CHAPTER 24 Preanesthesia, Anesthesia, Analgesia, and Euthanasia Paul Flecknell, MA, VetMB, PhD, DECLAM, DLAS, DECVA, (Hon) DACLAM, (Hon) FRCVSa, Jennifer L.S. Lofgren, DVM, MS, DACLAMb, Melissa C. Dyson, DVM, DACLAMb, Robert R. Marini, DVMc, M. Michael Swindle, DVMd and Ronald P. Wilson, VMD, MSe aComparative Biology Medicine, Newcastle University, Newcastle Upon Tyne, UK bUnit for Laboratory Animal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA cDivision of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts dDepartment of Comparative Medicine, Medical University of South Carolina, Charleston, SC, USA ePenn State College of Medicine, Department of Comparative Medicine, Hershey, PA, USA

OUTLINE

I. Introduction 1136 IV. Ferrets 1156 A. Introduction 1156 II. Rodents 1136 B. Preoperative Assessment and Preparation 1157 A. Introduction 1136 C. Intraoperative 1158 B. Preoperative Assessment and Preparation 1136 D. Intraoperative Monitoring and Support 1160 C. Agents 1137 E. Postoperative Recovery 1160 D. Intraoperative Monitoring and Support 1143 F. Acute and Chronic Analgesic Therapy 1160 E. Special Anesthetic Considerations 1144 G. Special Anesthetic Considerations 1161 F. Analgesic Therapy 1145 G. Species Considerations 1147 V. Swine 1162 H. Euthanasia 1148 A. Introduction 1162 B. Preoperative Assessment and Preparation 1162 III. Rabbits 1149 C. Intraoperative Anesthesia 1163 A. Introduction 1149 D. Intraoperative Monitoring and Support 1166 B. Preoperative Assessment and Preparation 1149 E. Special Anesthetic Considerations 1166 C. Intraoperative Anesthesia 1150 F. Postoperative Recovery 1167 D. Intraoperative Monitoring and Support 1154 G. Acute and Chronic Analgesic Therapy 1167 E. Special Anesthetic Considerations 1154 H. Euthanasia 1168 F. Acute and Chronic Analgesic Therapy 1155

Laboratory Animal Medicine, Third Edition DOI: http://dx.doi.org/10.1016/B978-0-12-409527-4.00024-9 1135 © 20122015 Elsevier Inc. All rights reserved. 1136 24. Preanesthesia, Anesthesia, Analgesia, and Euthanasia

VI. Small Ruminants 1168 C. Intraoperative Anesthesia 1182 A. Introduction 1168 D. Intraoperative Monitoring and Support 1184 B. Preoperative Evaluation and Preparation 1169 E. Special Anesthesia Considerations 1185 C. Postoperative Recovery and Pain Management 1178 F. Postoperative Recovery 1186 D. Euthanasia 1180 G. Euthanasia 1187 VII. Nonhuman Primates 1180 References 1187 A. Introduction 1180 B. Preoperative Assessment and Preparation 1180

I. INTRODUCTION analgesia for rodents are rapidly evolving. The reader is urged to consult laboratory animal anesthesia texts The purpose of this chapter is to provide practical (Gaertner et al., 2008; Flecknell, 2009) and journals for advice for administering anesthetics, analgesics, and more complete and timely information. euthanasia agents to the most commonly used labora- tory animal species. It is not meant to be an exhaustive review of all anesthetic and analgesic protocols used in B. Preoperative Assessment and Preparation these species. More detailed information is available in 1. Preanesthetic Evaluation both specialist laboratory animal anesthesia texts, (e.g., No anesthetic regimen is universally safe or appro- Fish, 2008; Kohn et al., 1997; Flecknell, 2009), and general priate. Anesthetic protocols that are satisfactory for veterinary anesthesia texts (e.g., Tranquilli et al., 2007). healthy animals can severely compromise or be fatal The most commonly used rodent species, rabbits, to unhealthy ones. A vigilant rodent health surveil- ferrets, pigs, small ruminants, and nonhuman primates lance program is the first line of defense but cannot be were selected for inclusion in this chapter. These species expected to detect alterations in husbandry or experi- encompass the overwhelming majority of laboratory ani- mental manipulations that may affect anesthesia out- mals that are currently utilized. References for in-depth comes. Preanesthetic considerations should include an discussion of anesthetic and analgesic protocols for each assessment of appearance, behavior, and bodyweight species are included within its particular section. for evidence of abnormality as well as for establishing doses for injectable agents. The history of the group of II. RODENTS animals should be examined to identify experimental, housing, and strain-specific features that might affect A. Introduction choice of anesthesic agents and techniques. In addi- tion, sex, age, and specific procedures may be expected Rodents, especially rats and mice, are the most to alter the effects of anesthesia, often to a surprising numerous and arguably the most important group of degree. A summary of anesthetic and analgesic varia- animals used in research. Among the factors that dif- tions among mice and rat stocks and strains is available ferentiate anesthetic techniques used in rodents from in the chapter on Laboratory Rodents in the second edi- those more commonly employed in larger species are tion of the text Anesthesia and Analgesia in Laboratory the small size of the animals, the perceived and often Animals (Gaertner et al., 2008). Unless the anesthetic real difficulties of vascular and airway access, and the regimen is known to be safe and effective in the animals frequent need to anesthetize large numbers of animals to be used, a pilot study should be conducted to assess in a relatively short time. Trends that have combined and verify the effects of the proposed anesthetic and to increase attention on the safe and appropriate anes- analgesic protocol (Flecknell, 2009). In all cases, it is thetic and analgesic delivery and monitoring for rodents important to allow adequate time for newly arrived ani- include: humane concerns; scientific recognition of the mals to become acclimated to changes in housing, diet, effects of pain, anesthesia, and analgesia on experimen- and husbandry conditions and to recover from shipping tal design; and the expense of generation or purchase stress. An acclimation period of at least 24–72 h is rec- of genetically altered rodents. This section will briefly ommended for rodents (Capdevila et al., 2007; Obernier discuss common anesthetic techniques for mice and rats, and Baldwin, 2006). Fasting is not usually necessary or with comments on hamsters, gerbils, and guinea pigs. recommended before anesthesia and surgery in rodents , techniques, and approaches to anesthesia and (Gaertner et al., 2008).

LABORATORY ANIMAL MEDICINE II. Rodents 1137

2. Choice of Anesthetic Techniques TABLE 24.1 selected Anesthetic and Analgesic Doses for Micea

All anesthetics have undesirable properties, so a pri- Dose Route Duration mary goal must be to select an anesthetic technique that has the least adverse effect on the animal and on the ANESTHESTICS research. Thus, the goals of the research and the limita- tions this places on selection of the anesthetic agents must 40–50 mg/kg IP 20–40 min be taken into account in formulating an anesthetic pro- tocol. For example, when prolonged stable anesthesia is EMTU (inactin) 80–mg/kg IP 60–240 min required, the choice might favor an inhalation or continu- agents and combinations ous infusion method to avoid the variations that tend to occur with repeated bolus administration of drugs. A pro- + 100 mg/kg IP 20–30 min longed recovery period can further stress the animal and acepromazine 5 mg/kg the resources of the facility, favoring selection of agents Ketamine+ 50–75 mg/kg IP 20–30 min that have rapid recovery characteristics or agents that can medetomidine 1–10 mg/kg easily be reversed, such as α2-agonists, , and some . Finally, some techniques that meet all Ketamine+ 80–100 mg/kg IP 20–30 min of the preceding criteria may be too technically demand- 5–10 mg/kg ing or simply too expensive for the proposed research. Ketamine+ 80–100 mg/kg IP 30–40 min Inhalation anesthesia with modern agents requires the Xylazine+ 5–10 mg/kg proper equipment and training. Precise intravenous infu- sion methods may require an infusion pump, as well as acepromazine 3 mg/kg vascular access and experience with the technique. In Neuroleptanesthetics many cases, only brief anesthesia may be needed, such as procedures that may not be extremely painful but require -fluanisone (hypnorm) adequate restraint for the safety of the animal and the b operator (e.g., bleeding, injections, or sampling of small + 10.0 ml/kg IP 30–40 min amounts of tissue). Brief exposure to an inhalation agent Fentanyl-fluanisone 0.4 ml/kg is often selected to meet these needs. (hypnorm) In summary, selection of a suitable anesthetic tech- + 5 mg/kg IP 30–40 min nique must include professional and humane consid- Inhalant agents erations, scientific requirements and restrictions, and recognition of technical and personnel limitations. 1–4%, to effect Inhalant

3. Preanesthetic Other agents Preanesthetic drugs are not commonly used in 12–26 mg/kg IV 5–10 min rodents. However, the advantages of sedation, analge- 125–300 mg/kg IP 15–45 min sia, and reduced doses of the general anesthetic agent or agents apply equally well to rodents. The principal ANALGESICS disadvantage of using a preanesthetic agent in rodents Buprenorphine 0.05–0.1 mg/kg SC, IP 6–12 h is the need to restrain and thus stress the animals twice Butorphanol 1–5 mg/kg SC, IP 4 h rather than once for the induction of anesthesia. Drugs commonly used as preanesthetic agents in other species 2.5 mg/kg SC, IP 2–4 h are frequently incorporated into anesthetic combina- Carprofen 5 mg/kg SC, IP 24 h tions for rodents. However, if postprocedural analgesia Meloxicam 5 mg/kg SC, IP 24 ht is needed following very short procedures, or if pre- Bupivacaine Do not exceed Local 1–2 ht emptive analgesia is desired, then some means of pre- 2 mg/kg infiltration anesthetic administration must be used. Preanesthetic administration of tranquilizers, including the phenothi- MISCELLANEOUS azine derivatives, benzodiazepines, and pote-nt analge- 0.04 mg/kg SC, IP t min sics will all tend to substantially reduce the required Yohimbine 0.2 mg/kg IV – dose of the principal anesthetic agent or agents. 0.5 mg/kg IM C. Anesthetic Agents Atipamezole 0.1–1 mg/kg IM, IP, SC, IV – aDoses adapted with modifications fromGaertner et al. (2008) and Flecknell (2009). A summary of commonly used drug doses is pro- bSee Flecknell (2009) for mixing instructions. vided in Tables 24.1–24.5. These dosages should be used tUncertain duration.

LABORATORY ANIMAL MEDICINE 1138 24. Preanesthesia, Anesthesia, Analgesia, and Euthanasia

TABLE 24.2 rats: Drug Dosesa TABLE 24.3 drug Dosesa for Hamsters

Drug Dose Route Duration Drug Dose Route Duration

ANESTHESTICS ANESTHESTICS

Barbiturates Dissociative agents and combinations

Pentobarbital 30–60 mg/kg IP 20–60 min Ketamine+ 150 mg/kg IP 45–120 min EMTU (inactin)* 80–100 mg/kg IP 60–240 min acepromazine 5 mg/kg Dissociative agents and combinations Ketamine+ 75–100 mg/kg IP 30–60 min

Ketamine+ 75 mg/kg IP 20–30 min medetomidine 0.25–1 mg/kg acepromazine 2.5 mg/kg Ketamine+ 80–200 mg/kg IP 30–60 min Ketamine+ 60–75 mg/kg IP 20–30 min xylazine 5–10 mg/kg medetomidine 0.5 mg/kg -+ 20–30 mg/kg IP 10–30 min Ketamine+ 40–80 mg/kg IP 20–40 min xylazine 10 mg/kg xylazine 5–10 mg/kg Neuroleptanesthetics Ketamine+ 40–50 mg/kg IP 20–30 min Fentanyl-fluanisone Xylazine+ 2.5–8 mg/kg (hypnorm) acepromazine 0.75–4 mg/kg +midazolam 4.0 ml/kgb IP 20–40 min Neuroleptanesthetics Fentanyl-fluanisone 1 ml/kg (hypnorm) Fentanyl-fluanisone (hypnorm) +diazepam 5.0 mg/kg IP 20–40 min b +midazolam 2.7 ml/kg IP 30–40 min Inhalant agents Fentanyl-fluanisone 0.4–0.6 ml/kg (hypnorm) Isoflurane 1–4%, to effect Inhalant +diazepam 2.5 mg/kg IP 20–40 min Other agents

Inhalant agents α- 80–100 mg/kg IP 3–4 h (hypnosis only)* Isoflurane 1–4%, to effect Inhalant Urethane* 1000–2000 mg/kg IP 6–8 h Other agents ANALGESICS Propofol 7.5–10 mg/kg IV 5–10 min

ANALGESICS Buprenorphine 0.01–0.05 mg/kg SC, IP 6–12 h Butorphanol 1–5 mg/kg SC, IP 4 h Buprenorphine 0.01–0.05 mg/kg SC, IP 8–12 h Morphine 2.5 mg/kg SC, IP 2–4 h Butorphanol 2 mg/kg SC, IP 4 h Meloxicam 1–2 mg/kg SC, IP 24 h Morphine 2.5 mg/kg SC, IP 2–4 h Carprofen 5 mg/kg SC, IP 24 h Carprofen 5 mg/kg SC, IP 24 h Ketoprofen 5 mg/kg SC, IP 24 ht Ketoprofen 5 mg/kg* SC, IP 24t t Meloxicam 1–2 mg/kg SC, IP 24t Bupivacaine Do not exceed Local 1–2 h 2 mg/kg infiltration Bupivacaine Do not exceed Local 1–2 ht 2mg/kg infiltration MISCELLANEOUS

MISCELLANEOUS Atropine 0.04 mg/kg SC

Atropine 0.05 mg/kg SC Yohimbine 0.2 mg/kg IV – Atipamezole 0.1–1 mg/kg IP, SC 0.5 mg/kg IM Yohimbine 0.2 mg/kg IV Atipamezole 0.1–1 mg/kg IM, IP, – SC, IV 0.5 mg/kg IM aDoses adapted with modifications fromGaertner et al. (2008) and Flecknell (2009). aDoses adapted with modifications fromGaertner et al. (2008) and Flecknell (2009). bSee Flecknell (2009) for mixing instructions. bSee Flecknell (2009) for mixing instructions. *Terminal use only. *Use caution, reported to cause gastric ulceration at therapeutic doses (Lamon et al., tUncertain duration. 2008; Shientag et al., 2012). tUncertain duration. II. Rodents 1139

TABLE 24.4 drug Dosesa for Gerbils TABLE 24.5 drug Dosesa for Guinea Pigs

Drug Dose Route Duration Drug Dose Route Duration

ANESTHESTICS ANESTHESTICS

Dissociative agents and combinations Barbiturates

Ketamine+ 75 mg/kg IP 60–90 min Pentobarbital 15–40 mg/kg IP 60–90 min acepromazine 3 mg/kg Dissociative agents and combinations Ketamine+ 75 mg/kg IP 20–30 min Ketamine+ 100–125 mg/kg IM/IP 45–120 min medetomidine 0.5 mg/kg acepromazine 5 mg/kg Ketamine+ 50 mg/kg IP 20–50 min Ketamine+ 40 mg/kg IP 30–40 min xylazine 2 mg/kg medetomidine 0.5 mg/kg Neuroleptanesthetics Ketamine+ 40 mg/kg IP 30 min

Fentanyl-fluanisone xylazine 5 mg/kg (Hypnorm) Neuroleptanesthetics +midazolam 8.0 ml/kgb IP 20 min Fentanyl- Fentanyl-fluanisone 0.3 ml/kg fluanisone (Hypnorm) (hypnorm) +diazepam 5.0 mg/kg IP 20 min +midazolam 8.0 ml/kgb IP 45–60 min Inhalant agents Fentanyl- 1 ml/kg Isoflurane 1–4%, to effect Inhalant fluanisone (hypnorm) Other agents +diazepam 2.5 mg/kg IP 45–60 min Fentanyl+ 75 mg/kg IP 20–30 min Inhalant Agents medetomidine 0.5 mg/kg Isoflurane 1–4%, to effect Inhalant Tribromoethanol 250–300 mg/kg IP 15–30 min ANALGESICS ANALGESIC Buprenorphine 0.01–0.05 mg/ SC, IP 6–12 h Buprenorphine 0.01–0.05 mg/kg SC, IP 6–12 h kg Butorphanol 1–5 mg/kg SC, IP 4 h Butorphanol 0.2–2 mg/kg SC, IP 4 h Meloxicam 1–2 mg/kg SC, IP 24 h Codeine 25–40 mg/kg SC, IP 4 h Morphine 2.5 mg/kg SC, IP 2–4 h Morphine 2–5 mg/kg SC, IP 2–4 h Carprofen 5 mg/kg SC, IP 24 h Meloxicam 0.1–0.3 mg/kg SC, PO, IP 24 h Ketoprofen 5 mg/kg SC, IP 24 ht Carprofen 4 mg/kg SC, IP 24 h Bupivacaine Do not exceed Local 1–2 ht Ketoprofen 1 mg/kg SC, IP 12–24 h 2 mg/kg infiltration Bupivacaine Do not exceed Local 1–2 ht MISCELLANEOUS 2 mg/kg infiltration

Atropine 0.04 mg/kg SC MISCELLANEOUS Yohimbine 0.2 mg/kg IV – Atropine 0.05 mg/kg SC 0.5 mg/kg IM Yohimbine 0.2 mg/kg IV – Atipamezole 0.1–1 mg/kg IM, IP, SC, – 0.5 mg/kg IM IV Atipamezole 0.1–1 mg/kg IM, IP, SC, IV – aDoses adapted with modifications fromGaertner et al. (2008) and Flecknell (2009). bSee Flecknell (2009) for mixing instructions. aDoses adapted with modifications fromGaertner et al. (2008) and Flecknell (2009). tUncertain duration. bSee Flecknell (2009) for mixing instructions. tUncertain duration.

LABORATORY ANIMAL MEDICINE 1140 24. Preanesthesia, Anesthesia, Analgesia, and Euthanasia as guidelines, not as firm recommendations. Although rodents (Stokes et al., 2009). Ketamine produces a degree guidelines are useful starting points, final determina- of analgesia and immobility without muscle relaxation. tion of optimum doses depends on careful observation Used alone, it is generally considered to be adequate of the animal’s responses, consideration of experimental for restraint but not for anesthesia. For this reason, and needs, and judicious fine tuning. because of its wide margin of safety, ketamine is often combined with various tranquilizers and to 1. Injectable Anesthetics make a wide variety of anesthetic cocktails. The most Injectable agents, used singly or in combination, are commonly utilized combinations include ketamine with common choices for rodent anesthesia. Their ease of xylazine (± acepromazine) or (see use and apparent simplicity tend to conceal complex further details in anesthetic adjuvant section). The anal- actions and side effects, but when correctly used, inject- gesic effects of ketamine can be influenced by a number able agents are safe, effective, and convenient. of factors but may be present 1–4 h after administra- Many drugs used in rodent anesthesia are formu- tion providing some short term postoperative pain relief lated for use in larger species, with the result that their (Wixson et al., 1987; Qian et al., 1996; Koizuka et al., 2005; concentration is often too high for accurate and con- Minville, 2010). Ketamine can be given orally, intramus- venient administration to rodents. Measurement errors cularly (IM), intravenously (IV), and subcutaneously that might be inconsequential in larger patients can have (SC) but, for operator convenience and animal comfort, significant effects in small ones. Further, many drug mix- is usually given IP. Typical ketamine mixtures are given tures are compounded extemporaneously by the user, in the dose tables and mentioned under the specific spe- sometimes without taking into account issues of mea- cies discussions following this section. surement and dosing. These difficulties are addressed Tiletamine (Telazol) is available only in combination by diluting drugs to easily measurable concentrations. with zolazepam, a . Telazol has been At the same time, final volumes can be adjusted to yield used alone or in combination with xylazine or butor- convenient dose rates, such as 0.2 ml per 100 gm body phanol to produce anesthesia in rats and mice (Arras weight for a rat, or 0.10 ml per 10 gm body weight for et al., 2001; Saha et al., 2007). Aside from a longer dura- a mouse. Thoughtful dilution and concentration adjust- tion of effect, there seem to be few reasons to favor this ment increase not only the convenience but also the more expensive combination in rodents over the various safety of drug administration in small rodents. Suitable ketamine-based formulations (Saha et al., 2007). diluents are sterile water for injection, U.S.P. (United States Pharmacopeia) or saline for injection, U.S.P. When b. Barbituates producing these mixtures, it is important to establish Once the dominant drugs for rodent anesthesia, bar- that the combined components are compatible, stable, biturates have waned in popularity (Stokes et al., 2009). and safe. At sufficient doses these produce Intraperitoneal (IP) injection is a commonly utilized general anesthesia with muscle relaxation, loss of con- route because of the relatively large potential space for sciousness, and failure to respond to noxious stimuli. injection and the ease and rapidity with which it can be As a class, they have minimal analgesic effect indepen- carried out. It is important to consider the composition dent of their ability to affect consciousness; therefore, of injection to prevent irritation of abdominal contents if used for invasive recovery procedures, analgesia due to pH or other factors. Additionally, this method should also be provided. They also cause dose-related requires training to ensure proper technique to prevent respiratory and cardiovascular depression, which wors- administration into abdominal organs or adipose tissues ens with time. As in other species, repeated doses of that could injure the animal or result in variable drug short-acting barbiturates are cumulative and will result effects (Morton et al., 2001). Even with reduced volumes, in prolonged recovery. Barbiturates tend to have a nar- intramuscular injection is unreliable and usually painful row therapeutic window. In some species, such as ham- in conscious rodents, and intravenous injection is often sters and gerbils, the window is so narrow as to be difficult, with the possible exception of rat, and perhaps a crevice, and considerable experience and finesse are mouse, tail veins. Subcutaneous injection is also rela- required for safe use. tively straightforward and useful for some drugs and Sodium pentobarbital is the most frequently used for perioperative fluid support. for rodent anesthesia, and it is usually given IP. If suitably diluted, it can be given IV in inter- a. Dissociative Agents mittent boluses or as a continuous infusion. Whereas Ketamine and tiletamine are the two representatives dose requirements are somewhat predictable for rats, of this class, for which is the parent com- the effect of a given dose of pentobarbital in mice can pound. Drug combinations containing ketamine are the be highly variable, depending on strain, sex, age, and most commonly used injectable protocols in laboratory housing conditions (Lovell, 1986a,b,c). Recovery can be

LABORATORY ANIMAL MEDICINE II. Rodents 1141 lengthy, especially with longer surgical procedures, and analgesic, albeit at different concentrations. In human is further prolonged by hypothermia. Anesthetic formu- practice these agents used alone would be termed neu- lations of pentobarbital are currently not available in the roleptanalgesics. To produce neuroleptanesthesia, addi- USA and many other countries. tional agents, such as , would be added. In provides brief anesthesia follow- fact, the recommendations for Hypnorm in rats include ing a single dose. It is probably best given IV, as in larger the addition of a benzodiazepine, either diazepam or species, in which case other and safer agents would ordi- midazolam. The combination of hypnorm and a ben- narily be preferred. Like pentobarbital, thiopental is not zodiazepine is reported to reliably produce adequate to currently available in the USA and other countries. good anesthesia in mice, rats, and hamsters (Green, 1975; Inactin (EMTU, or ethylmalonyl urea) has a longer Flecknell and Mitchell, 1984; Richardson and Flecknell, duration of effect than pentobarbital and is usually 2005). reserved for procedures requiring more than 3 h anes- thesia in the rat (Buelke-Sam et al., 1978). The primary f. Miscellaneous Injectables advantage of inactin is a greater duration of stable The drugs listed below are rarely used in mod- anesthesia. ern rodent anesthesia as there are usually much safer and effective drugs available. However, there may be c. Alkylphenol Derivative: Propofol instances where these drugs or drug combinations are Propofol is an alkylphenol derivative that should be still being used for scientific or historical reasons. administered IV. It produces respiratory and cardiovas- α-Chloralose As a sedative that provides cular depression if administered too rapidly or if used minimal analgesia, α-chloralose is often used for studies as the sole means of achieving surgical anesthesia (Glen involving autonomic reflexes. It is recommended that and Hunter, 1984; Brammer et al., 1993). The vehicle painful manipulations, such as surgical procedures, supports microbial growth well, so careful aseptic tech- should be carried out under a more effective anesthetic, nique is essential if an opened vial or bottle is to be kept after which α-chloralose can be substituted to produce for more than a brief period of time. In use, propofol prolonged stable study conditions (Wixson and Smiler, resembles rapid-acting barbiturates, such as thiopental, 1997; Flecknell, 1996, 2009; Silverman and Muir, 1993). It with the important exception that recovery from propo- is not considered suitable for survival procedures. fol is relatively rapid, even following repeated boluses Hydrate As a sedative hypnotic, chloral or infusion. hydrate at doses of 400 mg/kg IP can produce anesthesia for about 1–2 h in rats, causing respiratory, cardiovascu- d. Tribromoethanol (Avertin) lar, and thermoregulatory depression. Used properly, tribromoethanol is an adequate anes- has been associated with peritonitis and adynamic thetic for short surgical procedures in mice when given ileus, apparently related more to concentration than to IP. For safety, it is essential that it be prepared, stored, and total dose. (Silverman and Muir, 1993; Field et al., 1993; used properly. Tribromoethanol is not stable if stored at Vachon et al., 1999). room temperature or if exposed to light and the resulting and Metomidate and eto- products of decomposition are irritant and toxic, result- midate are 5-carbonic acid derivatives. Given ing in significant morbidity and mortality Papaioannou( IV, they produce rapid loss of consciousness, minimal and Fox, 1993; Zeller et al., 1998). The continued use of analgesia, and good cardiovascular stability. Only eto- tribromoethanol has been questioned given the safety midate (amidate) is available in the United States. When and effectiveness of alternative drugs or combinations used in rodents, they are usually combined with a potent (isoflurane or ketamine/xylazine combinations). Some (Wixson and Smiler, 1997; Flecknell, 2009; Green, recommend that it only be utilized for terminal proce- 1975). dures when given IP (Meyer and Fish, 2005), although Urethane Ethyl , or urethane, provides there are anesthetic situations or procedures in which greater analgesia than α-chloralose, as well as prolonged recovery procedures may be appropriate (see Special and relatively stable anesthesia in rats (Field et al., 1993). Anesthetic Considerations). It is sometimes used in conjunction with α-chloralose, to gain the advantage of increased analgesia. Urethane is e. Neuroleptics carcinogenic and is unsuitable for recovery anesthesia. These drugs are combinations of potent opioids and butyrophenone tranquilizers. The two that have 2. Inhalation Anesthetics seen widespread use, Innovar-Vet (droperidol) and Inhalation anesthesia circumvents many of the dif- Hypnorm (fluanisone), are not available for use in the ficulties associated with injectable agents. Because the United States, but may be compounded by a pharmacy agents are used to effect, issues of dose calculation and if necessary. Both products use fentanyl as the opioid variations in response do not arise. These agents are

LABORATORY ANIMAL MEDICINE 1142 24. Preanesthesia, Anesthesia, Analgesia, and Euthanasia not controlled substances and also escape the burden sedation prior to brief procedures. In this setting animals of detailed record keeping required for barbiturates, must be closely monitored and removed from the cham- opioids, benzodiazepines, and ketamine. Isoflurane and ber rapidly upon loss of consciousness. Diluting isoflu- are the most widely used of the inhalant rane with propylene glycol can reduce the concentration anesthetics for rodents. Ether is sympathomimetic and is of isoflurane vapor in an open circuit/nose cone setting not recommended because it is highly irritant and causes and create a somewhat safer and more stable anesthetic increased respiratory secretions. It also forms explosive without the use of a precision vaporizer. This method mixtures with air and oxygen, resulting in storage and has been utilized in short-term surgical procedures with safety concerns. Pharmaceutical grade preparations of success (Itah et al., 2004). Fume hoods or scavenging sys- ether are not currently available. In general, the rec- tems must be used to prevent environmental contamina- ommended inhalant agents are characterized by rapid tion with these techniques (Flecknell, 2009). induction and recovery. To varying degrees, all inhala- Nitrous oxide is sometimes used to reduce the tion anesthetics cause dose-related cardiovascular and amount of potent inhalation anesthetic agent needed respiratory depression, but these effects are frequently and to minimize respiratory and cardiovascular depres- less severe than equipotent doses of injectable agents. sion, but it is inadequate as a sole anesthetic. As with No currently produced inhalation agents are analge- all inhalation agents, adequate scavenging is essential sic at subanesthetic doses. Currently, isoflurane prob- to avoid potential human health hazards. Scavenging ably possesses the best combination of properties in systems that rely on activated charcoal are not effective terms of expense and safety of personnel and patient. for removing nitrous oxide. Sevoflurane is expensive but can provide a smoother and more rapid induction and recovery than isoflurane. 3. Anesthestic Adjuvants There have been concerns about nephrotoxic effects in a. Local Anesthetics rats exposed to a product formed from sevoflurane in the presence of soda lime, called ‘Compound A,’ effects Lidocaine, bupivacaine, and other local anesthetics have been seen in low flow anesthesia when rats are are often used to supplement light levels of general exposed to high levels of sevoflurane (Kharasch et al., anesthesia, as well as to provide some degree of post- 2005; Morio et al., 1992). operative analgesia. In rodents they are usually given The equipment needed for rodent anesthesia is rela- by local infiltration or topical application prior to an tively simple, comprising of a flowmeter, a vaporizer, an invasive procedure. With care to assure adequate general induction chamber, a delivery circuit, and some means anesthesia and to avoid toxicity, both short-acting (lido- of waste gas disposal. Face masks are commonly used in caine) and longer-acting (bupivacaine) local anesthetics rodents, although endotracheal intubation, if mastered, are useful. Discomfort from injection of local anesthetics permits assisted or controlled ventilation. The means of in conscious animals can be reduced by buffering and waste gas disposal varies with the facilities available. warming the solution. Additional analgesia should be Because of increased interest in inhalation anesthesia used if pain is expected to last more than a few hours due for rodents, new equipment and innovative designs that to the relatively short duration of action of these agents. more specifically address the needs of rodent anesthesia are becoming commercially available. Basic equipment b. Opioids and techniques for administration, including endo- As anesthetic adjuvants, opioid agonists provide tracheal intubation, are described in Flecknell (2009); potent analgesia and lower the required doses of anes- Watanabe et al. (2009); Hamacher et al. (2008); Molthen thetic agents. Buprenorphine, a partial mu-agonist, is (2006); and Turner et al. (1992). the preferred opioid for rodents as it is more potent than Bell jar or anesthetic chambers may be used for induc- morphine and has a longer duration of action. In addi- tion of anesthesia with inhalant agents by allowing the tion to buprenorphine, morphine, methadone, trama- anesthetic to vaporize within the ambient air into a dol, butorphanol, fentanyl, and have been closed jar or induction chamber. This ‘open drop’ or found to reduce the isoflurane and sevoflurane minimum ‘open circuit’ method was developed when ether was alveolar concentration (MAC) requirements in rats and the most widely used anesthetic agent. This type of mice (Criado and Gómez de Segura, 2000, 2003; Abreu administration is dangerously imprecise and requires et al., 2012; Smith et al., 2004). Opioids can be accompa- careful monitoring due to the uncontrolled amount of nied by bradycardia and respiratory depression, which gas to which the animal is exposed. Delivery of isoflu- may be balanced by lowering the dose of anesthetics. rane in this way, for example, results in anesthetic con- Additionally, atropine or glycopyrrolate can be used to centrations of 20–25%, which can be rapidly lethal. It is counteract bradycardia but should be used with caution, not a suitable method for surgical anesthesia when using if at all, when medetomidine is also a component of the modern inhalant agent, however it is sometimes used for anesthesia protocol, as correcting the bradycardia can

