Response to Incremental Replacement of Palm Oil with Fish Oil in Starter Diet on Growth Performance, Plasma Metabolites, Ruminal Fermentation, and Behavior of Dairy Calves.

The objective of this study was to evaluate the effects of the incremental levels of n-3 fatty acids (FA) in starter feed (SF) on growth and metabolic performance of milk-fed calves. From day 3 of age, 30 female calves (39.4 ± 3.1 kg of body weight) were randomly assigned to one of three dietary treatments: (1) SF supplemented with 3.3% palm fatty acids (PO), (2) SF supplemented with 1.7% of PO and 1.9% fish oil (PFO), or (3) SF supplemented with 3.9% fish oil (FO). Chopped straw (7.5% of DM) was included in the SF of all treatments as total mixed ration (TMR). Diets had similar energy and protein contents. Total n-3 FA (% of total FA) and n-6/n-3 of PO, PFO, and FO were 1.90, 6.80, and 11.8 and 15.5, 4.50, and 2.70, respectively. The BW was greater for calves receiving FO (60.2 ± 0.3 kg) compared with PFO (58.7 ± 0.3 kg; p = 0.007) and tended to be greater for calves receiving FO vs. PO (59.0 ± 0.3 kg; p = 0.050). Because there was no interaction effect between diet × week of experiment, the greater BW of FO could not be attributed to the dietary treatment. Accordingly, average daily gain, total dry matter intake (DMI), starter DMI, and gain to intake ratio (G:FI) did not differ among dietary treatments during the entire period of the study (p > 0.05). Dietary treatments did not impact body size parameters such as body length, body girth, withers height, heart girth, hip height, and width (p > 0.05). Neither ruminal fermentation parameters nor blood variables were influenced by supplementing the types of oil at different time points. Calves' behavioral parameters, such as standing, lying, eating, and ruminating, were not influenced by different dietary treatments (p > 0.05). The number of days with abnormal fecal score was not different among dietary groups (p > 0.05). Overall, our findings suggest that changing the n-6/n-3 ratio in starter feed by incremental replacement of palm fatty acid with fish oil at a moderate supplemental level of ~3% of DM may not affect the growth and metabolic performance of young calves under non-challenged conditions.

Effects of isoflurane anaesthesia depth and duration on renal function measured with [99mTc]Tc-mercaptoacetyltriglycine SPECT in mice.

BACKGROUND: The influence of anaesthetic depth and the potential influence of different anaesthetic beds and thus different handling procedures were investigated in 86 severe combined immunodeficient (SCID) mice using semi-stationary dynamic single photon emission computed tomography (SPECT) for kidney scintigraphy. Therefore, isoflurane concentrations were adjusted using respiratory rate for low (80-90 breath/min) and deep anaesthesia (40-45 breath/min). At low anaesthesia, we additionally tested the influence of single bed versus 3-mouse bed hotel; the hotel mice were anaesthetized consecutively at ~ 30, 20, and 10 min before tracer injections for positions 1, 2, and 3, respectively. Intravenous [99mTc]Tc-MAG3 injection of ~ 28 MBq was performed after SPECT start. Time-activity curves were used to calculate time-to-peak (Tmax), T50 (50% clearance) and T25 (75% clearance). RESULTS: Low and deep anaesthesia corresponded to median isoflurane concentrations of 1.3% and 1.5%, respectively, with no significant differences in heart rate (p = 0.74). Low anaesthesia resulted in shorter aortic blood clearance half-life (p = 0.091) and increased relative renal tracer influx rate (p = 0.018). A tendency toward earlier Tmax occurred under low anaesthesia (p = 0.063) with no differences in T50 (p = 0.40) and T25 (p = 0.24). Variance increased with deep anaesthesia. Compared to single mouse scans, hotel mice in position 1 showed a delayed Tmax, T50, and T25 (p < 0.05 each). Furthermore, hotel mice in position 1 showed delayed Tmax versus position 3, and delayed T50 and T25 versus position 2 and 3 (p < 0.05 each). No difference occurred between single bed and positions 2 (p = 1.0) and 3 (p = 1.0). CONCLUSIONS: Deep anaesthesia and prolonged low anaesthesia should be avoided during renal scintigraphy because they result in prolonged blood clearance half-life, delayed renal influx and/or later Tmax. Vice versa, low anaesthesia with high respiratory rates of 80-90 rpm and short duration (≤ 20 min) should be preferred to obtain representative data with low variance.

The type of lipid supplement has crucial implications for forage particle size in calf starter diets.

