Oxygen Supplemented via Face Mask Counteracts Hypoxemia in Capuchin Monkeys (Sapajus spp.) under Butorphanol-Ketamine-Midazolam Restraint

Abstract

This prospective, randomized, complete crossover study evaluated the effects of supplemental oxygen on arterial oxygenation and recovery quality in adult capuchin monkeys (Sapajus spp.; n = 8; 6 males and 2 females) chemically restrained with intramuscular butorphanol (0.5 mg/kg), ketamine (15 mg/kg), and midazolam (1 mg/kg). Each monkey underwent 2 anesthetic events, separated by a 2-week interval: one breathing room air (G_AIR) and one receiving oxygen via face mask at 3 L/min (G_OXY), both conducted during dorsal recumbency. Heart rate (HR), respiratory rate (RR), peripheral oxygen saturation (SpO₂), and rectal temperature were registered every 10 minutes for 45 minutes. Arterial blood gases were analyzed at 10 and 30 minutes postrestraint, with oxygen supplementation in G_OXY initiated after the 10-minute time point and maintained for 35 minutes. Recovery was continuously video-recorded for later assessment of time to standing and recovery quality by one blinded and one nonblinded observer. At 10 minutes, 14 of 16 monkeys exhibited hypoxemia (partial pressure of arterial oxygen [PaO₂] range: 48-81 mm Hg). By 30 minutes, PaO₂ and arterial oxygen saturation (SaO₂) increased significantly in G_OXY, reaching 298-458 mm Hg and 100%, respectively, whereas G_AIR animals remained hypoxemic (47-70 mm Hg and ≤92%, respectively). Pulse oximetry-derived SpO₂ consistently overestimated saturation at low SaO₂ values. The partial pressure of carbon dioxide (PaCO₂) rose significantly in G_OXY, with hypercapnia (PaCO₂ range: 33-57 mm Hg) documented in 6/8 individuals at 30 minutes. In contrast, no cases of hypercapnia were observed in G_AIR (PaCO₂ range: 22-45 mm Hg) at the same time point. No significant between-group differences were detected in SpO₂, HR, RR, or temperature over time. Time to standing and recovery quality were similar between groups. Supplemental oxygen via face mask effectively corrected hypoxemia in Sapajus spp. chemically restrained with butorphanol, ketamine, and midazolam.

Introduction

Capuchin monkeys (Sapajus spp.) are nonhuman primates (NHPs) of medium body size broadly distributed across Central and South America. As a result of habitat fragmentation driven by anthropogenic pressures and the impacts of illegal trafficking, capuchin monkeys account for a substantial portion of the primates admitted to wildlife screening and rehabilitation centers in Brazil.¹ Veterinary management of capuchins also extends to zoos, conservation breeding facilities, and research institutions worldwide. Importantly, they occupy a significant role in biomedical and behavioral research, serving as experimental models owing to their advanced cognitive skills, complex social behavior, and physiologic similarities to humans.^2,3 Within this context, as with other primate taxa, chemical restraint constitutes an essential tool for the safe handling and care of Sapajus spp., especially when short-term immobility is required.

In clinical practice, ketamine is routinely used for primate chemical restraint because it is rapidly absorbed when administered intramuscularly and has a wide therapeutic index,⁴ an important consideration when body weight is estimated. Although ketamine has been reported as a sole agent for chemical restraint in primates,^5,6 its use alone has been associated with adverse effects such as muscle rigidity, spontaneous movements, and excessive salivation; therefore, it is more commonly administered alongside muscle relaxants and/or sedatives to enhance immobilization quality.⁴ Multidrug anesthetic combinations incorporating dissociative anesthesia with agents such as opioids and benzodiazepines are widely employed during short-term handling procedures such as physical examination, biologic sample collection, and dental prophylaxis.^7–9 However, when used in multidrug protocols, dissociative anesthesia is not without risks. Hypoxemia has been reported in tufted capuchin monkeys (Sapajus apella) breathing room air under both ketamine-dexmedetomidine and ketamine-methadone-midazolam.⁷ A comparable pattern has been observed in common marmosets (Callithrix jacchus) subjected to ketamine-based protocols, where hypoxemia was confirmed via blood gas analysis¹⁰ or inferred from low peripheral oxygen saturation (SpO₂ < 95%).¹¹ These findings underscore the need for respiratory support to maintain adequate arterial oxygen levels in small- to medium-sized primates, even after single-dose chemical restraint: a point particularly relevant because hypoxemia can lead to tissue and myocardial hypoxia.¹² Moreover, hypoxemia has been implicated in poorer recovery outcomes following chemical restraint of wild ruminant species, including North American elk (Cervus canadensis manitobensis),¹³ reindeer (Rangifer tarandus),¹⁴ and moose (Alces alces).¹⁵

