Division of Anaesthesiology and 2Division of Clinical Veterinary Research, Department of Clinical Veterinary Medicine, University of Bern Accepted for publication in the Schweizer Archiv fr Tierheilkunde, December 2006
7 Introduction Anaesthesia and analgesia for piglet castration is an animal welfare imperative and is currently required by law in Norway and pain elimination will be necessary according to Swiss law by 2009. The technique used should result in significant reduction or elimination of pain and stress for the piglets and should also be short acting, economical, ecologic, residue free, easy to use and have a large therapeutic range. Several investigations have been undertaken to find an ideal method. Intramuscular ketamine and azaperone is effective but prolonged recovery remains a problem (Kmiec, 2005). Local anaesthetic techniques are performed routinely in Norway since 2002, but do not completely block the nociceptive response (Haga and Ranheim, 2005). Inhaled carbon dioxide provides reliable unconsciousness, but unwanted side effects including hyperventilation, agitation and gasping make this technique unacceptable (Kohler et al., 1998). Isoflurane, via a special double mask to reduce pollution of the environment and exposure to the staff, has been shown to be a fast, safe and practical method (Walker et al., 2004). However, handling, equipment costs and exposure concerns have to date limited its acceptance. Intranasal delivery systems are widely used today in human paediatric medicine to deliver medication in a pain- and stress-free manner. Intranasal ketamine and midazolam, in combination, have been used successfully for sedation of children (Louon and Reddy, 1994). An intranasal system would be needle-less making it more desirable for on the farm use, as needle injury to both administrator and piglet can be avoided. The objectives of this study were 1) to compare the use of intranasal versus intramuscular administration techniques and 2) evaluate cardiovascular effects, degree of nociception, body temperature change and recovery time when ketamine, climazolam and azaperone are used in combination for castration of four to seven day old piglets.
8 Animals, Material and Methods Animals All experiments were performed at the same commercial Bio-Suisse organic swine facility over a 6-month period from July 2005 to January 2006. Forty purebred Large White male piglets, aged 4 to 7 days, from 10 different litters were included in the study. Numbers are presented as medians with upper and lower 95% confidence intervals (CI). The median body weight of all animals was 2.24 (2.1; 2.65) kg and the median age was 5 (5; 6) days. All animals were deemed healthy based on clinical examination, had normal testicular anatomy and no concurrent treatments. The Committee for Animal Experimentation of the Kanton Bern of Switzerland approved the study. Experimental Design A randomized and blinded experimental study design, using matched pairs for litter and weight, was used. Ketamine 15 mg kg-1, climazolam 1.5 mg kg-1and azaperone 1.0 mg kg-1 were administered in combination either intramuscularly (IM) or intranasally (IN) prior to the castration of the piglets. Just prior to the experiment, the male piglets were sorted and weighed unfasted. Pairs assigned were of the same litter and had less than 10% difference in body weight. Each pair received anaesthesia on the same day, sequentially. The first member of each pair was randomly assigned by a computer generated random number chart to receive either the IM or IN administration route, while the second member of each pair received the other. Anaesthesia Ketamine (Ketamin-HCl-Lsung 173 mg ml-1; Dr. E. Graeub AG, Bern, Switzerland), climazolam (Climazolam-Lsung 30 mg ml-1; Dr. E. Graeub AG, Bern, Switzerland) and azaperone (Stresnil 40 mg ml; Janssen Pharmaceutica, Beerse, Belgium) were combined in saline to give a final concentration that yielded ketamine 15 mg kg-1, climazolam 1.5 mg kg-1 and azaperone 1 mg kg-1 when dosed at 0.2 ml kg-1. The drugs were mixed less than 12 hours prior to the start of the experiments and stored at room temperature. Administration of drugs commenced 10 minutes prior to castration. IM administration was performed with a 1 ml syringe (Injekt F; Braun, Melsungen, Germany) and a 0.6 x 25 mm needle (Sterican; Braun, Melsungen, Germany) into the right neck muscle. A human nasal spray device, which dispensed 0.1 ml per spray, (AccuPump, Saint-Gobain Calmar GmbH, Hemer, Germany) was used for IN administration into both nostrils. For administration, the piglets were held in a sternal position by the investigator with the head fixed by hand at a 45 angle. Postadministration piglets were placed into a heated straw box for observation. Five

