Hematologic profile of Amazon river dolphins Inia geoffrensis and its variation during acute capture stress.

Hematological values are of primary importance when investigating the health and physiological status of populations as they reflect the biological equilibrium of aquatic ecosystems. The objectives of this study are to produce baseline values for hematological parameters of the Amazon River dolphin (Inia geoffrensis), as well as to investigate significant variations according to sex, age, reproductive status and stress level. One-hundred-and-ten dolphins from Mamirauá Sustainable Development Reserve (3°3'S, 64°51'W), Central Amazon, Brazil, were live captured and sampled in November 2004 and 2005. Further, the means, standard deviations, minimum and maximum values and reference values (90% CI) were calculated. Correlations were performed to assess the relationships among blood values and cardiac rate (CR), respiratory frequency (RF), handling time and level of stress. No significant differences were found between sexes. Also, no differences occurred among pregnant and non-pregnant females, pregnant females and adult males or non-pregnant females and adult males. Calves had a higher white blood cell (WBC) count, and the neutrophil and lymphocyte absolute counts were significantly higher in calves than adults. The level of stress determined by empirical observation positively correlated with the WBC, neutrophil, lymphocyte and monocyte absolute counts and CR and RF. It was found that less stressed animals tend to present lower platelet counts and lower CR. The handling time of the dolphins was positively correlated with hematocrit (Hct), red blood cells (RBC) and Hb level. The hematological and physiological parameters varied according to time of handling and proved to be a good bioindicator of acute stress in Amazon River dolphins. The data provided here can complement long-term monitoring and identify the early warning indicators of health problems at the population level.

Introduction

Methods and techniques that advance the understanding of the physiological responses to the environment are becoming commonplace in the ecological studies of wild animals [1]. The health status of a population can be assessed by blood analysis of wild individuals [2,3]. The baseline data could then be used to evaluate the impact of environmental or anthropogenic health threats; e.g., pollutants [4]; firestorms [5]; algal toxins and cold temperatures [6] and noise stress [7]. Health assessments have been carried out on netted open ocean wild populations of dolphins [8], as well as in capture-release programs on coastal species [3,9,10] and have successfully provided blood parameter baselines for different species. The applicability of this kind of study can be perceived in a study where blood alterations were observed in bottlenose dolphins (Tursiops truncatus) after an oil spill which occurred due to the explosion of an offshore platform [11]. Blood values had been previously established for the species in Sarasota Bay population, Florida, USA, and had served as a reference to evaluate the Barataria Bay, Louisiana, USA, health population [12][13].

Distinct blood values can be observed in dolphins that endure different physiological states. Younger cetaceans may present different blood values than older individuals [9,14], including higher white blood cell (WBC) counts [15] [16]. Females may present higher haematocrit values than males [9], while pregnant females may have lower hematocrit (Hct) than males and non-pregnant females [3]. Alterations in blood values may also occur due to stress caused by transportation. The transportation and handling of wild animals may involve different steps from capturing to sampling, and the influence/duration of each step may be reflected in distinct blood alterations [8]. Given the significant differences that may occur in blood values according to sex, age, occurrence of pregnancy [15] and stress level, these potential differences should be investigated before establishing the baseline blood parameters.

The Amazon River dolphin or boto (Inia geoffrensis) is endemic to the Amazonian biome [17] and has been recently classified as an endangered by the International Union for Conservation of Nature’s Red List of Threatened Species [18]. These dolphins have been actively fished in different regions of Amazon to serve as bait for piracatinga (Calophysus macropterus) fishery, which led to a significant population decline [19,20]. Although some studies have analyzed captive Amazon River dolphins [21,22], no comprehensive health assessments have been conducted in the vast area of Amazonia so far. It is likely that Amazon River dolphins (I. geoffrensis), as a top predator, may reflect the effects of natural or anthropogenic stressors. Being long-lived and long-term residents of the Mamirauá Sustainable Development Reserve (RDSM) in the Amazon River, these dolphins can serve as important sentinels of an area that had very limited human interference in the past.

The current study was designed primarily to establish baseline values for the conventional hematological parameters of Amazon River dolphins. Further, variations in certain blood parameters according to gender, age, reproductive status and stress level are explored. The study also relates stress level and capture time to the variation in the hematological parameters. These data may be used to complement the long-term monitoring efforts of the dolphin population at Mamirauá [23,24] and to identify early warning indicators of potential long-term health problems at the population level.

Materials and methods

Animal ethics

The study was approved by the Committee on the Ethics of Animal Experiments of the National Institute for Amazonian Research. Every possible effort was made to ensure the safety and health of animals and personnel throughout the capture, examination and blood sampling procedure. No anesthesia, euthanasia or surgical procedure was used in this study. The dolphins were constantly provided sufficient shade and were kept wet while their behavioral and respiratory patterns were being closely monitored.

Study site

The study was conducted at RDSM (3°3’S, 64°51’W), an area of 11,000 km2 that is located between the Solimões and Japurá Rivers in the central Amazon region of Brazil. The specific localities from which the animals were captured include the Boca do Mamirauá and Paraná do Apara. A laboratory was set up at a floating research station, which was equipped with a gasoline-powered generator and was supplied with all the necessary equipment and reagents.

Animal capture and handling

The dolphins were captured using a previously perfected seining technique and then moved to a shallow beach [25] before being transported by boat to a nearby floating laboratory. The total time of capture and handling were registered from the moment the animals were encircled and covered with the small mesh net to their release in the proximity of the floating research station after the sampling procedures. The number and behavior of the encircled animals varied according to environmental conditions. A net of 12 meters in height was used to completely block a channel (maximum 10 meters depth), and the isolated area was subdivided by the deployment of more nets. The dolphins were brought together by seining them into a gradually shelving shoreline with a separate small mesh net.

The animals that exhibited any external signs of ill health were excluded from subsequent analyses. Age classes were determined according to animal’s total body length and sexual maturity [26]. Body color, scarring and skin lesions were also used to identify the age class of the 54 males and 56 females of the present study [27] (Table 1). Pregnancy was determined through ultrasound examinations (SonoSite Vet180plus, SonoSite Inc., Bothell, Washington, 98021–3904, USA) that were conducted on 33 adult females.

Table 1

Amazon River dolphins captured and released at RDSM, Brazil, in November 2004 and 2005.

Gender and age class	N	Min length (cm)	Max length (cm)
Adult male	42	190	248
Adult female	43	171	212
Juvenile male	6	169	189
Juvenile female	7	151	166
Male calf	6	119	152
Female calf	6	110	143
Total	110	110	248

This work was carried out after successive permits were granted to Projeto Boto by IBAMA/SISBIO (Brazilian National Environmental Agency) number 13157–1 and after it satisfied its criteria for ethics and animal welfare.

Sampling and blood analysis

Most of blood samples were collected from 110 relatively calm dolphins while they were manually restrained at the floating research station. Around 15 ml of blood was drawn from the superficial caudal peduncle vessel using a 19-gauge, 1.9 cm long butterfly catheter into a 20 ml syringe (Becton Dickinson Indústrias Cirúrgicas Ltda., São Paulo, São Paulo, 04717–004, Brazil) and was transferred to 5ml ethylenediaminetetracetic acid (EDTA) Vacutainer tubes (Becton Dickinson Indústrias Cirúrgicas Ltda., São Paulo, São Paulo, 04717–004, Brazil). After filling the tubes, they were gently rocked and then refrigerated within a maximum period of 20 mins following initial blood collection. The remaining blood was transferred into tubes with no anticoagulant and allowed to clot in ambient temperature to be used in other studies.

