Intranasal oxygen reverses hypoxaemia in immobilised free-ranging capybaras (Hydrochoerus hydrochaeris).

Capybara (Hydrochoerus hydrochaeris) is the main host of tick-borne pathogens causing Brazilian spotted fever; therefore, controlling its population is essential, and this may require chemical restraint. We assessed the impact of chemical restraint protocols on the partial pressure of arterial oxygen (PaO2) and other blood variables in 36 capybaras and the effect of different flows of nasal oxygen (O2) supplementation. The capybaras were hand-injected with dexmedetomidine (5 μg/kg) and midazolam (0.1 mg/kg) and butorphanol (0.2 mg/kg) (DMB, n = 18) or methadone (0.1 mg/kg) (DMM, n = 18). One-third of the animals were maintained in ambient air throughout the procedure, and one-third were administered intranasal 2 L/min O2 after 30 min whereas the other third were administered 5 L/min O2. Arterial blood gases, acid-base status, and electrolytes were assessed 30 and 60 min after drug injection. The DMB and DMM groups did not vary based on any of the evaluated variables. All animals developed hypoxaemia (PaO2 44 [30; 73] mmHg, SaO2 81 [62; 93] %) 30 min before O2 supplementation. Intranasal O2 at 2 L/min improved PaO2 (63 [49; 97] mmHg and SaO2 [92 [85; 98] %), but 9 of 12 capybaras remained hypoxaemic. A higher O2 flow of 5 L/min was efficient in treating hypoxaemia (PaO2 188 [146; 414] mmHg, SaO2 100 [99; 100] %) in all the 12 animals that received it. Both drug protocols induced hypoxaemia, which could be treated with intranasal oxygen supplementation.

Introduction

The capybara (Hydrochoerus hydrochaeris) is the world’s largest rodent, and its population can grow exponentially with a large food supply and in the absence of natural predators [1]. In Brazil, the capybara is an important host for the transmission cycle of Brazilian spotted fever [2]. Seven hundred and thirty-six human cases of the disease have been confirmed only in São Paulo state from 2011 to 2020, with a mortality rate of 63.5% [3]. It is, therefore, imperative to control the capybara population by removing individuals [4] or using contraceptive measures [5]; these are strategies that may require capture procedures [5-8].

Several anaesthetic protocols for captive or free-ranging capybaras have been previously described [6-12], but no study has assessed the impact of these protocols on the arterial partial pressure of oxygen (PaO2). Hypoxaemia is a concern during the chemical restraint of wild animals [13] as it can lead to myopathy and myocardial hypoxia [14, 15]. Thus, oxygen (O2) supplementation has been used to prevent or treat hypoxaemia during the immobilisation of wild animals [13, 16–19]. Furthermore, chemical restraint protocols in capybaras have been based on dissociative anaesthesia (ketamine or tiletamine) in combination with other drugs (xylazine, romifidine, midazolam, zolazepam, levomepromazine, medetomidine, or dexmedetomidine) [7-12], and may be associated with muscle spasticity, nystagmus, disorientation, increased heart rate, and blood pressure [9]. In general, if the state of ‘dissociation’ persists during recovery, it can cause disorientation, anxiety, stress, myoclonus, catalepsy, and abnormal behaviour in the targeted animal [20].

Reversible chemical restraint combination protocols have been used in free-ranging animals, seeking to avoid the side effects observed with dissociative anaesthesia [18, 19, 21, 22]; the advantages of their use include animals returning to their normal pattern of activities quicker and safer, preventing them from becoming vulnerable to predators or accidents due to the disorientation related to anaesthetic recovery. Similarly, they can given drug reversal agents if a dose higher than the recommended dose is administered or if the animal experiences unwanted side effects.

The objectives of this study were to assess the impact of fully reversible chemical restraint protocols on free-ranging capybaras and the effects of different O2 supplementation flows. Based on the studies carried out with other species [18, 19, 23] as well as the authors’ experience, we hypothesised that nasal oxygen flows of either 2 or 5 L/min would reverse hypoxaemia in chemically restrained capybaras.

Materials and methods

The study was approved by the Chico Mendes Institute for Biodiversity Conservation (protocol 58028) and the Animal Ethics Committee of the Faculty of Animal Science and Food Engineering, University of São Paulo (protocol 9796180717). The animals were captured between May 2018 and October 2019 in southeast Brazil (21°59ʹ46ʺS; 47°25ʹ33ʺW) at 627 m above sea level during rainless nights with an average temperature of 21.8 ± 3.5°C.

To capture the capybaras, two portable corral-style traps (4 and 25 m2, respectively) with an iron fence or metal mesh walls were used and baited with corn or sugarcane in automatic closing platforms. If more than one animal was caught at once, only one animal was immobilised, and the others were released. Capybaras were excluded if they were very wet, weighed less than 20 kg, had severe injuries, or did not allow manipulation after 20 min after the drug injection. Thirty-six of 49 capybaras that were captured were included in the study; these included 30 females and 6 males weighing 48.9 ± 17.8 kg (actual body weight).

After the capybaras were caught in the corral-style trap, they were physically restrained with a one-metre diameter net for drug administration. They were given one of two drug combinations by intramuscular hand injection in the lateral side of the hind limb using a syringe and a 20 G needle. The drug combinations used were 5 μg/kg dexmedetomidine (Dexdomitor® 0.5 mg/mL, Zoetis, São Paulo, SP, Brazil) + 0.1 mg/kg midazolam (Dormire® 5 mg/mL, Cristalia, Itapira, SP, Brazil) + 0.2 mg/kg butorphanol (Torbugesic® 10 mg/mL, Fort Dodge Animal Health, Fort Dodge, IA, USA) (DMB, n = 18) or 5 μg/kg dexmedetomidine + 0.1 mg/kg midazolam + 0.1 mg/kg methadone (Mytedom® 10 mg/mL, Cristalia, Itapira, SP, Brazil) (DMM, n = 18). The doses were calculated based on the estimated body weights. The animals were weighed during immobilisation, after which they were kept in right lateral recumbency.

