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Journal of Biodiversity & Endangered Species
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Blood Rheology in Cape Fur Seal and Bottlenose Dolphin: Implications for Muscle Perfusion

Ursula B Windberger1*, Roland Auer2 and Malcolm J Smale3

1Department of Biomedical Research, Medical University of Vienna, Vienna, Austria

2Biomedical Research Center, University of Pretoria, South Africa

3Port Elizabeth Museum at Bayworld, Port Elizabeth, South Africa and Department of Zoology, Nelson Mandela Metropolitan University, Port Elizabeth, South Africa

*Corresponding Author:
Ursula B Windberger
Department of Biomedical Research
Medical University of Vienna
Waehringerguertel, Vienna, Austria
Tel: +43(0)1-4040052230
E-mail: ursula.windberger@meduniwien.ac.at

Received date: January 08, 2015; Accepted date: March 06, 2015; Published date: March 13, 2015

Citation: Windberger UB, Auer R, Smale M (2015) Blood Rheology in Cape Fur Seal and Bottlenose Dolphin: Implications for Muscle Perfusion. J Biodivers Endanger Species 3:149. doi:10.4172/2332-2543.1000149

Copyright: © 2015 Windberger UB, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Blood O2-store in diving animals is increased by great numbers of large erythrocytes which carry enhanced hemoglobin contents. In seal and dolphin, resting hemato (HCT) and/or Red Blood Cell (RBC) indices were elevated (HCT: seal: 50 (47/51); dolphin: 45/50); MCH: seal: 35.8 (34.4/37.3); dolphin: 41/40); MCHC: seal: 33.9 (33.4/36.0); dolphin: 35.6/36.6), and RBC volume was increased (seal: 101 (99/110); dolphin: 115/110 fL) compared to terrestrial animals and man. However, WBV increases in parallel with the HCT. In seal, we therefore calculated the range in which the desired HCT effect is not weakened by an increase of WBV; we plotted the theoretical oxygen transport capacity (WBV*HCT-1) against the HCT. A quadratic regression showed that the resting HCT was higher than the optimal HCT value (at 11.6 s-1: 31%; 40.5 s-1: 37%; 267 s-1: 45%; all: p<0.05). This may facilitate blood stasis during a dive, when HCT is further increased through splenic release of RBC. Flow curves of seals showed shear thinning (11.6/500: seal: 1.98 (1.87/2.11), indicating that the blood texture changes with shear rates. RBC deformability in seals was pronounced (elongation index (ectacytometry) at 60Pa: females: 32, 29; males: 24, 24), but aggregability was low (aggregation indices M0: 3.15 (1.6/4.8); M1: 13.3 (8.7/18.8)), and therefore intermediate regarding Weddell seal (high aggregability) and ringed seal (nil). Considering the importance of aggregability on the cell free layer width in arterioles, feeding of RBC into subsequent vessels at bifurcations may be enhanced.

Keywords

Blood viscosity; Hemorheology; RBC aggregation; RBC deformability; Marine mammals; Seal; Dolphin

Introduction

Evolutionary theory would predict that any mechanism that extends the aerobic dive limit of marine animals [1] should be selected. This can principally be achieved by the enhancement of the body O2 stores, by optimization of their utilization, and by the reduction of the cellular O2 demand during the dive. Apart from the specialized respiratory and myogenic O2 storage capacity in the body, blood O2 storage connects the two others by the species-specific O2-affinity of hemoglobin [2], and by the continuous movement of blood through the vasculature. The rate of this movement depends on hemodynamic and geometric cardiovascular variables, and on the fluidity of blood and plasma. The fluidity of blood itself is influenced by quantitative and qualitative properties of Red Blood Cells (RBC) and the plasma composition, which in combination determine Whole Blood Viscosity (WBV). The viscosity of blood is low due to the ability of RBC to change their shape according to the hemodynamic forces and by the rotation of the phospholipid membrane around the cytoplasm. The ability of mammalian RBC to aggregate and deform essentially provide the viscoelastic behavior of blood, although plasma colloids may play some role as well [3]. In vitro flow curves of blood show a higher WBV value at low shear rate (<10 s-1), and a decrease of WBV when shear rates are raised (>100 s-1). Thus, thinning of blood occurs with the shear, and as a result, blood viscosity will be different in the vascular compartments. Shear thinning depends on the extent of RBC aggregation and disaggregation at low and intermediate shear rates, and on the RBC deformation at higher shear rates. The speciesspecificity of this property is surprising, and is assumed to be a fingerprint for a certain species [4].