BACKGROUND: Forage inclusion in starters of young dairy calves has become an acceptable strategy in the last decade. To compensate for the lower energy provided by forage, concurrent lipid supplementation can be proposed. However, ruminal microbial activity and forage digestibility may be decreased by lipid supplementation. We hypothesized that the composite effect of forage and lipid supplements may be dependent on forage particle size and the type of lipid supplement. Therefore, we evaluated the effect of long (LP; geometric mean, 4.97 mm) vs. short alfalfa hay particle sizes (SP; geometric mean, 1.26 mm) with either soybean oil (SBO) or palm fatty acids (PLF) as lipid source in a 2 × 2 factorial design with treatments SP-SBO, SP-PLF, LP-SBO, and LP-PLF. Treatments (n = 13 with 6 males and 7 females each) were offered to Holstein calves (3 days old) with equal amounts of lipid (25 g/kg DM) throughout the experimental period. The milk offering scheme (d 1 to 53) was equal for all groups. Data collection continued until 20 d post-weaning. RESULTS: Interaction between forage particle size and lipid supplement was significant for the following readouts: the highest and lowest starter intakes during the pre-weaning period occurred in LP-PLF and LP-SBO, respectively. This was associated with similarly contrasting changes in average daily gain (ADG) during the post-weaning period, body weight at the end of experiment, withers height, digestibility of organic matter and neutral detergent fiber, and blood serum concentrations of glucose, beta-hydroxybutyrate, and insulin during the pre-weaning period. During both pre- and post-weaning periods, the highest and lowest urinary excretion of allantoin and total purine derivatives, representing microbial protein synthesis, were observed in LP-PLF and LP-SBO, respectively, indicating that those diets were most and least favorable for rumen development. Irrespective of forage particle size, supplemental SBO vs. PLF increased serum malondialdehyde as an oxidative stress indicator across periods, increased blood urea nitrogen and feed efficiency in the pre-weaning period, and reduced hip height during the post-weaning period. CONCLUSIONS: It can be concluded that feeding a rumen-inert, mostly saturated fatty acid source with alfalfa hay as long particle size is recommended with view on performance, whereas a combination soybean oil rich in unsaturated fatty acids should not be provided to milk-fed Holstein calves together with long particle forage. Feeding soybean oil and alfalfa hay as long particles is not advisable mainly due to lower starter consumption and impaired development of ruminal function. If dietary supplementation of soybean oil is applied, incorporation of forage as small particles should be preferred to support rumen development.

Acid tolerance of lactate-utilizing bacteria of the order Bacteroidales contributes to prevention of ruminal acidosis in goats adapted to a high-concentrate diet.

The rapid accumulation of organic acids, particularly lactate, has been suggested as the main cause of ruminal acidosis (RA) for ruminants fed high-concentrate diets. Previous research has shown that a gradual shift from low-to high-concentrate diets within 4 to 5 weeks effectively reduces the risk for RA. However, the mechanisms remain unknown. In this study, 20 goats were randomly allocated into four groups (n = 5) and fed with a diet containing a weekly increasing concentrate portion of 20%, 40%, 60%, and 80% over 28 d. At d 7, 14, 21, and 28, one group (named C20, C40, C60, and C80 according to the last concentrate level that they received) was killed and the ruminal microbiome was collected. Ruminal acidosis was not detected in any of the goats during the experiment. Nonetheless, ruminal pH dropped sharply from 6.2 to 5.7 (P < 0.05) when dietary concentrate increased from 40% to 60%. A combined metagenome and metatranscriptome sequencing approach identified that this was linked to a sharp decrease in the abundance and expression of genes encoding nicotinamide adenine dinucleotide (NAD)-dependent lactate dehydrogenase (nLDH), catalyzing the enzymatic conversion of pyruvate to lactate (P < 0.01), whereas the expression of two genes encoding NAD-independent lactate dehydrogenase (iLDH), catalyzing lactate oxidation to pyruvate, showed no significant concomitant change. Abundance and expression alterations for nLDH- and iLDH-encoding genes were attributable to bacteria from Clostridiales and Bacteroidales, respectively. By analyzing the gene profiles of 9 metagenome bins (MAG) with nLDH-encoding genes and 5 MAG with iLDH-encoding genes, we identified primary and secondary active transporters as being the major types of sugar transporter for lactate-producing bacteria (LPB) and lactate-utilizing bacteria (LUB), respectively. Furthermore, more adenosine triphosphate was required for the phosphorylation of sugars to initiate their catabolic pathways in LPB compared to LUB. Thus, the low dependence of sugar transport systems and catabolic pathways on primary energy sources supports the acid tolerance of LUB from Bacteroidales. It favors ruminal lactate utilization during the adaptation of goats to a high-concentrate diet. This finding has valuable implications for the development of measures to prevent RA.

Low Magnesium Concentration Enforces Bone Calcium Deposition Irrespective of 1,25-Dihydroxyvitamin D3 Concentration.

