**9. Animal ethics and handling**

From an ethical viewpoint, animal experiments involving pregnant animals do not differ from other types of animal experimentation, but the ethical aspects of using fetuses for experiments should be considered. The 3Rs should be considered in all other types of animal experimentation [75], including the options for replacement, which is the first R. One obvious alternative option is to use the placenta from women who have just given birth [76]. The maternal and the fetal circulations are re-established with a pump system, and this option has been used to study the passage of chemical substances from mother to fetus [77]. In addition to the fact that this alternative can replace the use of live animals, it also has the advantage of avoiding problems related to species differences in the maternal-fetal barrier. However, it can only represent transport in the last part of the third trimester. The second R, reduction, should not only include the number of mothers, but also the number of fetuses. The studies should be conducted so that no more experimental animals are used than is necessary to obtain statistically safe results [75]. The third R, refinement, should importantly include the accommodation, feeding and care of the experimental animals based on the specific requirements of the pregnant animals.

#### **9.1. Physiological effects of gestation**

The physiology of pregnancy can create stress problems in animals which therefore pose special requirements for their handling, care and anesthesia. Most studies on this topic have been performed in ewes, but the same changes are expected in other species. When the size of the fetus increases, more energy and blood must flow to the uterus, and therefore maternal blood volume, cardiac output and contractility approach their maximum [78]. Additionally, the lungs should be able to deliver an increased amount of oxygen while the uterus presses on the diaphragm so that the thoracic cavity volume is decreased. As a result, respiratory rate increases, but at the same time the risk of hypoxia increases even during short-term apnea. The expanding uterus further delays gastric emptying and decreases the esophageal sphincter tone, which increases the risk of regurgitation and aspiration pneumonia during anesthesia. Special conditions apply to sheep and other ruminants, as the last part of the gestation period is characterized by very little space in the abdominal cavity, which may limit the volume of the fermenting compartment so that feed intake is limited. Furthermore, pregnancy may also prolong plasma half-lives of anesthetics and other drugs, and the high level of progesterone will have a sedative effect, and therefore anesthesia doses must be reduced to prevent overdosing [79]. The fasting period prior to anesthesia should be minimized to prevent metabolic disturbances.

#### **9.2. Feeding pregnant animals**

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].

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

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

From an ethical viewpoint, animal experiments involving pregnant animals do not differ from other types of animal experimentation, but the ethical aspects of using fetuses for experiments should be considered. The 3Rs should be considered in all other types of animal experimentation [75], including the options for replacement, which is the first R. One obvious alternative option is to use the placenta from women who have just given birth [76]. The maternal and the fetal circulations are re-established with a pump system, and this option has been used to study the passage of chemical substances from mother to fetus [77]. In addition to the fact that this alternative can replace the use of live animals, it also has the advantage of avoiding problems related to species differences in the maternal-fetal barrier. However, it can only represent transport in the last part of the third trimester. The second R, reduction, should not only include the number of mothers, but also the number of fetuses. The studies should be conducted so that no more experimental animals are used than is necessary to obtain statistically safe results [75]. The third R, refinement, should importantly include the accommodation, feeding and care of

the experimental animals based on the specific requirements of the pregnant animals.

period prior to anesthesia should be minimized to prevent metabolic disturbances.

The physiology of pregnancy can create stress problems in animals which therefore pose special requirements for their handling, care and anesthesia. Most studies on this topic have been performed in ewes, but the same changes are expected in other species. When the size of the fetus increases, more energy and blood must flow to the uterus, and therefore maternal blood volume, cardiac output and contractility approach their maximum [78]. Additionally, the lungs should be able to deliver an increased amount of oxygen while the uterus presses on the diaphragm so that the thoracic cavity volume is decreased. As a result, respiratory rate increases, but at the same time the risk of hypoxia increases even during short-term apnea. The expanding uterus further delays gastric emptying and decreases the esophageal sphincter tone, which increases the risk of regurgitation and aspiration pneumonia during anesthesia. Special conditions apply to sheep and other ruminants, as the last part of the gestation period is characterized by very little space in the abdominal cavity, which may limit the volume of the fermenting compartment so that feed intake is limited. Furthermore, pregnancy may also prolong plasma half-lives of anesthetics and other drugs, and the high level of progesterone will have a sedative effect, and therefore anesthesia doses must be reduced to prevent overdosing [79]. The fasting

working with animal pregnancies.

**9. Animal ethics and handling**

**9.1. Physiological effects of gestation**

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

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

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.

#### **9.4. Anesthesia of pregnant animals**

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 can induce decreased uterine blood flow and fetal acidosis. Both sevoflurane, isoflurane and nitrous oxide are safe to use. Caution should be taken when using opioids as these are only slowly eliminated in the fetus. Xylazine and other alpha-2-agonists are also problematic as they have major depression effects on the fetus. During cesarean section, anticholinergic drugs should be given for inhibition of vagal tone during uterus traction, and ketamine can be used in combination with other drugs, such as thiopental, as long as ketamine is administrated in low doses. Propofol induces a rapid anesthetic phase and is rapidly cleared from the neonates blood circulation [79].

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