Short communication: Predicting blood plasma non-esterified fatty acid and beta-hydroxybutyrate concentrations from cow milk-addressing systematic issues in modelling.

Negative energy status in early lactation is linked to a variety of metabolic disorders, reduced fertility, and decreased milk production. To improve the energy status of cows by breeding and management, the identification of negative energy status is crucial. While biomarkers such as non-esterified fatty acid (NEFA) concentration and beta-hydroxybutyrate (BHB) in blood plasma could be used to identify a negative energy state, measuring them directly from blood is both invasive and expensive. In this work, we developed prediction equations for blood plasma NEFA and BHB levels based on mid-IR spectral measurements of milk. The models were fitted using partial least squares regression and evaluated using both cross-validation and independent-herd validation. A total of 3 183 spectral records from 606 lactations originating from three different herds were utilised. R2 values of 0.53 (RMSE = 0.206 mmol/l, RMSE of cross-validation (RMSECV) 0.217 mmol/l) for NEFA and 0.63 (RMSE = 0.326 mmol/l, RMSECV = 0.353 mmol/l) for BHB were obtained. Furthermore, relatively similar prediction accuracies were found for BHB (RMSE of prediction (RMSEP) 0.411 mmol/l and 0.422 mmol/l) and NEFA (RMSEP = 0.186 mmol/l and 0.221 mmol/l) when model training was done using two herds and validated on the third herd. The results from the model fits confirm that it is possible to build blood plasma BHB and NEFA models based on mid-IR spectra that are sufficiently accurate for practical use.

Mid-infrared spectroscopic analysis of raw milk to predict the blood nonesterified fatty acid concentrations in dairy cows.

In high-yielding dairy cattle, severe postpartum negative energy balance is often associated with metabolic and infectious disorders that negatively affect production, fertility, and welfare. Mobilization of adipose tissue associated with negative energy balance is reflected through an increased level of nonesterified fatty acids (NEFA) in the blood plasma. Earlier, identification of negative energy balance through detection of increased blood plasma NEFA concentration required laborious and stressful blood sampling. More recently, attempts have been made to predict blood NEFA concentration from milk samples. In this study, we aimed to develop and validate a model to predict blood plasma NEFA concentration using the milk mid-infrared (MIR) spectra that are routinely measured in the context of milk recording. To this end, blood plasma and milk samples were collected in wk 2, 3, and 20 postpartum for 192 lactations in 3 herds. The blood plasma samples were taken in the morning, and representative milk samples were collected during the morning and evening milk sessions on the same day. To predict plasma NEFA concentration from the milk MIR spectra, partial least squares regression models were trained on part of the observations from the first herd. The models were then thoroughly validated on all other observations of the first herd and on the observations of the 2 independent herds to explore their robustness and wide applicability. The final model could accurately predict blood plasma NEFA concentrations <0.6 mmol/L with a root mean square error of prediction of <0.143 mmol/L. However, for blood plasma with >1.2 mmol/L NEFA, the model clearly underestimated the true level. Additionally, we found that morning blood plasma NEFA levels were predicted with significantly higher accuracy using MIR spectra of evening milk samples compared with MIR spectra of morning samples, with root mean square error of prediction values of, respectively, 0.182 and 0.197 mmol/L, and R2 values of 0.613 and 0.502. These results suggest a time delay between variations in blood plasma NEFA and related milk biomarkers. Based on the MIR spectra of evening milk samples, cows at risk for negative energy status, indicated by detrimental morning blood plasma NEFA levels (>0.6 mmol/L), could be identified with a sensitivity and specificity of, respectively, 0.831 and 0.800. As this model can be applied to millions of historical and future milk MIR spectra, it opens an opportunity for regular metabolic screening and improved resilience phenotyping.

Publisher Correction: The composition of the perinatal intestinal microbiota in cattle.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Prepartal high-energy feeding with grass silage-based diets does not disturb the hepatic adaptation of dairy cows during the periparturient period.