Manual hematological techniques were employed by a single trained individual for all the measurements. The red blood cell (RBC) and WBC counts were determined using Hayem and Turck (acetic acid and gentian violet) reagents [28], respectively (Newprov Produtos para Laboratório Ltda., Pinhais, Paraná, 83.323–020, Brazil). Subsequently, the platelet counts were determined by using a formaldehyde in sodium citrate solution [28], a binocular microscope (Nikon E-200, Nikon Instruments Inc., Melville, New York, 11747–3064, USA.), and a Neubauer improved counting chamber (LO—Laboroptik GmbH, Friedrichsdorf, Hessen, 61381, Germany). The microhematocrit capillary tubes were centrifuged at 12,850 X G for 10 minutes and then inspected against a standard calibration to determine the packed cell volume (PCV). The samples used in the hemoglobin (Hb) analyses were prepared by pipetting 20 μl of whole blood into 5 ml of Drabkin’s reagent (Newprov Produtos para Laboratório Ltda., Pinhais, Paraná, 83.323–020, Brazil) for subsequent photometric determination [28]. The erythrocyte cell indices were calculated by mean cell volume (MCV) (fl) = Hct (l) x 10/RBC (1012/L) and by mean cell hemoglobin concentration (MCHC) (g/L) = [Hb concentration (g/L) /PCV (l)] x 100. Blood smears were treated with Wright’s staining solution (Laborclin Produtos para Laboratório Ltda., Pinhais, Paraná, 83.321–210, Brazil) to conduct differential counts of 100 WBC per slide. All the parameters could not be determined for all the individuals due to lack of sample availability.

Stress level

The stress level of each animal that was handled at the research station was estimated by assigning a score of 1–4, where 1 represents the absence of visible stress and 4 represents a high level of stress (Table 2). The cardiac rate (CR, beats/min) was recorded just after blood collection for a minimum period of 1 min. The respiratory frequency (RF, breaths/min) was monitored throughout the handling period and was recorded just after the CR was noted.

Table 2

Four different levels of stress were attributed to Amazon River dolphins based on their behavior during handling time.

Level of stress	n	Behavior
Stress 1 (unstressed)	14	Appropriate responsiveness to external stimuli; no resistance to handling and immobilization; no vocalizations; animal is clearly relaxed.
Stress 2	19	Some, but not excessive, responsiveness to external stimuli; little resistance to handling and restraint; no vocalization.
Stress 3	14	Moderate responsiveness to external stimuli; mild resistance to handling and immobilization; occasional vocalization.
Stress 4	19	Extreme responsiveness to external stimuli; high resistance to handling and immobilization; frequent vocalization; temporary apnea.

The influence of time on the blood values was evaluated by correlating the total handling time of the Amazon River dolphins–combination of specific events (Table 3)–with the hematological and physiological parameters (CR and RF). The neutrophil to lymphocyte ratio (N:L) was calculated for 24 individuals and were separated in two time categories– 18 to 31 mins of manipulation and 32 to 45 mins of manipulation–in order to study the chronology of acute stress responses. CR and RF were determined for 26 and 33 dolphins, respectively.

Table 3

Specific events of the Amazon River dolphins handling during the capture-release program at RDSM in November 2004 and 2005.

	Small Mesh Net(min)	Boat(min)	Research Station(min)	Total Time(min)
Mean	10	11	10	30
Median	10	10	11	31
Min	4	6	3	18
Max	16	16	16	45

Statistical analysis

Descriptive statistics were calculated for the hematological values and 90% confidence limits were used to define the reference intervals (RI) for Gaussian distributed data following the guidelines by the American Society for Veterinary Clinical Pathology for sample sizes 40 ≤ x ≤ 120 [29]. The means, standard deviations (SD), minimum and maximum values and 90% RI were estimated. Outliers were defined by the Horn’s algorithm using Tukey’s interquartile fences and were discarded from the analyses. Levene’s test was used to assess the homogeneity of variance. The differences between the means of 2 groups (e.g., males and females or pregnant and non-pregnant females) were assessed using a Student’s t-test. Comparisons among calves, juveniles and adults, as well as among adult pregnant females, adult non-pregnant females and males, were carried out using a one-way analysis of variance (ANOVA) or Kruskal-Wallis for non-homoscedastic variances. Post hoc, Tukey’s test was performed when significant differences were found. All the statistical analyses were performed using the software package Statistica version 7.0 (StatSoft, Tulsa, Oklahoma, 74104, USA) at a p ≤ 0.05 level of significance.

All of the possible comparisons could not be made because of small or non-existent samples for a particular age-class or gender. As such, the sample size of each comparison may vary as it reflects the number of animals with complete data for that specific outcome. To determine the extent to which stress-related changes in hematological elements, cardiac and respiratory rates may covary with time, the strength of Pearson correlation coefficients (very weak, weak, moderate, strong, very strong) were determined [30].

Results

One-hundred-and-ten Amazon River dolphins were successfully captured and sampled during 2 separate expeditions in November 2004 and 2005. The duration of small mesh net seining varied from 4 to 16 minutes, according to the environmental conditions and the behavior of the animals. The handling process at the research station was always performed within a predetermined safety time range at an average of 10 minutes and never exceeding 16 minutes. The combined time of handling in the small mesh net and blood collection had a mean of 30 mins and varied from 18 to 45 mins (Table 3).

Reference ranges

Using a normal distribution, 90% double sided RI were determined after excluding the calves and the outliers through box plot and histogram analyses. The calves were not included in the RI calculation as they presented a significantly higher WBC than the adults and subadults (Table 4).

Table 4

Hematological values for Inia geoffrensis captured and released at RDSM, Brazil, in November 2004 and 2005.

		Adults and subadults								Calves
	SI Units	n	Mean	SD	Min	Max	Reference values			n	Mean	SD	Min	Max
Hct	L/L	97	0,40	0,03	0,31	0,46	0,39	0,40		13	0,39	0,03	0,34	0,43
RBC count	1012/L	92	3,91	0,65	2,84	5,93	3,80	4,02		13	3,69	0,43	2,95	4,55
Hemoglobin	g/L	66	155,97	31,41	122,00	390,00	149,52	162,42		6	159,13	14,28	133,00	175,00
MCV	fL	92	104	17	71	136	101	107		13	109	12	95	136
MCHC	g/L	66	386,8	36	298	469	379,4	394,1		6	400	49,1	332	471
WBC count	109/L	92	16,62*	4,61	8,15	27,45	15,81	17,44		13	24,54*	6,02	11,60	37,00
Band neut	%	58	1,06	1,11	0,00	4,00	0,83	1,30		9	0,89	1,05	0,00	3,00
Band neut	109/L	63	0,19	0,23	0,00	1,01	0,14	0,24		9	0,20	0,20	0,00	0,50
Neutrophil	%	58	48,00	8,30	32,00	63,00	46,16	49,65		9	45,67	8,83	35,00	60,00
Neutrophil	109/L	63	8,28	2,90	3,33	16,22	7,64	8,91		9	10,92	2,63	8,28	14,80
Lymphocytes	%	58	35,08	7,94	10,00	50,00	33,41	36,75		9	40,67	12,67	20,00	57,00
Lymphocytes	109/L	63	6,30	2,81	0,48	14,83	5,68	6,91		9	10,59	4,98	2,97	17,76
Monocyte	%	58	2,86	1,82	0,00	7,00	2,47	3,24		9	3,00	2,12	0,00	6,00
Monocyte	109/L	63	0,69	1,56	0,00	12,00	0,35	1,04		9	0,80	0,70	0,00	1,80
Eosinophils	%	58	13,06	5,73	4,00	30,00	11,86	14,27		9	9,67	6,42	0,00	19,00
Eosinophils	109/L	63	2,42	1,71	0,62	11,90	2,04	2,79		9	2,13	1,25	0,00	4,49
Basophils	%	58	aND							9	aND
Basophils	106/L	63	aND							9	aND
Platelets	109/L	87	297,00	111,95	78,00	556,00	277,08	316,99		13	296,46	69,57	194,00	420,00
ESR	mm/hour	88	50,69	4,95	20,00	58,00	49,82	51,57		10	48,90	3,45	44,00	56,00

(a)ND = Not determined

Significant difference with *P < 0.001

Gender and age variation in hematology

In order to generate accurate reference values, statistical analyses were performed by separating groups according to gender, age class and reproductive status. No significant difference was found between the hematologic values of males and females. Also, no differences were observed between the pregnant (n = 10) and non-pregnant females (n = 23), pregnant females and adult males or non-pregnant females and adult males.

