Compared to Fe concentration, Ht, and Hb or RBC counts, individual serum ferritin concentration is regarded as a more suitable parameter to determine and evaluate iron-deficiency anemia because of its direct correlation with the iron storage of an organism [21, 26]. Fe concentrations underlie age-dependent fluctuations [21, 27,28,29], but hematological parameters and serum ferritin are thought to be influenced by a variety of factors, such as housing and nutrition [30], hydration, health status [31], or sex [20, 22, 32]. Therefore, it is unsurprising that the literature contains conflicting reference ranges and cutoff values for serum ferritin caused by differing analytical methods used to determine serum ferritin levels. Based on these data, a preliminary study used a cutoff value for serum ferritin of < 15 µg/L to monitor iron deficiency in calves of four different herds [33]. Although this was a preliminary study, the results showed that serum ferritin diagnosed anemia more reliably and earlier than Fe and Hb levels. Therefore, we elucidated and compared the temporal courses of the known parameters, Ht, RBC count, Hb concentration, erythrocyte indices, and Fe, with the serum ferritin concentrations in dairy calves within the first 10 days of life to evaluate their possible suitability as a more sensitive diagnostic tool for early indication of deficiencies. A possible influence of prophylactic iron supplementation and neonatal diseases, such as diarrhea and omphalitis, was evaluated.
However, the results of our study suggest that serum ferritin levels are not suitable as early diagnostic markers for neonatal calves. There were no significant correlations between ferritin levels and previously validated diagnostic markers of anemia in calves or adult cattle. Nevertheless, the interpretation of hematological parameters in neonates is complicated and sometimes misleading.
In neonatal calves, some physiological changes occur in hematological parameters, erythrocyte indices, and acid-base balance and should not be misinterpreted as pathological changes [7, 32, 34]. Within the first weeks of life, fetal Hb is replaced by adult Hb, Fe shows marked changes, and a reduction in MCV is observed without an underlying iron deficiency. Our observation of an elevation in serum ferritin within the first day of life aligns with previous studies in piglets and calves [21, 35] and might result from physiological alteration processes associated with neonatal erythrocytosis.
Interestingly, serum ferritin concentrations in our study were not influenced by the administration of iron via an intramuscular injection of iron dextran on the first day of life. In contrast, iron dextran injections (100 mg Fe) in nursing piglets resulted in a peak in serum ferritin at 1 week of age, which declined until 3 weeks of age [35]. In calves, iron dextran injections on day 3 and at the age of 2 weeks also led to an increase in serum ferritin concentrations within 1 week [21]. The observed differences between the aforementioned studies and our study might be due to a difference in serum ferritin levels at birth between the calves in the treatment and control groups in our study. However, concentration-dependent effects are plausible. In our study, the dosage consisted of 10 mg/kg iron dextran on day 1 of life, representing an often-used treatment pattern (injection of 5 mL of iron dextran solution per calf). A previously published study used comparable dosages, but not on the first day of life. Due to the different enzyme settings in neonates, treatment on the first day of life may not have the same results as treatment on day 3 [36]. Therefore, interference with the physiological increase in ferritin up to day 3 of life and possible pharmacodynamic differences between days 1 and 3 might have influenced the resorption of iron dextran, thus leading to a supplementation-independent elevation of serum ferritin. Therefore, a direct comparison between previous studies and the present study is critical. All the aforementioned studies reported marked inter-individual variances for serum ferritin, which might further complicate the interpretation of the presented results. However, the effects of the iron injection were observed in the treatment group. Fe levels were much higher in the treated animals than in the control animals, although the effect of iron supplementation was only visible until day 4.
A recent study examined the effects of different iron supplementation strategies (oral versus injection) on iron homeostasis in calves [37]. Although this study was conducted at a local research farm with its offspring and used a standardized amount of 1,000 mg of iron, our study investigated the effects of low-dose iron dextran supplementation (10 mg/kg i.m.) in an animal group that originated from different farms. These farms had below-average hygiene standards and were chosen to mimic the field conditions in our study, although the calves were transferred to the clinic. A low dose of iron dextran was used to examine the effect of the standard prophylactic treatment often used in the field to boost neonatal calves on the first day of their lives.
