Respiratory Rates and Arterial Blood-Gas Tensions in Healthy Rabbits Given Buprenorphine, Butorphanol, Midazolam, or Their Combinations

Abstract

The objective of this study was to evaluate the respiratory effects of buprenorphine, butorphanol, midazolam, and their combinations in healthy conscious rabbits. Six adult female New Zealand white rabbits were anesthetized briefly with isoflurane by mask to allow placement of a catheter into the central ear artery. After a 60-min recovery period, a baseline arterial sample was obtained. Animals then were injected intramuscularly with either 0.9% NaCl (1 mL), buprenorphine (0.03 mg/kg), butorphanol (0.3 mg/kg), midazolam (2 mg/kg), buprenorphine + midazolam (0.03 mg/kg, 2 mg/kg), or butorphanol + midazolam (0.3 mg/kg, 2 mg/kg). Arterial blood gases were evaluated at 30, 60, 90, 120, 180, 240, and 360 min after drug administration. All drug treatments caused significant decreases in respiratory rate, compared with saline. Buprenorphine and the combinations of midazolam–butorphanol and midazolam–buprenorphine resulted in statistically significant decreases in pO₂. No significant changes in pCO₂ pressure were recorded for any treatment. Increases in blood pH were associated with administration of butorphanol, midazolam, and the combinations of midazolam–butorphanol and midazolam–buprenorphine. In light of these results, buprenorphine and the combinations of midazolam–buprenorphine and midazolam–butorphanol result in statistically significant hypoxemia in rabbits that breathe room air. The degree of hypoxemia is of questionable clinical importance in these healthy subjects. Hypoxemia resulting from these drug combinations may be amplified in rabbits with underlying pulmonary or systemic disease.

Companion animal practitioners and academic anesthesiologists advocate many different combinations of sedatives and opioids to achieve balanced anesthesia. The benefits of premedicating with an opioid–sedative combination is that patient stress is reduced, animal handling is easier, preemptive analgesia can be attained, and inhalant anesthetic requirements are reduced, resulting in less cardiovascular and respiratory depression. In laboratory animals and pet exotic animals, such as rabbits, balanced anesthesia is often foregone in preference to simple mask induction with inhalant agents. In addition, analgesia in these species is often inadequate due to concern about adverse respiratory effects of opioids. Because obtaining a controlled airway in these species can be challenging, pronounced respiratory depression from opioids is an even greater concern. As such, the mixed agonist–antagonist opioids (for example, butorphanol) or partial-agonist opioids (for example, buprenorphine) are often chosen as analgesics or premedications in laboratory animal species, because these classes of opioids may induce less respiratory depression than others.

The majority of the medical literature, both human and veterinary, points to a ‘ceiling effect’ in respiratory depression with agonist–antagonist or partial-agonist opioid drugs as compared with the dose-dependent respiratory depression that may be observed with pure μ-agonist opioids, such as hydromorphone.^6,7,24,26,27 There are conflicting data in the literature, however, regarding the clinical significance of respiratory depression associated with the partial-agonist and mixed agonist–antagonist opioids. One study that examined the respiratory and cardiovascular effects of buprenorphine in rabbits reported mild hypoxemia associated with its administration.²² Another study similarly found that administration of buprenorphine to anesthetized rabbits resulted in hypoventilation but no change in the ventilatory response to hypoxia.⁸ Buprenorphine caused a moderate decrease in respiratory rate in rabbits, but significant respiratory depression as evidenced by respiratory rate was apparent only at dosages higher than those used clinically.¹¹ Butorphanol has not been as extensively studied as a sole agent in rabbits. However, butorphanol and butorphanol combinations are known to result in statistically significant respiratory depression in rabbits, dogs, sheep, horses, and humans.^{15,23-25,27,28} In another study, dogs given butorphanol postoperatively had no change in pO₂ or pCO₂.⁵ Discrepancies in the literature regarding buprenorphine's and butorphanol's effects on respiratory drive likely relate to differences in methodology, sampling, and doses studied. The clinical significance of any changes in blood-gas tensions that occur after buprenorphine or butorphanol in most species is questionable in healthy animals, and most studies conclude that these 2 opioid drugs do not result in clinically unacceptable respiratory depression in most species.