The diving response is characterized by an increase of peripheral resistance and a reduction in heart rate [5]. Since venous return matches the peripheral resistance, cardiac output is decreased and enables a physiologic systemic perfusion pressure [6]. The reduction of cardiac output is associated with the reduction of muscle perfusion. For instance, in seals during forced submersion, muscle perfusion is close to nil, and heart rate and cardiac output are low. During sleep apnoea of animals on the surface, muscle perfusion is present, although reduced, and heart rate is not that low compared to forced submersion [7,8]. Complete isolation of the muscles from the circulation to prevent lactate washout during forced or prolonged dives would sequester a certain blood volume in the entire vascular network of those muscles for a period of several minutes. This raises the question of whether hemorheological parameters play a role in the generation of blood stasis. At the same shear stress in a given vascular segment, blood stasis could occur earlier when RBC aggregation and haematocrit are increased.

Our in vitro measurements of blood from Cape fur seal, Arctocephalus pusillus puscillus indicate that blood stasis may even be facilitated in specific vascular regions, which would foster RBC sequestering in areas of low flow during a prolonged dive. A stated recently [9], the advantage of increasing the volume of O2 that can be carried by the blood may more critical to the animal than the efficiency at which it can be delivered.

Materials and Methods

Animals

Blood was collected during routine health checks from 2 bottlenose dolphins (Tursiops aduncus , 1 male, 1 female) and 6 Cape fur seals (3 male, 3 female) kept in the Bayworld aquarium, Port Elizabeth, Republic of South Africa. Four of these seals (seal 1, 3, 5, 6) had been rescued as black pups from a beach when they were washed off the breeding colony in Algoa Bay during storms. The other two seals (seal 2 and 4) were born in captivity. The two dolphins were second and third generation born in captivity, respectively. Blood was withdrawn by vacutainer systems from the pin veins. Blood was put into EDTAtubes and placed on ice. All the animals, except one of the dolphins, were clinically healthy at the time of the blood collection. One of the dolphins showed clinical signs of gastritis. However, the routine blood chemistry was within the reference range.

Laboratory measurements

Hematology was performed by Advia 2120 Haematology Analyser (Siemens, Berlin, Germany) to obtain red blood cell count (RCC, in cells 109μL-1), Mean Cellular Volume (MCV, in fL), mean cellular hemoglobin concentration (MCHC, in g dL-1), and White Blood Cell Count (WCC, in cells 106μL-1). A routine blood chemistry profile was performed to check the health status (NEXT/VETEX and ACE Alera; Alfa Wassermann B.V., Woerden, The Netherlands). Hematocrit (HCT) was measured by centrifugation (JOUAN “Hema C”- centrifuge, Hawksley & Sons, West Sussex, Great Britain).

The hemorheological measurements included Whole Blood Viscosity (WBV, in mPa.s), Plasma Viscosity (PV, in mPa.s), RBC aggregation (aggregation indices: M0, M1) and RBC deformability (Elongation Index: EI).

WBV (η) was analyzed by the strain-controlled Physica MCR301 rheometer (Paar, Graz, Austria). Shear rates between 11.6 and 500 s-1 were adjusted for data processing. Isothermal runs at 37°C were conducted with a logarithmic shear rate ramp. To show the shear thinning of blood, the ratio of WBV at the lowest (η11.6) and the highest measured shear rate (η500) was calculated [10].

PV was analyzed by the rolling ball viscometer AMVn (Paar, Graz, Austria). Plasma was tested at a 60° capillary angle (capillary diameter 0.9 mm; stainless steel ball diameter 0.794 mm) at 37°C.

RBC aggregation was analyzed by the Myrenne Aggregometer MA1 (Myrenne, Roetgen, Germany). Aggregation indices M0 and M1 were obtained for each sample (native HCT) at room temperature (22°C). At least five readings were taken for each individual value to obtain mean values for M0 and M1.

RBC deformability was checked by laser diffractometry by the Rheodyn SS (Myrenne, Roetgen, Germany) at room temperature (22°C). The device displays an Elongation Index (EI), expressed as percentual elongation, at eight different shear stresses (τ) between 0.3 and 60 Pa.

