**8. Imaging diagnostics of the pregnant animal**

Ultrasound imaging is widely used in small animal practice for the evaluation of the pregnancy and determination of number of fetuses, and it is also used to monitor abnormal pregnancies, such as poorly fetal development for gestational age and to identify pregnancies in which there is embryonic resorption or fetal abortion. In the placenta, ultrasoundbased Doppler is the first-line technique for the evaluation of uteroplacental blood flow. The Doppler technology is based on analysis of the change in frequency or intensity of ultrasound waves when they are reflected by a moving target such as erythrocytes. Ultrasound exposure is considered harmless; and in fact, animal experiments subjected to fetal ultrasound imaging in various mammalian species showed no pathological effects for the embryo, no congenital malformations or adverse neurobehavioral effects [66]. The technique is often combined with simultaneous administration of a sonographic contrast agent, resulting in an enhanced gray scale or color Doppler signal, facilitating visualization of microvascular structures down to the microvascular perfusion. The mean diameter of micro-bubbles ranges from 2 to 10 μm, less than that of a red blood cell but sufficiently large to be trapped within the vascular space [67]. Thus, ultrasound imaging allows discrimination between fetal and maternal circulatory systems by imaging the intervillous space alone, and it could be used to diagnose the abnormalities of placental blood flow [68] (**Figure 3A**).

MRI is another non-invasive method for diagnostic information. MRI uses the body's natural magnetic properties to produce detailed images from any part of the body. For imaging purposes, the hydrogen nucleus is used because of its abundance in water and fat. What makes MRI so powerful is the exquisite soft tissue and anatomic details. MRI has been increasingly used for detailed visualization of the fetus *in utero* as well as placental structures. While small rodents have fetal sizes that are difficult to investigate with most MRI systems, recent development in very high-field MRI systems now allows visualization of fetal anatomical structures down to a mouse fetus. Wu et al. demonstrated how embryonic mice brain structures could be delineated *in vivo* at embryonic day 17 using an 11.7 T MRI system. *In utero*, 3D MRI has been extensively used in larger animals in clinically available MRI systems. As an example, high-resolution MRI of the inner ear structures of fetal sheep *in vivo* has been demonstrated [69]. Pregnant domestic pigs, on the other hand, are too large to fit in a standard MRI machine bore, precluding MRI as a diagnostic tool in this animal model. Non-brain investigations of the fetus have been increasingly performed using MRI.

the intrinsic T1-relaxation times of various soft tissues where the contrast agent accumulates. For example, Mourier et al. have evaluated placental blood flow with MRI in a rabbit model before and after injection of a paramagnetic contrast agent [68] (**Figure 3B**–**C**). Animal studies have shown that small-size gadolinium agents cross the placenta and are extracted by the fetal kidneys into the amniotic fluid [70] [71]. Mikkelsen et al [12] revealed a high signal of [1-13C]-pyruvate and its derivative [1-13C]-lactate in the chinchilla placenta using hyperpolarized MRI; a non-harmful imaging modality using non-ionizing endogenous substrates for interrogating accumulation and metabolic pathways. In parallel, Friesen-Waldner et al. examined noninvasively the fetoplacental metabolism and transport of pyruvate in guinea pigs using the same technique [72]. The relation between maternal oxygen challenge and fetal oxygenation has recently become possible to study using the blood oxygen-level dependent (BOLD) MRI sequence; a non-invasive technique for evaluating organ tissue oxygenation that requires no contrast exposure (**Figure 3D**). Studies in sheep fetuses have shown that changes in cotyledon and fetal BOLD MRI signals are closely related to changes in fetal oxygenation estimated by fetal arterial hemoglobin saturation [73]. MRI

(A–C are reprinted from ref. [66]; D is reprinted from ref. [71]).

**Figure 3.** Placental blood flow mapping with discrimination of fetal and maternal circulation using ultrafast ultrasound Doppler in the rabbit model. With this technique, the pulsatility of each placental vessel is analyzed. Discrimination between maternal and fetal blood flows is performed using advanced analysis (A). Evaluation of placental blood flow with magnetic resonance imaging (MRI) in the rabbit model before (B) and after (C) injection of contrast product. Blood oxygen level-dependent MRI showing an axial view of the fetus with the fetal liver selected as the region of interest (D).

