Introduction
Studies1,2,3,4,5,6,7 of laparoscopic procedures in dogs and cats have demonstrated decreases in surgical stress and pain relative to open laparotomy on the basis of pain scores, physiologic variables (eg, heart rate, respiratory rate, and blood glucose and plasma or serum glucocorticoids concentrations), and behavioral variables (eg, posture, response to palpation, and activity level). Dogs undergoing gastropexy in a randomized controlled trial3 had significantly lower plasma C-reactive protein and cortisol concentrations when a laparoscopic versus open surgical approach was used, suggesting a difference between approaches in the amounts of postoperative inflammation and distress. In another trial,8 dogs undergoing laparoscopic versus open splenectomy had lower serum C-reactive protein concentrations and pain scores. Other studies4,6,9 have shown that pain scores assigned within 24 hours after surgery for cats undergoing LapOVE and dogs undergoing laparoscopic ovariohysterectomy are lower than those for animals undergoing open surgery; however, observers in those studies were not always blinded to surgical approach when assigning scores. Postoperative activity as measured by accelerometry has also been reported to be higher in dogs following LapOVE versus OVE.10 In addition to causing less pain or stress in dogs, laparoscopic and laparoscopic-assisted procedures result in shorter hospitalization times and fewer postoperative complications than do open surgical procedures.11,12,13,14
Laparoscopic surgical techniques have been described for rabbits (Oryctolagus cuniculus).15,16,17,18,19,20,21 Nevertheless, the advantages or disadvantages over conventional open surgical approaches have not been well explored.22,23 As a prey species, rabbits have cryptic pain behaviors.24,25 Facial grimace scales and ethograms have been developed to evaluate pain or discomfort in rabbits.26,27,28 Rabbits can also develop gastrointestinal stasis from stress or pain,29 so laparoscopic approaches may be beneficial if they could reduce the amount of pain and morbidity following surgical procedures. Furthermore, the unique anatomic and behavioral characteristics of rabbits may influence the response to pneumoperitoneum and surgical technique.
Potential advantages of laparoscopic surgery for rabbits have been described17 but not formally investigated. The purpose of the study reported here was to determine the effects of LapOVE versus OVE on several intraoperative and postoperative variables, including behaviors associated with pain expression, in rabbits. We hypothesized that LapOVE would result in less postoperative morbidity and, hence, fewer postoperative physiologic and behavioral manifestations of pain or discomfort.
Materials and Methods
Animals
Two trials were conducted: a preliminary uncontrolled trial and then a randomized controlled trial. In the preliminary trial, six 5-month-old sexually intact female New Zealand White rabbitsa were used to determine the most efficient surgical technique for LapOVE. For the randomized controlled trial, 12 specific pathogen–free 4− to 5-month-old sexually intact female New Zealand White rabbits weighing between 2.8 and 3.3 kg were used. An animal use protocol for both trials was reviewed and approved by the Animal Care Committee of the University of Guelph in accordance with guidelines set by the Canadian Council on Animal Care.
Housing
All rabbits were individually housed in a room with floor pens and allowed to acclimate to their environment for 7 days prior to the study period. The housing setup allowed them to see and hear neighboring rabbits; however, physical barriers prevented mixing of fecal pellets and food. A 12-hour light cycle was provided. Rabbits were fed a commercial pelleted diet and water from bottles. Litter boxes with pine shavings and enrichment items (1 clear plastic hide box and toys) were provided. Items were arranged in an identical orientation within the pen for each rabbit.
Preliminary trial
For the preliminary trial, food was withheld from the 6 rabbits for 1 hour and then hydromorphone hydrochlorideb (0.2 mg/kg) and midazolam hydrochlorideb (1 mg/kg) were administered IM. A lateral auricular vein was catheterized with a 22-gauge catheterc for IV administration of crystalloid fluidd at a rate of 10 mL/kg/h. Propofole (8 mg/kg, IV, with topping up to effect) was administered to induce anesthesia. Endotracheal intubation was performed with a 4-mm uncuffed endotracheal tube, and anesthesia was maintained with 2% to 3.5% isofluranef with oxygen delivered at a rate of 1.5 L of oxygen/min via a mechanical volume-cycled ventilator in a circle circuit.
A 3-port LapOVE was performed in each rabbit, as described elsewhere.18 Different port positions were tested for placement of the endoscope, grasping forceps, and VSD.g Cefazolinh (20 mg/kg, IV) was administered before surgery, and flumazenilb (0.012 mg/kg, IV and 0.012 mg/kg, SC) was provided after surgery. Meloxicami (1 mg/kg, IV) was administered on recovery from anesthesia and then daily (1 mg/kg,j PO) for 3 days after surgery unless additional analgesics were required (as described for the second trial).
Randomized controlled trial
Design—For the randomized controlled trial, statistical softwarek was used to randomly assign 12 rabbits to undergo OVE or LapOVE (n = 6/group). Rabbits in both surgical groups were housed in pens equidistant from the door for the purpose of behavior monitoring. Surgery was performed on 1 rabbit from each group each day, and pre− and postoperative variables were measured at the same time of day.
Anesthesia—Anesthesia was performed as described for the preliminary trial. Food was withheld from rabbits for 30 to 90 minutes prior to premedication. A physical examination was performed, and a blood sample was obtained for measurement of PCV and plasma total solids concentration (as measured via refractometry). Anesthesia was induced with propofol 25 to 45 minutes after premedication was performed, then maintained with isoflurane. Tidal volume was maintained at 15 to 20 mL/kg, with a maximum ventilatory pressure of 15 mm Hg and rate of 14 breaths/min. During surgery, all rabbits were monitored by use of an esophageal thermometer, ECG, capnography, and pulse oximetry.
On recovery from anesthesia, flumazenil (0.025 mg/kg, IV and 0.025 mg/kg, SC, simultaneously) was administered to partially reverse the sedative effects of midazolam so that rabbits could be returned to the animal facility with less need for supervision. After surgery, rabbits received meloxicam (1 mg/kg, IV initially, then 1 mg/kg, PO, once daily for 3 days) and buprenorphine hydrochloridel (0.05 mg/kg, SC, 4 to 5 hours after premedication).
Surgical approach—Surgeries were performed by a board-certified veterinary surgeon with Founding Fellow status in minimally invasive surgery and considerable experience with laparoscopic surgery in other species of companion animals (AS) and a zoological companion animal resident (CK). The roles of primary and secondary surgeon were randomly assigned for any given surgical day.