LABORATORY ANIMAL MEDICINE II. Rodents 1143 result in a dangerous degree of hypertension. Opioid fentanyl significantly reduced behaviors associated with agonists can be effectively reversed by specific antago- distress during induction and reduced sevoflurane MAC nists, such as naloxone, as well as by some partial ago- by one-third (Cesarovic et al., 2012). The effects of these nists and mixed-function agonist–antagonists, such as agents can be quickly terminated by the reversal agent, butorphanol, nalbuphine, and buprenorphine, which are flumazenil, however the effects of the antagonist are typically utilized for postoperative analgesia. relatively brief, and repeated dosing may be needed. c. α2-Adrenergic Agonists e. Phenothiazine Tranquilizers Members of this group commonly used in rodent Acepromazine, and to a lesser extent promazine, are anesthesia include xylazine and medetomidine (or the most common phenothiazine tranquilizers utilized dexmedetomidine) (Stokes et al., 2009). These drugs for rodent anesthesia. Phenothiazines produce sedation are anxiolytic, analgesic, and provide muscle relax- and potentiate the effects of anesthetics but do not pro- ation. Another benefit of these agents is their ability vide analgesia. They also cause vasodilation, with some to provide a brief period of analgesia (15–30 min, if depression of blood pressure and body temperature, co-administered with ketamine) beyond the period of and should not be used in dehydrated or hemorrhaging surgical anesthesia (Gaertner et al., 2008). They also patients. cause hyperglycemia, bradycardia, peripheral vasocon- In mice, acepromazine combined with ketamine striction, hypothermia, and diuresis of variable sever- and xylazine (KXA) has been shown to result in longer ity in different species and with varying doses and duration of anesthesia and immobility, as well as more drug mixtures (Hsu et al., 1986; Gaertner et al., 2008). stable physiologic parameters compared to ketamine/ Even so, the combination of ketamine and xylazine is xylazine alone (Arras et al., 2001; Buitrago et al., 2008). generally reliable, safe, and convenient in laboratory The KXA combination has also been demonstrated to be rodents and enjoys wide popularity (Green et al., 1981; effective for surgical anesthesia in rats (Welberg et al., Gaertner et al., 2008; Stokes et al., 2009). Ketamine can 2006). also be combined with medetomidine to provide simi- lar effects (Nevlainen et al., 1989). Some studies have f. Neuromuscular Blocking Agents noted increased anesthetic complications and death Paralytics may be required for specific experimental when using buprenorphine as a pre-medicant to ket- protocols, but their use must be justified as no anes- amine/medetomidine combinations (Hedenqvist et al., thesia or analgesia is provided, and once the animal 2000b). If redosing is necessary for extended anesthetic is paralyzed, it is difficult to assess depth of anesthe- effect, it is recommended that only ketamine be pro- sia. For this reason the anesthetic protocol should be vided as additional dosing of xylazine or dexmedetomi- demonstrated to be adequate for performance of the dine can cause bradycardia and cardiac arrest (Gaertner study procedures humanely without the use of neuro- et al., 2008). The sedation produced by α2-adrenergics muscular blocking (NMB) agents. Controlled ventila- can be effectively antagonized by a number of drugs. tion must be provided to paralyzed rodents and careful Yohimbine is often used to reverse the sedation, diuresis, monitoring of blood pressure, heart rate, and end tidal and cardiovascular effects produced by xylazine (Hsu CO2, while imperfect indicators, may assist in assur- et al., 1986). Atipamezole was developed to antagonize ing the patient is adequately anesthetized. Neostigmine α2-adrenergics, especially medetomidine, more specifi- and physostigmine may be used to reverse the effects cally and rapidly (Flecknell, 2009). The ability to reverse of nondepolarizing NMB agents. The use of NMB the effects of these drugs can be used to advantage to agents in research has been reviewed by Gaertner et al. shorten recovery time, as well as to rescue the occa- (2008). sional patient that displays severe adverse reactions to the drugs. However, it should be noted that reversal D. Intraoperative Monitoring and Support of the effects of xylazine and dexmedetomidine also reduce the analgesic effect of the drugs, so additional Monitoring anesthesia in rodents is often limited to analgesics may be necessary following reversal. observation of respiratory rate and character, color of the skin and mucus membranes (if the animal is an albino), d. Benzodiazepines and response to surgical stimulus. Depth of anesthesia Diazepam and midazolam are often used as adju- may be estimated by pedal withdrawal response and vants to anesthesia. They have similar effects, including eye reflexes but is probably most reliably indicated by anxiolysis, sedation, and muscle relaxation but are not response to surgical stimulus (Flecknell, 2009). While analgesic. Midazolam is shorter-acting and is water- heart and respiratory rate, as well as oxygen saturation soluble, making it preferable for mixtures intended to are more easily monitored in larger rodents, such as provide short-term anesthesia. Midazolam paired with rats and guinea pigs, pulse oximeter options are now

LABORATORY ANIMAL MEDICINE 1144 24. Preanesthesia, Anesthesia, Analgesia, and Euthanasia commercially available for use in mice and sensitive and therefore are unable to experience conscious pain enough to be utilized for hypoxia research (Chodzyński until after birth (Murrell et al., 2008). et al., 2013). Electrocardiography is primarily used only Overall, suitability of an anesthetic protocol will as a research tool in rodents. Hypothermia will occur depend to some extent on the stage of pregnancy and with all anesthetic protocols in rodents, and it is there- the species. Late pregnancy and the associated increased fore important that body temperature is measured, and abdominal pressure may impede adequate respiration; means of warming the animal are employed. Temperature therefore positioning the animal on its side, as well as can be monitored by using small rectal and surface provision of supplemental oxygen and possibly ventila- probes. Thermal support utilizing circulating hot-water tion may be necessary (Flecknell, 2009). If surgical access pads, homeothermic blankets, infrared warming pads, to the fetus is required, inhaled anesthetics are preferred heating lamps, space blanket, or even bubble wraps are as they cause uterine relaxation and facilitate surgical recommended. Fluid support in the form of warmed manipulation of the uterus. Alpha-2 adrenergic ago- subcutaneous fluids is useful and simple, especially with nists, on the other hand, reduce uterine blood flow and prolonged procedures. As rodents do not close their eyes increase uterine contractility making them a less favor- while anesthetized, lubrication to prevent corneal drying able choice for fetal surgery (Murrell et al., 2008). Local is an important step for all anesthetic events lasting more and regional analgesia, as well as preoperative adminis- than a few minutes. Another undesirable side-effect of tration of opioid analgesics can significantly reduce the anesthesia, particularly with injectable agents, is modest anesthetic requirements (Murrell et al., 2008; Flecknell, to severe hypoxia and hypercapnia. Moderate hypercap- 2009). General principles and approaches to anesthesia nia can be tolerated by healthy animals for reasonable and analgesia in the pregnant dam are given in Murrell periods of time but if progressive may be difficult to et al. (2008) and Flecknell (2009). correct without mechanical ventilation. Hypoxia, on the other hand, is often easily and inexpensively corrected 2. Neonatal Anesthesia by providing a low flow of oxygen by mask during Inhalant anesthesia, primarily sevoflurane or isoflu- anesthesia and surgery. rane, is the preferred method of neonatal anesthesia due to the rapid induction and recovery; of note, neonates E. Special Anesthetic Considerations may require a higher concentration of gas anesthetic than adult rodents (Flecknell, 2009). Hypothermia can also be 1. Anesthesia for Transgenic Production and an effective anesthetic for altricial neonates when cooled Pregnant Rodents to 5–10°C; though there is some debate as to possible While appropriate preparation and storage are discomfort during cooling and rewarming (Janus and required, tribromoethanol has a long-standing history of Golde, 2014; Gaertner et al., 2008; Flecknell, 2009). Care successful use in transgenic mouse production. However, must be taken not to expose neonates directly to ice to its use is waning as safer anesthetic options, ketamine/ avoid tissue damage. Fentanyl plus fluanisone have also xylazine and isoflurane, have been shown to have equiv- been used successfully for neonatal anesthesia (Clowry alent minimal effects on fetal growth and pregnancy and Flecknell, 2000). Significant concerns with neonatal rates in mice as tribromoethanol (Thaete et al., 2013; Bagis anesthesia include rapid development of hypoglycemia et al., 2004). Similarly, the number of live mouse embryos, and hypothermia, which can contribute to subsequent pups born and weaned from dams administered maternal rejection or cannibalization. Where possible, buprenorphine, flunixin meglumine, or a combination analgesia for invasive procedures should be considered of buprenorphine and carprofen post-embryo transfer as unalleviated pain in the rodent neonate can affect surgery were not significantly impacted by analgesic response to pain and stress later in life (LaPrairie and administration (Krueger and Fujiwara, 2008; Goulding Murphy, 2010; Sternberg et al., 2005; Victoria et al., 2013a,b, et al., 2010; Parker et al., 2011). Providing supplemental 2014). These alterations can be attenuated if preemptive heat overnight after embryo transfer surgery was shown analgesia is provided (LaPrairie et al., 2008; Walker et al., to significantly increase rates of pregnancy and fetal 2009), however there is little objective data on appropri- implantation (Bagis et al., 2004). In rat dams undergoing ate dose rates of analgesics for neonatal rodents. embryo transfer, use of isoflurane and morphine resulted in significantly greater pregnancy rates and faster induc- 3. Stereotactic and Neurosurgery tion and recovery times compared to ketamine and xyla- Anesthesia for stereotactic surgery has usually relied zine; additionally, both anesthetic regimens resulted in on the same anesthetics used for other purposes. Because comparable numbers of live pups (Smith et al., 2004). placement of ear bars for fixation of the head is often more Regarding surgical manipulation of the fetus during ges- painful than the surgery itself, a relatively deep plane of tation, the literature largely suggests that fetuses in utero anesthesia is needed. Lidocaine creams placed on the ear are maintained in a sleeplike state of unconsciousness bars may reduce some of this discomfort. Removal of the

LABORATORY ANIMAL MEDICINE II. Rodents 1145 ear bars following surgery can result in an abrupt increase small-bore infusion lines can be used to deliver inject- in the apparent depth of anesthesia (Gardiner and Toth, able agents. For inhalation techniques, rodents can be 1999). Face-mask adapters, used in place of the incisor bar, placed in plastic cylinders with fresh gas entering at one allow for more convenient use of inhalation techniques. end of the cylinder and exiting at the opposite end to the Alternatively, intranasal cannulas with scavenging by scavenging system. If gating is necessary, the animal will snorkel or downdraft may facilitate delivery of gas anes- likely need to be ventilated. Anesthetic monitoring may thesia when use of a face-mask is not possible (Flecknell, be limited to intermittent visual observations, but MRI- 2009). Heat support can be challenging if the rodent is not compatible pulse oximeters and capnographs may be in contact with a heated surface due to the stereotaxic posi- used. Full commercial suites of MRI-compatible rodent tioning, thus possible options for preventing hypothermia monitoring, gating, and heating systems are available. include wrapping the animal in bubble wrap or pairing Alternatively, a simple means of monitoring respiration reflective foil with a thermogenic gel or circulating warm rate is a thermistor placed near the nares, ET tube or water blanket (Flecknell, 2009, Caro et al., 2013). Lastly, face mask, which is sensitive for animals weighing more craniotomies and manipulation of the muscles of mas- than 300 g, or a pressure sensor (baby respiration sensor) tication may result in post-procedural discomfort when to detect respiratory movements for animals weighing eating or drinking; early nutritional and fluid support has as little as 30 g (Flecknell, 2009, Tremoleda et al., 2012). been demonstrated to significantly improve recovery after Heat support that will not interfere with the image, such stereotactic craniotomy surgery (Hampshire et al., 2001). as warm air or warmed fluid bags or gels, may be use- For minimally invasive nonsurvival studies involving ful. A thorough review of anesthesia and physiological measurement of autonomic reflexes,α -chloralose, with or monitoring of in vivo imaging of rodents can be found without urethane, is sometimes used (Gaertner et al., 2008). in Tremoleda et al. (2012) and Gargiulo et al. (2012a,b).

4. Anesthesia for Imaging Procedures F. Analgesic Therapy Imaging is an increasingly important tool for in vivo research, facilitating noninvasive longitudinal studies. The ethical and scientific value of relieving pain and Anesthesia plays a crucial role in reducing movement distress in experimental animals is well accepted in artifacts but can present challenges for monitoring, principle but remains a challenging and often contro- maintaining adequate anesthetic depth and preventing versial topic in practice. Discussions regarding scien- hypothermia. Bioluminescent imaging modalities fre- tific justification for withholding of analgesics should be quently feature a heated stage and gas anesthetic deliv- weighed with the behavioral, physiological, and immu- ery via face-masks. Other modalities, such as computed nological sequelae of untreated pain. In some models tomography (CT); micropositron emission tomography the untreated pain may be a greater variable than the (MicroPET); single-photon emission CT (SPECT); and analgesic (Piersma et al., 1999; Page, 2002; Page and Ben- ultrasound permit use of a range of anesthetic agents, Eliyahu, 2002; Franchi et al., 2007; Kolstad et al., 2012). thermal support approaches, and associated monitor- Ideally the impact of analgesia versus unalleviated pain ing. A shared complication is the often-long acquisition should be evaluated within each model to fully appreci- times requiring either inhalant or long acting injectable ate their individual contributions as variables on scien- anesthesia. Anesthetic effect on tracer uptake and other tific outcomes. physiological variables that may influence image quality should be considered (Alstrup and Smith, 2013). Where 1. Assessment of Pain/Discomfort possible, monitoring should include basic respiratory Species-specific signs of acute and chronic pain, parameters such as rate, depth and character, heart rate, approaches to pain recognition and to providing and temperature, mucous membrane color, muscle tone, and monitoring analgesia are described in Karas and Cadillac reflexes. If pre-imaging fasting is required, withholding (2008), Flecknell (2009), ACLAM Task Force Members food for up to 6 h prior to imaging is sufficient for the (Kohn et al., 2006), ILAR Committee on Recognition and stomach to clear ingesta (Tremoleda et al., 2012). Alleviation of Pain in Laboratory Animals (National Magnetic resonance imaging (MRI) of rodents presents Research Council, 2009) as well as emerging behavioral unique difficulties in terms of anesthetic administration, assessments such as pain faces (grimace scores), burrow- thermal support, and monitoring. For short procedures, ing and nest building behaviors, behavioral ethograms any injectable method compatible with the study can and even affective state (Matsumiya et al., 2012; Sotocinal be used. For longer procedures, inhalation anesthesia is et al., 2011; Jirkof et al., 2010, 2013a,b; Cesarovic et al., the method of choice, but in its absence infusion tech- 2011; Roughan and Flecknell, 2003a; Wright-Williams niques are also satisfactory. Although infusion pumps et al., 2007, 2013; Roughan et al., 2009; Flecknell, 2010; and conventional inhalation equipment must be kept Langford et al., 2010; Leach et al., 2012; Makowska et al., at a safe distance, both can be effectively used. Long, 2013; Rock et al., 2014). Carefully constructed scoring

LABORATORY ANIMAL MEDICINE 1146 24. Preanesthesia, Anesthesia, Analgesia, and Euthanasia systems, specifically adapted to the conditions of the pro- 2012). Due to their small size and high metabolic rate, tocol, are most useful in recognizing pain and monitor- rodents may require a buprenorphine dose frequency of ing analgesia in rodents. When planning for assessment 2–4 times daily (Gades et al., 2008, 2000; Deng et al., 2000; and alleviation of postoperative and post-procedural Roughan and Flecknell, 2002; Yu et al., 2006). While lower pain, consider all sources of discomfort such as hypo- doses are not associated with significant side effects, in thermia, prolonged body positioning, ear bars, potential the rat, pica and possibly long-term weight reductions nausea, disorientation, etc. have been reported at higher doses or with prolonged dosing schedules ((Schaap et al., 2012; Bomzon, 2006). 2. Nonpharmacologic Methods Strain and sex of rodents can also modulate response Almost invariably, the term analgesia is associated to opiates (Jablonski et al., 2001; Avsaroglu et al., 2007, with drugs. But drugs cannot be substituted for meticu- 2008; Cotroneo et al., 2012; Wright-Williams et al., 2013). lous surgical technique, nor for excellent nursing and Although usually given as an injection, in some stud- husbandry. Comfortable bedding, ability to control light ies orally administered buprenorphine was efficacious exposure and temperature with a shelter and/or appro- in alleviating postoperative pain, however, its utility priate nesting material, supplemental heat provided to may be limited in duration of analgesia and by reliable a portion of the cage, easy access to palatable food and consumption of the food stuffs or water into which it is water, attention to wound care, as well as addressing compounded (Flecknell et al., 1999a; Martin et al., 2001; issues of social deprivation will greatly improve the Jablonski and Howden, 2002; Thompson et al., 2004, quality of postoperative and post-procedural recovery 2006; Jessen et al., 2007; Goldkuhl et al., 2010a,b; Leach (Jirkof et al., 2012; Van Loo et al., 2007; Pham et al., 2010; et al., 2010b; Kalliokoski et al., 2011; Abelson et al., 2012). Gabriel et al., 2009, 2010a,b). Of note, cryoanalgesia was Additionally, sustained release and transdermal formu- recently found to provide effective local anesthesia for lations of buprenorphine are emerging as potentially tail-tip biopsy in mice older than 21 days and may be a viable and practical opiate delivery systems (Park et al., valuable alternative to isoflurane anesthesia; however 2008; Foley et al., 2011; Carbone et al., 2012a). Another cryoanalgesia was not recommended prior to toe clip- opioid gaining attention is tramadol, particularly as it ping in mouse pups (Matthias et al., 2013; Paluch et al., is not a controlled drug in some states. Data evaluating 2014). Electroacupuncture has been demonstrated to be the therapeutic benefit of tramadol over buprenorphine analgesic in several rodent pain models but veterinary or NSAID therapy is mixed (Cannon et al., 2010; Zegre use in rodents has yet to be explored (Kim et al., 2010, Cannon et al., 2011; McKeon et al., 2011; Rätsep et al., 2011; Jiang et al., 2013; Wang et al., 2013). 2013) and high dose tramadol may be associated with increased mortality in some mouse models of sepsis 3. Pharmacologic Methods (Hugunin et al., 2010). The major drugs used for analgesia include opioids, nonsteroidal anti-inflammatory drugs (NSAIDs), and b. Non-Steroidal Anti-Inflammatory Drugs local anesthesia. Optimal analgesia may be best achieved NSAIDs are sometimes preferable to opiates as they with a multimodal approach incorporating preemptive are less likely to induce behavioral changes, provide a analgesia (Gaertner et al., 2008; Zegre Cannon et al., 2011; longer duration of analgesia, and are not regulated con- Flecknell, 2009; Penderis and Franklin, 2005; Schaap trolled substances (Matsumiya et al., 2012; Roughan and et al., 2012). As with all drugs used in anesthesia and Flecknell, 2004; Roughan et al., 2004; Bourque et al., 2010; analgesia, these agents are generally safe and effective Brennan et al., 2009). NSAIDs such as meloxicam, car- but are not completely free of adverse effects. profen, ketoprofen, flunixin and indomethacin are potent and effective for moderate pain when injected subcuta- a. Opioids neously or intraperitoneally (Stewart and Martin, 2003; For severe pain, pure opioid agonists, such as Tubbs et al., 2011; Roughan and Flecknell, 2001, 2004; morphine, are preferred, despite a relatively brief Matsumiya et al., 2012; Cooper et al., 2005, 2008; Blaha period of effect (Gaertner et al., 2008; Flecknell, 2009). and Leon, 2008). Carprofen but not meloxicam was found Oxymorphone and hydromorphone also provide signifi- to reach therapeutic plasma levels if placed in drinking cant analgesia in mice and rats, but will likely only be water 12–24 h prior to a painful procedure (Ingrao et al., useful for clinical applications if provided in sustained 2013). However, neither carprofen nor ketoprofen admin- release formulations (Gillingham et al., 2001; Krugner- istered orally in jelly resulted in significant decreases in Higby et al., 2003; Smith et al., 2006; Clark et al., 2004; postoperative pain (Flecknell et al., 1999). A carprofen- Leach et al., 2010a; Schmidt et al., 2011). For pain of infused diet gel is commercially available but has not moderate or lower intensity, partial opioid agonists and been evaluated in the peer-reviewed literature at this mixed-function agonist–antagonists, such as buprenor- time. Acetaminophen was shown by Cooper et al. (1997) phine are often used (Curtin et al., 2009; Guarnieri et al., to be effective in the rat when delivered intraperitoneally

LABORATORY ANIMAL MEDICINE II. Rodents 1147 but was not efficacious when given SC (Matsumiya et al., agents and even inhalation agents. A pilot study is the 2012). Acetaminophen had limited evidence of efficacy best way to establish an acceptable anesthetic protocol when administered in drinking water, predominantly due with new techniques or agents. Published studies con- to neophobia and resulting decrease in fluid consump- cerning anesthetic techniques in hamsters, gerbils, and tion (Cooper et al., 1997; Speth et al., 2001; Bauer et al., guinea pigs are few compared with those for mice and 2003; Mickley et al., 2006; Dickinson et al., 2009). Sustained rats, reflecting their relative numbers as research ani- release NSAID formulations are on the horizon (Khurana mals. To an even greater degree, information concerning et al., 2013). When administering NSAIDs, attention must analgesia efficacy is scarce. Commonly suggested agents be paid to the patient’s hydration status, blood pressure, and doses are given below and in dose tables, but the and any other drugs being administered concurrently, as phrases ‘to effect’ and ‘as needed’ assume particular sig- they may increase risk for renal tubular necrosis and/ nificance with these species Tables( 24.1–24.4). or gastric ulceration (Lamon et al., 2008; Shientag et al., 2012; Jaquenod et al., 1998; Kumar et al., 2010). COX-2 1. Mice specific NSAIDs such as Carprofen or Meloxicam offer Table 24.1 lists selected anesthetics and analgesia analgesic efficacy with fewer renal and gastric side-effects doses for mice. Both induction and maintenance with (Flecknell, 2009; King et al., 2009). gas anesthesia, primarily isoflurane, produces the safest and most titratable anesthetic option. While endotra- c. Local Anesthesia cheal intubation requires training, a nose cone works Infiltration of incision sites can be used to decrease well when paired with appropriate waste gas scavenging postoperative pain and topical application of a local anes- and air exchanges. Combinations of ketamine and xyla- thetic cream may minimize pain associated with needle zine, or dexmedetomidine, with or without aceproma- sticks (Flecknell et al., 1990; Leach et al., 2012; Mert and zine, may produce surgical anesthesia for up to 90 min, Gunes, 2012; Kolesnikov et al., 2004; Jones et al., 2012). although depth of anesthesia may be variable (Burnside However, the duration of effect is relatively brief and et al., 2013). Reversal of xylazine or dexmedetomidine may be more useful pre- or intraoperatively than in the with atipamezole can significantly shorten the recov- postoperative period. The degree of analgesia provided ery period. Where available, fentanyl-fluanisone plus by a local block alone is insufficient to control for visceral midazolam can be an excellent choice. Historically, tri- pain associated with laparotomy and therefore should bromoethanol has served as the anesthetic of choice in be included as part of a multimodal analgesic approach transgenic production facilities, yielding effective surgi- (Hayes and Flecknell, 1999; Liles and Flecknell, 1993). cal anesthesia for at least 15 min when properly used Spinal and epidural administration of local anesthetics (Meyer and Fish, 2005; Papaioannou and Fox, 1993). Use have not been reported for clinical use in rodents but of pentobarbital, where available, as an anesthetic in efficacy data have been demonstrated in research evalua- mice is decreasing with time but is still sometimes used tions (Tseng et al., 2013; Sato et al., 2008; Kroin et al., 2012; for terminal procedures (Stokes et al., 2009). Heat support Wada et al., 2007; Bar-Yosef et al., 2001; Morimoto et al., and lubrication of the eyes remain important consider- 2001). Similarly, extended release formulations are being ations for all anesthetic events lasting more than a few explored (Ickowicz, 2013). minutes. Commonly used analgesics include buprenor- phine and/or NSAIDs via multiple routes including d. Gabapentin some oral, topical, and sustained release formulations, Originally developed as an antiepileptic, gabapentin as well as local anesthesia. is increasingly utilized to treat neuropathic pain and non-neuropathic pain from surgery, cancer, and arthritis 2. Rats in both human and veterinary patients. In rats, gabapen- Inhalation anesthesia has many advantages in the rat tin decreased acetic acid-induced writhing and signifi- and is the preferred anesthetic for most research needs. cantly attenuated paw incision-induced c-Fos activation, While endotracheal intubation via transillumination or indicating reduced post-surgical stress (Feng et al., 2003; other visual techniques is common, blind oral endo- Kazi and Gee, 2007). When combined with tramadol, tracheal intubation techniques and novel devices such gabapentin provided superior relief from footpad inci- as a face mask and a supraglottic airway device have sional pain than tramadol alone; however it did not out- recently been described (Cheong et al., 2010, 2013; Wang perform buprenorphine (McKeon et al., 2011). et al., 2012). Ketamine with xylazine, or fentanyl-flua- nisone with midazolam, if available, are the most com- G. Species Considerations monly used injectable anesthetics in rats (see Table 24.2). Ketamine-xylazine intramuscular injection resulted in Even within species, rodents manifest surprising significant muscle necrosis; to a lesser degree lesions variability in response to ‘standard’ doses of injectable were identified in the abdominal musculature after IP

