Changes in some Blood Micronutrients, Leukocytes and Neutrophil Expression of Adhesion Molecules in Periparturient Dairy Cows
© The Author(s) 2001
Received: 14 August 2000
Accepted: 20 October 2000
Published: 31 March 2001
Dairy cows are highly susceptible to infectious diseases, like mastitis, during the period around calving. Although factors contributing to increased susceptibility to infection have not been fully elucidated, impaired neutrophil recruitment to the site of infection and changes in the concentrations of some micronutrients related with the function of the immune defence has been implicated. Most of the current information is based on studies outside the Nordic countries where the conditions for dairy cows are different. Therefore, the aim of the study was to evaluate changes in blood concentrations of the vitamins A and E, the minerals calcium (Ca), phosphorous (P), and magnesium (Mg), the electrolytes potassium (K) and sodium (Na) and the trace elements selenium (Se), copper (Cu) and zinc (Zn), as well as changes in total and differential white blood cell counts (WBC) and expression of the adhesion molecules CD62L and CD18 on blood neutrophils in Swedish dairy cows during the period around calving. Blood samples were taken from 10 cows one month before expected calving, at calving and one month after calving. The results were mainly in line with reports from other countries. The concentrations of vitamins A and E, and of Zn, Ca and P decreased significantly at calving, while Se, Cu, and Na increased. Leukocytosis was detected at calving, mainly explained by neutrophilia, but also by monocytosis. The numbers of lymphocytes tended to decrease at the same time. The mean fluorescent intensity (MFI) of CD62L and CD18 molecules on blood neutrophils remained constant over time. The proportion of CD62L+ neutrophils decreased significantly at calving. The animals were fed according to, or above, their requirements. Therefore, changes in blood levels of vitamins, minerals and trace elements were mainly in response to colostrum formation, changes in dry matter intake, and ruminal metabolism around calving. Decreased levels of vitamins A and E, and of Zn at calving might have negative implications for the functions of the immune defence. The lower proportion of CD62L+ neutrophils at calving may result in less migration of blood neutrophils into the tissues, and might contribute to the increased susceptibility to infections at this time.
Mjölkkor är mycket känsliga för infektionssjukdomar,som mastit, under perioden runt kalvningen. Det är inte helt klarlagt vilka faktorer som bidrar till den ökade infektionskänsligheten men försämrad rekrytering av neutrofiler till infektionsplatsen och förändringar i koncentrationen av vissa mikroelement har troligen betydelse. De flesta befintliga uppgifter baseras på studier gjorda utanför Norden där förhållandena för mjölkkor är annorlunda. Syftet med denna studie var därför att undersöka förändringar i blodkoncentrationen av vitaminerna A och E, mineralerna kalcium (Ca), fosfor (P) och magnesium (Mg), elektrolyterna kalium (K) och natrium (Na) samt spårämnena selen (Se), koppar (Cu) och zink (Zn) hos svenska mjölkkor under perioden runt kalvning. Dessutom undersöktes förändringar i antalet vita blodkroppar och uttrycket av adhesionsmolekylerna CD62L och CD18 på blodneutrofiler. Resultaten stämde huvudsakligen med tidigare rapporterfrån andra länder. Koncentrationen av vitaminerna A och E, Zn, Ca och P minskade signifikant vid kalvning medan Se, Cu och Na ökade. Leukocytos sågs vid kalvning huvudsakligen orsakat av neutrofili men också till viss del monocytos. Antalet lymfocyter tenderade att minska vid samma tidpunkt. Medelvärdet av fluorescensintensiteten för CD62Loch CD18-molekylerna på blodneutrofiler var konstant över tiden. Andelen CD62L+ neutrofiler minskade dock signifikant vid kalvningen. Djuren utfodrades i enlighet med eller över deras behov varför förändringarna i blodnivåerna av vitaminer, mineraler och spårämnen huvudsakligen var ett resultat avkolostrumproduktion, samt förändringar i torrsubstansintag och våmmetabolism runt kalvningen.Minskade nivåer av vitaminerna A och E och av Zn vid kalvning kan ha negativ effekt på immunförsvarets funktion. Den lägre andelen CD62L+ neutrofiler vid kalvning kan resultera i en försämrad mi-.
