Processes regulating the number of circulating RBCs. Our data clearly indicate that RBC number in blood of mullets changed during annual cycle. The differences between the highest and the lowest number of circulating RBCs were about 22–23% that corresponded to 0,46 − 0,47 (106) cells µl− 1. Interestingly, these results argue with data obtained on Tilapia zilli (Ezzat et al., 1974) and other teleosts (Sharma, Joshi, 1985; Joshi, 1989) where annual dynamics of RBC number in circulating blood was associated with water temperature. The highest number of circulating RBCs (2,35 − 2,55 (106) cells µl− 1) was observed at the end of autumn.
Several processes may participate in annual changes in number of circulating RBCs of mullet: (1) shifts in productive and destructive processes in blood, release of cells from depot-organs, hydration or dehydration of plasma (Soldatov, 2005). RBC number in mullet blood increased when erythroid hemopoietic line in pronephros was active and decreased with inhibition of erythropoiesis and the determination coefficient (R2) between these parameters was 0.781. Head kidney is a major site of erythropoiesis in teleosts and other hemopoietic organs are less important for RBC production (Verde et al., 2011; Soldatov, 2005). Therefore, active formation of RBCs in pronephros may determine the balance in fish blood towards productive processes. The monocyclic pattern of erythropoiesis in mullet found in the present work is in line with the duration of lifespan period estimated for teleostean RBCs (270–310 days) (Zolotova, 1987; Fischer et al., 1998).
Spleen is considered as secondary erythropoietic organ (Soldatov, 2005; Witeska, 2013) and immature RBCs are observed there when the activity of erythropoiesis in pronephros reached maximal levels. Spleen was shown to produce mainly lymphoid, granular cells and thrombocytes (Soldatov, 2005; Mahabady et al., 2012; Witeska, 2013). Furthermore, the key function of spleen in higher vertebrates is elimination and degradation of aged RBCs by melanomacrophage centers (Borgioli, Frangioni, 1997; Galindez., Aggio, 1998; Kurtović et al., 2008). Spleen also contains reticular tissue that ensures organ contraction and release of RBCs into circulating blood. This process leads to an increase of blood oxygen capacity during stress, acute hypoxia and other factors that influence aerobic metabolism (Soldatov, 2005; Witeska, 2013). The increase of RBC number in circulating blood due to spleen restriction may reach 30% and is more common for pelagic fishes compared to benthic species. However, release of RBCs from spleen was unlikely involved into changes in RBC number observed in the present work as aged RBCs cannot circulate in blood for long period of time. Moreover, stress caused by blood sampling was reduced as we used urethane narcosis.
The relative RBC number in circulating blood also depends on plasma hydration (dehydration), but these processes are usually observed during salinity fluctuations or hypoxia (Freire, Prodocimo, 2007; Sudesh, Sabhlok, 2014). Therefore, we may exclude the influence of these processes on the annual dynamics of RBC number in blood of mullet.
Spawning and the state of hematopoietic tissue. Reproduction is an essential part of animal life cycle. Spawning is associated with drastic shifts of energy sources towards generative tissue (Shulman, Love, 1999). Rearrangements of energy in the organism influence functional state of various physiological systems including blood. Some teleosts demonstrate anemia during spawning period as the number of RBCs and hemoglobin content decreases (Jawad etal., 2004). These processes are accompanied with suppression of locomotion and feeding (Shulman, Love, 1999). Interestingly, that cultivated fish do not demonstrate anemia and disturbances of feeding at spawning.
Pre-spawning anemia may result from partial destruction of aged RBCs. We found that RBC formation was enhanced at post-spawning period as the number of immature RBCs increased in both pronephros and head kidney during 2–3 months. Taking to account duration of life-span period of teleostean RBCs we may suppose that circulating blood contain large number of aged RBCs at pre-spawning and spawning period. These cells are less elastic comparing to young erythrocytes and are eliminated by spleen for further destruction (Soldatov, 2005a; Chu et al., 2008; Witeska, 2013).
In aged RBCs antioxidant capacity is suppressed resulting in enhanced lipid peroxidation (Phillips et al., 2000). Shifts in hormonal activity associated with spawning may also influence this process in teleosts. This hypothesis is in line with previously reported loss of circulating RBC number observed in fish following the injection of extracts of carp pituitary gland, gonadotropin, estrogen, releasing-factors (Ochiai et al., 1975; Hilge, Klinger, 1978).
Furthermore, RBC senescence may be also associated with methemoglobinemia. Respiratory pigments of teleostean RBCs are more vulnerable to oxidation comparing to hemoglobin of higher vertebrates (Maestre et al., 2009; Blair et al., 2020). RBCs possess NADH-diaphorase that is responsible to maintaining hemoglobin in ferry-form (Schoore et al., 1995; Saleh, McConkey, 2012). This process is also ensured by glutathione (GSH), ascorbic acid, tocopherol (Krishna, Venkataramana, 2007). This complex may be at least partly suppressed in aged RBCs by increasing the content of ferry-form and decreasing blood oxygen capacity. For example, in Gadus morhua methemoglobin concentration in blood 27% was not associated with any toxic impact (Graham, Fletcher, 1986).
Our data clearly indicate that circulating blood of mullet contain large number of aged RBCs during pre-spawning period, and these cells are intensively eliminated and degraded in spleen. These processes are accompanied with anemia and methemoglobinemia and likely induce production of erythropoietin for stimulation of erythropoiesis in hemopoietic tissue (Houston et al., 1996; Rothmann et al., 2000). Erythropoietin was identified in circulating blood by immunochemical analysis and the highest concentration was observed in the head kidney (Wickramasinghe 1993; Moritz et al. 1997; Lai et al. 2006). Structure of erythropoietin has been annotated for Japanese puffer (Takifugu rubripes) (Chou et al., 2004). Erythropoietin concentration in the head kidney was shown to correlate with blood testosterone concentration (Pottinger, Pickering, 1987). These results are in line with annual variation of RBC number in mullet blood found in the present work.
Thus, in the present work we found that red blood system state of mullet changes during annual cycle. The increase of number of immature RBCs in circulating blood and the head kidney indicate that RBC formation in hemopoietic tissue occurs at post-spawning period and lasts for 2–3 months. This process is accompanied with an increase of RBC number in blood. During the rest months erythropoiesis is suppressed.