There is a critical need for safe and effective analgesic treatments to address pain resulting from surgical husbandry procedures in livestock. Piglet castration results in acute pain and stress to the animal; however, it is performed globally on millions of piglets annually, often without any analgesia what‐so‐ever. Tri‐Solfen® (Animal Ethics Pty Ltd, Yarra Glen, Victoria, Australia) is a combination local anaesthetic and antiseptic formulation which, applied topically to wounds, has proven effective, and is registered for use to alleviate pain associated with castration (and other wounds) in lambs and calves in Australia and New Zealand. It is also reported to be effective to reduce pain in piglets following castration.
This randomised, blinded, placebo‐controlled study examined the safety and efficacy of the formulation, administered via an adapted wound instillation method, to control pain both during and following piglet castration.
Piglets received Tri‐Solfen or placebo, instilled to the wound immediately following skin incision. A 30 s wait period was then observed prior to completing castration. Pain mitigation was assessed by grading nociceptive resistance movements and piglet vocal response during castration, as well as by grading response to mechanical sensory stimulation of the wound (von Frey and needlestick) following castration.
There was a significant reduction in nociceptive motor and vocal response during castration and in response to mechanical sensory wound stimulation up to and including 2 h following castration. There were no adverse events.
Administered via this method, Tri‐Solfen is effective to mitigate acute peri‐operative castration pain in piglets.
Sutherland et al16 reported that ‘lying without contact’ (considered a behavioural sign of pain in piglets) was reduced in Tri‐Solfen‐treated piglets, as compared with untreated piglets in the first 180 min following castration. However, there was no evidence of a significant impact on pain during the procedure itself, based on vocal response and cortisol levels. This finding is not unexpected as topical anaesthetics cannot be expected to mitigate procedural pain if applied only immediately prior to the procedure. Nevertheless, the question arises whether, if a longer dwell time is provided, Tri‐Solfen may be effective to mitigate the acute procedural pain that occurs when subsequently severing the cords. Applied topically to mucosal tissues, lidocaine has an onset time between 20 and 30 s23 suggesting that a relatively short increase in Tri‐Solfen dwell time may be effective to anaesthetise the cords. This could be highly beneficial, as applying traction and severing the cords are reported to be the most painful parts of the procedure based on motor and vocal responses.11, 13 This study thus examines whether, in the on‐farm setting, topical wound anaesthesia with Tri‐Solfen is effective to provide procedural as well as postoperative pain mitigation for piglets undergoing castration if a longer wait time (minimum 30 s) is implemented between applying the dose and severing the spermatic cords.
We hypothesised that topical wound anaesthesia (with Tri‐Solfen) applied with a 30 s dwell time would be effective to mitigate pain both during and following subsequent piglet castration. Our aims were to examine evidence of peri‐operative pain mitigation.
At present, there is no single validated parameter for measuring pain in piglets. It is generally accepted, however, that piglets react to pain in a number of ways including: physiologically, behaviourally and through resistance movements and vocalisation. An extensive literature review was performed to identify the optimal parameters for assessing pain in piglets during and following castration. This identified:
Based on these findings, nociceptive motor and vocal responses to castration were chosen as primary and secondary efficacy variables for assessment of pain mitigation during castration, and nociceptive response to wound sensory stimulation was chosen as a primary efficacy variable to assess pain mitigation following castration. A second efficacy variable for pain mitigation following the procedure (postoperative painrelated behaviour) has also been examined (and confirmed) in a large multi‐centre complementary field trial which is reported separately. (This was unable to be assessed in the same cohort of piglets undergoing wound sensory testing, as this involves frequent re‐handling, and hence disturbance of piglet behaviour.)
The study was conducted in December 2018 and complied with the following national and international standards: VICH GL9 Good Clinical Practice (issued June 2000); APVMA Data Guidelines – Efficacy and target animal safety general guidelines (Part 8, 01 July 2014), and in compliance with University of New England Animal Ethics Committee approval no. 18‐100.
The study was a placebo‐controlled clinical efficacy study, using a randomised blocked design based on body weights measured pretreatment. The experimental unit was the individual animal and the statistical animal was the treatment group. Male piglets (commercial hybrid) were confirmed suitable for enrolment into the study if they were between 3 and 7 days of age, in good health. These were individually identified (uniquely numbered via ear tags), weighed and ranked (heaviest to lightest) and the heaviest and lightest piglets were excluded. The remaining piglets (n = 40, mean weight 2.2 kg, range [1.6–3.0 kg]) were sequentially grouped into blocks of two animals and randomly allocated to Groups 1 and 2 from within each block, such that each of Groups 1 and 2 contained 20 male piglets and had similar group mean and range of body weights, (as assessed and confirmed using Statistix 10.0 [Analytical Software Inc., Tallahassee, FL, USA 2010]). On Day 0 after treatment, animals numbered 20, 26 and 41 were removed from the study due to herniation and subsequently replaced with animals numbered 61, 62 and 17, respectively. This re‐allocation was statistically assessed confirming that the group mean body weights were similar across treatment groups.
