Anaesthesia for pigs,sheep,goats and cattle involved in biomedical research: FELASA working group guidelines—Parts I–IV

Introduction

Introducing farm animals to the laboratory environment raises several challenges ⁶ but, importantly, produces adverse physiological and psychological effects which vary in severity and duration and may be collectively termed as ‘stress’, i.e. ‘the sum of biological reactions to adverse physical, mental, or emotional, internal or external stimuli, that tends to disturb the homeostasis of an organism’. ⁷ These biological reactions are highly undesirable as they are likely to: (a) confound data; (b) complicate day-to-day animal handling ⁸ ; and (c) may have adverse health consequences such as decreased immunity and thus disease susceptibility. ⁹ The stress of introducing farm animals to the laboratory arises from (a) transport and (b) the new (unfamiliar) environment.

Animal sourcing and transport

Farms or other facilities supplying ruminants and pigs to laboratories should meet criteria set by the destination laboratory and confirmed by periodic inspection by the facility’s NVS or DV. Recommended criteria are: demonstration of high health status ⁶ and high welfare status; high levels of animal–stockperson interaction; common environmental and husbandry features (with laboratory); brief transit time to facility. Such criteria are necessary for making accurate accumulative severity assessments ¹⁰ for animals obtained outwith the facility. These, in turn, are required for informed decision-making with respect to the animals’: (i) allocation to studies of different severities; (ii) re-use; or (iii) scheduled killing. For these reasons, obtaining inexpensive cull animals from public markets is strongly discouraged.

The effects of transport on pigs, sheep and calves are well documented. ¹¹ In general, transport raises cortisol and body temperature and invokes cardiovascular and behavioural changes.^12,13 Identified transport stressors are duration of transport, separation from familiar conditions, container and vehicle design, food and water supply and driver attitude. ¹¹ Therefore, transport stress can be minimized by limiting transport time, minimizing animal numbers per transport, considering species-related environmental conditions and ensuring that all staff are properly trained. ¹¹ Transport stress can be avoided by using facility-bred animals with laboratory adjacency. Numerous features of the laboratory environment will be entirely unfamiliar to most animals sourced outwith research facilities (except, perhaps, for purpose-bred minipigs). Animals will have been reared under conditions ranging from intensive, densely stocked indoor systems (pigs) to extensive outdoor ranges where animals (sheep) will have had little human contact. This contrasts with the regulated, usually indoor, laboratory conditions in which most environmental features are legally prescribed, closely managed, but nevertheless unfamiliar: the new environment, diet, bedding, lighting regimes, temperature and humidity, drinking facilities, pen- or room-mates and the (human) care and scientific staff with which they will interact.

Acclimatization

Acclimatization allows out-sourced farm animals to adapt to their new environment, achieving physiological, psychological and nutritional stabilization. Its specific purpose is to allow time for the neuro-endocrinological changes arising from transport stress and neophobia to resolve, so that animals may be enrolled on study as soon as possible. During acclimatization some baseline physiological and behavioural variables such as food and water intake can be determined; relevant biological samples can be collected; and animal health confirmed before study. Importantly, successful acclimatisation is a prerequisite to subsequent familiarization, habituation and training, if necessary. Successful habituation and training, in turn, may result in animals allowing some procedures to be conducted with minimal physical or pharmacological restraint. (In these recommendations, the term ‘pharmacological restraint’ is used to mean any drug or drug combination, e.g. tranquilizers, anxiolytics, sedative-hypnotics, anaesthetics and analgesics, that are used to reduce, ideally preclude, the need for (and stress of) physical restraint and allow scientific procedures to be carried out.). When sedation or general anaesthesia are unavoidable, minimal stress levels are still necessary because anxiety is a lethal risk factor in medical anaesthesia. ¹⁴ Consequently, acclimatization, habituation and training can be seen as major contributors to experimental refinement, which is in accordance with European legislation protecting animals in research. ¹ Finally, the ARRIVE 2.0 guidelines (Animal Research: Reporting of In Vivo Experiments) ask for a description of ‘Welfare-related assessments and interventions that were carried out prior to, during, or after the experiment’ in the Essential 10 list, Section 9c. This supports the notion that reporting details of acclimatisation, habituation and training contributes to experimental reproducibility. ¹⁵

Acclimatization methods significantly affect study outcomes ⁷ so must be carefully considered. Its fundamental advantages, i.e. stress reduction, optimized animal welfare and data quality, outweigh the main disadvantages (increased costs from delayed study onset and increased time commitment from scientists and animal facility staff) and support the recommendation that all animals undergo acclimatization.^7,16 However, methods and duration will depend on numerous factors, with ‘source’, along with species, age, sex, breed and function, probably being the most important. It seems intuitive that laboratory-bred minipigs will require less acclimatization than freshly weaned growers from a commercial herd, while a laboratory-bred sheep will not require the intense, possibly futile attempts to acclimatize an aged ewe which has never been housed. Other factors determining the elements of acclimatization include the nature and duration of the experimental work (experimental outputs) and recovery versus non-recovery procedures. The feasibility of acclimatisation depends on the laboratory infrastructure and the experience of the personnel involved. People competent in acclimatizing and/or restraining animals are important in minimizing the stress associated with human interaction ¹⁷ and so represent experimental refinement. Therefore, they should be recruited as a matter of priority. Acclimatization also allows the collection and analysis of baseline blood, urine and other samples to facilitate the later interpretation of treatment effects. Acclimatisation should last until its pre-defined objectives are met and there is confidence that the study begins with each animal being biologically comparable and all animals being at similarly low levels of stress.

Ideally, animals would be acclimatized, habituated and trained as promptly as possible, to undergo all necessary procedures without physical restraint or the need for drugs affecting scientific outcomes. This idyll is unrealistic and so study planning must establish beforehand the extent to which physical and/or pharmacological restraint will be necessary (practically) and permissible (ethically, legally and scientifically). While the primary goal of acclimatization is always to reduce stress to levels which will not obfuscate scientific outcomes, the methods used and periods they are applied for will depend on the predicted cost: benefit ratio of using options involving physical and/or pharmacological restraint. In the final analysis, habituation and especially training are time-consuming and expensive, while results may be variable and not always sustainable; adverse (noxious) experiences can rapidly reverse an animal’s procedural cooperativity.^18,19

Familiarization and socialization

Familiarization and socialization are important elements of acclimatisation. Familiarization involves exposing animals to all personnel which they will interact with while on study. This includes those involved in cleaning and feeding, as well as those conducting procedures. The goal is to reduce fear, stress and anxiety and make animals comfortable with the proximity and actions of humans without necessarily altering their social behaviours. The advantages of familiarization are fear reduction, improved handling and improved welfare. Familiarization is achieved by exposing animals to frequent positive interactions, i.e. repeated gentle contact, gentle stroking, talking softly and offering food rewards.

Socialization, an extension of familiarization, actively encourages animals to engage socially with relevant laboratory personnel to produce affiliative behaviours and interactions. Socialization seeks to promote positive social behaviours in animals and develop relationships and interactions that go beyond mere tolerance. Human–animal socialization is particularly desirable in studies which may have aversive physiological, e.g. pain, or psychological outcomes. Subtle post-procedural behavioural changes are likely to be more apparent in socialized animals, which will facilitate pain (and severity) assessment.

Habituation

Habituation describes an animal’s behavioural adaptation to repeated or constant stimuli, resulting in a diminished response over time. This form of non-associative learning enables animals to filter irrelevant information and allocate cognitive resources more efficiently. Unlike acclimatization, which is primarily physiological, habituation is a behavioural process that allows animals to ignore non-threatening stimuli or those irrelevant to survival. Habituating animals to non-aversive activities such as thoracic auscultation, rectal temperature monitoring or ultrasonic examination, while time-consuming, will yield more representative data than the same examinations conducted using physical and/or pharmacological restraint. Habituation is necessary when minimizing the effects of human presence is critical, e.g. for unbiased observations of natural behaviours. It should be noted that animals will not habituate to extreme treatments. ‘Flooding’, i.e. where an individual is acutely exposed to potentially aversive stimuli at full intensity, without any gradual introduction, can be highly distressing and has no place in laboratory animal habituation.

Training

Training is an intentional, structured process in which animals associatively learn to enact specific behaviours through repeated exposure to cues and rewards. Unlike habituation, which involves a generalized reduction in response, training focuses on eliciting specific, project-focused behaviours. Positive reinforcement, using, for example, high-quality food rewards, is commonly used in training to reinforce the learning of behaviours that are beneficial for both the animal and laboratory staff. While animal training is a specialist activity and beyond the scope of this document ²⁰ information is available elsewhere for training laboratory pigs and sheep.^14,21

Separation or isolation studies

Isolation, or single housing of animals after periods of group housing, causes agitation, anxiety and is extremely stressful in the social species, i.e. pigs, ²² sheep, ²³ goats, ²³ calves ²⁴ and cattle ²⁵ so must be avoided unless there are compelling scientific, veterinary or practical justifications. Sexually mature boars are naturally solitary animals making single housing less important.