9 minutes after administration, piglets were assessed for relaxation and recumbency. If recumbency was not achieved by 10 minutes after administration, the animals were redosed with half of the original volume and left undisturbed for 10 more minutes. Number of animals requiring redosing was recorded. Castration Castration was performed 10 minutes after the last administration of anaesthetic agents by the same investigator. Animals were placed in dorsal recumbency and the hind legs were fixed cranially with a velcro strap. The electrocardiogram was monitored throughout the procedure using a standard base-apex lead configuration (Datex S5, Datex-Ohmeda, Finland). The scrotom was washed with a dilute chlorhexadine scrub (Lifoscrub, B-Braun-Medical AG, Emmenbrcke, Switzerland) and the castration was performed with a scalpel blade (Packagon #10 sterile single use scalpel, Lance Paragon LTD, Sheffield, England). A small skin incision was made in the scrotum directly over each testicle. Pressure was applied to each testicle, until it emerged from the incision. The testicle was then gently pulled from the incision and the spermatic cord and vessels were cut. The entire castration was recorded on video for later castration scoring. Antibacterial spray (Chlor-tetracyclin Spray, Werner Stricker AG, Zollikofen, Switzerland) was then applied to the wound and the piglets were immediately placed back on straw underneath heat lamps and were disturbed only to record measurements. Measurements Three different blinded individuals judged the reaction of the piglets to the castration using a scoring system previously described by Wenger et al. (2002). A score from 0 to 4 (Tab. 1) was assigned four times for each castration, one for the reaction to each skin incision and cord transection. These scores were then added together and the scores of the 3 judges averaged to obtain one final score (0 to 16). A low score is associated with no movement and anaesthesia and a high score with vocalization and struggling. Heart rate (HR) and respiratory rate (RR) were determined by palpation and observation of thoracic wall excursions and were recorded prior to manipulation (timepoint (TP) 10); 5 (TP -5) and 1 (TP 1) minute prior to castration; during castration (TP 0) and 10 (TP +10), 20 (TP +20), 30 (TP +30) and 60 (TP +60) minutes after castration. Rectal temperature was recorded at TP 10, +30 and +60 with a rectal digital thermometer (Microlife MT 1961, Migros, Zrich, Switzerland). Percent oxygen saturation (SpO2) using a pulse oximetry probe (Datex S5, Datex-Ohmeda, Finland) placed on the base of the tail and was recorded at timepoints -5, -1, 0, +10 and +20. Systolic blood pressure (SBP) was measured using a Doppler blood pressure probe (Ultrasonic

10 Doppler Flow Detector, Parks Medical Electronics, Inc., Aloha, OR) placed on the left radial artery at timepoints -1, 0 and +10. Room temperature was determined with a digital thermometer (MTR102, OregonScientific, Oregon, USA). Recovery Sarmazenil (Sarmasol 1 mg ml-1, Dr. E. Grub AG, Bern, Switzerland) at a dose of 0.2 mg kg-1 was administered, using the same route as for the prior agents, 30 minutes after castration for antagonization of climazolam. Time to walking from drug administration was recorded when the piglets were able to stand on their own and walk without falling and with minimal ataxia. Time to walking was judged by the same observer. Piglets were returned to the sow when walking and all measurements were recorded. Statistical Analysis Descriptive and comparative statistical analyses for all data were performed using NCSS software (www.ncss.com, Kaysville, UT). Cardiovascular data (HR, RR, SBP, SpO2), castration score, rectal temperature change and time to walking were compared between groups using a paired Wilcoxon Signed-Rank Test for difference in medians, with the pairs defined by matching. A Wilcoxon Rank-Sum test for difference in medians was used to evaluate the castration scores at different room temperatures within each anaesthesia group. The alpha level for statistical significance was set to 0.05.
11 Results As data was not normally distributed, results are reported as medians with upper and lower 95% confidence intervals (CI). Twenty pairs were analyzed for the study. There are several individuals with missing cardiovascular data due to inability to get accurate measurements because of movement or equipment malfunction. Table 2 lists the number of animals analyzed for each data point. There were no deaths during the study period. Most animals achieved recumbency 4 - 5 minutes after receiving the anaesthetic agents, except for 5 animals in the IN group that required additional dosing to achieve recumbency. The actual time to castration from the induction time was significantly higher (P=0.015) in the IN group (median 13 minutes, 95% CI 11; 16) compared to 11 (10; 12) minutes for the IM group because of the 5 animals in the IN group that required additional anaesthesia. The median administration volume per piglet was 0.4 (0.4; 0.5) ml of both the initial anaesthetic agents and also the sarmazenil for reversal. The IN group had a significantly higher (P=0.0070) castration score (7.7 (0.7; 10) compared to the IM group (1 (0.3; 5) (Fig. 1). Room temperature over the study period was over a wide range as we performed the study over a 6 month period 21.5 (10.1; 21.5). C When both groups were sub-divided according to the actual room temperature categories, a marked association between castration score and room temperature was detected in the IN group. Room temperatures below 18 C were associated with significantly higher castration scores (12.9 (10; 16) than temperatures above 18 (4.3 C (0; 9.3) (Fig. 2, upper graph). Two of 5 IN animals that required redosing were in the high room temperature group and 3 in the low. This association was not present in the IM group although the scores were uniformly lower with higher room temperatures (Fig. 2, lower graph). There were no significant differences between groups at all time points for SpO2 or SBP (Tab. 2). HR was significantly higher 10 minutes after castration in the IN group (180 (165; 208) compared to the IM group (165 (155; 193) (P=0.013). RR was significantly higher in the IM group at time points -5, -1, 10, 20 and 30 (Tab. 2). The decrease in rectal temperature 60 minutes after castration was significantly more (P=0.0011) in the IM group (2.74 (1.9; 3,47) compared to the IN group (1.5C (0.6; 1.7). Timing of C administration of the reversal agent sarmazenil was similar in both groups: 34.5 (32; 41) minutes in the IM group and 37 (35; 46) minutes in the IN group. The IN group was walking 50.5 (46; 58) minutes after induction which was significantly faster than the IM group 80.5 (66; 132) minutes (P=0.0001).

