Hindgut fermenters such as guinea pigs (Cavia porcellus) are prone to gastrointestinal stasis when undergoing pain, stress, or surgery.1,2 This can complicate the animal’s recovery and even result in death. Therefore, it is crucial to provide appropriate analgesia and ensure pain is minimized. In small animal medicine, there is increasing interest in laparoscopy and laparoscopy-assisted procedures, as some studies3–6 have shown that they can be associated with less pain compared to open surgery. Although guinea pigs have been used as a model for laparoscopic procedures in humans,7–11 information regarding laparoscopic surgery applications for pet guinea pigs is very limited at this time.12,13
During laparoscopic surgery, gas or fluid is usually instilled into the body cavity in order to increase working space for the endoscope and endosurgical instruments and allow equipment triangulation. One of the most commonly used gases for this purpose is carbon dioxide (CO2), due to several beneficial properties (unlikely to cause gas embolism, noncombustible, and inexpensive).14–16 However, the CO2 insufflation itself can be a source of pain or nausea, which could potentially lessen the benefits obtained from performing a minimally invasive procedure.17
In addition, the guinea pig has a voluminous gastrointestinal tract, with a very large cecum,18 which is compressed during increased pneumoperitoneal pressure. There is therefore a significant chance that induction of pneumoperitoneum may lead to postoperative ileus or other adverse gastrointestinal effects. There is a need to assess the effects of pneumoperitoneum on postoperative gastrointestinal function and general pain in guinea pigs, in order to comprehensively and critically evaluate the safety of laparoscopic surgery over open surgery in this species.
The purpose of the study was to compare stress markers (blood glucose and fecal cortisol), markers of gastrointestinal motility (appetite and fecal production), and behavioral indicators of pain between guinea pigs undergoing abdominal insufflation with CO2 and guinea pigs undergoing abdominal catheter placement without insufflation (control). Our hypothesis was that there would be no significant difference in stress markers, gastrointestinal motility, and signs of pain between the 2 groups.
Materials and Methods
Animals
The study was approved by the Animal Care Committee of the University of Guelph (AUP no. 4282). Fourteen specific pathogen-free intact female Hartley guinea pigs (Cavia porcellus) were obtained from a commercial vendor (Charles River Laboratories) for this study. The guinea pigs were approximately 4 to 5 months of age at the start of the study. They were apparently healthy on the basis of a physical examination. Prior to the study procedures, they were housed in 2 groups of 7 guinea pigs in large pens with kiln-dried pine as shavings. A variety of enrichment items were provided, including plastic huts, paper bags, and chew toys. The diet consisted of autoclaved hay and guinea pig pellets (Teklad Global High Fiber Guinea Pig Diet 2041; Envigo). The treatment (insufflation or not) was randomized to guinea pigs as well as order of procedures within this guinea pig cohort. An equal number of insufflated and noninsufflated guinea pigs were assigned to each study day. Randomization was performed using statistical software (R version 4.0.4, 2021; R Foundation for Statistical Computing).
Procedures
The animals were fasted at 4:00 am to 5:00 am prior to procedures. Procedures were performed between 9:00 am and 2:00 pm on 4 days over the course of 2 weeks. Body weight, rectal temperature, heart rate (HR), and respiratory rate (RR) were measured preoperatively (T0). Each guinea pig was sedated with midazolam (1.5 mg/kg; Midazolam Sandoz 5 mg/mL; Sandoz Canada) and buprenorphine hydrochloride (0.2 mg/kg; Vetergesic Multidose 0.3 mg/mL; Champion Alstoe) IM, with a single injection in the semimembranosus or semitendinosus muscles. General anesthesia was then induced with 5% isoflurane delivered in 100% oxygen via face mask and connected to a nonrebreathing pediatric circle system. The animal was placed on a tabletop rabbit and rodent dentistry table and intubated endoscopically with a 16-gauge, 8.3-cm IV catheter without the stylet using a 30° scope (Karl Storz Endoscopy) and a previously described technique.13 Lidocaine 1 mg/kg (lidocaine HCl injection with preservative; Teligent OU) was used topically near the glottis to help facilitate intubation. Anesthesia was maintained with isoflurane delivered in 100% oxygen at a flow rate range of 0.5 to 1 L/min. The isoflurane percentage varied between 3.5% and 2.5% during the experiment to maintain an adequate anesthetic depth. The animals were mechanically ventilated during the procedure (Datex Ohmeda Aestiva/5 with 7900 SmartVent; GE Healthcare). The RR was maintained at 12 breaths/min. Anesthetic monitoring equipment included a pulse oximeter (Model 2500A Vet; Nonin Medical), a rectal thermometer, and a multiparametric monitor (Datex-Ohmeda S/5; GE Healthcare) to monitor ECG, end-tidal carbon dioxide, peak inspiratory pressure, and tidal volume. In addition, an ultrasonic Doppler flow detector (Ultrasonic Doppler Flow Detector Model 811-B; Parks Medical Electronics) was used to measure indirect blood pressure, with the probe placed over the medial carpus and with a No. 1 neonatal blood pressure cuff placed over the proximal forelimb and connected to a sphygmomanometer. Heat support was provided with a rodent heating pad (Far Infrared Warming Pad; Kent Scientific Corporation) and warmed oat bags. The guinea pigs’ eyes were lubricated with an ophthalmic liquid gel.
