Cryorecovery of Mouse Sperm by Different IVF Methods Using MBCD and GSH

Abstract

Different protocols incorporating methyl-β-cyclodextrin (MBCD) and reduced glutathione (GSH) have been reported to improve IVF recovery of cryopreserved mouse sperm on a C57BL/6 (J and N) genetic background. However, it is not clear which IVF protocol is most appropriate when using the various methods to cryorecover sperm with different sperm quality and sample volumes. Therefore, in the present study we correlated sperm motility with fertilization rate and compared the efficiency of different IVF methods using various sperm samples so as to establish general guidelines for mouse sperm cryorecovery by IVF. High linear correlation between sperm fertilization rate and progressive motility was found, R2 was 0.9623 and 0.9993 for pre-freezing and post-thaw progressive motility, respectively. High amounts of cryoprotective agent (CPA) were observed to impair both sperm capacitation and fertilization. Moreover, the presence of a large number of immotile sperm in the sperm-oocyte co-incubation drop was found to reduce IVF success which could be partially reversed by supplementation using monothioglycerol (MTG) during centrifugation. It was concluded that the efficiency of IVF using cryorecovered mouse sperm in media containing MBCD and GSH can be predicted from sperm progressive motility. High concentrations of CPA and immotile sperm should be mitigated prior to IVF. The optimum IVF method should be selected based on sperm sample volume and sperm parameters.

The impact of five years storage/biobanking at −80°C on mouse spermatozoa fertility, physiology, and function

“…As previously reported, 12 IVF was performed using the protocol described by Nakagata et al 13 and modified by Li et al 14 Before thawing, the straws stored in liquid nitrogen (control) or a −80°C freezer were transferred into a LN 2 Dewar flask for at least 10 min. That was performed to prove that spermatozoa can be transferred back to LN 2 for permanent storage.…”

DNA fragmentation index (DFI) as a measure of sperm quality and fertility in mice

“…Sperm count was calculated by sperm concentration (millions/mL) × 0.5 mL. Sperm total motility (% of sperm displaying any type of movement) and progressive motility (% of progressively swimming sperm with average path velocity ≥50 μm/s and straightness ratio ≥50%) were analyzed in chambers with depth 80 μm (2X-cel slides, Hamilton Thorne) by reading 8-10 fields (2000-3000 sperm) in two counting chambers at 37 °C as described previously 24,37 .…”

“…For ICSI, oocytes were dissociated from cumulus cells by treatment with 300 U/ml bovine testis hyaluronidase in M2 medium at 37 °C, and then washed and cultured in pre-equilibrated KSOMaa medium at 37 °C in humidified 5% CO 2 in air. www.nature.com/scientificreports www.nature.com/scientificreports/ IVF was performed in 5.5% CO 2 in humidified air at 37 °C using a modification of a previously published MBCD-GSH IVF method 24,37 . Briefly, thawed sperm were mixed and distributed evenly into 3 pre-equilibrated 120-µL long-flat MBCD medium drops (10 µL sperm per drop), and then incubated for 50-60 min before IVF.…”

“…As a result, management of these burgeoning mouse numbers is increasingly reliant upon sperm cryopreservation and recovery by ART such as IVF and ICSI followed by embryo transfer 23 . Examinations used today to assess sperm quality in mice before and after cryopreservation include an evaluation of sperm count, motility and morphology 24 . However, as in humans, conventional sperm parameters cannot reliably be used to predict male reproductive success following ART.…”

Graphene Oxide Improves in vitro Fertilization in Mice With No Impact on Embryo Development and Preserves the Membrane Microdomains Architecture

“…All the chemicals were purchased from Sigma Aldrich (Saint Louis, MO, United States) and were analytical grade. Human tubal fluid (hTF) was purchased from Millipore (Merck, KGaA, Darmstadt, Germany) and modified with the addition of CaCl 2 to increase the Ca 2+ concentration from 2.04 mM (regular concentration) to 5.14 mM (high concentration) (Li and Glass, 2016). For sperm capacitation, TYH medium (containing 1 mg/ml PVA) was prepared in-house (Choi and Toyoda, 1998).…”