Efficient coordination between Mg2+ and vitamin D maintains adequate Ca2+ levels during lactation. This study explored the possible interaction between Mg2+ (0.3, 0.8, and 3 mM) and 1,25-dihydroxyvitamin D3 (1,25D; 0.05 and 5 nM) during osteogenesis using bovine mesenchymal stem cells. After 21 days, differentiated osteocytes were subjected to OsteoImage analysis, alkaline phosphatase (ALP) activity measurements, and immunocytochemistry of NT5E, ENG (endoglin), SP7 (osterix), SPP1 (osteopontin), and the BGLAP gene product osteocalcin. The mRNA expression of NT5E, THY1, ENG, SP7, BGLAP, CYP24A1, VDR, SLC41A1, SLC41A2, SLC41A3, TRPM6, TRPM7, and NIPA1 was also assessed. Reducing the Mg2+ concentration in the medium increased the accumulation of mineral hydroxyapatite and ALP activity. There was no change in the immunocytochemical localization of stem cell markers. Expression of CYP24A1 was higher in all groups receiving 5 nM 1,25D. There were tendencies for higher mRNA abundance of THY1, BGLAP, and NIPA1 in cells receiving 0.3 mM Mg2+ and 5 nM 1,25D. In conclusion, low levels of Mg2+ greatly enhanced the deposition of bone hydroxyapatite matrix. The effect of Mg2+ was not modulated by 1,25D, although the expression of certain genes (including BGLAP) tended to be increased by the combination of low Mg2+ and high 1,25D concentrations.

Multilineage Differentiation Potential of Equine Adipose-Derived Stromal/Stem Cells from Different Sources.

The investigation of multipotent stem/stromal cells (MSCs) in vitro represents an important basis for translational studies in large animal models. The study's aim was to examine and compare clinically relevant in vitro properties of equine MSCs, which were isolated from abdominal (abd), retrobulbar (rb) and subcutaneous (sc) adipose tissue by collagenase digestion (ASCs-SVF) and an explant technique (ASCs-EXP). Firstly, we examined proliferation and trilineage differentiation and, secondly, the cardiomyogenic differentiation potential using activin A, bone morphogenetic protein-4 and Dickkopf-1. Fibroblast-like, plastic-adherent ASCs-SVF and ASCs-EXP were obtained from all sources. The proliferation and chondrogenic differentiation potential did not differ significantly between the isolation methods and localizations. However, abd-ASCs-EXP showed the highest adipogenic differentiation potential compared to rb- and sc-ASCs-EXP on day 7 and abd-ASCs-SVF a higher adipogenic potential compared to abd-ASCs-EXP on day 14. Osteogenic differentiation potential was comparable at day 14, but by day 21, abd-ASCs-EXP demonstrated a higher osteogenic potential compared to abd-ASCs-SVF and rb-ASCs-EXP. Cardiomyogenic differentiation could not be achieved. This study provides insight into the proliferation and multilineage differentiation potential of equine ASCs and is expected to provide a basis for future preclinical and clinical studies in horses.

Growth performance, nutrient digestibility, and ruminal fermentation of dairy calves fed starter diets with alfalfa hay versus corn silage as forage and soybean oil versus palm fatty acids as fat source.

The present study was intended to evaluate the effect of forage source (alfalfa hay; ALF vs. corn silage; CS) along with a supplemental fat source (soybean oil; SO vs. rumen-inert palm fatty acids; PF) on growth performance, nutrient digestibility, and ruminal fermentation in dairy calves. Forty-eight new-born Holstein female calves (3 d old) were assigned to one of 4 treatments: (1) alfalfa hay with soybean oil (ALF-SO); (2) alfalfa hay with palm fatty acids (ALF-PF); (3) corn silage with soybean oil (CS-SO); (4) corn silage with palm fatty acids (CS-PF). Starter diets had equal amounts of forage (100 g/kg dry matter; DM) and fat source (30 g/kg DM). Calves were fed a constant amount of milk (d 1 to 63) and had ad libitum access to water and starters (d 1 to 83). The lowest and greatest starter intakes during the preweaning period occurred in ALF-SO and CS-PF, respectively. This coincided with forage × fat source interaction for average daily gain (ADG) during preweaning. The forage source affected total DM intake and ADG over the entire period, body weight (BW) at weaning, and final BW with greater values in calves that received CS compared with ALF. The concentrations of total short-chain fatty acids and butyrate were increased, whereas concentration of acetate and acetate:propionate ratio were decreased in the rumen of calves fed CS compared with ALF. Feeding CS increased urinary excretion of allantoin and, as a trend, total purine derivatives (PD) and estimated microbial protein synthesis in comparison with ALF. The fat source affected starter intake, ADG, and BW postweaning with the highest values in PF. The digestibility of neutral detergent fiber, crude protein and, as a trend, organic matter were higher in calves fed PF compared with SO. Calves fed PF had lower ruminal ammonia-N concentration and urinary N excretion and greater urinary excretion of allantoin and total PD. Calves receiving SO had a lower ruminal protozoa population. In conclusion, supplementing starter diets with CS and PF is superior to ALF and SO. Interaction of the positive effects of CS and PF on performance underlines that concurrent supplementation of CS with PF is especially recommendable in young calves before weaning.

