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The progression from gestation into lactation represents the transition period, and it is accompanied by marked physiological, metabolic, and inflammatory adjustments. The entire lactation and a cow's opportunity to have an additional lactation are heavily dependent on how successfully she adapts during the periparturient period. Additionally, a disproportionate amount of health care and culling occurs early following parturition. Thus, lactation maladaptation has been a heavily researched area of dairy science for more than 50 yr. It was traditionally thought that excessive adipose tissue mobilization in large part dictated transition period success. Further, the magnitude of hypocalcemia has also been assumed to partly control whether a cow effectively navigates the first few months of lactation. The canon became that adipose tissue released nonesterified fatty acids (NEFA) and the resulting hepatic-derived ketones coupled with hypocalcemia lead to immune suppression, which is responsible for transition disorders (e.g., mastitis, metritis, retained placenta, poor fertility). In other words, the dogma evolved that these metabolites and hypocalcemia were causal to transition cow problems and that large efforts should be enlisted to prevent increased NEFA, hyperketonemia, and subclinical hypocalcemia. However, despite intensive academic and industry focus, the periparturient period remains a large hurdle to animal welfare, farm profitability, and dairy sustainability. Thus, it stands to reason that there are alternative explanations to periparturient failures. Recently, it has become firmly established that immune activation and the ipso facto inflammatory response are a normal component of transition cow biology. The origin of immune activation likely stems from the mammary gland, tissue trauma during parturition, and the gastrointestinal tract. If inflammation becomes pathological, it reduces feed intake and causes hypocalcemia. Our tenet is that immune system utilization of glucose and its induction of hypophagia are responsible for the extensive increase in NEFA and ketones, and this explains why they (and the severity of hypocalcemia) are correlated with poor health, production, and reproduction outcomes. In this review, we argue that changes in circulating NEFA, ketones, and calcium are simply reflective of either (1) normal homeorhetic adjustments that healthy, high-producing cows use to prioritize milk synthesis or (2) the consequence of immune activation and its sequelae.
Early lactation is a unique physiological state in which nutrient consumption often does not meet maintenance and milk production costs, creating a negative energy balance (NEB;
). Milk energy output increases more rapidly than the increase in consumed energy. The magnitude of NEB varies, but nadir usually occurs within the first 10 DIM, and cows return to calculated positive energy balance between 30 and 100 DIM (
Effects of somatotropin and dietary calcium soaps of fatty acids in early lactation on milk production, dry matter intake, and energy balance of high-yielding dairy cows.
). To support milk synthesis during NEB, significant alterations in carbohydrate, lipid, protein, and mineral metabolism are implemented.
A thorough appreciation of how important glucose is to milk synthesis is required to understand why these changes (energetics in particular) occur. Glucose is the precursor for lactose synthesis, and lactose is the primary osmoregulator driving milk volume (
). During established lactation, hepatic glucose output is exquisitely orchestrated to precisely meet peripheral tissue (e.g., mammary, muscle, adipose, central nervous system) glucose requirements (
). However, inadequate feed intake during the periparturient period means that the contribution of diet-derived gluconeogenic precursors to hepatic glucose output is insufficient to meet the mammary gland's increasing requirement, as more than 90% of glucose made by the liver is utilized by the mammary gland in early lactation (
). Consequently, multiple tissues coordinate efforts in an attempt to compensate for the dietary shortage by becoming insulin resistant, a hormonal scenario that allows for tissue catabolism and mobilization of AA and glycerol (gluconeogenic precursors) from skeletal muscle and adipose tissue, respectively (
In addition to providing gluconeogenic building blocks, both adipose tissue and skeletal muscle coordinate metabolism during the transition period to increase the supply of and reliance upon lipid fuel. During NEB, somatotropin (increased during NEB;
) promotes nonesterified fatty acid (NEFA) export from adipose tissue by accentuating the lipolytic response to β-adrenergic signals and by blunting insulin-mediated lipogenesis and glucose utilization (
), which represent a substantial energy source for both peripheral tissues (skeletal muscle in particular) and the mammary gland. However, some tissues (i.e., the brain) and cell types are unable to oxidize NEFA and thus require the energy within fatty acids to be converted into ketones.