Six animals from each drug protocol group were maintained in breathing air (21% O2) (DMB-Air and DMM-Air) throughout the procedure, whereas six animals received O2 supplementation at 2 L/min (DMB-2L and DMM-2L) and six others received O2 supplementation at 5 L/min (DMB-5L and DMM-5L) for 30 min after the initial drug injection. This design was proposed to understand which treatment might reverse a possible hypoxaemia condition, when PaO2 < 80 mmHg. Oxygen was provided from a 5-L portable cylinder connected to a number 16 bladder probe (outer diameter 5.3 mm) lubricated with 2% lidocaine (Xylestesin jelly 20 mg/mL, Cristalia, Itapira, SP, Brazil) and introduced into one of the nostrils up to the medial canthus of the eye. Oxygen supplementation was provided for 30 min and discontinued 60 min after the drug injection. Afterward, the immobilisation was reversed with an intramuscular injection of 2 μg/kg flumazenil (Flumazenil 0.1 mg/mL, Cristalia, Itapira, SP, Brazil) + 5 μg/kg atipamezole (Antisedan 5 mg/mL, Zoetis, São Paulo, SP, Brazil) + 4 μg/kg naloxone (Narcan 0.4 mg/mL, Cristalia, Itapira, SP, Brazil), mixed in the same syringe, and the animals were released from the traps 30 min later. The treatment order was assigned as a block randomisation design, using a website (www.sorteador.com.br).

Respiratory rate (RR) was monitored by observing chest movements, and heart rate (HR) was measured by auscultation of the heart. Arterial blood samples were withdrawn from a branch of the femoral artery with a heparinised 1-mL syringe and a 22 G needle for blood gas analysis and the analysis of other blood variables. The first sample was withdrawn 30 min after the injection, when all the animals were breathing air, and the second sample was withdrawn 60 min after the injection. The samples were immediately processed with a portable blood gas analyser (I-Stat®1 and CG8+ Cartridges, Abbott Point of Care, IL, USA). pH, PaO2, partial pressure of arterial CO2 (PaCO2), plasma ionised calcium (Ca2+), potassium (K+), sodium (Na+), and glucose concentrations were measured, and the bicarbonate (HCO3-) and haemoglobin oxygen saturation (SaO2) were calculated. The results were corrected for the rectal temperature of the animal. The ratio of arterial oxygen partial pressure to fractional inspired oxygen (PaO2/FiO2) was also calculated.

Plasma lactate was measured with another handheld device (Accutrend® Plus—Roche Diagnostics, Mannheim, Germany). The device had a lower limit of detection of 0.8 mmol/L, and values below that were considered rounded to 0.7 mmol/L for statistical comparison.

The alveolar-arterial O2 gradient [P(A-a)O2], PaO2, and partial pressure of inspired O2 (PiO2) were estimated at a standard temperature of 37°C, respiratory coefficient (RQ) of 1, barometric pressure (PB) of 707.5 mmHg, fraction of inspired O2 (FiO2) of 0.21, and saturated water vapour pressure (PH2O) of 47 mmHg.

Based on the principle that [P(A-a)O2] < 10 mmHg, the minimum expected normal value for PaO2 was calculated for animals breathing ambient air.

The statistical analysis was performed using GraphPad Prism Software (San Diego, CA, USA). The variables (mean ± standard deviation) were considered parametric if they were normally distributed according to the Shapiro–Wilk test and had a coefficient of variation of less than 0.2; otherwise, they were considered nonparametric data (median [interquartile range]). For parametric data, a one-tailed unpaired t-test was used for comparison of values at 30 and 60 min, and analysis of variance (ANOVA) with Tukey’s post-test was performed for comparing data among the treatments. For non-parametric data, Wilcoxon test and Kruskal–Wallis test with Dunn’s post-test were used. Pearson’s correlation analysis was used to assess the correlation between the impact of O2 supplementation and blood gas variables at 60 min while comparing the animals of the Air group with those of the 2 L/min and 5 L/min groups. Differences were considered statistically significant at p < 0.05.

Results

The capybaras remained quiet after they were caught in the trap. Both DMM (9.2 ± 3.0 min) and DMB (10.5 ± 3.7 min) produced an equally fast induction, and there were no deaths during the study. No statistically significant differences were observed between the DMB and DMM groups. Thus, the animals from the DMB and DMM were grouped together for a more robust data analysis.

HR was approximately 70–80 beats/min and RR was about 26–36 breaths/min throughout the procedure. There was no statistical difference in these variables among the treatments. The same was observed in the arterial blood pH, over time. All animals that were breathing air only developed hypoxaemia at 30 min after drug injection, and remained hypoxaemic at 60 min. PaO2 was lower than the PaO2minimum expected, i.e., higher than 83 ± 4 mmHg while breathing air, resulting in a P(A-a)O2 of 49 [16; 65] mmHg and PaO2/FiO2 ratio of 209 [190; 228]. After O2 supplementation at a flow rate of 2 L/min, there was an increase in SaO2 and PaO2 compared with that at 30 min when breathing only air (p < 0.0001; p = 0.0002, respectively), but 9 of the 12 capybaras remained hypoxaemic, and there was no significant difference in both variables compared with the air group at 60 min (Table 1). A flow rate of 5 L/min reversed the hypoxaemia in all 12 animals (PaO2, p < 0.0001) (Table 1).

