Gastrointestinal permeability and inflammatory status of preweaning dairy calves in response to decreasing the ratio of n-6 to n-3 fatty acid in milk replacer.

The ratio of n-6 to n-3 fatty acid (FA) is between 2 and 10 times higher in milk replacer (MR) than in whole milk, which may promote inflammation and compromise the integrity of the intestinal epithelium. To evaluate how decreasing the n-6:n-3 FA ratio of MR affects gastrointestinal (GIT) permeability and inflammatory status, 30 dairy calves (2.8 ± 1.06 d of age; mean ± standard deviation) were randomly assigned to be fed an MR with an n-6:n-3 FA ratio of 40:1 (CON; 29.3% crude fat of DM; n = 15) or 6.5:1 (n-3; 29.1% crude fat of DM; n = 15). Calves were fed 7.0 L/d in 2 meals. Calves were weighed and fecal consistency was analyzed weekly. On d 22, calves were administered Cr-EDTA, lactulose, and d-mannitol to assess GIT permeability. Blood and total urine were sequentially collected for 6 and 24 h, respectively, and analyzed for marker content. Whole blood collected 4 h after the meal was subjected to an ex vivo lipopolysaccharide (LPS) challenge to evaluate cytokine secretion from blood cells. Calves were euthanized on d 25 for collection of intestinal tissue samples. Tissue samples were processed to assess FA composition by gas chromatography, histomorphology by bright-field microscopy, and gene expression of tight junction proteins, lipid metabolism enzymes, and immune molecules by real-time quantitative PCR. Data were analyzed using PROC GLIMMIX in SAS (version 9.4, SAS Institute Inc.). Growth performance and fecal consistency were unaffected. Calves fed MR with a lower ratio of n-6 to n-3 FA had 2-fold higher n-3 FA contents and 2-fold lower ratios of n-6 to n-3 FA in proximal jejunum and ileum tissues. Total urinary recovery (0-24 h relative to marker administration) and plasma concentrations of the markers were unaffected. Expression of TJP1 tended to be higher in proximal jejunum tissue and lower in ileum tissue of n-3 calves. The expression of TLR4 and TNFA tended to be higher and CD14 was higher in ileum tissue of n-3 calves. Plasma concentrations of interleukin-4 were decreased in response to the ex vivo LPS challenge in n-3 calves. Histomorphology and GIT permeability were largely unaffected by treatment. Furthermore, the inclusion of linseed and algae oil may promote inflammation, as suggested by greater concentrations of the acute-phase proteins haptoglobin and serum amyloid A postprandially, demonstrating that fat sources should be evaluated for their suitability for MR formulations. Understanding how MR composition affects dairy calf health may improve nutritional strategies on farm.

High dietary Ca and microbial phytase reduce expression of Ca transporters while enhancing claudins involved in paracellular Ca absorption in the porcine jejunum and colon.

Expression levels of genes (RT-qPCR) related to Ca and P homeostasis (transporters and claudins (CLDN)) were determined in porcine jejunal and colonic mucosa. Forty growing pigs (BW 30.4±1.3 kg) received a low and high Ca content (2.0 and 9.6 g/kg, respectively) diet with or without microbial phytase (500 FTU/kg) for 21 days. Dietary Ca intake enhanced serum Ca and alkaline phosphatase concentration and reduced P, 1,25(OH)2D3, and parathyroid hormone concentration. Jejunal TRPV5 mRNA expression was decreased (32%) with phytase inclusion only, while colonic transient receptor potential vanilloid 5 (TRPV5) mRNA was reduced by dietary Ca (34%) and phytase (44%). Both jejunal and colonic TRPV6 mRNA expression was reduced (30%) with microbial phytase. Calbindin-D9k mRNA expression was lower in colonic but not jejunal mucosa with high dietary Ca (59%) and microbial phytase (37%). None of the mRNAs encoding the Na-P cotransporters (NaPi-IIc, PiT-1, PiT-2) were affected. Jejunal, but not colonic expression of the phosphate transporter XPR1, was slightly downregulated with dietary Ca. Dietary Ca downregulated colonic CLDN-4 (20%) and -10 (40%) expression while CLDN-7 was reduced by phytase inclusion in pigs fed low dietary Ca. Expression of colonic CLDN-12 tended to be increased by phytase. In jejunal mucosa, dietary Ca increased CLDN-2 expression (48%) and decreased CLDN-10 (49%) expression, while phytase slightly upregulated CLDN-12 expression. In conclusion, compared to a Ca deficient phytase-free diet, high dietary Ca and phytase intake in pigs downregulate jejunal and colonic genes related to transcellular Ca absorption and upregulate Ca pore-forming claudins.

The effect of adjuvant chemotherapy on survival in patients with FIGO stage I high-grade serous ovarian cancer.