The susceptibility of dairy cows to infectious diseases, like mastitis, is higher during the period around calving than any other time. Host resistance mechanisms are usually depressed from approximately 3 weeks before calving until 3 weeks after calving . Underlying mechanisms and factors have not been fully explained. However, many metabolic and hormonal changes take place during this period, which may contribute to the impaired immune defence [41, 46, 22]. Changes in white blood cell counts are observed around parturition, for example an increase in the numbers of circulating neutrophils (e.g. [11, 21]). Neutrophils are considered the first line of cellular defence against pathogens. However, at calving, important neutrophil functions, like migration and phagocytosis, are impaired [16, 21, 37]. Reduced migration of blood neutrophils can be explained by a lower expression of the adhesion molecules CD62L (L-selectin) and CD11/CD18, which are of vital importance for their migration to the site of inflammation [29, 24].
The nutritional status of the animals has been associated with the ability to resist infections. Reports have shown a depression in the blood levels of calcium (Ca), zinc (Zn), magnesium (Mg), phosphorous (P), potassium (K), selenium (Se), vitamins A and E during the periparturient period [18, 9, 49, 6, 53]. Several of these nutrients are important for the immune system. Increased incidence of mastitis was reported at calving when the concentrations of vitamins A and E were decreased [3, 27, 32, 43]. Selenium plays an important role in preventing impaired function of the immune response . Neutrophils from Se-deficient animals were less capable of intracellular killing of mastitis pathogens [12, 43]. Cu deficiencies have been shown to result in lowered bactericidal activities of blood leukocytes in cattle and sheep [19, 52]. Moreover,  reported a higher proportion of uninfected quarters during the peripartum period in Holstein heifers after additional Cu supplementation. Zinc sufficiency has also been linked to proper immune functions, whereas deficiencies were related with irregular immunological profiles [17, 34].
Most of the available information in this field is based on studies outside the Nordic countries where the conditions for dairy cows are different, for example in housing systems, feeding, climate and management. Therefore, the aim of this study was to evaluate leukocyte numbers and the expression of the adhesion molecules CD62L and CD18 on blood neutrophils, as well as blood vitamins A and E, the minerals Ca, P and Mg, the electrolytes K and Na, and the trace elements Se, Cu and Zn, during the periparturient period in Swedish dairy cows. This would also give baseline data for future studies in which different management routines could be compared.
Materials and methods
Diet composition and estimated dry matter intake (DMI) of 10 dairy cows one month before expected calving, at calving, and one month after calving, expressed in kilograms of dry matter and in percentage (%) of the total diet.
Nutrient requirements according to NRC (National Research Council, 1989) and approximate daily allotments to ten dairy cows one month before expected calving (-1), at calving (0), and one month after calving (+1), calculated on a body weight of 600 kg, and an average milk production of 30 l/day one month after calving.
Daily requirements (NRC)
From each cow, jugular blood samples were collected in the morning, using Vacutainer® tubes (Becton Dickinson Vacutainer Systems, Meylan, France), one month before estimated calving, at calving (within 24 hours after calving) and one month after calving. Before sampling, the skin was cleaned with Milli-Q-water (Milli-Q, Millipore Corp., Bedford, MA, USA). Blood collected in a Zn-free vacutainer tube without additives was used for serum analyses of Zn, Cu, Ca, P, K, Na and Mg. Heparinized blood was used for separation of plasma and erythrocytes which was analysed for Se. Blood without additives was taken for serum vitamin E and vitamin A analysis. The tubes were centrifuged at 1500 g for 35 min to get plasma or serum, which was frozen at -20°C until analysis of the nutrients. Blood samples with EDTA added were taken for neutrophil immunostaining of CD18 and CD62L adhesion molecules, and for total and differential white blood cell counts.
Total and differential leukocyte counts were determined within 2 h using a Cell-DynR3500 (Abbott diagnostics, Abbott Laboratories, Abbott Park, IL, USA) according to standard procedures at the Department of Clinical Chemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden.
Polymorphonuclear leukocyte immunostaining and flow cytometry analysis
For immunostaining with monoclonal antibodies (mAb), erythrocytes were lysed with ammonium chloride (NH4Cl) before the staining procedure, and washed three times with phosphate buffered saline (PBS) without Ca and Mg. A double staining procedure was used to identify CD45+ leukocytes bearing the other markers of interest as described by . The cell suspensions were labeled for flow cytometry with CD45 (clone CACTB51A, Veterinary Medical Research and Development (VMRD), Pullman, WA, USA), and either CD18 (clone BAQ30A, VMRD) or CD62L (clone BAQ92A, VMRD). Two secondary antibodies, goat anti-mouse IgG1 FITC (Caltag Laboratories, Burlingame, CA, USA), and goat anti-mouse IgG2a PE (Caltag), were used. The following controls were performed, blood without antibodies and blood with primary monoclonal antibodies CD45 (clone CACTB51A, VMRD) and a negative IgG1 isotype control (clone DAK-G01, DAKO, Glostrup, Denmark). Finally, the cell pellet was fixed in 200 μl of 1% paraformaldehyde in PBS and was stored in darkness at 4°C and analysed within a week. Before analysis, cells were washed twice and resuspended in PBS.
