Sedation Efficacy of Midazolam in Conjunction with Ketamine and Alfaxalone in Female Laboratory Guinea Pigs (Cavia porcellus)
Guinea pigs have been integral as models used in biomedical research, making significant contributions to nutritional, auditory, immunologic, and hypersensitivity studies, and necessitating the routine need for sedation in laboratory settings. The ketamine-xylazine (KX) combination has been the standard sedation protocol for decades. However, due to the adverse effects and abuse potential of xylazine, this study explores the possibility of substituting xylazine with midazolam and examines the combined use of midazolam with ketamine and alfaxalone in female laboratory guinea pigs. Our findings indicate that KX facilitates the fastest induction and longest duration of sedation compared with other sedatives, including ketamine-midazolam (KM), which, despite its rapid induction, results in significantly shorter sedation durations. KX also ensures a deeper anesthetic depth and greater odds of loss of withdrawal and inguinal reflexes, in contrast to KM and alfaxalone-midazolam (AM), under which only 15% of the animals lost these reflexes. In terms of cardiopulmonary function, KM led to an increased heart rate attributed to elevated sympathetic activity. All 4 sedative protocols lead to respiratory depression, except KM, which causes minimal reduction. Adverse events varied, with 75% of animals experiencing injection site reactions after KX administration and 67% exhibiting regurgitation post-KM administration. No adverse events were reported for the AM combination, suggesting its safer profile. In conclusion, while KX remains the superior protocol for sedation due to its efficiency, reliability, and minimal impact on physiologic parameters, midazolam is not a preferable alternative to replace xylazine. Its increased sympathetic tone, hyperesthesia, and shorter action duration, coupled with a higher potential for adverse events, limit its suitability to combine with ketamine in guinea pig sedation. However, when midazolam is used in conjunction with safer alternatives like alfaxalone, it presents a viable sedation strategy, emphasizing the need for further research into optimizing sedative combinations for laboratory guinea pigs.Abstract
Introduction
In the field of laboratory animal medicine, especially with small mammals such as guinea pigs, selecting the right sedative is crucial for ensuring the animals’ welfare during experiments or medical procedures. Sedating guinea pigs is particularly challenging due to their distinctive anatomic and physiologic characteristics, along with their increased risk of stress-related complications.10,30 It is recommended to use injectable sedative agents that are short acting, have a broad safety margin, and are reversible. A common approach for sedating laboratory animals, including guinea pigs, involves subcutaneous or intramuscular injections, especially when inhalation anesthesia is not feasible or intubation is difficult due to the animal’s small size and narrow oral cavity.10,11
Ketamine is a commonly used anesthetic for guinea pigs, particularly for minor procedures, and as a component of combination anesthesia regimens to achieve a safe and effective sedation level. Complementing ketamine, xylazine, an α2-adrenergic agonist, provides additional muscle relaxation, sedation, and analgesia.3,5,14 However, use of xylazine is not without concerns, as it can induce significant cardiovascular issues such as bradycardia, arrhythmias, and respiratory depression, although its effects are reversible with α2-adrenergic antagonists like atipamezole, enabling swift recovery.3,14–16,31,37 In addition, the administration of a ketamine-xylazine (KX) mixture in rodents has led to significant hypothermia and conditions like dry eye (keratoconjunctivitis sicca) and hyperglycemia.10,16,18 Muscle necrosis has also been reported in many small animals, such as guinea pigs, following intramuscular injections.10,27,37 While the KX combination offers some advantages, its association with adverse effects underscores the need for safer sedative alternatives. An additional complicating factor is that the potential for abuse of xyalzine threatens its availability.12 The growing awareness of its misuse and association with illicit use could prompt regulatory bodies to enforce stricter controls on xylazine. Such restrictions, while aimed at preventing abuse, could inadvertently hinder veterinarians’ access to this valuable medication, known for its sedative, analgesic, and muscle relaxant properties.
Amid these challenges, benzodiazepines, such as midazolam, are a viable alternative to xylazine, offering a better safety profile, reduced likelihood of adverse reactions, and a low risk of misuse.23,24 Midazolam and diazepam exert their effects via the γ-aminobutyric acid (GABA) neurotransmitter system and have been effectively used in rodents and guinea pigs in combination with other sedatives.1,10,22,27 Midazolam is noted for its quick onset of action, leading to rapid sedation, whereas diazepam provides a more prolonged effect due to its high lipid solubility and extended half-life.10,23,24 Compared with diazepam, midazolam is associated with less discomfort and irritation since it is more water soluble. The absorption of diazepam following intramuscular administration is slow and unpredictable, affected by factors such as the muscle mass involved, limiting its use to intraperitoneal routes in small animals.10
Midazolam’s effectiveness as a sedative is highlighted by its substantial anxiolytic, muscle relaxant, and anticonvulsant benefits, making it invaluable for preprocedural sedation, anesthesia induction, and seizure control in veterinary and laboratory settings.10,22,24,34 Its rapid onset and short action duration, paired with the reversibility offered by flumazenil, establish midazolam as a flexible and manageable option for a variety of clinical scenarios.10,20,23,24 In research models involving rodents, rabbits, and pigs, the combination of midazolam with sedatives like ketamine has been effective in rapidly inducing a calm state crucial for short-term procedures, leading to swift recovery and minimal cardiopulmonary complications.10,22,34 This underscores the advantages of opting for midazolam over xylazine, especially in terms of quick sedation onset, ease of sedation depth management, and a better safety profile for maintaining respiratory and cardiovascular stability across different laboratory animal sedations.