Supplemental oxygen is advocated by many researchers as a prerequisite during wildlife immobilization and has been shown to effectively prevent hypoxemia in several large mammals such as white-tailed deer (Odocoileus virginianus),¹⁶ brown bears (Ursus arctos),¹⁷ capybaras (Hydrochoerus hydrochaeris),¹⁸ North American elk,¹³ and moose.¹⁵ Although the reference textbook on wildlife anesthesia recommends administering supplemental oxygen via a face mask in chemically restrained primates,⁴ its efficacy in mitigating hypoxemia in Neotropical primates has, to the authors’ knowledge, been assessed only in a preliminary trial involving marmosets.¹⁰ In that study, data from 3 animals immobilized with ketamine-xylazine-atropine showed a pronounced increase in the partial pressure of arterial oxygen (PaO₂) following supplemental oxygen administration (from 48-67 to 303-338 mm Hg), although hypercapnia developed concurrently. Additional parameters of the marmosets’ acid-base status were not documented, nor was the impact of oxygen supplementation on recovery.¹⁰

The aim of this study was to evaluate the effects of face mask oxygen supplementation on blood gas variables during chemical restraint in capuchin monkeys, with a secondary aim of determining its influence on recovery quality. It was hypothesized that administration of butorphanol-ketamine-midazolam would induce hypoxemia and that oxygen supplementation would mitigate this effect and improve recovery.

Materials and Methods

Ethical review.

This study was approved by the Animal Care and Use Committee of the School of Veterinary Medicine and Animal Science of the University of São Paulo. In addition, all research procedures were conducted with approval from the “Departamento de Fauna, Secretaria do Meio Ambiente” (DeFau/SMA), the governmental agency responsible for overseeing the care of primates at the facility where the study was carried out.

Animals.

Nine capuchin monkeys (Sapajus spp.), 7 males and 2 females, classified as adults based on permanent dentition and tooth wear, were enrolled in the study. Animals were eligible for inclusion if (1) medical records indicated an unremarkable clinical history; (2) no life-threatening abnormalities were documented during prior immobilizations; and (3) current health status was confirmed by physical examination, feeding behavior, coproparasitological analyses, and complete blood count and plasma biochemical profiles.¹⁹ Animals were excluded from the study if they recovered before completion of the final data-collection time point or if adequate chemical restraint could not be achieved, as indicated by insufficient muscle relaxation during the procedure.

The animals were part of the primate colony of The Technical Division of Veterinary Medicine and Management of Wild Fauna (DEPAVE-3, from the Portuguese acronym), a wildlife screening center where they had been temporarily maintained for at least 3 months while awaiting release or transfer to an appropriate facility. The capuchins at this facility comprised black capuchins (Sapajus nigritus), black-striped capuchins (Sapajus libidinosus), and hybrids; accordingly, they are reported herein at the genus level only. The monkeys were housed in semiopen enclosures, either in pairs or in groups of 3, and were fed once daily with a diet of fruits, vegetables, eggs, and commercial pellets (Nutral). Water was provided ad libitum. Enclosures (4 m × 5 m × 3 m) were equipped with perches, nest boxes, wooden branches, and hanging tires and were exposed to daily temperature fluctuations under a natural photoperiod. Husbandry was carried out by the same group of 4 trained personnel. Individuals were identified using a subcutaneous microchip (AnimallTAG, Des Plaines, IL) inserted between the scapulae, as well as by their external features when observed from a distance by the animal keepers.

Study design.

This was a prospective, randomized, complete crossover study with partial blinding; the main investigators (MEF, AAJ, and SRCG) were aware of the treatment groups. Chemical restraint was administered on 2 separate occasions, either with animals receiving supplemental oxygen via a face mask (G_OXY) or with animals breathing room air (G_AIR). Treatments were randomized by a paper draw. For each animal, the 2 anesthetic episodes were separated by a 2-week interval. All data were collected in June-July 2025, with anesthetic procedures performed during daytime hours (8:00 am to 2:00 pm).

Chemical restraint and variables monitored.

Animals were fasted overnight, with food withheld on the morning of the anesthetic procedure. After being captured with a hand net, the capuchin was administered a combination of 0.5 mg/kg butorphanol (Torbugesic; Zoetis, Parsippany, NJ), 15 mg/kg ketamine (Ketalex; Dechra, Overland Park, KS), and 1 mg/kg midazolam (Dormire; Cristália, São Paulo, Brazil) into the lateral thigh using a 30 × 0.7-mm hypodermic needle attached to a 3-mL syringe. Only one animal was immobilized at a time. For drug preparation, the most recent weight available in the medical records was used. This combination was selected to provide reliable immobilization and muscle relaxation in accordance with routine practice at the study facility for short (<1 hour) clinical and diagnostic procedures such as blood sampling, imaging examinations, and minor wound management. Doses in the protocol described herein are also consistent with published studies in Neotropical primates.^20–23