Stained cells were analysed on a FACStar Plus flow cytometer (Becton Dickinson Immunocytometry systems, Mountain View, CA, USA) with standard optical equipment using an argon ion laser at 200 mW tuned to 488 nm. The data were acquired with a FACstation, with the software Cellquest, version 1.2.2 (Becton Dickinson Immunocytometry Systems). Thirty thousand events were collected. The following parameters were obtained: forward light scatter (FSC), orthogonal light scatter (SSC), FITC fluorescence (FL1), and PE fluorescence (FL2). Leukocytes were identified by their expression of CD45, while their size (FSC) and granularity (SSC) identified polymorphonuclear leukocytes (PMNL). PMNL were gated to identify the proportions of CD18+ and CD62L+ cells. The discrimination between positive and negative cells was set using the isotype control. The mean fluorescent intensity (MFI) of each cell in FL1 was determined using quantum beads (Flow Cytometry Standards Corporation, San Juan, Puerto Rico).
Analysis of vitamins, minerals and trace elements
Vitamin A and E were extracted from the serum samples with hexan. The separation was done by High Performance Liquid Chromatography (HPLC) on a C18 colonn. Vitamin A and E were determined by using ultraviolet and fluorescence detection, respectively according to standard procedures at the Department of Chemistry, National Veterinary Institute, Uppsala, Sweden.
Serum samples were diluted (1:10) with ultrapure water (Milli-Q). The determination of Ca, Cu, K, Mg, Na and Zn was performed using inductively coupled plasma emission spectrometry (ICP-AES, Jobin Yvon 238 emission-spectrometer, Instruments S.A., Division Jobin Yvon, Longjuemeau, France) with set-up and conditions according to the method accredited by SWEDAC (Swedish Board for Accreditation and Conformity Assessment). Serum inorganic phosphate (P) was determined according to standard procedures at the Department of Clinical Chemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden.
The Se concentrations in the plasma and erythrocyte fractions were determined with flow injection hydrid generation atomic absorption spectrometry (FI-HG-AAS) after wet digestion of the biological material with a mixture of oxidizing acids . Selenium content in whole blood was calculated from plasma Se and erythrocyte Se assuming an average hematocrit content of 35% .
Analyses of variance for the concentrations of nutrients and leukocytes, and the proportions and MFI for the neutrophil adhesion molecules were done using the General Linear Model (SAS Institute Inc., Cary, NC, USA). The effects of cow and period were included in the model. Mean fluorescent intensity for CD18 and CD62L were log-transformed. The results are presented as least square means ± standard error of the mean (LSM ± SEM). Probabilities less than 0.05 were considered significant.
Total and differential blood leukocytes
Neutrophil adhesion molecules
Vitamins A and E
Minerals, electrolytes and trace elements
The Na concentrations were 138.3 ± 1.2, 142 ± 1.2 and 138 ± 1.2 mmol/l before, at and after calving, respectively. The value at calving was significantly (p < 0.05) higher than at the other time points. The Mg concentrations remained fairly constant over time, at approximately 1.05 ± 0.05 mmol/l.
In agreement with earlier studies (e.g. [11, 21]), we detected a significant increase in the numbers of WBC at calving. This was mainly due to an increase in the numbers of circulating neutrophils, and to a less extent, an increase in monocytes. At calving, the levels of corticosteroids are elevated [41, 11]. Corticosteroids induce neutrophilia by an increased output of neutrophils from the bone marrow, by neutrophil demargination from the blood vessel wall, or by a combination of the two [36, 24]. According to , the neutrophil expression of CD18 increases, while the expression of CD62L decreases at calving. Such changes were not observed in this study as the expression of CD62L and CD18 remained constant over time. However, we observed a depression in the proportion of CD62L+ neutrophils at calving, in accordance with . Fewer cells expressing this molecule means that the marginating pool of neutrophils, rolling along the vessel wall, will shift to the main blood flow stream contributing to the leukocytosis. As a result, fewer neutrophils are able to migrate into the tissues. In agreement with , we found that the numbers of blood lymphocytes were reduced at calving.  presented the hypothesis that lymphocytes migrate in a different manner than neutrophils, suggesting that the high levels of cortisol detected at calving do not affect the adhesion molecules of lymphocytes and therefore they can migrate into the tissues.