Table 1

Heart rate, respiratory rate, blood gas analysis, rectal temperature, glucose, lactate, plasma ionised calcium (Ca2+), sodium (Na+) and potassium (K+) plasma concentrations in free-ranging capybaras (Hydrochoerus hydrochaeris) chemically restrained with dexmedetomidine and midazolam, and either butorphanol (DMB) or methadone (DMM)§.

All animals (n = 36) were breathing air when the 30-min sample was collected (pre-treatment) and supplemented nasally or not with oxygen after that until the 60-min sample was collected. Values expressed as mean ± standard deviation or median [interquartile range].

Variable	Pre-treatment	30 min	Treatment	60 min
HR	Air (n = 36)	79 ± 8.8	Air (n = 12)	76 ± 5.8
			2L (n = 12)	74 ± 10.9
			5L (n = 12)	73 ± 4.5
RR	Air (n = 36)	36 [20; 55]	Air (n = 12)	30 [25; 38]
			2L (n = 12)	34 [25; 47]
			5L (n = 12)	26 [21; 32]
pH	Air (n = 36)	7.40 ± 0.04	Air (n = 12)	7.42 ± 0.04
			2L (n = 12)	7.40 ± 0.04
			5L (n = 12)	7.40 ± 0.02
PaO2	Air (n = 36)	44 [40; 47]	Air (n = 12)	51 [45; 56]A
(mmHg)			2L (n = 12)	63 [56; 86]*A
			5L (n = 12)	188 [171; 286]*B
SaO2	Air (n = 36)	81.6 ± 6.1	Air (n = 12)	85.7 ± 6.0A
(%)			2L (n = 12)	92.4 ± 4.2*A
			5L (n = 12)	99.8 ± 0.4*B
PaCO2	Air (n = 36)	45 ± 4	Air (n = 12)	45 ± 4A
(mmHg)			2L (n = 12)	46 ± 7A
			5L (n = 12)	54 ± 4*B
HCO3 -	Air (n = 36)	29 [28; 31]	Air (n = 12)	30 [26; 32]A
(mEq/L)			2L (n = 12)	29 [26; 31]A
			5L (n = 12)	35 [34; 36]*B
Rectal temp.	Air (n = 36)	36.5 [35.7; 36.8]	Air (n = 12)	36.6 [36.2; 37.2]A
			2L (n = 12)	36.2 [35.6; 36.8]A
			5L (n = 12)	35.2 [35.0; 35.7]*B
Glucose	Air (n = 36)	197 [169; 214]	Air (n = 12)	206 [162; 224]A
(mg/dL)			2L (n = 12)	188 [180; 191]A
			5L (n = 12)	273 [245; 301]*B
Lactate	Air (n = 36)	0.75 [0.70; 2.07]	Air (n = 12)	2.10 [0.70; 3.65]
(mmol/L)			2L (n = 12)	0.85 [0.70; 2.58]
			5L (n = 12)	0.70 [0.70; 0.77]*
Ca2+	Air (n = 36)	0.95 [0.86; 1.09]	Air (n = 12)	0.85 [0.65; 0.96]*A
(mEq/L)			2L (n = 12)	0.91 [0.70; 1.06]AB
			5L (n = 12)	1.15 [0.95; 1.19]*B
Na+	Air (n = 36)	135.5 ± 1.96	Air (n = 12)	135.92 ± 1.38
(mEq/L)			2L (n = 12)	135.22 ± 3.16
			5L (n = 12)	136.17 ± 1.75
K+	Air (n = 36)	3.9 ± 0.43	Air (n = 12)	4.18 ± 0.60
(mEq/L)			2L (n = 12)	4.33 ± 0.61*
			5L (n = 12)	3.93 ± 0.36

HR, heart rate; RR, respiratory rate; pH, potential hydrogen; PaO2, partial pressure of arterial of oxygen; SaO2, haemoglobin oxygen saturation; PaCO2, partial pressure of arterial of carbon dioxide; HCO3-, bicarbonate; Ca2+, plasma ionised calcium; K+, plasma potassium; Na+, plasma sodium; mmHg, millimetres of mercury; mEq/L, milliequivalents per litre; mmol/L, millimoles per litre; mg/dL, milligrams per decilitre; Air, animals without oxygen supplementation; 2L, animals which received 2L/min oxygen supplementation after 30 min; 5L, animals which received 5L/min oxygen supplementation after 30 min.

* indicates a significant difference between the times (30 and 60 min), and different superscript letters indicate differences between the treatments (p < 0.05). pH, PaO2 and PaCO2 values were corrected to the rectal temperature of the animal.

§ The animals from the DMB and DMM were grouped together for a more robust data analysis since there were no statistical differences between the groups.

The PaCO2, HCO3-, and plasma glucose levels increased significantly over time (from 30 min pre-O2 to 60 min) in the 5 L/min group (p = <0,0001) (Table 1). There was difference in plasma lactate over time only in the 5 L/min group (p = 0.0365). Values above 2 mmol/L were measured in six animals in the Air group, four animals in the 2 L/min group, and none in the 5 L/min group. Rectal temperature decreased over time in the 5 L/min group (p < 0.0001) (Table 1). Regarding electrolytes, ionised Ca2+ concentration slightly decreased in the Air group between 30 and 60 min (p = 0.0449) and increased in the 5L/min group (p = 0.030), K+ concentration increased in the 2L/min group (p = 0.0161), while Na+ did not vary over time in any group (Table 1).