Each rabbit was positioned in dorsal recumbency, and the abdomen was clipped of hair and aseptically prepared. Laparoscopic ovariectomy was performed with 3 ports as supported by results of the preliminary trial (Figure 1). A skin incision was made 1 cm caudal to the umbilicus by use of a No. 15 scalpel blade. Two stay sutures were placed on either side of the linea alba with 4-0 polydioxanone.m Using a modified Hasson technique, the primary surgeon made an initial incision with a No. 15 blade and inserted a 3.9-mm-diameter threaded graphite cannulan into the abdomen. Carbon dioxide insufflation was applied to a pressure of 8 mm Hg, which was the optimal pneumoperitoneal pressure for rabbits in previous studies.30,31 A 2.7-mm-diameter 30° rigid endoscopen with a 3.5-mm examination sheathn was used to visualize cannula placement. A second incision was made along the midline approximately 3 cm cranial to the first port. A 3.5-mm-diameter threaded graphite cannulan with a pyramidal-tip metal trocar was inserted for positioning of fenestrated grasping forcepsn (Figure 2). A final incision was made approximately 3 cm caudal to the first port along the midline. A 6-mm-diameter plastic cannulao with a smooth trocar was placed for passage of the VSD.
Illustration (A) and photograph (B) showing the placement of ports for performance of LapOVE in rabbits. A—One port, consisting of a 3.9-mm-diameter subumbilical cannula, was placed for passage of the 2.7-mm-diameter 30° sheathed rigid endoscope (b); the second port, a 3.5-mm-diameter cannula, was placed for passage of the grasping forceps (a); and the third port, a 6-mm-diameter plastic cannula, was placed for passage of the VSD (c).
Citation: American Journal of Veterinary Research 82, 3; 10.2460/ajvr.82.3.237
Laparoscopic images showing performance of LapOVE in a rabbit by use of the 3-port approach described in Figure 1. Using laparoscopic visualization from the subumbilical port, the primary surgeon placed a 3.5-mm-diameter graphite cannula (A) and 6-mm-diameter plastic cannula (B) cranially and caudally, respectively, along the midline. The left ovary was visualized (C) and lifted with the grasping forceps, and the ovarian pedicle was sealed and transected with the VSD inserted through the caudal port (D). The right ovary was similarly visualized from its position laterodorsal to the cecum (E) and again was grasped (F) prior to sealing and transection of the ovarian pedicle.
Citation: American Journal of Veterinary Research 82, 3; 10.2460/ajvr.82.3.237
The rabbit's body was tilted 45° to the right to access the left ovary (Figure 2). Standing on the right side of the rabbit, the secondary surgeon controlled the laparoscope while the primary surgeon manipulated the grasping forceps and VSD to seal and transect the ovarian pedicle and oviduct. The shaft of the VSD was retracted up the cannula, and the ovary was gently removed through the 5-mm caudal incision. The procedure was repeated for the right ovary with the surgeons standing on the left and a second monitor placed across from the primary monitor. The right ovary was more difficult to access owing to its position under the cecum. After both ovaries had been removed, the cannulas were removed and manual compression was applied to release the insufflated gas. Closure of the abdominal fascia was performed with 4-0 polydioxanone in a simple interrupted pattern, and the skin was apposed with 4-0 poliglecaprone 25p in a continuous intradermal pattern.
Open ovariectomy was performed by use of a No. 15 scalpel blade and midline skin incision that extended caudally from the umbilicus. Metzenbaum scissors were used to open the abdomen over the linea alba. The left ovary was grasped with hemostatic forceps, and the ovarian pedicle and oviduct were sealed and transected with the VSD. The same procedure was repeated on the right side. Closure was performed as described for LapOVE.
Surgery time for each surgical approach was measured in minutes from the point the first incision began to the point closure was completed. Anesthesia time was measured from intubation to extubation. Incision lengths were measured after sutures were placed.
Postoperative observations
To monitor food ingestion by rabbits, food pellets were weighed twice daily (morning and night) with a gram scale the day before surgery and for 7 days after surgery. At the same time points, fecal pellets were counted and weighed. Rabbits were weighed once daily and physiologic variables (heart rate, respiratory rate, and rectal temperature) were measured twice daily, starting the day before surgery and ending 7 days after surgery.
Blood samples were collected from a lateral saphenous vein in the morning, starting before surgery and ending 3 days after surgery. Blood glucose concentration was measured with a hand-held glucometer.q Serum was harvested from the remaining portion of the blood sample within 1 hour after collection and was frozen at −20°C pending submission to the Animal Health Laboratory, University of Guelph, for measurement of cortisol concentration with a chemiluminescence assay.
Abdominal palpation was performed twice daily for 7 days after surgery. Muscle tension or decreased cecal fill, suggestive of discomfort or decreased food consumption, was evaluated, and the palpation findings were categorized as normal or abnormal. Incision sites were examined twice daily for signs of infection, dehiscence, or swelling.
For behavior monitoring, rabbits were video recordedr for 45 to 60 minutes twice daily (7 am and 6 pm) starting the day before surgery and continuing for 3 days after surgery. No people were allowed access to the room during filming. The first 10 minutes of video footage were excluded to allow the rabbits’ behaviors to settle. One observer (SD), who was unaware of surgical group assignment and was trained to recognize and score behaviors consistently, viewed the next 30 minutes of footage. Surgical incisions could not be identified from the recordings. Behaviors were measured as duration of time displayed or number of occurrences (Appendix). These behaviors were part of an ethogram previously developed to assess postoperative pain in rabbits.32,33 Similar behaviors were combined into composite groups on the basis of previously established associations32 to increase statistical power. For behaviors measured in numbers, morning and evening observations were combined to remove variation associated with time of day.
Facial grimace scale scoring26,34 was performed at the start and end of every video. As described elsewhere,34 scores were assigned on a scale of 0 (not present) to 2 (obviously present) for 5 facial action units, and these scores were summed to generate the FGS score (maximum possible score, 10). Assigned FGS scores for morning and evening video sequences were averaged to obtain a daily score.
Statistical analysis
Data were analyzed by use of freely available statistical software.k Figures were created with a data visualization package.35
Primary surgeon assignment and surgical approach (LapOVE or OVE) were simultaneously assessed for an influence on surgery time, anesthesia time, and incision length by use of multiple linear regression. Mixed-effects linear regression was used to compare continuous postoperative variables, including behaviors measured in duration, between surgical approaches. Behaviors measured in numbers (ie, count data) were compared between surgical approaches by means of Poisson regression; if overdispersion was identified, negative binomial regression was applied. Individual rabbit was included in the linear, Poisson, and negative binomial mixed models as a random effect. Surgical approach, time of day (morning vs evening), time from first video-recorded assessment, and the interval relative to surgery (before vs after) were included as fixed effects when they improved the fit of the model and were significantly associated with the outcome variables. If variables were identified as being strongly correlated with each other as shown by the Fisher exact test, Pearson correlation, or ANOVA, the variable with the lesser association with the outcome was removed from the model. Analysis of variance was used to determine P values for each predictor variable, and Tukey post hoc adjustment was applied.
