O R I G I N A L R E S E A R C H
Erythrocyte osmotic fragility and select hematologic variables in
postparturient mares and their foals
Francesca Arfuso, Marco Quartuccio, Marilena Bazzano, Francesco Fazio, Giuseppe Piccione
Department of Veterinary Sciences, Polo Universitario Annunziata, University of Messina, Messina, Italy
Key Words
Erythrocyte osmotic resistance, red blood cells, hemolysis, neonatal period
Correspondence
G. Piccione, Department of Veterinary Sciences, Polo Universitario Annunziata, University of Messina, Messina 98168, Italy E-mail: giuseppe.piccione@unime.it
DOI:10.1111/vcp.12344
Background: Knowledge of hematologic function in postparturient mares
and foals is crucial for the monitoring of their health status and for the prompt diagnosis of pathologic conditions.
Objectives: The aim of this study was to evaluate erythrocyte osmotic fra-gility (EOF) and select hematologic variables in mares and their foals during the first month after foaling.
Methods: Blood samples were collected from each animal every 3 days
from day 1 until day 30 after foaling, and tested for RBC count, HGB, HCT, MCV, and EOF. Two-way repeated measures analysis of variance (ANOVA) and Bonferroni post hoc comparison test were applied to determine the effects of time and age (mares vs foals). Pearson correlation coefficients were computed for EOF and time or MCV values.
Results: There was a significant effect of time in RBC, HGB, HCT, and EOF (P < .001). Statistically significant differences were observed between mares and foals for RBC, MCV, and EOF (P < .05) throughout the experi-mental period. Significant correlations between EOF and both time and MCV (P < .05) were found in mares as well as in foals.
Conclusion: These results contribute to the understanding of perinatal
changes in hematologic variables in mares and foals. In addition, the data support the relevance of specific reference intervals for postparturient mares and foals. The findings also provide useful information that could help clinicians to better interpret clinical data and diagnose equine disease.
Introduction
A successful transition from the fetal to the neonatal state involves remarkable physiologic adaptations on the part of the newborn foal and the mare.1 Specifi-cally, the foaling process and the early postpartum per-iod represent stressful conditions for both the mare and the foal.2In foaling mares, the dynamics of hor-mones leading to the delivery and the beginning of lac-tation drastically change energy metabolism.1,3 In newborn foals, crucial adaptive processes to the extrauterine environment occur; these include the transition from fetal to adult circulation, the onset of pulmonary respiration, and enteral nutrition.4 It is well known that, for the entire perinatal period, mares and newborn foals are physiologically unstable and more susceptible to infections and metabolic diseases. In particular, periparturient mares may develop hyper-lipidemia, endometritis, subclinical hepatopathy, and
reproductive disorders caused by malnutrition.3,5,6 The main diseases occurring in neonatal foals are omphalitis, diarrhea, pneumonia, and immunodefi-ciency due to failure of passive transfer of colostral immunity.3,5,6
The evaluation of hematologic variables is one of the most common investigations performed in the clin-ical practice. However, the interpretation of the results can be difficult because hematologic variables in the equine species exhibit variations with age, breed, sex, and reproductive status.7Within the hematologic pro-file, erythrocyte variables represent a valuable tool for the assessment of the severity of a number of patho-logic conditions. For instance, the flexibility of the ery-throcyte membrane can be compromised.7,8 Therefore, tests of the integrity and elasticity of the RBC membrane have value for the assessment of the proper function of RBC.9One method used to deter-mine the membrane elasticity of RBC is the
erythro-cyte osmotic fragility (EOF) test.10This test measures the degree of resistance of erythrocytes to osmotic swelling and lysis in saline solutions with increasing degrees of hypotonicity.11
In the last few years, several authors addressed some aspects of the physiology of mares and foals during the peripartum period, focusing on endo-crine1, metabolic12,13, biochemical3,12, hemato-logic3,14,15, and hemostatic16,17profiles. Some studies included measurements of hematologic variables on periparturient mares and foals.3,14,18 However, no research on EOF in healthy mares and foals have been reported during the first month after foaling. Therefore, the aim of this study was to evaluate how select hematologic variables and EOF changed in postparturient mares and their foals over the first 4 weeks after foaling.