The time from drug administration until the monkey stopped struggling with the net and no longer responded to external stimuli, assessed at 30-second interval, was recorded as the latency period. Once chemically restrained, the animals were transported in a crate (80 cm × 30 cm) to the ambulatory care unit, located approximately 100 m from their original enclosure. Actual body weights were measured using a digital scale, and the animals were then positioned in dorsal recumbency on a rubber mat with their heads slightly elevated on a cushioned platform. Individual identification was confirmed via microchip. Blindfolds were placed over the animals’ eyes, and gauze was inserted into their ears to reduce external sensory stimulation. A forced-air warming device (Bair Hugger; Arizant Healthcare, Eden Prairie, MN) was used to maintain rectal temperatures between 37.0 and 38.5 °C, as measured with a digital thermometer. Heart rate (HR) was determined by auscultation, and respiratory rate (RR) was determined by observing chest wall movements. A pulse oximeter (Nellcor NPB-600, Medtronic, Minneapolis, MN) was attached to the vaginal mucosa in females or the penile mucosa in males for continuous SpO₂ monitoring. The above variables were recorded at 5 minutes after chemical restraint and at 10-minute intervals thereafter up to 45 minutes.

In G_OXY, oxygen supplementation was initiated 10 minutes after the onset of chemical restraint and maintained until the final time point of cardiorespiratory monitoring, for a total duration of 35 minutes. For blood gas and electrolyte measurements, 0.5 mL of blood was collected from a femoral artery branch using a 1-mL heparinized syringe and a 20 × 5.5-mm hypodermic needle. The medial aspect of the distal hindlimb was clipped and aseptically prepared, and the artery was identified by palpating its pulsatile flow. The first sample was obtained just before 10 minutes of chemical restraint, when animals in both groups were breathing room air. The second was collected 20 minutes later, by which time G_OXY animals were receiving supplemental oxygen while G_AIR animals remained on room air (Figure 1). For each animal, consecutive samples were drawn from alternating hindlimbs. The samples were immediately processed by a portable blood gas analyzer (i-STAT System; Abbott Point of Care, Princeton, NJ). Cartridges (CG8+ Test Cartridge; Abbott Point of Care, Princeton, NJ) were used to measure pH, PaO₂, partial pressure of carbon dioxide (PaCO₂), bicarbonate (HCO₃⁻), base excess (BE), arterial hemoglobin oxygen saturation (SaO₂), plasma ionized calcium (Ca²⁺), potassium (K⁺), sodium (Na⁺), and glucose concentration. All measurements were performed at 37 °C, with pH, PaO₂, and PaCO₂ automatically corrected for the animal’s body temperature according to i-STAT algorithms. Lactate concentration was determined from a single drop of blood using a handheld analyzer (Accutrend Plus; Roche Diagnostics, Indianapolis, IN). The device’s detection limit was 0.8 mmol/L, so readings below this threshold were rounded to 0.7 mmol/L for statistical analysis.²⁴ Cardiorespiratory monitoring was discontinued after 45 minutes of chemical restraint, and the animals were allowed to recover.

Figure 1.

Timeline of Procedures Performed to Investigate the Effects of Face Mask Oxygen Supplementation on Blood Gases and Recovery in Capuchin Monkeys (Sapajus spp.) Chemically Restrained with Butorphanol-Ketamine-Midazolam. G_OXY indicates the group of monkeys supplemented with oxygen.

Oxygen supplementation.

In G_OXY, oxygen supplementation was provided using a portable 5-L aluminum oxygen cylinder. The cylinder was connected to a pressure-reducing valve, a flowmeter-equipped regulator, and a humidifier reservoir. A flexible tube from the flowmeter led to a nonrebreathing circuit consisting of an Ayre T-piece and a 0.5-L reservoir bag. The circuit was attached to a pediatric, silicone-based inflatable face mask, which the experimenter held by hand over the primate’s face (Figure 2). Oxygen was supplemented at a flow rate of 3 L/min.²⁵

Figure 2.

Capuchin Monkey (Sapajus sp.) in Dorsal Recumbency during Chemical Restraint with Butorphanol, Ketamine, and Midazolam, Receiving Oxygen Supplementation via a Pediatric Silicone Face Mask Connected to a Nonrebreathing Circuit. The head is turned laterally to illustrate mask fit.

Recovery.

After 45 minutes of cardiorespiratory monitoring, animals were returned to the transport crate in right lateral recumbency on a blanket. From that point onward, all primates were maintained on room air irrespective of treatment group and continuously video-recorded (Powershot SX50 HS; Canon, Melville, NY) without disturbance or direct eye contact from the research team. Time to standing (quadrupedal position) was determined from the video recordings. The recordings were subsequently edited by one investigator (MEF) into 3-minute segments capturing the transition from lateral recumbency to standing. Behaviors associated with recovery quality were later independently scored by 2 investigators (MEF and BRL), one of whom (BRL) was blinded to treatment group. The scoring system was based on Valverde et al²⁶ and was modified to reflect the behaviors exhibited by capuchins while recovering from anesthesia in a transport crate (Table 1). Four recovery behaviors were observed and scored on a 1-3 scale. The scores were then summed, and the total recovery score was classified as excellent (4-5), good (6-7), fair (8-9), or poor (10-12). Once animals were deemed fully awake (vocalizing and moving around the transport crate without ataxia), they were reunited with their social groups.