The exact mechanisms regulating all aspects of hepatic ketogenesis remain unclear, especially in ruminants (
). However, it is likely that 2 biochemical sequences of events partially control ketone production simultaneously. First, fatty acid β-oxidation generates large quantities of NADH and reduced flavin adenine dinucleotide (
), a scenario that presumably meets (in part) the hepatocyte's ATP requirements and thus decreases key tricarboxylic acid (TCA) enzymes (isocitrate dehydrogenase and ketoglutarate dehydrogenase). This would slow the cycle and create a buildup of acetyl CoA. Second is the salient explanation put forth by Sir Hans Krebs more than 55 yr ago. Ketone synthesis is enhanced when the TCA cycle intermediate oxaloacetate (OAA) supply is limited. Increased gluconeogenesis in early lactation causes cataplerosis (removal from the TCA cycle) of OAA to support phosphoenolpyruvate production (an early step in gluconeogenesis). Simultaneously, a large amount of acetyl CoA originates from β-oxidation of adipose-derived NEFA (
). When OAA is plentiful, it combines with acetyl CoA to make citrate, and the TCA cycle progresses. The unavailability of OAA is now the metabolic crossroad between carbohydrate and lipid metabolism, and accumulated acetyl CoA enters into ketogenesis (
). The aforementioned changes effectively partition glucose toward the mammary gland because glucose's contribution as a fuel source to extramammary tissues is markedly decreased (
). These metabolic adjustments essentially create a coordinated unidirectional glucose flow from the liver to the mammary gland. Ultimately, the normal homeorhetic adaptations described above empower “metabolic flexibility” (
). Eucalcemia is typically under tight homeostatic control via the action of the calcitropic hormones parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D. The parathyroid gland detects hypocalcemia and secretes PTH, which increases renal Ca reabsorption (i.e., reduces urinary Ca loss), increases osteocytic and osteoclastic bone Ca release, and stimulates renal production of 1,25-dihydroxyvitamin D (also known as calcitriol). Calcitriol acts synergistically with PTH at the kidney and bone and also increases active transport of dietary Ca across the intestinal epithelium (as reviewed by
). It has long been hypothesized that the mammary gland's sudden Ca demand is so extensive and acute that it often exceeds these homeostatic mechanisms, resulting in clinical or subclinical hypocalcemia (SCH;
In the 1980s, it was demonstrated that precalving metabolic alkalosis predisposed cows to milk fever via diminishing tissue responsiveness to PTH, and adding dietary anions markedly reduced the incidence of clinical milk fever (
). Mechanisms by which metabolic acidosis improves Ca homeostasis have not been fully elucidated but may include improved tissue responsiveness to PTH (
Calcium and magnesium physiology and nutrition in relation to the prevention of milk fever and tetany (dietary management of macrominerals in preventing disease).
Vet. Clin. North Am. Food Anim. Pract.2014; 30 (25245611): 643-670
). Implementing these dietary strategies has successfully reduced rates of clinical hypocalcemia; however, SCH remains common, afflicting ∼25% of primiparous and ∼50% of multiparous cows (
). Thus, the inability to strictly maintain Ca homeostasis continues to occur in the early postpartum period.
METABOLIC DISORDERS AND INFECTIOUS DISEASE: TRADITIONAL DOGMAS
Maintaining cow health and productivity during the transition period represents a significant obstacle to the dairy industry. Coinciding with the changes mentioned above in energetic and Ca homeostasis is an increased risk of metabolic disorders and infectious diseases such as ketosis, fatty liver, milk fever, displaced abomasum (DA), retained placenta (RP), mastitis, and metritis (
A field study to determine the prevalence, dairy herd management systems, and fresh cow clinical conditions associated with ketosis in western European dairy herds.
). Not surprisingly, a disproportionate amount of culling occurs early in lactation. This animal welfare issue has profound implications for farm profitability, the social license to operate, and industry sustainability.
