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5.2.1.1 Animals

Twenty four mixed breed dogs undergoing routine ovariohysterectomy were recruited for the trial. The mean (±SE) body weight of the dogs was 19.3 (±4.7) kg, with a range from 4.5 to 34.5 kg. Age of the dogs ranged from six months to six years with a mean (±SE) of 1.9 ± 0.6 years. All the dogs were clinically normal.

5.2.1.2 Anaesthesia and analgesia

After routine physical examination, each dog was assessed for the presence of pain, using the Glasgow CMPS-SF. The dog was then randomly allocated to one of the three treatment groups described below.

5.2.1.2.1 Pre-anaesthetic medication

Dogs were given pre-anaesthetic medication with acepromazine 0.05 mg kg-1 and atropine 0.04 mg kg -1 SC, 30-45 minutes before anaesthetic induction. This is our VTH standard pre- anaesthetic medication of dogs.

5.2.1.2.2 Analgesia

Dogs were randomly allocated to one of the three treatment groups (eight dogs per treatment group). Test drugs were given at the same time as the pre-anaesthetic medication. Dogs in control group received morphine0.5 mg kg-1 SC, those in tramadol group received tramadol 3 mg kg-1 SC and dogs in low-dose morphine group administered low-dose morphine 0.1 mg kg-1 SC. Dogs in this group were given tramadol 3 mg kg-1 IV at extubation postoperatively. Each dogs’ sedation was assessed using the VAS just prior to induction of anaesthesia (figure 14).

0 100 No sedation fast asleep

Figure 14: The visual analogue scale used for assessing dogs’ sedation in the perioperative period

In this system, the level of sedation was assessed by observing dog’s posture, mental alertness, and its ability to stand and walk. At each assessment, a mark was made on a 100 mm scale, on which 0 corresponds to ‘no sedation’ and 100 corresponds to ‘fast asleep’ (Lascelles et al. 1994). The distance from 0 to the marked point on the scale was later converted to numerical form for statistical analysis.

5.2.1.2.3 Anaesthesia

Anaesthesia was induced with IV thiopentone sodium (7.1±0.4 mg kg-1 mean±SE) to effect and maintained with halothane delivered in 100% oxygen via a circle system. The

concentration of halothane was adjusted to keep the dog at a suitable plane of surgical anaesthesia. As soon as the dog was anaesthetised and its airway, breathing and circulation had been checked, a pulse oximeter and Doppler transducer with cuff was attached so that SpO2 of the haemoglobin and blood pressure were monitored non-invasively. Dogs were given Hartmann’s solution IV to maintain systolic arterial blood pressure above 100 mm Hg throughout the anaesthetic period. Intra-operatively, fentanyl (fentanyl injection; 500 g in 100 mL; Mayne Pharma Pty Ltd, Victoria, Australia)1 g kg -1 IV was given by the

anaesthetist as needed, to control any tachycardia and tachypnoea which occurred in response to surgery. Respiratory rate, end-tidal halothane tension (ET HAL)and end–tidal CO2 tension

(ETCO2) were also monitored. All these parameters, and the signs of depth of anaesthesia,

were recorded every five minutes until the end of surgery. 5.2.1.3 EEG and electrocardiogram (ECG) recording

The EEG was recorded in a three electrode montage as described in chapter 3. The EEG recording (figure 15) was started as soon as the dog was stabilised under halothane

anaesthesia (SA). During ovariohysterectomy, EEG data from 100-second blocks were taken at each surgical time point (figure 16). The 100-second blocks immediately preceding the skin incision were taken as the baseline (BL) period and during the skin incision were taken as skin incision period (SI). Data from 100-second blocks following clamping through ligation of the suspensory ligament of each ovary were used for ovary 1(O1) and ovary 2 (O2). The 100-second block data following clamping through ligation of body of uterus were described as the uterine body (UB). The 100-second block data during skin closure was taken skin closure (SC). EEG recording was stopped at the end of anaesthesia (EA).

Figure 15: Position of the EEG electrodes on a dog undergoing ovariohysterectomy

For castration, the 100-second blocks immediately preceding the skin incision were taken as the baseline period. Data from 100-second blocks following clamping through ligation of the spermatic cord of each testicle were taken as testicle 1 and testicle 2. Data were averaged over the 100-second blocks at each surgical variable for statistical comparison.

BL EEG SI EEG O1 EEG O2 EEG UB EEG SC EEG

EEGamplifier Non-inverting electrode

Inverting electrode Ground electrode

SA SI O1 O2 UB SC EA

Figure 16: Diagram of the EEG recording pattern for different surgical time points in dogs undergoing ovariohysterectomy

SA = stabilisation of anaesthesia; BL EEG = baseline EEG; SI EEG = EEG recorded during skin incision; O1EEG = EEG recorded during ligation of ovary 1; O2 EEG = EEG recored during ovary 2 ligation; UB EEG = EEG recorded during ligation of body of uterus; SC EEG = EEG recorded during skin closure; EA = end of anaesthesia

The raw EEG (figure 17) was analysed using spectral analysis programme as described in chapter 3. The derived spectral variables (F50, Ptot and SEF) were used for statistical comparison.

Figure 17: Raw EEG recorded in an anaesthetised dog undergoing surgery

The ECG was recorded continuously in a lead II electrode configuration, using an Apple Macintosh personal computer installed with Chart 5.2.2 recording software and connected to Power lab 4/20 data recording system (PowerlabTM data acquisition system®, AD Instruments Ltd, Sydney, Australia). The ECG was recorded at a rate of 1 kHz. The recorded data were analysed off-line using the rate meter function of the Chart 5.2.2 to obtain heart rate. 5.2.1.4 Surgery

Final year veterinary students under the supervision of a veterinary anaesthetist and surgeon performed the ovariohysterectomy with a routine ventral midline approach. The mean±SE surgery time was 87.9±6.7 minutes.

5.2.1.5 Postoperative pain assessment

Before recovery from anaesthesia dogs were moved to a cage in the recovery ward and the endotracheal tube was removed when their laryngeal reflexes were restored. Each dog’s pain was assessed by the same person using the Glasgow CMPS-SF, at 1, 3, 6 and 9 hours

postoperatively. Sedation was assessed at the same postoperative time points.

If a dog appeared to be in an unacceptable pain (CMPS-SF score 6 out of 24) rescue analgesia was given with morphine 0.3 mg kg-1 intramuscularly and fentanyl 1 µg kg-1 IV. The dogs were also observed for side effects, if any, of the administered analgesics.