Weaning transition is a critical phase in calf development, marked by economic, physiological and management challenges (Hulbert et al., Reference Hulbert, Cobb, Carroll and Ballou2011). The high costs of liquid diets and labor necessitate efficient weaning strategies. While intensive suckling accelerates growth, it can delay rumen development, complicating the transition to solid feed compared with conventionally fed calves. Indicators such as adequate starter intake and weight gain, are essential for ensuring proper growth and disease resistance (Bittar et al., Reference Bittar, Gallo, Silva, De Paula, Poczynek and Mourão2020).
Weaning induces stress through dietary changes, housing adjustments and social interactions, often leading to reduced feed intake, gastrointestinal issues, behavioral changes and weakened immunity (Hulbert and Moisá, Reference Hulbert and Moisá2016). Additional stressors such as transport, dehorning and animal grouping further suppress immune function and decrease disease resistance (Ballou et al., Reference Ballou, Hanson, Cobb, Obeidat, Sellers, Pepper-Yowell, Carroll, Earleywine and Lawhon2015). Haematological studies have shown age-related immune development, with high neutrophil counts at birth reflecting immune immaturity and a gradual shift to lymphocyte predominance as adaptive immunity develops (Santos et al., Reference Santos, Santarosa, Dal Más, Silva, Guirro and Gomes2023). Although innate immunity provides the initial pathogen defense, it is less robust in newborn calves and improves with age through enhanced phagocytosis and cytokine production (Lynch et al., Reference Lynch, Earley, McGee and Doyle2010). Weaning typically causes transient neutrophilia, suppresses neutrophil function and reduces disease resistance, all of which is influenced by pre-weaning nutrition (Ballou, Reference Ballou2012). This study investigated the impact of weaning on haematological parameters and innate immune responses in Holstein calves managed under an intensive milk program. Although weaning is known to induce physiological changes, its effects on calf health, especially inflammation and immunity, require further study across different ages and management systems. This study focused on evaluating haematological profiles, systemic inflammatory indicators (total serum protein, iron,and haptoglobin), and neutrophil functions, including phagocytosis and ROS production, after stimulation with Staphylococcus aureus, Escherichia coli, and Mannheimia haemolytica. These findings highlight the suppression of innate immune responses during weaning, emphasizing the need for improved nutritional and stress reduction practices.
Material and methods
This study was approved by the Ethics Committee on the Use of Animals (CEUA n. 1658230119) of the College of Veterinary Medicine and Animal Science (FMVZ) at the University of São Paulo (USP), Brazil. The study monitored 188 Holstein calves over seven months (from May to October 2017), all from the same commercial herd in Araras, São Paulo State (latitude 22°21ʹ25″S, longitude 47°23ʹ03″W). This farm is one of Brazil's largest milk producers, averaging 2,100 lactating Holstein cows and producing approximately 96,000 liters of milk per day, following Brazil's definition of good management practices.
Experimental design
Forty-seven Holstein calves were included in the study. The inclusion factors and nutritional management are detailed in the online Supplementary materials & methods and Supplementary Figure S1. Calf age at weaning ranged from 69 to 85 days, since the weaning decision was based on size and body weight. Prior to the weaning phase each calf received 8 l/d of milk replacer, split into two feeds. Commencing six days before weaning (D-6) this was reduced to 5, then 4 and then 3 l/d for 2d each such that on the two days before complete weaning only 3 l/d was offered. Water and pellet feed (Rumileite LS® Guabi Nutrition and Animal Health, Brazil; Supplementary Tables 1 and 2) were provided ad libitum throughout. Each calf was assessed at six, four and two days before weaning (D-6, D-4 and D-2, respectively) as well as on the weaning day (D0), totaling 188 blood samples. Blood was collected from the calves by jugular vein puncture and drawn into three vacuum tubes: one containing sodium heparin, one with no anticoagulant, and one containing ethylenediaminetetraacetic acid (EDTA). A heparin tube was used to collect white blood cells for neutrophil phagocytosis assays. The tube without anticoagulant was allowed to clot at room temperature, after which the serum was collected by centrifugation at 2000 × g for 15 min, aliquoted and stored at −20°C for up to 3 months. Serum was used to analyze iron, total protein and haptoglobin concentrations (Ramos et al., Reference Ramos, Madureira, Silva, Bosco, Morita, Guimarães and Gomes2021). EDTA samples were refrigerated at 4°C, transported to the laboratory and analyzed within 3 h of collection for blood cell counts using a haematology system (ADVIA 2120i®, Siemens Healthcare Diagnostics, Tarrytown, NY, USA), as previously described by Morita et al. (Reference Morita, Martin, Silva, Woolums, Hurley and Gomes2022).