Effects of Oil Supplements on Growth Performance, Eating Behavior, Ruminal Fermentation, and Ruminal Morphology in Lambs during Transition from a Low- to a High-Grain Diet.

The objectives of this study were to investigate the effect of a maximum recommended oil supplementation on growth performance, eating behavior, ruminal fermentation, and ruminal morphological characteristics in growing lambs during transition from a low- to a high-grain diet. A total of 21 Afshari male lambs with an initial body weight (BW) of 41.4 ± 9.1 kg (mean ± SD) and at 5−6 months of age were randomly assigned to one of three dietary treatments (n = 7 per group), including (1) a grain-based diet with no fat supplement (CON), (2) CON plus 80 g/d of prilled palm oil (PALM), and (3) CON plus 80 g/d soybean oil (SOY); oils were equivalent to 50 g/kg of dry matter based on initial dry matter intake (DMI). All lambs were adapted to the high-grain diet for 21 d. In the adaptation period, lambs were gradually transferred to a dietary forage-to-concentrate ratio of 20:80 by replacing 100 g/kg of the preceding diet every 3 d. Thereafter, lambs were fed experimental diets for another 22 days. Fat-supplemented lambs had greater DMI, body weight (BW), and average daily gain (ADG), with a lower feed to gain ratio (p < 0.05), compared to CON lambs. The highest differences of DMI between fat-supplemented and CON-lambs were observed in week 3 of the adaptation period (p = 0.010). PALM- or SOY-supplementation lowered DM and NDF digestibility compared with CON (p < 0.05), and SOY caused the lowest organic matter (OM) digestibility compared with CON and PALM lambs (62.0 vs. 67.6 and 66.9; p < 0.05). Ruminal pH was higher for PALM and SOY compared with CON (p = 0.018). Lambs in SOY tended to have the highest ammonia-N concentrations (p = 0.075), together with a trend for higher concentrations of propionic acid, at the expense of acetic acid in ruminal fluid, on the last day of the adaptation period (diet × time, p = 0.079). Fat-supplemented lambs had lower isovaleric and valeric acid concentrations compared with CON on d 40 (diet × time, p < 0.05). PALM and SOY-fed lambs had a longer eating time (min/d and min/kg of DMI), chewing activity (min/d), meal frequency (n), and duration of eating the first and second meals after morning feeding (p < 0.05), and the largest meal size (p < 0.001). Fat supplemented lambs had greater ruminal papillary length (p < 0.05) and width (p < 0.01), and thicker submucosal, epithelial, and muscle layers, compared with the CON (p < 0.01). Blood metabolites were not influenced by dietary treatments (p > 0.05). The results from this study suggest that fat supplementation to high-grain diets may improve the development of ruminal epithelia and modify ruminal fermentation via optimized eating behavior or the direct effect of oils on the ruminal environment, resulting in better growth performance in growing lambs.

Comparison of Sources and Methods for the Isolation of Equine Adipose Tissue-Derived Stromal/Stem Cells and Preliminary Results on Their Reaction to Incubation with 5-Azacytidine.

Physiological particularities of the equine heart justify the development of an in vitro model suitable for investigations of the species-specific equine cardiac electrophysiology. Adipose tissue-derived stromal/stem cells (ASCs) could be a promising starting point from which to develop such a cardiomyocyte (CM)-like cell model. Therefore, we compared abdominal, retrobulbar, and subcutaneous adipose tissue as sources for the isolation of ASCs applying two isolation methods: the collagenase digestion and direct explant culture. Abdominal adipose tissue was most suitable for the isolation of ASCs and both isolation methods resulted in comparable yields of CD45-/CD34-negative cells expressing the mesenchymal stem cell markers CD29, CD44, and CD90, as well as pluripotency markers, as determined by flow cytometry and real-time quantitative PCR. However, exposure of equine ASCs to 5-azacytidine (5-AZA), reportedly inducing CM differentiation from rats, rabbits, and human ASCs, was not successful in our study. More precisely, neither the early differentiation markers GATA4 and NKX2-5, nor the late CM differentiation markers TNNI3, MYH6, and MYH7 were upregulated in equine ASCs exposed to 10 µM 5-AZA for 48 h. Hence, further work focusing on the optimal conditions for CM differentiation of equine stem cells derived from adipose tissue, as well as possibly from other origins, are needed.

Paracellular intestinal permeability of chickens induced by DON and/or C. jejuni is associated with alterations in tight junction mRNA expression.