LABORATORY ANIMAL MEDICINE 1148 24. Preanesthesia, Anesthesia, Analgesia, and Euthanasia injection (Smiler et al., 1990; Wellington et al., 2013). In Successful anesthesia and recovery require careful moni- comparison to ketamine-xylazine, ketamine-dexmedeto- toring and perioperative support, as well as appropriate midine resulted in better local tissue tolerance when agents, dose and dosing routes (see Table 24.5). Over-the- administered IP, while maintaining similar anesthetic endoscope or stylet, transillumination of the neck, use properties (Wellington et al., 2013). Pentobarbital, where of an otoscope speculum, and even use of a purpose- available, is less reliable but can be useful for non-recov- made laryngoscope to facilitate intubation of guinea pigs ery applications. Buprenorphine is frequently employed have all been described (Johnson, 2010; Flecknell, 2009). for analgesia in rats. Its side-effects, such as pica, can be In the absence of intubation, inhalation anesthesia is usu- minimized by altering the dose schedule and utilizing ally maintained by mask, using isoflurane or sevoflurane. a multimodal approach (Schaap et al., 2012). NSAIDs, Upon exposure to high concentrations of isoflurane in an including meloxicam, carprofen, and ketoprofen, are induction box, guinea pigs frequently exhibit excessive also beneficial (Flecknell et al., 1999b; Flecknell, 2009) lacrimation and salivation, which may be partially allevi- but can be associated with gastric ulceration (Shientag ated by preemptive application of sterile eye lubrication. et al., 2012; Cooper et al., 2008; Lamon et al., 2008). Local Laboratory guinea pigs often have a considerable amount anesthesia is a useful addition to multimodal analgesia of pasty feed in their mouths, which can contribute to air- (Wen et al., 2012). way obstruction. This residue can be removed or reduced by gently rinsing the mouth with 10–20 ml of water before 3. Hamsters induction or swabbing gross debris out of the mouth with Inhalation anesthesia by nosecone or endotracheal cotton swabs after induction. intubation is safe and effective with the equipment and Compared to alfaxolone/alfadolone (Saffan), pento- techniques used for other rodents (Gebhardt-Henrich either as a sole agent or in combination with et al., 2007; Picazo et al., 2009; Flecknell, 2009). As with droperidol and fentanyl (Innovar Vet), ketamine with rats, ketamine with xylazine or dexmedetomidine, or xylazine had the least respiratory effects fentanyl-fluanisone with midazolam, are safe and reli- in guinea pigs (Schwenke and Cragg, 2004). When able for procedures of moderate duration (see Table 24.3) given IP it will result in a surgical plane anesthesia for (Payton et al., 1993; Flecknell, 2009). A combination of about 30 min but may require supplementation with an Telazol and xylazine given IP was reported to produce inhalant (Cetin et al., 2005; Dang et al., 2008). Fentanyl- surgical anesthesia (Forsythe et al., 1992). Pentobarbital fluanisone with midazolam is also effective for a simi- is reported to have a narrow margin of safety in ham- lar period of time (Flecknell, 2009). A combination of sters and should be used with caution (Flecknell, 2009). tiletamine/zolazepam, xylazine, and butorphanol pro- Analgesics used in mice and rats can be similarly uti- vided deep surgical, long-duration anesthesia in guinea lized in hamsters. pigs (Jacobson, 2001). In contrast, medetomidine and medetomidine in combination with ketamine was found 4. Gerbils to sedate but not anesthetize guinea pigs (Dang et al., Inhalation anesthesia using isoflurane or sevoflurane 2008; Nevlainen et al., 1989). However, the combination is the safest and most reliable means of anesthetizing of tiletamine-zolazepam and medetomidine was shown these small rodents, though gerbils may require a lower to induce a surgical plane of anesthesia (Buchanan et al., concentration of anesthetic gas than mice or rats (Henke 1998). Intramuscular administration of anesthetics in et al., 2004; de Segura et al., 2009). Additionally, anes- guinea pigs has been reported to cause self-mutilation thesia in gerbils can be produced using a combination and should be avoided (Leash et al., 1973; Newton et al., of fentanyl and metomidate, (see Table 24.4) (Flecknell, 1975). Judicious use of local anesthetics can reduce the 2009; Pérez-García et al., 2003). Alternatively, Telazol risks of deep anesthesia and the dangers associated with given IP resulted in surgical anesthesia but with a pro- repeated doses of injectable agents. Like other rodents, longed recovery time (Hrapkiewicz and Smiler, 1989). buprenorphine, meloxicam, and carprofen, as well as Pentobarbital should be used with caution. Gerbils are local anesthesia, are frequently utilized to reduce post- particularly sensitive to hypothermia during anesthesia procedural pain (Flecknell, 2009; Aguiar et al., 2013). and upon recovery may take several days to regain nor- mal circadian rhythms (Weinandy et al., 2005). Analgesics H. Euthanasia used in mice and rats can be similarly utilized in gerbils. Euthanasia methods must meet both experimental 5. Guinea Pigs and humane criteria; a comprehensive review can be Guinea pigs are considered to be among the more dif- found in the Report of the ACLAM Task Force on Rodent ficult laboratory animals to safely anesthetize. Guinea pigs Euthanasia and the AVMA Guidelines for the Euthanasia can be quite neophobic, have few accessible peripheral ves- of Animals (2013) (Artwohl et al., 2006; Leary et al., 2013). sels, unusually tough skin, and can be difficult to intubate. Prior to euthanasia, activities that contribute to distress

LABORATORY ANIMAL MEDICINE III. Rabbits 1149 should be minimized; this may include euthanizing ani- defined health status from quality vendors, this reputa- mals in their home cage to maintain established scent tion has been dispelled. Although rabbits continue to marks and not combining unfamiliar animals (Leary et al., be an anesthetic challenge because of individual varia- 2013). Injectable anesthetic overdose using a combina- tion in drug response and their timid nature, there now tion of xylazine, ketamine, or diazepam or a barbiturate, exist numerous methods for safe and effective induction barbituric acid derivative, or barbiturate combination and maintenance of anesthesia in the rabbit (Flecknell, are acceptable methods of euthanasia of rodents (Leary 2009; Lipman et al., 1997, 2008; Suckow et al., 2012). The et al., 2013). Overdose with inhalant anesthetics or carbon purpose of this section is to provide a basic guide to dioxide are also acceptable, as long as death is confirmed commonly used techniques. by examination or via an adjunctive physical method. Carbon dioxide, the most common means of euthanasia for small rodents, should be administered from a com- B. Preoperative Assessment and Preparation pressed cylinder at a rate that will displace 10–30% of the 1. Preoperative Evaluation cage volume per minute and not via prefilled chamber (Leary et al., 2013). Additionally, altricial neonates require Rabbits should be purchased specific pathogen-free for Pasteurella multocida and other infectious agents that an extended period of CO2 exposure, which may need to be followed by a physical adjunctive method of eutha- might influence research. The two breeds most commonly nasia (Klaunberg et al., 2004; Pritchett-Corning, 2009). used for research in the United States are the New Zealand Automated carbon dioxide euthanasia systems have been White and the Dutch Belted. Rabbits that specifically validated and are commercially available (McIntyre et al., model certain diseases, such as the Watanabe heritable 2007). There remains controversy regarding the recom- hyperlipidemia rabbit, a model for familial hypercho- lesterolemia, may present additional challenges to the mended administration rate of CO2 and the potential ben- efit for administration of isoflurane anesthesia or other anesthetist. Rabbits within a particular study should be gases or even injectable tranquilizers, sedatives, or anes- purchased from a single vendor as genetic differences will exist among stocks maintained by different vendors. thetics prior to, or in combination with, CO2 (Hackbarth et al., 2000; Niel and Weary, 2006, 2007; Makowska et al., Rabbits should be allowed acclimatization periods of a 2008, 2009; Chisholm et al., 2013; Wong et al., 2013; Leach minimum of 72 h prior to use in a research project. Testing et al., 2002a,b, 2009; Valentine et al., 2012; Makowska and during the acclimatization period will be determined by Weary, 2009a,b; Makowska et al., 2012; Niel et al., 2008a,b; the health status of the vendor colony, the duration of Thomas et al., 2012a). Guinea pigs may struggle exces- use, and the demands of the specific protocol. sively when exposed to carbon dioxide. Pre-sedation, There is no consensus on the requirement for overnight concurrent use of an inhalant anesthetic such as isoflu- fasting for rabbits (Flecknell, 2009; Rees-Davies and Rees- rane or sevoflurane, and application of sterile eye lubri- Davies, 2003). Proponents argue that there is more con- sistent anesthesia and that the reduction in intragastric cant may reduce distress associated with CO2 euthanasia in guinea pigs. Cervical dislocation and decapitation of volume allows more effective diaphragmatic excursion mice and rats less than 200 g can be rapid and humane if with consequent improvements in ventilation. Opponents carried out by experienced operators, who were prefer- argue that coprophagia precludes complete gastric emp- ably trained on anesthetized animals, and as approved tying and that the inability of the rabbit to vomit makes by the IACUC (Carbone et al., 2012b; Cartner et al., 2007; food withdrawal unnecessary. Rabbits under 3 kg should Golledge, 2012; Roustan et al., 2012; Leary et al., 2013). not have more than 12 h of food withdrawal; these rabbits may develop metabolic acidosis and a decline in blood glucose concentration (Bonath et al., 1982).

III. RABBITS 2. Choice of Anesthetic Technique A. Introduction Anesthetic regimens used in companion animal prac- tice may differ from those used in the laboratory due The rabbit remains an important research animal by to differences in objective and patient population. The virtue of its convenient size, ease of handling, defini- choice of anesthetic technique is determined by the tion in a number of experimental and teaching models, desired duration of anesthesia, the nature of the surgi- and availability at various ages and reproductive sta- cal stimulus, the physiologic effects of the technique, tus. Rabbits have been considered difficult to anesthe- their potential impact on the variables studied, and the tize safely, a reputation earned when pentobarbital was age and preoperative status of the animal. For example, in wide usage and rabbits were of questionable health the hyperglycemic effect of α2-agonists such as xylazine, status. With the development of newer anesthetic drugs should be considered in studies where glucose concen- and techniques, and with the availability of rabbits with tration is important (Gleed, 1987).

LABORATORY ANIMAL MEDICINE 1150 24. Preanesthesia, Anesthesia, Analgesia, and Euthanasia

3. Preoperative Medications TABLE 24.6 drug Dosesa in Rabbits

The use of preoperative medications may reduce the Drug Dosage Route anesthetic requirement for maintenance agents, relieve patient apprehension, and facilitate handling. These agents ANTICHOLINERGICS may suffice for restraint when subjecting animals to non- Atropine 0.04–2.0 mg/kg IM, SC invasive procedures like phlebotomy or mask induction (0.5 mg/kg with inhalants. Anticholinergics may be used to prevent commonly vagal reflexes and to reduce salivary and tracheobronchial recommended) secretion that may compromise the airway during anes- Glycopyrrolate 0.1 mg/kg IM, SC thesia. Atropine use may be ineffective in some rabbits SEDATIVES/TRANQUILIZERS because of high levels of atropinesterase, an enzyme that degrades atropine into inactive products (Ecobichon and Diazepam 5–10 mg/kg IM Comeau, 1974). The enzyme may be present in up to 50% of New Zealand White rabbits, is highly variable by breed, 1–2 mg/kg IM, IV and is heritable (Harrison et al., 2006). Consequently, some Midazolam 2 mg/kg IP, IV authors have recommended very high doses of atropine, Acepromazine 0.75–10.0 mg/kg IM such as 1–2 mg/kg (Hall and Clarke, 1991), with frequent (0.75–1.0 mg/kg redosing, as often as every 15–20 min (Sedgwick, 1986). most frequently Glycopyrrolate is an effective parasympatholytic agent in used) the rabbit and produces 60 min of elevation of heart rate, Xylazine 3–9 mg/kg IM prevention of ketamine/xylazine-associated bradycardia, Medetomidine 0.25 mg/kg IM and an antisialogogue effect when administered at a dose Dexmedetomidine 20–80 g/kg IM, IV of 0.1 mg/kg IM (Olson et al., 1994). μ Tranquilizers used in rabbits include phenothiazines BARBITURATES (principally acepromazine), benzodiazepines (diazepam Thiopental 15–30 mg/kg IV (1% solution) GTEb and midazolam), opioids, and α2-agonists (xylazine and dexmedetomidine). Dexmedetomidine has greater affin- 50 mg/kg IV (2.5% solution) GTE ity and selectivity for α2-receptors than does xylazine sodium 15 mg/kg IV (1% solution) GTE (Hellebrekers et al., 1997). These agents may be used as 29 mg/kg IV (2% solution) GTE sole agents in minor procedures such as echocardiogra- phy, radiology, physical examination, and phlebotomy. EMTU 47.5 mg/kg IV GTE Benzodiazepine, opioids, and the α2-agonists have spe- 5–10 mg/kg IV (1% solution) GTE cific antagonists that provide the anesthetist greater Pentobarbital 20–60 mg/kg IV GTE control over duration of sedation or anesthesia. Drug dosages are listed in Table 24.7. Ketamine 20–60 mg/kg IM C. Intraoperative Anesthesia Ketamine+ 10 mg/kg IV xylazine 3 mg/kg IV In this section, the most commonly used anesthetic agents will be described. Dosages are given in Tables Ketamine+ 22–50 mg/kg IM 24.6 and 24.7. xylazine (reverse with 2.5–10 mg/kg yohimbine) 0.2 mg/kg IV 1. Injectable Anesthetics Acepromazine+ 0.75–1.0 mg/kg SC Injectables are useful for induction of anesthesia in preparation for the use of inhalants, as maintenance ketamine+ 35–40 mg/kg IM agents in short procedures, or as continuous infusions in xylazine (preop. 3–5 mg/kg balanced anesthetic techniques. Additional increments with atropine, 0.04 mg/kg IM) of the initial dosage may be used to prolong anesthesia. The popularity of injectables may be attributed to their Ketamine+ 75 mg/kg IM ease of administration, predictability, and reasonable acepromazine 5 mg/kg IM (given 30 min prior efficacy and safety. Many injectable combinations cause to ketamine) hypoxemia and supplemental oxygen should be used for Ketamine+ 60–80 mg/kg IM those combinations that cause hypoxemia (Hellebrekers et al., 1997; Peeters et al., 1988). (Continued)

LABORATORY ANIMAL MEDICINE III. Rabbits 1151

TABLE 24.6 d(Continued)rug Dosesa in Rabbits TABLE 24.7 bolus or Infusion Regimensa in Rabbits

Drug Dosage Route Drug Dosage Route

-Chloralose + 60 mg/kg diazepam 5–10 mg/kg IM (given 30 min prior α urethane to ketamine) 400 mg/kg followed Ketamine+ 25 mg/kg IM by 1–3 ml medetomidine 0.5 mg/kg SC 1% α-chloralose IV NEUROLEPTANALGESICS q30–50 min

α-Chloralose + 40–60 mg/kg Fentanyl-fluanisone 0.2–0.6 ml/kg IM, SC urethane Diazepam + 1.5–5 mg/kg, IM, IV, IP 800 mg/kg, followed fentanyl-fluanisone 0.2–0.5 ml/kg by 3–4 ml/h 1% (administer IM, SC α-chloralose diazepam 5 min prior to IP, IV SEDATION fentanyl-fluanisone) Ketamine+ 35 mg/kg IM Midazolam + 2 mg/kg, IM fentanyl-fluanisone 0.3 ml/kg xylazine 5 mg/kg IM (administer midazolam MAINTENANCE 5 min prior to fentanyl-fluanisone) Ketamine+ 1 mg/min Continuous IV infusion Propofol 7.5–15 mg/kg IV xylazine 0.1 mg/min Medetomidine + 0.25 mg/kg followed IM Ketamine+ 25 mg/kg IV propofol in 5 min by 4 mg/kg IV xylazine 5 mg/kg One-third bolus dose medetomidine+ 0.25 mg/kg IM over 1 min, remainder over 4 min midazolam+ 0.5 mg/kg IM SEDATION propofol 2 mg/kg IV

α-Chloralose + 32 mmol (10 gm)/ Propofol 1.5 mg/kg IV bolus urethane liter in saline MAINTENANCE at dose of 258 μmol (80 mg)/kg Propofol 0.2–0.6 mg/kg/min Continuous IV infusion 400–500 mg/kg IV (slowly) (5.61 mmol/kg) in INDUCTION 1 liter of saline (2.81 mol/liter) Ketamine+ 25 mg/kg IM Urethane 1–1.6 gm/kg IP xylazine 15 mg/kg IM 1.5 gm/kg IV MAINTENANCE

REVERSAL AGENTS Propofol 0.6 mg/kg/min Continuous IV infusion α2-Antagonists Fentanyl 0.48 mg/kg/min Yohimbine 0.2–1.0 mg/kg IM, IV Vecuronium 0.003 mg/kg/min Atipamezole 0.2–0.35 mg/kg IV Adapted from Lipman et al. (2008). Benzodiazepine 0.1 mg/kg SC aIntermittent bolus or continuous IV infusion regimens. antagonist: flumazenil Opioid antagonist: 0.001–0.1 mg/kg IV naloxone 2. Dissociatives aAdapted from Lipman et al. (2008) and Wixson (1994). As a sole agent, ketamine can be used for restraint bGTE, given to effect. during noninvasive procedures. Endotracheal intuba- tion can be achieved with ketamine combined with other agents (Lindquist, 1972; Green et al., 1981). Ketamine has been used with xylazine, medetomi- dine, dexmedetomidine, diazepam or midazolam, and

LABORATORY ANIMAL MEDICINE 1152 24. Preanesthesia, Anesthesia, Analgesia, and Euthanasia myriad other agents. Ketamine/xylazine may be aug- Mitchell, 1984). It may be used for restraint or for sedation mented with acepromazine or butorphanol to provide prior to administration of inhalation agents. The use of anesthesia of greater duration and depth (Lipman et al., this agent after IV or IP diazepam or midazolam produces 1990; Marini et al., 1992). These agents provide approxi- anesthesia of moderate duration; increments of hypnorm mately 30–45 min of loss of the pedal withdrawal reflex. IM or diazepam IV will prolong anesthesia. Others admin- Physiologic effects of these combinations include depres- ister fentanyl-fluanisone intramuscularly and follow with sion of respiratory rate, hypoxemia, hypercarbemia, intravenous midazolam (Benato et al., 2013). Premedication hypotension, and bradycardia. A constant-rate infusion with parasympatholytics is important when using opi- (CRI) technique has also been described (Wyatt et al., oids. Other regimens that have been investigated include 1989). Procedures of moderate surgical stimulus inten- medetomidine-midazolam-fentanyl (Baumgartner et al., sity (such as carotid or iliac endarterectomy) may be per- 2010), and fentanyl-propofol (Baumgartner et al., 2009). formed with ketamine/xylazine. Accidental perineural Significant cardiovascular and respiratory effects have injections of these combinations may lead to self-trauma. been noted with both these combinations, making them Medetomidine/ketamine and dexmedetomidine/ket- less suitable for animals with impaired cardiovascular amine combinations are useful regimens for rabbit anes- function. thesia (Lipman et al., 2008). The physiologic effects of Naloxone, doxapram, and various mixed agonist/ medetomidine/ketamine are similar to those observed antagonist opioids may be used to reverse fentanyl- with ketamine/xylazine; moderate hypoxemia, brady- induced respiratory depression. Use of mixed agonist/ cardia, and respiratory rate reduction occur (Hellebrekers antagonist opioids for this purpose provides fentanyl et al., 1997). Arterial blood pressure is better preserved by reversal while preserving analgesia of various duration the medetomidine combination in most studies. Reversal (DeCastro and Viars, 1968). When buprenorphine was of medetomidine with atipamezole at doses once or twice used in this fashion to reverse hypnorm, it provided that of medetomidine reduces arousal time and reverses 420 min of analgesia while reversing depression of respi- physiologic changes dramatically (Kim et al., 2004). ratory rate and effecting normalization of oxygenation Premedication of rabbits with buprenorphine (0.03 mg/ and carbon dioxide elimination (Flecknell et al., 1989). kg SC) one hour before SC administration of ketamine (15 mg/kg)/medetomidine (0.25 mg/kg), significantly a. Barbiturates increased duration of anesthesia with only transient Pentobarbital was widely used in the past but has but physiologically benign depression of respiratory become prohibitively expensive and is no longer available rate (Murphy et al., 2010). Similarly, use of butorphanol as an anesthetic in some parts of the world. Physiologic (0.4 mg/kg SC) in ketamine/medetomidine-administered effects may include respiratory depression or arrest, rabbits prolonged the duration of the loss of the ear pinch decreased arterial blood pressure, peripheral vasodila- response and exacerbated respiratory rate depression but tion, decreased cardiac output, depression of the vaso- without influence on arterial blood gases Hedenqvist( pressor response to hemorrhage, and preservation of or et al., 2002). Dexmedetomidine administered intrave- increase in heart rate (Korner et al., 1968; Warren and nously as a 10 min infusion at three different dosages Ledingham, 1978; Morita et al., 1987; Borkowski et al., (20, 80, or 320 μg/kg) caused dose-dependent depression 1990). Pentobarbital may influence research variables. of PaO2 and heart and respiratory rates, and dose-depen- For example, it diminishes myocardial damage after dent increase in sedation and PaCO2 (Zornow, 1991). No coronary artery ligation when compared with significant change was observed in mean arterial blood or α-chloralose (Chakrabarty et al., 1991) and reduces pressure. Dexmedetomidine administered IM with sub- plasma potassium ion concentration with consequent sequent IV ketamine produces approximately 20 min of elevation of plasma renin and aldosterone concentra- surgical anesthesia. tions (Robson et al., 1981). The combination agent Telazol (Parke-Davis, Morris Thiamylal, thiopental, methohexital, and ethylmalo- Plains, New Jersey), which contains the dissociative nyl urea (EMTU) are other barbiturates that have been tiletamine and the benzodiazepine zolazepam, has been used in the rabbit. As with pentobarbital, these agents shown to be nephrotoxic in the rabbit and is best avoided should be used as dilute solutions and injected slowly. (Doerning et al., 1992). Tiletamine is the offending sub- stance in this combination. b. Propofol The intravenously administered hypnotic agent pro- 3. Neuroleptanesthesia–Neuroleptanalgesia pofol has been evaluated in several studies, the sum of The most useful agent of the neuroleptanesthesia–neu- which suggests that as a sole agent it is suitable only roleptanalgesia class is a combination of fentanyl and for induction and noninvasive procedures (Blake et al., fluanisone (Hypnorm; Vetapharma, United Kingdom) 1988; Ko et al., 1992a,b; Aeschbacher and Webb, 1993a,b). available in Europe (Flecknell et al., 1983; Flecknell and Degree of sedation or anesthesia, as well as alteration of

LABORATORY ANIMAL MEDICINE III. Rabbits 1153 physiologic variables, depends upon infusion rate and has historically been used by physiologists because of time of day of administration. Ko used medetomidine/ its reputation for preservation of baroreceptor reflexes atropine and medetomidine/midazolam/atropine pre- (Sebel and Lowdon, 1989). prior to propofol induction and found the combination useful for anesthesia induction sufficient 4. Inhalation Anesthesia to achieve endotracheal intubation. Pedal withdrawal The inhalant agents are especially useful in rabbit reflexes and preanesthetic levels of heart rate, respira- anesthesia because of their reliability, efficacy, ease of tory rate, mean arterial pressure, and end tidal CO2 manipulation of anesthetic depth, and reduction in were preserved (Ko et al., 1992). Propofol infusion in recovery time when compared with many injectable unsedated animals can be facilitated by catheterization agents. Invasive manipulations, especially those requir- of a marginal ear vein on which a lidocaine and prilo- ing prolonged surgical time, are best achieved through caine ointment (EMLA cream, Astra, Westborough, MA) the use of inhalants. Isoflurane and sevoflurane are most has been applied. Allweiler et al. (2010) describe such commonly used. They may be administered via face- administration to unsedated, pre-oxygenated rabbits. mask or endotracheal tube. Rabbits should be sedated An initial infusion of 10 mg/kg IV over 60 s was fol- prior to face-mask inductions because the animals may lowed by incremental doses (1–2 mg/kg) until relaxation struggle vigorously in this setting. adequate for endotracheal administration was achieved. Endotracheal tubes of 3.0–4.0 mm internal diam- The mean dosage required for intubation tolerance was eter can be used in most rabbits. Both blind and visual 16 ± 5 mg/kg propofol; apnea did not occur. Rabbits orotracheal intubation techniques have been described were then maintained on sevoflurane for ovariohyster- and are summarized elsewhere (Lipman et al., 2008). ectomy. Excellent recovery and extubation within 2 min A novel supraglottic device has been described as an of termination of sevoflurane characterized this regi- alternative to endotracheal intubation (van Zeeland and men. Pharmacokinetics and pharmacodynamics of pro- Schoemaker, 2012). Another alternative is nasotracheal pofol depend upon the time-of-day of administration. intubation using an endotracheal tube (inner diam- The degree of anesthesia achieved with 5 mg/kg pro- eter, 2.0–2.5 mm; length, 14.5 cm; Mallinckrodt Medical, pofol administered at different times-of-day, in order of St Louis, MO, and Rusch, Duluth, GA) inserted through greatest to least was 10:00 h, 22:00 h, and 16:00 h (Bienert the nares into the ventral nasal meatus. Factors facilitating et al., 2011). Medetomidine administered IM and fol- placement and maintenance of such tubes are adequate lowed by IV propofol provides approximately 11 min of anesthesia (complete relaxation), positioning the animal surgical anesthesia with clinically acceptable preserva- in dorsal recumbency but with dorsiflexion of the head tion of physiologic variables (Hellebrekers et al., 1997). during intubation, and securing the catheter to the dor- Electroencephalographic features of propofol infusions sum of the nose by butterfly tape and sutures Stephens( in rabbits have been described (Silva et al., 2011). DeValle, 2009). Anesthesia in rabbits may be maintained Propofol CRI has been evaluated in rabbits in two sep- by using a chosen inhalant with oxygen as the carrier arate studies. Baumgartner et al. (2009) used propofol at gas delivered via a Bain or other non-rebreathing circuit 1.2–1.3 mg/kg/min IV after induction of anesthesia with at 2–2.2 times the minute respiratory volume (Flecknell, 4–8 mg/kg IV. Intraosseus and intravenous infusion of 2009; Lipman et al., 1997, 2008). Inhalational anesthesia propofol were evaluated by Mazaheri-Khameneh et al. with isoflurane, , or halothane has been shown (2012) who found both routes of administration to be to protect the ischemic rabbit myocardium from infarc- similar with regard to physiologic variables and recov- tion when compared with anesthesia with pentobarbital, ery times. Induction in this latter study was achieved propofol, or ketamine/xylazine (Cope et al., 1997). with 12.5 mg/kg (1% propofol) and maintained with 1 mg/kg/min for 30 min. a. Isoflurane Isoflurane is currently the most commonly used c. Urethane inhalant for rabbit anesthesia. Cardiac safety, rapid Urethane continues to be used alone and in combina- induction and recovery, minimal hepatic transforma- tion with other agents (e.g., Tanaka et al., 2012). Among tion, and attendant reduction in viscerotoxicity are all its characteristics are a long duration of action, excellent features of this agent (Blake et al., 1991). Disadvantages muscle relaxation, numerous endocrine effects, hemo- of isoflurane include breath holding at first exposure, lysis, prolonged recovery, carcinogenic potential, and hypotension, and respiratory depression. Isoflurane has reduced response of vascular smooth muscle to nor- also been shown to decrease serum calcium and potas- epinephrine (Bree and Cohen, 1965; Maggi et al., 1984). sium concentration, and increase serum triglyceride, These features require its regulation by institutional phosphorus and chloride concentrations (Gonzalez Gil safety personnel and restrict its use in animals to nonsur- et al., 2010). The MAC of isoflurane in rabbits is 2.05± vival procedures. Chloralose combined with urethane 0.18%, 1.39 ± 0.32% for halothane, and 2.86 ± 0.18% for

LABORATORY ANIMAL MEDICINE 1154 24. Preanesthesia, Anesthesia, Analgesia, and Euthanasia enflurane Drummond,( 1985). In another study evalu- Supplemental heat sources should be used judi- ating the MAC-sparing effects of butorphanol, with or ciously both intraoperatively and postoperatively to without meloxicam in rabbits, the MAC of isoflurane reduce hypothermia and attendant changes in metabo- was determined to be 2.49 ± 0.07% (Turner et al., 2006). lism of injectable drugs and MAC reduction of inhal- Meloxicam did not have a direct sparing effect and did ants. Drapes, circulating hot-water blankets, hot-water not influence the MAC-sparing effect of butorphanol. bottles, air blanket, fabric warmers, and use of warm IV Both tramadol (4.4 mg/kg IV) and lidocaine (2 mg/kg and irrigation fluids should be considered, depending IV followed by a CRI of 50 μg/kg/min) were shown in on the circumstances. separate experiments to be MAC-sparing in the isoflu- Conventional veterinary monitoring equipment may rane-anesthetized rabbit (Egger et al., 2009; Schnellbacher be used to monitor such cardiopulmonary variables as et al., 2013). The physiologic effects of 1.3 MAC isoflu- heart rate and rhythm, direct arterial blood pressure, oxy- rane in rabbits include increased heart rate, preservation gen saturation, and pulse rate. End tidal CO2 may be of hepatic blood flow, and reduction in cardiac out- evaluated but better reflects PaCO2 when measured at the put, respiratory rate, mean arterial blood pressure and pulmonary tip of the endotracheal tube (Rich et al., 1990). renal blood flow Blake( et al., 1991). Isoflurane produces Arterial blood pressure measurement is facilitated by significantly less depression of myocardial contractility the presence of two large and percutaneously accessible than does halothane of equivalent MAC concentration (1 peripheral arteries, the central auricular and saphenous MAC) (Marano et al., 1997). Nitrous oxide may be used as a arteries. Pulse oximetry is best performed using transmis- carrier gas at a ratio of 2:1 (N2O:O2), but concerns over sion clips on the tongue or reflectance probes intrarectally. waste gas pollution and recreational abuse have reduced Fluid infusion rate for most procedures of short to mod- the use of this agent. To avoid diffusion hypoxia when erate duration is 10 ml/kg/h. The rate for neurosurgical the technique includes N2O, 10 min of pure O2 should be procedures is 4 ml/kg/h (Hindman et al., 1990). administered at the completion of anesthesia. Bispectral index (BIS) has been evaluated in rab- bits anesthetized for laparotomy either by sevoflurane b. Sevoflurane (1 MAC) or CRI (8 mg/kg IV over 60 s followed by Sevoflurane use in the rabbit has now been well- 0.6 mg/kg/min IV) of propofol (Martín-Cancho et al., characterized (Scheller et al., 1988; Allweiler et al., 2010; 2006). Both agents reduced the BIS from a mean in con- Flecknell et al., 1999). The MAC for this agent in rabbits scious animals of approximately 98, to a value of 49.3 ± as determined by tail clamp is 3.7% (Scheller et al., 1988). 2.2 for sevoflurane and 69.1± 6.0 for propofol CRI. All In rabbits being ovariohysterectomized, propofol induc- animals were adequately anesthetized as judged by tra- tion followed by sevoflurane maintenance (4.0± 0.5%) ditional criteria. BIS of rabbits induced with thiopental provided surgical anesthesia with cardiopulmonary sta- has also been evaluated and found to correlate with loss bility and a return to sternal recumbency within 8 min of of righting and other reflexes Kazemi( et al., 2011). sevoflurane termination (Allweiler et al., 2010). A num- ber of studies have characterized the hemodynamic and E. Special Anesthetic Considerations ventilatory response of sevoflurane-anesthetized rabbits to infusion of various sedative-hypnotic (propofol, mid- 1. Spinal Anesthesia azolam) and analgesic (remifentanyl, dexmedetomidine) Spinal anesthesia of the rabbit has been described agents (Nishizawa et al., 2012; Chang et al., 2009; Sazuka in both clinical and research settings (Kero et al., 1981; et al., 2012; Koshika et al., 2011). Hughes et al., 1993). It has been used as a model for evaluating the pharmacology and toxicology of spinal anesthesia and analgesia. Both epidural and subarach- D. Intraoperative Monitoring and Support noid catheterization procedures have been described Intraoperative monitoring and support of rabbits (Langerman et al., 1990; Jensen et al., 1992; Madsen et al., are similar to those of other animals of similar size. 1993; Yamashita et al., 2003). Although subarachnoid space Monitoring of reflexes, body temperature, and cardio- cannulation requires exposure of the lumbar spinal col- pulmonary variables should be performed by trained umn and incision of the ligamentum flavum, both surgi- personnel. The reflexes, ranked in descending order of cal and percutaneous techniques have been described for usefulness and accuracy for determination of depth of epidural catheterization (Taguchi et al., 1996; Malinosky anesthesia, are pinna, pedal withdrawal, corneal, and et al., 1997). The lumbosacral anatomy of the rabbit spi- palpebral (Borkowski et al., 1990; Hellebrekers et al., nal cord makes inadvertent intrathecal administration 1997). Other indices of anesthetic depth, such as mus- of agents possible. In this regard, the spinal cord ends cle tone, jaw tone, vocalization, and gross purposeful within the sacrum in the rabbit, and the epidural space is movement in response to surgical stimuli, may be used approximately 1 mm in width; even a short beveled spi- (Hellebrekers et al., 1997). nal needle can pierce the dura. Moreover, a ‘dry’ tap does