OBJECTIVE: The benefit of adjuvant chemotherapy for FIGO stage I, high-grade serous ovarian cancer (HGSOC) after optimal staging is a matter of debate. We investigated the effect of adjuvant chemotherapy on recurrence-free survival (RFS) and overall survival (OS) in a population-based cohort study. METHODS: All patients diagnosed in the Netherlands between 2002 and 2014 with FIGO stage I HGSOC who underwent surgical staging were included. Data on clinical characteristics, histopathology, completeness of staging and survival were collected from the Netherlands Cancer Registry and Dutch Pathology Registry. Recurrence data was collected from hospital files. We used Kaplan-Meier methods to estimate RFS and OS and Cox-proportional hazard analyses to control for differences in baseline characteristics between patients who did or did not receive chemotherapy. RESULTS: We identified 223 patients who underwent optimal staging procedures including lymph node sampling. Events of disease recurrence occurred in 21 of the 101 patients (21%) who received adjuvant chemotherapy and in 46 of the 122 patients (38%) who did not (multivariable hazard ratio (HR), 0.37; 95%CI 0.22-0.64; p < 0.01). Five-year RFS was 81% after staging plus chemotherapy and 59% after staging only. At a median follow-up of 105 months, 21 patients (21%) in the chemotherapy group and 38 patients (31%) in the no-chemotherapy group had died (multivariable HR 0.50; 95%CI 0.28-0.89; p = 0.02). Ten-year OS was 78% with chemotherapy and 62% without chemotherapy. CONCLUSIONS: Adjuvant chemotherapy improves long-term RFS and OS in patients with FIGO stage I HGSOC after optimal staging.

Short communication: Relationship between lysine/methionine ratios and glucose levels and their effects on casein synthesis via activation of the mechanistic target of rapamycin signaling pathway in bovine mammary epithelial cells.

The synthesis of protein requires the availability of specific AA and a large supply of energy in bovine mammary epithelial cells (BMEC). Whether an interaction exists between Lys/Met ratio and glucose level on milk protein synthesis and its potential regulatory mechanism is unclear. We investigated the effects of different Lys/Met ratios and glucose levels on casein synthesis-related gene expression in BMEC to elucidate the underlying molecular mechanisms. Primary BMEC were subjected to 4 treatments for 36 h, arranged in a 2 × 2 factorial design with Lys/Met ratios of 3:1 (1.2:0.4 mM, LM3.0; total AA = 8.24 mM) and 2.3:1 (1.4:0.6 mM, LM2.3; total AA = 8.64 mM) and glucose levels of 17.5 mM (high glucose level) and 2.5 mM (low glucose level). No interactions between Lys/Met ratio and glucose level on cell viability, cell cycle progression, mRNA, or protein expression levels were found. High glucose level increased cell proliferation and promoted cell cycle transition from intermediate phase (G1 phase) to synthesis (S phase) by approximately 50%, whereas Lys/Met ratio had no effect. Both mRNA and protein abundance of αS1-casein and ß-casein were positively affected by LM3.0, whereas a high glucose level increased protein abundance of αS1-casein and ß-casein and increased gene expression of CSN1S1 but not of CSN2. Furthermore, high glucose increased the mRNA abundance of ELF5 and decreased that of GLUT8, enhanced protein expression of total and phosphorylated mechanistic target of rapamycin (mTOR), and decreased phosphorylated AMP-activated protein kinase (AMPK) levels. Treatment LM3.0 had a stimulatory effect on total and phosphorylated mTOR but did not affect AMPK phosphorylation. The mRNA levels of JAK2, ELF5, and RPS6KB1 were upregulated and mRNA levels of EIF4EBP1 were downregulated with LM3.0 compared with LM2.3. Our results indicate that casein synthesis was regulated by Lys/Met ratio via JAK2/ELF5, mTOR, and its downstream RPS6KB1 and EIF4EBP1 signaling. In contrast, glucose regulated casein synthesis through promoting cell proliferation, accelerating cell cycle progression, and activating the ELF5 and AMPK/mTOR signaling pathways. Within the range of substrate levels in the present study, a change in Lys/Met ratio had a stronger effect on abundance of αS1-casein and ß-casein than a change in glucose level.

Minor amputation does not negatively affect health-related quality of life as compared with conservative treatment in patients with a diabetic foot ulcer: An observational study.

BACKGROUND: Health-related quality of life (HRQoL) is poor in patients with persistent diabetic foot ulcers and poor HRQoL predicts worse outcomes in these patients. Amputation is often considered a treatment failure, which is why conservative treatment is generally preferred over amputation. However, it is unclear whether minor amputation negatively affects HRQoL compared with conservative treatment in patients with diabetic foot ulcers. METHODS: In the cohort of the multicenter, prospective, observational Eurodiale study, we determined difference in change of HRQoL measured by EQ-5D between patients with a diabetic foot ulcers that healed after conservative treatment (n = 676) and after minor amputation (n = 145). Propensity score was used to adjust for known confounders, attempting to overcome lack of randomization. RESULTS: Baseline HRQoL was not significantly different between patients treated conservatively and undergoing minor amputation. In addition, there was no difference in the change of HRQoL between these groups. In patients who healed 6 to 12 months after the first visit, HRQoL on the anxiety/depression subscale even appeared to improve more in those who underwent minor amputation. CONCLUSIONS: Minor amputation was not associated with a negative impact on HRQoL in patients with a diabetic foot ulcers. It may therefore not be considered treatment failure in terms of HRQoL but rather a viable treatment option. A randomized controlled trial is warranted to further examine the influence of minor amputations on health-related quality of life.