The liver of dairy cow naturally undergoes metabolic adaptation during the periparturient period in response to the increasing demand for nutrients. The hepatic adaptation is affected by prepartal energy intake level and is potentially associated with inflammatory responses. To study the changes in the liver function during the periparturient period, 16 cows (body condition score = 3.7 ± 0.3, mean ± standard deviation; parity = second through fourth) were allocated to a grass silage-based controlled-energy diet (104 MJ/d) or a high-energy diet (135 MJ/d) during the last 6 wk before the predicted parturition. Liver samples were collected by biopsy at 8 d before the predicted parturition (-8 d) and at 1 and 9 d after the actual parturition (1 and 9 d). The lipidomic profile of liver samples collected at -8 and 9 d was analyzed using ultra performance liquid chromatography-mass spectrometry-based lipidomics. Liver samples from all the time points were subjected to microarray analysis and the subsequent pathway analysis with Ingenuity Pathway Analysis software (Ingenuity Systems, Mountain View, CA). Prepartal energy intake level affected hepatic gene expression and lipidomic profiles prepartum, whereas little or no effect was observed postpartum. At -8 d, hepatic lipogenesis was promoted by prepartal high-energy feeding through the activation of X receptor/retinoid X receptor pathway and through increased transcription of thyroid hormone-responsive (THRSP). Hepatic inflammatory and acute phase responses at -8 d were suppressed (z-score = -2.236) by prepartal high-energy feeding through the increase in the mRNA abundance of suppressor of cytokine signaling 3 (SOCS3) and the decrease in the mRNA abundance of interleukin 1 (IL1), nuclear factor kappa B 1 (NFKB1), apolipoprotein A1 (APOA1), serum amyloid A3 (SAA3), haptoglobin (HP), lipopolysaccharide-binding protein (LBP), and inter-α-trypsin inhibitor heavy chain 3 (ITIH3). Moreover, prepartal high-energy feeding elevated hepatic concentrations of C18- (7%), C20- (17%), C21- (26%), C23-sphingomyelins (26%), and total saturated sphingomyelin (21%). In addition, cows in both groups displayed increased lipogenesis at the gene expression level after parturition and alterations in the concentration of various sphingolipids between the first and last samplings. In conclusion, prepartal high-energy feeding promoted lipogenesis and suppressed inflammatory and acute phase responses in the liver before parturition, whereas only minor effects were observed after parturition.

Dietary supplement of conjugated linoleic acids or polyunsaturated fatty acids suppressed the mobilization of body fat reserves in dairy cows at early lactation through different pathways.

To investigate the metabolic changes in the adipose tissue (AT) of dairy cows under milk fat depression (MFD), 30 cows were randomly allocated to a control diet, a conjugated linoleic acid (CLA)-supplemented diet, or a high-starch diet supplemented with a mixture of sunflower and fish oil (2:1; as HSO diet) from 1 to 112 d in milk. Performance of animals, milk yield, milk composition, energy balance, and blood metabolites were measured during lactation. Quantitative PCR analyses were conducted on the AT samples collected at wk 3 and 15 of lactation. The CLA and HSO diets considerably depressed milk fat yield and milk fat content at both wk 3 and 15 in the absence of significant changes in milk protein and lactose contents. In addition, the HSO diet lowered milk yield at wk 15 and decreased dry matter intake of cows from wk 3 to 15. Compared with the control, both CLA and HSO groups showed reduced body weight loss, improved energy balance, and decreased plasma concentrations of nonesterified fatty acids and ß-hydroxybutyrate at early lactation. The gene expression analyses reflected suppressed lipolysis in AT of the CLA and HSO groups compared with the control at wk 3, as suggested by the downregulation of hormone-sensitive lipase and fatty acid binding protein 4 and the upregulation of perilipin 2. In addition, the HSO diet promoted lipogenesis in AT at wk 15 through the upregulation of 1-acylglycerol-3-phosphate O-acyltransferase 2, mitochondrial glycerol-3-phosphate acyltransferase, perilipin 2, and peroxisome proliferator-activated receptor γ. The CLA diet likely regulated insulin sensitivity in AT as it upregulated the transcription of various genes involved in insulin signaling, inflammatory responses, and ceramide metabolism, including protein kinase B2, nuclear factor κ B1, toll-like receptor 4, caveolin 1, serine palmitoyltransferase long chain base subunit 1, and N-acylsphingosine amidohydrolase 1. In contrast, the HSO diet resulted in little or no change in the pathways relevant to insulin sensitivity. In conclusion, the CLA and HSO diets induced a shift in energy partitioning toward AT instead of mammary gland during lactation through the regulation of different pathways.

The composition of the perinatal intestinal microbiota in cattle.