LABORATORY ANIMAL MEDICINE III. Rabbits 1155 not guarantee placement in the epidural space after loss F. Acute and Chronic Analgesic Therapy of resistance. In a study highlighting these issues, the use of minimum electrical threshold with an insulated spinal Preemptive administration of analgesia should be needle was not able to distinguish the epidural from the considered as part of the complete program of anal- intrathecal space in the rabbit (Otero et al., 2012). To the gesia in rabbits subjected to surgery (see Table 24.8). authors’ knowledge, the use of analgesics administered In a report assessing analgesic administration to rab- through these routes has not been rigorously evaluated bits used in experimental surgery, Coulter et al. (2011) for the clinical setting in the rabbit. identified three areas in which perioperative care might be improved: pre- or peri-operative administration of 2. Hypnosis agents in contrast to postoperative administration only; Hypnosis, or the immobility response or tonic immo- the use of multimodal technique in procedures likely to bility state, describes a constellation of physiologic produce moderate or severe pain; and increasing the use and behavioral changes in rabbits effected by physical of NSAIDs and adjunctive methods of analgesia (e.g., manipulation with or without incantation and reduced epidural analgesia). In their survey, the most commonly lighting (Danneman et al., 1988; Klemm, 2001). Gentle used agent was buprenorphine and opioid use exceeded head and neck traction appears to be commonly, but not NSAID use in two time periods evaluated (1995–1997 uniformly, employed in this technique. Rabbits can be and 2005–2007). used as an animal model to study pain attenuation and Use of local infiltrative techniques in association with other features of hypnosis of humans (Castiglioni et al., general anesthesia may also be used to enhance postop- 2009). The tonic immobility state in this latter report erative analgesia. Lidocaine and bupivacaine are com- simply places animals in the supine position and immo- monly used. A liposomal formulation of bupivacaine bilizes them for 15 min with gentle pressure. Hypnotized has been evaluated for, and found safe to use in perineu- rabbits exhibit miosis, analgesia, increased depth of res- ral application in the rabbit, but efficacy data were not piration, and reduced respiratory rate, heart rate, and reported (Richard et al., 2012). Use of these formulations blood pressure. The immobility response is not inhibited are associated with prolonged duration of nerve block by naloxone. Although fascinating to the experimental- in other species. Morphometric evaluation of the New ist, variation in response among rabbits limits the use- Zealand White rabbit skull can assist in localization of fulness of this technique. It should not be considered a nerves in regional anesthesia (Monfared, 2013). In oph- suitable surrogate for analgesia or anesthesia. thalmic procedures, topical and intracameral anesthetics should be considered as adjuncts to general anesthesia 3. Long-Term Anesthetic Preparations (Zemel et al., 1995; Barquet et al., 1999). During ocu- Long-term anesthesia in the setting of nonsurvival lar manipulations in ophthalmic surgery, use of topical surgery in the rabbit requires rigorous attention to hydration, body temperature, adequacy of anesthetic TABLE 24.8 Analgesicsa for Rabbits depth, and monitoring of physiologic variables (Lipman et al., 2008). For those techniques that include paralyt- Analgesic Dosage ics, guidelines described in the Guidelines for the Care Aspirin 100 mg/kg per os and Use of Mammals in Neuroscience and Behavioral Ibuprofen 10–20 mg/kg, IV 4 h Research should be consulted (National Research coun- cil, 2003). A common technique is for surgery to be per- Buprenorphine 0.01–0.05 mg/kg SC, IV q 6–12 hourly formed without paralytics, so that adequacy of analgesia Butorphanol 0.1–0.5 mg/kg IV, 4 hourly may be determined; paralytics are then used in conjunc- Carprofen 5 mg/kg SC or PO q 24 hourly tion with anesthesia during the period of data collection. Flunixin 1.1 mg/kg IM, q 24 hourly The use of paralytics with a high ‘autonomic margin of safety’ allow the experimentalist to use heart rate Piroxicam 0.2 mg/kg per os, 8 hourly and blood pressure as indices of depth of anesthesia. Meloxicam 0.3–0.6 mg/kg SC or PO q 24 hourly Increases in these variables, suggestive of a sympatho- Meperidine 5–10 mg/kg SC, 2–3 hourly adrenal response to anesthesia, should prompt admin- Morphine 2.5 mg/kg SC, 2–4 hourly istration of additional anesthetic. A typical regimen may include the following: induction and initial administra- Fentanyl 5–20 μg/kg, IV bolus tion using an injectable technique, followed by mainte- 15 μg/kg, continuous infusion over 2 h nance with an inhalant or total IV infusion technique and Nalbuphine 1–2 mg/kg IV, 4–5 hourly pancuronium or vecuronium (Mills et al., 1987; Hindman et al., 1990; De Mulder et al., 1997). These techniques have Pentazocine 5 mg/kg IV, 2–4 hourly been reviewed by Lipman et al. (1997, 2008). aAdapted from Lipman et al. (2008) and Wixson (1994).

LABORATORY ANIMAL MEDICINE 1156 24. Preanesthesia, Anesthesia, Analgesia, and Euthanasia anesthetics in rabbits anesthetized with propofol blocked Butorphanol (0.1–0.5 mg/kg), a mixed agonist/antagonist the oculocardiac reflex Singh( et al., 2010). In a study agent, is useful in providing short-term analgesia in rab- of peribulbar and retrobulbar anesthesia, Zhang et al. bits (4 h). Butorphanol may be added to acepromazine or (2010) showed that full-strength bupivicaine can cause xylazine to effect both sedation and analgesia (Lipman myonecrosis and degeneration of extraocular muscles et al., 1997). It has a MAC sparing effect in the isoflurane- in rabbits. Half- and quarter-strength formulations had anesthetized rabbit (Turner et al., 2006). fewer or no acute changes and were unassociated with untoward long-term effects. Finally, EMLA cream (Astra, 4. Non-Steroidal Anti-Inflammatory Drugs Westborough, Massachusetts), a mixture of prilocaine Numerous NSAIDs have been advocated for rab- and lidocaine that is administered topically, is useful for bits, but rigorous data are lacking. Some of the agents vasodilation and analgesia during venotomy and arte- that have been advocated are aspirin (10 mg/kg SQ), riotomy procedures (Flecknell et al., 1990; Hellebrekers acetaminophen-codeine at 1 ml drug/100 ml water, et al., 1997; Keating et al., 2012). meloxicam, and carprofen (Lipman et al., 1997; 2008). In an experimental fracture model, both piroxicam and 1. Assessment of Pain and Discomfort flunixin were shown to reduce limb swelling, but anal- The most frequently recognized signs of pain and gesic action was not independently evaluated (More discomfort in rabbits include inappetance, an unkempt et al., 1989). The cyclooxygenase inhibitor ketorolac tro- appearance due to a failure to groom, and reduced activ- methamine (Toradol; Roche Laboratories, Nutley, New ity. Postoperative evaluation for pain is greatly assisted Jersey) has been evaluated in the rabbit at various doses by preoperative assessment of the demeanor of indi- because of its antithrombotic effect (Shufflebarger et al., vidual rabbits and their food and water consumption. 1996; Delaporte-Cerceau et al., 1998). Unfortunately, Behavioral changes and facial expressions thought likely analgesic activity independent of investigation of these to be associated with pain in rabbits have been described antithrombotic and anti-inflammatory effects appears (Keating et al, 2012). lacking. In another study using a rabbit model of acute temporomandibular joint inflammation, intraperitoneal 2. Methods of Analgesic Drug Delivery and intra-articular administration of 50 μg of ketorolac Parenteral analgesics may be administered IM, IV, SC, decreased the generation of the inflammatory media- and epidurally or intrathecally (Table 24.8). Intravenous tors, prostaglandin E2, and bradykinin (Swift et al., 1998). administration of μ-agonist opioids has been associ- The drug is well absorbed with no untoward effects ated with muscle rigidity, opisthotonus, and oculogy- related to ophthalmic, IM, or intranasal administration ric effects (Borkowski et al., 1990; Marini et al., 1993). (Rooks et al., 1985; Santus et al., 1993). The COX-2 inhibi- Consequently, these drugs are best administered only tor meloxicam was evaluated for MAC-altering effects after sedation with appropriate sedatives or as part of a in isoflurane-anesthetized rabbits with or without con- neuroleptanesthetic or analgesic combination (Lipman current butorphanol administration. No independent et al., 1997, 2008). Oral administration, although used in MAC-altering effects were observed, but meloxicam rabbits, has not been fully characterized for efficacy to at 0.3 or 1.5 mg/kg augmented butorphanol-associated the authors’ knowledge. Patch administration of analge- MAC reduction (Turner et al., 2006). sics in rabbits has been described for the agents fentanyl (Foley et al., 2001) and buprenorphine (Park et al., 2008), but no efficacy data were presented. IV. FERRETS 3. Opioids A. Introduction The opioid of choice in rabbits is buprenorphine, a partial agonist/antagonist with a long duration of action The ferret is easily and reliably anesthetized using a (8–12 h) (Flecknell, 2009; Lipman et al., 1997, 2008). This variety of regimens (Ko and Marini, 2008, 2014). With agent has only mild respiratory depressant properties, in practice, anesthetists can safely and reliably catheterize contrast to the pure μ-agonists. A dose of 0.02–0.05 mg/ peripheral vessels, establish an airway by orotracheal kg IV, SQ, or IM produced 10 h of analgesia when a intubation, and monitor anesthesia with instruments thermal stimulus was used (Flecknell and Liles, 1990). common in companion animal practice. Ferrets should be Because onset of action is 30 min, this agent should be purchased from a quality vendor, be vaccinated against administered intraoperatively or preoperatively. distemper and rabies, and be allowed an acclimation Other opioid drugs that may be useful include mor- period of at least 3–7 days, depending on whether they phine, which provides 2–4 h of potent analgesia at doses of will be used acutely or chronically. Facilities receiving 2–5 mg/kg SC or IM (Wixson, 1994). It may cause sedation, ferrets during warm weather should recognize the lim- respiratory depression, and moderate histamine release. ited heat tolerance of this species. Carriers and receivers

LABORATORY ANIMAL MEDICINE IV. Ferrets 1157 should not allow animals to linger during transit, and if prolonged, may cause estrogen-induced pancytopenia animal technicians and veterinarians should be espe- and a tendency towards hemorrhage. Complete blood cially vigilant for signs of hyperthermia (e.g., prostra- counts will help quantify risk in this context. BUN helps tion, panting etc.; Marini, 2014). determine if higher hematocrits are associated with dehy- Quarantine testing should include, at a minimum, body dration (pre-renal azotemia), and blood glucose determi- weight, physical examination, fecal endoparasite flota- nation helps identify the presence of an insulin-secreting tion, and rectal culture for Salmonella spp., Campylobacter pancreatic tumor (insulinoma), a very common disease spp., E. coli, and other pathogens. Additional testing in ferrets. Clinicians should consider screening ferrets such as influenza virus status, complete blood count, over 18 months of age for both insulin-secreting islet and serum chemistry analysis is determined by experi- cell-tumors and hyperadrenocorticism (adrenal-associ- mental usage. Investigators studying vision and hearing ated endocrinopathy). Bilaterally symmetric alopecia is a should avoid the use of albino and white-headed ferrets hallmark of the latter in ferrets. Vulvar enlargement in a in that these animals have deficits in both these senses spayed jill or prostatic enlargement in a hob may also be (Hupfeld et al., 2006; Morgan et al., 1987; Moore and signs of this disease. Kowalchuk, 1988). The sedated or anesthetized ferret is very prone to hypothermia due to its small size and correspondingly large surface area to body weight ratio. Great care is B. Preoperative Assessment and Preparation required to preserve body temperature, especially dur- ing preparation for surgery. Use of supplemental heating 1. Preoperative Evaluation devices such as conductive fabric warmers, circulating Ferrets used in biomedical research are likely to have hot-water blankets, forced air systems, and heated sur- been purchased from breeding facilities with good ani- gery or exam tables immediately upon onset of sedation mal health and conditioning standards. Young, experi- or anesthetic induction helps to reduce heat loss. Ferrets mentally naïve ferrets are good anesthetic subjects for also have short gastrointestinal transit times of approxi- which there are many acceptable regimens for anes- mately 3–4 h. Traditional fasting periods used in dogs thesia induction, maintenance, and monitoring. A thor- and cats are far too long for ferrets and may result in ough physical examination should be performed prior hypoglycemia. Ferrets to be used in procedures sched- to anesthesia. Techniques for conscious restraint and uled for late morning should therefore have food with- physical examination have been reviewed elsewhere drawn from early morning and not the previous evening. (Ko and Marini 2008; Marini, 2014). Normal physiologic variables evaluated during the physical examination 2. Choice of Anesthetic Technique include a heart rate of 180–300 bpm, a respiratory rate As in other species, experimental objectives, age and of 30–40 breaths per minute, and a body temperature condition of the subject, and investigator experience and of 100–104°F (37.7–40°C). Struggling may increase tem- expertise will help determine the choice of technique. perature. Particular attention should be paid to the pre- Relatively short procedures of mild to moderate surgi- anesthetic size of the spleen, both because splenomegaly cal intensity can be done with intermediate acting inject- is common in ferrets but also because the ferret spleen ables, while longer, more complicated procedures should enlarges during anesthesia with isoflurane, and presum- be done under inhalant anesthesia or a balanced tech- ably other inhalants as well. Clinicians must recognize nique. An example of the latter is a continuous infusion that splenic sequestration of red blood cells and altera- of propofol and ketamine in ferrets pre-medicated with tion of hematologic variables occurs during isoflurane medetomidine/atropine/buprenorphine/meloxicam. and ketamine/xylazine anesthesia in ferrets (Jackson This regimen was designed and implemented as a con- et al., 1992; Marini et al., 1997). Arrhythmias are common sequence of severe, unresponsive bradycardia observed in ferrets, and can be appreciated during auscultation. in ferrets undergoing bilateral cochlear implants main- The most common arrhythmias are second and third tained with other regimens (Hartley et al., 2010), and degree heart block (Malakoff et al., 2012). These animals might be suitable for a wide range of surgical proce- are best precluded from use. dures. Glycemic management in ferrets subjected to Minimal preoperative blood work should include long procedures is critical; the authors typically use 5% complete blood cell (CBC), blood glucose, and BUN. dextrose in lactated Ringer’s solution as a crystalloid Adult ferrets have higher hematocrits (44–55%) than maintenance fluid, infused at 10 ml/kg/h. Simple pro- other laboratory animals. Blood transfusion should be cedures in debilitated animals, and physical examination considered in anemic ferrets with hematocrits less than or sample collection in uncooperative animals can be 20–25%. The lack of discernable blood groups in ferrets achieved through face mask induction with isoflurane obviates the need for cross matching prior to transfusion. or sevoflurane. Ferrets will salivate copiously during Vulvar enlargement in jills is indicative of estrus, which, mask induction, and should be premedicated with

LABORATORY ANIMAL MEDICINE 1158 24. Preanesthesia, Anesthesia, Analgesia, and Euthanasia glycopyrrolate (0.01 mg/kg IM) or atropine (0.04 mg/kg precluded or mitigated by preadministration with atro- IM). Pre-induction sedation is recommended for ferrets pine or glycopyrrolate. Medetomidine and dexmedeto- before mask induction. Chamber induction is associated midine can produce hypertension in association with with violent struggling and digging behaviors and is best bradycardia. avoided in the ferret. Opioids can be used for pre-emptive analgesia, post- operative analgesia, or in combination with a neurolep- 3. Preoperative Medications tic in neuroleptanalgesia. Morphine, hydromorphone, The use of preoperative medications may reduce the buprenorphine, and butorphanol have been recommended anesthetic requirement for maintenance agents, relieve as analgesics for use in ferrets (Ko and Marini, 2014). patient apprehension, and facilitate handling. Injectable These agents may cause bradycardia and should be used pre-anesthetic and anesthetic agents can be injected SC, with anticholinergics. Opioid use can also provide an IM, or IV. The typical site for subcutaneous injection anesthetic sparing effect (Murat and Housmans, 1988). is interscapular; those for intramuscular administration Morphine (0.5–0.75 mg/kg SC, IM) or hydromor- are the lateral thigh and epaxial musculatures. Most phone (0.05–0.1 mg/kg SC, IM) provide analgesia and fully conscious ferrets will not tolerate the placement reduced resistance to manipulation in ferrets subjected of an intravenous catheter, so premedicants and induc- to procedures such as bandage change and intravenous tion agents tend to be administered by other routes. In catheterization. Morphine doses lower than 0.5 mg/kg sedated ferrets, those anesthetized with injectable com- induce emesis in ferrets. binations, and those induced with inhalant agents via The opioid most commonly administered to ferrets face-mask, 22–24 gauge, over the needle catheters can is the partial μ agonist buprenorphine. It has a rela- be placed in cephalic, lateral saphenous, or lateral tail tively slow onset (30 min) but a prolonged duration of veins. Piercing the skin with a 20-gauge needle facilitates action, the latter presumably as a result of the slow dis- intravenous catheterization of ferrets. sociation of this agent from μ receptors (Stoelting, 1987). Premedication and anesthetic induction and main- Buprenorphine is only mildly sedative and is commonly tenance have recently been reviewed (Ko and Marini, used in postoperative pain management. 2008; Ko and Marini, 2014). This section will present only a select number of those available. As in other spe- C. Intraoperative Anesthetics cies, ferrets may be premedicated with anticholinergics, analgesics, and neuroleptics of different classes. Among In this section, the most commonly used sedatives, acepromazine and the α2-agonists are the most anesthetic agents will be described. Dosages are given useful and commonly used. Acepromazine (0.01 mg/kg in Table 24.9. IM) produces rapid onset sedation, lateral recumbency, 1. Injectable Anesthetics and immobilization adequate for minor procedures for approximately 40 min. Lower doses are unreliable while a. Dissociatives higher doses prolong recovery. Endotracheal intubation Dissociative anesthetic combinations are effective cannot be achieved with acepromazine alone and hypo- anesthetic induction regimens and can be used both thermia and hypotension may occur due to α-adrenergic to induce and maintain anesthesia in procedures of blockade. Acepromazine has no analgesic effect and mild to moderate surgical intensity and short duration. should only be used in young, healthy, well-hydrated Ketamine and xylazine produce lateral recumbency ferrets. and anesthesia adequate for endotracheal intubation, The α2-agonists, xylazine, medetomidine, and dex- gastrointestinal endoscopy, and most minor proce- medetomidine are useful agents in ferrets. Xylazine dures. Atropine pre-medication helps preclude cardiac (2 mg/kg IM) and dexmedetomidine (40 μg/kg IM) arrhythmias. A combination of ketamine and medeto- produce rapid immobilization, analgesia, and muscle midine or dexmedetomidine produces immobiliza- relaxation sufficient for minor procedures but not for tion, analgesia, and excellent muscle relaxation for endotracheal intubation. Without reversal, xylazine 60 min (Ko et al., 1997). Atipamezole administration and dexmedetomidine immobilization lasts 40–70 min will reverse these effects. Another regimen providing and 150–200 min, respectively. Xylazine reversal can be 60–80 min of surgical anesthesia and intubation toler- achieved with yohimbine (0.5 mg/kg IM; Sylvina et al., ance is ketamine, medetomidine (or dexmedetomidine), 1990); dexmedetomidine by atipamezole (400 μg/kg and butorphanol. IM; Ko et al., 1997). While heart rates of acepromazine- Telazol/xylazine, telazol/xylazine/butorphanol, and sedated ferrets remain within normal limits, those of a telazol combination in which 2.5 ml of medetomidine α2-agonist-sedated animals are significantly reduced. (1 mg/ml) (or dexmedetomine; 0.5 mg/ml) and 2.5 ml Xylazine causes hypotension, heart block, and ven- of butorphanol (10 mg/ml) are used as diluents for the tricular arrhythmias in ferrets. These effects can be Telazol powder (Ko et al., 1998) are commonly used.

LABORATORY ANIMAL MEDICINE IV. Ferrets 1159

TABLE 24.9 Common Agents and Anesthetic Combinations and their Effects in Ferrets

Time from Duration of Time from injection injection to lateral Duration of dorsal endotracheal to complete Drug combinations (IM) recumbency (min) recumbency (min) intubation (min) mobilization (min)

Medetomidine (80 μg/kg) 3 ± 1 >120a 16 ± 14 >120 Medetomidine (80 mg/kg), butorphanol (0.1 mg/kg) 3 ± 1 >120a 91 ± 8a >120 Medetomidine (80 mg/kg), butorphanol (0.1 mg/kg), 2 ± 0.5 >180a 95 ± 0a >180 ketamine (5 mg/kg)

Xylazine (2 mg/kg) 2 ± 0.9 68 ± 20 35 ± 17 71 ± 19 Xylazine (2 mg/kg), butorphanol (0.2 mg/kg) 2 ± 0.6 82 ± 4 69 ± 5 86 ± 9 Xylazine (2 mg/kg), butorphanol (0.2 mg/kg), 1 ± 1 94 ± 13 81 ± 19 106 ± 13 ketamine (15 mg/kg)

Diazepam (3 mg/kg) 3 ± 1 43 ± 8 0 51 ± 12 Diazepam (3 mg/kg), butorphanol (0.2 mg/kg) 3 ± 1 79 ± 11 4 ± 9 85 ± 12 Diazepam (3 mg/kg), butorphanol (0.2 mg/kg), 4 ± 5 75 ± 34 20 ± 25 95 ± 48 ketamine (15 mg/kg)

Acepromazine (0.1 mg/kg) 5 ± 3 49 ± 11 0 56 ± 12 Acepromazine (0.1 mg/kg), butorphanol (0.2 mg/kg) 5 ± 1 79 ± 11 16 ± 19 85 ± 12 Telazol (3 mg/kg), xylazine (3 mg/kg) 1.5 ± 0.9 103 ± 3 26 ± 29 117 ± 20 Xylazine (3 mg/kg)

Adapted from Ko and Marini, 2014. aIf not reversed with atipamezole. Medetomidine in this table can be replaced with 40 μg/kg of dexmedetomidine for the similar effects.

A dose of 0.03–0.04 ml/kg of the latter cocktail enables intubation. Assistants may prefer to restrain the maxilla intubation and provides a surgical plane of anesthesia with roll gauze placed behind the incisor teeth. This for 30–50 min. Oxygen (>40%) should be provided when makes operator injury less likely if the animal is too using these dissociative combinations, either by mask, lightly anesthetized. Depressing the base of the tongue intubation, or nasal catheter. by downward displacement of the laryngoscope blade helps provide visualization of the glottis. Inhalants may b. Propofol also be administered in oxygen by face-mask, either Propofol can be used for induction, typically after for creating adequate depth to allow intubation or for sedation and establishment of vascular access, and must anesthetic maintenance in the absence of endotracheal be given intravenously. The induction dose of 6–8 mg/kg intubation. Glycopyrrolate or atropine premedication should be reduced to 1–3 mg/kg in the sedated animals. prevents hypersalivation associated with induction with Apnea, oxygen desaturation, and decreased myocardial either face-mask or dissociative agent. contractility can all be observed, especially with rapid Inhalants are administered via non-rebreathing sys- administration; rapid intubation and oxygen should be tems such as the co-axial (Bain) circuit or Ayre’s T-piece provided (Cook and Housmans, 1994). Hartley et al., with Jackson-Rees modification. Flow rates of 2.2 × the (2010) describe maintenance of anesthesia in bilateral minute respiratory volume will preclude re-breathing. cochlear implant surgery using a CRI of propofol and Lower flows can be used in conjunction with capnogra- ketamine in 5% glucose/saline solution. phy or capnometry to assure non-rebreathing. A surgi- cal plane of anesthesia can be achieved in most ferrets 2. Inhalation Anesthesia with 2–2.25% isoflurane or with 2.5–4.5% sevoflurane. The inhalant anesthetics isoflurane and sevoflurane Both agents reduce peripheral vascular resistance and are the agents most often used for longer procedures can therefore predispose the animal to hypothermia of moderate to high levels of invasiveness in ferrets. and hypotension (MacPhail et al., 2004); sevoflurane bet- Inhalants are best administered to the intubated ferret. ter preserves blood pressure in closely-related species Most adult ferrets can be intubated successfully with (Gaynor et al., 1997). Moreover, sevoflurane has been 2.5–3.0 mm endotracheal tubes and a straight Miller 0 associated with reduction of cardiac work and myocar- or curved Macintosh 1 laryngoscope blade. Dorsiflexion dial oxygen consumption, leading to increased cardiac of the head and moderate lingual traction facilitate efficiency at high doses.

