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 (*).

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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, B6CBAF1, C3H, and C57BL/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.