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Supporting healthy immune function in transition dairy cows

Published: September 13, 2021
By: Stephen LeBlanc / Professor of Ruminant Health Management, Population Medicine, University of Guelph, ON.

There is a high risk of clinical or subclinical reproductive tract disease in the postpartum period in dairy cows. An integrated process of adaptive events should occur synchronously, including a robust but well-regulated immune response in the uterus. Failure of this process may result in reproductive tract inflammatory disease. 

Neutrophils are the most abundant type of inflammatory cell and the main line of defence against infection in the uterus. Avoidance of clinical disease (metritis and purulent vaginal discharge) depends in large part on how effective the immune response is at limiting the burden and effects of bacterial pathogens, while the occurrence of subclinical endometritis is more a function of avoiding excessive or persistent inflammation.

Glucose supply, hypocalcemia, lipid mobilization from body fat, ketosis, and the flux of proinflammatory cytokines influence immune response and change rapidly and variably among individual cows. It is clear that hypocalcaemia is associated with reproductive tract disease, but the mechanisms remain unclear (i.e., direct effects of calcium on innate immune cell function vs. indirect effects of hypocalcemia through shared pathways related to feed intake or behaviour). Therefore, the importance of prevention or early treatment of subclinical hypocalcemia as a means to improve immune function and prevent reproductive tract disease is also unclear. Effective but well-regulated inflammatory response will be favoured by best management practices for transition cows, but specific interventions to modulate immune response to prevent uterine disease remain developmental.

Dairy cows experience massive metabolic demands to support lactation. They adapt their metabolism to do so, including uncoupling of the somatotropic axis (Baumgard et al., 2017) with peripheral insulin resistance and increased lipolysis to fuel milk production. However, cows are challenged by a transient decrease in feed intake resulting in negative energy and protein balance in early lactation, and short-duration but substantial hypocalcemia around calving. Concurrently, there is impairment of innate immune function and regulation of inflammation which is central to the development of mastitis (Ballou et al., 2012) and uterine diseases (Sheldon et al., 2019). The mechanisms linking these changes and metabolic challenges are only partially understood. 
Markers of aspects of adaptation to negative energy balance (e.g., serum concentrations of nonesterified fatty acids (NEFA) and β-hydroxybutyrate (BHB)) are associated with the risk of many metabolic and infectious diseases, in part through their associations with suppressed immune function and excessive inflammation (Ingvartsen and Moyes, 2013). Approximately 35% of peripartum cows have NEFA and 45% have BHB above thresholds associated with metabolic disease or compromised production or reproduction (McArt et al., 2013). In a large dataset, 44% of cows had at least 1 disease condition in early lactation, and of these, 39% had 2 or more separate diseases (Santos et al., 2010). Impaired innate immune function appears to have an important place in this web of metabolic health and disease.
This paper provides a brief narrative review of selected important determinants of health in dairy cows in the transition period around calving. Specifically, the focus is on management and social stressors, markers of adaptation to negative energy balance, and hypocalcemia, and their associations with neutrophil function. 
General neutrophil function
Neutrophils marginate, and adhere to and ‘crawl’ along endothelial cells, then perform diapedesis to move from blood vessels into tissue (Kolackowska and Kubes, 2013). Once in tissues, neutrophils interact with damaged cells or bacteria to remove foreign cells through a variety of mechanisms (Figure 1) including phagocytosis and intracellular digestion by oxidation (e.g., oxidative burst in lysosomes), extra-cellular release of oxidants from neutrophil granules (e.g., myeloperoxidase; Figure 1), or casting neutrophil extra-cellular traps (NET’s) of DNA (Nauseef and Borregaard, 2014; Liew and Kubes, 2019).
Figure 1. Schematic summary of the mechanisms by which neutrophils kill pathogens, such as bacteria as illustrated here. Neutrophil granules contain pro-inflammatory proteins including myeloperoxidase, lactoferrin, gelatinase, and matrix metalloproteinase 9. Following phagocytosis, encapsulated pathogens are killed intracellularly by reactive oxygen species or proteins from granules that fuse with the phagosome. Neutrophil extracellular traps consist of extruded DNA and cargo from extracellular granules.