Comparing the data from the capybaras belonging to the Air and 2 L/min groups at 60 min, positive correlations were found between increased flow and SaO2 and PaO2. Comparing the air and 5 L/min groups at 60 min, positive correlations were observed between the flow and SaO2, PaO2, PaCO2, HCO3-, Ca2+, and glucose. The increase in the O2 flow rate was negatively correlated with lactate (Fig 1).

Fig 1

Pearson’s correlation coefficient (r) and significance (p) of blood variables with different O2 flows during immobilisation of free-ranging capybaras (Hydrochoerus hydrochaeris).

pH, potential hydrogen; SaO2, haemoglobin oxygen saturation; PaO2, partial pressure of arterial of oxygen; PaCO2, partial pressure of arterial of carbon dioxide; HCO3-, bicarbonate; Ca2+, plasma ionised calcium; Na+, plasma sodium; K+, plasma potassium; Glu, glucose; 2L, animals which received 2L/min oxygen supplementation after 30 min until 60 min; 5L, animals which received 5L/min oxygen supplementation after 30 min until 60 min. Vertical axis represents r values. In the bright area, the closer to the edge, the greater the positive correlation. In the dark area, the closer to the centre, the greater the negative correlation. Markers represent a significant correlation (p <0.05).

Discussion

The chemical restraint of the free-ranging capybaras is an important step in population management, and optimal maintenance of respiratory capacity is indispensable in choosing the anaesthetic protocol. Thus, we assessed the changes in the respiratory variables and electrolytes in the capybaras after the administration of two different drug combinations, based on a benzodiazepine, an alpha2 adrenergic agonist, and an opioid. We also assessed the effectiveness of two O2 supplementation flows and the factors involved in this process.

Although blood gas analysers use algorithms based on human haemoglobin, these values have commonly been used in species for which results validation studies have not been carried out [17–19, 24]. Thus, the data obtained here for capybaras can help in interpreting the results and variations observed due to the change in oxygen flow.

Arterial blood gases did not differ when methadone or butorphanol was included in the drug protocol. Due to the agonist function of methadone at the μ-opioid receptors, it was expected that methadone would induce greater respiratory depression than butorphanol, since the latter acts as an antagonist at the μ-opioid receptors [25]. However, our results are consistent with those of another study that reported no difference in sheep sedated with a combination of dexmedetomidine and butorphanol or methadone [26].

Blood gas analysis is crucial for detecting hypoxaemia in capybaras. All animals developed hypoxaemia at 30 min, even in the absence of evident clinical signs. Although the portable blood gas analyser used was based on human-determined algorithms, it was and is, together with the co-oximetre, one of the available tools for measuring oxygen levels for field conditions [27]. In addition to the parameters of an ordinary blood gas analysis, there are also easy-to-calculate indices that allow a better understanding of respiratory dynamics and provide clinically useful prognostic information. It was found that the values of [P(A-a)O2] were higher than 10 mmHg and the PaO2/FiO2 ratio was less than 300 in animals breathing air. This was also observed during the chemical restraint of other wild animals, indicating that changes may have occurred in the integrity of the alveolar capillary, causing hypoxaemia due to intrapulmonary factors, such as the formation of shunts and ventilation/perfusion imbalance [17, 28, 29].

Although the intranasal flow of 2 L/min O2 improved the PaO2 in all animals, nine animals remained hypoxaemic. In comparison, the low flow rate of 2 L/min O2 was adequate to prevent hypoxaemia in cheetahs (Acinonyx jubatus) sedated with dexmedetomidine, butorphanol, and midazolam [18] and revert hypoxaemia in brown bears (Ursus arctos) immobilised with the medetomidine/tiletamine-zolazepam combination [23].

Hypercapnia (PaCO2 > 45 mmHg) was observed in six animals that were breathing air, six in the 2 L/min group, and all the 12 in the 5 L/min group at 60 min. Hypercapnia may have occurred due to poor ventilation, but its increase in the 5 L/min group may be due to the inhibition of hypoxic pulmonary vasoconstriction. This compensatory mechanism is activated in response to low PAO2 and diverts blood flow to better-ventilated areas of the lung [30-32]. Thus, the CO2 produced goes through the functional alveoli before it is eliminated. When hypoxaemia was reversed with O2 supplementation, hypoxic pulmonary vasoconstriction was inhibited, leading to impaired CO2 elimination.

The Haldane effect can also be involved in this process, as higher levels of O2 acidify haemoglobin, which impairs the binding of CO2 and increases PaCO2 [33]. Although the FiO2 of animals was not measured in this study, it is known that high FiO2 rates are associated with the appearance of atelectasis, and if they are provided to individuals with low ventilation/perfusion ratio, the lungs may collapse because alveolar gas passes into the blood at a higher flow rate than that at which it is inspired [34].

High values of plasma glucose were expected because glycogenolysis occurs to provide energy to meet the increased energy demand during the increase in cardiac frequency during stressful conditions, such as capture [35]. In addition, dexmedetomidine decreases the levels of insulin [36], which may lead to an increase in plasma glucose within a few minutes after drug administration [37]. However, plasma glucose increased over time only in the 5 L/min group. Anaerobic glycolysis was probably present in some animals in the air and 2 L/min groups due to low O2 levels. Despite the highest level of lactate (9.9 mmol/L) recorded in a capybara that was breathing air at 60 min, lactic acidosis was not observed.

