Impacts of chronic and increasing lipopolysaccharide exposure on production and reproductive parameters in lactating Holstein dairy cows.

Lipopolysaccharide (LPS) administration causes immunoactivation, which negatively affects production and fertility, but experimental exposure via an acute bolus is unlikely to resemble natural infections. Thus, the objectives were to characterize effects of chronic endotoxemia on production parameters and follicular development in estrous-synchronized lactating cows. Eleven Holstein cows (169 ± 20 d in milk; 681 ± 16 kg of body weight) were acclimated to their environmental surroundings for 3 d and then enrolled in 2 experimental periods (P). During P1 (3 d) cows consumed feed ad libitum and baseline samples were obtained. During P2 (7 d), cows were assigned to continuous infusion of either (1) saline-infused and pair-fed (CON-PF; 40 mL/h of saline i.v.; n = 5) or (2) LPS infused and ad libitum fed (LPS-AL; Escherichia coli O55:B5; 0.017, 0.020, 0.026, 0.036, 0.055, 0.088, and 0.148 µg/kg of body weight/h i.v. on d 1 to 7, respectively; n = 6). Controls were pair-fed to the LPS-AL group to eliminate confounding effects of dissimilar nutrient intake. Infusing LPS temporally caused mild hyperthermia on d 1 to 3 (+0.49°C) relative to baseline. Dry matter intake of LPS-AL cows decreased (28%) on d 1 of P2, then progressively returned to baseline. Relative to baseline, milk yield from LPS-AL cows was decreased on d 1 of P2 (12%). No treatment differences were observed in milk yield during P2. Follicular growth, dominant follicle size, serum progesterone (P4), and follicular P4 and 17ß-estradiol concentrations were similar between treatments. Serum 17ß-estradiol tended to increase (115%) and serum amyloid A and LPS-binding protein were increased (118 and 40%, respectively) in LPS-AL relative to CON-PF cows. Compared with CON-PF, neutrophils in LPS-AL cows were initially increased (45%), then gradually decreased. In contrast, monocytes were initially decreased (40%) and progressively increased with time in the LPS-AL cows. Hepatic mRNA abundance of cytochrome P450 family 2 subfamily C (CYP2C) or CYP3A was not affected by LPS, nor was there a treatment effect on toll-like receptor 4 or LBP; however, acyloxyacyl hydrolase and RELA subunit of nuclear factor kappa B tended to be increased in LPS-AL cows. These data suggest lactating dairy cows become tolerant to chronic and exponentially increasing LPS infusion in terms of production and reproductive parameters.

Effects of continuous and increasing lipopolysaccharide infusion on basal and stimulated metabolism in lactating Holstein cows.

Experimental objectives of this study were to characterize the systemic and intracellular metabolic response to continuous lipopolysaccharide (LPS) infusion in mid-lactation Holstein cows (169 ± 20 d in milk; 681 ± 16 kg of body weight). Following 3 d of acclimation, cows were enrolled in 2 experimental periods (P). During P1 (3 d), cows were fed ad libitum and baseline data were collected. In P2 (8 d), cows were assigned to 1 of 2 treatments: (1) saline-infused and pair-fed (CON-PF; i.v. sterile saline at 40 mL/h; n = 5) or (2) LPS-infused and fed ad libitum (LPS-AL; Escherichia coli O55:B5 at 0.017, 0.020, 0.026, 0.036, 0.055, 0.088, 0.148, and 0.148 µg/kg of body weight per hour for d 1 through 8, respectively; n = 6). During P2, CON-PF cows were pair-fed to LPS-AL cows to eliminate confounding effects of dissimilar nutrient intake. Blood samples were collected on d 1 and 2 of P1 and d 1, 3, 5, and 7 of P2. Following the P2 d 7 a.m. milking, adipose tissue, skeletal muscle, and liver biopsies were collected for reverse transcription quantitative PCR and Western blot analysis. To assess whole-body nutrient trafficking, an i.v. glucose tolerance test (GTT) was performed following the a.m. milking on P2 d 8; 4 h after the GTT, cows received an epinephrine challenge. During P2, there were no treatment differences in circulating glucose. Relative to P1, CON-PF cows had or tended to have decreased plasma ß-hydroxybutyrate and insulin (29 and 47%, respectively) during P2, whereas neither variable changed in LPS-AL cows, leading to an overall increase in ß-hydroxybutyrate and insulin (41 and 140%, respectively) relative to CON-PF cows. Circulating nonesterified fatty acids were increased from d 1 to 3 and subsequently decreased from d 3 to 7 in cows from both treatments. Blood urea nitrogen gradually decreased in CON-PF cows and increased in LPS-AL cows from d 1 to 5 of P2, resulting in an overall 25% increase in LPS-AL versus CON-PF cows. In response to the GTT, the glucose and insulin area under the curve were increased 33 and 56%, respectively, in LPS-AL compared with CON-PF cows; changes reflective of whole-body insulin resistance. However, protein abundance of insulin signaling markers within muscle, liver, and adipose tissue were similar between treatments. There were no observable treatment differences in the glucose or nonesterified fatty acids response to the epinephrine challenge. No treatment differences were observed in hepatic mRNA abundance of key gluconeogenic or lipid export enzymes. In conclusion, chronic LPS exposure altered multiple parameters of basal and stimulated metabolism, but did not appear to affect the molecular machinery evaluated herein.

