Evolutionary differences in the circulatory systems of vertebrates are paralleled by functional variations in red blood cell (RBC) morphology, numbers, and biophysical properties [1, 2]. Mammalian RBCs are characterized by enucleation of erythroid cells towards the end of their maturation [3]. Interspecies differences in mammalian RBCs are observed in RBC cell size, aggregation, metabolism, membrane lipid composition, and the overall proteome [4, 5, 6, 7, 8]. These variations dictate differences in hemorheological properties and the RBC’s ability to adapt to various extracellular environmental challenges in the circulation [9, 10, 11, 12]. RBC lifespan varies across mammalian species with bovine RBCs known to circulate longer (~160 days) than human RBCs (~120 days) [13]. Premature cell death of circulating RBCs prior to completion of their expected lifespan in vivo has been demonstrated in a wide range of human diseases associated with anemia, as well as in mouse models of different clinical conditions [14]. However, little is known about this phenomenon in cattle.

During their lifespan, RBCs accrue a wide range of physical injuries together with enzymatic alterations leading to their senescence and subsequent clearance from the circulation by phagocytic catabolism [13, 14, 15]. In the context of various systemic diseases in humans, RBCs may be afflicted by oxidative injuries leading to reduced survival [16]. Other pathophysiologic cell stressors, such as fever-range hyperthermia, extracellular tonicity alterations, and energy starvation, can trigger RBC dysfunction and premature cell death [14]. When excessive loss of RBCs is not compensated by enhanced erythropoiesis in the bone marrow, anemia ensues [14]. Apoptotic cell death in RBCs, also known as eryptosis, is characterized by the breakdown of the phospholipid asymmetry in the cell membrane leading to the flipping of the aminophospholipid, phosphatidylserine (PS), towards the exterior leaflet of the cell membrane [14, 17]. While a wide array of mechanisms are operative during RBC cell death, Ca${}^{2+}$-mediated signaling has been considered as a canonical pathway regulating this process in human RBCs [14].

Structurally, ruminant RBCs exhibit anomalies in their cytoplasmic membrane phospholipid architecture as compared to RBCs from other mammalian species such as the presence of N-acylated aminophospholipids [18]. Bovine RBCs are deficient in phosphatidylcholine and possess high levels of sphingomyelin in the cell membrane [19]. Strikingly, unlike human RBCs, the outer leaflet of bovine RBC cell membrane is reported to have a significantly lower proportion of phosphatidylethanolamine (2% versus 20%) [19]. Differences in composition of the lipid membranes of bovine and human RBCs have been linked with differential RBC membrane fluidity [20] and water permeability [21] between the two species. Despite the unique lipid organization in the bovine RBC membrane PS is found entirely in the inner leaflet of the lipid bilayer of intact cells, like human RBCs [19].

In bovine RBCs, the possibility of PS translocation to the outer leaflet in response to proapoptotic signals, as in human and murine RBCs, remains elusive. One important clue on the clinical significance of apoptotic cell death in stressed bovine RBCs comes from a previous study showing increased RBC PS expression in anemic cattle infected with the intraerythrocytic parasite, Theileria sergenti [22]. Infected cattle have been reported to display enhanced clearance, hemolysis, and morphological changes in RBCs [22, 23, 24]. Systemic bovine diseases involving RBC dysfunction and anemia can influence the overall health and productivity of livestock. Thus, characterization of the apoptotic cell death process in bovine RBCs would provide novel insights into the pathogenesis of bovine anemia resulting from accelerated loss of circulating RBCs.

Firstly, we examined the effects of artificially enhancing intracellular Ca${}^{2+}$ content in human and bovine RBCs using ionomycin, a well-established Ca${}^{2+}$ ionophore. As shown in Fig. 1A–C, ionomycin treatment similarly enhanced Ca${}^{2+}$-dependent Fluo 4 fluorescence in both bovine and human RBCs. While extracellular Ca${}^{2+}$ uptake responses are similar for RBCs from both species, PS expression in bovine RBCs was achieved with a higher ionomycin concentration as compared to human RBCs, potentially indicating differences in downstream enzymatic activation in RBCs from the two species (Fig. 1D–F).

