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Cryorecovery of Mouse Sperm by Different IVF Methods Using MBCD and GSH.
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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.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.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.
Affiliation: Mouse Biology Program, University of California, Davis, CA 95618, United States.
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, R(2) 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. No MeSH data available. |
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Figure 5: Comparison of IVF rates of Rescue MBCD-GSH IVF and Rescue IVF in MBCD using sperm samples with high concentration and low motility (n=4, P<0.05). Mentions: We also compared the fertilization rates of the Rescue MBCD-GSH IVF and Rescue IVF in MBCD methods using sperm samples from 4 knockout mouse lines with high sperm concentration but low post-thaw motility (total motility 9–25% and progressive motility 5–9%) to determine if immotile sperm affect sperm capacitation and fertilization. The final sperm concentration was 5–8 × 106 cells/mL in the 100-µL MBCD drop (sperm capacitation drop) for the Rescue MBCD-GSH IVF method and 2.5–4 × 106 cells/mL in the 200-µL MBCD drop (for both sperm capacitation and fertilization) for the Rescue IVF in MBCD method. The results summarized in Figure 5 show that the IVF rate of the Rescue MBCD-GSH IVF, in which sperm were pre-incubated in the MBCD medium drop and only motile sperm with minimal immotile sperm were used for insemination in the fertilization drop (GSH medium drop), was significantly higher than that of the Rescue IVF in MBCD, in which all sperm (dead, immotile alive, and motile) were present in the fertilization drop (MBCD medium drop). These results indicate that the presence of a high number of immotile sperm significantly inhibits the fertilization after capacitation in MBCD medium, but does not significantly affect sperm capacitation in MBCD medium. |
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Affiliation: Mouse Biology Program, University of California, Davis, CA 95618, United States.
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, R(2) 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.
No MeSH data available.
