Decontamination of rat embryos and transfer to specific pathogen-free recipients for the production of a breeding colony

When animals are introduced to a specific pathogen-free (SPF) facility, care must be taken to avoid the possibility of disease transmission to the local colony. This study investigated the application of a combination of reproductive biotechnologies to establish a new disease-free colony of two rat strains, DarkAgouti(Da/Pit) and Wistar Furth(WF/Pit), from a stock known to be chronically infected with the following pathogens: Mycoplasma pulmonis, Kilham's rat virus, sialodacryoadenitis/coronavirus, and reovirus type 3. To eliminate the pathogens and optimize the use of animals, superovulation, embryo washing and trypsinization, and embryo transfer were used. Donors (DA/Pit and WF/Pit) were treated as follows: the mature females were synchronized by subcutaneous (s.c.) injection with 40 micrograms luteinizing hormone-releasing hormone agonist/animal on day 4. All immature and mature females were induced to superovulate by continuous s.c. infusion with a commercial porcine follicle-stimulating hormone (FSH) preparation (3.4 or 6.8 mg NIH-FSH-P1 units per day, respectively), beginning on the morning of day-2. On the afternoon of day 0, the animals received 30 IU human chorionic gonadotropin injected intraperitoneally and mated. From a total of 213 ova flushed from the oviducts of 16 programmed donors, 195 transferrable two-cell embryos were recovered. Two outbred strains of SPF rats, Long-Evans (LE) and Wistar (W), were used as recipients. These mature females (LE and W) were synchronized by using luteinizing hormone-releasing hormone agonist as described and made pseudopregnant by cervical stimulation. Two-cell embryos (DA/Pit and WF/Pit) were washed and trypsinized, then transferred to the oviducts of the pseudopregnant recipients (LE and W). From a total of 195 embryos transferred, 57 pups were born (29.2% of embryos transferred.) All offspring tested negative for the viruses infecting the donors as long as they were kept under strict quarantine. The combination of those three techniques provides an efficient alternative to the traditional derivation by caesarean section.