Differences within and between surgical groups in abdominal palpation findings (normal vs abnormal) were assessed by use of mixed-effects logistic regression, with individual rabbit as a random effect and surgical approach and postoperative day as fixed effects. Differences within and between surgical groups in mean daily FGS scores were assessed by means of mixed-effects ordinal logistic regression, with rabbit as a random effect and surgical approach and time relative to surgery as fixed effects.
For all regression models, applicable assumptions were evaluated and outliers were removed when appropriate. Interactions between predictor variables were assessed for significance in models. Effects were considered significant if the P value was < 0.05.
Results
Rabbits
All 6 rabbits completed the preliminary uncontrolled trial to ensure that LapOVE could be safely and reproducibly performed by the surgical team with a 3-port technique. During the randomized controlled trial, a loop of small intestine was inadvertently grasped with a stay suture in 1 rabbit in the LapOVE group before the surgeon entered the abdomen, and the small intestine was perforated during subsequent placement of the initial optical cannula. This rabbit was euthanized in accordance with the animal use protocol after the abdomen was explored to ensure no anatomic variation or other abnormalities were present. Consequently, 5 rabbits in the LapOVE group and 6 in the OVE group were included in statistical analyses for all variables.
Intraoperative variables
Surgery time was significantly (P = 0.001) greater for LapOVE (n = 5) than for OVE (6; Table 1). Primary surgeon assignment had no significant (P = 0.21) association with this variable. Surgery time for LapOVE was further broken down into the times required for port placement (mean ± SD, 10.4 ± 4.3 minutes), ovaries removal (14.2 ± 2.9 minutes), and incision closure (16.4 ± 3.0 minutes). Anesthesia time was also significantly (P = 0.007) longer for LapOVE than for OVE, with no significant (P = 0.42) difference between primary surgeons. Incision length was significantly (P = 0.001) longer in the OVE group than in the LapOVE group but had no significant (P = 0.78) association with primary surgeon assignment. Incisions in the LapOVE group had mean ± SD lengths of 4.8 ± 0.4 mm for the cranial incision, 12.2 ± 4.0 mm for the middle incision, and 7.0 ± 2.0 mm for the caudal incision.
Summary statistics for intraoperative variables for rabbits undergoing LapOVE (n = 5) or OVE (6).
| LapOVE | OVE | |||
|---|---|---|---|---|
| Variable | Mean ± SD | 95% CI | Mean ± SD | 95% CI |
| Surgery time (min) | 43.2 ± 9.5* | 34.9–51.5 | 21.7 ± 5.8 | 17.0–26.3 |
| Anesthesia time (min) | 76.2 ± 14.7* | 63.4–89.0 | 48.8 ± 10.5 | 40.4–57.2 |
| Incision length (mm) | 24.0 ± 3.5* | 20.9–27.1 | 41.5 ± 10.6 | 33.0–50.0 |
Value differs significantly (P < 0.007) from the OVE value for this variable.
Postoperative variables
No significant differences were identified between surgical approaches in objectively measured postoperative variables, including weight of food consumed (P = 0.25), number and weight of feces produced (P ≥ 0.84), change in body weight (P = 0.31), heart rate (P = 0.26), respiratory rate (P = 0.65), rectal temperature (P = 0.86), and blood glucose concentration (P = 0.63). A decrease in respiratory rate was observed in rabbits of both surgical groups the evening after surgery (all P < 0.05; decrease of 39 to 67 breaths/min), compared with respiratory rates on the other days.
Time of day (morning or evening) and time in the study from first evaluation had a significant influence on values of postoperative variables. Except for the first 48 hours after surgery when feces production was consistently low relative to before surgery, an interaction effect was evident such that feces production was lower during the day than at night (P ≤ 0.003; Figure 3). A similar pattern was observed for food consumption (P < 0.001). Blood glucose concentrations increased slightly over the 4 days, except for the morning after surgery (P < 0.001; Supplementary Figure S1, available at: avmajournals.avma.org/doi/suppl/10.2460/ajvr.82.3.237). Blood cortisol concentrations were all < 28 nmol/L (approx 1 μg/dL, the lower quantitation limit of the laboratory assay) except for a single time point in 1 rabbit. Therefore, no difference between surgical groups could be assessed statistically.
Mean feces production (A) and food consumption (B) the day before (0 hours) and for 7 days after rabbits underwent LapOVE (circles; n = 5) or OVE (triangles; 6; time of surgery indicated by vertical dashed line). Feces production and food consumption were lower during the day than at night. However, during the first 48 hours after surgery, consistently lower feces production and food consumption relative to presurgical values were observed in both surgical groups. Error bars represent SD.
Citation: American Journal of Veterinary Research 82, 3; 10.2460/ajvr.82.3.237
Of all abdominal palpation results, the proportion of normal results did not differ significantly (P = 0.12) between LapOVE (49/74 [66%]) and OVE (44/89 [49%]; OR = 2.52; 95% CI, 0.63 to 10.75). The odds of a normal (vs abnormal) palpation result increased by 48% (OR, 1.48; 95% CI, 1.25 to 1.79; P < 0.001) for every additional day after surgery (Figure 4).
Number of abdominal palpation results for the rabbits of Figure 1 that were normal (black bars) or abnormal (gray bars) by day relative to surgery (on day 0). The odds of a normal palpation result increased by 48% (OR, 1.48; 95% CI, 1.25 to 1.79) for every additional day after surgery.
Citation: American Journal of Veterinary Research 82, 3; 10.2460/ajvr.82.3.237
Postoperative complications
Complications after LapOVE included subcutaneous emphysema in one rabbit that resolved without treatment 4 days after surgery and mild edema or seroma around 1 incision for 2 days in another rabbit. With both trials considered, superficial incisional dehiscence and minor opening was noted in 2 of the 11 surviving rabbits that had undergone LapOVE (1 from the preliminary trial and 1 from the randomized controlled trial) and 2 of the 6 rabbits that had undergone OVE. These lesions were treated with a combination of topical cleaning, topical 1% silver sulfadiazine ointmentt application (2/4 rabbits), Elizabethan collar placement (3/4 rabbits), enrofloxacinu (10 mg/kg, q 12 h for 10 days; 1/4 rabbits), and skin-staple placement to reappose skin edges (2/4 rabbits). Additional meloxicam treatment was provided for the 3 rabbits that required Elizabethan collars.