Conclusion

Both drug protocols quickly induced chemical immobilisation in the capybaras, but they also caused hypoxaemia, regardless of the opioid used. Furthermore, oxygen supplementation should be provided to prevent hypoxaemia during chemical restraint, when these drug combinations are used. Based on the results of this study, a flow rate of 5 L/min is recommended because the lower evaluated flow rate of 2 L/min did not correct the hypoxaemia in all the animals. However, further studies are needed to determine if 3 or 4 L/min is adequate.

9 Jul 2021

PONE-D-21-16924

Intranasal oxygen reverses hypoxaemia in immobilised free-ranging capybaras (Hydrochoerus hydrochaeris)

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Reviewer #1: 1. Line 26 : Mention the scientific name

2. Line 90 : Describe the recumbency in which the animals were maintained

3. Line 90 : ...... maintained in breathing air.... (Mention the percentage of oxygen for better understanding)

4. Line 93: Oxygen was provided ............ (Mention the percentage of oxygen for better understanding)

5. Line 96:..... height of medial corner of the eye (Consider using the term medial canthus of eye)

6. Line 98 to 102: Was the reversal agents mixed in a single syringe ?? (Describe it as in separate syringes)

Reviewer #2: Dear authors,

this study aims to verification of hypoxaemia prevention in capybaras by oxygen supplementation during anesthesia. As any anesthetic protocol for wild animals the presented findings may be of use in wildlife population control as well as for treatment of animals in captivity. This makes it a worth publishing. On the other hand, the paper is kept very technical. Without any additional contribution to the field and I do not find it suitable for publication in high ranking journal in its current state. I am missing a review of so far published anesthetic protocols in capybaras and I do not like the experimental design. I would understand if the design was governed by other study/procedure and this study was a by product, but there is no indication for that. Moreover the most basic physiological parameters - breathing rate and hearth rate - were not measured. Apart from this, I have also following specific comments:

45 "it can" -> "it's population"

54 there are a multiple anesthesia protocols missing. Eg https://doi.org/10.29374/2527-2179.bjvm107220, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7136804/ or https://www.jstor.org/stable/3784559.

55-58 Please show why is this a concern with capybaras. Do other protocols result in hypoxaemia as well? Even anecdotic observations could be informative. It would be good if you could justify why you used a different anesthetic protocol than those already published. Are there any practical or theoretical advantages or disadvantages of your protocol or of the published protocols?

59-62 Please explain the choice of oxygen flow rates. Did you have some preliminary data?

Was any other treatment done on the capybaras (eg sterilization) during the anesthesia or was the restraint only because of this study?

Materials and methods

78 please indicate the sex in the raw data and check the (rounding of) mean weight. My calculation suggest that the average mass is 49kg.

82-88 Why did you choose these exact doses? Are these doses recommended by the manufacturer for rodents/caviomorphs/capybaras?

88 Was weight estimated or measured? If estimated, please add the dose of anesthetic in the raw data.

90-93 Please justify the design. I do not see apparent advantage in your design. It seems to me that it would be better if you started started with 3 groups, which would either be air, 2L/min and 5L/min oxygen supplementation immediately after onset on anesthesia. Such design would allow more direct comparison by paired tests and would also be more linked with the planned use of the anesthesia protocol. I do not expect anyone to first wait 30 min and then apply oxygen to follow your protocol. If you aimed to show that hypoxaemia is reversible, please state this clearly.

103 "all the groups ..." please be more specific here. I do not understand the sentence, what repetition you mean?

106 "25x7 mm" Please specify this in more detail or remove the information.

111 Is there any support for the assumption that the cartridges measure/calculate SaO2 and other parameter correctly? I doubt that there is data for this in capybaras and as such this should be clearly admitted. I do not find your statement in discussion too helpful. You could for example estimate the possible error or put emphasis on the non-calculated parameters. What is the precision of measurement by CG8+ cartridges?

113 How was the Tb measured?

116 I believe that this is not correct. Do you have any justification for this approach?

120 Why didn't you use the measured body temperature?

120 RQ=1 - Why? Normally assumed RQ is 0.85.

122 pH2O=47 mmHg - please justify this assumption. It seems to me that you assume that the temperature of the expired air has temperature of 37°C and saturated water vapour content at that temperature. I doubt this is correct. The maxilloturbinals in the nasal cavity will for sure preserve some heat and water, resulting in lower expired air temperature and lower water vapor pressure in the expired air. Also please explain all abbreviations. PAO2 and PiO2 are not explained.

136 Why did you use one-tailed paired t-test? For single tailed test you need to state the hypothesis first (greater/lower). What do you mean by intragroup?

Results

146 "The capybaras remained quiet after they were caught in the trap." How do you know this? Were the traps observed?

146 "The small size of the traps facilitated physical restraint with the net for drug administration by hand injection." This belongs to methods.

148 not meaningful to pool the two protocols at this moment. You explain the pooling below. Please check the mean, my calculations show 9.9min mean induction time.

149-150. missing Test statistics, p levels, df. Pleas provide this data and the tests for the revision process or even better place them in an appendix.

153 Please define hypoxaemia in methods. What is the threshold for hypoxaemia?

154 This information is not shown in Table 1. Only means are presented in Table 1. Please reformulate the sentence.

154 check/remove the ">" sign.

157 difference in what?