The ventral abdomen was shaved and aseptically prepared. A 0.5- to 1-cm preumbilical skin incision was made and the abdomen was entered using a modified Hasson technique.13 An 18-gauge IV catheter was placed for insufflation through the linea alba mid abdomen. The catheter was secured with sutures (5-0 polydioxanone) and a transparent film dressing (Tegaderm Film; 3M Health Care).
Half (n = 7) of the guinea pigs were assigned to receive insufflation. This group received insufflation with standard-temperature CO2 to a pressure of 6 mm Hg. The animal was maintained under general anesthesia as the pressure was held for 30 minutes. The intra-abdominal pressure (IAP) of 6 mm Hg was chosen based on a previous pilot study13 on laparoscopic ovariectomy in guinea pigs. The control group (n = 7) had an abdominal catheter placed but no insufflation. The control animals were likewise maintained under anesthesia for 30 minutes.
After 30 minutes, the abdomen was desufflated if needed and the insufflation catheter was removed. The skin was closed with 5-0 polydioxanone suture in a simple interrupted pattern.
Postoperative care and monitoring
After the surgery was completed, isoflurane was discontinued and the animal was extubated once breathing spontaneously. Subcutaneous crystalloid fluids (30 mL; PlasmaLyte-A; Baxter) were provided at the conclusion of anesthesia. Flumazenil (0.05 mg/kg) was given IM to hasten recovery (flumazenil, 0.1 mg/mL injectable; Sandoz). Meloxicam (1.5 mg/kg) was given SC after the procedure for analgesia (Metacam, 5 mg/mL; Boehringer Ingelheim). An additional dose of buprenorphine (0.2 mg/kg IM)19 was given for additional analgesia the evening after surgery (between 8 and 10.5 hours after the initial dose). Meloxicam was given orally20 the evening after surgery (1.5 mg/kg, PO; Metacam, 1.5 mg/mL; Boehringer Ingelheim) and continued twice per day for another 3 days. Marbofloxacin (5 mg/kg, PO, once daily) was given following the procedure for infection prophylaxis. Each guinea pig was kept in an individual cage adjacent to the one of the larger pens for 2 days after surgery. Each guinea pig’s attitude, weight, and incision healing were monitored for 7 days after surgery. Vital parameters (rectal temperature, HR, and RR) were monitored the evening postoperatively (T0.5) and then twice daily for 2 days after surgery (T1, T1.5, T2, and T2.5 days). At the conclusion of the research study, the guinea pigs were placed up for adoption.
Gastrointestinal motility evaluation
Fecal production and appetite for pellets were evaluated at T0.5, T1, T1.5, T2, and T2.5 days. Guinea pigs were offered ad libitum pellets and hay. The amount of pellets consumed was measured using a scale with a 0.5-gram precision. Hay consumed was not measured, but hay was available to ensure sufficient fiber and abrasiveness in the diet to stimulate gastrointestinal motility and tooth wear. Eating behavior, including both hay and pellet consumption, was included in the ethogram in order to account for hay intake. The fecal production was quantified both by number of fecal pellets and by weight in grams. Sample collection occurred at approximately 8:00 am to 10:45 am and 7:00 pm to 9:15 pm. During the postoperative period, guinea pigs were provided with plastic trays and towels as substrate rather than shavings to facilitate sample collection. Plastic trays were also used to separate the guinea pigs from the adjacent large pen to ensure that feces from the group-housed guinea pigs were not accidentally counted.
Stress marker evaluation
Blood glucose was measured at T1 and T2 via a single glucometer (Contour Next; Bayer) and a 29-gauge needle to obtain drop of blood from the pinna.