Phagocytosis of S. aureus and E. coli was measured in vitro using pHrodo™ Red-labeled bacteria and flow cytometry, while ROS production was assessed through the conversion of dihydrorhodamine 123 to Rhodamine-123, following established methods (Morita et al., Reference Morita, Martin, Silva, Woolums, Hurley and Gomes2022). The assay details are described in the Supplementary Material and Methods section.
Statistical analysis
All data were analyzed using Statistical Analysis System for Windows (version 9.2, SAS Institute Inc., Cary, NC, USA). Quantitative variables (blood count, total protein, haptoglobin, iron concentration, and immune response) were subjected to descriptive analysis (mean ± standard error) and assessed for distribution relative to the Gaussian curve using a Guided Data Analytical function. Variables that did not follow a normal distribution were transformed using log10, square root or inverse functions to achieve data normalization. Outliers identified through command-guided data analysis were evaluated individually. After correcting for any typing errors in the raw data, these outliers were included in the statistical analysis. PROC ANOVA was performed to determine the overall effect of the time points on each variable. Differences among the four sample times were analyzed using Tukey's test and are reported as the untransformed mean ± standard error. Qualitative data such as diarrhea, bovine respiratory disease (BRD), hyperthermia (≥ 39.4 °C) and haptoglobin values (≥ 2 mg/dL) are presented as frequencies which were compared using the chi-square analysis. Statistical significance was set at P ≤ 0.05.
Results and discussion
Prevalences of diarrhea, BRD and fever are shown in Table 1. The incidence of diarrhea and BRD was relatively low throughout the weaning phase (0 to 3 calves from 47) whereas that of fever was higher, up to 9 calves, with the highest values close to or at weaning day. The rectal temperature of the calves was assessed as hyperthermic (febrile) when it was 39.4 °C or above.
Incidence of disease in calves at days relative to weaning (D0)
Values are number and (percentage) of calves displaying symptoms, n-47 calves. BRD, bovine respiratory disease.
ANOVA revealed significantly different values (P < 0.05) for some red blood cell (RBC) and platelet variables from six days before (D-6) to the final day of weaning (D0: Figure 1 and Supplementary Table S3). Iron concentration increased from D-6 until D-2 (P = 0.002), followed by an abrupt decrease at the end of milk offering (D0), and seemed to have an effect on four other specific variables (cell haemoglobin concentration mean, haemoglobin width, reticulocyte number and mean haemoglobin content of reticulocytes). Qualitative interpretation of RBC parameters revealed only a single case of anemia at D-6 based on intervals of references described by Morita et al. (Reference Morita, Martin, Silva, Woolums, Hurley and Gomes2022). Similarly, only one calf had hypoferremia at D0, according to the cut-off of 5.7 µmol/L (Martin et al., Reference Martin, Smfn, Costa, Baccili, Silva, Hurley and Gomes2021). Cell haemoglobin concentration means and haemoglobin width oscillated significantly (both P < 0.01) in the short period of weaning management, and both variables expressed anisochromia, often due to variations in hemoglobin content (Morita et al., Reference Morita, Martin, Silva, Woolums, Hurley and Gomes2022). The same was observed for the mean hemoglobin content of reticulocytes, in addition to an increase in the number of reticulocytes, which represents the amount of hemoglobin in the reticulocyte population. These indicators are useful for assessing the functional iron available for erythropoiesis during the previous three or four days (Morita et al., Reference Morita, Martin, Silva, Woolums, Hurley and Gomes2022). There was no difference in platelet count during the weaning period, however, the mean platelet volume did vary (P < 0.001), peaking two days before weaning (D-2). This is a measure of the average size of platelets in the blood and can be an indicator of platelet production and function. A higher value could indicate an increased production of platelets as part of the inflammatory process, explained by the stressful period during weaning (Hulbert et al., Reference Hulbert, Cobb, Carroll and Ballou2011).
Blood cell count (mean ± SEM) of ADVIA 2120i® equipment (A–K), serum total protein (L), haptoglobin (M), and iron (N) concentrations in pre-weaned Holstein calves during the weaning period: 6 (D-6), 4 (D-4) and 2 (D-2) days before weaning and on the weaning day (D0).