12 Discussion In the current study, administration of ketamine, climazolam and azaperone in combination provided a consistent and reliable anaesthesia for castration of newborn piglets when administered IM. IN administration provided advantages including minor temperature loss and shorter recovery times, but had significantly higher castration scores, indicating that the anaesthesia was less effective. All of the scores that were 10 or greater (on a scale of 16) were associated with room temperatures of less than 18 C, therefore further study could prove the IN technique to be more reliable under controlled environmental conditions. Veterinarians have limited access to anaesthetic agents suitable for anaesthetizing piglets, as few of these drugs have specified meat withdrawal times. Ketamine and azaperone are 2 agents, which are approved for use in swine in Switzerland and in the European Union. Ketamine is a dissociative anaesthetic that provokes sedation, immobility and analgesia but can cause muscle rigidity and tremors in swine when used alone. No such side effects were apparent with the combination of a benzodiazepine (climazolam) and a butyrophenone derivative (azaperone). Azaperone causes tranquilization and sedation in swine and has also been used to control aggressiveness when regrouping weanling or feeder pigs. It causes slight respiratory stimulation and is known to reduce the arterial blood pressure due to vasodilation after intramuscular injection in pigs (Clarke 1969). Its duration of action can be as long as 2-3 hours. In our study, the IM group had a significantly higher RR at several time points, decreased rectal temperature and longer recovery when compared to the IN group. Possibly this could be explained by poor IN absorption of azaperone, making the side effects of azaperone more prominent in the IM group: increased respiration, heat loss due to vasodilation and longer duration of action leading to a longer recovery time. The combination of ketamine and azaperone intramuscularly for castration of newborn piglets was studied extensively (Kmiec, 2005), but was associated with undesirable side effects. In a pilot study, (S. Axiak, unpublished data), climazolam was added to reduce the required doses of ketamine and azaperone and potentiate their effects. Climazolam is a benzodiazepine tranquilizer marketed in Switzerland. It is very similar to the benzodiazepine midazolam, which has been found to be effective in swine for induction of anaesthesia in combination with ketamine and azaperone (Clutton et al., 1997). In the study by Kmiec (2005), a dose combination of 2 mg kg-1 azaperone and 25 mg kg-1 ketamine IM was found to be most effective in the initial trial, which compared 6 different dosing regimens. Recovery time in the 2 mg kg-1 azaperone 25 mg kg-1 ketamine group ranged between 198 and 234 minutes. The