Fecal samples were collected at T0.5, T1, T1.5, T2, and T2.5 for fecal cortisol analysis. Samples were placed in sterilized plastic bags (Whirl-Pak) and frozen at −80 °C. Feces were dried at 37 °C for 24 h. Dried feces were pulverized, and 0.25 g was placed in sterile glass tubes. Ethanol (2.5 mL of 90% ethanol) was added into each tube and vortexed for 1 minute followed by 30 minutes of orbital shaking. Samples were centrifuged at 4,696 X g for 15 minutes, and then 1 mL of supernatant was transferred into a clean 2-mL single-use polypropylene tube (Eppendorf Tube; Eppendorf) for evaporation using a vacuum concentrator (Savant SpeedVac Concentrator; ThermoFisher Scientific). Dried extracted samples were kept frozen at –20 °C until the day of analysis. Fecal cortisol was quantified in duplicates using a commercially available cortisol enzyme immune assay kit (StressXpress; StressMarq Biosciences). Following the manufacturer’s instructions, extracted samples were dissolved with 100 μL of 90% ethanol and diluted to reach an ethanol concentration below 5%. Samples or standards were pipetted into each well (50 μL/well), followed by 25 μL of cortisol conjugate and 25 μL of cortisol antibody. After 1 hour of incubation at room temperature on an orbital shaker, wells were washed 4 times. TMB substrate was added (100 μL/well) and incubated for 30 minutes at room temperature with shaking. Stop solution was added (50 μl/well), and optical density was measured using a microplate reader at 450 nm (Synergy HT; Biotek). The detection range of the kit was 100 to 3,200 pg/mL.
Behavior evaluation
Videos were obtained to monitor the guinea pigs’ behavior at T0.5, T1, T1.5, T2, and T2.5 days. The videos were taken at 6:45 am to 8:00 am and 5:45 pm to 7:00 pm each day. The animals were filmed with wide-angle HD video cameras (HERO 7 and HERO8; GoPro) that were clipped to the bars at the top of the pen (Supplementary Figure S1). Plastic hides were removed from the small pens to facilitate visualization of the guinea pigs’ behavior. Instead, a towel was placed across part of the top of the pen to provide a sense of security to the animals while allowing them to be visible for recording. Video recording took place for 1 hour during each session, and 30 minutes of the video were evaluated. The first 15 minutes of video footage were discarded to avoid the influence of the stress of an observer on the guinea pigs’ behavior. No personnel entered the room during recording.
The videos were evaluated for behavioral indicators of pain based on previously described ethograms.21,22 In particular, the guinea pigs were evaluated for passive behaviors, which increase with pain, and active behaviors, which decrease with pain21 (Appendix 1; Supplementary Figure S1). Ingestive and grooming behaviors were also monitored.
Hiding was not originally included in the ethogram, but was added to due to several occurrences of the subject not being visible for the length of the video due to hiding under the towel. Lying down was also not part of the original ethogram but was added after a number of guinea pigs were noted to display this behavior. There behaviors were categorized as “other” as they were not previously validated in the described ethograms.
The videos were scored by a blinded observer who was not aware of whether the guinea pigs had received insufflation or not. Videos were analyzed using an event-logging software for behavioral observations (Behavioral Observation Research Interactive Software [BORIS]); Olivier Friard and Marco Gamba).23 Each behavior in the ethogram was denoted a key and each guinea pig was denoted a subject number. The video was analyzed using the keys associated with each behavior. If the behavior was performed by the guinea pig, the respective key was hit by the observer, recording the associated behavior, time of occurrence, and subject. Behaviors in the BORIS program can be categorized as a point event or state event.23 For point events, the number of times a behavior occurs is measured, while for state events the amount of time over which the behavior takes place is measured. For each behavior on the ethogram, the number of occurrences of the behavior was documented, and several of the behaviors (Appendix) were also considered state events.
Statistical analysis
Outcome physiologic and behavioral variables that were continuous (eg, HR, RR, fecal output in grams) were analyzed using linear mixed models with treatment (insufflation or not), time, and interaction as fixed effects and individual guinea pigs as a random effect. Assumptions of the model (normality of the residuals, homogeneous variances, linearity, and lack of outliers) were assessed graphically on residual plots. An ANOVA was performed on fixed effects, and post hoc analysis was performed using a Tukey adjustment if needed.
Outcome behavioral count data (eg, number of active movements, number of times the guinea pig drank) and physiologic count data (eg, fecal output in number of fecal pellets) were analyzed using loglinear (Poisson) mixed models with treatment, time, and interaction as fixed effects and individual guinea pigs as a random effect. When a significant fixed effect was detected on the interaction term or a multilevel factor (time), the different levels of the factors were compared by changing the reference level and repeating the loglinear models. Assumptions were evaluated on residual plots. Overdispersion of the models was also checked.