Behavioral variables
No significant (P > 0.30) differences were identified between the 2 surgical approaches in the time and frequency with which any of the monitored behaviors were observed. Behaviors observed slightly (but not significantly) more often in one group versus the other included frequency and duration of abdominal pressing (increased in the LapOVE group; P ≥ 0.07), frequency of inactive pain composite behaviors (increased in the LapOVE group; P = 0.07), time spent eating (increased in the OVE group; P = 0.09), and frequency of yawning incidents (increased in the LapOVE group; P = 0.15).
Time variables were significantly associated with the duration or frequency with which behaviors were observed (Supplementary Figures S2–S7; Supplementary Table S1, available at: avmajournals.avma.org/doi/suppl/10.2460/ajvr.82.3.237). Rabbits spent more time exploring and displaying the composite movement behaviors (hopping and shuffling) in the morning, and alternatively were recumbent for longer in the evening. The following behaviors were observed less frequently in the 24 to 48 hours following surgery than prior to surgery: interacting, hopping, partial hopping, rearing, stretching, and alerting. The composite inactive pain behaviors were seen more frequently for 24 hours following surgery than at other times. Times spent drinking and displaying the composite movements were lower after (vs before) surgery, whereas times spent huddling and with eyes closed were greater after surgery.
No digging or stomping behaviors were observed for any rabbit. Twitching was observed in only 2 rabbits; consequently, this variable was not analyzed on its own but was included in analyses concerning the composite inactive pain behaviors.
For FGS scoring, the facial action unit whisker position could not be determined by the observer from most of the video recordings and the recordings in which it could be determined were scored as 0. Therefore, whisker position was excluded from the FGS score. Daily FGS scores were not significantly (P = 0.83) associated with surgical approach (OR, 0.89; 95% CI, 0.30 to 2.64). The odds of the mean FGS score increasing by 0.25 points were 17.81 times as great after (vs before) surgery (95% CI, 4.18 to 75.84; P < 0.001; Figure 5).
Box-and-whisker plots comparing mean daily FGS score before and after surgery between rabbits that underwent LapOVE (n = 5) or OVE (6). Central horizontal lines within boxes represent the median, boxes represent the interquartile (25th to 75th percentile) range, and whiskers represent the range. Although these scores did not differ between surgical groups (P = 0.83), they were significantly (P < 0.001) lower before versus after surgery.
Citation: American Journal of Veterinary Research 82, 3; 10.2460/ajvr.82.3.237
Discussion
To the authors’ knowledge, the present study was the first in which intraoperative and short-term postoperative variables, including postoperative pain expression, were compared between rabbits undergoing LapOVE and those undergoing OVE. The only significant differences between surgical approaches were with respect to surgery time, anesthesia time, and incision length. This is in contrast to other evaluations of laparoscopic versus open surgery that revealed improvements in postoperative activity in dogs10; less pain expression and morbidity in dogs and cats2,3,4,6,8,9; lower incidence of infection or adhesions in dogs, cats, and rabbits14,22,36,37,38; and shorter hospitalization times in dogs.11,12
The lack of differences in the present study may have been attributable to any of several factors. Pain expression in rabbits is poorly understood, and changes in behavior and FGS scores can be subtle. As a result, type II error may have existed, and a larger sample size may improve the ability to detect differences if they truly exist. On the other hand, any real difference in the amount of postoperative pain between LapOVE and OVE may be small, in which case the clinical relevance of such a difference may be negligible. This needs to be verified separately for more complex surgical procedures that benefit from less tissue handling and magnification when performed laparoscopically.
In the authors’ experience, ovariectomy is a simple procedure that requires less training and minimal patient handling even when performed with a conventional open approach. Ovariectomy was selected for the present study because it is commonly performed in rabbits up to 6 months of age to help minimize the risk of future uterine abnormalities and adenocarcinoma.39,40 Little difference was noted in postoperative variables when the resident with less experience, as opposed to the board-certified surgeon, was assigned to be primary surgeon.
The shorter anesthesia time for OVE versus LapOVE in the present study would benefit rabbits, which have a greater potential for complications than do other companion animals.41,42 Shortening the anesthetic period could be expected to decrease the risk of complications, especially for older patients or those with cardiovascular instability.
In the study reported here, OVE in rabbits took on average half the time required for LapOVE; however, surgeon efficiency during laparoscopic surgery can improve with experience performing LapOVE in rabbits.18,43,44 Approximately 24% of the LapOVE time (mean, 10.4 minutes of the total 43.2 minutes) involved placement of the 3 ports. Single-port and 2-port entry approaches, which may provide quicker access than the 3-port approach, have been described for rabbits and other companion animals.15,20,45 Port placement should be a focus of training for veterinarians who are new to laparoscopic surgery. Additionally, approximately 38% (mean, 16.4 minutes) of the surgery time for LapOVE was spent closing the incisions. Modification of the suture patterns or use of tissue adhesives could allow for shorter surgery times. Moreover, we speculate that given the size of most companion rabbit breeds, the use of a smaller VSD would allow shorter incisions and easier closure. Thus, surgery time could potentially become more comparable between LapOVE and OVE with slight modifications to the approach and closure.
To objectively assess postoperative pain in the rabbits of the present study, we used body function variables (ie, food consumption and feces production), physiologic variables (ie, vital parameters and blood glucose and serum cortisol concentrations), and behavioral variables. No differences between surgical approaches were identified for any of these variables. This may have been due to provision of appropriate postoperative analgesia, although the analgesic protocol used was fairly conservative: 12 hours of opioid analgesia and 3 days of NSAID treatment. Furthermore, if analgesics had been sufficient to mask the differences in pain between rabbits in the 2 surgical groups, we should not have seen a difference in variables measured immediately after surgery versus before surgery or several days later. A decrease was observed in food consumption, feces production, and glucose measurements during the first 48 hours after surgery. Although these differences may have been due to postoperative pain, they may also have reflected changes in gastrointestinal motility as a result of anesthesia or analgesia. For future research of this type, inclusion of a nonsurgical treatment group should be considered to allow assessment of the effects of anesthesia alone.46,47 Medical intervention (eg, promotility agents, additional analgesics, and supportive feeding) was not required, and food consumption and feces production returned to presurgical levels by 2 days after surgery.