Schematic summary of the mechanisms by which neutrophils kill pathogens, such as bacteria as illustrated here. Neutrophil granules contain pro-inflammatory proteins including myeloperoxidase, lactoferrin, gelatinase, and matrix metalloproteinase 9. Following phagocytosis, encapsulated pathogens are killed intracellularly by reactive oxygen species or proteins from granules that fuse with the phagosome. Neutrophil extracellular traps consist of extruded DNA and cargo from extracellular granules.
It is now recognized that there are important links of systemic metabolism (especially adipose metabolism and insulin resistance) with immune function and inflammation in humans and laboratory animals (Hotamisligil and Erbay, 2006; Osborn and Olefsky, 2012) as well as in dairy cows (Bradford et al., 2015). Both obese people and periparturient dairy cows are characterized by elevated circulating NEFA, insulin resistance, hepatic lipid accumulation and systemic inflammation, and there is great interest in the phenomenon of metabolic or sterile inflammation in dairy cows (LeBlanc, 2014). NEFA, particularly saturated fatty acids that predominate in transition cows, may also impair neutrophil function (Ingvartsen and Moyes, 2013). Briefly, certain NEFA activate Toll-like receptor 4 (TLR4), a main receptor for lipopolysaccharide (LPS), which activates Nuclear Factor κβ (NF-κβ) and leads to secretion of Tumour Necrosis Factor α (TNFα), Interleukin 1 (IL-1) and IL-8. TNFα and IL-1 act on intracellular messengers to up-regulate inflammation and increase insulin resistance. Based on work in mice and humans, local, classical neutrophil actions may feed into chronic, systemic, sterile inflammation (Buck et al., 2017) under conditions that may include lipolysis, ketosis, and lack of supply of substrates for immune cells – all of which are common in dairy cows in the transition period. Host microbiome(s) are starting to be recognized as having important interactions with regulation of inflammation well beyond the gut (Kubes, 2018). However, there are also important differences between cows in early lactation and people with ‘metabolic syndrome’ or non-alcoholic fatty liver disease.
Neutrophil function in dairy cows
Classic work by Kehrli et al. (1989) demonstrated impairment of elements of innate immune function around calving, although the temporal changes may have been confounded to some extent by clinical or subclinical mastitis in 5 of the 8 primiparous animals studied. Most measures of function in neutrophils (except random migration the week before) were increased in the weeks preceding calving, followed by a nadir in week 1 after calving, particularly for their 3 measures of oxidative burst function. Each of retained placenta, metritis, purulent vaginal discharge, endometritis, and mastitis (which collectively affect at least 25% of dairy cows in early lactation) is strongly associated with impairments of one or more aspects of neutrophil function (Gunnink 1984; Cai et al., 1994; Kimura et al., 2002; Hammon et al., 2006; Ballou et al., 2012). While some reports document differences in neutrophil migration capacity between cows that subsequently have disease or remain healthy, the bulk of evidence points to impairment of neutrophil killing function (i.e., reduction of measures of oxidative burst such as iodination (myeloperoxidase activity), or cytochrome C reduction (generation of superoxide anion)). There is less indication of impairment of ingestion (phagocytic capacity) by neutrophils. The changes and differences in neutrophil function precede detection of clinical disease (Hammon et al., 2006) and may precede calving (Gunnink 1984; Cai et al., 1994; Kimura et al., 2002). 
It is important to be specific when discussing neutrophil function, because not all aspects are typically impaired in transition cows, and different variables affect particular elements of neutrophil function.
In the transition period, there are inevitable substantial changes in circulating hormone concentrations in late pregnancy and the postpartum period and numerous profound endocrine adaptations to support lactation. These are compounded by imposed changes applied with good intentions but not always good effect (such as diet and pen or social group changes), as well as variable changes that may result from the foregoing or be somewhat independent of it, such as the degree of reduction in feed intake. Finally, other variables such as heat stress, competition for feeding or lying space, the quality of resting places, feed and water quality and availability, and the extent of social turmoil may combine to abate or exacerbate the inevitable challenges of the periparturient period. 