Table 1.

Recovery Quality Scoring System Used in Capuchin Monkeys Subjected to Chemical Restraint with Butorphanol, Ketamine, and Midazolam

Behavior	Score	Description
Attemp to attain quadrupedal position	1	The animal is able to assume and maintain a quadrupedal position with minimal ataxia
	2	The animal displays ataxia and/or is unable to maintain the quadrupedal position and falls
	3	The animal falls and strikes the bottom or side of the transport crate abruptly
Moving inside the crate	1	The animal moves within the crate with no or minimal ataxia, and without falling
	2	The animal moves with ataxia but manages to complete its movements without falling
	3	The animal shows severe ataxia and falls while moving
Interaction with the environment	1	Active animal, grasping the grid, manipulating the blanket (kneading/biting), and peeking through the holes of the crate (showing all types of interactions)
	2	Animal shows mild interest in the environment, grasping the grid and/or manipulating the blanket (kneading/biting) and/or peeking through the holes of the crate (showing one or 2 types of interaction)
	3	Animal indifferent to the environment, not interacting with the grid, blanket (kneading/biting), and/or holes of the crate
Attempt to attain bipedal position	1	The animal uses the grid to stand in a bipedal position and is able to maintain the posture
	2	The animal uses the grid to stand in a bipedal position but cannot maintain it for long and falls
	3	The animal does not attempt to stand in a bipedal position

Statistical analysis.

Assuming a PaCO₂ of 40 mm Hg, a respiratory quotient (RQ) of 1, a barometric pressure (P_B) at the study site of 690 mm Hg, a fraction of inspired oxygen (FiO₂) of 0.21, and a saturated vapor water pressure (P_H2O) of 47 mm Hg at 37 °C, the alveolar oxygen tension (PAO₂) was estimated according to the following equation:¹⁶

$${\text{PAO}}_2=\left[{\text{FiO}}_2\times \left({\text{P}}_{\text{B}}-{\text{P}}_{\text{H}2\text{O}}\right)\right]-\left(\frac{{\text{PaCO}}_2}{\text{RQ}}\right)$$

$${\text{PAO}}_2=\left[0.21\times \left(690-47\right)\right]-\left(\frac{40}{1}\right)$$

$${\text{PAO}}_2=95 \text{mm}\hspace{0.17em}\text{Hg}$$

Based on a normal alveolar-to-arterial [P(A-a)O₂] gradient of 15 mm Hg,¹⁶ the calculated normal PaO₂ for capuchins breathing room air was 80 mm Hg (95 to 15 = 80 mm Hg). Capuchins with a PaO₂ below this value on room air were considered hypoxemic. The P(A-a)O₂ was not calculated following oxygen supplementation, because the FiO₂ was then undetermined.

The sample size was calculated using an online tool (https://estatistica.bauru.usp.br/calculoamostral/), with PaO₂ as the primary outcome variable. The G_AIR and G_OXY groups were considered paired, and the expected mean difference in PaO₂ was estimated at 60 mm Hg. This estimate was based on the premise that administering 3 L/min of supplemental oxygen via face mask (as in this study) can increase PaO₂ to approximately 140 mm Hg⁸ (140 to 80 = 60 mm Hg). In the absence of variability data, the within-individual standard deviation of differences was set equal to the expected effect size, representing a high-variability (conservative) scenario. Considering an α error of 0.05 and a β error of 0.2, a sample size of 9 animals was required.

Statistical analyses were performed using GraphPad Prism version 8.0.2 (GraphPad Software, San Diego, CA) and SAS version 9.4 (SAS Institute, Cary, NC). Continuous data were assessed for normality visually through histograms and Q-Q plots and were also subjected to the Shapiro-Wilk test. When required, individual data points were log transformed to meet the assumptions of a normal distribution for parametric analysis. Data were expressed as mean and SD (if normally distributed) or as median and IQR (if nonnormally distributed). For PaO₂ and PaCO₂, observed minimum and maximum values were in addition reported to provide descriptive detail, despite a normal data distribution. Within-group cardiorespiratory data were compared across time points using a one-way repeated measures ANOVA, followed by the Tukey test if the data were normally distributed; otherwise, the Friedman test with the Dunn multiple comparisons was used. Between-group cardiorespiratory variables were analyzed with a 2-way ANOVA followed by the Sidak test for normally distributed data, while the Wilcoxon test was applied to nonnormal data. Blood gases and electrolytes were compared between time points and groups using paired t test or Wilcoxon test, depending on data distribution. Variables that were transformed for analysis were back-transformed to their original units for reporting. A paired t test was also applied to compare recovery scores between the 2 evaluators after confirming normal distribution.

Agreement between recovery scores assigned by a blinded and a nonblinded evaluator was assessed using a weighted κ statistic, which accounts for the extent of disagreement. Kappa values are interpreted as indicating poor (<0.20), fair (0.21-0.40), moderate (0.41-0.60), good (0.61-0.80), or very good (0.81-1.00) agreement between observers.²⁷ For all analyses, differences were considered statistically significant at P < 0.05.