Research characterizing periparturient disorders by alterations in a single circulating metabolite began as early as the 1920s. Milk fever was identified by decreased circulating Ca (
). Associations between increased NEFA, hyperketonemia, and hypocalcemia and the incidence of disease became a topic of intensive investigation beginning in the 1980s (
A field study to determine the prevalence, dairy herd management systems, and fresh cow clinical conditions associated with ketosis in western European dairy herds.
), and hypocalcemia was later considered a gateway disorder leading to ketosis, mastitis, metritis, DA, impaired reproduction, and decreased milk yield (
Epidemiology of subclinical hypocalcemia in early-lactation Holstein dairy cows: The temporal associations of plasma calcium concentration in the first 4 days in milk with disease and milk production.
Association of immediate postpartum plasma calcium concentration with early-lactation clinical diseases, culling, reproduction, and milk production in Holstein cows.
A common observational approach in the aforementioned research is to obtain blood samples from cows during the transition period and retrospectively classify them according to health status. Once retroclassified, differences in circulating metabolites, minerals, and hormones can be evaluated between groups (e.g., diseased vs. healthy, high vs. low performers, pregnant vs. open, high NEFA vs. low NEFA). Another common method is to simply correlate circulating variables with a performance metric or health variable. Despite not using traditional intervening or controlled experimentation, increased NEFA, hyperketonemia, and hypocalcemia are presumed to have a causal relationship with poor transition cow success (Figure 1;
Evaluation of nonesterified fatty acids and β-hydroxybutyrate in transition dairy cattle in the northeastern United States: Critical thresholds for prediction of clinical diseases.
Associations of prepartum plasma cortisol, haptoglobin, fecal cortisol metabolites, and nonesterified fatty acids with postpartum health status in Holstein dairy cows.
Figure 1Traditional mechanisms by which hypocalcemia and increased nonesterified fatty acids (NEFA) and hyperketonemia are thought to cause poor transition cow health and performance. DA = displaced abomasum; RP = retained placenta.
We believe that there are multiple flaws in the theory connecting NEFA, ketones, and Ca with negative outcomes in the postpartum dairy cow. In addition to not having causal substantiation and having limited biological plausibility, many of the theory's principles counter evolutionary adaptations associated with milk synthesis, reproduction, and species survival. Below, we outline the inadequacies of the rationale for causation and provide evidence demonstrating that changes in circulating NEFA, ketones, and Ca are not responsible for negative outcomes but rather are simply reflective of either normal metabolic changes that healthy cows enlist to achieve high production or the metabolic downstream consequences of immune activation-induced hypophagia.
Correlation Does Not Equal Causation
Causality and correlation are incorrectly interchanged when an observational relationship between 2 events is claimed to be inevitable rather than coincidental. Dozens of peer-reviewed articles have demonstrated an association between metabolites and transition cow problems, but importantly numerous inconsistencies exist. For example, a variety of papers indicate no relationship between NEFA, ketones, and Ca and negative outcomes (
Effect of trace mineral supplementation on selected minerals, energy metabolites, oxidative stress, and immune parameters and its association with uterine diseases in dairy cattle.
Elevated non-esterified fatty acid and β-hydroxybutyrate in transition dairy cows and their association with reproductive performance and disorders: A meta-analysis.
). The consistency of an effect is crucial when making causal inference from observational and field research. Second, as already mentioned, these tenets are largely based on associations and not cause-and-effect relationships garnered from controlled and intervening experimentation. Even from a relationship perspective, assessing the strength or robustness of the associations is difficult due to variability in analysis and statistical methods. In particular, different metabolite thresholds are set for different outcomes and time points (e.g., pre- vs. postpartum, wk 1 vs. wk 2) within observational studies. In addition, inconsistent association metrics (e.g., odds ratio, relative risk, hazard ratio) are used to assess the relationship. A partial summary of the association studies was recently compiled by
. Although these reports illustrate the large number of studies demonstrating a relationship of the metabolites (NEFA, BHB, Ca) with health and performance, they also indicate substantial variability in metabolite thresholds and association strength. For example, the association (as measured by odds ratios) between postpartum BHB and DA incidence ranged from 1.1 to 27.6 across studies (
). Interestingly, several reports demonstrated both a negative association of elevated NEFA and ketones with health outcomes and a positive association with milk yield (
Associations of elevated nonesterified fatty acids and β-hydroxybutyrate concentrations with early lactation reproductive performance and milk production in transition dairy cattle in the northeastern United States.
