Abstract (Applied Research) The generation of novel genetically modified rat models has greatly accelerated with the availability of rat embryonic stem cells and the advent of nuclease-based genome-editing technologies such as ZFNs, TALENs, and CRISPR/Cas9. There is a need for reliable and cost-effective methods of cryopreservation to archive these models and ensure their future availability. Sperm cryopreservation, which is straightforward and inexpensive to perform, is ideal for preserving genetically modified rat models; yet, the limited methods for successful cryorecovery using frozen rat sperm employ methods, such as intracytoplasmic sperm injection (ICSI) that are beyond the means of most research laboratories or transgenic cores and are challenging even for experienced facilities like the RRRC. The ability to perform in vitro fertilization (IVF), which is a common and highly successful method of cryorecovery in the mouse, would be ideal for cryorecovery in the rat. Unfortunately, the limited published protocols for rat IVF have not been reliably repeatable by others, including the RRRC. Based on preliminary data suggesting amino acid supplementation has a beneficial impact on sperm motility, we propose to test the addition of various amino acid combinations to media to measure the outcome of this supplementation on sperm motility, capacitation and in vitro fertilization. The ability of amino acid supplementation to improve post-thaw sperm motility and increase in vitro fertilization success rates when frozen/thawed sperm is used will also be evaluated. These studies will be performed with spermatozoa from both outbred stocks and inbred strains. The data generated will guide optimization of rat sperm cryopreservation and rat IVF, thus enhancing the RRRC’s ability to archive and reanimate rat models in a cost-effective and efficient manner.