Use of real-time ultrasonography as an alternative method for early detection, confirmation and evaluation of rat pregnancy.

Researchers sometimes face difficulties in the diagnosis of pregnancy and assessment of embryonic development. Ultrasonography (US) is a non-invasive imaging method with minimal side effects on the subjects or operators. It provides real-time evaluation of the physiology of rapidly moving structures (i.e., heart) and facilitates evaluation of fetal tissue development. US discerns tissues based on composition, making it the imaging method of choice for abdominal examination. In this study we used real-time US as an alternative method for early diagnosis of pregnancy in rats. Sixty-four Wistar rats aged 16-20 wk were examined, and day 8 was the earliest point at which pregnancy could be detected. We constructed a detailed timeline of embryonic features detectable by US on days 8 to 19. We trust this index will be a valuable tool. More refined work toward a more detailed "atlas" will help to reduce animal sacrifice during embryonic development studies.

Introduction

Rats have been the backbone of many biomedical research studies (Aitman et al. 2008) and are one of the most common models in toxicity and developmental studies during gestation. Early and precise diagnosis of pregnancy, confirmation of the number and age of fetuses and evaluation of embryonic development are prominent in these studies (Krinke 2000). Pregnancy can be determined traditionally by the presence of a copulatory plug after mating, an enlarged lower abdomen, manual palpation (Agematsu et al., 1983, Bennet and Vickery, 1970), hair loss around nipples and nipple enlargement (Bennet and Vickery, 1970, Bivin, 1986) and finally, monitoring of weight gain.

Ultrasonography, although widely used in human pregnancy, is not as common in laboratory animal studies. Until recently, the use of ultrasonography was limited to larger non-rodent species (Beccaglia and Luvoni, 2006, Carr et al., 2012, Lenard et al., 2007, Lindahl, 1966, Quintela et al., 2012, Zambelli and Prati, 2006). Because of advances in ultrasound imaging technology, commercially available ultrasound systems now have the spatial and temporal resolution to obtain accurate images of rat and mouse hearts, kidneys and other organs (Coatney, 2001, Golden et al., 2012, Kobayashi et al., 2001, Srinivasan et al., 1998, Stypmann, 2007). For this reason, ultrasound is a practical method for the diagnosis of pregnancy in agouti mice (Sousa et al. 2012), staging of gestation and monitoring of intrauterine growth in rats (Brown et al., 2006, Mu et al., 2008, Nguyen et al., 2012, Pallares and Gonzalez-Bulnes, 2009, Ypsilantis et al., 2009).

The aim of the present study was to develop a method that could be used in conjunction with the standard pregnancy detection setup in routine experiments. Additionally, this method had to be non-invasive and comply with the “3 Rs”— replacement, reduction and refinement—in animal research (Russell and Burch 1959). In this article, we describe the results of monitoring pregnancy in Wistar rats using real-time ultrasound and share the results collected relative not only to the early determination of pregnancy, but also to the assessment of fetal development at different embryonic stages.

Methods

Ethical statement

The study was performed in the animal facility of the Centre for Experimental Surgery of the Biomedical Research Foundation of the Academy of Athens, and was evaluated and authorized by the Veterinary Service of the Prefecture of Athens, as mandated by Greek legal requirements for animal experimentation. The facility is registered as a “breeding” and “experimental” facility according to Greek Presidential Decree 160/91, which harmonizes national legislation with European Community Directive 86/609/EEC on the Protection of Animals Used for Experimental and Other Scientific Purposes.

Study design and experimental procedures

The animals were weighed before mating and for the duration of pregnancy. Consumption of food and water was observed during experimentation.

The first ultrasound examination was performed on the seventh day (D7) post-coitum in all animals. Thereafter, pregnant animals were examined ultrasonographically daily by the same operator (G.M.), between 1100 and 1300 h. The last ultrasound was performed on day 19. The ultrasonographer was blinded to the day the animals became pregnant.