Fig. 1.

Ionophore-mediated Ca${}^{\mathrm{2}\mathrm{+}}$ influx and cell death in bovine and human red blood cells (RBCs). Representative histogram (A,D; Black: 0 µM ionomycin; Red and Blue: 20 µM ionomycin). Means $\pm$ standard error of mean (SEM) of Fluo 4 mean fluorescence intensity (MFI) (B,C; n = 4) and percentage of phosphatidylserine (PS) expression (E,F; n = 4) in bovine and human RBCs following 1-hour incubation in Ringer’s solution containing 0–50 µM ionomycin at 37 °C. *, ** and *** indicate significant difference (p 0.05, p 0.01, and p 0.001, respectively) from the absence of ionomycin treatment.

Oxidative damage to human RBCs is paralleled by increased cation channel activation leading to increased Ca${}^{2+}$ influx [28]. Acute exposure of RBCs to the oxidant, TBOOH, elicited significantly enhanced Fluo 4 fluorescence in both bovine (Fig. 2A,B) and human RBCs (Fig. 2A,C). We then interrogated the extent to which TBOOH-induced Ca${}^{2+}$ increases translated into increases in cell membrane PS exposure. As illustrated in Fig. 2D–F, oxidant treatment significantly upregulated PS expression in both bovine and human RBCs. We further tested RBC viability after oxidant treatment by determining intracellular esterase activity using quantification of Calcein fluorescence. Human RBCs exposed to oxidant treatment showed reduced Calcein fluorescence confirming a recent report [27]. However, bovine RBCs did not show decreases in Calcein fluorescence (data not shown) possibly due to reduced cellular uptake of this dye.

Fig. 2.

Oxidative stress stimulates Ca${}^{\mathrm{2}\mathrm{+}}$ entry into and cell death in bovine and human RBCs. Representative histogram (A,D; Black: Control; Red and Blue: 0.2 mM tertbutyl-hydroperoxide (TBOOH)). Means $\pm$ SEM of Fluo 4 MFI (B,C; n = 4) and percentage of PS expression (E,F; n = 4–6) in bovine and human RBCs following 30-minute incubation in Ringer’s solution containing 0.2 mM TBOOH at 37 °C. * and *** indicate significant difference (p 0.05 and p 0.001, respectively) from the absence of TBOOH (Control).

Next, we evaluated bovine and human RBC survival under the conditions of fever-grade hyperthermia and glucose deprivation. As shown in Fig. 3A–C, a 72-hour incubation of bovine RBCs in Ringer’s solution, glucose-devoid conditions, or exposure to 41 °C temperature upregulated intracellular Ca${}^{2+}$ levels in human and bovine RBCs as compared to intracellular Ca${}^{2+}$ levels in refrigerated RBCs (baseline). Due to large data scatter, the effects were not statistically significant in bovine RBCs. We then sought to elucidate whether enhanced cytosolic Ca${}^{2+}$ levels translated into breakdown of cell membrane phospholipid asymmetry. In contrast to human RBCs, bovine RBCs displayed only a subtle increase in PS expression in response to energy starvation and hyperthermia (Fig. 3D–F).

Fig. 3.

Effects of hyperthermia and energy starvation on cytoplasmic Ca${}^{\mathrm{2}\mathrm{+}}$ levels and PS expression in bovine and human RBCs. Representative histogram (A,D; Dotted: Baseline; Black: Control; Red: Energy-depleted; Blue: Hyperthermia). Means $\pm$ SEM of Fluo 4 MFI (B,C; n = 4) and percentage of PS expression (E,F; n = 4) in bovine and human RBCs following 72-hour incubation in Ringer’s solution without glucose (energy-depleted) at 37 °C or in Ringer’s solution at 41 °C (hyperthermia). Baseline readings were determined in refrigerated RBCs. *, ** and *** indicate significant difference (p 0.05, p 0.01, and p 0.001, respectively) from baseline values.