Administration of midazolam alone has proven problematic, as it led to an exaggerated sensitivity to touch and noise in laboratory guinea pigs.33 Yet, when combined with alfaxalone, it effectively produced gentle sedation suitable for minor procedures in pet guinea pigs.1 Alfaxalone, a neuroactive steroid that enhances the action of γ-aminobutyric acid A receptors, leading to muscle relaxation and anesthesia, has gained favor for its use in sedating and anesthetizing laboratory animals.4,7,10,32,36 However, since alfaxalone lacks analgesic properties, using it alongside α2-adrenoceptor agonists and/or opioids can enhance anesthesia quality, prolong analgesia, and minimize the alfaxalone dose and volume needed.7,10,25 While alfaxalone by itself offers only mild sedation to guinea pigs, its use in combination with benzodiazepines and an opioid significantly extends the sedation duration and immobility without inducing general anesthesia.2,7,17,19 Our prior research highlighted the sedation quality and physiologic benefits of this mix in laboratory guinea pigs.2 Nonetheless, information on using midazolam as an alternative to the traditional KX regimen or in combination with alfaxalone in laboratory guinea pigs is scarce. Our goal was to conduct a systematic evaluation of these sedative combinations to discover more effective and safer methods for sedation. Therefore, this study was designed to compare the efficacy of a sedation protocol combining midazolam with ketamine and alfaxalone against the traditional KX mix for sedating female laboratory guinea pigs. By making this comparison, we aim to improve animal welfare and the reliability of experimental outcomes in laboratory settings involving guinea pigs.
Methods
Animals.
In our study, we used total of 32 healthy female Dunkin-Hartley guinea pigs each aged 10 to 11 mo and weighing an average of 876.5 ± 114.22 g, obtained from Charles River (Charles River Laboratories, Montreal, Canada). Before their arrival, these guinea pigs underwent extensive screening at the supplier to certify their absence from a broad spectrum of pathogens and parasites. This evaluation included, but was not limited to, Sendai virus, reovirus, lymphocytic choriomeningitis virus, as well as both ectoparasites and endoparasites. The guinea pigs were housed in caging systems designed for guinea pigs (Allentown, Allentown, NJ), maintaining a group size of 3 to 5 per cage. This setup adhered to Yale University’s housing standards. The diet consisted of Teklad global high-fiber guinea pig food (Inotiv, Boston, MA), supplemented daily with hay, fresh fruits, and vegetables, and unrestricted access to water. Environmental conditions were controlled to a 12:12-h light-dark cycle, with temperatures held at 70 ± 2 °F (21 ± 22 °C) and humidity levels between 30% and 70%. Enrichment was provided via plastic tunnels.
Before this study, the selected guinea pigs were part of an IACUC-approved long-term research project centered on immunization against tick proteins, which included exposure to pathogen-free nymphs. The preference for female guinea pigs is attributed to their docile nature and minimal aggression, advantageous for studies involving prolonged cohabitation and ensuring a more controlled, safer research environment. These guinea pigs, initially slated for euthanasia, were reassigned to our study under a new protocol, thereby extending their contribution to scientific research. Our study’s experimental protocols were approved by Yale University’s IACUC, affirming our adherence to AAALAC standards and federal guidelines governing animal research. All animals used to generate data for both the pilot and main studies were from the same source during the same time period.
Animal Preparation and Sedation
Animal sedation.
On the day of the sedation procedure, the guinea pigs’ weights were determined using a Tanita digital scale ([KD-321 Digital Kitchen Scale], Arlington Heights, IL). The animals underwent a fasting period of 1 h before sedation, during which their mouths were cleaned with a 10-mL saline rinse (normal saline; Hospira, Lake Forest, IL). A combination dose of ketamine (Ketathesia; Covetrus, Dublin, OH), xylazine (Anased; Akorn, Lake Forest, IL), alfaxalone (Alfaxan Multidose; Jurox), and midazolam (Heritage Pharmaceuticals, Eatontown, NJ) was administered subcutaneously (A and AM) using a 27-G needle into the multiple sites of the dorsal neck or intramuscular (KX and KM) into the right or left semimembranosus muscles. The drugs were administered in their undiluted forms, with the total injection volume ranging between 0.4 and 3 mL, depending on the specific drug combination used and the animal’s weight. The current formulation of alfaxalone, with doses of 20 mg/kg (A20) to 40 mg/kg (A40), exceeds the permissible volume for intramuscular injection (up to 3 mL). Therefore, we administered alfaxalone via the subcutaneous route.
To accurately measure the induction time, each guinea pig was sedated individually, ensuring a stress-reduced environment for the sedation process. The animals were isolated in separate cages and distanced from their companions, and the cages were covered to minimize sensory exposure, thus reducing the impact of tactile, auditory, and visual stimuli. The period from the administration of the sedative until the guinea pig could no longer right itself, making no effort to return to a normal stance, was recorded as the induction time.
Sedation quality.
