57BL/6 backgrounds. RESULTS: Glutamine at 100 mM inhibited, but MTG at 477 µM protected, fresh sperm motility significantly (P < 0.05). Sperm cryopreserved in R18S3+MTG had significantly better (P < 0.05) post-thaw progressive motility and IVF rate than when cryopreserved in R18S3 alone, R18S3+Glu (100 mM), or RSGlu87 (15.7% raffinose, 2.6% skim milk, and 87 mM L-glutamine). There was no significant difference in IVF rates among sperm cryopreserved with R18S3+MTG in cryovials or in cryostraws (P > 0.05). Sperm from 63 knockout mouse lines on C57BL/6 backgrounds cryopreserved using R18S3+MTG in cryovials were all recovered successfully to genotypically-confirmed offspring. CONCLUSION: Mouse sperm on C56BL/6 backgrounds can be successfully cryopreserved in cryovials using R18S3+MTG.

Investigations of motility and fertilization potential in thawed cryopreserved mouse sperm from cold-stored epididymides.

Cold transport of epididymides from genetically modified mice is an efficient alternative to the shipment of live animals between research facilities. Mouse sperm from epididymides cold-stored for short periods can maintain viability. We previously reported that cold storage of mouse epididymides in Lifor® perfusion medium prolonged sperm motility and fertilization potential and that the sperm efficiently fertilized oocytes when reduced glutathione was added to the fertilization medium. Cryopreservation usually results in decreased sperm viability; an optimized protocol for cold storage of epididymides plus sperm cryopreservation has yet to be established. Here, we examined the motility and fertilization potential of cryopreserved, thawed (frozen-thawed) sperm from previously cold-stored mouse epididymides. We also examined the protective effect of sphingosine-1-phosphate (S1P) on sperm viability when S1P was added to the preservation medium during cold storage. We assessed viability of frozen-thawed sperm from mouse epididymides that had been cold-transported domestically or internationally and investigated whether embryos fertilized in vitro with these sperm developed normally when implanted in pseudo-pregnant mice. Our results indicate that frozen-thawed sperm from epididymides cold-stored for up to 48 h maintained high fertilization potential. Fertilization potential was reduced after cold storage for 72 h, but not if S1P was included in the cold storage medium. Live pups were born normally to recipients after in vitro fertilization using frozen-thawed sperm from cold-transported epididymides. In summary, we demonstrate an improved protocol for cold-storage of epididymides that can facilitate transport of genetically engineered-mice and preserve sperm viability after cryopreservation.

High osmolality vitrification: a new method for the simple and temperature-permissive cryopreservation of mouse embryos.

Procedures for cryopreserving embryos vary considerably, each having its specific advantages and disadvantages in terms of technical feasibility, embryo survival yield, temperature permissibility and species- or strain-dependent applicability. Here we report a high osmolality vitrification (HOV) method that is advantageous in these respects. Cryopreservation by vitrification is generally very simple, but, unlike slow freezing, embryos should be kept at a supercooling temperature (below -130°C) to avoid cryodamage. We overcame this problem by using an HOV solution containing 42.5% (v/v) ethylene glycol, 17.3% (w/v) Ficoll and 1.0 M sucrose. This solution is more viscous than other cryopreservation solutions, but easy handling of embryos was assured by employing a less viscous equilibration solution before vitrification. Most (>80%) embryos cryopreserved in this solution survived at -80°C for at least 30 days. Normal mice were recovered even after intercontinental transportation in a conventional dry-ice package for 2-3 days, indicating that special containers such as dry shippers with liquid nitrogen vapor are unnecessary. The HOV solution could also be employed for long-term storage in liquid nitrogen, as with other conventional cryoprotectants. Finally, we confirmed that this new vitrification method could be applied successfully to embryos of all six strains of mice we have tested so far. Thus, our HOV method provides an efficient and reliable strategy for the routine cryopreservation of mouse embryos in animal facilities and biomedical laboratories, and for easy and cheap transportation.

Safety, efficacy and efficiency of laser-assisted IVF in subfertile mutant mouse strains.

In the present report we studied the safety, efficacy and efficiency of using an infrared laser to facilitate IVF by assessing fertilization, development and birth rates after laser-zona drilling (LZD) in 30 subfertile genetically modified (GM) mouse lines. We determined that LZD increased the fertilization rate four to ten times that of regular IVF, thus facilitating the derivation of 26 of 30 (86.7%) GM mouse lines. Cryopreserved two-cell stage embryos derived by LZD-assisted IVF were recovered and developed to blastocysts in vitro at the same rate as frozen-thawed embryos derived by regular IVF. Surprisingly after surgical transfer to pseudopregnant recipients the birth rate of embryos derived by LZD-assisted IVF was significantly lower than that of embryos derived by regular IVF. However this result could be completely mitigated by the addition of 0.25 M sucrose to the culture medium during LZD which caused the oocyte to shrink in volume relative to the perivitelline space. By increasing the distance from the laser target site on the zona pellucida, we hypothesize that the hyperosmotic effect of sucrose reduced the potential for laser-induced cytotoxic thermal damage to the underlying oocytes. With appropriate preparation and cautious application, our results indicate that LZD-assisted IVF is a safe, efficacious and efficient assisted reproductive technology for deriving mutant mouse lines with male factor infertility and subfertility caused by sperm-zona penetration defects.