Results

One monkey was excluded from the study due to poor quality of chemical restraint, characterized by restlessness and lack of adequate muscle relaxation in both treatment groups. Data from the remaining 8 monkeys were analyzed, and all statistical analyses were performed using only these animals. The dose of drugs used for chemical restraint (mg/kg, based on actual body weight) and the latency time did not differ between treatment groups (P > 0.382 and P = 0.372, respectively). Across both groups (n = 16 anesthetic events), doses of butorphanol, ketamine, and midazolam were 0.46 ± 0.03 mg/kg, 14.1 ± 1.1 mg/kg, and 0.9 ± 0.07 mg/kg, respectively, and the latency time was 113 ± 32 seconds (Table S1).

Body temperature and HR were higher 5 minutes after chemical restraint compared with later time points in both treatment groups (P < 0.001-0.023 and P < 0.001-0.037, respectively). At minute 5, SpO₂ < 95% occurred in 2 of 8 G_AIR and 5 of 8 G_OXY monkeys. No statistically significant differences in SpO₂ over time were observed in either G_AIR (P ≥ 0.113) or G_OXY (P ≥ 0.398). However, values below 95% were recorded in 1 of 8 to 5 of 8 capuchins at various time points in G_AIR, whereas all capuchins maintained SpO₂ levels above 98% in G_OXY following the onset of oxygen supplementation. An increase in RR was observed in minute 45 in G_AIR compared with minutes 25 (P = 0.019) and 35 (P = 0.023), whereas in G_OXY, a similar increase was seen in minute 5 compared with minutes 15 (P = 0.026) and 25 (P = 0.015). None of the cardiorespiratory variables differed significantly between G_AIR and G_OXY (Table 2).

Table 2.

Body Temperature, HR, SpO₂, and RR in 8 Capuchin Monkeys (Sapajus sp.) Chemically Restrained with Butorphanol-Ketamine-Midazolam under Room Air (G_AIR) or Oxygen Supplementation via Face Mask (G_OXY) in a Crossover Design

Variable	Group	Time after chemical restraint (min)
		5	15	25	35	45
Temperature, ºC	GAIR	38.3 ± 0.4a	37.4 ± 0.5b	37.3 ± 0.4b	37.4 ± 0.5b	37.4 ± 0.5b
	GOXY	38.1 ± 0.3a	37.4 ± 0.3b	37.2 ± 0.4b	37.2 ± 0.7a,b	37.6 ± 0.7a,b
HR, beats/min	GAIR	215 ± 29a	185 ± 35b	179 ± 48a,b	169 ± 28b	179 ± 36b
	GOXY	216 ± 24a	176 ± 45a,b	170 ± 46b	180 ± 44a,b	180 ± 39b
SpO2, %	GAIR	97 (90-99)	97 (92-99)	92 (92-99)	97 (94-100)	98 (95-100)
	GOXY	90 (89-100)	100 (100-100)	100 (99-100)	100 (99-100)	100 (100-100)
RR, breaths/min	GAIR	68 (55-86)a,b	58 (42-70)a,b	48 (48-58)b	54 (49-69)b	64 (53-80)a
	GOXY	76 (54-86)a	48 (44-73)b	44 (41-74)b	46 (41-79)a,b	56 (52-63)a,b

Normal data are reported as mean ± SD, and nonnormal data as median (IQR).

Abbreviations: HR, heart rate; RR, respiratory rate; SpO₂, peripheral oxygen saturation.

^a,bSignificant difference (P < 0.05) between time points within the same treatment group (a > b).

At 10 minutes postrestraint, hypoxemia was observed in all animals in G_AIR (PaO₂ range: 53-72 mm Hg) and in 6 of 8 animals in G_OXY (PaO₂ range: 48-81 mm Hg) (Table 3; Table S1). The P(A-a)O₂ at this time point did not differ between G_AIR and G_OXY (P = 0.690), being well above 15 mm Hg in both groups. At 30 minutes, all monkeys without supplemental oxygen remained hypoxemic (PaO₂ range: 47-70 mm Hg), whereas PaO₂ increased significantly in G_OXY (P < 0.001), with values well above the hypoxemic threshold (PaO₂ range: 298-458 mm Hg). Similarly, SaO₂ remained below 92% across treatment groups at the first blood gas analysis but increased significantly from 10 to 30 minutes only in G_OXY (P < 0.001). Both PaO₂ and SaO₂ were higher in G_OXY than in G_AIR at 30 minutes (P < 0.001 and P = 0.007). The calculated P(A-a)O₂ at 30 minutes in G_AIR persisted above 15 mm Hg and did not differ from the value at 10 minutes (P = 0.887). At 10 minutes, hypercapnia (PaCO₂ > 45 mm Hg) was documented in a single animal in each group (G_AIR: PaCO₂ range, 27-47 mm Hg; G_OXY: PaCO₂ range, 35-49 mm Hg). PaCO₂ values rose significantly in G_OXY after supplemental oxygen administration (P = 0.001), with hypercapnia observed in 6 of 8 animals at minute 30 (PaCO₂ range: 33-57 mm Hg). By comparison, hypercapnia was absent in all monkeys breathing room air at this time point (PaCO₂ range: 22-45 mm Hg). A concomitant rise in HCO₃⁻ and BE was also observed in G_OXY at 30 minutes compared with 10 minutes (P = 0.033 and P = 0.046, respectively). In G_AIR, pH was higher at 30 minutes than at 10 minutes (P = 0.027) and was also higher than in G_OXY at the same time point (P = 0.005). In both groups, blood glucose and lactate levels were higher at 10 minutes than at 30 minutes (P ≤ 0.006 and P ≤ 0.031, respectively). No significant differences in electrolyte concentrations were detected between groups or time points (Table 3).

