Time elapsed between ovulation and insemination determines the quality of fertilized rat oocytes.

Genetically modified rats are valuable models in human disease research. We recently developed an improved system for rat sperm cryopreservation and in vitro fertilization (IVF) that facilitates the efficient production and preservation of genetically modified rats. In the IVF procedure performed using frozen-thawed rat sperm, the IVF schedule is fixed to ensure timely hormone administration and oocyte collection. To enhance the flexibility of the IVF schedule, possible periods of postovulated rat oocytes with normal fertility and developmental abilities should be determined. Therefore, in this study, we examined the fertilization and developmental ability of incubated oocytes 1-13 h after oocyte collection at 9:00 AM. The fertilization rate decreased 7 h after oocyte collection, and abnormally fertilized oocytes appeared 10 h after oocyte collection. The developmental rate also decreased 7 h after oocyte collection; however, live pups were obtained from oocytes 12 h after oocyte collection. In summary, ovulated rat oocytes exhibited a high developmental ability after IVF for up to 4 h after oocyte collection.

High-concentration bovine serum albumin enhances fertilization ability of cold-stored rat sperm.

Cold transport of the cauda epididymides is a useful technique for shipping laboratory rat sperm. Cold transport of rat sperm avoids potential risks of microbiological infection, animal escape or death, and animal welfare issues. Previously, we reported that a cold-storage solution containing dimethyl sulfoxide and quercetin maintained the fertility of cold-stored rat sperm. However, cold-stored rat sperm exhibited a decreased fertilization rate after 24-h storage. To recover the fertility of cold-stored sperm, we focused on the effects of bovine serum albumin (BSA), a cholesterol acceptor that induces sperm capacitation. We sought to determine the optimal concentration of BSA in fertilization medium based on the fertility of cold-stored rat sperm. High concentrations of BSA (40 mg/ml) enhanced the fertilization rate of cold-stored rat sperm and maintained sperm fertility for 144 h. Embryos derived from cold-stored and BSA-treated sperm normally developed into pups after embryo transfer. In summary, high BSA concentrations enhanced the fertility of cold-stored rat sperm and prolonged the storage period to 144 h, thereby expanding the transportable region for genetically engineered rats.

In vivo fertilization improved the cryotolerance and developmental ability of vitrified-warmed rat fertilized oocytes.

The cryopreservation of rat embryos is useful for efficiently archiving rat resources in bioresource repositories. The cryopreserved fertilized oocytes can be quickly reanimated to rats with homozygous mutations using embryo transfer. In addition, cryopreserved rat fertilized oocytes are easier to transport than live animals. Before cryopreservation, fertilized oocytes are obtained by mating or in vitro fertilization. However, it is not clear which fertilized oocytes are most suited to cryopreservation. In this study, we performed a systematic comparison of the fertilizing ability, cryotolerance, and developmental ability of cryopreserved fertilized oocytes at the pronuclear stage produced either by mating (in vivo) or in vitro fertilization (in vitro) in SD and F344 rats. In vivo-fertilized oocytes had higher cryotolerance and developmental ability than in vitro-fertilized oocytes in SD and F344 rats. Furthermore, the fertilization ability, cryotolerance, and developmental ability of vitrified-warmed fertilized oocytes differed between SD and F344 rats. In conclusion, our study suggests that in vivo-fertilized rat oocytes were more suitable for cryopreservation. Our protocol provides an optimized system for the management of rat colonies using fertilized oocytes cryopreservation and contributes to the 3Rs principle by reducing the number of animals used for research.

Observation of the in vitro fertilization process in living oocytes using frozen-thawed sperm in rats.

Previous studies have observed the fertilization process in rats using whole-mount preparation at different time-points after insemination. However, very few reports have described the various events during the fertilization process using an inverted microscope without whole-mount. Moreover, to the best of our knowledge, no reports have described the observation of changes in sperm motility associated with sperm penetration into oocytes. In this study, in vitro fertilization was performed using frozen-thawed sperm in various rat strains (SD, Wistar, LE, F344, and BN) and oocytes from the SD strain, and the process of sperm penetration into the oocytes and the subsequent development were observed. The sperm motility was assessed, and the correlation between the process of sperm penetration into the oocytes and sperm motility over time was examined. The motility of frozen sperm from the SD, Wistar, LE, and F344 increased at 2-3 h after thawing, at which time the sperm attached themselves to the zona pellucida. Sperm penetration into the zona pellucida occurred after 3-5 h, and pronuclei were formed in the cytoplasm of oocytes 5-9 h after insemination. The fertilities of frozen-thawed sperm from the SD, Wistar, LE, and F344 were 92.7%, 90.0%, 90.7%, and 68.7%, respectively. However, no increase in motility was observed after thawing of frozen sperm from the BN, and the fertility was only 21%. In addition, very few polyspermic oocytes were observed with use of frozen-thawed sperm of all strains. In summary, rats are suitable animals for the observation of sperm penetration into the oocytes, and we determined the timing of fertilization events in IVF using frozen-thawed rat sperm.

Optimized protocols for sperm cryopreservation and in vitro fertilization in the rat.

Laboratory rats have been used in biomedical research for over 170 years. Recently, genome editing technology has facilitated the generation of genetically modified rats worldwide. This development has increased the demand for efficient preservation and production of rat resources. Sperm cryopreservation is the most efficient and robust means to archive genetic resources, and this technique reduces the number of animals required for colony management. Previously, we have reported a protocol for rat sperm cryopreservation and in vitro fertilization using frozen-thawed sperm. Here we describe an improved in vitro fertilization protocol to enhance the fertilization rate of cryopreserved sperm in major strains of rats. In this optimized protocol, treatment of frozen-thawed rat sperm with a high concentration of bovine serum albumin (40 mg/ml) results in a high in vitro fertilization rate. This protocol consists of three main steps: preparation of cryopreserved sperm, in vitro fertilization using cryopreserved sperm and embryo transfer. This process takes approximately 1 month to produce live pups from cryopreserved sperm. This protocol can be easily implemented by researchers and technicians with experience in reproductive engineering technology; it can also be used, albeit with some practice, by researchers and technicians who have no experience in reproductive techniques. This sperm cryopreservation and in vitro fertilization protocol for rats will provide an efficient system for the archiving and production of genetically modified rats for the transgenic community.

Quercetin-treated rat sperm enables refrigerated transport with motility and fertility for five days.

Shipment of laboratory rats between animal facilities is frequently performed using special containers. However, the shipment of live animals is associated with potential risks of infectious diseases, escape and death during shipment and animal welfare issues. The transport of cold-stored sperm avoids such risks; however, there have been no reports on cold storage of rat sperm. We previously reported that dimethyl sulfoxide (DMSO) and quercetin maintained the motility and fertilising abilities of cold-stored mouse sperm stored for 10 days. The present study investigated the efficacy of DMSO and quercetin in the cold storage of rat sperm. Quercetin maintained motility and fertility of cold-stored rat sperm stored for 5 days. After in vitro fertilisation using cold-stored sperm, pronuclear and two-cell embryos developed normally to pups following embryo transfer. Therefore, we demonstrated that live pups could be obtained from sperm transported using the cold-storage system. We conclude that cold storage of rat sperm may provide an efficient system for transporting rat resources as an alternative to shipping live animals.