We continuously monitored the onset and depth of sedation every 5 min, adopting the standardized scale to assess sedation depth (Table 1).2,27 This scale ranged from 0, indicating normal behavior, to 5, which represents a complete lack of movement and response. We assessed the highest sedation level achievable for each drug combination using the scoring chart. Only the drug-dose combination that achieved a sedation score of 5, at the highest sedation level, was considered an appropriate sedative.
| Criteria evaluated | Sedation depth score |
|---|---|
| Normal coordinated movement with immediate sternal recumbency | 0 |
| Coordinated movement but takes 5–10 s to regain sternal recumbency. Decrease response to manipulation (movement/noise) | 1 |
| Uncoordinated movement with attempted, but failed efforts to regain recumbency. Drowsy, floppy and response to manipulation Pedal/inguinal reflex present | 2 |
| Lateral recumbency. Animal does not attempt to reposition and some response to manipulation. Infrequent and uncoordinated movement. Pedal/inguinal reflex present | 3 |
| Dorsal recumbency. Infrequent, weak, uncoordinated movements with minimum response to manipulation Pedal/inguinal reflex present | 4 |
| No movement observed, lies in dorsal recumbency and is unresponsive to manipulation. Pedal/inguinal reflex present or absent | 5 |
Experimental Design
Pilot study.
To determine the initial dose for sedative use, we conducted a pilot study to identify the most effective sedative combination, allocating 2 animals to each group. The doses for this pilot study were derived from both our published data and that of other researchers, as well as from commonly used sedative practices in pet guinea pig care across Europe (Table 1).1,2,6,7,21 We explored various doses of the KM combination and AM, in addition to alfaxalone alone, to ascertain the optimal mix (Table 2). We adopted the widely used KX dose of ketamine at 40 mg/kg and xylazine at 4 mg/kg (KX) as our benchmark for comparison against other combinations.5,10 This regimen is known to yield 30 to 90 min of dependable sedation with minimal adverse effects. Among the tested drug combinations, we selected those that most closely matched the KX sedation duration and minimized side effects. The inclusion of an alfaxalone-alone group allowed us to evaluate the potential benefits of adding midazolam to alfaxalone in terms of sedation quality.
| Drug combination (n = 2) | Induction time (min) | Sedation depth score | Reflexes (pedal + inguinal) | Time to recover (min) |
|---|---|---|---|---|
| A20 | 12 ± 2 | 4 | + | 82.5 ± 10.6 |
| A40 | 5 ± 1 | 4 | + | 118.5 ± 10.6 |
| A15M2 | 18 ± 3.5 | 4 | + | 106.8 ± 13.8 |
| A20M2 | 14 ± 1.53 | 4 | + | 137.5 ± 5.5 |
| K15M0.5 | 5 ± 1 | 4 | + | 42.5 ± 4.2 |
| K15M1 | 5 ± 1 | 5 | + | 90 ± 15.5 |
| K20M2 | 4 ± 1 | 5 (n = 1:euth*) | + | 160.32 ± 5.4 |
Each dosage was administered to 2 animals. Recovery time was determined from the moment of loss of righting reflexes until the animal regained its righting reflex twice and was able to ambulate normally. A20, 20 mg/kg alfaxalone; A40, 40 mg/kg alfaxalone; A15M2, 15 mg/kg alfaxalone and 2 mg/kg midazolam; A20M2, 20 mg/kg alfaxalone and 2 mg/kg midazolam. K15M0.5, 15 mg/kg ketamine and 0.5 mg/kg midazolam; K15M1, 15 mg/kg ketamine and 1 mg/kg midazolam; K20M2, 20 mg/kg ketamine and 2 mg/kg midazolam. *One animal from K20M2 was euthanized, and another was substituted to complete the study.
Initial findings revealed that the ketamine at 15 mg/kg and midazolam at 1 mg/kg (K15M1) combination closely resembled the KX sedation effects but led to tachycardia and regurgitation in all subjects, requiring the administration of supplemental oxygen and oral cavity cleaning, thus deeming it unsuitable for our comparative study. The ketamine at 15 mg/kg and midazolam at 0.5 mg/kg (K15M0.5) showed no adverse effects, yet the sedation lasted approximately 45 min. In contrast, the ketamine at 20 mg/kg and midazolam at 2 mg/kg (K20M2) resulted in a fatality due to aspiration, and an additional animal was substituted to complete the study. There were no adverse reactions observed with any alfaxalone dosages or their combinations, with A20, A40 and 20 mg/kg alfaxalone and 20 mg/kg alfaxalone and 2 mg/kg midazolam (A20M2) showing similar sedation characteristics to KX in terms of recovery time and depth. Therefore, we selected K15M0.5 and A20M2 to be compared against the standard KX regimen, and A40 against A20M2, focusing on assessing sedation timing, duration, quality, and physiologic impacts to identify the optimal sedative protocol for laboratory female guinea pigs.
Sample size.
Preliminary calculations for determining the necessary sample size were conducted using G*Power software (version 3.1.9.7; Heinrich-Heine University Düsseldorf, Düsseldorf, Germany). Sample size analysis using time to sedation as a variable, it was determined that each group should consist of 7 to 8 animals, totaling 28 to 32 animals to achieve an 80% power level. Therefore, we selected a total of 32 animals randomly divided into 8 animals per group for the 4 sedative combinations used. Some of these animals had already been used in the pilot study, and they were given a washout period of at least 7 d before being included in the main study. The process of randomization was meticulously handled to prevent bias; the investigator was blind to the drug combinations, which were numerically labeled by another individual tasked with drug preparation but not involved in evaluating the sedation effects.