Tri‐Solfen Batch No.:181031–1. Composition: 50 g/L Lignocaine hydrochloride, 5 g/L Bupivacaine hydrochloride, 0.048 g/L Adrenaline (as acid tartrate), 5 g/L Cetrimide.
Blue food dye was added to 0.9% saline solution (Baxter, Batch: W47P5, Expiry: April 2020) at the rate of 2.5 to 250 mL saline (matching the investigational veterinary product colour and hence indistinguishable postapplication).
Investigational veterinary product and placebo products were administered via a custom designed 1 mL Tri‐Solfen applicator with ball‐point tip fitted (Prodigy Instruments Pty Ltd, Mount Kuring‐Gai, NSW, Aust), which was verified prior to use. Treatment administration (total dose) was: piglets 1–<2 kg – 1 mL; piglets >2–4 kg – 2 mL.
Study animals were housed in farrowing pens (2.1 m × 2.1 m) with their dam and litter mates, and routine management practices were followed. Piglets had constant suckling access to their dam and ad libitum access to potable water. On the day of the study, piglets were removed from their dams and castration and treatment was performed as detailed in the following. Video and sound recording were performed during piglet castration, and wound sensory testing was performed 1 min following the procedure. Following this, piglets were returned to their dams. Clinical observations of animals were performed concurrent with postoperative pain assessments at 1 min, thence 1, 2, 4, 8, 12 and 24 h posttreatment.
Method of castration and treatment:
A video‐camera recording device (iPhone 8 – Apple Inc., Cupertino, CA, USA) was fixed in position above the cradle, and used to record from time of application of the topical wound treatment to approximately 5 s following the severance of the second spermatic cord (i.e. completion of the castration procedure). Each piglet was clearly identified by placing a number label next to the piglet cradle within the camera view. The behavioural response to castration was assessed off‐line, by a blinded assessor, using an NRS intensity scale of 0 to 2 at each of four time points: (1) traction on first testis, (2) cutting first spermatic cord, (3) traction on second testis and (4) cutting second spermatic cord. The nociceptive motor response was graded as: 0 = no motor response, 1 = mild motor response, such as a short‐lived leg extension or kicking but no major body resistance movement in the cradle, 2 = marked motor response, such as prolonged leg movements and/or marked body resistance movement in the cradle. Assessment thus documented the handling of each teste (traction) and cutting of each spermatic cord, giving a score (0–2) for each event, and a combined total score (0–8) for the castration procedure.
Sound was recorded with a Zoom H2n Handy Recorder (Zoom North America, Hauppauge, NY, USA), mounted on a stand 50 cm from the snout. Audio recording was time and date stamped to correlate with video data. The recording period commenced 30 s following treatment application to the castration wound, which was marked with a verbal cue, and finished with the removal of the second testicle and settling of the piglet. Sound files were analysed off‐line by a sound consultant who was blinded to piglet treatment. For analysis, sound files were downloaded from the Zoom recorder and imported to Pro Tools® (Avid technology Inc., Burlington, MA, USA). They were then placed on the Pro Tools timeline at their time stamp position and synchronised with audio from the video recordings to allow precise identification of cutting points. To allow isolation and comparative quantification of vocal output of piglets during the procedure, the time of commencing traction, and time of cord severance were annotated from the time stamp (correlated with the video data). Screen shots were generated with the same time duration window (x axis) and signal scale (y axis). The Area Under the dB/time (waveform) Curve (AUC) was then calculated (in pixels) using image analysis software (Image‐J® U. S. National Institutes of Health, Bethesda, MD, USA), for each of the following periods: (1) from commencing traction until severance of the first spermatic cord, (2) from severance of the first spermatic cord until settled at the end of the procedure, and (3) total recorded procedure (Figure 1).