All animals were anesthetized for ultrasound examinations. Anesthesia was induced by administration of isoflurane 5% (Wenger, 2012, Flecknell, 2009) via a face mask (VME Small Animal Anesthesia Machine, MDS Matrix, Orchard Park, NY, USA). Induction of anesthesia was confirmed by loss of the pedal withdraw reflex. Ultrasonography examination commenced immediately after loss of the pedal withdraw reflex of the animal. Anesthesia was maintained by administration of isoflurane 2.5% (Medical Supplies & Services International, Keighley, West Yorkshire, UK) simultaneously with oxygen 1 to 1.2 L/min.

Ultrasonography was conducted at the operating room of the facility where the vaporizer is located. All animals were examined in the supine position. The hair of the abdomen was thoroughly clipped and shaved immediately after anesthesia induction, to achieve full contact of the probe with the skin in the region concerned. Acoustic gel (Skintact, Leonard Lang, Innsbruck, Austria) was liberally applied. In this way, the phenomenon of “grimy acoustic shadow” created by the air, which holds the animal dander, was reduced to a minimum and the resolution of ultrasound was greatly improved.

We used the Vivid I ultrasound machine (GE Medical Systems, Tirat Carmel, Israel) with the linear transducer probe 12 L-RS (GE Yokogawa Medical Systems, Tokyo, Japan), which has a variable frequency of 5–13 MHz. To achieve detailed imaging of the reproductive tract of rats and embryos, we used a frequency of 13 MHz, with one focal zone set at a depth of 0.5–2 cm. The total duration of the examination was approximately 10 min.

Immediately after the end of each study, administration of isoflurane was interrupted; all animals were receiving oxygen and recovering smoothly when returned to their cages. Use of a volatile anesthetic at the precise dosage for the vaporizer is considered a tolerable method of anesthesia (Flecknell, 2009, Kohn et al., 1997). Specialized personnel (E.B.) handled the animals during vaginal smear collection and anesthesia induction and there were no adverse effects.

Experimental animals and sample size

A total of 64 female Wistar rats (HsdOla:WI) aged 18 ± 1.56 wk were studied. These animals were obtained from the breeding colony of the animal facility and were being used for the first time in reproductive studies; their estrous cycles were confirmed as normal with vaginal smears. The animals were caged in pairs in H-Temp polysulfone type III cages 425 mm long × 266 mm wide × 185 mm high (Tecniplast, Varese, Italy).

Husbandry

Two female Wistar rats were introduced into the cage of a male rat of proven fertility where they remained until the day sperm was detected on the vaginal smear. All cytologic examinations were performed by the same person (M.S.). The day on which sperm was detected was designated as embryonic day 0 (D0), and the female was returned to its cage.

Housing (food, water and bedding)

All cages were kept in the same animal room with a HEPA-filtered air supply, 15 ACH, at a room temperature of 24 ± 2°C, relative humidity of 55 ± 10%, 12 h:12 h light/dark cycle (0700/1900), light intensity of 300 lx, as measured 1 m above the floor in the middle of the room, and positive air pressure of 0.6 Pa within the room. All animals were free of a wide range of pathogens including Kilham rat virus, rat parvovirus, Toolan's H-1 virus, Sendai virus, pneumonia virus of mice, reovirus type III, murine encephalomyelitis virus, sialodacryoadenitis virus, rat min virus, Hantaan virus, lymphocytic choriomeningitis virus, CAR bacillus, MAD 1 and 2, rat rotavirus, rat coronavirus, Mycoplasma pulmonis, Clostridium piliforme, Bordetella bronchiseptica, Pasteurella spp, fur mites and pinworms.

All Wistar rats had ad libitum access to filtered tap water in drinking bottles and pelleted chow, which contained 18.5% protein, 5.5% fat, 4.5% fiber, 6% ash (irradiated vacuum packed, 2918, Harlan, Italy). Each cage contained ∼140 g of corncob bedding (Rehofix MK 2000, J. Rettenmaier & Soehne, Rosenberg, Germany). Cages were cleaned once a week.

Results

Presence of an embryonic sac and ability to distinguish embryo, embryonic organs and placenta were evaluated. Findings were correlated with the overall development of the fetus, as previously described by other authors (Theiler, 1989, Witschi, 1972, Ypsilantis et al., 2009). The characteristic features unique for each day were noted and are summarized in Table 1 .

