Sheep Models in Translational Surgery Open Access

In fundamental or preclinical research, large animal models are required for several reasons. According to the available literature, amongst the large animal models, the sheep might be, in certain medical fields, the one having the most resemblance to humans [1‒3]. This can lead to increased translational value. Several characteristics make the sheep a preferred model: its size (similar to humans), its availability, the possibility of using both sexes, its hormonal status (intact vs. castrated vs. menopausal), its affordable cost, the cost and ease of housing, the ease of handling, the few zoonotic diseases transmitted by sheep, and finally its social acceptability. This chapter will summarize the different fields in which the sheep is used preferentially in research involving surgical models. We will outline the advantages, disadvantages, and justification of using the sheep in experimental surgery. Furthermore, the differences or particularities of analgesic/anaesthetic protocols of the ovine model will be outlined.

The section editors chose not to present the sheep as a translational model in the digestive, urinary, and respiratory systems as they are not the most used. Moreover, the sheep’s physiology is different than humans, factors which negatively influence the selection of the ovine model in the aforementioned research fields. For those systems, other animal models (especially pig and non-human primates) will be used and discussed in their respective sections.

Sheep are the state-of-the-art test model for cardiovascular research and safety studies required for approval of cardiovascular implantable devices [4‒6]. The main reasons are as follows:

• The sheep’s heart valves’ mechanical and hemodynamic parameters are very similar to humans [7].

• Heart size and cardiac muscle structure resemble humans [7, 8].

• Prosthetic leaflets are known to calcify very quickly in sheep [4].

• Sheep are widely available, easy to handle, and do not outgrow prosthetics during long-term studies [4].

• The blood coagulation profile resembles that of humans and has been used in numerous thrombogenicity studies [4].

• Cardiovascular procedures in the ovine have a high success rate with few postoperative complications [4].

• Cardiac and vascular catheterization techniques in the sheep are similar to the techniques used in humans. Standard human diagnostic equipment and interventional supplies can be used [4].

• Sheep are an excellent model for ventricular or valvular dysfunction [9, 10].

• Ovine models are well-established and widely accepted by regulatory agencies for preclinical safety studies [11].

Nevertheless, there are some limitations to this model. A non-extensive list of differences is as follows: thorax shape and volume (less space available to accommodate a pump in the thorax in a sheep), a more conical shape of the ovine heart (resulting in less fitting accuracy of devices and delivery systems), a much shorter ascending aorta (making aortic surgery more challenging), the absence of a true aorto-mitral “curtain” [7], risk of impinging the anterior leaflet of the mitral valve when performing aortic surgery, significantly thinner and more frail leaflets and chordae, which may lead to abrasion or rupture, and a more oval mitral annulus. Sheep lack many human cardiovascular diseases (especially atherosclerosis) that require artificial disease models, thus may not fully replicate human pathophysiology.

As to coronary and peripheral vessels, there are some notable anatomic differences between humans and sheep, but this does not preclude the use of sheep for coronary or peripheral vascular procedures, quite the contrary. Sheep are less prone to arrhythmia than pigs and in the relevant weight and size range do not outgrow devices [4]. Their coagulation system is also closer to that of humans than in pigs. Therefore, for cardiovascular applications, whether they be structural heart related, surgical, or interventional, for electrophysiology or ventricular assist devices, for healthy or disease models, or even for foetal cardiovascular surgery [12], as well as for vascular devices, the ovine model is one of the models of choice.

The pregnant sheep constitutes a robust model for studying neonatal adaptation and placental physiology due to its physiological similarities with humans. Its placental structure supports efficient gas exchange, resembling human oxygen transfer mechanisms [13]. Unlike humans, sheep have a cotyledonary placenta rather than a discoid placenta. While the chorionic villi in sheep are more extensively branched, providing a larger exchange surface, the overall pattern of placental vascularization closely resembles that of humans. Furthermore, sheep have comparable foetal haemoglobin adaptations, enhancing oxygen affinity for effective transplacental exchange [14].

Thus, the sheep model has been used to evaluate foetal physiology and neonatal adaptation in normal or pathological context. We give here three main examples:

• 1.

  Adaptation at birth: the lamb’s pulmonary development, including surfactant production and structural maturation, closely resembles humans, making it ideal for studying neonatal respiratory conditions and adaptation to extra-uterine life [15, 16]. The pulmonary vascular resistance (PVR) decreases significantly after birth in both lambs and humans, facilitating the transition from foetal to neonatal circulation. In humans, PVR drops rapidly within minutes to hours postpartum due to lung expansion, oxygenation, and the release of vasodilators. Similarly, lambs exhibit a comparable reduction in PVR driven by similar mechanisms. This experimental model enables a better understanding of the underlying adaptive mechanisms and facilitates the development of therapeutic strategies.