Since no significant differences were detected between the sexes for any parameter (p > 0.05), the data were pooled for age class comparisons. Significant differences in the blood leukocyte counts were shown in Fig 1. Calves had higher WBC (mean = 24.54; SD = 6.02) than juveniles (mean = 18.85; SD = 5.6) (df = 102; p = 0.03) and adults (mean = 17.24; SD = 5.2) (df = 102; p = 0.0003).

Fig 1

WBC count for calves (n = 13), juveniles (n = 13) and adults (n = 80) of Inia geoffrensis from the RDSM, Amazonas, Brazil.

SE = standard error, CI = confidence interval. * Significant difference p ≤ 0.001.

Within relative WBC counts, age also affected the lymphocyte and neutrophil absolute values. The lymphocyte count was significantly higher in calves (mean = 10.59; SD = 4.98) than adults (mean = 6.20; SD = 2.70) (df = 64; p = 0.0009). In the same manner, the neutrophils count was observed to be higher in calves (mean = 10.92; SD = 2.63) than adults (mean = 8.12; SD = 2.79) (df = 64; p = 0.02) (Fig 2).

Fig 2

Relative WBC count for juveniles (n = 13) and adults (n = 80) of I. geoffrensis from the RDSM, Amazonas, Brazil.

* Significant difference p ≤ 0.05; ** Significant difference p ≤ 0.001.

Pregnant and non-pregnant females

The blood values of 33 adult females were assessed by ultrasound exams; 10 of these were pregnant and 23 were not. No statistically significant differences (p > 0.05) were found in any of the hematological parameters between the two groups. Although similar, this valuable information is worth noting given the scarce opportunity to examine pregnant individuals in the wild, particularly dolphins. The sample sizes, means, SD and minimum and maximum values for pregnant females are shown in the S1 Table.

Acute capture stress influences on cardiac rate, respiratory frequency and blood values

The minimum and maximum values of CR and RF for each age class are given in Table 5. The relative stress levels were estimated for 66 individuals. Furthermore, the duration of handling time was recorded for 26 Amazon River dolphins, which was positively correlated with Hct (r = 0.61) (p = 0.002); RBC (r = 0.45) (p = 0. 04) and Hb (r = 0.54) (p = 0.01) (Fig 3).

Table 5

Cardiac rate and respiratory frequency of the Amazon River dolphins during handling time at the research station in Amazonas, Brazil.

	Cardiac rate		Respiratory frequency
	(beats/min)		(breaths/min)
	(n = 26)		(n = 33)
	Min	Max	Min	Max
Calves	78	127	8	14
Juveniles	88	110	3	18
Adults	60	113	3	18

Fig 3

Correlation of total handling time of Amazon River dolphins with blood values at RDSM in Amazonas, Brazil.

A positive correlation was found between the total handling time and Hematocrit (Hct) (A), RBC count (B) and Hb level (C).

No correlation was observed between the total handling time and estimated stress level. However, the stress level was positive and moderately correlated with the lymphocyte level (r = 0.44) (p = 0.004) and CR (r = 0.57) (p = 0.03) and positive and weakly correlated with WBC (r = 0.29) (p = 0.02), absolute neutrophil (r = 0.38) (p = 0.01), and monocyte (r = 0.34) (p = 0.03) counts, and RF (r = 0.35) (p = 0.04) (Fig 4).

Fig 4

Correlation of stress level with blood and physiological parameters of captured-released I geoffrensis at RDSM in Amazonas, Brazil.

The stress level was positively correlated with WBC (A), neutrophils (B), lymphocytes (C), monocytes (D), cardiac rate (E) and respiratory frequency (F).

Other significant correlations were noted among of the blood variables. Some of the estimates were expected to be strongly or very strongly correlated, such as neutrophil with lymphocyte percentages (r = - 0.78) (p < 0.001); WBCs with neutrophil absolute values (r = 0.85) (p = 0.000); CR and RF (r = 0.45) (p = 0.021) and Hb and MCHC (r = 0.55) (p = 0.003), which were moderately correlated since one variable was for the most part determined or influenced by the other variable. Other comparisons have revealed some more apparently dynamic interactions with moderate correlations, particularly for WBC and CR (r = 0.43) (p = 0.031), WBC and RF (r = 0.56) (p = 0.003) and neutrophils absolute values (r = 0.44) (p = 0.039) (Fig 5).

Fig 5

Correlation of blood values and physiological parameters of captured-released I geoffrensis at RDSM, Amazonas, Brazil.

Positive correlation was found between WBC with CR (A) and RF (beats/min) (B) and neutrophils and RF (C).

Most stressed animals (level 4) presented significantly higher lymphocyte (mean = 8.27; SD = 3.31) (df = 3; p = 0.03) counts than the animals at level 1–3 (mean = 4.99, 5.35, 7.32, respectively), whereas the least stressed animals (level 1) presented significantly less platelet counts (mean = 227; SD = 106) (df = 3; p = 0.008) than level 2 (mean = 349), level 3 (mean = 313) and level 4 animals (mean = 319).Additionally, the level of CR in the least stressed animals (mean = 67; SD = 4) (df = 3; p = 0.03) was less than those at levels 2–4 (mean = 80, mean = 86, mean = 96, respectively).

The neutrophil to lymphocyte ratio N:L was calculated with the time of capture for 24 individuals. The total time (sum of intervals 1, 2 and 3) was divided into 2 parts– 18 to 31 mins of capture and 32 to 46 mins of capture–and it was observed that the proportion of neutrophils to lymphocytes had increased from 1.4:1 to 1.7:1.

Discussion

The goal of the estimation and use of reference values and prediction intervals for hematology data is to establish baseline values for healthy populations against which perturbations can be judged in putative non-healthy populations or individuals [10]. The basis for selecting healthy populations is clearly very difficult in wild animals, but the data reported here is assumed to have been collected from healthy individuals with no obvious clinical signs of ill health. The only previously available data on the blood values of I. geoffrensis is from that of 22 animals that were kept in aquariums in the USA in the 1970s [31]. The Hb concentration of the free-ranging Amazon River dolphins that were examined in this study were higher (15.51 g/dl) than those of the captive individuals (13.85 g/dl) [31], which is probably a consequence of the lack of space and the shallow depth of water that these captive animals were subjected to. Also, the wild I. geoffrensis had higher WBC counts (18.74 x 103/mm3) than the captive ones (13.38 x 103/mm3) [31]. A major reason for the increased antigenic stimulation that was observed in the free-ranging dolphins may be due to parasitic infections, which consequently increase eosinophil counts [32]. Although it has not been documented yet, the probable higher degree of parasitism experienced by wild Amazon River dolphins as compared to the captive ones may lead them to have a higher percentage of eosinophil (5% and 13%, respectively). Besides, the captive dolphins were also less likely to be stressed than the wild dolphins, which has also affected the WBC count. Subclinical bacterial, fungal, protozoal and viral infections should also be considered as factors for elevated WBC in wild populations as compared to the managed populations [33].

Erythrocyte sedimentation rate (ESR) is a nonspecific indicator of inflammation acute phase response and is used as a monitoring tool in veterinary and human medicine [3]. Although it is not commonly cited in studies conducted on the blood values of marine mammals, the significance and application of ESR measurements in dolphins is likely to be correlated with diseases and other sources of inflammation and may be a useful prognostic indicator [3]. While ESR values are very species-specific and tend to vary considerably among marine mammals (e. g. 16–52 mm/hour for short-finned pilot whale Globicephala macrorhynchus; 3–29 mm/hour for false killer whale Pseudorca crassidens) [34], the CI 90% found in the 102 animals that were sampled in the present study (50–51 mm/h) indicated a very narrow range, which may represent a reliable parameter to monitor infections in river dolphins, as all the animals included in the analysis were apparently healthy. However, no validation was done on the animals with inflammation and the response to it regarding the ESR.