To our knowledge, no studies have examined the concurrent use of opioids and benzodiazepines and their effects on arterial blood gases in rabbits, despite the fact that, when premedication is used, benzodiazepines and butorphanol or buprenorphine are often combined for use in these animals.⁹ Various studies show that benzodiazepines, such as midazolam, cause mild to moderate hypoxemia in rabbits and humans,^5,13,18 but the clinical significance of any additive effect of benzodiazepine-induced respiratory depression when these drugs are combined with butorphanol or buprenorphine is unknown. The combination of benzodiazepines and opioids in rabbits can cause significant sedation.¹⁷ The purpose of the current study was to assess changes in arterial blood-gas values in healthy rabbits given midazolam, butorphanol, or buprenorphine, alone or in combination. We hypothesized that administration of butorphanol, buprenorphine, midazolam, or midazolam–opioid combinations would cause statistically significant changes in respiratory rate and arterial blood gases.

Materials and Methods

Animals.

Six female SPF (Pasteurella multocida, Salmonella spp., Treponema spp., and Tyzzer disease virus excluded) New Zealand white rabbits (weight, 2.8 to 4.8 kg; age, 1 to 3 y) were obtained from Charles River Laboratories (Wilmington, MA). Rabbits were housed in individual stainless steel cages throughout the study, maintained in a controlled environment with a 12:12-h light:dark cycle, and fed ad libitum (Rabbit Chow, Purina Mills, St Louis, MO). Rabbits were acclimated to their housing for 3 wk prior to the beginning of experimentation. The experimental protocol was approved by the Research Animal Care and Use Committee at the University of Wisconsin-Madison, an AALAC-accredited facility. All experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals¹⁶ and complied with federal animal use guidelines.

Study design.

Prior to experimental days, rabbits were assigned randomly (www.randomizer.org) to receive 1 of 6 treatments on each of 6 experimental days. Therefore, each animal received each experimental treatment once in randomized order and served as its own control. Rabbits were transported to the laboratory the morning of experiments and allowed approximately 30 min until induction of anesthesia. Anesthesia was induced with isoflurane (IsoFlo, Abbott Laboratories, North Chicago, IL) in 100% oxygen at 3 L/min by face mask and maintained for 5 to 10 min, depending on the difficulty in arterial cannulation. The central ear artery was cannulated aseptically by using an over-the-needle catheter (22-gauge, Tyco Healthcare, Marshfield, MA) and the catheter securely taped into the ear. The ear used for arterial cannulation was alternated between experiments to allow healing. Rabbits were allowed to recover from the brief anesthesia for a minimum of 1 h prior to experimental treatments described following. Each rabbit had at least a 6-d ‘washout period’ between experimental days.

Experimental procedure.

After complete recovery from isoflurane anesthesia, rabbits were given 1 of 6 drug treatments. All drug combinations were administered intramuscularly in the caudal epaxial musculature. Drug combinations were: 0.9% NaCl, 1 mL; midazolam (Abbott Laboratories, North Chicago, IL), 2 mg/kg; butorphanol (Hospira, Lake Forest, IL), 0.3 mg/kg; buprenorphine (Abbott Laboratories), 0.03 mg/kg; midazolam (2 mg/kg)–butorphanol (0.3 mg/kg); and midazolam (2 mg/kg)–buprenorphine (0.03 mg/kg). All dosages were within ranges recommended by the American College of Laboratory Animal Medicine for use in rabbits.⁹ Volumes of drug injectate ranged from 0.3 to 2.4 mL; no attempt was made to standardize the volume. Temperature, respiratory rate, and arterial blood gas data were recorded at baseline (0 min; prior to drug administration) and at 30, 60, 90, 120, 180, 240, and 360 min after drug administration. In an attempt to minimize changes in respiratory rate and blood-gas parameters associated with handling stress, respiratory rate was assessed by observation first, followed by sampling of arterial blood with minimal restraint and, last, measurement of rectal temperature under manual restraint. All measurements and sampling were performed by 1 of 2 staff members. No attempt was made to measure arterial blood pressure or cardiovascular parameters in experimental animals. Arterial blood samples were removed from the arterial catheter with plastic syringes and transferred into heparinized hematocrit tubes (Chase Scientific Glass, Rockwood, TN). Arterial blood gases were measured by using a calibrated blood-gas analyzer with body temperature correction (ABL800, Radiometer Medical, Brønshøj, Denmark). Samples were held at room temperature until analysis, which occurred within 5 to 20 min of sampling. The temperatures of the laboratory animal housing facility and laboratory where samples were drawn were approximately the same, although no attempts to standardize temperatures were made.