Values of P < 0.05 were considered statistically significant. Available software was used for statistical analysis (R, version 4.1.2, 2021; R Foundation for Statistical Computing) and for graphs (R package ggplot2; R Foundation for Statistical Computing).24
Results
Physiologic variables
The guinea pigs had a mean ± SD weight of 731 ± 63.3 g at T0. The mean T0 RR was elevated for the species25 (Table 1). RR significantly decreased in both groups after T0 at all time points (all P > 0.05) by 24 to 31 breaths/minute depending on time point (Figure 1). The mean rectal temperature was elevated for the species25,26 at T0.5, T1, and T1.5. The mean rectal temperature was normal, albeit at the high end of normal, by T2. Temperature values were not statistically different over time or between treatments.
Selected physiologic parameters (mean ± SD) over time in guinea pigs (Cavia porcellus) after having undergone pneumoperitoneum at 6 mm Hg CO2 (treatment group, n = 7) or anesthesia and abdominal catheter placement alone (control group, 7).
| T0 | T0.5 | T1 | T1.5 | T2 | T2.5 | Reference interval25 | |
|---|---|---|---|---|---|---|---|
| Rectal temperature (°C) | 39 ± 0.3 | 39.9 ± 0.5 | 40.0 ± 0.4 | 39.7 ± 0.4 | 39.4 ± 0.5 | 39.3 ± 0.3 | 37.5–39.5 |
| Heart rate (beats/min) | 254 ± 36 | 245 ± 47 | 261 ± 27 | 265 ± 41 | 255 ± 31 | 240 ± 28 | 230–380 |
| Respiratory rate (breaths/min) | 130 ± 25a | 98.9 ± 17b | 106 ± 12b | 102 ± 18b | 101 ± 19b | 102 ± 27b | 40–120 |
| Blood glucose (mmol/L) | 6.8 ± 1.1 | 6 ± 1.2 | 3.3–6.9 | ||||
| Fecal cortisol (μg/g) | 2.43 ± 3.3 | 5.49 ± 9.51 | 3.27 ± 2.46 | 2.02 ± 1.25 | 3.21 ± 2.78 |
Different superscript letters for values in a row indicate a significant difference.
Rectal temperature, heart rate, and respiratory rate were measured before the procedure (T0), and approximately 12 hours (T0.5 days), 24 hours (T1 day), 36 hours (T1.5 days), 48 hours (T2 days), and 60 hours (T2.5 days) after the procedure. Blood glucose via hand-held glucometer and fecal cortisol were measured at selected time points. Blank cells indicate that measurements were not taken at that time point. There was no significant difference in these parameters between treatment groups, so results from control and insufflated guinea pigs are combined. Respiratory rate significantly decreased in both groups after T0 at all time points. There were no other significant differences in parameters over time.
Line plots of respiratory rates in guinea pigs (Cavia porcellus) that underwent anesthesia and abdominal catheter placement alone (control group; gray; n = 7) or with pneumoperitoneum at 6 mm Hg CO2 for 30 minutes (treatment group; black; 7). Respiratory rate was measured before the procedure (T0), and approximately 12 hours (T0.5 days), 24 hours (T1 day), 36 hours (T1.5 days), 48 hours (T2 days), and 60 hours (T2.5 days) after the procedure. For each error bar, the solid circle represents the mean and the whiskers represent the SE. For each group, a line connects the mean respiratory rates over time. The mean T0 respiratory rate was elevated for the species, and significantly decreased in both groups after T0 at all time points. Respiratory rates were not significantly different between groups.
Citation: American Journal of Veterinary Research 83, 8; 10.2460/ajvr.22.01.0001
Only 1 guinea pig (insufflation group) had returned to its presurgical weight within 7 days postoperation. The remainder of the animals had a lower body weight at T7 (mean ± SD, 688 ± 53.2 g) compared to T0.
There was no difference between groups and over time for rectal temperature, HR, fecal output in grams, food pellets consumed, blood glucose, and fecal cortisol (all P > 0.05; Table 1). For the fecal output by number, there was a significant time-treatment interaction effect in the loglinear mixed model (P = 0.012). Guinea pigs that underwent CO2 insufflation had significantly higher fecal output in fecal pellet numbers at T1.5 than control guinea pigs by 2.1-fold (P < 0.001; Figure 2). No other significant differences were observed between treatments for other time points.