While the blood values were similar among age classes, some differences were noted. Calves presented higher WBC counts, which was followed by juveniles and adults. Higher leukocyte indices may be due to the developing immune systems of the younger animals as they mount immunologic responses to a broader range of largely novel antigenic stimuli [35]. The influence of age on blood values in aquatic mammals has been documented in previous studies [3,9,36,37]. Older animals tended to have lower WBC counts than juveniles and calves of the false killer whale (Orcinus orca), bottlenose dolphin and beluga whale (Delphinapterus leucas) [16,35]. In the WBC differential count, calves had a higher absolute value of lymphocytes as compared to juveniles and adults, while neutrophils were more pronounced in adults.

Consistent studies of blood values in wild pregnant female dolphins are very scarce, which is mostly because of the major logistical difficulties in obtaining samples from free-ranging cetaceans. Here, the blood values of 33 adult females that had their reproductive status assessed by an ultrasound exam showed no difference between pregnant and nonpregnant animals. Similar results were reported on bottlenose dolphins from the Indian River Lagoon, Florida, USA [9]. Alternatively, pregnant bottlenose dolphins from Charleston, South Carolina, USA, had statistically lower Hct as compared to both the non-pregnant females and males, and the authors attributed this observation to the physiologic hemodilution caused by pregnancy [3]. The physiological demands of the fetus and pregnancy itself on the mother varies according to the stage of pregnancy in cetaceans [38]. As they were free ranging animals, the sampled female I. geoffrensis in this study presented varied pregnancy stages, which can also explain the lack of blood value differences between pregnant and non-pregnant females.

Handling and transportation are known to be stressful for wild terrestrial mammals, and it is likely that the same holds true in aquatic mammals; however, few studies have reported this occurrence in dolphins [39]. A significant challenge of studying stress in marine mammals, or any wild species for that matter, is obtaining the baseline data that represents an unstressed state of being. Pursuit, capture, restraint and sampling procedures are obvious stressors that can influence analyses, sometimes within minutes [34]. Projeto Boto [25], a long-term project that involved the capture and release of freshwater dolphins in the Amazon region of Brazil, granted us a very good opportunity to take the first steps in understanding the physiological changes in the blood parameters of Amazon River dolphins under different degrees of stress.

Induced alterations in blood values driven by stressors can be due to different stimuli in mammals’ bodies. Leukocytosis and neutrophilia can be induced by epinephrine mobilization of these cells from the bone marrow [40]. The handling of the dolphins here as a potential source of stress, showed a positive correlation in the duration of handling to the RBC, Hb concentration and Hct. Similar results of higher Hb and platelet counts were observed in pantropical spotted dolphins (Stenella attenuata) that were under stress during an encirclement by a tuna purse seine operation in Mexican waters [8]. Increase in RBC counts, Hb concentration and Hct were associated with the stress-mediated decrease in calculated plasma in women under acute stress. The plasma volume decreased secondarily to a rise of circulating total cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol, and triglyceride concentrations [41]. Further investigation on these plasma elements would be necessary to confirm the cause of RBC, Hb and Hct rise in dolphins under stress.

Besides blood value alterations, acute stress conditions can be visually detected through behavioral alterations. Here, the WBC, absolute counts of neutrophils, lymphocytes and monocytes showed a positive correlation with the stress level, as well as with the CR and RF. This indicates that the empirical observations of stress and its classification/hierarchy based on the degree of stress can be used to assess alterations in the blood cell values and physiological response to it. It is likely that WBC may increase under stressful conditions, as it was documented in the case of pantropical spotted dolphins in the southern coast of Mexico where the time of being chased and captured was correlated with an increase in WBC during tuna fishing [8].

The positive correlation between the RF and WBC is probably due to a more pronounced increase of neutrophils than other types of leucocytes during stress. The increase in the proportion of N:L reinforces the finding that the occurrence of a neutrophil increases during acute stress. However, it should also be pointed out that although both observations could be considered indicative of acute stress, the relationships found here could also represent a normal physiological state than a signal of stress, especially for immature dolphins. Younger animals can have a naturally higher percentage of WBCs due to their developing immune systems, while their RF tends to be higher because of a normally elevated metabolism [42]. This combination may have led to some error in the correlation analysis between RF and WBC.

Given the very clear effect of stress related glucocorticoid hormones on leukocyte profiles, an increase in the N:L ratios is an expected observation of stress response. The N:L ratio has been shown to increase in a variety of mammals following transportation, including horses, goats, swine and cattle [43], as well as the Amazonian manatee (Trichechus inunguis) [44], dolphins [39] and black rhinoceros (Diceros bicornis) [45]. “Typical stress leukogram” was observed during a mass stranding of 17 striped dolphins (Stenella coeruleoalba) along with mild relative neutrophilia and absolute lymphopenia and eosinopenia [46]. Despite the low number of animals in this specific analysis, a similar increase in the N:L occurred in I. geoffrensis in proportion to the duration of capture and handling. Also, the relative white blood cell counts appear to be an efficient method of assessing acute stress in free-ranging river dolphins.

Conclusions

The baseline values for the hematological parameters of an apparently healthy river dolphin population of Amazon River dolphin (I. geoffrensis) have been described for the first described. Calves had higher neutrophil and lymphocyte absolute counts, which was reflected in a higher overall WBC count. Empirical observations of stress based on its intensity/degree can be used to assess alterations in the blood cell values and physiological response to it. Variations in Hct, Hb, N:L ratio, WBC, platelet counts, neutrophils, lymphocytes and monocytes absolute counts proved to be a good bioindicator of acute stress in Amazon River dolphins. CR and RF also showed a positive correlation to the level of stress in these animals. The baseline values are of crucial importance to efficiently monitor the health of wild populations, and the data provided here represents a major contribution on this matter. In addition, the hematological blood parameters of such populations from an area with low human impact could serve as a trustworthy baseline for other dolphin populations in the Amazon region.

Hematological values for adult female Inia geoffrensis.

(XLSX)

Click here for additional data file.

6 Nov 2019

PONE-D-19-17847

Hematologic profile of Amazon river dolphins Inia geoffrensis and its variation during acute capture stress

PLOS ONE

Dear Dr. Magalhães Drummond de Mello

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Both reviews think this manuscript is an important contribution to the veterinary field, particularly since it increases knowledge on a rare an vulnerable species, the Amazon river dolphin.  However, as you will see, one reviewer wants more clarity regarding which individuals and in which way you conducted the statistical analyses.  Both reviewers also suggest you to have a native english speaker helping you with some of the grammar of the manuscript.

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Reviewer #2: Yes

**********

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Reviewer #1: Overall Comment:

The data presented here represent important contributions to the literature regarding blood reference ranges for the understudied boto population. I do have some reservations regarding its publication at this time, and request a revision for the following reasons.

I am concerned about the inclusion of animals with high stress scores in the creation of baseline values/reference ranges. Ideally, animals with high stress scores would be excluded from the reference range creation since they are not “normal.” I see this paper as having two separate purposes, 1) to establish hematological reference ranges for minimally stressed boto by age class and 2) to evaluate the effect of stress on hematological parameters in this species. I think it would be a stronger manuscript if organized as such. If you choose not to split the study out in this manner, there should be some mention of this as a study limitation in the discussion (the inclusion of stressed animals when establishing baselines).

I was unclear whether or not all the data were tested for a normal or Gaussian distribution. Especially for some of the parameters with relatively low sample sizes, this is extremely important with regards to the question on whether rigorous statistical methods were applied. I answered “I don’t know” because this is a pending question I have for the authors in my review.

While the authors in general did a good job of translating to English, the language is still awkward in many places and there are still some confusing statements and grammatical errors that appear to be associated with translation. I tried to assist and interpret where I could, but I recommend a native English speaker edit the manuscript prior to resubmission.

Abstract:

Line 30, 36: Change “gender” to “sex” throughout text as sex refers to the biological differences between male and female, and gender refers to the individual’s identity and is therefore not suitable for use with animals.

Lines 36-40: Since reference ranges are often created according to sex, reproductive status, and age class, I would recommend moving these findings up to line 33 before the sentence starting “Means…” You have essentially created age-class specific reference ranges in this study since those were the groups with statistically significant differences – and I would clarify that in the abstract.

Lines 38-39: I recommend deleting “as a result of the still-developing immune system.” There are other potential explanations for calves having higher WBCs (higher lung worm burden in younger animals - which has been observed in other dolphin populations, or higher stress level than older animals). Given these alternate possible explanations and the fact that this is not a common finding in all populations of dolphins, I do not recommend being so definitive in explaining its cause.