Data analysis.

Mean values of respiratory rate, HCO₃^–, base excess, and temperature-corrected pO₂, pCO₂, and pH were compared with those of the saline control in repeated-measures ANOVA (SigmaStat 4, Aspire Software International, Ashburn, VA). One-way ANOVA was used to compare time points within treatments. The Student Newman–Keuls test was used for posthoc comparisons. Statistical significance was defined as a P value of less than 0.05.

Results

All 6 rabbits recovered unremarkably from their brief isoflurane anesthesia and were alert, responsive, moving, and eating small offerings of hay by 30 min after the discontinuation of isoflurane. Intraarterial catheter placement was associated with relatively few complications, although some of the rabbits developed scarring over the site, and subsequent catheterizations in the same ear became more difficult. Some rabbits developed small hematomas after the catheters were removed, but none of the rabbits developed ear-tip necrosis.

All treatments resulted in noticeable sedation. Signs indicative of sedation included muscle relaxation, sternal or lateral recumbency, lack of resistance to manipulation, closed eyes, and lowering of the head. The administration of butorphanol, buprenorphine, or midazolam alone produced mild sedation for 30 to 60 min, whereas the combination of midazolam and butorphanol resulted in moderate sedation lasting 60 to 90 min. The combination of midazolam and buprenorphine resulted in marked sedation lasting 90 to 120 min. At the 360-min time point, all animals in all treatment groups were sufficiently awake to engage in eating, drinking, and grooming behaviors, with minimal residual sedation.

Measured parameters were evaluated by comparing values with those of saline control groups and with baseline data. This practice allowed rabbits to serve as their own controls for comparing with saline-induced values and analyzing the significance of changes over time when compared with baseline.

Ambient temperature and atmospheric pressure in the laboratory were held constant throughout the experimental period. Baseline rectal temperature of all rabbits ranged from 37.9 to 39.5 °C. Rectal temperatures were significantly (P < 0.05) decreased compared with the saline control values at 120, 180, and 240 min after administration in both the midazolam–buprenorphine and midazolam–butorphanol groups. Temperatures were significantly (P < 0.05) decreased compared with the group baseline value within that group at 120, 240, and 360 min in rabbits treated with midazolam–buprenorphine. Individual nadirs for rectal temperatures were 37.1 °C in the midazolam–butorphanol group and 36.4 °C for the midazolam–buprenorphine group. No statistically significant decreases in temperature were found in other treatment groups.

Baseline respiratory rates ranged from 120 to more than 200 breaths per minute. When compared with values for saline controls, respiratory rate was significantly (P < 0.05) decreased at multiple time points in all treatment groups (Figure 1). Respiratory rates were significantly (P < 0.05) lower than baseline values for multiple time points in the midazolam, buprenorphine, and midazolam–buprenorphine treatment groups (Figure 1 A and B).

Figure 1.

Respiratory rate (breaths per minute; mean ± SE) recorded from 6 rabbits sedated with various sedative drugs as compared with saline controls. +, significant (P < 0.05) difference from value for saline; *, significant (P < 0.05) difference from baseline value. (A) Butorphanol, buprenorphine, and midazolam treatment groups. (B) Midazolam–buprenorphine and midazolam–butorphanol treatment groups.

When compared with saline control values, pO₂ did not differ over time in any treatment group. Baseline pO₂ ranged from 76.2 to 117.0 mm Hg in all groups. pO₂ was significantly (P < 0.05) decreased compared with baseline values at multiple time points in the buprenorphine, midazolam–butorphanol, and midazolam–buprenorphine treatment groups (Figure 2 A and B), with nadirs of 72.4, 73.8, and 74.6 mm Hg, respectively, all occurring at the 30-min time point.

Figure 2.

pO₂ (mm Hg; mean ± SE) recorded from 6 rabbits sedated with various sedative drugs as compared with saline controls. +, significant (P < 0.05) difference from value for saline; *, significant (P < 0.05) difference from baseline value. (A) Butorphanol, buprenorphine, and midazolam treatment groups. (B) Midazolam–buprenorphine and midazolam–butorphanol treatment groups.

pCO₂ did not differ significantly at any time point for any treatment group when compared with saline control or baseline. The pCO₂ of the saline group had a tendency to decrease over the experimental period, whereas that of drug treatment groups had a tendency to remain static or increase over the experimental period. Baseline pCO₂ among all groups ranged from 28.8 to 42.4 mm Hg. At the 30- and 60-min time points, the range of pCO₂ observed among all treatment groups was 25 to 44 and 29 to 46 mm Hg, respectively (Figures 3 A and B). At all times and treatment groups, measured carbon dioxide tensions were highly variable.