Additional experiments were conducted to assess whether fever-grade hyperthermia or energy starvation induced oxidative stress in bovine and human RBCs. As shown in Fig. 4A–C, DCF fluorescence, indicating increased ROS levels, was significantly enhanced in both bovine and human RBCs after a 72-hour incubation, under glucose-devoid conditions or incubation in hyperthermic conditions (41 °C), suggesting that despite starvation- and hyperthermia-associated redox imbalances in RBCs from both species, bovine RBCs displayed a minimal increase in membrane PS expression.

Fig. 4.

Effects of hyperthermia and energy starvation on reactive oxygen species (ROS) production in bovine and human RBCs. Representative histogram (A; Dotted: Baseline; Black: Control; Red: Energy-depleted; Blue: Hyperthermia). Means $\pm$ SEM of dichlorodihydrofluorescein (DCF) MFI (B,C; n = 4) in bovine and human RBCs following a 72-hour incubation in Ringer’s solution without glucose (energy-depleted) at 37 °C or in Ringer’s solution at 41 °C (hyperthermia). * and ** indicate significant difference (p 0.05 and p 0.01, respectively) from baseline values.

Deviations in extracellular tonicity are known to trigger ionic and fluid shifts in RBCs. Hemolysis in bovine RBCs was significantly higher at 30–50% reductions in osmolality as compared to human RBCs (Fig. 5A). Hypertonicity (addition of 600 mM of sucrose into the medium) significantly enhanced PS expression in human RBCs after 4 hours (Fig. 5B,E). In contrast, a modest but significant increase in PS exposure was observed in bovine RBCs after a 48-hour, but not 4-hour, incubation in hypertonic Ringer solution (Fig. 5B–D) suggesting that bovine RBCs are relatively more resistant to hypertonic stress-induced cell death as compared to human RBCs.

Fig. 5.

Impact of extracellular osmolality changes on bovine and human RBCs. Means $\pm$ SEM of relative hemolysis (n = 4; 405 nm absorbance; A) in bovine RBCs (red line) and human RBCs (blue line) after exposure to different extracellular osmolalities. *, ** and *** indicate significant difference (p 0.05, p 0.01, and p 0.001, respectively) from human RBC at the respective osmolality. Representative histogram (Black: Control; Red and Blue: hypertonic; B) and means $\pm$ SEM of percentage of PS expression in bovine RBCs (C,D; n = 4–6) and human RBCs (E; n = 4) following incubation in Ringer’s solution containing 600 mM sucrose for 4 hours or 48 hours at 37 °C. ** indicates significant difference (p 0.01) from Control.

Clinically, the pathogenesis of anemia in cattle has been ascribed to various factors such as immune-mediated RBC destruction [29], parasitic infections [30, 31, 32], and nutrient deficiency [33, 34]. In vitro and in vivo studies provide some mechanistic evidence that increased erythrophagocytosis and extravascular RBC catabolism are responsible for Trypanosoma-elicited anemia, a common parasitic infection in cattle [35, 36]. Despite the morphological and compositional peculiarities of bovine RBCs, our in vitro data indicate that bovine RBCs undergo a cell death process with some mechanistic similarities to that observed in human [14], murine [14], and canine RBCs [37].

