Fig 1.
Comparison of the Ostermeier et al., CARD Set and SEcuRe protocols.
CPA = cryoprotective agent; MTG = monothioglycerol; L-Glu = L-glutamine; RVF medium = Research Vitro Fert fertilization medium; mRVF medium = modified RVF fertilization medium with elevated calcium concentration (5.14 mM) and GSH supplementation.
Fig 2.
Scheme of the SEcuRe protocol.
(A) Sperm cryopreservation, (B) in vitro fertilization using frozen-thawed sperm. gCPA = cryoprotective agent supplemented with L-glutamine; RVF medium = Research Vitro Fert fertilization medium; mRVF = modified RVF fertilization medium with elevated calcium concentration (5.14 mM) and GSH supplementation.
Fig 3.
Validation of the SEcuRe protocol.
(A) Comparison of the median fertilization rates obtained in the IVF procedures applying the SEcuRe, CARD Set and Ostermeier et al. protocols. Frozen-thawed sperm of C57BL/6 mutant males and oocytes of wild-type C57BL/6N or C57BL/6J females were used for the IVF procedures. Thick lines in the violin plots indicate median fertilization rates and dotted lines the first and the third quartile. Points represent individual experiments (IVF procedures). **** p < 0.0001; * p = 0.031; ns = non-significant (p > 0.99) (Kruskal–Wallis test). (B) Comparison of the average fertilization rates and blastocyst formation rates in the IVF procedures where either FERTIUP® PM or c-TYH was used as a sperm preincubation medium. The data was collected from three independent experiments. Frozen-thawed sperm of wild-type C57BL/6N males and oocytes of wild-type C57BL/6N females were used in these experiments. Data are shown as means ± standard deviation. The blastocyst formation rate shows the percentage of 2-cell embryos obtained in the IVF procedures that reached the blastocyst stage after 72 hours of in vitro culture. N indicates the total number of fertilized oocytes (fertilization rate) or 2-cell stage embryos (blastocyst formation rate). ns = non-significant (p ≥ 0.41) (Student’s t-test).
Table 1.
In vivo development of 2-cell stage embryos produced with the SEcuRe, CARD Set and Ostermeier et al. protocols.