In 1963, Yanagimachi and Chang first succeeded in fertilizing golden hamster eggs in vitro without any contributions from the female genital tract [1]. This is the first success of 'In Vitro Fertilization (IVF)' in mammals. After this success, the protocol of IVF has been established in most of mammalian species studied up to date. At last, the Nobel Prize in Physiology or Medicine 2010 was awarded to Dr. Robert G. Edwards who reported the first success of IVF in human with Dr. Patrick C. Steptoe [2]. Also in experimental animals, IVF is a powerful tool for production of offspring. Alternatively, intracytoplasmic sperm injection (ICSI) is now available for the purpose; however, ICSI requires a great deal of skill and cannot be used for many oocytes at the same time. Thus, especially in the production of rodent offspring at the facility, IVF is a more simple and general-purpose method. Moreover using frozen-thawed spermatozoa for IVF can decrease the number of males to be sacrificed for collection of epididymal spermatozoa. However, it has been reported that frozen-thawed spermatozoa from several strains even in mice contribute to low fertility after IVF. Since fresh sperm can fertilize to the oocytes, the process during freezing and thawing seems to affect serious damage of the sperm. Recently, some recent reports have demonstrated significant improvement of IVF using cryopreserved sperm.

C57BL/6 is a common inbred strain of laboratory mice. C57BL/6 is one of the most widely used for genetically modified (transgenic, knock-in and knock-out) mice for use including human disease models. Even though motility of the sperm after freezing and thawing was quite similar to that of fresh sperm, the sperm is fertilized to oocytes with low rate. Takeo et al. [3] demonstrated that methyl-beta-cyclodextrin (MBCD) induced efflux of cholesterol in the sperm plasma membrane, resulted in the improvement of freezing ability of C57BL/6 mouse sperm. They also reported that combined treatment of MBCD and L-glutamine (in sperm freezing medium) [4] or reduced glutathione (in IVF medium) [5]. Positive effects of reduced glutathione on fertility after IVF were shown in not only C57BL/6 but also 129S1, FVB and C3H frozen-thawed sperm [6]. Alternatively, it has been reported that only increase of raffinose concentration can improve fertility of frozen-thawed sperm derived from C57BL6 after IVF [7]. On the other hand, it has been demonstrated that depletion of calcium in medium dramatically reduces damaged acrosomal membrane of frozen-thawed C57BL/6 sperm, improves fertility of the sperm [8]. These results suggest that the damage of the sperm during freezing and thawing can be rescued by some chemicals directly or indirectly. In rats, it is known that low fertility of frozen-thawed sperm after IVF is more serious problem. Since no one has succeeded in IVF using cryopreserved sperm even in closed colony until very recently, artificial insemination is used as a general-purpose method to produce gene modified rats using cryopreserved sperm [9]. Treatment with a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) improved fertility after IVF using frozen-thawed sperm [10]. In the study, IBMX directly improved induction of capacitation of frozen-thawed rat sperm but not MBCD. This result suggests that process of cryopreservation seems to give more serious damage to rat sperm than mouse sperm. Application of rat IVF protocol to inbred strain is in progress. Such chemicals as reduced glutathione and higher concentration of raffinose may be also effective on IVF in rats.