Material and Methods
Animals
Nine Italian Saddle mares (8–12 years old; body weight 400 50 kg, mean values standard devia-tion) and their foals (5 females and 4 males, body weight at birth 39.00 3.50 kg) were enrolled in the study with the owner’s informed consent. All deliver-ies occurred between March and April 2014. Each mare included in the study delivered a viable full-term foal spontaneously without human assistance, and shed a normal and intact placenta within 2 h.
Mares were subjected to a clinical examination within 12 h after foaling. During the postpartum iod, transrectal ultrasound examinations were per-formed weekly to ensure the normal involution of the uterus. Mares were fed 3 times a day (7 AM, 12 AM, and 5 PM) and water was available ad libitum. The diet consisted of 5.5 0.5 kg/day dried grass hay (crude protein 9%, crude fiber 35%, Ca 0.4%, P 0.23%) and 4.5 0.5 kg/day concentrate (crude protein 16%, crude fat 6%, crude fiber 7.35%, ash 10.09%, sodium 0.46%, lysine 0.85%, methionine 0.35%, and omega-3 0.65%). All animals were stationed at the same breeding center in Sicily (latitude 38.18°N; longitude 15.55°E). Foals were kept in the box stall with their dams under natural environmental conditions at night and were moved to paddocks (with no grass available) during the day (10 AM–4 PM).
At birth, each foal was subjected to a complete physical examination performed by the same veteri-narian, and routine hematology and plasma biochemistry profiles were determined. The foals included in the study were considered healthy at
clinical examination. All treatments, housing and animal care were carried out in accordance with the standards recommended by the EU Directive 2010/ 63/EU for animal experiments.
Data Collection and Experimental Design
Blood was sampled from each mare and foal every 3 days from day 1 until day 30 after foaling. Blood samples were collected by the same operator by jugu-lar venipuncture into 3-mL vacutainer tubes (Ter-umo Corporation, Leuven, Belgium) containing ethylenediamine tetraacetic acid (EDTA). All EDTA whole blood samples were delivered to the laboratory and processed within 2 h. Red blood cell count, HCT, HGB, and MCV were assessed using an automated hematology analyzer (HeCo Vet C, SEAC, Florence, Italy).
For the determination of EOF, a sodium chloride (NaCl) solution was prepared as described previ-ously.19 Ten plastic tubes without anticoagulant or other additives (Nunc, Thermo Scientific, Wyman Street Waltham, MA, USA) were prepared with 10 mL of serially increasing concentrations of NaCl solution (0.0% up to 0.9%) at pH 7.4, checked by a pH meter (HI 3220, Hanna instruments, Milan, Italy). An aliquot of 0.02 mL of whole EDTA blood was transferred to each tube. After gentle mixing, the tubes were main-tained at room temperature (26–27°C) for 30 min, mixed once more, and centrifuged at 327g for 15 min. The supernatant was transferred to a glass cuvette and the concentration of HGB in the supernatant solution was assessed spectrophotometrically at 540 nm (auto-mated analyzer UV Spectrophotometer, model Slim SEAC, Florence, Italy). For each tube, hemolysis was expressed as percentage, with 100%/maximum after hemolysis in straight distilled water (0.0% NaCl con-centration). The percentage of hemolysis was calcu-lated according to the formula19:
Hemolysisð%Þ : ðOD of test=OD of distilled waterÞ 100
where OD is optical density.
The EOF curve was obtained by plotting the per-cent of hemolysis at each saline conper-centration.
Statistical Analysis
Statistical analysis was performed using a commer-cially available software package (STATISTICA 7.5 Stat Software Inc., Tulsa, Oklahoma, USA). Two-way
repeated measures analysis of variance (ANOVA), fol-lowed by Bonferroni post hoc comparison test, were applied to determine the statistical significance of the effect of time on RBC, HCT, HGB, MCV, and EOF in mares and foals during the first month after foaling, and to evaluate statistical differences on these variables between mares and their foals. Pearson correlation coefficients were computed for EOF and time, and for EOF and MCV. In all cases, a P< .05 was considered statistically significant.
Results
There was a statistically significant decrease of RBC, HGB, and HCT (P< .001) with increasing number of days after foaling in mares and foals, while there were no significant changes in MCV (Table 1). In addition, RBC and MCV were statistically significantly higher in mares than in foals (P< .05) between days 1 and 30 post foaling.
Erythrocyte osmotic fragility significantly and gradually decreased up to day 21 after foaling in both mares and foals (P< .0001) (Figure 1), but remained unchanged thereafter. In addition, EOF in foals was significantly higher compared to their mares (P< .05)
at NaCl concentrations 0.1%, through 0.5% from day 1 up to 18.