13 recovery time defined in Kmiecs study was from the raising of the head until first independent suckle on the teat. Our study defined the recovery period to be from induction of anaesthesia to full consciousness and walking. Despite this difference, it is evident that the present recovery times were much shorter; 50.5 (46; 58) minutes after induction for the IN group and 80.5 (66; 132) minutes for the IM group. This can be attributed to the lower dosages required of ketamine and azaperone and the addition of climazolam, which is reversible by sarmazenil. In pilot studies (Axiak, unpublished data), we found that the recovery times were excessive without reversal of the climazolam and it was our goal to achieve the shortest possible recovery with these agents. Further studies are warranted though, comparing the recovery times with and without sarmazenil, as the cost and additional handling with sarmazenil could limit the usefulness of this technique in practice. When compared though to isoflurane anaesthesia, the recovery times are much longer. Piglets were standing with a mean of 126 seconds after discontinuation of isoflurane (Walker et al., 2004). Piglets nurse approximately one time an hour (McGlone and Hellman, 1988), and it can be estimated that two nursings are missed due to the prolonged recovery from the combination of ketamine, climazolam and azaperone. Longevity studies (i.e. weight at weaning, morbidity and mortality) would need to be studied to determine the significance of this. The most clinically relevant discrepancy between the two groups was the difference in the castration score. The IN group had a higher castration score indicating that the anaesthesia was less effective in this group and some of these animals showed strong movements. There are several possible explanations for this. In humans, a nasal cycle occurs as a phenomenon in which the nose goes through regular periods of congestion and decongestion. During periods of congestion the nose has decreased mucociliary clearance and this in turn can have an effect on drug absorption (Soane, et al., 2001). The nasal cycle has been documented in swine by Campbell and Kern (1981). Other factors that can effect the effectiveness of the intranasal administration technique are: viscosity of the drug delivered, the ability of the nasal spray device to generate an aerosol, the pump design and the actuation force of the administrator on the pump (Dayal et al., 2004). Large volumes administered too quickly can lead to swallowing of the drug and then subsequent delayed uptake from the gastrointestinal system (Visweswaraiah et al., 2002). Finally sneezing, a symptom that was observed in this study in all piglets can also decrease the amount of drug available. Further study using increased dosing in the intranasal group is warranted, taking these facts into account. Although the scores were better in the IM group, the median score was 1,

14 indicating that most animals showed a slight reaction, whereas data from the isoflurane studies indicate that isoflurane provided more reliable anaesthesia as 15 of 24 piglets showed no reaction at all (Walker et al., 2004). Likely this is because isoflurane can be administered to effect and this is not possible with an IM injection. Another and probable cause of the discrepancies between the two groups is simply that the solution was too cold. All of the scores above 10 were associated with room temperatures less than 18 C. There are several reasons why temperature could affect the usefulness of this technique. First, cold temperatures (i.e. the air temperature and the solution) cause vasoconstriction. Landis et al. (2003) reported that intranasal fluid nebulization induced a sympathetic mediated, transient vasoconstrictor response at a solution temperature of 22 When the fluid was warmed to 37 C this response C. was abolished. Although in our study, solution temperature was not measured, room temperature was. Likely, the solution temperatures mirrored the room temperature as the agents were left out at room temperature. Further study of IN administration of these agents warmed to body temperature is warranted. We hypothesize that consistently lower castration scores will then be obtained. In our study, temperature had no effect on the castration score of the IM group. In a study by Clutton et al. (1998) effect of drug temperature on intramuscular ketamine and azaperone sedation in pigs was studied at three different temperature groups ranging from 6 to 40.5 Mean time C. to recumbency was shortest with the warmest injections but not statistically significant. The observed difference in temperature loss and recovery times between the IM and IN group in this study may be due to absorption and elimination characteristics of the administration route. Likely the drugs were absorbed and distributed much faster from the IN route. In a study in pigs, it was shown that IN administration of titrated progesterone resulted in an increased concentration in the blood supplying the brain than in other arterial blood, indicating a local vascular transfer from the nasal mucosa to the arterial blood of the brain (Skipor et al., 2003). In children, ketamine and norketamine plasma concentrations were studied after venous, nasal and rectal administration of ketamine at a dose of 9 mg kg-1 and IN administration induced a more rapid and greater plasma concentration when compared to rectal administration (Malinovsky et al. 1996). Norketamine also appeared later with the IN route indicating a lack of first past metabolism and an almost two-fold greater bioavailability. In contrast, another study, which compared IN and IM administration of midazolam in children, found no significant differences between the two methods in the onset and degree of sedation and response of the children to venipuncture (de Santos et al., 1991). The cardiovascular variables were similar in both groups, no major side effects were

