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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.


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Comparison of IVF rates of Rescue IVF in MBCD using sperm centrifuged in the presence (RVF+MTG) or absence of MTG (RVF only). n=3, P<0.05.
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Figure 6: Comparison of IVF rates of Rescue IVF in MBCD using sperm centrifuged in the presence (RVF+MTG) or absence of MTG (RVF only). n=3, P<0.05.

Mentions: Centrifugation is a step of the Rescue MBCD-GSH IVF and Rescue IVF in MBCD methods necessary for the removal of cryoprotectants and adjustment of sperm concentration. However, mouse sperm are sensitive to mechanical stress induced by centrifugal force, and during centrifugation sperm can undergo oxidative stress. To determine if the addition of MTG to a frozen-thawed sperm suspension protects sperm fertility during centrifugation, the fertilization rates of Rescue IVF in MBCD procedures were compared after sperm were centrifuged at 300×g for 3, 4, or 5 min in 1.2 mL RVF medium in the presence or absence of 239 µM MTG. To readily determine the effect of MTG, a low sperm concentration (final 1 × 105 cells/mL) with low post-thaw progressive motility (3%) taken from the same pool of cryopreserved C57BL/6N sperm (mixture of sperm from 3 males) was used in each of the 3 paired IVF experiments. The IVF rates obtained in the presence of MTG for all 3 centrifugation times (3, 4, and 5 min) were higher than that for sperm centrifuged for the same times in the absence of MTG. Data analysis shows that this difference is significant (P<0.05, Figure 6), which indicates that MTG can protect sperm fertilization ability during post-thaw centrifugation.


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)

Comparison of IVF rates of Rescue IVF in MBCD using sperm centrifuged in the presence (RVF+MTG) or absence of MTG (RVF only). n=3, P<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Comparison of IVF rates of Rescue IVF in MBCD using sperm centrifuged in the presence (RVF+MTG) or absence of MTG (RVF only). n=3, P<0.05.
Mentions: Centrifugation is a step of the Rescue MBCD-GSH IVF and Rescue IVF in MBCD methods necessary for the removal of cryoprotectants and adjustment of sperm concentration. However, mouse sperm are sensitive to mechanical stress induced by centrifugal force, and during centrifugation sperm can undergo oxidative stress. To determine if the addition of MTG to a frozen-thawed sperm suspension protects sperm fertility during centrifugation, the fertilization rates of Rescue IVF in MBCD procedures were compared after sperm were centrifuged at 300×g for 3, 4, or 5 min in 1.2 mL RVF medium in the presence or absence of 239 µM MTG. To readily determine the effect of MTG, a low sperm concentration (final 1 × 105 cells/mL) with low post-thaw progressive motility (3%) taken from the same pool of cryopreserved C57BL/6N sperm (mixture of sperm from 3 males) was used in each of the 3 paired IVF experiments. The IVF rates obtained in the presence of MTG for all 3 centrifugation times (3, 4, and 5 min) were higher than that for sperm centrifuged for the same times in the absence of MTG. Data analysis shows that this difference is significant (P<0.05, Figure 6), which indicates that MTG can protect sperm fertilization ability during post-thaw centrifugation.

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.


Related in: MedlinePlus