Toxins, antigens, and harmful pathogens continuously challenge the intestinal mucosa. Therefore, regulation of the intestinal barrier is crucial for the maintenance of mucosal homeostasis and gut health. Intercellular complexes, namely, tight junctions (TJs), regulate paracellular permeability. TJs are mainly composed of claudins (CLDN), occludin (OCLN), tight junction associated MARVEL-domain proteins (TAMPS), the scaffolding zonula occludens (ZO) proteins and junction-adhesion molecules (JAMs). Different studies have shown that a Campylobacter infection can lead to a phenomenon so-called "leaky gut", including the translocation of luminal bacteria to the underlying tissue and internal organs. Based on the effects of C. jejuni on the chicken gut, we hypothesize that impacts on TJ proteins play a crucial role in the destructive effects of the intestinal barrier. Likewise, the mycotoxin deoxynivalenol (DON) can also alter gut permeability in chickens. Albeit DON and C. jejuni are widely distributed, no data are available on their effect on the tight junctions' barrier in the broiler intestine and consequences for permeability. Therefore, the aim of this study was to analyze the interaction between DON and C. jejuni on the gut barrier by linking permeability with gene expression of TJ proteins and to determine the relationships between the measurements. Following oral infection of birds with C. jejuni NCTC 12744 at 14 days of age, we demonstrate that the co-exposure with DON has considerable consequences on gut permeability as well as on gut TJ mRNA expression. Co-exposure of DON and C. jejuni enhanced the negative effect on paracellular permeability of the intestine, which was also noticed for the bacteria or the mycotoxin alone by the Ussing chamber technique at certain time points in both jejunum and caecum. Furthermore, the increased paracellular permeability was associated with significant changes in TJ mRNA expression in the small and large intestine. The actual study demonstrates that co-exposure of broiler chickens to DON and C. jejuni resulted in a decreased barrier function via up-regulation of pore-forming tight junctions (CLDN7 and CLDN10), as well as the cytosolic TJ protein occludin (OCLN) that can shift to various paracellular locations and are therefore able to alter the epithelial permeability. These findings indicate that the co-exposure of broiler chickens to DON and C. jejuni affects the paracellular permeability of the gut by altering the tight junction proteins. Furthermore, analysing of correlations between TJs revealed that the mRNA expression levels of most tight junctions were correlated with each other in both jejunum and caecum. Finally, the findings indicate that the molecular composition of tight junctions can be used as a marker for gut health and integrity.

Dietary methionine source alters the lipidome in the small intestinal epithelium of pigs.

Methionine (Met) as an essential amino acid has key importance in a variety of metabolic pathways. This study investigated the influence of three dietary Met supplements (0.21% L-Met, 0.21% DL-Met and 0.31% DL-2-hydroxy-4-(methylthio)butanoic acid (DL-HMTBA)) on the metabolome and inflammatory status in the small intestine of pigs. Epithelia from duodenum, proximal jejunum, middle jejunum and ileum were subjected to metabolomics analysis and qRT-PCR of caspase 1, NLR family pyrin domain containing 3 (NLRP3), interleukins IL1ß, IL8, IL18, and transforming growth factor TGFß. Principal component analysis of the intraepithelial metabolome revealed strong clustering of samples by intestinal segment but not by dietary treatment. However, pathway enrichment analysis revealed that after L-Met supplementation polyunsaturated fatty acids (PUFA) and tocopherol metabolites were lower across small intestinal segments, whereas monohydroxy fatty acids were increased in distal small intestine. Pigs supplemented with DL-HMTBA showed a pronounced shift of secondary bile acids (BA) and sphingosine metabolites from middle jejunum to ileum. In the amino acid super pathway, only histidine metabolism tended to be altered in DL-Met-supplemented pigs. Diet did not affect the expression of inflammation-related genes. These findings suggest that dietary supplementation of young pigs with different Met sources selectively alters lipid metabolism without consequences for inflammatory status.

Characterization and differentiation potential of mesenchymal stem cells isolated from multiple canine adipose tissue sources.

BACKGROUND: Mesenchymal stem cells (MSCs) are undifferentiated cells that can give rise to a mesoderm lineage. Adipose-derived MSCs are an easy and accessible source for MSCs isolation, although each source of MSC has its own advantages and disadvantages. Our study identifies a promising source for the isolation and differentiation of canines MSCs. For this purpose, adipose tissue from inguinal subcutaneous (SC), perirenal (PR), omental (OM), and infrapatellar fat pad (IPFP) was isolated and processed for MSCs isolation. In the third passage, MSCs proliferation/metabolism, surface markers expression, in vitro differentiation potential and quantitative reverse transcription PCR (CD73, CD90, CD105, PPARγ, FabP4, FAS, SP7, Osteopontin, and Osteocalcin) were evaluated. RESULTS: Our results showed that MSCs derived from IPFP have a higher proliferation rate, while OM-derived MSCs have higher cell metabolism. In addition, MSCs from all adipose tissue sources showed positive expression of CD73 (NT5E), CD90 (THY1), CD105 (ENDOGLIN), and very low expression of CD45. The isolated canine MSCs were successfully differentiated into adipogenic and osteogenic lineages. The oil-red-O quantification and adipogenic gene expression (FAS, FabP4, and PPARγ) were higher in OM-derived cells, followed by IPFP-MSCs. Similarly, in osteogenic differentiation, alkaline phosphatase activity and osteogenic gene (SP7 and Osteocalcin) expression were higher in OM-derived MSCs, while osteopontin expression was higher in PR-derived MSCs. CONCLUSION: In summary, among all four adipose tissue sources, OM-derived MSCs have better differentiation potential toward adipo- and osteogenic lineages, followed by IPFP-MSCs. Interestingly, among all adipose tissue sources, MSCs derived from IPFP have the maximum proliferation potential. The characterization and differentiation potential of canine MSCs isolated from four different adipose tissue sources are useful to assess their potential for application in regenerative medicine.