In a bold experiment, researchers at the US National Animal Disease Center attempted to separate the effects of late pregnancy and calving from those of lactation by mastectomizing 10 cows in early to mid-pregnancy and comparing 3 markers of innate immune function to 8 intact cows that calved and lactated (Kimura et al., 1999). A caveat is that the effects of lactation may be confounded by the fact that all intact cows had milk fever and 3 of the 8 had ketosis and displaced abomasum. Expression of L-selectin on the surface of neutrophils (necessary for initial (rolling) endothelial adhesion) decreased in both groups at calving but recovered within 1 to 3 days. The decrease was likely caused by inhibition of L-selectin (CD62) by glucocorticoids (Burton et al., 1995) i.e., cortisol released as part of parturition. Neutrophil surface expression of 2-integrins (needed for final adhesion for diapedesis) was actually greater before calving and to 3 d postpartum in the cows that lactated. However, myeloperoxidase activity (a measure of oxidative burst capacity) declined in both groups from 3 weeks before to 3 days after calving, but then quickly and fully recovered in the mastectomized cows while remaining at the parturient trough level until the end of the study at 21 d postpartum in the lactating cows. On the available evidence, oxidative burst is the most consistently impaired element of neutrophil function in dairy cows after calving, and these unique data support the inference that factors related to the demands of lactation sustain but do not initiate this impairment.
Factors affecting neutrophil function in dairy cows
There are numerous factors that are likely to affect neutrophil functional capacity in dairy cows, the headlines of which are illustrated in Figure 2. Elaboration is provided below.
Figure 2. Factors that influence the functional response capacity of neutrophils in dairy cows. The scheme is simplified because there are likely interactions among these known factors, and others including genetics. Nutritional formulation and feeding management determine the potential supply of immune system inputs, with additional variability imposed by social group and competitive pressures, as well as heat stress, and the comfort of the lying space. 
Factors that influence the functional response capacity of neutrophils in dairy cows. The scheme is simplified because there are likely interactions among these known factors, and others including genetics. Nutritional formulation and feeding management determine the potential supply of immune system inputs, with additional variability imposed by social group and competitive pressures, as well as heat stress, and the comfort of the lying space.
Management and social stress effects 
Lesser feed intake up to 2 weeks before calving is a risk factor for metritis (Hammon et al., 2006; Huzzey et al., 2007). That finding makes sense because lower nutrient intake would plausibly reduce the availability of fuel and substrates for neutrophil function and so diminish immune response, which in turn is a risk factor for metritis (Cai et al., 1944; Hammon et al, 2006). The reasons for which some cows consume less than others before calving and before the onset of visible disease are not well understood. In the study by Huzzey et al. (2007), competitive behaviour at the feed bin was studied, and cows that were more socially submissive (had fewer interactions in which they displaced another cow for feed access) were more likely to have lower intake and metritis. That study showed that cows apparently have a strong drive to eat at the same time, most acutely after fresh feed delivery, and that more submissive animals do not compensate their intake by eating at off-peak times. That is consistent with empirical observations that crowding or competition for space at the feed bunk (i.e., < 75 cm of feed bunk per cow or > 80% cows to headlocks) during the transition period is a risk factor for postpartum disease (Nordlund et al., 2006). Observations of the adverse effects on feed intake and lying time of pen moves and social group changes led to the logical suggestion that stable social groups and fewer movements of cows to new pens during the transition period (Cook and Nordlund, 2004), along with adequate feeding space, should reduce the incidence of postpartum disease. That ought to reduce stressors and/or improving feed intake for cows at the submissive end of the social spectrum, and so improve metabolic status and thereby immune function. Endogenous cortisol has a transient but substantial increase at calving, which is associated with decreased expression of glucocorticoid receptor on neutrophils and increased circulating neutrophil counts (Preisler et al., 2000). Both elevated endogenous cortisol concentrations and administration of dexamethasone decreased expression of L-selectin on neutrophils, leading to neutrophilia but likely contributing to decreased functional capacity of the neutrophil system for 1 to 2 days (Weber et al., 2004).