Disruption of female reproductive function by endotoxins.

Endotoxemia can be caused by obesity, environmental chemical exposure, abiotic stressors and bacterial infection. Circumstances that deleteriously impact intestinal barrier integrity can induce endotoxemia, and controlled experiments have identified negative impacts of lipopolysaccharide (LPS; an endotoxin mimetic) on folliculogenesis, puberty onset, estrus behavior, ovulation, meiotic competence, luteal function and ovarian steroidogenesis. In addition, neonatal LPS exposures have transgenerational female reproductive impacts, raising concern about early life contacts to this endogenous reproductive toxicant. Aims of this review are to identify physiological stressors causing endotoxemia, to highlight potential mechanism(s) by which LPS compromises female reproduction and identify knowledge gaps regarding how acute and/or metabolic endotoxemia influence(s) female reproduction.

Effect of chromium on bioenergetics and leukocyte dynamics following immunoactivation in lactating Holstein cows.

Activated immune cells are insulin sensitive and utilize copious amounts of glucose. Because chromium (Cr) increases insulin sensitivity and may be immunomodulatory, our objective was to evaluate the effect of supplemental Cr (KemTrace Cr propionate, 20 g/d; Kemin Industries Inc., Des Moines, IA) on immune system glucose utilization and immune system dynamics following an intravenous endotoxin challenge in lactating Holstein cows. Twenty cows (320 ± 18 d in milk) were randomly assigned to 1 of 4 treatments: (1) pair-fed (PF) control (PF-CON; 5 mL of saline; n = 5), (2) PF and Cr supplemented (PF-Cr; 5 mL of saline; n = 5), (3) lipopolysaccharide (LPS)-euglycemic clamp and control supplemented (LPS-CON; 0.375 µg/kg of body weight LPS; n = 5), and (4) LPS-euglycemic clamp and Cr supplemented (LPS-Cr; 0.375 µg/kg of body weight LPS; n = 5). The experiment was conducted serially in 3 periods (P). During P1 (3 d), cows received their respective dietary treatments and baseline values were obtained. At the initiation of P2 (2 d), either a 12-h LPS-euglycemic clamp was conducted or cows were PF to their respective dietary counterparts. During P3 (3 d), cows consumed feed ad libitum and continued to receive their respective dietary treatment. During P2, LPS administration decreased dry matter intake (DMI; 40%) similarly among diets, and by experimental design the pattern and magnitude of reduced DMI were similar in the PF cohorts. During P3, LPS-Cr cows tended to have decreased DMI (6%) relative to LPS-CON cows. Relative to controls, milk yield from LPS-challenged cows decreased (58%) during P2 and LPS-Cr cows produced less (16%) milk than LPS-CON cows. During P3, milk yield progressively increased similarly in LPS-administered cows, but overall milk yield remained decreased (24%) compared with PF controls. There were no dietary treatment differences in milk yield during P3. Circulating insulin increased 9- and 15-fold in LPS-administered cows at 6 and 12 h postbolus, respectively, compared with PF controls. Compared with LPS-CON cows, circulating insulin in LPS-Cr cows was decreased (48%) at 6 h postbolus. Relative to PF cows, circulating LPS binding protein and serum amyloid A from LPS-administered cows increased 2- and 5-fold, respectively. Compared with PF cows, blood neutrophil counts in LPS-infused cows initially decreased, then gradually increased 163%. Between 18 and 48 h postbolus, the number of neutrophils was increased (12%) in LPS-Cr versus LPS-CON cows. The 12-h total glucose deficit was 220 and 1,777 g for the PF and LPS treatments, respectively, but glucose utilization following immune activation was not influenced by Cr. In summary, supplemental Cr reduced the insulin response and increased circulating neutrophils following an LPS challenge but did not appear to alter the immune system's glucose requirement following acute and intense activation.