• 2.

  Foetal physiology during labour: most of the knowledge on foetal adaptation on hypoxia during labour is from experimental studies [17]. Different models of cord occlusion have been proposed to mimic uterine contractions [18]. Those models highlighted the main role of autonomic nervous system to keep foetal homeostasis [19‒21].

• 3.

  Congenital diaphragmatic hernia (CDH): CDH is a critical pathology characterized by lung hypoplasia, pulmonary hypertension, and cardiac dysfunction. We highlight the relevance of lambs for understanding CDH pathophysiology and potential interventions [22]. This model, compared to healthy lambs, provided insights into intact cord resuscitation, a technique involving initiating resuscitation before clamping the umbilical cord [23].

Compared to smaller species, lambs offer unmatched anatomical and physiological similarities, particularly in lung structure and foetal weight, making them essential for translational studies bridging experimental research and foetal/neonatal medicine.

The sheep is frequently used to evaluate therapeutic modalities for the alleviation of clinical signs of osteoarthritis (OA). The surgically induced models of OA are preferred in the sheep. The stifle joint is commonly used for OA induction. Several methods are used, but the most common ones are anterior cruciate ligament transection (ACLt) or the partial or complete medial meniscectomy (pMMX/tMMX). The sheep model is preferred over other large animal models because of the size and accessibility of the joint, which is similar to humans, and the ease of manipulation of the animal [24]. The surgical induction of OA can be performed through an arthrotomy or with arthroscopy [2, 25‒27]. The sheep has also been used as a model for knee joint sports surgery [1].

For the evaluation of efficacy of therapeutic modalities, the administration of parenteral or intra-articular drugs is preferred. However, the oral route is not indicated because the sheep is a polygastric animal that ruminates.

The outcome measures are aligned with the ones used in humans: goniometry, kinetics, kinematics, imaging (radiography, CT, and MRI), arthroscopic grading, synovial fluid analysis (physical, cytological, cytokines, etc.), and biomarkers (synovial fluid, serum, urine). Furthermore, the post-mortem evaluation can entice histological evaluation on decalcified or calcified tissues. The sheep has also been used in cartilage resurfacing studies: filling gaps with matrix/gels, osteochondral grafts, or hemi-joint arthroplasties.

The sheep model is used in several orthopaedic preclinical models. Here is a non-exhaustive list:

• Critical bone defect: these defects lead to the development of non-unions. They are a challenge to address, for which large animal models have been developed to evaluate different therapeutic strategies. The sheep critical-sized tibia defect model is preferred because of the weight of the animals and the size of their medullary canal [28, 29]. A metatarsal critical defect model has also been used [30, 31].

• Cancellous bone healing: the gold standard for replacing cancellous bone defects is fresh autologous cancellous bone graft. This comes with associated morbidities and limitations. To palliate these, the development of cancellous bone replacement materials is a thriving discipline. The sheep is a preferred model because of its size and similarity to humans. In a validated drill hole model, up to 8 sites can be implanted with autologous cancellous bone graft or synthetic replacement graft material [32].

• Osteomyelitis: the sheep has similar bone structures to humans. In a systematic review of preclinical bone infection research [33], the sheep has proven its translational value. It can be used in studies where osteomyelitis is associated with bone defects, at several sites (tibia, femur, mandible, or iliac crest), either at cortical, medullar, or trabecular locations. Fixation material is also used; it mimics the clinical situation, potentiates the formation of a biofilm, and protects bone against catastrophic failures. The sheep is an animal that exhibits low morbidity to the development of osteomyelitis and that mimics the pathobiology of the disease reported in humans.

• Joint replacement surgeries: the sheep has been used extensively as a model for total joint replacement of the hip or the knee joints [34]. The advantage of using the sheep is that prosthetic components designed for humans can be directly used in sheep. Several aspects of total joint replacement can be evaluated in the sheep: surgical components (geometry, surface), biological augmentation (stem cells, graft material), implant loosening [35], surgical techniques (robotic surgery, intraoperative navigation).

• Bone healing in osteoporotic models: the occurrence of fractures in osteoporotic patients is highly prevalent. The sheep has been validated and used extensively to investigate several aspects of the disease and its treatment [36, 37].