Line plots of fecal output in terms of the number of fecal pellets produced by the guinea pigs in the control group (gray) and treatment group (black) described in Figure 1. For each error bar, the solid circle represents the median and the whiskers represent the interquartile (25th to 75th percentile) range. For each group, a line connects the median fecal output over time. Guinea pigs that underwent CO2 insufflation had significantly higher fecal output in fecal pellet numbers on day 1.5 than control guinea pigs by 2.1-fold (P < 0.001). No other significant differences were observed between treatments for other time points.
Citation: American Journal of Veterinary Research 83, 8; 10.2460/ajvr.22.01.0001
Behavioral variables
During 4 out of the 70 videos, the guinea pig was hidden for the entire video, which precluded measuring any other behaviors. During 3 videos, the guinea pig was hidden for a portion of the video, with 10.4 minutes (35%), 19.2 minutes (64%), and 24.4 minutes (81%) of the 30 minutes of video missing.
Several behaviors were expressed similarly between groups and over time such as the number of body turns, incomplete movement (initiating coprophagy or grooming behavior but stopping), rearing, number of lying episodes, lying time, drinking, hiding episodes, and hiding time (all P > 0.05). Most other behaviors were differentially expressed over time but not between treatments (Supplementary Figure S2).
The number of active movements increased over time with significance compared to T0.5 starting at T2 onward (all P < 0.001, increased by at least 3.5-fold). The amount of time spent actively moving increased over time with significance starting at T2 onward (all P < 0.03). The number of head and neck movements was increased compared to T0.5 at all time points (all P < 0.012). The number of coprophagy events differed between treatments over time (significant interaction term, P < 0.001). There were less coprophagy behaviors in the insufflated group than in the noninsufflated groups by 5.26-fold at T0.5 (P = 0.008), but the number of coprophagy behaviors were similar at other time points. The number of summed active behaviors significantly changed over time, with significance starting at T1 onward (all P < 0.001). Behaviors increased in number compared to T0.5 at T1 and T2 (all P < 0.001) but decreased at T1.5 (P = 0.001).
The number of squinting behaviors significantly increased over time, with significance starting at T1.5 onward (all P < 0.001). For the time spent squinting, there was a significant time-treatment effect (P = 0.010). On post hoc analysis, there was only a difference between treatment at the last time point (T2.5), at which point insufflated guinea pigs spent less time squinting (P = 0.011). The number of weight shifting behaviors, subtle body movements, and summed passive behaviors significantly decreased over time, with significance starting at T1 onward (all P < 0.01). No piloerection was observed.
The number of eating behaviors significantly decreased initially over time at T1 and T1.5 (all P < 0.012), was not significantly different from T0.5 at T2 (P = 0.58), and then was significantly higher at T2.5 (P = 0.012). The number of grooming behaviors significantly increased over time (P < 0.001). However, due to failure for the loglinear models to converge with time as a factor, comparisons between time points could not be performed. The amount of time spent grooming also increased over time (P = 0.014). Post hoc analysis could also not be performed as when time was used as a factor, and no difference between time points was detected. The number of episodes of licking the incision or pulling at sutures increased over time and was only significant at T2.5 (P = 0.001).
All guinea pigs survived anesthesia, the procedures, and the postoperative monitoring period. Several of guinea pigs (6 of 7 in insufflation group, 3 of 7 in control group) developed pigmenturia, which grossly appeared consistent with hematuria rather than porphyrinuria, after the procedure. Because of a history of Corynebacterium renale cystitis outbreaks in guinea pigs from the same source, all guinea pigs, including those that did not show signs, were treated with marbofloxacin at 5 mg/kg, PO every 24 hours following the procedure, and the clinical signs resolved in all animals. The length of treatment varied between animals depending on the presence or absence of clinical signs. One guinea pig (insufflation group) developed suspect burns on the perineum and hind feet, likely secondary to an overheated warming bag. The animal was managed with analgesics, antimicrobials, and wound care, and the wounds were fully healed by 8 weeks after the procedure. This guinea pig was not excluded as the data did not appear to be an outlier aside from fecal cortisol. Removing this animal’s data from the fecal cortisol results did not change the significance of the results so the data were not excluded. Another guinea pig (insufflation group) was noted to have dehiscence of the incision 3 days after the procedure (after the monitoring of vital parameters, appetite, fecal output, fecal cortisol, blood glucose, and behavior had been completed). The incision was debrided and repaired and also healed fully.