LABORATORY ANIMAL MEDICINE 1160 24. Preanesthesia, Anesthesia, Analgesia, and Euthanasia D. Intraoperative Monitoring and Support trained as kits or adolescents to lick these pastes from a tongue depressor. If the animal objects to the taste of Intraoperative monitoring of body temperature, medication in the paste, it can be administered by plac- depth of anesthesia, and cardiovascular and respiratory ing it on the end of a Popsicle stick or longitudinally function should be performed as in other species. Depth split tongue depressor, and scraping the medicated paste of anesthesia is reflected by palpebral and pedal with- from the stick onto the lingual surface of the maxillary drawal reflexes, eyelid aperture, ventral rotation of the incisors. Ferrets restrained by the scruff typically yawn eyeballs, reaction to surgical stimuli, muscle tone, and widely, and clinicians can use this phenomenon to time cardiorespiratory variables. Imai et al. (1999) showed their drug administration or gavage. that PaCO2, eyelid aperture, and pupillary diameter all increased with increasing isoflurane dose. In addition to traditional methods of observation and F. Acute and Chronic Analgesic Therapy auscultation, cardiovascular and respiratory monitoring 1. Assessment of Pain and Discomfort can be achieved in ferrets by indirect or direct blood pres- sure evaluation, ECG, pulse oximetry, blood gas analysis, Activity level, food or water consumption, production and capnometry. Indirect blood pressure is most easily of feces and urine, heart rate, respiratory rate and character, measured with an infant cuff (size 1) and an automated and incisional characteristics like integrity, touch-elicited oscillometric device (e.g., BO-AccuGuard) placed on pain and signs of inflammation, all inform the clinician the distal fore or hind limb or base of the tail. A mean of the adequacy of pain management. Ko has adapted arterial pressure of >60 mm Hg should be maintained. the use of the Dynamic Interactive Visual Analog Scale Electrocardiographic monitoring can be achieved with flat- (DiVas) used in dogs (Ko et al., 2011) to ferrets. Observation tened ECG clips or with adhesive pediatric electrode pads. of ferrets in their enclosure and then as they interact with A traditional lead II or base apex lead with the RA and LL the observer, as well as food and water consumption, and leads on the right side of the neck, and the LA lead on the fecal and urine production, are combined in this system left side of the thorax caudal to the heart can be used. The with palpation of potentially painful sites. The use of a base apex lead is visualized using Lead I or Lead III of hand-held ‘palpometer’ for pressure-controlled palpation the monitor. Oxygenation can be monitored using pulse provides an objective measure of incisional or regional oximetry probes placed on the tongue, paw, ear, or tail tip; pain. Each observation and site palpation is assigned a the authors find an intrarectal probe to provide the most score from zero (no pain) to 10 (severe pain), and the total stable measurements. Capnography can be used to evalu- score is used to direct clinical decisions. ate ventilation (Olin et al., 1997; Ko and Marini, 2008, 2014). 2. Methods of Analgesic Drug Delivery The current standard of care is the use of pre-emptive, E. Postoperative Recovery multimodal pain management. Opioids, α2-agonists, Ferrets are robust surgical subjects, but their small size local anesthetics, and NSAIDs can all be used (Johnston, requires clinicians to be exceptionally mindful of man- 2005; van Oostrom et al., 2011). The following are specific agement of heat, energy, and hydration. Intraoperative comments pertaining to the use of these agents in ferrets. devices used to provide heat can be used postoperatively Dosages are contained in Table 24.10. until recovery from anesthesia or normothermia has 3. Non-Steroidal Anti-Inflammatory Drugs occurred. During postoperative recovery, ferrets can be provided with fabric tubes (snooze tubes) and will crawl Non-steroidal anti-inflammatory drugs should be into them upon recovery from anesthesia. Energy require- used with caution in ferrets because they are deficient ments can be managed by oral gavage of gruel in ferrets in the glucuronidation pathway (Lichtenberger, 2006; that are reluctant to eat and fluids can be provided either Lichtenberger and Ko, 2007) and may manifest signs per os or by subcutaneous administration in ferrets with- of toxicity (renal failure and gastrointestinal tract ulcer- out intravenous or intraosseus catheters. Ferrets object ation). One or more doses of the COX-2 inhibitors car- vigorously to subcutaneous administration of fluids. The profen or meloxicam can be used safely, however, and authors prefer to administer warm crystalloids by placing improves the quality and duration of analgesia when the ferret into a deep sink and using a 12-inch butterfly combined with opioids or other analgesics. Always infusion set with 21- to 25-gauge needles through which ensure adequate hydration in ferrets to which these to inject the fluids. This protects the clinician and facili- agents are administered. tates the process. Traditional methods of evaluating dehy- dration and replacement or maintenance volumes apply. 4. Epidural Analgesia Oral medications can be administered in nutritive Both epidural anesthesia and analgesia have been pastes such as Nutrical or Ferret-Cal. Ferrets can be described for use in ferrets (Sladsky et al., 2000; Eshar

LABORATORY ANIMAL MEDICINE IV. Ferrets 1161

TABLE 24.10 Analgesic Agents Used in Ferrets

Dose/route Effects Duration Indications

OPIOIDS Butorphanol 0.2–0.8 mg/kg, Analgesia/sedation 1–2 h For mild-to-moderate degree of pain SC, IM or IV Morphine 0.25 – 1 mg/kg, Analgesia/sedation, may vomit, 3–4 h For mild-to-severe degree of pain SC, IM, or IV bradycardia may occur with doses higher than 0.5 mg/kg Hydromorphone 0.025–0.1 mg/kg, Analgesia/sedation, occasionally vomit, 1–2 h For mild-to-severe degree of pain SC, IM, or IV bradycardia and respiratory depression may occur

Fentanyl 4–10 μg/kg, Analgesia; bradycardia and respiratory 30 min For immediate relief of severe pain IM or IV depression may occur Buprenorphine 0.01–0.02 mg/kg, Analgesia, slow onset of effect 6–8 h SC, IM, or IV

ALPHA-2 AGONISTS

Medetomidine 0.02–0.04 mg/kg, Analgesia–moderate sedation 30–60 min Need sedation with analgesia SC, IM, or IV Dexmedetomidine 0.01–0.03 mg/kg SC, IM, or IV Xylazine 1–2 mg/kg Analgesia–moderate sedation 30–50 min Need sedation with analgesia

NSAID

Ketoprefen 1–2 mg/kg, SC, Analgesia, anti-inflammation 24 h In combination with opioids for severe IM, IV, or PO pain with longer-lasting analgesia effect Caprofen 2–4 mg/kg, SC, Analgesia, anti-inflammation 24 h In combination with opioids for severe IM, IV, or PO pain with longer lasting analgesia effect Meloxicam 0.2 mg/kg, SC, Analgesia, anti-inflammation 24 h In combination with opioids for severe IM, IV, or PO pain with longer lasting analgesia effect

Adapted from Ko and Marini (2014). and Wilson, 2010; Lichtenberger and Ko, 2007; Kleine preserve the function of interest to the investigator. An and Quandt, 2012). The spinal cord of the ferret typi- example of such a regimen was published by Liu et al. cally ends cranial to the site of administration, the lum- (2008) who were able to preserve visually-evoked activ- bosacral space, and so intrathecal injection is unlikely. ity in ferrets used in functional MRI, by using a nitrous Nonetheless, if intrathecal injection is suspected, the oxide/oxygen carrier gas mix with isoflurane (0.8–1.5% dose rate of the agents used should be reduced. isoflurane with a 2N O to O2 balance of 1:1.4). Other regi- mens include continuous infusions of short-acting intra- G. Special Anesthetic Considerations venous anesthetics and these may also be combined with the use of NMB agents. Select regimens for prolonged 1. Long-Term Anesthetic Preparations anesthesia are found in Table 24.11. The use of ferrets in neuroscience and physiology research has necessitated the development of regimens 2. Neonatal Anesthesia for prolonged anesthesia. These regimens often involve Ferrets are used in neuroplasticity studies because balanced anesthetic techniques designed to maintain much of neural development occurs postnatally (Sharma some critical physiologic function in an animal that has and Sur, 2014). In such studies, the normal course of either just had surgery or is being imaged, and which development is altered for the purpose of evaluating therefore must remain anesthetized. The challenge is to hypothesized outcomes. A safe and efficacious method maintain homeostasis, monitor and record anesthesia for providing anesthesia to neonates up until approxi- adequately and in accordance with approved animal mately day 5 is hypothermia. Kits are wrapped in moist use protocols, insure adequate anesthetic depth, and gauze or placed in a latex sleeve (made from the finger

LABORATORY ANIMAL MEDICINE 1162 24. Preanesthesia, Anesthesia, Analgesia, and Euthanasia

TABLE 24.11 select Anesthetic Regimens for Prolonged Anesthesia

Induction Maintenance Comments

Pentobarbital 40 mg/kg IP Pentobarbital 5 mg/kg/h IV Auditory study, drug administered in ‘dextrose-electrolyte’ solution. Breathing unassisted Ketamine 25 mg/kg IM Pentobarbital 1.5–2 mg/kg/h Vision research Xylazine 1.5 mg/kg IM Gallamine triethiodide 10 mg/kg/h CRI of a 1:1 mixture of 5% dextrose and lactated Atropine 0.04 mg/kg IM Ringer’s solution

Ketamine 40 mg/kg IM Isoflurane in 2O Vision research Acepromazine 0.4 mg/kg IM Vecuronium 0.2 mg/kg/h Isoflurane 2% Ketamine 10 mg/kg IM Isoflurane 1% in 1:1 Vision research Medetomidine 0.08 mg/kg IM N2O:O2 Atropine 0.15 mg/kg IM Pancuronium initial dose 0.15 mg/kg followed by 6 μg/kg/h Ketamine 10 mg/kg IM Isoflurane 0.5% in 1:1 Vision research Medetomidine 0.08 mg/kg IM N2O:O2 Atropine 0.1 mg/kg IM Gallamine triethiodide 6 mg/kg/h IV Alphaxalone/alphadalone Alphaxalone/alphadalone Rate adjusted to pedal withdrawal reflex (Saffan) 6 mg/kg IV (Saffan) 6–8 mg/kg/h Alphaxalone/alphadalone 5 ml/h of medetomidine (0.022 mg/kg/h) and CRI of 0.5 mg/kg/h dexamethasone and 0.06 mg/kg/h (Saffan) 2 ml/kg IM ketamine (5 mg/kg/h) in saline and 5% glucose) atropine. Auditory study Medetomidine 0.08 mg/kg IM Propofol 1 mg/kg/h Cochlear implantation Atropine 0.1 mg/kg IM Ketamine 5 mg/kg/h Ventilated with air/O2 mix Buprenorphine 0.05 mg/kg IM Isoflurane 1% in 1:1 Isoflurane 1% in 1:1 Vision research N2O:O2 N2O:O2 Pancuronium 0.6 mg/kg/h

Adapted from Ko and Marini, 2014. of a surgical glove), and then placed in a container of selection criteria for the various protocols specific to crushed ice until the cessation of spontaneous move- laboratory swine (Flecknell, 2009; Smith and Swindle, ment and respiration. Anesthesia is maintained in the 2008; Swindle, 2007). Veterinary-oriented textbooks also anesthetized kit by placing it on a bed of crushed ice or contain useful information on anesthesia in swine, but a cold glass plate. The use of dry ice leads to frostbite care must be taken to consider the physiologic effects of and is unacceptable. Kits must be adequately warmed the anesthetics on the research protocol (Riebold et al., before return to the jill. 1995; Thurmon and Benson, 1986). Information on han- Two recent studies have departed from the use of dling and selection of swine for surgical protocols is hypothermia in kits (Mao and Pallas, 2012; Borrell, 2010). included in Chapter 16 in this text as well as in a text- These studies used isoflurane from 1–5% delivered via book by Swindle (2007). The purpose of this section is customized face mask, to anesthetize kits from 12 h to 6 to provide an abbreviated guide to the most commonly days of age. Warm SC saline and the respiratory stim- used techniques in a research setting. ulant doxapram (2 mg/kg SC) can promote survival. A more thorough discussion of issues pertaining to anes- B. Preoperative Assessment and Preparation thesia and analgesia of neonates can be found in Ko and 1. Preoperative Evaluation Marini (2014). Swine should be selected from sources with a known health status and standardized preventive health pro- V. SWINE gram. They should be stabilized and/or conditioned in the research institution for 5–7 days prior to perform- A. Introduction ing anesthesia for survival surgery. Stabilization should include a physical exam as a minimum and, depending Research-oriented textbooks contain complete on the source and purpose of the research, laboratory descriptions of anesthetic techniques and appropriate tests, including a fecal exam, CBC count, and blood

LABORATORY ANIMAL MEDICINE V. Swine 1163 chemistry determination. Vaccination against common 2007; Smith et al., 1991). Drug dosages are provided in diseases may be appropriate for animals on long-term Table 24.12. projects. A judgment should also be made in advance of the protocol on whether a particular breed or age of C. Intraoperative Anesthesia pig should be selected. The criteria for selection of swine for research projects and the differences between minia- In this section, the appropriate selection of anesthetics ture and domestic farm breeds of swine are discussed used in research protocols is discussed. The drug dos- in Chapter 16, as well as in Swindle (2007). Diseases of ages are included in Table 24.12. swine and their potential complications to research are also discussed in Chapter 16. 1. Injectable Anesthetics Injectable anesthetics may be useful to induce anes- 2. Choice of Anesthetic Technique thesia prior to administering an inhalant agent or for The choice of anesthetic technique should be based short-term procedures. Protocols that infuse these agents on the physiologic effects of the anesthetic protocol may be indicated in cases where inhalant anesthesia is and the potential complications that a particular pro- not appropriate or is unavailable. Most injectable com- tocol may have on the research being conducted. Many binations only provide 20–30 min of surgical anesthesia. swine are used in cardiovascular research; therefore, Consequently, it is preferable to administer continuous stable hemodynamics, which may be greatly influ- infusions of these injectable agents when longer anes- enced by the anesthetic protocol, are important for thetic periods are required. Repeated bolus injections of many projects. The basic criteria for selection of anes- these agents result in an unstable plane of anesthesia thetics for swine are similar to those for other species. (Smith and Swindle, 2008; Swindle, 2007). Malignant hyperthermia, discussed in Chapter 15, is a unique genetic condition in certain breeds of domestic a. Dissociatives swine. Swine herds can be prescreened for this poten- The dissociative agents, when administered alone, are tial complication, which is triggered by many inhalant not sufficient to provide surgical anesthesia or relaxation and injectable anesthetic agents, particularly halothane. for endotracheal intubation. Ketamine and tiletamine- Swine may also have congenital heart defects, such zolazepam (telazol) are the two most widely used agents as ventricular septal defect (VSD) and patent foramen in swine. They are frequently combined with phenothi- ovale (PFO) (Swindle et al., 1992). Auscultation of swine azine derivatives, benzodiazepines, and α2-agonists. prior to anesthesia is useful for detecting these con- Combinations include ketamine/acepromazine, ket- ditions, as well as determining whether the animals amine/azaperone, ketamine/diazepam, ketamine/ have chronic respiratory diseases, which are common midazolam, ketamine/xylazine, ketamine/medetomi- in some herds of domestic swine. dine, and telazol/xylazine (Thurmon et al., 1988; Swindle, 2007; Smith and Swindle, 2008; Ko et al., 1997; Flecknell, 3. Preoperative Medications 1997, 2009; Portier and Slusser, 1985). These combina- Preoperative medications may be appropriate to tions typically require the addition of other agents, such relieve anxiety and decrease the amount of general anes- as barbiturates, or the administration of inhalant agents thetic to be administered. Anticholinergics may also be via a face mask in order to provide enough relaxation for useful in preventing the vagal reflex that may occur endotracheal intubation. All of the combinations provide during endotracheal intubation and manipulation of 20–30 min of restraint when administered IM or SC. The the cardiovascular and pulmonary systems. Both atro- combinations of ketamine/xylazine, ketamine/medeto- pine and glycopyrrolate have been utilized successfully midine, and telazol/xylazine are sufficient to provide as anticholinergics in swine. Atropine is also useful to anesthesia for minor surgical procedures. Ketamine/ counteract bradycardia associated with some protocols, medetomidine may provide up to 45 min of chemical such as those that use high-dose opioids. restraint as a single injection but rapidly induces hypo- Tranquilizers are mainly used to reduce anxiety, thermia. None of the combinations are suitable for visceral facilitate handling, and reduce the dosage of general analgesia unless provided as a continuous IV infusion. anesthetics. The phenothiazine derivatives, especially An infusion of ketamine/xylazine/guiafenesin is useful acepromazine, have been widely used for this pur- for providing stable hemodynamics for cardiovascular pose. Benzodiazepine agents, such as diazepam and protocols (Thurmon, 1986). Ketamine/medetomidine is midazolam, are also used in swine for this purpose. preferred when an α2-agonist is indicated for a proto- Midazolam is used as a sole agent to provide approxi- col, because it has less deleterious cardiovascular effects mately 20 min of relaxation to perform cardiovascular than xylazine. Telazol and telazol/xylazine may also be imaging techniques, such as echocardiography, with more cardiopressive than other dissociative combina- minimal effects on cardiovascular parameters (Swindle, tions. Their usage in research protocols in swine should

LABORATORY ANIMAL MEDICINE 1164 24. Preanesthesia, Anesthesia, Analgesia, and Euthanasia

TABLE 24.12 drug Dosagesa in Swine TABLE 24.12 (Continued) drug Dosagesa in Swine

Drug Dosage Route Drug Dosage Route

DISSOCIATIVE AGENTS AND COMBINATIONS azaperone+ 1 mg/lb followed in 20 min by Ketamineb 11–33 mg/kg IM, IV ketamine+ 10 mg/lb 10–33 mg/kg/h IV infusion morphine 1 mg/lb Ketamine+ 22–33 mg/kg IM Propofol 0.83–1.66 mg/kg IV Acepromazineb 1.1 mg/kg 12–20 mg/kg/h Continuous Ketamine+ 15 mg/kg IM IV infusion b Diazepam 2 mg/kg ANALGESICS Ketamine+ 20 mg/kg IM Fentanyl 0.02–0.05 mg/kg IM q2 h xylazine 2 mg/kg 30–100 mg/kg/h IV drip Ketamine+ 15 mg/kg IM Sufentanilb 5–10 μg/kg IM q2 h azaperone 2 mg/kg 10–30 mg/kg/h IV drip Ketamine+ 2 mg/kg IV (2 × dose Buprenorphineb 0.05–0.1 mg/kg IM q8–12 h for IM) Butorphanol 0.1–0.3 mg/kg IM q4–6 h xylazine+ 2 mg/kg Meperidine 2–10 mg/kg IM q4 h oxymorphone 0.075 mg/kg Oxymorphone 0.15 mg/kg IM q4 h Ketamine+ 20 mg/kg IM Pentazocine 1.5–3.0 mg/kg IM q4 h 5–1.0 mg/kg Phenylbutazone 10–20 mg/kg PO ql2 h Tiletamine-zolazepam (Telazol) 4–6 mg/kg IM Aspirin 10 mg/kg PO q4 h Tiletamine-zolazepam (Telazol)+ 4–6 mg/kg IM Carprofenb 2.0–3.0 mg/kg PO BID xylazine 2.2 mg/kg MISCELLANEOUS BARBITURATES Antiarrhythmics Pentobarbital 20–40 mg/kg IV Bretylium tosylate 3.0–5.0 mg/kg IV q30 min 5–40 mg/kg/h Continuous IV infusion Lidocaine 2–4 mg/kg IV Thiopental 6.6–30 mg/kg IV 50 μg/kg/min Continuous IV infusion 3–30 mg/kg/h Continuous Calcium channel blocker: 2–4 mg/kg PO TID IV infusion diltiazem Thiamylal 6.6–30 mg/kg IV Paralytic agents 3–30 mg/kg/h Continuous IV infusion Pancuronium 0.02–0.15 mg/kg IV

MISCELLANEOUS INJECTABLE RESTRAINT AGENTS 5–6 μg/kg/min Continuous IV infusion Azaperone 2–8 mg/kg IM Vercuronium 1.0 mg/kg IV α-Chloralose 55–100 mg/kg IV Succinylcholine 1.1 mg/kg IV b Etomidate 4–8 mg/kg IV Coronary vasorelaxant: 200 μg diluted in Infused Etorphine/acepromazine 0.245 mg/10 kg nitroglycerin 2 ml saline slowly into (Imobilon)+ coronary sinus Diprenorphine (Revivon) 0.3 mg/kg Anticholinergic: atropine 0.05 mg/kg IM Ketamine xylazine glyceryl 1 ml/kg/h See text for 0.02 mg/kg IV guaiacolate mixture Malignant hyperthermia treatment 5 mg/kg IV Midazolamb 100–500 g/kg IM μ and prophylaxis: Dantrolene Metomidate 4 mg/kg IV aSee text for references. Only commonly used agents are listed here. The reference Meperidine+ 1 mg/lb books cited in Section V, A provide complete information on drug dosages and administration techniques. (Continued) bMost commonly recommended anesthetics and analgesics. V. Swine 1165 be limited to single-dose administrations for chemical IV infusion first. The opioids induce bradycardia, espe- restraint or for protocols in which cardiovascular depres- cially when administered as IV boluses for induction sion is unimportant. These agents have been shown to of anesthesia. This bradycardia is transient but may be produce prolonged cardiovascular depression following counteracted by atropine. For invasive surgical proce- a single injection (Lefkov and Mussig, 2007). Ketamine/ dures, administration of low doses of inhalant agents is medetomidine and ketamine/midazolam have a pro- necessary during surgical manipulation. The analgesia tectant effect against cardiac arrhythmias and provide provided by continuous infusions of these agents is ade- stable hemodynamics when administered as continuous quate to provide a long-term stable plane of anesthesia IV infusions (Swindle, 2007; Smith and Swindle, 2008). and/or chemical restraint for cardiovascular measure- ments after the surgical manipulations are completed b. Propofol (Swindle, 2007). Propofol is administered as a continuous IV infusion. Its use in research in swine is limited because of its e. Etomidate minimal analgesic effects and significant cardiovascular Etomidate does not provide any advantage over other depression in higher dosages. It must be administered in agents in research protocols in swine. It is relatively inef- combination with analgesics or other agents in order to fective as a sole agent for any purpose other than short- provide visceral analgesia. However, it may be useful as term chemical restraint and it is a safe agent to use when a continuous infusion for nonsurvival teaching protocols cardiovascular compromise exists in the pig. It may be (Ramsey et al., 1993; Foster et al., 1992). combined with azaperone or ketamine to provide anes- thesia suitable for minor surgery (Worek et al., 1988; c. Barbiturates Smith and Swindle, 2008). The effects of the barbiturates in swine are similar to those in other species. Barbiturates are administered f. α2-Agonists IV as bolus injections to facilitate endotracheal intuba- Xylazine, dexmedetomidine, detomidine, and meto- tion or as continuous IV infusions for general anesthe- midine are the most commonly used -agonists in sia. Tranquilizers may be utilized as preanesthetics to α2 swine. These agents are associated with blockage of reduce the IV dosage by one-third to one-half. The bar- the cardiac conduction system and with cardiovascular biturates are potent respiratory in swine. depression. Medetomidine has the fewest cardiovascu- Thiobarbiturates, such as thiopental, are less potent than lar effects of this class of agents in swine. These agents pentobarbital and are shorter-acting; consequently, they are useful in combination with dissociative agents for are easier and safer to control. The thiobarbiturates are short-term surgical analgesia or for inclusion in combi- minimally metabolized by the liver, unlike pentobarbital, nation IV infusion protocols. They have minimal usage and are mainly excreted by the kidneys. The recovery time as sole agents for chemical restraint (Smith and Swindle, from thiobarbiturates may be as short as 20 min, whereas 2008; Swindle, 2007; Vainio et al., 1992; Riebold et al., it may be hours for pentobarbital. For longer protocols 2007; Flecknell, 2009). the barbiturates should be administered as continuous IV infusions. They are most useful for nonsurvival teaching g. Miscellaneous Anesthetics protocols, but thiobarbiturate infusions may be useful for providing a stable plane of cardiovascular hemodynamics α-Chloralose was promoted in the past as an agent when other agents are contraindicated (Smith et al., 1997; to provide anesthesia for cardiovascular hemodynamic Swindle, 2007). Availability of these agents has greatly measurements. However, it must be used at a high dos- decreased and the cost has increased in recent years. age or in combination with other agents in swine to pro- vide analgesia, which minimizes its effectiveness for this d. Opioids purpose. α-Chloralose may be replaced by other IV infu- Opioids may be used as analgesic adjuncts to other sion protocols, such as high-dose opioids or ketamine/ anesthetics to provide balanced anesthesia or may be xylazine/guiafenesin, that provide adequate analgesia administered in high-dose infusions for cardiovascular for these protocols (Silverman and Muir, 1993; Swindle, protocols. They have minimal effects on cardiac contrac- 2007; Thurmon, 1986). tility and coronary blood flow when administered for those purposes. The opioids administered most com- 2. Inhalational Anesthesia monly for cardiovascular protocols are fentanyl, sufent- Administration of general anesthesia using inhala- anil, and . The latter two agents are more potent tional agents is the preferred method for swine for most than fentanyl and consequently require lesser volumes protocols. However, proper administration of these agents for infusion; however, their potency may induce mus- requires an investment in equipment both for administra- cular rigidity, which can be controlled by starting the tion of the agents as well as monitoring of physiological

LABORATORY ANIMAL MEDICINE 1166 24. Preanesthesia, Anesthesia, Analgesia, and Euthanasia parameters. Personnel must also be properly trained in d. and Sevoflurane the techniques involved in this form of anesthesia. Flow Desflurane and sevoflurane have physiologic effects rates for gas delivery are variable between units, but as a similar to those of isoflurane. They are not commonly general guideline 5–15 ml/kg/min is usually sufficient. used in swine, because of the increased expense Nitrous oxide, halothane, and have (Weiskopf et al., 1992). Desflurane requires specialized potential human health hazards when the vapors are equipment not commonly found in the research setting. inhaled on a chronic basis. No long-term health effects have been described for the minimally metabolized agents, such as isoflurane, desflurane, enflurane, and D. Intraoperative Monitoring and Support sevoflurane. However, arbitrary limits have been set Intraoperative monitoring and support of swine for environmental exposure to these agents as well. An are similar to those of other large animal species. In evacuation system to control waste gases and periodic order to monitor homeostasis, the following param- monitoring of the operating room to check for leaks in eters should be monitored: muscular reflexes, ECG, the circuits are essential components of a program that heart rate, blood pressure, blood gas saturation values, uses inhalational anesthetics. Maintenance of equipment, and core temperature. Swine are susceptible to hypo- including cleaning and calibration of the vaporizers and thermia because of their relatively hairless skin; con- flowmeters as well as leak testing of anesthesia circuits, sequently, the use of circulating hot-water blankets or is also essential. The two primary inhalant agents used heated air wraps and protection from stainless steel in swine are isoflurane and sevoflurane. surfaces should be considered. Animals should be com- a. Nitrous Oxide pletely covered by drapes during surgery to prevent heat loss. Animals may be monitored by a variety of Nitrous oxide is ineffective as a sole agent for anesthe- mechanical means. Pulse oximeters can monitor oxy- sia in swine. However, it may be administered in combi- gen saturation and pulse rates. Surgical monitors may nation with oxygen to reduce the amount of inhalational be obtained that monitor ECG, rectal temperature, and agent that must be administered. This reduces the blood pressures. Blood pressure may be monitored dose-dependent cardiovascular depression associated either by invasive intravascular techniques or by use with inhalational anesthetics in swine. Nitrous oxide is of blood-pressure cuffs on the tail, medial saphenous administered as an adjunct to oxygen in a 33–66% com- artery, or radial artery. Pulse oximetry finger cuffs can bination. Nitrous oxide–oxygen (2:1) provides blood gas be attached to the tongue, ear, tail, or dewclaw. There measurements similar to those in unanesthetized swine will be some variability in the ability of the pulse oxim- and minimizes the amount of inhalant that must be etry cuff to function, depending on skin pigmentation administered (Swindle, 2007; Smith and Swindle, 2008). and thickness of the body part. IV fluid maintenance Diffusion hypoxia and the potential for abuse of the rates are 5–10 ml/kg/h (Smith and Swindle, 2008; agent by personnel are considerations. Swindle, 2007). b. Halothane Halothane has a MAC of 0.91–1.25% (Tranquilli et al., E. Special Anesthetic Considerations 1983; Smith et al., 1997). It is more cardiodepressant than the newer agents discussed below. Halothane is metabo- 1. Cardiac Anesthesia lized by the liver as well as being eliminated by the lungs. Anesthesia for cardiac surgery requires that the This agent is not widely available and its use should be protocol minimize effects on cardiovascular function. discontinued in favor of the newer inhalant agents. Parameters that need to be considered when selecting a protocol include cardiac contractility, cardiac output, c. Isoflurane blood pressure, myocardial oxygen consumption, and Isoflurane is the least cardiodepressent of the inhala- prevention of cardiac arrhythmias. Isoflurane or sevoflu- tional agents commonly used in swine. Isoflurane has a rane delivered in nitrous oxide–oxygen (2:1) minimizes MAC value of 1.58% (Smith and Swindle, 2008; Eisele cardiodepressant effects during surgery. If an inhalant et al., 1985). Less than 1% of this agent is metabolized by agent is contraindicated, then high-dose opioid infu- the liver. Isoflurane is generally used at concentrations sions may be used, especially if there is a requirement of 2–4% for induction and 0.5–2.0% for maintenance of to maintain myocardial contractility and coronary blood general anesthesia. When using nitrous oxide–oxygen flow. The physiologic effects of the various anesthetics 1:1 or 2:1 for delivery, the isoflurane concentration may should be reviewed prior to performing complex cardiac be reduced to 0.5–1.0%. Isoflurane in nitrous oxide is surgeries (Swindle, 2007). commonly used for maintaining long-term anesthesia Amiodarone and lidocaine infusions may be necessary with physiological measurements. as preventives for fatal cardiac arrhythmias. Paralytic

LABORATORY ANIMAL MEDICINE V. Swine 1167 agents, such as pancuronium and vercuronium, will 5. Anesthesia for Imaging Procedures be necessary to paralyze the diaphragm during cardiac (MRI and PET) manipulation and may be useful for providing increased Specialized imaging procedures require that a long exposure when using a lateral thoracotomy. Paralytic duration of anesthesia be provided without equip- agents should not be administered until there is an ment that may be affected by the imaging equipment. assurance that adequate analgesia has been obtained. Depending on the equipment and duration of the imaging This can be ascertained by performing skin and mus- procedure, the short-term (20–30 min) injectable proto- cular incisions prior to their administration. Heart rate cols described above may be adequate for the procedure. and blood pressure should be monitored during surgical Complete relaxation without paralysis is necessary to manipulation in a paralyzed animal to assure adequate obtain clear images. The combinations of ketamine/mid- anesthesia (Swindle, 2007). azolam and ketamine/medetomidine provide the best Performing cardiopulmonary bypass (CPB) and extra- relaxation for these procedures. Infusion protocols with corporeal membrane support (ECMO) procedures in thiobarbiturates may be used if having an IV infusion is swine is more difficult than in most species. In order to not contraindicated (Swindle, 2007). perform these techniques as survival procedures, a mul- tidisciplinary team of personnel competent in these pro- cedures is necessary. It is beyond the scope of this chapter F. Postoperative Recovery to describe CPB and ECMO procedures; however, they Postoperative recovery procedures for swine are simi- are described elsewhere in a detailed stepwise fashion, lar to those for other species. A complete description of which should be adequate for most research facilities to these procedures and the emergency procedures for car- perform them successfully (Swindle, 2007; Smith, 1997). diopulmonary distress have been published. Extubation can be performed when the pig is moving into a sternal 2. Pediatric Anesthesia position and struggling against the endotracheal tube. It is Neonatal dosage rates are frequently different from best to let the air out of the cuff of the tube first and allow dosage rates for adults. The dosage ranges given in the pig to stabilize prior to removing the tube. Apnea this manuscript are safe for swine of all ages in our frequently occurs when the tube is removed, and com- experience. Control of hypothermia during anesthesia pressive manipulation of the chest or stimulation of the is especially important in the neonate, particularly in pharynx and epiglottis may have to be instituted to start the first week postnatally, when they are incapable of spontaneous respiration. In some cases, the pig may have controlling their own body temperature adequately to be reintubated and respirated with a handheld respira- (Swindle et al., 1996). tory bag. The apnea is more likely to occur with injectable anesthetics, such as the barbiturates, than with inhalants. 3. Neuroanesthesia Monitoring of cardiopulmonary function during Swine have not been used very often in neurosurgi- recovery can be provided by pulse oximetry and appro- cal research, and specific anesthetic protocols for neuro- priate countermeasures taken when hypoventilation or surgery have not been published. The general principles cardiac emergencies occur. If pulse oximetry is not avail- of neuroanesthesia for other species should be applied. able, then the pulse and respiratory rates should be mon- For instance, all inhalant anesthetics increase blood flow itored either by auscultation or observation (Swindle, to the brain in swine, and swelling of the tissue may 2007; Smith, 1997). result postsurgically after manipulation; therefore, tech- niques to reduce brain swelling, such as the use of diuret- G. Acute and Chronic Analgesic Therapy ics and hypertonic glucose solutions, should be applied. Ventilation rates and volumes may also influence the oxy- If surgical procedures are performed, it is best to gen supply to the central nervous system (Swindle, 2007). provide preemptive analgesia prior to making the skin incision or at least prior to removing the animal from 4. Obstetrics and Gynecology general anesthesia if intraoperative administration of Most anesthetic agents will cross the epitheliochorial these agents is not possible (Smith, 1997; Swindle, 2007). placentation of swine to affect the fetus. Transport across the placenta is enhanced if the agent is lipophilic, and 1. Assessment of Pain and Discomfort some of these agents may reach a higher concentration Swine are generally sedentary animals that respond in the fetus than in the sow. Tocolytic agents, such as to the presence of humans only during manipulation or terbutaline, may be useful if uterine contractions under feeding activity. Pigs that are hyperactive and vocalizing anesthesia need to be controlled. Reviews of the special tend to be in distress postsurgically. Incisional pain and considerations and detailed protocols for performing fetal abnormal posture are other reliable indicators of pain or surgery in swine have been published (Swindle et al., 1996). distress. Pigs will readily eat, even after major surgical