Effect of supplemental concentrate during the dry period or early lactation on rumen epithelium gene and protein expression in dairy cattle during the transition period.

We previously reported 2 experiments with rumen-cannulated Holstein-Friesian dairy cows showing that during the transition period, rumen papillae surface area, and fractional absorption rate of volatile fatty acids (VFA) increase after calving. However, supplemental concentrate during the dry period and rate of increase of concentrate allowance during lactation affected papillae surface area, but not VFA absorption. Here we report the changes in gene and protein expression in rumen papillae related to tissue growth and VFA utilization. The lactation experiment treatment consisted of a rapid [RAP; 1.0 kg of dry matter (DM)/d; n = 6] or gradual (GRAD; 0.25 kg of DM/d; n = 6) increase of concentrate allowance (up to 10.9 kg of DM/d), starting at 4 d postpartum (pp). The dry period experiment treatment consisted of 3.0 kg of DM/d of concentrate (n = 4) or no concentrate (n = 5) during the last 28 d of the dry period. Real-time quantitative PCR analysis of rumen papillae showed that the expression of apoptosis-related genes was neither affected by day nor its interaction with treatment for both experiments. Expression of epithelial transporter genes was not affected by day or treatment in the lactation experiment, except for NBC1. In the dry period experiment, expression of MCT1, NBC1, DRA, NHE2, NHE3, and UT-B generally decreased after calving. A day and treatment interaction was observed for ATP1A1 in the dry period experiment, with greater expression at 18 and 8 d antepartum for concentrate than no concentrate. Generally, expression of VFA metabolism-related genes was not affected by day or its interaction with treatment. In the lactation experiment, immunoblotting of 5 selected genes showed that protein expression of DRA and PCCA was greater at 16 d pp compared with 3 and 44 d pp. Expression of NHE2 was greater, and that of ATP1A1 lower, at 16 and 44 d pp compared with 3 d pp, suggesting alterations in intracellular pH regulation and sodium homeostasis. Both MCT1 and PCCA protein were upregulated by RAP from 3 to 16 d pp, indicating modulations in VFA metabolism. Our data suggests that VFA absorption and metabolic capacity changed little per unit of surface area during the transition period, and suggests that a change in mitosis rate rather than apoptosis rate is associated with the increased ruminal VFA production, resulting in tissue growth. A significant but weak correlation between the examined gene and protein expression levels was observed only for PCCA, indicating that care must be taken when interpreting results obtained at either level.

Upregulation of miRNA-143, -145, -192, and -194 in esophageal epithelial cells upon acidic bile salt stimulation.

Barrett's esophagus (BE) is a metaplastic condition of the distal esophagus that occurs because of chronic gastroesophageal reflux. Previous studies have identified BE-specific microRNAs (miRNAs) in comparison with normal squamous epithelium (SQ). We hypothesized that BE-specific miRNAs could be induced in esophageal SQ cells by exposure to acid and/or bile salts. We aimed to determine whether BE-specific miRNAs are upregulated in an esophageal SQ cell line (Het-1A) in an environment with acid and/or bile salts and whether this is nuclear factor-κB (NF-κB) dependent. Acid and/or bile salt incubations were performed in Het-1A cells. Experiments were performed with or without inhibiting the NF-κB pathway. Quantitative reverse transcriptase polymerase chain reaction was performed to determine expression of miRNA-143, -145, -192, -194, cyclo-oxygenase-2 (COX2), mucin 2 (MUC2), and sex determining region Y-box 9. For validation, we determined levels of these miRNAs in biopsies from patients with reflux esophagitis and normal SQ. Significantly increased expression levels of miRNA-143 (2.7-fold), -145 (2.6-fold), -192 (2.0-fold), -194 (2.2-fold), COX2, MUC2, and sex determining region Y-box 9 were found upon acidic bile salt incubation, but not upon acid or bile salt alone. NF-κB pathway inhibition significantly decreased miRNA-143, -192, -194, COX2, and MUC2 expression. Additionally, miRNA-143, -145 and -194 expression was increased in reflux esophagitis biopsies compared with normal SQ, but no changes were found in miRNA-192 expression. Our findings suggest that upregulation of BE-specific miRNAs by acidic bile may be an early event in the transition of SQ to BE and that their expression is partly regulated by the NF-κB pathway.

Influence of copper source and dietary inclusion level on growth performance of weaned pigs and expression of trace element related genes in the small intestine.