Genetic parameters of blood β- hydroxybutyrate predicted from milk infrared spectra and clinical ketosis, and their associations with milk production traits in Norwegian Red cows.
). The conflicting relationships described above exemplify the dogma's limitations and highlight the boundaries of retrospective classification and epidemiology. Additionally, emphasis on association metrics (e.g., odds ratios, relative risks) can lead to a non sequitur (
Arguably, the best line of evidence in support of the dogma is extrapolated from the purported role of elevated NEFA, hyperketonemia, and hypocalcemia in immunosuppression and its predisposing role in disease (
). For example, in vitro incubation of isolated circulating neutrophils with increasing NEFA and BHB concentrations negatively affects leukocyte function, such as neutrophil oxidative burst (
). Additionally, chemotaxis and myeloperoxidase activity were impaired in neutrophils isolated from periparturient cows with elevated NEFA and ketones (
Phagocytic response of bovine polymorphonuclear leukocytes to different incubation conditions and following exposure to some effectors of phagocytosis and different anticoagulants in vitro.
). Consequently, the metabolic and mineral profile dominating the periparturient period is presumed to adversely affect immune function, and the resulting immune suppression predisposes cows to a variety of disorders and diseases (
reported reduced neutrophil reactive oxygen species production but no change in neutrophil phagocytosis when incubated with increasing NEFA concentrations in vitro. Incidentally, most ex vivo research evaluating increasing NEFA concentrations on leukocyte function uses very low levels of albumin and thus are not replicating in vivo conditions. Similarly,
observed no difference in blood mononuclear cell proliferation or interferon-γ production with BHB concentrations ≥1.0 mmol/L and no effect on oxidative burst up to 10 mmol/L. Further, no relationship was observed between BHB concentrations and neutrophil killing ability (
). Rodent studies have even shown that ketone bodies may have a protective effect and limit reactive oxygen species-induced damage during bacterial inflammation (
). In addition to the aforementioned discrepancies, extending in vitro results to whole-animal biology has obvious limitations, and this is especially pertinent when considering the immune system. For example, most leukocyte function is integrally dependent on an intracellular metabolic shift from oxidative phosphorylation to aerobic glycolysis (discussed below;
), and it is highly unlikely that in vitro conditions can mimic the extracellular endocrine and energetic milieu accompanying normal immune activation. Additionally, we now realize that almost all periparturient dairy cows (even the seemingly healthy ones) experience some degree of immune activation and inflammation (discussed more below;
). This is particularly important when considering neutrophils because they continue to mature while in circulation, and this aging can affect their functional properties (
). Even more concerning is that inflammation causes the bone marrow to release immature and incompetent neutrophils, including neutrophil progenitor cells (
), and this would very likely influence ex vivo neutrophil function metrics. Consequently, it is not clear whether ex vivo function assays during the transition period reflect immunosuppression or simply the pathology and leukocyte footprint associated with normal immune activation. In other words, some arms may appear immunosuppressed, whereas others are activated. Continued research into the immune system consistently reveals how little we know, how complex the interactions are (especially with metabolism), and how oversimplified our interpretation may have been.