Animal Models of Fetal Medicine and Obstetrics http://dx.doi.org/10.5772/intechopen.74038 359

Similar to the ultrasound-based contrast-enhanced method, an excellent soft tissue image contrast can be obtained by MRI contrast agents; usually a paramagnetic (gadolinium) molecule that alters

macrosomia regardless of fetal genotype. An important factor is that the diabetic phenotype of the "db/+" mouse is not present prior to gestation, making this model more transferable to

With genetic models of diabetic pregnancy, it is important to remember the genetic predisposition to diabetes in the fetus. Embryo transfer can be used to study the influence of maternal diabetes separately from the fetal genotype [65]. Many genes affecting β cell function in preg-

Ultrasound imaging is widely used in small animal practice for the evaluation of the pregnancy and determination of number of fetuses, and it is also used to monitor abnormal pregnancies, such as poorly fetal development for gestational age and to identify pregnancies in which there is embryonic resorption or fetal abortion. In the placenta, ultrasoundbased Doppler is the first-line technique for the evaluation of uteroplacental blood flow. The Doppler technology is based on analysis of the change in frequency or intensity of ultrasound waves when they are reflected by a moving target such as erythrocytes. Ultrasound exposure is considered harmless; and in fact, animal experiments subjected to fetal ultrasound imaging in various mammalian species showed no pathological effects for the embryo, no congenital malformations or adverse neurobehavioral effects [66]. The technique is often combined with simultaneous administration of a sonographic contrast agent, resulting in an enhanced gray scale or color Doppler signal, facilitating visualization of microvascular structures down to the microvascular perfusion. The mean diameter of micro-bubbles ranges from 2 to 10 μm, less than that of a red blood cell but sufficiently large to be trapped within the vascular space [67]. Thus, ultrasound imaging allows discrimination between fetal and maternal circulatory systems by imaging the intervillous space alone, and it could be used to diagnose the abnor-

MRI is another non-invasive method for diagnostic information. MRI uses the body's natural magnetic properties to produce detailed images from any part of the body. For imaging purposes, the hydrogen nucleus is used because of its abundance in water and fat. What makes MRI so powerful is the exquisite soft tissue and anatomic details. MRI has been increasingly used for detailed visualization of the fetus *in utero* as well as placental structures. While small rodents have fetal sizes that are difficult to investigate with most MRI systems, recent development in very high-field MRI systems now allows visualization of fetal anatomical structures down to a mouse fetus. Wu et al. demonstrated how embryonic mice brain structures could be delineated *in vivo* at embryonic day 17 using an 11.7 T MRI system. *In utero*, 3D MRI has been extensively used in larger animals in clinically available MRI systems. As an example, high-resolution MRI of the inner ear structures of fetal sheep *in vivo* has been demonstrated [69]. Pregnant domestic pigs, on the other hand, are too large to fit in a standard MRI machine bore, precluding MRI as a diagnostic tool in this animal model. Non-brain investigations of

Similar to the ultrasound-based contrast-enhanced method, an excellent soft tissue image contrast can be obtained by MRI contrast agents; usually a paramagnetic (gadolinium) molecule that alters

nancy can be mutated in mice to induce a diabetic phenotype [56].

358 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

**8. Imaging diagnostics of the pregnant animal**

malities of placental blood flow [68] (**Figure 3A**).

the fetus have been increasingly performed using MRI.

GDM than many other models [56].

**Figure 3.** Placental blood flow mapping with discrimination of fetal and maternal circulation using ultrafast ultrasound Doppler in the rabbit model. With this technique, the pulsatility of each placental vessel is analyzed. Discrimination between maternal and fetal blood flows is performed using advanced analysis (A). Evaluation of placental blood flow with magnetic resonance imaging (MRI) in the rabbit model before (B) and after (C) injection of contrast product. Blood oxygen level-dependent MRI showing an axial view of the fetus with the fetal liver selected as the region of interest (D). (A–C are reprinted from ref. [66]; D is reprinted from ref. [71]).

