Non-surgical Embryo Transfer In Mice Is An Easy, Effective, and Ethical Replacement For Surgery Kendra Steele, Barbara Stone, James Hester, Angelika Fath-Goodin ParaTechs Corporation, Lexington, Kentucky Abstract Surgical embryo transfer (ET) is an effective method to deposit embryos into the uterine horn of mice. However, surgery is expensive, time-consuming, and requires technical expertise. Surgery is also a stressful procedure for the mouse, which has to be anesthetized and treated with an analgesic. We have developed a simple, brief procedure for ET using a non-surgical device, and our hypothesis is that this non-surgical procedure is less stressful for the mouse, as effective as surgical ET, and the procedure can be repeated on the same mouse. In order to compare the effectiveness between the non-surgical and surgical procedures, we performed side-by-side comparisons with 20 mice per method and repeated the experiment using four different strains. Pregnancy rate is higher in mice that have undergone non-surgical ET than mice subjected to surgery, and litter size and birth rate from the two procedures are similar. We then performed the non-surgical method up to two more times on individual CD-1 mice, each time allowing the mouse to recover for at least 20 days post-partum before becoming pseudopregnant again. The data demonstrate that the non-surgical ET procedure can be used multiple times on a mouse, but there is a reduced pregnancy rate. Since non-surgical ET does not require sedation, opening of the inner body cavity, or use of an analgesic, we hypothesized that this procedure is less stressful for the mouse than surgery. We used electrocardiography (n=11) and fecal corticosterone ELISA (n=15) to monitor pseudopregnant mice that underwent anesthesia only, non-surgical ET with and without anesthesia, or surgery. Our results show that the non-surgical procedure without anesthesia does not affect heart rate or alter the levels of the stress biomarker fecal corticosterone, whereas surgery and anesthesia alone lower heart rate for at least one hour after administration and increase levels of fecal corticosterone. Responsible animal scientists are required to follow Russell and Burch’s “3Rs” of animal research: to replace, reduce, and refine. The non-surgical procedure refines the ET method by minimizing animal stress, and can reduce the number of mice needed for experiments, offering an advantageous alternative to surgical transfer. Conclusions. • Surgical and non-surgical NSET procedures results in the same pregnancy rate, birth rate, and litter sizes. • The anesthesia associated with surgery increases physiological stress on mice, while the NSET procedure does not. • Mice can be reused employing the non-surgical NSET procedure, whereas mice cannot if they underwent surgery. Future Directions. • Evaluate the efficiency of surgical versus non- surgical embryo transfer using additional mouse strains. • Evaluate the efficiency of the two procedures using morula and/or 1.5 dpc pseudopregnant mice. • Determine if the NSET device can be used for artificial insemination. Acknowledgements. We thank Mouse Specifics for their generous support regarding the ECGenie instrument and for their blinded analysis of the heart rate data. Research reported in this publication was supported in part by the Office of the Director, NIH, under award numbers 2R44RR025737-02 and 8R44OD010958. In addition, this technology was supported in part by an award from the Kentucky Cabinet for Economic Development, Office of Commercialization and Innovation, under the grant agreement KSTC-184- 512-11-115 with the Kentucky Science and Technology Corporation. Figure 1. Embryo transfer using a NSET device is more simple and requires less equipment than using surgery. Figure 2. Mice that underwent the NSET procedure had similar pregnancy rates than mice that underwent surgery. Figure 3. Surgical and non-surgical procedures have similar birth rate and litter sizes. Figure 4. Heart rate decreases dramatically in response to anesthesia. Figure 5. Fecal corticosterone levels are higher in response to surgery then to insertion of the NSET device. Figure 6. While the NSET device is able to be reused, the procedure is less successful in subsequent attempts. A wire-topped cage allows the mouse to grip the bars. The NSET device is a small, tapered catheter Small and large specula are placed sequentially into the vagina to open and expose the cervix. An elaborate anesthesia machine and technical expertise is required for surgery. P256 NSET: vs. SURGERY: Imagine • Innovate • Integrate Heart rate of animals in beats per minute are graphed at various hours (h) post procedure for 2.5 dpc pseudopregnant CD-1 mice. The raw data was analyzed by Mouse Specifics using their EzCG analysis software. The data presented are means and standard deviations for readings of 11 mice per time point. Statistical significance (P ≤ 0.05) for the comparison of the control group versus the experimental groups is represented by an asterisk (*). Time post procedure Fold increase in corticosterone levels 3 h 6 h 10 h 24 h 48 h 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 No Procedure NSET NSET with Anesthesia Anesthesia Control Surgery with Anesthesia * * * * * Pseudopregnant mice (2.5 dpc) were randomly assigned into the five experimental groups, with 15 mice per group. Fecal samples were then collected at -24, 0, 3, 6, 10, 24, 48, 72, 96, and 120 hours (h) after the procedure. Samples were collected by transferring each mouse to a clean, empty cage where the subject was given 30 min to defecate or until 3 pellets were collected. Each sample was stored at -80°C until processed. A commercially available ELISA assay (Cayman Chemical Company, Ann Arbor, MI) was used to measure corticosterone concentrations in each fecal sample in accordance with the manufacturer’s directions. The NSET procedure was performed on pseudopregnant (2.5 dpc) female CD-1 mice (N=42), and each mouse was cared for during a 3 week gestation period followed by a 3 week post-partum period if necessary. The recipient CD-1 females were then mated again with a vasectomized CD-1 male to become pseudopregnant. If she became pseudopregnant, she underwent the NSET procedure again. We were able to test the efficiency of the NSET procedure three times on a single female in 18 recipient mice, and the data from these 18 recipients are evaluated here. Surgical (N=20) and non-surgical NSET (N=20) embryo transfers were performed on pseudopregnant (2.5 dpc) female mice of strains CD-1, Swiss Webster, B 6 CBAF 1 , C3H, and C 57 BL/6. Pregnancies were carried full-term, and a successful litter was evaluated after birth. Surgical (N=20) and non- surgical NSET (N=20) embryo transfers were performed on pseudopregnant (2.5 dpc) female mice of various strains. Ten to 15 embryos were transferred during each procedure. Birth rate is determined by number of pups born from the number of embryos specifically implanted in each mouse. After 21- 23 days of gestation, the number of pups delivered were counted. The average number of pups per litter for each procedure is illustrated here. The NSET catheter is then inserted through the large speculum, past the cervical opening, and into the uterine horn. Biotechniques 2009, 47:919-924.