The sheep is mainly used in axial spinal surgery because of the similarity of the ovine to human vertebrae [11]. There are several differences, but small ruminants seem to be the best compromise between size and ease of manipulation. The other large animal model used in spinal surgery is the pig, which can weigh up to 100 kg for minipigs (up to 300 kg in non-minipigs) rendering their size as a negative factor in model selection. The dog had been used in earlier spinal surgery research but has been replaced by the sheep mainly because of ethical concerns but also because of size. The ovine model is used for the development and validation of medical devices or in the evaluation of cancellous bone graft replacement for spinal fusion. Medical devices that have been studied are numerous: pedicle screws, rods, cages, anterior plates/screws. These devices have been evaluated in the treatment of fractures and in cervical/lumbar spinal fusion studies, using or not cancellous bone graft substitutes that were inert or bioactive [38‒40]. The outcome measures have been adapted to the sheep. For several diseases, the sheep is a model used in either open or percutaneous vertebroplasty as a model of osteoporotic fractures or in vertebral tumours [41, 42].

Challenges encountered in ruminant anaesthesia are mainly in relation with their digestive physiology that creates a high risk of regurgitation during anaesthesia and a hindrance of diaphragm/respiratory movements due to rumen repletion and meteorism [43, 44]. Strategies to limit the likelihood of regurgitation, bloating, airway contamination, and obstruction are summarized in Table 1. Various fasting recommendations for decreasing rumen content and tympany have been made [43, 44]. To limit its impact on rumen flora and motility as well as the starving sensation, adaptative fasting regimen includes tailoring fasting duration to rumen size and function, withholding fermentable feed for 24–48 h prior to anaesthesia and hay only overnight. Maintaining straw bedding and water may reduce animal frustration but may not be possible with prolonged fasting. Water withholding during fasting can result in hypovolemia and warrant adjustment of intravenous fluid rate during anaesthesia. Preserving social contact during period of fasting and recovery may help reducing overall stress linked to the experimentation. Small pen designs while allowing to separate animals should allow visual, olfactory, and if possible nose-to-nose contact.

The anaesthetic protocol is designed to provide optimal chemical restraint and plan of anaesthesia to decrease handling stress, optimize gain of upper airway control, and prepare for a fast return of consciousness and motor function while maintaining stability of anaesthesia and cardiovascular function. Use of less soluble inhalant anaesthetics like sevoflurane and desflurane yields faster recovery compared to isoflurane [44]. Controlled ventilation with the application of positive end expiratory pressure counteracts V/Q mismatch, especially during dorsal recumbency, and warrants a steady plan of anaesthesia. Pre-emptive pain management during anaesthesia is tailored to the level of surgical trauma and estimated postoperative pain with the aim of allowing a fast return from sternal recumbency to standing, moving, eating, resting, and ruminating. Intraoperative constant rate infusions of lidocaine and ketamine in combination with an opioid provide intense analgesia and have a sparing effect on inhalant anaesthetics [45]. Locoregional anaesthesia techniques also provide profound analgesia, but persistence of a motor blockade in early recovery may not be desirable. Long-acting local anaesthetics like bupivacaine and ropivacaine or long-acting formulation (liposomal or encapsulated) can be used to reduce surgical wound pain. Available pharmacokinetics data in sheep gear to administer injectable meloxicam twice a day rather than once (0.5 mg/kg BID) and showed a good oral bioavailability (72%) at 1 mg/kg/day [45]. Recent advances in pain assessment have made available facial expression and composite pain scales that are good tools to document postoperative pain and response to pain management [46‒48].

The quest for the perfect model in all domains of biomedical research is universal. As it does not exist, the best we can do is try to propose models that seem the most applicable for certain fields.

The sheep has become a popular model because of its size, availability, robustness, cost, and ease of handling. Furthermore, as a preclinical model, the same validated objective outcome measures used to measure success in humans can apply to sheep.

Prof. Thomas Hubert was a member of the journal’s Editorial Board at the time of submission. The authors have no conflicts of interest to declare.

This study was not supported by any sponsor or funder.

B.L., L.B., N.B., I.B., C.G., L.G., K.D., D.S., L.S., G.T.J., J.W., and T.H. did the bibliographic research, wrote the article, and contributed substantially to its revision. G.T.J. and B.L. conceived Table 1. T.H. coordinated the writing of the manuscript. All authors take responsibility for the paper as a whole.

All authors have equally contributed to the chapter.