Monitoring and Recovery
Following the loss of their righting reflex, the guinea pigs were carefully positioned on a veterinary heating system equipped with a 50-W heating pad preheated to 40 °C (105 °F) (Hot Dog Warming; Augustine Surgical, Eden Prairie, MN), and they remained there throughout their recovery. A fleece towel was in addition wrapped around the animals for extra warmth. To protect their eyes during sedation, eye lubricant (Dechra, Overland Park, KS) was administered. Monitoring equipment was attached within 5 min following the loss of righting reflex to keep track of cardiopulmonary functions and body temperature, which were checked every 15 min. Respiratory rates were visually assessed by counting breaths per minute. To monitor oxygen saturation levels (SpO2) and heart rate, a mouse pulse oximeter probe (MouseSTAT Jr; Kent Scientific, Torrington, CT) was affixed to the interdigital area of a forelimb. Core body temperature was measured using a lubricated fiberoptic probe inserted rectally. Blood pressure was gauged noninvasively using a size one cuff placed on the right thoracic limb, connected to an oscillometric measuring device (Jorvet, Loveland, CO). The same individual monitored sedation parameters to maintain consistency and prevent variability. The withdrawal reflex was evaluated by clamping a mosquito forceps to a toe at the interphalangeal junction level, while inguinal reflexes were assessed by applying digital pressure to the inguinal region, as previously described.2,7 This monitoring was consistently carried out by the same individual until the guinea pigs regained their righting reflex. Following the procedure, we ensured the guinea pigs were kept warm and monitored until they fully recovered. The time to recover is defined as the period until the guinea pig can right itself on 2 separate occasions, as previously detailed.2
Statistical Analysis Method
For all the statistical analyses, we used the Matlab R2023b distribution of the Statistics and Machine Learning Toolbox (The MathWorks, Natick, MA). For the comparison between sedation and recovery times, we considered the samples drawn from the 4 treatment methods to be independent from each other. All the continuous variables that were compared between the groups take only positive values, and each treatment was administered for less than 10 animals. Therefore, the sedation and recovery data were assumed to take a skewed distribution. We tested this assumption using boxplots and the Shapiro-Wilk test whenever appropriate. We also tested the homogeneity of variance using the Levene test to choose an appropriate ANOVA test. With the skewed distributions, we employed the Kruskal-Wallis test to test the null hypothesis that the distribution underlying the samples from the treatment groups was the same, against the alternative that the distributions were stochastically different. When we considered categorical variables (reflexes) and the sample size of any outcome was small, we used the Fisher exact test. Otherwise, we use the χ2 test.
Whenever we collected measurements over time points, treating the animals in each treatment group as repeated measurements, we used repeated measures analysis of variance to check whether there was a significant effect of time or significant treatment-time interactions among the measurements. Even though measurements were collected over 150 min for 3 groups, one group had a shorter duration before recovery. Therefore, we used only the measurements up to the 75-min mark to ensure that there were sufficient data points for each time interval and had full column rank for the repeated measures ANOVA test. We used the Mauchly test for sphericity to check the null hypothesis that the sphericity assumption holds for each treatment group. If the compound symmetry assumption failed, we used Greenhouse-Geisser or Huynd-Feldt corrections to adjust the P values. We simply indicated this as the adjusted P value described in the section Statistical Analysis Results. When we failed to reject the null hypothesis for the ANOVA test (P > 0.05), we explicitly stated the result to conclude the relevant analysis. Otherwise, we did not explicitly provide the preceding test results (Levene test, Mauchly test, or repeated measures ANOVA test). Instead, we reported the comparison test results as explained below.
Whenever we rejected the null hypothesis for the treatment-time interactions, we used pairwise comparison results with either the Games-Howell procedure or Tukey honestly significant difference procedure with unequal sample sizes (Tukey-Kramer) to identify the pairwise distinctions. If the homogeneity of variances or the sphericity assumptions failed, we used the Games-Howell procedure, and otherwise, we used the Tukey-Kramer procedure. When we compared 2 samples from different groups with a specified direction, we used a one-tailed Wilcoxon rank-sum test. Whenever there was a significant effect of time, we took paired samples from the same group to check time dependency using the Wilcoxon signed-rank test.
Results
Sedation and duration.
The average induction times were similar between the KX and KM groups, with 3.14 ± 0.64 min and 3.13 ± 0.83 min, respectively. The AM and A groups had longer average induction times, 6 ± 2.67 min and 5.38 ± 1.3 min, respectively. Using the Kruskal-Wallis test, we concluded that the distribution underlying the samples from the different treatments was stochastically different (P = 0.0004). The Games-Howell procedure indicated that there was no distinction between KX and KM or AM and A (P = 1 and 0.9324, respectively). Using one-tailed Wilcoxon rank-sum tests, we concluded that the distributions underlying KX and KM are stochastically less than the distributions of both AM and A (Figure 1). KX and KM produce rapid induction compared with the other 2 methods.
