Introduction and considerations
Whether you have frozen sperm in the NTS biobank or want to import sperm from another research institution, NTS may assist in the process of obtaining living animals.
Import of genetically modified animals from another facility or vendor poses a risk to the whole facility. Import of frozen sperm followed by in vitro fertilization and implantation into pseudo pregnant foster mothers, will often be a better alternative than import of living genetically modified mice for temporal housing in the quarantine unit, prior to rederivation. Frozen sperm do not pose the same risk to the facility as living animals, and a health report will not be required for imports of sperm.
In cases where the in vitro fertilization procedure is performed with sperm received from other institutes, the service must include pathogen testing of the foster mother and litter. Animals will be delivered with a health certificate tested for the absence of any pathogens listed by FELASA.
NTS also offers recovery from living animals (rederivation) or embryos (embryo recovery).
Benefits
- Recovery completed within 9-12 weeks.
- Mice produced are SOPF and will come with the appropriate health report.
Provided by the customer
The paperwork involved for import of embryos or sperm are different for import of living animals. The customer will be responsible for the administration as well as all costs for transport of embryos. NTS may assist in the process, but will then charge the customer an extra administrative fee.
Requirements and time frame
- Satisfactory health report for the sperm is not required.
- Available for most strains. Cost will be higher for strains known to be difficult to superovulate, fertilize and/or implant.
- NTS requires at least three straws of sperm.
- Female mice will be superovulated and unfertilized oocyttes will be harvested.
- Sperm will be thawed and used to fertilize oocyttes (IVF).
- Resulting embryos will be collected and implanted into pseudo pregnant foster mothers. Offspring will be separated at 3 weeks of age.
- Biopsies will be taken and provided to customer. NTS expect confirmation of genotyping within a reasonable time (1 week).
- Foster mother and offspring will be tested against the presence of pathogens according to FELASA standards. Contact NTS for a complete list of screened pathogens.
- The project is typically completed after 9-12 weeks.
Technical service performed by NTS
- NTS is responsible for the animal procedure permit (FOTS application).
- Planning and coordination of the project.
- Superovulation of donor females, dissection of oviducts and collection of unfertilized oocytes.
- Thawing of sperm and fertilization of oocytes (IVF).
- Implantation of embryos into pseudo pregnant foster mothers, separation of offspring and collection of biopsies.
- Customer will receive biopsies in house. If personal pick-up is not an option, transport will be charged customer.
- Sample collection and coordination of pathogen screening.
Guarantee
- Under certain conditions, such as when fewer than three offspring are born, NTS will repeat the procedure once for free.
Service Fees
Estimated costs for the above service can be found on our Service Fees page.
Extended description of the in vitro fertilization procedure (IVF)
Planning and considerations
The standard arrangement uses embryo donors from a commercial supplier. Please contact NTS to discuss details if this appears not to be the preferred option for you. Alternative arrangements must be planned in close collaboration with NTS staff to assure success. In vitro fertilization can be performed with either hetero-, hemi- and homozygote transgenic sperm. NTS will order wild-type females from a commercial vendor, superovulate these and collect oocyttes the next day.
Collection of unfertilized oocyttes and in vitro fertilization
The procedure is roughly as follows. Young female mice (donors) are treated with hormones to induce superovulation. Following two rounds of hormone administration (PMS followed by HCG), females will release a large number of unfertilized oocyttes that can be collected the day following the last hormone administration. The reproductive tracts from the mice are aseptically isolated in the morning of day 0.5 dpc. Freshly collected or frozen sperm are mixed with the oocyttes to produce embryos (IVF). Obtained embryos will be cultured until the next day and implanted at the 2-cell stage.
Embryo implantation and pathogen testing
Depending on the number of embryos collected, embryos will be introduced into the oviduct of one/several pseudopregnant foster mothers. Embryos will develop to term (~20 days). Offspring will be separated at three weeks of age and biopsies taken.
Whenever health monitoring is required for transport of the animlas, samples for pathogen testing will be taken from both foster mothers and offspring and sent to a commercial health monitor provider. Animals will not be delivered to the investigator before the health monitoring is completed and animals proven to be without pathogens.
The embryo implantation procedure is occasionally unsuccessful. If three or fewer offspring are recovered, NTS will repeat the procedure without additional cost (only applies if sperm quality was acceptable).
Service provided by NTS included in the cost estimate
- Approval for the procedure (FOTS application).
- Ordering of donor females (cost is included in the cost estimate).
- Housing of vasectomized males and foster mothers.
- Provide pseudopregnant foster mothers (plug mating).
- Purchase of required hormones.
- Superovulation to obtain unfertilized oocytes.
- Dissect out oviducts and isolate unfertilized oocytes.
- Mix sperm and oocytes (IVF).
- Culture embryos overnight and transfer 2-cell stage embryos to pseudo pregnant foster mothers.
- Separate foster mother and offspring.
- Take ear biopsies from offspring.
- Organize transport of animals.
Costs not included
- Detailed pathogen testing of the line.