Experimental protocol

Due to technical reasons, measurements were performed at two time points. Hematology and blood chemistry was performed on each time point from each individual. Hemorheological measurements needed a larger amount of blood and were split to both time points. On the first time point, RBC deformability and plasma viscosity was checked (only 4 seals were present at that time) at Bayworld immediately after withdrawal of blood. On the second time point, RBC aggregability was checked at Bayworld, but for the measurement of whole blood viscosity, seal blood had to be transported from Port Elizabeth to Johannesburg by airfreight. The samples were transported in insulated bags below 10°C and reached the laboratory after a six hour time interval. The blood was checked for hemolysis or clot and suspicious samples were removed. All measurements were finished within 12 hours following the withdrawal of blood.

To measure the range, at which HCT is “optimal”, blood from the seals was centrifuged for HCT adjustment. Concentrated blood cells and autologous plasma were carefully re-mixed to obtain HCT values between 15% and 94%, and WBV was analyzed subsequently.

Statistical analysis

Descriptive statistics were performed in the SPSS software (PASW Statistics 17) showing median, 25%, and 75% percentile of parameters in seals. For the 2 dolphins the individual values are presented.

Theoretical oxygen transport capacity through the formula HCT·WBV-1 was calculated in seals only. HCT·WBV-1 was plotted on the linear axis against its respective HCT value. A regression analysis was used to test whether HCT has quadratic influence on HCT·WBV-1.

Results and Discussion

In comparison to terrestrial mammals [11-13], HCT was elevated in our Cape fur seals and bottlenose dolphins. The high volume of the RBC allows higher intracellular O2 concentrations.

The concentration of hemoglobin in the RBC was also increased (Table 1). This result is in accordance with other findings [5,14]. Compared to other seals [9], hematological values were low. Our captive seals could not experience the high level of exercise undertaken by free-living animals. Generally, they lived at the surface with short dives and ad libitum swimming in shallow water (<3 m deep). Since the spleen releases RBC during the dive [15], the life style of our animals - being at the surface or swimming in shallow water - might have resulted in a constant sequestering of RBC in the spleen. The two seals born in captivity (seal 2, seal 4) exhibited no difference in HCT, MCV, MCH, and MCHC compared to the other four seals that were born in the wild. Possibly, the phenotype of the animals in regard to hematology had not yet changed. There is the possibility that the HCT in free living Cape fur seal increases during a prolonged deep dives. An increase of HCT impacts blood fluidity through the viscosity increase. The transport of O2 to the organs may be limited as a result.

  Cape fur seal Bottlenose dolphin
HCT (in %) 50 (47/51) 45 / 50
RCC (in109 cells/μL) 5.2 (5.0/5.4) 3.72 / 4.36
MCV (in fL) 101 (99/110) 115 / 110
MCH (in pg) 35.8 (34.4/37.3) 41.0 / 40.3
MCHC (in g/dL) 33.9 (33.4/36.0) 35.6 / 36.6
TP (in g/dL) 7.8 (7.5/8.1) 7.4 /7.9
PV (in mPa.s) 1.32 (1.27/1.33) 1.19 / 1.22
M0 3.15 (1.6/4.8) 4.1 / 3.8
M1 13.3 (8.7/18.8) 15.1 / 16.5
h11.6 / h500 1.98 (1.87/2.11) -

Table 1: Hematology, total protein concentration as well asaggregation indices (M0, M1) at native hematocrit and plasma viscosity (PV) of Cape fur seal and bottlenose dolphin. The viscosity ratio (η11.6 / η500) shows the degree of shear thinning of blood. Data of seals are expressed as median and 25% and 75% percentile in parentheses.