In a previous study,27 plasma cortisol concentration was measured to assess stimulation of the hypothalamic-pituitary-adrenal axis in rabbits in response to pain. Lower plasma cortisol concentrations as well as plasma epinephrine and tumor necrosis factor-α concentrations were identified in rabbits within 24 hours after laparoscopic ovariohysterectomy versus open surgery in another study.23 This may indicate a lower amount of surgical stress with the laparoscopic approach; however, evaluation was not performed beyond 24 hours after surgery. Although plasma cortisol concentration could not be appropriately measured in the present study, all rabbits consistently had a concentration < 28 nmol/L (approx 1 μg/dL). Observed values were lower than plasma cortisol concentrations reported for rabbits 48 hours after ovariohysterectomy in another study,27 suggesting that neither surgical group in our study had substantial postoperative stress.
Release of corticosterone and cortisol into circulation is known to respond to circadian rhythms in rabbits, with peak concentrations occurring in the afternoons and nadirs in the early morning hours.48 Higher plasma cortisol concentrations may have been observed in the present study had the blood samples been collected in the afternoon. An alternative approach for assessing postoperative stress responses would be to measure fecal or urine cortisol concentrations. Such concentrations could be presumed to be influenced less by daily fluctuations and stress induced by handling of rabbits.
Similarly, blood glucose concentration can change rapidly in response to stress, and samples collected during manual restraint may not be reflective of glucose concentrations at rest. Postoperative blood glucose concentrations in the present study were higher than before surgery, apart from the morning after surgery. The initial decrease may have been due to rabbits having an appetite reduction over the previous 12 to 24 hours; that decrease did not differ significantly between surgical groups. Subsequent increases in blood glucose concentration may have indicated postoperative pain or anticipatory stress for rabbits handled for their third and fourth venipuncture. Use of indwelling catheters for blood sample collection should be considered for future studies to minimize rabbit handling. Despite the limitations of using blood analyte concentrations for assessment of pain, glucose and glucocorticoids may still be valuable objective analytes for measurement of metabolic and systemic stress after surgery.
Abdominal palpation findings were used as a subjective assessment of postoperative well-being in the rabbits of the present study. Rabbits with abnormal palpation findings were presumed to be uncomfortable, having an altered appetite, or experiencing changes in intestinal motility. Although no significant difference was identified between surgical groups, more rabbits in the OVE group had abnormal palpation findings than did those in the LapOVE group. Nevertheless, it must be emphasized that the evaluator was not blinded to surgical group assignment when assessing this particular variable. Open ovariectomy resulted in longer incisions and more tissue manipulation during surgery, possibly contributing to a decrease in gastrointestinal motility and increase in postoperative pain. Abdominal palpation abnormalities were transient and returned to presurgical findings during the follow-up period, with no additional intervention required.
Facial grimace scales and ethograms can be used to assess postoperative pain in rabbits and other species. The grimace scale has been validated in rats, and performance of biopsies by means of laparoscopy (vs laparotomy) has been associated with lower postoperative scores.49 Facial grimace scale scores are particularly useful in a clinical setting; however, important facial action units may be missed from a distance or on video monitors. Ethograms and FGS scores are also susceptible to observer bias and may be influenced by use of sedatives or analgesics. Behaviors need to be clearly defined and assessed consistently by blinded observers. When assigning FGS scores, it is important that rabbits be undisturbed by people, the environment remain unchanged, each pen be similar in layout, and rabbits be consistently assessed at the same time of day.27,32 Time of day significantly affects the frequency and duration with which some behaviors are observed.32 In the present study, the rabbits were acclimated to 12-hour light cycles and had less feces production and food consumption during the day versus night. They also displayed more exploratory behavior in the morning and more time being recumbent in the evening.
In previous studies27,28,32,33 on postoperative pain assessment, rabbits were less active and had decreased rearing, hopping, and jumping after surgery; spent more time sitting, huddling, abdominal pressing, and grooming the abdomen or flank; and had a greater incidence of wincing, flinching, and shuffling or truncated gaits. Duration of movements (eg, hopping and shuffling) and frequency of inactive pain behaviors (eg, twitching, drawing back, staggering, full-body flexing, abdominal pressing, and hind limb shuffling) are composite behaviors indicative of postoperative pain in rabbits.39 Despite the lack of significant differences in behaviors between the 2 ovariectomy groups in the present study, it is interesting to note that abdominal pressing and the frequency of inactive pain behaviors were slightly (but not significantly) higher after LapOVE versus OVE. Rabbits that underwent LapOVE may have had greater discomfort owing to morbidities such as subcutaneous emphysema, incisional swelling, and dehiscence. Again, type II error cannot be excluded as a reason no behavioral differences were identified between the surgical groups in some situations.
Incisional dehiscence was a postoperative complication for both OVE and LapOVE in the present study. In affected rabbits, only the skin layer opened, presumably owing to discomfort from sutures. However, one would expect that the smaller incision size with a laparoscopic approach would decrease the incidence or extent of dehiscence, postoperative herniation, or incisional infection, as reported for other veterinary species and humans.13,36,50,51,52 In a retrospective study1 of postoperative complications in dogs after LapOVE, 5% (7/132) had opening of skin incisions. Similarly, in another study13 comparing LapOVE with OVE, superficial surgical site infections were identified in 3.2% and 4.7% of dogs, respectively. No dogs that underwent LapOVE were noted to have dehiscence, but 3.7% of dogs had dehiscence after OVE. Rabbits of the present study were assessed twice daily but not monitored more closely for the remainder of the day. Dehiscence could be avoided by diligent monitoring of patients for overgrooming of the abdomen and by maintaining a cleaner environment to prevent infection.
Complications reportedly unique to laparoscopic surgeries include subcutaneous emphysema and organ trauma during abdominal entry. Emphysema can occur as a result of improper cannula placement and high intra-abdominal pressures during insufflation.53,54,55,56,57 Reported incidences in dogs range from approximately 6% to 12%58,59,60. Emphysema is a source of potential discomfort after surgery but is otherwise a minor concern that resolves within a few days. Organ trauma, on the other hand, can be serious. In the present study, 1 rabbit had to be euthanized after the small intestine was perforated during placement of the first cannula. Trauma to intestines, including the cecum, is an important risk in rabbits because of the position of these organs under the initial abdominal incision. It is important that the surgeon elevate the linea alba with forceps as much as possible before placing stay sutures and entering the abdomen. Intestinal perforation is a serious complication that could result in peritonitis. In dogs, the greatest risk of trauma is to the spleen, with a reported incidence of 7% to 18%.9,53,55,58,59,60 If organ trauma is suspected during laparoscopic surgery, conversion to an open technique may be necessary to improve access and resolve the problem. In 1 study,61 5 of 49 dogs undergoing laparoscopic-assisted gastropexy had access-related laceration of the spleen or urinary bladder and 3 dogs required conversion to an open surgical approach. Careful abdominal entry with visualization of cannulas is recommended to minimize access-related complications.
