In this study, postpartum dairy cows diagnosed with metritis, clinical endometritis, or subclinical endometritis had higher serum concentrations of adiponectin, TNF-α, IL-1β and IL-6 compared to normal cows. In addition, adiponectin concentrations were higher for cows with low BCS compared to cows with high BCS throughout the study period. Cows with persistent uterine inflammation had higher serum concentrations of adiponectin, TNF-α, IL-1β and IL-6 compared to normal and recovered cows. Furthermore, cows with metritis or clinical endometritis had lower or lost body condition compared to those with subclinical endometritis or normal cows during the study period. Additionally, cows with persistent uterine inflammation lost body condition.
In the early stages of inflammation, blood concentrations of proinflammatory cytokines are increased . Weight loss caused dramatic decreases in IL-6 and TNF-α and increased adiponectin [14, 30]. The physiology of appetite and feeding behavior is very complex and involves neuroanatomical, neurophysiological and neurochemical pathways. Evidence suggests that cytokines IL-1, IL-6, TNF-α and interferon suppress appetite. Conditions such as inflammation, infection and cancer are usually associated with increased cytokine production, have also been associated with anorexia and weight loss . In addition, in cows with lower body condition, it is reasonable to expect high adiponectin concentrations as a cause-effect relationship due to depletion of tissue fat depots. In the present study, there were higher concentrations of adiponectin, TNF-α, IL-1β and IL-6 in cows with uterine inflammatory conditions that also had lower BCS.
Circulating IL-6 concentrations were elevated in dairy cows before calving, but they decreased to baseline by 8 d after calving . During the postpartum interval, IL-6 is expressed in the bovine endometrium in a time-related manner, with a significant peak on Day 17 postpartum, perhaps representing a mucosal immune response in the uterus . In addition, Fischer et al. (2010) reported IL-6 mRNA expression was not influenced by inflammation between 21 and 27 d postpartum , and Galvão et al. (2011) reported increased IL-6 gene expression in the seventh week postpartum in cows with subclinical endometritis . It should be noted that in acute inflammation there is an increase peripheral cytokines in association with increased tissue cytokine expression. Interestingly, in the present study, IL-6 increased during Weeks 1 and 2 in cows with metritis, and during Weeks 4 and 5 in cows with clinical and subclinical endometritis. Further, the IL-6 also increased in persistently infected cows and cows with lower BCS. It is noteworthy that IL-6 has lipolytic activity . Perhaps increased IL-6 concentrations during disease conditions and during weight loss as caused lipolysis, which not only provided energy but also provided necessary adipokines to increase resistance to disease. In response, Toll-like receptors 2 and 4 in the adipocyte respond to bacterial pathogens [37, 38]. Cows that recover from a negative energy balance are able to restore body condition and have a more functional immune system. In contrast, increased IL-6 concentrations immediately after calving in this and other studies [32–35], supported the notion that altered cytokines mediated body condition loss, and body condition loss mediated alterations in anti- and pro-inflammatory cytokines which acted serially to prolong uterine inflammation in dairy cows.
Pro-inflammatory cytokines act on several targets that may exacerbate body condition loss of immunologically challenged animals. Muscle protein degradation and lipolysis are mediated by IL-1β, IL-6, and TNF-α. Furthermore, IL-1β inhibits the anabolic effects of insulin on skeletal muscle . Consequently, IL-1β, IL-6, and TNF-α are part of a network that links muscle protein degradation and lipolysis with hepatic acute phase protein synthesis . This clearly represents an integrated host response to inflammatory stimuli. Furthermore, many of the metabolic effects of peripheral immunological stress are mediated by the actions of cytokines in the brain. In that regard, IL-1β, IL-6, and TNF-α from activated leukocytes suppress the CNS, decrease appetite and feed intake, thereby causing weight loss. IL-1β, IL-6, and TNF-α were elevated in persistently infected cows and in cows with low BCS.
Interleukin 1β is secreted by mononuclear cells, including monocytes and macrophages, in response to infections . In the present study, serum concentrations were higher in cows with metritis, and clinical or subclinical endometritis compared to normal cows. It is known that IL-1β acts centrally to induce anorexia (by acting on neurotransmitters) and peripherally it inhibits gastric motility, gastric emptying, and gastric acid secretion . It also causes alterations in the endocrine system, including corticotrophin-releasing factor, cholecystokinin, glucagon, and insulin . These central and peripheral actions of leukocytic cytokines can decrease feed intake by more than 50% during the acute phase of the disease. Perhaps cytokine-mediated neuroendocrine changes have a direct effect in cows with metritis and overt systemic signs, and they have an indirect effect in cows with clinical or subclinical endometritis without systemic involvement.