To estimate the significance of the elevated HCT value on blood flow, we used the following approach [16]: We calculated the theoretical oxygen transport capacity of blood - which is described in a simplified way by the HCT to WBV ratio out of a series of WBVmeasurements at incremental HCT values. When HCT.WBV-1 is put on a linear y-axis, it generally yields a curve concave to the HCT-axis (on the linear x-axis). The maximum of this curve reflects a theoretical value indicating a range where the benefit of RBC in the bloodstream is not reversed by a concomitant increase of viscosity. Due to the availability of larger amounts of blood, which are necessary for the measurements, we performed them for seals only. Regression coefficients R2 were: 0.476 for 11.6 s-1 (significance of coefficient: p<0.05), 0.612 for 40.5 s-1 (p<0.01), and 0.704 for 267 s-1 (p<0.001). At 40.5 s-1 and 267 s-1 the regression curve had its maximum at 37.5% and 45% HCT, which was lower than the resting HCT of the animals. At 11.6 s-1, this maximum shifted further towards a lower HCT (31%) (Figure 1). This approach, however, has limitations. For instance, the formula cannot predict O2-delivery, since hemoglobin O2-affinity and –dissociation, or RBC transit time in the microvasculature [17] are not included. Furthermore, the formula is not designed to consider vascular geometries or hemodynamic parameters. It merely focuses on the two main parameters that estimate oxygen transport on the blood level. Despite that, we think it may be useful to suggest the range in which the HCT of an individual may be “optimal”. The application of this formula on our data denotes that the resting HCT value in our seals is higher than its theoretical “optimal” range up to shear rates, normally postulated in peripheral arteries [18]. It can be seen from the tests that resting HCT moves towards its “optimal” range, when the WBV at higher shear rates (267 s-1) is entered into the formula. This would indicate that O2 transport is suboptimal in vascular regions with low shear rate, and that higher intravascular shear rates are needed to facilitate blood flow. Low shear rates are present in large vessels at low blood flow. Typical low flow vessels are the post capillary venules and the capacitance vessels. During submergence further low flow vessels should be present in association with the individual diving response of the animal. The second finding was that RBC flexibility in our Cape fur seals was comparably high, which should facilitate capillary transit of the RBC. Comparison is provided in (Figure 2) with species-specific data obtained in our laboratory [19,20]. This result is, however, weakened by the low number of measurements in seals. RBC deformability in seals may be sex dependent: female seals had higher EI than the male individuals. However, data are too few to make a definite conclusion.

biodiversity-endangered-species-Cape-fur-seal

Figure 1: Theoretical “optimal HCT” plots (HCT.WBV-1) on the yaxis versus HCT on the x-axis) of Cape fur seal at various shear rates (A: 11.6 s-1; B: 40.5 s-1; C: 267s-1). Theoretical “optimal” hematocrit increases with the shear rate. Regression coefficients R2 were 0.48 for 11.6 s-1 (p<0.05), 0.61 for 40.5 s-1 (p<0.01), and 0.70 for 267 s-1 (p<0.001)

biodiversity-endangered-species-shear-stress-curves

Figure 2: Elongation index vs. shear stress curves in 4 seals (2 male, 2 females; seals 2 and seal 4 were born in captivity), and 2 dolphins (1 male, 1 female) measured by Laser diffractometry. At shear stresses above 3 Pa, RBC elongation in the 2 female seals is higher than in the 2 male seals. References values are from sheep and cow, two terrestrial species with remarkable differences in RBC deformability. Although cow RBC show good elongation during shear, the indices of seals and dolphins were even higher. This is also the result of the large volume of seal and dolphin RBC

The third finding was that RBC aggregability in Cape fur seals was in a low range, compared to other mammals [4]. Compared to other pinnipeds, it was much lower than in Weddell seal [14], but higher than in ringed seal [21]. Instead, RBC aggregability of our seals had a comparable quality to dogs. Flow curves of native seal blood indicated that blood had its typical shear thinning property, although RBC aggregability was low (Figure 3). There may be no gender difference in blood viscosity. Generally, we are aware that our results are rather tentative rather than conclusive due to the small number of animals available. It has to be kept in mind that body mass, body movement, and dive duration of Cape fur seal are quite different from most phocid seals [22]. Therefore the species cannot be directly compared. In the two bottlenose dolphins, RBC aggregability was comparable to our seals.

biodiversity-endangered-species-whole-blood-native

Figure 3: Flow curves of whole blood at native HCT in Cape fur seals (3 male, 3 female) at shear rates between 11.6 and 500 s-1. The typical shear thinning behavior of blood is generated by the reversible formation and break-up of RBC aggregates when the shear rates change. Seal 2 and 4 were born in captivity in contrast to the other four seals

These findings may have implications on blood flow. First, blood stasis may be facilitated, because the resting HCT was higher than its suggested optimal value. Second, capillary passage is not reduced through RBC rigidity. Third, endothelial shear stress as the result of the low RBC aggregability may be enhanced. This point will be discussed below.