15 observed and no deaths occurred. IM administration was reliable and IN administration was inconsistent, but all of the scores in the IN group that were 10 or greater (on a scale of 16) were associated with room temperatures of less than 18 C, therefore further study could prove the IN technique to be more reliable with a solution warmed to body temperature.
16 Acknowledgements This study was supported by ProSchwein, Switzerland. I would like to thank U. Schatzmann and N. Jggin for all of their support and guidance, not just for this project, but as well for my anesthesia residency and professional development. A big thanks also to V. Gerber, P. Larenza, C. Tessier and S. Di Concetto for their assistance, friendship and support.
17 References Campbell W., Kern E.: The nasal cycle in swine. Rhinology 1981, 19:127-148. Clarke K.: Effect of azaperone on the blood pressure and pulmonary ventilation in pigs. Vet. Rec. 1969, 85:648-51. Clutton R., Blisset K., Bradley A., Camburn M.: Comparison of three injectable anaesthetic techniques in pigs. Vet. Rec. 1997, 141:140-146. Clutton R., Bracken J., Ritchie M.: Effect of muscle injection site and drug temperature on pre-anaesthetic sedation in pigs. Vet. Rec. 1998, 142:718-21. Dayal P., Shaik M. S., Singh M.: Evaluation of different parameters that affect dropletsize distribution from nasal sprays using the Malvern Spraytec. J. Pharm. Sci. 2004, 93:1725-1742. de Santos P., Chabas E., Valero R., Nalda M.: Comparison of intramuscular and intranasal premedication with midazolam in children. Rev. Esp. Anestesiol. Reanim. 1991, 38:12-15. Haga H.A., Ranheim B.: Castration of piglets: the analgesic effects of intratesticular and intrafunicular lidocaine injection. Vet. Anaesth. Analg. 2005, 32:1-9. Kmiec M.: Die Kastration von Saugferkeln ohne und mit Allgemeinanaesthesie (Azaperone-Ketamine): Praktikabilitaet, Wohlbefinden und Wirtschaftlichkeit. Dissertation, Freie Universitt Berlin, 2005. Kohler I., Moens Y., Busato A., Blum J., Schatzmann U.: Inhalation anaesthesia for the castration of piglets: CO2 compared to halothane. Zentralbl. Veterinrmed. A. 1998, 45:625-633. Lacoste L., Bouquet S., Ingrand P., Caritez J. C., Carretier M., Debaene B.: Intrasnasal midazolam in piglets: pharmacodynamics (0.2 vs. 0.4 mg/kg) and pharamcokinetics (0.4 mg/kg) with bioavailability determination. Lab. Anim. 1999, 34:29-35. Landis B. N., Beghetti M., Morel D.R., Giger R., Rimensberger P. C., Lacroix J.: Somato-sympathetic vasoconstriction to intranasal fluid administration with consecutive decrease in nasal nitric oxide. Acta Physiol. Scand. 2003, 177:507-515. Louon A., Reddy V.: Nasal midazolam and ketamine for paediatric sedation during computerised tomography. Acta Anaesthesiol. Scand. 1994, 38:259-261. Malinovsky, J. M., Servin F., Cozian A., Lepage J. Y., Pinaud M.: Ketamine and norketamine plasma concentrations after i.v., nasal and rectal administration in children. Br. J. Anaesth. 1996, 77:203-207. McGlone J.J., Hellman J. M.: Local and general anesthetic effects on behaviour and performance of two- and seven-week-old castrated and uncastrated piglets. J. Anim. Sci. 1988, 66:3049-3058.