There was a significant negative correlation between EOF, indicating increasing osmotic resistance of RBC with time after foaling in both mares and foals, as demonstrated in a linear regression model (Table 2, Figures 2 and 3). There was also a positive correlation between EOF and MCV in mares (Figure 4), while in foals the correlation between EOF and MCV was nega-tive, indicating increasing osmotic resistance with higher MCV (Figure 5).
Discussion
For a meaningful interpretation of hematologic vari-ables, the comparison to an established reference inter-val is needed that is species-specific and also specific for the current physiologic state, including sex, age, and parturition.18 Breed, age, sex, and reproductive status are important factors that can affect the hemato-logic profile in the equine species.7
Knowledge of hematologic function in postpar-turient mares and foals is crucial for the monitoring of their health status and for the prompt diagnosis of pathologic conditions. In the current study, RBC, HGB,
Table 1. Means SD of RBC, HGB, and HCT in 9 mares and their foals on postnatal days 1 through 30. Animals Days After Foaling
Hematologic Variables RBC (106/lL) HGB (g/dL) HCT (%) MCV (fL) Mares 1 8.66 0.86 14.32 1.59 41.14 3.59e 49.82 3.94 3 7.37 0.57c 13.22 0.95 37.29 3.49e 49.64 3.07 6 7.62 0.66 13.92 0.79 37.62 1.93g 49.52 1.64 9 7.64 0.81 13.38 1.71 37.18 2.97g 48.78 1.31 12 7.30 0.71c 13.11 0.81 34.74 2.95 47.63 1.31 15 7.13 0.30c 13.08 0.45 33.93 1.85c 47.60 2.12 18 7.25 0.80c 13.03 1.29 34.77 3.89c 47.95 1.26 21 6.96 0.27c 12.93 1.57 33.07 2.89d 47.07 3.23 24 6.96 0.51c 12.16 0.51 32.01 3.95cd 46.18 4.65 27 7.07 0.40c 11.89 1.51 32.50 2.54cd 46.03 3.72 30 7.02 0.40c 11.59 1.49c 32.06 1.23d 45.77 3.18 Foals 1 9.58 0.49ab 14.71 1.00 39.70 2.71 41.40 1.15a 3 9.15 0.60ab 13.91 7.75 37.33 3.51g 40.72 1.40a 6 9.08 0.79ab 13.81 1.79 36.94 2.13 40.82 2.16a 9 8.92 0.60a 13.50 2.04 36.51 1.72 41.03 2.81a 12 8.54 0.98a 13.06 2.53 36.36 3.31 41.20 2.05a 15 8.55 1.13a 12.81 1.61 36.10 2.14 42.00 3.97 18 8.68 1.26a 12.63 1.66 36.03 2.30 42.17 3.59 21 8.84 0.91a 12.28 2.01 36.97 1.86 42.17 3.17 24 8.36 0.83a 12.26 1.32 36.27 0.63 42.29 3.90 27 7.86 1.16 12.26 1.94 30.41 3.53g 43.30 2.92 30 7.71 0.56 12.20 1.30c 30.37 5.20g 42.45 3.95
Significance of time (P< .05):avs mares on the same day.
