Abstract. Cryopreservation of mouse sperm is useful for maintaining various strains. However, fertility generally decreases after freezing. In particular, the fertility of cryopreserved C57BL/6J sperm is very low. To improve the fertility of frozen sperm, we examined the efficiencies of various media used for sperm preincubation (SP) and in vitro fertilization (IVF) in frozen C57BL/6J sperm. In this study, SP medium was examined for efficiency of fertility with respect to content, especially calcium (Ca 2+ ), phosphate (PO4 3-) and lactate. In all media containing no Ca 2+ , including medium lacking Ca 2+ , lacking Ca 2+ and PO4 3-, lacking Ca 2+ and lactate and lacking Ca 2+ , PO4 3-and lactate, high IVF rates were obtained (79, 69, 76 and 71%, respectively). On the other hand, the rates for media containing Ca 2+ were significantly lower (30-38%, P<0.05). After transfer, 41-50% of newborns were obtained in all media containing no Ca 2+ .In conclusion, preincubation of thawed sperm in medium containing no Ca 2+ markedly improved the fertility of cryopreserved C57BL/6J sperm. These results indicate that the present method of IVF using medium with no Ca 2+ is practical for use in cryopreserved C57BL/6J sperm. Key words: C57BL/6, Calcium, Cryopreservation, In vitro fertilization, Sperm (J. Reprod. Dev. 55: [386][387][388][389][390][391][392] 2009) ryopreservation of mouse sperm is useful because it is simple, rapid and inexpensive. A large number of sperm can be frozen immediately after collection from a male. Females are required only when the frozen sperm are thawed for fertilization. Successful cryopreservation of mouse sperm was reported in 1990 using raffinose with glycerol [1], ME2SO [2] or skim milk [3]. Since then, cryoprotectant solutions and freezing methods for mouse sperm have been investigated [4][5][6][7][8][9][10][11]. At present, the 18% raffinose plus 3% skim milk method [12] is relied upon in most laboratories. However, with this technique, the fertility of cryopreserved sperm is far less than that of fresh sperm, especially with inbred mice [13][14][15][16]. In particular, fertility decreases markedly in C57BL/6J, a principal inbred strain used for transgenesis studies. Frozen C57BL/6J sperm exhibit acrosome damage with loss of contents caused by the freeze-thaw procedure [17], and almost no sperm can penetrate the zona pellucida. When partial zona pellucida nicking [18,19] or intracytoplasmic sperm injection (ICSI) [20,21] is performed to increase fertility, post-thawed C57BL/6J sperm can fertilize a high percentage of eggs. Improved IVF rates with intact oocytes have rarely been reported using motile sperm separated from thawed suspension [16,22] or methyl-beta-cyclodextrin (MBCD) stimulating cholesterol efflux from thawed sperm [23]. Successful IVF with fresh sperm depends on the media used. Various IVF media have been developed, and the effects of media on capacitation and IVF have been investigated. However, the effects of media on frozen mouse sperm have not been investigated.In this s...

Aim: Cryopreservation of mouse sperm commonly uses raffinose, which is a trisaccharide, plus 3% skim milk. Because of the present lack of knowledge of the effectiveness of any other saccharides, we examined the cryoprotective effects of various saccharides on the viability of mouse sperm from various strains to determine which saccharides are the best cryoprotectants for mouse sperm.Methods: Sperm from the caudae epididymides of mature C57BL/6J mice were frozen with monosaccharides (fructose, glucose, rhamnose, xylose), disaccharides (lactose, maltose, sucrose, trehalose) or trisaccharides (melezitose, raffinose) in a range of concentrations (4-33%). After thawing, the optimal concentration was determined to be the concentration in which there was the highest proportion of motile sperm. In addition, sperm of inbred and hybrid mice were frozen with the saccharides at the optimal concentrations and used for in vitro fertilization.Results: The optimal concentration was 12% for the disaccharides and 18% for the trisaccharides. The fertility of all strains, except C57BL/6J, showed the best cryoprotective effects with maltose, melezitose and raffinose when compared with fresh sperm. Conclusion:Maltose, melezitose and raffinose have the best effects when used as a protectant for cryopreservation of mouse sperm. (Reprod Med Biol 2007; 6: 229-233)