Recent research suggests that the microbial colonization of the mammalian intestine may begin before birth, but the observations are controversial due to challenges in the reliable sampling and analysis of low-abundance microbiota. We studied the perinatal microbiota of calves by sampling them immediately at birth and during the first postnatal week. The large size of the bovine newborns allows sampling directly from rectum using contamination-shielded swabs. Our 16S rDNA data, purged of potential contaminant sequences shared with negative controls, indicates the existence of a diverse low-abundance microbiota in the newborn rectal meconium and mucosa. The newborn rectal microbiota was composed of Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes. The microbial profile resembled dam oral rather than fecal or vaginal vestibular microbiota, but included typical intestinal taxa. During the first postnatal day, the rectum was invaded by Escherichia/Shigella and Clostridia, and the diversity collapsed. By 7 days, diversity was again increasing. In terms of relative abundance, Proteobacteria were replaced by Firmicutes, Bacteroidetes and Actinobacteria, including Faecalibacterium, Bacteroides, Lactobacillus, Butyricicoccus and Bifidobacterium. Our observations suggest that mammals are seeded before birth with a diverse microbiota, but the microbiota changes rapidly in the early postnatal life.

Endothelial Fas-Ligand in Inflammatory Bowel Diseases and in Acute Appendicitis.

Fas-mediated induction of apoptosis is a major factor in the selection of lymphocytes and downregulation of immunological processes. In the present study, we have assessed endothelial Fas-ligand (FasL) expression in normal human ileum, appendix, and colon, and compared the expression levels with that in inflammatory bowel disease and in acute appendicitis. In a normal appendix, endothelial FasL levels were constant in almost half of the mucosal vessels; but, in the normal ileum and colon, endothelial FasL was practically restricted to areas in close proximity to lymphatic follicles, and was expressed mainly in the submucosal aspect of the follicles in the vessels with high endothelium. In samples from subjects with either Crohn's disease or ulcerative colitis, the extent of endothelial FasL expression was elevated in the submucosa and associated with an elevated number of lymphoid follicles. In inflammatory bowel disease, ulcers and areas with a high density of mononuclear cells expressing FasL also showed an elevated density of blood vessels with endothelial FasL expression. Although the function of endothelial FasL remains unclear, such a specific expression pattern suggests that endothelial FasL expression has a role in the regulation of lymphocyte access to the peripheral lymphoid tissues, including the intestinal mucosa.

Relationships among Body Condition, Insulin Resistance and Subcutaneous Adipose Tissue Gene Expression during the Grazing Season in Mares.

Obesity and insulin resistance have been shown to be risk factors for laminitis in horses. The objective of the study was to determine the effect of changes in body condition during the grazing season on insulin resistance and the expression of genes associated with obesity and insulin resistance in subcutaneous adipose tissue (SAT). Sixteen Finnhorse mares were grazing either on cultivated high-yielding pasture (CG) or semi-natural grassland (NG) from the end of May to the beginning of September. Body measurements, intravenous glucose tolerance test (IVGTT), and neck and tailhead SAT gene expressions were measured in May and September. At the end of grazing, CG had higher median body condition score (7 vs. 5.4, interquartile range 0.25 vs. 0.43; P=0.05) and body weight (618 kg vs. 572 kg ± 10.21 (mean ± SEM); P=0.02), and larger waist circumference (P=0.03) than NG. Neck fat thickness was not different between treatments. However, tailhead fat thickness was smaller in CG compared to NG in May (P=0.04), but this difference disappeared in September. Greater basal and peak insulin concentrations, and faster glucose clearance rate (P=0.03) during IVGTT were observed in CG compared to NG in September. A greater decrease in plasma non-esterified fatty acids during IVGTT (P<0.05) was noticed in CG compared to NG after grazing. There was down-regulation of insulin receptor, retinol binding protein 4, leptin, and monocyte chemoattractant protein-1, and up-regulation of adiponectin (ADIPOQ), adiponectin receptor 1 and stearoyl-CoA desaturase (SCD) gene expressions in SAT of both groups during the grazing season (P<0.05). Positive correlations were observed between ADIPOQ and its receptors and between SCD and ADIPOQ in SAT (P<0.01). In conclusion, grazing on CG had a moderate effect on responses during IVGTT, but did not trigger insulin resistance. Significant temporal differences in gene expression profiles were observed during the grazing season.

Serum and tissue CD23, IL-15, and FasL in cow's-milk protein-sensitive enteropathy and in coeliac disease.