The hypothesis of social stress (mediated by acute or chronic elevations of cortisol or catecholamines and/or effects on feed intake or behaviour) leading to impaired immune function or health has generally not been supported in controlled studies. Huzzey et al. (2012) provided 4 groups of 10 cows in late gestation with 67 cm of feed rail space and 1 freestall per cow or 34 cm of feeding space and 2 cows per stall for 14 days per treatment in a crossover design. On average, feed intake was greater in the overstocked group, particularly in the second week of the treatment. There were significantly but modestly greater plasma NEFA and fecal cortisol metabolite concentrations, and lower plasma glucose concentrations in overcrowded primiparous animals, and a lesser insulin response to a glucose tolerance test in overcrowded cows. However, it is unclear that the magnitude of the effects observed would contribute to disease risk. 
In an experiment, 756 Jersey cows were assigned 26 d before expected calving to separate pens for heifers or parous cows, stocked at either 80% cows to headlocks and 80% cows to freestalls, or approximately 100% cows to headlocks and stalls. There were more competitive displacements at the feed bunk in the crowded groups, but only small differences in mean feeding and lying times (Lobeck- Luchterhand et al., 2015). There were no differences between treatments in the incidence of retained placenta, metritis, purulent vaginal discharge, or culling, nor in plasma concentrations of NEFA or BHB, energy-corrected milk yield to 155 days of lactation, or probability of pregnancy at the first 2 inseminations (Silva et al., 2014). In a subset of 48 cows per treatment per parity, measures of innate and adaptive immune function and chronic stress were assessed from 2 to 3 weeks before, to 2 to 3 weeks after calving (Silva et al., 2016). There were no differences between treatments in neutrophil phagocytosis or oxidative burst function, expression of L-selection or cluster of differentiation (CD)18, production of immunoglobulin (Ig)G against ovalbumin, serum haptoglobin concentrations, or concentrations of cortisol in blood or hair. 
To assess the effect of stable versus dynamic social group (pen populations) in the 4 weeks before calving, Silva et al. (2013a) assigned a total of 567 Jersey cows to groups of 44 cows per freestall pen in 6 replicates of stable (“all-in-all-out”; no additions to the pen) or dynamic (weekly additions of new animals to replace cows that left for calving) social groups. That resulted in differences in social stability as well as average stocking density (72 and 87%, respectively, but with much greater variance in the stable groups). There were no differences between treatments in plasma concentrations of NEFA or BHB, the incidence of retained placenta, metritis, endometritis, lameness, or early culling, the prevalence of persistent anovulation, pregnancy at first or second insemination, or energy-corrected milk yield. In a subset of 34 to 40 cows per treatment, there were no differences between treatments in neutrophil phagocytosis or oxidative burst function or expression of L-selection or CD18, production of IgG against ovalbumin, serum hapoglobin concentrations, or concentrations of cortisol in blood (Silva et al., 2013b).
Miltenburg et al. (2018a) assigned 48 cows in groups of 6 to 10 to either of two space allowances for the 3 weeks before calving: 90 cm of feeding rail space per cow and 80% cows to freestalls, or 45 cm of feeding space and 120% cows to stalls. Lying time was reduced by 2 h/day in the overcrowded group but was still > 12 h/day on average. As seen by Silva et al. (2014), there were more competitive displacements at the feed bunk in overcrowded groups, although that was confounded with a greater number of such interactions among multiparous cows generally. Overcrowded cows tended to have greater liver fat content at week 3 postpartum. However, during the treatment period and to 5 weeks postpartum, there were no differences between treatments in serum BHB, NEFA, glucose, insulin, insulin-like growth factor 1, aspartate aminotransferase, bilirubin, or haptoglobin concentrations and no differences in neutrophil phagocytosis or oxidative burst function. One interesting finding was that when cows were ranked by successful displacements of other cows at the feed bunk, in under-crowded groups, medium and high success index animals had greater neutrophil oxidative burst function than low success cows or any cows in overcrowded groups. This suggests that greater space allowance may have an effect on innate immune function, but it is not to improve function for the most submissive cows. Similarly, Chebel et al. (2016) found that the cows in the 90th percentile of social rank (most dominant) had modestly but significantly greater neutrophil phagocytosis and oxidative burst capacity, particularly at calving. Also interestingly, and in contrast to Huzzey et al. (2007) compilation of data from several large experiments on space and social group stability indicated that, counter-intuitively, more dominant cows (based on displacements at the feed bunk) were at greater risk of retained placenta or metritis (Chebel et al., 2016). These authors hypothesize that such cows may spend more time in aggressive interactions but fail to use their apparent advantage to consume more feed or achieve greater health (at least with respect to uterine disease). We concluded from our study (Miltenburg et al., 2018a) and the published data on the effects of crowding on metabolic health and innate immune function that the evidence does not refute the potential importance of space allowances under field conditions, but feeding and lying space alone are not the critical determinants of immune function in transition dairy cows. The minimal and optimal amounts of feeding and lying space likely depend on other variables, including whether primiparous and multiparous animals are commingled in the prepartum period (Chebel et al., 2016) 
Nutrient supply for neutrophil function 
Mounting an immune or inflammatory response consumes meaningful quantities of nutrients, and is notably energetically costly. In an elegant experiment, Kvidera et al., (2017) employed an IV LPS challenge with a euglycemic clamp technique to estimate the minimal requirement of glucose to mount an acute inflammatory response, which was approximately a net of 1 kg of glucose in 12 h. The glucose requirement for immune response appears to be consistent among dairy and beef cattle at 0.7 to 1.0 g/kg body weight0.75 per hour.