Table 3.

Arterial Blood Gases and Electrolytes Recorded in 8 Capuchin Monkeys (Sapajus sp.) Chemically Restrained with Butorphanol-Ketamine-Midazolam under Room Air (G_AIR) or Oxygen Supplementation via Face Mask (G_OXY) in a Crossover Design

Variable	Group	Time after chemical restraint (min)
		10	30
pH	GAIR	7.25 (7.21 to 7.27)a	7.29 (7.28 to 7.32)b
	GOXY	7.26 (7.18 to 7.29)	7.24 (7.21 to 7.30)
PaO2, mm Hg	GAIR	62 ± 7	63 ± 7b
	GOXY	64 ± 13a	397 ± 55
P(A-a)O2, mm Hg	GAIR	33 ± 6.5	33 ± 7.5
	GOXY	31 ± 13	—
PaCO2, mm Hg	GAIR	38 ± 6	38 ± 8b
	GOXY	41 ± 5a	49 ± 8
HCO3−, mEq/L	GAIR	16.5 (14.5 to 17.0)	20.1 (19.4 to 21.1)
	GOXY	16.9 (10.0 to 21.3)a	21.8 (19.6 to 23.4)
BE, mEq/L	GAIR	−10.5 (−13.3 to −10.0)	−6 (−7 to −4.5)
	GOXY	−10.0 (−13.0 to −4.7)a	−5.5 (−7.7 to −4.0)
SaO2, %	GAIR	86 (84 to 87)	88 (85 to 91)b
	GOXY	85 (77 to 92)a	100 (100 to 100)
Lactate, mmol/L	GAIR	3.9 ± 2.1a	1.6 ± 1.2
	GOXY	4.9 ± 4.7a	1.6 ± 1.4
Sodium, mmol/L	GAIR	145 (144 to 147)	147 (143 to 148)
	GOXY	145 (145 to 146)	146 (144 to 146)
Potassium, mmol/L	GAIR	2.7 ± 0.3	2.8 ± 0.5
	GOXY	2.8 ± 0.5	2.9 ± 0.6
Calcium, mmol/L	GAIR	0.9 (0.7 to 1.0)	0.9 (0.9 to 1.0)
	GOXY	1.0 (0.7 to 1.0)	0.9 (0.8 to 1.0)
Glucose, mg/dL	GAIR	69 ± 6a	60 ± 10
	GOXY	77 ± 13a	63 ± 15

Normal data are reported as mean ± SD and nonnormal data as median (IQR).

Abbreviations: BE_ecf, extracellular base excess; HCO₃⁻, bicarbonate concentration; P(A-a)O_2, calculated alveolar-to-arterial oxygen; PaCO₂, arterial partial pressure of carbon dioxide; PaO₂, arterial partial pressure of oxygen; SaO₂, arterial hemoglobin oxygen saturation.

^a Significant difference between time points within the same treatment group (P ≤ 0.05).

^b Significant difference between G_AIR and G_OXY at a given time point (P ≤ 0.05).

Time to standing was comparable between groups (67 ± 21 minutes in G_AIR compared with 62 ± 25 minutes in G_OXY; P = 0.625). Interrater agreement for total recovery scores was good (weighted κ value = 0.740), and recovery quality was similar between treatment groups. The blinded investigator assigned scores of 7.6 ± 2.3 for G_OXY and 8.3 ± 1.5 for G_AIR (P = 0.303), while the unblinded investigator assigned scores of 8.2 ± 2.3 and 8.1 ± 1.5, respectively (P = 0.897). In G_OXY, the blinded investigator rated recovery as excellent in 1 of 8 animals, good in 2 of 8, fair in 2 of 8, and poor in 3 of 8, whereas the unblinded investigator rated 2 of 8 animals in each category (excellent, good, fair, and poor). For G_AIR, the blinded observer rated 4 of 8 capuchins as having good recoveries, 2 of 8 as fair, and 2 of 8 as poor, while the unblinded observer rated 3 of 8 as good, 3 of 8 as fair, and 2 of 8 as poor. All animals recovered uneventfully and rejoined their social group the same day.