Citation: Journal of the American Association for Laboratory Animal Science 63, 5; 10.30802/AALAS-JAALAS-24-000028
The average time for recovery showed an increase in the following order: KM (44.9 ± 8.7 min), A (104.8 ± 23.2 min), AM (108.8 ± 42.0 min), and finally KX (137.8 ± 26.0 min). Using the Kruskal-Wallis test, we concluded that the distribution underlying the samples from the different treatments is stochastically different (P = 0.0002). The Games-Howell procedure indicates that there was no distinction between AM and A (P = 0.9958). Pairwise comparisons using one-tailed Wilcoxon rank-sum tests showed that the distribution underlying the recovery duration for KX is stochastically greater than the other methods and the recovery duration for KM is stochastically less than the others (Figure 1). Faster induction and longer sedation duration were achieved using KX. While KM produces a fast induction, it was unable to provide a long sedation duration.
Loss of reflexes.
Every sedative combination successfully reached a sedation score of 5 for all animals at the highest level of sedation. All animals, with one exception (88%), subjected to the KX regimen lost both withdrawal and inguinal reflexes as evaluated by the methods previously described. On average, they remained in a state of anesthesia for approximately 60 ± 23.4 min. In contrast, animals treated with the KM regimen retained their withdrawal and inguinal reflexes, demonstrating localized responses to pressure applied to the toe or inguinal area and exhibited hypersensitivity to touch and noise. Due to their reactive state during sedation, it was not feasible to apply the pressure cuff, yet they maintained a sedation score of 3 or 4 throughout the period. Only 12% of the animals experienced a brief loss of reflexes for about 15 min under the AM sedation and none under A regimens. Thrity-seven percent of the animals under AM sedation showed intermittent vocalization and twitching of the hind legs. To analyze the outcomes related to the loss of reflexes, we used a right-tailed the Fisher exact test. With P < 0.0001, it was concluded that subjects under the KX regimen had significantly higher odds of losing reflexes compared with those in other groups during sedation.
SpO2 and temperature.
SpO2 ranged from 89% to 100% at all time points. This did not significantly differ from each other with any of the sedative agents (P = 0.539 and P = 0.621).
The KX group exhibited lower body temperature during the sedated period (36.93 ± 1.36 °C) compared with the others (37.46 ± 0.92 °C for KM, 37.46 ± 1.47 °C for AM, and 37.57 ± 0.77 °C for A). Using the Games-Howell procedure, we concluded that there was a significant difference between KX and the other groups (P = 0.0053 with KM, P = 0.0464 with AM, and P = 0.0016 with A), and there was no significant difference between any other pair combination (P > 0.8) (Figure 2). Using minute 5 observations as the baseline, we observed that the mean body temperature of the KX group was reduced by about 0.3 °C during the first 75 min (from 37.4 to 37.1 °C). This period coincides with most of the observations of no reflexes. In contrast, the mean body temperatures of both the KM and AM groups increased, by 1.6 and 0.6 °C, respectively, and stayed the same for the A group. After the first 75 min, we observed that there was no significant difference between body temperature for any pairwise comparison between KX, AM, and A (P > 0.6). KX resulted in a marginal reduction of mean body temperature, whereas KM was more likely to increase mean body temperature.
Citation: Journal of the American Association for Laboratory Animal Science 63, 5; 10.30802/AALAS-JAALAS-24-000028
Heart rates and respiratory rates.
Mean heart rates were higher for the KM group (327.7 ± 44.7 beats/min) compared with the other groups: 187.1 ± 12.2 beats/min for KX, 232.1 ± 32.7 beats/min for AM, and 259 ± 31.1 beats/min for A. However, the mean heart rates stayed relatively constant from one time measurement to another (Figure 3). In fact, the adjusted P values for repeated measures ANOVA indicate that there was neither a significant effect of time on heart rate measurements (P = 0.0944), nor a significant treatment-time interaction (P = 0.0629). The evidence failed to suggest that the heart rates have any explicit relationship to the sedatives.
Citation: Journal of the American Association for Laboratory Animal Science 63, 5; 10.30802/AALAS-JAALAS-24-000028
The mean respiratory rates were highest for KM (81.8 ± 6.8 breaths/min) and lowest for AM (67.7 ± 10.2 breaths/min) compared with the other groups (74.8 ± 4.8 breaths/min for KX and 74.1 ± 7.2 breaths/min for A). We conclude that the KM group indeed had a statistically higher respiratory rate (Games-Howell: P = 0.0011 with KX, P = 0.0007 with AM, and P = 0.0012 with A) and AM had a statistically lower respiratory rate than the other groups (Games-Howell: P = 0.0014 with KX, P = 0.0007 with KM, and P = 0.0013 with A). During the first 15 min, respiratory rates were similar for all the sedatives. However, the distributions were stochastically different by the 30-minute observations. Using right-tailed signed rank tests, we had sufficient evidence to establish that KX, AM, and A all decrease respiratory rates during the first hour (see Table 2 for P values). The administration of sedatives reduced the respiratory rates; however, that reduction was minimal in KM, with a mean reduction from 83.75 to 81.43 breaths/min during the first 30 min, and the reduction is maximal for AM, with a mean reduction from 77.88 to 61.88 breaths/min. KX and A exhibited a 5.5 and 10.5 breaths/min reduction, respectively, during the same period. Both AM and A reductions were much more significant compared with others.