Non-recovery procedures

The need for acclimatization may be challenged when animals reared ‘off-site’ are required for non-recovery studies in which stress variables are unimportant. In these circumstances, anaesthetizing animals as soon as they reach the facility avoids problems with alternative stressors, e.g. prolonged solitary housing or mixing with ‘local’ animals. Mixing unfamiliar animals (of any species) upon arrival at research facilities should be avoided because the ensuing disruption of established social hierarchies causes stress.^26,27 Whether such animals are delivered with a travelling ‘companion’ demands consideration. The benefits of pairing, which are likely to be greatest when long distances are involved, will be negated if the second animal is superfluous to study requirements, because it must either be killed or undergo the stress of being single. This option should only be considered when animals are obtained from trusted farms with high health status and reliable health records, which are located within brief transit times from the facility and when the experiment’s scientific outcomes are unaltered by transit stress. The risk of anaesthesia complications in animals with transport stress may be reduced by ensuring pre-anaesthetic medication contains anxiolytic as well as sedative components and delaying induction until their effects are apparent.

Telemetry

Telemetry allows the continuous collection of physiological data from numerous animals simultaneously, e.g. control and treatment animals, which is unaffected by the confounding effects of human presence. Invasive (surgically implanted) and non-invasive (collars; jackets) technologies are available for pigs, sheep and calves. The pros and cons of telemetry in laboratory (but not farm) animals have been reviewed elsewhere.^40,41

Thermometry

Microchip and infrared thermometry can be used to measure body temperature in pigs, provided baseline readings and correction factors are established. The former can also be used in sheep and goats. Both methods provide consistent measurements in individual animals and populations over time. ⁴²

Metabolism cages

Metabolism cages have been used for qualitative and quantitative studies requiring the simultaneous collection of urine and faeces while monitoring food and water intake. However, they severely limit mobility and invariably require animal isolation for extended periods (>12 hours). These conditions are highly stressful and while this may be ameliorated by acclimatization, they may nevertheless compromise data quality. The advantages of metabolism cages probably do not justify these disadvantages. Alternatives to metabolism cages that require individual housing but allow for olfactory, visual, acoustic and tactile contact are available and should be used in preference where animal isolation is complete.

Vascular access ports

Surgically implanted vascular access ports (VAPs) greatly facilitate repeated blood sample collection, blood pressure monitoring and infusions. Their use as a safe and efficient method for obtaining reliable chronic vascular access in farmed animal species has been described.^{43 -46} Vascular access buttons achieve similar goals but obviate the need for (potentially painful) skin puncture.

Urine collection

Collecting free-flowing urine samples with a paper dish is facilitated by training and species-specific manoeuvres. For example, activating the nipple on drinkers can induce urine flow in recalcitrant pigs, ⁴⁷ while inducing transient apnoea promotes urination in sheep.

Cerebrospinal fluid sampling

Cerebrospinal fluid (CSF) sampling can be achieved on a single occasion by lumbar puncture in sedated animals ⁴⁸ or repeatedly using surgically implanted intrathecal catheters in the cisterna magna or cerebral ventricles.^49,50

Behavioural studies

Digital photography and/or video recording provide data for behavioural studies in free-moving animals in familiar environments and are additionally useful for postoperative observation. ⁵¹

Behavioural preparation

Pigs are intelligent and have advanced problem-solving abilities so are good candidates for behavioural preparation. They learn from their environment and readily adapt to new situations. They can manipulate objects to obtain food or solve challenges; cognitive stimulation is important for their well-being. ⁵² Training should be initiated during the acclimatization period and continued throughout project-specific treatments for the study’s duration.^26,36,50 A two-week, four-step habituation and socialization process facilitating urine and blood sampling (via implanted cannulae) and abdominal ultrasonography has been reported. ⁴⁷

While behavioural preparation is facilitated by optimized environmental conditions, the difference between basic animal needs and environmental enrichment must be recognized. Inadequate bedding can cause hyperactivity, aggression, excessive use of drinkers and the repetitive banging of pen panels; adequate bedding is, therefore, a necessity. ⁵³ Deep bedding satisfies the pig’s drive to burrow (root), prevents stress-related stereotypic behaviours and facilitates handling, but does not necessarily constitute enrichment. ⁵⁴ Formalized methods of scoring the progress of acclimatization, familiarization, habituation, socialization and training have been described in pigs. ⁵⁵

Isolation

Pigs with procedural accoutrement, e.g. external cannulae and stomata, will attract destructive investigation by pen-mates, so must be separated. Under these circumstances, animals should be habituated to both implants and separation beforehand. When individual housing is required, it is important to ensure that visual, olfactory, auditory and limited tactile access to conspecifics is allowed. This may be achieved using bar-separated pens, ‘snout holes’ in (ideally) transparent, i.e. Perspex walls and/or periodic supervised access to walkways allowing ‘visits’ to adjacent occupied pens. Regular and frequent rewarding human contact can serve as a substitute for social housing.^34,39 Pleasant interactions, such as positive reinforcement, e.g. patting, scratching, rubbing and offering high-quality food rewards, should be implemented during this period. Environmental enrichment stimulating exploratory behaviours, i.e. new objects, manipulation (prehensible ‘toys’) and burrowing (deep straw) must be present in ‘separation’ pens.

Physical restraint

Pigs can resist physical restraint energetically although this depends on the method and the animal’s age, breed and training. ⁵⁶ Pigs can remember human behaviour in terms of negative ⁵⁷ or positive events. ⁵⁸ This influences their reactions to humans and so affects their welfare. Older, habituated pigs react less violently than younger, less well-handled animals. Unsympathetic physical restraint has been reported to cause traumatic liver lesions in neonatal Göttingen minipigs.^38,39,59 Pigs communicate through different vocal and olfactory signals and body language. Grunting, squealing and other vocal expressions convey emotions and intentions. It is highly recommended that these are taken to guide the proper use of restraint methods in pigs.

Common physical restraint methods in pigs, e.g. ‘snares’, ‘snitches‘ or ‘snatches’, should only be used when absolutely necessary because they may cause greater pain than the scheduled procedure as well as acute and chronic stress.^19,39,60,61 The person snaring should be trained and competent and the snare should be purpose-designed. Enforced recumbency in ‘V-troughs’ is similarly condemned.

Limiting movement in laboratory pigs for innocuous, very brief procedures, e.g. rectal thermometry and intramuscular injections, is best achieved using familiar devices, e.g. weighing crates or (for penned animals) pig (driving) boards with or without positive reinforcement (offered food also provides a useful distraction). Pigs can be distracted with ‘forking’: firmly scraping the animal’s back with a fork or a back scratcher elicits pleasure responses.

For similar procedures in animals <10 kg, capture and brief suspension by the pelvic limb followed rapidly by thoracic support allows the horizontal animal to be wrapped in towel and embraced firmly with the lower jaw supported, i.e. as for dogs.^62,63 However, the duration of the suspension stage must be minimized: it places the rectus abdominis muscle under tension promoting umbilical herniation. ⁶⁴ Göttingen minipigs weighing 8 kg have been habituated successfully to slings for blood sampling. ⁶⁵

Larger pigs can be restrained in proprietary purpose-built slings^34,38,39,63 or crushes ⁶⁶ although device selection is important: poorly designed or ‘generic’ devices have caused epistaxis, bleeding gums and bruising and must be avoided in pigs with bleeding disorders. ⁶⁷ Complications are less likely with equipment purposefully designed for immobilization and procedures such as blood sampling, physical examination and substance administration (intravenous and oral). ³⁹ Devices for the humane immobilization of pigs should support the head and neck, be of robust construction and be readily cleaned and disinfected. Slings must allow access to specific body areas when physiological instrumentation is in situ, for special procedures such as abdominal ultrasonography, blood sampling or the administration of medications. The design must consider the animal’s height, length and weight, its limbs, the external genitalia of male animals and the animal’s need to urinate and defaecate. It is highly recommended that pharmacological restraint be used for noxious procedures in pigs of all ages.

