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 β-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.

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.

Bacterial infection of the uterus causes clinical endometritis in 15 to 20% of postpartum dairy cows and reduces fertility, even after the resolution of disease. However, it is difficult to disentangle the mechanisms linking reduced fertility with endometritis because cows have multiple confounding postpartum conditions. The aim of the present experiment was to develop an in vivo model of clinical endometritis in Holstein heifers using pathogenic Escherichia coli and Trueperella pyogenes. Estrous cycles of heifers were synchronized using a 5-d Co-Synch protocol, and subsequently received exogenous progesterone to elevate circulating progesterone at the time of uterine infusion. Endometrial scarification was performed before uterine infusion of live pathogenic Escherichia coli and Trueperella pyogenes, or sterile vehicle. Effects of infusion were evaluated by measuring rectal temperature, plasma haptoglobin, hematology, grading pus in the vaginal mucus, quantifying 16S rRNA in vaginal mucus, and transrectal ultrasonography. Bacterial infusion increased the median vaginal mucus to grade 2 by d 3 postinfusion, and to grade 3 from d 4 to 6 postinfusion. Control heifers maintained a median vaginal mucus grade ≤1 from d 1 to 6. Transrectal ultrasound revealed the accumulation of echogenic fluid in the uterus of heifers following bacterial infusion, which was absent in control heifers. Total 16S rRNA in vaginal mucus was elevated in bacteria-infused heifers compared with control heifers at d 5. Rectal temperature was increased in bacteria-infused heifers. Plasma haptoglobin, general health, and appetite did not differ between groups. As indicated by increased vaginal mucus grade after bacterial infusion and absence of systemic signs of illness, this model successfully induced symptoms resembling clinical endometritis in virgin Holstein heifers. The model allows the isolation of effects of uterine disease on fertility from confounding factors that can occur during the postpartum period in dairy cows.

Study objectives were to determine the effects of zinc (Zn) amino acid complex (Availa Zn, Zinpro Corporation, Eden Prairie, MN) on metabolism, biomarkers of leaky gut, and inflammation during and following heat stress (HS) and nutrient restriction. Crossbred gilts (n = 50; 50 ± 2 kg BW) were blocked by initial BW and randomly assigned to one of five treatments: 1) thermoneutral (TN) and ad libitum fed a control diet (TNCtl), 2) TN and pair-fed a control diet (PFCtl), 3) TN and pair-fed a Zn-supplemented diet (PFZn), 4) HS and ad libitum fed a control diet (HSCtl), and 5) HS and ad libitum fed a Zn-supplemented diet (HSZn). The study consisted of 3 experimental periods (P): during P1 (7 d), all pigs were fed their respective diets ad libitum and housed in TN conditions (20.84 ± 0.03 °C, 47.11 ± 0.42% relative humidity). During P2 (7 d), HSCtl and HSZn pigs were exposed to progressive cyclical HS conditions (27 to 30 °C, 41.9 ± 0.5% relative humidity), while TNCtl, PFCtl, and PFZn pigs remained in TN conditions and were fed ad libitum or pair-fed to their respective HSCtl and HSZn counterparts. During P3 (5 d; "recovery phase"), all pigs were housed in TN conditions and fed ad libitum. Pigs exposed to HS had overall increased rectal temperature, skin temperature, and respiration rate (0.33 °C, 3.76 °C, and 27 bpm, respectively; P < 0.01). Relative to TN controls, HS decreased ADFI and ADG (28 and 35%, respectively; P < 0.05), but these variables were unaffected by dietary treatment. Additionally, circulating insulin did not differ between HS and TN pigs (P = 0.41), but was decreased in PF relative to TN pigs (P < 0.01). During recovery, no differences were observed in rectal temperature or respiration rate across treatments, but HSZn pigs had decreased skin temperature relative to TN, PF, and HSCtl pigs (P < 0.01). During P3, no Zn effects were observed in production parameters; however, PF pigs had increased ADFI and ADG relative to TN and HS treatments (P < 0.01). During P3, circulating insulin was increased in pigs that were HS relative to TN and PF pigs (75%, P < 0.05). Interestingly, tumor necrosis factor alpha (TNFα) levels were decreased during P3 (P = 0.04) in Zn relative to Ctl-fed pigs. Circulating lipopolysaccharide-binding protein was not different among periods (P > 0.10). In summary, Zn reduced TNFα (regardless of HS), and the stimulatory effect of HS on insulin secretion is amplified during HS recovery.