Copper is routinely supplemented to weanling pig diets at concentrations above nutritional requirements to enhance growth performance. We hypothesised that this effect depends on the source of Cu and its dietary concentration. We tested this in weaned pigs (26 d of age) over a 35-d period using a 2 × 3 factorial arrangement with two Cu-sources (CuSO4 and Cu2O, monovalent copper oxide, CoRouge®) and three supplementary dietary Cu-levels (15, 80 and 160 mg Cu/kg) as respective factors. Increasing Cu level linearly increased (P < 0.001) final BW and daily gain. These effects tended (P = 0.09) to be greater with Cu2O than CuSO4. Feed conversion ratio decreased linearly (P < 0.001) with increasing dietary Cu content, independent of Cu source. Plasma Cu, Zn and Fe levels were unaffected, whereas liver Cu content increased quadratically (P < 0.001) with increasing dietary Cu content, with a larger increase (P < 0.001) with CuSO4 than Cu2O. Bile Cu content increased quadratically (P = 0.025) with increasing Cu content, irrespective of Cu source. RT-qPCR analysis revealed that increasing Cu content quadratically (P = 0.009) increased duodenal but not ileal metallothionein 1A (MT1A) mRNA, with greater effect (P = 0.010) of CuSO4. Regardless of the Cu source, increasing Cu dose linearly increased (P = 0.006) duodenal DMT1/SLC11A2 mRNA but decreased ZIP4/SLC39A4 mRNA in duodenum (P < 0.001) and ileum (P < 0.005). ZnT10/SLC30A10 mRNA was significantly (P = 0.021) and numerically (P = 0.061) greater with Cu2O compared to CuSO4, in duodenum and ileum, respectively. Copper content quadratically modulated duodenal but not ileal transferrin receptor (P = 0.029) and ferric reductase CYBRD1 mRNA (P = 0.022). In hypothalamus, high Cu dose (P = 0.024) and Cu2O as source (P = 0.028) reduced corticotropin-releasing hormone (CRH) mRNA. Low versus high CuSO4 increased corticotropin-releasing hormone receptor (CRHR2) mRNA, while low Cu2O had the opposite effect (P = 0.009). In conclusion, incremental Cu intake enhanced growth performance, with a tendency for a greater effect of Cu2O. The lower increase in duodenal MT1A mRNA and liver Cu content indicates that less Cu from Cu2O was absorbed by gut and sequestered in liver. Thus, high Cu absorption is not essential for its growth-promoting effect and dietary Cu may affect intestinal Fe and Zn absorption via the active, transcellular route. The effects on hypothalamic CRH and CRHR2 expression indicate a role for the hypothalamus in mediating the effects of Cu on growth performance.

Importance of factors determining the low health-related quality of life in people presenting with a diabetic foot ulcer: the Eurodiale study.

AIM: To identify the factors responsible for the low health-related quality of life associated with foot ulcers and the relative importance of these factors. METHODS: A total of 1232 patients with a new foot ulcer, who presented at one of the 14 centres in 10 European countries participating in the Eurodiale study, were included in this cross-sectional study. Patient and ulcer characteristics were obtained as well as results from the Euro-Qol-5D questionnaire, a health-related quality of life instrument with five domains (mobility, self-care, usual activities, pain/discomfort and anxiety/depression). To analyse the relative importance of comorbidities and ulcer- and patient-related factors for health-related quality of life, linear regression models were used to calculate the relative contributions of each factor to the fit (R(2) ) of the model. RESULTS: Patients reported poor overall health-related quality of life, with problems primarily in the mobility and pain/discomfort domains. Among the comorbidities, the inability to stand or walk without help was the most important determinant of decreased health-related quality of life in all five domains. Among ulcer-related factors, ulcer size, limb-threatening ischaemia and elevated C-reactive protein concentration also had high importance in all domains. The clinical diagnosis of infection, peripheral arterial disease and polyneuropathy were only important in the pain/discomfort domain. CONCLUSIONS: The factors that determine health-related quality of life are diverse and to an extent not disease-specific. To improve health-related quality of life, treatment should not only be focused on ulcer healing but a multifactorial approach by a specialized multidisciplinary team is also important.

Effect of rumen-protected choline supplementation on liver and adipose gene expression during the transition period in dairy cattle.

We previously reported that supplementation of rumen-protected choline (RPC) reduces the hepatic triacylglycerol concentration in periparturient dairy cows during early lactation. Here, we investigated the effect of RPC on the transcript levels of lipid metabolism-related genes in liver and adipose tissue biopsies, taken at wk -3, 1, 3, and 6 after calving, to elucidate the mechanisms underlying this RPC-induced reduction of hepatic lipidosis. Sixteen multiparous cows were blocked into 8 pairs and randomly allocated to either 1 of 2 treatments, with or without RPC. Treatments were applied from 3 wk before to 6 wk after calving. Both groups received a basal diet and concentrate mixture. One group received RPC supplementation, resulting in an intake of 14.4 g of choline per day, whereas controls received an isoenergetic mixture of palm oil and additional soybean meal. Liver and adipose tissue biopsies were taken at wk -3, 1, 3, and 6 to determine the mRNA abundance of 18 key genes involved in liver and adipose tissue lipid and energy metabolism. Milk samples were collected in wk 1, 2, 3, and 6 postpartum for analysis of milk fatty acid (FA) composition. The RPC-induced reduction in hepatic lipidosis could not be attributed to altered lipolysis in adipose tissue, as no treatment effect was observed on the expression of peroxisome proliferator-activated receptor γ, lipoprotein lipase, or FA synthase in adipose tissue, or on the milk FA composition. Rumen-protected choline supplementation increased the expression of FA transport protein 5 and carnitine transporter SLC22A5 in the liver, suggesting an increase in the capacity of FA uptake and intracellular transport, but no treatment effect was observed on carnitine palmitoyl transferase 1A, transporting long-chain FA into mitochondria. In the same organ, RPC appeared to promote apolipoprotein B-containing lipoprotein assembly, as shown by elevated microsomal triglyceride transfer protein expression and apolipoprotein B100 expression. Cows supplemented with RPC displayed elevated levels of glucose transporter 2 mRNA and a reduced peak in pyruvate carboxylase mRNA immediately after calving, showing that supplementation also resulted in improved carbohydrate metabolism. The results from this study suggest that RPC supplementation reduces liver triacylglycerol by improved FA processing and very-low-density lipoprotein synthesis, and RPC also benefits hepatic carbohydrate metabolism.