Urine collection

Foley catheters placed under deep sedation can be used for repeated urine sampling in female pigs, but the pig must be kept in an individual cage until the end of the sampling period to avoid interference from other pigs. ⁶⁸

Behavioural preparation

● Highly recommended: Mixing unfamiliar pigs disrupts social hierarchies and results in post-mixing aggression. If mixing is unavoidable, it should be phased and involve pigs of minimal age and size differential as soon as possible following legal weaning times, i.e. 3 weeks. It should be carried out early morning allowing trained personnel to be present (and to intervene) during the highest risk period or in the evening (before lighting is discontinued). Mixing pens should be large, contain visual barriers (allowing escape space for subordinate animals) and enriched, i.e. toys and straw. The use of olfactory distractants and/or anxiolytic drugs, e.g. azaperone or acepromazine, may be considered.

Physical restraint

● Highly recommended: The use of snitches (‘snares’ or ‘snatches’), V-troughs, ear suspension or prolonged limb suspension (not >5 seconds) must not be used in laboratory pigs.

● Highly recommended: Limiting movement in laboratory pigs for innocuous, brief procedures, should be achieved using familiar devices, e.g. weighing crates or pig boards with or without positive reinforcement.

● Recommended: Pigs should be habituated to slinging if these are to be used post-procedurally. Slings for heavier pigs should be customized to meet individual pig and procedural requirements.

● Recommended: Younger (smaller) animals and minipigs should be wrapped in towels and embraced, with the lower jaw supported, or held firmly in a manner like that in dogs.

Pharmacological restraint

● Highly recommended: Pharmacological restraint should be used for noxious procedures in pigs of all ages. Pigs should be sedated for all potentially stressful non-invasive, e.g. radiography (in untrained, non-standing animals), or minimally invasive, e.g. venous cannulation, procedures.

Behavioural preparation

Being flock animals, sheep have a strong instinct to form cohesive groups, which provides a sense of security, and under natural conditions, aids in protection against predators. Flock behaviours, i.e. a tendency to follow a leading animal or flee en masse, are seen in groups of four or more animals, and account for sheep becoming distressed when isolated or separated from companions, although this depends on breed and age.^{69 –74}

Minimizing stress in laboratory sheep requires that aversive procedures are avoided until animals are acclimatized. ⁷² Animals obtained from commercial, extensively husbanded flocks in particular require habituation to human contact and handling. ¹⁹ Laboratory sheep benefit from environmental enrichment which promotes the diversity of natural behaviours while reducing stereotypies and aggressive behaviours. ²⁷ The strong escape proclivity of sheep can be reduced by space restriction, although this itself requires habituation. ⁷⁵

Enforced separation from the flock or familiar conspecifics is ameliorated by the presence of at least one other familiar animal. The effects of repeated blood sampling on haematological variables in sheep, which are well-recognized ⁷⁶ are reduced by providing ad libitum food and water and the presence of a social partner.^18,77

Sheep are readily trained: positive anticipation training using food rewards to conditioning behaviour is particularly useful. ⁷⁸ Sheep can be trained to voluntarily enter a restraint device when they can move as a group and procedures are repeatedly imposed.^35,72 Positive events, e.g. handling or wool brushing reduces wariness of humans, although this effect may not be transferable to all situations. ⁷⁹

Reviews of methods assessing behavioural reactions to handling and restraint in sheep^18,26,72 generally affirm that the degree and signs of evoked fear depend on how the animal perceives handling (or transport). ¹⁹ This may be useful in quantifying the success of acclimatization before animals are enrolled in experiments. An interaction between oxytocin release, sheep–human bonding and stress responses has been identified.^80,81 This supports the pre-experimental acclimatization of sheep. ¹⁸

Physical restraint

Under farm conditions, many potentially aversive procedures, e.g. shearing, ‘drenching’ or foot trimming, are performed with the animal restrained manually by being forcibly positioned upon its tuber ischii or ‘pin-bones’. Less commonly, restraining booths may be used. These and other procedures, e.g. dipping, are less aversive when conducted briskly and en masse. ⁸²

In certain circumstances, minor procedures can be conducted in standing sheep with minimal physical restraint and without drugs. Accessing intrathecal catheters at the lumbar reservoir or the cisterna magna for CSF sampling is possible with mild physical restraint alone.^77,83 Access to implanted sampling devices is also facilitated in non-sedated animals when they are confined in a familiar, but size-restricted pen. ⁸⁴

Most sheep are accustomed to enforced restraint upon their ‘pin-bones’. Consequently, injections can be made, and blood samples taken without sedation in animals accustomed to and restrained in this way. ⁸⁵

Sheep may be restrained in canvas slings, the height of which allow the hooves to be in contact with or suspended above the floor. ⁷⁷ Slings may be useful postoperatively in supporting recovery from painful or prolonged surgery as they facilitate breathing and intestinal movement until the animal is able to stand unaided. Habituation to slinging is advisable to avoid adverse reactions, e.g. anxiety and struggling.^86,87

Commercial sheep chairs are available and make non-invasive imaging techniques, e.g. ultrasonography, possible without sedation^82,88 although the results may be confounded by the accompanying tachycardia, tachypnoea, vocalization and/or regurgitation.^82,89,90 Refined methods for cardiac ultrasonography in standing sheep have been described and suggest an abducted limb is more acceptable than standing with the thoracic limb extended. ⁸⁸ Restraining booths may be used in laboratory sheep, but care is necessary when the risk of accidental de-instrumentation exists.

Introduced in the 1980s, proprietary electro-immobilization devices proved to be more aversive in sheep than physical restraint. ¹⁸ They have no place in laboratory animal restraint, and their use is banned in the UK and EU member states.

Telemetry

Telemetric monitoring of foetal haemodynamic variables in pregnant sheep allows unrestricted movement in pens equipped with several telemetry receivers. ⁴⁵

Similarly, respiration chambers allow free animal movement during experiments of several days’ duration.

Metabolism cages

The combination of respiration chambers with metabolism cages allows sampling without the need for further restraint although prior acclimatization is required^{91 –94} and absolute isolation is condemned.

Behavioural preparation

● Recommended: Heavily fleeced sheep should be sheared to reduce risk of inexpert physical restraint (see below) and heat stress and to improve clinical observation, e.g. breathing rate and pattern.

Physical restraint

● Highly recommended: Sheep may be physically restrained in a standing position or on their tuber ischii. Sheep must not be restrained by holding and/or pulling the fleece.

● Recommended: Physical restraint with local anaesthesia is suitable for blood sampling and short-term venous cannula placement.

Miscellaneous

● Highly recommended: Heavily fleeced animals should be sheered on admission to the facility if they are to be housed indoors.

Behavioural preparation

Most bovidae involved in research are young, i.e. calves. In these, separation from the dam, and later, conspecifics are major psychological stressors. ¹⁹ Early weaning and individual housing have profound effects on the calf’s physical and psychological development. ⁹⁵ Environmental enrichment of individual housing has minor or insignificant benefits for calves, compared with the presence of other calves. ⁹⁶ Paired housing systems promote greater daily feed intake and if calves are joined in pairs from birth, rather than a few days later, they exhibit fewer behavioural disruptions caused by early weaning. ⁹⁷

Calves are easier to handle and become less stressed if habituated and trained, i.e. handling occurs during acclimatization. Calves accustomed to handling have lower cortisol levels after restraint than those that have less-frequent human contact. ⁹⁸ Younger, bottle-fed calves acquired from the dairy herd at 2–2.5 months of age are easier to habituate than older calves. ⁹⁹

Physical restraint

Physical restraint of the standing calf, with or without halters or manual head restraint, is appropriate in adequately acclimatized and habituated calves for minor, brief non-invasive procedures such as physical examination, postoperative wound care, blood sampling from permanent central venous cannulae, etc. For more invasive procedures, the use of local or systemic analgesia decreases acute pain and is strongly recommended.

Commercially available devices are available for prolonged restraint. One ¹⁰⁰ involves a mobile calf pen with a hydraulic lifting device. In this, calves wear a sling of reinforced nylon webbing with padded openings for the extremities and an adjustable central opening to prevent pressure on the abdomen. The pen is designed so that calves can stand or lie down at will or be restrained in an upright position when necessary.

Older male bovidae are seldom involved in research but require particular care when they are. They often develop aggressive tendencies, especially if entire, and despite behavioural preparation. Experienced staff and purpose-built handling facilities are necessary. More robust restraint methods, i.e. crushes will be required in these to reduce the risk to personnel conducting even innocuous procedures.

While young cattle are best housed in groups their play and exploratory behaviours may result in displacement of unprotected external instrumentation, e.g. central venous cannulae.

Telemetry

Non-contact monitoring does not affect cattle behaviour or induce stress, so is particularly useful for diagnosing respiratory disease. ¹⁰¹ Telemetry has been used in calves in hemodynamically complex diseases such as pulmonary arterial hypertension. ¹⁰²

Behavioural preparation

● Highly recommended: Growing calves should be habituated to physical restraint. Adult cattle should be habituated to head-yolks and ‘crushes’.