LABORATORY ANIMAL MEDICINE 1168 24. Preanesthesia, Anesthesia, Analgesia, and Euthanasia procedures, if they are comfortable. Consequently, swine initial dosage BID, depending upon the clinical condi- that are not resting comfortably and responding to tion of the animal. Buprenorphine may be combined feeding are probably in pain or distress postsurgically with NSAIDs (see Section V, G, 3), such as ketoprofen, (Swindle, 2007; Castel et al., 2014). carprofen, meloxicam, or ketorolac, to reduce the dosage and get a synergistic effect. 2. Methods for Analgesic Drug Delivery Fentanyl and have been used for both bal- Parenteral analgesics are usually administered IM anced anesthesia and as high-dose opioid infusions for or SC in the neck. IV administration is generally given cardiac surgery. These agents have a short half-life and by using indwelling catheters in the ear vein or one of are not good for postoperative analgesia when admin- the other cannulated vessels intraoperatively. Swine can istered as bolus injections. Anecdotal accounts indicate readily be induced to take oral medication when the that dermal patches of fentanyl may be used in swine, substance is placed in a food treat. Canned dog and cat but a controlled study with blood levels of the agent food, apples, chocolate syrup, and sweets are usually has not been published to date. Effectiveness of fentanyl successful when using this technique (Swindle, 2007). patches can be variable, depending upon housing condi- tions and such factors as moisture and heat. In our expe- 3. Non-Steroidal Anti-Inflammatory Drugs rience, it is possible to get analgesia using 50- to 100-pg The newer-generation NSAIDs, such as ketorolac, patches, but animals may show signs of overdosage and ketoprofen, meloxicam, and carprofen, have been uti- have to be monitored. Until such time as a controlled lized successfully in postoperative analgesia protocols study is available, the use of these patches should be either by injection or per os. For some procedures they considered experimental in swine. have been effective in BID dosages as sole agents, but usually they are combined with buprenorphine (see Section V, G, 5) in the lower dose range of each agent to H. Euthanasia maximize the effects of opioids and NSAIDs (Flecknell, Most of the injectable forms of euthanasia utilized 2009; Swindle, 2007). in other large animal species are suitable for swine. Pentobarbital overdose (>150 mg/kg) is the preferred 4. Local, Regional, and Epidural Anesthesia form of parenteral euthanasia. It is acceptable to admin- Local and regional anesthesia has not been commonly ister KCl injections or perform exsanguination while used in research settings and is typically used as an swine are under general anesthesia (Swindle, 2007). adjunct to analgesia rather than as the sole anesthetic (Smith and Swindle, 2008; Thurmon, 1986; St. Jean and Anderson, 1999). The most common areas are dorsal VI. SMALL RUMINANTS nerve root blocks in the intercostal spaces or lumbar regions as an adjunct to anesthesia and analgesia for A. Introduction dorsal–ventral surgical incisions, such as thoracotomies. Infiltration of the incision with local anesthetics is also According to the American Association of Small performed prior to making the initial incision as a form Ruminant Practitioners, the term small ruminants encom- of preemptive analgesia. Xylazine (2 mg/kg diluted in passes sheep, goats, camelids, elk, deer, and related spe- saline), xylazine (1 mg/kg) plus lidocaine (10 ml 10% cies (AASRP, 2010). Sheep, goats, and calves are most solution), and medetomidine (0.5 mg/kg diluted in often encountered in research, testing, or training, thus saline) have been utilized epidurally to provide analge- this review will be limited to those species. These ani- sia during general surgery (St. Jean and Anderson, 1999; mals are docile, adapt well to frequent handling, restraint, Ko et al., 1992a; Royal et al., 2013). and chronic instrumentation that may be dictated by the research needs. They are readily available either 5. Opioids as purpose-bred or farm-raised, conditioned animals. Buprenorphine is generally considered to be the opi- Historically, these species have been used in a number of oid analgesic of choice postoperatively. Preemptive anal- areas of investigation including cardiovascular research, gesia with this agent preoperatively or intraoperatively medical device implantation and testing, orthopedic reduces the course of postoperative analgesia and the research, fetal surgery, and pulmonary studies. Sheep and dosage that may have to be given. There is a wide range goats are often used for production of various reagents of therapeutic effectiveness, depending on the procedure used in experimentation including red blood cells, sera and time of administration. For major surgical proce- and antibodies. dures, such as thoracotomies or visceral transplantation, Relief of pain is a scientific imperative for any species a dosage of 0.05–0.1 mg/kg may be needed in the initial used in biomedical research (NRC, 2011). Recognition stages. The dosage may be reduced by 50–75% of the and relief of pain is required by the Animal Welfare

LABORATORY ANIMAL MEDICINE VI. sMALL Ruminants 1169 Regulations when these species are used in biomedical B. Preoperative Evaluation and Preparation research (USDA, 2008). Furthermore, use of anesthetics and analgesics for routine veterinary practices such as 1. Preoperative Evaluation castration and dehorning, that in the past were often Prior to any surgical procedure, the ruminant ani- performed without the benefit of analgesics is now mal should have a complete physical examination with strongly encouraged (AVMA, 2012a,b). There is a grow- special attention to the respiratory and gastrointestinal ing body of literature on anesthesia, analgesia, and pain systems to determine if the animal is a suitable surgical management specific to small ruminants Gray( and candidate and to provide baseline data with which to McDonell, 1986a, b; Carroll and Hartsfield, 1996; Lee and compare the postoperative clinical condition. Laboratory Swanson, 1996; Carroll et al., 1998b; Lin and Pugh, 2002; diagnostics should include at a minimum, a hematocrit Swindle et al., 2002; Greene, 2003; Riebold, 2007; and measurement of blood urea and creatinine Abrahamsen, 2009a,c, 2013; Valverde and Doherty, 2009; levels. Additional tests, including CBC, chemistry panel Coetzee, 2013). In the research setting, anesthetic tech- and evaluation of fecal sample for ova and parasites niques and analgesic protocols often differ from those are advisable, especially if the animals have been main- used in the field setting common to clinical practice and tained on pasture. certain experimental surgical procedures may require complex anesthetic and analgesic regimens. For some 2. Injection Sites and Venous Access procedures, empirical use of anesthetics and analge- The preferred area for IM or SC injections in ruminants sics reportedly used in humans, companion animals, is a triangular area of the neck bordered by the nuchal or other species may be adopted and modified for the ligament dorsally, the cervical vertebrae ventrally and small ruminant. Cardiovascular studies in particular may the shoulder caudally (Diffay et al., 2002; Radositits et al., require use of cardiopulmonary bypass which is beyond 2007). Other sites that may be used for IM injections the scope of clinical practice (Collan, 1970; Gerring and include the epaxial muscles in the lumbar region, the Scarth, 1974; Schauvliege et al., 2006; Carney et al., 2009). quadriceps femoris, and the triceps (Diffay et al., 2002). The attending veterinarian should be consulted for assis- The semimembranosus/semitendinosus muscle group tance in developing specific anesthetic protocols to meet can be used with caution to avoid injecting irritating study objectives. drugs close to the sciatic nerve. The axillary region and The use of many anesthetic and analgesic drugs chest wall may be used for SC injections in sheep and in small ruminants may constitute ‘extra-label’ use. goats (Diffay et al., 2002). Currently there are no analgesic drugs approved for The jugular vein is often used for administering drugs the alleviation of pain in livestock in the United States IV or blood collection. In sheep and goats the cephalic (Coetzee, 2013; Smith, 2013). Only one anesthetic drug, vein is readily available for IV administration and is eas- 2% lidocaine, is approved for use in cattle in the US ily accessed by having an assistant ‘set up’ the sheep and one NSAID, flunixine meglumine, approved for on its rump and hold off the vein while venipuncture is use in livestock for the relief of pyrexia and inflam- made (Diffay et al., 2002). The saphenous vein may also mation, but not pain (Smith and Modric, 2013; Smith, be used for IV injection in the anesthetized or sedated 2013). Extra-label use of these drugs and its ramifica- animal. tions should be taken into consideration if there is the Intravenous catheters can be placed in the jugular vein potential for return of ruminants used in research into to provide continuous access for IV administration of the food supply through practices such as adoption, fluids, drugs, or total intravenous anesthesia (TIVA). A resale, or rendering. 14-gauge, 5½ inch catheter can be used in most ruminants Attention to the unique anatomical and physiologic (Abrahamsen, 2009a). Smaller catheters can be used in characteristics of the ruminant and taking steps to mini- the cephalic or saphenous veins or in lambs and kids. mize potential adverse effects these differences may Jugular vein catheters should be placed in the cranial have on anesthesia, surgery and recovery is paramount one-third of the jugular furrow to avoid interference to a successful anesthetic and surgical outcome in these with catheter function by the valves found in the distal species. The unique challenges of anesthesia and surgery jugular vein (Divers and Peek, 2008). The author often in ruminants not encountered in monogastric species is places double- or triple-lumen central venous catheters discussed below after which commonly used drugs and using the Seldinger technique after anesthesia induction. protocols for anesthesia and analgesia are presented (see The additional lumens provide access for multiple infu- also Table 24.13). Detailed information on all aspects of sions and for monitoring central venous pressure. The anesthesia and analgesia for ruminants in the research flexible design of most multi-lumen catheters leads to setting is beyond the scope of this section, however, greater longevity and less complications if catheters are readers are referred to the excellent review of this topic maintained long-term. Rigid catheters are more likely to by Valverde and Doherty (2008). fail, especially at the junction of the catheter with the

LABORATORY ANIMAL MEDICINE 1170 24. Preanesthesia, Anesthesia, Analgesia, and Euthanasia

TABLE 24.13 drug Dosages in Small Ruminantsa

Drug Dose Route Comments

ANTICHOLINERGICS

Atropine 0.02 mg/kg IV Not recommended as a routine premedication. Indicated for treatment of bradyarrhythmias Glycopyrrolate 0.005–0.01 mg/kg IV

SEDATIVE/TRANQUILIZERS

Acepromazine 0.02 mg/kg SC, IM, IV Xylazine 0.01–0.02 mg/kg; 0.02–0.04 mg/kg IV IM Medetomidine 0.01–0.03 mg/kg IV, IM Dexmedetomidine 0.05 mg/kg IV Sheep Diazepam 0.25–0.5 mg/kg IV Midazolam 0.1–0.3 mg/kg IV, IM

α2-ADRENERGIC ANTAGONISTS

Yohimbine 0.1 mg/kg (cattle) IV 1.0 mg/kg (sheep)

Atipamezole 20–60 μg/kg IV, IM Tolazoline 0.5–2.0 mg/kg IV

INDUCTION AGENTS

Thiopental 10–16 mg/kg IV Methohexital 3–5 mg/kg IV Propofol 4–6 mg/kg IV Continuous rate infusion (0.3–0.5 mg/kg/min IV) for TIVA (see text).

Xylazine + ketamine 0.05–0.1 mg/kg X, followed by IV or IM IV Xylazine and ketamine can be combined and 3–5 mg/kg K administered concurrently.

Xylazine + ketamine 0.22 mg/kg X + 11 mg/kg K IM Medetomidine + ketamine 0.005–0.01 mg/kg + 2 mg/kg K IV or IM Dexmedetomidine + ketamine 0.005–0.015 mg/kg + 2 m/kg K IV or IM Ketamine + diazepam 5 mg/kg K + 0.3–0.5 mg/kg D IV Premedicate with xylazine, 0.03–0.05 mg/kg IM Ketamine + midazolam 4 mg/kg K + 0.4 mg/kg M IV Midazolam can be administered IM followed by ketamine IV

ANALGESICS

Morphine 0.1 mg/kg IV

Fentanyl 2.5–5 μg/kg IV Transdermal 50 μg/h Butorphanol 0.05–0.1 mg/kg Q 4–6 h SC, IM, IV Buprenorphine 0.005–0.01 mg/kg Q 4–6 h SC, IM, IV Flunixin meglumine 1.1–2.2 mg/kg BID Limit to total of 4 doses Phenylbutazone 2–5 mg/kg IV Do not use chronically 4–8 mg/kg PO Carprofen 4 mg/kg Q 24 h SC, IM Meloxicam 0.5 mg/kg BID 1 mg/kg Q 24 h IM, IV, (sheep) PO aSee text for references and discussion. VI. sMALL Ruminants 1171 luer-lock fitting from repeated flexion with movement key to successful anesthetic management and surgery of the animal. of the small ruminant is preparation of the animal and taking preventive measures to minimize the potential for 3. Common Complications to Anesthesia and regurgitation and aspiration of stomach contents, prevent Prevention bloating and ensure adequate ventilation during anesthe- The anatomy of the ruminant stomach coupled with sia and surgery. the normal physiological functions of salivation, eruc- tation, and regurgitation present unique challenges for a. Fasting anesthesia and surgery of the small ruminant. Common Withholding food and water prior to surgery may complications encountered in anesthetizing ruminants decrease rumen volume, decreases the rate of fermen- are directly associated with the effects of the digestive tation and risk of regurgitation (Swindle et al., 2002). system on adequate ventilation and include regurgita- Recommendations on the duration of fasting prior to tion and aspiration, inadequate oxygenation, and bloat- surgery vary widely ranging from a few hours to 48 h. ing. Addressing these potential problems by proper Excessive fasting may lead to alterations in the rumen preparation of the animal and preventive measures is flora, reduced motility and rumen stasis resulting in the key to a successful surgical outcome irrespective of a negative energy balance and complications during the anesthetic regimen used. the postoperative period (Abrahamsen, 2009a, 2013). The stomach consisting of the rumen, reticulum, oma- Furthermore, fasting may have adverse effects on acid sum, and abomasum is unique to ruminant species and is base status sufficient to cause cardiac arryhythmias the site of production of volatile fatty acids, the primary (Abrahamsen, 2009a, 2013). In cattle, a 48-h fast pro- energy source, through microbial fermentation (Leek, duced 20–30% reduction in heart rate which persisted 2004). The ruminant stomach occupies approximately for 48 h following recovery (Bednarski and McGuirk, 75% of the abdominal cavity, filling most of the left half 1986; McGuirk et al., 1990; Riebold, 2007). Fasting from of the cavity and extending into the right half of the food for 24–48 h and withholding water for 12–24 h in abdomen (Habel, 1975). The relative size of the four com- healthy sheep and goats resulted in better ventilation, partments of the stomach develop and change with age less tympany and reduced incidence of regurgitation of the animal. In the newborn calf, the ruminoreticulum (Carroll and Hartsfield, 1996). Other authors recom- contains less than half the volume of the abomasum and mend shorter periods of no more than 12–18 h fasting remains functionless while the animal is on a milk diet from food and either not withholding water or with- (Nickel et al., 1973; Habel, 1975). The capacity of the rumi- holding for only 4–6 h (Swindle et al., 2002, Abrahamsen, noreticulum is approximately equal to the abomasum by 2009a, 2013). In the author’s experience withholding food 8 weeks of age, double the capacity of the abomasum by and water for 8–12 h before surgery and supporting fluid 12 weeks, and in the adult the capacity is approximately balance with intravenous maintenance fluids is sufficient 9:1 that of the abomasum (Habel, 1975). In lambs, the while avoiding the complications of prolonged fasting. stomach represents 22% of total gastrointestinal wet tis- Young animals should not be fasted for longer than 12 h sue mass, but increases to 49% in adult sheep (Valverde as at this age, they are transitioning from a functional and Doherty, 2008). In cattle, the volume of the stomach monogastric to ruminant (Carroll and Hartsfield, 1996). is approximately 115–150 l while in sheep and goats stom- ach volume is 15–18 l (Habel, 1975; Valverde and Doherty, b. Intubation 2008). The size and volume of the ruminant stomach can Endotracheal intubation of the ruminant under gen- impede respiration and ventilation in the anesthetized eral anesthesia is essential to protect the airway and animal by interfering with diaphragmatic excursion assist in ventilating the animal. The long, narrow oral resulting in a reduction in functional residual capacity cavity of the ruminant requires use of laryngoscopes of the lung, thus interfering with effective pulmonary with longer blades than typically used in human or gas exchange (Lee and Swanson, 1996; Greene, 2003). veterinary anesthesia. Cuffed, silicone endotracheal Positioning of the anesthetized ruminant may further tubes in this size range are available for large animal exacerbate hypoventilation as recumbency shifts the applications (Cook, Surgivet, Pointe). Endotracheal rumen mass leading to displacement of the diaphragm tubes of 9.0–14.0 mm internal diameter (i.d.), 35–55 cm into the thoracic cavity. Cattle placed in lateral or dor- in length are used in adult sheep 50–80 kg body weight sal recumbency developed significant hypoxemia and and tubes of 12.0–16.0 mm i.d., 50–75 cm in length are hypercapnea (Wagner et al., 1990; Jorgensen and Cannedy, used in calves 50–100 kg body weight. The airway of 1996). Furthermore, the displaced rumen may interfere goats is typically smaller than sheep and endotracheal with venous return, predisposing to decreased cardiac tubes of 7.5–9.0 mm i.d. are recommended for goats of output and low blood pressure (Jorgensen and Cannedy, 50–70 kg body weight (Valverde and Doherty, 2008). For 1996; Valverde and Doherty, 2008). For these reasons a sheep and goats, a laryngoscope with a long, straight

LABORATORY ANIMAL MEDICINE 1172 24. Preanesthesia, Anesthesia, Analgesia, and Euthanasia blade, such as a Miller #4 (20 cm in length) can be used an assistant can apply gentle external pressure to the to assist intubation; while the longer Rowson (35–45 cm cricoid area of the neck to further align the laryngeal in length) may be needed for larger sheep and calves opening with the oropharynx. A plastic-coated mallea- (Carroll and Hartsfield, 1996). The entrance to the lar- ble wire, or plastic, metal, or wooden rod can be used ynx is positioned obliquely and faces rostrodorsally as a stylet to stiffen the endotracheal tube and assist relative to the oropharynx making intubation difficult intubation (Carroll and Hartsfield, 1996). Application of (Fig. 24.1) (Valverde and Doherty, 2008). Positioning lidocaine gel or liquid to the arytenoids to obtund the the animal in sternal recumbancy with the head and laryngeal reflex will aid smooth intubation. Benzocaine- neck extended and flexed dorsally will bring the lar- based sprays such as Cetacaine® should be avoided as ynx in line with the oral cavity (Fig. 24.2). In addition, they are reported to cause methemoglobinemia in sheep and goats (Lagutchik et al., 1992; Carroll and Hartsfield, 1996). Care should be taken in passing the endotracheal tube as the sharp points of the molar teeth can eas- ily damage the tube. Once in place and intratracheal positioning confirmed, the endotracheal tube is secured with adhesive or cloth tape.

c. Preventing Regurgitation and Bloat Regurgitation in the anesthetized ruminant may be either an active or passive process. Active regurgitation is most likely to occur due to inadequate or light anesthe- sia, whereas passive regurgitation results from increased transluminal pressure gradients and relaxed esopha- geal sphincters (Steffey, 1986; Jorgensen and Cannedy, 1996). In addition to fasting prior to anesthesia, induc- tion techniques that quickly eliminate the gag reflex and FIGURE 24.1 Lateral view of the head of a sheep showing the oral positioning the animal in sternal recumbancy with the and nasal cavities. Note the rostrodorsal positioning of the epiglottis head elevated reduces the risk of regurgitation during (1), which hinders the entrance to the laryngeal cavity making orotra- cheal intubation difficult.Valverde and Doherty (2008). intubation (Abrahamsen, 2009a, 2013). Intubation with an appropriately sized endotracheal tube with the cuff inflated will protect the airway if regurgitation occurs during surgery. In the ruminant animal, gases in the form of carbon dioxide (60%) and methane (30–40%) are produced by the fermentation process in the rumen (Leek, 2004). The amount of gas production in adult cattle has been esti- mated to peak at a rate of 40 l/h, 2–4 h following a meal and accumulation of gas is normally eliminated by eruc- tation which occurs every 1–2 min (Leek, 2004). Heavy sedation or general anesthesia inhibits ruminoreticular motility and impairs eructation (Valverde and Doherty, 2008). Placement of an orogastric tube into the rumen at the time of anesthesia induction will minimize accumu- lation of gas. A tube with an inflatable cuff such as a foal urethral tube will assist in positioning the end of the tube at the gas–liquid interface in the rumen so that primarily gas and not rumen fluid will be suctioned off Swindle( et al., 2002). Removal of large amounts of liquid from the rumen will not eliminate gas production and is likely to result in a dry mass of ingesta that can impair return to normal digestive function in the postoperative period. The orogastric tube may on occasion become clogged FIGURE 24.2 Endotracheal intubation of an adult sheep. Positioning of the sedated animal in sternal recumbency with the head with ingesta or the wall of the rumen sucked on to the and neck extended aligns the oropharnynx, larynx, and trachea for end of the tube causing it to no longer work. The authors easier intubation. have found that use of low pressures and intermittent

LABORATORY ANIMAL MEDICINE VI. sMALL Ruminants 1173 vacuum is sufficient to minimize gas accumulation and i. PHENOTHIAZINE TRANQUILIZERS Acepro­ avoid the problems of clogging or occlusion. If the tube mazine maleate is the most commonly used phenothi- ceases to function and gas accumulates during surgery, azine tranquilizer in veterinary medicine. For sedation the gas cap can be cannulated percutaneously with a prior to anesthesia, a dose of 0.02 mg/kg SC, IM, or IV large bore (14–18 gauge) intravenous catheter connected provides a slow onset of sedation with a slight decrease to a sterile vacuum hose. The use of oral antibiotics such in respiratory rate but no change in heart rate (Valverde as neomycin prior to surgery to reduce fermentation will and Doherty, 2008). Acepromazine has a sparing effect not significantly reduce the potential for regurgitation on inhalant anesthetics, in one study reducing the MAC or bloating, can lead to problems with return to normal 36–45% in goats (Doherty et al., 2002a). It may also pro- gastrointestinal function following surgery, and is not tect against the arryhthmogenic effects of anesthetics as recommended. observed in other species. Acepromazine (0.5 mg/kg, IV) inhibited epinephrine-induced arrhythmias in thio- 4. Preoperative Medications pental–halothane anesthetized sheep (Rezakhani et al., a. Anticholinergics 1977). Pre-anesthetic medication with anticholinergics is ii. α2-ADRENERGIC AGONISTS AND ANTAGONISTS controversial. Some authors recommend against their Xyalzine, medetomidine, dexmedetomidine, detomi- routine use as premedicants (Riebold, 2007; Abrahamsen, dine, and have all been used in small rumi- 2009a). Others recommend pretreatment with anticho- nants as sole agents or in combination with other drugs linergics when administering xylazine to counteract its for sedation, analgesia, or anesthesia. All α2-adrenergics brachycardic effects (Plumb, 2011). Anticholinergics do produce rapid, dose-dependent sedation in ruminants not consistently reduce salivary secretions unless given (Valverde and Doherty, 2008). A biphasic blood pressure at high doses which may cause tachycardia (Short, 1986; response characterized by an initial hypertension due et al Carroll and Hartsfield, 1996; Ahern ., 2010). Atropine to increased vascular resistance is followed by hypoten- has a shorter duration of action in ruminants compared sion secondary to decreased release of norepinephrine to other species (Short, 1986). Sheep and goats require (Valverde and Doherty, 2008). Hypoxemia secondary to large and repeated doses of atropine to decrease saliva- pulmonary edema has been reported in cattle, goats and tion (Gray and McDonell, 1986a; Carroll and Hartsfield, sheep but is most severe in the latter species (Kumar 1996). Atropine reduces the aqueous fraction of secre- and Thurmon, 1979; Celly et al., 1997, 1999). Xylazine, tions making any respiratory secretions more vis- romifidine, detomidine and medetomidine can produce cous and difficult to clear, potentially causing airway hypoxemia without a concomitant hypercapnia (Celly obstruction (Short, 1986; Carroll and Hartsfield, 1996; et al., 1997). Dexmedetomidine-induced hypoxemia with Abrahamsen, 2009a). Furthermore, atropine reduces evidence of pulmonary edema and pulmonary vascular gastrointestinal motility in ruminants (Abrahamsen, congestion has been reported in sheep (Kästner et al., 2009a). The anticholinergics atropine (0.02 mg/kg IV) 2007). For the purpose of this review, further discus- and glyocpyrrolate (0.005–0.01 mg/kg IV) are indicated sion of the α2 adrenergic agonists will be limited to when bradyarrhythmias occur (Carroll and Hartsfield, the more commonly used xylazine, medetomidine and 1996; Riebold, 2007). Goats appear to require a higher dexmedetomidine. dose of glycopyrrolate (0.01 mg/kg IV) than other spe- Xylazine is 10–20 times more potent in ruminants cies (Carroll and Hartsfield, 1996). than other species with some breeds, e.g., the Brahman and Hereford breeds requiring only 1/10th the normal b. Sedatives and Tranquilizers bovine dose for sedation (Greene and Thurmon, 1988; Sedatives may be used prior to anesthesia to minimize Kästner, 2006; Riebold, 2007). The difference in intraspe- stress and anxiety and facilitate induction. Reducing cies sensitivity is likely due to G-protein binding affinity stress and anxiety permits a greater portion of cardiac in ruminants compared to other species (Torneke et al., output to be directed to vital organs thus more of the 2003). Within the small ruminants, the spectrum of sen- induction agent is directed to the central nervous system sitivity to the effects of xyalzine is goats > cattle> sheep rather than skeletal muscle (Abrahamsen, 2009a). This (Riebold, 2007). Low doses of xylazine (0.01–0.02 mg/kg allows for smoother induction with less induction agent. IV or 0.02–0.04 mg/kg IM) produce standing sedation in Depending on the drug used, sedatives may also reduce cattle suitable for short diagnostic or therapeutic proce- the amount of anesthetic agent necessary for mainte- dures (Abrahamsen, 2013). Higher doses (0.1–0.2 mg/ nance. Sedatives can be used for their calming and anx- kg) of xylazine will induce heavy sedation, and possibly iolytic effects when regional anesthesia is used for minor light planes of anesthesia (Riebold, 2007; Abrahamsen, procedures, and may be combined with analgesics for 2008). Intramuscular administration of xylazine will typ- pain management (Swindle et al., 2002). ically double the duration of effect (Abrahamsen, 2013).

LABORATORY ANIMAL MEDICINE 1174 24. Preanesthesia, Anesthesia, Analgesia, and Euthanasia

Xylazine may be used alone or combined with an opi- arrest, and hypotension (Riebold, 2007). Both tolazoline oid drug (i.e., butorphanol, morphine) as a premedicant (2.2 μg/kg IV) and atipamezole (20–60 μg/kg IV or IM) prior to anesthesia, or administered with ketamine or were effective in reversing medetomidine–ketamine- tiletamine-zolazepam for anesthesia induction. induced sedation in calves (Raekallio et al., 1991; Lin Medetomidine is a sedative-analgesic labeled for use et al., 1999). Re-sedation following atipamazole reversal in dogs that also produces dose-dependent analgesia of medetomidine, presumably due to redistribution or of a degree similar to morphine in sheep (Muge et al., slower elimination from the central nervous system has 1994). It is an equal mixture of two optical enantiomers, been reported in dairy calves but not sheep (Ranheim dexmedetomidine, the active drug, and levomedeto- et al., 1998, 2000a). Intramuscular administration of midine, the inactive drug, with a high affinity for α2- the antagonists is preferred because it reduces the risk adrenergic receptors (Lamont, 2009). Sedation produced of CNS excitement or cardiovascular complications by medetomidine is more rapid and longer lasting than (Abrahamsen, 2008). Dividing the dose of the rever- xylazine (Carroll et al., 2005; Rioja et al., 2008). Its effects sal agent and administering a portion IM followed by on the cardiovascular system are variable depending IV can produce a more rapid recovery while avoiding on species, the route of administration and concur- return of sedation (Abrahamsen, 2008). rent administration with other drugs. Heart rate, mean arterial blood pressure, and pulmonary arterial blood iii. BENZODIAZEPINES Diazepam and midazolam pressure all increased following IV or IM administra- are the most commonly used benzodiazepine sedatives in tion of medetomidine in calves (0.03 mg/kg IV), sheep small ruminants. For sedation, diazepam (0.25–0.5 mg/ (0.01 mg/kg IV or 0.03 mg/kg IM), and goats (0.02 mg/ kg IV) or midazolam (0.1–0.3 mg/kg, IV or IM) can be kg IV) (Kästner et al., 2003; Carroll et al., 2005; Rioja et al., administered to small ruminants (Swindle et al., 2002; 2008). Medetomidine caused cardiovascular depres- Valverde and Doherty, 2008). Diazepam or midazolam sion when administered epidurally or with midazolam are most often combined with other drugs, for example, (Raekallio et al, 1991; Mpanduji et al., 2000). In contrast to ketamine or α2-agonists, for the purposes of sedation, other species, medetomidine has significant effects on the injectable anesthesia or anesthesia induction. Another stress response and tends to increase cortisol and glucose benzodiazepine, zolazepam, combined with tiletamine levels in ruminants (Carroll et al., 1998a, 2005; Ranheim is a component of the injectable sedative/anesthetic ® et al., 2000b). Telazol . As a group, the benzodiazepines have mini- Dexmedetomidine, being the single enantiomer prep- mal, transient effects on the cardiopulmonary systems aration of the dextrarotary form of medetomidine, is and can be used alone for mild sedation and restraint of twice as potent as medetomidine (Plumb, 2011). As a small ruminants (Valverde and Doherty, 2008). Diazepam sedative in sheep, 5 μg/kg IV of dexmedetomidine is is irritating to tissues and poorly absorbed following IM equipotent to 10 μg/kg IV of medetomidine (Kästner administration (Valverde and Doherty, 2008; Plumb, et al., 2001b). As with other α2-agonists, dexmedeto- 2011). It should only be given by slow IV administration midine causes cardiopulmonary depression and may to avoid thrombogenesis (Plumb, 2011). Midazolam in produce moderate to severe hypoxemia (Kästner et al., contrast, is water soluble and can be administered by the 2001b, Kästner et al., 2005, Kästner et al., 2007a, Kästner IV or IM routes (Plumb, 2011). Midazolam is reported to et al., 2007b). In sevoflurane-anesthetized goats, the have antinociceptive properties in a sheep model, pre- adverse pulmonary effects may be mitigated by use of sumably mediated by gamma-aminobutyric acid (GABA) a CRI without loading dose (Kästner et al., 2007a). As receptors at the spinal level (Kyles et al., 1995; Valverde with xylazine, medetomidine or dexmedetomidine may and Doherty, 2008). be used alone or combined with other drugs (e.g. ket- amine) for sedation or anesthesia induction. iv. OPIOIDS In contrast to the α2-adrenergic ago- An advantage of the α2-adrenergic agonists drugs is nists and benzodiazepines, opioids are less commonly the ability to reverse sedation and some of their adverse used in ruminant species for the purpose of sedation and effects with the α2-adrenergic antagonists, yohimbine, premedication prior to general anesthesia. Butorphanol tolazoline, atipamezole and idazoxan. Yohimbine (0.05–0.1 mg/kg IV or IM) or morphine (0.05–0.1 mg/kg (0.12 mg/kg IV) has variable efficacy in cattle for IV or IM) can be co-administered with an α2-agonist to reversing α2-adrenergic, while a higher dose (1.0 mg/kg improve sedation and analgesia while reducing the total IV) is necessary to reverse xylazine sedation in sheep dose of the α2-agonist, thereby minimizing unwanted (Thurmon et al., 1989; Riebold, 2007). Tolazoline (0.5– side effects (Riebold, 2007; Abrahamsen, 2008, 2013). 2.0 mg/kg IV) reverses xylazine-induced sedation in Intramuscular administration of butorphaonol is pre- calves more rapidly than yohimbine (Thurmon et al., ferred as ataxia and dysphoria has been reported in 1989; Young et al., 1989). At higher doses, tolazoline can sheep when the drug was given IV (Waterman et al., cause hyperesthesia and transient bradycardia, sinus 1991a; Riebold, 2007).