No long-term postoperative outcomes were assessed in the present study, and this would be an area for additional research. Previous studies22,37,38 involving rabbits have shown a lower risk of adhesion formation with laparoscopic surgery versus laparotomy. However, pneumoperitoneum with a high intra-abdominal pressure and high CO2 flow rate have been associated with desiccation and adhesion of abdominal organs in rabbits.46 Recent approaches to laparoscopy involving abdominal lifting techniques may help to eliminate complications attributable to insufflation and should be evaluated in more detail.47 Other findings have also suggested that greater surgeon experience and shorter surgery times may decrease the risk of adhesion formation.43
Results of the study reported here indicated that LapOVE in rabbits provided no benefit over standard OVE with respect to intraoperative and short-term postoperative variables. Although the shorter incision length associated with LapOVE might contribute to lower postoperative infection and herniation rates, those outcomes were not evaluated. Open surgical techniques are advised for patients in which anesthesia time needs to be minimized and the effects of CO2 insufflation for laparoscopy may be deleterious. Even for routine laparoscopic surgeries, the option should be reserved to convert to an open approach if complications such as organ trauma occur. Further research comparing LapOVE and OVE outcomes in rabbits is warranted, and modified surgical techniques, long-term outcomes such as adhesion formation and infection rate, and larger sample sizes than in the present study for pain assessment should be considered.
Acknowledgments
This manuscript represents a portion of a thesis submitted by Dr. Kabakchiev to the Department of Clinical Studies, Ontario Veterinary College, University of Guelph as partial fulfillment of the requirements for a Doctor of Veterinary Science degree.
Funded by a grant from OVC Pet Trust. Additional financial support was provided by a University of Guelph undergraduate work-study program.
The authors declare that there were no conflicts of interest. Funding sources did not have any involvement in the study design, data analysis and interpretation, or writing and publication of the manuscript.
The authors thank Megan Freedman for her assistance measuring postoperative variables and Medtronic Canada for lending the VSD equipment used for laparoscopic surgeries.
Abbreviations
| FGS | Facial grimace scale |
| LapOVE | Laparoscopic ovariectomy |
| OVE | Open ovariectomy |
| VSD | Vessel-sealing device |
Footnotes
Charles River Laboratories, Saint-Constant, QC, Canada.
Sandoz Canada Inc, Boucherville, QC, Canada.
BD Canada, Mississauga, ON, Canada.
Plasmalyte-A, Baxter Healthcare, Deerfield, Ill.
Pharmascience Inc, Montreal, QC, Canada.
IsoFlo, Zoetis Canada Inc, Kirkland, QC, Canada.
Ligasure, Medtronic (Covidien) Canada, Saint-Laurent, QC, Canada.
Fresenius Kabi Canada Ltd, Toronto, ON, Canada.
Metacam injectable (20 mg/mL), Boehringer Ingelheim, Burlington, ON, Canada.
Metacam oral suspension (1.5 mg/mL), Boehringer Ingelheim, Burlington, ON, Canada.
R, version 3.5.1, R Core Team, R Foundation for Statistical Computing, Vienna, Austria.
Vetergesic, Sogeval UK Ltd, Sheriff Hutton, York, England.
PDS II, Ethicon, Johnson & Johnson Medical Products, Somerville, NJ.
Karl Storz Endoscopy America Inc, El Segunda, Calif.
VersaOne, Covidien Canada, Saint-Laurent, QC, Canada.
Monocryl, Ethicon, Johnson & Johnson Medical Products, Somerville, NJ.
Contour glucometer, Bayer Inc, Mississauga, ON, Canada.
1080P HD wireless IP camera, Floureon, Shenzhen Shi, Guangdong Sheng, China.
Flamazine, Smith and Nephew Inc, Mississauga, ON, Canada.
Compounded by Ontario Veterinary College Pharmacy, Guelph, ON, Canada.
References
- 1. ↑
Binder C, Katic N, Aurich JE, et al. Postoperative complications and owner assessment of single portal laparoscopic ovariectomy in dogs. Vet Rec 2018;183:745.
- 2. ↑
Hancock RB, Lanz OI, Waldron DR, et al. Comparison of postoperative pain after ovariohysterectomy by harmonic scalpel-assisted laparoscopy compared with median celiotomy and ligation in dogs. Vet Surg 2005;34:273–282.
- 3. ↑
Haraguchi T, Kimura S, Itoh H, et al. Comparison of postoperative pain and inflammation reaction in dogs undergoing preventive laparoscopic-assisted and incisional gastropexy. J Vet Med Sci 2017;79:1524–1531.
- 4. ↑
Gauthier O, Holopherne-Doran D, Gendarme T, et al. Assessment of postoperative pain in cats after ovariectomy by laparoscopy, median celiotomy, or flank laparotomy. Vet Surg 2015;44(suppl 1):23–30.
- 5. ↑
McClaran JK, Skerrett SC, Currao RL, et al. Comparison of laparoscopic-assisted technique and open laparotomy for gastrointestinal biopsy in cats. Vet Surg 2017;46:821–828.
- 6. ↑
Devitt CM, Cox RE, Hailey JJ. Duration, complications, stress, and pain of open ovariohysterectomy versus a simple method of laparoscopic-assisted ovariohysterectomy in dogs. J Am Vet Med Assoc 2005;227:921–927.
- 7. ↑
Freeman LJ, Rahmani EY, Al-Haddad M, et al. Comparison of pain and postoperative stress in dogs undergoing natural orifice transluminal endoscopic surgery, laparoscopic, and open oophorectomy. Gastrointest Endosc 2010;72:373–380.
- 8. ↑
Stedile R, Beck CAC, Schiochet F, et al. Laparoscopic versus open splenectomy in dogs. Pesqui Vet Bras 2009;29:653–660.
- 9. ↑
Davidson EB, Moll HD, Payton ME. Comparison of laparoscopic ovariohysterectomy and ovariohysterectomy in dogs. Vet Surg 2004;33:62–69.
- 10. ↑
Culp WT, Mayhew PD, Brown DC. The effect of laparoscopic versus open ovariectomy on postsurgical activity in small dogs. Vet Surg 2009;38:811–817.
- 11. ↑
Mayhew PD, Culp WT, Hunt GB, et al. Comparison of perioperative morbidity and mortality rates in dogs with noninvasive adrenocortical masses undergoing laparoscopic versus open adrenalectomy. J Am Vet Med Assoc 2014;245:1028–1035.
