We read with interest the paper by Brijs et al. (2020) regarding the ‘blood-boosting’ properties apparently exhibited by the Antarctic notothenioid fish (Pagothenia borchgrevinki). Although the data provide additional insights into the physiology of an extreme cold-adapted fish, we believe the authors have drawn erroneous conclusions about the mechanisms involved with this ‘blood-boosting’ phenomenon. The authors conclude that the spleen sequesters enough red blood cells (RBCs) to increase the haematocrit (Hct) and, therefore, blood oxygen carrying capacity in fed and exercise states. Further, the authors posit the spleen holds these RBCs in reserve to reduce blood viscosity until additional oxygen is needed to support increases in metabolic rate. In our view, the increases in Hct in P. borchgrevinki can be primarily explained by an alternative mechanism that the authors did not consider in their analysis: elevated blood pressure increases plasma efflux from the vascular to the interstitial space, thus increasing the fraction of RBCs in the vascular space (i.e. Hct).

We present two arguments against a role for the spleen in providing a significant contribution to increased Hct in P. borchgrevinki. Our first argument is based on the principle of conservation of mass. Brijs et al. (2020) used uninstrumented fish to examine changes in spleen volume at rest, after feeding and following enforced exercise. The comparisons are partly confounded by significant differences in body mass between groups. We have plotted the authors' data for unfed and fed animals in resting and exercised states to show the significant relationships between spleen mass and body mass (Fig. 1). If we compare a 74 g fish for both groups, spleen mass for resting fish is 0.365 g and is 0.213 g after exercise (Δ spleen mass=0.152 g). Can this change in spleen mass account for the changes in Hct observed by the authors? If we assume a blood volume of 5% of body mass, for a 74 g fish blood volume is 3.7 g (=3.7 ml, assuming blood has a density of 1 g ml−1). The average Hct was 15.8% and 27.1% for resting and exercised fish, respectively. The mass of RBCs for this blood volume is therefore 0.585 g (rest) and 1.003 g (exercise) with a difference of 0.418 g (ml). It is clear that changes in spleen volume cannot account for the mass of RBCs added to the vascular space during exercise. The changes in spleen mass account for approximately 36% of the change in RBC mass with the remaining 64% of RBC unaccounted for; with unfed fish, Hct changes from 8.6% to 25.1% and the non-splenic contribution is larger (76%). We also note that for resting fish (unfed vs fed), Hct increased from ∼9% to ∼21%, but absolute spleen mass increased, rather than decreased, a further indication that non-splenic mechanisms account for the increased Hct.

Fig. 1.

The relationship between spleen mass (g) and body mass (g) for resting and exercised P. borchgrevinki plotted fromBrijs et al. (2020) . Both resting and exercised animals are combined data from fed and unfed groups. For resting animals, Y=0.0046+0.03; r2=0.61; F1,20=31.4; for exercised animals, Y=0.0049–0.15; r2=0.60; F1,16=24.2.

Fig. 1.

The relationship between spleen mass (g) and body mass (g) for resting and exercised P. borchgrevinki plotted fromBrijs et al. (2020) . Both resting and exercised animals are combined data from fed and unfed groups. For resting animals, Y=0.0046+0.03; r2=0.61; F1,20=31.4; for exercised animals, Y=0.0049–0.15; r2=0.60; F1,16=24.2.

If the spleen volume change cannot fully account for the change in RBC mass and changes in Hct, what mechanistic explanation is responsible for these changes? Our second argument is based on the balance of hydrostatic and oncotic forces at the capillary (i.e. Starling forces). These forces, in conjunction with the capillary filtration coefficient, dictate fluid flux across the capillary. Fish capillaries are highly permeable to protein and have a high transvascular fluid flux (Olson et al., 2003); thus a traditional Starling analysis is inappropriate for fishes. An alternative approach for analyzing the balance of forces for transvascular fluid flux was previously developed for the whole animal and incorporates values for hydrostatic pressures and compliances of the vascular and interstitial spaces, along with a whole-body filtration coefficient (Tanaka, 1979); these relationships can be reduced to:
formula
(1)
where Fc is the whole-body filtration coefficient, Pvas is blood pressure and Pint is the hydrostatic pressure of the interstitial space (Tanaka, 1979). Because Fc is high in fish, any increase in Pvas relative to Pint will result in efflux of plasma from the vascular space. This leads to the prediction that Hct should increase with elevated Pvas because plasma efflux is not accompanied by loss of RBCs from the vascular space (see also Hillman et al., 2010). This is precisely what the authors observed in their study: Hct was linearly and significantly related to blood pressure in both splenectomized and sham-operated fish during exercise (see table 1 and fig. 5B in Brijs et al., 2020). This is entirely consistent with plasma efflux from the vascular space causing an increase of Hct. We note that previous work with fishes has also demonstrated that increased Hct can result from potentially three different factors: splenic contraction, RBC swelling and plasma efflux. For example, Pearson and Stevens (1991) showed that RBC swelling and splenic contraction each accounted for about 25% of the increased Hct with exercise and air exposure stress in rainbow trout, with the remaining 50% due to plasma efflux. The relatively small contribution by the spleen is in the same range that we have estimated for P. borchgrevinki.

An unknown factor in this analysis is blood volume for P. borchgrevinki. Blood volume estimates in fish are highly variable and subject to considerable error as techniques typically use plasma protein labeling (see Olson et al., 2003; Hillman et al., 2010) and lead to overestimates in BV. We have assumed a BV of 5% of body mass (a typical vertebrate value), but for the authors' conclusion to be correct, BV would have to be approximately 1.5% of body mass. This low value seems unlikely. Increased Hct would increase blood O2 carrying capacity and contribute to O2 transport during exercise (Hedrick et al., 2015), but may also be detrimental to O2 transport with increased viscous resistance and/or limited venous return. What the authors characterize as a unique blood-boosting role for the spleen as an adaptation to a low temperature environment, we, instead, view the Hct changes as an expected consequence of the vascular properties of fish capillaries in general; the role of the spleen for boosting Hct during elevated metabolic states appears to be significantly overstated.

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