![]() We have combined the best of our knowledge and experience in rat reproductive technology. In the current study, we addressed the problem of poor reproducibility of rat sperm cryopreservation and in vitro fertilisation. However, since then, there have been no papers showing that frozen sperm can retain sufficient sperm motility to produce pups via in vitro fertilisation and embryo transfer. Their data showed that when isobutylmethylxanthine (IBMX)-treated frozen/thawed sperm were used for in vitro fertilisation, the rates of pronuclear formation and blastocyst formation were significantly higher (pronuclear formation: 50%, blastocyst formation: 20%, birth rate: 49%) than what they achieved when frozen/thawed sperm were used without IBMX treatment. It was 2009 before the first account of successful in vitro fertilisation using frozen Wistar rat sperm, which was reported by Seita et al. in 1974 14, there were no successful cases of in vitro fertilisation using frozen sperm for many years after that. The current study is the first report to show that transgenic rat spermatozoa can be successfully cryopreserved and that the oocytes fertilised by cryopreserved spermatozoa can develop into normal offspring after in vitro fertilisation and transfer of the fertilised oocyte.Īlthough the success of in vitro fertilisation using fresh sperm was demonstrated by Toyoda et al. In this paper, we restrict ourselves to a description of the detailed procedure routinely used for freezing rat sperm. Recently, we established a rat sperm cryopreservation method that results in high fertilisation rates via in vitro fertilisation using frozen/thawed sperm. Meanwhile, satisfactory and acceptable protocols for rat sperm cryopreservation and subsequent IVF have not yet been established. However, only one of those papers has reported the successful production of pups derived from embryos that were obtained via in vitro fertilisation using frozen rat sperm 5. Since the publication in 2001 of the first successful report concerning the cryopreservation of rat sperm 2, many papers on the topic have been published 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13. Thus, sperm freezing is a much simpler, more efficient and more economical alternative to embryo freezing for preserving genetically modified rat strains. On the other hand, a large number of spermatozoa can be cryopreserved immediately after being collected from the epididymides of males. Moreover, if only a few genetically modified males are available to be used for mating, it would take from 2 to 4 months to obtain sufficient embryos for cryopreservation because the number of females used for mating at any one time is limited and the males have to mate with females multiple times. Using the conventional embryo freezing method, 400–500 embryos per strain are required from the oviducts of 30–50 mated females. In general, embryo freezing is a reliable method for preserving rat strains. Frozen embryos and sperm from many rat strains have been preserved in rat resource centres worldwide, including the National BioResource Project - Rat, Kyoto University, Japan the Rat Resource & Research Center, University of Missouri, USA and the Gene Editing Rat Resource Center, Medical College of Wisconsin, USA. ![]() Consequently, the cryobanking of gametes and embryos from these strains is rapidly becoming an important issue. Over the past ten years, genome-editing techniques have been used to produce many genetically modified rat strains 1.
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