- 12. ↑
Singh A, Hoddinott K, Morrison S, et al. Perioperative characteristics of dogs undergoing open versus laparoscopic-assisted cystotomy for treatment of cystic calculi: 89 cases (2011–2015). J Am Vet Med Assoc 2016;249:1401–1407.
- 13. ↑
Charlesworth TM, Sanchez FT. A comparison of the rates of postoperative complications between dogs undergoing laparoscopic and open ovariectomy. J Small Anim Pract 2019;60:218–222.
- 14. ↑
Shariati E, Bakhtiari J, Khalaj A, et al. Comparison between two portal laparoscopy and open surgery for ovariectomy in dogs. Vet Res Forum 2014;5:219–223.
- 15. ↑
Coleman KA, Monnet E, Johnston M. Single port laparoscopic-assisted ovariohysterectomy in three rabbits. J Exot Pet Med 2018;27:21–24.
- 16. ↑
Mehler SJ. Minimally invasive surgery techniques in exotic animals. J Exot Pet Med 2011;20:188–205.
- 17. ↑
Divers SJ. Clinical technique: endoscopic oophorectomy in the rabbit (Oryctolagus cuniculus): the future of preventative sterilizations. J Exot Pet Med 2010;19:231–239.
- 18. ↑
Divers SJ. Endoscopic ovariectomy of exotic mammals using a three-port approach. Vet Clin North Am Exot Anim Pract 2015;18:401–415.
- 19. ↑
Divers SJ. Exotic mammal diagnostic endoscopy and endosurgery. Vet Clin North Am Exot Anim Pract 2010;13:255–272.
- 20. ↑
Proença LM. Two-portal access laparoscopic ovariectomy using ligasure atlas in exotic companion mammals. Vet Clin North Am Exot Anim Pract 2015;18:587–596.
- 21. ↑
Proença LM, Camus M, Nemeth N, et al. Diagnostic quality of percutaneous fine-needle aspirates and laparoscopic biopsy specimens of the liver in rabbits (Oryctolagus cuniculus). J Am Vet Med Assoc 2015;246:313–320.
- 22. ↑
Telles de Sousa ID, Candido dos Reis FJ, Brito Garcia S, et al. Adhesion formation after uterine horn resection in rabbit model: comparative study of laparoscopic procedure vs laparotomy. Gynaecol Endosc 2002;11:365–369.
- 23. ↑
Chaniotakis I, Antoniou E, Kostomitsopoulos N, et al. Stress response to ovariohysterectomy in rabbits: role of anaesthesia and surgery. J Obstet Gynaecol 2018;38:697–701.
- 24. ↑
Mayer J. Use of behavior analysis to recognize pain in small mammals. Lab Anim (NY) 2007;36:43–48.
- 25. ↑
Weary DM, Niel L, Flower FC, et al. Identifying and preventing pain in animals. Appl Anim Behav Sci 2006;100:64–76.
- 26. ↑
Keating SC, Thomas AA, Flecknell PA, et al. Evaluation of EMLA cream for preventing pain during tattooing of rabbits: changes in physiological, behavioural and facial expression responses. PLoS One 2012;7:e44437.
- 27. ↑
Schnellbacher RW, Divers SJ, Comolli JR, et al. Effects of intravenous administration of lidocaine and buprenorphine on gastrointestinal tract motility and signs of pain in New Zealand White rabbits after ovariohysterectomy. Am J Vet Res 2017;78:1359–1371.
- 28. ↑
Weaver LA, Blaze CA, Linder DE, et al. A model for clinical evaluation of perioperative analgesia in rabbits (Oryctolagus cuniculus). J Am Assoc Lab Anim Sci 2010;49:845–851.
- 29. ↑
Lichtenberger M, Lennox A. Updates and advanced therapies for gastrointestinal stasis in rabbits. Vet Clin North Am Exot Anim Pract 2010;13:525–541.
- 30. ↑
Kabakchiev CM, zur Linden AR, Singh A, et al. Effects of intra-abdominal pressure on laparoscopic working space in domestic rabbits (Oryctolagus cuniculus). Am J Vet Res 2020;81:77–83.
- 31. ↑
Kabakchiev C, Valverde A, Singh A, et al. Cardiovascular and respiratory effects of carbon dioxide pneumoperitoneum in the domestic rabbit (Oryctolagus cuniculus). Can J Vet Res 2020;84:108–114.
- 32. ↑
Leach MC, Allweiler S, Richardson C, et al. Behavioural effects of ovariohysterectomy and oral administration of meloxicam in laboratory housed rabbits. Res Vet Sci 2009;87:336–347.
- 33. ↑
Farnworth M, Walker J, Schweizer K, et al. Potential behavioural indicators of post-operative pain in male laboratory rabbits following abdominal surgery. Anim Welf 2011;20:225–237.
- 34. ↑
Hampshire V, Robertson S. Using the facial grimace scale to evaluate rabbit wellness in post-procedural monitoring. Lab Anim (NY) 2015;44:259–260.
- 36. ↑
Mayhew PD, Freeman L, Kwan T, et al. Comparison of surgical site infection rates in clean and clean-contaminated wounds in dogs and cats after minimally invasive versus open surgery: 179 cases (2007–2008). J Am Vet Med Assoc 2012;240:193–198.
- 37. ↑
Luciano AA, Maier DB, Koch EI, et al. A comparative study of postoperative adhesions following laser surgery by laparoscopy versus laparotomy in the rabbit model. Obstet Gynecol 1989;74:220–224.
- 38. ↑
Jorgensen JO, Lalak NJ, Hunt DR. Is laparoscopy associated with a lower rate of postoperative adhesions than laparotomy? A comparative study in the rabbit. Aust N Z J Surg 1995;65:342–344.
- 39. ↑
Klaphake E, Paul-Murphy J. Disorders of the reproductive and urinary systems. In: Quesenberry KE, Carpenter JW, eds. Ferrets, rabbits, and rodents: clinical medicine and surgery. St Louis: Elsevier, 2012;217–231.
- 40. ↑
Bertram CA, Muller K, Klopfleisch R. Genital tract pathology in female pet rabbits (Oryctolagus cuniculus): a retrospective study of 854 necropsy examinations and 152 biopsy samples. J Comp Pathol 2018;164:17–26.
- 41. ↑
Lee HW, Machin H, Adami C. Peri-anaesthetic mortality and nonfatal gastrointestinal complications in pet rabbits: a retrospective study on 210 cases. Vet Anaesth Analg 2018;45:520–528.