The cytokine TNF-α is produced by immune, epithelial, glandular epithelial and endothelial cells in the stromal layer of the bovine uterus [43–45]; in addition to regulating immunologic, inflammatory or reparative responses, it also controls prostaglandin (PG) synthesis in the bovine endometrium . In the present study, serum TNF-α concentrations were higher in subclinical cows compared to normal cows, and higher in cows with metritis or clinical endometritis cows compared to those with subclinical endometritis or normal cows. PGF metabolites were higher in cows with postpartum uterine disorders [47–49], possibly due to TNF-α controlled endometrial PG synthesis. Galvão et al. (2011) evaluated modulation of TNF-α gene expression during the postpartum period in dairy cows and reported that expression was lower in endometritis cows compared to healthy cows 1 week postpartum; furthermore, there was a trend for significant interaction between endometritis and weeks postpartum on TNF-α gene expression . Perhaps cows in that study were undergoing body condition loss, which caused an adipocyte-induced increase in cytokines. In contrast, in the present study, TNF-α concentrations were decreased during first 5 weeks, but reached precalving concentrations during Weeks 6 and 7 in normal cows and those with subclinical endometritis, respectively, whereas TNF-α was increased during the first 3 weeks postcalving and reached precalving concentrations during Weeks 4, 5 and 6 in cows with metritis. Concentrations of TNF-α were reduced during Week 7 in cows with metritis or clinical endometritis.
Based on metabolic alterations triggered by acute inflammation, we inferred that circulating cytokines may induce weight loss. Several studies have shown an increase in adiponectin expression after weight loss [50, 51]. Weight loss may increase adiponectin receptor 1 (adipoR1) expression . The mRNA expression of adiponectin is negatively correlated with body mass index and expression of the pro-inflammatory cytokines IL-6 and TNF-α, demonstrating that postpartum weight loss is a clear shift in adipokine profile [50–53]. Cytokines rapidly produce several systemic effects if the infection becomes generalized and/or prolonged [54, 55]. In hepatocytes, cytokines up-regulate production and release of acute phase proteins . In addition, exogenous IL-1β and TNF-α did not induce anorexia individually, but did so when administered simultaneously [56–58] by causing neuroendocrine changes. Interestingly, the increased ACTH induced by lipopolysaccharide (LPS) or the combination of IL-1β and TNF-α, was inhibited by pretreatment with a monoclonal antibody to IL-6, suggesting the ACTH profile in serum induced by LPS is actually the collective result of at least three cytokines [56–58].
Leptin has significant influence on energy balance and both innate and adoptive immunity. Together with IL-1, IL-6 and TNF-α, leptin acts as acute phase reactant during inflammation [21–23]. In natural immunity, leptin stimulates chemotaxis, phagocytosis and release of oxygen radicals in PMNs. In adaptive immunity, leptin stimulates proliferation of native T cells and promotes the secretion of T helper 1 cytokines. Dairy cows experience dramatic changes in energy metabolism and feed intake during the periparturient period . Feed intake is reduced in the last trimester of pregnancy, and particularly during the last 2 to 3 weeks prepartum despite growing energy needs for fetus and mammary development. Generally, leptin is unlikely to contribute to reduced appetite at that time because plasma concentration decreases during the last 1 to 2 weeks preceding parturition. It remains possible that changes in plasma leptin at earlier times during pregnancy modulate maternal food intake. After parturition, the lower concentration of plasma leptin could promote a faster increase in voluntary feed intake . Studies have observed that the IGF-1 and leptin levels of metritis-affected cows tended to decline compared to normal cows, and remained very low for weeks during postpartum, and also their body condition loss was more severe . In this study the leptin concentrations were high in both metritis and clinical endometritis cows whereas IGF-1 concentrations were lower in other cow groups. Higher leptin levels in metritis and clinical endometritis cows in this study possibly explain reduced feed intake and loss in body condition. It should also be noted that the leptin concentrations decreased in cows with persistent uterine inflammatory condition and increased in normal and recovered cows during 2 to 3 weeks. The leptin concentrations reversed, they were decreased in normal and recovered cows and increased in persistently inflamed cows starting from 4 weeks postcalving. However, the leptin levels were remained high from weeks 1 to 5 in cows with high BCS.