Comparison between stearoyl-CoA desaturase expression in milk somatic cells and in mammary tissue of lactating dairy cows.

Stearoyl-CoA desaturase (SCD) is an important enzyme in the bovine mammary gland, where it inserts a cis-double bond at the Δ9 position in a wide range of fatty acids. Investigating SCD expression in the bovine mammary gland generally requires invasive biopsy to obtain mammary tissue. The aim of this study was to evaluate the use of milk somatic cells as a non-invasive alternative to biopsy for measuring mammary SCD expression in dairy cows. Both milk somatic cells and mammary tissue were collected from 14 Holstein-Friesian cows and used for analysis of SCD expression by real-time PCR. The SCD5 mRNA levels in mammary tissue compared with SCD1 were low, and for several milk somatic cell samples, SCD5 expression was even below the limit of detection. A significant relationship was found between SCD1 expression in milk somatic cells and in mammary tissue. In addition, SCD1 expression in milk somatic cells was significantly related to Δ9-desaturase indices in milk, which are commonly used as an indicator of SCD1 activity within the mammary gland. Our study showed that milk somatic cells can be used as a source of mRNA to study SCD1 expression in dairy cows, offering a non-invasive alternative to mammary tissue samples obtained by biopsy.

Dietary effects of linseed on fatty acid composition of milk and on liver, adipose and mammary gland metabolism of periparturient dairy cows.

During the transition period in dairy cows, drastic adaptations within and between key tissues and cell types occur in a coordinated manner to support late gestation, the synthesis of large quantities of milk and metabolic homoeostasis. The start of lactation coincides with an increase of triacylglycerols in the liver, which has been associated with several economically important diseases in dairy cows (i.e. hepatic lipidiosis, mastitis). The polyunsaturated fatty acids have been used to improve liver metabolism and immune function in the mammary gland. Therefore, the effects of dietary linseed supplementation on milk quality and liver, adipose and mammary gland metabolism of periparturient dairy cows were studied in 14 cows that were randomly assigned to control or linseed supplementation. Animals were treated from 3 weeks antepartum until 6 weeks post-partum. Linseed did not modify dry matter intake, but increased milk yield and lactose yield, and decreased milk fat concentration, which coincided with lower proportion of C16 and higher proportions of stearic acid, conjugated linoleic acid and α-linolenic acid in milk fat. Linseed supplementation did not significantly change the expression of key lipid metabolism genes in liver and adipose tissues, except of glucose transporter 2 (GLUT2) in liver, which was increased in cows supplemented with linseed, suggesting that more glucose was secreted and probably available for lactose synthesis compared with cows fed control diet. Large adaptations of transcription occurred in the mammary gland when dairy cows were supplemented with linseed. The main affected functional modules were related to energy metabolism, cell proliferation and remodelling, as well as the immune system response.

Mucosal expression of Ca and P transporters and claudins in the small intestine of broilers is altered by dietary Ca:P in a limestone particle size dependent manner.

High calcium (Ca) intake and fine limestone reduces precaecal phosphorus (P) absorption independently of P solubility in broilers. This study aimed to determine whether dietary total Ca: total P ratio (Ca:P) and limestone particle size (LPS) affect gene expression of P transporters in the small intestine. A total of 384 one-day-old Ross 308 male broiler chickens received diets low (0.50), medium (1.00) or high (1.75) in Ca:P containing either fine (160 µm) or coarse (1062 µm) limestone, in a 3×2 factorial arrangement. Expression of Ca- and P-related genes were determined using real-time quantitative PCR (RT-qPCR) in duodenum and jejunum. Increasing dietary Ca:P decreased duodenal calcium-sensing receptor (CaSR), calbindin-D28k (CaBP-D28k), plasma membrane Ca-ATPase 1 (PMCA1) and sodium-coupled P cotransporter type IIb (NaPi-IIb), but not transient receptor potential canonical 1 (TRPC1) mRNA. This effect was greater with fine limestone when Ca:P increased from low to medium, but greater with coarse limestone when increased from medium to high. A similar inhibitory effect was observed for jejunal CaBP-D28k expression where increasing dietary Ca:P and fine limestone decreased CaSR mRNA, while dietary Ca:P decreased TRPC1 mRNA only for coarse limestone. It also decreased jejunal NaPi-IIb mRNA irrespective of LPS. Dietary treatments did not affect jejunal PMCA1 mRNA expression or that of inorganic phosphate transporter 1 and 2 and xenotropic and polytropic retrovirus receptor 1 in both intestinal segments. Dietary Ca increase reduced mucosal claudin-2 mRNA in both segments, and jejunal zonula occludens-1 (ZO-1) mRNA only for coarse limestone. In conclusion, increasing dietary Ca:P reduced expression of duodenal P transporters (NaPi-IIb) in a LPS dependent manner, hence Ca induced reduction in intestinal P absorption is mediated by decreasing P transporters expression. Dietary Ca reduces Ca digestibility by downregulating mRNA expression of both Ca permeable claudin-2 and Ca transporters (CaBP-D28k, PMCA1).