Our results have shown a marked decline at calving in serum concentrations of vitamins A and E, and in Zn in agreement with earlier reports [18, 9, 49, 53]. A drop in the serum concentrations of these nutrients is associated with impaired immune functions and a higher incidence of diseases, like mastitis [18, 34, 27, 43].
The drop in serum concentrations of vitamins A and E is largely due to colostrum formation , but can also be due to changes in dry matter intake and ruminal metabolism . Moreover, storage and season can have negative effects on the amount of vitamins A and E in the feedstuffs [9, 28, 33]. Dry matter intake (DMI) can drop remarkably during the week before calving [2, 10]. As a result, reduced blood concentrations of nutrients can be expected, especially as the nutrient demands to initiate milk synthesis is increasing. However,  reported less reduction in DMI before calving in Swedish dairy cows fed high quality feedstuffs. Ruminal metabolism has been implicated in the destruction of vitamin E , but others have suggested that ruminal vitamin E metabolism is essentially nil [25, 51]. Vitamin E in blood is present mainly as a component of lipoproteins. As parturition approaches, the liver secretion of lipoproteins decreases. As a consequence, its transport capacity of vitamin E is lowered . However, the ruminal destruction of vitamin A can be substantial and increases as the level of concentrates in the diet is elevated [35, 51].
The significant drop in serum Zn concentration reported at calving, is most likely a consequence of colostrum formation  and increased stress e.g. in association with an acute phase response due to inflammatory reactions in the uterus. Stress induces synthesis of metallothionein, a protein associated with Zn distribution. As a consequence, Zn is redistributed from blood to other tissues, such as the liver [44, 53]. Physiological fluctuations occur immediately before and after calving in the blood levels of Ca, P, K and Na . Blood levels of Ca and P is expected to decrease at calving due to the large demand of colostrum and milk production. In agreement with , we detected a reduced blood P concentration one month after calving. There is an inverse relationship between milk production and plasma P concentration . The K values were also depressed one month after calving, which might be related with K being the major cation secreted into the milk of cattle .
The blood Cu status undergoes several changes during the periparturient period. The lower value before calving could be due to the drainage by the fetal liver . In contrast to other reports [15, 53], an increased blood level of Cu was detected at calving in this study.  suggest that cattle undergoing stressful periods have increased blood levels of Cu and ceruloplasmin, as Cu transport protein. Ceruloplasmin is considered an acute phase protein and its concentration increase in response to injury, infections and inflammation . This might be one reason for the increased blood level of this nutrient, as calving is considered a stressful period with tissue damages for example in the uterus.
There is a relationship between the Se status of the animals around parturition and the functions of the immune system and disease resistance [12, 43]. From these studies it can be concluded that beneficial effects of Se supplementation occur only when the animals are Se deficient. Whole blood Se levels in the range of 0.1–0.2 mg/l could be considered optimal from immunological standpoint [23, 20]. In this study, whole blood Se concentrations were in the range of 0.167–0.180 mg/kg, i.e. according to recommendations.  hypothesised that the increase in the level of Se at calving may be related to the high fragility of the red blood cells detected at calving.
In conclusion, the results obtained under Swedish conditions were mainly in line with earlier reports. At calving, leukocytosis due to neutrophilia and monocytosis was detected. A lower proportion of CD62L+ neutrophils at calving suggests that fewer of these cells can migrate into the tissues with negative consequences for the defence against infections. Moreover, reduced concentrations of vitamins A and E, and the trace element Zn, were observed at this time. This can also have negative effects on the functions of the immune system resulting in increased susceptibility to diseases, such as mastitis. Despite the fact that vitamin A was fed according to recommendations, and vitamin E above recommendations, the levels of both nutrients decreased at calving. Vitamin A levels dropped below the normal reference value, while vitamin E levels remained within the normal range. Several authors [42, 27, 48] reported improvements in milk production, immune functions and mammary gland health when additional vitamins A and E were given compared with NRC recommendations. Results from the present study, in combination with aforementioned data, suggest that the NRC vitamin A and E recommendations may not be adequate, at least not around calving. However, further studies are needed to evaluate whether the low blood values reflect a true body deficiency and the importance of decreased absorption of vitamins during this period.
The authors thank Inga-Lena Örde-Öström, Gabor Sellei, and Anna Stepinska, National Veterinary Institute (SVA), Uppsala, Sweden for skilful technical assistance.
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