Physical restraint

● Highly recommended: Calves should be restrained in the standing position for pain-free procedures such as clinical examination. Adult cattle should be restrained using head-yolks or ‘crushes’. In bovidae of all ages, physical restraint with local anaesthesia should be used for vascular port sampling, blood sampling and short-term venous cannula placement.

Behavioural preparation

Goats are renowned for their exploratory and climbing ability (with the latter reflecting the former) arising from their tendency to forage rather than graze. Consequently, goats are regarded as more curious, quicker-to-learn ¹⁰³ and easier to handle than sheep and so are easier to acclimatize although their behaviour may be less predictable.^104,105 Laboratory goats are usually group-housed under environmentally enriched conditions ¹⁰⁶ which promotes their learning performance and confers other behavioural benefits. ¹⁰⁷ Mixing unfamiliar goats can promote stress and impair welfare for up to 5 days. ¹⁰⁸ Horned animals require special attention because of the risk of injuries. ¹⁰⁸ Visual, auditory and tactile contact with group members should be maintained when goats are physically separated from pen-mates in order to facilitate reintroduction. ¹⁰⁹ The behaviour and well-being of goats depends on their relationship with animal care technicians and upon previous experiences with human beings.^105,110,111 Goats readily habituate to experimental conditions, e.g. the wearing of belts for body function measurements^103,112 although learning experimental procedures in a group, or introducing an experimental setup into the animals’ familiar environment can limit anxiety and excitement. ¹¹²

Physical restraint

Restraint methods commonly used in sheep, e.g. physical restraint by hand, harness or holster, are suitable for standing goats. ¹¹³ In studies in which human presence is ideally minimal, goats are restrained when necessary by familiar handlers. ¹¹⁴ Some studies, e.g. respiratory gas measurements, can be completed without restraint although animals should be acclimatized to measurement conditions for 2–3 days beforehand. ¹¹⁴ Cardiac ultrasonography can be conducted on unsedated standing animals after habituation to the experimental conditions. ¹¹⁵ Access to previously implanted sampling devices, e.g. intrathecal cannulae, is possible in unsedated standing goats when repeated sampling of CSF fluid is required. ¹¹³ Slings can be used to facilitate husbandry procedures such as hoof trimming. The successful use of a sling is reported in a study on hip arthroplasty when the sling was used for preventing goats from lying down. ¹¹⁶

Telemetry

Implantable telemetry devices allowed the 24-hour recording of heart rate and blood pressure in the same unrestrained animals and to identify the effects of routine manipulations and reproductive cycle. ¹¹⁷

Behavioural preparation

See all species (above).

Physical restraint

● Highly recommended: Goats can be restrained in the standing position for examination. Physical restraint with local anaesthetics should be used for blood sampling and short-term venous cannula placement.

Anatomy

The superficiality of jugular veins makes central vein cannulation straightforward in unsedated calves and sheep compared with pigs. Differences in lumbosacral spinal cord anatomy will affect techniques for extradural and spinal injection in farmed animal species. The presence of a right accessory bronchus in pigs demands attention to tracheal tube length.

Ethology

Pigs react to physical restraint more violently than small ruminants and calves, which justifies the use of intramuscular sedative drugs before attempted venepuncture.

Physiology

The functional rumen which develops in growing calves, lambs and kids creates problems, e.g., ptyalism, tympany, regurgitation-aspiration, that are seldom encountered in monogastric species. Preoperative food and water deprivation requires greater consideration in ruminants than in suidae.

Pharmacology

Inter-species differences in drug sensitivity and dosing are drug-dependent. The minimum alveolar concentration (MAC) of volatile anaesthetics varies marginally when compared with responses to α₂ agonists. ²⁰⁰ There are also absolute differences: paracetamol causes methaemo-globinaemia in pigs, but not sheep. ²⁰¹ There are major differences between sheep and pigs in their response to neuromuscular blocking agents (NMBAs). ²⁰²

Age

Very young or aged animals are generally believed to be more sensitive to fixed drug doses because of a reduced clearance capacity. Age-related changes in body composition introduce similar pharmacokinetic variability. The central nervous effects of anaesthetics are age-dependent in other species.^204,205 Anaesthetic equipment designed for adult human use which is suitable for size-relevant pig and sheep models may cause critical problems if used in minipigs, piglets or lambs.

Sex

Sex differences in drug biotransformation and pharmacokinetics have been identified in sheep ²⁰⁶ and pigs ²⁰⁷ but their relevance to anaesthesia is unknown. Catheterization of the urinary bladder via the urethra is not possible in male sheep nor male pigs, so direct bladder catheterization is required if urine output is to be measured.

Temperament

Animals acclimatized to the laboratory will generally be more sensitive to fixed anaesthetic doses compared with non-acclimatized animals.

Size

Allometric principles are well-recognized and applied in small laboratory animal species ²⁰⁸ although their clinical relevance in farmed animals is less well-known. However, basic thermodynamic principles predict that heat loss during anaesthesia is an inverse function of body size so anaesthetics promoting this will have a disproportionately greater effect on body temperature in smaller individuals. Consequently, ambient (laboratory) temperatures are of greater importance when anaesthetizing lambs, piglets and minipigs compared with adult or larger animals.

Breed

Breeding affects temperament in sheep and pigs, but whether this extends to anaesthetic sensitivity is unknown. Ethnicity affects anaesthetic requirement in human beings ²⁰⁹ while sheep breeds differ in their sensitivity to xylazine^210,211 which may have a genetic basis. It seems possible that the inbred docility of some laboratory breeds, e.g. the Göttingen minipig, renders them more sensitive to sedatives than less well-handled commercial pig breeds. Breed predisposition of pigs to malignant hyperthermia, a condition triggered by stress, as well as specific anaesthetics, is a well-recognized pharmacogenetic problem in veterinary anaesthesia.

Health status

Unhealthy animals, including genetically modified forms with morbid phenotypes, are seldom involved in studies because the underlying pathology may confound study outcomes. When this is not the case, greater care is needed in anaesthetizing sick animals because anaesthetic risk is increased according to the nature and extent of the disease present.

Habitus

Extremely obese or lean animals should not be enrolled on study unless these states are induced as a deliberate study feature. The pharmacokinetic behaviour of anaesthetics may be affected by extremes of subject habitus and will have secondary effects on dosing and/or drug selection.

Medication

Seemingly innocuous medications, e.g. antibiotics, may adversely interact with anaesthetics and must be considered when techniques are selected. ²¹²

Pregnancy

The potentially abortifacient effects of α₂ agonist drugs ²¹³ and glucocorticoids ²¹⁴ should be considered in studies involving pregnant animals. The use of non-steroidal anti-inflammatory drugs (NSAIDs) may also affect experimental results in pregnant animals. ²¹⁵

Source

Whether an animal was obtained from a commercial or institutional farm, a laboratory animal breeder, or was ‘home’ (laboratory) bred probably plays an understated role on the animal’s responses to anaesthetics. Laboratory-bred animals will be more familiar with the environment than animals from commercial sources and so less anxious, making them more sensitive to drugs with sedative properties. ⁶

Duration

Ceteris paribus, brief, non-invasive procedures justify the use of both short-acting drugs and non-invasive, rapidly applied (though less accurate) methods of physiological monitoring. The exclusion of some support measures, e.g. intravenous fluids, may also be justified. In procedures of indeterminable duration such as certain types of imaging, drugs allowing rapid recovery despite prolonged administration should be chosen, e.g. volatile anaesthetics.

Invasiveness

The extent of procedural tissue damage is a determinant of anaesthetic risk, postoperative pain and severity level estimation. More invasive procedures justify more extensive physiological monitoring during anaesthesia and recovery, there being a greater need for awareness of nocistimulation during anaesthesia (and pain thereafter), haemorrhage and volume replacement, and thermoregulation. The actual tissue damage incurred during a procedure determines the frequency and degree of postoperative care required.

Precision

Unexpected movement and/or haemorrhage complicates precision surgery. Irregular breathing movements may be controlled with positive pressure (lung) ventilation (PPV). In general, and when permitted, NMBAs are used to prevent reflex movement and muscle tone. ²¹⁶ Measures taken to minimize haemorrhage in precise procedures conducted under surgical microscopy are based on surgical site positioning and in special circumstances, hypotensive anaesthetic techniques.