Table 1

Embryonic features detectable by ultrasonography during gestational days 8–19

	D8	D9	D10	D12	D12	D13	D14	D15	D16	D17	D18	D19
Embryonic sac	+	+	+	+	+	+	+	+	+	+	+	+
Placenta		+	+	+	+	+	+	+	+	+	+	+
Amniotic fluid				+	+	+	+	+	+	+	+	+
Umbilical cord					+	+	+	+	+	+	+	+
Embryo			Visible embryo	Visible embryo	Visible embryo	Distinct embryos; recogniz-able body ends
Posture							Snout lifts off chest; embryos bent dor-sally; vis-ible body ends	Clear separation between head and body; body uncoils
Tail						Appears
Heart			Embryonic heart pulse appears; HR not yet measurable; BFI signal present	Heart visible using B-mode; no measure-ments when PWD is used	Clear imaging of cardiac function and estimation of HR (using both B-mode and PWD	All four heart chambers visible				Thoracic and abdominal aorta visible	Heart ventricles visible
Liver/gallbladder/kidneys/urinary bladder								Urinary bladder visible	Liver visible	Gall-bladder visible	Liver distinguish-able	Kidneys visible
Brain/cranium/spine							Head-body distinction; medullary tube visible	Onset of cranial ossification	Eye sockets/orbits visible	Brain ventricles visible; spine visible	Brain ventricles distinct/recognizable; spine visible	Visible jaw bone diff-erentiation relative to rest of skull
Ribs									Ribs start develop-ing	Ossified ribs
Extremities									Bone tissue appears at body ends; discrimi-nation of phalan-ges	Ossified phalanges

BFI = blood flow imaging; HR = heart rate; PWV = pulse wave Doppler.

With D0 considered the day of detection of sperm in the vaginal smear, ultrasound examination was performed daily starting from D7. The pregnancy was first detected by ultrasound on D8 (Fig. 1 a). The only visible structure was the embryonic sac, which appeared as a round to oval structure filled with anechoic to isoechoic fluid. There was no evidence of the embryo. The placenta was first evident on D9 (Fig. 1b), as a hyperechoic zonar structure attached in the inner surface of the gestational sac. On D10, the fetus became distinguishable and the heart was also obvious with color Doppler signal (Fig. 1c). The cardiac pulse appeared on the same day, but the heart rate could not be measured with pulse wave Doppler. On D11, the heart was clearly visible with B-mode imaging, but heart rate could not be estimated (Fig. 1d). The next day D12, the amniotic fluid was visible and cardiac function was evaluated with pulse wave Doppler (Fig. 1e). The tail could be distinguished and the fetal extremities were apparent on D13 (Fig. 1f). The four chambers of the heart and the umbilical cord were also visible on D13.

Fig. 1

Representative ultrasound images of a rat uterus on different days of gestation: In some of the images, white crosses mark the start and end points of measurements (1, 2) of the longitudinal and transverse diameters, the values of which are given (in cm) in the inset at the top left of the image. (a) Day 8 of gestation: The embryonic sac is visible as a round or oval structure filled with anechoic to isoechoic fluid. (b) Day 9: The developing placenta can also be recognized as a hyperechoic structure attached to the inner surface of the gestational sac. (c) Day 10: The fetus is distinguishable. (d) Day 11: The heart can be imaged by B-mode. (e) Day 12: Cardiac function is detectable using both B-mode and pulse wave Doppler as imaging techniques. (f) Day 13: The extremities are apparent. (g) Day 14: The body and head can be distinguished. (h) Day 15: The body uncoils and the head is distinct from the body. (i) Day 16: The lungs appear as hyperechoic structures surrounding the heart in the thoracic cavity. (j) Day: The urinary bladder is visualized as a cystic structure filled with anechoic fluid (urine), in the abdominal cavity. Ossification of ribs is obvious. Brain ventricles are detectable as hypoechoic discontinuities in the cranial outline. (k) Day 18: The ribs are ossified and visible along with the urinary bladder. (l) Day 19: Brain ventricles are bisected from the ossified joint of the frontal cranial bones. Cranial ossification is more intense and the jawbone can be differentiated.