Homeorrhetic adaptions in support of lactation are oriented around partitioning nutrients to the mammary gland, notably increasing the supply of glucose for milk synthesis by sparing its use by other tissues, primarily by inducing peripheral insulin resistance. This situation likely contributes to reduction of immune function in early lactation. Activated immune cells appear to be obligate users of glucose and to increase their consumption of glucose (Kvidera et al., 2017). The fuels used by bovine neutrophils are not well characterized but glucose appears to be crucial (Ingvartsen and Moyes, 2013). Conversely, in vitro supplementation of glucose in neutrophils from early- or mid-lactation dairy cows modestly increased phagocytic capacity but mostly did not meaningfully affect PMN function (Garcia et al., 2015). However, the basal concentration of glucose in the cell media (7.2 mmol/L) used by Garcia et al. was considerably greater than in circulation in cows of either stage of lactation (~ 3.2 mmol/L), or even in dry cows. Once migrated, neutrophils likely must rely on stores of glycogen to function. Galvao et al. (2010) showed that there were lesser glycogen stores in neutrophils in circulation at calving in cows that 3 to 7 days later had metritis. That is consistent with Hammon et al. (2006), who showed that circulating neutrophils had lower oxidative burst capacity in cows that 1 week later had metritis, or 3 to 4 weeks later had endometritis. In that study, one explanatory variable was that neutrophils from cows in the lowest quartile of feed intake through the 3 weeks before calving (despite ad libitum availability) had (in relative terms) 50% lesser oxidative capacity from 1 week before to 3 weeks after calving than cows in the top quartile of feed intake. In summary, it seems that the availability of glucose to fuel neutrophil function may be a contributing factor to the impaired capacity observed in the transition period.
The supply of anti-oxidants (e.g., selenium and vitamin E) is important to contain the potent oxygen free radicals generated within neutrophils as part of their killing function. If there is insufficient selenium or sulfur-containing amino acids, glutathione peroxidase may not be able to detoxify hydrogen peroxide in the cytosol. Similarly, insufficient vitamin E may allow hydroxyl radicals to initiate a chain reaction of cell membrane peroxidation. Either could result in neutrophils conducting a short-lived suicide mission rather than a more sustained response with numerous iterations of ingestion and degradation of bacteria.
There is an abundance of large-scale observational studies that demonstrate associations of elevated serum concentrations of NEFA and/or BHB (hyperketonemia or ketosis) with increased risk of infectious and metabolic disease (summarized in McArt et al., 2013). Briefly, serum NEFA > 0.3 mmol/L in the 1 to 2 weeks before expected calving is associated with increased risk of retained placenta, metritis, or displaced abomasum, decreased milk production, and worse reproductive performance. Serum or blood BHB ≥ 1.2 mmol/L in the first 2 weeks after calving is associated with increased risk of displaced abomasum, endometritis, prolonged anovulation, and early culling, decreased milk yield in early lactation (conditional on the concentration of BHB and the timing of onset of ketosis), and decreased reproductive performance (McArt et al., 2013). Elevated serum NEFA concentrations and hyperketonemia are indicators of some degree of maladaptive response to the demands of lactation, probably in some measure reflect the availability of glucose to fuel neutrophils, and more generally are correlated with a greater degree of negative energy balance and perhaps heightened systemic inflammation, all of which are intertwined. In the whole animal, it is difficult to distinguish whether these markers are indictors of other complex processes, or NEFA or BHB might have direct effects on innate immune function. Several studies provide insight into this question, with inconsistent results.