Intentionally induced intestinal barrier dysfunction causes inflammation, affects metabolism, and reduces productivity in lactating Holstein cows.

Study objectives were to evaluate the effects of intentionally reduced intestinal barrier function on productivity, metabolism, and inflammatory indices in otherwise healthy dairy cows. Fourteen lactating Holstein cows (parity 2.6 ± 0.3; 117 ± 18 d in milk) were enrolled in 2 experimental periods. Period 1 (5 d) served as the baseline for period 2 (7 d), during which cows received 1 of 2 i.v. treatments twice per day: sterile saline or a gamma-secretase inhibitor (GSI; 1.5 mg/kg of body weight). Gamma-secretase inhibitors reduce intestinal barrier function by inhibiting crypt cell differentiation into absorptive enterocytes. During period 2, control cows receiving sterile saline were pair-fed (PF) to the GSI-treated cows, and all cows were killed at the end of period 2. Administering GSI increased goblet cell area 218, 70, and 28% in jejunum, ileum, and colon, respectively. In the jejunum, GSI-treated cows had increased crypt depth and reduced villus height, villus height-to-crypt depth ratio, cell proliferation, and mucosal surface area. Plasma lipopolysaccharide binding protein increased with time, and tended to be increased 42% in GSI-treated cows relative to PF controls on d 5 to 7. Circulating haptoglobin and serum amyloid A concentrations increased (585- and 4.4-fold, respectively) similarly in both treatments. Administering GSI progressively reduced dry matter intake (66%) and, by design, the pattern and magnitude of decreased nutrient intake was similar in PF controls. A similar progressive decrease (42%) in milk yield occurred in both treatments, but we observed no treatment effects on milk components. Cows treated with GSI tended to have increased plasma insulin (68%) and decreased circulating nonesterified fatty acids (29%) compared with PF cows. For both treatments, plasma glucose decreased with time while ß-hydroxybutyrate progressively increased. Liver triglycerides increased 221% from period 1 to sacrifice in both treatments. No differences were detected in liver weight, liver moisture, or body weight change. Intentionally compromising intestinal barrier function caused inflammation, altered metabolism, and markedly reduced feed intake and milk yield. Further, we demonstrated that progressive feed reduction appeared to cause leaky gut and inflammation.

Molecular identification of an N-type Ca2+ channel in saccular hair cells.

We report new molecular evidence for the presence of an N-type (Ca(v)2.2, alpha1B) voltage-gated Ca(2+) channel in hair cells of the saccular epithelium of the rainbow trout. The Ca(v)2.2 amino-acid sequence shows 68% and 63% identity compared with chick and human Ca(v)2.2, respectively. This channel reveals features that are characteristic of an N-type Ca(2+) channel: an omega-conotoxin GVIA binding domain, G(betagamma) binding regions, and a synaptic protein interaction site. Immunohistochemical studies with a custom antibody show that immunoreactivity for the Ca(v)2.2 is concentrated in the basolateral and apical regions of hair cells. Whereas trout brain and saccular macula express an 11-amino-acid insert in the second G(betagamma) binding domain of the Ca(v)2.2 I-II loop, isolated hair cells appear not to express this variant. We constructed fusion polypeptides representing portions of the I-II loop, beta1 and beta2a auxiliary subunits, the II-III loop, and syntaxin, and examined their intermolecular interactions via immunoprecipitation and surface plasmon resonance. The I-II loop polypeptides bound both beta1 and beta2a subunits with a preference for beta1, and the II-III loop exhibited Ca(2+)-dependent syntaxin binding. We demonstrated syntaxin immunoreactivity near afferent endings in hair cells, at hair-cell apices, and in efferent endings on hair cells, the former two sites consistent with binding of syntaxin to Ca(v)2.2. The present molecular characterization of the Ca(v)2.2 channel provides novel biochemical evidence for an N-type channel in hair cells, and details molecular interactions of this channel that reflect hair-cell function, such as spontaneous activity and vesicular trafficking. The current work, to our knowledge, represents the first demonstration of a putative N-type channel in hair cells as documented by tissue-specific antibody immunoreactivity and hair-cell-specific cDNA sequence.