Differences in minor amputation rate in diabetic foot disease throughout Europe are in part explained by differences in disease severity at presentation.

OBJECTIVES: The incidence of minor amputation may vary significantly, and determinants of minor amputation have not been studied systematically. We evaluated minor amputation rate, the determinants of minor amputation and differences in amputation rate between European centres. METHODS: In the Eurodiale study, a prospective cohort study of 1232 patients (1088 followed until end-point) with a new diabetic foot ulcer were followed on a monthly basis until healing, death, major amputation or up to a maximum of 1 year. Ulcers were treated according to international guidelines. Baseline characteristics independently associated with minor amputation were examined using multiple logistic regression modelling. Based on the results of the multivariable analysis, a disease severity score was calculated for each patient. RESULTS: One hundred and ninety-four (18%) patients underwent a minor amputation. Predictors of minor amputation were depth of the ulcer (odds ratio 6.08, confidence interval 4.10-9.03), peripheral arterial disease (odds ratio 1.84, confidence interval 1.30-2.60), infection (odds ratio 1.56, confidence interval 1.05-2.30) and male sex (odds ratio 1.42, confidence interval 0.99-2.04). Minor amputation rate varied between 2.4 and 34% in the centres. Minor amputation rate in centres correlated strongly with disease severity score at the moment of presentation to the foot clinic (r=0.75). CONCLUSIONS: Minor amputation is performed frequently in diabetic foot centres throughout Europe and is determined by depth of the ulcer, peripheral arterial disease, infection and male sex. There are important differences in amputation rate between the European centres, which can be explained in part by severity of disease at presentation. This may suggest that early referral to foot clinics can prevent minor amputations.

Influence of nutrition on feline calcium oxalate urolithiasis with emphasis on endogenous oxalate synthesis.

The prevalence of calcium oxalate (CaOx) uroliths detected in cats with lower urinary tract disease has shown a sharp increase over the last decades with a concomitant reciprocal decrease in the occurrence of struvite (magnesium ammonium phosphate) uroliths. CaOx stone-preventative diets are available nowadays, but seem to be marginally effective, as CaOx urolith recurrence occurs in patients fed these diets. In order to improve the preventative measures against CaOx urolithiasis, it is important to understand its aetiopathogenesis. The main research focus in CaOx formation in cats has been on the role of Ca, whereas little research effort has been directed towards the role and origin of urinary oxalates. As in man, the exogenous origin of urinary oxalates in cats is thought to be of minor importance, although the precise contribution of dietary oxalates remains unclear. The generally accepted dietary risk factors for CaOx urolithiasis in cats are discussed and a model for the biosynthetic pathways of oxalate in feline liver is provided. Alanine:glyoxylate aminotransferase 1 (AGT1) in endogenous oxalate metabolism is a liver-specific enzyme targeted in the mitochondria in cats, and allows for efficient conversion of glyoxylate to glycine when fed a carnivorous diet. The low peroxisomal activity of AGT1 in cat liver is compatible with the view that felids utilised a low-carbohydrate diet throughout evolution. Future research should focus on understanding de novo biosynthesis of oxalate in cats and their adaptation(s) in oxalate metabolism, and on dietary oxalate intake and absorption by cats.

Effect of rumen-protected choline on performance, blood metabolites, and hepatic triacylglycerols of periparturient dairy cattle.

The effects of a dietary supplement of rumen-protected choline on feed intake, milk yield, milk composition, blood metabolites, and hepatic triacylglycerol were evaluated in periparturient dairy cows. Thirty-eight multiparous cows were blocked into 19 pairs and then randomly allocated to either one of 2 treatments. The treatments were supplementation either with or without (control) rumen-protected choline. Treatments were applied from 3 wk before until 6 wk after calving. Both groups received the same basal diet, being a mixed feed of grass silage, corn silage, straw, and soybean meal, and a concentrate mixture delivered through transponder-controlled feed dispensers. For all cows, the concentrate mixture was gradually increased from 0 kg/day (wk -3) to 0.9 kg of dry matter (DM)/d (day of calving) and up to 8.1 kg of DM/d on d 17 postcalving until the end of the experiment. Additionally, a mixture of 60 g of a rumen-protected choline supplement (providing 14.4 g of choline) and of 540 g of soybean meal or a (isoenergetic) mixture of 18 g of palm oil and 582 g of soybean meal (control) was offered individually in feed dispensers. Individual feed intake, milk yield, and body weight were recorded daily. Milk samples were analyzed weekly for fat, protein, and lactose content. Blood was sampled at wk -3, d 1, d 4, d 7, d 10, wk 2, wk 3, and wk 6 and analyzed for glucose, nonesterified fatty acids, and ß-hydroxybutyric acid. Liver biopsies were taken from 8 randomly selected pairs of cows at wk -3, wk 1, wk 4, and wk 6 and analyzed for triacylglycerol concentration. We found that choline supplementation increased DM intake from 14.4 to 16.0 kg/d and, hence, net energy intake from 98.2 to 109.1 MJ/d at the intercept of the lactation curve at 1 day in milk (DIM), but the effect of choline on milk protein yield gradually decreased during the course of the study. Choline supplementation had no effect on milk yield, milk fat yield, or lactose yield. Milk protein yield was increased from 1.13 to 1.26 kg/d at the intercept of the lactation curve at 1 DIM, but the effect of choline on milk protein yield gradually decreased during the course of the study. Choline supplementation was associated with decreased milk fat concentration at the intercept of the lactation curve at 1 DIM, but the effect of choline on milk fat concentration gradually decreased as lactation progressed. Choline supplementation had no effect on energy-corrected milk yield, energy balance, body weight, body condition score, and measured blood parameters. Choline supplementation decreased the concentration of liver triacylglycerol during the first 4 wk after parturition. Results from this study suggest that hepatic fat export in periparturient dairy cows is improved by choline supplementation during the transition period and this may potentially decrease the risk for metabolic disorders in the periparturient dairy cow.