While Ht, Hb, and MCV are known to decline within the first weeks of life in young calves [7], neither their development nor that of other hematologic parameters have been evaluated within the first days of life, especially considering health status. RBC counts have been reported to be higher in young calves than in adult cattle, and a reduction in RBC count, Ht, Hb, and MCV confirms the diagnosis of anemia [38]. In our study, only four calves showed a reduction in RBC count, Ht, and Hb levels below their reference ranges. However, concerning the timely course, a mild reduction in these parameters was observed in all calves within the first 7 days of their lives. As previously shown in older calves [33], a significant positive correlation was observed.
In contrast to other studies [27, 34], we observed increasing concentrations of Fe independent of iron supplementation after birth. These differences may be the result of differences in the feeding habits of suckling calves. The animals in our study were nourished on a commercial milk substitute containing 150 mg/kg DM iron and forage, whereas Bostedt et al. [34] used calves fed whole milk only. Atyabi et al. [27] did not comment on the nutritional premises of their products. The possible oral iron intake of the calves in our study was higher than that of calves fed whole milk only, thus resulting in an elevation of Fe levels independent of supplementation within the first days of life. This is consistent with the results of Golbeck et al. [37]. The calves used in the present study also showed an increase in estimated transferrin saturation at the end of the first week of life. We concluded that this increase resulted from feeding with a conventional milk substitute.
While our results suggest a negative effect of iron supplementation on the development of Ht, Hb, and RBC, we must consider a possible bias due to the adverse distribution of calves between the treatment and control groups. Although the calves were randomly allocated to the groups, the calves in the treatment group had lower average values of nearly all parameters on day 1 compared to those in the control group. Timely developments in Ht, Hb, and RBC counts did not differ between the groups. Consequently, the statistically significant influence of iron supplementation on these parameters was not apparent clinically. Therefore, in line with other studies, this warrants a critical evaluation of the necessity for iron supplementation [37, 39, 40].
In contrast, the effects of diseases were not biased because equal numbers of animals in both groups developed signs of disease during the study protocol. As observed in previous studies [9, 12, 13], severely diseased animals had reduced Fe concentrations compared to healthy animals. This physiological metabolic pathway may be seen as a counter-regulation of the body against pathogens [8]. The effects of diseases on the hematological parameters, Ht, Hb, and RBC, were consistent. Animals with moderate signs of disease showed higher levels of Ht, Hb, and RBC. However, the necessity of fluid therapy in severely diseased animals may explain these effects as dilution effects [41].
Another important aspect that warrants mention is that iron absorption is impaired in intestinal inflammation [42] resulting in hypoferremia at an early stage of infection [43]. Siderophilic bacteria, such as Salmonella, Clostridia, and some pathogenic E.coli, further influence iron absorption, because these enteric pathogens compete for Fe2+, the absorbable oxidation state of iron [44]. Modulatory innate defense mechanism and iron deprivation via bacterial infection might have contributed to the reduced serum iron concentrations in the present study. Hence, the results of the present study must be considered limited, due to the fact that no microbiological examinations were performed that could have validated or eradicated the presence of the aforementioned siderophilic enteropathogenic bacteria.
In general, Fe and serum ferritin are reportedly influenced by inflammation [45], resulting in reduced suitability as iron deficiency indicators. However, serum ferritin is still considered the most reliable biomarker of total body iron stores in cases of minor inflammation [45]. In the present study, serum ferritin concentrations showed no dependence on the presence of disease, although various inflammatory diseases, such as diarrhea, thrombophlebitis, and omphalitis, occurred during the course of the study protocol. As serum ferritin itself is a positive, acute-phase protein [46], it was assumed that the inflammatory status in the animals of the present study was rather mild, and therefore, no effects of the disease could be demonstrated. However, correlation analyses targeting regression corrections were not performed. In preschool children, this approach resulted in a 25% higher estimated prevalence of iron deficiency based on serum ferritin levels [46]. Unfortunately, no data interpreting iron indicators using inflammatory biomarkers in neonatal calves are available; this should be assessed in future studies.
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