Figure 3.

pCO₂ (mm Hg; mean ± SE) recorded from 6 rabbits sedated with various sedative drugs as compared with saline controls. +, significant (P < 0.05) difference from value for saline; *, significant (P < 0.05) difference from baseline value. (A) Butorphanol, buprenorphine, and midazolam treatment groups. (B) Midazolam–buprenorphine and midazolam–butorphanol treatment groups.

Significant (P < 0.05) increases in blood pH were evident when compared with values from saline controls in the midazolam, midazolam–butorphanol, and midazolam–buprenorphine groups (Table 1) and compared with baseline values in the butorphanol, midazolam, and midazolam–buprenorphine treatment groups (Table 1).

Table 1.

Acid–base data (mean ± SE) collected from 6 rabbits given saline, butorphanol, buprenorphine, midazolam, or their combinations

		Time after administration (min)
		0	30	60	90	120	180	240	360
Saline
	pH	7.37 ± 0.0	7.38 ± 0.03	7.40 ± 0.02	7.39 ± 0.01	7.40 ± 0.03	7.39 ± 0.04	7.40 ± 0.03	7.42 ± 0.01
	HCO3–	20.4 ± 1.8	21.5 ± 1.3	20.8 ± 2.0	21.1 ± 2.6	20.8 ± 2.5	19.8 ± 3.6	21.0 ± 4.0	22.6 ± 1.7
	Base excess	−3.0 ± 1.8	−2.0 ± 1.6	−2.3 ± 2.0	−2.1 ± 2.3	−2.2 ± 2.1	−3.3 ± 3.8	−2.1 ± 3.8	−0.3 ± 1.3
Butorphanol
	pH	7.37 ± 0.04	7.41 ± 0.03a	7.40 ± 0.02	7.42 ± 0.02a	7.42 ± 0.01a	7.42 ± 0.01a	7.41 ± 0.02a	7.42 ± 0.03a
	HCO3−	19.8 ± 2.5	22.0 ± 3.8	22.4 ± 3.0	23.6 ± 1.1	23.9 ± 1.7	22.9 ± 2.0	21.8 ± 1.8	20.6 ± 3.3
	Base excess	−3.5 ± 2.9	−1.2 ± 3.7	−0.8 ± 2.6	−0.5 ± 2.6	0.88 ± 1.3	−0.07 ± 1.6b	−1.2 ± 1.8	−1.9 ± 2.7
Buprenorphine
	pH	7.38 ± 0.05	7.37 ± 0.03	7.42 ± 0.09	7.39 ± 0.03	7.40 ± 0.02	7.4 ± 0.02	7.42 ± 0.03	7.47 ± 0.03
	HCO3−	19.4 ± 3.1	20.6 ± 2.6a	25 ± 6.5a,b	22.8 ± 1.7a	23.8 ± 2.4a	22.8 ± 1.8a	23.4 ± 1.8a	21.4 ± 2.6a
	Base excess	−4.1 ± 3.5	−3.3 ± 2.1	1.6 ± 6.4	−0.89 ± 1.5	0.06 ± 2.0	−0.6 ± 1.5b	0.16 ± 1.6	−1.8 ± 2.3
Midazolam
	pH	7.38 ± 0.03	7.41 ± 0.04	7.43 ± 0.03a	7.44 ± 0.03a,b	7.43 ± 0.03a	7.41 ± 0.03	7.40 ± 0.04	7.38 ± 0.04
	HCO3−	19.4 ± 3.1	20.6 ± 2.6	25 ± 6.5	22.8 ± 1.7	23.8 ± 2.4	22.8 ± 1.8	23.4 ± 1.8	21.4 ± 2.6
	Base excess	−2.0 ± 2.2	0.1 ± 1.9	1.1 ± 1.1	1.1 ± 3.0	0.5 ± 1.9	0.4 ± 1.6b	−2.0 ± 1.6	−3.5 ± 1.5
Midazolam–butorphanol
	pH	7.38 ± 0.03	7.40 ± 0.02	7.42 ± 0.01	7.43 ± 0.02	7.43 ± 0.01	7.45 ± 0.02b	7.43 ± 0.06	7.42 ± 0.04b
	HCO3−	20.7 ± 2.4	24.3 ± 1.2	24.9 ± 2.0a	24.4 ± 2.8a	25.6 ± 2.1a,b	25.4 ± 3.0a,b	24.9 ± 4.1a,b	22.9 ± 2.8a
	Base excess	−2.8 ± 2.2	0.6 ± 1.0a	1.4 ± 1.6a	1.3 ± 2.0a	2.0 ± 1.5a,b	2.4 ± 2.5a,b	1.5 ± 4.0a	−0.4 ± 2.9
Midazolam–buprenorphine
	pH	7.38 ± 0.02	7.39 ± 0.02	7.40 ± 0.02	7.40 ± 0.01	7.43 ± 0.03	7.44 ± 0.02	7.44 ± 0.0a	7.43 ± 0.02a
	HCO3−	20.4 ± 1.2	22.5 ± 2.0	23.6 ± 2.6a	24.8 ± 0.7a	24.5 ± 1.0a,b	26.4 ± 1.1a,b	24.9 ± 1.4a,b	24.5 ± 3.3
	Base excess	−3.0 ± 1.3	−1.2 ± 1.8	−0.2 ± 2.0a	0.8 ± 0.8a	1.0 ± 1.4a	2.6 ± 1.2a,b	1.6 ± 1.2a	1.4 ± 2.3a