160 Please list all abbreviation in the table caption. Table should be able to stand alone. Valid for Tab. 2 as well.

Tab 1 3rd column. Group does not seem to be a good name. Moreover this column is the same in whole table and therefore redundant. Please remove. Same for table 2

Please list the parameters by subgroups as well. Add subgroup term in methods, you call call them groups there You may still keep the grand mean as well. You are showing statistical significant differences for the paired test and we do not see the first value of the pair. Moreover, it seems that you are using paired t-test, which has an assumption of normal distribution for data which you tested distribution and claim non-normal for a different test.

SaO2 It seems that you compared percentages by parametric test without any transformation. Please justify, the usual apprach is to perform arcsine transformation prior to statistical comparison.

PaO2/FiO2 please add to methods how was this calculated. Add a unit. And consider removing from the table as it is not used in the second part of the table.

P(A-a)O2 Consider removing from the table or filling the other part of the table.

175 this wording suggest gradual increase, but only two measurements do not allow this statement.

Table 2

Why is this table separate from table 1? Consider merging the tables.

Unify the units throughout the text. Please express the units per Liter or justify. I also suggest to use SI units, ie mol/L everywhere where possible.

Lactate: please check if median really equals lower boundary of interquartile range. The raw data suggest median of 1 and not 0.7 in the 30 min group. Please check the other values as well. Moreover the numbers do not seem to be correct because of the assumption on line 116.

Na+ and K+ data missing for one animal in the raw data and the methods state that animals with missing data were excluded. Please adjust.

200 Fig 1. Consider erasing the figure and putting the correlations in the table1/2. "BE" not explained anywhere in the text, please delete it from the figure. In the image legend on the right change the decimal comma to decimal point. Remove the legend bellow the figure and put the text in figure caption.

204 Was the p-value adjusted for multiple tests? if yes, please add this information to methods. If not, consider doing so. With 22 test the probability of false positive is very high.

222 patients?

226 But how reliable and comparable with reality these figures are?

228-230. Ratios do not have dimension. Additionally the sentence does not have verb and is meaningless, please reformulate.

234 It seems to me that PaO2 increased in ALL animals in 2L group. Moreover I suggest not to mix PaO2 and hypoxaemic. Please be careful in formulations.

239 "animals that were breathing air in the 2L group" is not clear. Two meanings are possible. Either delete the breathing air or 2L group.

259 why however?

260 please check the units. You are using mmol/L here, but in table 2 you are using mg/dL with the same parameter. One of these is probably faulty.

265 If no long-term adverse effect of the anesthesia on the animals was observed, how necessary is to prevent hypoxaemia then?

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22 Sep 2021

REVIEWER 1

- Line 26: Mention the scientific name.

- Line 90: Describe the recumbency in which the animals were maintained.

- Line 90: ...... maintained in breathing air.... (Mention the percentage of oxygen for better understanding).

- Line 93: Oxygen was provided ............ (Mention the percentage of oxygen for better understanding).

Answer: Thank you for this remark. Information has been added (lines 26, 103-111).

- Line 96: “height of medial corner of the eye (Consider using the term medial canthus of eye)

Answer: Thank you for this remark. Information has been included (lines 114-115).

- Line 98 to 102: Was the reversal agents mixed in a single syringe?

Answer: Thank you for the comment. Capybaras were given a mixture of the reversal agents. This information was added in this revised version in lines 119-120.

REVIEWER 2

- This study aims to verification of hypoxaemia prevention in capybaras by oxygen supplementation during anesthesia. As any anesthetic protocol for wild animals the presented findings may be of use in wildlife population control as well as for treatment of animals in captivity. This makes it a worth publishing. On the other hand, the paper is kept very technical. Without any additional contribution to the field and I do not find it suitable for publication in high ranking journal in its current state. I am missing a review of so far published anesthetic protocols in capybaras and I do not like the experimental design. I would understand if the design was governed by other study/procedure and this study was a by product, but there is no indication for that. Moreover the most basic physiological parameters - breathing rate and hearth rate - were not measured.

Answer: We thank the Reviewer for the comments and to fit this paper as a technical study. It was one of our propose. The authors have been studying about chemical restraint techniques in exotics and their impact on this procedure in different species. It is well-known hypoxaemia is the main issue in exotics capture, which is poorly identified and treated as well. Thus, the main focus of this study was to deliver a total reversible chemical restraint protocol, safer and avoiding hypoxaemia during the procedure. Indeed, the HR and the RR were measured throughout the procedure. They were not described in the original version because we have been writing down another paper, comparing cardiorespiratory monitoring, induction time, immobilization and recovery observed with both protocols. However, we agree with de Reviewer about the importance to describe HR and RR. Thus, as requested, we decided to add them as one mean/median in this revised version (lines 122-123, 173-174). The Reviewer comments certainly enriched our study.

- line 45 "it can" -> "it's population"

Answer: Thank you for this remark. The text has been changed in this revised version (lines 46-47).

- line 54 there are a multiple anesthesia protocols missing. Eg https://doi.org/10.29374/2527-2179.bjvm107220, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7136804/ or https://www.jstor.org/stable/3784559.

Answer: Thank you for the comment. We would like to point out the first paper was published 13 days before we had submitted our MS. So, it was not possible to add it to that original version. The second one is a review, not a study, and the last one focused on methods of capture, with poor description about drugs, monitoring and other physiological information. Thus, we have decided to add only the Rosenfield et al study in this revised version (ref #12, line 55).

- lines 55-58 Please show why is this a concern with capybaras. Do other protocols result in hypoxaemia as well? Even anecdotic observations could be informative. It would be good if you could justify why you used a different anesthetic protocol than those already published. Are there any practical or theoretical advantages or disadvantages of your protocol or of the published protocols?