OBJECTIVES: The aim of the study was to explore pathogenesis and find new serum markers for cow's-milk-sensitive enteropathy (CMSE) and coeliac disease (CD). We assessed the intestinal expression and serum concentration of CD23, IL-15, and FasL. We hypothesised that the serum levels of CD23, a protein expressed in the lymphoid follicles, would be associated with lymphonodular hyperplasia (LNH), a feature characteristic of CMSE. We also presumed that interleukin (IL)-15 and FasL, functionally connected with proliferation and apoptosis of the intraepithelial lymphocytes (IELs), would relate with the increased numbers of IELs present in both CMSE and CD. METHODS: Twenty-three children with CMSE, 20 with untreated CD, and 14 controls were studied for CD3, α/ß- and γ/δ-expressing IELs, and for duodenal and ileal expression of CD23, FasL, and IL-15 by immunohistochemistry, and for serum concentration of sCD23, sFasL, and sIL-15 by enzyme-linked immunosorbent assay. RESULTS: There was a trend for increase in sCD23 serum levels in untreated CMSE and in CD (P = 0.074; P = 0.077). CD23 was expressed in the mucosal germinal centres, but sCD23 was not related to presence of LNH. In CMSE, there was a trend for increase in serum sFasL (P = 0.07) and high levels associated with LNH (P = 0.025) and correlated with the IEL numbers (P < 0.05). Mucosal high endothelial venules adjacent to lymphoid follicles showed an intensive FasL expression. CONCLUSIONS: Serum sCD23 shows a trend of increment in CMSE and CD, and in the latter, sCD23 level may provide information about the severity of villous atrophy. In CMSE, high serum sFasL indicates both LNH and an increase of IELs, suggesting importance of FasL-mediated mechanisms in the pathogenesis of these features characteristic of CMSE. Further studies are necessary to evaluate whether intensive FasL expression in mucosal high endothelial venules presents a regulatory element in mucosal immunity.

Fas/Fas ligand-mediated apoptosis in different cell lineages and functional compartments of human lymph nodes.

We have optimized an immunohistochemical double-staining method combining immunohistochemical lymphocyte lineage marker detection and apoptosis detection with terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling. The method was used to trace Fas-mediated apoptosis in human reactive lymph nodes according to cell lineage and anatomical location. In addition to Fas, we also studied the expression of Fas ligand (FasL), CD3, CD20, CD19, CD23, and CD68 of apoptotic cells. The presence of simultaneous Fas and FasL positivity indicated involvement of activation-induced death in the induction of paracortical apoptosis. FasL expression in the high endothelial venules might be an inductor of apoptosis of Fas-positive lymphoid cells. In addition to B-lymphocyte apoptosis in the germinal centers, there was often a high apoptosis rate of CD23-expressing follicular dendritic cells. In summary, our double-staining method provides valuable new information about the occurrence and mechanisms of apoptosis of different immune cell types in the lymph node compartments. Among other things, we present support for the importance of Fas/FasL-mediated apoptosis in lymph node homeostasis.

High endothelial venules of the lymph nodes express Fas ligand.

Fas (CD95, APO-1) is widely expressed on lymphatic cells, and by interacting with its natural ligand (Fas-L), Fas induces apoptosis through a complex caspase cascade. In this study we sought to survey Fas-L expression in vascular and sinusoidal structures of human reactive lymph nodes. Immunohistochemical Fas-L expression was present in all paracortical high endothelial venules (HEVs), in cells lining the marginal sinus wall, and in a few lymphocytes, but only occasionally in non-HEV vascular endothelium. In the paracortical zone over 60% of all vessels and all paracortical HEVs showed Fas-L expression, whereas in the medullary zone less than 10% of the blood vessels were stained with Fas-L. Normal vessels outside lymph nodes mostly showed no Fas-L expression. We show that in human reactive lymph nodes Fas-L expression is predominantly present in HEVs. Because the circulating lymphocytes gain entry to nodal parenchyma by transendothelial migration through HEVs, the suggested physiological importance of Fas-L expression in these vessels lies in the regulation of lymphocyte access to lymph node parenchyma by possibly inducing Fas/Fas-L mediated apoptosis of activated Fas-expressing lymphoid cells. The Fas-L expressing cells in the marginal sinus might have a similar function for cells accessing the node in afferent lymph.