LABORATORY ANIMAL MEDICINE VI. sMALL Ruminants 1175

5. Induction Techniques Hartsfield, 1996; Riebold, 2007; Valverde and Doherty, a. Mask Induction 2008). Premedication of the animal with an α2-agonist or fentanyl, reduces the dose of propofol necessary for Anesthesia can be induced in tractable small rumi- induction (Kästner et al., 2006; Dzikiti et al., 2009). Unlike nants weighing less than 100 kg with isoflurane or sevo- barbiturates, propofol is non-cumulative and does not flurane delivered through a large dog mask (Swindle depend on redistribution of the drug from body stores et al., 2002; Riebold, 2007). In the opinion of the authors, for elimination; therefore, it can be given by CRI for main- mask induction is a less desirable method of inducing tenance of anesthesia (Prassinos et al., 2005; Valverde and anesthesia in ruminants because it does not provide Doherty, 2008). In sheep, induction with propofol (6 mg/ rapid control of the animal’s airway and contamina- kg IV) followed by a CRI (0.5 mg/kg/min IV) resulted in tion of the immediate work environment with waste stable, light anesthesia followed by recovery to standing anesthetic gases will occur. Some authors recommend within approximately 15 min (Lin et al., 1997). Because the use of nitrous oxide with the inhalant agent, taking propofol has minimal analgesic properties, when it is advantage of the ‘second gas effect’ to hasten induc- used as the sole anesthetic agent, appropriate analge- tion, however, nitrous oxide should be used with caution sic drugs should be administered when the potential due to its propensity to diffuse into gas-filled spaces for intraoperative or postoperative pain exists (Plumb, including the gastrointestinal tract causing distention 2011). Carroll et al. reported that TIVA with propofol and ruminal tympany (Trim, 1987; Borkowski and Allen, (induction, 3–4 mg/kg IV followed by 0.3 mg/kg/min 1999; Riebold, 2007). IV infusion) in goats premedicated with detomidine and butorphanol provided adequate planes of surgical b. Barbiturates anesthesia for carotid artery translocation, castration or Ultra-short acting barbiturates are often used for rapid ovariectomy (Carroll et al., 1998b). induction of anesthesia, however availability of the drug can be problematic. Of the thiobarbiturates, thiamyl is no d. Ketamine longer available and thiopental is currently not available Ketamine is commonly used as an induction agent in in the United States at the time of writing. Thiopental small ruminants, and in combination with other sedatives (10–16 mg/kg IV) has been recommended for induction or tranquilizers, may be suitable for short procedures and intubation followed by maintenance with an inhal- (Riebold, 2007; Abrahamsen, 2009a). Ketamine induces ant agent (Carroll and Hartsfield, 1996; Lin and Pugh, a dissociative anesthetic state characterized by increased 2002; Swindle et al., 2002). Methohexital sodium is a muscle tone and retention of peripheral reflexes, in par- non-sulfur containing, ultra-short acting oxybarbiturate ticular, upper airway reflexes which may make intubation that has been used as an induction agent in calves and difficult Valverde( and Doherty, 2008). Because of these sheep (Stewart, 1965; Collan, 1970; Carney et al., 2009). properties, ketamine should not be used as the sole agent A dose of 3–5 mg/kg IV is sufficient for induction of for induction or short-term anesthesia and is most com- anesthesia and endotracheal intubation in calves, sheep, monly used in conjunction with α2-agonists and benzo- and goats (Thurmon and Benson, 1986). In the authors’ diazepines. Ketamine and xylazine or the more selective experience, methohexital permits rapid induction and α2-agonists, medetomidine and dexmedetomidine have intubation with a minimum of upper airway secretions been used successfully for induction or short-term anes- in comparison to ketamine combinations (Carney et al., thesia in small ruminants (Raekallio et al., 1991; Caulkett 2009). Currently, methohexital is only available in the et al., 1996; Lin et al., 1997; Kästner et al., 2001a; Swindle U.S. Barbiturates especially sulfur-containing drugs (i.e., et al., 2002; Gogoi et al., 2003; Valverde and Doherty, thiopental) are not recommended in ruminants under 2008; Singh et al., 2010). The sympathomimetic effects of 2–3 months of age (Trim, 1987; Carroll and Hartsfield, ketamine counteract the negative cardiovascular effects 1996). Due to the high alkalinity of the barbiturates, they of xylazine and other α2-agonists (Abrahamsen, 2009a). should only be administered IV, preferably through a Xylazine (0.05–0.1 mg/kg, IV or IM) is administered ini- pre-placed catheter to avoid perivascular necrosis if tially, followed by ketamine (3–5 mg/kg IV or 5–10 mg/ extravasation occurs (Swindle et al., 2002). kg IM) when the animal becomes sedated or recum- bent (Swindle et al., 2002; Valverde and Doherty, 2008; c. Propofol Abrahamsen, 2009a). Alternatively, xylazine (0.22 mg/kg) Propofol is a non-barbiturate, non-steroidal hypnotic can be combined with ketamine (11 mg/kg) in the same agent labeled for use in cats and dogs (Plumb, 2011). It syringe and administered IM (Swindle et al., 2002). rapidly induces anesthesia in sheep, goats and calves Medetomidine (0.005–0.01 mg/kg, IV or IM) or dexme- (Waterman, 1988; Alves et al., 2003; Prassinos et al., 2005). detomidine (0.005–0. 015 mg/kg, IV or IM) can be sub- A dose of 4–6 mg/kg IV is recommended for induction stituted for xylazine, however all three α2-agonist can of anesthesia in ruminants (Reid et al., 1993; Carroll and cause significant respiratory depression and hypoxemia

LABORATORY ANIMAL MEDICINE 1176 24. Preanesthesia, Anesthesia, Analgesia, and Euthanasia especially when administered intravenously (Kästner minimize adverse effects on the lungs (Carney et al., et al., 2001a; Valverde and Doherty, 2008). 2009; Weiss et al., 2012). For mechanical ventilation, The combination of ketamine with the benzodiaze- respiratory rates of 6–10 breaths/min, tidal volumes of pines, diazepam, or midazolam, mitigates the increased 10–22 ml/kg and peak pressures of 20–30 cm of water are muscle tone produced by ketamine alone with the typically used to minimize the effects of positive pres- advantage of minimal effects on the cardiorespira- sure ventilation on the cardiovascular system (Swindle tory system (Valverde and Doherty, 2008). Due to poor et al., 2002; Riebold, 2007). absorption from tissues, diazepam is administered IV The MAC, i.e., the concentration of the inhalant agent followed by ketamine, although midazolam may be at the alveolus that inhibits purposeful movement in administered IM (Stegmann, 1998; Swindle et al., 2002). 50% of anesthetized animals in response to a noxiuous Equal volumes of ketamine (100 mg/ml) and diazepam stimulus for the inhalant anesthetics in small ruminants (5 mg/ml) can be combined and the resultant mixture is presented in Table 24.14. All inhalant anesthetics pro- administered IV at the rate of 1 ml/18–22 kg body weight duce dose-dependent decreases in cardiac output, stroke (Abrahamsen, 2009a). Midazolam (0.4 mg/kg IV or IM) volume, blood pressure, tidal volume and respiratory can be substituted for diazepam and given with ket- rate, and increases in PaCO2 (Valverde and Doherty, amine (4 mg/kg IV) (Stegmann, 1998). A classic induc- 2008). The negative hemodynamic effects of inhalant tion protocol for small ruminants is to administer a low agents may be exacerbated by intermittent positive dose (0.03 mg/kg) of xylazine IM, followed 10–15 min pressure ventilation (IPPV) because mechanical ventila- later by induction with ketamine (5 mg/kg) mixed with tion suppresses venous return and cardiac function dur- diazepam (0.3–0.5 mg/kg) given IV to effect (Valverde ing the positive pressure phase (Valverde and Doherty, and Doherty, 2008). Sedation with a low dose of xyla- 2008; Abrahamsen, 2009a). Drugs used for premedica- zine prior to induction reduces anxiety in the ruminant tion typically lower the MAC (see below) thus lessening patient directing a greater proportion of cardiac output the dose-dependent cardiorespiratory depression of the to vital organs instead of skeletal muscle, thus intensi- inhalant agents. fying and extending the effects of the induction bolus Both sevoflurane and desflurane are less soluble in (Abrahamsen, 2009a). blood than isoflurane. Therefore, induction, change in depth of anesthesia and recovery is more rapid than with isoflurane. There is little difference in cardiovascular and 6. Anesthesia Maintenance respiratory effects between the three agents (Hikasa a. Inhalant Anesthetics Anesthesia in small ruminants is most often main- tained with inhalant agents especially if the animals are TABLE 24.14 reported Minimum Alveolar Concentration of intubated. Inhalant anesthetics provide the benefit of Inhalant Anesthetics in Small Ruminants rapid adjustment of anesthetic depth and relatively rapid smooth recovery from anesthesia. Older inhalant anes- Inhalant agent Cattle Sheep Goat thetics, methoxyflurane and halothane are off market and no longer available in most countries. New agents, Halothane 0.76% 0.69% 0.96 ± 0.12% including isoflurane, sevoflurane, and desflurane have (Valverde and (Valverde (Hikasa et al., 1998) Doherty, 2008) and Doherty, 1.3 0.1% been used successfully in small ruminants (Hikasa et al., 2008) ± 1998; Greene et al., 2002; Mohamadnia et al., 2008; Sellers (Antognini and et al., 2013). Eisele, 1993) For small ruminants <100 kg, anesthesia machines Isoflurane 1.14 ± 0.01% 1.19–1.53% 1.31 ± 0.03% designed for humans or companion animals can be (Cantalapiedra (Valverde (Doherty et al., 2002b) et al., 2000) and Doherty, used. This equipment may not be able to support ani- 1.29 0.11% 2008) ± mals above 100 kg body weight. Anesthesia machines (Hikasa et al., 1998) specifically designed for larger animals are available 1.5 ± 0.3% commercially and can provide the tidal volume and (Antognini and flow rates necessary to meet the ventilator require- Eisele, 1993) ments of these larger animals. High oxygen flow rates Desflurane Not reported 9.5% Not reported (5–10 L/min) are initially used to flush nitrogen from (Lukasik the circuit and animal and promote uptake of the inhal- et al., 1998a) ant agent (Abrahamsen, 2009a). Flow rates of 7–10 ml/ Sevoflurane Not reported 3.3% 2.33 ± 0.15% kg (semi-closed system) or 2 ml/kg (closed system) are (Lukasik (Hikasa et al., 1998) used for maintenance (Abrahamsen, 2009a). Very young et al., 1998b) animals may benefit from breathing 60–70% oxygen to MAC values for calves are assumed to be similar to adult cattle.

LABORATORY ANIMAL MEDICINE VI. sMALL Ruminants 1177 et al., 1998, 2002; Greene et al., 2002; Mohamadnia et al., et al., 2006; Doherty et al., 2007). In calves, infusion of 2008; Sellers et al., 2013). Because sevoflurane is approxi- lidocaine at 50 μg/kg/min resulted in a 16.7% reduction mately seven times the cost of isoflurane and a higher in isoflurane MAC required for umbilical surgery Vesal( MAC is required for a surgical plane of anesthesia, any et al., 2011). Because small ruminants appear to clear advantages of this agent may be outweighed by the cost lidocaine more rapidly than other species, Valverde and per volume of inhalant needed (Sellers et al., 2013). More Doherty recommend higher infusion rates (150–200 μg/ rapid recovery of the animal anesthetized with sevoflu- kg/min) preceded by a loading dose of 2.5 mg/kg rane may be advantageous in specific situations such as administered over 5 min (Valverde and Doherty, 2008). anesthesia of pre-weanling lambs (Vettorato et al., 2012; Clutton et al., 2014). 7. Monitoring Isoflurane, sevoflurane, and desflurane provide little As with any other species, monitoring of small rumi- to no post-recovery analgesia, therefore pain manage- nants is critical to ensure the appropriate level anes- ment must be provided by other drugs and techniques. thesia and quickly detect and respond to any insult to Premedication or intra-operative use of analgesic drugs the cardiovascular, respiratory, and other body systems. with inhalant anesthetics has the added benefit of reduc- Due to the nature of anesthetic and surgical procedures ing the amount of inhalant necessary to provide a surgical typically encountered in biomedical research, the level plane of anesthesia. Tiletamine-zolazepam administered of monitoring will often be more extensive than in the as a premedicant or induction agent reduced the con- clinical setting. Most research facilities will have the centration of isoflurane needed to maintain anesthesia equipment and capabilities to measure heart rate and in goats (Doherty et al., 2002b). Similarly, acepromazine, rhythm, blood pressure, oxygenation, and end-tidal CO2 but not butorphanol when administered as premedi- levels. The American College of Veterinary Anesthesia cants reduced isoflurane MAC 36–45% in anesthetized and Analgesia (ACVAA) recommends routine assess- goats (Doherty et al., 2002a). CRIs of lidocaine alone or in ment of circulation, oxygenation, ventilation, and body combination with ketamine during anesthesia reduced temperature on a routine basis every 5–10 min during the concentration of isoflurane necessary to maintain anesthesia (ACVAA, 2009). Guidelines for monitoring anesthesia in goats and calves (Doherty et al., 2007; Vesal of small animal and equine patients can be adopted for et al., 2011). monitoring of small ruminants. Anesthetic depth is best assessed using several param- b. Total and Partial Intravenous Anesthesia eters, palpebral reflex, eye location, jaw tone, changes TIVA refers to maintenance of an anesthetic plane in ventilation, and response to surgical stimulation. by a combination of injectable anesthetic, sedative, and Palpebral reflex decreases as anesthetic depth increases, tranquilizer drugs most often administered by inter- being moderately brisk at lighter planes, obtunded at mittent boluses or CRI. These injectable techniques are surgical planes and absent at deep planes of anesthesia often used in the field where vaporizers and ventilators (Riebold, 2007; Abrahamsen, 2009a). Position of the eye are not available or practical. Specific drug and dose as an indicator of anesthetic depth can be misleading as recommendations for field anesthesia are provided in the globe is centrally located on induction, moves ven- the published works of Abrahamsen (2008, 2009c). In trally as the plane of anesthesia deepens, moves from the research setting, TIVA may be used for anesthetic ventral toward a central location when a surgical plane maintenance where inhalant agents cannot be used, for is reached, and returns toward the ventral position at example, heart–lung bypass procedures or MRI imaging deep planes of anesthesia (Riebold, 2007; Abrahamsen, studies. ‘Triple drip,’ i.e., adding xylazine (50 mg) and 2009a). Eye position is not a reliable indicator of anes- ketamine (1–2 g) to 1 l of 5% guiafenesin and administer- thetic depth in sheep and goats (Riebold, 2007). ing the drug combination at a rate of 1–2 ml/kg/h has Cardiovascular function can be determined by moni- long been used for TIVA of ruminants (Lin et al., 1993; toring electrocardiogram (ECG), pulse pressure and Greene, 2003). An advantage of this drug combination measurement of arterial pressure. Standard ECG limb is the ability to partially reverse anesthesia with atipa- leads (I, II, and III) are useful for measuring heart rate mezole (Yamashita et al., 1996). and detecting rhythm disturbances. Pulse pressure and Partial intravenous anesthesia (PIVA) is the admin- quality can be determined by palpation of the com- istration of anesthetic, analgesic, and sedative drugs by mon digital, auricular, radial and saphenous arteries, CRI to supplement analgesia and reduce the inspired and the facial artery in young calves (Riebold, 2007). concentration of the inhalant, thereby lessening cardiore- Direct measurement of blood pressure is most accurate. spiratory depression (Valverde and Doherty, 2008). CRI of Non-invasive blood pressure monitoring is unreliable in low doses of ketamine (25–50 μg/kg/min) with or with- small ruminants (Aarnes et al., 2013; Trim et al., 2013). For out lidocaine (100 μg/kg/min) reduced the MAC of iso- direct blood pressure monitoring, the auricular, saphe- flurane by approximately 30% in goats Queiroz-Castro( nous, and common digital arteries can be catheterized

LABORATORY ANIMAL MEDICINE 1178 24. Preanesthesia, Anesthesia, Analgesia, and Euthanasia

during controlled ventilation (Riebold, 2007). End-tidal CO2 in the mechanically ventilated animal should be in the range of 30–40 mmHg (4–5.3 kPa) (Valverde and Doherty, 2008). Furthermore, gas analyzers, especially those which measure the low infrared spectrum may erroneously report elevated inhalant agent concentra- tions due to detection of low levels of methane within the same spectrum (Moens and Gootjes, 1993; Dujardin et al., 2005; Turner et al., 2008). Arterial blood gas analysis provides the most accurate measure of the partial pres- sures of oxygen and carbon dioxide in the animal, and values in small ruminants are similar to other species (Riebold, 2007; Valverde and Doherty, 2008).

FIGURE 24.3 Catheterization of the common digital artery in the C. Postoperative Recovery and Pain foreleg of an adult sheep for invasive blood pressure monitoring. Management In addition to assessing respiration, heart or pulse rate, and temperature, ruminants must be monitored in most ruminant patients (Riebold, 2007). The median closely in the immediate postoperative period for regur- auricular branch of the rostral auricular artery located gitation and bloating. If intubated, small ruminants on the external surface of the ear can be easily can- should not be extubated until they exhibit full return nulated with a 22- or 20-gauge over-the-needle Teflon of swallowing and upper airway reflexes. It is best to catheter (Valverde and Doherty, 2008; Abrahamsen, maintain the animal in sternal recumbency until it is 2009a). In animals for which the auricular artery cannot able to stand. Bolsters or even hay bales can be placed be catheterized, the common digital artery in the foreleg alongside the animal to maintain the sternal position. is an alternative site for direct blood pressure measure- If bloating occurs a stomach tube can be passed or in ment (Fig. 24.3). The artery runs between the dewclaws, an emergency, a trocar placed percutaneously in the left crossing over the palmer surface of the medial branch dorsolateral fossa into the gas cap. of the superficial flexor tendon, coursing rostrally to the medial side of the forelimb (Habel, 1978). An elongated 1. Assessment of Pain and Distress pulsating bulge can be easily seen just proximal to the Many small ruminants are docile and often stoic and dewclaws and cannulated with a 20- or 18-gauge over- may only exhibit subtle signs of pain. Pain is most often the-needle Teflon catheter. Central venous pressure is assessed in these species by observation and interpreta- easily measured from a jugular catheter and the normal tion of changes from normal behavior. (Stasiak et al., range is 5–10 cm H2O (3–7 mmHg) (Riebold, 2007). 2003; Radositits et al., 2007; Anderson and Edmondson, Inhalant anesthetics cause dose-dependent respira- 2013; Plummer and Schleining, 2013). Classic behav- tory depression. In contrast to other species, respira- ioral indicators of pain in large animals include rolling, tory rate is higher (20–40 breaths per min) and tidal pawing, crouching, moaning, grunting, or grinding of volume lower in spontaneously breathing anesthetized teeth (bruxism or odontoprisis) (Radositits et al., 2007). ruminants (Riebold, 2007). Respiratory rate and/or tidal Bellowing by cattle or bleating in sheep and goats may volume will decrease with deeper planes of anesthesia also suggest pain and distress in these species (Radositits (Abrahamsen, 2009a). Hypoventilation is further exacer- et al., 2007). However, more subtle signs such as subdued bated by pressure from the abdominal viscera reducing behavior, more time lying down, decreased appetite, and diaphragmatic excursions in the recumbent ruminant altered rumination may be the only signs of pain exhib- (Valverde and Doherty, 2008). It is preferable to support ited by small ruminants (Anderson and Edmondson, ventilation with intermittent positive pressure ventila- 2013; Plummer and Schleining, 2013). Physiologic tion (IPPV) in anesthetized ruminants. For small rumi- responses to pain include increased heart rate, rapid nants, a tidal volume of 10–15 ml/kg at a rate of 8–12 shallow respirations and in extreme cases, dilated pupils breaths/min with a peak pressure not to exceed 30 cm (Swindle et al., 2002; Radositits et al., 2007). Assessment H2O is used (Valverde and Doherty, 2008). of small ruminants for pain following surgery should Pulse oximetry provides a continuous estimation of be frequent with comparison of behaviors observed in oxygen saturation and should be >90%. Capnography the postoperative period with normal behavior of the in spontaneously breathing animals may not accurately animal. Scoring systems have been developed for rumi- reflect alveolar gas concentrations but is more accurate nants (Stasiak et al., 2003).

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2. Opioids in small ruminants (Abrahamsen, 2009b; Plummer and Opioids can be administered preoperatively to pro- Schleining, 2013). vide pre-emptive analgesia, or administered intraopera- Buprenorphine (0.005–0.01 mg/kg) is effective in calves tively or postoperatively to small ruminants. Opioids and sheep, although its duration of action appears to be with μ receptor agonist activity, such as morphine can shorter in ruminants compared to other species and may be used in ruminants, but with caution due to adverse need to be dosed as frequently as every 4–6 h (Swindle effects on the GI system and behavioral side effects et al., 2002; Ahern et al., 2009). Antinociception to thermal due to CNS stimulation. For these reasons, lower doses but not mechanical stimuli was observed for up to 3 h (0.1 mg/kg IV or IM) of morphine are recommended following a single IV dose (6 μg/kg) of buprenorphine in ruminants (Abrahamsen, 2009b). Although GI side in sheep (Nolan et al., 1987; Waterman et al., 1991b). effects are minimized, rumen motility and contraction, In one study, in sheep undergoing orthopedic surgery, and fecal output should be closely monitored and mor- buprenorphine provided equally effective analgesia phine discontinued if either parameter is decreased or compared to piritramide, a μ receptor agonist, however absent. in another report, transdermal fentanyl provided more Fentanyl, a potent μ-receptor agonist can be used effective analgesia than intermittent IM buprenorphine in ruminants by either the IV or transdermal route of (Otto et al., 2000; Ahern et al., 2009). Buprenorphine may administration. The duration of activity when fentanyl is not be suitable for use as an analgesic in goats. Goats given IV is extremely short (about 20 min) and the half- became agitated and ceased rumination after receiving life in sheep is 3 h and 1.2 h in goats (Carroll et al., 1999; IV or IM buprenorphine (Ingvast-Larsson et al., 2007). Ahern et al., 2010). For these reasons, IV administered 3. Non-Steroidal Anti-Inflammatory Drugs fentanyl is most appropriately used intra-operatively or in combination with other drugs as a CRI for manage- Flunixin meglumine (1.1–2.2 mg/kg) although labeled ment of postoperative pain. In the authors’ experience, as an antipyrexic agent in cattle is often used effectively fentanyl has had little effect on gastrointestinal function for control of postoperative pain. To minimize potential and rumen motility. adverse effects such as renal toxicity and gastric hemor- For postoperative pain relief, fentanyl can be adminis- rhage, it should be given as needed every 12–24 h not tered transdermally using a fentanyl patch, but their use to exceed 4 doses (Swindle et al., 2002). Other NSAIDs has only been reported in sheep and goats (Dowd et al., including phenylbutazone (2–6 mg/kg PO, IV), ketopro- 1998; Carroll et al., 1999; Ahern et al., 2009). Application fen (2–3 mg/kg PO, IV), and aspirin (100 mg/kg PO) of transdermal fentanyl patches (50 μg/h) to the skin of have been used for alleviation of pain following sur- sheep prior to general anesthesia resulted in sustained gery in small ruminants, but none are approved for use plasma levels of fentanyl for 40 h (Ahern et al., 2010). In in these species (Valverde and Doherty, 2008; Anderson contrast, variable, potentially ineffective plasma concen- and Edmondson, 2013; Plummer and Schleining, 2013). trations of fentanyl were found in goats after applica- Both meloxicam and carprofen have been reported to tion of transdermal fentanyl patches (Carroll et al., 1999). provide effective analgesia in sheep and calves following In sheep undergoing orthopedic surgery, postopera- husbandry procedures such as castration and dehorning tive pain management with transdermal fentanyl was (Heinrich et al., 2007; Paull et al., 2007; Stilwell et al., judged to be superior to either oral phenylbutazone or 2012; Glynn et al., 2013). In sheep, meloxicam is dosed IM buprenorphine (Dowd et al., 1998; Ahern et al., 2009). at 0.5 mg/kg IV or 1.0 mg/kg PO, while in goats the Butorphanol, a synthetic κ- and σ-receptor agonist, μ recommended dose is 0.5 mg/kg IV, IM, or PO (Plummer receptor antagonist is three- to five-times more potent and Schleining, 2013). Carprofen (4 mg/kg IM) was used than morphine and has fewer GI and respiratory sys- effectively in combination with other analgesics for pain tem side effects (Abrahamsen, 2009b, Plummer and management in sheep following thoracotomy and aortic Schleining, 2013). Butorphanol (0.05–0.1 mg/kg IV, IM, valve implantation (Schauvliege et al., 2006). or SC) will relieve mild-moderate pain in small rumi- 4. Local Anesthetics nants but duration is limited to 4–6 h (Abrahamsen, 2009b). Intravenous administration of butorphanol has The use of lidocaine and other local anesthetics for been reported to cause negative behavioral side effects in regional anesthesia and for blockade of specific nerves goats and sheep (Waterman et al., 1991a; Doherty et al., are well described and illustrated elsewhere (Skarda and 2002a). Intramuscular or SC administration results in Tranquilli, 2007). Used in conjunction with other analge- lower peak serum concentrations than IV administration sic drugs such as opioids and NSAIDs, local or regional and less negative side effects (Plummer and Schleining, anesthesia provides an effective regimen of multimodal 2013). Administration of an NSAID with butorpha- analgesia (Schauvliege et al., 2006; Carney et al., 2009). nol provides greater analgesia than either drug alone Following thoracotomy in sheep and calves, the authors and is efficacious in treating moderate levels of pain have used a diffusion catheter (Mila International) to