The concentration of insulin remained high in normal and subclinical endometritis cows compared to clinical endometritis and metritis cows. In normal and subclinical endometritis cows, the insulin concentrations started to increase around 4 weeks, peaked at 5 weeks postpartum and decreased abruptly during 6 and 7 weeks postpartum, even though the change in insulin concentrations in clinical endometritis followed similar pattern as in normal and subclinical endometritis cows the concentration was lower than the normal and subclinical endometritis cows. The insulin level was lower in the metritis cows compared to other groups. However the insulin concentration increased at 3 weeks postcalving to the level as clinical endometritis and decreased abruptly to a lower concentration at 7 weeks. Cows with metritis that had continuous hyperketonemia during postpartum, the insulin level was decreased at first week postcalving starting from day 2 and the level of insulin was increased at 4 to 5 weeks to same concentrations in metritis cows . However in this study, the insulin concentration was decreased during 4 to 7 weeks post calving. It may be expected that hyperketonemic cows with severe clinical signs of uterine inflammatory conditions should have stronger evidence of insulin resistance as a result of an additional effect of cytokine production on insulin unresponsiveness. Severe inflammatory conditions like metritis and subclinical endometritis with intensive release of cytokines potentially further depress insulin secretion of pancreatic β-cells and whole-body insulin responsiveness in dairy cows. In the present study, cows with persistent uterine inflammation had lower insulin during the study period.
In the present study, concentrations of IGF-1 in normal and subclinical endometritis cows remained high, whereas in cows with clinical endometritis and metritis concentrations of IGF-1 were low. Additionally, cows with persistent uterine inflammation had lower IGF-1 during the study period. Therefore, the current study provided evidence for an ongoing inflammatory response in the uteri of cows with low IGF-1. An antagonistic relationship between the proinflammatory cytokines and IGF generally occurs during disease conditions . It is plausible that poor energy status indicated by low leptin and IGF-1 may therefore inhibit the ability of the cow to raise an effective immune response to the bacterial challenge after calving and also delay the general repair process within the endometrium, thus prolonging uterine involution. In comparison, cows with a positive energy balance or those that recovered from their energy deficit 2 weeks after calving, as evidenced by the increase in circulating IGF-1 concentrations, undergo uterine involution at a more rapid rate. In support of this, IGF may antagonize proinflammatory activity by decreasing expression of the IL-receptor and via suppression of cytokine signaling proteins .
The adipokines, insulin and IGF-1 were different between persistent and normal recovered cows during the study period. The insulin and IGF-1 were remained increased in cows with high BCS except for weeks 2, 3 and 4. The leptin levels were remained high from weeks 1 to 5 in cows with high BCS. It should be noted that there were BCS by uterine inflammatory condition, BCS by uterine inflammatory condition persistence categories and BCS by weeks in postpartum interactions indicating that BCS changed during the study period and that the BCS influenced the adipokines and metabolic biomarkers studied.
Uterine infections increase expression of mRNA transcripts in the endometrium that encode molecules associated with inflammation, such as the cytokines IL-1β and IL-6, and the chemokine IL-8 . The impact of Gram-negative infections on tissues and the immune defense against these bacteria are highly dependent on recognition of LPS by TLR4. The endometrial cells secrete cytokines and chemokines in response to the LPS of Gram-negative bacteria via TLR4/ MYD88-dependent pathways, and inhibition of MAPK3/1 counters the proinflammatory response . Adipose tissue contributes up to 35% of the circulating IL-6 and is critical to counter endometrial damage caused by pathogens where endometrial production of cytokines is limited . Impairment in endometrium cytokine production could be due to decreased production or failure of obtaining the necessary stimulation or the endometrium could produce inhibitors of proliferation [62–65]. In cows where endometrial production of cytokines is impaired, the cow might depend on cytokines from adipose tissue. However, postpartum dairy cows that have impairment in both endometrial and adipose tissue production of cytokines suffer from persistent uterine inflammatory condition (Figure 2).