Neutrophils were collected from 8 Holstein cows in mid-lactation and incubated with mixtures of NEFA to resemble concentrations from low to typical in the days after calving (2-fold increments from 0.06 to 1 mmol/L) to extremely high (2 mmol/L) (Scalia et al., 2006). Phagocytic function was not affected at any concentration of NEFA, and oxidative burst function was only affected at 2 mmol/L, where it was substantially increased, but in conjunction with massive neutrophil necrosis (49% vs. < 1% at lower concentrations), though not increased apoptosis. It is unclear if brief in vitro exposure of circulating neutrophils to moderately elevated concentrations of NEFA replicates the direct or indirect effects of maturation and circulation in the milieu of a peripartum cow with elevated blood NEFA concentrations, which likely also includes increased concentrations of pro-inflammatory cytokines and other modulators of neutrophil function. Ster et al. (2012) also mixed neutrophils from mid-lactation cows with a mixture of NEFA at 0, 0.1, 0.25, 0.5, or 0.75 mmol/L to mimic concentrations in early postpartum cows. They observed a dose-dependent reduction in oxidative burst function, with significant and substantial impairment at the two higher concentrations. They did not measure other aspects of neutrophil function with the spiked NEFA approach, but they did demonstrate inhibition of proliferation of peripheral blood mononuclear cells (PMBC) with NEFA as low as 0.13 mmol/L. Hammon et al. (2006) found a significant but modest correlation between in vivo plasma NEFA concentration and neutrophil oxidative burst activity in vitro (R2 = 0.2). Taken together, these studies support a possible direct and rapid effect of elevated concentrations of NEFA on the functional capacity of circulating neutrophils, but encourage more investigation into the mechanisms by which perhaps specific fatty acids modulate neutrophil functions and whether such effects are more relevant during maturation or in circulation.
Greater severity of clinical mastitis has been observed in cows with ketosis (Kremer et al., 1993). Hoeben et al. (1997) isolated neutrophils from 7 high-producing (presumably non-ketotic) cows and employed several assays to assess oxidative burst function in samples with BHB added at 0.01, 0.05, 0.1, 1.0, or 2.5 mmol/L. They showed impairment of generation of hydrogen peroxide specifically, through not of superoxide anion or myeloperoxidase activity, with BHB ≥ 1.0 mmol/L. In an experiment with only 3 to 6 cows per group with naturally-occurring differences in blood BHB concentration, neutrophil chemotaxis was reduced in cows with BHB > 1.6 mmol/L (Suriyasathaporn et al., 1999). Neutrophils from these same cows were then incubated with different combinations of ketone bodies (BHB alone at 1.0 or 4.8. mmol/L; acetoacetate or acetone alone; or a combination of all three at high or low concentrations). BHB alone did not consistently impair chemotaxis, but the other ketones and the combination of all did in all cases. This experiment has the advantage of using neutrophils that had natural exposure to ketotic cows (presumably not only elevated BHB, although the cows were at 5 to 10 weeks in lactation, so not the complex metabolic and endocrine milieu of the transition period). Ster et al. (2006; details above) demonstrated that adding BHB up to 1 mmol/L had no effect on blood mononuclear cell proliferation or interferon-γ production, and up to 10 mmol/L (i.e., extremely high) had no effect on neutrophil oxidative burst function. Similarly, Hammon et al. (2006) found no association of blood BHB concentrations with neutrophil killing ability. As with NEFA, the effects of ketone bodies are inconsistent, but there is sufficient evidence to suggest a potential effect on migration and killing functions of neutrophils. It is unclear if BHB alone is a sufficient cause of impaired neutrophil function.