18 Skipor J., Grzegorzewski W., Einer-Jensen N., Wsowska B.: Local vascular pathway for progesterone transfer to the brain after nasal administration in gilts. Reprod. Biol. 2003, 3:143-159. Soane R., Carney A., Jones N., Frier M., Perkins A., Davis S., Illum L.: The effect of the nasal cycle on mucociliary clearance. Clin. Otolaryngol. 2001, 26:9-15. Walker B., Jaeggin N., Doherr M., Schatzmann U.: Inhalation anaesthesia for castration of newborn piglets: experiences with isoflurane and isoflurane/N2O. J. Vet. Med. A Physio. Pathol. Clin. Med. 2004, 51:150-154. Wenger S., Jaeggin N., Doherr M., Schaztmann U.: Die Halothananaesthesie zur Kastration des Saugesferkels. Machbarkeitsstudie und Kosten-Nutzen-Analyse. Tierrztl. Prax. 2002, 30:164-170. Visweswaraiah A., Novotny L., Hjemdahl-Monsen E., Bakaletz L., Thanavala Y.: Tracking the tissue distribution of marker dye following intranasal delivery in mice and chinchillas: a multifactorial analysis of parameters affecting nasal retention. Vaccine 2002, 20:3209-3220.
Table 1: Numerical scoring system for assessment of castration. Score 4 Criteria No reaction or vocalization Movement of one limb, no vocalization Movement of two limbs, no vocalization Movement of all limbs with or without vocalization Strong movements of all limbs and vocalization
Table 2: Cardiovascular variables and rectal temperature just prior to (time -10) and following administration of ketamine 15 mg kg , climazolam 1.5 mg kg and azaperone 1.0 mg kg to piglets either intramuscularly (IM) or intranasally (IN)

1,2,3 -1 -1 -1

. Time points in minutes relative to castration (time 0)

Group Parameter IM

240 (228; 252)

206 (174; 214) [19]

182 (154; 200)

189 (157; 200) [19]

165 (155; 193)*

178 (162; 198)

211 (192; 252)

235 (228; 258)

80 (70; 96)

99 (84; 132)***

90 (90; 102)*

82 (78; 100)

98 (90; 120)*

90 (78; 120)**

100 (70; 108)** [19]

75 (66; 80)

95 (93; 96) [18]
93 (88; 97) [18] 82 (70; 95) [19]
95 (87; 97) [17] 90 (81; 108) [19] ---
95 (91; 97) [19] 66 (60; 77) [19] ---

97.5 (95; 99) [14] ---

39.3 (39.0; 39.4)

36.2 (35.8; 36.9)*

36.5 (35.7; 37.8)**
HR 251 (234; 270) 196.5 (176; 206) RR 66 (54; 100) 72 (60; 80)*** 178.5 (162; 187) 72 (70; 100)* 181 (171; 191) [19] 84 (72; 96) [19] SpO2 --94 (85; 95) [19] SBP ----76 (67; 86) [18] Temp 39.3 (39.0; 39.4) ------91.5 (89; 95) 94 (92; 98) [17] 78 (65; 100) [15] --95.5 (92; 97) [16] 76 (51; 79) [16] --36.7 (35.9; 37.4)* 37.9 (37.5; 38.3)** --96.5 (93; 98) [14] --------76 (66; 100)* 72 (60; 80)** 71 (60; 72)** 180 (165; 208)* 190 (168; 206) 231 (189; 240) 240 (234; 270) [19] 74 (54; 90) [19]

Values are reported as medians with upper and lower 95% confidence intervals in parentheses
Observations: 20 piglets in the intramuscular administration group and 20 in the intranasal group unless indicated IM = intramuscular administration group; IN = intranasal administration group HR = heart rate (minute
differently by [brackets]
); RR = respiratory rate (minute ); SpO2 = hemoglobin oxygen saturation (%); SBP = systolic
blood pressure (mm Hg), Temp = rectal temperature (C) *Indicates significant difference between the medians of the IM and IN groups (P < 0.05) based on pairing *Indicates significant difference between the medians of the IM and IN groups (P < 0.01) based on pairing ***Indicates significant difference between the medians of the IM and IN groups (P < 0.001) based on pairing

21 Figure 1

Figure 1: Castration score of piglets anesthetized with ketamine, climazolam, and azaperone either intramuscularly (IM) or intranasally (IN). Castration scores were significantly higher (*P=0.007) in the IN group when compared to the IM group.

22 Figure 2

Figure 2: Castration score of the intranasal group (IN) (top panel) and intramuscular group (IM) (bottom panel) taking into account room temperature at the time of anaesthesia and castration. The IN group had a significantly better (*P=0.0006) castration score when the room temperature was greater than 18 C. There was no significant difference between temperature divisions in the IM group but there was less variability in the score (0.7 (0; 3.3) when the temperature was greater than 18 than C when it was less than 18 (5.5 (0.3; 9). Thirteen pairs were analyzed in the high C temperature group and 6 pairs in the low temperature group.