Expression of proposed methionine transporters along the gastrointestinal tract of pigs and their regulation by dietary methionine sources.

BACKGROUND: Given the key role of methionine (Met) in biological processes like protein translation, methylation, and antioxidant defense, inadequate Met supply can limit performance. This study investigated the effect of different dietary Met sources on the expression profile of various Met transporters along the gastrointestinal tract (GIT) of pigs. METHODS: A total of 27 pigs received a diet supplemented with 0.21% DL-Met, 0.21% L-Met, or 0.31% DL-2-hydroxy-4-(methylthio)butanoic acid (DL-HMTBA). Changes in mRNA expression of B0AT1, ATB0,+, rBAT, ASCT2, IMINO, LAT4, y+LAT1, LAT2, and SNAT2 were evaluated in the oral mucosa, cardia, fundus, pylorus, duodenum, proximal jejunum, middle jejunum, ileum, cecum, proximal colon, and distal colon, complemented by protein expression analysis of B0AT1, ASCT2, LAT2, and LAT4. RESULTS: Expression of all investigated transcripts differed significantly along the GIT. B0AT1, rBAT, y+LAT1, LAT2, and LAT4 showed strongest mRNA expression in small intestinal segments. ASCT2, IMINO, and SNAT2 were similarly expressed along the small and large intestines but expression differed in the oral mucosa and stomach. ATB0,+ showed highest mRNA expression in large intestinal tissues, cardia, and pylorus. In pigs fed DL-Met, mRNA expression of ASCT2 was higher than in pigs fed DL-HMTBA in small intestinal tissues and mRNA expression of IMINO was lower than in pigs fed L-Met in large intestinal tissues. Dietary DL-HMTBA induced a stronger mRNA expression of basolateral uptake systems either in the small (LAT2) or large (y+LAT1) intestine. Protein expression of B0AT1 was higher in the middle jejunum and ileum in pigs fed DL-Met when compared with the other Met supplements. LAT4 expression was higher in pigs fed DL-HMTBA when compared with DL-Met (small intestine) and L-Met (small intestine, oral mucosa, and stomach). CONCLUSION: A high expression of several Met transporters in small intestinal segments underlines the primary role of these segments in amino acid absorption; however, some Met transporters show high transcript and protein levels also in large intestine, oral mucosa, and stomach. A diet containing DL-Met has potential to increase apical Met transport in the small intestine, whereas a diet containing DL-HMTBA has potential to increase basolateral Met transport in the small intestine and, partly, other gastrointestinal tissues.

The Combined Influence of Magnesium and Insulin on Central Metabolic Functions and Expression of Genes Involved in Magnesium Homeostasis of Cultured Bovine Adipocytes.

At the onset of lactation, dairy cows suffer from insulin resistance, insulin deficiency or both, similar to human diabetes, resulting in lipolysis, ketosis and fatty liver. This work explored the combined effects of different levels of magnesium (0.1, 0.3, 1 and 3 mM) and insulin (25, 250 and 25,000 pM) on metabolic pathways and the expression of magnesium-responsive genes in a bovine adipocyte model. Magnesium starvation (0.1 mM) and low insulin (25 pM) independently decreased or tended to decrease the accumulation of non-polar lipids and uptake of the glucose analog 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-6-deoxyglucose (6-NBDG). Activity of glycerol 3-phosphate dehydrogenase (GPDH) was highest at 25 pM insulin and 3 mM magnesium. Expression of SLC41A1 and SLC41A3 was reduced at 0.1 mM magnesium either across insulin concentrations (SLC41A1) or at 250 pM insulin (SLC41A3). MAGT1 expression was reduced at 3 mM magnesium. NIPA1 expression was reduced at 3 mM and 0.1 mM magnesium at 25 and 250 pM insulin, respectively. Expression of SLC41A2, CNNM2, TRPM6 and TRPM7 was not affected. We conclude that magnesium promotes lipogenesis in adipocytes and inversely regulates the transcription of genes that increase vs. decrease cytosolic magnesium concentration. The induction of GAPDH activity by surplus magnesium at low insulin concentration can counteract excessive lipomobilization.

Changes in the Relationship between Ionized and Total Calcium in Clinically Healthy Dairy Cows in the Period around Calving.