Intracellular calcium signaling is a key element in the activation of neutrophils. Calcium acts as a second messenger for intracellular signal transduction for a variety of cell-surface receptors (Vig and Kinet, 2009). Neutrophils treated in vitro with EDTA, an extracellular calcium ion chelator, had severely reduced phagocytosis capacity (Ducusin et al., 2001). Therefore, hypocalcemia around calving may contribute to immune cell dysfunction. Neutrophils collected from cows with clinical milk fever had lower intracellular calcium concentrations and impaired phagocytosis compared to cows without parturient paresis (Ducusin et al., 2003). Kimura et al. (2006) isolated peripheral blood mononuclear cells (PMBC) from 27 cows through the transition period, 8 of which had developed clinical milk fever, and assessed the quantity and release of intracellular calcium stores. They showed that the stimulated flux of intracellular ionized calcium (iCa) was reduced at calving, and lower in cows that developed milk fever days from 12 days before the onset of clinical signs. Intracellular iCa response of PMBC was low at the time of milk fever, but doubled following treatment with intravenous calcium, indicating prompt response of PMBC intracellular iCa to increased plasma concentrations of total calcium. The releasable intracellular iCa store decreased before calving and was correlated with blood calcium concentration and with intracellular iCa flux in response to stimulation. The authors suggested that intracellular stores of iCa may be diminished before calving as there is next efflux of iCa in an attempt to maintain blood calcium concentration, likely contributing to impaired PMBC function by decreasing the magnitude of intracellular iCa flux available to activate cell function.
Recent work demonstrates the calcium ‘cost’ of mounting an inflammatory response to an acute lipopolysaccharide (LPS) challenge. Using a model analogous to Kvidera et al. (2017) for glucose, Horst et al., (2018) showed that in the 12 h after challenge with LPS, blood calcium concentration was reduced by 32%, and maintenance of eucalcemia during that time required infusion of 12 g of Ca, or somewhat more than the typical deficit (8 to 10 g) in a cow recumbent with milk fever.
Subclinical hypocalcemia is highly prevalent among periparturient cows and is associated with increased risk of displaced abomasum (Chapinal et al., 2011) and milk production losses (Chapinal et al., 2012) and increased culling risk in early lactation (Roberts et al., 2012). Cows classified at high risk for metritis (having one or more of dystocia, twins, stillbirth or retained placenta) that were able to maintain serum calcium concentrations above 2.15 mmol/L had one-half and one-third the incidence of metritis and puerperal metritis, respectively, when compared to low metritis risk cows that were below this cut-point at least once in the first 3 days postpartum (Martinez et al., 2012). That study showed reduced total circulating neutrophil number, neutrophil phagocytosis and neutrophil oxidative burst capacity in cows with blood calcium < 2.15mmol/L through the first 3 days postpartum. Based on the variables measured in the study, at least two-thirds of the cases of metritis were estimated to be attributable to having blood calcium below 2.15 mmol/L in the first 3 days postpartum. Even if that is an overestimate if more variables in more herds were considered, it points to sub-optimal calcemia contributing meaningfully to the occurrence of metritis, mediated at least in part by impairment of neutrophil function. 
The same research group explored this association through experimental induction of hypocalcemia with a 24-hour infusion of an selective iCa chelator (Martinez et al., 2014). They used 10 mature, nonpregnant, non-lactating cows in a crossover design. By about 4 h after the start of treatment, steady-state plasma concentrations of ~0.75 mmol/L iCa and ~1.75 mmol/L total calcium were maintained for 20 hours; therefore, the model replicated blood calcium levels in a hypocalemic (but not milk fever) cow in the day after calving. Feed intake (~ 5 vs. 10 kg DM/day), blood glucose (~4.2 vs 4.4 mmol/L), and insulin concentrations decreased and NEFA increased during treatment, so the effects of treatment may not all be directly attributable to calcium. Neutrophil phagocytosis and oxidative burst function decreased at the end of the infusion and continued to diverge negatively from the controls until 3 d after the end of the infusion. The data patterns for both measures of neutrophil function were similar to those in their field study (Martinez et al., 2012). Similar to Kimura et al (2006), experimentally-induced hypocalcemia decreased the stimulated intracellular iCa flux (Martinez et al., 2014). The data from the latter study support earlier experimental and observational data that transient (≤ 1 day) hypocalcemia contributes to impaired neutrophil function and consequently to disease risk.