Repeats

Repeating surgery at the same site has two consequences for anaesthetic management: (i) scar tissue formation and (ii) peripheral and central pain sensitization. The presence of scar tissue and adhesions will complicate and prolong subsequent surgeries with corresponding demands on anaesthetic management. This, and the possibility that nociceptive pathways have been ‘sensitized’ during previous operations, will necessitate increasingly aggressive perioperative pain management on subsequent interventions. Repeatedly mismanaged anaesthetics and/or postprocedural pain will contribute to major cumulative changes in an animal’s extended welfare assessment grid. ¹⁰

Operation site

The operation site affects anaesthetic management in specific and general ways. For example, thoracic procedures require PPV, while endocardial procedures will require ECG monitoring and the availability of anti-arrhythmic drugs. Visceral exposure during laparotomy and thoracotomy will predispose to hypothermia. Unintentional surgical damage to large blood vessels may necessitate rapid volume restoration while inadvertent nerve traction may initiate unexpected motor responses, e.g. movement or parasympathetic nervous activity in the case of direct vagal stimulation. Regarding analgesia, tissues vary in the level and type of sensory innervation which may explain why certain procedures are associated with a greater risk of developing chronic post-surgical pain than others. ²¹⁷ Certain body areas are amenable to complete desensitization using local anaesthetic techniques, e.g. the distal antebrachium is anaesthetized after brachial plexus block.

Procedure

The effects of procedure on the pharmacodynamic/pharmacokinetic (PK/PD) profiles of anaesthetics must be considered and modifications made as necessary. Normal doses of NMBAs that rely on renal or hepatic clearance may prove excessive after transplantation procedures, which reduce renal or hepatic function.

Non-recovery procedures

While adequate anaesthesia and intraoperative analgesia must always be prioritized the need to prepare for acute and/or chronic post-procedural pain is unnecessary in animals scheduled to be terminated before recovery occurs.

Pain control

The broad goals are to minimize pain, suffering and distress using general and/or local anaesthesia and/or analgesia (or, enigmatically, other ‘appropriate’ methods). Anaesthesia must be used in procedures involving serious [sic] injuries that may cause severe pain, and analgesics must be used if postprocedural pain is likely after recovery from anaesthesia. Disconcertingly, these mandates are conditional with scenarios given where anaesthesia and/or analgesia may be withheld.

Conditions of use

Anaesthetics need not be used when: (i) anaesthesia is judged to be more traumatic [sic] to the animal than the procedure itself; or (ii) anaesthesia, and postoperative analgesia are incompatible with procedural objectives. A strong scientific and ethical argument is required to withhold anaesthesia and/or analgesia. The Directive also requires that drugs stopping or restricting an animal’s capacity to show pain, presumably NMBAs, must not be used unless adequate levels of anaesthesia or analgesia are assured and that a scientific justification and details of the anaesthetic or analgesic regime have been provided. The A(SP)A 1986 is more meticulous in defining conditions for NMBA use (see below).

Staff competence

This relates to the requirements of both authorities that staff shall be adequately educated and trained before they design and/or carry out procedures on animals and/or take care of animals. The Directive of National Competent Authorities for the implementation of Directive 2010/63/EU ¹ specifies learning outcomes that are instrumental in defining both theoretical and practical competences and skills as well as the species-specific knowledge needed to perform these functions. Before carrying out these functions, staff must be supervised until they have demonstrated the requisite competence. This process will normally involve animal-free training in the theory and fundamentals of anaesthesia followed by adequate periods of supervised practical application in the defined species. In conjunction with continuing professional development, acquired skills can develop if they are applied frequently and revised, when necessary, after critical evaluation. The A(SP)A also mandates that staff possess personal licenses (PILs) which are granted on completion of formal modular training designed to meet the requirements of six procedural categories, three of which are defined in terms of the necessity for sedation, analgesia or general anaesthesia. Both A(SP)A and the Directive recognize that training and supervision are ongoing processes that require regular review, especially before implementing new knowledge such as the use of new anaesthetic or analgesic agents or methods.

However, these legal requirements have two limitations. First, they fail to allow for training in the management of unexpected, relatively uncommon but potentially fatal incidents which may occur in the course of anaesthetising sufficient numbers of ‘normal’ animals. Second, they do not accommodate anaesthetic challenges arising, for example, when new genotypes are to be anaesthetized for the first time, or when studies involving unfamiliar procedures or treatments are planned. In medical anaesthetic practice, simulation training plays a major role in resolving the first limitation, and the Royal College of Anaesthetists (RCoA) sponsor advanced initiatives in this area. Nothing equivalent exists in veterinary anaesthesia, although elements of the RCoA’s initiatives could be adapted to train uncommonly needed but life-saving skills in laboratory animal anaesthesia. The second problem, albeit uncommon, poses a conundrum: the law requires staff to be adequately educated and trained before they conduct procedures, i.e. to anaesthetize animals, but does not explain how this is accomplished when those procedures, being experimental, have never been conducted before. Similarly, one cannot be adequately trained to anaesthetize genetically modified animals if that phenotype has not previously been anaesthetized. This conundrum is solved, i.e. competency is gained, and failures minimized, by adopting an iterative process managed by highly qualified staff applying early endpoints (when needed) to ensure animal welfare. Learning through a ‘trial-and-error’ training programme with unqualified supervision is irresponsible and condemned.

In all circumstances, it is a legal imperative that measures are taken to eliminate the gap between the skills available and the skills required. The measures required depend on the size of the skill differential, the magnitude of the challenge and the availability (and cost) of qualified assistance.

Depth of anaesthesia

Monitoring the ‘depth’ of anaesthesia through the evaluation of cranial nerve reflexes, ocular position and other signs assists in making general estimates about: (i) the animal’s likely response to surgical stimulation; and (ii) the level of physiological depression present. However, measuring important physiological signs directly, e.g. arterial blood pressure and minute volume of ventilation, are the definitive indicators of the need for support, rather than the presence and briskness, or absence of specific, potentially arbitrary reflexes. DOA monitoring in pigs, sheep, goats and cattle is described in most authoritative anaesthesia textbooks, e.g. Hall, Clarke and Trim’s Veterinary Anaesthesia.^{125 -127}

Modern multiparameter anaesthetic monitors incorporate modules measuring inspired and expired concentrations of anaesthetic vapour. End-tidal concentrations do not directly represent anaesthetic depth but are useful in comparing anaesthetic uptake with the vaporiser settings. Knowledge of the MAC of an inhaled agent is essential when interpreting this information and is especially useful: (i) when partial intravenous anaesthesia is used to decrease the requirement for inhaled agents; and (ii) when NMBAs are used.

DOA monitors transforming surface electroencephalographic (EEG) signals or auditory evoked potentials have become available, although their role has yet to be established in recommending monitoring standards for human subjects. This holds true in animals, where monitoring central nervous system depression directly and accurately is difficult. The EEG, or EEG-based derivates of cortical activity, e.g. the bispectral index (BIS), are of limited use in animals because the human-based algorithms do not accommodate, amongst other things, the variation in calvaria anatomy, skin thickness and hair coverage found in animals. That said, the value of BIS monitoring increases when neuromuscular blockade obviates the use of reflexes commonly used to assess anaesthetic depth, e.g. the palpebral reflex. The influence of anaesthetics on the EEG (and derived outputs) in pigs, sheep, goats and cattle have not been fully characterized.

Vital signs

Vital signs, i.e. circulation, ventilation, oxygenation and body temperature, can be continually evaluated using observation, palpation and auscultation. Continuous monitoring, which is strongly recommended, necessitates constant anaesthetist–subject contact, which may be impractical. Electronic monitors are capable of continuous monitoring and recording, but (thus far, and unlike anaesthetists) largely incapable of integrating information from the most important organ systems, i.e. the nervous, cardiovascular and respiratory systems.

Heart rate

The heart and/or pulse rate can be: (i) observed and palpated as an apex beat; (ii) calculated on auscultation; (iii) counted electronically from the ECG and/or pulse oximeter; or (v) from non-invasive or invasive methods of blood pressure monitoring. Of these, only the ECG gives no indication of pulsatile blood flow so does not necessarily reflect cardiac output. The heart rate should be determined continuously by palpating suitable arteries as this provides additional information, i.e. pulse rate, rhythm and quality (pulse pressure). Differences between heart and pulse rate indicate cardiac contractions with reduced stroke volume. Associated arrhythmias and/or circulating volume loss may be identified on the ECG or by recording low central venous pressures (CVPs; see below) respectively.

Pulse quality

The continual palpation of a peripheral pulse is highly recommended in sedated and anaesthetized animals because it assesses the pulse pressure, i.e. the difference between the systolic and diastolic pressures, which allows stroke volume estimation. The pulse pressure can also be estimated from the amplitude of pulse plethysmographs. During invasive procedures, blood pressure can be inferred by observing surgical haemorrhage and pulsations in palpable arteries.