We aimed to establish a model for prediction of iCa from tCa, using multivariable regressions with diverse blood constituents. Blood was taken from 14 cows at days -2, 0, 2, 4, 7, and 14 relative to parturition. Cows were clinically healthy, and no hypocalcaemia prophylaxis and treatment were applied. Total calcium and further parameters were determined from frozen serum. Ionized calcium, blood gases, and electrolytes were determined from heparin-stabilized blood samples. Linear regression between iCa and tCa was estimated. Precision improved only slightly using a multivariable model. Best precision was achieved when estimating the iCa:tCa ratio from other blood constituents. To identify the reason behind the poorly predictive value of tCa for iCa, the relative changes of iCa and tCa around calving were calibrated to the respective values of day -2 (=100%) for each cow. An increase in the iCa:tCa ratio was observed from 0.43 at day -2 to 0.48 at day 0, followed by a gradual decrease towards 0.43 at day 7. We conclude that routine measurement of iCa should be implemented in the diagnosis of hypocalcaemia. An optimized estimate of iCa from tCa with non-esterified fatty acids (NEFA), beta-hydroxybutyric acid, cholesterol, and phosphorous as co-predictors is still poorly satisfying.

Establishment of a novel probe-based RT-qPCR approach for detection and quantification of tight junctions reveals age-related changes in the gut barriers of broiler chickens.

Tight junctions (TJs) play a dominant role in gut barrier formation, therefore, resolving the structures of TJs in any animal species is crucial but of major importance in fast growing broilers. They are regulated in molecular composition, ultrastructure and function by intracellular proteins and the cytoskeleton. TJ proteins are classified according to their function into barrier-forming, scaffolding and pore-forming types with deductible consequences for permeability. In spite of their importance for gut health and its integrity limited studies have investigated the TJs in chickens, including the comprehensive evaluation of TJs molecular composition and function in the chicken gut. In the actual study sequence-specific probes to target different TJ genes (claudin 1, 3, 5, 7, 10, 19, zonula occludens 1 (ZO1), occludin (OCLN) and tricellulin (MD2)) were designed and probe-based RT-qPCRs were newly developed. Claudin (CLDN) 1, 5, ZO1 and CLDN 3, 7, MD2 were engulfed in multiplex RT-qPCRs, minimizing the number of separate reactions and enabling robust testing of many samples. All RT-qPCRs were standardized for chicken jejunum and caecum samples, which enabled specific detection and quantification of the gene expression. Furthermore, the newly established protocols were used to investigate the age developmental changes in the TJs of broiler chickens from 1-35 days of age in the same organ samples. Results revealed a significant increase in mRNA expression between 14 and 21days of age of all tested TJs in jejunum. However, in caecum, mRNA expression of some TJs decreased after 1 day of age whereas some TJs mRNA remained constant till 35 days of age. Taken together, determining the segment-specific changes in the expression of TJ- proteins by RT-qPCR provides a deeper understanding of the molecular mechanisms underpinning pathophysiological changes in the gut of broiler chickens with various etiologies.

Effects of dietary oil sources (sunflower and fish) on fermentation characteristics, epithelial gene expression and microbial community in the rumen of lambs fed a high-concentrate diet.

The feeding of high-concentrate diets commonly results in lowered pH and ruminal dysbiosis which cause shifts in uptake dynamics of short-chain fatty acids (SCFA) and altered epithelial function. Therefore, the current study evaluated the effect of dietary polyunsaturated fatty acids (PUFA) on ruminal fermentation products, gene expression in the ruminal epithelium and the associated changes in ruminal microorganisms in lambs fed a high-concentrate diet. Twenty-six Afshari lambs adapted to a high-concentrate diet during a completely randomised design were fed with a basal diet supplemented with 100 g oil supplement (OS; 60 g sunflower oil and 40 g fish oil) for 10 (OS10), 20 (OS20) and 30 (OS30) d, respectively (n = 6). Lambs with no oil supplementation (OS0, n = 8) were considered as control and slaughtered at d 0 of the experiment, and the remaining lambs were slaughtered at 10, 20 and 30 d on feed. After slaughter, ruminal digesta was collected for evaluating fermentation and microbial community. Ruminal papillae were taken for assessment of epithelial gene expression. Compared with OS0 lambs, supplemental PUFA in OS30 lambs tended to decrease total SCFA concentration with decreased acetic and increased propionic acid concentrations. Acetate:propionate ratios were decreased and ruminal pH was increased in OS20 and OS30 lambs compared to OS0. All groups with included OS had decreased concentrations of iso-valeric and valeric acids compared to OS0. Relative mRNA abundance of monocarboxylate transporter isoforms 1 and 4, insulin-like growth factor binding protein 3, sterol regulatory element-binding proteins 1 and 2 decreased with increasing OS duration. The relative abundance of 3-hydroxy-3-methylglutaryl-CoA synthase 1 mRNA transcript was higher for OS10 and OS20 lambs relative to OS0 lambs. OS20 and OS30 showed a decrease of lipopolysaccharide binding protein mRNA expression compared with OS0. Feeding supplemental PUFA decreased Ciliate protozoa and increased Butyrivibrio fibrisolvens in OS20 and OS30 lambs, whereas Megasphaera elsdenii was increased in OS30 lambs. In conclusion, combined supplementation of sunflower and fish oil to a high-concentrate diet affects the ruminal microbial community with prominent decreases in ruminal ciliate protozoa and increases in B. fibrisolvens and M. elsdenii. These results lead to a more stabilised ruminal pH and a fermentation shift towards more propionate generation. Consideration of nutrients digestion will help to fully understand the benefits of feeding PUFA with a high-concentrate diet.