HCT, and MCV values were within published reference intervals for adult horses and foals.6–8In addition, our results showed significantly higher RBC and lower MCV in foals on days 1 through 30 after foaling. These findings reflect the effect of age (mares vs foals) on hematologic variables7,20,21and are in agreement with studies by other authors.22,23 Furthermore, the pro-gressive decrease in RBC, HGB, and HCT observed in the mares from day 1 through 30 is in agreement with previous studies on mares14,18, dairy cows24, and sows.25 This decreased red blood cell mass might be related to an increase in water consumption (up to 50– 70% above that required for maintenance) at the beginning of lactation.25,26The increased water intake is likely due to a combination of factors, including fluid loss associated with milk production, and increased food consumption to support milk production. This could lead to mild overhydration and hemodilution.27
A gradual decrease in RBC, HGB, and HCT val-ues was also found in the foals from day 1 to 30, which was previously described in young foals3 and newborn donkeys28,29, and might represent the
physiologic adjustments to early postnatal develop-ment and growth.12 This could include an acute stress/catecholamine release-related rise in RBC, HGB, and HCT during the first 24 hours after birth.7 In addition, an extended attachment of the umbilical cord can result in transfer of up to 1500 mL of blood from the placenta during the first several minutes following birth.7
Furthermore, the decline in RBC, HGB, and HCT during the first week of life in these foals can be related to the adjustment of fluid balance as a result of the osmotic effect of intestinal absorption of colostral immunoglobulins.7The ongoing decline in red blood cell mass can also be attributed to a shorter RBC life span, and/or reduced intestinal iron uptake and subse-quent delivery to bone marrow, as well as decreased erythropoietin production due to higher oxygen satu-ration of HGB.3,7No change was found in MCV values of foals during the experimental period. This could be due to the concurrent presence of micro- and macro-cytes. A mild anisocytosis is a typical finding in young foals.7
Figure 1. Erythrocyte hemolysis curves (means SD) with serial sodium chloride concentrations in whole blood of mares (n = 9) and their foals (n = 9) on days 1 through 30 after foaling. Statistically significant differences between mares and foals are indicated by*; statistically significant differences among time points are indicated by** (vs all days); and *** (vs days 3, 6, 9, 12, 15, and 18).
Figure 2. Linear regression between the erythrocyte osmotic fragility (EOF) test and time in postparturient mares (n= 9) from day 1 through 30 after foaling. Continuous lines indicate regression lines; dotted lines indicate 95% confidence intervals; and ◊ are values of EOF.
Figure 3. Linear regression between the erythrocyte osmotic fragility (EOF) test and time in newborn foals (n= 9) from day 1 through 30 after foaling. Continuous lines indicate regression lines; dotted lines indicate 95% confidence intervals; and ◊ are values of EOF.
Figure 4. Linear regression between the erythrocyte osmotic fragility (EOF) test and MCV values in postparturient mares (n= 9) from day 1 through 30 after foaling. Continuous lines indicate regression lines; dotted lines indicate 95% confidence intervals; and open circles represent individual values of EOF.
Figure 5. Linear regression between erythrocyte osmotic fragility (EOF) test and MCV values in newborn foals (n= 9) from day 1 through 30 after foal-ing. Continuous lines indicate regression lines; dotted lines indicate 95% confidence intervals; and open circles represent values of EOF.
Foals had a significantly higher EOF than mares at NaCl concentrantions of 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% from day 1 until day 18. The EOF in hypo-tonic solution has been studied in artiodactyls9, humans10, donkeys30,31, camels31, pigs32, and horses affected by immune-mediated hemolytic anemia.11It has been shown that EOF is related to the morpho-logic characteristics and, particularly, to the size of the erythrocytes and their cell membrane integrity.9 In our study, increasing EOF was correlated with increasing MVC in mares, and decreasing MCV in foals. These contrasting results might reflect the physiologic changes during the neonatal period nec-essary for an adaptation of the foal to extrauterine life.4,8,33 For instance, the initiation of pulmonary respiration causes changes in blood pH34 that could explain the higher osmotic fragility of the foal’s ery-throcytes. This is in accordance with previous studies carried out on donkeys30, domestic fowl35, cattle, pigs, rats, and rabbits36that found a significant influ-ence of blood pH on EOF. Some authors37,38 have concluded that the decrease in pH accelerates EOF by affecting the potassium–chloride co-transporter on the red cell membrane in vitro. Furthermore, the high levels of catecholamines, associated with the hypoxic stress of delivery, are typical of newborns in the early postnatal period39 and might be an addi-tional cause for the highest EOF in newborn foals. Indeed, these hormones cause intracellular calcium to increase by stimulating the adrenergic receptors, resulting in reduced erythrocyte deformability and an increased EOF.40
Conclusion
During the neonatal and postpartum phase, substan-tial physiologic adjustments take place in foals and mares.41To the best of our knowledge, no previous
studies investigated the EOF in healthy postparturi-ent mares and neonatal foals. Our results documpostparturi-ent the dynamics of select hematologic variables in mares and foals during the first month after foaling. They point to the importance of establishing specific reference values for hematologic variables and EOF in postparturient mares and newborn foals, and pro-vide useful information that could help clinicians to better interpret clinical data in the diagnosis of equine diseases.
Disclosure: The authors have indicated that they have no affiliations or financial involvement with any organization or entity with a financial interest in, or in financial competi-tion with, the subject matter or materials discussed in this article.
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