Respiratory rate and volume

Ventilation must be assessed near-continuously in anaesthetized and sedated animals because respiratory arrest is a life-threatening event. Respiration can be evaluated in terms of rate, volume, rhythm and the inspiratory:expiratory effort; all are assessed by observing thoracic excursions or the reservoir bag of the anaesthetic breathing system (when observation is obscured by surgical drapes). Oesophageal stethoscopy can also be used to assess lung sounds and respiratory rate.

In all animals, but particularly ruminants, monitoring respiration (and upper airway patency) must be more meticulous when the trachea is not intubated; ruminants are at especial risk from the aspiration of saliva or regurgitated rumen contents. (Consequently, not intubating the trachea of animals deprived of protective upper airway reflexes is not defensible.)

Respirometers attached to the ABS measure the volume of gas inspired and expired on a breath-for-breath basis, and when measured over 60 seconds yields the minute volume. Electronic respiratory monitors operating on the temperature difference between inspired (cold) and expired (warm) gas do not indicate ventilatory adequacy. More complex respiratory monitors (capnometers, capnographs, spirometers) are discussed in the following.

Temperature

Hypothermia is a common complication of general anaesthesia and while core temperature rarely changes rapidly, it should be monitored continuously. Accurate thermistor probes are freely available, and rapidly positioned within the oesophagus (for measuring aortic blood temperature) or the rectum. It is recommended that core temperature be monitored during all anaesthetics, but particularly when considerable heat loss is likely, e.g. when prolonged and major surgery (laparotomy, thoracotomy) is conducted on smaller, younger animals under conditions of low ambient temperatures. If active warming devices are used to prevent hypothermia, continuous monitoring of body temperature is required to prevent unintended hyperthermia.

Monitoring core temperature is highly recommended in pigs with a familial propensity to hypermetabolic disorders, i.e. porcine stress syndrome, pale soft exudative pork and malignant hyperthermia.^258,259

Complementary vital signs

Monitoring variables related to the vital signs, e.g. perfusion, allows a more complete and accurate assessment of cardiopulmonary function and is highly recommended in sedated or anaesthetized animals. Perfusion is the fundamental objective of cardiovascular activity but, being difficult to measure objectively, is estimated by the integration of information from the following methods or sources: CRT, mucous membrane colour, surgical ‘ooze’, core–periphery temperature gradients and urine output (UO).

Capillary refill time

This is estimated by blanching the oral mucous membrane and timing the return of colour. Normally this is less than 2 seconds. Intense vasoconstriction and/or hypotension prolongs CRT. Ceteris paribus, prolonged CRT indicates poor perfusion. It is recommended that the CRT be recorded every 5 minutes, the core–periphery temperature gradient every 1 hour and the UO, if measured, every 2–3 hours.

Mucous membrane colour

Perfused oral mucous membranes in pigs should be pink; in sheep, calves and goats they may be pigmented, light grey or brown. Perfused vulval, rectal or conjunctival mucous membranes are usually pink in all species. They become bright pink in hypercapnia (or vasodilatation due to other causes) and endotoxaemia, and blue with cyanosis (haemoglobin desaturation). In anaemic animals, cyanosis may not be evident despite desaturation. When blood pressure falls and blood vessels in the oral mucosa constrict, they become pale.

Capillary ooze

Examination of the surgical site may reveal capillary ‘ooze’. Bright red blood at the surgical site indicates good perfusion. Darkly coloured or slowly oozing blood reflects poor perfusion.

Increased core–periphery temperature gradient differences

Worsening perfusion is characterized by falls in peripheral (cutaneous) temperatures and an increased core–periphery temperature gradient difference. Cold ears and extremities may indicate that the animal is hypothermic or that warm blood is being retained in the ‘core’, i.e. being withheld from non-essential tissue. In all species, increased core–periphery temperature gradient differences are quantified by the simultaneous use of two calibrated thermistors placed at appropriate sites, e.g. over the heart base (via the oesophagus) and on the ear tip.

Urine output

UO measurement reflects renal perfusion and in conjunction with blood and urine analysis, quantifies renal function. It coincidentally prevents bladder overdistension during prolonged procedures which, if unrelieved, contributes to postoperative discomfort. Non-invasive bladder catheterization is facilitated in female sheep and goats using an appropriately sized colposcope. It is possible, though not straightforward, in female pigs. ²⁶⁰ In male pigs, sheep, goats and cattle, the bladder can only be catheterized surgically. Catheterising the urinary bladder and measuring the collected urine volume at 1–3 hourly intervals allow UO, a surrogate measure of perfusion, to be calculated. A value in excess of 1.0 ml kg⁻¹ h⁻¹ is held to represent adequate renal (and other vital organ) perfusion (see below).

Pulse oximetry

The technology measuring oxygen (O₂) saturation of haemoglobin in arterial blood (SaO₂) using pulse oximetry yields SpO₂ data. The pulse oximeter is a readily applied non-invasive measure which provides information on pulmonary function (oxygenation) and in conjunction with pulse plethysmography (and other estimates of cardiac output) O₂ delivery to peripheral tissue. Their use involves applying the pulse oximeter probe to the animal’s tongue (sheep, goat, cattle and pig), ear (sheep and pig; not in case of lower blood pressure) or tail (pig). ²⁶¹ The vulva can also be used.

Pulse plethysmography quantifies and displays the blood volume flowing between the transmitter and sensor yielding an oscillating signal whose amplitude is proportional to the pulse pressure. In conjunction with oximetry this simultaneously monitors three important physiological variables: (i) SpO₂; (ii) pulsatile blood through a tissue bed, i.e. mechanical cardiac activity; and (iii) the pulse rate.

Pulse oximeters are most useful: (i) in revealing an animal’s capacity to maintain Hb saturation when room-air breathing resumes at the end of anaesthesia; (ii) when ‘weaning’ animals off ventilators, when minute volumes should be as low as is consistent with SpO₂ values >0.92; and (iii) in any procedure in which blood oxygenation may be reduced. When pulse oximeters are used, the variable pitch pulse tone and the low threshold alarm should be audible to the anaesthetist.

Capnography

Capnography non-invasively measures (capnometry) inspired and expired carbon dioxide (CO₂) on a breath-by-breath basis while generating a CO₂ concentration/time waveform (capnogram). The values generated include fr and expired and inspired CO₂. The capnogram identifies partial or complete airway obstruction and the effectiveness of CO₂ removal between breaths. The end-tidal CO₂ value usually reflects CO₂ levels in arterial blood and is a useful indication of adequacy of ventilation. The CO₂ level is directly related to minute ventilation. Capnography is essential during controlled or mechanical ventilation. Capnography provides indirect information about cardiac output as it reflects pulmonary and systemic perfusion. Cardiac arrest or pulmonary thromboembolism can also be diagnosed with capnography. The use of capnography is highly recommended when anaesthetizing pigs at risk of hypermetabolic reactions.

Non-invasive blood pressure

Arterial blood pressure (AP) measurement provides indirect information on tissue perfusion, cardiac output and arterial elasticity. NIBP monitoring using automated oscillotonometry, i.e. the application of a suitably sized inflatable cuff to a suitable appendage is rapidly applied and used properly, offers a reasonably accurate method for intermittently monitoring systolic (SAP), mean (MAP) and diastolic (DAP) arterial blood pressure. When blood flow distal to an occlusive cuff is recorded using an ultrasonic Doppler probe, then SAPs alone are registered. Oscillotonometry can be used safely on conscious, free-roaming animals, i.e. during recovery from anaesthesia. Monitoring NIBP is highly recommended during brief procedures, when there is little justification for invasive monitoring, and whenever continuous pulse palpation is impractical. Agreement between NIBP and the gold-standard, IBP has been examined in pigs,^262,263 sheep,^{264 -266} goats ²⁶⁵ and cattle ²⁶⁵ and is fair when IBP is within accepted ranges.

Electrocardiography

The ECG provides continuous heart rate and rhythm information but does not provide assurance that the observed electrical activity represents myocardial contractility, i.e. cardiac output. Therefore, the ECG should not be the sole monitor of cardiovascular function under any circumstances. Its use is highly recommended during arrhythmogenic procedures, e.g. cardiac catheterization.

Spirometry

Many modern multiparameter anaesthesia monitors incorporate spirometry modules, which simultaneously measure airway pressures, flows and volumes allowing pressure–volume plots to be displayed on a breath-by-breath basis. The interpretation of ‘spirometry loops’ enables rapid detection of changes in lung and breathing system mechanics and verifies ventilator performance. ²⁴⁰

Central venous pressure

Passing suitable catheters via the jugular vein (ruminants) or cranial vena cava (pig) to the cranial vena caval–right atrial junction before connection to a water manometer (zeroed to the level of the manubrium sternae) allows CVP to be measured continuously. In briefest terms the CVP represents the balance between inflow (venous return or right heart preload) and outflow (right ventricular output or left atrial preload) in subjects with normal cardiac function. ²⁶⁸ It is lowered in hypovolaemia, and raised in cardiac failure, pulmonary embolism, fluid over-transfusion, PPV and cardiac tamponade. It is useful when aggressive volume restoration is likely under conditions of cardiac dysfunction. Values for CVP vary considerably between individuals but within the same animal, CVP is useful for monitoring circulating volume trends during prolonged anaesthesia.