NEB and BW Loss During Lactation Are Normal
Adipose tissue mobilization to support lactation is a highly conserved response (
). Interestingly, in certain mammals such as bears, seals, dolphins, and baleen whales (i.e., the blue whale), lactation occurs concurrently with a prolonged fast; consequently, these mammals rely almost entirely on adipose tissue reserves to meet their energy demands (
). In fact, baleen whales will sustain a 6- to 7-mo lactation without eating and will mobilize ∼33% of their fat stores, which is equivalent to 16 tons of BW (
). Evolutionarily closer to the cow, deer go through periods of insufficient intake after parturition and rely on reserves to support lactation, even during ad libitum feeding (
). Regardless, the species-conserved reliance on NEFA to support lactation further exemplifies the importance of this strategy. In fact, the extent to which cows incorporate adipose tissue mobilization during early lactation pales compared with many other species (
in: Wiseman J. Bradley R. Yield of Farmed Species: Constrains and Opportunities in the 21st Century. Proc. 61st Easter School, Nottingham, England. Nottingham University Press,
2005: 351-377
). Consequently, interpreting BW loss and tissue mobilization outside the bounds of proper biological context could lead to a pessimistic judgment.
NEFA and BHB Do Not Directly Inhibit Feed Intake
Regulation of feed intake is an extremely complex topic, exemplified by the fact that pharmaceutical interventions to reduce human caloric consumption have yet to be successful. Theories attempting to explain ruminant appetite control include energy requirements (
). Pertinent to this review, the detrimental effects of elevated NEFA and hyperketonemia on health and performance are partially attributed to their alleged suppressive effect on feed intake (
Feeding- and management related diseases in the transition cow: Physiological adaptations around calving and strategies to reduce feeding related diseases.
Performance and metabolic and endocrine changes with emphasis on glucose metabolism in high-yielding dairy cows with high and low fat content in liver after calving.
). This is an especially prevalent mindset in veterinary medicine as clinicians often anecdotally claim that ketones depress periparturient cow feed intake. However, this purported effect is largely based on association (see above) and is in contrast with the normal biology accompanying a healthy and successful transition (high circulating NEFA and BHB). Furthermore, results of several infusion studies suggest appetite is largely unaffected by ketones and lipids. In an elegant series of controlled experiments, it was demonstrated that intravenous BHB infusion did not affect feed intake (
). This type of experimentation needs to be interpreted within homeostatic and homeorhetic context because administering a fuel would intuitively decrease energy consumption when the animal is in positive energy balance (
). Regardless, from an evolutionary perspective, it is bioenergetically difficult to hypothesize why NEFA and BHB would decrease appetite. Adipose tissue mobilization and partial conversion of NEFA into ketones is a key metabolic strategy animals use to conserve skeletal muscle and ultimately survive NEB (
). The importance of ketogenesis to surviving malnutrition is highlighted by the fact that mutations in the gene regulating ketone synthesis (mitochondrial HMG-CoA synthetase) result in hypoglycemic-induced coma within days (
). Thus, even the simplest of life forms have been utilizing these basic and uncomplicated ancient fuels (NEFA and ketones) since the beginning of time. If NEFA and ketones actually blunted the urge to eat, a starving animal would be anorexic, a scenario that would hasten their demise. In summary, animals have ebbed and flowed into and out of NEB (because of, e.g., food insecurity, hibernation, migration, and lactation) for eons, and oxidizing NEFA and ketones is absolutely essential to survival.
High-Producing Cows Are Hypoinsulinemic
A key strategy (maybe the most integral part) to successfully initiating lactogenesis and sustaining galactopoiesis is the development of insulin resistance in both skeletal muscle and adipose tissue and the decrease in pancreatic insulin secretion (
). As already mentioned, this allows adipose tissue mobilization and the exiting NEFA to be used by most cell types and tissues as a way to spare glucose for milk synthesis. Thus, it is not surprising that (1) higher producing cows are more hypoinsulinemic than their lower producing herdmates throughout lactation (
Endocrine control of energy metabolism in the cow: Comparison of the levels of hormones (prolactin, growth hormone, insulin, and thyroxine) and metabolites in the plasma of high- and low-yielding cattle at various stages of lactation.
Changes of mammary vein concentrations of glucose and free fatty acids induced by exogenous insulin and glucose, and relation to mammary gland function in Saanen goats.