Effects of feeding rapeseed oil, soybean oil, or linseed oil on stearoyl-CoA desaturase expression in the mammary gland of dairy cows.

Stearoyl-CoA desaturase (SCD) is an important enzyme in the bovine mammary gland, and it introduces a double bond at the Δ(9) location of primarily myristoyl-, palmitoyl-, and stearoyl-CoA. The main objective of this study was to compare the effects of various fatty acids (FA) typically present in dairy cow rations on the expression of SCD1 and SCD5 in the mammary gland of dairy cows. Twenty-eight Holstein-Friesian cows were randomly assigned to 1 of 4 dietary treatments. The dietary treatments were a basal diet supplemented (dry matter basis) with 2.7% rapeseed oil as a source of C18:1 cis-9; 2.7% soybean oil as a source of C18:2 cis-9,12; 2.7% linseed oil as a source of C18:3 cis-9,12,15; or 2.7% of a 1:1:1 mixture of the 3 oils. The oil supplements were included in the concentrate, which was fed together with corn silage and grass silage. In addition, cows were grazing on pasture, consisting mainly of perennial ryegrass, during the day. Biopsies from the mammary gland were taken and analyzed for mRNA expression of SCD1 and SCD5 by using quantitative real-time PCR. Milk yield as well as milk protein and fat contents did not differ among the 4 dietary treatments. Dietary supplementation with rapeseed oil and linseed oil increased proportions of C18:1 cis-9 and C18:3 cis-9,12,15 in blood plasma, respectively, compared with the other treatments. Supplementation with soybean oil and linseed oil increased milk FA proportions of C18:2 cis-9,12 and C18:3 cis-9,12,15, respectively, but supplementation with rapeseed oil did not increase C18:1 cis-9 in milk. Mammary SCD1 expression was reduced by supplementation of soybean oil compared with rapeseed oil and linseed oil. In contrast, SCD5 expression did not differ among the 4 treatments. The C16 and C18 desaturation indices, representing proxies for SCD activity, were lower for the soybean oil diet compared with the diet supplemented with a mixture of the 3 oils. In conclusion, our study shows that mammary SCD1 expression is significantly downregulated in dairy cows by feeding unprotected soybean oil compared with rapeseed oil or linseed oil, and this is partially reflected by the lower desaturase indices in the milk. Furthermore, mammary SCD5 expression appears to be differently regulated than expression of SCD1.

Low-calcium diets increase duodenal mRNA expression of calcium and phosphorus transporters and claudins but compromise growth performance irrespective of microbial phytase inclusion in broilers.

The hypothesis that dietary inclusion of microbial phytase improves apparent calcium (Ca) digestibility thereby allowing a lower dietary Ca inclusion without compromising growth performance was tested. One-day-old male Ross 308 broilers (25 birds/pen, 9 pens/treatment) were assigned to 8 experimental diets containing one of 4 dietary Ca to retainable P (rP) ratios (1.3, 1.8, 2.3, and 2.8) with (1,000 FTU/kg) or without microbial phytase. On d 21 to 23, digesta from different intestinal segments of 8 birds per pen were collected to determine apparent Ca and P digestibility. Mid duodenal mucosa was collected for expression of Ca (CaBP-D28k, PMCA1) and P (NaPi-IIb, PiT-1, PiT-2, and XPR1) transporters by RT-qPCR. Dietary phytase inclusion in low Ca/rP diets increased Ca digestibility in the distal ileum (Pinteraction = 0.023) but not the proximal or distal jejunum. Broilers receiving the lowest Ca/rP displayed the lowest body weight gain, highest feed conversion ratio (P < 0.001), and lowest tibia strength, irrespective of dietary phytase inclusion. Incremental dietary Ca/rP linearly reduced P digestibility to a greater extent in the absence of phytase in the distal jejunum and ileum (Pinteraction = 0.021 and 0.001, respectively). Incremental dietary Ca/rP linearly reduced serum P more in phytase-free diets (Pinteraction < 0.001), and lowered duodenal expression of P transporters NaPi-IIb, PiT-2, and XPR1 (P = 0.052, 0.071 and 0.028, respectively). Incremental dietary Ca/rP linearly increased (P < 0.001) serum Ca irrespective of phytase inclusion, accompanied by a lower (P < 0.001) duodenal expression of Ca transporters CaSR, CaBP-D28k and PMCA1 and Ca-pore forming claudins CLDN-2 and CLDN-12. Dietary phytase increased (P = 0.026) NaPi-IIb but reduced (P = 0.029) CLDN-2 expression. Incremental Ca/rP reduced Ca and P digestibility, increased serum Ca, lowered serum P and inhibited mRNA levels of Ca and P-related transporters, indicating that these transporters and CLDN contribute to the observed effect of dietary Ca and phytase on Ca and P absorption. Despite the improvement in Ca digestibility, dietary phytase did not restore the compromised growth performance and tibia strength of broilers fed a Ca-deficient diet, leading to rejection of the hypothesis.