^aSignificantly (P < 0.05) different from baseline value.

^bSignificantly (P < 0.05) different from value for saline.

When compared with saline control data, bicarbonate was elevated at various time points in the buprenorphine, midazolam–butorphanol, and midazolam–buprenorphine groups (Table 1). Statistically significant increases in bicarbonate were evident compared with baseline values at multiple time points in the buprenorphine, midazolam–butorphanol, and midazolam–buprenorphine treatment groups (Table 1).

Statistically significant (P < 0.05) increases in base excess were found in all treatment groups after comparison with values from saline controls (Table 1). Multiple time points in the midazolam–butorphanol and midazolam–buprenorphine treatment groups showed significantly (P < 0.05) increased base excess when compared with baseline data (Table 1).

Discussion

The combination of a benzodiazepine and opioid for premedication in domestic species is a widely accepted practice for providing sedation and analgesia prior to general anesthesia. Due to concerns for respiratory depression in species such as rabbits, in which ventilatory support may be particularly challenging, these combinations often are avoided in favor of inhalant induction without premedication. The goal of the current study was to examine the changes incurred in arterial blood gas tensions when combinations of a benzodiazepine, midazolam; a partial agonist opioid, buprenorphine; and a mixed agonist–antagonist opioid, butorphanol, were given to healthy New Zealand white rabbits.

In the current study, rabbits given the combination of midazolam–buprenorphine showed marked sedation, whereas slightly less profound sedation was seen in rabbits given the combination of midazolam–butorphanol. Both of these combinations resulted in a return to normal behavior by the end of the study period. However, these 2 potent drug combinations were accompanied by significant decreases in body temperature at later time points. The temperature in the laboratory was held constant, but we made no attempt to provide temperature support. Likely due to decreased locomotor activity over the experimental period, the decreases in body temperature were mild to moderate and seemed well tolerated by study animals. When using these drug combinations, however, it would be prudent to monitor temperature and provide external temperature support as needed. Furthermore, if animals will not tolerate prolonged and profound sedation, such as we observed in the midazolam–buprenorphine group, reversal agents may be necessary. In our experience, flumazenil is highly effective in reversing the sedation of this combination while preserving the analgesic effects. The marked sedation may be avoided by decreasing the dosage of midazolam, especially in compromised patients.

