We previously reported that high concentrations (≥3.42 mM) of calcium during in vitro fertilization (IVF) disturbed the extrusion of the second polar body (PBII) in C3H/He inbred mice. In this study, the substrain specificity of this phenomenon was examined under 1.71–6.84 mM calcium concentration in ova from six C3H/He mouse commercially available substrains in Japan. PBII extrusion in ova from J substrains was not affected by calcium concentrations (<10% at any calcium level), but was grossly disturbed at high calcium levels in the ova of other substrains. This result has practical applications for the efficient production of normal zygotes by IVF, therefore contributing to the reduction in the numbers of donor animals for further zygote or embryo manipulation. Care must be taken in choosing IVF medium for particular strains and substrains.

Successful in vitro fertilization (IVF) of all inbred strains of laboratory mice has not yet been accomplished. We have previously shown that a high calcium concentration improved IVF in various inbred mice. However, we also found that in cumulus-free ova of C3H/He mice such IVF conditions significantly increased the deficiency of extrusion of the second polar body (PBII) in a dose-dependent manner (2% at 1.71 mM and 29% at 6.84 mM, P < 0.05) and that PBII extrusion was affected by high calcium levels at 2–3 h post-insemination. While developmental competence of ova without PBII extrusion to blastocysts after 96 h culture was not affected, a significant reduction in the nuclear number of the inner cell mass was observed in blastocyst fertilized under high calcium condition. We also examined how high calcium concentration during IVF affects PBII extrusion in C3H/He mice. Cumulus cells cultured under high calcium conditions showed a significantly alleviated deficient PBII extrusion. This phenomenon is likely to be specific to C3H/He ova because deficient PBII extrusion in reciprocal fertilization between C3H and BDF1 gametes was observed only in C3H/He ova. Sperm factor(s) was still involved in deficient PBII extrusion due to high calcium concentrations, as this phenomenon was not observed in ova activated by ethanol. The cytoskeletal organization of ova without PBII extrusion showed disturbed spindle rotation, incomplete formation of contractile ring and disturbed localization of actin, suggesting that high calcium levels affect the anchoring machinery of the meiotic spindle. These results indicate that in C3H/He mice high calcium levels induce abnormal fertilization, i.e. deficient PBII extrusion by affecting the cytoskeletal organization, resulting in disturbed cytokinesis during the second meiotic division. Thus, use of high calcium media for IVF should be avoided for this strain.

Mature porcine oocytes containing first polar bodies (Pb I) were obtained by in vitro culture of follicle oocytes from ovaries obtained from a local abattoir, and zygotes with second polar bodies (Pb II) were grown after in vitro fertilization of the mature oocytes. Extrusion, biological activity and morphology of Pb I and Pb II were statistically analysed. Polar bodies were isolated and collected from oocytes by enzyme digestion or micromanipulation. Their vigour under different preservation conditions was analysed and evaluated using a Trypan blue staining method. The results showed that 66.7% of the oocytes extruded Pb I after 40 h of in vitro mature culture of oocytes, and 49.7% of the zygotes extruded Pb II 20 h after in vitro fertilization. The efficiency of isolation of Pb II by micromanipulation significantly exceeded that by enzyme digestion, the Pb I and Pb II isolated by micromanipulation presenting with good vigour and normal morphology (95.3% versus 58.9%). The survival rates of Pb I and Pb II were 63.3% and 93.1% for 4 h at 39°C, 85.0% and 72.9% for 40 h at 4°C, and over 95.0% and 84.6% for less than 7 days at −20°C. In comparison with the above preservation conditions for Pb I and Pb II, the results for cryopreservation were best, with rates of survival as high as 89.1% for Pb I and 87.9% for Pb II for preservation periods of over a month, and rates of normal morphology of 97.8% and 95.7%, respectively. The Pb I and Pb II could be isolated and preserved effectively, for use in further research on the recombination of oocytes and zygotes.