Introduction:

Line 52: Change “accessed” to “assessed”

Line 55: Recommend changing “health status approaches” to “health assessments”

Line 56-57: All these studies can be referred to as “capture-release” studies, I am unclear why the distinction is being made for the St. Aubin study as “netted wild population” vs capture release programs. Please clarify.

Line 57: Change semicolon to comma and “parameters” to “parameter” and “baseline” to “baselines”

Lines 58-62: This sentence is confusing the way it is written, please reword. With reference #11 – there had not been a baseline study prior to the oil spill on that particular population (although baseline studies existed for other Tt populations that were used for comparison). I would be more explicit in explaining how this particular citation makes the authors’ point. I also think the reference to seasonal variation in the Sarasota dolphins is confusing since you do not address seasonality at all in the remainder of the paper (even though it is controlled for with your sample timing).

Line 64: delete “aged”

Line 65: This is not necessarily a common finding among the capture-release sampled dolphins in various locations. Given the age of this citation, I would recommend either striking this comment or finding additional more recent citations to make a stronger case.

Line 67: Change “alteration on” to alterations in”

Line 69: change “englobe” to “involve”

Line 70: change “may reflect” to “may be reflected in”

Line 71: change “gender” to “sex”

Line 73: I believe you mean “before establishing baseline blood parameters.” Ideally you are establishing these reference ranges with animals that are minimally stressed and removing the outliers from your “normal” sampling population.

Line 76: include citation for the most recent IUCN report

Line 76-77: Recommend changing “under intentional catch” (which is vague) to “actively fished commercially”

Line 78: Recommend changing “leading to significant populations decrease” to “leading to a significant population decline”

Lines 83-84: delete comma after “reserve” and replace with “in the”

Line 87: Why single out the ESR here? Seems unnecessary – either please justify or delete.

Materials and Methods:

Line 109: Recommend spelling out the RDSM acronym in the figure caption

Line 116: Change “proximities of research floating station” to “proximity of the floating research station”

Lines 116-117: Recommend changing the sentence starting with “It varied” to: “The number and behavior of encircled animals varied according to environmental conditions.”

Line 118: If you are going to indicate the depth of the channel it would also be helpful to know the depth of your capture net (s).

Lines 119-121: You state that the usual method was to seine the dolphins to the shoreline. That leads me to believe that there were other methods used to capture the dolphins in some instances. If so, please describe those methods as well, if not, please clarify the text here.

Line 126: What were your criteria for determining pregnancy? Identification of a fetus? Recommend explicitly stating that.

Line 139: Did you actually use a catheter or a needle? Was the blood drawn directly into the EDTA vacutainer tube or was it drawn into a syringe and then transferred to the tubes? The way this is written, it sounds like the blood from the EDTA tubes was transferred to a tube with no anticoagulant and allowed to clot, which is confusing. Please clarify your methods.

Line 146: How were these manual methods standardized? Were they consistently done by a single trained individual or were multiple individuals trained on standardized methods?

Line 171: For stress levels 1 & 2 behavior, perhaps you mean no/little excessive response to external stimuli? Or appropriate response to external stimuli? Relaxed dolphins should be alert and responsive to external stimuli, but not hyper-responsive. By immobilization do you mean restraint? Recommend re-wording these behavioral descriptions.

Line 175: I think you mean “the neutrophil to lymphocyte ratio” ?

Lines 176-177: 12 individuals in each category? Are samples from the same individual compared between the two time categories or are the comparisons done between different animals? Please clarify the methods here.

Line 177: insert ‘of’ between ‘chronology’ and ‘acute’

Line 181: Recommend first discussing the tests for significant differences in age class, reproductive status, and sex, then discuss the methods for creating reference ranges based on those outcomes. Were all the data tested for normality (Gaussian distribution)? Especially with some of the parameters that had smaller sample size, normal distribution cannot be assumed and should be tested.

Line 200: Strength of correlation (weak, moderate, or strong according to r value) should be stated here. You can find references in the literature to describe correlation strength.

Line 195: How were multiple comparisons controlled for in order to prevent Type I error (false positives)? You compared a lot of variables and should either control for these multiple comparisons or justify why you did not do so.

Line 214: Since Table 4 is cut off, I cannot see if you stated 90% RI for the calves. I am assuming you did not due to small sample size. I would explicitly state what you are presenting for the calf data here and why there are not reference ranges established. I would also recommend removing the moderate and severely stressed dolphins from the reference range calculation.

Line 220: Formatting in the version I have cuts off the left (parameter) and right (last column is calves min) margins of this table. Cannot fully evaluate data presented. Recommend including more details regarding the reference values in the table caption (90% double sided reference intervals)

Line 221: In the methods, it stated the level of significance was set at p < 0.05 but here it states p < 0.001. Why the discrepancy?

Line 234: In Fig 2: Recommend adding sample sizes (n) above each age class box plot

Line 242: Again, recommend including sample sizes in Fig 3 (either in the caption or in the legend)

Line 251: I do not see medians in table S1, delete reference to them here.

Line 252: These are not reference intervals, recommend deleting ”RI” and just referring to the data in table S1 as summary statistics.

Line 263: Fig 4: Recommend including r and p values either on the figure or in the caption. Same comment for Figs 5 and 6 as well.

Line 254: Recommend spelling out cardiac rate and respiratory frequency in this section title as these are not common abbreviations.

Line 268: Correlation should be categorized as weak, moderate or strong based on the r values.

Discussion:

Line 300: change “comes” to “come” – “data” is a pleural noun (singular “datum”)

Line 301: change to “in the 1970’s”

Lines 304-310: Captive boto were also likely less stressed than the wild-caught boto, which would also affect the WBC count. Subclinical bacterial, fungal, protozoal, and viral infections should also be included in the differentials for elevated WBC in wild vs managed populations.

Line 320: change “an” to “a” Also, without demonstrating a correlation between ESR and inflammation, I would caution against evaluating this parameters utility here. While the narrow range MAY indicate that this could be a useful indicator IF it does in fact change with inflammation, you have not proven the latter yet.

Lines 318-320: Was there any correlation between ESR and WBC count? If not, this makes it more likely that the WBC/neut elevations were due to stress rather than inflammation/infection/antigenic stimulation.

Lines 321-330: Also need to include stress as a differential for higher WBC counts in juveniles. And a note that this is not a consistent finding among all cetacean populations.

Line 335: change “was” to “were”

Line 340: Change “towards” to “on”

Line 342: Can you present data in the results regarding the sample sizes according to each trimester if available to help elucidate this point?

Line 343: change “values” to “value” and “towards non-pregnant females” to “between pregnant and non-pregnant females”

Lines 356-358: Due to the spleen’s small size relative to the dolphin’s overall body mass, my understanding is that unlike in other species, splenic contraction is not considered a significant differential for elevated RBC, HCT, Hb in cetaceans.

Line 359: Recommend using the term “epinephrine” rather than “adrenalin”

Line 369: change “access” to “assess”

Line 373-374: Also cannot rule out subclinical pulmonary disease as contributing to this trend, and this should be included as a differential for that finding.

Lines 376-378: Please reword this sentence for clarity.

Line 379: Add “can” between ‘animals’ and ‘have’ – as this is not always a consistent finding in cetaceans

Lines 384-394: More clarification in your discussion of the different types of stress leukograms is warranted – since you have the expected neutrophilia with a corticosteroid stress response, but not the associated lymphopenia (as with the “typical stress leukogram” that you cite). Lymphocytosis would be associated with an epinephrine-induced response. While you mention both of these hormone-induced pathways, your explanation of the lymphocytosis is incomplete.

Line 400: change ‘access’ to ‘assess’

Line 403: change “ad” to “and”

Reviewer #2: I attach minor corrections per line.

The title identifies the study subject as Amazon river dolphin, also known as boto. But the interchange of this common name in the manuscript may be confusing to the reader. I would recommend being consistent in using one. Mention both names once, but after that be consistent in using one.