Emerging data (McArt - see elsewhere in this issue) indicate that the pattern and duration of reduced blood calcium concentrations in the 4 days after calving are more predictive of disease risk and milk yield than the nadir concentration or single point measurements in the first 24 h after calving. This new approach to classifying hypocalcemia should be applied to study its effects on neutrophil function. 
We evaluated (Miltenburg et al., 2018) whether administration of an injectable calcium supplement product at time of calving increased neutrophil oxidative burst or phagocytosis capacity. Cows (n = 27) from 4 farms were blocked by parity and randomly assigned to receive either a commercial injectable calcium supplement or a placebo within 12 hours after calving and again 24 hours later. In a separate study with the same protocol (Miltenburg et al., 2016), treatment increased serum total calcium at 24 hours postpartum, conditional on calcium concentration before treatment. Total serum calcium concentration (tCa), neutrophil oxidative burst and neutrophil phagocytosis capacity were measured from coccygeal blood samples before and 72 hours after the first treatment. The study animals were 23 first parity heifers and 6 multiparous cows. There was no effect of treatment on oxidative burst or phagocytosis. Therefore, despite plasma calcium concentration being associated with neutrophil function as described above, this study does not support the ability of supplemental calcium, as given to low-parity parturient cows soon after calving, to improve oxidative burst or phagocytosis capacity of neutrophils.
Martinez et al. (2018) used 80 cows in a 2 × 2 factorial experiment of positive or negative (-130 mEq/kg DM) DCAD with different dietary sources of vitamin D fed for 4 weeks before calving to assess a variety of health outcomes. The negative DCAD treatment increased plasma iCa and tCa at calving and 1 d later. Regarding neutrophil function, there were no effects of treatments on phagocytosis, and no interactions of the effects of DCAD and source of vitamin D on neutrophil function. Overall, cows fed calcidiol had better oxidative burst function postpartum than those fed cholecalciferol. Among multiparous cows, there was a modest effect of the negative DCAD diet to improve phagocytosis function before calving and oxidative burst function after calving. Therefore, improving maintenance of calcium homeostasis through dietary prevention approaches holds some promise for support of neutrophil function.
There is a body of evidence to support that each of glucose supply, blood concentrations of calcium, NEFA, and BHB are associated with the overall capacity of neutrophil responses in dairy cows in the 1 to 2 weeks after calving. Most commonly, the aspect of neutrophil function reported to be affected relates to oxidative burst, although that may be skewed because it is the most studied. There are fewer assessments of migration capacity, and still little in the bovine on the role of neutrophils in downregulating inflammation after the initial response. Notably, none of the effects of these factors influencing neutrophil function has been consistent among studies. Furthermore, controlled studies of socially-competitive environments do not reproduce the effects on immune function or related clinical diseases that would be expected from empirical observations. These inconsistencies may be partially attributable to differences in the study populations or the methods of analysis of neutrophil function, but it seems more likely that the interactions of these known factors (and probably others) are the key determinants of effective innate immune function and inflammatory response. Future research should assess the interactions among markers of energy supply and metabolism, and of those with calcium supply, and investigate the effects of the timing and duration of these effects. It would also be particularly relevant in cattle to pursue the question of whether neutrophils behave or are regulated differently in infection, injury, and sterile inflammation (also known as metabolic inflammation). It would be of practical importance to understand whether glucose and calcium supply, or exposure to NEFA or BHB are critical during myelopoiesis, in circulation in blood, or both. The present state of scientific evidence is consistent with the notion that best management practices to support adaptive metabolism and prevent excessive negative energy balance or hypocalcemia should plausibly be beneficial for innate immune function in transition dairy cows. However, there are insufficient data to make specific recommendations that would consistently enhance neutrophil function or reduce the incidence of diseases understood to be consequences of impaired or dysregulated immune function.
Published in the proceedings of the Animal Nutrition Conference of Canada 2020. For information on the event, past and future editions, check out https://animalnutritionconference.ca/.

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Stephen LeBlanc
University of Guelph
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