Although not directly focusing on insulin per se, evaluating how feeding controlled-energy diets (low-quality forage) before calving affects energetic metabolism and production provides additional conceptual framing on how important metabolic flexibility is to normal lactation. Prepartum low-energy diets successfully reduced postcalving NEFA, ketones, and liver fat content, but this was unsurprisingly accompanied by a substantial reduction in ECM or FCM yield (
). Additionally, regardless of diet, cows that had increased postcalving circulating ketones (1.2–2.9 mmol/L) produced more milk (>3 kg/d) than cows whose ketone concentrations were considered healthy (<1.2 mmol/L;
Relationships between body condition score change, prior mid-lactation phenotypic residual feed intake, and hyperketonemia onset in transition dairy cows.
). Clearly, mobilizing adipose tissue and converting NEFA into ketones is a physiological adaptation that mammals utilize to prioritize milk synthesis, and attempts to blunt or intervene with this homeorhetic process predictably come at the expense of milk yield.
The Confusing Insulin Status of Ketosis
Given insulin's incredibly potent regulation of intermediary metabolism, high milk production associated with associated with excessive adipose tissue mobilization-induced ketosis should be accompanied by severe hypoinsulinemia (
). This is a peculiar pathological endocrine profile as insulin would normally prevent ketosis on multiple levels: (1) blunting adipose tissue mobilization, (2) reducing hepatic gluconeogenesis and thus minimizing depletion of the TCA cycle's OAA pool, (3) decreasing fatty acid transport into the mitochondria via carnitine palmitoyltransferase 1 (CPT1) downregulation, (4) negatively governing the rate-limiting enzyme of ketone synthesis (HMG-CoA synthase), and (5) increasing peripheral tissue ketone utilization (
). Incidentally, despite inappetence, immune activation is also characterized by acute hyperinsulinemia (discussed below). As a result, there are numerous metabolic and endocrine footprints clearly associated with ketosis, a controversial concept originally proposed by
Given hyperketonemia's purported crucial role in transition cow pathophysiology, it stands to reason that clinical intervention should increase productivity. In fact, administering propylene glycol to subclinical hyperketonemic cows did increase milk yield in some instances (
Effects of propylene glycol supplementation on blood indicators of hepatic function, body condition score, milk fat-protein concentration and reproductive performance of dairy cows.
GplusE Consortium Only few benefits from propylene glycol drench in early lactation for cows identified as physiologically imbalanced based on milk spectra analyses.
). Explanations for the inconsistencies are not clear; however, one explanation for a positive effect may be that the additional endogenous glucose produced with propylene glycol administration temporarily alleviated the glucose burden of a transition dairy cow that is simultaneously inflamed. A reason for not observing an effect on milk yield is that the cows were healthy and the hyperketonemia was a crucial adjustment they were using to prioritize milk synthesis. Additionally, ketones blunt adipose tissue mobilization (in a negative feedback loop;
); therefore, therapeutically reducing ketones during subclinical ketosis could do more harm than good. Regardless, the collective body of evidence does not fully support the notion that medically treating hyperketonemia benefits milk synthesis. Incidentally, using steroids as part of a regimen to remediate ketosis needs a thorough re-examination, considering their role in immunosuppression.
In summary, transition cow health problems, suboptimal milk production, premature culling, and poor reproduction remain key hurdles to profitable dairy farming. During the last 50 yr, dairy scientists have increasingly viewed elevated circulating NEFA and ketones and hypocalcemia as pathological and causal toward negative outcomes. This tenet is largely based on observational studies, epidemiology, correlations, and ex vivo immune cell function assays. However, it is becoming more evident that periparturient diseases and disorders cannot be explained by the severity of changes in these simple metabolites. Interpreting biomarkers as causal agents of metabolic disorders deviates from the purpose of epidemiological studies. We believe that the postcalving changes to energetic and Ca metabolism reflect normal biological processes that healthy cows use to maximize milk synthesis or severe dysregulation of these processes arising from inflammation-induced changes enlisted to prioritize health (Figure 2).