- 42. ↑
Brodbelt DC, Blissitt KJ, Hammond RA, et al. The risk of death: the confidential enquiry into perioperative small animal fatalities. Vet Anaesth Analg 2008;35:365–373.
- 43. ↑
Ordoñez JL, Domínguez J, Evrard V, et al. The effect of training and duration of surgery on adhesion formation in the rabbit model. Hum Reprod 1997;12:2654–2657.
- 44. ↑
Pope JF, Knowles TG. Retrospective analysis of the learning curve associated with laparoscopic ovariectomy in dogs and associated perioperative complication rates. Vet Surg 2014;43:668–677.
- 45. ↑
Coisman JG, Case JB, Shih A, et al. Comparison of surgical variables in cats undergoing single-incision laparoscopic ovariectomy using a LigaSure or extracorporeal suture versus open ovariectomy. Vet Surg 2014;43:38–44.
- 46. ↑
Yesildaglar N, Koninckx PR. Adhesion formation in intubated rabbits increases with high insufflation pressure during endoscopic surgery. Hum Reprod 2000;15:687–691.
- 47. ↑
McCracken BD, Beths T, Herbert S, et al. Comparison of isobaric and insufflated laparoscopy-assisted ovariohysterectomy in the common rabbit (Oryctolagus cuniculus). J Exot Pet Med 2019;28:193–204.
- 48. ↑
Szeto A, Gonzales JA, Spitzer SB, et al. Circulating levels of glucocorticoid hormones in WHHL and NZW rabbits: circadian cycle and response to repeated social encounter. Psychoneuroendocrinology 2004;29:861–866.
- 49. ↑
Préfontaine L, Helie P, Vachon P. Postoperative pain in Sprague Dawley rats after liver biopsy by laparotomy versus laparoscopy. Lab Anim (NY) 2015;44:174–178.
- 50. ↑
Richards C, Edwards J, Culver D, et al. Does using a laparoscopic approach to cholecystectomy decrease the risk of surgical site infection? Ann Surg 2003;237:358–362.
- 51. ↑
Varela JE, Wilson SE, Nguyen NT. Laparoscopic surgery significantly reduces surgical-site infections compared with open surgery. Surg Endosc 2010;24:270–276.
- 52. ↑
Kössler-Ebs JB, Grummich K, Jensen K, et al. Incisional hernia rates after laparoscopic or open abdominal surgery—a systematic review and meta-analysis. World J Surg 2016;40:2319–2330.
- 53. ↑
Case JB, Marvel SJ, Boscan P, et al. Surgical time and severity of postoperative pain in dogs undergoing laparoscopic ovariectomy with one, two, or three instrument cannulas. J Am Vet Med Assoc 2011;239:203–208.
- 54. ↑
Lee DW, Kim MJ, Lee YK, et al. Does intraabdominal pressure affect development of subcutaneous emphysema at gynecologic laparoscopy? J Minim Invasive Gynecol 2011;18:761–765.
- 55. ↑
Kim YK, Park SJ, Lee SY, et al. Laparoscopic nephrectomy in dogs: an initial experience of 16 experimental procedures. Vet J 2013;198:513–517.
- 56. ↑
Murdock CM, Wolff AJ, Van Geem T. Risk factors for hypercarbia, subcutaneous emphysema, pneumothorax, and pneumomediastinum during laparoscopy. Obstet Gynecol 2000;95:704–709.
- 58. ↑
Mayhew PD. Complications of minimally invasive surgery in companion animals. Vet Clin North Am Small Anim Pract 2011;41:1007–1021, vii–viii.
- 59. ↑
Dupré G, Fiorbianco V, Skalicky M, et al. Laparoscopic ovariectomy in dogs: comparison between single portal and two-portal access. Vet Surg 2009;38:818–824.
- 60. ↑
Mayhew PD, Brown DC. Prospective evaluation of two intracorporeally sutured prophylactic laparoscopic gastropexy techniques compared with laparoscopic-assisted gastropexy in dogs. Vet Surg 2009;38:738–746.
- 61. ↑
Loy Son NK, Singh A, Amsellem P, et al. Long-term outcome and complications following prophylactic laparoscopic-assisted gastropexy in dogs. Vet Surg 2016;45(S1):O77–O83.
Appendix
Descriptions of video-recorded behaviors of rabbits measured by a blinded observer before and after LapOVE or OVE.
| Behavior | Description | Duration (min) | No. of incidents |
|---|---|---|---|
| Activity | |||
| Activity level | Being active and moving around the pen | × | — |
| Exploring | Exploring environment | × | — |
| Eating | Eating from food dishes | × | — |
| Drinking | Drinking from water bottles | × | — |
| Time at front | Located in front half of pen | × | — |
| Time at back | Located in back half of pen | × | — |
| Time in hut | Located in clear plastic hut | × | — |
| Digging | Digging action on floor | — | × |
| Jumping | Jumping straight up | — | × |
| Interacting | Interacting with objects in the pen | — | × |
| Locomotion | |||
| Hopping | Hopping around the pen | × | × |
| Shuffling* | Walking slowly around the pen | × | — |
| Movement composite* | Combined duration of time spent hopping and shuffling | × | — |
| Hind limb shuffle* | Slowly shuffling hind limbs after stretching forelimbs forward | — | × |
| Partial hop* | Extending forelimbs forward, but not completing hop with hind limbs | — | × |
| Stomping | Stomping 1 foot on the floor | — | × |
| Grooming | |||
| General grooming | Grooming head, feet, or body | × | — |
| Grooming abdomen or flank | Grooming abdomen or flank* | × | — |
| Posture | |||
| Recumbency | Lying in sternal or lateral recumbency | × | — |
| Rearing | Rearing or standing on hind limbs | — | × |
| Stretching | Stretching various parts of the body | — | × |
| Alerting | Stopping suddenly with ears erect | — | × |
| Yawning | Yawning by the rabbit | — | × |
| Pain | |||
| Huddling* | Sitting with back arched | × | — |
| Eyes closed | Eyes closed or semiclosed | × | — |
| Abdominal pressing* | Arching back and pressing abdomen to the floor | × | × |
| Stagger* | Falling or losing balance | — | × |
| Drawing back* | Wincing or rapidly, briefly drawing back with a grimace or blink | — | × |
| Full-body flex* | Flinching or rapidly flexing body upward | — | × |
| Twitching* | Rapidly moving skin or subcutaneous muscles | — | × |
| Composite of inactive pain behaviors* | Combined counts for hind limb shuffling, abdominal pressing, staggering, drawing back, full-body flex, and twitching | — | × |