In Spanish and perhaps in Portuguese, the names of rivers begin with capital letters, but the word river itself does not begin with capital letters, i.e., río Amazonas. But in English, both the river name and the word river begin with capital letters, i.e., Amazon River. Thus, in the title and throughout the document, it should be Amazon River dolphin.

All animals, except common animals (horses, goats, swine, cattle), should be first introduced with a common name followed by its scientific name. Afterwards, I recommend using just the common name.

Add the common names for Globicephala macrorhynchus, Pseudorca crassidens, Orcinus orca, Delphinapterus leucas, Stenella coeruleoalba

Add the scientific names for Amazonian manatee, rhinoceros

31: 110 write out as One-hundred-and-ten

38: Delete The before calves

40: should be lymphocytes

42: should be neutrophils, lymphocytes and monocytes

44: define Hct and then put in parenthesis. Red Blood Cells should be red blood cells. Define Hb and then put in parenthesis.

65: should be white blood cells (WBC)

66: should be hematocrit (Hct)

74: should be The Amazon river dolphin or boto (Inia geoffrensis)

76: cite where the IUCN lists the species as endangered.

76: instead of “under intentional catch” use affected by intentional capture

81: delete or boto (I. geoffrensis)

83: should be Mamirauá Sustainable Development Reserve (RDSM) and Amazon River.

103: delete Mamirauá Sustainable Development Reserve and delete the parenthesis in RDSM

144: Comment: the red top tubes were allowed to clot at ambient temperature, room temperature or in the refrigerator? Please specify.

147: delete white blood cell and delete delete the parenthesis in WBC

167: should be Cardiac rate (CR, beats/minute)

168: should be respiratory frequency (RF, breaths/minute)

174: Should be (Table 3)

178: should be CR and RF were

202: 110 write out as One-hundred-and-ten

202-203: Delete by Projeto Boto,

214-217: box plot and histogram analyses (Table 4). Then delete line 215, 216, 217.

231: change white blood cells counts for WBC

234: Delete Blood cell (and the other ).

270: cardiac rate should be CR, and respiratory frequency should be RF

277: understandably/expected should be understandably and expected

279: cadiac rate and respiratory frequency should be CR and RF

280: put a comma (,) after (p=0.003)

282: respiratory frequency should be RF

283: respiratory frequency should be RF

287: respiratory frequency should be RF

288: cadiac rate should be CR

301: by the 1970s. It does not 1970’s because it is not possessive. It is plural, so it should be 1970s

305: Where it says I. geoffrensis, change for ones

317: use common name before Globicephala macrorhynchus and Pseudorca crassidens

327: use common names for Orcinus orca, T. truncatus, Delphinapturus leucas in conjunction with scientific name

356: Hb instead of hemoglobin

362: the scientific name for the pantropical spotted dolphin is Stenella atteanuata. Stenella tropicalis does not exist.

373: should be RF for respiratory frequency

375, 385, 386, 391: Explain first what is N:L before using it.

387: follow the scientific name for Amazonian manatee

388: rhinos is a popular way to refer to rhinoceros. However, which species of rhinoceros are you referring to? White rhinoceros (Ceratotherium simum), black rhinoceros (Diceros bicornis), Indian rhinoceros (Rhinoceros unicornis), Javan rhinoceros (Rhinoceros sondaicus) or Sumatran rhinoceros (Dicerorhinus sumatrensis). Or is it to species of the Rhinocerotidae?

389: common name for Stenella coeruleoalba

397: instead of boto, use Amazon River dolphin

401: use Hct and Hb

403: use CR and RF. The and is missing an n.

Figure 1: Legends inside figure should be in English or the same terms used in the text.

Very good clinical work, and much needed for this species.

Congratulations in this accomplishment.

**********

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Reviewer #1: No

Reviewer #2: Yes: Antonio A. Mignucci-Giannoni

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Submitted filename: PONE-D-19-17847 Review.docx

Click here for additional data file.

Submitted filename: PONE-D-19-17847 Review.docx

Click here for additional data file.

29 Nov 2019

Dear Reviewers,

Please find my responses to all of your corrections, suggestions and comments. I believe the modifications suggested by you were every important to enhance the manuscript quality. Despite all comments have been answered, I would like to point out some modifications. The manuscript has been reviewed by a native English speaker. Figure 1 was removed from the text and some information was included on figures 3-5. The permit number was included.

Again, I would like to thank for all your valuable suggestions.

Best regards,

Daniela.

PONE-D-19-17847

Hematological profile of Amazon river dolphins Inia geoffrensis and its variation during acute capture stress.

de Mello & da Silva

• The title identifies the study subject as Amazon river dolphin, also known as boto. But the interchange of this common name in the manuscript may be confusing to the reader. I would recommend being consistent in using one. Mention both names once, but after that be consistent in using one.

A: The term “boto” was replaced by Amazon river dolphins throughout the text, with exception of “Projeto Boto”

• In Spanish and perhaps in Portuguese, the names of rivers are begin with capital letters, but the word river itself does not begin with capital letters, i.e., río Amazonas. But in English, both the river name and the word river begin with capital letters. Thus, in the title and throughout the document, it should be Amazon River dolphin.

A: Thank you for your observation, the word river was modified throughout the manuscript.

• All animals, except common animals (horses, goats, swine, cattle), should be first introduced with a common name followed by its scientific name. Afterwards, I recommend using just the common name.

• Add the common names for Globicephala macrorhynchus, Pseudorca crassidens, Orcinus orca, Delphinapterus leucas, Stenella coeruleoalba

A: Attended

• Add the scientific names for Amazonian manatee, rhinoceros

• A: Attended

Lines:

31: 110 write out as One-hundred-and-ten

A: Attended

38: Delete The before calves

A: Attended

40: should be lymphocytes

A: Attended

42: should be neutrophils, lymphocytes and monocytes

A: Attended

44: define Hct and then put in parenthesis. Red Blood Cells should be red blood cells. Define Hb and then put in parenthesis.

A: Attended

65: should be white blood cells (WBC)

A: Attended

66: should be hematocrit (Hct)

A: Attended

74: should be The Amazon river dolphin or boto (Inia geoffrensis)

A: Attended

76: cite where the IUCN lists the species as endangered.

A: Attended

76: instead of “under intentional catch” use affected by intentional capture

A: Attended

81: delete or boto (I. geoffrensis)

A: Attended

83: should be Mamirauá Sustainable Development Reserve (RDSM) and Amazon River.

A: Attended

103: delete Mamirauá Sustainable Development Reserve and delete the parenthesis in RDSM

A: Attended

144: Comment: the red top tubes were allowed to clot at ambient temperature, room temperature or in the refrigerator? Please specify.

A: Ambient temperature, information included.

147: delete white blood cell and delete delete the parenthesis in WBC

A: Attended

167: should be Cardiac rate (CR, beats/minute)

A: Attended

168: should be respiratory frequency (RF, breaths/minute)

A: Attended

174: Should be (Table 3)

A: Attended

178: should be CR and RF were

A: Attended

202: 110 write out as One-hundred-and-ten

A: Attended

202-203: Delete by Projeto Boto,

A: Attended

214-217: box plot and histogram analyses (Table 4). Then delete line 215, 216, 217.

A: Attended

231: change white blood cells counts for WBC

A: Attended

234: Delete Blood cell (and the other ).

A: Attended

270: cardiac rate should be CR, and respiratory frequency should be RF

A: Attended

277: understandably/expected should be understandably and expected

A: Attended

279: cadiac rate and respiratory frequency should be CR and RF

A: Attended

280: put a comma (,) after (p=0.003)

A: Attended

282: respiratory frequency should be RF

A: Attended

283: respiratory frequency should be RF

A: Attended

287: respiratory frequency should be RF

A: Attended

288: cadiac rate should be CR

A: Attended

301: by the 1970s. It does not 1970’s because it is not possessive. It is plural, so it should be 1970s

A: Attended

305: Where it says I. geoffrensis, change for ones

A: Attended

317: use common name before Globicephala macrorhynchus and Pseudorca crassidens

A: Attended

327: use common names for Orcinus orca, T. truncatus, Delphinapturus leucas in conjunction with scientific name

A: Attended

356: Hb instead of hemoglobin

A: Attended

362: the scientific name for the pantropical spotted dolphin is Stenella atteanuata. Stenella tropicalis does not exist.