Systolic, diastolic, and mean arterial pressure.
The blood pressure and mean arterial pressure (MAP) measurements were evaluated only for the KX and AM groups. The mean values were 84.2 ± 12.6 and 81.8 ± 12.2 mmHg for systolic pressure, 29.5 ± 9.6 and 28.0 ± 11.3 mmHg for diastolic pressure, and 47.3 ± 13.4 and 46.8 ± 10.4 mmHg for MAP, for KX and AM, respectively. None of the measurements significantly differ between the 2 groups. In fact, the adjusted P values for repeated measures ANOVA indicated that there is neither a significant effect of time on measurements, nor a significant treatment-time interaction for systolic pressure (P = 0.8205 and 0.8230), diastolic pressure (P = 0.8863 and 0.2929), and MAP (P = 0.3028 and 0.512).
Adverse events.
All animals were observed daily for 2 wk after the procedure. A significant injection site reaction was noted in 75% of the animals that received KM via intramuscular injections as visually evidenced by thickening, swelling, or scabbing at the injection site and resolved within 7 to 10 d. All animals sedated with the KM combination exhibited hypersensitivity to touch and noise, while still under sedation, often vocalizing and displaying sporadic involuntary movements in their hind legs. Approximately 37% of the animals undergoing AM sedation experienced occasional vocalization and twitching of the hind limbs. Despite a fasting period of 1 h and oral cavity cleaning, 62% of the animals under the KM regimen regurgitated. All animals required supplemental oxygen due to SpO2 levels falling briefly to between 89% and 95% postregurgitation. Nonetheless, these animals managed to return to normal oxygen saturation and recovered without any adverse respiratory effects, as verified through auscultation. The regurgitated material was clear and frothy, lacking any food particles. Remarkably, none of the animals under the KM regimen developed severe respiratory distress necessitating euthanasia, and all recovered well, maintaining good health a month after the study.
Discussion
The process of sedating guinea pigs for laboratory procedures demands a comprehensive understanding of the induction times, durations, and physiologic impacts of various sedative combinations. We investigated the efficacy of using Midazolam as an alternative to xylazine in traditional KX formulations, comparing it against KM and AM combinations. KX sedation led to increased sensitivity, involuntary movements, and a significant incidence of regurgitation, although without associated severe respiratory complications. On the other hand, AM demonstrated a longer induction time but minimal cardiopulmonary effects compared with KX, with KM notably leading to increased heart rate with a rapid recovery. The selected sedative protocol profoundly influenced recovery times, with KX and AM combinations facilitating prolonged sedation, which is advantageous for more extended procedures.
Our analysis revealed that the KX and KM drug combinations led to faster induction in comparison to the A and AM combinations. This discrepancy can be attributed to a variety of factors, such as dosage, method of administration, individual animal variability, and the specific sedatives used. A significant factor contributing to the differences in induction times in our study is the administration route; alfaxalone-based combinations required subcutaneous administration due to the larger volumes needed for effective sedation in guinea pigs.7,9,25 Ketamine’s lipid and water solubility facilitates its straightforward preparation in aqueous solutions, suitable for various administration routes, ensuring prompt sedation.10,31,37 Our guinea pigs showed an induction time of 3 to 4 min with ketamine-based combination drugs given intramuscularly, highlighting the efficiency of these formulations in quickly inducing sedation. Similarly, in rodents and rabbits, ketamine, whether used alone or in combination, can act within 2 to 5 min postintramuscular injection.10,11,13,15,16
In contrast, alfaxalone, a neuroactive steroid anesthetic, does not possess inherent water solubility and is commonly formulated in a cyclodextrin-based solution to improve its solubility. Although this modification enhances absorption at the site of injection, it does not inherently guarantee quicker induction times.25 In our study, guinea pigs demonstrated induction times with alfaxalone and AM administered subcutaneously within the 5- to 8-min range, compared with pet guinea pigs where it ranges from 2.4 to 5 min.1 This variation could stem from individual animal responses and differences in induction methods (subcutaneous compared with intramuscular), as well as environmental acclimation, noting that pet guinea pigs, more accustomed to handling, may exhibit less stress than laboratory-raised guinea pigs, affecting sedative efficacy. Our findings suggest that for rapid sedation onset in guinea pigs, KX and KM combinations are more effective, despite the potential side effects associated with KM. This result reinforces the importance of choosing sedation protocols carefully, taking into account the specific responses of different species and the requirements of the procedure while considering factors such as rapid onset, duration of sedation, and minimal physiologic impact to ensure the welfare and safety of guinea pigs in laboratory settings.