Dietary Mg2+ Intake and the Na+/Mg2+ Exchanger SLC41A1 Influence Components of Mitochondrial Energetics in Murine Cardiomyocytes.

Cardiomyocytes are among the most energy-intensive cell types. Interplay between the components of cellular magnesium (Mg) homeostasis and energy metabolism in cardiomyocytes is poorly understood. We have investigated the effects of dietary Mg content and presence/functionality of the Na+/Mg2+ exchanger SLC41A1 on enzymatic functions of selected constituents of the Krebs cycle and complexes of the electron transport chain (ETC). The activities of aconitate hydratase (ACON), isocitrate dehydrogenase (ICDH), α-ketoglutarate dehydrogenase (KGDH), and ETC complexes CI-CV have been determined in vitro in mitochondria isolated from hearts of wild-type (WT) and Slc41a1-/- mice fed a diet with either normal or low Mg content. Our data demonstrate that both, the type of Mg diet and the Slc41a1 genotype largely impact on the activities of enzymes of the Krebs cycle and ETC. Moreover, a compensatory effect of Slc41a1-/- genotype on the effect of low Mg diet on activities of the tested Krebs cycle enzymes has been identified. A machine-learning analysis identified activities of ICDH, CI, CIV, and CV as common predictors of the type of Mg diet and of CII as suitable predictor of Slc41a1 genotype. Thus, our data delineate the effect of dietary Mg content and of SLC41A1 functionality on the energy-production in cardiac mitochondria.

Erratum to "The GadX regulon affects virulence gene expression and adhesion of porcine enteropathogenic Escherichia coli in vitro" [Vet. Anim. Sci. 3C (2017) 10-17].

[This corrects the article DOI: 10.1016/j.vas.2017.04.001.].

Dietary Supplementation of dl-Methionine Potently Induces Sodium-Dependent l-Methionine Absorption in Porcine Jejunum Ex Vivo.

BACKGROUND: Methionine is an essential amino acid (AA) with many fundamental roles. Humans often supplement l-Met, whereas dl-Met and dl-2-hydroxy-4-(methylthio)butanoic acid (dl-HMTBA) are more frequently used to supplement livestock. OBJECTIVES: The study aimed to investigate whether dietary Met source alters the absorptive capacity for Met isomers in the small intestine of piglets. METHODS: A total of 27 male 10-wk-old piglets in 3 feeding groups received a diet supplemented with 0.21% dl-Met, 0.21% l-Met, or 0.31% dl-HMTBA to meet the Met + cystine requirement. After ≥10 d, absorptive fluxes of d-Met or l-Met were measured at a physiological concentration of 50 µM and a high concentration of 5 mM in duodenum, middle jejunum, and ileum ex vivo. Data were compared by 2-factor ANOVA. RESULTS: Across diets, fluxes of both Met isomers at both tested concentrations increased from duodenum to ileum by a factor of ∼2-5.5 (P < 0.05). Pigs supplemented with dl-Met had greater (P < 0.085) absorptive fluxes at 50 µM l-Met (0.50, 2.07, and 3.86 nmol · cm-2 · h-1) and d-Met (0.62, 1.41, and 1.19 nmol · cm-2 · h-1) than did pigs supplemented with dl-HMTBA (l-Met: 0.28, 0.76, and 1.08 nmol · cm-2 · h-1; d-Met: 0.34, 0.58, and 0.64 nmol · cm-2 · h-1) in duodenum, jejunum, and ileum, respectively. Only in jejunum of dl-Met-fed pigs, fluxes at 50 µM l-Met were reduced by the omission of luminal Na+ (from 3.27 to 0.86 nmol · cm-2 · h-1; P < 0.05) and by a cocktail of 22 luminal AAs (to 1.05 nmol · cm-2 · h-1; P < 0.05). CONCLUSIONS: Dietary supplementation of dl-Met increases the efficiency of l-Met and d-Met absorption at physiologically relevant luminal Met concentrations along the small intestine of pigs, including a very prominent induction of an Na+-dependent transport system with preference for l-Met in the mid-jejunum. Dietary supplementation with dl-Met could be a promising tool to improve the absorption of Met and other AAs.