Discussion
In this study, there was no significant difference between insufflated and control guinea pigs in regards to postoperative vital parameters, appetite, fecal output in grams, blood glucose, and fecal cortisol. There were limited behavioral parameters that were significantly different at single time points, including less coprophagy in the insufflated group at T0.5 and more time squinting in the noninsufflated group at T2.5. The clinical significance of these changes is not known but is likely minimal given that the majority behaviors did not follow these trends. Therefore, the results reflect that insufflation of CO2 at 6 mm Hg for 30 minutes in guinea pigs is not associated with significant increases in observable pain or stress or decreases in gastrointestinal motility compared to control.
The elevated preoperative RR was likely due to the stress of handling. The elevated postoperative rectal temperature was likewise suspected to be due to stress, but a drug reaction or an underlying inflammatory or infectious condition cannot be excluded.
Guinea pigs had lower weights than their preoperative weight at almost all of the time points, and only 1 guinea pig had returned to the preoperative weight within 7 days. Although weight loss can be related to decreased appetite from pain or stress, drug administration can also contribute. In another study,21 guinea pigs treated with anesthesia and extended-release buprenorphine without surgery demonstrated ≥10% body weight loss. Given that both groups in our study received buprenorphine and meloxicam, it is unknown to what extent drug administration contributed to weight loss.
Appetite and fecal production in grams were not significantly different between the groups. Guinea pigs that underwent insufflation did produce a significantly greater number of fecal pellets at T1.5. However, it is possible that the guinea pigs could have produced a greater number of smaller fecal pellets, as the weight of feces produced was not significantly different between groups at any time point. In addition, the groups were not significantly different at other time points in regard to the number of fecal pellets produced. Similarly, mice undergoing CO2 insufflation to an IAP of 6 to 8 mm Hg for 15 minutes had no significant difference in their fecal production compared to untreated controls.27 In contrast, those receiving a laparotomy had significantly reduced fecal production.27 In rabbits undergoing laparoscopic or open ovariectomy, the number and weight of feces produced and weight of food consumed postoperatively were not significantly different.28 However, those in the open ovariectomy group spent more time eating, but not significantly so.28
A recent study29 in guinea pigs found that the time from anesthesia until the start of defecation and spontaneous feeding were longer in the pneumoperitoneum group in comparison to the anesthesia group. However, these changes were only seen when the procedure was carried out for ≥60 minutes. In contrast to the present study, guinea pigs in that study29 were insufflated with 12 mm Hg CO2 via a Veress needle while the control group received anesthesia alone. In the present study, we compared guinea pigs undergoing abdominal catheter placement under anesthesia to those receiving anesthesia, abdominal catheter placement, and insufflation. This was an attempt to remove the pain associated with abdominal catheter placement as a potential confounding factor and ensure equal anesthetic times. We used an open (modified Hasson technique) rather than a closed (Veress needle) technique to reduce the risk of organ perforation.30 The lack of major differences in appetite and fecal output in our study compared to Badarudeen et al29 could be secondary to the shorter length of insufflation, the lower IAP used, or differences in anesthesia and analgesia protocols.
Blood glucose concentration was not significantly different between the 2 groups. In this study, blood glucose was used as a marker of stress31 associated with the procedures. The blood glucose concentration as measured by glucometer was found not to be significantly different in rabbits undergoing laparoscopic or open ovariectomy.28 Likewise, no significant differences were found in glucose concentration between dogs undergoing laparoscopic versus open ovariohysterectomy32 or in cats undergoing laparoscopic-assisted ovariectomy versus open ovariohysterectomy.33 In contrast, another study4 in dogs found significantly increased blood glucose levels at 2, 4, and 6 hours postoperatively in the open surgery group but not the laparoscopic-assisted group. It is possible that more frequent glucose monitoring may have elicited some differences in glucose levels.