The KM combination resulted in the shortest recovery times, averaging 44.9 ± 8.7 min. In stark contrast, the KX combination leads to the longest duration of sedation, with recovery times averaging 137.8 ± 26.0 min. The half-life of a drug, indicating the time required for the drug’s concentration in the bloodstream to halve, is a key pharmacokinetic parameter that influences both the duration of its action and the recovery time postsedation. This parameter varies with factors such as dose, species, individual metabolism, route of administration, and the specific drug combinations used.10 In guinea pigs, the effects of alfaxalone and its combinations with midazolam (AM) are dose dependent. In our study, intermediate recovery times were noted for both the AM and A regimens, averaging 108.8 ± 42.0 min and 104.8 ± 23.2 min, respectively, when administered at doses of 40 mg/kg for alfaxalone and 20 mg/kg for alfaxalone combined with midazolam. Conversely, a dose of 20 mg/kg alfaxalone administered subcutaneously resulted in a shorter recovery period of approximately 75 min in laboratory guinea pigs, indicating its appropriateness for shorter procedures.7 Pet guinea pigs exhibited even quicker recovery times, with 36.70 ± 10.80 min for A and 62 ± 15.70 min for AM, using a lower dose of 5 mg/kg alfaxalone.1,7,9 These findings highlight the impact of dosage variations on sedation outcomes. On the other hand, xylazine, with a generally longer half-life compared with the shorter-acting midazolam, results in prolonged sedation periods. In rodents and rabbits, the KM combination typically yields quicker recovery times than KX, likely due to midazolam’s shorter half-life facilitating a faster return to normal function compared with xylazine’s extended sedative effects.10,13 Therefore, KM sedation allows for faster recovery than KX, beneficial for research or procedures requiring minimal downtime. However, the KM combination in guinea pigs, despite its shorter sedation duration, warrants careful consideration of potential side effects related to an increased sympathetic tone.
Previous studies have noted that midazolam can induce excitatory behavior in guinea pigs when used as a sole sedative agent, leading to excessive responses to noise and touch. However, this hypersensitivity was not evident when Midazolam was combined with other drugs such as alfaxalone, medetomidine, or fentanyl.1,2,27,28 Ketamine’s antidepressant properties, attributed to its ability to enhance excitability by diminishing GABAergic inhibition and boosting nonNMDA transmission through increased endogenous glutamate release, could stimulate the motor cortex.8 Midazolam has been known to cause paradoxical behaviors like excitation, panting, pacing, restlessness, and vocalization in dogs and some people, suggesting guinea pigs might exhibit similar reactions when midazolam is used alone or in combination with ketamine.30,35 All animals on the KX regimen displayed hypersensitivity to touch and noise while remaining sedated, often vocalizing and showing occasional involuntary movements in their hind legs. In dogs, behavioral changes were less pronounced when midazolam was administered following alfaxalone induction.38 Similarly, in our study these effects were not observed with alfaxalone, except for occasional vocalization and hind limb twitching in about 37% of the animals under AM sedation. The synergistic effect observed when combining ketamine with midazolam presents an interesting avenue for further investigation.
The response of guinea pigs to the KX regimen has been found to be both unpredictable and, at times, inadequate for reducing reflex responses and reaching a surgical plane of anesthesia.5,10 Nevertheless, in our study, 88% of the animals exposed to the KX protocol experienced a loss of both withdrawal and inguinal reflexes for an average of 60 min. In contrast, under AM sedation, only 15% of the animals showed a loss of both reflexes, lasting for approximately 15 min. However, without an actual surgical incision, it remains uncertain if the loss of pedal and inguinal reflexes accurately indicates an adequate depth of anesthesia in this species.2,20 None of the animals subjected to the KM regimen experienced a loss of reflexes; instead, they exhibited exaggerated sensitivity reactions to touch. Furthermore, 62% of the animals regurgitated despite having fasted for 1 h and receiving mouth cavity cleaning. Ketamine is known to induce nausea and vomiting, symptoms similar to those of vestibular disturbances, in some dogs and humans, a reaction that has also been observed with midazolam administration.23,24,30,35 It remains uncertain whether the regurgitation observed in guinea pigs is related to this phenomenon synergically affecting them or is a result of hyperesthesia leading to vomiting. Considering that midazolam typically possesses antiemetic properties, which help prevent postoperative vomiting, further research is necessary to determine whether incorporating an antiemetic into the KM mixture could prevent regurgitation in these animals.
Respiratory depression was noted in animals sedated with the KX combination, attributable to xylazine’s respiratory depressant effects.29 Xylazine decreases respiratory rate and can change respiration patterns by reducing the brain’s respiratory center sensitivity to carbon dioxide.4 While xylazine significantly depresses respiratory function, this effect may be exacerbated by ketamine.3,10 However, in small mammals, the impact is less pronounced due to their rapid metabolism and the smaller doses used.10 Midazolam offers a safer alternative with respect to respiratory effects, with our study revealing differing impacts when combined with ketamine or alfaxalone. KM combination unexpectedly increased respiratory rate, which might be explained by regurgitation events increasing respiratory rate and effort or by a cumulative excitement effect. In contrast, animals administered the AM combination showed decreased respiratory rates, possibly because midazolam enhanced alfaxalone’s respiratory depressant effect in a dose-dependent manner.