the intrinsic T1-relaxation times of various soft tissues where the contrast agent accumulates. For example, Mourier et al. have evaluated placental blood flow with MRI in a rabbit model before and after injection of a paramagnetic contrast agent [68] (**Figure 3B**–**C**). Animal studies have shown that small-size gadolinium agents cross the placenta and are extracted by the fetal kidneys into the amniotic fluid [70] [71]. Mikkelsen et al [12] revealed a high signal of [1-13C]-pyruvate and its derivative [1-13C]-lactate in the chinchilla placenta using hyperpolarized MRI; a non-harmful imaging modality using non-ionizing endogenous substrates for interrogating accumulation and metabolic pathways. In parallel, Friesen-Waldner et al. examined noninvasively the fetoplacental metabolism and transport of pyruvate in guinea pigs using the same technique [72]. The relation between maternal oxygen challenge and fetal oxygenation has recently become possible to study using the blood oxygen-level dependent (BOLD) MRI sequence; a non-invasive technique for evaluating organ tissue oxygenation that requires no contrast exposure (**Figure 3D**). Studies in sheep fetuses have shown that changes in cotyledon and fetal BOLD MRI signals are closely related to changes in fetal oxygenation estimated by fetal arterial hemoglobin saturation [73]. MRI has also shown promises in the fetal heart. As example, Yamamura et al. have demonstrated the applicability of MRI for future evaluation of fetuses with complex congenital heart defects [74].

**9.2. Feeding pregnant animals**

Many females will have a temporary loss of appetite in the first part of the pregnancy, and an increased appetite later. In general, the nutrition requirements are the same as for nonpregnant females. However, in the last part of the gestation period, the fetus growth will increase dramatically, and so will the nutrition requirements for the mother [80]. For the species with largest litters and heaviest fetuses, the need for energy will increase most. Pregnant rodents should typically be fed with a special breeding mix, which has a slightly higher content of proteins, vitamins and minerals. They are typical feed *ad libitum*, so the increased amount of feed is not observed, but for *restricted* fed animals, like pigs, the amount should be adjusted. Pregnant sows should be fed individually so that they maintain a normal weight and body mass, and their energy needs will specially increase during the last 4 weeks [81]. The composition of the sow feed does not need to be changed once it has been ensured that it contains sufficient amino acids; this is particularly important in young sows. Sheep are fed normal maintenance diet during the first 2/3 of the gestation period, and it should be ensured that they maintain a normal weight and body mass. In the last 1/3 of the pregnancy, the feed requirement increases, and as the space in the abdominal cavity is limited, it is important to

Animal Models of Fetal Medicine and Obstetrics http://dx.doi.org/10.5772/intechopen.74038 361

Pregnancy poses special requirements for the handling of experimental animals. Generally, pregnant animals tolerate less stress than non-pregnant animals, and should be transported as little as possible during the first and last part of the pregnancy. At the beginning of pregnancy, the implantation process of ovarian eggs is sensitive to stress, and ultimately in pregnancy, the mothers are physiologically stressed and therefore have a low threshold of stress tolerance. Mice and rats are bred in monogamous (one male and one female) or polygamous mating systems (one male and two to six females). In guinea pigs, the polygamous mating system can be practiced with 1 male to 10 females. In polygamous systems, the females are removed from the male before they give birth. For pigs, the gilts will go into estrus after contact with a boar, and after mating, the pregnant sows are group housed. The sheep differs by being seasonally polyestrous. Ewes are typically paired in autumn so that they lamb in the spring. If the animals should give birth, they must have access to pre-birth material during the last days of gestation.

The anesthesia risk is higher in the pregnant than in non-pregnant animals due to physiological alterations described above [79]. In general, anesthetics can cross the blood-brain barrier and will usually cross placenta. Therefore, in some species, local anesthesia, such as epidural anesthesia, is preferred due to their minimal systemic effects; this applies especially to cows, sheep and other ruminants where general anesthesia furthermore can lead to tympanitis. In most other species, it is necessary to use general anesthesia. The choice of anesthetics depends on the animal species, but drugs generally have to be selected for their minimum effects on cardiac output, renal blood flow and fetus physiology [79]. Drugs with major depression effects on the fetus should be avoided. Inhalation drugs can be used, but as the degree of neonatal depression depends on the maternal anesthesia depth, higher doses should be avoided. Furthermore, they

feed them with a high quality feed that does not overload the rumen.

**9.3. Handling and care of pregnant animals**

**9.4. Anesthesia of pregnant animals**

Moreover, more sophisticated MRI sequences, such as diffusion-weighted imaging (DWI), MR spectroscopy and diffusion tensor imaging allow for visualization of inherent structural, metabolic, cellular and microvascular characteristics. While these techniques have potential applications in fetal imaging, the familiarity with fetal MRI is still limited within researchers working with animal pregnancies.