Figure 2Potential downstream consequences of immune activation. In this model, decreased feed intake, hypocalcemia, excessive nonesterified fatty acids (NEFA), hyperketonemia, and hepatic lipidosis are not causative of poor transition cow performance and health but rather reflect prior immune stimulation. DA = displaced abomasum.
). Immune activation appears to be a double-edged sword, as a proper amount is required to healthfully navigate the periparturient period. In part, an active immune system is a normal constituent of dry-off and parturition arising from nonpathogenic sources such as tissue damage and remodeling (i.e., sterile homeostatic inflammation). Examples include mammary gland involution (
). In these situations, the immune system is activated via molecular patterns of nonpathogenic origin with the primary goal of remodeling tissue to support a new physiological state. It is unclear how much these nonpathogenic sources of inflammation contribute to systemic inflammation observed in poorly transitioning dairy cows. Cows are exposed to a myriad of physiological, environmental, and psychological stressors between dry-off and the early postpartum period that disrupt barrier integrity at epithelial interfaces (e.g., uterine, mammary, intestinal, and lung), which are constantly exposed to pathogens and colonized by commensal microorganisms. When microorganisms breach the epithelial barrier, underlying immune cells and tissues react quickly to prevent further infection. Immune cells respond after recognizing pathogen-associated molecular patterns (PAMP) via pathogen recognition receptors (PRR). These PRR are present on leukocytes and other cells, including adipocytes (
Grain challenge affects systemic and hepatic molecular biomarkers of inflammation, stress, and metabolic responses to a greater extent in Holstein than Jersey cows.
Regional and age dependent changes in gene expression of Toll-like receptors and key antimicrobial defence molecules throughout the gastrointestinal tract of dairy calves.
). Immune activation can be experimentally modeled via administrating LPS, the antigenic component of gram-negative bacteria, which is recognized by the toll-like receptors (TLR;
). Other models utilizing specific pathogens or PAMP also exist (i.e., live bacteria, lipoteichoic acid), and much of what we know about immune system effects on metabolism stem from these well-controlled and repeatable models. However, it is important to remember that the source of inflammation underlying these responses in practical situations arises from a wide variety of immunogenic and pathogenic components at 3 prominent sources in the transition cow: the uterus, mammary gland, and gastrointestinal tract.
Sources of Pathogenic Inflammation in the Transition Cow
Uterus
Bacteria present within the uterine lumen were originally thought to originate exclusively from contamination with environmental pathogens during and after parturition (
). Both bacteria adapted to the uterus (part of the existing microbiome before parturition) and bacteria originating from the environment contribute to metritis (
). Infiltration of environmental microorganisms is restricted by anatomical barriers, including the vulva, vagina, and cervix; however, dilation of these structures during and after parturition reduces their ability to prevent pathogen entry. Tight junction (TJ) proteins connect adjacent uterine epithelial cells separating the apical and basolateral components of the endometrium and prevent bacteria from penetrating the underlying stroma (
). Interestingly, both apical and basolateral PRR activation triggers cytokine secretion apically, and this aids in immune cell recruitment to the infection site (
During parturition, the protective uterine epithelium is often physically injured. Damaged or dying cells release damage-associated molecular patterns, which activate the immune system (independently of bacterial infiltration) to help clear unhealthy tissue. This damaged tissue creates an opportunity for bacteria to access the underlying stroma. Bacterial infiltration of the stroma induces cell damage and cytolysis, stimulating further release of damage-associated molecular patterns (
). As alluded to above, the act of parturition independently triggers inflammation, and the severity of dystocia likely predisposes cows to a higher risk of pathogen entry into local and systemic circulation. In summary, both the act of parturition and bacterial contamination can contribute to local and systemic inflammation in dairy cows.