Blood-gas analysis

Intermittent arterial blood sampling for blood-gas analysis can be used to ‘calibrate’ capnography and pulse oximetry. Modern analysers are ‘point-of-care’ devices, i.e. small, hand-held devices using specific cassettes to meet different clinical requirements. In addition to blood gases, those used during anaesthesia also analyse sodium, potassium, chloride, bicarbonate, blood urea nitrogen, creatinine and lactate levels. Such analyses are useful in the management of prolonged anaesthetics or whenever pulmonary dysfunction is possible.

Cardiac output

Cardiac output can be measured in several ways, each having specific advantages and disadvantages, equipment and instrumentation requirements. In general, invasive techniques are more accurate than non-invasive techniques. Cardiac output measurement provides valuable information including heart rate, stroke volume, peripheral vascular resistance and circulating blood volume. These variables can be derived simply and non-invasively by pressure-recording analytic methods (PRAMs), i.e. high-frequency sampling and PPC analysis. PRAM methods examined in pigs showed promising results. ²⁶⁹

Rumen pressure

Ruminal tympany is a potentially lethal complication of ruminant anaesthesia. Ruminal gas accumulation, which depends on time, the volume and type of recently ingested feed, progressively ‘splints’ the diaphragm impairing ventilation. High pressures may reduce venous return to the heart, and if unrelieved, are painful in the recovering animal.

Tympany is identified by periodically observing and palpating the abdomen, specifically the left sub-lumbar concavity (fossa) which becomes convex with time. Decreasing blood pressure coincidental with increased heart rate and inspiratory pressure (during PPV) are also indicative. (In spontaneously breathing animals, the fr increases as Vt falls.)

‘Bloat’ should be treated if systemic effects are present and the procedure time is prolonged. Peroral placement of a gastric tube or percutaneous trocarisation of the rumen can be performed during anaesthesia. The intraoperative relief of ruminal pressure requires precautionary measures to prevent interference with aseptic surgical technique (contamination of the surgical field, instruments and/or the surgical suite).

Oropharyngeal fluid

Ruminants often salivate profusely during anaesthesia, and there is a risk of passive regurgitation whenever ruminal pressure is high and rumen contents are fluid. Both increase the risk of fluid accumulation in the oropharynx. This may result in the pulmonary aspiration of foreign material when the trachea is unprotected and the head inappropriately positioned. Drainage of fluid by careful positioning of the animal’s head and neck is not always effective but should be attempted in all cases.

Limb position and muscle compression

In large animals (cattle and adult pigs) the position of, and tension in limbs, joints and muscles require periodic attention, particularly with respect to the surface upon which they lie. In prolonged procedures periodic limb repositioning may be needed to avoid ischaemic muscle damage and/or postoperative myalgia. Similar attention must be paid to nerves at risk of positional trauma.

Integumentary damage

The eyes should be checked periodically and lubricated with ophthalmic ointment to ensure the cornea remains moist and protected from injury. Tapes, chords or ropes maintaining animals in an operating position must be checked periodically to ensure cutaneous blood supply is not impaired and that underlying structures are not being damaged. The same applies to bandages or tapes used to secure the ETT in situ.

Degree of monitoring

It must be appreciated, despite the recommendations enunciated previously, that numerous factors dictate: (i) the range of physiological variables monitored; (ii) the frequency each variable is monitored; and (iii) the accuracy of measurement (and recording) required for each variable. The most important determinants are: (i) the procedure, its (ii) duration; and (iii) invasiveness; (iv) species; (v) age; (vi) the experimental objectives; and (vii) whether the study is terminal or requires the animal to recover. Additional, usually more advanced and invasive monitoring methods, e.g. intracranial pressure or Qt, are required when more complicated, invasive and/or prolonged recovery procedures are scheduled. A greater range of variables should be monitored, more frequently, when unfamiliar drugs or techniques are being used, or when unfamiliar genotypes are being anaesthetized. In specific models it may be necessary to monitor biochemical variables for animal management rather than scientific purpose, e.g. blood glucose in diabetic pigs. Before studies begin, it is highly recommended that the anaesthetist and research group meet to discuss which variables (if any) are to be monitored for scientific (rather than management) purposes. ³ In the final analysis, the ‘level of monitoring’ will be influenced by equipment availability and the ability to use it.

Monitoring is necessary in non-recovery procedures to ensure that physiological changes caused by anaesthesia do not corrupt the scientific value of collected data or cause intraoperative complications.

It is recommended that the variables monitored are limited to those that are necessary. Measuring, recording, analysing and responding to every possible variable may divert attention from critical changes especially when manual record keeping is employed.

Sedation with or without local anaesthetic techniques

Perioperative morbidity–mortality studies have not been conducted on sedated pigs, sheep, goats or cattle, with or without additional local anaesthetics. In contrast, studies on procedurally sedated humans (with or without local anaesthesia) have justified establishing monitoring standards for this subpopulation. ²⁴⁰ Similar guidelines are required in animals because there’s a general belief amongst many that work with animals that physiological monitoring is only required in unconscious subjects. Based on the (mistaken) belief that sedatives cause less physiological perturbation than general anaesthetics, sedated animals usually do not (or cannot) benefit from a secured airway, intravenous access and the closer attention, i.e. more extensive physiological monitoring, unconscious animals receive. While movement and/or intolerance of even non-invasive devices, e.g. pulse oximetry and the ECG, limits the ability to fully assess function in sedated animals, an underestimation of risk justifies the high recommendation that the DOA, vital and complementary signs are continually monitored in these by an assigned anaesthetist.

Closer attention to sedated ruminants is warranted given their propensity to ruminal tympany. The possibility of regurgitation and/or the accumulation of oropharyngeal saliva further justifies the need to continuously monitor airway patency when it is unprotected.

There is limited information on morbidity and mortality in pigs, sheep, goats and calves receiving local anaesthetics. However, NIBP is suggested when local anaesthetics are injected into the lumbosacral extradural space because this can cause fatal hypotension. ²⁷⁰

Maintaining airway patency and security

When the trachea is not intubated in anaesthetized or profoundly sedated animals, a reduction or loss of airway patency will eventually become manifest as deteriorating SpO₂ values and changes in other indices of hypoxia and/or hypercapnia. However, upper airway obstruction in depressed or unconscious animals is an emergency and justifies the high recommendation for continual monitoring of airway patency in these circumstances. This involves detecting observable and audible signs of dyspnoea, i.e. altered, usually a strenuous, inspiratory effort accompanied by upper airway ‘gurgling’ sounds. Such vigilance is particularly important in ruminants which are at greater risk of airway obstruction caused by saliva or regurgitated rumen contents.

When ETTs are used, their correct positioning must be verified by auscultating similar lung sounds over both hemi-thoraces or (less preferably): (i) by detecting gas expulsion from the ETT upon thoracic percussion; or, during manual lung inflation, (ii) by observing symmetrical thoracic wall movement while appreciating ‘normal’ elastic lung resistance during reservoir bag compression; or (iii) by observing ‘normal’ capnograms during capnography.

Monitoring lung ventilation

Positive pressure ventilation by manual or mechanical means aims to optimize gas exchange while minimizing risks of ventilator-induced lung injury. Mechanical ventilator designs vary, but the simultaneous control and measurement of airway pressure, Vt and fr are minimal requirements. Control of inspiratory:expiratory time ratios and end-expiratory pressures are highly desirable. These settings are adjusted to maintain normocapnia, i.e. Pe^/CO₂ values between 5.0 and 5.7 kPa (38–42 mmHg) using the lowest inflation pressures possible.

It is recommended that ventilator settings are confirmed by measuring ventilatory variables independently, e.g. using spirometry. Modules providing information on peak, plateau, mean and end-expiratory airway pressures, ideally as a displayed waveform, provides breath-by-breath information on thoracic wall and lung mechanics.

Monitoring blood pressure is recommended whenever PPV is likely to significantly reduce Qt.

Measuring inspired oxygen levels

Medical authorities recommend ⁵ or highly recommend ²⁴⁰ that inspired O₂ concentration (FiO₂) of oxygen in the patient breathing system be measured continuously by an O₂ analyser with an audible low limit alarm always set for use. (The measurement of expired O₂ concentrations (Fe^/O₂) while useful, is less important.) The positioning of the sampling port, which depends on the breathing system used, must be placed in such a position that the composition of the gas mixture delivered to the patient is monitored continuously.