Uterine infection is associated with infertility in women and dairy cows, even after the resolution of infection. However, the mechanisms causing this persistent infertility are unclear. Here, we hypothesized that induced endometritis in non-lactating dairy cows would reduce the developmental competence of oocytes. Non-lactating Holstein cows received an intrauterine infusion of endometrial pathogenic bacteria (Escherichia coli and Trueperella pyogenes; n = 12) or vehicle control (n = 11) on day 2 of the estrous cycle. Bacterial infusion increased expression of endometrial inflammatory mediators, and a mucopurulent discharge in the vagina confirmed the establishment of endometritis. Oocytes were collected by transvaginal ultrasound-guided ovum pickup on days 2, 24, 45, and 66 following infusion and subjected to in vitro fertilization and embryo culture. Bacterial infusion resulted in fewer cleaved oocytes developing to morulae compared to vehicle-infused controls (30.7 versus 45.0%), with the greatest effect observed in oocytes collected on day 24. Development to morula was inversely correlated with endometrial expression of IL6 on day 6. The expression of genes associated with embryo quality did not differ significantly between morulae from bacteria-infused and control cows. Artificial insemination 130 days after intrauterine infusion resulted in normal, filamentous embryos that produced interferon tau 16 days after conception in both infusion groups. This model of experimentally induced uterine infection successfully resulted in endometritis and a reduction in the proportion of oocytes that developed to morulae following in vitro fertilization. In conclusion, endometritis reduced the capacity of oocytes to develop to morulae.

Mechanisms underlying obesity-associated reproductive impairment are ill defined. Hyperinsulinemia is a metabolic perturbation often observed in obese subjects. Insulin activates phosphatidylinositol 3-kinase (PI3K) signaling, which regulates ovarian folliculogenesis, steroidogenesis, and xenobiotic metabolism. The impact of progressive obesity on ovarian genes encoding mRNA involved in insulin-mediated PI3K signaling and xenobiotic biotransformation [insulin receptor (Insr), insulin receptor substrate 1 (Irs1), 2 (Irs2), and 3 (Irs3); kit ligand (Kitlg), stem cell growth factor receptor (Kit), protein kinase B (AKT) alpha (Akt1), beta (Akt2), forkhead transcription factor (FOXO) subfamily 1 (Foxo1), and subfamily 3 (Foxo3a), microsomal epoxide hydrolase (Ephx1), cytochrome P450 family 2, subfamily E, polypeptide 1 (Cyp2e1), glutathione S-transferase (GST) class Pi (Gstp1) and class mu 1 (Gstm1)] was determined in normal wild-type nonagouti (a/a; lean) and lethal yellow mice (KK.CG-Ay/J; obese) at 6, 12, 18, or 24 weeks of age. At 6 weeks, ovaries from obese mice had increased (P < 0.05) Insr and Irs3 but decreased (P < 0.05) Kitlg, Foxo1, and Cyp2e1 mRNA levels. Interestingly, at 12 weeks, an increase (P < 0.05) in Kitlg and Kit mRNA, pIRS1Ser302, pAKTThr308, EPHX1, and GSTP1 protein level was observed due to obesity, while Cyp2e1 mRNA and protein were reduced. A phosphoramide mustard (PM) challenge increased (P < 0.05) ovarian EPHX1 protein abundance in lean but not obese females. In addition, lung tissue from PM-exposed animals had increased (P < 0.05) EPHX1 protein with no impact of obesity thereon. Taken together, progressive obesity affected ovarian signaling pathways potentially involved in obesity-associated reproductive disorders.

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Environmental conditions that impede heat dissipation and increase body temperature cause heat stress (HS). The study objective was to evaluate impacts of HS on the follicular phase of the estrous cycle. Postpubertal gilts (126.0 ± 21.6 kg) were orally administered altrenogest to synchronize estrus, and subjected to either 5 d of thermal-neutral (TN; 20.3 ± 0.5 °C; n = 6) or cyclical HS (25.4 - 31.9 °C; n = 6) conditions during the follicular phase preceding behavioral estrus. On d 5, blood samples were obtained, gilts were euthanized, and ovaries collected. Fluid from dominant follicles was aspirated and ovarian protein homogenates prepared for protein abundance analysis. HS decreased feed intake (22%; P = 0.03) and while plasma insulin levels did not differ, the insulin:feed intake ratio was increased 3-fold by HS (P = 0.02). Insulin receptor protein abundance was increased (29%; P < 0.01), but insulin receptor substrate 1, total and phosphorylated protein kinase B, superoxide dismutase 1, and acyloxyacyl hydrolase protein abundance were unaffected by HS (P > 0.05). Plasma and follicular fluid 17β-estradiol, progesterone, and lipopolysaccharide-binding protein concentrations as well as abundance of steroid acute regulatory protein, cytochrome P450 19A1, and multidrug resistance-associated protein 1 were not affected by HS (P > 0.05). HS increased estrogen sulfotransferase protein abundance (44%; P = 0.02), toll-like receptor 4 (36%; P = 0.05), and phosphorylated REL-associated protein (31%; P = 0.02). Regardless of treatment, toll-like receptor 4 protein was localized to mural granulosa cells in the porcine ovary. In conclusion, HS altered ovarian signaling in postpubertal gilts during their follicular phase in ways that likely contributes to seasonal infertility.