To optimize IVF conditions for BALB/c mice, which are known to have poor in vitro fertilizability, the requirements for sperm–ova interaction were studied by use of modified simplex optimization medium (mKSOM) as a basic medium. Modified human tubal fluid (mHTF) was used for sperm preincubation and acted as a positive control. When the two media were compared, neither capacitation nor fertilization was supported in mKSOM. Increasing the calcium concentration in mKSOM to 5 mM or more during sperm: ova coincubation improved zona penetration but not male pronuclear (MPN) formation to the same level as those cells incubated in mHTF. When medium osmolarity was varied from 230–305 mOsmol by NaCl at 5 mM CaCl2, MPN formation improved at 280 mOsmol or higher osmolarity to the same level as that found when using mHTF. When NaCl equivalent to 25–75 mOsmol was substituted with trehalose, no significant reduction in fertilization was observed. Substitution of NaCl equivalent to 75 mOsmol with other osmotic reagents (sucrose, choline chloride and sorbitol) resulted in similar levels of fertilization as found with mHTF, except for sorbitol, which reduced fertilization significantly caused by its detrimental effect on sperm viability. At isotonic osmolarity (305 mOsmol), maximum fertilization was observed at 5 mM CaCl2; lower or higher concentrations of CaCl2 resulted in reduced fertilization. Calcium and osmolarity, therefore, are important for sperm : ova interaction in BALB/c mice and the increases in calcium to 5 mM and osmolarity to 305 mOsmol are optimal for BALB/c sperm to penetrate through the zona and to form MPN.

Inbred BALB/c mice are one of the most difficult inbred strains to fertilize in vitro. In this study we examined the abilities of various media used for mouse in vitro fertilization (IVF) to support capacitation and sperm penetration through the zona pellucida (ZP) of inbred BALB/c spermatozoa. Media examined were TYH, M16, CZB, mWhitten medium, T6, modified Tyrode's solution (mTyrode's), mKSOM, MEM and TCM199. Modified human tubal fluid (mHTF) was used as a control medium. When sperm were capacitated and inseminated in the same medium, mHTF showed the best fertilization (approximately 80%) scored by male pronuclear formation (<26%) at 5 h post-insemination (PI). When sperm were capacitated in various media and inseminated in mHTF, sperm capacitated in TYH solution (93%) but no other media (<45%) showed a significantly higher level of sperm nuclear decondensation (SND) than mHTF at 2 h PI (approximately 65%). When sperm were capacitated in mHTF and inseminated in various media, only mTyrode's (52%) was not significantly lower than mHTF (66%) in terms of SND at 2 h PI (<49%). Sperm capacitation also was examined by chlortetracycline (CTC) staining. Sperm capacitated in TYH solution showed a significantly higher percentage of capacitation (46%) than those treated in HTF (28%) and other media (<24%). These results indicate that the best approach for IVF in the BALB/c strain is capacitation in TYH and insemination in mHTF. Poor fertilization of BALB/c may result from suboptimal conditions of sperm capacitation and insemination, and overall IVF success may differ depending on strains used.