Fecal cortisol was not significantly different between the 2 groups. In dogs undergoing laparoscopic or open ovariohysterectomy, blood cortisol was significantly higher in the open group 2 hours postoperatively, but returned to normal by 6 hours postoperatively.32 Another study4 in dogs found significantly increased cortisol levels at 1 and 2 hours postoperatively in the open surgery group but not the laparoscopic-assisted group. In rabbits undergoing open or laparoscopic ovariectomy, every postoperative blood cortisol value except 1 was below the quantification for the assay, preventing it from being used to compare the groups.28 In the present study, it was elected to measure fecal rather than blood cortisol. Obtaining sufficient volumes of blood to measure cortisol in guinea pigs would require prolonged restraint or anesthesia, both of which can induce stress. Fecal glucocorticoid metabolite levels reflect long-term, rather than short-term, stress.34,35 While a gut passage time of 18 of 20 hours for glucocorticoid metabolites in guinea pigs was previously reported, a more recent study34 demonstrated a 6- to 8-hour gut passage time. Diurnal variations in fecal glucocorticoid metabolites have been noted in some species, but guinea pigs are not thought to have such fluctuations.34 Peak fecal corticosterone levels were found to be higher in mice that underwent a vasectomy and received saline, 5 mg/kg meloxicam, or 10 mg/kg meloxicam, SC, compared to control mice. Those receiving the highest meloxicam dose (20 mg/kg) had a peak fecal corticosterone levels that were not statistically different from the control (nonsurgery) group.36
Video recording was used in this study to remotely monitor signs of pain, and the first 15 minutes of video recording were removed to avoid the influence of an observer on behaviors. A cageside ethogram was found to be insensitive to detect postoperative pain compared to a video ethogram in a previous report,21 likely due to the stoic nature of guinea pigs and their ability to hide signs of pain. In a different study,22 cageside monitoring of time-to-consumption of a treat was also ineffective in evaluating pain, further highlighting the fact that remote monitoring is required to accurately assess pain in guinea pigs. A study37 comparing behavioral changes associated with postorchiectomy pain in saline- versus analgesia-treated groups concluded that a behavior-based pain scoring system may have limited value in guinea pigs as behavioral changes after surgery were subtle.
Overall, there were minimal significant differences in behavioral indicators of pain between the groups. Coprophagy occurred less often in the insufflated versus noninsufflated group at T0.5, but there was no significant difference at other time points. It is possible that this decrease in coprophagy may indicate greater pain21 in the insufflation group at that time point. However, given that other pain-associated behaviors were not significantly different at that time point, this cannot be confirmed. Interestingly, at T2.5 postprocedure, insufflated guinea pigs spent less time squinting. Squinting is a passive behavior, which increases with pain.21 Given that there is no discernable reason for the noninsufflated guinea pigs to be experiencing greater pain, this result is likely not clinically relevant.
While insufflation with CO2 can be associated with peritoneal irritation,38 CO2 pneumoperitoneum in humans is associated with a lower risk of abdominal discomfort and shoulder pain compared to insufflation of room air.39 However, a study28 in rabbits found no significant differences between laparoscopic and open ovariectomy in terms of behavioral variables. Interestingly, the laparoscopic group had a nonsignificant increase in abdominal pressing and inactive pain composite behaviors.28 This may indicate pneumoperitoneum can be a source of mild pain. In contrast, our study did not show that a short period of pneumoperitoneum was a significant source of pain in guinea pigs. The rabbits undergoing laparoscopic surgery had a mean ± SD surgery time of 43.2 ± 9.5 and anesthesia time of 76.2 ± 14.7 min,28 so it is possible that longer periods of insufflation could result in significant changes to signs of pain in guinea pigs. The temperature of the insufflation gas may have an impact on postoperative discomfort, as dogs insufflated with warmed, humidified CO2 were more likely to elevated pain scores compared to dogs insufflated with standard-temperature CO2.38
Summed active behaviors were not significantly different between the 2 groups. A study21 found that summed active behaviors were not significantly different between anesthesia and analgesia alone compared to hysterectomy in guinea pigs, but forward or backward movement at 48 h was significantly decreased following surgery. Movement was not significantly different after orchiectomy in guinea pigs compared to anesthesia alone.22 Similarly, another study37 found that the composite active behavior score was not significantly different between analgesic and control groups postorchiectomy. A study29 in guinea pigs undergoing pneumoperitoneum compared to anesthesia alone showed that arousal times and time to ambulation were prolonged in the pneumoperitoneum group. The authors29 posited that the central depressant effect of the CO2 contributed to this finding.
Passive behaviors were not significantly different between the 2 groups. As noted above, rabbits undergoing laparoscopic ovariectomy had a nonsignificant (P = 0.07) increase in inactive pain composite behaviors.28 An increase in summed passive behaviors was noted in guinea pigs 2 h posthysterectomy.21 Similarly, subtle body movements were found to increase in guinea pigs at 2 and 8 h postorchiectomy.22 The lack of increased passive behaviors in the pneumoperitoneum group likely reflects that there was not significantly greater pain in this group compared to controls.