Discussion

This study reveals a high prevalence of hypoxemia in capuchin monkeys subjected to short-term (<60 minutes) chemical restraint with a commonly used drug combination, as PaO₂ values below the expected minimum of 80 mm Hg were recorded in nearly all (14 of 16) anesthetic events. Oxygen supplementation via face mask at 3 L/min improved arterial oxygenation and effectively counteracted hypoxemia; however, no significant effects on time to recovery and recovery characteristics were observed when compared with capuchins breathing room air.

Although statistical differences were not observed, SpO₂ in G_OXY showed an increasing trend over time, consistent with SaO₂ values and reflecting the effect of oxygen therapy. Conversely, pulse oximetry did not reliably detect hypoxemia in immobilized capuchins maintained on room air. In the first blood gas sample, all animals had SaO₂ ≤ 92%, with values as low as 80%-90% in most cases, whereas SpO₂ values were ≥95%, indicative of normoxemia, at 10 minutes in most anesthetic procedures. In the capuchins examined in this study, attaching the pulse oximeter probe to the finger yielded inconsistent readings, and occasional tongue movements compromised measurements at this site. In contrast, placement of the probe on the animals’ well-developed genitalia yielded high-quality readings. While the contribution of tissue thickness and skin pigmentation on measurement errors²⁸ at this site remains unclear, similar discrepancies between actual and pulse oximeter-derived oxygen saturation at lower SaO₂ levels have been reported in tufted capuchins when the probe was positioned on the interdigital region.⁷ This finding reinforces the poor sensitivity of pulse oximetry compared with arterial blood gas analysis and highlights the potential for unrecognized hypoxemia when pulse oximetry is the sole method used to assess oxygenation in this group of primates.

Hypoxemia is a multifactorial condition that may arise from hypoventilation, ventilation-perfusion mismatch, right-to-left anatomic shunts, or diffusion impairment, either individually or in combination.²⁹ Although anesthetic drug selection and dosing may influence respiratory function, PaCO₂ values at the initial blood gas assessment exceeded the hypercapnia threshold (>45 mm Hg)⁵ in only one animal per group, and no cases of hypercapnia were observed at the second blood gas analysis in G_AIR, indicating that overt hypoventilation was unlikely to be a major contributor to the mild-to-severe hypoxemia observed. This interpretation is noteworthy given the immobilization protocol used. Butorphanol has been shown to exert significant respiratory-depressant effects in rhesus monkeys (Macaca mulatta),³⁰ and midazolam administered intravenously at doses of 0.05-0.2 mg/kg has been associated with an approximately 40% reduction in tidal volume in humans, without a clear dose–response relationship.³¹ Despite these known effects, the PaCO₂ profile documented herein closely resembles that reported in capuchins receiving ketamine-dexmedetomidine and ketamine-midazolam-methadone, in which hypoxemia occurred in the absence of consistent hypercapnia.⁷ When considering alternative explanations, although comprehensive assessments of pulmonary and cardiovascular function, such as computed tomography and echocardiography, were not conducted, it is unlikely that the capuchins in this study had preexisting parenchymal disease impairing diffusion or a vascular abnormality diverting unoxygenated blood away from the lungs. Accordingly, disruption of normal matching between alveolar ventilation and pulmonary perfusion represents a plausible contributing mechanism. In humans, transition from an upright to the supine (dorsal recumbent) position reduces functional residual capacity due to upward displacement of the diaphragm by the abdominal viscera.³² This reduction in lung volume provides context for the greater decline in oxygen saturation observed in the supine position, relative to others, among individuals breathing room air.³³ Given the anatomic and physiologic similarities between humans and NHPs, and the fact that all animals in this study were maintained in dorsal recumbency during chemical restraint, posture-related venous admixture may have contributed to the observed hypoxemia. Impaired oxygen exchange, likely resulting from dorsal atelectasis and associated ventilation–perfusion mismatch, is further supported by the increased P(A-a)O₂ (>15 mm Hg) observed in capuchins breathing room air and by similarities to previously reported PaO₂ values in supine, immobilized tufted capuchins.⁷ Should this interpretation hold true, dorsal recumbency may predispose immobilized capuchins to hypoxemia; however, further investigation is warranted to determine the magnitude of this effect.

The present findings add to the existing body of evidence from medium-sized Old World monkeys,^4,8 by further characterizing the benefits of oxygen supplementation on arterial oxygenation in immobilized primates managed with multidrug anesthetic protocols. This recommendation applies even to brief immobilization periods, as hypoxemia developed within 10 minutes of drug administration. Oxygen was administered via face mask to resemble typical clinical practice,⁴ using flow rates described in earlier reports.^8,25 Notably, the supplemented capuchins exhibited higher PaO₂ values (298-458 mm Hg) than immobilized Japanese macaques (Macaca fuscata) maintained on 1-3 L/min oxygen via face mask (140-270 mm Hg).⁸ This higher-than-expected PaO₂ response reflects a larger effect size than initially estimated, which, in turn, offsets the potential loss of statistical power resulting from the exclusion of one individual (final n = 8). The use of a human-designed face mask may have provided a better fit on the capuchin’s face, enriching the FiO₂ more effectively than the cone-shaped veterinary masks commonly used in chemically restrained primates.⁴ It is therefore reasonable to assume that much lower oxygen flow rates are sufficient to increase FiO₂ and prevent hypoxemia in healthy capuchin monkeys undergoing ketamine-based protocols, while also minimizing unnecessary and wasteful oxygen use.