From D14 onward, the body and head became increasingly distinguishable as the embryo was bent dorsally, the snout lifted from the chest and the medullary tube was visible (Fig. 1g). The body uncoiled and there was clear separation between the head and body at D15, along with cranial ossification. The urinary bladder was also visible on D15 (Fig. 1h). On D16, osseous tissue started to develop, especially at the body ends, with clear discrimination of the phalanges. The ribs started to develop, and eye sockets were visible. D16 was also the first day the liver became visible (Fig. 1i). Brain ventricles became detectable on D17, along with the thoracic and abdominal aorta. Ossification of the spine and ribs was in progress, whereas the phalanges were clearly ossified (Fig. 1j). On D18, the heart ventricles became evident, while ossification of the spine and ribs continued. The liver and other abdominal organs were also distinguishable (Fig. 1k). Finally, on D19, the jaw bone could be differentiated from the rest of the skull and the kidneys were also discernible (Fig. 1l).

Discussion

Because of advances in ultrasound imaging technology, commercially available ultrasound systems now have the spatial and temporal resolution to obtain accurate images of rat and mouse hearts, kidneys and other target tissues (Coatney, 2001, Golden et al., 2012, Stypmann, 2007). The primary aim of this study was to evaluate the use of real-time ultrasound examination in the accurate detection of rat pregnancy. According to the data obtained, D8 is the earliest day on which pregnancy could be detected, and it was based on the observation of the embryonic sacs. Detection of pregnancy is just as important as detection of non-pregnancy; it is crucial for researchers in terms of time management and planning, reducing the number of mother animals sacrificed and waiting time between experiments. Such information might prove vital to decision making in each case. In addition, ultrasound imaging of the reproductive tract during pregnancy provides valuable information relative to the developmental stage of embryos, embryo viability and progression of fetal development.

Figure 1 is a sequence of representative images of embryonic development. Information on fetal age and development may be gathered from both somatometric measurements and indexing of other detectable features, such as the heart rate on each day of pregnancy (Zambelli and Prati 2006). Ultrasonographic detection of the heart rate at day 12 was also considered a clear indicator of embryonic age. Days 14 and 15 were milestones with respect to anatomic changes in the fetus, as fetal skeletal morphology could easily be distinguished and evaluated. Furthermore, another useful finding was the detection of fetal abdominal organs from day 15 onward.

We created an index as a reference for researchers in their everyday laboratory animal science praxis. The results obtained by ultrasonography (Table 1) were in accordance with anatomic descriptions reported in textbooks (Theiler, 1989, Witschi, 1972), can be non-invasively obtained by ultrasound examination and may prove to be a valuable tool for future studies.

The limitations of the present study must, however, be recognized. Only one person performed the ultrasound examinations using a single ultrasound scanner. The intrinsic spatial resolution of the imaging system and the absence of reproducibility analysis may limit the generalizability of our results. Further work is necessary to expand the dataset obtained from this study.

Ultrasonography is an examination method with minimal biological or other side effects for the patient, the operator and assisting personnel (Shaw et al. 1999). Furthermore the interventions used in the present study were in compliance with the principle of refinement of the 3 Rs for humane use of animals in research. Furthermore, this effort may be of benefit in future experimentation in reducing the numbers of animals used or sacrificed, specifically in protocols concerning pregnancy or embryo transfer. Finally, it is worth mentioning that use of ultrasonography in embryonic development studies provides the unique opportunity to monitor the same pregnant animal throughout the whole gestational period, in parallel with real-time monitoring of the viability and somatometric characteristics of the fetuses.

Conclusions

By exploiting the high sensitivity and specificity of ultrasonography in examining soft tissue, we were able to detect rat embryos from gestational day 8. In conjunction with the vaginal smear technique, a profile of the estrous cycle and development of the fetus can be obtained. Such data, along with the corresponding gestational days, are invaluable in studies concerning pregnancy, fetal development and embryo transfer.