A: Thanks for your observation. “S. tropicalis” was funny…

373: should be RF for respiratory frequency

A: Attended

375, 385, 386, 391: Explain first what is N:L before using it.

A: Information added to the material and methods section.

387: follow the scientific name for Amazonian manatee

A: Attended

388: rhinos is a popular way to refer to rhinoceros. However, which species of rhinoceros are you referring to? White rhinoceros (Ceratotherium simum), black rhinoceros (Diceros bicornis), Indian rhinoceros (Rhinoceros unicornis), Javan rhinoceros (Rhinoceros sondaicus) or Sumatran rhinoceros (Dicerorhinus sumatrensis). Or is it to species of the Rhinocerotidae?

A: Black rhinoceros, information included

389: common name for Stenella coeruleoalba

A: information included

397: instead of boto, use Amazon River dolphin

A: Attended

401: use Hct and Hb

A: Attended

403: use CR and RF. The and is missing an n.

A: Attended

Figure 1: Legends inside figure should be in English or the same terms used in the text.

A: Figure 1 was removed from the manuscript.

Very good clinical work, and much needed for this species.

Congratulations in this accomplishment.

Thank you :D

Review of “Hematologic profile of Amazon river dolphins Inia geoffrensis and its variation during acute capture stress”

Overall Comment:

The data presented here represent important contributions to the literature regarding blood reference ranges for the understudied boto population. I do have some reservations regarding its publication at this time, and request a revision for the following reasons.

I am concerned about the inclusion of animals with high stress scores in the creation of baseline values/reference ranges. Ideally, animals with high stress scores would be excluded from the reference range creation since they are not “normal.” I see this paper as having two separate purposes, 1) to establish hematological reference ranges for minimally stressed boto by age class and 2) to evaluate the effect of stress on hematological parameters in this species. I think it would be a stronger manuscript if organized as such. If you choose not to split the study out in this manner, there should be some mention of this as a study limitation in the discussion (the inclusion of stressed animals when establishing baselines).

A: Yes, the study had two main goals: first to establish reference blood values for the species e second to see with there is any variation on these values regarding age, sex, stress level… I understand your point, but still I believe all animals (with exception to outliers) should be included in the reference ranges since there is no other way to capture and restraint wild animals without causing any stress. As we have included values from both situations - less and more stressed animals – we have created a range of blood values that represent healthy individuals under different degrees of stress. This will be better explained in the discussion. Also, I have included in the manuscript comparisons of blood values among different stress level.

I was unclear whether or not all the data were tested for a normal or Gaussian distribution. Especially for some of the parameters with relatively low sample sizes, this is extremely important with regards to the question on whether rigorous statistical methods were applied. I answered “I don’t know” because this is a pending question I have for the authors in my review.

A: I understand your concern about the normality of the data. Same occurred to one reviewer when I have presented my masters study (same as this one), I had to show him my raw data and all analyses I had performed. If there is any particular data you want to have a look, I am happy to send you. Or if there are any specific values that you believe I should test again, please let me know. Besides the relatively low number of individuals, the variation of the blood values was not very pronounced, I believe that’s the reason the distribution was normal and the variances were homocedastic for most parameters and comparisons. The normality was first observed by histogram graphs, outliers were identified and excluded from the analysis which promoted a subsequent adjustment of the Gaussian curve.

While the authors in general did a good job of translating to English, the language is still awkward in many places and there are still some confusing statements and grammatical errors that appear to be associated with translation. I tried to assist and interpret where I could, but I recommend a native English speaker edit the manuscript prior to resubmission.

A: The manuscript has been reviewed by a native English speaker.

Abstract:

Line 30, 36: Change “gender” to “sex” throughout text as sex refers to the biological differences between male and female, and gender refers to the individual’s identity and is therefore not suitable for use with animals.

A: Attended

Lines 36-40: Since reference ranges are often created according to sex, reproductive status, and age class, I would recommend moving these findings up to line 33 before the sentence starting “Means…” You have essentially created age-class specific reference ranges in this study since those were the groups with statistically significant differences – and I would clarify that in the abstract.

A: Considering that the main goal of my study was to establish reference intervals for wild individuals of the species, I believe this information would come before the variations found among different categories. Besides, not much differences were found among the groups, with exception of WBC. For these reasons I believe the results would be better present with the information about the RI coming before any variation found.

Lines 38-39: I recommend deleting “as a result of the still-developing immune system.” There are other potential explanations for calves having higher WBCs (higher lung worm burden in younger animals - which has been observed in other dolphin populations, or higher stress level than older animals). Given these alternate possible explanations and the fact that this is not a common finding in all populations of dolphins, I do not recommend being so definitive in explaining its cause.

A: Attended

Introduction:

Line 52: Change “accessed” to “assessed”

A: Attended

Line 55: Recommend changing “health status approaches” to “health assessments”

A: Attended

Line 56-57: All these studies can be referred to as “capture-release” studies, I am unclear why the distinction is being made for the St. Aubin study as “netted wild population” vs capture release programs. Please clarify.

A: I meant that commonly the dolphins are captured in the coast (coastal species) many times along the year.; and in one occasion the capture happened in open ocean on open ocean species. I did some modification on the text, please check if it is appropriate now.

Line 57: Change semicolon to comma and “parameters” to “parameter” and “baseline” to “baselines”

A: Attended

Lines 58-62: This sentence is confusing the way it is written, please reword.

A: Attended

With reference #11 – there had not been a baseline study prior to the oil spill on that particular population (although baseline studies existed for other Tt populations that were used for comparison). I would be more explicit in explaining how this particular citation makes the authors’ point.

A: The sentence was re written.

I also think the reference to seasonal variation in the Sarasota dolphins is confusing since you do not address seasonality at all in the remainder of the paper (even though it is controlled for with your sample timing).

A: The sentence regarding seasonality and the reference Hall et al (2007) were removed from this part of the manuscript.

Line 64: delete “aged”

A: Attended

Line 65: This is not necessarily a common finding among the capture-release sampled dolphins in various locations. Given the age of this citation, I would recommend either striking this comment or finding additional more recent citations to make a stronger case.

A: Nabi et al (2017) also found higher WBC in younger Yangtse finless porpoises “In terms of lymphocyte and WBCs counts, age class and reproductive states were a significant variable: both were significantly higher in either calves or juveniles compared to adults in both populations.” This reference was added to the sentence.

Line 67: Change “alteration on” to alterations in”

A: Attended

Line 69: change “englobe” to “involve”

A: Attended

Line 70: change “may reflect” to “may be reflected in”

A: Attended

Line 71: change “gender” to “sex”

A: Attended

Line 73: I believe you mean “before establishing baseline blood parameters.” Ideally you are establishing these reference ranges with animals that are minimally stressed and removing the outliers from your “normal” sampling population.

A: “baseline” was added to the sentence. Outliers were removed from the statistical analysis.

Line 76: include citation for the most recent IUCN report

A: Attended

Line 76-77: Recommend changing “under intentional catch” (which is vague) to “actively fished commercially”

A: “actively fished” was added to the sentence. The botos are not being sold so I did not include the word “commercially”.

Line 78: Recommend changing “leading to significant populations decrease” to “leading to a significant population decline”

A: Attended

Lines 83-84: delete comma after “reserve” and replace with “in the”

A: Attended

Line 87: Why single out the ESR here? Seems unnecessary – either please justify or delete.

A: The sentence was deleted.

Materials and Methods:

Line 109: Recommend spelling out the RDSM acronym in the figure caption

A: Attended

Line 116: Change “proximities of research floating station” to “proximity of the floating research station”

A: Attended

Lines 116-117: Recommend changing the sentence starting with “It varied” to: “The number and behavior of encircled animals varied according to environmental conditions.”

A: Attended

Line 118: If you are going to indicate the depth of the channel it would also be helpful to know the depth of your capture net (s).