The response of each piglet to sensory testing of the wound site was assessed with a von Frey filament (300 g filament) and thence pin‐prick (18G, 1.5 inch needle) at 1 min, thence 1, 2, 4, 8, 12 and 24 h posttreatment, by trained staff, blind to piglet treatment. Each assessment was scored on a scale of 0–3, based on Lomax et al22: 0 = no motor response; 1 = there was a mild local motor response – including a local muscle twitch, flick of the tail or puckering of the anus; 2 = there was a partial lower body withdrawal response – including lifting the rump from the cradle and/or prominent movements (e.g. extension or escape movements) of the rear legs; 3 = there was a full body withdrawal response – including lifting the rump from the cradle and thoracic movements and/or vigorous movement of both the front and rear legs. Animals were restrained similarly to that used for the castration procedure in the piglet cradle for these assessments. Points of application of the von‐Frey or needle tip for each site included: Cut edge; both lateral aspects of castration wound – 2 sites. Intact skin: dorsal and ventral aspects of the castration wound ~3 mm from cut edge – 2 sites. This gave a maximum score of 12 for each test method (von Frey or needle) at each time point.
Raw data were entered into Microsoft EXCEL 2016 using double‐entry techniques. Summary tables were prepared in EXCEL while summary figures were prepared using EXCEL and TIBCO Spotfire S+ 8.2, 2010® (TIBCO software Inc. Palo Alto, CA, USA). Video (nociceptive motor response NRS scores) and Audio AUC data were compared using both parametric t‐tests and the equivalent nonparametric test (Wilcoxon rank‐sum tests); statistical comparisons were performed using S+®. Mechanical sensory response score data were collated by treatment, location (cut edge and intact skin), time point and method of stimulus (von Frey filaments and 18‐gauge needles). Total scores for von Frey, needle‐stick and overall were calculated. Scores to 4 h posttreatment were compared by treatment, method and over time using Repeated‐Measures Analysis of Variance and Statistix 10.0® (2013, Analytical Software, Tallahassee, FL, USA). Suitability of the statistical model was checked via sphericity assumptions, assumptions of covariance and residuals plots. Sphericity assumptions were met and residual plots were generally acceptable although assumptions of covariance were generally not met. Data were therefore also aggregated up to ‘response to stimuli/no response to stimuli’ based on total score criteria (>2, >3, >4 and >5) and the proportion of piglets responding to stimuli at each time calculated to 8 h posttreatment. Proportions were then compared using Chi‐Squared tests and Statistix 10.0.
Tri‐Solfen‐treated piglets demonstrated lower nociceptive motor response scores associated with traction on each teste and cutting of each spermatic cord (Table 1). The total motor response score and the responses to traction on testes (combined) and cutting of spermatic cords (combined) were significantly lower in Tri‐Solfen‐treated piglets (P = 0.000; P = 0.000; P = 0.004, respectively) than in placebo piglets (Figure 2).
|Group||Treatment||Traction onfirst testis (/2)||Cut first spermatic cord (/2)||Traction on 2nd testis (/2)||Cut second spermatic cord (/2)||Total Motor Response score (/8)|
Treatment with Tri‐Solfen resulted in a significant reduction in vocal response of piglets to castration as compared with placebo treatment as measured by AUC (mean ± SD of 5070 ± 5667 versus 12,109 ± 9270 pxls, respectively) during the first recorded time period (from commencing traction until severance of the first spermatic cord), (P = 0.007, Figure 3). Numerically lower mean AUC values were recorded in Tri‐Solfen‐treated piglets, throughout the remainder, or whole of the procedure; however, these differences were not statistically significant, due to wider variability (Figure 4). (A sample size of 20 per group predicted a significant [P < 0.05] reduction in phase 1 AUC with 83% power, versus 10% power for the whole of procedure AUC assessment.)
Group mean wound sensitivity scores by test and time point are presented in Table 2 and Figure 5. Placebo‐treated animals were more sensitive to both the von Frey filament and needle and at both the cut skin edge and intact skin adjacent to the wound, at 1 min following treatment through to and including 2 h posttreatment. These differences were statistically significant at 1 min and 1 h as shown in Table 3. At 2 h posttreatment, a greater proportion of placebo‐treated piglets had wound pain response scores greater than 4 (P = 0.04) or greater than 5 (P = 0.01) than Tri‐Solfen‐treated piglets. There were no significant differences between groups at 4–24 h following treatment.
|Site||Test||Group||Treatment||1 min||1 h||2 h||4 h||8 h||12 h||24 h|
|Cut edge||Von Frey||1||Placebo||0.41||0.48||0.85||1.05||0.85||0.45||1.05|
|Intact skin||Von Frey||1||Placebo||0.73||0.43||0.50||1.05||0.60||0.60||0.89|