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

Li MW, Glass OC, Zarrabi J, Baker LN, Lloyd KC - J Fertili In Vitro (2016)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

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.


Effect of high CPA concentration on IVF rates of MBCD-GSH IVF and Rescue MBCD-GSH methods (n=4, P<0.05).
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Figure 4: Effect of high CPA concentration on IVF rates of MBCD-GSH IVF and Rescue MBCD-GSH methods (n=4, P<0.05).

Mentions: To determine if a high concentration of CPA inhibits sperm capacitation and fertilization, MBCD-GSH IVF and Rescue MBCD-GSH IVF methods were compared in pairs using the same amount of cryopreserved sperm from the same males of 4 knockout mouse lines. For each MBCD-GSH IVF procedure, a high volume (20 µL) of thawed sperm in CPA was mixed with 100 µL of MBCD medium (final concentration raffinose 3% and skim milk 0.5%, w/v), and for each Rescue MBCD-GSH IVF, 20 µL of thawed sperm were washed by centrifugation to remove CPA and then resuspended in 120 µL of MBCD medium for capacitation. The results summarized in Figure 4 show that the IVF rate of the Rescue MBCD-GSH IVF was significantly higher (P<0.05) than that of the MBCD-GSH IVF, indicating that the presence of a high concentration of CPA (3% raffinose and 0.5% skim milk) in the MBCD medium significantly inhibits the ability of MBCD to enhance sperm capacitation.


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

Li MW, Glass OC, Zarrabi J, Baker LN, Lloyd KC - J Fertili In Vitro (2016)

Effect of high CPA concentration on IVF rates of MBCD-GSH IVF and Rescue MBCD-GSH methods (n=4, P<0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4940049&req=5

Figure 4: Effect of high CPA concentration on IVF rates of MBCD-GSH IVF and Rescue MBCD-GSH methods (n=4, P<0.05).
Mentions: To determine if a high concentration of CPA inhibits sperm capacitation and fertilization, MBCD-GSH IVF and Rescue MBCD-GSH IVF methods were compared in pairs using the same amount of cryopreserved sperm from the same males of 4 knockout mouse lines. For each MBCD-GSH IVF procedure, a high volume (20 µL) of thawed sperm in CPA was mixed with 100 µL of MBCD medium (final concentration raffinose 3% and skim milk 0.5%, w/v), and for each Rescue MBCD-GSH IVF, 20 µL of thawed sperm were washed by centrifugation to remove CPA and then resuspended in 120 µL of MBCD medium for capacitation. The results summarized in Figure 4 show that the IVF rate of the Rescue MBCD-GSH IVF was significantly higher (P<0.05) than that of the MBCD-GSH IVF, indicating that the presence of a high concentration of CPA (3% raffinose and 0.5% skim milk) in the MBCD medium significantly inhibits the ability of MBCD to enhance sperm capacitation.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

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.