Mammary Gland
The mammary gland is highly susceptible to bacterial infections, making it a prominent source of pathogen infiltration in the transition period. Intramammary infections are most prevalent during early involution (i.e., dry-off) and colostrogenesis (
). Abrupt milking cessation at dry-off engorges the udder with milk, increasing intramammary pressure and disrupting physical defense mechanisms within the streak canal (i.e., the keratin plug;
). Regardless, bacterial infections often remain quiescent throughout the dry period and clinical disease is not observed until the periparturient period (
). Interestingly, a previous report estimated that approximately 65% of early-lactation clinical coliform mastitis cases originated during the dry period (
Bovine mammary epithelial cells synthesize and secrete milk while simultaneously maintaining a semipermeable barrier between blood and milk components. Integrity of the blood–milk barrier is reliant on TJ proteins, which connect adjacent epithelial cells (
). Lipopolysaccharide, released during gram-negative bacterial proliferation within the teat and gland cistern, is recognized by resident leukocytes and mammary epithelial cells via TLR4 (
). Proinflammatory cytokines, produced in response to TLR4 activation, signal recruitment of effector leukocytes into the mammary gland and disrupt TJ integrity (
). Altogether, these changes can disrupt the blood–milk barrier, resulting in systemic inflammation and potentially both endotoxemia and bacteremia; this occurs in an alarming number of gram-negative bacterial infections (
). Consequently, the mammary gland is a likely culprit in immune activation both after and before parturition.
Gastrointestinal Tract
The intestinal epithelium serves a dual purpose of nutrient absorption and protection from pathogens and other antigens present within the gastrointestinal tract. The importance of proper barrier function cannot be overstated as the intestine is continuously exposed to potential pathogens and toxins and has an enormous surface area (∼400 m2 in humans;
). Microbial exposure is certainly more extensive in ruminants due to pregastric fermentation and the relative size of the alimentary tract. The stratified squamous epithelium lining the reticulorumen and omasum is composed of 4 distinct strata that serve both metabolic and barrier integrity roles. In contrast to the reticulorumen and omasum, the lower gut is composed of a simple columnar epithelium, which consists of both absorptive epithelial cells and a myriad of immune-related cells with extensive defense mechanisms to protect the epithelial barrier (the intricate details of which are reviewed by
Early weaning stress induces chronic functional diarrhea, intestinal barrier defects, and increased mast cell activity in a porcine model of early life adversity.
). In response to stress, the hypothalamic-pituitary-adrenal axis is activated, which in turn stimulates nervous system and peripheral tissue production of corticotropin-releasing factor (CRF) and subsequent release of adrenocorticotropin hormone from the anterior pituitary gland (
). Receptors for CRF are widely expressed in both the central and peripheral nervous system, where they interact with enteric neurons and epithelial immune cells (
). The negative consequences of CRF on the epithelium seem to be mediated by intestinal resident mast cell degranulation and release of histamine, proteases, and cytokines, which negatively affect intestinal barrier function (
). In addition to hypothalamic release, CRF is produced and released by intestinal cells (including immune and enterochromaffin cells), and the localized production can also affect intestinal epithelial function (
). Stress-mediated effects on the gut barrier may explain why so many seemingly unrelated situations (e.g., heat stress, cold stress, weaning, acidosis, feed restriction) share a common consequence of leaky gut and systemic inflammation.
Hepatic Response to Inflammation
The liver is the first organ to filter blood from the portal-drained viscera, intimately tying it with any gut-derived inflammatory challenges. During inflammation, the liver shifts priority from metabolism to defense as it is a critical organ in the immune response. This change is known as the acute phase protein (APP) response, and it involves reduced synthesis of proteins integral in normal liver metabolism (e.g, albumin, cholesterol, retinol-binding protein, transferrin, and paraoxonase) and increased synthesis of proteins, which aid in the immune and detoxification response (
); circulating positive APP increase in response to inflammation, whereas negative APP concomitantly decrease. In coordination with APP production, the liver plays a key role in detoxifying bacterial components and excreting them via bile. Interestingly, more than 60% of intravenously infused bacteria are hepatically sequestered within 10 min of infusion (
) and can be further classified as minor, moderate, or major depending on the magnitude of increase observed following immune activation. Common positive APP evaluated in ruminants include serum amyloid A (SAA), haptoglobin (Hp), and LPS-binding protein (LBP;
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