Whilst adequate oxygenation must be assured during anaesthesia in all species, monitoring FiO₂ during anaesthesia in pigs, sheep, goats and cattle has been recommended, not highly recommended, in these guidelines, because in veterinary anaesthesia: (i) low-flow anaesthesia is not commonly used; (ii) the inclusion of medical air or nitrous oxide in inspired gas is less prevalent; (iii) the neurological consequences of hypoxia are more severe in human than animal subjects; (iv) animals with hypoxia-induced neurological injury can be legitimately euthanatized.

The use of NMBAs

Using NMBAs during anaesthesia makes it possible for conscious animals to undergo invasive surgery without showing normal signs of nociception, i.e. movement and tachypnoea. This intolerable situation, which may not be uncommon in laboratory pigs ²⁷¹ can be avoided by monitoring (and ensuring adequate) anaesthetic delivery while closely evaluating autonomic nervous activity. Ensuring end-tidal concentrations of volatile anaesthetics (when used) are in excess, e.g. 1.1–1.3 times the reported (species- and agent-specific) values of MAC goes some way to ensuring insensibility is present.

Paralysed yet conscious animals usually respond to nocistimulation with signs of sympathetic nervous stimulation, e.g. mydriasis, tachycardia, hypertension, arrhythmias, sweating and lacrimation. However, this is not always the case in paralysed conscious humans in which the sudden appearance of pallor, hypotension, bradycardia and other signs of ‘vasovagal syncope’ also indicate inadequate anaesthesia. In any paralysed animal in which an obvious change in heart rate can be linked with intense surgical stimulation and/or a failure of anaesthetic delivery, and which is not associated with haemorrhage, hypotension, hypercapnia or hypoxaemia, should raise the suspicion of consciousness, especially when accompanied with parallel changes in blood pressure, salivation, lacrimation and ECG patterns associated with rising plasma catecholamine levels, e.g. ST-segment changes leading to premature ventricular depolarizations and ventricular tachycardia. ²¹⁶ The use of NMBAs clearly justifies the need for more meticulous, e.g. IBP rather than NIBP and the more frequent monitoring of signs of autonomic nervous activity. When NMBAs are used, it is highly recommended that the alarm thresholds on all relevant monitoring devices be set to detect slight, rather than major changes, and that the audible signal volumes are set to ‘high’.

Achieving surgical levels of neuromuscular blockade (NMBA) almost always paralyses the respiratory muscles and so PPV must be imposed (and appropriately monitored). The need to meticulously measure ventilatory adequacy arises during recovery from NMBA because such information defines criteria when full, and then partial ventilatory support is ended. Observing the range and pattern of diaphragmatic and thoracic wall movement is subjective, so while the trachea remains intubated, the use of spirometry or respirometry to measure Vt and Vm is highly recommended. Inspiratory manometry is also recommended. The combined use of respirometry with both pulse oximetry and capnography reveals whether ventilatory variables are associated with adequate lung function and provides more substantive information on the need (or otherwise) for continued ventilatory support. The usefulness of these variables to determine adequate ventilatory function is greatly increased if baseline values are established before NMBAs are given.

Medical authorities are unanimous in recommending methods of monitoring NMBA in human patients because: (i) residual blockade continues to be responsible for critical complications in medical anaesthetic practice; and (ii) there is considerable information linking the strength of evoked activity in the ulnaris–adductor pollicis nerve–muscle unit (NMU) and the ability to overcome upper airway resistance and ventilate adequately. This NMU (which adducts the thumb) only exists in Homo sapiens so similar information for pigs, sheep, goats and cattle does not exist. Thus, monitoring neuromuscular transmission in, for example, the N.facialis–M.levator nasolabialis, or the N.ulnaris–M.carpi flexorum NMUs (which are both feasible in pigs, sheep and goats) reflects muscle force in those units, but not the ability to breathe adequately. Given the considerable variation in sensitivity of different NMUs to NMBAs, it is possible to record full muscle function in a peripheral NMU, whilst the respiratory musculature remains paralysed. For this reason, the use of mechanomyography or acceleromyography cannot be used alone to judge recovery of ventilatory function after NMBA use.

These points are particularly important in sheep whose response to NMBAs differs markedly from those in other domesticated species. ²⁰²

The increased complexity of monitoring DOA, ventilatory adequacy and neuromuscular transmission in paralysed animals, coupled with the dire consequences of ineptitude justifies the need for specialized training and ensuring the prescriptions of Appendix H of Guidance on the Operation of the Animals (Scientific Procedures) Act 1986 are met. ²⁵⁵

Monitoring anaesthetic delivery, support and monitoring equipment

The assigned anaesthetist must check all equipment before use and set all appropriate audible alarm settings. Monitoring equipment should always be used in conjunction with careful clinical observation by the anaesthetist, as there are circumstances in which equipment may not detect unfavourable clinical developments.

When the monitors are in use, the alarms (visual and audible) must be enabled. The audible component of the alarm system should be easily heard by the anaesthetist.

The continued functioning of all anaesthetic delivery and support devices, including anaesthetic workstations, infusion controllers, syringe drivers, heater blankets, mechanical ventilators and ABSs should be periodically examined.

It is highly recommended that the anaesthetic workstation be checked every 15–30 minutes for: cylinder and pipeline pressures; the anaesthetic level in the vaporizer; and the vaporizer temperature (by palpation). It is highly recommended that the flow meter(s) and vaporizer settings and the emergency O₂ valve position are checked continually (at least every 5 minutes).

It is highly recommended that when anaesthetics or anaesthesia adjunct drugs are being delivered by infusion controllers or syringe drivers, settings must be checked at least every 10 minutes and re-adjusted if necessary. The volume to be infused must be checked. (This also applies to intravenous fluid therapy.)

The functioning of the ABSs should be checked periodically. It is highly recommended that: (i) the degree of reservoir bag distension; (ii) the breathing system pressure; (iii) the position of the adjustable pressure limiting valve; and (iv) the ABS–ETT connection are checked at 5- to 10-minute intervals. It is recommended that the CO₂ absorbent colour is checked every 30 minutes. It is suggested that the connection of the ABS to the common gas outlet is examined every hour.

Monitoring the procedure and operators

It is recommended that assigned anaesthetists should evaluate the ongoing procedure at appropriate intervals and if this is not possible, e.g. for reasons of sterility, then the operator must supply any information requested. Knowing the proximity of the procedure to the airway, large blood vessels and/or nerves allows the prediction of postoperative problems with airway patency, haemorrhage and pain (and function), respectively. Anticipating ‘stimulating’ parts of the procedure allows the preparation of ‘top-ups’ or the deepening of anaesthesia. A general appraisal of procedural trauma, including the degree of traction exerted should be conducted, as this is likely to influence postoperative analgesic requirements. Haemorrhage should be continuously assessed: bloody swabs should be retrieved and weighed. Observation of arterial pulsations at the surgical site provides information on cardiovascular performance. During thoracotomy procedures, the adequacy or otherwise of lung inflation and the development of atelectasis should be noted and corrected when appropriate. Similarly, during laparotomy procedures, e.g. for gut loop creation, bowel colour should be noted.

Duration of monitoring and clinical responsibility

It is highly recommended that the assigned anaesthetist should observe animals unobtrusively, ideally by CCTV, from the moment pre-anaesthetic medication is given. In poorly designed pens, pigs may entrap limbs or their nares, while ruminants may hypersalivate and/or regurgitate, necessitating prompt intervention.

It is highly recommended that the anaesthetist remains with the animal throughout anaesthesia and dedicates their attention to monitoring anaesthesia. When necessary, the anaesthetist may delegate responsibility to another competent individual for brief periods.

In recovery procedures, it is highly recommended that the anaesthetist maintains constant supervision until vital activity is restored. If necessary, another competent individual may be assigned this responsibility after being fully appraised of the animal’s condition. Recovery is a high-risk period accounting for 50–60% of anaesthetic-related deaths in companion animals ²⁵¹ and 66–100% in horses. ²⁷²

The physiological and behavioural criteria for the restoration of vital activity are detailed described (Part II: General principles); put simply, when the animal can stand, breathe normally, ambulate, prehend food and drink.

It is highly recommended that ruminants are known to have eructated and ideally, begun ruminating and judged capable of maintaining sternal recumbency, preferably the standing position, before close surveillance is ended.

Additional staff should be available in proximity to the recovery area to assist in the event of emergencies. This is particularly important when adult cattle and large pigs are involved: physical assistance may be required, for example, to reposition entrapped or ‘cast’ animals.