Significant decreases in respiratory rate were recorded in all drug treatment groups in the current study. These rates were greater than those observed during isoflurane anesthesia for catheter placement, but nadirs were lower than those reported in a study evaluating propofol–sevoflurane anesthesia for ovariohysterectomy.² Another study demonstrated that respiratory rate provided an estimation of respiratory depression.^10,12 Although pO₂ was decreased significantly at some time points that correlated with significant decreases in respiratory rate, the current studys did not demonstrate a strong correlation between respiratory rate and hypoxemia as evidenced by arterial blood gas tensions. It is accepted that rabbits normally demonstrate a rapid and irregular respiratory pattern in the laboratory setting.^10,12,21,22 This scenario is probably due largely to a stress response and does not adequately reflect the animals’ normal resting respiratory rate. Although we obtained respiratory rates while the animals were at rest and before attempting temperature or arterial blood gas sampling, the unfamiliar setting of the laboratory may have caused a significant increase in resting respiratory rate. The decrease in respiratory rates demonstrated by drug treatments may be more a reflection of sedation rather than true respiratory depression. In the current study, minimal attempts were made to desensitize the rabbits to the laboratory setting. We felt that the manipulation associated with bringing animals to the laboratory without prior desensitization would more properly mimic the clinical setting of rabbits coming into a veterinary hospital or treatment facility. However, adequate desensitization may have allowed for less variability in respiratory rates and, potentially, blood-gas tensions.

No treatment groups experienced significant decreases in pO₂ when compared with saline control groups. However, decreases in pO₂ over time were evident when compared with baseline at multiple time points in the buprenorphine and midazolam–buprenorphine groups. Only at the 30-min time point was there a significant decrease in pO₂ in the midazolam-butorphanol group. No significant changes in pO₂ were evident in the midazolam or butorphanol groups, and there was no change in pO₂ in the saline group over time. Although there were significant decreases in pO₂ in multiple groups_, only in the buprenorphine group were they consistent with moderate hypoxemia. Because there were no significant increases in pCO₂ in any treatment groups, it is unclear whether this hypoxemia was due to alveolar hypoventilation. This uncertainty echoes the results of a previous study, in which ventilation–perfusion mismatching was the cause of hypoxemia evident after buprenorphine administration.²² However, why the combination of midazolam–buprenorphine would result in only mild hypoxemia whereas the sole administration of buprenorphine would result in more significant hypoxemia is unknown. These rabbits did not demonstrate any clinical evidence of respiratory disease throughout the experimental period. It is unknown why ventilation–perfusion mismatching would have occurred after drug administration. The animals were anesthetized for 5 to 10 min at 1 h prior to the experimental period for arterial cannulation, but it is unlikely that the very brief period of general anesthesia would have contributed to any significant atelectasis of the lung. Furthermore, the severity of hypoxemia did not correlate with the degree of sedation. The midazolam–buprenorphine group experienced a period of recumbency, often lasting 60 to 90 min, which could have contributed to lung atelectasis, but the buprenorphine treatment group had only mild to moderate sedation with no recumbency.

One possible source of error is the handling of the arterial samples. Samples were removed and taken immediately for analysis at room temperature. To ensure accurate pO₂ values, samples of equine blood held at ambient temperature are recommended to be analyzed within 10 min.²⁰ Over time, pO₂—but not pCO₂—values decrease when not held on ice.²⁰ Arterial blood gas samples held on ice are better preserved due to a decrease in blood-cell metabolism.²⁰ In the field of human medicine, the American Association for Respiratory Care recommends that blood gas samples should not be stored at ambient temperature for longer than 30 min.¹⁴ In the current study, samples were analyzed within 20 min, and most were analyzed within 10 min of sampling. This methodology was held consistent throughout all treatments and, although a potential source of error, it would have affected all of the data to the same degree.

Despite the observed decreases in respiratory rates, no statistically significant changes in pCO₂ occurred in any treatment group. Although an acceptable range of pCO₂ specifically for rabbits has not been published, pCO₂ greater than 45 mm Hg is a general guideline for hypoventilation.²⁹ None of the measured values of pCO₂ approached or exceeded this value. Multiple studies have demonstrated a decrease in respiratory rate associated with the administration of both butorphanol and buprenorphine in rabbits.^{9,11,13,15,21,22} However, only one study²² documented a concurrent increase in pCO₂ after the administration of intravenous and subcutaneous buprenorphine. A comparison of buprenorphine and its metabolite, norbuprenorphine, in rats revealed that administration of buprenorphine did not result in respiratory depression as evidenced by either respiratory rate or arterial carbon dioxide tensions.¹⁹ Perhaps the decrease in arterial oxygen tension that we observed was not a result of hypoventilation but a result of ventilation–perfusion mismatching and the observed decrease in respiratory rate may have been compensated by an increase in tidal volume. However, rabbit respiratory rates are highly variable, and pCO₂ can change in each breath. The current study attempted to account for this variability by performing arterial blood gas sampling in duplicate. The additional handling time required for withdrawal of further blood samples over multiple minutes may have added another element of stress, introducing even more variability into the results. Again, desensitization of animals may have affected the results and avoided some of the variability encountered. Another explanation is that of study size. There was a statistical trend toward increased pCO₂, but more data points might have resulted in statistical significance.