LABORATORY ANIMAL MEDICINE 1180 24. Preanesthesia, Anesthesia, Analgesia, and Euthanasia deliver bupivacaine (0.25%, 5–12 ml q 8 h) at the thora- risk of physical injury, some primate species may be cotomy incision (Carney et al., 2009; Weiss et al., 2012). infected with potentially dangerous zoonotic infections. Although CRI may be used with this method of drug For example, macaques in many facilities may carry delivery, intermittent boluses of local anesthetics deliv- Macacine herpesvirus 1 (B virus), and personnel working ered through a diffusion catheter appear to provide with these animals need be provided with appropriate more effective local anesthesia (Hansen et al., 2013). personal protective equipment (PPE), including gloves and face-shields. Anesthesia can pose an increased risk because of use of needles, production of aerosols of secre- D. Euthanasia tions (e.g., during endotracheal intubation), and the risk Most of the injectable euthanasia methods used in of bites and scratches during induction and recovery. other species, with proper dosing are effective in small a. Preoperative Evaluation ruminants. An overdose (>150 mg/kg IV) of sodium pentobarbital or sodium pentobarbital-based euthana- Preoperative assessment is an important starting sia solutions is the preferred form of euthanasia in these point that helps formulate the anesthetic plan. It usu- species. To assist with IV administration of the euthana- ally includes history of previous use, physical examina- sia drug, reduce anxiety of the animal, and provide for tion, and pertinent laboratory data. Despite limitations the safety of personnel, sedation of the animal prior to associated with performing a thorough physical exami- euthanasia is highly recommended. It is acceptable to nation in nonhuman primates, signs of illness such as administer an overdose of potassium chloride or mag- unusual posture, anorexia, and abnormal feces or uri- nesium sulfate or perform exsanguination while small nary output can be readily identified. Laboratory ani- ruminants are under general anesthesia (AVMA, 2013). mal handlers who have regular contact with individual animals can provide even more insight into an animal’s unique characteristics that warrant more detailed eval- VII. NONHUMAN PRIMATES uation. Routine laboratory tests should be performed while nonhuman primates are in quarantine and later A. Introduction as part of a preventive medicine program. It is usually not necessary nor is it practical to perform preoperative Nonhuman primates are important models for a wide clinical laboratory testing in healthy animals. However, variety of biomedical and behavior research because the nature of the surgical procedure should be taken into of their phylogenetic proximity to humans. The range consideration. A baseline hematocrit may be desirable of species used include New World primates such as for animals undergoing surgical procedures that may marmosets, tamarins, capuchins and squirrel monkeys; produce vascular volume deficiencies. Old World primates such as macaques and baboons; Preoperative fasting is an accepted practice for and hominoids such as chimpanzees. The wide range of nonhuman primates undergoing surgical procedures. body sizes and weight of these different species needs Although it is conventional practice to fast primates for to be considered when selecting anesthetic regimens, as at least 12 h, members of the Callitrichidae (marmosets does species variation in response to different agents. and tamarins) and other small species should be fasted Extrapolation of anesthetic and analgesic doses from one only for 4–6 h, to avoid perioperative hypoglycemia. In primate species to another should be done with caution. situations requiring emergency surgery or in pregnant The aim of this section is to provide a general guide to animals with delayed gastric emptying, inclusion of his- anesthesia and analgesia of nonhuman primates, indi- tamine 2 antagonists (cimetidine 10 mg/kg, ranitidine cate how this should be integrated into an overall plan 1.5 mg/kg) may reduce the risk of aspiration pneumonia of perioperative care, and highlight some of the specific (Popilskis et al., 1992). Metoclopromide (0.2 mg/kg SC or issues arising when working with these species. Drug IM) can reduce the risk of vomiting and increase gastric dosages are listed in Table 24.15. emptying. It is always advisable to have suction avail- able to clear the pharynx and airway during anesthetic B. Preoperative Assessment and Preparation induction and recovery, even in animals that have been fasted, since they may have ingested material from their 1. Preparations for Anesthesia cage environment. Larger primates pose a specific risk of physical injury when handling, and anesthetic regimens frequently 2. Methods of Anesthetic Delivery incorporate initial immobilization with a sedative/tran- Intramuscular injection in nonhuman primates is quillizer. In addition, the ability to handle the animal commonly done into the caudal muscle of the thigh. safely during both anesthetic induction and the recovery For repeated IM injections, it is advisable to alternate period need be considered carefully. In addition to the the leg used to reduce the possibility of muscle or nerve

LABORATORY ANIMAL MEDICINE VII. Nonhuman Primates 1181

TABLE 24.15 drug Dosesa in Nonhuman Primates TABLE 24.15 d(Continued)rug Dosesa in Nonhuman Primates

Drug Dosage Route Drug Dosage Route

OLD WORLD NONHUMAN PRIMATES (MACACA AND Isoflurane 1 MAC = 1.28% PAPIO SPECIES) Sevoflurane 1 MAC = 2% Dissociative agents and combinations Analgesics Ketamine 5–20 mg/kg IM Alfentanil 0.01–0.06 mg/kg/h IV in combination with Ketamine+ 7–10 mg/kg IM a volatile anesthetic or xylazine 0.15–0.5 mg/kg IM infusion of propofol

Ketamine+ 5–10 mg/kg IM Fentanyl 5–10 μg/kg IV in combination with a volatile anesthetic or diazepam 0.3–1 mg/kg IM infusion of propofol Ketamine+ 5 mg/kg IM Fentanyl 10–25 μg/kg/h (as above) medetomidine 0.05 mg/kg IM Morphine 0.1–2 mg/kg IM, SC Ketamine+ 5–10 mg/kg IM Morphine 0.1 mg/kg Epidurally Dexmedetomidine 0.01–0.03 mg/kg IM Oxymorphone 0.15 mg/kg IM Tiletamine- 2–6 mg/kg IM uprenorphine 0.005–0.03 mg/kg IM, SC zolazepam (Telazol) Β Opioid antagonist: 0.1–0.2 mg IV as needed α2-antagonist 0.3–0.75 mg/kg naloxone antipamezole Local anesthetic: Barbiturates Bupivacaine 1–2 mg/kg Tissue infiltration or Thiopentol 5–7 mg/kg IV (induction) local nerve block 15–17 mg/kg/h IV infusion Lidocaine 4 mg/kg As above (maintenance) NSAIDs Pentobarbital 25–30 mg/kg IV Carprofen 2–4 mg/kg SC, IM Other injectable anesthetics Meloxicam 0.1–0.2 mg/kg SC, PO Alphaxolone- 18 mg/kg IM (induction) alphadolone (Saffan) Miscellaneous

Alphaxalone 1–3 mg/kg IV (after sedation with Antiarrhythmic: 1–2 mg/kg IV Continuous IV infusion ketamine) lidocaine 20–50 μg/kg/h Propofol 2–6 mg/kg IV (after sedation with Vasoactive drugs and ketamine) inotropes

0.04–0.12 mg/kg/ IV for maintenance Dopamine 1–10 μg/kg/min Continuous IV infusion min Norepinephrine 0.05–0.2 μg/kg/ Continuous IV infusion Etomidate 0.5 mg/kg IV min

0.2 mg/kg boluses IV (repeated boluses) Dobutamine 2–10 μg/kg/min Continuous IV infusion every 6–12 min Phenylephrine 1–2 μg/kg IV bolus Anticholinergics 0.5–1.0 μg/kg/min Continuous IV infusion Atropine 0.02–0.05 mg/kg IM NEW WORLD NONHUMAN PRIMATES (SAIMIRI AND Glycopyrrolate 0.005–0.01 mg/kg IM CALLITHRIX SPECIES)

MUSCLE RELAXANTS Dissociative agents and combinations

Pancuronium 0.04–0.1 mg/kg IV Ketamine 15–25 mg/kg IM Vecuronium 0.04–0.06 mg/kg IV Ketamine+ 10–25 mg/kg IM xylazine 0.5–1 mg/kg IM Inhalational anesthesia Ketamine+ 15–20 mg/kg IM Halothane 1 MAC = 0.89–1.15% diazepam 1 mg/kg IM (Continued) (Continued)

LABORATORY ANIMAL MEDICINE 1182 24. Preanesthesia, Anesthesia, Analgesia, and Euthanasia

TABLE 24.15 d(Continued)rug Dosesa in Nonhuman Primates Seldinger technique is practicable. Whenever the femo- ral artery is used, manual pressure must be applied after Drug Dosage Route blood sampling, for 1–2 min or longer, after removal of a Tiletamine- 2–5 mg/kg IM percutaneous catheter, to prevent hematoma formation, zolazepam (Telazol) especially if anticoagulants have been administered as part of the study protocol. Other injectable anesthetics

Alphaxolone- 10 mg/kg IM 3. Preoperative Medication alphadolone (Saffan) Anticholinergic drugs are used to diminish salivary Alphaxalone 2–5 mg/kg IV and bronchial secretions and prevent vagally induced 0.16–0.18 mg/kg/ IV infusion for bradycardia. These agents have a number of other min maintenance effects, including predisposing animals to tachyarryth- mias. For this reason they are now rarely used routinely Propofol 2–8 mg/kg IV in anesthesia, unless there is a particular risk of brady- 0.3–0.6 mg/kg/min IV infusion for cardia, for example, when high doses of opioids are used maintenance as part of the anesthetic regimen. If required, atropine Medetomidine 100 μg/kg IM, SC (0.02–0.05 mg/kg) or glycopyrrolate (0.01 mg/kg) can be Atipamezole 200 μg IV administered. Fentanyl-fluanisone 0.3 ml/kg IM, SC (Hypnorm) C. Intraoperative Anesthesia Anticholinergics 1. Injectable Anesthetics Atropine 0.04 mg/kg IM a. Dissociatives Analgesics Ketamine has a wide margin of safety in many spe- cies of nonhuman primates, and it is widely used as an Opioids agent for chemical restraint and induction for subsequent Morphine 0.1–2 mg/kg IM, SC administration of other injectable or gaseous anesthet- Oxymorphone 0.075 mg/kg IM ics, particularly in Old World primates. At the dose of Buprenorphine 0.005–0.03 mg/kg IM, SC 5–20 mg/kg IM, onset of action is rapid and chemical restraint is provided for 15–30 min that is sufficient for Butorphanol 0.02 mg/kg SQ minor procedures. Complete recovery occurs within 1 h, Opioid antagonist: 0.01–0.02 mg/kg IV as needed depending on the dosage used. The bite reflex is lost even naloxone at lower doses of ketamine, but laryngeal and pharyn- aSee text for references. geal reflexes are retained, even at high dose rates. This can be advantageous in providing protective reflexes should vomiting or regurgitation occur, but requires irritation by drugs with low pH, such as ketamine. In other agents to be administered to allow endotracheal nonhuman primates, SC injections into lateral thigh or intubation. Ketamine is usually administered by intra- dorsolateral sites in the back can be done with the help muscular injection, but lower doses can be administered of ‘squeeze-back’ cages. The uptake of drugs from SC intravenously to prolong the period of immobilization. injection, and to lesser degree IM injection, can be vari- The agent can also be administered orally if intramuscu- able and influenced by the rate of hydration and local lar injection is not possible. Oral ketamine is most rapidly perfusion. Chemical restraint is usually needed in pri- absorbed if applied to the mucus membranes, and higher mates for IV injections or blood withdrawal although doses are needed to produce sedation. When injected animals can also be trained, using positive reinforce- into food items to sedate escaped animals, the effects are ment methods, to accept these procedures. The cephalic very unpredictable, and it is usually preferable to employ and saphenous veins can be used both for venipunc- another technique, such as a blow-pipe or dart pistol to ture and administration of drugs, although in marmo- deliver the drug. Ketamine administration in marmosets sets the tail vein may be more accessible. The femoral can be associated with muscle damage because of the low vein and artery are commonly used for the withdrawal pH (3–4), relatively small muscle mass, and the relatively of relatively large blood volumes. The femoral artery high volume of drug needed in this species. also offers a site for insertion of an indwelling catheter The influence of ketamine on hematologic, bio- (20- to 22-gauge) to monitor direct blood pressure and in chemical, and hormonal values should be considered in larger primates percutaneous catheter placement using a interpreting experimental data. Ketamine consistently

LABORATORY ANIMAL MEDICINE VII. Nonhuman Primates 1183 reduces total leukocyte and absolute numbers of lym- Saimiri sciureus. Rapid induction and uneventful recov- phocyte and monocytes (Bennett et al., 1992; Fernie ery accompany Saffan anesthesia. However, respiratory et al., 1994). The red blood cell count, hemoglobin, and depression and hypothermia are also noted (Logdberg, hematocrit are also significantly lower when ketamine is 1988). In macaques, effective surgical anesthesia can be administered. When comparing the effects of ketamine produced with an IM injection of Saffan at 18 mg/kg, fol- on gender, higher values for lactate dehydrogenase lowed by 6–12 mg/kg IV as needed (Box and Ellis, 1973). (LDH) and creatine kinase (CK) are noted for males. In This agent is no longer available commercially, however contrast, females tend to have higher values for amylase a new formulation consisting of alphaxalone (Alfaxan, and (Fernie et al., 1994). Ketamine does not Jurox) is marketed in Europe, Australia, and the USA. appear to alter the magnitude of endocrine responses The single agent has similar effects to the combined even after multiple injections, thereby making it a suit- steroid product. able anesthetic for studies on hormonal change (Castro et al., 1981; Malaivijitnond et al., 1998). d. Propofol Combining the α2-agonists xylazine, medetomidine Propofol provides a smooth induction with adequate or dexmedetomidine, with ketamine provides muscu- muscle relaxation sufficient for procedures of short dura- lar relaxation and analgesia sufficient for minor surgical tion. Because rapid clearance of propofol contributes to procedures. These combinations may cause bradycardia relatively fast awakening, repeated IV boluses of 2–5 mg/ and occasional heart-block, but as in other species, it is kg, or a continuous infusion IV can be administered to not advisable to attempt to correct this with atropine extend the duration of anesthesia without greatly delay- (Lewis, 1993; Vie et al., 1998). Recovery times can be ing recovery. At 2.5 mg/kg IV, propofol provides good reduced by administration of atipamezole (300–750 μg/ muscle relaxation sufficient for laparoscopy, with mini- kg) which is a more specific α2-antagonist than yohim- mal effects on cardiovascular and respiratory functions bine, and has fewer side-effects. in Macaca fascicularis (Sainsbury et al., 1991) and has also A combination of ketamine (5–10 mg/kg) and diaz- been used successfully in marmosets and chimpanzees epam (0.2–0.4 mg/kg) can be used to induce sedation (Ludlage and Mansfield 2003; Sleeman, 2007). It can and avoid perioperative excitement in Old World pri- cause transient apnea if given rapidly, but this can be mates. In Saimiri sciureus and Callithrix jacchus the dose avoided by administering the initial dose slowly, over of diazepam required is higher (1 mg/kg IM). Although about 60 s. Supplemental oxygen via endotracheal tube the duration of sedation is short, this combination is or face mask is advisable, as with any injectable anes- effective for minor procedures requiring muscle relax- thetic regimen. The other potential problem associated ation (Woolfson et al, 1980). Midazolam may cause less with the infusion of propofol is a moderate hypoten- pain on injection than diazepam, and absorption may be sion. The hypotension is dose-dependent and can be more reliable. The shorter elimination half-life of mid- partially corrected by adjusting the propofol infusion azolam makes it suitable for use as an infusion (0.05– and by providing intravenous fluids and is rarely a sig- 0.15 mg/kg) in combination with ketamine (15 mg/kg nificant problem in healthy animals. IM) for various imaging techniques in Macaca mulatta and Chlorocebus aethiops (Jacobs et al., 1993). e. Etomidate Etomidate is an intravenous short-acting hypnotic b. Tiletamine-Zolazepam (Telazol) agent that has been used in neuroanesthesia to monitor Telazol at 4–6 mg/kg IM has been reported to be motor-evoked potentials after transcranial stimulation useful as an anesthetic for minor procedures for about (Ghaly et al., 1990). Because it is very short-acting, an ini- 45–60 min in various species of macaques (Cohen and tial dose of 0.5 mg/kg IV, followed by repeated boluses Bree, 1978). New World primates require a higher dose of etomidate 0.2 mg/kg every 6–12 min, is needed to of Telazol to produce immobilization; in Saimiri sciu- maintain anesthesia in Macaca fascicularis. reus the dose is 10 mg/kg, and in Alouatta seniculus a dose of 22–30 mg/kg IM produces light to moderate f. Barbiturates anesthesia sufficient for restraint in wildlife conditions Thiopental provides short periods of anesthesia (Agoramoorthy and Rudran, 1994). (5–10 min) and was widely used for anesthetic induc- tion prior to intubation and maintenance of anesthesia c. Miscellaneous Anesthetics with inhalational agents. One-half of the calculated dos- Although not available in the United States, alphaxo- age of 5–10 mg/kg IV is given as a bolus, followed by lone-alphadolone (Saffan) has been reported to produce additional drug to effect. The dosage is reduced if the effective anesthesia in New World primates. A single IM animal has received ketamine. Slow infusion of thio- injection of Saffan at 11.5–15.5 mg/kg provides up to 1 h pental at 15–17 mg/kg/h provides satisfactory chemical of surgical anesthesia with good muscle relaxation in restraint with stable physiological values for 90 min in

LABORATORY ANIMAL MEDICINE 1184 24. Preanesthesia, Anesthesia, Analgesia, and Euthanasia

Papio ursinus. Animals were reported to recover unevent- decrease in blood pressure because it reduces sys- fully within 20 min after discontinuation of the infusion temic vascular resistance. Hypotension is especially (Goosen et al., 1984), however like most barbiturates, pronounced during mask induction or when animals thiopental has greater cumulative effects than agents are maintained at or above 2%. Inclusion of fentanyl such as propofol and alphaxalone. or another opioid allows the concentration of isoflu- Despite its long history of use in research animal rane to be reduced, which attenuates hypotension. anesthesia, pentobarbital is now less frequently used Isoflurane produces direct dose-related cerebral vaso- as an anesthetic for survival procedures and commer- dilatory effects. Although isoflurane-induced hypoten- cially available anesthetic formulations of this agent are sion reduces cerebral blood flow, cerebral oxygenation currently unavailable in the United States. Pronounced is maintained (Enlund et al., 1997). Isoflurane anesthesia respiratory depression at the dosages required to pro- does not influence the binding of dopamine receptor duce adequate anesthesia, inability to modulate the ligands, making it suitable for various positron emis- depth of anesthesia, and a long period of recovery are sion tomography studies (Nader et al., 1999). Isoflurane’s additional shortcomings. Pentobarbital is better replaced effect on calcium metabolism in cynomolgus monkeys by other agents, except for terminal (non-recovery) pro- has been studied. Unexpectedly, isoflurane lowered ion- cedures such as perfusion–fixation, when the depressant ized calcium, with secondary increases in parathyroid effects of this drug are considered unimportant. The dose hormone and osteocalcin concentrations (Hotchkiss of pentobarbital in nonhuman primates is 25–30 mg/ et al., 1998). A cautious approach may be needed with kg IV; however, in animals that have been chemically respect to the use of isoflurane for studies of osteopo- restrained with ketamine, the dose is reduced by about rosis and the effects of various antiosteoporotic agents. one-third to one-half. c. Sevoflurane g. Opioids Sevoflurane has a lower blood/gas partition coeffi- Opioids have been used in nonhuman primates to cient than isoflurane, making it useful for rapid induc- reduce the requirement for inhalational or injectable tion and fast recovery from anesthesia. Because of its anesthetics and to enhance intraoperative analgesia dur- pleasant smell and lack of respiratory irritant properties, ing balanced anesthesia. Fentanyl, alfentanil, sufentanil, mask induction is a feasible alternative to other inhal- and remifentanil can all be used as anesthetic adjuncts, ant agents. However, it is degraded by carbon diox- with infusion rates varying depending upon the dose ide absorbents into an haloalkane known commonly as rates of other agents that are administered (Table 24.1). ‘compound A.’ Compound A at high doses has been Care must be taken to monitor and support respira- shown to be nephrotoxic in nonhuman primates, causing tory function, as respiratory depression is common in proximal tubular necrosis (Kharasch, 1998). In common nonhuman primates even after relatively small doses of with other inhalational anesthetics, sevoflurane causes opioids. Opioid administration can also cause a marked a dose-dependent cardiovascular depression. During bradycardia, and if this results in a fall in blood pressure, sevoflurane anesthesia, cerebral blood flow is generally then it should be corrected by administration of atropine. preserved; however, at 3% sevoflurane there was a clear inhibition of autoregulation of cerebral blood flow in 2. Inhalational Anesthesia rhesus monkeys (Yoshikawa et al., 1997). Regardless, a. Nitrous Oxide high concentrations of sevoflurane maintained oxygen Unlike the potent volatile anesthetics, nitrous oxide consumption and delivery throughout the brain. has high MAC (= 200%), preventing its use as a complete surgical anesthetic. Nitrous oxide has been used in com- D. Intraoperative Monitoring and Support bination with inhalational anesthetics such as isoflurane or sevoflurane because it allows for a lower concentra- Although primates can be maintained on a face mask, tion of these agents. This in turn results in a less pro- endotracheal intubation is advisable because of the high nounced circulatory depression, which may accompany risk of vomiting or regurgitation, even for short proce- the sole administration of isoflurane or sevoflurane. dures. Visualization of the larynx is relatively straight- Administration of nitrous oxide should be terminated forward using an appropriately-sized curved or straight and 100% oxygen should be administered 10 min prior blade, and for small primates, rodent intubation equip- to extubation. This will preclude the development of ment can be used (Thomas et al., 2012b). It is advisable diffusion hypoxia. to spray the vocal cords with local anesthetic to prevent laryngospasm, and an introducer can be used to aid tube b. Isoflurane placement if this proves difficult. Isoflurane has a MAC of 1.28% in Macaca fascicu- As in other species, anesthetic monitoring is impor- laris (Tinker et al., 1977). It produces a dose-dependent tant even during brief periods of anesthesia. A range of

LABORATORY ANIMAL MEDICINE VII. Nonhuman Primates 1185 electronic monitoring devices can be used successfully associated with imaging are due to the restricted access in larger primates. In marmosets, tamarins, and squirrel for clinical monitoring and intervention, and the lack of monkeys, the small body size of the animal may limit direct view of the animal. Use of electronic monitoring the use of some instruments, but specialist devices that devices can help resolve these problems, but specially function successfully in these smaller animals are now designed devices are required for MRI. Some instru- available. Maintenance of body temperature is particu- ments, such as side-stream capnographs, can be placed larly important and can be achieved using a range of a safe distance from the magnet. However, this requires different warming devices. In larger primates, forced air use of extensions to sampling tubing which can lead to warming systems are particularly effective. Whichever instrument malfunction due to a loss of signal quality. method of maintaining temperature is employed, its effi- If delivering intravenous fluids, or maintaining anes- cacy should be monitored using electronic temperature thesia using continuous intravenous infusions, long probes. extensions to the infusion tubing can increase resistance Pulse oximetry is a quick and easy means of both and cause pump failure due to activation of an occlusion detecting hypoxemia and monitoring heart rate, and alarm. Maintaining body temperature can be a particu- should be routine in nonhuman primate anesthesia. larly challenging problem during imaging, and warm Both main-stream and side-stream capnographs can be air blowers and magnet-compatible heating devices, used in all species, although the accuracy of these instru- coupled with use of insulating materials, will be needed ments in detecting end-tidal carbon dioxide is reduced to maintain normothermia. in small animals (<1 kg) which have low tidal volumes. Blood pressure can be measured non-invasively in non- 2. Neuroscience Procedures human primates, with a suitable sized cuff placed on Nonhuman primates are used in a range of neurosci- the upper or lower limb, or on the tail in marmosets. ence and neurosurgical studies, and surgical procedures During prolonged procedures, it is advisable to remove may require placement in a stereotaxic frame. Although the cuff, massage the limb, and replace or reposition on it is always advisable to intubate animals, care must be the contralateral limb, to avoid partial constriction of taken when positioning the animal, as flexing the neck limb circulation. Invasive blood pressure measurement may result in occlusion of the endotracheal tube. For is possible in all species, with percutaneous placement non-recovery procedures, a tracheostomy can be carried of catheters practicable in larger animals (>3–4 kg). In out which avoids this problem. Use of an armored endo- smaller animals, surgical exposure of the vessel may be tracheal tube (providing the animal will not be imaged needed. in an MRI) prevents any risk of kinking of the tube. Anesthetic depth can be assessed using standard Pain caused by pressure from the ear bars of the stereo- somatic reflex responses, coupled with loss of muscle taxic frame can be reduced by filling the ear canals with tone (most easily assessed by evaluating jaw tone). The local anesthetic cream (e.g., EMLA, Astra), and NSAIDs palpebral reflex is lost as anesthesia deepens, but in should be administered to reduce tissue inflammation many species the eye remains relatively central rather (see below). than rotating downwards. Anesthetic depth can also be assessed by evaluating the EEG, or by use of bispectral 3. Obstetric Anesthesia index (BIS) monitors. Both techniques require careful Physiologic changes during pregnancy (such as interpretation. EEG changes are greatly influenced by increased cardiac output and decreased requirements the particular anesthetic agent used, and commercially for inhaled anesthetics) can directly influence anesthesia available BIS monitors use an algorithm derived from management for obstetric surgery. Maintenance of anes- human data. However there are reports of successful thesia for obstetric procedures is usually achieved with use of this technology in nonhuman primates (Izrailtyan isoflurane or sevoflurane. Low inspired concentrations et al., 2004). of either agent, supplemented with at least 50–60% of inspired nitrous oxide, minimize cardiovascular depres- E. Special Anesthesia Considerations sion. It has been shown that maintaining pregnant macaques at around 1.5% halothane reduces maternal 1. Imaging blood pressure and cardiac output with consequential Non-invasive imaging introduces a number of com- reduction in uterine blood flow Eng( et al., 1975; Sanders plications to anesthetic regimens in all species, and prob- et al., 1991). This decreases fetal perfusion, resulting in lems may become greater when the imaging process fetal acidosis and hypoxia. Placing pregnant animals in is prolonged. Nonhuman primates can be trained to a supine position may contribute to the hypotension remain immobile for imaging procedures, but frequently because of compression of the caudal vena cava and anesthesia is necessary to reduce the stress of pro- abdominal aorta by the gravid uterus. Therefore the ani- longed immobility. The main anesthetic complications mal should be tilted to the left by placing a wedge under

LABORATORY ANIMAL MEDICINE 1186 24. Preanesthesia, Anesthesia, Analgesia, and Euthanasia the right hip. Adequate fluid therapy is also indicated of adults, even moderate blood loss can result in a pre- to prevent hypovolemia and hypotension. Placement of cipitous decline in blood pressure. Blood loss should an urinary catheter allows one to monitor the effective- be immediately replaced with three- to four-times the ness of fluid therapy. Because hypotension is the most volume of crystalloid solution. common complication encountered during anesthesia, monitoring blood pressure should be routine during F. Postoperative Recovery experimental procedures in pregnant nonhuman pri- mates. If maternal hypotension persists despite hydra- Although extubation is performed after the nonhu- tion and positioning, consideration should be given man primate regains the swallowing reflex, it may lead to vasopressor therapy. Ephedrine (2.5–5.0 mg) can be to gagging or vomiting. If vomiting occurs, the animal used to correct maternal hypotension. Because of its pre- should be placed in a prone position with its head low- dominant β-adrenergic activity, ephedrine will maintain ered to avoid aspiration of the vomitus, and the oro- arterial perfusion by increasing maternal cardiac output pharynx should be suctioned. Gentle suctioning of the and restoring uterine blood flow without uterine arterial trachea prior to recovery from anesthesia is important vasoconstriction. in smaller primates because respiratory passages can be Maternal normocapnia should be maintained dur- easily obstructed with bronchial secretions. Monitoring ing the intraoperative period because the effects of of cardiopulmonary functions in the immediate post- hypoventilation may be associated with premature ven- operative period will depend on the extent and type tricular contraction in macaques (Sanders et al., 1991). of procedure. Monitoring of arterial pressure is often Accordingly, controlled ventilation is the most efficient needed until the nonhuman primate regains sufficient way of maintaining normal PCO2. consciousness after cardiac or neurosurgical procedures. Maintaining a femoral arterial catheter helps to deter- 4. Pediatric Anesthesia mine the status of arterial blood gases and avoid hypox- There are important physiologic differences between emia in the immediate postoperative period. adults and neonates. Neonatal cardiac output is heart Postoperative hypothermia is frequently encoun- rate-dependent. Therefore, one of the main goals of tered during and after surgical procedures, particularly preanesthesia and induction is avoidance of heart rate in young and small nonhuman primates. Hypothermia reduction. Administration of ketamine and atropine and associated shivering can be treated effectively with provides protection against possible reduction in heart warming devices such as a Bair Hugger. Administering rate during the initial stages of anesthesia in pediatric warm fluids will help avoid pronounced hypothermia. primates. Although mask induction is an accepted technique in 1. Analgesic Therapy pediatric animals, it must be recognized that isoflurane Postoperatively, nonhuman primates often show is pungent and produces airway irritation, which could little reaction to surgical procedures, and it should be possibly result in laryngospasm during induction, so recognized that the signs of pain may not be evident use of sevoflurane is preferable. Smooth induction can until the animal is in severe pain. Therefore, treating be easily achieved with the IV propofol at 2–4 mg/kg. postoperative pain in a nonhuman primate in an effec- Because neonatal primates are usually allowed to nurse tive and timely matter can be difficult to achieve. Many until anesthesia time, assisted ventilation during mask IACUCs may require the administration of analgesics induction may lead to insufflation of the stomach with before the surgical stimulus, i.e., in a pre-emptive fash- gases and put neonates at risk of aspiration. Cuffed or ion. Analgesics available for use in other species can uncuffed tracheal tubes may be used in intubation of be used safely and effectively in nonhuman primates pediatric primates. Postintubation laryngeal edema is (Table 24.15). However since there are no reliable means unlikely if the size of the tube in the trachea is such that of pain assessment in these species, determining which slightly audible air leaks occur around it. Some operat- analgesics are effective in the individual animal can ing theater pollution is inherent in this approach. Small be challenging. At present, attempts should be made tidal volume in pediatric primates requires the use of to develop clinical scoring systems using well-defined nonrebreathing systems such as modified Jackson-Rees signs that all personnel involved in the animal’s care and Bain. The fresh gas flow of twice minute volume can recognize. Repeated assessment, particularly after (estimated as 7–10 ml/kg × respiratory rate) is adequate analgesic administration and analgesic withdrawal, can to prevent rebreathing during spontanous ventilation. help focus on those signs which are most related to an Because isoflurane and sevoflurane depress spontane- individual’s pain. ous ventilation, controlled or assisted ventilation will Based on the invasiveness of the procedure, surgeons prevent CO2 accumulation. Because vasoconstrictive should administer opioid analgesics such as buprenor- responses of neonates to hemorrhage are less than those phine, either alone, or preferably in combination with

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