Answer: Thank you for this remark. It is well-known hypoxaemia is the main issue in exotics chemical restraint procedures. As mentioned in the paper, no studies aimed at blood gas analysis in capybaras, neither one that assesses hypoxaemia and oxygen supplementation. The disadvantages of ordinary protocols, based on dissociative anaesthesia, and the advantages of the reversible chemical restraint protocols have now been highlighted in the introduction (lines 59-73).

- lines 59-62 Please explain the choice of oxygen flow rates. Did you have some preliminary data?

Answer: Thank you for this remark. The information was added in the revised version (lines 76-77).

- Was any other treatment done on the capybaras (eg sterilization) during the anesthesia or was the restraint only because of this study?

Answer: No. They were caught only for this study.

- line 78: please indicate the sex in the raw data and check the (rounding of) mean weight. My calculation suggest that the average mass is 49kg.

- line 88: Was weight estimated or measured? If estimated, please add the dose of anesthetic in the raw data.

Answer: Thank you for the remark. Sex was added in the raw data. We apologize about the mean weight. We added one digit after decimal number. This information was corrected in this revised version (line 94).

- lines 82-88: Why did you choose these exact doses? Are these doses recommended by the manufacturer for rodents/caviomorphs/capybaras?

Answer: Thank you for the comment. The doses were adjusted from pilot studies.

- lines 90-93: Please justify the design. I do not see apparent advantage in your design. It seems to me that it would be better if you started with 3 groups, which would either be air, 2L/min and 5L/min oxygen supplementation immediately after onset on anesthesia. Such design would allow more direct comparison by paired tests and would also be more linked with the planned use of the anesthesia protocol. I do not expect anyone to first wait 30 min and then apply oxygen to follow your protocol. If you aimed to show that hypoxaemia is reversible, please state this clearly.

Answer: Thank you for this remark. This suggestion could be done, indeed, but we were seeking the impact of the O2 supplementation to reverse the hypoxemia. Thus, we must let them breathing air, to evaluate if they developed a low PaO2, and then, give them the treatment. The text was rewritten in this revised version (lines 106-111).

- line 103: "all the groups ..." please be more specific here. I do not understand the sentence, what repetition you mean?

Answer: The text was rewritten in this revised version (lines 120-121).

- line 106: "25x7 mm" Please specify this in more detail or remove the information.

Answer: Thank you for this remark. The text was rewritten in this revised version (line 125).

- line 111: Is there any support for the assumption that the cartridges measure/calculate SaO2 and other parameter correctly? I doubt that there is data for this in capybaras and as such this should be clearly admitted. I do not find your statement in discussion too helpful. You could for example estimate the possible error or put emphasis on the non-calculated parameters. What is the precision of measurement by CG8+ cartridges?

Answer: Although blood gas analysers use algorithms based on human haemoglobin, these values have commonly been used in species for which results validation studies have not been carried out.

- line 113 How was the Tb measured?

Answer: We sorry about that but what does Tb mean?

- line 116: I believe that this is not correct. Do you have any justification for this approach?

Answer: The measurement of serum lactate under field conditions was one of the limitations of the study. According to the manufacturer's specifications, the lower limit of the measuring range of the device is 0.8 mmol/L (https://diagnostics.roche.com/global/en/products/instruments/accutrend-plus.html#productSpecs). Therefore, to run statistical analysis, we decided to adopt the value of 0.7 in the measurements that showed the message 'LOW', even knowing that the real value could be lower than this. Although this limitation, we understand the lactate serum data is quite important to show the reader the capture was fast and not stressful.

- line 120: Why didn't you use the measured body temperature? 122 pH2O=47 mmHg - please justify this assumption. It seems to me that you assume that the temperature of the expired air has temperature of 37°C and saturated water vapour content at that temperature. I doubt this is correct. The maxilloturbinals in the nasal cavity will for sure preserve some heat and water, resulting in lower expired air temperature and lower water vapor pressure in the expired air.

Answer: The capybaras’ body temperature was measured by rectal route (line 132-133 and Table 1) but it was assumed 37°C for [P(A-a)O2] calculation, since it is not possible to obtain the real temperature in the alveoli. This methodology has been used in other studies as Fahlman Å, Woodbury M, Duke-novakovski T, Wourms V. Low flow oxygen therapy from a portable oxygen concentrator or an oxygen cylinder effectively treats hypoxemia in anesthetized white-tailed deer (Odocoileus virginianus). J Zoo Wildl Med. 2014;45(2):272–7

- line120: RQ=1 - Why? Normally assumed RQ is 0.85.

Answer: Thank you for the question. RQ = 1 was adopted because it concerns values of a strictly herbivorous species.

- Also please explain all abbreviations. PAO2 and PiO2 are not explained.

Answer: We apologize about that. All of the abbreviations have been explained in this revised version.

- line 136: Why did you use one-tailed paired t-test? For single tailed test you need to state the hypothesis first (greater/lower).

- lines 149-150: missing Test statistics, p levels, df. Pleas provide this data and the tests for the revision process or even better place them in an appendix.

Answer: Significant p levels were added in this revised version. We used one-tail because we increased the O2 flow so, we would expect only an increase in the PaO2. In this way, a one-tail test is the best choice to do. An important Reviewer’s remark was about paired or unpaired data. Indeed, the corrected test is unpaired, which we have already applied in this paper. Thank you very much for this remark. This issue occurred because we did statistical analysis considering three groups at 30 min in the draft version. However, we understood all of the capybaras were given the same condition at 30 min and then, we decided to put them all together at that moment. We re-run the statistical analysis, applying the “unpaired” test now. We apologise for this mistake.