The changes that we recorded in pH, bicarbonate, and base excess were unexpected. Over the course of the experimental period, pH increased in butorphanol, midazolam, midazolam–butorphanol, and midazolam–buprenorphine treatment groups. Because these changes did not result in clinically significant alkalosis, it is a trend that is difficult to explain. The observed increases in both bicarbonate and base excess suggest that the increases in pH are due to a metabolic rather than a respiratory component. Statistically significant increases in bicarbonate were observed over time in the buprenorphine, midazolam–buprenorphine, and midazolam–butorphanol treatment groups. These 3 treatment groups also demonstrated significant decreases in pO₂. All treatment groups showed an increase in base excess at the 180-min time point, and midazolam–butorphanol and midazolam–buprenorphine treatment groups showed significant increases in base excess at nearly all time points. Although there were no statistically significant increases in pCO₂, arterial blood gas analysis demonstrated trends toward increased pCO₂ in buprenorphine, midazolam–butorphanol, and midazolam–buprenorphine treatment groups, possibly resulting in respiratory acidosis. The increase in bicarbonate might be explained by compensatory metabolic alkalosis. However, this explanation seems somewhat implausible, given the short time course as well as the fact that metabolic compensation would not result in increased pH but rather would bring the pH back toward normal. A similar study evaluating serial arterial blood gases after the administration of buprenorphine to rabbits did not reveal increases in pH during the 90-min study period.²² In the current study, the increases in bicarbonate and base excess suggest that a metabolic process accounts for the alkalosis. Another possible explanation for the observed alkalosis is the interaction between stress, anorexia, and the rabbit gastrointestinal tract. Although we did not intentionally fast the rabbits during the preexperimental or experimental time period, most of the animals did not eat much while in the laboratory, regardless of treatment. The midazolam–buprenorphine and midazolam–butorphanol groups were sedated sufficiently to consume less food than the other groups, but the administration of buprenorphine alone only resulted in mild sedation, which was inadequate to modify observed eating habits compared with those of the saline group. The muscle pH of fasted rabbits is known to be less than that of nonfasted rabbits⁴—a finding inconsistent with the current data. However, fasting decreased the amount of lactic acid in the stomach contents of rabbits.² How this effect influences arterial pH in the short-term is unknown. To the best of our knowledge, no study evaluating the effects of mild anorexia on the arterial pH or bicarbonate of rabbits is ongoing. The effects of sedation and accompanying anorexia on arterial pH in rabbits over time may need further evaluation and research.

The combinations of midazolam–buprenorphine and midazolam–butorphanol resulted in significant sedation in healthy rabbits, suggesting that these drugs, when used at the doses studied here, would provide excellent preanesthetic sedation. These combinations resulted in decreased respiratory rate but only mild hypoxemia and no hypercapnia, as evidenced by arterial blood gas analyses. The sole administration of buprenorphine resulted in more pronounced hypoxemia, which is difficult to explain in light of the data we obtained with buprenorphine and midazolam combined. This uncertainty is possibly due to the limitations of the study, where a relatively small number of animals were used and where analysis of some of the arterial samples may have been slightly delayed. Furthermore, lack of desensitization of the rabbits to the laboratory may have resulted in variability that could have influenced the results. In summary, combinations of midazolam with butorphanol and buprenorphine provided mild to moderate sedation in healthy rabbits with minimal changes in arterial blood gas tensions. Although cardiovascular effects were not evaluated, the sedation and mild respiratory depression seemed to be clinically well tolerated by these healthy animals. This study supports the use of premedications in rabbits. The combinations of midazolam–butorphanol and midazolam–buprenorphine provide the best sedation which, in our experience, smoothes anesthetic induction and is sufficient to perform a variety of noninvasive diagnostic procedures. Caution is warranted, however, when using these combinations in rabbits with compromised respiratory function, overt or occult respiratory disease, or general systemic disease, as in these animals the sedative effects of any drug may be more pronounced.