Cardiovascular parameters generally showed no significant changes across different drug combinations in the studies described here. However, the KM group exhibited a notably higher heart rate compared with other groups, and animals administered KX demonstrated the lowest heart rates. Animals administered alfaxalone and AM maintained heart rates closer to the average baseline resting heart rate of similar weight, laboratory female guinea pigs.26 Midazolam’s potential for inducing peripheral vasodilation can lead to decreased blood pressure and variably affect heart rate, potentially causing it to decrease, increase, or remain unchanged, depending on the species and dosage.23,24 Conversely, ketamine usually increases both blood pressure and heart rate through its sympathomimetic effects, stimulating sympathetic outflow and inhibiting norepinephrine reuptake.10,32,37 The combination of these drugs tends to stabilize cardiovascular profiles more than when each is used alone, with midazolam’s vasodilatory effects potentially counteracting ketamine’s cardiovascular stimulatory effects.10 The observed higher heart rate in the KM group may result from the midazolam’s sensitivity and excitement effects in this species, synergistically elevating the heart rate. On the other hand, xylazine, known for its potent cardiovascular depressive effects, leads to a lower heart rate when combined with ketamine, mirroring observations from other guinea pig studies.3,14,27,29 However, alfaxalone, which does not significantly depress cardiovascular responses in these animals, maintains or slightly increases heart rate at resting levels. When combined with midazolam, this mixture did not significantly alter heart rate, keeping it just above the basal resting rate of female guinea pigs.26
Blood pressure measurements were conducted exclusively for the KX and AM regimens, as the animals sedated with the KM combination displayed hypersensitivity to touch, rendering the application of the blood pressure cuff challenging. Nevertheless, there was no significant difference observed between the blood pressure readings of the KX and AM groups. Overstimulation or excitement induced by KM regimen may have also increased the core body temperature in these animals. However, core body temperature was maintained across all groups with adequate thermal support, except for those animals in the KX group, which experienced slight hypothermia. This discrepancy can also be attributed to the KX combination achieving a greater surgical plane of anesthesia more than the other sedative regimens. The KX regimen’s tendency to marginally lower body temperature emphasizes the need for extra monitoring and possible interventions to preserve thermal balance during prolonged procedures.10,31,37
A notable injection site reaction was observed in 75% of the animals receiving KM through intramuscular injections, a known adverse effect of this drug combination in rodents and guinea pigs.6,10,37 This suggests an advantage for administering this combination via the intraperitoneal route.6 However, the relatively large cecum of guinea pigs makes the intraperitoneal injection method challenging and increases the risk of improper injections, thereby supporting the intramuscular route as a safer approach. In contrast, the alfaxalone or AM combinations did not produce any adverse reactions, indicating these as safer options among the tested drug combinations.
The significance of our findings highlights the differential response of guinea pigs to sedatives, a phenomenon that has been observed in both female and male guinea pigs subjected to various sedation and anesthetic protocols.10,11 Including male animals in our study cohort could have provided additional valuable insights into these differences. However, our study design focused on the female guinea pigs already present at our institution and scheduled for euthanasia. This approach allowed us to repurpose these animals and avoid recruiting additional subjects, aligning with the 3R (Replacement, Reduction, and Refinement) principle aimed at minimizing animal usage. By doing so, we not only ensured the ethical use of animals but also made practical and efficient use of available resources.
In conclusion, our research aimed to explore the potential for substitution of xylazine with midazolam in the well-established KX sedation regimen that has been used in guinea pig sedation for over two decades. Our results indicate that replacement of xylazine with midazolam in the KX mix falls short as an optimal sedation strategy for reducing pain perception and responsiveness to external stimuli in laboratory guinea pigs. Combining midazolam with alfaxalone effectively extends both the duration of sedation and muscle relaxation in female laboratory guinea pigs. However, the absence of analgesic properties in both drugs represents a limitation, necessitating the need for combination with an opioid as an approach to comprehensive pain management. Despite its adverse side effects, including cardiovascular, thermoregulatory, and injection site reactions, KX continues to stand out as the superior sedative agent for guinea pig sedation.
Boxplots for induction time (A) and recovery time (B) measurements (in minutes) for the 4 groups: ketamin-xylazine (KX), ketamin-midazolam (KM), alfaxalone-midazolam (AM), and alfaxalone (A). The average induction time between KX and KM was not significantly different. However, the induction time was significantly lower in KX compared to AM or A. Similarly, the induction time between AM and A was not significantly different, but it was significantly lower in AM compared to M. The P values for one-tailed Wilcoxon rank-sums tests comparing underlying distributions are given. P < 0.05 was considered significant.
Mean and SD for temperature (°C) and SpO2 (%), over time, showing the overall trends for different drug combinations used in 4 groups: ketamine-xylazine (KX), ketamine-midazolam (KM), alfaxalone-midazolam (AM), and alfaxalone (A). (A) There were no significant differences observed for SpO2 at any time for the different drug combinations used. (B) The KX group exhibited a lower body temperature during the sedated period (36.93 ± 1.36 °C) compared to the other combinations. There was a significant difference between KX and the other groups: *, P < 0.05.
Boxplots for heart rate (in beats per minute) and respiratory rate measurements (in breaths per minute) for the 4 groups: ketamine-xylazine (KX), ketamine-midazolam (KM), alfaxalone-midazolam (AM), and alfaxalone (A) at 5 min (t = 5), 15 min (t = 15), 45 min (t = 45), and 60 min (t = 65). The mean heart rates were higher for the KM group compared to the other groups and lowest for the KX group, but these differences were not statistically significant between treatment groups or at different time points. There is no significant difference between the distributions for respiratory rates at the 5- and 15-min measurements. However, the KM group had a statistically higher respiratory rate and AM had a statistically lower respiratory rate than the other groups which was more apparent for the 30-, 45-, and 60-min measurements: *, P < 0.05.
Contributor Notes