A: Information included

Lines 119-121: You state that the usual method was to seine the dolphins to the shoreline. That leads me to believe that there were other methods used to capture the dolphins in some instances. If so, please describe those methods as well, if not, please clarify the text here.

A: There are no other methods, the sentence was rewritten.

Line 126: What were your criteria for determining pregnancy? Identification of a fetus? Recommend explicitly stating that.

A: Yes, identification of a fetus. Information included.

Line 139: Did you actually use a catheter or a needle? Was the blood drawn directly into the EDTA vacutainer tube or was it drawn into a syringe and then transferred to the tubes? The way this is written, it sounds like the blood from the EDTA tubes was transferred to a tube with no anticoagulant and allowed to clot, which is confusing. Please clarify your methods.

A: I have used a catheter and the blood was drawn into a syringe and then transferred to EDTA tubes. The sentence was modified.

Line 146: How were these manual methods standardized? Were they consistently done by a single trained individual or were multiple individuals trained on standardized methods?

A: I did perform all the blood analyses. The sentence was modified.

Line 171: For stress levels 1 & 2 behavior, perhaps you mean no/little excessive response to external stimuli? Or appropriate response to external stimuli? Relaxed dolphins should be alert and responsive to external stimuli, but not hyper-responsive. By immobilization do you mean restraint? Recommend re-wording these behavioral descriptions.

A: Thank you for your observations. The behavioral descriptions were re-worded.

Line 175: I think you mean “the neutrophil to lymphocyte ratio” ?

A: Yes, thank you.

Lines 176-177: 12 individuals in each category? Are samples from the same individual compared between the two time categories or are the comparisons done between different animals? Please clarify the methods here.

A: Comparison done between different animals, and yes 12 on each category (24 animals in total). Please let me know if it is clearer now.

Line 177: insert ‘of’ between ‘chronology’ and ‘acute’

A: Attended

Line 181: Recommend first discussing the tests for significant differences in age class, reproductive status, and sex, then discuss the methods for creating reference ranges based on those outcomes. Were all the data tested for normality (Gaussian distribution)? Especially with some of the parameters that had smaller sample size, normal distribution cannot be assumed and should be tested.

A: I believe the establishment of the reference ranges is the most important result of this study, that´s why is comes before the differences found between the categories.

As I stated before, the normality was first observed by histogram graphs, outliers were identified and excluded from the analysis. This have caused a subsequent adjustment of the Gaussian curve.

Line 200: Strength of correlation (weak, moderate, or strong according to r value) should be stated here. You can find references in the literature to describe correlation strength.

A: Attended

Line 195: How were multiple comparisons controlled for in order to prevent Type I error (false positives)? You compared a lot of variables and should either control for these multiple comparisons or justify why you did not do so.

A: What I meant here is that not all comparisons were made, for example, male (n = 2) vs female calves (n = 4) for hemoglobin, MCHC, …Type I error was prevented by the observation of the histograms and Gaussian curves. If you believe these tests were not sufficient to make an accurate analysis and If you have any suggestion on how I should proceed I would appreciate to hear.

Line 214: Since Table 4 is cut off, I cannot see if you stated 90% RI for the calves. I am assuming you did not due to small sample size. I would explicitly state what you are presenting for the calf data here and why there are not reference ranges established. I would also recommend removing the moderate and severely stressed dolphins from the reference range calculation.

A: I am sorry for this table layout, but the Journal requires it goes like this for review. If the Journal allows me, I can send you the table 4 in a different format.

The calves were not included on the reference interval because they presented statistically significant higher WBC than adults and subadults. As you said, I did not calculate RI for calves due to the small sample size, but I did include the min and max values.

I have included in the text the explanation of why blood values from calves are in a different column.

Line 220: Formatting in the version I have cuts off the left (parameter) and right (last column is calves min) margins of this table. Cannot fully evaluate data presented. Recommend including more details regarding the reference values in the table caption (90% double sided reference intervals).

A: Same situation as previous comment.

Line 221: In the methods, it stated the level of significance was set at p < 0.05 but here it states p < 0.001. Why the discrepancy?

A: Because the minimum of significance was 0.05, but here it was found as 0.001.

Line 234: In Fig 2: Recommend adding sample sizes (n) above each age class box plot

A: Number of each age class was included in the figure caption.

Line 242: Again, recommend including sample sizes in Fig 3 (either in the caption or in the legend)

A: Number of each age class was included in the figure caption.

Line 251: I do not see medians in table S1, delete reference to them here.

A: Thank you for your observation, medians was deleted.

Line 252: These are not reference intervals, recommend deleting ”RI” and just referring to the data in table S1 as summary statistics.

A: Thank you for your observation, RI was deleted.

Line 263: Fig 4: Recommend including r and p values either on the figure or in the caption. Same comment for Figs 5 and 6 as well.

A: Attended

Line 254: Recommend spelling out cardiac rate and respiratory frequency in this section title as these are not common abbreviations.

A: Attended

Line 268: Correlation should be categorized as weak, moderate or strong based on the r values.

A: Attended

Discussion:

Line 300: change “comes” to “come” – “data” is a pleural noun (singular “datum”)

A: Attended

Line 301: change to “in the 1970’s”

A: Attended

Lines 304-310: Captive boto were also likely less stressed than the wild-caught boto, which would also affect the WBC count. Subclinical bacterial, fungal, protozoal, and viral infections should also be included in the differentials for elevated WBC in wild vs managed populations.

A: I agree with you. These information were added.

Line 320: change “an” to “a” Also, without demonstrating a correlation between ESR and inflammation, I would caution against evaluating this parameters utility here. While the narrow range MAY indicate that this could be a useful indicator IF it does in fact change with inflammation, you have not proven the latter yet.

A: Attended.

I agree and the text was modified.

Lines 318-320: Was there any correlation between ESR and WBC count? If not, this makes it more likely that the WBC/neut elevations were due to stress rather than inflammation/infection/antigenic stimulation.

Lines 321-330: Also need to include stress as a differential for higher WBC counts in juveniles. And a note that this is not a consistent finding among all cetacean populations.

Line 335: change “was” to “were”

A: Attended

Line 340: Change “towards” to “on”

A: Attended

Line 342: Can you present data in the results regarding the sample sizes according to each trimester if available to help elucidate this point?

Line 343: change “values” to “value” and “towards non-pregnant females” to “between pregnant and non-pregnant females”

A: Attended

Lines 356-358: Due to the spleen’s small size relative to the dolphin’s overall body mass, my understanding is that unlike in other species, splenic contraction is not considered a significant differential for elevated RBC, HCT, Hb in cetaceans.

A: Thank you for your observation. Another explanation was presented as a potential source of Hb, HCT and RBC rise in dolphins under acute stress.

Line 359: Recommend using the term “epinephrine” rather than “adrenalin”

A: Attended

Line 369: change “access” to “assess”

A: Attended

Line 373-374: Also cannot rule out subclinical pulmonary disease as contributing to this trend, and this should be included as a differential for that finding.

Lines 376-378: Please reword this sentence for clarity.

Line 379: Add “can” between ‘animals’ and ‘have’ – as this is not always a consistent finding in cetaceans

A: Attended

Lines 384-394: More clarification in your discussion of the different types of stress leukograms is warranted – since you have the expected neutrophilia with a corticosteroid stress response, but not the associated lymphopenia (as with the “typical stress leukogram” that you cite). Lymphocytosis would be associated with an epinephrine-induced response. While you mention both of these hormone-induced pathways, your explanation of the lymphocytosis is incomplete.

Line 400: change ‘access’ to ‘assess’

A: Attended

Line 403: change “ad” to “and”

A: Attended

Submitted filename: Response to reviewers.docx

Click here for additional data file.

11 Dec 2019

Hematologic profile of Amazon river dolphins Inia geoffrensis and its variation during acute capture stress

PONE-D-19-17847R1

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Additional Editor Comments (optional):

Thank you for working on all the comments and suggestions raised by the reviewers

Reviewers' comments:

16 Dec 2019

PONE-D-19-17847R1

Hematologic profile of Amazon river dolphins Inia geoffrensis and its variation during acute capture stress

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