The observed rise in PaCO₂ following oxygen treatment, although unexpected, has now been documented across a broad spectrum of wild species,^{13,15–18,34} including marmosets.¹⁰ One leading theory is that elevated PaO₂ levels reduce stimulation of the oxygen-sensitive chemoreceptors in the aortic and carotid bodies, thereby suppressing ventilatory drive.^13,15 This phenomenon aligns with the marked increase in PaO₂ observed at 30 minutes with supplemental oxygen, although it appears inconsistent with the comparable respiratory rates observed between treatment groups. However, alveolar ventilation in G_OXY may still have decreased due to reductions in tidal volume, irrespective of respiratory rate. Improved oxygenation may also have diminished the capacity for carbon dioxide transport, owing to a shift in hemoglobin’s affinity for carbon dioxide when oxygen is bound (ie, the Haldane effect),³⁵ leading to an overall elevation in PaCO₂ as PaO₂ improves. Alternatively, the mask itself could have contributed to rebreathing of expired gases by increasing dead space. However, this likely played only a minor role in the observed increase in PaCO₂, as hypercapnia has also been widely reported in animals receiving oxygen via intranasal cannula, that is, without any imposed dead space.^15–18,34 Although the magnitude of the PaCO₂ increase was considerably less pronounced than in the aforementioned studies, respiratory acidosis was probably involved in a compensatory metabolic response. This is evidenced by elevated HCO₃⁻ and BE at 30 minutes, preventing concomitant decreases in pH in G_OXY. Regardless of the underlying mechanism, clinicians should be mindful that, as in many species, PaCO₂ may increase during oxygen supplementation in capuchins: a change that may be exacerbated in the presence of pulmonary disease or respiratory distress, in which case intubation and ventilatory support may be required.

The increase in pH observed between sequential blood samples in G_AIR may be interpreted as a return to normal values after an initial period of metabolic acidosis and anaerobiosis induced by the stress of physical restraint. Although induction of anesthesia was rapid (<2 minutes) and smooth, some degree of struggling is inevitable during net capture. At this early time point, PaCO₂ values remained below 45 mm Hg, suggesting that the acidosis was not respiratory in origin. Evidence further supporting a metabolic component includes the hyperlactatemia and elevated blood glucose levels observed in both groups at 10 minutes postrestraint, as well as higher HR values and body temperatures early in the immobilization period. A similar transient peak in lactic acid levels following immobilization, with a subsequent decrease in plasma concentration over time, has been documented in wild mammals and attributed to strenuous muscle activity associated with restraint and induction of anesthesia.^13,15 In G_OXY, a similar pH increase between sequential samples may have been blunted by the parallel rise in PaCO₂, as previously discussed.

The inability to determine whether hypoxemia resumed after discontinuation of oxygen supplementation represents the main limitation of this study. In immobilized white-tailed deer, PaO₂ returned to presupplementation hypoxemic levels within 10 minutes after oxygen therapy was interrupted,¹⁶ supporting the notion that improvements in arterial blood gases persist only for the duration of oxygen administration. This observation may partially explain the comparable recoveries between treatment groups in the capuchins studied here, as hypoxemia might have been equally present during recovery. From a clinical standpoint, oxygen should be continuously provided to capuchins until the face mask is no longer tolerated, unless future studies indicate otherwise. Another possible explanation for the absence of recovery differences between groups is that the degree of experienced hypoxemia may not have been sufficient to impair recovery quality. Support for this hypothesis comes from reports in reindeer, where worsened recovery following chemical restraint with etorphine and xylazine was noted only in individuals with PaO₂ values below 35 mm Hg, indicating life-threatening hypoxemia.¹⁴ Furthermore, the recovery scoring system, although tailored to the species and conditions of this study, may not have been sufficiently sensitive to detect behavioral signs of hypoxemia.

In conclusion, hypoxemia commonly occurred in dorsally recumbent capuchin monkeys chemically restrained with butorphanol, ketamine, and midazolam, but it was effectively treated with oxygen supplementation via face mask. Substantial increases in PaO₂ were achieved at an oxygen flow rate of 3 L/min, suggesting that lower flow rates may be sufficient to maintain normal arterial oxygenation.

Supplementary Materials

Table S1. ^{(15.2KB, docx)} Raw PaO₂ and PaCO₂ data for verification.

Conflict of Interest

The authors have no conflicts of interest to declare.

Funding

This study was funded by grant no. 2024/14429-3, São Paulo Research Foundation (FAPESP).