RBC aggregability is associated with NO bioavailability and vascular diameter. At physiologic conditions, which imply a regular endothelial cell phenotype, vessel diameters are adjusted by the shear stress stimulus [23,24]. It is important for the organisms that shear stress is controlled in order to prevent pro-atherogenic cell phenotypes [25]. Experimental studies showed that eNOS expression and phosphorylation in endothelial cells could be modulated by changes in RBC aggregation [26]. There is a cell free zone at the marginal fluid layers in arteries that shows spatial variations with the blood flow partitioning at bifurcations [27,28]. The association of that Cell Free layer Width (CFLW) with the shear stress response of the endothelial cell has been evidenced [29]. The CFLW is the result of the bulk blood structure in a specific vessel and associated with RBC aggregability. If RBC aggregation is present, the axial migration of RBC reduces the viscosity on the outer fluid layers. Through this shear stress reduction the NO production in the endothelial cell is diminished. In our Cape fur seals, RBC aggregability was present, but low. Endothelial wall stress and NO production may be enhanced for that reason, and feeding of RBC into smaller arterioles at the bifurcations may be more effective by the Fahraeus effect. For the efficiency of the blood O2 storage during the dive, the micro-vascular HCT in the muscles is important. Hemorheological parameters may be effective on that level, since they can modify this value. For instance, RBC aggregability modulated the intramyocardial HCT gradient in guinea pigs [30].

What might happen during submergence from the hemorheological point of view? It is known that metering from hepatic sinuses or splenic contraction increase the HCT [31,32] and by that way the WBV, too. The decrease of cardiac output by the decrease of heart rate [8] diminishes the blood flow velocity, and intravascular shear rates should be reduced if vessel radius is not balanced essentially. This can turn WBV to still higher values. Muscle flow varies between a complete and a more reduced cutout, depending on the motive of breath hold [33] to result in areas with sufficient flow together with areas of low to zero flow [34]. Specifically, blood with a high viscosity (HCT release and reduction of blood flow velocity) may be pumped through a mosaic of more or less restricted vascular geometries. A high WBV is often displayed clinically as “hyper-viscosity syndrome”. Bleeding of mucous membranes, retinopathy, and neuropathy are typical signs of long-term hyper-viscosity that need to be treated by plasmapheresis in man. Why do diving seals not experience such symptoms? If areas with zero flow exist in the body of seals, shear stress is zero as well there, and the stimulus to dilate the vessels should be restrained. In this situation, shear stress might be lower than the yield stress of blood resulting in blood stasis. In other areas, like in brain, heart, and locomotory muscles, shear stress may be increased. To prevent high capillary pressures and the “hyper-viscosity syndrome”, shear stresses in the micro-vascular network must be reduced. This can be accomplished by plasma skimming with the aid of intravascular phase separation and the formation of a CFLW of relevant magnitude. Indeed, RBC aggregation might be physiologically relevant during diving: (1) Hydrostatic pressures raise RBC aggregation [35], and human volunteers were found to have an increased RBC aggregability when diving to a depth of 300 feet of seawater [36]. (2) In a study comparing free-ranging and captive seals, the free-ranging animals had a higher HCT and RBC aggregability than the captive animals [37]. Our suggestions are hypothetical. It is clear that the importance of RBC aggregability during submerge has to be clarified in a further study.

In conclusion, of the three O2-stores that are utilized during a dive, blood is unique. Unlike the respiratory and the muscular O2-store, blood is not stationary, but intermediates exchanges between the two other compartments by its continuous movement. Based on the mechanical properties of the blood cells – which is high deformability and low aggregability of RBC in our Cape fur seals –, blood changes its texture by the intravascular shear forces. It has been thoroughly investigated that the self-assembly of RBC modulates arteriolar diameters through the NO bioavailability. During a dive, the peripheral perfusion may be fine-tuned by this mechanism to prevent a “hyper-viscosity syndrome”. However, our data suggest that blood stasis may be facilitated within specific vascular regions. Our next step will encompass the investigation of the suspension stability of blood at very low shear stresses and the kinetic costs for blood flow. This approach will provide information on the behavior of seal blood during stasis and on the energy demand during the dive from the hemorheological point of view. Preliminary data indicate that the suspension stability of blood is improved in Otaria flavescens compared to other mammals (please see the Supplementary File, Figure A).

Acknowledgements

The authors are indebted to the Bayworld husbandry teams, especially to Robyn Greyling and Steven Warren for upkeep the animals and maintaining their health and training. We would like to thank Dr. Lucy Runall, Robyn Greyling, and Dr. Ross Kobus for the blood collection. We also thank Dr. Roberto Plasenzotti for his technical assistance at the first visit in Bayworld.

Funding

The National Research Foundation of South Africa supported this work, Program: Knowledge and Collaboration, 2008.

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