We would like to thank Hedrick and colleagues for their thoughtful comments and for the opportunity to further elaborate on the splenic blood-boosting strategy of Pagothenia borchgrevinki (Brijs et al., 2020). Hedrick et al. (2020) argue that the reduction in spleen mass observed in our study cannot account for the exercise-induced increase in haematocrit, and that the elevated haematocrit is instead primarily explained by an elevated blood pressure increasing plasma efflux from the vascular to the interstitial space. In our study, we show that the exercise-induced increase in haematocrit is mainly due to the splenic release of erythrocytes, yet, we discussed that cell swelling and haemoconcentration via plasma efflux are also involved. Therefore, we are in agreement with Hedrick et al. regarding the underlying mechanisms and it seems that the disagreement merely concerns the relative contribution of each individual mechanism.

Before we proceed to demonstrate why we have not significantly overstated the role of the spleen for boosting blood oxygen carrying capacity in P. borchgrevinki, we would like to clarify that in contrast to what was stated in the correspondence (Hedrick et al., 2020), none of the comparisons made in our study were confounded by differences in body mass. This is because body mass was included as a covariate in all analyses where it was linearly related to the dependent variable. The correspondence also stated that absolute spleen mass was higher in resting fed fish than in resting unfed fish, which is not the case when corrected for body mass (0.330 versus 0.411 g, respectively, for a 74 g fish).

In our study, we experimentally demonstrated the splenic contribution of erythrocytes during exercise by comparing the exercise-induced increases in haematocrit of fish that were able to eject erythrocytes into circulation from the spleen (sham-operated) to fish that were unable to do so (splenectomised). Haematocrit of sham-operated fish increased from 16.1 to 25.9% in response to exercise, whereas splenectomised fish were only able to elevate haematocrit from 13.1 to 15.3% (Fig. 1A). More importantly, this splenic release of erythrocytes during exercise was associated with substantial metabolic benefits (aerobic scope was ∼103% higher in sham-operated fish) and cardiovascular trade-offs [ventral aortic blood pressure (PVA) and cardiac workload were ∼12% and ∼30% higher, respectively, in sham-operated fish]. Furthermore, the performance-enhancing benefits of this strategy are demonstrated by the fact that splenectomised fish could not complete an exercise regime and fatigued twice as fast as sham-operated fish (Franklin et al., 1993).

Fig. 1.

Exercise-induced changes in haematocrit, ventral aortic (PVA) and dorsal aortic blood pressure (PDA) in Pagothenia borchgrevinki, as well as an overview of the splenic blood-boosting strategy of this species. Exercise-induced changes in (A) haematocrit (adapted from table 1 in Brijs et al., 2020) and (B) PVA and PDA of P. borchgrevinki (adapted from fig. 2 in Axelsson et al., 1994). Significant differences (P<0.05) within and between groups are represented by * and ‡, respectively, in A, while differences in ventral and dorsal aortic blood pressure before and after exercise are represented by * in B. (C,D) Overview of the splenic blood-boosting strategy during (C) rest or (D) exercise. In response to exercise, the spleen contracts to eject erythrocytes into circulation (i), which may subsequently swell in size (ii). This increases blood viscosity, which consequently increases cardiac workload (iii) and PVA (iv). This potentially increases plasma efflux at the gills (v). However, because PDA decreases during exercise (vi), plasma efflux may simultaneously be lower in the systemic vasculature (vii). The width of the arrows illustrate the relative differences in PVA (blue arrows), PDA (red arrows) and plasma efflux (light blue arrows) between fish (C) at rest and (D) during exercise.

Fig. 1.

Exercise-induced changes in haematocrit, ventral aortic (PVA) and dorsal aortic blood pressure (PDA) in Pagothenia borchgrevinki, as well as an overview of the splenic blood-boosting strategy of this species. Exercise-induced changes in (A) haematocrit (adapted from table 1 in Brijs et al., 2020) and (B) PVA and PDA of P. borchgrevinki (adapted from fig. 2 in Axelsson et al., 1994). Significant differences (P<0.05) within and between groups are represented by * and ‡, respectively, in A, while differences in ventral and dorsal aortic blood pressure before and after exercise are represented by * in B. (C,D) Overview of the splenic blood-boosting strategy during (C) rest or (D) exercise. In response to exercise, the spleen contracts to eject erythrocytes into circulation (i), which may subsequently swell in size (ii). This increases blood viscosity, which consequently increases cardiac workload (iii) and PVA (iv). This potentially increases plasma efflux at the gills (v). However, because PDA decreases during exercise (vi), plasma efflux may simultaneously be lower in the systemic vasculature (vii). The width of the arrows illustrate the relative differences in PVA (blue arrows), PDA (red arrows) and plasma efflux (light blue arrows) between fish (C) at rest and (D) during exercise.

The significant contribution of the spleen in P. borchgrevinki is further highlighted by the observed changes in haematocrit and spleen mass of uninstrumented fish following exercise and/or feeding. The exercise-induced increases in haematocrit coincided with decreases in both absolute spleen mass (unfed fish: from 0.411 to 0.214 g, fed fish: from 0.330 to 0.224 g, values corrected for 74 g fish) and relative spleen mass (fig. 3A–C in Brijs et al., 2020). In fact, linear regression analyses revealed that relative spleen mass explained 55.5% and 41.5% of the variation in haematocrit of unfed and fed fish, respectively. Furthermore, relative spleen mass can significantly predict the haematocrit of unfed (y=–44.798x+37.290, P<0.001) and fed P. borchgrevinki (y=−21.256x+31.469, P=0.001), where y represents haematocrit and x represents relative spleen mass.

With regards to the argument pertaining to the principle of conservation of mass, the relative contribution of the ejected erythrocytes towards the increase in haematocrit calculated by Hedrick et al. (2020) is incomplete, as they have not taken into account the additional contributions of cell swelling and haemoconcentration via plasma efflux. Interestingly, a previous study by Franklin et al. (1993) attributed >60% of the exercise-induced increase in haematocrit of P. borchgrevinki to the splenic release of erythrocytes, with the remainder mainly attributed to cell swelling (e.g. >30% decrease in mean corpuscular haemoglobin concentration), as the effects of haemoconcentration via plasma efflux were considered to be relatively minor. The relatively minor contribution of the latter towards the exercise-induced increase in haematocrit is most likely because elevations in PVA of P. borchgrevinki during exercise have been shown to coincide with simultaneous decreases in dorsal aortic blood pressure (PDA, Fig. 1B; Axelsson et al., 1994; Sandblom et al., 2008). Thus, the relatively higher level of plasma efflux from the branchial circulation during exercise will be partially counteracted by the relatively lower plasma efflux occurring in the systemic circulation (illustrated by changes in the relative thickness of the light blue arrows between resting and exercising fish in Fig. 1C,D). In light of our findings and those referenced in this response, we now summarize the splenic blood-boosting strategy and its consequences for P. borchgrevinki (Fig. 1C,D).

In conclusion, P. borchgrevinki clearly have a splenic reservoir of erythrocytes that can be released during metabolically demanding situations to substantially elevate blood oxygen carrying capacity. This provides them with an extraordinary facultative aerobic scope that enables an active lifestyle in the extreme Antarctic marine environment, while minimizing the energetic and physiological costs of transporting highly viscous blood during times of reduced energetic demand.

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