Supraphysiologic Ca${}^{2+}$ overload in human RBCs induces metabolic reprogramming, ion channel modulation, posttranslational protein modifications, and affects the RBC cytoskeleton [14, 38]. Remarkably, bovine RBCs showed higher resistance to Ca${}^{2+}$ ionophore-induced cell death as compared to human RBCs suggesting a potentially higher molecular threshold for RBC cell death in bovine diseases. Unlike humans and other species, physiological levels of ionized Ca${}^{2+}$ in bovine RBCs remain unknown [39]. It may be surmised that possible interspecies variations in basal Ca${}^{2+}$ concentrations may dictate differences in downstream signaling responses to acute Ca${}^{2+}$ elevations. Removal of extracellular Ca${}^{2+}$ mitigates metabolic alterations in human RBCs such as changes in glutathione synthesis and lipid remodeling [38]. Oxidative stress is an important activator of Ca${}^{2+}$-permeable cation channels in human RBCs leading to their dysfunction and eventually cell death [16, 40]. Our data showed enhanced ROS levels in bovine RBCs after incubation in glucose-depleted medium, despite very low membrane PS expression. A potentially plausible explanation for this observation comes from a recent study showing several metabolic anomalies in bovine RBCs during prolonged refrigerated storage [4]. Bovine RBCs were reported to display reduced levels of glycolytic metabolites, S-adenosylmethionine, adenosine triphosphate (ATP), and glutathione, and enhanced purine oxidation products as compared to RBCs from other mammalian species, suggesting slow energy metabolism and high oxidative stress during storage [4].

Heat stress in cows can affect their physiological homeostasis and reduce livestock productivity [41]. Cattle suffering from some types of infectious febrile conditions such as anaplasmosis and trypanosomiasis, often display anemia [42, 43]. Normal bovine body temperatures have been reported to vary between 38–39.5 °C depending on age and breed [44]. Our data show that exposure of bovine and human RBCs to hyperthermia increased ROS levels. However, bovine RBCs showed a minimal increase in PS expression despite prolonged hyperthermia exposure, suggesting that bovine RBCs potentially have a higher acute adaptability, possibly underpinned by interspecies metabolic variations, to hyperthermic challenge. Interestingly, exposure of bovine RBCs to extracellular hypertonic stress for relatively shorter periods did not elicit increased PS exposure as compared to human RBCs and only a prolonged incubation of bovine RBCs modestly enhanced PS expression. However, in contrast to human RBCs, bovine RBCs displayed markedly increased hemolysis in response to reduction in extracellular osmolality. Indeed, RBCs from cows have previously been reported to display higher levels of hemolysis in response to various challenges compared to RBCs from various mammalian species [5, 45]. A limitation of the present study is that the relatively small sample size did not permit us to determine the effects of biological variables such as age, sex, and cow breed. Notably, age, sex, and genetic heterogeneity have been documented to influence RBC phenotype in other mammalian species [46, 47, 48]. Nevertheless, the strength of our study is that it provides the first published evidence of the cell death process in bovine RBCs and paves the way for follow-up studies in the future, relevant to veterinary medicine and the livestock industry.

From our data and previous findings taken together, it may be conjectured that structural and metabolic variations between human and bovine RBCs confer differential responses to stress-induced cell death patterns. Accordingly, further studies are required to fully discern the cell death machinery in bovine RBCs, which may potentially be an important mechanism underlying bovine anemia.

Data supporting the findings of this study are available from the corresponding author upon reasonable request.

BK and SMQ designed the experiments. BK, SM, and TM performed the acquisition and analysis of data. BK, SM, and SMQ wrote the manuscript and prepared the figures. RB, TES, HJT, JGJ, and SMQ advised on the research, provided resources, and/or interpreted the data. All authors have sufficiently participated in the research, reviewed the manuscript draft, edited the changes, approved the final version, and agreed to take responsibility for this work.

Use of bovine and human blood for research was cleared by Ontario Tech University’s Biosafety committee (permit# BSC28). RBC concentrates were supplied by CBS Blood4Research (CBS REB #2022.034).

The authors thank Masoomeh Eghtedari and Shivani Patel for their technical support, and Windcrest Meat Packers Ltd. staff (Port Perry, ON) for providing bovine blood samples.

SMQ received research funding from the Natural Sciences and Engineering Research Council of Canada (NSERC; RGPIN-2022-04382). RB was supported by a grant from the German Research Foundation (BI 2149/2-1). SM and TM were recipients of the NSERC Undergraduate Student Research Award.

The authors declare no conflict of interest.