- line 136: What do you mean by intragroup?

Answer: This part was rewritten in this revised version (lines 157-161).

- line 146: "The capybaras remained quiet after they were caught in the trap." How do you know this? Were the traps observed?

Answer: Yes, we followed animals behaviour throughout the procedure.

- line 146: "The small size of the traps facilitated physical restraint with the net for drug administration by hand injection." This belongs to methods.

Answer: Thank you for the comment. This part was removed from the revised version.

- line 148: not meaningful to pool the two protocols at this moment. You explain the pooling below. Please check the mean, my calculations show 9.9min mean induction time.

Answer: Sorry about that. This part was rewritten in this revised version, pointing out the induction times from the DMM and DMB protocols (lines 167-169).

- line 153: Please define hypoxaemia in methods. What is the threshold for hypoxaemia?

Answer: Thank you for this remark. Hypoxaemia definition was added in the revised version (line 110-111).

- line 154: This information is not shown in Table 1. Only means are presented in Table 1. Please reformulate the sentence. Check/remove the ">" sign.

- line 157: difference in what?

Answer: This part was written in this revised version (lines 177-179).

- line 160: Please list all abbreviation in the table caption. Table should be able to stand alone. Valid for Tab. 2 as well.

- Table 2: Why is this table separate from table 1? Consider merging the tables.

Answer: Thank you for this suggestion. We have made them, as suggested.

- Tab 1 3rd column. Group does not seem to be a good name. Moreover this column is the same in whole table and therefore redundant. Please remove. Same for table 2

Answer: We changed “sub-group” for “treatments”. This information was added in this revised version.

- SaO2: It seems that you compared percentages by parametric test without any transformation. Please justify, the usual approach is to perform arcsine transformation prior to statistical comparison.

Answer: The SaO2 was considered parametric data, according to the statistical analysis.

- PaO2/FiO2 please add to methods how was this calculated. Add a unit. And consider removing from the table as it is not used in the second part of the table.

- P(A-a)O2 Consider removing from the table or filling the other part of the table.

175 this wording suggest gradual increase, but only two measurements do not allow this statement.

Answer: Thank you for the question. We have made them, as suggested.

- Unify the units throughout the text. Please express the units per Liter or justify. I also suggest to use SI units, ie mol/L everywhere where possible.

Answer: Thank you for the question. We have made them as suggested.

- Lactate: please check if median really equals lower boundary of interquartile range. The raw data suggest median of 1 and not 0.7 in the 30 min group. Please check the other values as well. Moreover the numbers do not seem to be correct because of the assumption on line 116.

Answer: Thank you for this remark. The serum lactate median (0.75 mmol/L) was added in the new version of the raw data and was corrected in this revised version (Table 1). We understand the other comment has been answered aforementioned.

- Na+ and K+ data missing for one animal in the raw data and the methods state that animals with missing data were excluded. Please adjust.

Answer: Thank you for this remark. This information has been added in this revised version (raw data).

- line 200 Fig 1. Consider erasing the figure and putting the correlations in the table1/2. "BE" not explained anywhere in the text, please delete it from the figure. In the image legend on the right change the decimal comma to decimal point. Remove the legend bellow the figure and put the text in figure caption.

Answer: Thank you for this suggestion. We have made them as suggested.

- line 204: Was the p-value adjusted for multiple tests? if yes, please add this information to methods. If not, consider doing so. With 22 test the probability of false positive is very high.

Answer: No, it was not.

- line 222: patients?

Answer: Sorry about that. The text has been changed in this revised version (line 257).

- line 226: But how reliable and comparable with reality these figures are?

Answer: Although the calculated indices use reference values and not the actual value, the data indicate that an impairment of oxygen exchange may have occurred. Thus, it might explain the low PaO2 values observed at 30 minutes. Probably due to hypoventilation, as neither altitude nor barometric pressure was high. There was no reason to suspect intrapulmonary causes, as the animals were clinically well before and after capture.

- lines 228-230: Ratios do not have dimension. Additionally the sentence does not have verb and is meaningless, please reformulate.

Answer: Thank you for this suggestion. This part was rewritten in this revised version (line 264-265).

- line 234: It seems to me that PaO2 increased in ALL animals in 2L group. Moreover I suggest not to mix PaO2 and hypoxaemic. Please be careful in formulations.

Answer: Thank you for this remark. This information was corrected in this revised version (lines 270-271).

- line 239: "animals that were breathing air in the 2L group" is not clear. Two meanings are possible. Either delete the breathing air or 2L group.

Answer: This part was rewritten in this revised version (line 276-277).

- 259 why however?

Answer: Sorry about that. This part was rewritten in this revised version (lines 298-299).

- line 260: please check the units. You are using mmol/L here, but in table 2 you are using mg/dL with the same parameter. One of these is probably faulty.

Answer: Lactate plasma concentration was measured as mmol/L, indeed. This information was corrected throughout the text.

- line 265: If no long-term adverse effect of the anesthesia on the animals was observed, how necessary is to prevent hypoxaemia then?

Answer: Thank you for this remark. The text was rewritten in this revised version (line 304).

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

10 Nov 2021

Intranasal oxygen reverses hypoxaemia in immobilised free-ranging capybaras (Hydrochoerus hydrochaeris)

PONE-D-21-16924R1

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18 Nov 2021

PONE-D-21-16924R1

Intranasal oxygen reverses hypoxaemia in immobilised free-ranging capybaras (Hydrochoerus hydrochaeris)

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