Neutrophil counts also fluctuated between sampling days (P < 0.05), with the highest mean observed at (D-2: Figure 1). However, the absolute values still remained within the normal range proposed in the literature for the same age group (4.11 ± 0.37 × 103/µL: Morita et al., Reference Morita, Martin, Silva, Woolums, Hurley and Gomes2022). The number of basophils decreased rapidly (P < 0.01), reaching its lowest value on D-4 and remaining low for the rest of the weaning transition period. By contrast, other authors have reported no changes in eosinophil and basophil counts during the weaning period (Lynch et al., Reference Lynch, Earley, McGee and Doyle2010; O'loughlin et al., Reference O'loughlin, McGee, Waters, Doyle and Earley2011). The myeloperoxidase intracellular index/myeloperoxidase content was stable from D-6 to D0. Myeloperoxidase is an enzyme produced by neutrophils and is activated when these cells respond to infection or inflammation.
The absolute number of monocytes had an opposite profile to that of neutrophils, with the lowest value detected at D-2 (P < 0.01). A similar profile was observed for the changes registered in the large unstained cells, which are observed in the circulation of calves and include a variety of cell types, such as immature granulocytes (precursors to mature neutrophils, eosinophils and basophils) and immature monocytes (Morita et al., Reference Morita, Martin, Silva, Woolums, Hurley and Gomes2022). During periods of stress, infection or inflammation, the body may release more immature cells, including monoblasts, into the bloodstream. This could lead to an increase in both large unstained cells and monocyte counts as the body attempts to respond to the challenge (Brun-Hansen et al., Reference Brun-Hansen, Kampen and Lund2006), such as calves that suffer during the weaning period.
Lobularity index changes registered during the weaning period had the same profile as neutrophil count, which correlated with the white blood cell (WBC) count in the peroxidase-positive channel. This offers a value for the WBC count that includes neutrophils, eosinophils and monocytes, but not peroxidase-negative cells such as lymphocytes, basophils and large unstained cells (Morita et al., Reference Morita, Martin, Silva, Woolums, Hurley and Gomes2022). These parameters are reported here for the first time in relation to the weaning period.
Somewhat surprisingly, the neutrophil:lymphocyte (N:L) ratio demonstrated no significant statistical association with the weaning transition period (P > 0.05, online Supplementary Table S3). This ratio reflects the impact of stress on the innate immune system, as it is affected by cortisol release. Other studies have showing shown decreased lymphocyte counts concomitant with elevated WBC and neutrophil levels following stress induced by abrupt dietary changes (Lynch et al., Reference Lynch, Earley, McGee and Doyle2010) but in our case there was little or no change in lymphocyte count or percentage (online Supplementary Table S3) and so changes in the N:L ratio were too small to achieve significance. Nevertheless, the N:L ratio is generally considered to be a valuable indicator for assessing stress and immune function in calves.
The total protein (TP) and haptoglobin (Hp) concentrations were used to monitor the inflammatory profile of the calves. We anticipated that both TP and Hp concentrations would increase during weaning due to the immunosuppression associated with the transition to solid feed and the heightened risk of infectious diseases, which could drive up the inflammatory profile. However, both showed a significant decrease (P < 0.01 and P < 0.05, respectively, online Supplementary Table S3, Figure 1). Considering individual values, 9 of the 47 calves (19.15%) had Hp ≥ 2 mg/dL (Ramos et al., Reference Ramos, Madureira, Silva, Bosco, Morita, Guimarães and Gomes2021) early in the weaning phase (D-6 and D-4), decreasing to 7 and 1 at D-2 and D0, respectively (significantly different, P < 0.05, chi-square test). If the evaluation had been conducted after weaning, higher values might have been observed once the calves no longer had access to their liquid diet, similar to the findings of a study with 15 Holstein calves (Kim et al., Reference Kim, Yang, Upadhaya, Lee, Yun and Ha2011), which reported increased Hp levels at three- and five-days post-weaning compared to two days before weaning. A different group (Kim et al., Reference Kim, Lee, Kim, Son, Kim, Joo, Park, Park, Lee and Kim2020) investigated other components of the immune system to assess the impact of weaning on 18 Holstein calves, such as pro-inflammatory cytokines and acute-phase proteins (APP). They described higher levels of pro-inflammatory cytokines on the third day post-weaning, which coincided with the elevation of APP. We did not observe any increase in Hp (a major component of APP), contrary to the initial hypothesis. This difference may have arisen because the management was gradual in our study. However, these authors did report a higher neutrophil count and N:L ratio two days prior to weaning, which is in part consistent with our findings.