Intestinal epithelium integrity after delayed onset of nutrition in broiler chickens.

Fasting older broiler chickens (>7 d of age) enlarges the intestinal tight junction (TJ) pore size, resulting in high paracellular intestinal permeability. Broiler chickens often do not receive feed and water (nutrition) directly after hatch, which may result in fasting up to 72 h of age. Whether perinatal fasting affects intestinal permeability is minimally studied. We therefore investigated whether delayed access to nutrition after hatch increases intestinal permeability, compared with broilers receiving early access to nutrition. Therefore, 432 hatched broilers received nutrition 72 h after hatch (delayed nutrition [DN]) or directly after hatch (early nutrition [EN]) and were reared under similar conditions until 14 d of age. Two hours after application of an oral pulse dose (3.85 mg) of fluorescein isothiocyanate-dextran (4000 Da) at 4, 10, and 14 d of age, blood plasma concentrations of the marker were measured in 24 to 36 broilers per treatment and time point. Marker concentration in plasma did not differ between DN and EN broilers at any age. The villus width measured in at least 8 broilers per treatment was smaller in DN than in EN broilers at 4 d for both the ileum (92 ± 3 µm vs. 121 ± 4; P < 0.001) and colon (100 ± 3 vs. 120 ± 4; P < 0.01). Real-time quantitative PCR revealed that the expression of TJ protein claudin 3 in the ceca was elevated in DN, compared with EN broilers at 4 d of age, whereas that of zonula occludens 1 in the ileum was reduced. Expression of host defense-related genes was reduced in DN, compared with EN broilers, in the ileum (cyclo-oxygenase 2, mucin 2) and ceca (interleukin 1ß, cyclo-oxygenase 2). We conclude that 72-hour DN reduced the BW up to 14 d of age, coinciding with transient effects on the villus width in the ileum and colon, and divergent expression of genes involved in TJ formation and host defense. These effects likely reflect the delayed onset of intestinal and immune development in DN, compared with EN broilers, while DN does not fundamentally alter intestinal permeability.

Coarse limestone does not alleviate the negative effect of a low Ca/P ratio diet on characteristics of tibia strength and growth performance in broilers.

The hypothesis was tested that an increased digestion of coarse compared with fine limestone can alleviate the negative effects of a low dietary Ca/P ratio on the growth performance and characteristics of tibia strength (CTS) in broilers. A total of 1,152 Ross 308 broiler chickens received a standard commercial starter feed from day 0 to 13. From day 14 onward, birds received 1 of 12 diets containing 1 of 6 Ca/P ratios (0.50, 0.75, 1.00, 1.25, 1.50, and 1.75) and 1 of 2 limestone particle sizes (<500 [fine] and 500 to 2,000 [coarse] µm) in a study with a 6 × 2 factorial arrangement of treatments. Total P content was fixed at 5.5 g/kg for all treatment diets. Each treatment was replicated 6 times with 16 birds per replicate pen. On day 20 and 21, twelve birds per pen were randomly selected from 4 of the 6 replicate pens for tibia analysis and digesta collection from different gut segments. The apparent Ca digestibility was higher for fine than coarse limestone in the jejunum (P = 0.043). However, this difference in Ca digestibility disappeared for the low, whereas it remained for the high Ca/P ratios in the proximal (Pinteraction = 0.067) and distal (Pinteraction = 0.052) ileum. In addition, coarse limestone improved apparent P digestibility in the proximal and distal ileum (P < 0.001) but not in the jejunum (P = 0.305). Regardless of limestone particle size, reducing dietary Ca/P ratio linearly improved apparent Ca and P digestibility in the proximal and distal ileum (P < 0.001). Moreover, decreasing dietary Ca/P ratio linearly (P < 0.001) and quadratically (P < 0.046) reduced the CTS. Reducing dietary Ca/P ratio linearly (P < 0.003) and quadratically (P ≤ 0.006) decreased body weight gain and increased feed conversion ratio. For both fine and coarse limestone, the optimal Ca/P ratio was 1.00 to 1.25 to optimize apparent Ca and P digestibility while maintaining growth performance and CTS. Reducing Ca/P ratio from 1.75 to 1.00 improved distal ileal Ca and P apparent digestibility from 36.6 to 53.7% and 48.0 to 58.3%, respectively. In conclusion, coarse limestone is equally digestible with fine limestone at a low Ca/P ratio but is less digestible at a high Ca/P ratio, and the optimal Ca/P ratio in the diet is 1.00 to 1.25 for both fine and coarse limestone.