Lying down was not included in the original ethogram as it was mainly adapted from one previously developed.21 The study37 that compared postorchiectomy pain behaviors in control versus treated guinea pigs found no significant difference in the total duration of lying down or lying down with legs underneath the body between groups. However, the control group did spend more time lying down with hind legs to the side or extended backwards 1 hour postoperatively compared to the analgesia-treated group.37 We did not specifically evaluate leg positioning associated with recumbent position in our study. Another study21 found nonsignificant differences in behaviors such as lying down and grooming between different analgesic conditions.21 In our study, there was no significant change in lying down over time or between groups.
One of the animals developed incisional dehiscence. The dehiscence was noted after the measurements for fecal output, appetite, blood glucose, fecal cortisol, and behavior had been completed, so there was likely not a major impact on those results. Although the animals were apparently healthy at the start of the study and were specific-pathogen free for several organisms, several of them developed urinary signs following the procedures. Given that both the insufflation and control groups were affected, it is not thought that this finding had a major influence on the results.
One limitation of this study is the fact that the insufflation period was only 30 minutes. It is possible that more significant changes may have been seen with a longer insufflation period.29,40 This timeframe was chosen to reflect a typical insufflation period that may be performed in a guinea pig in clinical practice. We previously found that the amount of time it takes to perform a laparoscopic procedure in a guinea pig decreases once experience is gained with the procedure.13 Due to their higher risk of anesthetic death compared to dogs, cats, and rabbits,41 prolonged anesthesia is generally avoided in guinea pigs. In addition, the differences between the 2 groups may have been more significant at higher pressure, but a pressure of 6 mm Hg was chosen because higher pressures may have deleterious cardiorespiratory effects and are likely not necessary in guinea pigs in a clinical setting. Another limitation is that the procedures did not all take place at the exact same time during the day, resulting in some of the guinea pigs having slightly less time to recover prior to postoperative measurements. However, this was accounted for by the order of randomization. Another potential weakness is that baseline behaviors at T0 prior to anesthesia were not evaluated. It is unlikely that the inclusion of baseline behaviors would have impacted the results of the study, however.
In conclusion, insufflation did not significantly affect appetite, fecal output in grams, blood glucose, fecal cortisol, and behavior in guinea pigs for the vast majority of parameters and time points. Pneumoperitoneum at an IAP of 6 mm Hg for 30 minutes did not have any major adverse effects on the guinea pigs in terms of pain, stress, or gastrointestinal motility. The use of pneumoperitoneum at 6 mm Hg could be reasonable for laparoscopic surgeries in guinea pigs. However, it is possible that longer periods of insufflation could have different effects. Future studies are indicated to compare postoperative variables in guinea pigs undergoing laparoscopic versus open surgeries and to evaluate for the impact of longer periods of pneumoperitoneum.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org
Acknowledgments
This study was funded by a grant from the Ontario Veterinary College Pet Trust.
The authors declare that there were no conflicts of interest.
The authors wish to thank Monica Baquero for performing the fecal cortisol testing.
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Appendix
Ethogram of behaviors21 monitored by video recording in guinea pigs having undergone pneumoperitoneum at 6 mm Hg CO2 or anesthesia and abdominal catheter placement alone.
| Behavior | Definition | Numbers of occurrences monitored | Duration monitored |
|---|---|---|---|
| Active Behaviors | |||
| Active movement | Walking in any direction | X | X |
| Body turn | Changing head or body orientation by more than 180° | X | |
| Head or neck movement | Flexing or extending the head or neck | X | |
| Rearing | Standing on hind feet with forefeet off the ground | X | |
| Coprophagy | Moving head toward anus to ingest feces | X | |
| Passive Behaviors | |||
| Eyes closed or squinting | Eyelids closed for longer than blink or eyes not open a normal amount | X | X |
| Piloerection | Bristling of hairs affecting >50% of body surface caudal to ears | X | |
| Weight shifting | Movement of 1 or more feet without gaining distance | X | |
| Subtle body movement | Abdominal contraction, back arching, twitching, movement of jaw, or movement of thorax; behavior occur independent of movement of extremities | X | |
| Incomplete movement | Begins act of coprophagy or grooming but abruptly stops | X | |
| Ingestive | |||
| Eating | Animal picks up and ingests food (hay or pellets) | X | |
| Drinking | Animal drinks water from a bottle | X | |
| Grooming | |||
| Grooming | Guinea pig grooms any part of body other than incision | X | X |
| Licking incision | Guinea pig licks at lower abdomen or pulls at sutures | X | |
| Other | |||
| Lying down | Guinea rests with ventrum on floor of enclosure | X | X |
| Hiding | Guinea pig is hidden under towel and out of view of the camera | X | X |