Overall, polymorphonuclear leukocyte phagocytosis activity induced by S. aureus (%) and E. coli (% and medium fluorescence intensity) decreased from D-6 to D-2, indicating persistence in the low phagocytosis rate for S. aureus (Figure 2 and Supplementary Table S4). ROS production was constant for all bacterial stimulations (S. aureus, E. coli, and M. haemolytica) from D-6 to D-2, followed by a sharp increase on D0. However, only unstimulated cells showed a reduction during the weaning transition period (P < 0.01, Supplementary Table S5). The response ratio (RR) provided a clearer view of ROS production by stimulated versus unstimulated cells during weaning transition. PMA, E. coli, and M. haemolytica stimulation showed increasing RR values, peaking on D0 (day of full weaning). In contrast, S. aureus stimulation resulted in a steady decline in RR values throughout the weaning period (Figure 2 , Supplementary Table S6).
Phagocytosis rate (%) and median fluorescence intensity (MFI - mean ± SEM) of E. coli (A, B) and S. aureus (C); response ratio (RR, mean ± SEM) of reactive oxygen species (ROS) production in cells challenged by PMA (D), E. coli (E), S. aureus (F), pure M. haemolytica (G), and M. haemolytica 1:5 (H) of pre-weaned Holstein calves during the weaning period: 6 (D-6), 4 (D-4) and 2 (D-2) days before weaning and on the weaning day (D0).
Weaning influenced neutrophil phagocytosis and ROS production following stimulation with E. coli, S. aureus, and M. haemolytica, common pathogens in dairy systems. M. haemolytica is a primary cause of bovine respiratory disease (Credille et al., Reference Credille, Capik, Credille, Crossley, Blanchard, Woolums and Lehenbauer2023), E. coli is a major pathogen in calf diarrhea and septicemia (Gomes et al., Reference Gomes, Barros, Castro-Tardón, Martin, Santos, Knobl, Santarosa, Padilha and Hurley2023), and S. aureus causes contagious mastitis (Exel et al., Reference Exel, Gerritsen, Spaninks, Duim, Koop and Benedictus2023), which could potentially contaminate rearing calves. We observed a different result of neutrophil phagocytosis and ROS production against the pathogens tested (online Supplementary Table S4) which indicated that calf immunocompetence differs between Gram-positive and Gram-negative bacteria (Supplementary table 4). Despite compromised neutrophil phagocytosis during weaning, oxidative burst activity remained effective, allowing internalized pathogens to be successfully degraded, even with reduced uptake of Gram-positive bacteria.
The same decline in the innate immune response was observed by Lynch et al. (Reference Lynch, Earley, McGee and Doyle2010) in beef cattle and by Hulbert et al. (Reference Hulbert, Cobb, Carroll and Ballou2011) in dairy cattle, both of which investigated abrupt weaning. Lynch et al. (Reference Lynch, Earley, McGee and Doyle2010) noted a decrease in neutrophil phagocytosis two days after weaning compared to the day of abrupt weaning. Moreover, weaned calves exhibited a lower percentage of neutrophils engaged in phagocytosis than non-weaned (control) calves. Nevertheless, this transient decline in phagocytosis was short-lived, with cells recovering this function by 7 d post-weaning. In contrast, the oxidative burst activity remained unchanged throughout the sampling period. Consequently, these authors reported that weaning affects neutrophil surveillance and phagocytic activity, as observed in our results. Unlike our results, these authors reported that the bactericidal activity remained unaffected.
In conclusion, we have demonstrated that the weaning transition significantly influences the innate immune response and haematological parameters in calves. While our gradual weaning process was intended to mitigate stress, fluctuations in neutrophil phagocytic activity, ROS production and leukogram changes (such as increased neutrophilia and slightly altered N:L ratios), highlight the immunosuppressive effects of transitioning from a liquid to a solid diet. These effects occurred despite the provision of adequate nutrition, as indicated by normal RBC and hemoglobin levels, suggesting that the observed immune changes were a direct consequence of weaning. The study's limitations, including the lack of post-weaning data, standardized weaning age and cortisol measurements, restricted a comprehensive understanding of the long-term impacts and stress-related effects during this critical phase. Future research should explore adaptive immune responses, such as T and B cell activities, while addressing current limitations through larger sample sizes, standardized weaning protocols and extended monitoring.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0022029925000330.
Acknowledgements
The authors thank Colorado Farm for the animal use for this experiment and the Coordination of Superior Level Staff Improvement (CAPES, Brazil, process n. 88887.503498/2020-00) for the master´s degree scholarship (KNS) and the resources received from Bayer Animal Health.
